Note: Descriptions are shown in the official language in which they were submitted.
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1
EXPRESSION CASSETTE COMPRISING AN ARABIDOPSIS THALIANA
TRIOSE PHOSPHATE TRANSLOCATOR PROMOTER
The invention relates to expression cassettes and vectors which
contain constitutive promoters of plants and to the use of said
expression cassettes or vectors for transgenic expression of
nucleic acid sequences, preferably selection markers, in
organisms, preferably in plants. The invention further relates to
transgenia plants which have been transformed with said
expression cassettes or vectors, to cultures, parts or
propagation material derived therefrom and also to the use of
same for the production of food- and feedstuffs, seed,
pharmaceuticals or fine chemicals.
The aim of biotechnological studies on plants is the preparation
of plants having improved properties, for example to increase
agricultural productivity. The preparation of transgenic plants
is a fundamental technique in plant biotechnology and thus an
indispensible prerequisite for basic research on plants in order
for the preparation of plants having improved novel properties
for agriculture, for improving the quality of_foodstuffs or for
the production of particular chemicals or pharmaceuticals
(Dunwell JM, J Exp Bot. 2000;51 Spec No:487-96). The natural
defence mechanisms of the plant, for example against pathogens,
are often inadequate. The introduction of foreign genes from
plants, animals or microbial sources can enhance the defence.
Examples are the protection against insects feeding on tobacco by
expression of the Bacillus thuringiensis endotoxin under the
control of the 35 S CaMV promoter (Vaeck et al. (1987) Nature
328:33-37) and the protection of tobacco against fungal infection
by expression of a chitinase from beans under the control of the
CaNV promoter (Broglie et al. (1991) Science 254:1194-1197). It
is furthermore possible to achieve resistance to herbicides by
introducing foreign genes, thereby optimizing the cultivation
conditions and reducing crop losses (Ott KB et al. (1996) J Mol
Biol 263(2):359-368). The quality of the products may also be
improved. Thus it is possible, for example, to increase the shelf
CA 02454127 2010-07-29
la
life and storability of crop products by inactivating particular
maturation genes. This was demonstrated, for example, by
inactivating polygalacturonase in tomatoes (Hamilton AJ et
al.(1995) Curr Top Microbiol Immunol 197:77-89).
A basic prerequisite for transgenic expression of particular
genes in plants is the provision of plant-specific promoters.
Various plant promoters are known. It is possible to distinguish
between constitutive promoters which enable expression in various
parts of a plant, which is only slightly restricted in terms of
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2
location and time, and specific promoters which allow expression
only in particular parts or cells of a plant (e.g. root, seeds,
pollen, leaves, etc.) or only at particular times during
development. Constitutive promoters are used, for example, for
expressing "selection markers". Selection markers (e.g.
antibiotic or herbicidal resistance genes) permit filtering the
transformation event out of the multiplicity of untransformed but
otherwise identical individual plants.
Constitutive promoters active in plants have beenm written [sic]
relatively rarely up to now. Promoters to be mentioned are the
=
Agrobacterium tumefaciens, TR double promoter, the promoters of
the vacuolar ATPase subunits or the promoter of a proline-rich
wheat protein (WO 91/13991) and also the Ppcl promoter
Mesembryanthemum cryctallinum (Cushman et al. (1993) Plant Mol
Biol 21:561-566).
The constitutive promoters which are currently the predominantly
used promoters in plants are almost exclusively viral promoters
or promoters isolated from Agrobacterium. In detail, these are
the nopaline synthase (nos) promoter (Shaw et al. (1984) Nucleic
Acids Res. 12(20):7831-7846), the mannopine synthase (mas)
promoter (Comai et al. (1990) Plant Mol Biol 15 (3):373-381) and
the octopine synthase (ocs) promoter (Leisner and Gelvin (1988)
Proc Natl Acad Sci USA 85(5):2553-2557) from Agrobacterium
tumefaciens and the CaMV35S promoter from cauliflower mosaic
virus. The latter is the most frequently used promoter in
expression systems with ubiquitous and continuous expression
(Odell et al.(1985) Nature 313:810-812; Battraw and Hall (1990)
Plant Mol Biol 15:527-538; Benfey et al. (1990) EMBO J
9(69):1677-1684; US 5,612,472). However, the CaMV 35S promoter
which is frequently applied as constitutive promoter exhibits
variations in its activity in different plants and in different
tissues of the same plant (Atanassova et al. (1998) Plant Mol
Biol 37:275-85; Battraw and Hall (1990) Plant Mol Biol
15:527-538; Holtorf et al. (1995) Plant Mol Biol 29:637-646;
Jefferson et al. (1987) EMBO J 6:3901-3907). A further
disadvantage of the 35S promoter is a change in transgene
expression in the case of an infection with cauliflower mosaic
virus and its typical pathogenic variants. Thus, plants
expressing the BAR gene under the control of the 35S promoter are
no longer resistant after infection with the virus which
typically occurs in nature (Al-Kaff et al. (2000) Natur
Biotechnology 18:995-99).
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From the range of viral promoters, the sugarcane bacilliform
badnavirus (ScBV) which imparts an expression pattern similar to
that Of CamV has been described as an alternative to the CaMV 35S
promoter (Schenk et al. (1999) Plant Mol Biol 39(6):1221-1230).
The activity of the ScBV promoter was analyzed in transient
expression analyses using various dicotyledonous plants,
including Nicotiana tabacum and N. benthamiana, sunflower and
oilseed rape, and monocotyledonous plants, here in the form of
banana, corn and millet. In the transient analyses in corn, the
ScBV promoter-mediated expression level was comparable to that of
the ubiquitin promoter from corn (see below). Furthermore, the
ScBV promoter-mediated rate of expression was assayed in
transgenic banana and tobacco plants and displayed in both plant
species essentially constitutive expression.
=
Common promoters for expressing selection markers in plants are
especially the nos promoter, or else the mas promoter and ocs
promoter, all of which have been isolated from Agrobacterium
strains.
The use of viral sequences is often met with great reservations
on the part of the consumer. These doubts are fed not least by
studies which question the safety of the 35S CaMV promoter, owing
to a possible horizontal gene transfer due to a recombination hot
spot (Ho MW et al. (1999) Microbial Ecology in Health and Disease
11:194-197; Cummins J et al. (2000) Nature Biotechnology 18:363).
It is therefore an aim of future biotechnological studies on
plants to replace viral genetic elements by plant regulatory
elements in order to keep as closely as possible to the plant
system.
Owing to the prevailing doubts with regard to viral promoters,
there are extensive efforts to replace said promoters by plant
promoters. However, a promoter of plant origin, which is
comparable to the viral elements, has not been described as yet.
What has been described, is a plant ubiquitin promoter from
Arabidopsis thaliana (Callis et al.(1990) J Biol Chem 265:12486-
12493; Holtorf S et al. (1995) Plant Mol Biol 29:637-747).
Contrary to the findings in the articles mentioned, some studies
revealed that the Arabidopsis ubiquitin promoter is unsuitable
for expressing selection marker genes and that, for this reason,
its general applicability must be called into question (see
comparative examples 1 and 3).
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The expression pattern mediated by the corn ubiquitin promoter
has been described for the Ubi-1 and Ubi-2 promoters from corn
(Christensen et al. (1992) Plant Mol Biol 18(4):675-689). While
the Ubi-1 promoter has good expression activity in corn and other
monocotyledonous plants, it exhibits in dicotyledonous tobacco
plants only 10% of the activity which had been achieved in
comparable experiments using the viral 35S promoter. It was
furthermore shown that the corn Ubi-1 promoter is suitable for
overexpression of genes in monocotyledonous plant systems and, in
addition, is sufficiently strong in order to mediate a herbicidal
resistance via the expression of selection markers (Christensen
and Quail (1996) Transgenic Res 5(3):213-218). The Ubi-1 promoter
proved unsuitable for dicotyledonous expression systems.
A comparison of the organ specificity and strength of various
constitutive promoters was carried out by Holtorf (Holtorf et al.
(1995) Plant Mol Biol 29(4):637-646) on the basis of stably
transformed Arabidopsis plants. The study comprised, interalia,
the CaMV35S promoter, the leaf-specific thionine promoter from
barley and the Arabidopsis ubiquitin promoter (UBQ1). The CaMV35S
promoter exhibited the highest rate of expression. On the basis
of using an additional translational enhancer (TMV omega
element), it was possible to increase the rate of expression of
the promoter by a factor of two to three with unchanged organ
specificity. The leaf-specific thionine promoter from barley was
inactive in the majority of transformed lines, while the UBQ1
promoter from Arabidopsis resulted in medium rates of expression.
McElroy and colleagues reported a construct for transforming
monocotyledonous plants, which is based on the rice actin 1
(Actl) promoter (McElroy et al. (1991) Mol Gen Genet
231:150-160). Overall, it was concluded from the afore-described
studies that the Actl promoter-based expression vectors are
suitable for controlling a sufficiently strong and constitutive
expression of foreign DNA in transformed cells of
monocotyledonous plants.
Another constitutive promoter which has been described is the
promoter of an S-adenosyl-L-methionine synthetase (WO 00/37662).
A disadvantage here is especially a dependence of the strength of
expression on the methionine concentration (see WO 00/37662; Fig.
7).
WO 99/31258 describes chimeric constitutive plant promoters which
are composed of various elements of various promoters with
complementary expression patterns so that combination of
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individual tissue specificities additively results in a
constitutive expression pattern.
Ferredoxin NADPH oxidoreductase (FNR) is a protein of the
5 electron transport chain and reduces NADP+ to NADPH. Experiments
in spinach using the spinach FNR promoter fused to the GUS gene
hint at a light-inducible element in the FNR promoter (Oelmuller
et al. (1993) Mol. Gen. Genet. 237:261-72). Owing to its
function, a strictly leaf-specific expression pattern would have
been expected for the promoter. Owing to the tissue-dependent
expression pattern, the promoter would be poorly suited to
expressing selection markers. Here, a selection in all tissue
parts, if possible, is required in order to ensure efficient
selection.
Owing to its function during photosynthesis, the promoter of the
triose phosphate translocator (TPT) should be mainly
leaf-specific. The cDNAs from potato (Schulz et al. (1993) Mol
Gen Genet 238:357-61), cauliflower (Fischer et al. (1997) Plant
Cell 9:453-62), oilseed rape (WO 97/25346) and corn Kammerer B
(1998) The Plant Cell 10:105-117) have been described. Kammerer
et al. demonstrate that TPT mRNA expression in corn is strong in
the leaves and the stamen. In contrast, no expression was
observed in the stem or in the roots. Owing to the
tissue-dependent expression pattern, the promoter would be poorly
suited to expressing selection markers. Here, a selection in all
tissue parts, if possible, is required in order to ensure
efficient selection.
The "constitutive" promoters described in the prior art have one
or more of the following disadvantages:
1. Inadequate homogeneity of expression:
The known "constitutive" promoters frequently display a
different level of expression, depending on the type of
tissue or cell. Moreover, the expression property is often
highly dependent on the site of insertion into the host
genome. As a consequence of this, the effects to be obtained
by heterologous expression cannot be achieved to the same
extent homogeneously in the plant. Under or over dosages may
occur. This may have an adverse effect on plant growth or
plant value.
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2. Inadequate time profile:
The "constitutive" promoters known in the prior art often
exhibit a nonconsistent activity during the development of a
tissue. As a result, it is not possible, for example, to
achieve desirable effects (such as selection) =in the early
phase of somatic embryogenesis which would be advantageous,
especially here, due to the sensitivity of the embryo to in
vitro conditions and stress factors.
3. Inadequate applicability to many plant species:
The "constitutive" promoters described in the prior art are
often not active in the same way in all species.
4. If a plurality of expression cassettes with in each case the
same "constitutive" promoter are present in an organism,
interactions between said expression cassettes and even
switching-off (gene silencing) of individual expression
cassettes may occur (Mette et al. (1999) EMBO J. 18:241-248).
5. Promoters of viral origin may be influenced by virus
infections of the transgenic plant and may then no longer
express the desired property (Al-Kaff et al. (2000) Natur
_ Biotechnology 18:995-99).
6. The public acceptance toward the use of promoters and
elements from plant systems is higher than for viral systems.
7. The number of promoters suitable for expressing selection
markers in plants is low and said promoters are usually of
viral or bacterial origin.
8. Pollen/anther expression: The promoters mentioned (such as,
for example, 35S CaMV) exhibit strong activity in the pollen
or in the anthers. This may have disadvantageous effects on
the environment. Thus, unspecific expression of Bacillus
thuringiensis endotoxins resulted not only in the desired
effect on feeding insects due to expression in the root but
also, due to expression in the pollen, in considerable damage
in the population of the monarch butterfly which feeds
predominantly on the pollen (Losey JE et al. (1999) Nature
399, 214).
An ideal constitutive promoter should have as many of the
following properties as possible:
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a) a gene expression which is as homogeneous as possible with
regard to location and time, i.e. an expression in as many
cell types or tissues of an organism as possible during the
various phases of the developmental cycle. Furthermore, an
5 efficient selection in differentiated cells (various callus
phases) from a tissue culture and other developmental stages
suitable for tissue culture is desired.
b) An applicability to various plant species, which is as broad
10 as possible, and applicability to species in which it is not
possible to achieve any expression using the "constitutive"
promoters known to date.
C) In order to combine a plurality of transgenes in one plant,
15 it is desirable to carry out a plurality of transformations
in succession or to use constructs with a plurality of
promoter cassettes, but without generating silencing effects
due to the multiple use of identical regulatory sequences.
20 d) A plant origin in order to avoid problems of acceptance by
the consumer and possible problems of future approval.
e)_ Secondary activities of a promoter in the anthers/pollen are
undesirable, for example in order to avoid environmental
25 damage (see above).
It is an object of the present invention to provide regulatory
sequences of plants, which fulfill as many of the abovementioned
properties as possible and which mediate especially a ubiquitous
30 and development-independent (constitutive) expression of a
nucleic acid sequence to be expressed which preferably codes for
a selection marker. Despite various plant promoters for which a
constitutive expression at least in individual species is
claimed, no promoter having the desired properties listed above
= 35 has been described up to now. It was therefore the object to
identify appropriate promoters.
We have found that this object is achieved by providing
expression cassettes based on the promoters of a putative
40 ferredoxin gene (pFD "putative ferredoxin" hereinbelow) from
Arabidopsis thaliana, of the ferredoxin NADI'S oxidoreductase (FNR
hereinbelow) gene from Arabidopsis thaliana and of the triose
phosphate translocator (TPT) gene from Arabidopsis thaliana:
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1.) Promotor of a putative ferredoxin (pFD) from Arabidopsis
thaliana
During analysis of the Arabidopsis genome, the ORF of a
putative ferredoxin gene was identified. The isolated 836 bp
5'- flanking sequence fused to the Glucuronidase gene
surprisingly exhibited a constitutive expression pattern in
transgenic tobacco. The sequence corresponds to a sequence
section on Arabidopsis thaliana chromosome 4, as it has been
deposited at GenBank under Acc. No. Z97337 (Version Z97337.2;
base pair 85117 to 85952; the gene starting from bp 85953 is
annotated "strong similarity to ferredoxin [2Fe-2S] I, Nostoc
=
muscorum"). (The gene is not to be confused with the A.
thaliana gene for preferredoxin annotated under GenBank
Acc-NbAcc. No: X51370; Vorst 0 et al. (1990) Plant Mol Biol
14(4):491-499).
Only a weak activity was detected in the anthers/pollen of
the closed flower buds and no activity whatsoever was
detected in mature flowers. Contrary to the reservations,
derived from the findings in the literature, toward a
suitability of the promoter for effective expression of
selection markers (for example, owing to the suspected leaf
_
specificity or function in the photosynthetic electron
transport), it was possible to demonstrate a highly efficient
selection by combination with, for example, the homolog
resistance gene (nptII).
2.) Ferredoxin NADPH oxidoreductase (FNR) promoter from
Arabidopsis thaliana
Starting from the information on FNR-encoding cDNA from N.
tabacum (GenBank Acc. No.: Y14032) the Arabidopis data base
was screened for a homologous gene. Primers were synthesized
according to said sequence information. The promoter
amplified via PCR from Arabidopsis thaliana genomic DNA
(635 bp), of which a leaf-specific expression was expected,
exhibited in transgenic tobacco plants a surprisingly
ubiquitous and insertion site-independent expression.
The promoter sequence partly corresponds to a sequence
section on Arabidopsis thaliana chromosome 5, as it is
deposited at GenBank under Acc. No. AB011474 (Version
AB011474.1 from 12.27.2000; base pair 70127 to 69493; the
gene starting at bp 69492 is annotated with "ferredoxin-NADP+
reductase").
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No activity was detected in the pollen. Contrary to the
reservations, derived from the findings in the literature,
toward a suitability of the promoter for effective expression
of selection markers (for example, owing to the suspected
leaf specificity or function in the photosynthetic electron
transport), it was possible to demonstrate a highly efficient
selection by combination with, for example,
the phosphinothricin resistance gene (bar/pat).
The nondetectable activity of the FNR promoter in seeds
=
allows a use for the expression of genes whose gene products
are desired in other parts of the plant and are unwanted in .
the seeds. For example, pests can be repelled by expressing
appropriate toxins such as, for example, Bacillus
thuringiensis crystal proteins. Thus it is possible to
achieve in potatoes expression in the plant organs above the
ground (and thus, for example, a repulsion of pests such as
the potato beetle) without simultaneous expression in the
tuber which is used as food or animal feed, and this could
increase the suitability and acceptance.
3.) Triose phosphate translocator (TPT) promoter from Arabidopsis
thaliana
A 2038bp PCR fragment was amplified, starting from
Arabidopsis GenBank data of chromosome V, clone MCL19. The
promoter sequence partly corresponds to a sequence section on
Arabidopsis thaliana chromosome 5, as it is deposited with
GenBank under Acc. No. AB006698 (Version AB006698.1 from
12.27.2000; base pair 53242 to 55281; the gene starting at bp
55282 is annotated with "phosphate/triose-phosphate
translocator").
Surprisingly, transgenic tobacco plants exhibited not only a
high activity in numerous parts of the plant. No activity was
detected in the pollen. Contrary to the reservations, derived
from the findings in the literature, toward a suitability of
the promoter for effective expression of selection markers
(for example, owing to the suspected leaf specificity), it
was possible to demonstrate a highly efficient selection by
combination with, for example, the phosphinothricin
resistance gene (bar/pat).
The ubiquitous expression pattern, but especially also the
ability of the TPT promoter regarding the expression of
selection markers, comes as a great surprise for the skilled
worker, since the triosephosphate translocator is responsible
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for the exchange of C3 sugar phosphates between the cytosol
and the plastids in photosynthetic leaves. The TPT is located
in the inner chloroplast membrane. Colorless plastids
typically contain a hexose transporter via which C6-sugar
phosphates are exchanged. It is not to be expected that such
genes are active in the early callus and embryogenesis stages
(Stitt (1997) Plant Metabolism, 2nd ed., Dennis eds. Longman
Press, Harlow, UK, 382-400).
The pFD, FNR and TPT promoters proved to be sufficiently strong
in order to express nucleic acid sequences, in particular
selection marker genes, successfully. Furthermore, various
10 deletion variants of the abovementioned promoters, in particular
a truncated variant of the pFD promoter (699 bp) and of the TPT
promoter (1318 bp), proved suitable for ensuring the expression
of, for example, selection markers such as the homolog resistance
(nptII).
Furthermore, the Arabidopsis thaliana ubiquitin promoter (Holtorf
et al. (1995) Plant Mol Biol 29:637-646) and the squalene
synthase promoter (Kribii et al. (1997) Eur J Biochem 249:61-69)
were studied within the framework of the studies mentioned, both
of which, however, were surprisingly unsuitable for mediating
selection marker gene expression although the literature data of
the ubiquitin promoters from monocotyledons (see above) had led
to the assumption that in particular the ubiquitin promoter of a
dicotyledonous plant should have worked as a promoter of a
selection marker system (see comparative examples 1 and 3). A
similar statement applies to the squalene synthase promoter whose
characterization had led to the expectation that it would be
possible to achieve sufficiently high rates of expression for the
successful control of a selection marker gene (Del Arco and
Boronat (1999) 4th European Symposium on Plant Isoprenoids,
21.-4.23.1999, Barcelona, Spain) (see comparative examples 2 and
3).
The present invention relates to an expression cassette for transgenic
expression of
nucleic acids, comprising:
CA 02454127 2013-11-27
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10a
a) a promoter according to SEQ ID NO: 3 or
b) functional equivalents selected from the group consisting of:
i) Arabidopsis thaliana triose phosphate translocator promoter,
amplifiable with primers of SEQ ID NO: 17 and 18 or SEQ ID NO: 18
and 26;
ii) sequences hybridizing to SEQ ID NO: 3 or the nucleic acid sequence
complementary thereto under conditions consisting of 0.2 x SSC at
50 C, and
iii) the equivalent fragment of a), specified by SEQ ID NO: 27 and
iv) sequences which are at least 80% homologous over a length of at least
500 basepairs,
which possess the same promoter activities as a),
a) or b) being functionally linked to a nucleic acid sequence to be expressed
transgenically and mediating ubiquitous and development-independent
expression thereof.
The present invention relates to an expression cassette for transgenic
expression of
a nucleic acid, comprising:
a) a promoter specified by SEQ ID NO: 3 or
b) a promoter having a sequence which is:
i) specified by SEQ ID NO: 27; or
ii) a sequence which is at least 95% identical with the promoter of
a), or bi),
wherein any one of a) or b) promoter is functionally linked to a nucleic acid
sequence
to be expressed transgenically which is operably linked to and heterologous in
relation to said promoter and wherein said promoter has constitutive promoter
activity.
The present invention relates to a vector comprising the expression cassette
as
defined herein.
CA 02454127 2013-11-27
10b
As well the object of the present invention is a use for transgenic expression
of
nucleic acids, wherein a nucleic acid sequence which is functionally linked
to:
a) a promoter according to SEQ ID NO: 3 or
b) functional equivalents selected from the group consisting of:
i) Arabidopsis thaliana triose phosphate translocator promoter,
amplifiable with primers of SEQ ID NO: 17 and 18 or SEQ ID NO: 18
and 26;
ii) sequences hybridizing to SEQ ID NO: 3 or the nucleic acid sequence
complementary thereto under conditions consisting of 0.2 x SSC at
50 C, and
iii) the equivalent fragment of a), specified by SEQ ID NO: 27 and
iv) sequences which are at least 80% homologous over a length of at least
500 basepairs,
which possess the same promoter activities as a), is expressed transgenically.
As well the object of the present invention is a use for transgenic expression
of a
nucleic acid for preparing transformed cells, wherein the expression cassette
defined
herein is expressed transgenically.
As well the object of the present invention is a use of selected transformed
cells,
wherein a nucleic acid sequence coding for a selection marker, which is
functionally
and transgenically linked to
a) a promoter according to SEQ ID NO: 3, or
b) functional equivalents selected from the group consisting of:
i) Arabidopsis thatiana triose phosphate translocator promoter,
amplifiable with primers of SEQ ID NO: 17 and 18 or SEQ ID NO: 18
and 26;
ii) sequences hybridizing to SEQ ID NO: 3 or the nucleic acid sequence
complementary thereto under conditions consisting of 0.2 x SSC at
50 C, and
CA 02454127 2013-11-27
10c
iii) the equivalent fragment of a), specified by SEQ ID NO: 27 and
iv) sequences which are at least 80% homologous over a length of at least
500 basepairs,
which possess the same promoter activities as a), is introduced into an
organism, the
selection marker is expressed and a selection is carried out.
As well the object of the present invention is a use of selected transformed
cells, for
the production of foodstuff, feedstuff, a seed, a pharmaceutical, or a fine
chemical,
wherein the pharmaceutical is an antibody, an enzyme or a pharmaceutically
active
protein, wherein a nucleic acid sequence codes for a selection marker, which
is
functionally and transgenically linked to:
a) a promoter according to SEQ ID NO: 3 or
b) a promoter having a sequence which is:
i) an Arabidopsis thaliana triose phosphate translocator promoter,
amplifiable with primers of SEQ ID NO: 17 and 18 or SEQ ID NO: 18
and 26;
ii) a sequence hybridizing to SEQ ID NO: 3 or the nucleic acid sequence
complementary thereto under conditions consisting of 0.2 x SSC at
50 C,; and
iii) a promoter, specified by SEQ ID NO: 27; or
iv) a sequence which is at least 90% identical to any one of a), bi), bii)
or
biii) over a length of at least 500 basepairs,
wherein any one of a) or b) promoter is functionally linked to a nucleic acid
sequence
to be expressed transgenically which is operably linked to and heterologous in
relation to said promoter and wherein said promoter has constitutive promoter
activity.
As well the object of the present invention is a transgenic cell transformed
with the
expression cassette as defined herein or with the vector as defined herein.
CA 02454127 2012-12-21
1 Od
As well the object of the present invention is the use of a transgenic cell as
defined
herein or of cell cultures, for the production of food- and feedstuffs, seed,
pharmaceutical, or a fine chemical, wherein the pharmaceutical is an antibody,
enzyme or pharmaceutically active protein.
As well the object of the present invention is a method for preparing a
pharmaceutical, or a fine chemical, in transgenic cells as defined herein or
in cell
cultures, which comprises growing the transgenic organism and isolating the
desired
pharmaceutical or the desired fine chemical, wherein the pharmaceutical is an
antibody, enzyme or pharmaceutically active protein.
The present invention therefore relates firstly to expression
cassettes for transgenic expression of nucleic acids, comprising
a) a promoter according to SEQ ID No: 1, 2 or 3,
b) a functional equivalent or equivalent fragment of a), which
essentially possesses the same promoter activity as a),
a) or b) being functionally linked to a nucleic acid sequence to
be expressed transgenically.
0817/00024 CA 02454127 2004-01-09
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11
The invention further relates to methods for transgenic
expression of nucleic acids, wherein a nucleic acid sequence
which is functionally linked to
a) a promoter according to SEQ ID NO: 1, 2 or 3 or
b) a functional equivalent or equivalent fragment of a) which
essentially possesses the same promoter activities as a),
is expressed transgenically.
Expression comprises transcription of the nucleic acid sequence
=
to be expressed transgenically but may also include, in the case
of an open reading frame in sense orientation, translation of the
transcribed RNA of the nucleic acid sequence to be expressed
transgenically into a corresponding polypeptide.
An expression cassette for transgenic expression of nucleic acids
or a method for transgenic expression of nucleic acids comprises
all those constructions produced by genetic methods or methods in
which either
a)_
a. promoter according to SEQ ID No: 1, 2 or 3 or a functional
equivalent or equivalent fragment thereof, or
b) the nucleic acid sequence to be expressed, or
C) (a) and (b)
are not present in their natural genetic environment (i.e. at
their natural chromosomal locus) or have been modified by genetic
methods, and said modification may be, by way of example, a
substitution, addition, deletion, inversion or insertion of one
or more nucleotide residues.
The expression cassettes of the invention, vectors derived from
them or the methods of the invention may comprise functional
equivalents of the promoter sequences described under SEQ ID No:
1, 2 or 3. Functionally equivalent sequences also comprise all
those sequences which are derived from the complementary counter
[sic] strand of the sequences defined by SEQ ID NO: 1, 2 or 3 and
which have essentially the same promoter activity.
Functional equivalents with respect to the promoters of the
invention means in particular natural or artificial mutations of
the promoter sequences described under SEQ ID No: 1, 2 or 3 and
0817/00024 CA 02454127 2004-01-09
12
of the homologs thereof from other plant genera and species,
which furthermore have essentially the same promoter activity.
A promoter activity is essentially referred to as identical, if
the transcription of a particular gene to be expressed under the
control of a particular promoter derived from SEQ ID NO: 1, 2 or
3 under otherwise unchanged conditions has a location within the
plant, which is at least 50%, preferably at least 70%,
particularly preferably at least 90%, very particularly
preferably at least 95%, congruent with that of a comparative
expression obtained using a promoter described by SEQ ID NO: 1, 2
or 3. In this connection, the expression level may deviate both
downward and upward in comparison to a comparative value. In this
connection, preference is given to those sequences whose
expression level, measured on the basis of the transcribed mRNA
or the subsequently translated protein, differs quantitatively by
not more than 50%, preferably 25%, particularly preferably 10%,
from a comparative value obtained using a promoter described by
SEQ ID NO: 1, 2 or 3, under otherwise unchanged conditions.
Particular preference is given to those sequences whose
expression level, measured on the basis of the transcribed mRNA
or of the subsequently translated protein, is quantitatively more
than 30%, preferably 100%, particularly preferably 500%, very
particularly preferably 1000%, higher than a comparative value
obtained with the promoter described by SEQ ID NO: 1, 2 or 3,
under otherwise unchanged conditions. The comparative value is
preferably the expression level of the natural mRNA of the
particular gene or of the natural gene product. A further
preferred comparative value is the expression level obtained
using a random but particular nucleic acid sequence, preferably
those nucleic acid sequences which code for readily quantifiable
proteins. In this connection, very particular preference is given
to reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol.
1999; 13(1):29-44) such as the green fluorescence protein (GFP)
(Chui WL et al., Curr Biol 1996, 6:325-330; Leffel SM et al.,
Biotechniques. 23(5):912-8, 1997), chloramphenicol transferase, a
luciferase (Millar et al., Plant Mol Biol Rep 1992 10:324-414) or
P-galactosidase, and very particular preference is given to
p-glucuronidase (Jefferson et al. (1987) EMBO J. 6:3901-3907).
Otherwise unchanged conditions means the expression initiated by
one of the expression cassettes to be compared is not modified by
a combination with additional genetic control sequences, for
example enhancer sequences. Unchanged conditions further means
that all basic conditions such as, for example, plant species,
developmental stage of the plants, growing conditions, assay
0817/00024 CA 02454127 2004-01-09
13
conditions (such as buffer, temperature, substrates, etc.) are
kept identical between the expressions to be compared.
Mutations comprise substitutions, additions, deletions,
inversions or insertions of one or more nucleotide residues.
Thus, for example, the present invention also includes those
nucleotide sequences which are obtained by modification of a
promoter of SEQ ID NO: 1, 2 or 3. The aim of such a modification
may be the further narrowing down of the sequence comprised
therein or else, for example, the introduction of further
restriction enzyme cleavage sites, the removal of excess DNA or
the addition of further sequences, for example of further =
regulatory sequences.
