U.S. patent application number 10/849939 was filed with the patent office on 2005-01-20 for genetic transformation using a parp inhibitor.
This patent application is currently assigned to Bayer BioScience NV. Invention is credited to De Block, Marc.
Application Number | 20050014268 10/849939 |
Document ID | / |
Family ID | 8221514 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050014268 |
Kind Code |
A1 |
De Block, Marc |
January 20, 2005 |
Genetic transformation using a PARP inhibitor
Abstract
The invention concerns a process for producing transgenic plant
cells, which comprises: contacting a culture of plant cells with an
inhibitor of poly-(ADP-ribose) polymerase, prior to transformation,
for a period of time sufficient to reduce the response of the
cultured cells to stress and to reduce their metabolism. The
untransformed cells are then contacted with foreign DNA comprising
at least one gene of interest under conditions in which the foreign
DNA is taken up by the untransformed cells and the gene of interest
is stably integrated in the nuclear genome of the untransformed
cells to produce the transgenic cells. The transgenic plant cells
are recovered from the culture. The invention further concerns a
process for increasing the frequency of obtaining transgenic plant
cells, via Agrobacterium-mediated transformation, which comprises:
contacting a culture of plant cells with an inhibitor or
poly(ADP-ribose) polymerase prior to transformation for a period of
approximately 1 to 2 days or culturing transgenic plant cells after
transformation in a medium containing an inhibitor of
poly(ADP-ribose) polymerase for a period of time of approximately 1
to 14 days.
Inventors: |
De Block, Marc; (Merelbeke,
BE) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Bayer BioScience NV
|
Family ID: |
8221514 |
Appl. No.: |
10/849939 |
Filed: |
May 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10849939 |
May 21, 2004 |
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09461416 |
Dec 16, 1999 |
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09461416 |
Dec 16, 1999 |
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08817188 |
May 15, 1997 |
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6074876 |
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Current U.S.
Class: |
435/468 ;
435/419; 800/278; 800/287; 800/320.3 |
Current CPC
Class: |
C12N 15/8201 20130101;
G01N 33/5097 20130101; C12N 15/8289 20130101; C12N 15/79 20130101;
C12N 15/8213 20130101; G01N 33/5091 20130101 |
Class at
Publication: |
435/468 ;
800/278; 800/287; 435/419; 800/320.3 |
International
Class: |
C12N 015/82; C12N
015/87; A01H 005/00; C12N 005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 1995 |
GB |
95401844.6 |
Jul 31, 1996 |
WO |
PCT/EP96/03366 |
Claims
1. (Cancelled).
2. (Cancelled).
3. (Cancelled).
4. (Cancelled).
5. (Cancelled).
6. (Cancelled).
7. (Cancelled).
8. (Cancelled).
9. (Cancelled).
10. (Cancelled).
11. (Cancelled).
12. (Cancelled).
13. (Cancelled).
14. (Cancelled).
15. (Cancelled).
16. (Cancelled).
17. (Cancelled).
18. (Cancelled).
19. (Cancelled).
20. (Cancelled).
21. (Cancelled).
22. (Cancelled).
23. (Cancelled).
24. A method for assessing in vitro the agronomical fitness of a
plant as measured by its seed yield, comprising the steps of: a)
subjecting an explant of said plant to a stress condition; b)
measuring the electron flow in the mitochondrial electron transport
chain to assess agronomical fitness in cells of said explant of
said plant; c) comparing said measurement to that of explants of
control plants or control plant material, under the same conditions
as for said explants of said plant, wherein the greater the amount
of electron flow the fitter said plant.
25. The method of claim 24, wherein said electron flow in the
mitochondrial electron transport chain is determined by measuring
the capacity of said explant subjected to said stress condition to
reduce 2,3,5-triphenyltetrazolium chloride.
26. The method of claim 24, wherein said electron flow in the
mitochondrial electron transport chain is determined by measuring
the capacity of said explant subjected to said stress condition to
reduce 3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyl-2H-tetrazolium.
27. The method of claim 24, wherein said stress condition is
selected from salt stress, osmotic stress, stress by incubation in
the presence of an inhibitor of poly-ADP-ribose polymerase, stress
from extreme temperatures, stress by treatment with sublethal doses
of chemicals, stress by treatment with sublethal doses of
herbicides, stress by treatment with sublethal doses of heavy
metals, or stress by irradiation with UV.
28. The method of claim 24, wherein said stress condition is salt
stress.
29. The method of claim 28, wherein said salt stress is induced by
incubation in K-phosphate buffer comprising between 10 mM and 80 mM
K-phosphate.
30. The method of claim 24, wherein said stress condition is
osmotic stress.
31. The method of claim 30, wherein said osmotic stress in induced
by incubation in a buffer comprising about 2% sucrose.
32. The method of claim 24, wherein said stress condition is
incubation in the presence of an inhibitor of poly-ADP-ribose
polymerase.
33. The method of claim 32, wherein said inhibitor of
poly-ADP-ribose polymerase is selected from niacinamide,
picolinamide, 5-methyl nicotinamide, methylxanthine, thymidine,
benzamide, 3-methoxybenzamide, 3-aminobenzamide, 2-aminobenzamide,
pyrazinamide, theobromine and theophylline.
34. The method of claim 32, wherein said inhibitor is present in a
concentration from about 100 mg/L to about 1,000 mg/L.
35. The method of claim 24, wherein said explant is selected from
callus, hypocotyl explants, shoots, leaf disks or whole leaves.
36. The method of claim 24, wherein said plant is a transgenic
plant.
37. The method of claim 24, wherein said plant is a Brassica plant.
Description
[0001] This invention is related to tissue culture of eucaryotic
cells and improved techniques to obtain genetically transformed
eucaryotic cells and organisms, such as transgenic plant cells or
plants, by lowering the stress reaction of cultured eucaryotic
cells prior to contacting the cells with foreign DNA, particularly
by specific inhibition of poly-(ADP-ribose) polymerase.
BACKGROUND TO THE INVENTION
[0002] Over the years many techniques for the genetic
transformation of higher organisms (animals and plants) have been
developed. In these techniques it is the ultimate goal to obtain a
transgenic organism, e.g. a plant, in which all cells contain a
foreign DNA comprising a gene of interest (the so-called transgene)
stably integrated in their genome, particularly their nuclear
genome.
[0003] Transformation is a complex process which always involves
the contacting of starting cells with a DNA, usually a DNA
comprising foreign gene(s) of interest. The contacting of the cells
with the DNA is carried out under conditions that promote the
uptake of the DNA by the cells and the integration of the DNA,
including the gene(s) of interest into the genome of the cell.
[0004] Starting cells for transformation are usually cells that
have been cultured in vitro for some time. After contacting the
cells with the DNA, the transformed cells generally need to be
cultured in vitro for a certain period in order to separate the
transformed cells from the non-transformed cells and, in the case
of plants, to regenerate transformed plants from the transformed
cells. Indeed, complete plants can be regenerated from individual
transformed cells thus ensuring that all cells of the regenerated
plant will contain the transgene.
[0005] In many plants, genetic transformation can be achieved by
using the natural capacity of certain Agrobacterium strains to
introduce a part of their Ti-plasmid, i.e. the T-DNA, into plant
cells and to integrate this T-DNA into the nuclear genome of the
cells. It was found that the part of the Ti-plasmid that is
transferred and integrated is delineated by specific DNA sequences,
the so-called left and right T-DNA border sequences and that the
natural T-DNA sequences between these border sequences can be
replaced by foreign DNA (European Patent Publication "EP" 116718;
Deblaere et al, 1987 Meth.Enzymol. 153:277-293).
[0006] Certain plant species have proven to be recalcitrant to
Agrobacterium mediated transformation and in these species, as well
as in animals, genetic transformation has been achieved by means of
direct gene transfer by which DNA is inserted into the cells by
physical and/or chemical means, such as by electroporation, by
treatment of the cells with polyethyleneglycol (PEG), by
bombardment of the cells with DNA-coated microprojectiles, etc. (WO
92/09696; Potrykus et al, 1991, Annu.Rev.Plant Physiol.Plant
Mol.Biol. 42:205-225).
[0007] Genetic transformation of eucaryotic cells is generally a
random event, i.e. the transgene is integrated in the genome at
random positions. Often several copies (or parts of copies) of the
transforming DNA are integrated in a single position, and/or at
different positions, resulting in a transformed cell containing
multiple copies of the transgene.
[0008] The expression of the transgene is known to be influenced by
its position in the genome. For instance, a foreign DNA, when
introduced in a plant cell appears to integrate randomly in the
plant genome. Examination of independently transformed plants has
shown a high degree of variability (up to 100-fold) in the
expression level of the introduced gene. Several studies have shown
no correlation between this "between-transformant variability" and
the copy number of the introduced DNA at a given locus. It has been
suggested that some of the variability in expression of introduced
genes in transgenic plants is a consequence of "position effects"
caused by influences of adjacent plant genomic DNA. Other factors
that could contribute to the variability in expression are
physiological variability of the plant material, differences in the
number of independent T-DNA loci in different transformants or the
inhibitory effects of certain T-DNA structures on gene expression.
Between-transformant variability in expression has been observed
for the majority of introduced genes in transgenic plants. The
variability in expression of many introduced genes in independent
transgenic plants necessitates large numbers of transgenic plants
to be assayed to accurately quantitate the expression of the gene.
It would be of great importance if the amount of
between-transformant variability could be reduced (Dean et al,
1988, NAR 16:9267-9283).
[0009] If the transgene is under the control of a tissue-specific
promoter, with the expectation that it will be expressed in
selected tissues of the transformed organisms, the position effects
can lead, at least in some transformants, to loss of specificity of
the promoter and expression of the transgene in undesired tissues,
e.g. in tissue cultured in vitro.
[0010] Factors that are known to influence the efficiency and
quality of the genetic transformation process are the method of DNA
delivery, specific tissue culture conditions, the physiological and
metabolic state of the target cells etc. Direct gene transfer
methods for instance are generally known to result in transformed
organisms with a high copy number of the transgene.
[0011] Many of these factors are not under the control of man.
SUMMARY OF THE INVENTION
[0012] This invention provides a process for producing transgenic
eucaryotic cells, particularly plant cells. The process comprises
contacting a culture of untransformed cells with an inhibitor of
poly-(ADP-ribose) for a period of time sufficient to reduce the
response of the cultured cells to stress and to reduce the
metabolism of the cultured cells, particularly to reduce the
electron flow in the mitochondrial electron transport chain. The
untransformed cells are then contacted with foreign DNA comprising
at least one gene of interest under conditions in which the foreign
DNA is taken up by the untransformed cells and the gene of interest
is stably integrated in the nuclear genome of the untransformed
cells to produce the transgenic cells which are recovered from the
culture.
[0013] The process may preferably comprise contacting untransformed
eucaryotic (e.g.) cells with foreign DNA comprising at least one
gene of interest under conditions in which the foreign DNA is taken
up by the untransformed cells and the gene of interest is stably
integrated in the nuclear genome of the untransformed cells to
produce the transgenic cells. The untransformed cells are cultured
in vitro in a culture medium containing an inhibitor of
poly-(ADP-ribose) polymerase, preferably niacinamide, preferably
for at least 2 to 3 days, particularly for at least 4 days (e.g.
4-5 days), before the contacting of the untransformed cells with
the foreign DNA. The inhibitor can in addition also be applied to
cultured cells that are being contacted or that have been contacted
the foreign DNA.
DESCRIPTION OF THE INVENTION
[0014] The present invention is based on the observations that
poly-(ADP-ribose) polymerase (PARP) is an enzyme that is involved
in regulating the general metabolic state of an eucaryotic cell and
that inhibition of this enzyme can be used to influence the
metabolic state of cells which are targeted for transformation (or
which are being transformed) to increase the efficiency and/or
quality of transformation.
[0015] In mammalians, PARP is a monomeric nuclear Zn-finger protein
of about 116 kD that is closely associated with nuclear DNA,
particularly with actively transcribed euchromatic regions (Shah et
al, 1995, Anal.Biochem. 227:1-13). The protein is normally an
inactive enzyme but is known to be activated by nicked or otherwise
damaged DNA. Active PARP transfers the ADP-ribose moiety of NAD+ to
various nuclear proteins to synthesize a polymer of ADP-ribose
bound to these proteins which include PARP itself, polymerases,
histones, endonuclease etc. The proteins on which such a ADP-ribose
polymer is synthesized become biologically inactive (de Murcia et
al, 1994, TIBS 19:172-176; Cleaver et al, 1991, Mutation Res.
257:1-18).
