Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
0 WO 97/06267 2200496 PCT/EP96/03366
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GENETIC TRANSFORMATION USING A PARP INHIBITOR
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
io 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.
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.
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.
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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).
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).
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.
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
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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).
is 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.
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.
Many of these factors are not under the control of man.
CA 02200496 2008-10-08
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Summary of the Invention
This invention provides a process for producing transgenic eucaryotic cells,
particularly plant cells. The process comprises contacting a culture of
untransfoimed 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.
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
untransfoimed 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
with the foreign DNA.
More particularly, the invention provides 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
of
about 2 days to about 28 days; contacting said plant cells with foreign DNA
comprising at least one gene of interest under conditions in which said
foreign
DNA is taken up by said plant cells and said gene of interest is stably
integrated
in the nuclear genome of said plant cells to produce said transgenic plant
cells;
and recovering said transgenic plant cells from said culture.
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More particularly, the invention also provides a process for increasing the
frequency of obtaining transgenic plant cells which comprises: contacting
plant
cells with foreign DNA comprising at least one gene of interest under
conditions
in which said foreign DNA is taken up by said plant cells and said gene of
interest is stably integrated in the nuclear genome of said plant cells to
produce
said transgenic cells; contacting said transgenic plant cells with an
inhibitor of
poly-(ADP-ribose); and further culturing said transgenic cells in a medium
containing said inhibitor for a period of time of approximately 1 to 14 days.
Description of the Invention
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
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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.
~ 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).
is The biological function of PARP is largely unknown but the enzyme has been
implicated in :
- enhancement of DNA repair (Satoh et al, 1992, Nature 356:356-358; Satoh
et al, 1993, J.Biol.Chem. 268:5480-5487),
- 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),
- 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);
- reducing the amount of available NAD+ (and by consequence its precursor
ATP) : this results in a general slowing down of cell metabolism (Lazebnik
WO 97/06267 2 2 0 0 4 9 6 PCT/EP96/03366
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et al, '1994, 371:346-347; Gaal et al, 1987, TIBS 12:129-130; Cleaver et al,
u ra
It is known that PARP can be efficiently inhibited by a number of compounds
s (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
lo 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 x10"5, particularly lower than 1 x10~6 . Generally
it is
15 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,
su ra . Generally it is also preferred that cells cultured in medium
containing
the PARP inhibitor retain their capacity of DNA repair.
20 Particularly preferred PARP inhibitors are those listed above and
especially
niacinamide (nicotinamide), picolinamide, 5-methyinicotinamide, 2-
aminobenzamide, pyrazinamide, theobromine and theophylline. Particularly
niacinamide is believed to be a useful inhibitor for the purpose of this
invention.
25 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
WO 97/06267 2 2 0 0 4 9 6 PCT/EP96/03366
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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.
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.
In many conventional procedures for genetic transformation of eucaryotic
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
is 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.
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.
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,
WO 97/06267 22004 9 6 PCT/EP96/033660
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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/I
(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/I (about 4 mM) and 1000
mg/i (approx. 8 mM). On the other hand, when nicotinamide is used in
combination with plant material derived from com, the concentration of
nicotinamAe should preferably be 100 mg/I. Likewise, a concentration of 100
mg/I 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
ls inhibition (as measured by its Ki and/or by its percentage inhibition of
PARP
under standard conditions - Sims et al, su ra . It was found for instance that
the optimal concentration for nicotinamide is approximately 250 mg/I (i.e.
about
2 mM) but it is believed that concentrations up to 1000 mg/I (approx. 8 mM)
and as low as 150 mg/I (approx. 1.25 mM), even as low as 100mg/I can be
used to good effect. Preferably the nicotinamide concentration should be
between 200 and 300 mg/(, i.e. between approximately 1.5 mM and 2.5 mM. In
similar coriditions, 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.
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).
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For other PARP inhibitors optimal concentrations can be easily established by
experimentation in accordance with this invention.
- ,:
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
is 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.
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
WO 97/06267 220UA96 PCT/EP96/03366
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of cells contacted with foreign DNA, amount of delivered DNA, type and
conditions of DNA delivery, general culture conditions etc.).
Therefore it is preferred that the invention is used with transformation
procedures that already have a high efficiency, such as Acarobacterium
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
lo 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.
In anothei- 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
altematively 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
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of lesser importance since Aarobacterium mediated transformation, being a
biological process, already results in a generally low copy number of the
transgene in the transformed plant cells.
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.
The cell of a plant, particularly a plant capable of being infected with
Aarobacterium such as most dicotyledonous plants (e.g. Brassica napus) and
i s some monocotyledonous plants, can be transformed using a vector that is a
disarmed Ti-plasmid containing the gene(s) of interest and carried by
Aarobacterium. This transformation can be carried out using conventional
procedures (EP 0,116,718; Deblaere et al, supra; Chang et al, 1994, The Plant
Joumal 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 US patent 4,684,611), plant RNA virus-
mediated transformation (as described, for example, in EP 0,067,553 and US
patent 4,407,956) and liposome-mediated transformation (as described, for
example, in US patent 4,536,475). Cells of monocotyledonous plants such as
the major cereals including corn, rice, wheat, barley, and rye, can be
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transformed (e.g. by electroporation) using wounded or enzyme-degraded
intact tissues capable of forrning 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 5
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/7echnology 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).
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.