Where insertions, deletions or substitutions such as, for
example, transitions and transversions are suitable, techniques
known per se, such as in vitro mutagenesis, "primer repair",
restriction or ligation, may be used. Manipulations such as, for
example, restriction, chewing-back or filling-in of protruding
ends to give blunt ends can provide complementary fragment ends
for ligation. Similar results can be obtained using the
polymerase chain reaction (PCR) using specific oligonucleotide
primers.
Homology between two nucleic acids means the identity of the
nucleic acid sequence over the in each case entire length of the
sequence, which is calculated by comparison with the aid of the
GAP program algorithm (Wisconsin Package Version 10.0, University
of Wisconsin, Genetics Computer Group (GCG), Madison, USA), with
the parameters set as follows:
Gap Weight: 12 Length Weight: 4
Average Match: 2.912 Average Mismatch:-2.003
By way of example, a sequence which is at least 50% homologous at
the nucleic acid level with the sequence SEQ ID NO: 1, 2 or 3
means a sequence which is at least 50% homologous when compared
to the sequence SEQ ID NO. 1, 2 or 3 according to the above
program algorithm using the above set of parameters.
Functional homologs to the abovementioned promoters for use in
the expression cassettes of the invention preferably include
those sequences which , are at least 50%, preferably 70%,
preferentially at least 80%, particularly preferably at least
90%, very particularly preferably at least 95%, most preferably
99%, homologous over a length of at least 100 base pairs,
0817/00024 CA 02454127 2004-01-09
14
preferably at least 200 base pairs, particularly preferably at
least 300 base pairs, very particularly preferably at least 400
base pairs and most preferably of at least 500 base pairs.
Further examples of the promoter sequences employed in the
expression cassettes or vectors of the invention can readily be
found, for example, in various organisms whose genomic sequence
is known, such as, for example, Arabidopsis thaliana, Brassica
napus, Nicotiana tabacum, Solanum tuberosum, Helianthium anuus,
Linum sativum by comparing homologies in databases.
Functional equivalents further means DNA sequences which
hybridize under standard conditions with the nucleic acid
sequence coding for a promoter according to SEQ ID NO:1, 2 or 3
or with the nucleic acid sequences complementary to it and which
have essentially the same properties. Standard hybridization
conditions has a broad meaning and means both stringent and less
stringent hybridization conditions. Such hybridization conditions
are described, inter alia, in Sambrook J, Fritsch EF, Maniatis T
et al., in Molecular Cloning - A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57) or in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(19_894, 6.3.1-6.3.6.
The conditions during the washing step may be selected by way of
example from the range of conditions limited by those of low
stringency (with approximately 2X SSC at 50 C) and those with high
stringency (with approximately 0.2X SSC at 50 C, preferably at
65 C) (20X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). In
addition, the temperature may be raised during the washing step
from low stringency conditions at room temperature, approximately
22 C, to higher stringency conditions at approximately 65 C. Both
parameters, salt concentration and temperature, may be varied
simultaneously, and it is also possible to keep one of the two
parameters constant and to vary only the other one. Denaturing
agents such as, for example, formamide or SDS may also be
employed during hybridization. In the presence of 50% formamide,
hybridization is preferably carried out at 42 C. Some exemplary
conditions for hybridization and washing are listed below:
(1) Hybridization conditions with, for example,
a) 4X SSC at 65 C, or
b) 6X SSC, 0.5% SOS, 1011q/m1 denatured, fragmented salmon
sperm DNA at 65 C, or
0817 /00024 CA 02454127 2004-01-09
C) 4X SSC, 50% formamide, at 42 C, or
d) 6X SSC, 0.5% SDS, 10g/ml denatured, fragmented salmon
sperm-DNA, 50% formamide at 42 C, or
5
e) 2X or 4X SSC at 50 C (low stringency condition), or
f) 2X or 4X SSC, 30 to 40% formamide at 42 C (low stringency
condition).
g) 6x SSC at 45 M@C, or,
h) 50% formamide, 4xSSC at 42 C, or
i) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1%
Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate
buffer pH 6.5, 75mM NaC1, 75 mM sodium citrate at 42 C,
or
j) 0.05 M sodium phosphate buffer pH 7.0, 2 mM EDTA, 1% BSA
and 7% SDS.
(2) Washing steps with, for example,
a) 0.1X SSC at 65 C, or
b) 0.1X SSC, 0.5% SDS at 68 C, or
C) 0.1X SSC, 0.5% SDS, 50% formamide at 42 C, or
d) 0.2X SSC, 0.1% SDS at 42 C, or
e) 2X SSC at 65 C (low stringency condition), or
f) 40 mM sodium phosphate buffer pH 7.0, 1% SDS, 2 mM EDTA.
Methods for preparing functional equivalents of the invention
preferably comprise introducing mutations into a promoter of SEQ
ID NO: 1, 2 or 3. A mutagenesis may be random and the mutagenized
sequences are subsequently screened with respect to their
properties according to a trial-by-error [sic] procedure.
Examples of particularly advantageous selection criteria are an
increased resistance to a selection marker and the level of the
resulting expression of the introduced nucleic acid sequence.
0817/00024 CA 02454127 2004-01-09
. .
16
As an alternative, it is possible to delete non-essential
sequences of a promoter of the invention without substantially
impairing said properties. Such deletion variants are
functionally equivalent fragments of the promoters described by
SEQ ID NO: 1, 2 or 3. Examples of such deletion mutants or
functionally equivalent fragments, which may be mentioned, are
the truncated pFD promoter sequence (pFDs) according to SEQ ID
NO: 4 and the truncated TPT promoter sequence according to SEQ ID
NO: 27 which, as functionally equivalent parts of their
respective source promoters, are expressly included.
The narrowing-down of the promoter sequence to particular
essential regulatory regions may also be carried out with the aid
of search routines for searching for promoter elements.
Particular promoter elements are often present in increased
numbers in the regions relevant for promoter activity. Said
analysis may be carried out, for example, by computer programs
such as the program PLACE ("Plant Cis-acting Regulatory DNA
Elements") (K. Higo et al., (1999) Nucleic Acids Research 27:1,
297-300) or the BIOBASE data bank "Transfac" (Biologische
Datenbanken GmbH, Brunswick)
Methods for mutagenizing nucleic acid sequences are known to the
skilled worker and include, by way of example, the use of
oligonucleotides having one or more mutations in comparison with
the region to be mutated (for example, within the framework of a
site-specific mutagenesis). Typically, primers with from
approximately 15 to approximately 75 nucleotides or more are
employed, preferably from approx. 10 to approx. 25 or more
nucleotide residues being located on both sites of the sequence
to be modified. Details and the procedure of said mutagenesis
methods are familiar to the skilled worker (Kunkel et al.,
Methods Enzymol, 154:367-382, 1987; Tomic et al. (1990) Nucl
Acids Res 12:1656; Upender, Raj, Weir (1995) Biotechniques
18(1):29-30; US 4,237,224). A mutagenesis may also be carried out
by treating, for example, vectors comprising one of the nucleic
acid sequences of the invention with mutagenizing agents such as
hydroxylamine.
The nucleic acid sequences which are comprised in the expression
cassettes of the invention and which are to be expressed
transgenically may be functionally linked to further genetic
control sequences, in addition to one of the promoters of the
invention.
0817/00024 CA 02454127 2004-01-09
17
A functional linkage means, for example, the sequential
arrangement of a promoter, of the nucleic acid sequence to be
expressed transgenically and, where appropriate, of further
regulatory elements such as, for example, a terminator in such a
way that each of the regulatory elements can carry out its
function in the transgenic expression of said nucleic acid
sequence, depending on the arrangement of the nucleic acid
sequences with respect to sense or antisense RNA. This does not
absolutely necessitate a direct linkage in the chemical sense.
Genetic control sequences such as, for example, enhancer
sequences may exert their function on the target sequence also
from relatively distant positions or even from other DNA
molecules. Preference is given to arrangements in which the
nucleic acid sequence to be expressed transgenically is
positioned downstream of the sequence functioning as promoter so
that both sequences are covalently linked to one another. The
distance between the promoter sequence and the nucleic acid
sequence to be expressed transgenically is preferably less than
200 base pairs, particularly preferably less than 100 base pairs
and very particularly preferably less than 50 base pairs.
A functional linkage may be prepared by means of common
recombination and cloning techniques, as are described, for
example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and
L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et
al., Current Protocols in Molecular Biology, Greene Publishing
Assoc. and Wiley Interscience (1987). It is also possible to
position further sequences between said two sequences, which
have, for example, the function of a linker with particular
restriction enzyme cleavage sites or of a signal peptide.
Likewise, the insertion of sequences may lead to the expression
of fusion proteins.
The term "genetic control sequences" has a broad meaning and
means all those sequences which influence the generation or
function of the expression cassette of the invention. For
example, genetic control sequences modify transcription and
translation in prokaryotic or eukaryotic organisms. The
expression cassettes of the invention preferably comprise
5'-upstream of the particular nucleic acid sequence to be
expressed transgenically one of the promoters of the invention
and 3'-downstream a terminator sequence as an additional genetic
control sequence and also, where appropriate, further common
0817/00024 CA 02454127 2004-01-09
18
regulatory elements which are in each case functionally linked to
the nucleic acid sequence to be expressed transgenically.
Genetic control sequences also include further promoters,
promoter elements or minimal promoters which may modify the
expression-controlling properties. Thus, for example, genetic
control sequences can effect tissue-specific expression
additionally depending on particular stress factors.
Corresponding elements have been described, for example, for
water stress, abscisic acid (Lam E and Chua NE (1991) J Biol Chem
266(26): 17131-17135) and heat stress (Schoffl F et al., (1989)
Molecular & General Genetics 217(2-3):246-53).
Further promoters which make possible expression in further plant_
tissues or in other organisms such as, for example, in E.coli
bacteria may furthermore be functionally linked to the nucleic
acid sequence to be expressed. Suitable plant promoters are in
principle all of the above-described promoters. It is
conceivable, for example, that a particular nucleic acid sequence
is transcribed as sense RNA via one promoter (for example one of
the promoters of the invention) in one plant tissue and is
translated into the corresponding protein, while the same nucleic
acid sequence is transcribed to antisense RNA via another
promoter having a different specificity in another tissue and the
corresponding protein is down-regulated. This may be carried out
via an expression cassette of the invention by positioning the
one promoter upstream of the nucleic acid sequence to be
expressed transgenically and the other promoter downstream of
said sequence.
Genetic control sequences furthermore also include the
5'-untranslated region, introns or the noncoding 3'-region of
genes, preferably of the pFD, FNR or TPT-genes. It has been
demonstrated that these genes may have a substantial function in
the regulation of gene expression. Thus it was shown that
5r-untranslated sequences can enhance transient expression of
heterologous genes. They may furthermore promote tissue
specificity (Rouster J et al.(1998) Plant J. 15:435-440).
Conversely, the 5'-untranslated region of the opaque-2 gene
suppresses expression. A deletion of the corresponding region
leads to an increase in gene activity (Lohmer S et al. (1993)
Plant Cell 5:65-73). The nucleic acid sequence indicated under
SEQ ID NO:1, 2 or 3 contains the pFD, FNR or TPT-gene section
which represents the promoter and the 5'-untranslated region up
to the ATG start codon of the respective protein.
0817/00024 CA 02454127 2004-01-09
= . 19
McElroy and colleagues (McElroy et al. (1991) Mol Gen Genet
231(1):150-160) reported a construct for transforming
monocotyledonous plants, which is based on the rice actin 1
(Actl) promoter. The use of the Actl intron in combination with
the 35S promoter leads in transgenic rice cells to-a ten times
higher rate of expression compared to the isolated 35S promoter.
Optimization of the sequence surrounding the translation
initiation site of the reporter gene (GUS) resulted in a
four-fold increase of GUS expression in transformed rice cells. A
combination of optimized translation initiation site and Actl
intron resulted in a 40-fold increase in GUS expression via the
CaMV35S promoter in transformed rice cells; similar results were =
achieved on the basis of transformed corn cells. Overall, it was
concluded from the above-described studies that the expression
vectors based on the Actl promoter are suitable for controlling a
sufficiently strong and constitutive expression of foreign DNA in
transformed cells of monocotyledonous plants.
The expression cassette may advantageously contain one or more
"enhancer sequences" which are functionally linked to the
promoter and which enable an increased transgenic expression of
the nucleic acid sequence. It is possible to insert additional
advantageous sequences such as further regulatory elements or
terminators at the 3'-end of the nucleic acid sequences to be
expressed transgenically, too. Any of the expression cassettes of
the invention may contain one or more copies of the nucleic acid
sequences to be expressed transgenically.
Control sequences furthermore means those which enable homologous
recombination or insertion into the genome of a host organism or
which allow the removal from the genome. In homologous
recombination, for example, the natural promoter of a particular
gene may be replaced with one of the promoters of the invention.
Methods such as the cre/lox technology allow tissue-specific,
specifically inducible removal of the expression cassette from
the genome of the host organism (Sauer B. (1998) Methods.
14(4):381-92). In this case, particular flanking sequences are
attached to the target gene (lox sequences), which later enable a
removal by means of the cre recombinase.
The promoter to be introduced may be placed upstream of the
target gene to be expressed transgenically by means of homologous
recombination by linking the promoter to DNA sequences which are,
for example, homologous to endogenous sequences upstream of the
reading frame of the target gene. Such sequences are to be
understood as genetic control sequences. After a cell has been
transformed with the appropriate DNA construct, the two
0817/00024 CA 02454127 2004-01-09
= 20
homologous sequences can interact and thus place the promoter
sequence at the desired position upstream of the target gene so
that said promoter sequence is now functionally linked to said
target gene and forms an expression cassette of the invention.
The selection of the homologous sequences determines the
insertion point of the promoter. In this case, the expression
cassette can be generated by homologous recombination by means of
a simple or a doubly-reciprocal recombination. In the case of the
singly-reciprocal recombination, only a single recombination
sequence is used and the entire introduced DNA is inserted. In
the case of the doubly-reciprocal recombination, the DNA to be
introduced is flanked by two homologous sequences and the flanked -
region is inserted. The latter method is suitable for replacing,
as described above, the natural promoter of a particular gene
with one of the promoters of the invention and thus modifying the
location and time of expression of this gene. This functional
linkage represents an expression cassette of the invention.
The selection of successfully homologously recombined or else
transformed cells normally requires the additional introduction
of a selectable marker which imparts to the successfully
recombined cells a resistance to a biocide (for example a
herbicide), a metabolism inhibitor such as 2-desoxyglucose
6-phosphate (WO 98/45456) or to an antibiotic. The selection
marker permits selection of the transformed cells from the
untransformed cells (McCormick et al., Plant Cell Reports 5
(1986), 81-84).
Homologous recombination is a relatively rare event in higher
eukaryotes, especially in plants. Random integrations into the
host genome predominate. One possibility of removing the randomly
integrated sequences and thus accumulating cell clones having a
correct homologous recombination is the use of a
sequence-specific recombination system as described in
US 6,110,736. This system consists of three elements: two pairs
of specific recombination sequences and a sequence-specific
recombinase. This recombinase catalyzes a recombination merely
between the two pairs of specific recombination sequences. One
pair of these specific DNA sequences is placed outside the DNA
sequence to integrated, i.e. outside the two homologous DNA
sequences. In the case of a correct homologous recombination,
these sequences are not cotransferred into the genome. In the
case of a random integration, they normally insert together with
the rest of the construct. Using a specific recombinase and a
construct comprising a second pair of said specific sequences,
the randomly inserted sequences can be excised or inactivated by
inversion, while the sequences inserted correctly via homologous
0817/00024 CA 02454127 2004-01-09
21
recombination remain in the genome. It is possible to use a
multiplicity of sequence-specific recombination systems and the
Cre/lox system of bacteriophage Pl, the FLP/FRT system of yeast,
the Gin recombinase of phase Mu, the E. coli Pin recombinase and
the R/RS system of the plasmid pSR1 are mentioned by way of
example. Preference is given to the bacteriophage P1 Cre/lox and
the yeast FLP/FRT system. Here the recombinase (Cre or FLP)
interacts specifically with its respective recombination
sequences (34bp lox sequence or 47bp FRT sequence) in order to
delete or invert the transiently stored sequences. The FLP/FRT
=
and cre/lox recombinase systems have already been applied to
plant systems (Odell et al.(1990) Mol. Gen. Genet., 223:369-378) .
Polyadenylation signals suitable as control sequences are plant
polyadenylation signals and, preferably, those which correspond
essentially to Agrobacterium tumefaciens T-DNA polyadenylation
signals, in particular of the T-DNA gene 3 (octopene synthase) of
the Ti plasmid pTiACHS (Gielen et al.,(1984) EMBO J. 3 :(1984),
835 if) or functional equivalents thereof.
In a particularly preferred embodiment, the expression cassette
contains a terminator sequence functional in plants. Terminator
sequences functional in plants means in general those sequences
which are capable of causing the termination of transcription of
a DNA sequence in plants. Examples of suitable terminator
sequences are the OCS (octopene synthase) terminator and the NOS
(nopaline synthase) terminator. However, particular preference is
given to terminator sequences of plants. Terminator sequences of
plants means in general those sequences which are part of a
natural plant gene. In this connection, particular preference is
given to the terminator of the potato cathepsin D inhibitor gene
(GenBank Acc. No.: X74985; terminator: SEQ ID NO: 28) or of [sic]
the terminator of the field bean storage protein gene VfLE1B3
(GenBank Acc. No.: Z26489; terminator: SEQ ID NO: 29). These
terminators are at least equivalent to the viral or T-DNA
terminators described in the prior art. The plasmid pSUN5NPTIICat
(SEQ ID NO: 24) contains the plant terminator of the potato
cathepsin D inhibitor gene.
The skilled worker knows a multiplicity of nucleic acids or
proteins whose recombinant expression which is controlled by the
expression cassettes or methods of the invention is advantageous.
The skilled worker further knows a multiplicity of genes whose
repression or elimination by means of expression of a
corresponding antisense RNA can likewise achieve advantageous
0817/00024 CA 02454127 2004-01-09
22
effects. Advantageous effects which may be mentioned by way of
example and not by way of limitation are:
- easier preparation of a transgenic organism, for example by
expression of selection markers .
- achieving a resistance to abiotic stress factors (heat, cold,
drought, increased humidity, environmental toxins, UV
radiation)
- achieving a resistance to biotic stress factors (pathogens,
viruses, insects and diseases)
- improvement of the properties of food- or feedstuffs
- improvement of growth rate or yield.
Some specific examples of nucleic acids whose expression provides
the desired advantageous effects are mentioned below:
1. Selection markers
_ Selection markers includes both positive selection markers
which impart a resistance to an antibiotic, herbicide or
biocide and negative selection markers which impart a
sensitivity to exactly these substances and also markers
which give a growth advantage to the transformed organism
(for example by expressing key genes of cytokine
biosynthesis; Ebinuma H et al. (2000) Proc Natl Acad Sci USA
= 94:2117-2121). In the case of positive selection, only those
organisms which express the appropriate selection marker
grow, while the same organisms die in the case of negative
selection. The preparation of transgenic plants prefers the
use of a positive selection marker. Furthermore, preference
is given to using selection markers which impart growth
advantages. Negative selection markers may be used
advantageously if particular genes or genome sections are to
be removed from an organism (for example in a crossing
process).
The selectable marker introduced with the expression cassette
imparts to the successfully recombined or transformed cells a
resistance to a biocide (for example a herbicide such as
phosphinothricin, glyphosate or bromoxynil), a metabolism
inhibitor such as 2-desoxyglucose 6-phosphate (WO 98/45456)
or to an antibiotic such as, for example, kanamycin, G 418,
bleomycin, hygromycin. The selection marker permits selection
0817/00024 CA 02454127 2004-01-09
23
=
of the transformed cells from the untransformed cells
(McCormick et al., Plant Cell Reports 5 (1986), 81-84).
Particularly preferred selection markers are those which
impart a resistance to herbicides. A large number of such
selection markers and the sequences coding therefor are known
to the skilled worker. Examples which may be mentioned by way
of example but not by way of limitation are the following:
i) Positive selection markers:
The selectable marker introduced with the expression cassette -
imparts to the successfully recombined or transformed cells a
resistance to a biocide (for example a herbicide such as
phosphinothricin, glyphosate or bromoxynil), a metabolism
inhibitor such as 2-desoxyglucose 6-phosphate (WO 98/45456)
or to an antibiotic such as, for example, tetracyclines,
ampicillin, kanamycin, G418, neomycin, bleomycin or
hygromycin. The selection marker permits selection of the
transformed cells from the Untransformed cells (McCormick et
al., Plant Cell Reports 5 (1986), 81-84). Particularly
preferred selection markers are those which impart a
resistance to herbicides. Examples of selection markers which
may be mentioned are:
- DNA sequences coding for phosphinothricin
acetyltransferases (PAT) which acetylate the free amino
group of the glutamine synthase inhibitor
phosphinothricin (PPT) and thus detoxify PPT (de Block et
al. 1987, EMBO J. 6, 2513-2518) (also referred to as
Bialaphos -Resistence gene (bar)). The bar gene coding
for a phosphinothricin acetyltransferase (PAT) may be
isolated, for example, from Streptomyces hygroscopicus or
S. viridochromogenes. Corresponding sequences are known .
to the skilled worker (from Streptomyces hygroscopicus
GenBank Acc. No.: X17220 and X05822, from Streptomyces
viridochromogenes GenBank Acc. No.: M 22827 and X65195;
US 5,489,520). Furthermore, synthetic genes, for example
for expression in plastids, have been described A3028212
[sic]. A synthetic Pat gene is described in Becker et al.
(1994), The Plant J. 5:299-307. Very particular
preference is likewise given to the expression of the
polypeptide according to SEQ ID NO: 5, for example
encoded by a nucleic acid sequence according to SEQ ID
NO: 4. The genes impart a resistance to the herbicide
Bialaphos or glufosinate and are frequently used markers
in transgenic plants (Vickers , JE et al. (1996). Plant
CA 02454127 2010-07-29
24
Mol. Biol. Reporter 14:363-368; Thompson CJ et al. (1987)
EMBO Journal 6:2519-2523).
- 5-Enolpyruvylshikimate 3-phosphate synthase genes (EPSP
synthasegenes) which impart a resistance to Glyphosat
(N-(phosphonomethyl)glycin). The molecular target of the
unselective herbicide glyphosate is
5-enolpyruvy1-3-phosphoshikimate synthase (EPSPS). This
enzyme has a key function in the biosynthesis of aromatic
amino acids in microbes and plants but not in mammals
(Steinrucken HC et al. (1980) Biochem. Biophys. Res.
Commun. 94:1207-1212; Levin JG and Sprinson DB (1964) J. =
Biol. Chem. 239: 1142-1150; Cole DJ (1985) Mode of action
of glyphosate; A literature analysis, p. 48-74. In:
Grossbard E and Atkinson D (eds.). The herbicide
glyphosate. Buttersworths, Boston). Preference is given
to using glyphosate-tolerant EPSPS variants as selection
markers (Padgette SR et al. (1996) J Nutr. 1996 Mar; 126(3):702-16). New
weed control opportunities: development of soybeans with a Roundup
ReadyTM gene. In: Herbicide Resistant Crops (Duke, S.O., ed.), pp. 53-84.
CRC Press, Boca Raton, FL; Saroha MK and Malik VS (1998) J Plant
Biochemistry and Biotechnology 7:65-72). The EPSPS gene of Agrobacterium
sp. strain CP4 has a natural tolerance for glyphosate, which can be
transferred
to appropriate transgenic plants. The CP4
EPSPS gene was cloned from Agrobacterium sp. strain CP4
(Padgette SR et al.(1995) Crop Science 35(5):1451-1461).
5-Enolpyruvylshikimate 3-phosphate synthases, which are
glyphosate-tolerant, as described, for example, in US
5,510,471; US 5,776,760; US 5,864,425; US 5,633,435; US
=5,627;061; US 5,463,175; EP 0 218 571, are preferred and
the sequences described in each case in the patents have
also been deposited with GenBank. Further sequences are
described under GenBank Accession X63374. The aroA gene .
is also preferred (M10947 S. typhimurium aroA locus
5-enolpyruvylshikimate-3-phosphate synthase (aroA
protein) gene).
CA 02454127 2010-07-29
24a
- the gox (glyphosate oxidoreductase) gene coding for the
Glyphosate-degrading enzyme. GOX (for example
Achromobacter sp. glyphosate oxidoreductase) catalyzes
the cleavage of a C-I' bond in glyphosate which is thus
converted to aminomethylphosphonic acid (AMPA) and
glyoxylate. GOX can thereby mediate a resistance to
glyphosate (Padgette SR et al. (1996) J Nutr. 1996
0817/00024 CA 02454127 2004-01-09
Mar;126(3):702-16; Shah D et al. (1986) Science 233:
478-481).
- the deh gene (coding for a dehalogenase which inactivates
5 Dalapon0), (GenBank Acc. No.: AX022822, AX022820 and
W099/27116)
- bxn genes which code for Bromoxyni16 -degrading nitrilase
enzyme. For example the Klebsiella ozanenae nitrilase.
10 Sequences can be found at GenBank, for example under Acc.
=
No: E01313 (DNA encoding bromoxynil-specific nitrilase)
and J03196 (K. pneumoniae bromoxynil-specific nitrilase =
(bxn) gene, complete cds).
15 - Neomycin phosphotransferases impart a resistance to
antibiotics (aminoglycosides) such as neomycin, G418,
hygromycin, paromomycin or kanamycin by reducing the
inhibiting action thereof by a phosphorylation reaction.
Particular preference is given to the nptII gene.
20 Sequences can be obtained from GenBank (AF080390
minitransposon mTn5-GNm; AF080389 minitransposon mTn5-Nm,
complete sequence). Moreover, the gene is already part of
numerous expression vectors and can be isolated therefrom
by using methods familiar to the skilled worker (such as,
25 for example, polymerase chain reaction) (AF234316
pCAMBIA-2301; AF234315 pCAMBIA-2300, AF234314
pCAMBIA-2201). The NPTII gene codes for an aminoglycoside
3'-0-phosphotransferase from E.coli, Tn5 (GenBank Acc.
No: U00004 position 1401-2300; Beck et al. (1982) Gene 19
327-336).
- the DOGR1-gene. The DOGR1 gene was isolated from the
yeast Saccharomyces cerevisiae (EP 0 807 836). It codes
for a 2-desoxyglucose 6-phosphate phosphatase which
imparts resistance to 2-DOG (Randez-Gil et al. 1995,
Yeast 11, 1233-1240; Sanz et al. (1994) Yeast 10:1195-
1202, Sequence: GenBank Acc. No.: NC001140 chromosome
VIII, Saccharomyces cervisiae position 194799-194056).
Sulfonylurea- and imidazolinone-inactivating acetolactate
synthases which impart a resistance to
imidazolinone/sulfonylurea herbicides. Examples of
imidazolinone herbicides which may be mentioned are the
active substances imazamethabenz-methyl, imazamox,
imazapyr, imazaquin, imazethapyr. Examples of
sulfonylurea herbicides which may be mentioned are
amidosulforon [sic], azimsulfuron, chlorimuronethyl,
0817/00024 CA 02454127 2004-01-09
26
chlorsulfuron, cinosulfuron, imazosulforon [sic],
oxasulforon [sic], prosulforon [sic], rimsulforon [sic],
sulfosulforon [sic]. Numerous further active substances
of said classes are known to the skilled worker. An
example of a suitable sequence is the sequence of
Arabidopsis thaliana Csr 1.2 gene deposited under the
GenBank Acc-No.: X51514 (EC 4.1.3.18) (Sathasivan K et
al. (1990) Nucleic Acids Res. 18(8):2188). Acetolactate
synthases which impart a resistance to imidazolinon
herbicides are furthermore described under GenBank Acc.
No.:
=
zsaias::::aar:LaS ::::::ate
synthase, complete cds, herbicide resistant biotype
ab: :::::::: h::::c:::
acetolactate synthase precursor (ALS) gene, complete
cds
C) X07645 Tobacco acetolactate synthase gene, ALS SuRB
(EC 4.1.3.18)
d) X07644 Tobacco acetolactate synthase gene, ALS SuRA
(EC 4.1.3.18)
e) A19547 Synthetic nucleotide mutant acetolactate
_ synthase
f) A19546 Synthetic nucleotide mutant acetolactate
synthase
g) A19545 Synthetic nucleotide mutant acetolactate
synthase
h) 105376 Sequence 5 from Patent EP 0257993
i) 105373 Sequence 2 from Patent EP 0257993
j) AL133315
Preference is given to expressing an acetolactate
synthase according to SEQ ID NO: 7, for example encoded
by a nucleic acid sequence according to SEQ ID NO: 6.
- Hygromycin phosphotransferases (X74325 P. pseudomallei
gene for hygromycin phosphotransferase) which impart a
resistance to the antibiotic hygromycin. The gene is part
of numerous expression vectors and can be isolated
therefrom by using methods familiar to the skilled worker
(such as, for example, polymerase chain reaction)
(AF294981 pINDEX4; AF234301 pCA14BIA-1380; AF234300
pCAMBIA-1304; AF234299 pCAMBIA-1303; AF234298
pCAMBIA-1302; AF354046 pC2 MBIA-1305.; AF354045
pCAMBIA-1305.1)
0817/00024 CA 02454127 2004-01-09
27
- Genes for resistance to
a) chloramphenicol (chloramphenicol acetyltransferase),
b) tetracycline, various resistance genes have been
described, for example X65876 S. ordonez genes class
D tetA and tetR for tetracycline resistance and
repressor proteins X51366 Bacillus cereus plasmid
pBC16 tetracycline resistance gene. The gene is also
already part of numerous expression vectors and can
be isolated therefrom by using methods familiar to
the skilled worker (such as, for example, polymerase =
chain reaction),
c) streptomycin, various resistance genes have been
described, for example under GenBank Acc.