[0016] The biological function of PARP is largely unknown but the
enzyme has been implicated in:
[0017] enhancement of DNA repair (Satoh et al, 1992, Nature
356:356-358; Satoh et al, 1993, J.Biol.Chem. 268:5480-5487),
[0018] recombination events: in general inhibition of PARP is
observed to inhibit illegitimate recombination and to increase
intrachromosomal recombination but it does apparently not affect
extrachromosomal recombination (Farzaneh et al, 1988, NAR
16:11319-11326; Waldman and Waldman, 1990, NAR 18:5981-5988;
Waldman and Waldman, 1991, NAR 19:5943-5947),
[0019] regulation of gene expression: inhibition of PARP is
observed to decrease gene expression (Girod et al, 1991, Plant
Cell, Tissue and Organ Culture 25:1-12);
[0020] reducing the amount of available NAD+ (and by consequence
its precursor ATP): this results in a general slowing down of cell
metabolism (Lazebnik et al, 1994, 371:346-347; Gaal et al, 1987,
TIBS 12:129-130; Cleaver et al, supra)
[0021] It is known that PARP can be efficiently inhibited by a
number of compounds (Durkacz et al, 1980, Nature 283:593-596; Sims
et al, 1982, Biochemistry 21:1813-1821). Examples of such compounds
are certain pyridine analogs such as nicotinamide analoques,
including niacinamide, picolinamide, and 5-methyl nicotinamide;
purine analogs like methylxanthines; thymidine; pyrazinamide
analogs and many aromatic amides such as many benzamide analogs
including benzamide, 3-methoxybenzamide and 3-aminobenzamide. For
the purpose of this invention a PARP inhibitor is generally
understood as any specific inhibitor of poly-(ADP-ribose)
polymerase which can be taken up by a eucaryotic cell, particularly
a plant cell, and which has a inhibition constant (Ki) which is
lower than 1.times.10.sup.-5, particularly lower than
1.times.10.sup.-6. Generally it is desired that the PARP inhibitor
used with this invention be a compound which in human lymphocytes,
cultured in medium containing the inhibitor at a concentration of 2
mM, results in a 80-90% inhibition of PARP (Sims et al, supra).
Generally it is also preferred that cells cultured in medium
containing the PARP inhibitor retain their capacity of DNA
repair.
[0022] Particularly preferred PARP inhibitors are those listed
above and especially niacinamide (nicotinamide), picolinamide,
5-methylnicotinamide, 2-aminobenzamide, pyrazinamide, theobromine
and theophylline. Particularly niacinamide is believed to be a
useful inhibitor for the purpose of this invention.
[0023] Basically the present invention provides a modification of
existing procedures for the genetic transformation of eucaryotic
cells, particularly plant cells, by including in the medium in
which such cells are cultured a PARP inhibitor such as niacinamide,
for a defined period of time. In particular the PARP inhibitor is
added to the culture medium at least 1 day prior to the moment (the
"contacting time") at which the cells are contacted with foreign
DNA comprising one or more genes of interest. However, depending on
the purpose, the PARP inhibitor may also be added to the culture
medium during and/or after the contacting time or even solely after
the contacting time.
[0024] In one aspect of this invention treatment of cultured cells,
tissues or organs with PARP inhibitors may be used to increase the
quality of transformation as measured by the copy number of the
transgene and by variation in transgene expression (quality and
quantity) in the transformed cells and in organisms obtained from
the transformed cells.
[0025] In many conventional procedures for genetic transformation
of eucaryotc cells, particularly plant cells, cultured cells,
tissues or organs will be used as starting material and cells in
such cultures will be contacted with foreign DNA comprising at
least one gene of interest (i.e. the transgene) under conditions
that will promote the uptake of the foreign DNA in the cells and
the ultimate integration of the foreign DNA into the genome of the
cells.
[0026] In one embodiment of the invention it is preferred that a
PARP inhibitor is added to the culture medium for a period of at
least 2-3 days, preferably at least about 3 days, prior to
contacting the cells with the foreign DNA. The exact period in
which the cultured cells are incubated in PARP inhibitor containing
medium is believed not to be critical but should probably not
exceed 4 weeks. It appears that 2-14 days, particularly 3-10 days,
is an optimal period and best results were obtained with an
incubation period of approximately 4 to 5 days prior to the
contacting time. Generally it is believed that 4 days is a useful
period for the PARP inhibitor to be added to the culture medium
prior to the contacting time.
[0027] The concentration of the PARP inhibitor in the medium is
also believed to have an effect on the inhibition of PARP, which
varies depending on the nature of the cells (species, tissue
explant, general culture conditions, etc.) However, within certain
concentration ranges, the effect is minimal, especially when the
cultured cells are not incubated for longer than 14 days. The
optimal concentration range of PARP inhibitor in the medium may
vary depending on the species from which the tissue, cell or cell
culture is derived, but 250 mg/l (about 2 mM) is believed to be a
suitable concentration for many purposes (e.g. for use with
material derived from wheat). However, when nicotinamide is used in
combination with plant material derived from rice, the
concentration of nicotinamide should preferably be between 500 mg/l
(about 4 mM) and 1000 mg/l (approx. 8 mM). On the other hand, when
nicotinamide is used in combination with plant material derived
from corn, the concentration of nicotinamide should preferably be
100 mg/l. Likewise, a concentration of 100 mg/l is already
effective for wheat-derived plant material, but higher
concentrations may be used. The optimal concentration will depend
on the nature of the specific PARP inhibitor used, particularly on
its strength of inhibition (as measured by its Ki and/or by its
percentage inhibition of PARP under standard conditions--Sims et
al, supra). It was found for instance that the optimal
concentration for nicotinamide is approximately 250 mg/l (i.e.
about 2 mM) but it is believed that concentrations up to 1000 mg/l
(approx. 8 mM) and as low as 150 mg/l (approx. 1.25 mM), even as
low as 100 mg/l can be used to good effect. Preferably the
nicotinamide concentration should be between 200 and 300 mg/l, i.e.
between approximately 1.5 mM and 2.5 mM. In similar conditions, the
optimal concentration for more potent PARP inhibitors such as
3-methoxybenzamide is about 0.5 mM, but it is believed that
concentrations up to 2 mM and as low as 0.1 mM can be used to good
effect. Similar concentrations apply to other PARP inhibitors.
[0028] If incubation times of longer than 14 days are used it is
believed that the PARP inhibitor concentration should be reduced
below 2 mM (e.g. between 0.5 mM and 1.5 mM and particularly
approximately 0.8 mM).
[0029] For other PARP inhibitors optimal concentrations can be
easily established by experimentation in accordance with this
invention.
[0030] During transformation it is not known whether the
integration of the DNA into the genome of the cell occurs
immediately after uptake of DNA by the cell. It may very well be
that the foreign DNA exists as free DNA within the cell for a
certain period after the contacting time. Therefore cultured cells
may be further incubated in medium containing a PARP inhibitor
during and, for a limited period of time after, contacting the
cells with the foreign DNA. Again the length of the incubation
period is not critical but is preferably 2-10 days, particularly
approximately 4 days. It is preferred that the inhibitor
concentration of the PARP inhibitor in the culture medium after the
contacting time should be below 2 mM, between 0.8 and 1 mM. If the
cells that are to be transformed are not obtained from a cell or
tissue culture (e.g. when intact tissue of an organism is contacted
directly with DNA, as for example described in WO 92/09696) the
PARP inhibitor may still be applied to the target cells prior to
the contacting time but the addition of the PARP inhibitor to the
culture of the transformed cells during or after the contacting
time is preferred.
[0031] As indicated above, PARP inhibitor treatment of cultured
cells for at least 2-3 days increases the quality of
transformation. Indeed the number of copies of the foreign DNA is
expected to be generally lower and variation in expression profile
(level--i.e. the quantity--of expression as well as spatial and
time distribution--i.e. the quality--of expression in the
transgenic organism) of the gene(s) of interest in the foreign DNA,
due to position effects, is decreased. However, at least in this
aspect of the invention, the efficiency of transformation can be
decreased. The efficiency of transformation as used herein can be
measured by the number of transformed cells (or transgenic
organisms grown from individual transformed cells) that are
recovered under standard experimental conditions (i.e. standardized
or normalized with respect to amount of cells contacted with
foreign DNA, amount of delivered DNA, type and conditions of DNA
delivery, general culture conditions etc.).
[0032] Therefore it is preferred that the invention is used with
transformation procedures that already have a high efficiency, such
as Agrobacterium mediated transformation of dicots and direct gene
transfer in monocots, particularly cereals (e.g. electroporation or
particle bombardment of compact embryogenic callus in cereals--see
WO 92/09696). Indeed these transformation procedures are generally
highly efficient but the quality of transformation is generally
poor. Position effects are large and, especially with direct gene
transfer, the copy number of the transgene is often exceptionally
high making analysis and selection of optimal transformants, as
well as further breeding with the transformants, difficult.
[0033] In another aspect of this invention treatment of cultured
plant cells for a short period of time (i.e. 1 day to maximally 2
days) prior to, or after contacting the cells with DNA may be used
to increase the efficiency of Agrobacterium mediated transformation
of plant species, such as many monocots, particularly the major
cereals such as wheat and corn, for which this method is generally
inefficient. It is believed that treatment of cultured plant cells
during the contacting time may result in a lower tranformation
efficiency, and might therefore not be suitable for this aspect of
this invention. Likewise, it is believed that for the purpose of
this aspect of the invention, the optimal treatment with a PARP
inhibitor is 1 day to maximally 2 days prior to the contacting
time, or alternatively 1 to maximally 2 days after the contacting
time. In this embodiment of the invention the contacting of the
plant cells with the DNA should of course be understood as
contacting the cells with an appropriate Agrobacterium strain
harboring an artificial T-DNA containing the foreign DNA with the
gene(s) of interest. In this embodiment of the invention the
quality of transformation is expected not to be affected but this
is generally deemed to be of lesser importance since Agrobacterium
mediated transformation, being a biological process, already
results in a generally low copy number of the transgene in the
transformed plant cells.
[0034] In accordance with this invention the addition of PARP
inhibitors, such as niacinamide, to the culture medium of
eucaryotic cells, can be used in combination with any known
transformation procedure that requires cells, tissues or organs
cultured in vitro as starting cells to be contacted with foreign
DNA. The process of this invention is thus generally identical to
existing conventional transformation methods except for the fact
that at some times during the tissue culture of the cells, a PARP
inhibitor is added to the culture medium.
[0035] The cell of a plant, particularly a plant capable of being
infected with Agrobacterium such as most dicotyledonous plants
(e.g. Brassica napus) and some monocotyledonous plants, can be
transformed using a vector that is a disarmed Ti-plasmid containing
the gene(s) of interest and carried by Agrobacterium. This
transformation can be carried out using conventional procedures (EP
0,116,718; Deblaere et al, supra; Chang et al, 1994, The Plant
Journal 5:551-558). Preferred Ti-plasmid vectors contain the
foreign DNA between the border sequences, or at least located to
the left of the right border sequence, of the T-DNA of the
Ti-plasmid. Of course, other types of vectors can be used to
transform the plant cell, using procedures such as direct gene
transfer (as described, for example, in EP 0,233,247), pollen
mediated transformation (as described, for example, in EP
0,270,356, PCT patent publication "WO" 85/01856, and U.S. Pat. No.
4,684,611), plant RNA virus-mediated transformation (as described,
for example, in EP 0,067,553 and U.S. Pat. No. 4,407,956) and
liposome-mediated transformation (as described, for example, in
U.S. Pat. No. 4,536,475). Cells of monocotyledonous plants such as
the major cereals including corn, rice, wheat, barley, and rye, can
be transformed (e.g. by electroporation) using wounded or
enzyme-degraded intact tissues capable of forming compact
embryogenic callus (such as immature embryos in corn), or the
embryogenic callus (such as type I callus in corn) obtained
thereof, as described in WO 92/09696. In case the plant to be
transformed is corn, other recently developed methods can also be
used such as, for example, the method described for certain lines
of corn by Fromm et al., 1990, Bio/Technology 8:833; Gordon-Kamm et
al., 1990, Bio/Technology 2:603 and Gould et al., 1991, Plant
Physiol. 95:426. In case the plant to be transformed is rice,
recently developed methods can also be used such as, for example,
the method described for certain lines of rice by Shimamoto et al.,
1989, Nature 338:274; Datta et al., 1990, Bio/Technology 8:736; and
Hayashimoto et al., 1990, Plant Physiol. 93:857; Hiei et al, 1994,
The Plant Journal 6:271-282).
[0036] The transformed cell can be regenerated into a mature plant
and the resulting transformed plant can be used in a conventional
breeding scheme to produce more transformed plants with the same
characteristics or to introduce the gene(s) of interest in other
varieties of the same related plant species. Seeds obtained from
the transformed plants contain the chimeric gene(s) of this
invention as a stable genomic insert. Thus the gene(s) of interest
when introduced into a particular line of a plant species can
always be introduced into any other line by backcrossing.
[0037] In animals pluripotent embryonic or somatic stem cells can
be used as target for transformation (Capecchi et al, 1989,
TIG:5:70-76).