No.:AJ278607 Corynebacterium acetoacidophilum ant
gene for streptomycin adenylyltransferase.
d) zeocin, the corresponding resistance gene is part of
numerous cloning vectors (e.g. L36849 cloning vector
pZEO) and can be isolated therefrom by using methods
_ _ familiar to the skilled worker (such as, for example,
polymerase chain reaction),
e) ampicillin (p-lactamase gene; Datta N, Richmond
MH.(1966) Biochem J. 98(1):204-9; Heffron F et al
(1975) J. Bacteriol 122: 250-256; the amp gene was
initially cloned for preparing the E. coli vectors
pBR322; Bolivar F et al. (1977) Gene 2:95-114). The
sequence is part of numerous cloning vectors and can
be isolated therefrom by using methods familiar to
the skilled worker (such as, for example, polymerase
chain reaction).
- Genes such as the [sic] isopentenyl transferase from
Agrobacterium tumefaciens (strain:P022) (Genbank Acc.
No.: AB025109). The ipt gene is [lacuna] a key enzyme of
cytokine biosynthesis. Its overexpression facilitates the
regeneration of plants (e.g. selection of cytokine-free
medium). The method for using the ipt gene has been
described (Ebinuma H et al. (2000) Proc Natl Acad Sci USA
94:2117-2121; Ebinuma, H et al. (2000) Selection of
Marker-free transgenic plants using the oncogenes (ipt,
rol A, B, C) of Agrobacterium as selectable markers, In
0817/00024 CA 02454127 2004-01-09
= , 28
Molecular Biology of Woody Plants. Kluwer Academic
Publishers).
Various other positive selection markers which impart to the
transformed plants a growth advantage over untransformed
plants and methods of their use are described, inter alia, in
EP-A 0 601 092. Examples which may be mentioned are
p-glucuronidase (in connection with, for example, cytokinine
glucuronide), mannose 6-phosphate isomerase (in connection
with mannose), UDP-galactose 4-epimerase (in connection with,
for example, galactose), mannose 6-phosphate isomerase in
connection with mannose being particularly preferred.
=
ii) Negative selection markers
Negative selection markers make possible, for example, the
selection of organisms in which sequences comprising the
marker gene have been successfully deleted (Koprek T et al.
(1999) The Plant Journal 19(6):719-726),In negative
selection, for example, a compound which otherwise has no
disadvantageous effect on the plant is converted to a
compound having a disadvantageous effect, due to the negative
selection marker introduced into the plant. Genes which have
a disadvantageous effect per se, such as, for example, TK
thymidine kinase (TK), and diphtheria toxin A fragment
(DT-A), the codA gene product coding for a cytosine deaminase
(Cleave AP et al. (1999) Plant Mol Biol. 40(2):223-35; Perera
RJ et al. (1993) Plant Mol. Biol 23(4): 793-799; Stougaard J;
(1993) Plant J 3:755-761), the cytochrom P450 gene (Koprek et
' al. (1999) Plant J. 16:719-726), genes coding for a
haloalkane dehalogenase (Naested H (1999) Plant J.
18:571-576), the iaaH gene (Sundaresan V et al. (1995) Genes
& Development 9:1797-1810) and the tms2 gene (Fedoroff NV &
Smith DL 1993, Plant J 3: 273-289) are also suitable.
The concentrations of the antibiotics, herbicides, biocides
or toxins, used in each case for selection, have to be
adapted to the particular assay conditions or organisms.
Examples which may be mentioned for plants are kanamycin (Km)
50 mg/1, hygromycin B 40 mg/1, phosphinothricin (Ppt) 6 mg/l.
It is furthermore possible to express functional analogs of
said nucleic acids coding for selection markers. Functional
analogs here means all those sequences which have essentially
the same function, i.e. which are capable of selection of
transformed organisms. In this connection, the functional
analog may quite possibly differ in other features. It may
0817/00024 CA 02454127 2004-01-09
29
have, for example, a higher or lower activity or else further
functionalities.
2. Improved protection of the plant against abiotic stress
factors such as drought, heat or cold, for example by
overexpression of antifreeze-polypeptides from Myoxocephalus
Scorpius (WO 00/00512), Myoxocephalus octodecemspinosus, of
Arabidopsis thaliana transcription activator CBF1, of
glutamate dehydrogenases (WO 97/12983, WO 98/11240),
calcium-dependent protein kinase genes (WO 98/26045),
calcineurins (WO 99/05902), farnesyl transferases
(WO 99/06580, Pei ZM et al., Science 1998, 282: 287-290), =
ferritin (Deak M et al., Nature Biotechnology 1999,
17:192-196), oxalate oxidase (WO 99/04013; Dunwell JM
Biotechnology and Genetic Engineering Reviews 1998, 15:1-32),
DREB1A-Factor (dehydration response element B 1A; Kasuga M
et al., Nature Biotechnology 1999, 17:276-286), of genes of
mannitol or trehalose synthesis, such as trehalose phosphate
synthase or trehalose phosphate phosphatase (WO 97/42326), or
by inhibition of genes such as trehalase (WO 97/50561).
Particular preference is given to nucleic acids which code
for the Arabidopsis thaliana transcription activator CBF1
(GenBank Acc. No.: U77378) or for the Myoxocephalus
octodecemspinosus antifreeze protein (GenBank Acc. No.:
AF306348) or functional equivalents of the same.
3. Expression of metabolic enzymes for use in the feed and food
sectors, for example expression of phytase and cellulases.
Particular preference is given to nucleic acids such as the
artificial cDNA coding for a microbial phytase (GenBank Acc.
No.: A19451) or functional equivalents thereof.
4. Achieving a resistance, for example to fungi, insects,
nematodes and diseases, by specific isolation or accumulation .
of particular metabolites or proteins in the embryonic
epidermis. Examples which may be mentioned are glucosinolates
(repulsion of herbivores), chitinases or glucanases and other
enzymes which destroy the cell wall of parasites,
ribosome-inactivating proteins (RIPs) and other proteins of
resistance and stress reactions of the plant, such as those
induced by injury or microbial infection of plants or
chemically by, for example, salicylic acid, jasmonic acid or
ethylene, lysozymes from sources other than plants, such as,
for example, T4 lysozyme or lysozyme from various mammals,
insecticidal proteins such as Bacillus thuringiensis
endotoxin, a-amylase inhibitor or protease inhibitors (cowpea
trypsine inhibitor), glucanases, lectins such as
0817/00024 CA 02454127 2004-01-09
* = 30
phytohemagglutinin, snowdrop lectin, wheat germ agglutinine,
RNases and ribozymes. Particular preference is given to
nucleic acids coding for chit42 endochitinase from
Trichoderma harzianum (GenBank Acc. No.: S78423) or for the
N-hydroxylating, multifunctional cytochrome P-450 (CYP79)
protein from Sorghum bicolor (GenBank Acc. No.: U32624) or
functional equivalents thereof.
What is known is the accumulation of glucosinolates in plants
of the genus of Cardales, in particular of oilseeds, for
protection against pests (Rask L et al.(2000) Plant Mol Biol
42:93-113; Menard R et al. (1999) Phytochemistry 52:29-35),
the expression of the Bacillus thuringiensis endotoxin under
the control of the 35 S CaMV promoter (Vaeck et al. (1987)
Nature 328:33-37) or the protection of tobacco against fungal
infection by expression of a bean chitinase under the control
of the CaMV promoter (Broglie et al. (1991) Science
254:1194-1197).
The expression of the snowdrop (Galanthus nivalis) lectin
agglutinine can achieve a resistance to pests such as
the rice pest Aq/aparvata lugens, for example in transgenic
rice plants (Rao et al. (1998) Plant J. 15(4):469-77.).
Nilaparvata lugens belongs to the phloem-sucking pests and,
in addition, acts as a transmitter of important virus-based
plant diseases.
The expression of synthetic cryIA(b) and cryIA(c) genes which
code for lepidoptera-specific delta-entotoxins from Bacillus
thuringiensis, can cause a resistance to insect pests in
various plants. Thus it is possible to achieve a resistance
in rice to two of the most important rice insect pests, the
striped stem borer (Chilo suppressalis) and the yellow stem
borer (Scirpophaga incertulas), (Cheng X et al. (1998) Proc
Natl Aced Sci USA 95(6):2767-2772; Nayak P et al. (1997) Proc
Natl Acad Sci USA 94(6):2111-2116).
5. Expression of genes which cause accumulation of fine
chemicals such as tocopherols, tocotrienols or carotenoids.
Phytoene desaturase may be mentioned as an example.
Preference is given to nucleic acids which code for Narcissus
pseudonarcissus phytoene desaturase (GenBank Acc. No.:
X78815) or functional equivalents thereof.
0817/00024 CA 02454127 2004-01-09
31
6. Production of nutraceuticals such as, for example,
polyunsaturated fatty acids such as, for example, arachidonic
acid or EP (eicosapentenoic acid) or DHA (docosahexaenoic
acid) by expressing fatty-acid elongases and/or desaturases
or by producing proteins having an improved nutritional value
such as, for example, a high proportion of essential amino
acids (e.g. the methionine-rich brazil nut albumingen [sic)).
Preference is given to nucleic acids coding for the
methionine-rich Bertholletia excelsa 2S albumin (GenBank Acc.
No.:AB044391), the Physcomitrella patens A6-acyllipid
desaturase (GenBank Acc. No.: AJ222980; Girke et al 1998, The
Plant Journal 15:39-48), the Mortierella alpina A6-desaturase =
(Sakuradani et al 1999 Gene 238:445-453), the Caenorhabditis
elegans A5-desaturase (Michaelson et al. 1998, FEBS Letters
439:215-218), the Caenorhabditis elegans AS-fatty-acid
desaturase (des-5) (GenBank Acc. No.: AF078796), the
Mortierella alpina AB-desaturase (Michaelson et al. JBC
273:19055 - 19059), the Caenorhabditis elegans A6-elongase
(Beaudoin et al. 2000, PNAS 97:6421-6426), the Physcomitrella
patens A6-elongase (Zank et al. 2000, Biochemical Society
Transactions 28:654-657) or functional equivalents thereof.
7. _ Production of fine chemicals (such as, for example, enzymes)
and pharmaceuticals (such as, for example, antibodies or
vaccines, as described in Hood EE, Jilka JM. (1999) Curr Opin
Biotechnol. 10(4):382-6; Ma JK, Vine ND (1999) Curr Top
Microbiol Immunol 236:275-92). For example, it was possible
to produce on a large scale recombinant avidin from egg white
and bacterial p-glucuronidase (GUS) in transgenic corn plants
(Hood et al. (1999) Adv Exp Med Biol 464:127-47. Review).
These recombinant proteins from corn plants are sold by Sigma
(Sigma Chemicals Co.) as high-purity biochemicals.
8. Achieving an increased storage capability in cells which
usually contain relatively few storage proteins or storage
lipids, with the aim of increasing the yield of said
substances, for example by expressing an acetyl-CoA
carboxylase. Preference is given to nucleic acids coding for
Medicago sativa acetyl-CoA carboxylase (accase) (GenBank Acc.
No.: L25042) or functional equivalents thereof.
Further examples of advantageous genes are mentioned, for
example, in Dunwell JM, Transgenic approaches to crop
improvement, J Exp Bot. 2000;51 Spec No; pages 487-96.
0817/00024 CA 02454127 2004-01-09
32
It is furthermore possible to express functional analogs of the
nucleic acids and proteins mentioned. Functional analogs here
means all those sequences which have essentially the same
function, i.e. which are capable of the same function (for
example substrate conversion or signal transduction) as the
protein mentioned by way of example. The functional analog may
quite possibly differ in other features. It may have, for
example, a higher or lower activity or else have further
functionalities. Functional analogs further means sequences which
code for fusion proteins comprising one of the preferred proteins
and other proteins, for example another preferred protein, or
else a signal peptide sequence.
The nucleic acids may be expressed under the control of the
promoters of the invention in any desired cell compartment such
as, for example, the endomembrane system, the vacuole and the
chloroplasts. Desired glycosylation reactions, particular
foldings, and the like are possible by utilizing the secretory
pathway. Secretion of the target protein to the cell surface or
secretion into the culture medium, for example when using
suspension-cultured cells or protoplasts, is also possible. The
required target sequences may both be taken into account in
individual vector variations and be introduced into the vector
together with the target gene to be cloned by using a suitable
cloning strategy. Target sequences which may be used are both
endogenous, if present, and heterologous sequences. Additional
heterologous sequences which are preferred for functional linkage
but not limited thereto are further targeting sequences for
ensuring subcellular localization in the apoplast, in the
vacuole, in plastids, in mitochondria, in the endoplasmic
reticulum (ER), in the nucleus, in elaioplasts or other
compartments; and also translation enhancers such as the
5'-leader sequence from tobacco mosaic virus (Gallie et al.,
Nucl. Acids Res. 15 (1987), 8693-8711) and the like. The method .
of transporting proteins which are per se not located in the
plastids specifically into said plastids has been described,
(Klosgen RB und Well al (1991) Mol Gen Genet 225(2):297-304; Van
Breusegem F et al. (1998) Plant Mel Biol. 38(3):491-496).
Preferred sequences are:
a) small subunit (SSU) of ribulose bisphosphate carboxylase
(Rubisco ssu) from pea, corn, sunflower
0817/00024 CA 02454127 2004-01-09
33
b) transit peptides derived from genes of fatty-acid
biosynthesis in plants, such as the transit peptide of the
plastid acyl carrier protein (ACP), stearyl-ACP desaturase,
13-ketoacyl-ACP synthase or acyl-ACP thioesterase.
c) the transit peptide for GBSSI ("granule bound starch
synthase I")
d) LHCP II genes.
The target sequences may be linked to other targeting sequences
which differ from the transit peptide-encoding sequences, in
=
order to ensure subcellular localization in the apoplast, in the
vacuole, in plastids, in the mitochondrion, in the endoplastic
= 15 reticulum (ER), in the nucleus, in elaioplasts or in other
compartments. It is also possible to use translation enhancers
such as the 5f-leader sequence from tobacco mosaic virus (Gallie
et al. (1987), Nucl. Acids Res. 15: 8693-8711) and the like.
The skilled worker further knows that there is no need for him to
express the above-described genes directly by using the nucleic
acid sequences coding for said genes or to repress them by
antiaense, for example. He may also use, for example, artificial
transcription factors of the zinc finger protein type (Beerli RR
et al. (2000) Proc Natl Acad Sci USA 97(4):1495-500). These
factors attach to the regulatory regions of the endogenous genes
to be expressed or repressed and cause expression or repression
of the endogenous gene, depending on the design of the factor.
Thus it is also possible to achieve the desired effects by
expressing an appropriate zinc finger transcription factor under
the control of one of the promoters of the invention.
It is likewise possible to use the expression cassettes of the
invention for suppressing or reducing the replication or/and
translation of target genes by gene silencing.
The expression cassettes of the invention may also be employed
for expressing nucleic acids which mediate "antisense" effects
and thus are capable of reducing the expression of a target
protein, for example.
Preferred genes and proteins whose suppression results in an
advantageous phenotype include by way of example but not by way
of limitation:
0817/00024 CA 02454127 2004-01-09
34
a) polygalacturonase for preventing cell degradation and
preventing plants and fruits, for example tomatoes, from
becoming "mushy". Preference is given to using for this
nucleic acid sequences such as that of the tomato
polygalacturonase gene (GenBank Acc. No.: X14074) or its
homologs from other genera and species.
b) reducing the expression of allergenic proteins, as described,
for example, in Tada Y et al. (1996) FEBS Lett 391(3):341-345
or Nakamura R (1996) Biosci Biotechnol Biochem
60(8):1215-1221.
c) modifying the color of flowers by suppressing the expression
of enzymes of anthocyane biosynthesis. Appropriate procedures
have been described (for example in Forkmann G, Martens S.
(2001) Curr Opin Biotechnol 12(2):155-160). Preference is
given to using for this nucleic acid sequences such as those
of flavonoid 3'-hydroxylase (GenBank Acc. No.: AB045593),
dihydroflavanol 4-reductase (GenBank Acc.- No.: AF017451),
chalcone isomerase (GenBank Acc. No.: AF276302), chalcone
synthase (GenBank Acc. No.: AB061022), flavanone
3-beta-hydroxylase (GenBank Acc. No.: X72592) and flavone
_ synthase II (GenBank Acc. No.: AB045592) and the homologs
thereof from other genera and species.
d) altering the amylose/amylopectin content in starch by
suppressing the branching enzyme Q which is responsible for
the a-1,6-glycosidic linkage. Appropriate procedures have
been described (for example in Schwall GP et al. (2000) Nat
Biotechnol 18(5):551-554). Preference is given to using for
this nucleic acid sequences such as that of the potato starch
branching enzyme II (GenBank Acc. No.: AR123356;
US 6,169,226) or its homologs from other genera and species.
An antisense nucleic acid first means a nucleic acid-sequence
which is completely or partially complementary to at least a part
of the sense strand of said target protein. The skilled worker
knows that it is possible to use, as an alternative, the cDNA or
the corresponding gene as starting template for corresponding
antisense constructs. Preferably, the antisense nucleic acid is
complementary to the coding region of the target protein or to a
part thereof. However, the antisense nucleic acid may also be
complementary to the noncoding region or to a part thereof.
Starting from the sequence information for a target protein, it
is possible to design an antisense nucleic acid in the manner
familiar to the skilled worker by taking into account the Watson
and Crick base pairing rules. An antisense nucleic acid may be
CA 02454127 2013-11-27
complementaryto the entire or to a part of the nucleic acid
sequence of a target protein. In a preferred embodiment, the
antisense nucleic acid is an oligonucleotide of, for example, 15,
20,.25,.30, 35, 40, 45 or 50 nucleotides in length.
In a preferred embodiment, the antisense nucleic acid comprises
a-anomric nucleic acid molecules. a-anomeric nucleic acid
molecules form particular double-stranded hybrids with
complementary RNA, in which, in contrast to the normal
P-units, the strands run parallel to one another (Gautier et al.
(1987) Nucleic Acids. Res. 15:6625-6641).
Likewise included is the use of the above-described sequences in
sense orientation, which may lead to cosuppression, as is
familiar to the skilled worker. It has been demonstrated in
tobacco, tomato and petunia that expression of sense RNA of an
endogenous gene can reduce or eliminate expression of said gene,
in a similar manner to what has been described for antisense
approaches (Goring at al. (1991) Proc. Nati Acad Sci USA,
88:1770-1774; Smith et al. (1990) No]. Gen Genet 224:447-481;
Napoli et al. (1990) Plant Cell 2:279-289; Van der Krol et al.
(1990) Plant Cell 2:291-299). The introduced construct may
represent the gene to be reduced completely or only partially.
The possibility of translation is not required.
Very particular preference is also given to the use of methods
such as gene regulation by means of double-stranded RNA
(double-stranded RNA interference). Relevant methods are known
to the skilled worker and have been described in detail (e.g.
Matzke MA et al. (2000) Plant Mol Biol 43:401-415; Fire A.
et al (1998) Nature 391:806-811; WO 99/32619; WO 99/53050;
10 WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035;
WO 00/63364). The simultaneous introduction of strand and
complementary strand causes here a highly efficient
suppression of native genes.
Advantageously, the antisense strategy may be coupled with a
ribozyme method. Ribozymes are catalytically active RNA, sequences
CA 02454127 2013-11-27
35a
which, coupled to the antisense sequences, catalytically cleave
the target sequences (Tanner NE. FEMS Microbiol Rev. 1999;
23 (3);257-75). This can increase the efficiency of an antisense
strategy. The expression of ribozymes in order to reduce
particular proteins is known to the skilled worker and is
described, for example, in EP-Al 0 291 533, EP-Al 0 321 201 and
EP-Al 0 360 257. Suitable target sequences and ribozymes may be
determined, for example, as described in Steinecke (Ribozymes,
0817/00024 CA 02454127 2004-01-09
36
Methods in Cell Biology 50, Galbraith et al eds Academic Press,
Inc. (1995), 449-460), by calculations of the secondary structure
of ribozyme RNA and target RNA and by the interaction thereof
(Bayley CC et al., Plant Mol Biol. 1992; 18(2):353-361; Lloyd AM
and Davis RW et al., Mol Gen Genet. 1994 Mar;242(6):653-657). An
example which may be mentioned is hammerhead ribozymes (Haselhoff
and Gerlach (1988) Nature 334:585-591). Preferred ribozymes are
based on derivatives of Tetrahymena L-19 IVS RNA (US 4,987,071;
US 5,116,742). Further ribozymes with selectivity for an L119
mRNA may be selected (Bartel D und Szostak JW (1993) Science
261:1411-1418).
In another embodiment, target protein expression may be reduced
using nucleic acid sequences which are complementary to
regulatory elements of the target protein genes and which form
together with said genes a triple-helical structure and thus
prevent gene transcription (Helene C (1991) Anticancer Drug Des.
6(6):569-84; Helene C et al. (1992) Ann NY Acad Sci 660:27-36;
Maher LJ (1992) Bioassays 14(12):807-815).
The expression cassette of the invention and the vectors derived
therefrom may contain further functional elements.
_
The term functional element has a broad meaning and means all
those elements which influence preparation, propagation or
function of the expression cassettes of the invention or of
vectors or organisms derived therefrom. Examples which may be
mentioned but which are not limiting are:
a) reporter genes which code for readily quantifiable proteins
and which ensure, via intrinsic color or enzyme activity, an
evaluation of the transformation efficiency and of the
location or time of expression. In this connection, very
particular preference is given to genes coding for reporter
proteins (see also Schenborn E, Groskreutz D. Mol Biotechnol.
1999; 13(1):29-44) such as
- green fluorescence protein (GFP) (Chui WL et al., Curr
Biol 1996, 6:325-330; Leffel SM et al., Biotechniques.
23(5):912-8, 1997; Sheen et al.(1995) Plant Journal
8(5):777-784; Haseloff et al.(1997) Proc Natl Acad Sci
USA 94(6):2122-2127; Reichel et al.(1996) Proc Natl Acad
Sci USA 93(12):5888-5893; Tian et al. (1997) Plant Cell
Rep 16:267-271; WO 97/41228).
=
0817/00024 CA 02454127 2004-01-09
37
- chloramphenicol transferase (Fromm et al. (1985) Proc.
Natl. Acad. Sci. USA 82:5824-5828),
- Luciferase (Millar et al., Plant Mol Biol Rep 1992
10:324-414; Ow et al. (1986) Science, 234:856-859);
allows bioluminescence detection.
P-galactosidase, coding for an enzyme for which various
chromogenic substrates are available.
- p-glucuronidase (GUS) (Jefferson et al., EMBO J. 1987, 6,
3901-3907) or the uidA gene which encodes an enzyme for =
various chromogenic substrates.
R-locus gene product: protein which regulates production
of anthocyanine pigments (red color) in plant tissue and
thus makes possible a direct analysis of the promoter
activity without the addition of additional auxiliary
substances or chromogenic substrates (Dellaporta et al.,
In: Chromosome Structure and Function: Impact of New
Concepts, 18th Stadler Genetics Symposium, 11:263-282,
1988).
_
- p-lactamase (Sutcliffe (1978) Proc Natl Acad Sci USA
75:3737-3741), enzyme for various chromogenic substrates
(e.g. PADAC, a chromogenic cephalosporin).
- xylE gene product (Zukowsky et al. (1983) Proc Natl Acad
Sci USA 80:1101-1105), catechol dioxygenase which can
convert chromogenic catechols.
- alpha-amylase (Ikuta et al. (1990) Bio/technol.
8:241-242).
tyrosinase (Katz et al.(1983) J Gen Microbiol
129:2703-2714), enzyme which oxidizes tyrosine to give
DOPA and dopaquinone which consequently form the readily
detectable melanine.
- aequorin (Prasher et al.(1985) Biochem Biophys Res Commun
I26(3):1259-1268), may be used in calcium-sensitive
bioluminescence detection.
0817/00024 CA 02454127 2004-01-09
38
b) replication origins which ensure a propagation of the
expression cassettes or vectors of the invention, for example
in E. co/i. Examples which may be mentioned are ORI (origin
of DNA replication), the pBR322 on or the P15A on (Sambrook
et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY,
1989).
c) elements, for example border sequences, which enable
agrobacteria-mediated transfer into plant cells for transfer
and integration into the plant genome, such as, for example,
the right or left border of T-DNA or the vir region. =
d) multiple cloning regions (mCS) allow and facilitate the
insertion of one or more nucleic acid sequences.
Various ways to achieve an expression cassette of the invention
are known to the skilled worker. An expression cassette of the
invention is prepared, for example, by fusing one of the
promoters of the invention (or a functional equivalent or
functionally equivalent part according to SEQ ID NO: 1, 2 or 3)
or a functional equivalent to a nucleotide sequence to be
expressed, where appropriate to a sequence coding for a transit
peptide, preferably a chloroplast-specific transit peptide, which
is preferably located between the promoter and the particular
nucleotide sequence, and also with a terminator or
polyadenylation signal. For this purpose, common recombination
and cloning techniques as described, for example, in T. Maniatis,
E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
(1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist,
Experiments with Gene Fusions, Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al.,(1987)
Current Protocols in Molecular Biology, Greene Publishing Assoc.
and Wiley Interscience are used.
However, an expression cassette means also those constructs in
which the promoter without having been functionally linked
beforehand to a nucleic acid sequence to be expressed, is
introduced into a host genome, for example, via specific
homologous recombination or random insertion and takes over there
regulatory control over nucleic acid sequences then functionally
linked to it and controls transgenic expression of said nucleic
acid sequences. Insertion of the promoter, for example by
homologous recombination, upstream of a nucleic acid coding for a
particular polypeptide results in an expression cassette of the
invention, which controls expression of the particular
0817/00024 CA 02454127 2004-01-09
39
polypeptide in the plant. Furthermore, the promoter may also be
inserted such that antisense RNA of the nucleic acid coding for a
particular polypeptide is expressed. As a result, the expression
of said particular polypeptide in plants is down-regulated or
eliminated.
Analogously, it is also possible to place a nucleic acid sequence
to be expressed transgenically downstream of the endogenous
natural promoter, for example by homologous recombination,
resulting in an expression cassette of the invention, which
controls expression of the nucleic acid sequence to be expressed
= transgenically in the cotyledons of the plant embryo.
The invention further relates to vectors which contain the
= 15 above-described expression cassettes. Vectors may be, by way of
example, plasmids, cosmids, phages, viruses or else agrobacteria.
The invention also relates to transgenic organisms transformed
= with at least one expression cassette of the invention or one=
vector of the invention and also to cells, cell cultures, tissue,
parts, such as, for example in the case of plant organisms,
leaves, roots, etc., or propagation material derived from such
organisms.
Organisms, starting or host organisms mean prokaryotic or
eukaryotic organisms such as, for example, microorganisms or
plant organisms. Preferred microorganisms are bacteria, yeasts,
= algae or fungi.
Preferred bacteria are bacteria of the genus Escherichia,
Erwinia, Agrobacterium, Flavobacterium, Alcaligenes or
cyanobacteria for example of the genus Synechocystis.
Preference is given especially to microorganisms which are
capable of infecting plants and thus transferring the cassettes
of the invention. Preferred microorganisms are those of the genus
Agrobacterium and, in particular of the species Agrobacterium
tumefaciens.
Preferred yeasts are Candida, Saccharomyces, Hansenula and
Pichia.
Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora,
Fusarium, Beauveria or other fungi described in Indian Chem
Engr. Section B. Vol 37, No 1,2 (1995) on page 15, Table 6.
0817/00024 CA 02454127 2004-01-09
=
Host or starting organisms preferred as transgenic organisms are
especially plants. Included within the scope of the invention are
all genera and species of the higher and lower plants of the
plant kingdom. The mature plants, seeds, shoots and seedlings and
5 also parts, propagation material and cultures, for example cell
cultures, derived therefrom are also included. Mature plants
means plants at any development stage beyond the seedling.
Seedling means a young immature plant in an early development
stage.
10 . .
Annual, perennial, monocotyledonous and dicotyledonous plants are
preferred host organisms for preparing transgenic plants. The
expression of genes is furthermore advantageous in all ornamental
plants, useful or ornamental trees, flowers, cut flowers, shrubs
15 or lawns. Plants which may be mentioned by way of example but not
by limitation are angiosperms, bryophytes such as, for example,
Hepaticae (liverworts) and Musci (mosses); pteridophytes such as
ferns, horsetail and club mosses; gymnosperms such as conifers,
cycades, ginkgo and Gnetalae; algae such as Chlorophyceae,
20 Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae,
Bacillariophyceae (diatoms) and Euglenophyceae.
Preference is given to plants of the following plant families:
Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae,
25 Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae,
Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae,
Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanacea,
Sterculiaceae, Tetragoniacea, Theaceae, Umbelliferae.
30 Preferred monocotyledonous plants are in particular selected from
the monocotyledonous crop plants, for example of the Gramineae
family, such as rice, corn, wheat, or other cereal species such
as barley, malt, rye, triticale or oats, and also sugar cane and
all grass species.
Preferred dicotyledonous plants are in particular selected from
the dicotyledonous crop plants, for example
Asteraceae such as sunflower, Tagetes or Calendula and
others,
Compositae, particularly the genus Lactuca, in particular the
species sativa (lettuce), and others,
Cruciferae, particularly the genus Brassica, very
particularly the species napus (oilseed rape), campestris
(beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y
0817/00024 CA 02454127 2004-01-09
41
(cauliflower) und oleracea cv Emperor (broccoli), and further
cabbage species; and the genus Arabidopsis, very particularly
the species thaliana, and also cress or canola, and others,
Cucurbitaceae such as melon, pumpkin or zucchini, and others,
Leguminosae particularly the genus Glyrine, very particularly
the species max (soyabean), soya and also alfalfa, pea, bean
plants or peanut, and others,
Rubiaceae, preferably of the subclass Lamiidae, such as, for
example, Coffea arabica or Coffea liberica (coffee bush), and =
others,
Solanaceae, in particular the genus Lycopersicon, very
particularly the species esculentum (tomato), and the genus
Solanum, very particularly the species tuberosum (potato) and
melongena (aubergine) and also tobacco or paprika, and
others,
Sterculiaceae, preferably of the subclass Dilleniidae, such
as, for example, Theobroma cacao (cacao bush) and others,
_
Theaceae, preferably of the subclass Dilleniidae, such as,
for example, Camellia sinensis or Thea sinensis (tea shrub)
and others,
Umbelliferae, preferably the genus Daucus, very particularly
the species carota (carrot), and Apium (very particularly the
species graveolens dulce (celery), and others; and the genus
Capsicum, very particularly the species annum (pepper), and
others,
and also linseed, soya, cotton, hemp, flax, cucumber, spinach,
carrot, sugarbeet and the various tree, nut and vine species, in
particular banana and kiwi fruit.
Also included are ornamental plants, useful and ornamental trees,
flowers, cut flowers, shrubs and lawns. Plants which may be
mentioned by way of example but not by limitation are
angiosperms, bryophytes such as, for example, Hepaticae
(liverworts) and Musci (mosses); pteridophytes such as ferns,
horsetail and club mosses; gymnosperms such as conifers, cycades,
ginkgo and Gnetalae, the Rosaceae families, such as rose,
Ericaceae such as rhododendrons and azaleas, Euphorbiaceae such
as poinsettias and croton, Caryophyllaceae such as pinks,
Solanaceae such as petunias, Gesneriaceae such as African violet,
0817/00024 CA 02454127 2004-01-09
42
Balsaminaceae such as catch-me-not, Orchidaceae such as orchids,
Iridaceae such as gladioli, iris, freesia and crocus, Compositae
such as marigold, Geraniaceae such as gerania, Liliaceae such as
dracaena, Moraceae such as ficus, Araceae such as sweetheart
plant, and others.
Most preference is given to Arabidopsis thaliana, Nicotiana
tabacum, Tagetes erecta, Calendula officinalis and Brassica napus
and to all genera and species which are used as food- or
feedstuffs, such as the cereal species described, or which are
suitable for preparing oils, such as oilseeds (e.g. oilseed
rape), nut species, soya, sunflower, pumpkin and peanut. =
Plant organisms for the purposes of this invention are
furthermore other organisms capable of photosynthetic activity,
such as, for example, algae or cyanobacteria, and also mosses.
Preferred algae are green algae such as, for example, algae of
the genus Haematococcus, Phaedactylum tricornatum, Volvox or
Dunaliella.
The preparation of a transformed organism or of a transformed
cell _requires introducing the appropriate DNA into the
appropriate host cell. A multiplicity of methods is available for
this process which is referred to as transformation (see also
Keown et al. 1990 Methods in Enzymology 185:527-537). Thus, by
way of example, the DNA may be introduced directly by
microinjection or by bombardment with DNA-coated microparticles.
The cell may also be permeabilized chemically, for example using
polyethylene glycol, so that the DNA can enter the cell via
diffusion. The DNA may also be performed [sic) via protoplast
fusion with other DNA-comprising units such as minicells, cells,
lysosomes or liposomes. Another suitable method for introducing
DNA is electroporation in which the cells are reversibly
permeabilized by an electric impulse.
In the case of plants, the methods described for transforming and
regenerating plants from plant tissues or plant cells are
utilized for transient or stable transformation. Suitable methods
are especially protoplast transformation by polyethylene
glycol-induced DNA uptake, the biolistic method using the gene
gun, the "particle bombardment" method, electroporation, the
incubation of dry embryos in DNA-comprising solution and
microinjection.
0817/00024 CA 02454127 2004-01-09
= 43
Apart from these "direct" transformation techniques, a
transformation may also be carried out by bacterial infection by
means of Agrobacterium tumefaciens or Agrobacterium rhizogenes.
These strains contain a plasmid (Ti or Ri plasmid) which is
transferred to the plant after Agrobacteria infection. A part of
this plasmid, denoted T-DNA (transferred DNA) is integrated into
the genome of the plant cell.
The Agrobacterium-mediated transformation is best suited to
dicotyledonous diploid plant cells, whereas the direct
transformation techniques are suitable for any cell type.
An expression cassette of the invention may be introduced
advantageously into cells, preferably into plant cells, by using
vectors.
In an advantageous embodiment, the expression cassette is
introduced by means of plasmid vectors. Preference is given to
those vectors which enable a stable integration of the expression
cassette into the host genome.
In the case of injection or electroporation of DNA into plant
cells, no particular demands on the plasmid used are made. It is
possible to use simple plasmids such as those of the pUC series.
If complete plants are to be regenerated from the transformed
cells, it is necessary for an additional selectable marker gene
to be present on the plasmid.
Transformation techniques have been described for various
monocotyledonous and dicotyledonous plant organisms. Furthermore,
various possible plasmid vectors which normally contain an origin
of replication for propagation in E.coli and a marker gene for
selection of transformed bacteria are available for introducing
foreign genes into plants. Examples are pBR322, pUC series, Ml3mp
series, pACYC184 etc.
The expression cassette may be introduced into the vector via a
suitable restriction cleavage site. The resultant plasmid is
first introduced into E.coli. Correctly transformed E.coli cells
are selected, cultivated and the recombinant plasmid is obtained
using methods familiar to the skilled worker. Restriction
analysis and sequencing may be used in order to check the cloning
step.
Transformed cells, i.e. those which contain the introduced DNA
integrated into the DNA of the host cell may be selected from
untransformed cells, if a selectable marker is part of the
0817/00024 CA 02454127 2004-01-09
=
44
introduced DNA. A marker may be, by way of example, any gene
which is capable of imparting a resistance to antibiotics or
herbicides. Transformed cells which express such a marker gene
are capable of surviving in the presence of concentrations of an
appropriate antibiotic or herbicide, which kill an untransformed
wild type. Examples are the bar gene which imparts resistance to
the herbicide phosphinothricin (Rathore KS et al., Plant Mol
Biol. 1993 Mar;21(5):871-884), the nptII gene which imparts
resistance to kanamycin, the hpt gene which imparts resistance to
hygromycin and the EPSP gene which imparts resistance to the
herbicide glyphosate.
Depending on the method of DNA introduction, further genes may be
required on the vector plasmid. If agrobacteria are used, the
expression cassette is to be integrated into specific plasmids,
either into an intermediate vector (shuttle vector) or a binary
vector. If, for example, a Ti or Ri plasmid is to be used for
transformation, at least the right border, in most cases,
however, the right and the left border, of the Ti or Ri plasmid
T-DNA is connected as flanking region with the expression
cassette to be introduced. Preference is given to using binary
vectors. Binary vectors can replicate both in E.coli and in
Agrobacterium. They normally contain a selection marker gene and
a linker or polylinker flanked by the right and left T-DNA border
sequences. They may be transformed directly into Agrobacterium
(Holsters et al.,Mol. Gen. Genet. 163 (1978), 181-187). The
selection marker gene permits selection of transformed
Agrobacteria; an example is the nptII gene which imparts a
resistance to kanamycin. The Agrobacterium which in this case
acts as the host organism should already contain a plasmid with
the vir region. This region is required for the transfer of T-DNA
into the plant cell. An Agrobacterium transformed in this way may
be used for transformation of plant cells.
The use of T-DNA for transformation of plant cells has been
intensely studied and described (EP 120516; Hoekema, In: The
Binary Plant Vector System, Off setdrukkerij Kanters B.V.,
Alblasserdam, Chapter V; Fraley et al., Crit. Rev. Plant. Sci.,
4:1-46 and An et al., EMBO J. 4 (1985), 277-287). Various binary
vectors are known and partly commercially available, such as, for
example, pBIN19 (Clontech Laboratories, Inc. U.S.A.).
The DNA is transferred into the plant cell by coculturing plant
explants with Agrobacterium tumefaciens or Agrobacterium
rhizogenes. Starting from infected plant material (e.g. leaf,
root or stem parts, but also protoplasts or plant cell
suspensions), it is possible to regenerate whole plants by using
0817/00024 CA 02454127 2004-01-09
a suitable medium which may contain, for example, antibiotics or
biocides for selection of transformed cells. The plants obtained
may then be screened for the presence of the introduced DNA, in
this case the expression cassette of the invention. As soon as
5 the DNA has integrated into the host genome, the corresponding
genotype is normally stable and the corresponding insertion is
also found again in subsequent generations. Normally, the
integrated expression cassette contains a selection marker which
imparts to the transformed plant a resistance to a biocide (for
10 example a herbicide), a metabolism inhibitor such as 2-DOG or an
antibiotic such as kanamycin, G 418, bleomycin, hygromycin or
phosphinothricin etc. The selection marker allows the selection =
of transformed cells from untransformed cells (McCormick et
al.,(1986) Plant Cell Reports 5: 81-84). The plants obtained may
15 be cultivated and crossed in the common manner. Two or more
generations should be cultured in order to ensure that the
genomic integration is stable and heritable.
The abovementioned methods are described, for example, in B.
20 Jenes et al., Techniques for Gene Transfer, in: Transgenic
Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung
and R. Wu, Academic Press (1993), pp.128 - 143 and in Potrykus
,(1991) Annu. Rev. Plant Physiol. Plant Molec. Biol. 42: 205 -
225). The construct to be expressed is preferably cloned into a
25 vector which is suitable for transforming Agrobacterium
tumefaciens, for example pBin19 (Bevan et al.,(1984) Nucl. Acids
Res. 12: 8711f.).
As soon as a transformed plant cell has been prepared, it is
30 possible to obtain a complete plant by using methods known to the
skilled worker. To this end, callus cultures are used as starting
point, by way of example. From these still undifferentiated cell
masses, it is possible to induce formation of shoot and root in
the known manner. The shoots obtained can be planted out and
35 cultivated.
The efficacy of expression of the nucleic acids to be expressed
transgenically can be determined, for example, in vitro by shoot
meristem propagation using one of the above-described selection
40 methods.
The invention further relates to cells, cell cultures, parts,
such as, for example, roots, leaves, etc. in the case of
transgenic plant organisms, and transgenic propagation material
45 such as seeds or fruits derived from the above-described
transgenic organisms.
0817/00024 CA 02454127 2004-01-09
46
Genetically modified plants of the invention, which can be
consumed by humans and animals, may also be used, for example
directly or after preparation known per se, as foodstuffs or
feedstuffs.
=
The invention further relates to the use of the above-described
transgenic organisms of the invention and of the cells, cell =
cultures, parts, such as, for example, roots, leaves, etc., in
the case of transgenic plant organisms, and transgenic
propagation material such as seeds or fruits for the production
of food- or feedstuffs, pharmaceuticals or fine chemicals.
Preference is further given to a method for the recombinant
=production of pharmaceuticals or fine chemicals in host
organisms, in which a host organism is transformed with one of
the above-described expression cassettes or vectors and said
expression cassette contains one or more structural genes which
code for the fine chemical of interest or catalyze the
biosynthesis of the fine chemical of interest, and the
transformed host organism is cultivated and the fine chemical of
interest is isolated from the cultivation medium. This method is
broadly applicable for fine chemicals such as enzymes, vitamins,
amino _acids, sugars, fatty acids, natural and synthetic
flavorings, aromatizing substances and colorants. Particular
preference is given to the production of tocopherols and
tocotrienols and also carotenoids. Cultivation of the transformed
host organisms and isolation from said host organisms or from the
cultivation medium are carried out by means of the methods known
to the skilled worker. The production of pharmaceuticals such as,
for example, antibodies or vaccines is described in Hood EE,
Jilka JM (1999). Curr Opin Biotechnol. 10(4):382-6; Ma JK, Vine
ND (1999) Curr Top Microbiol Immunol. 236:275-92.
Sequences
=
1. SEQ ID NO: 1
Promoter and 5'-untranslated region of the Arabidopsis
thaliana pFD promoter.
2. SEQ ID NO: 2
Promoter and 5'-untranslated region of the Arabidopsis
thaliana FNR promoter.
3. SEQ ID NO: 3
Promoter and 5'-untranslated region of the Arabidopsis
0817/00024 CA 02454127 2004-01-09
47
thaliana TPT promoter (2038 bp).
4. SEQ ID NO: 4
Promoter and 5'-untranslated region of the truncated
Arabidopsis thaliana pFDs promoter
5. SEQ ID NO: 5
Nucleic acids coding for a phosphinothricin
acetyltransferase.
6. SEQ ID NO: 6
Amino acid sequence coding for a phosphinothricin
acetyltransferase.
7. SEQ ID NO: 7
Nucleic acid coding for an acetolactate synthase.
8. SEQ ID NO: 8
Amino acid sequence coding for an acetolactate synthase.
9. SEQ ID NO: 9 - oligonucleotide primer pWL35
5'-GTC GAC GAA TTC GAG AGA CAG AGA GAC GG-3'
10. SEQ ID NO: 10 - oligonucleotide primer pWL36
5'-GTC GAC GGT ACC GAT TCA AGC TTC ACT GC-3'
11. SEQ ID NO: 11 - oligonucleotide primer pFD1
5'-GAG AAT TCG ATT CAA GCT TCA CTG C-3'
12. SEQ ID NO: 12 - oligonucleotide primer pFD2
5'-CCA TGG GAG AGA CAG AGA GAC G-3'
13. SEQ ID NO: 13 - oligonucleotide primer pFD3
5'-acggatccgagagacagagagacggagacaaaa-3'
14. SEQ ID NO: 14 - oligonucleotide primer pFD5
5'-gcggatccaacactcttaacaccaaatcaaca-3'
15. SEQ ID NO: 15 - oligonucleotide primer L-FNR ara
5'-GTCGACGGATCCGGTTGATCAGAAGAAGAAGAAGAAGATGAACT-31
16. SEQ ID NO: 16 - oligonucleotide primer R-FNR ara
5J-GTCGACTCTAGATTCATTATTTCGATTTTGATTTCGTGACC -3'
CA 02454127 2004-01-09
= ,
48
17. SEQ ID NO: 17 - oligonucleotide primer L-TPTara
5'-AAGTCGACGGATCCATAACCAAAAGAACTCTGATCATGTACGTACCCATT-3'
18. SEQ ID NO: 18 - oligonucleotide primer R-TPTara
5'-AGACGTCGACTCTAGATGAAATCGAAATTCAGAGTTTTGATAGTGAGAGC-3'
19. SEQ ID NO: 19 - oligonucleotide primer ubi5
5'-CCAAACCATGGTAAGTTTGTCTAAAGCTTA-3'
20. SEQ ID NO: 20 - oligonucleotide primer ubi3
5'-CGGATCCTTTTGTGTTTCGTCTTCTCTCACG-3'
21. SEQ ID NO: 21 - oligonucleotide primer sqs5
5'-GTCTAGAGGCAAACCACCGAGTGTT-3'
22. SEQ ID NO: 22 - oligonucleotide primer sqs3
5'-CGGTACCTGTTTCCAGAAAATTTTGATTCAG-3'
23. SEQ ID NO: 23
binary plasmid pSUN3 (Sungene GmbH & Co KGaA)
24. SEQ ID NO: 24
binary plasmid pSUN5NPTIICat (Sungene GmbH & Co KGaA)
25. SEQ ID NO: 25
binary plasmid pSUN3PatNos (Sungene GmbH & Co KGaA)
26. SEQ ID NO: 26 - oligonucleotide primer 5-TPTara
5'-AAGTCGACGGATCCTGATAGCTTATACTCAAATTCA7CAAGTTAT-3'
27. SEQ ID NO: 27
truncated promoter and 5'-untranslated region of the
Arabidopsis thaliana TPT-Promoters (1318 bp).
28. SEQ ID NO: 28
nucleic acid sequence of the terminator of the potato
cathepsin D inhibitor gene (GenBank Acc. No.: X74985)
29. SEQ ID NO: 29
nucleic acid sequence of the terminator of the field bean
storage protein gene VfLE1B3 (GenBank Acc. No.: Z26489).
Description of the figures
0817/00024 CA 02454127 2004-01-09
49
1. Figure la-c: The TPT and the FNR promoters show a comparable
expression pattern in green tissue and in flowers of tobacco
and potato. GUS-histochemical stains are formed. The
intensity of the GUS blue stain corresponds to the shades of
gray displayed. The figures show:
In Figure la:
A: Potato leaves with a homogeneous intensive stain over the
entire leaf region.
B: Tobacco petioles, intensive blue stain, especially on the
edges and in the vascular regions (see arrow)
=
In Figure lb:
C: Tobacco stems, intensive blue stain, especially on the
edges (see arrow)
D: Tobacco internodia
In Figure lc:
E: Tobacco flower; blue stain, especially in sepals and
petals
2. Figure 2a-b: The TPT promoter and the FNR promoter show a
_ different expression pattern in vegetative and germinative
storage tissue of tobacco and potato. While the TPT promoter
is active here, the FNR promoter shows no expression. GUS
histochemical stains of tobacco seeds and tobacco seedlings
and also of potato tubers are shown. However, both promoters
exhibit again a comparable activity in seedlings. The
intensity of the GUS blue stain corresponds to the shades of
gray displayed. The figures show:
In Figure 2a:
A: Tobacco seeds. In the case of the TPT promoter,
individual blue stained seeds are visible (see arrow). In.
the case of the FNR promoter, no stains are detectable.
B: Potato tubers. In the case of the TPT promoter, a
homogenous strong blue stain of the potato tuber is
visible. In the case of the FNR promoter, only a very
weak stain is detectable, if at all.
In Figure 2b:
C: Tobacco seedlings (10 days old). Both promoters show a
comparable blue stain (see arrow).
3. Expression cassettes for the expression of
kanamycin-resistance (nptII) and phosphinothricin-resistance
(pat) markers. Cassette A permits expression of kanamycin
0817/00024 CA 02454127 2004-01-09
resistance under the TPT or FNR promoter, in addition to a
phosphinothricin resistance under the NOS promoter. Cassette
B permits expression of phosphinothricin resistance under the
TPT or FNR promoter, in addition to kanamycin resistance
5 under the NOS promoter.
LB, RE: left and right border, respectively, of
Agrobacterium T-DNA
nosP: NOS promoter
10 pat: nucleic acid sequence coding for phosphinothricin
acetyltransferase (pat)
nptII: kanamycin resistance gene
(Neomycin -
phosphotransferase)
nosT: NOS terminator
15 FNR-P: FNR promoter
TPT-P: TPT promoter
4. Regeneration of transformed tobacco plumulae under kanamycin
selection pressure (100 mg/1 kanamycin). A: transformation
20 with an FNR promoter - nptII construct. 8: transformation
with a TPT promoter - nptII construct. A comparable efficient
regeneration of transformed tobacco plants was observed.
5. Germination of transformed tobacco plants from transgenic
25 tobacco seeds under phosphinothricin selection pressure
(10 mg/1 phosphinotricin).
A: transformed with an FNR promoter - pat construct.
B: transformed with a TPT promoter - pat construct.
30 C: control with untransformed tobacco seeds.
A comparably efficient germination of tobacco plants
transformed with the FNR promoter-pat construct and the
TPT promoter-pat construct was observed, while
untransformed tobacco plants treated in a corresponding .
35 manner had no resistance.
Examples
General methods:
The chemical synthesis of oligonucleotides may be carried out in
a manner known per se, for example according to the
phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press
New York, pages 896-897). The cloning steps carried out within
the framework of the present invention, such as, for example,
restriction cleavages, agarose gel electrophoreses, purification
of DNA fragments, transfer of nucleic acids to nitrocellulose and
CA 02454127 2011-11-17
51
nylon membranes, ligation of DNA fragments, transformation of E.
coli cells, cultivation of bacteria, propagation of phages and
sequence analysis of recombinant DNA, are carried out as
described in Sambrook et al. (1989) Cold Spring Harbor Laboratory
Press; ISBN 0-87969-309-6. Recombinant DNA molecules are
sequenced according to the method of Sanger (Sanger et al. (1977)
Proc Natl Acad Sci USA 74:5463-5467), using a laser fluorescence
DNA sequencer from ABI.
Example 1: Isolation of genomic DNA from Arabidopsis thaliana
(CTAB method)
Genomic DNA is isolated from Arabidopsis thaliana by grinding'
approx. 0.25 g of leaf material of young plants in the vegetative
state in liquid nitrogen to give a fine powder. The pulverulent
plant material is introduced together with 1 ml of 65 C CTAB I
buffer (CTAB: hexadecyltrimethylammonium bromide, also called
cetyltrimethylammonium bromide; Sigma Cat.-No.: H6269) and 20 pl
of p-mercaptoethanol into a prewarmed second mortar and, after
complete homogenization, the extract is transferred to a 2 ml
Eppendorf* vessel and incubated with careful regular mixing at 65 C
for 1 h. After cooling to room temperature, the mixture is
extracted with 1 ml of chloroform/octanol (24:1, equilibrated by
shaking with 1M Tris/HC1, pH8.0) by slowly inverting the vessel
and the phases are separated by centrifugation at 8,500 rpm
(7,500 x g) and room temperature for 5 min. Subsequently, the
aqueous phase is extracted again with 1 ml of chloroform/octanol,
centrifuged and carefully mixed with 1/10 volume of CTAB II
buffer prewarmed to 65 C by inverting the vessel. 1 ml of
chloroform/octanol mixture (see above) is then added with careful
agitation to the reaction mixture and the phases are again
separated by centrifugation at 8,500 rpm (7,500 x g) and room
temperature for 5 min. The aqueous lower phase is transferred to
a fresh Eppendorf vessel and the upper organic phase is again
centrifuged in a fresh Eppendorf vessel at 8,500 rpm (7,500 x g)
and room temperature for 15 min. The aqueous phase resulting
herefrom is combined with the aqueous phase of the previous
* Trademark
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centrifugation step and the entire reaction mixture is then mixed
with exactly the same volume of prewarmed CTAB III buffer. This
is followed by an incubation at 65 C until the DNA precipitates in
flakes. This may continue for up to 1 h or be effected by
incubation at 37 C overnight. The sediment resulting from the
subsequent centrifugation step (5 min, 2000 rpm (500 x g), 4 C) is
admixed with 250 1 of CTAB IV buffer prewarmed to 65 C, and the
mixture is incubated at 65 C for at least 30 min or until the
sediment has completely dissolved. The DNA is then precipitated
by mixing the solution with 2.5 volumes of ice-cold ethanol and
0817/00024 CA 02454127 2004-01-09
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incubating at ¨20 C for 1 h. As an alternative, the reaction
mixture is mixed with 0.6 volumes of isopropanol and, without
further incubation, immediately centrifuged at 8,500 rpm (7,500 x
g) and 4 C for 15 min. The sedimented DNA is washed twice with in
each case 1 ml of 80% strength ice-cold ethanol by inverting the
Eppendorf vessel, each washing step being followed by another
centrifugation (5 min, 8,500 rpm (7,500 x g), 4 C) and the DNA
pellet is then dried in air for approx. 15 min. Finally, the DNA
is resuspended in 100 !Al of TE comprising 100 g/ml RNase and the
mixture is incubated at room temperature for 30 min. After
another incubation phase at 4 C overnight, the DNA solution is
homogeneous and can be used for subsequent experiments.
=
= Solution for CTAB:
Solution I (for 200 ml):
100 mM Tris/HC1 pH 8.0 (2.42 g)
1.4 M NaC1 (16.36 g)
mM EDTA (8.0 ml of 0.5 M stock solution)
20 2 %(w/v) CTAB (4.0 g)
The following is added in each case prior to use: 2%
p-mercaptoethanol (20 1 for 1 ml of solution I).
Solution II (for 200 ml):
0.7 M NaC1 (8.18 g)
10 %(w/v) CTAB (20 g)
Solution III (for 200 ml):
50 mM Tris/HC1 pH 8.0 (1.21 g)
10 mM EDTA (4 ml 0.5 M of 0.5 M stock solution)
1 %(w/v) CTAB (2.0 g)
Solution IV (High-salt TE) (for 200 ml):
10.mM Tris/ HC]. pH 8.0 (0.242 g)
0,1 mM EDTA (40 1 of 0.5 M stock solution)
1 M NaCl (11.69 g)
Chloroform/Octanol (24:1) (for 200 ml):
192 ml of chloroform
8 ml of octanol
The mixture is equilibrated by shaking 2x with 1 M Tris/HC1 pH
8.0 and stored protected from light.
Example 2: Transformation of tobacco, oilseed rape and potato
CA 02454127 2010-07-29
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Tobacco was transformed via infection with Agrobacterium
tumefaciens. [sic] according to the method developed by Borsch
(Borsch et al. (1985) Science 227: 1229-1231). All constructs
used for transformation were transformed into Agrobacterium
tumefaciens by using the freeze/thaw method (repeated thawing and
freezing). The Agrobacterium colonies comprising the desired
construct were selected on mannitol/glutamate medium comprising
50 g/m1 kanamycin, 50 g/ml ampicilin and 25 g/ml rifampicin.
Tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were
transformed by centrifuging 10 ml of an Agrobacterium tumefaciens
overnight culture grown under selection, discarding the
supernatant and resuspending the bacteria in the same volume of
antibiotics-free medium. Leaf disks of sterile plants (approx.
1 cm in diameter) were bathed in this bacteria solution in a
sterile Petri dish. The leaf disks were then laid out in Petri
dishes on MS medium (Murashige und Skoog (1962) Physiol Plant
15:473ff.) comprising 2% sucrose and 0.8% Bacto agar. After
incubation in the dark at 25 C for 2 days, they were transferred
to MS medium comprising 100 mg/I kanamycin, 500 mg/I Claforan*
1mg/1 benzylaminopurine (BAP), 0.2 mg/1 naphthylacetic acid
(NAA), 1.6% glucose and 0.8% Bacto agar and cultivation was
continued (16 hours light / 8 hours dark). Growing shoots were
transferred to hormone-free MS medium comprising 2% sucrose,
250 mg/1 Claforan and 0.8% Bacto agar.
Oilseed rape was transformed by means of petiole transformation
according to Moloney et al. (Moloney mm, Walker JM & Sharma KK
(1989) Plant Cell Reports 8:238-242).
Potatoes (Solanum tuberosum) were transformed by infecting leaf
disks and internodia of in vitro plants with Agrobacterium
tumefaciens in liquid murashige Skoog medium for 20 minutes and
then coculturing them in the dark for 2 d. After coculturing, the
expIants were cultured on solid MS medium which contains instead
of sucrose 1.6% glucose (MG) and which has been supplemented with
5 mg/I NAA, 0.1 mg/I BAP, 250 mg/I Timentin* and 30 to 40 mg/I
*trademarks
CA 02454127 2010-07-29
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kanamycin (KIM), at 21 C in a 16h light/8h dark rhythm. After this
callus phase, the explants were placed on shoot induction medium
(SIM). SIM was composed as follows: MG + 2 mg/1 Zeatinriboside,
0.02 mg/1 NAA, 0.02 mg/1 GA3, 250 mg/1 Timentin, 30 to 40 mg/1
kanamycin. Every two weeks, the explants were transferred to
fresh SIM. The developing shoots were rooted on MS medium
comprising 2% sucrose and 250 mg/1 Timentin and 30 to 40 mg/1
kanamycin.
Example 3: Studies on the suitability of the putative ferredoxin
(pFD) promoter
a) Cloning of the pFD promoters from Arabidopsis thaliana
The putative ferredoxin promoter was amplified from genomic
Arabidopsis thaliana DNA by means of PCR using the primers pWL35
and pWL36. The primer pWL35 starts with the Sall and EcoRI
restriction cleavage sites which are located immediately upstream
of the coding region of the pFD gene and are highlighted in bold
type. The primer pWL36 starts with the Sail and Asp718
restriction cleavage sites highlighted in bold type.
Primer pWL35 (SEQ ID NO: 9)
5' GTC GAC GAA TTC GAG AGA CAG AGA GAC GG 3'
Primer pWL36 (SEQ ID NO: 10)
5' GTC GAC GGT ACC GAT TCA AGC TTC ACT GC 3'
Reaction mixture:
1 [11 Genomic Arabidopsis DNA (approx. 250 ng)
0.5 pl Tth* polymerase (2U/p1)
3 pl Mg (0Ac)2 (25 mM, final conc. 1.5 mM Mg2+)
15.2 pl 3.3 x buffer
4 pl dNTPs (2.5 mM each, Takara*, final concentration: 200 pM
each)
24.3 pl H20
* trademarks
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PCR conditions:
1 cycle at 95 C for 3 min
cycles at 94 C for 10 s, 50 C for 20 s and 72 C for 1
min.
cycles at 94 C for 10 s, 65 C for 20 s and 72 C for 1
min.
1. cycle at 72 C for 5 min.
followed by cooling to 4 C until further use.
b) Construction of the pFD promoter-GUS expression cassette
The PCR product of the pFD promoter was cloned into the pCRII
10 vector (Invitrogen) and subsequently isolated by means of the
Sail restriction cleavage sites introduced by the pair of primers
and purified by gel electrophoresis. For fusion with the GUS
gene, the approx. 850 bp pFD promoter fragment was cloned into
the Sail-cut binary vector pBI101.2 (Clontech Inc.) and the
orientation of the fragment was subsequently verified on the
0817/00024 CA 02454127 2004-01-09
basis of restriction analyses using the endonucleases BglII and
BamHI. The resulting plasmid pFD::GUS was transformed into
tobacco. The tobacco plants generated were denoted pFD:GUS.
5 c) Construction of the pFD promoter-nptII expression cassette
The putative ferredoxin promoter was amplified from genomic
Arabidopsis thaliana DNA by means of PCR. The primers were used
to add the restriction sites EcoRI and NcoI.
10=
Primer pFD1 (SEQ ID NO: 11)
5' GAG AAT TCG ATT CAA GCT TCA CTG C
Primer pFD2 (SEQ ID NO: 12)
15 5' CCA TGG GAG AGA CAG AGA GAC G
Reaction mixture:
37.5 1.41 H20
5.0 R1 10X reaction buffer (final concentration Mg2+ 1.5 mM)
20 4.0 1 dNTP mix (2.5 mM each)
1.0 R1 Primer pFD1 (10 RM)
1.0 R1 Primer pFD2 (10 RM)
_ Q,5 111 Tag polymerase (Takara, 2U/R1)
1.0 R1 genomic Arabidopsis DNA (approx. 250 ng)
PCR conditions:
1 cycle at 94 C for 3 min
10 cycles at 94 C for 10 s, 48 C for 20 s and 72 C for 1
min.
25 cycles at 94 C for 10 s, 65 C for 20 s and 72 C for 1
min.
1 cycle at 72 C for 5 min.
The PCR product was subcloned into the pCRII plasmid
(Invitrogen). The plasmid pCAMBIA 2300 (CAMBIA, GPO Box 3200,
Canberra ACT 2601, Australia; GenBank Acc. No: AF234315; Binary
vector pCAMBIA-2300, complete sequence; Hajdukiewicz P et al.