[0038] The transformed cells and organisms of any plant or animal
species, produced by the process of this invention, contain the
foreign DNA as a stable insert in their genome, particularly in
regions of the genome that remain transcriptionally active in the
untransformed cells that have been exposed to a PARP inhibitor in
accordance with this invention. As described above it is believed
that in cells treated with a PARP Inhibitor for at least 3 days,
particularly for at least 4 days, only a limited number of genomic
regions will remain transcriptionally active. In this regard the
transformed cells, obtained with this process of the invention,
will be characterized by having the foreign DNA integrated in a
limited number of genomic regions. That the transformed cell or
organism was obtained by this process of the invention can thus be
easily ascertained by 1) culturing transformed cells or tissues
under conditions that are similar as those in which the
untransformed cells or tissues were grown or incubated prior to the
integration of the foreign DNA in the genome (i.e. incubating in
medium containing 250 mg/l niacinamide for 4-5 days prior to the
contacting time), and 2) monitoring the expression of at least one
transgene in the foreign DNA that is expected to be expressed under
normal tissue culture conditions (i.e. a selectable marker gene
under the control of a promoter that directs expression in tissue
culture). Under the above conditions the transformed cells or
tissues of this invention express the relevant transgene in the
tissue culture at essentially the same levels whether or not a PARP
inhibitor is present in the culture medium. It is thus expected
that, for instance after 4-5 days of culturing of the transformed
cells in medium containing the PARP inhibitor, mRNA levels are not
signicantly decreased, i.e. do not become lower than 75%,
preferably not become lower than 90%, when compared to the mRNA
levels observed in cells cultured in medium not containing the
inhibitor. Indeed, if the relevant transgene is integrated in other
regions of the genome (i.e. in regions that are normally not
transcriptionally active in cells treated with PARP inhibitor
according to this embodiment of the invention), the expression of
the relevant transgene is considerably reduced after incubation of
the cells in medium containing the PARP inhibitor for at least 3
days, e.g. 4-5 days (i.e. mRNA levels will drop below 75%,
particularly below 50%, more particularly below 30%) when compared
to the mRNA levels observed in cells cultured in medium not
containing the inhibitor).
[0039] The method of the present invention can in principle be used
to transform eucaryotic cells with any foreign DNA. Generally the
foreign DNA comprises at least one gene of interest comprising 1) a
promoter region with a promoter capable of directing transcription
of DNA into a RNA in cells of the eucaryotic, e.g. plant, species
that is to be transformed and 2) a coding region coding for a RNA
or protein. Most often the gene of interest will also comprise 3) a
3' untranslated region of a eucaryotic gene containing a
polyadenylation signal. The promoter can be selected to direct
expression in selected tissues of the eucaryotic organism. Such a
tissue-selective promoter is not expected to direct expression in
other non-selected tissues. For instance promoters are known that
direct expression selectively in stamen tissues of a plant and such
promoters have been used to produce male sterile plants and other
plants useful for producing hybrids (EP 344029; EP 412911; WO
9213956; WO 9213957; Mariani et al, 1990, Nature 347:737-741;
Mariani et al, 1992, Nature 357:384-387).
[0040] It is believed that the method of the present invention is
particularly useful to transform eucaryotic cells with at least one
gene of interest comprising a tissue-selective promoter, such as a
stamen selective promoter, especially if expression of the gene of
interest in the organism, such as a plant, outside the selected
tissues (where the tissue-selective promoter is active, i.e.
directs expression) is undesired for example because the gene
product (for instance a protein such as a ribonuclease, e.g.
barnase) is capable of killing or disabling the cells in which they
are produced. In such cases expression of the gene of interest in
tissue culture, or in non-selected tissues of the organisms can
negatively affect the quality as well as the apparent efficiency of
transformation. When the method of this invention is used, the
overall efficiency of transformation may be reduced but the average
quality of transformation is expected to be significantly improved
because of lower copy number of the gene of interest in the genome
of the transformed cells and because of reduced position effects
i.e. the general integration of the gene of interest in the genomes
at locations that minimally affect the transcriptional properties
of the promoter of the transgene.
[0041] The foreign DNA used in the method of this invention
generally also comprises a selectable marker gene the expression of
which allows the selection of transformed cells (or organisms) from
non-transformed cells (or organisms). Such selectable marker gene
generally encodes a protein that confers to the cell resistance to
an antibiotic or other chemical compound that is normally toxic for
the cells. In plants the selectable marker gene may thus also
encode a protein that confers resistance to a herbicide, such as a
herbicide comprising a glutamine synthetase inhibitor (e.g.
phosphinothricin) as an active ingredient. An example of such genes
are genes encoding phosphinothricin acetyl transferase such as the
sfr or sfrv genes (EP 242236; EP 242246; De Block et al, 1987 EMBO
J 6:2513-2518).
[0042] The inventors also found that the initial reaction of cells,
particularly cells contacted with PARP inhibitors, is a stress
reaction which enhances free radical production by the cell.
However, this stress only lasts for a limited period of time after
which further contact with the PARP inhibitor causes a decrease in
cell metabolism, particularly a decrease in electron flow in the
mitochondrial electron transport chain. Therefore, the invention
also relates to a new method to assess the agronomical fitness of a
population of transformed plants to determine in which lines the
plants have a foreign DNA integrated in their genomes in a way that
agronomical performance is not or substantially not affected. The
assay is based on comparative reaction of transgenic cells and
corresponding untransformed controls to stress conditions.
[0043] The method comprises exposing the transgenic cells to stress
conditions which induce the production of free radicals in the
tissues or the cells, measuring the amount of free radicals
produced in the transgenic cells with the amount of free radicals
produced in control cells exposed to similar stress conditions.
Preferably the cells of the transgenic organism to be assayed are
exposed to stress conditions by being treated with a substance
which induces increasing osmotic and/ or salt stress on the
cells.
[0044] The properties of PARP inhibitors, such as niacinamide, to
enhance free radical production in cells incubated with the
inhibitor for not longer than 2 days, preferably not longer than 1
day, can be used to assay the (relative) fitness of a population of
transgenic eucaryotic organisms, particularly plants.
[0045] The term fitness used herein is intended to designate the
agronomical performance of a population of plants, as measured for
instance by its yield (e.g. its seed yield) as compared to a given
reference population. Agronomical performance is generally thought
to be correlated with the general resistance of the plants to a
range of stress conditions which are likely to be encountered in
the field locations where the plants are normally grown. For any
population of transformed plants (i.e. a transgenic line) the
relevant reference population is a population of untransformed
plants of the same variety.
[0046] It is known that in transformed plants and other organisms
transgene expression may be qualitatively and quantitatively
influenced by the genomic domain in which the transgene(s) are
integrated, that undesired transgene expression may interfere with
cell metabolism (e.g. when the transgene encodes a cytotoxic
protein), that mutations may be induced in the transformed organism
either by somaclonal variation or by insertional inactivation of
endogenous genes by the transgene(s), or that expression of
endogenous genes may be deregulated by sequences in the foreign
DNA. As a consequence many transformed lines may not be
agronomically useful. The assay of this invention will for example
allow to identify a line (i.e. a group of genetically similar
plants) of transformed plants that have the transgene(s) integrated
in regions that minimally affect the fitness of the plants, thus
avoiding the extensive laboratory, greenhouse and/or field
evaluations which are normally required to identify the
transformants with the best agronomical properties.
[0047] The assay in accordance with this invention essentially
comprises the incubation of cells or tissues of transformed plants
of a particular transgenic line (e.g. callus, hypocotyl explants,
shoots, leaf disks, whole leaves etc.) preferably with a PARP
inhibitor (although for some plant species this is not necessary)
under a range of conditions which induce the production of a
different amount of free radicals in the tissues. An incubation
time of approximately one day is normally sufficient to generate
the desired amount of free radicals. Appropriate controls, i.e.
untransformed tissues obtained from untransformed plants at the
same developmental stage and grown in the same conditions as the
transformed plant from which the transformed tissue was obtained,
are subjected to the same treatment. Preferably the untransformed
line is identical to the transgenic line except for the presence of
the transgene(s).
[0048] For each plant line (control or transformant) it is
preferred that a number of plants is assayed.
[0049] Useful conditions for the incubation of the untransformed
and transformed tissues are those which induce increasing osmotic
and salt stress in the incubated cells or tissues. For example a
series of buffers with different salt concentrations containing a
PARP inhibitor can be made. A useful buffer series is a K-phosphate
buffer containing 2% sucrose and 250 mg/l niacinamide in which the
K-phosphate concentration is increased from anywhere between 10 to
80 mM (e.g. in steps of 5 mM, i.e. 10, 20, 25, 30, 35, 40, 45, 50,
55, 60 mM). The K-phosphate concentrations will induce mild but
increasing salt and osmotic stress in plant cells. The niacinamide
in the medium further enhances radical production and stress on the
plant cells. The range of K-phosphate concentrations used will
depend on the natural sensitivity of the plant species (or if
desired the plant line) to the salt and osmotic stress. In
sensitive plant species, which will not tolerate high salt stress,
the maximum K-phosphate concentration may for instance be 50 mM, in
less sensitive species this maximum K-phosphate concentration can
be increased up to 70 or 80 mM or even higher. For each plant
species the minimum and particularly the maximum salt (e.g.
K-phosphate) concentration can be determined experimentally for an
untransformed line--the only requirement is that at all
concentrations used the plant tissue remains viable. Although the
addition of a PARP inhibitor to the medium, such as niacinamide, is
preferred it is not required for assaying plant species that are
very sensitive to salt and/or osmotic stress.
[0050] After the one day incubation the capacity of the transformed
and control tissues to reduce 2,3,5-triphenyltetrazolium chloride
(TTC) is measured e.g. by the following procedure which is modified
from Towill and Mazur (supra):
[0051] incubate the tissues for 1 to 4 hours in K-phosphate buffer
(pH 7.4) containing 10 mM TTC and 0.1% Tween20. As a control
similar plant material is incubated in the same buffer withour
TTC.
[0052] extraction of reduced TTC (e.g. freezing at -70.degree. C.
followed by thawing at 40.degree. C. and shaking the plant material
in ethanol for 45-60 minutes)
[0053] spectrophotometric quantification of reduced TTC at 485 nm
(optical density OD.sub.485; for chiorophyil poor plant material)
or 545 nm (OD.sub.545; for chlorophyll rich plant material). The
O.D. of the control extract is subtracted from the OD of the
TTC-reacted extracts. In the above conditions 0.1 mM reduced TTC
corresponds to an OD.sub.485 of 0.214 or OD.sub.545 of 1.025 (light
path 1 cm).
[0054] the reducing capacity of the transformed plant line is
compared to that of the control line.
[0055] The amount of reduced TTC is determined by the intensity of
the cytochromal and alternative respiratory pathways and the
radical concentration in the tissues which, in turn are determined
by the presence of mutations, the expression of genes affecting the
metabolic activity of the plant cells, the developmental stage and
the reaction of the tissue to external factors, such as stress
factors.
[0056] The TTC reducing capacity (as for instance measured by the
O.D. at 485 nm) for tissues incubated at high salt concentration
(TTC-high) is expressed as the percentage of the TTC reducing
capacity of the tissues incubated at a low salt concentration
(TTC-low); in other words a TTC-ratio value is calculated as
follows:
TTC-ratio=TTC/high.100/TTC.low.
[0057] The value of TTC-ratio is a measure of the fitness of a
plant line as compared to a control line.
[0058] The determination of TTC-low and TTC-high will depend on the
sensitivity of the plant species to the applied salt stress.
Usually TTC-low will correspond to a salt concentration between 10
and 25 mM K-phosphate, e.g. at 20 mM while TTC-high will correspond
to a salt concentration between 50 and 80 mM K-phosphate. The only
requirement is that TTC-high should be significantly lower than
TTC-low; preferably TTC-high should be lower than 50% of TTC-low,
particularly lower than 30% of TTC-low. For instance for Brassica
napus, TTC-low and TTC-high can be typically obtained from tissues
incubated at respectively 20 mM and 60 mM K-phosphate buffer
containing 250 mg/l niacinamide. TTC-high and TTC-low, for both the
transformed and untransformed line, will usually be an average
obtained from several measurements taken on a number of tissue
explants from a number of plants of each line. For instance for
each line of Brassica napus about 32 leaf discs (diameter 1 cm)
from 8 different plants (i.e. about four leaf discs per plant) can
be assayed to determine 32 TTC-high and 32 TTC-low values which are
averaged to obtain the TTC-high and TTC-low values used for the
calculation of TTC-ratio. Other examples of sample sizes which have
been used are 35 shoots from Arabidopsis thaliana,or 150 hypocotyl
explants derived from about 25 seedlings of Brassica napus.
[0059] Transformed lines with a value of TTC-ratio which does not
deviate more than 20%, preferably not more than 10% of the
TTC-ratio value of the control line are selected. These lines are
likely to have the transgene(s) integrated in regions that
minimally affect the fitness of the plants.
[0060] It is clear that additional information considering the
fitness of the plant material studied can be obtained by comparing
the TTC-reducing capacity of the plant material in absence of a
PARP-inibitor with the TTC-reducing capacity of the plant material
in the presence of a PARP-inibitor for each experimental point of
the buffer series mentioned above.
[0061] While the TTC-reduction assay is especially suitable for the
identification of transgenic plants, where transgenes are
integrated in regions that minimally affect the fitness of the
plants, this test can also be succesfully applied to discriminate
mutant plants, cells or cell lines from the wild-types.
[0062] The TTC-reducing assay can further be used in a modified way
to determine the quality and the fitness of plant material, for
example plant material to be used in transformation experiments
(i.e. whether particular plant material, e.g. explants, is suitable
as starting material). To this end the TTC-reducing assay can be
adapted for example in the following way:
[0063] 1. A sample of the plant material to be tested for its
suitability for transformation, is incubated for one day in plant
culture medium or a buffer containing 2% sucrose and a K-phosphate
concentration ranging between 10 and 80 mM, typically around 25 mM,
to which a suitable amount of a PARP inhibitor, such as niacinamide
has been added. For niacinamide, a preferred concentration to be
used is 250 mg/L, although concentrations as low as 100 mg/L and as
high as 1000 mg/L may be used. A comparable control sample of the
same plant material is incubated under similar conditions without
PARP inhibitor.