(1994) Plant Mol Biol 25(6):989-994) was cut with EcoRI/NcoI and
the pFD promoter fragment was cloned as EcoRI/NcoI fragment from
the pCRII plasmid into this vector. In the process, the 35S
promoter was removed from the Cambia vector. The resulting
plasmid was referred to as pFD promoter:NPTII and transformed
into tobacco.
d) Results of GUS analysis of the transgenic tobacco plants
. 0817/00024 CA 02454127 2004-01-09
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In the context of histochemical investigations, transgenic
pFD::GUS tobacco plants showed strong GUS staining in source
leaves and weak GUS staining in the tissues of all flower organs.
Strong staining in root tissue was only observed in in vitro
plants whose roots had been exposed to the illumination. Callus
growth was induced on the basis of leaf disks which had been
punched out of plants identified as pFD::GUS-positive. The callus
tissue and also the plant shoots developing therefrom showed GUS
staining whose intensity was comparable to that of the GUS
staining of CaMV35S::GUS (in pCambia 1304; CAMBIA, GPO Box 3200,
Canberra ACT 2601, Australia; GenBank Acc. No.: AF234300, Binary
vector pCAMBIA-1304, complete sequence, Hajdukiewicz P et al.
(1994) Plant Mol. Biol. 25(6):989-994) (1994)) transgenic plants.
The table listed below (Table 3) summarizes the data of
quantifying the GUS activity in the anthers and source leaves of
selected transgenic pFD::GUS tobacco plants.
Table 3: Quantification of GUS activity in anthers and source
leaves of selected transgenic pFD::GUS tobacco plants
GUS-Activity (pmol [4MU]fmg[protein]/min)
pFD :-: GUS Plant Anthers 'Source' leaves
no.
pFD5 275 3785
pFD11 174 6202
pFD14 362 2898
pFD15 57 2678
The anthers of the mature flowers display no promoter activity.
Said activity is weak in closed flowers.
e) Results of the analysis of kanamycin resistance of the
transgenic tobacco plants
In order to study the pFD promoter-assisted imparting of
resistance to kanamycin, the pFD promoter:NPTII plasmid was
transformed into tobacco. The tobacco plants were selectively
regenerated on kanamycin (100 mg/1). The plants regenerated from
the developing plumulae comprised kanamycin, demonstrating that
the pFD promoter had expressed the NPTII gene and thus made
selection possible. The results demonstrate that the isolated
nucleic acid sequence has the desired advantageous promoter
properties, i.e. it exhibits a promoter activity which is
suitable for expressing selection markers effectively and its
0817/00024 CA 02454127 2004-01-09
57
activity in the pollen is low. The activity in the anthers is
normally less than 10% of the activity in the source leaves.
f) Results of GUS analysis of the transgenic potato plants
The pFD:GUS plasmid (cf. Example 3 b) is transformed into
potatoes according to the method described in Example 2.
Result of functional studies: The pFD promoter is strongly
expressed in the leaves of the transgenic potato plants analyzed.
GUS staining was found to be stronger in the leaves of the potato
plants than in the leaves of the tobacco plants described. Weak -
staining of the flowers and no staining of the tubers indicated
low expression in the flowers and no expression in the tubers,
respectively.
The data demonstrate that this promoter has no activity in the
tubers of potato plants and is suitable for the expression of
genes, for example of insecticides, in the leaves and other
organs above the ground of plants, whose gene products are
unwanted in the storage organs.
g)_ Preparation of deletion variants of the pFD promoter
A further pFD promoter variant is the deletion pFD-short (pFds).
For this purpose, the pFD promoter section from base pairs 137
to 837 was amplified using the following primers:
pFD3 (SEQ ID NO: 13):
5'-acggatccgagagacagagagacggagacaaaa-3'
pFD5 (SEQ ID NO: 14):
5'gcggatccaacactcttaacaccaaatcaaca-3'
Reaction mixture:
37.5 111 R20
5.0 1 10X reaction buffer ("genomic PCR")
4.0 1 dNTP mix (2.5 mM each)
2.2 1 25 mM Mg(0Ac)2 (final concentration 1.1 mM)
1.0 1 Primer pFD3 (10 M)
1.0 1 Primer pFD5 (10 M.)
0.5 1 Pfu-turbo polymerase mix
1.0 1 Genomic Arabidopsis DNA (approx. 250 ng)
PCR conditions:
1 cycle at 95 C for 5 min
25 cycles at 94 C for 30 s, 50 C for 60 s and 72 C for
0817/00024 CA 02454127 2004-01-09
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1 min.
1 cycle at 50 C for 60 s, 72 C for 10 min,
followed by cooling to 4 C until further use
The primers comprised recognition sequences tor the restriction
enzyme BamHI. After BamHI cleavage, the PCR product was ligated
into the plasmid pGUSINT37 (see above) which had likewise been
cut with BamHI and had been dephosphorylated. Tobacco leaves were
bombarded with the resulting construct pFDsGUSINT by means of
Biolistics (BioRad). In this connection, microcarriers (25 g of
Gold, Heraeus 0.3 to 3 pm) were treated with 10 p..g of plasmid
DNA, 2.5 M CaCl2, and 0.1 M spermidine, washed with alcohol and =
fired at the leaves which were lying on MS medium under a vacuum
of 26 inches and a pressure of 1100 psi. The explants were then
incubated in MS medium comprising 2% sucrose for 24 h and then
histochemically stained with X-gluc. Blue spots indicated the
activity of the promoter.
h) Fusing the pFDs promoter to the NPTII gene
The pFDs promoter is excised as BamHI fragment from pFDsGUSINT
and its ends are rendered blunt by means of Klenow-÷Fill-In" .
The fragment obtained is cloned upstream of the NPTII gene of the
EcoRV-cut and dephosphorylated plasmid pSUN5NPTIICat (SEQ ID NO:
24). The plasmid pSUN5NPTII is a derivative of plasmid pSUN3 (SEQ
ID NO: 23), which contains, apart from nosP/Pat cassette, also a
promoterless NPTII gene. This construct makes it possible to
assay promoters on their ability to express NPTII. Selection on
phosphinothricin-comprising medium may be carried out in
parallel.
The resulting plasmid pSun5FdsNPTII is transformed into tobacco.
Regenerated and selected shoots showed that the pFDs promoter
allows selection for NPTII.
Example 4: Studies on the suitability of the ferredoxin NADPH
oxidoreductase (FNR) promoter
a) Cloning of the FNR promoter from Arabidopsis thaliana
The putative promoter region of the FNR gene was amplified from
genomic DNA by using the oligonucleotide primers L-FNRara and
R-FNRara, bypassing the ATG start codon of the FNR gene and
retaining four putative stop codons of the open reading frame
located upstream. Using the primers L-FNRara and R-FNRara, the
FNR promoter was amplified as a 635 bp fragment corresponding to
the section of the clone K2A18.15 from position 69493 to position
CA 02454127 2010-07-29
59
70127 (including these two nucleotides) from genomic Arabidopsis
thaliana DNA by means of PCR. The primer L-FNRara starts with th(
restriction cleavage sites Sail and BamHI highlighted in bold
type and is located upstream of the four stop codons of the gene
located upstream of the FNR promoter. The primer R-FNRara starts
with the Sall and XbaI restriction cleavage sites which are
located immediately upstream of the ATG start codon of the FNR
gene and are highlighted in bold type.
Primer L-FNR ara (44. mer) (SEQ ID NO: 15):
5' GTC GC GGA TCC GGT TGA TCA GAA GAA GAA GAA GAA GAT GAA CT 3'
Primer R-FNR ara (41 met) (SEQ ID NO: 16):
5' GTC GAC TCT AGA TTC ATT ATT TCG ATT TTG ATT TCG TGA CC 3'
The FNR promoter was amplified using a Ptouchdown" PCR protocol
with the use of the Advantage Genomic Polymerase Mix* (Clontech
Laboratories, Inc; Catalogue No. #8418-1). The above-mentioned
polymerase mix contains a thermostable DNA polymerase from
Thermus thermophilus (Tth DNA polymerase), mixed with a smaller
proportion of Vent proofreading 3'-5' polymerase, and the Tth
start antibody which makes hot-start PCR possible.
Reaction mixture:
36.8 1.11 H20
5 R1 10X reaction buffer ("genomic PCR")
1 R1 dNTP mix (10 mM each)
2.2 R1 25 mM Mg(0Ac)2.(fina1 concentration 1.1 mM)
1 R1 Primer L-FNR ara (10 RM)
1 R1 Primer R-FNR ara (10 p.m)
1 R1 50x polymerase mix
2 R1 Genomic Arabidopsis DNA (approx. 500 ng)
PCR conditions:
1 cycle at 94 C for 1 min.
10 cycles at 94 C for 30 s and 70 C for 3 min.
32 cycles at 94 C for 30 s and 65 C for 3 min.
1 ' cycle at 65 C for 4 min.,
followed by cooling to 4 C until further use.
b) Construction of the FNR promoter-GUS expression cassette
0817/00024 CA 02454127 2004-01-09
After gel-electrophoretic fractionation and purification from the
gel using the Quiagen PCR purification kit, the PCR product of
the FNR promoter was cloned into the pCRII vector (Invitrogen)
via TA cloning. The promoter fragment was then isolated from the
5 resulting plasmid pATFNR1 by digestion with Xbal/BamHI by means
of the XbaI and BamHI restriction cleavage sites introduced by
the pair of primers and purified by gel electrophoresis. For
fusion with the GUS gene, the approx. 600bp FNR promoter fragment
was cloned into the XbaI/BamHI-digested binary vector pBI101. The
10 correct insertion of the correct fragment in the resulting
plasmid pATFNR-Bi was then verified on the basis of a restriction
analysis using the endonuclease EcoRV. The plasmid pATFNR-Bi was -
used for transformation of tobacco.
15 For transformation in oilseed rape, the FNR promoter was cloned
as Sall fragment of plasmid pCR_ATFNR into the vector pS3NitGUS
cut with Sail and XhoI, thereby replacing the nitrilase promoter.
C) Construction of the FNR promoter-PAT expression cassette
In order to study the FNR promoter-assisted imparting of
resistance to phosphinothricin, the FNR promoter was cloned as
Sail fragment from plasmid pATFNR1 into the Sail-cut plasmid
pSUN3PatNos (SEQ ID NO: 25) upstream of the phosphinothricin
resistance gene.
d) Construction of the FNR promoter-NptII expression cassette
In order to impart resistance to kanamycin, the FNR promoter was
cloned as Sail fragment into the Xhar-cut dephosphorylated
plasmid pSUN5NptIICat (Sungene GmbH & Co KGaA, SEQ ID NO: 24)
upstream of the NPTII resistance gene. The resulting plasmid is
referred to as pS5FNRNptII and was transformed into tobacco and
oilseed rape.
e) Results of GUS analysis of the transgenic tobacco plants
Qualitative data:
Transgenic FNR::GUS-Tobacco plants displayed strong GUS
expression in all green tissues, especially in source leaves,
leaf stalks and internodia, and also in all flower organs of
fully developed flowers, (ovary, stigma, sepals and petals) with
the exception of pollen which showed no GUS activity; a low
staining intensity was detected in anthers. In the first analysis
of leaf disks of 80 in vitro plants, 70 plants displayed strong
GUS staining with low variation in staining intensity between the
0817/00024 CA 02454127 2004-01-09
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individual plants. This was regarded as an indication that the
FNR promoter contains an element which provides limited
positional effects. In the tissue culture plants, the GUS
activity of the FNR::GUS plants was markedly lower than the
activity of TPT::GUS plants. Transgenic oilseed rape plants
displayed the same staining pattern.
Seed material (Fl) of the lines FNR 13, FNR 45 and FNR 28 was
analyzed with respect to its GUS activity. It turned out that GUS
activity was detected neither in resting seeds nor in growing
seedlings (3.5 days after sowing).
In later seedlings stages (6 and 10 days after sowing), strong
GUS activity was detected in the cotyledons and in the upper
region of the seedling axis, whereas no GUS staining was detected
in the roots. In seedlings which had been cultivated in the dark,
GUS activity was limited to the cotyledons and was overall lower
than in the light-germinated seedlings.
Quantitative analysis of the GUS activity in FNR::GUS transgenic
tobacco plants (transformed with plasmidpATFNR-Bi) was analyzed
[sic] on the first fully developed leaves of tobacco plants 21
days _after transfer from the tissue culture to the greenhouse.
The data corresponds to the average of four independent
measurements.
Table 4: Quantification of GUS activity in leaf material of
selected transgenic FNR::GUS tobacco plants (transformed with
plasmidpATFNR-Bi)
=
Rank
FNR::GUS (x-strongest
GUS Activity
Standard .
Plant GUS activity
(pmol 4-MU/mg Protein/min) deviation
No. among 50
plants)
13 1 86491 2974
2 41726 1829
14 7 23951 2443
40 28 9 22148 401
17 10 21557 1157
30 20 13444 744
40 26 11972 1144
25 35 7662 519
45 35 39 5643 96
21 43 2858 194
C2-(WT) 49 28 4
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f) Result of analysis of phosphinothricin resistance of the
transgenic tobacco plants
The plasmid pSUN3FNRPat was used for transformation of tobacco by
using the Agrobacterium tumefaciens strain EHA101, as described
under 3. The tobacco plants are selectively regenerated either on
phosphinothricin (5 mg/1) or, as a control, on kanamycin
(100 mg/1). 97% of explants selected under kanamycin pressure
(nosP:NPTII) and 40% of explants selected under phosphinothricin
pressure (FNR:Bar) developed plumulae. The plants regenerated
under phosphinothricin pressure comprised both the kanamycin and
the phosphinothricin gene, demonstrating that the FNR promoter
had expressed the phosphinothricin acetyltransferase gene and
thus made selection possible. Seeds of the transgenic tobacco
plants were laid out on MS medium comprising 10 mg/1
phosphinothricin and the rate of germination was determined. In
contrast to the control of untransformed tobacco seeds, the
seedlings developed normally. The gene of phosphinothricin
acetyltransferase, which had been transferred and expressed via
the FNR promoter, was detected in the progeny of said lines by
means of PCR. The results demonstrated that the isolated nucleic
acid sequence has the desired advantageous promoter properties,
i.e.. It shows a promoter activity which is suitable for
expressing selection markers effectively and has no activity in
pollen.
g) Results of the analysis of kanamycin resistance of the
transgenic tobacco and oilseed rape plants
In order to study the FNR promoter-assisted imparting of
resistance to kanamycin, the FNR prompter was combined with the
NptII gene. The resulting construct pS5FNRNptII was transformed
into the Agrobacterium tumefaciens strain GV3101[mp90] for
transformation in tobacco and oilseed rape.
Seeds of the transgenic tobacco plants were laid out on MS medium
comprising 100 mg/1 kanamycin and the rate of germination was
determined. In contrast to the control of untransformed tobacco
seeds, the seedlings developed normally. The gene of neomycin
phosphotransferase (nptII), which had been transferred and
expressed via the FNR promoter, was detected in the progeny of
said lines by means of PCR.
The resulting strains have been used for transformation, as
described under Example 2. Selective regeneration was achieved in
the presence of 100 mg/1 (or 18 mg/1 in the case of oilseed rape)
kanamycin. The plants regenerated under kanamycin pressure
0817/00024 CA 02454127 2004-01-09
63
comprised both the kanamycin and the phosphinothricin gene,
demonstrating that the FNR promoter had expressed the NptII gene
and thus made selection of the plants possible.
=
The results demonstrated that the isolated nucleic acid sequence
has the desired advantageous promoter properties, i.e. it shows a
promoter activity which is suitable for expressing selection
markers effectively and has no activity in pollen.
h) Results of GUS analysis of the transgenic potato plants
The analysis of putatively transgenic potato plants showed in 20 =
lines a strong GUS staining in the leaves, comparable to the
expression pattern of tobacco plants. With the exception of 5
plants which showed a very weak staining in the potato tubers, no
FNR promoter expression was detected in the remaining plants.
The data demonstrate that this promoter has very weak, if any,
activity in the storage organs of potato plants and is suitable
for the expression of genes, for example of insecticides, in the
leaves and other organs above the ground of plants, whose gene
products are unwanted in the storage organs.
_
Example 5: Studies on the suitability of the triose phosphate
translocator (TPT) promoter
a) Cloning of the TPT promoter from Arabidopsis thaliana
The putative promoter region of the TPT gene from Arabidopsis
thaliana was isolated by amplification using the oligonucleotide
primers L-TPTara and R-TPTara, the ATG start codon of the TPT
gene being bypassed. Using the primers L-TPTara and R-TPTara, the
TPT promoter was amplified as a 2038 bp fragment from genomic
Arabidopsis thaliana DNA by means of PCR (SEQ ID NO: 3). The
primer L-TPTara starts with the Sail and Bamn restriction
cleavage sites highlighted in bold type. The primer R-TPTara
starts with the AatII, Sail and Xbal restriction cleavage sites
which are located immediately upstream of the ATG start codon of
the TPT gene and are highlighted in bold type.
Primer L-TPTara (SEQ ID NO: 17):
5' AAG TCG ACG GAT CCA TAA CCA AAA GAA CTC TGA TCA TGT ACG TAC
CCA TT 3'
Primer R-TPTara (SEQ ID NO: 18):
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5' AGA CGT CGA CTC TAG ATG AAA TCG AAA TTC AGA GTT TTG ATA GTG =
AGA GC 3'
The TPT promoter was amplified using a "touchdown" PCR protocol
with the use of the ,Advantage Genomic Polymerase Mix' (Clontech
Laboratories, Inc; Catalogue No. #8418-1). The above-mentioned
polymerase mix contains a thermostable DNA polymerase from
Thermus thermophilus (Tth DNA polymerase), mixed with a smaller
proportion of Vent proofreading 3'-5' polymerase, and the Tth
start antibody which makes hot-start PCR possible.
Reaction mixture:
36.8 pa H20
5 1 10X reaction buffer ("genomic PCR")
1 1 dNTP mix (10 mM each)
2.2 1 25 mM Mg(0Ac)2 (final concentration 1.1 mM)
1 1 Primer L-FNR ara (10 M)
1 1 Primer R-FNR ara (10 M)
1 1 50x polymerase mix
2 1 Genomic Arabidopsis DNA (approx. 500 ng)
PCR conditions:
1 cycle at 94 C for 1 min.
10 cycles at 94 C for 30 s and 70 C for 3 min.
32 cycles at 94 C for 30 s and 65 C for 3 min.
1 cycle at 65 C for 4 min.,
followed by cooling to 4 C until further use.
b) Construction of the TPT promoter-GUS expression cassette
After gel-electrophoretic fractionation and purification from the
gel using the Quiagen PCR purification kit, the PCR product of
the TPT promoter was cloned into the pCRII vector (Invitrogen)
via TA cloning. The promoter fragment was then isolated from the
resulting plasmid pATTPT by means of the Sail and XbaI
restriction cleavage sites introduced by the pair of primers and
purified by gel electrophoresis. For fusion with the GUS gene,
the approx. 2.0 kb TPT promoter fragment was cloned into the
Sa/I/XbaI-digested binary vector pBI101. The correct insertion of
the correct fragment in the resulting plasmid pATTPT-Bi was then
verified on the basis of a restriction analysis using the
endonuclease RindIII. The plasmid pATTPT-Bi was used for
transformation of tobacco.
0817/00024 CA 02454127 2004-01-09
For transformation in oilseed rape, the TPT promoter was cloned
as Sail fragment of plasmid pATTPT into the vector pS3NitGUS cut
with Sail and XhoI, thereby replacing the nitrilase promoter.
5 c) Construction of the TPT promoter-PAT expression cassette
In order to study the TPT promoter-assisted imparting of
resistance to phosphinothricin, the TPT promoter was cloned as
Sall fragment from plasmid pATTPT into the Sail-cut plasmid
10 pSUN3PatNos upstream of the phosphinothricin resistance gene. The
resulting' plasmid pSUN3TPTPat was used for transformation of
tobacco using the Agrobacterium tumefaciens strain EHA101. The
tobacco plants were selectively regenerated either on
phosphinothricin (5 mg/1) or, as a control, on kanamycin (100
15 mg/1).
d) Construction of the TPT promoter-NptII expression cassette
In order to impart resistance to kanamycin, the TPT promoter was
20 cloned as Sall fragment into the Xho/-cut dephosphorylated
plasmid pSUN5NptIICat (Sungene GmbH & Co KGaA, SEQ ID NO: 24)
upstream of the NPTII resistance gene. The resulting plasmid is
referred to as pS5TPTNptII and was transformed into tobacco and
oilseed rape.
e) Results of GUS analysis of the transgenic tobacco plants
Qualitative data
Transgenic TPT::GUS-tobacco plants displayed strong GUS
expression in all green tissues, especially in source leaves,
here in particular in the trichomes and the flower organs of
young and fully developed flowers. GUS activity in the flower
region was strongest in the ovaries and in the stigma; staining
of the sepals and petals was somewhat weaker. The GUS activity
was lowest in the anthers. No GUS activity was detected in the
pollen. Transgenic oilseed rape plants showed the same staining
pattern.
In the first analysis of leaf disks of 80 in vitro-plants, 22
plants showed no staining whatsoever and 22 plants showed strong
GUS staining after staining for only 3 hours. The remaining
plants displayed a good variety of GUS stainings of various
intensities in the individual plants. In the tissue culture
plants, the GUS activity of the TPT::GUS plants was markedly
stronger than that of the FNR::GUS plants. Seed material (F1) of
the lines TPT 55 and TPT 60 were analyzed with respect to their
0817/00024 CA 02454127 2004-01-09
.s
66
GUS activity. It turned out that strong, GUS activity was
detected both in resting seeds and in growing seedlings (3.5 days
after sowing). In later seedling stages (6 and 10 days after
sowing), the strongest GUS activity was detected in cotyledons
and in the upper region of the seedling axis and a weaker GUS
staining in the roots. Seedlings which had been cultivated in the
dark displayed an unchanged GUS staining pattern.
Quantitative data
Quantitative analysis of the GUS activity in TPT::GUS transgenic
tobacco plants (transformed with plasmidpAT-TPT-Bi) was carried
=
out on the first fully developed leaves of tobacco plants 19 days
after transfer from the tissue culture to the greenhouse. The
data correspond to the average of four independent measurements.
Table 5: Quantification of GUS activity in leaf tissue of
selected transgenic TPT::GUS tobacco plants (transformed with
plasmidpAT-TPT-Bi). WT2: controls from untransformed wild-type
plants.
Rank
TPT::GUS
(x-strongest GUS GUS Activity
Plant Standard
activity among (pmol 4-MU/mg Protein/min)
no. deviation
50 plants)
55 1st 62910 3576
15 2nd 58251 2533
10 5th 36759 1008
60 10th 19536 1783
56 11th 18876 1177
43 12th 18858 1404
27 35th 7390 233
44 59th 311 24
80-WT2 80th 5 13
f) Results of GUS analysis of the transgenic potato plants
The analysis of putatively transgenic potato plants showed in 28
lines strong GUS staining in the leaves, comparable to the
expression pattern of tobacco plants. A strong staining was
likewise detected in the potato tubers of the transgenic plants.
This demonstrates that the TPT promoter is expressed strongly and
ubiquitously in potatoes, too.
0817/00024 CA 02454127 2004-01-09
67
g) Results of the analysis of phosphinothricin resistance of the
transgenic tobacco plants
The plasmid pSUN3TPTPat was used for transformation of tobacco by
using the Agrobacterium tumefaciens strain EHA101, as described
under 3. The tobacco plants are selectively regenerated either on
phosphinothricin (5 mg/1) or, as a control, on kanamycin
(100 mg/1). 97% of explants selected under kanamycin pressure and
70% of explants selected under phosphinothricin pressure
developed plumulae. The plants regenerated under phosphinothricin
pressure comprised both the kanamycin and the phosphinothricin
gene, demonstrating that the TPT promoter had expressed the
phosphinothricin acetyltransferase gene and thus made selection
possible. Seeds of the transgenic tobacco plants were laid out on
MS medium comprising 10 mg/1 phosphinothricin and the rate of
germination was determined. In contrast to the control of
untransformed tobacco seeds, the seedlings developed normally.
The gene of phosphinothricin acetyltransferase, which had been
= transferred and expressed via the TPT promoter, was detected in
the progeny of said lines by means of PCR. The results
demonstrate that the isolated nucleic acid sequence has the
desired advantageous promoter properties, i.e. it shows a
pramater activity which is suitable for expressing selection
markers effectively and has no activity in pollen.
h) Results of the analysis of kanamycin resistance of the
transgenic tobacco and oilseed rape plants
In order to study the TPT promoter-assisted imparting of
resistance to kanamycin, the TPT promoter was combined with the
NptII gene. The resulting construct pS5TPTNptII was transformed
into the Agrobacterium tumefaciens strain GV3101[mp90] for
transformation in tobacco and oilseed rape.
The resulting strains have been used for transformation, as
described under Example 2. Selective regeneration was achieved in
the presence of 100 mg/1 (or 18 mg/1 in the case of oilseed rape)
kanamycin. The plants regenerated under kanamycin pressure
comprised both the kanamycin and the phosphinothricin gene,
demonstrating that the TPT promoter had expressed the NptII gene
and thus made selection of the plants possible.
Seeds of the transgenic tobacco plants were laid out on MS medium
comprising 100 mg/1 kanamycin and the rate of germination was
determined. In contrast to the control of untransformed tobacco
seeds, the seedlings developed normally. The gene of neomycin
0817/00024 CA 02454127 2004-01-09
68
phosphotransferase (nptII), which had been transferred and
expressed via the TPT promoter, was detected in the progeny of
said lines by means of PCR.
The results demonstrate that the isolated nucleic acid sequence
has the desired advantageous promoter properties, i.e. it shows a
promoter activity which is suitable for expressing selection
markers effectively and has no activity in pollen.
i) Cloning of the truncated TPT promoter (STPT)
The truncated putative promoter region of the Arabidopsis =
thaliana TPT gene (STPT) was isolated from the plasmid pATTPT
(SEQ ID NO: 27) by amplification by means of PCR using the primer
5-TPTara (SEQ-ID NO: 26) and R-TPTara (see above SEQ ID NO: 18).
Reaction mixture:
37.8 R1 1120
5 R1 10X Reaction buffer ("genomic PCR")
1 R1 dNTP mix (2.5 mM each)
2.2 R1 25 mM Mg(0Ac)2 (final concentration 1.1 mM)
_ l_R1 Primer 5-TPTara (10 RM)
1 R1 Primer R-TPTara (10 RM)
1 R1 50x polymerase mix ("Advantage Genomic Polymerase
Mix"; Clontech Lab., Inc.; Cat.-No.: #8418-1)
1 R1 pATTPT plasmid DNA (1 ng)
PCR conditions:
1 cycle at 94 C for 5 min
25 cycles at 94 C for 30 s and 52 C for 1 min.
1 cycle at 52 C for 4 min., followed by cooling to
4 C until further use.
Primer 5-TPTara (SEQ-ID NO: 26)
5'-AAG TCG ACG GAT CCT-GAT-AGC-TTA-TAC-TCA-AAT-TCA-ACA-AGT-TAT-3'
The 1.3 kb PCR product of the truncated TPT promoter was cloned,
after gel-electrophoretic fractionation and purification from the
gel, into the Sinai-cut and dephosphorylated vector pUC18, using
the SureClone Ligation Kit (Amersham Pharmacia Biotech; Code-No.:
27-9300-01). The resulting plasmid is referred to as pATSTPT. The
sequence was checked by means of sequencing.
j) Construction of the STPT promoter-NptII expression cassette
0817/00024 CA 02454127 2004-01-09
69
In order to impart resistance to kanamycin, the STPT promoter
(SEQ ID NO: 27) was cloned as Sall fragment into the Xho/-cut
dephosphorylated plasmid pSUN5NptIICat (Sungene GmbH & Co KGaA,
SEQ ID NO: 24) upstream of the NptII resistance gene. The
resulting plasmid is referred to as pS5STPTNptII and was
transformed into tobacco and oilseed rape.
k) Results of the analysis of kanamycin resistance of the
transgenic tobacco and oilseed rape plants
In order to study the STPT promoter-assisted imparting of
resistance to kanamycin, the plasmid pS5STPTNptII was transformed -
into the Agrobacterium tumefaciens strain GV3101[mp90] for
transformation into tobacco and oilseed rape. The resulting
strain has been used for transformation, as described under
Example 2. Selective regeneration was achieved in the presence of
100 mg/1 (or 18 mg/1 for oilseed rape) kanamycin.
The results demonstrate that the isolated nucleic acid sequence
has the desired advantageous promoter properties, i.e. it shows a
promoter activity which is suitable for expressing selection
markers effectively.
Example 6: Comparison of the transformation
efficiencies of the FNR and TPT promoters and of
the NOS promoter
In a comparative experiment, the efficiency of the transformation
of tobacco was determined using the FNR promoter (FNR-P), the TPT
promoter (TPT-P) and the Nos promoter (Nos-P). The promoters
were, as described, fused in each case to the NptII gene. After
the plumulae had formed and, respectively, the shoot had roots on
kanamycin-comprising medium, the resistant transformants were
counted and their numbers were compared. A PCR which was used to .
detect the NptII gene showed the high proportion of transgenic
plants.
NOS-? FNR-P TPT-P
Shoot formation 100 % 68 % 76 %
Rooted plants 80 % 81 % 80 %
Transgenic 92 % 100 % 100 %
plants
Table 6: Transformation efficiency
CA 02454127 2010-07-29
Comparative Example 1: Studies of the suitability of the
ubiquitin promoter
a) Cloning of the ubiquitin promoter from Arabidopsis thaliana
The ubiquitin promoter was amplified from genomic Arabidopsis
thaliana DNA by means of PCR using the primers ubi5 and ubi3.
ubi5 (SEQ ID NO: 19) :
5'-CCAAACCATGGTAAGTTTGTCTAAAGCTTA-3'
ubi3 (SEQ ID NO: 20):
10 5'-CGGATCCTTTTGTGTTTCGTCTTCTCTCACG-3'
Reaction mixture:
37.5 1 E20
5 4 10X reaction buffer ("genomic PCR")
4 1 .dNTP mix (2.5 mM each)
2.2 I 25 mM Mg(0Ac)2 (final concentration 1.1 mM)
1 1 Primer ubi3 (10 M)
1 1 Primer ubi5 (10 M)
0.5 pl Pfu*-turbo polymerase mix
1 pl Genomic Arabidopsis DNA (ca. 250 ng)
PCR conditions:
20 1 cycle at 94 C for 5 min.
25 cycles at 94 C for 30 s, 52 C for 1 min. and 72 C
= for 1 min.
1 cycle at 52 C for 1 min. and 72 C for 10 min.,
followed by cooling to 4 C until further use.
The resultant PCR fragment was cooled as HindIII/BamHI fragment
into the HindIII/BamHI-;cut plasmid pGUSINT37 (pUBI42GUS) and
verified by means of sequence analysis.
b) Cloning of the ubiquitin promoter upstream of the PAT gene
* trademark
CA 02454127 2010-07-29
70a
In order to study the ubiquitin promoter-assisted imparting of
resistance to phosphinothricin, the ubiquitin promoter was cloned
as BamHI/RindIII fragment into the BanHI/HindIII-cut plasmid
pSUN3PatNos upstream of the phosphinothricin resistance gene. The
resulting plaszld pSUN3UBIPat was used for transformation of
tobacco using the Agrobacterium tunefaciens strain EHA101. The
tobacco plants were selectively regenerated either on
0817/00024 CA 02454127 2004-01-09
71
phosphinothricin (5 mg/1) or, as a control, on kanamycin
(100 mg/1).
C) Results of the analysis of phosphinothricin resistance of the
transgenic tobacco plants
In contrast to selection on kanamycin, which was normal, no calli
or shoots were obtained under selection on phosphinothricin.
Thus, the ubiquitin promoter is unsuitable for expression of a
selective marker for the Agrobacterium tumefaci ens-mediated gene
transfer with subsequent regeneration of tissues.
Comparative Example 2: Studies on the suitability of the
squalene synthase (SQS) promoter
a) Cloning of the squalene synthase (SQS) promoter from
Arabidopsis thaliana
The squalene synthase promoter was amplified from genomic
Arabidopsis thaliana DNA by means of PCR using the primers sqs5
and sqs3.
sqs5 (SEQ ID NO: 21):
51-GTCTAGAGGCAAACCACCGAGTGTT-3'
sqs3 (SEQ ID NO: 22):
5i-CGGTACCTGTTTCCAGAAAATTTTGATTCAG-3'
Reaction mixture:
37.5 R1 H20
5 R1 10X Reaction buffer ("genomic PCR")
4 R1 dNTP mix (2.5 mM each)
2.2 ml 25 mm mg(oAc)2 (final concentration 1.1 mM)
1 R1 Primer sqs3 (10RM) (10 .LM)
1 R1 Primer sqs5 (10 RM)
0.5 1 Pfu-turbo polymerase mix
1 R1 Genomic Arabidopsis DNA (approx. 250 ng)
PCR conditions:
1 cycle at 94 C for 5 min.
25 cycles at 94 C for 30 s, 52 C for 1 min. and 72 C
for 1 min.
1 cycle at 52 C for 1 min. and 72 C for 10 min.,
followed by cooling to 4 C until further use.
0817/00024 CA 02454127 2004-01-09
72
The resultant PCR fragment was cloned as XbaII/BamHI fragment
into the XbaII/BamHI-cut plasmid pGUSINT37 (pSQSPGUS) and
verified by means of sequence analysis.
b) Cloning of the squalene synthase promoter upstream of the PAT
gene
In order to study the squalene synthase promoter-assisted
imparting of resistance to phosphinothricin, the squalene
synthase promoter was cloned as BamHI/SalI fragment into the
BamHI/SaIII-cut plasmid pSUN3PatNos upstream of the
phosphinothricin resistance gene. The resulting plasmid =
pSUN3SQSPat was used for transformation of tobacco using the
Agrobacterium tumefaciens strain EHA101. The tobacco plants were
selectively regenerated either on phosphinothricin (5 mg/1) or,
as a control, on kanamycin (100 mg/1).
C)
Results of the analysis of phosphinothricin resistance of the
transgenic tobacco plants
In contrast to selection on kanamycin, which was normal, no calli
or shoots were obtained under selection on phosphinothricin.
Thus,.. the ubiquitin [sic] promoter is unsuitable for expression
of a selective marker for the Agrobacterium tumefaciens-mediated
gene transfer with subsequent regeneration of tissues.
Comparative Example 3:
Promoter activity assay of the ubiquitin
and squalene synthase-promoter by means
of a particle gun
In order to assay the activity of the ubiquitin promoter and the
squalene synthase promoter, sterile tobacco leaves were bombarded
with plasmid DNA of plasmids pUBI42GUS, pSQSPGUS and pGUSINT37 by
means of the BioRad Biolistics particle gun. In this connection,
microcarriers (25 g of Gold, Heraeus 0.3 to 3 m) were treated
with 10 g of plasmid DNA, 2.5 M CaC12, and 0.1 M spermidine,
washed with alcohol and fired at the leaves which were lying on
MS agar medium under a vacuum of 26 inches and a pressure of
1100 psi. The explants were then incubated in MS medium
comprising 2% sucrose for 24 h and then histochemically stained
with X-gluc.
In contrast to the comparative construct pGUSINT37 in which the
GUS gene was expressed under the control of the 35S promoter, the
ubiquitin promoter and the squalene synthase promoter showed only
=
0817/00024 CA 02454127 2004-01-09
73
very few and very weak GUS-stained dots. This indicates that the
ubiquitin and squalene synthase promoter activities are
distinctly weaker than the CaMV35S promoter activity.
_
30
CA 02454127 2004-04-07
SEQUENCE LISTING
<110> SunGene GmbH & Co KGaA
<120> Expression cassettes for the transgenic expression of
nucleic acids
<130> 003230-3097
<140> 2.454.127
<141> 2002-07-05
<150> PCT/EP02/07527
<151> 2002-07-05
<150> DE 102 07 582.4
<151> 2002-02-22
<150> DE 101 59 455.0
<151> 2001-12-04
<150> DE 101 33 407.9
<151> 2001-07-13
<160> 29
<170> PatentIn Ver. 2.1
<210> 1
<211> 836
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> promoter
<222> (1)..(836)
<223> pFD promoter
<400> 1
gattcaagct tcactgctta aattcacaaa aagagaaaag taagaccaaa ggaataaatc 60
atcctcaaac caaaaacaca tcatacaaaa tcatcaaaca taaatctcca gatgtatgag 120
caccaatcca gttatacaac actcttaaca ccaaatcaac agatttaaca gcgaaataag 180
cttaagccca tacaattatc cgatccaaac aaatataatc gaaaccggca gaggaataag 240
caagtgaatc aaaaagtatg ggacgaggaa gaagatgata cctgaatgag aaagtcaata 300
accttgaccc gaatcgtttt gaagaaaatg gagaaaatcg gttgtatgga ataaaatctt 360
cgaatgatga gatatatgat ctctttggtg tcagtcacat ggcacacgct atcaatttag 420
aaaaacgcgg tggttggtca ccagaattac tacttctcgg tctgatttgg tcatatccgt 480
attaagtccg gttaatattt tccataactg gggtttgaac attcggtttc tttttttcag 540
ttagtccgat ttggagtttt gagtatggaa aaataatact gaatttattt gttcaaactg 600
ttttggaaaa aatatttccc ttaattacga atataattaa aattttaaaa ttcattttat 660
tagatcttgg ttaattcggt ttaatgcatt aatgaatttc ggtttaagtc ggttttcggt 720
ttttatgtcc caccactatc tacaaccgat gatcaacctt atctccgtat tcaccacaaa 780
cagtcatcac tctcacttga cacaaaaact cttttgtctc cgtctctctg tctctc 836
<210> 2
<211> 635
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> promoter
<222> (1)..(635)
<223> FNR promoter
Page 1
CA 02454127 2004-04-07
<400> 2
ttcattattt cgattttgat ttcgtgacca gcgaacgcag aataccttgt tgtgtaatac 60
tttacccgtg taaatcaaaa acaaaaaggc ttttgagctt tttgtagttg aatttctctg 120
gctgatcttt tctgtacaga ttcatatatc tgcagagacg atatcattga ttatttgagc 180
ttcttttgaa ctatttcgtg taatttggga tgagagctct atgtatgtgt gtaaactttg 240
aagacaacaa gaaaggtaac aagtgaggga gggatgactc catgtcaaaa tagatgtcat 300
aagaggccca tcaataagtg cttgagccca ttagctagcc cagtaactac cagattgtga 360
gatggatgtg tgaacagttt tttttttgat gtaggactga aatgtgaaca acaggcgcat 420
gaaaggctaa attaggacaa tgataagcag aaataactta tcctctctaa cacttggcct 480
cacattgccc ttcacacaat ccacacacat ccaatcacaa cctcatcata tatctcccgc 540
taatcttttt ttctttgatc tttttttttt tgcttattat ttttttgact ttgatctccc 600
atcagttcat cttcttcttc ttcttctgat caacc 635
<210> 3
<211> 2038
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> promoter
<222> (1)..(2038)
<223> TPT promoter
<400> 3
ataaccaaaa gaactctgat catgtacgta cccatttgcg tattccgccg ttgcggaatc 60
aaaaactgcc atagacttct ccacatcttt ttcagctcgc atcagtttga taacctgtga 120
aggcgtgatg ttctttgacc acttgaacat catcactttg ttacccatta ctaaacaatg 180
ataccttcca acaatagcaa agaatacacc tttttataga gaagaatctc agctacgcac 240
tacgtcgaac aggttgtgtg cataaacgat ttcgataggc ccaaaccaaa tgaaagaaac 300
acagaccaga aaaatcattt gatcttcaaa acatacgagt tccaaaagtg aaggaagcaa 360
caatgaaact cgttacgaac tagaaggtta atcaaattgc cggagaagaa tcgctcacca 420
gttttggcta gggtttatga aatgggagac tttagctgca aagagaagag tctctggacg 480
atttagaggg tgtctctcta ataggcaaca aagtacatat tattacagta ttaaccaaat 540
ttaaacgaat taagtgtcaa caaaagctta tataaaaaat ttaaagttta aaaattataa 600
aatatgtcaa caatatttta gtacttaaaa ttattatgcg aaatatttag atcaatggac 660
tactcatcta atatatttgc acctaatttt aaagtataaa ttcaaccaat aattagaaaa 720
tgatagctta tactcaaatt caacaagtta tatataaatg tatagatact acaatatcat 780
taacaaaagt caccttaaat aaatacacat atcttttatg ttctctattg ttttgcgtac 840
gctaacacaa tttctcatat gcaaaaggat gaatgagtaa caaattacct cataagaaca 900
atcatctttg cttacatact aatacaataa tcactcaatc aaccaataac atcaatcaca 960
taggtttaca tacaataatc actcaatcaa cttcataaga agaatcatgt ttacttaatt 1020
catcaattat ccccaaaaac accactatta agtataaact ataacatatt tgtagtgatg 1080
ggtcaacatt tttatcatat ttaaactcgg gttccctcaa atcgagaaat agtgaacatg 1140
taatattaat tttaaatcgc aattacagaa attaattgaa tttggtcaaa tggacagaat 1200
tttatagatt gggtggaact agaaaaaaaa aaaaaaagag tatagggtga attgagtaca 1260
tgaaagtaca tggtaatcct agttaaacgc ataatacatg tgggttcatt tgtatttttt 1320
tgtaacttac gagtaaactg gctacaacaa aaaaaattag aagatttttt tgttttgtag 1380
aaaaccctaa ttttagttat agttgtataa ctttgataaa attataaaat tgtattacga 1440
aaaaagtaat aagatattca aaaaagccta gaataacgta tatgactatg agcatgaaac 1500
tgcaagtcaa atgctgacag acaaccataa acaaaagaaa ttaaatagag atacctttaa 1560
aataagtaaa atttcattta taaaaaatct actttcttgt gaatctgtca cgttcaataa 1620
tttgaagacc actcaacata caaggtaaat aatgaaaaat aaaatctacc aaaatttcaa 1680
tcattattat cttccaaaaa aacaaaatta tacagatgat gatggtgata tggaacttcg 1740
attggctaat attcactgtg tctctaaaaa ccatccactt atcaagataa gatggaccct 1800
acactcatcc aatctaaacc agtatctcaa gattcttatc taattacatc attctctacc 1860
gttagatgaa attgaccatt aaccctacca taactccata caccgcgaga tactggatta 1920
accaaatcga gatcatcgta gccgtccgat caacaagtac catctcttga aatactcgaa 1980
atcctcataa gtccgtccct ctttgctctc actatcaaaa ctctgaattt cgatttca 2038
<210> 4
<211> 699
Page 2
CA 02454127 2004-04-07
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> promoter
<222> (1)..(699)
<223> FDS promoter
<400> 4
aacactctta acaccaaatc aacagattta acagcgaaat aagcttaagc ccatacaatt 60
atccgatcca aacaaatata atcgaaaccg gcagaggaat aagcaagtga atcaaaaagt 120
atgggacgag gaagaagatg atacctgaat gagaaagtca ataaccttga cccgaatcgt 180
tttgaagaaa atggagaaaa tcggttgtat ggaataaaat cttcgaatga tgagatatat 240
gatctctttg gtgtcagtca catggcacac gctatcaatt tagaaaaacg cggtggttgg 300
tcaccagaat tactacttct cggtctgatt tggtcatatc cgtattaagt ccggttaata 360
ttttccataa ctggggtttg aacattcggt ttcttttttt cagttagtcc gatttggagt 420
tttgagtatg gaaaaataat actgaattta tttgttcaaa ctgttttgga aaaaatattt 480
cccttaatta cgaatataat taaaatttta aaattcattt tattagatct tggttaattc 540
ggtttaatgc attaatgaat ttcggtttaa gtcggttttc ggtttttatg tcccaccact 600
atctacaacc gatgatcaac cttatctccg tattcaccac aaacagtcat cactctcact 660
tgacacaaaa actcttttgt ctccgtctct ctgtctctc 699
<210> 5
<211> 552
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence: codon
adapted sequence for
phosphinotricin-N-acetyltransferase
<220>
<221> CDS
<222> (1)..(549)
<223> phosphinotricin-N-acetyltransferase (PAT)
<400> 5
atg tct ccg gag agg aga cca gtt gag att agg cca gct aca gca gcc 48
Met Ser Pro Glu Arg Arg Pro Val Glu Ile Arg Pro Ala Thr Ala Ala
1 5 10 15
gat atg gcc gcg gtt tgt gac atc gtt aac cat tac att gag acg tct 96
Asp Met Ala Ala Val Cys Asp Ile Val Asn His Tyr Ile Glu Thr Ser
20 25 30
aca gtg aac ttt agg aca gag cca caa aca cca caa gag tgg att gat 144
Thr val Asn Phe Arg Thr Glu Pro Gln Thr Pro Gln Glu Trp Ile Asp
35 40 45
gac cta gag agg ttg caa gat aga tac cct tgg ttg gtt gct gag gtt 192
Asp Leu Glu Arg Leu Gln Asp Arg Tyr Pro Trp Leu Val Ala Glu val
50 55 60
gag ggt gtt gtg gct ggt att gct tac gct ggg ccc tgg aag gct agg 240
Glu Gly Val Val Ala Gly Ile Ala Tyr Ala Gly Pro Trp Lys Ala Arg
65 70 75 80
aac gct tac gat tgg aca gtt gag agt act gtt tac gtg tca cat agg 288
Asn Ala Tyr Asp Trp Thr Val Glu Ser Thr Val Tyr Val Ser His Arg
85 90 95
cat caa agg ttg ggc cta gga tct aca ttg tac aca cat ttg ctt aag 336
His Gln Arg Leu Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys
100 105 110
tct atg gag gcg caa ggt ttt aag tct gtg gtt gct gtt ata ggc ctt 384
Ser Met Glu Ala Gln Gly Phe Lys Ser Val val Ala Val Ile Gly Leu
115 120 125
cca aac gat cca tct gtt agg ttg cat gag gct ttg gga tac aca gcg 432
Page 3
CA 02454127 2004-04-07
Pro Asn Asp Pro Ser Val Arg Leu His Glu Ala Leu Gly Tyr Thr Ala
130 135 140
cgg ggt aca ttg cgc gcg gct gga tac aag cat ggt gga tgg cat gat 480
Arg Gly Thr Leu Arg Ala Ala Gly Tyr Lys His Gly Gly Trp His Asp
145 150 155 160
gtt ggt ttt tgg caa agg gat ttt gag ttg cca gct cct cca agg cca 528
Val Gly Phe Trp Gin Arg Asp Phe Glu Leu Pro Ala Pro Pro Arg Pro
165 170 175
gtt agg cca gtt acc cag atc tga 552
Val Arg Pro Val Thr Gln Ile
180
<210> 6
<211> 183
<212> PRT
<213> Artificial sequence
<220>
<223> Description of the artificial sequence: codon
adapted sequence for
phosphinotricin-N-acetyltransferase
<400> 6
Met Ser Pro Glu Arg Arg Pro Val Glu Ile Arg Pro Ala Thr Ala Ala
1 5 10 15
Asp Met Ala Ala Val Cys Asp Ile Val Asn His Tyr Ile Glu Thr Ser
20 25 30
Thr Val Asn Phe Arg Thr Glu Pro Gin Thr Pro Gin Glu Trp Ile AS
35 40 45
Asp Leu Glu Arg Leu Gin Asp Arg Tyr Pro Trp Leu Val Ala Glu Val
50 55 60
Glu Gly Val Val Ala Gly Ile Ala Tyr Ala Gly Pro Trp Lys Ala Arg
65 70 75 80
Asn Ala Tyr Asp Trp Thr Val Glu Ser Thr val Tyr val Ser His Arg
85 90 95
His Gin Arg Leu Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys
100 105 110
Ser Met Glu Ala Gin Gly Phe Lys Ser val Val Ala Val Ile Gly Leu
115 120 125
Pro Asn Asp Pro Ser val Arg Leu His Glu Ala Leu Gly Tyr Thr Ala
130 135 140
Arg Gly Thr Leu Arg Ala Ala Gly Tyr Lys His Gly Gly Trp His Asp
145 150 155 160
Val Gly Phe Trp Gin Arg Asp Phe Glu Leu Pro Ala Pro Pro Arg Pro
165 170 175
Val Arg Pro val Thr Gin Ile
180
<210> 7
<211> 2013
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (1)..(2010)
<223> acetolactate synthase
<400> 7
atg gcg gcg gca aca aca aca aca aca aca tct tct tcg atc tcc ttc 48
Met Ala Ala Ala Thr Thr Thr Thr Thr Thr Ser Ser Ser Ile Ser Phe
1 5 10 15
tcc acc aaa cca tct cct tcc tcc tcc aaa tca cca tta cca atc tcc 96
Page 4
CA 02454127 2004-04-07
Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser Pro Leu Pro Ile Ser
20 25 30
aga ttc tcc ctc cca ttc tcc cta aac ccc aac aaa tca tcc tcc tcc 144
Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn Lys Ser Ser Ser Ser
35 40 45
tcc cgc cgc cgc ggt atc aaa tcc agc tct ccc tcc tcc atc tcc gcc 192
Ser Arg Arg Arg Gly Ile Lys Ser Ser Ser Pro Ser Ser Ile Ser Ala
50 55 60
gtg ctc aac aca acc acc aat gtc aca acc act ccc tct cca acc aaa 240
Val Leu Asn Thr Thr Thr Asn Val Thr Thr Thr Pro Ser Pro Thr Lys
65 70 75 80
cct acc aaa ccc gaa aca ttc atc tcc cga ttc gct cca gat caa ccc 288
Pro Thr Lys Pro Glu Thr Phe Ile Ser Arg Phe Ala Pro Asp Gin Pro
85 90 95
cgc aaa ggc gct gat atc ctc gtc gaa gct tta gaa cgt caa ggc gta 336
Arg Lys Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg Gin Gly Val
100 105 110
gaa acc gta ttc gct tac cct gga ggt gca tca atg gag att cac caa 384
Glu Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His Gin
115 120 125
gcc tta acc cgc tct tcc tca atc cgt aac gtc ctt cct cgt cac gaa 432
Ala Leu Thr Arg Ser Ser Ser Ile Arg Asn Val Leu Pro Arg His Glu
130 135 140
caa gga ggt gta ttc gca gca gaa gga tac gct cga tcc tca ggt aaa 480
Gin Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser Gly Lys
145 150 155 160
cca ggt atc tgt ata gcc act tca ggt ccc gga gct aca aat ctc gtt 528
Pro Gly Ile Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu val
165 170 175
agc gga tta gcc gat gcg ttg tta gat agt gtt cct ctt gta gca atc 576
Ser Gly Leu Ala AS Ala Leu Leu Asp Ser Val Pro Leu Val Ala Ile
180 185 190
aca gga caa gtc cct cgt cgt atg att ggt aca gat gcg ttt caa gag 624
Thr Gly Gin Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gin Glu
195 200 205
act ccg att gtt gag gta acg cgt tcg att acg aag cat aac tat ctt 672
Thr Pro Ile val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu
210 215 220
gtg atg gat gtt gaa gat atc cct agg att att gag gaa gct ttc ttt 720
Val Met Asp Val Glu Asp Ile Pro Arg Ile Ile Glu Glu Ala Phe Phe
225 230 235 240
tta gct act tct ggt aga cct gga cct gtt ttg gtt gat gtt cct aaa 768
Leu Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Val Asp Val Pro Lys
245 250 255
gat att caa caa cag ctt gcg att cct aat tgg gaa cag gct atg aga 816
Asp Ile Gin Gin Gin Leu Ala Ile Pro Asn Trp Glu Gin Ala Met Arg
260 265 270
tta cct ggt tat atg tct agg atg cct aaa cct ccg gaa gat tct cat 864
Leu Pro Gly Tyr Met Ser Arg Met Pro Lys Pro Pro Glu Asp Ser His
275 280 285
ttg gag cag att gtt agg ttg att tct gag tct aag aag cct gtg ttg 912
Leu Glu Gin Ile Val Arg Leu Ile Ser Glu Ser Lys Lys Pro Val Leu
290 295 300
tat gtt ggt ggt ggt tgt ttg aat tct agc gat gaa ttg ggt agg ttt 960
Tyr val Gly Gly Gly Cys Leu ASt1 Ser Ser Asp Glu Leu Gly Arg Phe
305 310 315 320
gtt gag ctt acg ggg atc cct gtt gcg agt acg ttg atg ggg ctg gga 1008
Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly
325 330 335
tct tat cct tgt gat gat gag ttg tcg tta cat atg ctt gga atg cat 1056
Ser Tyr Pro Cys Asp Asp Glu Leu Ser Leu His Met Leu Gly Met HiS
340 345 350
ggg act gtg tat gca aat tac gct gtg gag cat agt gat ttg ttg ttg 1104
Gly Thr val Tyr Ala Asn Tyr Ala val Glu His Ser Asp Leu Leu Leu
Page 5
CA 02454127 2004-04-07
355 360 365
gcg ttt ggg gta agg ttt gat gat cgt gtc acg ggt aag ctt gag gct 1152
Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala
370 375 380
ttt gct agt agg gct aag att gtt cat att gat att gac tcg gct gag 1200
Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu
385 390 395 400
att ggg aag aat aag act cct cat gtg tct gtg tgt ggt gat gtt aag 1248
Ile Gly Lys Asn Lys Thr Pro His Val Ser Val Cys Gly Asp Val Lys
405 410 415
ctg gct ttg caa ggg atg aat aag gtt ctt gag aac cga gcg gag gag 1296
Leu Ala Leu Gin Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu
420 425 430
ctt aag ctt gat ttt gga gtt tgg agg aat gag ttg aac gta cag aaa 1344
Leu Lys Leu Asp Phe Gly Val Trp Arg Asn Glu Leu Asn val Gin Lys
435 440 445
cag aag ttt ccg ttg agc ttt aag acg ttt ggg gaa gct att cct cca 1392
Gin Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro
450 455 460
cag tat gcg att aag gtc ctt gat gag ttg act gat gga aaa gcc ata 1440
Gin Tyr Ala Ile Lys Val Leu Asp Glu Leu Thr Asp Gly Lys Ala Ile
465 470 475 480
ata agt act ggt gtc ggg caa cat caa atg tgg gcg gcg cag ttc tac 1488
Ile Ser Thr Gly Val Gly Gin His Gin Met Trp Ala Ala Gin Phe Tyr
485 490 495
aat tac aag aaa cca agg cag tgg cta tca tca gga ggc ctt gga gct 1536
Asn Tyr Lys Lys Pro Arg Gin Trp Leu Ser Ser Gly Gly Leu Gly Ala
500 505 510
atg gga ttt gga ctt cct gct gcg att gga gcg tct gtt gct aac cct 1584
Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val Ala Asn Pro
515 520 525
gat gcg ata gtt gtg gat att gac gga gat gga agc ttt ata atg aat 1632
Asp Ala Ile Val Val Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn
530 535 540
gtg caa gag cta gcc act att cgt gta gag aat ctt cca gtg aag gta 1680
val Gin Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys val
545 550 555 560
ctt tta tta aac aac cag cat ctt ggc atg gtt atg caa tgg gaa gat 1728
Leu Leu Leu Asn Asn Gin His Leu Gly Met Val Met Gin Trp Glu Asp
565 570 575
cgg ttc tac aaa gct aac cga gct cac aca ttt ctc ggg gat ccg gct 1776
Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu Gly Asp Pro Ala
580 585 590
cag gag gac gag ata ttc ccg aac atg ttg ctg ttt gca gca gct tgc 1824
Gin Glu Asp Glu Ile Phe Pro Asn Met Leu Leu Phe Ala Ala Ala Cys
595 600 605
ggg att cca gcg gcg agg gtg aca aag aaa gca gat ctc cga gaa gct 1872
Gly Ile Pro Ala Ala Arg Val Thr Lys Lys Ala Asp Leu Arg Glu Ala
610 615 620
att cag aca atg ctg gat aca cca gga cct tac ctg ttg gat gtg att 1920
Ile Gin Thr met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile
625 630 635 640
tgt ccg cac caa gaa cat gtg ttg ccg atg atc ccg aat ggt ggc act 1968
Cys Pro His Gin Glu His Val Leu Pro Met Ile Pro Asn Gly Gly Thr
645 650 655
ttc aac gat gtc ata acg gaa gga gat ggc cgg att aaa tac tga 2013
Phe Asn Asp Val Ile Thr Glu Gly Asp Gly Arg Ile Lys Tyr
660 665 670
<210> 8
<211> 670
<212> PRT
<213> Arabidopsis thaliana
Page 6
CA 02454127 2004-04-07
<400> 8
Met Ala Ala Ala Thr Thr Thr Thr Thr Thr Ser Ser Ser Ile Ser Phe
1 5 10 15
Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser Pro Leu Pro Ile ser
20 25 30
Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn Lys Ser Ser Ser ser
35 40 45
Ser Arg Arg Arg Gly Ile Lys Ser Ser Ser Pro Ser Ser Ile Ser Ala
50 55 60
val Leu Asn Thr Thr Thr Asn Val Thr Thr Thr Pro Ser Pro Thr Lys
65 70 75 80
Pro Thr Lys Pro Glu Thr Phe Ile Ser Arg Phe Ala Pro Asp Gin Pro
85 90 95
Arg Lys Gly Ala Asp Ile Leu val Glu Ala Leu Glu Arg Gin Gly val
100 105 110
Glu Thr val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His Gin
115 120 125
Ala Leu Thr Arg Ser Ser Ser Ile Arg Asn Val Leu Pro Arg His Glu
130 135 140
Gin Gly Gly val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser Gly Lys
145 150 155 160
Pro Gly Ile Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu val
165 170 175
Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Leu Val Ala Ile
180 185 190
Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gin Glu
195 200 205
Thr Pro Ile val Glu val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu
210 215 220
val met AS val Glu Asp Ile Pro Arg Ile Ile Glu Glu Ala Phe Phe
225 230 235 240
Leu Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Val Asp val Pro Lys
245 250 255
Asp Ile Gin Gin Gin Leu Ala Ile Pro Asn Trp Glu Gin Ala met Arg
260 265 270
Leu Pro Gly Tyr Met Ser Arg met Pro Lys Pro Pro Glu Asp Ser His
275 280 285
Leu Glu Gin Ile Val Arg Leu Ile Ser Glu Ser Lys Lys Pro Val Leu
290 295 300
Tyr val Gly Gly Gly Cys Leu Asn Ser ser Asp Glu Leu Gly Arg Phe
305 310 315 320
val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly
325 330 335
Ser Tyr Pro cys AS Asp Glu Leu Ser Leu His met Leu Gly Met His
340 345 350
Gly Thr Val Tyr Ala Asn Tyr Ala Val Glu His ser Asp Leu Leu Leu
355 360 365
Ala Phe Gly val Arg Phe Asp Asp Arg val Thr Gly Lys Leu Glu Ala
370 375 380
Phe Ala Ser Arg Ala Lys Ile val His Ile AS Ile Asp ser Ala Glu
385 390 395 400
Ile Gly Lys Asn Lys Thr Pro HiS val ser val Cys Gly Asp Val Lys
405 410 415
Leu Ala Leu Gin Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu
420 425 430
Leu Lys Leu Asp Phe Gly Val Trp Arg Asn Glu Leu Asn val Gin Lys
435 440 445
Gin Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro
450 455 460
Gin Tyr Ala Ile Lys val Leu Asp Glu Leu Thr Asp Gly Lys Ala Ile
465 470 475 480
Ile Ser Thr Gly Val Gly Gin His Gin Met Trp Ala Ala Gin Phe Tyr
485 490 495
Asn Tyr Lys Lys Pro Arg Gin Trp Leu Ser ser Gly Gly Leu Gly Ala
Page 7
CA 02454127 2004-04-07
500 505 510
Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val Ala Asn Pro
515 520 525
Asp Ala Ile val Val Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn
530 535 540
Val Gin Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys Val
545 550 555 560
Leu Leu Leu Asn Asn Gin His Leu Gly Met Val Met Gin Trp Glu Asp
565 570 575
Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu Gly Asp Pro Ala
580 585 590
Gin Glu Asp Glu Ile Phe Pro Asn Met Leu Leu Phe Ala Ala Ala Cys
595 600 605
Gly Ile Pro Ala Ala Arg Val Thr Lys Lys Ala Asp Leu Arg Glu Ala
610 615 620
Ile Gin Thr Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile
625 630 635 640
cys Pro His Gin Glu His Val Leu Pro Met Ile Pro Asn Gly Gly Thr
645 650 655
Phe Asn Asp Val Ile Thr Glu Gly Asp Gly Arg Ile Lys Tyr
660 665 670
<210> 9
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 9
gtcgacgaat tcgagagaca gagagacgg 29
<210> 10
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 10
gtcgacggta ccgattcaag cttcactgc 29
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 11
gagaattcga ttcaagcttc actgc 25
<210> 12
<211> 22
Page 8
CA 02454127 2004-04-07
_
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 12
ccatgggaga gacagagaga cg 22
<210> 13
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 13
acggatccga gagacagaga gacggagaca aaa 33
<210> 14
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 14
gcggatccaa cactcttaac accaaatcaa ca 32
<210> 15
<211> 44
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 15
gtcgacggat ccggttgatc agaagaagaa gaagaagatg aact 44
<210> 16
<211> 41
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 16
gtcgactcta gattcattat ttcgattttg atttcgtgac c 41
<210> 17
Page 9
CA 02454127 2004-04-07
<211> 50
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 17
aagtcgacgg atccataacc aaaagaactc tgatcatgta cgtacccatt 50
<210> 18
<211> 50
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 18
agacgtcgac tctagatgaa atcgaaattc agagttttga tagtgagagc 50
<210> 19
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 19
ccaaaccatg gtaagtttgt ctaaagctta 30
<210> 20
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 20
cggatccttt tgtgtttcgt cttctctcac g 31
<210> 21
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 21
gtctagaggc aaaccaccga gtgtt 25
Page 10
TT a6ed
09vz 66a66e6Dze 6p6ee6DDel TED1.1.1116D Dee6De66eD D66De66eDD eD6DDeee61
0017Z PAPPDPP66 U6PPDADPP blEP6P6D6 DA6D661DD D6De6DD66e 6DDe61.2ED6
OVEz De66e616DD 11DD6166D6 D66D66eDD6 6e6DDeDDD6 De616re66e 6666
08ZZ 1-1.0eD6D6DD e461DDDe6D 1D6DzeD616 D66aaD61De D61D66D66e Efeee63.6DDe
oaz D6DD66ee66 eDDe6Dle64 D6e6D6DDD6 3P36D6D1e6 PDPD66D3DD ED1D3DP1DD
ogiz D6DDDDD661 1.16ep616D6 6e6oDD66eu DD6DD66e6D 6D666D6e66 DDDPADDE6
OOTZ DIXDITP1DD D116D6P6D1. 1.5U6DDE011U D66)D6D116 1.6DDE61461 66aeD6DD66
OVOz D66D6DPD DPDDEll6D6 DPDDRDaPPR PDPAEolD6 PD66DDP6DD DD1DETDERD
0861 DETDARDDP DD6a3D1PlP 1UUDEODPDP 11a6111P2D 46D6UUlD1.6 116EellUal
OZ61 616leeD6DD 3.6De5eeela EDEZEIPD11P el161e66De EP31P16PPD E6U1441PED
0981 36PD1EPP63 1DPD61P6B1 eD6aeallla 613x6elleD 1611peleel 6611361pp].