[0064] 2. After one day of incubation the capacity of the plant
material incubated with PARP inhibitor and the control plant
material to reduce TTC is measured by the procedure described
above.
[0065] The TTC reducing capacity (as for instance measured by the
O.D. at 485 nm) for plant material incubated with PARP inhibitor
(TTC-INH) is compared with the TTC reducing capacity of the control
plant material incubated without PARP inhibitor (TTC-CON) and a
ratio (E) is calculated as follows:
E=TTC-INH/TTC-CON
[0066] The value E is a measure of the quality and fitness of the
plant material, for example explants to be transformed. It is
believed that those tissues, wherein the E value is larger than or
equals 1, are healthy tissues, which are particularly suitable as
starting material for transformation.
[0067] The modified TTC-procedure thus allows to select those types
of (cultured) plant material especially appropriate for use in a
transformation procedure, particularly the procedures of this
invention that include the use of a PARP inhibitor.
[0068] As the quality of plant material will also be affected by
the particular culture conditions used prior to transformation
(especially cells, tissues or explants derived from plants
recalcitrant to transformation)the assay of this invention is
further useful to identify suitable culture conditions to obtain
suitable starting plant material. Thus it has beeen found by the
inventor that, when culturing plant material from corn, it is
preferred to include proline, preferably at a concentration of
about 8 mM, simultaneously with the PARP inhibitor, in the culture
medium.
[0069] As already mentioned, incubation of cells or tissues in the
presence of a PARP inhibitor for longer than 1 to 2 days leads to a
general reduction in cell metabolism, particularly a reduction in
the electron flow in the mitochondrial electron transport chain
(after the initial increase, characteristic of healthy cells or
tissues, during the first day). The period of time required to
reduce the metabolism to an optimal level (for the purpose of
improving the qualitative aspect of transformation) is that period
after which a decrease in TTC-reducing capacity between 20% and
50%, preferably between 30% and 40%, particularly about 35%, is
achieved for plant material incubated with a PARP inhibitor (e.g.
niacinamide) when compared to control plant material incubated
without the PARP inhibitor (i.e. the period after which the E value
is between 0.5 and 0.8, preferably is between 0.6 and 0.7,
particularly is about 0.65).
[0070] It is clear that the assays of this invention can be readily
adapted by one skilled in the art of the field, for example to suit
the needs of the particular cell type, tissue or explant or of the
particular species from which the cells, tissues or explants are
derived. Furthermore the assay can be adapted to assay a peculiar
aspect of fitness of cells, tissue, explant or organism. For
instance, it is possible to apply a type of stress different from
osmotic or salt stress, such as stress brought about by extreme
temperatures, by sublethal treatment with chemicals (e.g.
herbicides, heavy metals) or by irradiation with UV. Furthermore,
other types of PARP inhibitors, as mentioned before may be used,
within the indicated concentration ranges. Although it is believed
that for the purpose of the assays defined here, TTC is the most
suited substrate, other indicator molecules, such as MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium) can be
used to measure the electron flow in the mitochondrial electron
transport chain downstream of the "ubiquinone pool".
[0071] Unless otherwise indicated all experimental procedures for
manipulating recombinant DNA were carried out by the standardized
procedures described in Sambrook et al., 1989, "Molecular Cloning:
a Laboratory Manual", Cold Spring Harbor Laboratory, and Ausubel et
al, 1994, "Current Protocols in Molecular Biology", John Wiley
& Sons.
[0072] The polymerase chain reactions ("PCR") were used to clone
and/or amplify DNA fragments. PCR with overlap extension was used
in order to construct chimeric genes (Horton et al, 1989, Gene
77:61-68; Ho et al, 1989, Gene 77:51-59).
[0073] All PCR reactions were performed under conventional
conditions using the Vent.TM. polymerase (Cat. No. 254L--Biolabs
New England, Beverley, Mass. 01915, U.S.A.) isolated from
Thermococcus litoralis (Neuner et al., 1990, Arch.Microbiol.
153:205-207). Oligonucleotides were designed according to known
rules as outlined for example by Kramer and Fritz (1968, Methods in
Enzymology 154:350), and synthesized by the phosphoramidite method
(Beaucage and Caruthers, 1981, Tetrahedron Letters 22:1859) on an
applied Biosystems 380A DNA synthesizer (Applied Biosystems B. V.,
Maarssen, Netherlands). In the examples MS medium means Murashige
and Skoog medium (Murashige and Skoog, 1962, Physiol. Plant
15:473-479).
[0074] In the following examples, reference will be made to the
following sequence listing and figures:
[0075] Sequence Listing
[0076] SEQ ID NO 1: T-DNA of plasmid pTHW107
[0077] SEQ ID NO 2: plasmid pTS172
[0078] SEQ ID NO 3: PT72 promoter contained in plasmid pTS772
[0079] SEQ ID No 4: plasmid pVE136
[0080] SEQ ID No 5: T-DNA of plasmid pTHW142
EXAMPLES
Example 1
Tissue Culture of Wheat Embryogenic Callus and Brassica napus
Hypocotyl Explants in Media Containing a PARP Inhibitor
[0081] Wheat embryogenic callus was cultured on W2 medium (see
Example 2). When niacinamide was added as PARP-inhibitor to the
medium at a concentration of 250 mg/l (approx. 2 mM) it was
observed that after 4 days the growth of the tissue was slowed down
considerably (to approximately 30% of the normal rate after 4
weeks) but the tissue remained viable for extended periods of time
(i.e. at least one month). If niacinamide was subsequently removed
from the medium the tissue started to grow normally again. It was
also observed that after 4-5 days of incubation of the plant tissue
with niacinamide, the TTC-reducing capacity (Towill and Mazur,
1975, Can J.Bot. 53:1097-1102) of the tissue was substantially
decreased probably indicating a reduction of the production of free
radicals and decreased mitochondrial electron transport.
[0082] Similar observations were made when Brassica napus hypocotyl
explants were cultured on A5 medium (see Example 3) containing 250
mg/l niacinamide. It was also observed that, in Brassica napus
tissue cultured on medium containing niacinamide, no anthocyanin
was produced; normally anthocyanin in tissue culture is produced in
stress conditions. In addition it was observed that after 4-5 days
of incubation of the plant tissue with niacinamide, the
concentrations of hydroxyl free radical and dehydroascorbate in the
explants were drastically decreased.
[0083] It was also observed that, after a 4 day incubation in
niacinamide containing medium, the percentage of cultured cells
that were in G2 phase of the cell cycle was considerably increased
(up to 45% of all cells in the culture).
[0084] The above observations are interpreted as indicating that
treating cultured cells with a PARP inhibitor for about 4-5 days
generally results in:
[0085] 1) a significant reduction of the response of the cultured
cells to stress as measured for instance by free radical and/or
anthocyanin production, and
[0086] 2) a reduction of the general metabolism of the cultured
cells to a very basic level as indicated by the fact that the
tissue growth was slowed down, and the TTC reducing capacity was
decreased while the tissue remained viable.
[0087] It is inferred that under these conditions many genes in
cells (e.g. cultured cells) that would normally be switched on in
response to stress (such as during transformation conditions) will
in fact no longer be induced. It is expected that in such cells
which only display a very basic metabolism, mainly general
"housekeeping genes", i.e. genes that are expressed in any cell
irrespective of its differentiated state or metabolic or
physiological condition, are expressed.
[0088] As it is believed that foreign DNA is preferably inserted in
portions of the genome that are transcriptionally active it follows
that treatment with PARP inhibitors will condition eucaryotic cells
to incorporate any foreign DNA preferentially in genomic regions
which are transcribed in all cells and not in regions of the genome
which would only be transcribed under certain conditions, i.e.
stress conditions, or during differentiation. This means that the
number of locations in which foreign DNA will be integrated, and
the concomitant variation in expression profile of the
transgene(s), will be reduced.
[0089] It is further believed that this will enhance integration of
foreign genes of interest in such locations which in turn will
result in a more reliable and faithful expression of these genes
which will be less affected by cell differentiation or cell
physiological and biochemical changes due to for instance
environmental conditions.
Example 2
Transformation of Wheat with a Barnase Gene Under the Control of a
Stamen-Specific Promoter Using the Particle Bombardment
[0090] The Wheat Spring variety Pavon is grown in a greenhouse or
conditioned room at 23-24.degree. C. during daytime and
18-20.degree. C. at night, with a photoperiod of 16 hours light and
8 hours dark. Developing seeds (white-greenish with white
semi-liquid endosperm) were harvested, sterilized by incubation for
1 minute in 70% ethanol followed by 15 minute incubation in 1.3%
NaOCl+0.1% Tween 20, and washed with sterile water. The sterilized
seeds were either used directly or were stored for one day at
4-7.degree. C.
[0091] Immature embryos of about 1 mm in size were isolated and
were placed, with the scutellum upwards, on callus inducing medium
W1 (MS medium supplemented with 3% sucrose, 40 mg/l
adenine.SO.sub.4, 0.5 mg/l thiamine.HCl, 0.5 g/l 2-[N-Morpholino]
ethane sulfonic acid (Mes) pH 5.8, 0.5% agarose, 0.5 to 2.5 mg/l
CuSO.sub.4.5H.sub.2O, 25 mg/l acetylsalicylic acid and 2 mg/l
2,4-dichlorophenoxyacetic acid (2,4-D)) and were incubated for 3
weeks at 27.degree. C. in the dark.
[0092] Embryogenic sections of the developing callus were isolated,
placed on callus maintenance medium W2 (W1 medium but without
acetylsalicylic acid and with only 0.5 mg/l CuSO.sub.4.5H.sub.2O
and 1 mg 2,4-D), and incubated for 3 weeks at 24-25.degree. C. in
the light (approx. 20 mEinsteins/s/m.sup.2 (with a photoperiod of
16 hours light and 8 hours dark).
[0093] About 2 weeks prior to bombardment the calli were cleaned up
by removal of non-morphogenic (i.e. the nonembryogenic and
nonmeristematic) parts and were subcultured on W2 medium.
[0094] For bombardment the calli were divided into small pieces
with an average maximum diameter of about 2-3 mm. These pieces were
placed at the center of a 9 cm Petridish containing W2 medium in a
circle with a diameter of approx. 0.5 cm. When required niacinamide
(250 mg/l) was added to the W2 medium and the tissue pieces were
maintained under these conditions for 4 days after they were
bombarded.
[0095] Bombardment was carried out using the Biolistic PDS-1000/He
apparatus (Bio-Rad). Preparation of the microcarriers (0.4-1.2 m)
and the coating of the microcarriers with DNA was essentially
carried out according to the manufacturer's instructions. The
Petridishes containing the calli were placed at level 2 of the
apparatus and the bombardment was done at 1550 psi.
[0096] For the transformation experiments the following plasmid DNA
was used.
[0097] plasmid pVE136, the sequence of which is given in SEQ ID No
4. This plasmid contains the following chimeric genes:
[0098] P35S-bar-3'nos
[0099] PCA55-barnase-3'nos
[0100] in which P35S is the 35S promoter of the Cauliflower, Mosaic
virus, bar is a DNA encoding phosphinothricin acetyltransferase (EP
242236), 3'nos is the 3' untranslated end of the Agrobacterium
T-DNA nopaline synthase gene, PCA55 is a stamen-specific promoter
from corn gene CA55 (WO 9213957), and barnase is a DNA encoding
barnase (Hartley, 1988, J.Mol.Biol.202:913-915)
[0101] plasmid pTS172 the sequence of which is given in SEQ ID No
2. This plasmid contains the following chimeric genes:
[0102] P35S-bar-3'g7
[0103] PE1-barnase-3'nos
[0104] in which in which P35S is the 35S promoter of the
Cauliflower Mosaic virus, bar is a DNA encoding phosphinothricin
acetyltransferase (EP 242236), 3'g7 is the 3' untranslated end of
the Aqrobacterium T-DNA gene 7, PE1 is a stamen-specific promoter
from rice gene E1 (WO 9213956), barnase is a DNA encoding barnase
(Hartley, 1988, J.Mol.Biol.202:913-915)- , and 3' nos is the 3'
untranslated end of the Agrobacterium T-DNA nopaline synthase
gene,
[0105] plasmid pTS772 which is identical to pTS172 except that the
region between nucleotides 2625-4313 of pTS172, containing PE1, is
replaced by the sequence of SEQ ID No 3 containing the PT72
promoter. Thus, plasmid pTS772 contains the following chimeric
genes:
[0106] P35S-bar-3'g7
[0107] PT72-barnase-3'nos
[0108] in which PT72 is a stamen-specific promoter from rice gene
T72 (WO 9213956)
[0109] The bombarded calli were transferred to selective medium W2
containing 2.5 mg/l phosphinothricin (PPT) and, if neccesary, 100
mg/l niacinamide. The calli that were placed on medium containing
niacinamide were transferred after 4 days to niacinamide-free W2
medium containing 2.5 mg/l PPT. The cells were cultured at
24-25.degree. C.