0081 e66eDe61.14 616eDlezDe eelal6eDla DADDD1116 D1611e6eD1 e6leDI.D6DD
OVLT ZDZITPlaDD Dlye616D D1.66666D
66DzeD16D6 DDD1611.366 DPPET16DRP
0891 ED3116eD1.6 aD1166D6z1 6DeeD3aDD1 3661.6E61A 6D6DDDeve6 PDD1DBPP11
OZ91 eD1D6e11D6 161ea6De61 D111.66leel eeD6D6Der6 1.111DP6D66 ellaDe616e
09S1 ae61D6e1D6 alauleeeft eDe611.111E D6e6616eD4 6Dee664ell lee66666D
00ST Yele661lee aD1De6e6le leu616e6e6 11e66111ea le6eD6166D DIT6eDDle6
OVVT Deee6D6leD aueD11614D leDDzeuD61 6D61DDee6e 66DD66D6ee DDDPDD1D1D
08E1 APZEPEIDD6 PaPD16EDDD 6a61.161Da6 lae6DD6eD6 e6eDleD66D 66DeDee66D
OZET D6eDe61D6D 61DDDD6D66 6D3PEEmPPP eDe6laDa66 D166uDe66D DeD666eD11
09ZT P31.16eD61D D16D1DD61.D 6D6DD6e1e6 DeDDETDD66 16D16DDD6D ep66eeD6D6
00ZT 4D6eDeD6e6 Dz6DeeDe61 6eD1.1D6DDD laDDD16PDD 6PD6P1PPDD DE01.1DP)66
OYU DDDD61DDIX 6e66eDe6ze 661.66D6 e66eD66D1D 111Dele661 ebleDDETD1
0801 PD611eD6DD 6DD6eD61e1 6666 DD6e166eD6 66 666
1.6614)6 1-1
OZOT 161e6D61e6 D1D6D1D615 Del6e6DDIE DD11D66DDe 6euDe6Dle6 1DDzeDle6e
096 DD16D11D1D 61e61DDDD6 e6D6D664D6 6D116eDee6 D661DD6e61 1DD6D6D61e
006 D666D16DD6 D1DDle6e6D e6DeD16661 eDD6DleD66 eD6eeD66D1 zele61PDDP
ovg DDlaaleDD6 6D6puge6eD Dlre64u6D1 6PDPDA6D) 6PDDDPORDD 6DD3.66D6e1
08L e63.DD161e1 D6DuEDD6u1 666DeDlele eD6rD11D1D 6euDD6DAD lleDDD6eD1
OZL 66666 DP36PPRZE0 Dele6D66D6 e666Dlee6D 61D6D61e6D 66ee6ele6D
ogg 66eu6eeD16 DaDee6e6p D1D6DDD166 6346PP1D4 E6PPAPAD UP6331X1PP
009 PPE0DPDD13P r666D661.61 Da666DDleD ze6666D1e6 Dee614461e eeDD614ell
OVS 1Deue6pula DITP311.P66 EDEEDMIDDP 6PPD6D1PDZ PP1P61PlE1 1PEBETDPPD
0817 11.eez6Deez 1.D6auDelze lauelableD elzeD6leDz 6DEPIXRPIX DaDZPDDDPP
OZV PPE1PD1Pel D13P666361 1UPI.P461EU ellE163631 elDa111611 laulelD6D6
09E 364116e1DD lellleele6 D6D6D6DDeD e64e6pleDe elbulDle6D DD66e66eDe
00E 611D6e6pD1 De6DpeDell 116D16DD66 1DeDllue6D 1.D6r6DDel6 6e6D1D666D
OVZ DD6Daule6D Dle6616ezD u6eaDaDE6D 1.66eD61.DD6 ZED611APP DDEOPZ1P61
081 DD 61D6 eDeee66e1.6 ulDze6upae eDe6Duee66 6D66eu61.De upaa46eleb
ou zDeDueeD16 3.Dpaulezee DD6DDDelll 66e1.4D1D11 1.1D1D6Deee leulDllell
09 P6DDITZPEP 1.141DDD6De D1114DDeee ze66Dep6De 661pueDele De66zeDD11
EZ <0017>
.104DDA ucqssaJcixa
lueLd :apuanbas
Lup1.4p.Ju a41. 4o uop.4.1Dsaa <m>
<OZZ>
aDuanbas Lep!.4p.Jv <m>
VN0 <ZTZ>
VSSL <TTZ>
EZ <OTZ>
TE 6
eplzu61z14 eeee6EDD14 161DDel.66D
a <00V>
Jawpd appoapnuo6qo
:aDuanbas Lpp!..41.1JE ap 4o uopdppsaa <Eu>
<M>
apuanbas Lep!.4p.iv <Ew>
vNa <ZTZ>
TE <FEZ>
ZZ <OW>
LO-VO-VOOZ LZTVSVZO VD
ZT aftd
00E9 166D6e1661 D6DDPDDUPP DPUPD66DDI. P611D4D6El 66116P6UPP UP66D11DDP
OVZ9 116PDAPP6 1D61.D1D6D6 1D1P16611.4 ul6pDp66ev 6u1DeDeaD6 EIDP4DPP1DD
08T9 666 laD116P6ED paD6166066 ea6ap166p6 D6E6up6pla e66pDepa66
OM aDPDDETD6P DE64DPDD6D 1P11DP6DPD P6PP166DDD PEDD16P6a1 316DaVaDPP
0909 166DDaPalp D6D61D6DDp 6DDDETD146 DDDDDDPP6D PD6161.6106 661D6PEDD1
0009 D6Daa6D166 ra6166D116 DD .66E 16aD6DuDaD 6papDaDala D6D6616)6p
0V6S P666DaaDDD apazaDD6DD 161Dpeap66 DppalD6DD6 appDp6DDal 6aDDaDaD6D
oggs 616DaDDDaD 6pp66aDDDD Dall6D66eD Duae6pEuap app66u3p6D DDpup6D661
OZ8S 66P6pDa6pp D1D6DP6DaP PPPRDEOlUD 5P6DP64DDD DDD6DD1D66 pappD1111.1
09LS EIDE61D611.6 DEIDDHETPP PlE0DPP66U DD66PPPPD6 EDDHIPPEED 6p6161pDpp
OOLS 6PPE66eD6D puar6666pD 1PEETDPDD1 P1166DP1UP 1.66D66PPPD aDeDaD6upa
OV9S P166D6p6D6 6D61D66Dal 6D166D1D6D 61D6DaDe61 DPDaD6D1D) llD5DD11Da
08SS DE066PDaRD 6DDPITPEP6 P5bEP16D61 P6PDEDE0DP 1PPP616166 D61PITDDEO
OZSS 616P6P64DP 1611PETDET 6PDITD66D6 1P1DEU11D6 666 P66D6P1P60
09VS ETa6DeDa6p DDDP61P)D6 PD6D6666D1 61666)6614 61.666D6PDa 6666P31.
00VS EIDDDETPDP6 PAP6b6D6 aP6606UP1.6 1D1611D6PD UD166DP6P6 6DDD1D6PD6
OtES TeDEDP6lD1 DDPUPP6166 DP6aP6166D 1116D6D6D1 DD61DDDED6 6PPD1PDPDD
ORS D6D66DD6DD P6DI.DPOD6D 16DD6DEIDD6 PPDP66D6D6 66PDDPIDIT PD66PDD66D
OZZS PaDD6666 1PPPPED1D6 DD664D6DD6 6D6D1PaDD6 6016D6D1.1D EIDD6DDD)63
091S P1DDDaD6D6 ap6D166Daa DDDP1DD6D6 PPPERD61D6 P6PP6DD6PD PD6D6PDD66
OOTS 1D161Dpeap D6161DD661 DDCIDDDPPUP 1.1D1DPPEE1 1P11DPPEU1 11D1DPPEP1
OVOS DDE0D1.4114 P6D66veppp u6p6peuule au6aDv6a6u u1601eDEDED 166DDEp666
086V 1.1PDea6DD6 UPUDDDPP66 611pDpa6DD Dpe66pDpe6 6611PDP16D D6PPPDDDPE
0Z6V 666aaPpea6 DeD6Elp66a 6aDDlaaDap 166puer6D1 66peepp666 6ED6paDDD6
098V 11PuPD666p 1D 6D6 66Dpa6appa DD1166DD6D Dapp6pp6D6 66E6)1.261D
008V DEED6DDP1D 6061PD16E1 DD1PEIDDD66 1D66PD6666 D66p66p66p r6allE6Dpa
ova, 6e66DD6aDD p6apuuD166 6Dau6aD6RE D6D616DDpD alabaplaft p6uppla6P6
089V P66DD6D6PD 6616PD6Dup 5opaDau6Dp 6puD11611.6 61p6pDD6ep 6pD66pPD66
OZ9V ED6DD6D6DD 6DpD661DD6 DDpaDapala 16DD66D1ED 66DDDle6Da P6alaaapPa
09SV 1P6DDDDuDa 1.66Dp6aD61 EIDP66DDDPP ETPDNUEET DPD1P6P6D6 DDP161P661
00St ar6aD6p1D6 u6Dap6p6D1 EDua6e66DD 660666pDpp e6D6p6pppa 66puppa
OVVV D6Dp6plau6 aaDD6pv6a6 66E6DDlua6 6DE61661DD 6DD66Dpe6e EDD66up6pu
08EV 6Dua6D6uD6 auDD6116Dp D6DEDDUDPP P1466D14ED 61DDPPPEIV1 6666
OZEV PPP6EDEIPPP 66D661e6DD 66666. DaaftpDaDu 16Dp66D6aa 6DPDPD316D
09Zt 311616D6DD 66DDDETPDP 6E666666 pp666DDpau 6DDPp6avDD 1PPE0DPPai
00Zt 1PD3D11166 DE061X61DP1 661DDP6DEZ 1P66616151 666.6 6DD66DD666
OVTV PD6DD11166 p6p16DpD66 6Dp6upplaD 6p6DeaD6DD ap6166E6D6 6666D
080V DP616D6pu6 Da6aDa6DDa aza6DD661.6 De661pDapD 6eD6Da6pap 6D6DDDED66
OZOV 6a6DuElaulD aD6ap6DDla 65 PDDEED6P6D P6D666PPDD ADD6PPE06P
096E alP6D16DD6 D6166DD6up 66DD6pDpeD 66DDDaep6p upD6DDapp6 Dap6aD6DD6
006E 6D6pED661.6 Dape6166DD DD6DeD6pe6 up66e6Daup 6DreD66D6E DDADD66ED
Ot8E 6D6DD66eu6 la6pp6u661 66appe61E6 16661D6D66 D6D66Daupu Dp166DDD66
08LE pplEDDPETD 6D166D6r61 6D66Dary66 PEIDDDE0PPDD DDDPP66;DP D661PPD61)
OM DA6DD6101 611.6661D6b DETP1E066U DD661a66D6 66Dee66u66 16161eDDDD
099E EfauP6616DD 6DE6DDeD66 PDDPPDPPft PD1PPER661 PD66D66P66 PPP1D66D6P
009E 111aPv6au6 paft6leppa PPE01PPPDET 61PPP16P6U DDE4D6PAD 6D1PDPITE6
OVSE 4D1P1D61D6 p6pDpalupp eftpD66ppD D6Dp166D6D pa6ap6pp61 DEoPPDDPDPD
08VE 1P66P66066 DD61.16PD11. 4DPPD1666D 6PP6lUDDE0P DDEIDEDE6PD 661DD6vDD6
OZVE 61.16DPuD61 D66pED6rD6 ED6DeD6D6p 6DDa6DD66D D616pulD6D PDPPEDEDET
09EE PPPD6u6app pp6p6ppul6 6p6apulaft 61.1aPpappu UPD1PPPlaP pp61D6DD66
00EE 106D66EDD1 66u6D6DDD6 1D6DUE0666 P6DDDPU6UD 1PPE014D116 DDPPDPD66D
0-17zE D6DADD6aD pD66eDDDua D6e6a6D6D6 pD6DpDapa6 DpDa6p6a1D laPDDD61D6
08TE u6Dua666DD 661D6D66u6 DD61166p61 66666 D6DPD66PPP D1P6D666D6
OZTE D1616D16a1 aDD66D6eeD PaD66pu661 r66Dup166E 611pD6D6pD 6pula661D6
090E v66166appv 6DD5DDPDD6 661eavDE6D PalDDAPPD APD61.66DD lar6aD616D
000E 11DPEIDAPD 66PUD1P6D6 DD161.61D66 14DP006D66D 66PDDDAD6 P66DE6ole6
0V6Z 16u16Daapp 6D6D66DD66 DappD66ep6 a6Dr6D6Dpu 614U6DPE0D ADDE6D1PD
088z 66D16116DD PE1ADDPUD ap6uu666D6 16DD66D666 lav6D6DDD6 16ED666pDD
OZ8Z DDav6Dpalp 6Da6pla6aD 146ap6DD66 66DD6DaDeu D6aDDD6D6D DD6EaDauDD
09LZ DPPDE01PDD PEIDP6PPD56 PD1666066P PE6PDDREll Dpa6aDOole la66Eu6lpp
00Lz 6Dep6666pp ADP1PPPDP PPITPE16P6 1p6D61e6ap lp16D61D6D 156AluD16
0.179Z D611D6PDEE uplEleblalp 16161e6166 PPETP1D1E0 ADD6D6P6D DPUP6PE610
08SZ 6DD66a1D6e DD66DD661D D66D661D6P PDD6au6aDa 641166DD66 appleur6ap
OZSZ D61D66D6a5 DDPE0101.6D PAADDD6D D6e6D11646 Dua666DD66 D6D1e61P6e
LO-VO-VOOZ LZTVSVZO VD
ET abud
0981 DDEED6EDEP zezDD66ee6 6666 ze6eDeezDD E61E31DD 6PEEDDD1E1
0081 6161.D6D6DD DDel6zeeD6 D6D6DD6eDD ze3.61.1D6ze 33ED31EDD6 zeDzeDeeDD
017LT eeeveDD611 1ODDleePED 13EED66136 B66P6611DD 666 61.Due16661.
0891 ple6eD1De6 Dzu6D166D1. P6PED6ED63 EV6331E1PE EP6DEDD1DP P666366161
OZ9T D1.666DDzeD ze6666Dze6 Dee61.1.1.6ze eeDD611e11. 1Deueftell. Dzeeplau66
09S1 EDERD66DDE 6PED6D1ED1 ueze6zezea zere6eDeeD zzeuz6Duel I.D6zeDezze
00ST zlee1.161eD ezleD6leD1 63PE1BEE1P D1D1P3DDPE PER1ED1PE1 D13E666)61
01717T zeezel6zue ezlea6D6DZ P131.1.1.1-614 laele4D6D6 D6111.6eaDD zealleele6
08ET 36363633PD E61E6P1EDE El6P1D1E6D 3366E66E3E 61136EDD6P 31DE6DEEDE
OZET 11146D16DD 661DeDzzee 6D1D6e6DDe 1.66e6DI.D66 6DDD6Dzele 6DDle6euuu
09Z1 leDzeeD1.16 1.1DzeDDzeu D616D61.DDe u6e66DD66D 6PEDDDEDD1 31036E1PE6
0OZT DD6eauD16e DDD6161461 D1614e6DD6 eD6e6eDzeD 66D66DeDeu 66DD6eDe63.
OTT 36D61DDDD6 D666DDee6 PPEEDE6113 1.66D1.66eDe 66DDeD666e DzleD116eD
0801 EaDD1.6D1DD 63.D6D6DD6e 1E6DEDD6PD 3661631633 ADVE66PED 6D61D6uDeD
OZOT 6E6D4.6DeeD e616eD14D6 DDDIaDDDI.6 E3D6E36E1E PDDD6011DE 366333361D
096 Dze6e66eDu 61.e6E61.66e eD6e66eD66 DI.D114Dele 661.E6zeDD6 eDzeD611eD
006 6DADD6PD6 zel6D6eeD1 e66DD6e166 eD666zee6D 1661661.1)6 D11.3.61e6D6
0178 ze6D1.D6D1D 66J 66 DaeDD14D66 30E6PEDE6D 1E61331E31 e6eDD16D34
08L D1D61.e61DD DD6e6D6D66 1D66D146eD ee6D661.DD6 e611DD6D6D 6zeD666D1.6
OZL DADZDDle6 e6De6DeD1.6 66zeDD6Dze D66eD6eeD6 6311E1E61e 3DEDD1111e
099 DD66D6eupe 6eDDlue6ze 6316E3E336 6336E33DED U336331663 6E1E613316
009 1P4D6DEEDD 6E16663E31 P1EE36PD11 D1D6PED363 36311E3336 eD166D6ee6
OVS 6e6DeD6eu 1.6DDele6D6 6D6e666Dze e6D61D6D61. u6D66ee6e1 e6D66ee6ee
0817 pluuuDDle6 61.6e1De6ez DI.De6D1.66D ez66666661. 6e66D666e1 61.6e6Delez
OZt PlezeD66p1. 1466eD6666 Due66e6lel EDED1E1EV1 eezeue6zee lEE1DDE1E1
09E 1-DzezDeall 6Dzellppla 1.D16zzueul eeD4D1.11.D6 61ED6e1.11.1 elelDleaDz
00E Ple166PE13 61D3PDPER1 PEEP161161 R1161E1161 P13E136E13 661)613361
OVZ Dp6e114161. B1DE1E1DE6 P1P6eD61DD 03331E6306 1E361136PP DD6Delle6z
081 eDDe61e1D6 epeee66e2.6 P1D1B6ED1P EDE6DEPP66 6D66ee61.DE ee1416eze6
OZT 1Depeuppl6 lopluzeleP DD6D3DE111 66E1131311 1131363EPP leelolzEla
09 E6331E1PEE 111133363E 31111DDEPE 1P663PE6DE 661PEEDE1E DP661E3311
VZ <00V>
JO1DaA ucqssaJdx .u[d
Aueupl :aDuanbas Lupw41JE auz 0 uop4JDsaa <Ea>
<OZZ>
aDuanbas Lep!_lpuv <ETZ>
VNG <ZTZ>
LZE8 <TTZ>
VZ <OTZ>
VSSL 6361
3E33633:ERE E61ED36PED eE6eDe EPEEPED3DD R161DP6E1P
00SL D66e6DDD61 e6611361D6 1136D6Deul 63663E33DR 6D1EDEEED1 EDEP1E3D1D
OVVL 51361a6Dle Deel6D61.D6 1DDD6a.De6D 666p1111.63. lappe14161. P61E333331
08EL 1D6DDEDI.E6 p66)11.pezp 6D16p6116p zu61D1Dpez DEPDADE61 26DaD6D663.
OZEL P6e6D1.166D 16DEPD6EDD 66DE161EPE D633666 3613E331PD D63)66E711
09ZL D663.61.61De 31E1UE31EE ED6EDDPE16 DDeD166Del 1DAPED1ED 1116116D6D
00ZZ 36D1D6PEE3 1E61163166 ppeppolal6 ee6DD6ve66 66p)D1.)131 36DI.D1pp6e
0.171L 66D6o6pDpz 3113E61661 PEDEEDED61 60163161E6 1PE660EDD6 Deezp661.D6
080L Pl1D6D6Dz1 6pD611.eeep ozplaeDD61. D6o611x31.6 1EPETED613 Deze6ee6D1.
OZOL 3661.36616D ze6D161.eeD zu6eDD6ple fteD6eD1.61. 1.14)61.1D1D
1.1.61el.D6De
0969 P366eP3DP1 DDE66136DD 633133416P 6EEPD1P663 Due66eD116 1DDI.e6elee
0069 Pol.D.D6D6p1 lapi1.11.66r p11.6D6uTeD D1.46E6D66D 666D1.6eDDD 62Do6D1ppl.
089 D63111EDE1 3BD6EP16DE EDE666361E UE33E16136 D613E11663 D63.111x6D6
08L9 D66D1aDDIx De6D6eD66D 16eDDD611e DDI.D6D66eD 66DD6661.De ze61D666De
0ZL9 baelbeeD11 D6E1316133 6PE1B6UEDD 1611311311 Dau636erpo 66p&6636
0999 103.e61.3eup DzIolleeeD e661.6e1.6De DIa1.DD6Dlo ze61661aDD eloe6DD63.1.
0099 lezzeDe6e1 D6e1Dllaer 6Due614366 Dz6D66D6ee 6D6DD6D6DD 6ee1.16e61.1.
01759 DEOPEZDZED 616E113616 zuazeeD6E6 4.61.D6611.1.6 plx61.Dop61 10E6PAPPE
08179 PlEB1EUEEP 11)63E761P 11E130666E 1616111eP3 30131E1E1P 56
mg 61.111.e666e P1163PD13E eve6Dee661 6eD1D6De61. Da6666DelD 11.34Dle614
09E9 lopze6Pe6u ED1D1P66PE EEPPE6P363 63E11E6E36 PAPED6111 6114111166
LO-VO-VOOZ LZTVSVZO VD
tT DbPd
OOLS PPDDDPP666 11PDP16DPD 6p1x661613 D111030466 pppu6D166p pupp6666pD
Ot9S 6P1.3D)611E pE0666e1D6 lu6pD6E66D pleapplopl 166DADDlp D6pp6D666p
08SS 6D1P61pDpu ADDE.I.D6D6 aupl6p1Dpa p6DDD661D6 6pD6666D66 e66p66ep6a
OZSS 11p6Dp16E6 6DD61pDp61 ppeD1666D1 p61D6ppp6D 616pDpD11.1. 61_plaftp6p
0917S P316E666 DD6D6PD661 6PD6DPP6DP 1D1P6DP6PE D11611661P 6EDD6PP6PD
00tS 66 666 DD6D6DADE D661.DADDu lolDaaal6D 366DIxD66D Dp1p6D1p61
ovEs llaapplap6 DDDDpD1466 Dp61D6a6Dp 66DDDEP6ET D66 6D
1p6p6D6DDE
08ZS 1_61p6611p6 1D6p1D6E6D 1p6p6Dappu 16p66DD66D 666Dpppp6p 6p6uppl6D1
OZZS P6PUDP1D6D D6P11P611D p6 61666p 6DD1p1.66DE 61661D6DD 66DEp6REDD
09TS 66pp6pp6Dp 16D6pD6app D6 6D6 PDDPDPPE11 66D11PD61D D6661.
OOTS DDP6DE6PPP 6PD6PPP66D 661P6DD6P6 D66DD61D11 6PPD1DP16D P66D6116DE
o'osDPDD16DD11 616D6DD66D DD6PPDP6P6 666666 66DDE1P6DD PE61PDD1PP
08617 ODDPP1D1PD DD11166D66 1E61DP1661 DDE6DP11P6 66161616PD D661pD66DD
OZ6t 66DD666pD6 Dp11466p6p 16DpD666Dp 666 Dp1D6DD1p6 1.66p6D661.3
09817 6p6Dp6DDp6 16D6pp6D16 I.D16DD1111 66666 Colepatobup 6D16p1p6D6
00817 DpDpD66616 Dp61p1D1D6 1p6DD11.6)1 1.1111.e6pD3 PPD6P6DP6D 666peppD6D
D6er66pllp 6D163D6D61 660D6PD663 D6DDPPD66D DD1PP6PUPD 6DDlup6plp
08917 61ADD66D6 ppD6616D1x P6166DDDD6 DED6PP6PD6 6P6D1PD6DP PD66D6PDDD
OZ9t 66666 DD66up611.6 pp6p661661 DD 666
61D6D66D6D 66DapppDp1
09S17 66DDD66D31 EDDEPPD6D1 666666 6olpp66p6D DAPPDDDDD PE661DPD66
00S17 IXED61DDD6 6)D61D1611. 6661D66D6e ea6D66ppD6 61166D6663 pp66p66161
Ott EfavDDDD6lp p66163D6Dp 6DpeD66upp PPDPP6PPD1 PPPP661PD6 66666PPE
08Et 1D66DETlaa lpp61p6ea6 E6leppapp6 /PEED6P61P PP16P6PDDP 1D6PD6D6D1
OZEt EDulpe6101 p1061o6p6D DP11P3DP6P ED66PPD6D el66D6Dpa6 au6e61D6r
09Zt PDDP3PD1E6 6P66D66DDE0 116PD111DP PD1666D6PP 61EDD6EDD6 DPDP6PD661
00Z17 DDETDD6611 6DPPD61D66 PPD6PD6ED6 DPD6D6P6DD 16DD66DD61 66DPDP
OtTt PPDPD6PEPP D6P61PPPP6 PEIPEP166E6 lppal6P61.1 1PD1DPRPPD lppulleep6
08017 1D6DD661D6 D66pDp166E 6D6DDD61D6 DP6D666P6D DDPP6PD1PP 6110116DDE
OZOV PDPD66DAD ADD61DED6 6PDDDP1D6P 616D6D6ED6 DPD1P16DDD 16.611011e
096E DDD6lo6p6p u16663D66. D6D66p6DD6 1466p6166D 66pleD6D6D eD66Epuolu
006E 6D666D6D16 1.6D46111DD 66D6evDelo 666666 proa66p611 PD6D6UD6PP
0t8E 11664D6p66 1664DDE6DD 6DDeDD6661. plppu6Dela DDAPPDAP D6166DD11P
08LE 610616D11D p6DDETD66p pplE6D6DD1 6161D6611D p66D66D66p DDDD6D6p66
OZLE Dp6pleblop 16Daape6Db D66DD66plu DD66RE616D p6D6pDp611 P6DP6DDD6D
099E DP6D1PD66D 16116DDPP1 D6DDPPD1P6 PP666D660 D66D66611P 6D6DD3616e
009E D666uDDDDa r6pplapopl 6rla61D116 ap6DD6666D D6D1DEPD61 DDD636DD36
OtSE P1D1EDDDEP D6D1EDDP6D P6PPD66VD1 666666 PDDPP11DP1 61D6D1P116
ostE Erep6aup6DE p6666upp6D P1PPPDPeE1 PUE16U61P6 D61P61P1P1 6D61D6D166
ozvE D61PD16D61 136PDPEEP1 6P6111P161 61E6166Epp PP1D16DAD D6D6P6DDPP
09EE u6pr61D6DD 6614D6EDD6 6DD661DD66 D661D6REDD 61polD1611 166DD661DD
00EE luPp61p361 D66D616Dpe upapl6DED6 ADDADD6P 6D 66D 666DD66D6D
OtZE ar6au6p66D 66p6ple6r6 PP6DDP11PD 11111.60DPP 6De66pop66 ou66epppD6
081E DDUPE61PD6 EUDPP66P6P PDD6DUP61P P6P6DD6DD6 6D661DDD6D U6DD66P6DD
OZTE P611PD63p6 6u616DDllo 36166D6)66 D66PDD66P6 DDPDDD6DE6 16pp66p6D6
090E eD6D6p611D ED6D6DDR16 zDDDE6D1D6 DTeD616)66 11D61DpD61 DE6D66p6pp
000E P616pDpAD D66pp66u3D p6D1p61D6u 6D6DDD6DED 6D6D4p6pDp D66DDDDPD1
Ot6Z DDDU1DDD6D DpoD661116 up616D66p6 DDD66pupp6 Dp66u6D6D6 66D6p66DDD
088Z PD6DDE6D1P D1PPIODD11. 6D6P6D116P 6DD611PD66 DD6D11616D DP61161661
OZ8Z P36DD66DD6 63D6DRDDRD DE116D6DPD DPD1PUPPDP D66D1D6PD6 6DDP6DDDD1.
09LZ D6PDPPDD6P DD6PDDPDD6 1DDlplelpe DEDDPDP111 6111PPD16D 666
OOLZ PEalP11616 1PPD6DD16D P6PPP11EDE 16PD11PU11 666DPPRD lploppDp6p
0179Z 1111PPDAP DlEEP6D1DU p61T6pluD6 au11114611 eftlapp161 lupapra661
08SZ 106applp66 ppp6111616 ED1P1DPRVa albuD11DD6 opp1116o16 11E6uplp61
OZSZ PD1D6DD1D1 1PP11DDD1D P616DPP1PD 166666D66D lvD16D6DDD 1611)66Dpe
0917Z PP16DPPRD3 116up161D1 166D6116Dp ppllop1066 l6e6lD66D6 DDDEUU6PDD
0017Z I.DPPP11ED1 D6P11D6161 U16DP61D11 1661PE1UPD 6D6DPP6111 1DP6D66E11
OVEZ DDP616E1E6 106E106111 ElPPP6UPDP SlaaaluD6p 666)16D e661vaalue
08ZZ 6666p6pDpa p6611EulD1 De6E6aplET 616p6p611E 666 e6D161.EDE6
OZZZ P66DDloaDD 1D166aDrup zplueloo66 1.p6e161D61 D66D1PlUDD 66D6DDPPED
09TZ PD161E6DPP 11661PE161 PPD1D16DE6 P161DPD116 EpulDDI.61D IA6161.116
OOTZ 1661614D1D UDD1UPD1PD 166P1D1D1D DETD611D1P 1D1P1666PP DDPPDDPPD6
OVOZ PD1DDPPD1D DDUDPPDEDD 6PDDP1PUD6 up1.6D6pDDD 666eppllop 6pappla6D6
0861 PE16D1PEDD 161DUUD1D1 DU16E3PPE1 6DPDP6161P 1.DD61P61.1l DDPEDDD6ET
0Z61 1DDIT6e161 pppu161.616 lpuppftell DP6P1PDD1D D6D611DDPP PP11DP6PDP
LO-VO-VOOZ LZTVSVZO VD
ST p5vd
0817 31v566631v 63Ev5111.61 vvv33611.v1. 413evvovv1 131vv311E6 6PDPPD66DD
OZ.17 P6UPD6D4PD ITP1E61Ple 11UPE6P3UP D11PP16)PP 11D61PDP11 elavv1161v
ogE 3v11E351v3 1.63Ev1vvv1 P34D1PDDDR PPPPIXDZET 1D1.3P666D6 41PP1P461P
00E vv11v16363 1E1.33.11161 111.v1E1363 636111.6v1.3 31v1.1.1vElv 636363633v
ovz 3E61e6E4e3 vv1.521.34E6 33366E65v) v61136v335 1v3511.36vv 3363v1.1v61
08T v33v61v136 P3PPRE06E16 P131P6PDIT PDP6DPPPE06 6D66PPEaDU PP1116U4P6
OZT 1DPDPPED16 1D31P1P1PE 3363D3P114 66P14D131.1. 1101363PPP luv1314P11.
og PEIDD1P1PEP 11.1133353v 31.11133evE le663vv5pv 661vev3v1v 3v661v3314
SZ <0017>
JOZDDA uo!_ssaJdxa lupLd
kipupq :aDuanbas Lpp!_44.1..1p aql lo uopAppsaa <Ezz>
<OZZ>
aDuanbas lypqp-lv <ETZ>
VNG <ZW>
117172 <TW>
SZ <OTZ>
LZE8 6D613PD
D633PEPP61 PDD6PPDPP1 PD16P3PPPP PEP3DDDP1E0
09z9 1DP6u1P366 P63DD61P66 11361D6a1D 6D5 .D6
63PDDDP631 P3PPPD1PDP
OZZ8 PaRD313613 511.631.E3vv 46361361.33 3613v63656 v1141.61413 pe11161P64
0918 UDD7DD11D6 DDPD1P6D66 311.DE1E6D1. 6P6116PaPn 1D1DDR13PP 3D6DP61P6D
0018 4363661.v6E 6314663163 PPAPDD663 P161PPEDP1 6D36E66761. DEDDITDAD
008 36666 16161DPD1P ITPDaPEP36 ED3PP16D3P Dl66DP11DD 6PPDITD11.1.
086L 6146363353 136vveplE6 14.63166vvE p331.116vv6 3366666P 331.313a363
0Z6L 131Ev6e663 636v3v1.311 3v61661ve3 vv3e361631 63161v6avu 663PDADEP
098L 1v65136v11 36363116v3 511vvv3313 11v3361363 61.1v316avv 6vv3EappEl.
008L P6vv631366 1.3661.631v6 3161vv31E6 e336v1v6vv 36v31.6111.1 361131.3116
otLL 1P1D6DPPD6 ETUDDP13DE 661.DE0D6D3 1D3116P6UP P3aP663DPP 66PDZI.61D3
089L 1.v6vzuvv34 33636v111v 314.156m4 636v1v331.1 6666666 31.6E3336v3
0Z9L 3631E31363 111v3E13E3 6ve453Eupv 666D6lEupp De161.D6D61 pv11663361.
09SL 111P6D6066 31.1.331eDe6 D636636e D3D614P3D1 D6D66ED66D D6661DPIT6
00SL 1D666Du61P 16.epplap6u 1D1.64DD6ve lu6pDp161 1D1431131p 6D6pED366p
01717L 6D6D6D61D1 e6apppppll plapeeDP66 1.6p16Dupla 1DD6D431e6 1661appplp
08EL P6DD6111E1 le3p6p1D6p 1314app6pp p61.1366D16 J663666 336D6DD6up
116P611D6D pplp1PD616 PlaD6161n lEpp6p61.61 D661416p1P 61DDP611DP
09ZL 6E36pPuuzp ElPPPPP1.13 6DP361PlaP 113666v161 6111vv3331 31.vavlv6lv
00ZL D61v316511 11v666vEll 6DED1DPPPP 6Due66z6up 1D6DE61D16 6663p1D111
OtTL 1D1P6111DD 1p6Pu6PEDI. DaP66puPPp Eu6pD6o6Dp 11x6pD6up6 epp611.1611
080L 111.1166166 36v456a363 DP3DPPPDPP PD66DDI.PE01 aDlD6P1661 16P6PPPPP6
OZOL EolaDDP416 pDp6uP6136 4D4D6D6aD1 P1.66111E16 ppuMpeElpl DU3P1366DU
0969 apppliD661 6616Pu611.3 116r6e3P13 666665 1u1.66e6D6E 666
0069 Ppee1661op 336p36e366 1.3v33531el. 43E63e3v6v u166oDoppp 316u611.31.6
089 DIT1DPE.166 DD1P14336D 613603P6D3 36PD116DDD DD3PPE0DED6 1616136661
08L9 D6vv331.353 1163166e16 166o11.6eD1 D1p166e161 DEoPpzp6u1 v313113.363
oug 6616D6pu66 6311DDD1D1. 1.1336DD164 DDE1P66DDP 1136DDEaDD 3v6331.1643
0999 D131363646 313331.36up 664333334; 1.5356E33v1 v6vev4v4pv 66upe6D3Dp
0099 Pr6D66166p 6p316ppplp 6Du6D1vpuu upupapp6p6 3U61DDDDDD 666PIT
017S9 3314141636 6106116363 365vEvev1.6 33vv66E336 6EPPRAUDD 6666
08179 161.P3PP6PP E66P6DPP1 U6666PD1PP 6PDPDDTell 6666 D66PPPD1DP
0Z1'9 31.36v31.E16 636v636636 136631453a 66D1D6D61.3 6D13v6lopp 1D6Dapplap
09E9 6DD14D136D 66pplppeop Pluree6E66 PP16361E6P 3PD6D)P1PP v616166361
00E9 Elv33v3616 v6v6a3E161 1v6e35E6up 1.v366351e1 3vv1136613 v1E1616v66
OVZ9 D6u1v635E1 53v316E333 E666 366663161.6 6635611616 6636v31636
02-E9 3666E31533 36vv3v6v36 p6563361E5 636Ev16131 614.36v3v31. 6666633
D1D6v361v3 P7P6101DDR PUP6166DE6 lE6166)141 6D6D631DD6 1333E366vv
0909 DIXDP3DAD 66DADDRE0 13PDAD163 DEIADD6PPD U66D6D666P 33v1.31.vv36
0009 6v33663v43 36613651EE vev31.36336 6136336536 31e1336634 6363113633
ov6s EIDD336DP13 DDI.D6D6106 3166D11DDD P1DAAPPE PED61D6U6P PE0D6PDP36
088S APD366aD1 61DueluD61 61DD661DA DDDUPEP110 aDPPPPllel lOPPPP1110
0?251DUPPP4DD6 3311.1.11e63 66vvvvve6v 6vvvElele6 13p616vE16 leDEopp166
09L5 DD 6664P DP16DDETPP DD3PP66611 PDP16DDDUP 6EIDDPP6661 IXDP463)6P
LO-VO-VOOZ LZTVSVZO VD
9T aftd
OZEt 6eD661DD6e DD66116Dee D61D66eeD6 eD6eD6DeD6 D6e6DD16DD 66DD616Eua
09Zi7 36DPDPPPDP D6UPPPD6P6 1PUPP6P6PP P166P6aPPZ 16P6111PD1 DPUUED1PPE
00ZI7 11PPP61D6D D661D6D66P DD 666 DD61D6DP6D 666P6DDDPP 6eDzee611D
OVTV 116DDUPDPD 66DD6DD6DD 6aDeD66eDD 3ulD6e616D 6D6ED6DeD1 el6DDD16e6
08017 11311eDDD6 1.D6e6De166 6DD661D6D6 6e6DD61.166 e6166066D1 eD6D6DeD66
OZOt PeeDae6D66 6D6D1.6a6Da 6111DD66D6 euDeaD66ee 661e66DeDl 66e611ED6D
096E 6E666 ZD6P661661 DDP6DD6DDP D36661ezeD e6DraaDDD6 eeDD6ED616
006E 666 16DzaDe6DD 6eD66eeDle 6D6DD16161 D6611De66D 66D66eDDDD
0-178E 6D6u66De6D le616e16D1 1De6D6D66D D66DleDD66 ee616De6D6 DDebale6De
08ZE 6DDD6DDe6D leD66D1.611 6DDeelD6Do eeDle6Ee66 6D616DD66D 66611e6D6D
OZLE DD616PD666 PDDDD1U6DD lae6D16ell 61D1161e6D D6666DD6D1 DPPD61.DDD6
oggE D6DDD6e1D1 PDDDPPD6D1 PDDE6DP6PP D66PD1.666D 66PPU6PDDP ellDel.61D6
009E D1P1466ee6 leD6Dee666 ETUD6DPITE PDPPPIXEU1 6P61P6D61E 6aelel6D6a
OVSE D6D166D6le Da6D611D6e Deeeel6e61 11e1616ze6 166eeeeelD 16DD6DD6D6
0817E P6DDPPP6PP 61D63D66l1 D6UDD66DD6 EaDD66D661 D6UPDD61P6 ZD16111660
OZVE D661DDaUPP 61UD61D66D 616DOPPD1D 1.6DPD6D6DD D6DD6P6D14 666663
09EE D66D6Dae61 u6e66D66e6 Dle6e6ee6D DelleD11.11 16DDee6De6 6eDD66De66
00E DDPD6DDPP P61PD6PPDP U66P6PPDD6 DET61UP6P6 DD6DD66766 1DDD6DP6DD
017ZE b6U6DDP6a1 PD6DP66P61 6DDIaDD616 6D6D66D66P DD66P6DDPD DD6De616ee
081E 66e6D6eD6D 6e611DeD6D 6DDel6;DDD u6D1D6DleD 616D6611D6 aDeD61D66D
orr 666E6.6 DDPD6DD66P P66PDDP6Da P61D6P6D6D DD63PD6D6D 1P6PDPD663
090E DD DP 1DDADDDDD 661116PP61 6D66e6DDD6 6eeDD6DD66 e6D6D666D6
000E E66DDDeD6D De6DluD1.ue 4DDDIa6D6u 6Dz16p6DD6 11ED66DD6D 11616DDe61
0176? 161661eD6D D6E0D66DD6 DPDDEDDPla 6D6DPDDED1 UPPPDPD66D aD6eD66DDe
088Z 6DDDD1D6ED PPDD6PDD6P DDPDDEaDD1 PITaPPDEDD upea11611.1 ueD16D6pel
OZ8Z D16116pull ea16161euD 6DD1.6De6ue elavDel6ED llee1161e6 66
09a PeDe6ellal PPDDETDIPP P6D1DPD61P 6P1PD61Pla 11.1611E6P1_ veD161.1uPa
00Zz ee1.6611D6a DDle66eDe6 11.1.63.6eDle aDeee1116e D11DD6DDD1 116Da611e6
0179Z Pple6leD1D 6DD1Daleel 1DDDaDe61.6 DpeluD1666 66D56pluD1 6DEopplbla
08SZ 366)Ppup16 pppp 116u Da61D11.66D 6116DeED11 DD1D6616e6 1.D66D6DDDe
OZSZ PV6PDD1DPP elaeD1D6e1 1D6161ea6D P61D14166; PPITED6D6D ue61111De6
0917? D66ellope6 16e4e61D6e aD6111eaee efteDe6111 aleD6e661.6 E66.
0017?Plzlee6666 e6DDele661. leulDI.Deft 61elee61.6e 6e611e6611. 1P41P6PD61.
017E? 66DDle6eDD ze6Deve6D6 leDleeD41.6 laDluDDITE D61.6D64DDe e6e66DD66D
08N ETPDDDUDD1 D1DD6P1PPEI DD6P1PD16P DDD6161161. D1611x6DD6 PD6P6PD1PD
OZU 66366DPDPP 66DDETDP61 D6D61.DDDD6 D6663DPP6P PEPUDE611D 166D1.66RDU
ogTz 66DDED666e DlapplabeD 61DD16D1DD 61D6D6DD6e le6DeDD6eD D6616D16DD
OOTZ D6Dee66euD 6D61D6eDeD 6e6D16DeeD e616eDlaD6 DDDIaDDDI.6 PDD6PD6P1P
OVOZ PDDD6D11DP D663DDD61.7 D1P6P66EDE 61e6e6166e eD6e66eD66 D1D111Dear
0861 6666 ED1PD6a1ED 6DD6DD6ED6 Tea6D6UPD1 P667D6P166 ED6661PP63
0Z61 166a6611D6 D11161x6D6 le6DaD6D1D 616Del6e6D DleDD11D66 DDPETPDP6D
0981 1u61DD1PD4 u6PDDa6Dal D1D61E61DD DDET6D6D66 1D66D116PD eu6D66aDD6
008T v6laDD6D6D 61eD666D16 DD6D1DDle6 u6De6DeD16 661eDD6Dle D66eD6euD6
OVLT Eollele6zu DDeDDIalle DD66D6eeee 6eDDleu6le 6D16eDeDD6 6DD6EDDDPD
0891 PDD6DD1.66D 6P1U61DDl6 1E1D6DPPOD 6P1666DPD4 P1PPD6VD11 D1D6PP3D6D
0Z91 D6Dl4eDDD6 eD466D6ee6 6e6DeD6eup 16DDele6D6 6D6e666Dle e6D61D6D6a
09S1 P6D66ee6ea u6D66eu6ee D16D1Deu6 6eD1D6DDD 1.66611D6e6 elDle6eD6
00ST PD6DEP6DD1 EIXPEP6DUD D1DPP666D6 6161D4666D DaeDle6666 D1r6Dee611
OVVT a6lueuDD61 le1.11Deve6 eelaDauppl. le66eDupD6 66D leDleele61
08E1 elelluee6e DueDllee46 DeealD6leD ellelluell 66 luDa6Duele
OZET PPIXD1DIXD DDPPEPP1PD 1PP1D1DP66 6D614uplel 6Ee6 D6D1e1D111
ogzT 161141e1-el D6D6D61416 elDD4ellle UlP6D6D6D6 DOPDP61P6P IMPP16P43
00Z1 le6DDD66e5 6PDP614D6P ETDI.DP6DPP DE1111E01.6 DD661YeDla up6D1D6r6D
OTT DP166P6D1D 666pDaEolp le6DDIT661 6e1De6e1D1 Du6D161eDe 6e66DD1DaD
0801 )101.661Dee DlDleuaDD6 61D6e161D6 aD66DaeleD D66D6DDeue DeD161e6DE
OZOT P1166leel6 leuDaD16De 6E164DeD11. beeeaDD161 DaD6616all 6166161aD1
096 DPDDITPD1X Dl66P1D1.D1 DDPPD611D1 P1D1P1666E EDDUPDDPPD 6PD13DPPD1.
006 DDDEDPPDPD D6PDDPIXED 6PE16D6PDD D666PDDIaD D6P1DDal6D 6pel6DleED
0178 D461DreD1D 1DP16PDEPE 1.6DUDE61.61 EaDDE0aP611 1DDPPDDD66 plpplr6p16
08L 1PPDP15161 61uueD6Pul 1DPETImpl Dp6D611opu uuulaDr6up UDDPPD6PDE
OZL v1P1DD66pu 661.116Dap6 61e6eppulD DupD61pD1D D6PPPDDD1P 16161D6D6D
099 DDDP161PPD 6D6D6DD6eD Dlua611D61 PDDPDDlUDD baRDIXDPUD DUPPPPDD61.
009 1.1DDDleeee D1DeeD661D 6e66p6611D D661DepaDD 661Dee1666 1Dle6eD1DE
OVS Eople6D166D lx6eeD6eD6 DUP6DDIX1P PPP6DPDD1D ee666D6616 1D1666DDle
LO-VO-VOOZ LZTVSVZO VD
LT aftd
0918 luDD6oD66p 0140551616 1DED1E1PED 1PEED6EDDE P1600E0166 DE11006PED
0018 1PD1116116 D6Do6D1D6p ppplp6116D 16ETETTDD1 1.16ppoppbe p6666epplp
0t08 ap1D6D1.Dlp P6e66D6D6p DP1plapp61 661PEDEEDE 0616016016 1E61EP660P
086L DD6Deulu66 1D6p11D6D6 0116E0611E EED01011ED D61.D6D6aap D161pp6ppD
OZ6L 61_DDelu6 66666
1601E60151 PED1E6E0D6 P1P6PE36ED 3.611110611
098L D1D1.161.eaD 6DpeD66Eup DelpDp661D 6DADD1DD1 16u6Eppplp 66DD-ep66pD
008L6
ll_aDD1u6e 1PEED10060 6E111E0111 66EE11606E 1E00116E60 660666015E
OLL D006E00604 ED4060111E DE1DED6EP1 6DEUDE6660 61PEEDDE16 1D6D6aDP11
089L 66DD613.11P 6D6D66D11D DluDu6D6pD 66D16EDDD6 11pDD1D6D6 666666
0Z9L 1Dp1p61066 6DE61u16pr D41D6p1Da6 1DD6pplp6p upD16z1D11 D1101p6D6p
09SL PDD66p6D6D 6D61D1E61D upDplaplaP ppDp6616E1 6DpD111DD6 D1Dze61.661
00SL 100E10E600 6111E11EDE 6E105E1011 1PE6DEE611 D660160660 6EE5060060
OVVL 6006EP116E 61106DEP10 lED616ulaD 6161plappD 6E6161.D661 116p1p61DD
08EL P611Dv6ED6 UPEElEE1PE upplaD6Dup 61e11PzaD6 66E1616111 EED00101E1
OHL Pau64ED6lp D1661111r6 66pE146Dup zDepue6Dup 6616PD1D6D e61D1.6666D
09ZL P1D1111D1P 6111DDle6 6puplplu6 6upuuupp6u D6D6Dvlze6 eD6up6puD6
00ZL 1-1161111l1 166166D6E1 661D6pDpDp EPEDPEED66 DD1p6aaD1D 66666
OtTL PPPpp66D11 DDpal6uDD6 pp61D61D1D 6D61D1p166 azapa6EDE,6 6EE6E1DEDE
080L 1D66DelDpp 1D3661.6616 pp611.Dla6p 6i 6.66
D66p161x16 66666
OZOL P11E66EDEP 1661DUDD6E 06E06610ED 0601P110E6 DEDE6EP166 DDDEED016E
0969 61_1D16D1p1 Dee166DDlp 11DD6D61D6 00E60006ED 116000000E p6DuD61616
0069 1D6661D6ET DD1D6D116D 166e16166D 1.16pD1D1p1 66p1.61.D6Dp DaD6papplD
01789 1a1D6D6646 06PE666011 D001011100 60016100E1 P6600E1136 D061000E50
08L9 D1161DD1D1 D6D616D1DD D1D6up661D DDDD1116D6 6EDDE1P6PE plelDp66PD
0ZL9 E6DDDEPE60 66166E6E01 6EED1060E6 01EPEPEDED 1E06E60E61 0003006001
0999 DE6PaPDDla zaa6D661D6 115060066E PEPE1600PE 66=66vue PD6eDD66pu
0099 ePD6v6161p DuE6uup66p D6Deuap666 6pp1.up6epp pplual66DE lpp166D66p
OtS9 PED1DED1D6 uplu166D6r 666666 D116D166D1 D6D61D6DaD p61DpD136D
08179 1D311D6Dpa 1D1D6D66eD ITADDelue up6E66pul6 D61p6uppD6 DDrzprr616
0z179 166061rapp DpD616E6p6 1Dp161.1p6p D6p6pDlED6 6D6zelDppl 1D661Delpz
09E9 616p66D6u1 p6D6v16DpD 16=Dv6lp DD6ED6D666 6D161666D6 61461666D6
00E9 PD16D6D666 upa6DDDErep Dr6eD6E666 DD61p66D6p el61D1614D 6rDeD166Du
OtZ9 6E66000106 E061PDPDP6 aDaDDuppp6 166Dp61p6z 66D1146D6D 6DaDD6aDDD
0219 eD66ppD1pD E000606600 600E601DED 0601600606 006EEDE660 6D666=
DlEP066E00 660E100661 0661EPPEED 1060066106 00660601E1 006601606D
0909 11D6DADDD D60E100010 6061060166 011000E10D 606PEUEED6 1D6p6uu6DD
0009 5
_EDED606ED D661D1.61Du EITD6161DD 661006000P EEP1401DEE PE11E110PE
0t6S PP11.301Dpp ppaDADD11 111E6066PP PEPE6E6PEE ElE1E610P6 16pp161pDp
088S DE0166DDRe 66611E0E16 006PEEDDDE P66611PDP1 600DEE6600 pp66611eDE
OZ8S 16006PEEDD DEE66611ED El6DP06E1E 5616100111 010166EEPE 60166UPPEE
09Ls 6666pD6p1D DD611PeeD6 66E1D61p6e D6p66Dpa6a pelDD1166D 06001ED6PE
OOLS 60666E601r 61DDEED6D3 elp6D6aupl 6uapple6DD D66666 6666666p
Ot9S 66pu6111x6 0E16E66006 100E61PEED 166601E610 6UP0606160 DED1116101
08SS 15EPETED11 6E6E560060 6E06616E06 DEP60E101E 60E6EED116 11661E6E00
OZSS 6EE6P066PE D66E060060 60060E0661 00603E101D alla6DD66D leD66DDDlu
0917S Eozp611111 Dullp6DDDD rDa166DE61 D646Dp66DD Dep6PeD66u v6rDvDap6u
00-17s 6D6Dpul6lv 6611p6aD6p 1D6p6D4E6p 6D1pDp16e6 6DD66D666D D'epe6D6E6E
(WES ET1.6D1x6up DE1D6DD6p1 1p611DD6up 6a666E6DD1 E166DE6166 1DADD66DE
E6PED066PE 6PE6DE1606 P061E00611 60E060PDDE DEPE116601 1E0610DEPE
OZZS ET166100E6 DE6PEE6PD6 PEE660661E 6006E60660 0610116PED 10E163E660
09's 6116DPDEDD 16DD11616D 6DD66aDD6E p3p6p666pp 666pu666DD v1P6Dpre61
OOTS P001PE6DDE paDluDDDla 166D661p61 De1661DDp6 Dpllp66616 4616pDD661
otos pD66DD66DD 666pD6DD11 166e6E16ip D666Dp6eDD 11D6p6Dvap 6pplp6166P
08617 6D661.D6p6D P6oDE616D6 er6D161D16 DD11116DD6 616Dp661eD lvD6pD6D16
0Z6t P1P60600DE 0666160E61 P101061E60 0116011111 1E6EDDEED6 e6Du6D666p
09817 P0006006PE 66E11E6016 D060616600 6E06600600 EU0660001E E6EEP06001
00817 Pe6D1p6106 DD66D6EED6 616D1up61.6 6000060E06 EU6E066E60 1ED6DEED66
OtLt 06E0006006 6E06060066 PE6116EE6E 66166100E6 au61.6661D6 D66D6D66D1
08917 PPEDE16600 066001E00E PED6016606 P61606601E E66E60006E U0000DEE66
0Z917 zDuD661peD 61DDD66DD6 1D16116661 D66D6pul6D 66pDD66116 6D666DEE66
095.17 P66161.61eD DpAlue661 60060E600E 066EDDEEDE E5PED1PEEP 661eD66D66
00S17 P66wep1D66 D6elllapp6 lubuz6p6lp pplpe6lpeu D6e64pup16 ubpDpElp6p
Ottt D6D6Dlepea pu61plelD6 1D6r6Dpul1. Ppop6ppD66 pepp6Dul.66 D6Dp1.61e6e
08E17 P5106EEDDE DED1E66E66 066006116E 01110PED16 6606EE61ED 06E0060E0E
LO-VO-VOOZ LZTVSVZO VD
CA 02454127 2004-04-07
ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga 8220
cgccaactac ctctgatagt tgagtcgata cttcggcgat caccgcttcc cccatgatgt 8280
ttaactttgt tttagggcga ctgccctgct gcgtaacatc gttgctgctc cataacatca 8340
aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata gactgtaccc 8400
caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc g 8441
<210> 26
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
oligonucleotide primer
<400> 26
aagtcgacgg atcctgatag cttatactca aattcaacaa gttat 45
<210> 27
<211> 1318
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> promoter
<222> (1)..(1318)
<223> TPT truncated promoter
<400> 27
tgatagctta tactcaaatt caacaagtta tatataaatg tatagatact acaatatcat 60
taacaaaagt caccttaaat aaatacacat atcttttatg ttctctattg ttttgcgtac 120
gctaacacaa tttctcatat gcaaaaggat gaatgagtaa caaattacct cataagaaca 180
atcatctttg cttacatact aatacaataa tcactcaatc aaccaataac atcaatcaca 240
taggtttaca tacaataatc actcaatcaa cttcataaga agaatcatgt ttacttaatt 300
catcaattat ccccaaaaac accactatta agtataaact ataacatatt tgtagtgatg 360
ggtcaacatt tttatcatat ttaaactcgg gttccctcaa atcgagaaat agtgaacatg 420
taatattaat tttaaatcgc aattacagaa attaattgaa tttggtcaaa tggacagaat 480
tttatagatt gggtggaact agaaaaaaaa aaaaaaagag tatagggtga attgagtaca 540
tgaaagtaca tggtaatcct agttaaacgc ataatacatg tgggttcatt tgtatttttt 600
tgtaacttac gagtaaactg gctacaacaa aaaaaattag aagatttttt tgttttgtag 660
aaaaccctaa ttttagttat agttgtataa ctttgataaa attataaaat tgtattacga 720
aaaaagtaat aagatattca aaaaagccta gaataacgta tatgactatg agcatgaaac 780
tgcaagtcaa atgctgacag acaaccataa acaaaagaaa ttaaatagag atacctttaa 840
aataagtaaa atttcattta taaaaaatct actttcttgt gaatctgtca cgttcaataa 900
tttgaagacc actcaacata caaggtaaat aatgaaaaat aaaatctacc aaaatttcaa 960
tcattattat cttccaaaaa aacaaaatta tacagatgat gatggtgata tggaacttcg 1020
attggctaat attcactgtg tctctaaaaa ccatccactt atcaagataa gatggaccct 1080
acactcatcc aatctaaacc agtatctcaa gattcttatc taattacatc attctctacc 1140
gttagatgaa attgaccatt aaccctacca taactccata caccgcgaga tactggatta 1200
accaaatcga gatcatcgta gccgtccgat caacaagtac catctcttga aatactcgaa 1260
atcctcataa gtccgtccct ctttgctctc actatcaaaa ctctgaattt cgatttca 1318
<210> 28
<211> 234
<212> DNA
<213> Solanum tuberosum
<220>
<221> terminator
<222> (1)..(234)
<223> terminator sequence of the cathepsin D
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CA 02454127 2004-04-07
Inhibitor gene from potato
<400> 28
cctgcagata gactatacta tgttttagcc tgcctgctgg ctagctacta tgttatgtta 60
tgttgtaaaa taaacacctg ctaaggtata tctatctata ttttagcatg gctttctcaa 120
taaattgtct ttccttatcg tttactatct tatacctaat aatgaaataa taatatcaca 180
tatgaggaac ggggcaggtt taggcatata tatacgagtg tagggcggag tggg 234
<210> 29
<211> 298
<212> DNA
<213> Vicia faba
<220>
<221> terminator
<222> (1)..(298)
<223> terminator of storage protein gene Vf1E1B3 from
Vicia faba
<400> 29
gatcctgcaa tagaatgttg aggtgaccac tttctgtaat aaaataatta taaaataaat 60
ttagaattgc tgtagtcaag aacatcagtt ctaaaatatt aataaagtta tggccttttg 120
acatatgtgt ttcgataaaa aaatcaaaat aaattgagat ttattcgaaa tacaatgaaa 180
gtttgcagat atgagatatg tttctacaaa ataataactt aaaactcaac tatatgctaa 240
tgtttttctt ggtgtgtttc atagaaaatt gtatccgttt cttagaaaat gctcgtaa 298
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