[0110] After two weeks the calli were subcultivated on W2 medium
and after a further two weeks the growing parts of the calli were
transferred to regeneration medium W4 (W1 medium but without
acetylsalicylic acid and with only 0.5 mg/l CuSO.sub.4.5H.sub.2O
and 0.5 mg/l 2,4-D). Calli were subcultivated every two weeks at
which time the nonmorphogenic parts of the calii were removed. When
the calii started to form shoots they were transferred to W5 medium
(W1 medium with half concentrated MS medium and only 0.5 mg/l
CuSO.sub.4.5H.sub.2O and without acetylsalicylic acid and 2,4-D,
but supplemented with 50 mg/l myo-inositol, 0.25 mg/l
pyridoxine.HCl and 0.25 mg/l nicotinic acid) containing 2.5 mg/l
PPT. For the rest of the procedure temperature was maintained at a
maximum of 24.degree. C. The calli were subcultivated every 3-4
weeks. Once the shoots started to elongate and small roots started
to form, the whole calli (or if possible individual shoots) were
transferred to 1 liter vessels with W6 medium (half-concentrated MS
medium supplemented with 1.5% sucrose, 50 mg/l myo-inositol, 0.25
mg/l pyridoxine.HCl, 0.25 mg/l nicotinic acid, 0.5 mg/l
thiamine.HCl, 0.7% agar (Difco) pH 5.8 and 0.5 mg/l
CuSO.sub.4.5H.sub.2O) containing 2.5 mg/l PPT. Once the shoots and
roots had grown out, individual shoots were separated from each
other and transferred to 1 I vessels containing W6 medium with 2.5
mg/l PPT. Well developed shoots are tested for PPT resistance by
means of the TLC assay (De Block et al, 1987, EMBO 6:2513-2518) or
by direct assay of ammonium production in the tissue (see e.g. De
Block et al, 1995, Planta 197: 619-626). Transformed shoots were
finally transferred to the greenhouse into soil.
[0111] For analysis of the results the transformed plants could be
subdivided according to the niacinamide treatment of the parent
calli during tissue culture. Thus the following groups were
distinguished:
1 Group Niacinamide treatment None No treatment Before 100 100 mg/l
niacinamide for four days prior to bombardment Before 250 250 mg/l
niacinamide for four days prior to bombardment Before/After 250
mg/l niacinamide for four days prior to bombardment plus 100 mg/l
niacinamide for four days after bombardment
[0112] Results of the experiments are presented in Tables 1, 2 and
3. Plants could be obtained only from bombarded calli that were
treated with niacinamide.
[0113] For the plants that were transformed with plasmid pTS172 it
was demonstrated that the foreign DNA, comprising the chimeric
PE1-barnase-3'nos and P35S-bar-3'g7, was stably incorporated in the
wheat genome in 2 to 3 copies on the average. The fact that
variation in expression profile (e.g. tissue-specificity) of the
transgenes, especially the chimeric barnase genes, was decreased in
transformed cells was evident from the fact that malesterile plants
that otherwise looked completely healthy could be obtained only
from bombarded calli treated with niacinamide. It is believed that
this is due to a more faithful expression characteristics (i.e.
lack of expression) of the integrated stamen-selective barnase gene
in these calli and shoots regenerated from these calli. In the
control calli undesired expression of the barnase gene in tissue
cultured cells might have prevented recovery of any transformed
plants from these calli. It is expected that to obtain the same
number of male-sterile wheat plants from control calli a much
larger number of calli would have to be bombarded.
[0114] Results of Wheat Transformation Experiments
2TABLE 1 Plasmid pTS172 Nr of PPT- Nr of PPT Nr of resistant
resistant Nr of MS bombarded calli plants plants Treatment calli
recovered recovered recovered None 60 30 1.sup.a) 0 Before 250 125
30 3 3.sup.b) .sup.a)This plant proved to be fertile and to be
transformed only with the chimeric bar gene .sup.b)The obtained
plants looked healthy and tillered vigorously
[0115]
3TABLE 2 Plasmid pTS772 Nr of PPT- Nr of PPT resistant resistant Nr
of MS Nr of bombarded calli plants plants Treatment calli recovered
recovered recovered None 250 22 0 0 Before 210 75 7 3.sup.a)b) 250
Before/After 210 45 6 3.sup.a .sup.a)The obtained plants looked
healthy and tillered vigorously .sup.b)Only six plants could be
analyzed for MS phenotype since one of the plants died
prematurely.
[0116]
4TABLE 3 Plasmid pVE136 Nr of PPT Nr of resistant Nr of MS
bombarded plants plants Treatment calli recovered recovered None
200 1 0 Before 800 8.sup.a) 8 100 .sup.a)The obtained plants looked
healthy and tillered vigorously
Example 3
Transformation of Oilseed Rape with a Barnase Gene Under the
Control of a Stamen-Specific Promoter Using Agrobacterium Mediated
Transformation.
[0117] Hypocotyl explants of Brassica napus were obtained, cultured
and transformed essentially as described by De Block et al, 1989,
Plant Physiol. 914:694-701 except for the following
modifications:
[0118] hypocotyl explants were precultured for 3 days on A2 medium
(MS, 0.5 g/l Mes (pH 5.7), 1.2% glucose, 0.5% agarose, 1 mg/l
2,4-D, 0.25 mg/l naphthalene acetic acid (NAA), 1 mg/l
6-benzylaminopurine (BAP)), and then transferred to the A2 medium
with or without niacinamide for another 4 days.
[0119] infection medium A3 was MS, 0.5 g/l Mes (pH 5.7), 1.2%
glucose, 0.1 mg/l NAA, 0.75 mg/l BAP, 0.01 mg/l giberellinic acid
(GA3)
[0120] selection medium A5 was 0.5 g/l Mes (pH 5.7), 1.2% glucose,
40 mg/l adenine.SO.sub.4, 0.5 g/l polyvinyl-polypyrrolidone (PVP),
0.5% agarose, 0.1 mg/l NAA, 0.75 mg/l BAP, 0.01 mg/l GA3, 250 mg/l
carbenicillin, 250 mg/l triacillin, 5 mg/l AgNO.sub.3.
[0121] regeneration medium A6 was MS, 0.5 g/l Mes (pH 5.7), 2%
sucrose, 40 mg/l adenine.SO.sub.4, 0.5 g/l PVP, 0.5% agarose,
0.0025 mg/l BAP, 250 mg/l triacillin.
[0122] healthy shoots were transferred to 1 liter vessels
containing rooting medium which was either A8 or A9; A8 consists of
100-130 ml half concentrated MS, 1% sucrose (pH 5.0), 1 mg/l
isobutyric acid (IBA), 100 mg/l triacillin added to 300 ml perlite
(final pH 6.2); A9 consists of half concentrated MS, 1.5% sucrose
(pH 5.8) solidified with agar (0.6%)
[0123] Hypocotyl explants (with or without niacinamide treatment)
were infected with Agrobacterium tumefaciens strain C58C1Rif
carrying T-DNA vector pTHW107 and a helper Ti-plasmid pMP90 (Koncz
and Schell, 1986, Mol.Gen.Genet. 204:383-396)(or a derivative
thereof).
[0124] Plasmid pTHW107 is a vector carrying a T-DNA comprising the
following chimeric genes:
[0125] PTA29-barnase-3'g7
[0126] PSSU-bar-3'nos
[0127] in which PTA29 is the promoter of the TA29 gene of tobacco
(EP 344029) and PSSU is the promoter of the gene of Arabidopsis
thaliana encoding the small subunit of Rubisco. The complete
sequence of the T-DNA of pTHW107 is presented in SEQ ID No 1.
[0128] Where required niacinamide (250 mg/l) was added to the media
for the last 4 days prior to infection with Agrobacterium. Plants
regenerated from transformed calli obtained on niacinamide cultured
cells were observed to have a low copy number as well as to display
less variation in the expression profile of the transgenes (results
summarized in Table 4). Five plants regenerated from the calli
obtained by transformation including niacinamide and five plants
regenerated from the calli obtained by conventional transformation
without niacinamide inclusion, were analyzed by Southern
hybridization to determine the copy number of the transgenes, and
were further analyzed for reproductive phenotype. In the
non-treated group, a substantial number of regenerated plants
proved: not to have a transgene integrated in their nuclear
DNA.
5TABLE 4 Vegetative Reproductive Copy No. of the Phenotype of
Treatment Id. No. phenotype.sup.a phenotype.sup.b transgenes.sup.c
the F1-progeny.sup.d no treatment 1 stressed sterile 3
stressed/sterile 2 stressed sterile 4-6 ND 3 stressed sterile 3
stressed/sterile 4 normal sterile 1 normal/sterile 5 stressed (bud
fall) ND ND Before 250 1 normal sterile 1 normal/sterile 2 normal
sterile 3 normal/sterile 3 normal sterile 1 ND 4 normal sterile 3
ND 5 normal sterile 2 ND .sup.aVegetatively stressed plants have a
small size and flower early, leaves are oblong and dark green.
.sup.bReproductive phenotype regards male sterility; in flowers
where the buds fell off prematurely this phenotype was not scored,
except where some buds resulted in flowers. .sup.cCopy number of
the transgenes was estimated by comparative Southern. ND: not
determined. .sup.dF1-progeny was obtained by pollinating the
transformed plants with pollen obtained from an untransformed
N90-740 line. F1-Progeny resistant to phosphinotricin was scored
for vegetative and reproductive phenotype.
Example 4
Agrobacterium-Mediated Transformation of Oilseed Rape Using
Niacinamide in the Culture Medium
[0129] Hypocotyl explants of Brassica napus were obtained as
described in Example 3. Four groups of 200 hypocotyl explants each,
were either not treated with niacinamide (indicated in table 4 as
NONE), treated with 250 mg/l niacinamide for 1 day prior to
infection with Agrobacterium (BEFORE), treated for 2 days during
the infection with 250 mg/l niacinamide (DURING), or treated for 1
day after the Agrobacterium infection with 250 mg/l niacinamide
(AFTER).
[0130] All hypocotyl explants were infected with Agrobacterium
tumefaciens strain C58C1Rif carrying T-DNA vector pTHW142 and a
helper Ti-plasmid pMP90 (Koncz and Shell, 1986 supra)(or a
derivative thereof). Plasmid pTHW142 is a vector carrying a T-DNA
comprising the following chimeric genes:
[0131] PSSU-bar-3'g7
[0132] p35S-uidA-3'35S
[0133] In which uidA is a DNA encoding b-glucuronidase (Jefferson
et al., 1986, Proc. Natl. Acad. Sci. USA 83, 8447-8451) and 3' 35S
is the 3' untranslated end of the cauliflower mosaic virus 35S
transcript.
[0134] The complete sequence of the T-DNA of pTHW142 is presented
in SEQ ID No 5.
[0135] After the Agrobacterium infection, hypocotyl explants were
transferred to selection medium A5, and if appropriate to A5 medium
containing 250 mg/ll niacinamide. The hypocotyl explants that were
placed on medium containing niacinamide were transferred after 1
day to niacinamide-free selection medium A5. After 5 weeks on
selective medium the number of transformed calli was scored.
b-glucuronidase expression was verified in the obtained calli using
established protocols (Jefferson et al., 1986). The results are
summarized in Table 5. Niacinamide treatment either before or after
the Agrobacterium infection significantly increase the
transformation efficiency.
6 TABLE 5 Transformation Treatment frequency.sup.a Remarks.sup.b
NONE 16% small, green calli BEFORE 32% large, green calli DURING
16% very small, light green calli large, green calli AFTER 29%
developing shoots .sup.aDetermined as the number of transformed
calli (PPT-resitant and GUS-positive) developing per 100 hypocotyl
explants .sup.bSize determination was as follows: very small:
callus diameter of approximately 1-2 mm small: callus diameter of
approximately 2-3 mm large: callus diameter of approximately 5
mm
[0136] All publications cited in this application are hereby
incorporated by reference.
Sequence CWU 1
1
5 1 4946 DNA Artificial Sequence T-DNA of plasmid pTHW107. 1
aattacaacg gtatatatcc tgccagtact cggccgtcga actcggccgt cgagtacatg
60 gtcgataaga aaaggcaatt tgtagatgtt aattcccatc ttgaaagaaa
tatagtttaa 120 atatttattg ataaaataac aagtcaggta ttatagtcca
agcaaaaaca taaatttatt 180 gatgcaagtt taaattcaga aatatttcaa
taactgatta tatcagctgg tacattgccg 240 tagatgaaag actgagtgcg
atattatgtg taatacataa attgatgata tagctagctt 300 agctcatcgg
gggatcctag acgcgtgaga tcagatctcg gtgacgggca ggaccggacg 360
gggcggtacc ggcaggctga agtccagctg ccagaaaccc acgtcatgcc agttcccgtg
420 cttgaagccg gccgcccgca gcatgccgcg gggggcatat ccgagcgcct
cgtgcatgcg 480 cacgctcggg tcgttgggca gcccgatgac agcgaccacg
ctcttgaagc cctgtgcctc 540 cagggacttc agcaggtggg tgtagagcgt
ggagcccagt cccgtccgct ggtggcgggg 600 ggagacgtac acggtcgact
cggccgtcca gtcgtaggcg ttgcgtgcct tccaggggcc 660 cgcgtaggcg
atgccggcga cctcgccgtc cacctcggcg acgagccagg gatagcgctc 720
ccgcagacgg acgaggtcgt ccgtccactc ctgcggttcc tgcggctcgg tacggaagtt
780 gaccgtgctt gtctcgatgt agtggttgac gatggtgcag accgccggca
tgtccgcctc 840 ggtggcacgg cggatgtcgg ccgggcgtcg ttctgggtcc
attgttcttc tttactcttt 900 gtgtgactga ggtttggtct agtgctttgg
tcatctatat ataatgataa caacaatgag 960 aacaagcttt ggagtgatcg
gagggtctag gatacatgag attcaagtgg actaggatct 1020 acaccgttgg
attttgagtg tggatatgtg tgaggttaat tttacttggt aacggccaca 1080
aaggcctaag gagaggtgtt gagaccctta tcggcttgaa ccgctggaat aatgccacgt
1140 ggaagataat tccatgaatc ttatcgttat ctatgagtga aattgtgtga
tggtggagtg 1200 gtgcttgctc attttacttg cctggtggac ttggcccttt
ccttatgggg aatttatatt 1260 ttacttacta tagagctttc ataccttttt
tttaccttgg atttagttaa tatataatgg 1320 tatgattcat gaataaaaat
gggaaatttt tgaatttgta ctgctaaatg cataagatta 1380 ggtgaaactg
tggaatatat atttttttca tttaaaagca aaatttgcct tttactagaa 1440
ttataaatat agaaaaatat ataacattca aataaaaatg aaaataagaa ctttcaaaaa
1500 acagaactat gtttaatgtg taaagattag tcgcacatca agtcatctgt
tacaatatgt 1560 tacaacaagt cataagccca acaaagttag cacgtctaaa
taaactaaag agtccacgaa 1620 aatattacaa atcataagcc caacaaagtt
attgatcaaa aaaaaaaaac gcccaacaaa 1680 gctaaacaaa gtccaaaaaa
aacttctcaa gtctccatct tcctttatga acattgaaaa 1740 ctatacacaa
aacaagtcag ataaatctct ttctgggcct gtcttcccaa cctcctacat 1800
cacttcccta tcggattgaa tgttttactt gtaccttttc cgttgcaatg atattgatag
1860 tatgtttgtg aaaactaata gggttaacaa tcgaagtcat ggaatatgga
tttggtccaa 1920 gattttccga gagctttcta gtagaaagcc catcaccaga
aatttactag taaaataaat 1980 caccaattag gtttcttatt atgtgccaaa
ttcaatataa ttatagagga tatttcaaat 2040 gaaaacgtat gaatgttatt
agtaaatggt caggtaagac attaaaaaaa tcctacgtca 2100 gatattcaac
tttaaaaatt cgatcagtgt ggaattgtac aaaaatttgg gatctactat 2160
atatatataa tgctttacaa cacttggatt tttttttgga ggctggaatt tttaatctac
2220 atatttgttt tggccatgca ccaactcatt gtttagtgta atactttgat
tttgtcaaat 2280 atatgtgttc gtgtatattt gtataagaat ttctttgacc
atatacacac acacatatat 2340 atatatatat atatattata tatcatgcac
ttttaattga aaaaataata tatatatata 2400 tagtgcattt tttctaacaa
ccatatatgt tgcgattgat ctgcaaaaat actgctagag 2460 taatgaaaaa
tataatctat tgctgaaatt atctcagatg ttaagatttt cttaaagtaa 2520
attctttcaa attttagcta aaagtcttgt aataactaaa gaataataca caatctcgac
2580 cacggaaaaa aaacacataa taaatttgaa tttcgaccgc ggtacccgga
attcgagctc 2640 ggtacccggg gatcttcccg atctagtaac atagatgaca
ccgcgcgcga taatttatcc 2700 tagtttgcgc gctatatttt gttttctatc
gcgtattaaa tgtataattg cgggactcta 2760 atcataaaaa cccatctcat
aaataacgtc atgcattaca tgttaattat tacatgctta 2820 acgtaattca
acagaaatta tatgataatc atcgcaagac cggcaacagg attcaatctt 2880
aagaaacttt attgccaaat gtttgaacga tctgcttcgg atcctctaga gccggaaagt
2940 gaaattgacc gatcagagtt tgaagaaaaa tttattacac actttatgta
aagctgaaaa 3000 aaacggcctc cgcaggaagc cgtttttttc gttatctgat
ttttgtaaag gtctgataat 3060 ggtccgttgt tttgtaaatc agccagtcgc
ttgagtaaag aatccggtct gaatttctga 3120 agcctgatgt atagttaata
tccgcttcac gccatgttcg tccgcttttg cccgggagtt 3180 tgccttccct
gtttgagaag atgtctccgc cgatgctttt ccccggagcg acgtctgcaa 3240
ggttcccttt tgatgccacc cagccgaggg cttgtgcttc tgattttgta atgtaattat
3300 caggtagctt atgatatgtc tgaagataat ccgcaacccc gtcaaacgtg
ttgataaccg 3360 gtaccatggt agctaatttc tttaagtaaa aactttgatt
tgagtgatga tgttgtactg 3420 ttacacttgc accacaaggg catatataga
gcacaagaca tacacaacaa cttgcaaaac 3480 taacttttgt tggagcattt
cgaggaaaat ggggagtagc aggctaatct gagggtaaca 3540 ttaaggtttc
atgtattaat ttgttgcaaa catggactta gtgtgaggaa aaagtaccaa 3600
aattttgtct caccctgatt tcagttatgg aaattacatt atgaagctgt gctagagaag
3660 atgtttattc tagtccagcc acccacctta tgcaagtctg cttttagctt
gattcaaaaa 3720 ctgatttaat ttacattgct aaatgtgcat acttcgagcc
tatgtcgctt taattcgagt 3780 aggatgtata tattagtaca taaaaaatca
tgtttgaatc atctttcata aagtgacaag 3840 tcaattgtcc cttcttgttt
ggcactatat tcaatctgtt aatgcaaatt atccagttat 3900 acttagctag
atatccaatt ttgaataaaa atagctcttg attagtaaac cggatagtga 3960
caaagtcaca tatccatcaa acttctggtg ctcgtggcta agttctgatc gacatggggt
4020 taaaatttaa attgggacac ataaatagcc tatttgtgca aatctcccca
tcgaaaatga 4080 cagattgtta catggaaaac aaaaagtcct ctgatagaag
tcgcaaagta tcacaatttt 4140 ctatcgagag atagattgaa agaagtgcag
ggaagcggtt aactggaaca taacacaatg 4200 tctaaattaa ttgcattcgc
taaccaaaaa gtgtattact ctctccggtc cacaataagt 4260 tattttttgg
cccttttttt atggtccaaa ataagtgagt tttttagatt tcaaaaatga 4320
tttaattatt tttttactac agtgcccttg gagtaaatgg tgttggagta tgtgttagaa
4380 atgtttatgt gaagaaatag taaaggttaa tatgatcaat ttcattgcta
tttaatgtta 4440 aaatgtgaat ttcttaatct gtgtgaaaac aaccaaaaaa
tcacttattg tggaccggag 4500 aaagtatata aatatatatt tggaagcgac
taaaaataaa cttttctcat attatacgaa 4560 cctaaaaaca gcatatggta
gtttctaggg aatctaaatc actaaaatta ataaaagaag 4620 caacaagtat
caatacatat gatttacacc gtcaaacacg aaattcgtaa atatttaata 4680
taataaagaa ttaatccaaa tagcctccca ccctataact taaactaaaa ataaccagcg
4740 aatgtatatt atatgcataa tttatatatt aaatgtgtat aatcatgtat
aatcaatgta 4800 taatctatgt atatggttag aaaaagtaaa caattaatat
agccggctat ttgtgtaaaa 4860 atccctaata taatcgcgac ggatccccgg
gaattccggg gaagcttaga tccatggagc 4920 catttacaat tgaatatatc ctgccg
4946 2 6548 DNA Artificial Sequence plasmid PTS172. 2 aattcaagct
tgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 60
ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
120 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
cgcccttatt 180 cccttttttg cggcattttg ccttcctgtt tttgctcacc
cagaaacgct ggtgaaagta 240 aaagatgctg aagatcagtt gggtgcacga
gtgggttaca tcgaactgga tctcaacagc 300 ggtaagatcc ttgagagttt
tcgccccgaa gaacgttttc caatgatgag cacttttaaa 360 gttctgctat
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 420
cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
480 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
tgataacact 540 gcggccaact tacttctgac aacgatcgga ggaccgaagg
agctaaccgc ttttttgcac 600 aacatggggg atcatgtaac tcgccttgat
cgttgggaac cggagctgaa tgaagccata 660 ccaaacgacg agcgtgacac
cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 720 ttaactggcg
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 780
gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
840 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
gccagatggt 900 aagccctccc gtatcgtagt tatctacacg acggggagtc
aggcaactat ggatgaacga 960 aatagacaga tcgctgagat aggtgcctca
ctgattaagc attggtaact gtcagaccaa 1020 gtttactcat atatacttta
gattgattta aaacttcatt tttaatttaa aaggatctag 1080 gtgaagatcc
tttttggctc gagtctcatg accaaaatcc cttaacgtga gttttcgttc 1140
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
1200 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
ttgtttgccg 1260 gatcaagagc taccaactct ttttccgaag gtaactggct
tcagcagagc gcagatacca 1320 aatactgtcc ttctagtgta gccgtagtta
ggccaccact tcaagaactc tgtagcaccg 1380 cctacatacc tcgctctgct
aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 1440 tgtcttaccg
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 1500
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
1560 ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc
ggacaggtat 1620 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
agcttccagg gggaaacgcc 1680 tggtatcttt atagtcctgt cgggtttcgc
cacctctgac ttgagcgtcg atttttgtga 1740 tgctcgtcag gggggcggag
cctatggaaa aacgccagca acgcggcctt tttacggttc 1800 ctggcctttt
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 1860
gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
1920 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
gcctctcccc 1980 gcgcgttggc ctgatcagaa ttcatatgca cgtgttcccg
atctagtaac atagatgaca 2040 ccgcgcgcga taatttatcc tagtttgcgc
gctatatttt gttttctatc gcgtattaaa 2100 tgtataattg cgggactcta
atcataaaaa cccatctcat aaataacgtc atgcattaca 2160 tgttaattat
tacatgctta acgtaattca acagaaatta tatgataatc atcgcaagac 2220
cggcaacagg attcaatctt aagaaacttt attgccaaat gtttgaacga tctgcttcgg
2280 aggttacctt atctgatttt tgtaaaggtc tgataatggt ccgttgtttt
gtaaatcagc 2340 cagtcgcttg agtaaagaat ccggtctgaa tttctgaagc
ctgatgtata gttaatatcc 2400 gcttcacgcc atgttcgtcc gcttttgccc
gggagtttgc cttccctgtt tgagaagatg 2460 tctccgccga tgcttttccc
cggagcgacg tctgcaaggt tcccttttga tgccacccag 2520 ccgagggctt
gtgcttctga ttttgtaatg taattatcag gtagcttatg atatgtctga 2580
agataatccg caaccccgtc aaacgtgttg ataaccggta ccatcgcgac ggcttgatgg
2640 atctcttgct ggacaccggg atgctaggat gggttatcgt ggccggcgtg
cgtgtgtggc 2700 ttttgtaggc gccggcgacg gcgggggcaa tgtggcaggt
gagtcacggt gcaagcgtgc 2760 gcaagtgact gcaacaacca aggacggtca
tggcgaaagc acctcacgcg tccaccgtct 2820 acaggatgta gcagtagcac
ggtgaaagaa gtgttgtccc gtccattagg tgcattctca 2880 ccgttggcca
gaacaggacc gttcaacagt taggttgagt gtaggacttt tacgtggtta 2940
atgtatggca aatagtagta aattttgccc ccattggtct ggctgagata gaacatattc
3000 tggaaagcct ctagcatatc ttttttgaca gctaaacttt gcttcttgcc
ttcttggtct 3060 agcaatgacg ttgcccatgt cgtggcaaac atctggtaag
gtaactgtat tcgtttgttc 3120 ccttcaacgg ctcaatcccc acaggccaag
ctatcctttc cttggcagta taggctcctt 3180 gagagattat actaccattt
ttaagtgctt ataaagacga tgctctctaa ccagatcgat 3240 cagaaacaca
aagttttagc agcgtaatat cccacacaca tacacacacg aagctatgcc 3300
tcctcatttt ccgagagatt ctgacagtga ccagaatgtc agaatgccat ttcatgggca
3360 caagtcgatc cacaagcttc ttggtggagg tcaaggtgtg ctattattat
tcgctttcta 3420 ggaaattatt cagaattagt gccttttatc ataacttctc
tctgagccga tgtggttttg 3480 gatttcattg ttgggagcta tgcagttgcg
gatattctgc tgtggaagaa caggaactta 3540 tctgcggggg tccttgctgg
ggcaacattg atatggttcc tgttcgatgt agtagaatac 3600 aatataattc
cgctcctttg ccagattgcc attcttgcca tgcttgtgat cttcatttgg 3660
tcaaatgccg caccactctt ggacaggtat tagctttatt tcctgtggag atggtagaaa
3720 actcagctta cagaaatggc atttcacgta gtataacgca agacattagg
tactaaaact 3780 caactaactg tttccgaatt tcagggcccc tccaaggatc
ccagaaatca tcatctctga 3840 acatgccttc agagaaatgg cattgaccgt
ccattacaaa ctaacgtaca ctgtatctgt 3900 tctttacgac attgcatgtg
gaaaggatct gaagagattt ctcctggtac ataataatct 3960 actcctttgc
tacgttaata agagatgtaa aaacatgcaa cagttccagt gccaacattg 4020
tccaaggatt gtgcaattct ttctggagcg ctaaaattga ccagattaga cgcatcagaa
4080 tattgaattg cagagttagc caataatcct cataatgtta atgtgctatt
gttgttcact 4140 actcaatata gttctggact aacaatcaga ttgtttatga
tattaaggtg gttggatctc 4200 tattggtatt gtcggcgatt ggaagttctt
gcagcttgac aagtctacta tatattggta 4260 ggtattccag ataaatatta
aattttaata aaacaatcac acagaaggat ctgcggccgc 4320 tagcctaggc
ccgggcccac aaaaatctga gcttaacagc acagttgctc ctctcagagc 4380
agaatcgggt attcaacacc ctcatatcaa ctactacgtt gtgtataacg gtccacatgc
4440 cggtatatac gatgactggg gttgtacaaa ggcggcaaca aacggcgttc
ccggagttgc 4500 acacaagaaa tttgccacta ttacagaggc aagagcagca
gctgacgcgt acacaacaag 4560 tcagcaaaca gacaggttga acttcatccc
caaaggagaa gctcaactca agcccaagag 4620 ctttgctaag gccctaacaa
gcccaccaaa gcaaaaagcc cactggctca cgctaggaac 4680 caaaaggccc
agcagtgatc cagccccaaa agagatctcc tttgccccgg agattacaat 4740
ggacgatttc ctctatcttt acgatctagg aaggaagttc gaaggtgaag gtgacgacac
4800 tatgttcacc actgataatg agaaggttag cctcttcaat ttcagaaaga
atgctgaccc 4860 acagatggtt agagaggcct acgcagcagg tctcatcaag
acgatctacc cgagtaacaa 4920 tctccaggag atcaaatacc ttcccaagaa
ggttaaagat gcagtcaaaa gattcaggac 4980 taattgcatc aagaacacag
agaaagacat atttctcaag atcagaagta ctattccagt 5040 atggacgatt
caaggcttgc ttcataaacc aaggcaagta atagagattg gagtctctaa 5100
aaaggtagtt cctactgaat ctaaggccat gcatggagtc taagattcaa atcgaggatc
5160 taacagaact cgccgtgaag actggcgaac agttcataca gagtctttta
cgactcaatg 5220 acaagaagaa aatcttcgtc aacatggtgg agcacgacac
tctggtctac tccaaaaatg 5280 tcaaagatac agtctcagaa gaccaaaggg
ctattgagac ttttcaacaa aggataattt 5340 cgggaaacct cctcggattc
cattgcccag ctatctgtca cttcatcgaa aggacagtag 5400 aaaaggaagg
tggctcctac aaatgccatc attgcgataa aggaaaggct atcattcaag 5460
atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa
5520 aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgacatc
tccactgacg 5580 taagggatga cgcacaatcc cactatcctt cgcaagaccc
ttcctctata taaggaagtt 5640 catttcattt ggagaggaca cgctgaaatc
accagtctct ctctataaat ctatctctct 5700 ctctataacc atggacccag
aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga 5760 catgccggcg
gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg 5820
taccgagccg caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta
5880 tccctggctc gtcgccgagg tggacggcga ggtcgccggc atcgcctacg
cgggcccctg 5940 gaaggcacgc aacgcctacg actggacggc cgagtcgacc
gtgtacgtct ccccccgcca 6000 ccagcggacg ggactgggct ccacgctcta
cacccacctg ctgaagtccc tggaggcaca 6060 gggcttcaag agcgtggtcg
ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca 6120 cgaggcgctc
ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa 6180
ctggcatgac gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt
6240 cctgcccgtc accgagatct gagatcacgc gttctaggat cccccgatga
gctaagctag 6300 ctatatcatc aatttatgta ttacacataa tatcgcactc
agtctttcat ctacggcaat 6360 gtaccagctg atataatcag ttattgaaat
atttctgaat ttaaacttgc atcaataaat 6420 ttatgttttt gcttggacta
taatacctga cttgttattt tatcaataaa tatttaaact 6480 atatttcttt
caagatggga attaacatct acaaattgcc ttttcttatc gaccatgtac 6540
gtatcgcg 6548 3 1601 DNA Artificial Sequence T72 promoter region. 3
cgccgtgagt gtcttctgcc gccgaggggc tctcgctcgt cgtcgatgcc tgcacggtgc
60 gtgcgtgtgt gtcgtggtgg tggtggcgat acgcgacgcg agctcgattt
ataggagggg 120 atcgaaggag gggagcgcgc gcggcgaggc ccgcgttgct
cacctacgcc gcgcgcatgc 180 ggcggacgcg cggtcggcgc ccgcgccggc
cgggaggacg agggcgcaag cgtgtgagcc 240 accgaacgcg cgcgcgcgcc
gcggcgcgaa ctctccatcg cgtcgcggcg agccgagagc 300 cgacgagagc
gtttcgcgcg cgcggttggg ccggcgacaa gatgggccgt agccctgggc 360
ctcgtgccat cttttttttt cttttttgcc ttttttggcc tggcaatttc tttttgtttt
420 tagtcttttt gtggtgataa tgtgtcgtct tccggtgaac taatttactc
gttgatcttt 480 ttgtgtccct tcgaatattc gcagtggtag aagatgacta
ctactaccag tagttgatct 540 cgaatggcaa cttttgtgca gaacttattc
cacggctatg tcagcttcca ctgtgactaa 600 aaaaactacg gccatctttt
ggacttgttc tatcttggaa ctgaacaaaa aggacgatcc 660 tgatgtacac
acggcatagt ttccagcact ggatgccaag ttgccaactg ttaccacgat 720
aatggaacga cgagatgaga tattatacaa gtccaatgga tcaagatcct gtgcagttgt
780 tattgtaact gtaacttaag ccgttaacat gtacatcaca tttcctactc
tatcaatgtc 840 ttgtgcgggt tgtttcaaaa aaacatgtac atcacatgat
ctagaacgga aggccaggat 900 atgaagtggt actgcagcaa aaacactgta
gcagagatgt actattatgc atgtactgta 960 gcagtcatct agagccgttg
gatctgaaaa cgaatggaca tgattgtgtg cagttgctat 1020 tgtgcagtta
caatagcaac tgcatttgat cttaatccaa gtccaataca tgcagaacag 1080
tagctacgag ctggaaagga tgcaaatctg ggtgacactg acagcaaccg tggaagaaca
1140 acagcagcaa agtcccagag ggatggcaat ttgaaggaat ttaaatactc
taatattact 1200 ccacccgtta aaaaaaacaa cttgctacgc ataatatatg
ttcggattta tagcgagaag 1260 ttaatttttc atgagaagaa gaatatatat
gtaatatgta ctaggagagt actcgcttca 1320 taaatataaa tattcataag
ttgtccagtg aagatagctt tagaaaaaac tagttatttt 1380 atttgtcaaa
ttttaaattt tgaagtagtt agattatctt tctagtagtt ctgattggtt 1440
gaaaatgttt agattttcat gtgttaagag ttccgtatcc taaaaatagt aatataattt
1500 taaatcatat atatatatat atatatatat atatatatat atatatatat
atatatatat 1560 tgttgaacgg tttgtgctct ggttgctatc ctgttctgtg g 1601
4 6291 DNA Artificial Sequence plasmid pVE136. 4 tcgcgcgttt
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg
aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc
acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420
cttcccgatc tagtaacata gatgacaccg cgcgcgataa tttatcctag tttgcgcgct
480 atattttgtt ttctatcgcg tattaaatgt ataattgcgg gactctaatc
ataaaaaccc 540 atctcataaa taacgtcatg cattacatgt taattattac
atgcttaacg taattcaaca 600 gaaattatat gataatcatc gcaagaccgg
caacaggatt caatcttaag aaactttatt 660 gccaaatgtt tgaacgatct
gcttcggatc ctctagagnn nnccggaaag tgaaattgac 720 cgatcagagt
ttgaagaaaa atttattaca cactttatgt aaagctgaaa aaaacggcct 780
ccgcaggaag ccgttttttt cgttatctga tttttgtaaa ggtctgataa tggtccgttg
840 ttttgtaaat cagccagtcg cttgagtaaa gaatccggtc tgaatttctg
aagcctgatg 900 tatagttaat atccgcttca cgccatgttc gtccgctttt
gcccgggagt ttgccttccc 960 tgtttgagaa gatgtctccg ccgatgcttt
tccccggagc gacgtctgca aggttccctt 1020 ttgatgccac ccagccgagg
gcttgtgctt ctgattttgt aatgtaatta tcaggtagct 1080 tatgatatgt
ctgaagataa tccgcaaccc cgtcaaacgt gttgataacc ggtaccatgg 1140
ctgcagctag ttagctcgat gtatcttctg tatatgcagt gcagcttctg cgttttggct
1200 gctttgagct gtgaaatctc gctttccagt ccctgcgtgt tttatagtgc
tgtacgttcg 1260 tgatcgtgag caaacagggc gtgcctcaac tactggtttg
gttgggtgac aggcgccaac 1320 tacgtgctcg taaccgatcg agtgagcgta
atgcaacatt ttttcttctt ctctcgcatt 1380 ggtttcatcc agccaggaga
cccgaatcga attgaaatca caaatctgag gtacagtatt 1440 tttacagtac
cgttcgttcg aaggtcttcg acaggtcaag gtaacaaaat cagttttaaa 1500
ttgttgtttc agatcaaaga aaattgagat gatctgaagg acttggacct tcgtccaatg
1560 aaacacttgg actaattaga ggtgaattga aagcaagcag atgcaaccga
aggtggtgaa 1620 agtggagttt cagcattgac gacgaaaacc ttcgaacggt
ataaaaaaga
agccgcaatt 1680 aaacgaagat ttgccaaaaa gatgcatcaa ccaagggaag
acgtgcatac atgtttgatg 1740 aaaactcgta aaaactgaag tacgattccc
cattcccctc cttttctcgt ttcttttaac 1800 tgaagcaaag aatttgtatg
tattccctcc attccatatt ctaggaggtt ttggcttttc 1860 ataccctcct
ccatttcaaa ttatttgtca tacattgaag atatacacca ttctaattta 1920
tactaaatta cagcttttag atacatatat tttattatac acttagatac gtattatata
1980 aaacacctaa tttaaaataa aaaattatat aaaaagtgta tctaaaaaat
caaaatacga 2040 cataatttga aacggagggg tactacttat gcaaaccaat
cgtggtaacc ctaaacccta 2100 tatgaatgag gccatgattg taatgcaccg
tctgattaac caagatatca atggtcaaag 2160 atatacatga tacatccaag
tcacagcgaa ggcaaatgtg acaacagttt tttttaccag 2220 agggacaagg
gagaatatct attcagatgt caagttcccg tatcacactg ccaggtcctt 2280
actccagacc atcttccggc tctattgatg cataccagga attgatctag agtcgacctg
2340 caggcatgca agctcctacg cagcaggtct catcaagacg atctacccga
gtaacaatct 2400 ccaggagatc aaataccttc ccaagaaggt taaagatgca
gtcaaaagat tcaggactaa 2460 ttgcatcaag aacacagaga aagacatatt
tctcaagatc agaagtacta ttccagtatg 2520 gacgattcaa ggcttgcttc
ataaaccaag gcaagtaata gagattggag tctctaaaaa 2580 ggtagttcct
actgaatcta aggccatgca tggagtctaa gattcaaatc gaggatctaa 2640
cagaactcgc cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca
2700 agaagaaaat cttcgtcaac atggtggagc acgacactct ggtctactcc
aaaaatgtca 2760 aagatacagt ctcagaagac caaagggcta ttgagacttt
tcaacaaagg ataatttcgg 2820 gaaacctcct cggattccat tgcccagcta
tctgtcactt catcgaaagg acagtagaaa 2880 aggaaggtgg ctcctacaaa
tgccatcatt gcgataaagg aaaggctatc attcaagatg 2940 cctctgccga
cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 3000
aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgacatctcc actgacgtaa
3060 gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa
ggaagttcat 3120 ttcatttgga gaggacacgc tgaaatcacc agtctctctc
tataaatcta tctctctctc 3180 tataaccatg gacccagaac gacgcccggc
cgacatccgc cgtgccaccg aggcggacat 3240 gccggcggtc tgcaccatcg
tcaaccacta catcgagaca agcacggtca acttccgtac 3300 cgagccgcag
gaaccgcagg agtggacgga cgacctcgtc cgtctgcggg agcgctatcc 3360
ctggctcgtc gccgaggtgg acggcgaggt cgccggcatc gcctacgcgg gcccctggaa
3420 ggcacgcaac gcctacgact ggacggccga gtcgaccgtg tacgtctccc
cccgccacca 3480 gcggacggga ctgggctcca cgctctacac ccacctgctg
aagtccctgg aggcacaggg 3540 cttcaagagc gtggtcgctg tcatcgggct
gcccaacgac ccgagcgtgc gcatgcacga 3600 ggcgctcgga tatgcccccc
gcggcatgct gcgggcggcc ggcttcaagc acgggaactg 3660 gcatgacgtg
ggtttctggc agctggactt cagcctgccg gtaccgcccc gtccggtcct 3720
gcccgtcacc gagatctgat ctcacgcgtc taggatccga agcagatcgt tcaaacattt
3780 ggcaataaag tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt
atcatataat 3840 ttctgttgaa ttacgttaag catgtaataa ttaacatgta
atgcatgacg ttatttatga 3900 gatgggtttt tatgattaga gtcccgcaat
tatacattta atacgcgata gaaaacaaaa 3960 tatagcgcgc aaactaggat
aaattatcgc gcgcggtgtc atctatgtta ctagatcggg 4020 aagatcctct
agagtcgacc tgcaggcatg caagcttggc gtaatcatgg tcatagctgt 4080
ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
4140 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
ttgcgctcac 4200 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
ttaatgaatc ggccaacgcg 4260 cggggagagg cggtttgcgt attgggcgct
cttccgcttc ctcgctcact gactcgctgc 4320 gctcggtcgt tcggctgcgg
cgagcggtat cagctcactc aaaggcggta atacggttat 4380 ccacagaatc
aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4440
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
4500 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
taaagatacc 4560 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
tccgaccctg ccgcttaccg 4620 gatacctgtc cgcctttctc ccttcgggaa
gcgtggcgct ttctcaatgc tcacgctgta 4680 ggtatctcag ttcggtgtag
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 4740 ttcagcccga
ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 4800
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
4860 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
aggacagtat 4920 ttggtatctg cgctctgctg aagccagtta ccttcggaaa
aagagttggt agctcttgat 4980 ccggcaaaca aaccaccgct ggtagcggtg
gtttttttgt ttgcaagcag cagattacgc 5040 gcagaaaaaa aggatctcaa
gaagatcctt tgatcttttc tacggggtct gacgctcagt 5100 ggaacgaaaa
ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 5160
agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
5220 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
tgtctatttc 5280 gttcatccat agttgcctga ctccccgtcg tgtagataac
tacgatacgg gagggcttac 5340 catctggccc cagtgctgca atgataccgc
gagacccacg ctcaccggct ccagatttat 5400 cagcaataaa ccagccagcc
ggaagggccg agcgcagaag tggtcctgca actttatccg 5460 cctccatcca
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 5520
gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
5580 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
cccatgttgt 5640 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
cagaagtaag ttggccgcag 5700 tgttatcact catggttatg gcagcactgc
ataattctct tactgtcatg ccatccgtaa 5760 gatgcttttc tgtgactggt
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 5820 gaccgagttg
ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 5880
taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
5940 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
gcatctttta 6000 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
aaatgccgca aaaaagggaa 6060 taagggcgac acggaaatgt tgaatactca
tactcttcct ttttcaatat tattgaagca 6120 tttatcaggg ttattgtctc
atgagcggat acatatttga atgtatttag aaaaataaac 6180 aaataggggt
tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 6240
ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c 6291 5
5560 DNA Artificial Sequence T-DNA of plasmid pTHW142. 5 aattacaacg
gtatatatcc tgccagtact cggccgtcga gtacatggtc gataagaaaa 60
ggcaatttgt agatgttaat tcccatcttg aaagaaatat agtttaaata tttattgata
120 aaataacaag tcaggtatta tagtccaagc aaaaacataa atttattgat
gcaagtttaa 180 attcagaaat atttcaataa ctgattatat cagctggtac
attgccgtag atgaaagact 240 gagtgcgata ttatgtgtaa tacataaatt
gatgatatag ctagcttagc tcatcggggg 300 atcctagacg cgtgagatca
gatctcggtg acgggcagga ccggacgggg cggtaccggc 360 aggctgaagt
ccagctgcca gaaacccacg tcatgccagt tcccgtgctt gaagccggcc 420
gcccgcagca tgccgcgggg ggcatatccg agcgcctcgt gcatgcgcac gctcgggtcg
480 ttgggcagcc cgatgacagc gaccacgctc ttgaagccct gtgcctccag
ggacttcagc 540 aggtgggtgt agagcgtgga gcccagtccc gtccgctggt
ggcgggggga gacgtacacg 600 gtcgactcgg ccgtccagtc gtaggcgttg
cgtgccttcc aggggcccgc gtaggcgatg 660 ccggcgacct cgccgtccac
ctcggcgacg agccagggat agcgctcccg cagacggacg 720 aggtcgtccg
tccactcctg cggttcctgc ggctcggtac ggaagttgac cgtgcttgtc 780
tcgatgtagt ggttgacgat ggtgcagacc gccggcatgt ccgcctcggt ggcacggcgg
840 atgtcggccg ggcgtcgttc tgggtccatg cagttaactc ttccgccgtt
gcttgtgatg 900 gaagtaatgt cgttgttagc cttgcgggtg gctgggaagg
cagcggagga cttaagtccg 960 ttgaaaggag cgaccatagt ggcctgagcc
ggagaggcaa ccatagtagc ggaagagagc 1020 atagaggaag ccattgttct
tctttactct ttgtgtgact gaggtttggt ctagtgcttt 1080 ggtcatctat
atataatgat aacaacaatg agaacaagct ttggagtgat cggagggtct 1140
aggatacatg agattcaagt ggactaggat ctacaccgtt ggattttgag tgtggatatg
1200 tgtgaggtta attttacttg gtaacggcca caaaggccta aggagaggtg
ttgagaccct 1260 tatcggcttg aaccgctgga ataatgccac gtggaagata
attccatgaa tcttatcgtt 1320 atctatgagt gaaattgtgt gatggtggag
tggtgcttgc tcattttact tgcctggtgg 1380 acttggccct ttccttatgg
ggaatttata ttttacttac tatagagctt tcataccttt 1440 tttttacctt
ggatttagtt aatatataat ggtatgattc atgaataaaa atgggaaatt 1500
tttgaatttg tactgctaaa tgcataagat taggtgaaac tgtggaatat atattttttt
1560 catttaaaag caaaatttgc cttttactag aattataaat atagaaaaat
atataacatt 1620 caaataaaaa tgaaaataag aactttcaaa aaacagaact
atgtttaatg tgtaaagatt 1680 agtcgcacat caagtcatct gttacaatat
gttacaacaa gtcataagcc caacaaagtt 1740 agcacgtcta aataaactaa
agagtccacg aaaatattac aaatcataag cccaacaaag 1800 ttattgatca
aaaaaaaaaa acgcccaaca aagctaaaca aagtccaaaa aaaacttctc 1860
aagtctccat cttcctttat gaacattgaa aactatacac aaaacaagtc agataaatct
1920 ctttctgggc ctgtcttccc aacctcctac atcacttccc tatcggattg
aatgttttac 1980 ttgtaccttt tccgttgcaa tgatattgat agtatgtttg
tgaaaactaa tagggttaac 2040 aatcgaagtc atggaatatg gatttggtcc
aagattttcc gagagctttc tagtagaaag 2100 cccatcacca gaaatttact
agtaaaataa atcaccaatt aggtttctta ttatgtgcca 2160 aattcaatat
aattatagag gatatttcaa atgaaaacgt atgaatgtta ttagtaaatg 2220
gtcaggtaag acattaaaaa aatcctacgt cagatattca actttaaaaa ttcgatcagt
2280 gtggaattgt acaaaaattt gggatctact atatatatat aatgctttac
aacacttgga 2340 tttttttttg gaggctggaa tttttaatct acatatttgt
tttggccatg caccaactca 2400 ttgtttagtg taatactttg attttgtcaa
atatatgtgt tcgtgtatat ttgtataaga 2460 atttctttga ccatatacac
acacacatat atatatatat atatatatta tatatcatgc 2520 acttttaatt
gaaaaaataa tatatatata tatagtgcat tttttctaac aaccatatat 2580
gttgcgattg atctgcaaaa atactgctag agtaatgaaa aatataatct attgctgaaa
2640 ttatctcaga tgttaagatt ttcttaaagt aaattctttc aaattttagc
taaaagtctt 2700 gtaataacta aagaataata cacaatctcg accacggaaa
aaaaacacat aataaatttg 2760 aattagcttg catgcctgca ggtcactgga
ttttggtttt aggaattaga aattttattg 2820 atagaagtat tttacaaata
caaatacata ctaagggttt cttatatgct caacacatga 2880 gcgaaaccct
ataagaaccc taattccctt atctgggaac tactcacaca ttattctgga 2940
gaaaaataga gagagataga tttgtagaga gagactggtg atttttgcgc cgggtaccga
3000 gctcggtagc aattcccgag gctgtagccg acgatggtgc gccaggagag
ttgttgattc 3060 attgtttgcc tccctgctgc ggtttttcac cgaagttcat
gccagtccag cgtttttgca 3120 gcagaaaagc cgccgacttc ggtttgcggt
cgcgagtgaa gatccctttc ttgttaccgc 3180 caacgcgcaa tatgccttgc
gaggtcgcaa aatcggcgaa attccatacc tgttcaccga 3240 cgacggcgct
gacgcgatca aagacgcggt gatacatatc cagccatgca cactgatact 3300
cttcactcca catgtcggtg tacattgagt gcagcccggc taacgtatcc acgccgtatt
3360 cggtgatgat aatcggctga tgcagtttct cctgccaggc cagaagttct
ttttccagta 3420 ccttctctgc cgtttccaaa tcgccgcttt ggacatacca
tccgtaataa cggttcaggc 3480 acagcacatc aaagagatcg ctgatggtat
cggtgtgagc gtcgcagaac attacattga 3540 cgcaggtgat cggacgcgtc
gggtcgagtt tacgcgttgc ttccgccagt ggcgaaatat 3600 tcccgtgcac
ttgcggacgg gtatccggtt cgttggcaat actccacatc accacgcttg 3660
ggtggttttt gtcacgcgct atcagctctt taatcgcctg taagtgcgct tgctgagttt
3720 ccccgttgac tgcctcttcg ctgtacagtt ctttcggctt gttgcccgct
tcgaaaccaa 3780 tgcctaaaga gaggttaaag ccgacagcag cagtttcatc
aatcaccacg atgccatgtt 3840 catctgccca gtcgagcatc tcttcagcgt
aagggtaatg cgaggtacgg taggagttgg 3900 ccccaatcca gtccattaat
gcgtggtcgt gcaccatcag cacgttatcg aatcctttgc 3960 cacgtaagtc
cgcatcttca tgacgaccaa agccagtaaa gtagaacggt ttgtggttaa 4020
tcaggaactg ttcgcccttc actgccactg accggatgcc gacgcgaagc gggtagatat
4080 cacactctgt ctggcttttg gctgtgacgc acagttcata gagataacct
tcacccggtt 4140 gccagaggtg cggattcacc acttgcaaag tcccgctagt
gccttgtcca gttgcaacca 4200 cctgttgatc cgcatcacgc agttcaacgc
tgacatcacc attggccacc acctgccagt 4260 caacagacgc gtggttacag
tcttgcgcga catgcgtcac cacggtgata tcgtccaccc 4320 aggtgttcgg
cgtggtgtag agcattacgc tgcgatggat tccggcatag ttaaagaaat 4380
catggaagta agactgcttt ttcttgccgt tttcgtcggt aatcaccatt cccggcggga
4440 tagtctgcca gttcagttcg ttgttcacac aaacggtgat acctgcacat
caccatgttt 4500 tggtcatata ttagaaaagt tataaattaa aatatacaca
cttataaact acagaaaagc 4560 aattgctata tactacattc ttttattttg
aaaaaaatat ttgaaatatt atattactac 4620 taattaatga taattattat
atatatatca aaggtagaag cagaaactta cgtacacttt 4680 tcccggcaat
aacatacggc gtgacatcgg cttcaaatgg cgtatagccg ccctgatgct 4740
ccatcacttc ctgattattg acccacactt tgccgtaatg agtgaccgca tcgaaacgca
4800 gcacgatacg ctggcctgcc caacctttcg gtataaagac ttcgcgctga
taccagacgt 4860 tgcccgcata attacgaata tctgcatcgg cgaactgatc
gttaaaactg cctggcacag 4920 caattgcccg gctttcttgt aacgcgcttt
cccaccaacg ctgatcaatt ccacagtttt 4980 cgcgatccag actgaatgcc
cacaggccgt cgagtttttt gatttcacgg gttggggttt 5040 ctacaggacg
gaccatgnnc ccggggatcc tctaganntt atagagagag agatagattt 5100
atagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact tccttatata
5160 gaggaagggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc
agtggagatg 5220 tcacatcaat ccacttgctt tgaagacgtg gttggaacgt
cttctttttc cacgatgctc 5280 ctcgtgggtg ggggtccatc tttgggacca
ctgtcggcag aggcatcttg aatgatagcc 5340 tttcctttat cgcaatgatg
gcatttgtag gagccacctt ccttttctac tgtcctttcg 5400 atgaagtgac
agatagctgg gcaatggaat ccgaggaggt ttcccgaaat tatcctttgt 5460
tgaaaagtct caatannnng tcgacctgca ggcatgcaag ctaattccgg ggaagcttag
5520 atccatggag ccatttacaa ttgaatatat cctgccgccg 5560
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