In animals pluripotent embryonic or somatic stem cells can be used as target
for transfonmation (Capecchi et al, 1989, TIG:5:70-76).
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 genorrie, particularly in regions of the genome that remain
transcriptionally
active in the untransformed cells that have been exposed to a PARP inhibitor
in
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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
io 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
WO 97/06267 2200496 PCT/EP96/03366=
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30%) when compared to the mRNA levels observed in cells cultured in medium
not contairiing the inhibitor).
The method of the present invention can in p(nciple be used to transform 5
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 transc(ption 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'
lo 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
15 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).
It is believed that the method of the present invention is particularly useful
to
20 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
25 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
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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.
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).
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.
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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 radicais produced in the transgenic cells with the amount of
free 5 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.
so 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.
ls 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
20 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.
It is known that in transformed plants and other organisms transgene
25 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 somacional variation or by insertional inactivation of
~ WO 97/06267 2200496 PCT/EP96/03366
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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.
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).
For each plant line (control or transformant) it is preferred that a number of
plants is assayed.
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
CA 02200496 2008-10-08
-18-
concentrations containing a PARP inhibitor can be made. A useful buffer series
is a K-phosphate buffer containing 2% sucrose and 250 mg/I 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
is 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.
After the one day incubation the capacity of the transformed and control
tissues
to reduce 2,3,5-triphenyltetrazolium chioride (TTC) is measured e.g. by the
following procedure which is modified from Towill and Mazur su ra :
- 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.
- extraction of reduced 'i"i'C (e.g. freezing at -70 C followed by thawing at
40 C and shaking the plant material in ethanol for 45-60 minutes)
* Trademark
WO 97/06267 2200496 PCT/EP96/03366
- 19 -
- spectrophotometric quantification of reduced TTC at 485 nm (optical
density OD485; for chlorophyll poor plant material) or 545 nm (OD545; 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
s reduced TTC corresponds to an OD485 of 0.214 or OD545 of 1.025 (light
path 1 cm).
- the reducing capacity of the transformed plant line is compared to that of
the control line.
The amount of reduced TTC is determined by the intensity of the cytochromal
and altemative 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 extemal factors, such as
stress factors.
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 (lTC-low); in other words a TTC-ratio value is calculated as
follows:
TTC-ratio = TTC/high.100/T7C.Iow.
The value of TTC-ratio is a measure of the fitness of a plant line as compared
to a control line. -
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-
WO 97/06267 2200496 PCT/EP96/03366~
- 20 -
low and TTC-high can be typically obtained from tissues incubated at
respectively 20mM and 60 mM K-phosphate buffer containing 250 mg/I
niacinamicle. TTC-high and TTC-low, for both the transformed and
untransfonrned 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 frorn Arabidopsis thaliana,or 150 hypocotyl explants derived from about
25 seedlings of Brassica napus.
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.
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.
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.
WO 97/06267 2200496 PCT/EP96/03366
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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:
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
lo 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.
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.
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
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.
The modified TTC-procedure thus allows to select those types of (cultured)
plant material especially appropriate for use in a transformation procedure,
WO 97/06267 2200496 PCT/EP96/03366
0
- 22 -
particularly the procedures of this invention that include the use of a PARP
inhibitor.
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 com, it is preferred to include proline, preferably at a
concentration of about BmM, simultaneously with the PARP inhibitor, in the
culture medium.
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
metabolisrn, 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).
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
WO 97/06267 2 2 0 0 4 9 6 PCT/EP96/03366
- 23 -
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".
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.
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).
All PCR reactions were performed under conventional conditions using the
Ventm polymerase (Cat. No. 254L - Biolabs New England, Beverley, MA
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,
WO 97/06267 2 2oo4 9 6 PCT/EP96/0336*
- 24 -
Netherlands). In the examples MS medium means Murashige and Skoog
medium (Murashige and Skoog, 1962, Physiol. Plant 15:473-479).
In the following examples, reference will be made to the following
sequence listing and figures:
Sequence Listin
SEQ ID NO I : T-DNA of plasmid pTHW107
SEQ ID NO 2: plasmid pTS172
SEQ ID NO 3: PT72 promoter contained in plasmid pTS772
SEQ ID No 4: plasmid pVE136
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 inhibftor.
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/I (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.
WO 97/06267 PCT/EP96/03366
2200496
- 25 -
Similar observations were made when Brassica napus hypocotyl explants were
cultured on A5 medium (see Example 3) containing 250 mg/I 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.
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).
The above observations are interpreted as indicating that treating cultured
cells
with a PARP inhibitor for about 4-5 days generally results in :
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
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.
It is inferred that under these conditions many genes in cells (e.g. cultured
cells) that would normally be swifched 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.
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
WO 97/06267 2 20 0 4 9 6 PCT/EP96/03366.
- 26 -
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 tha`
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.
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
The Wheat Spring variety Pavon is grown in a greenhouse or conditioned room
at 23-24 C during daytime and 18-20 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% NaOCI+ 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 C.
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/I adenine.S04, 0.5 mg/I thiamine.HCI,
0.5 g/I 2-[N-Morpholino] ethane sulfonic acid (Mes) pH 5.8, 0.5% agarose, 0.5
to 2.5 mg/I CuSO4.5H20, 25 mg/I acetylsalicylic acid and 2 mg/I 2,4-
dichlorophenoxyacetic acid (2,4-D)) and were incubated for 3 weeks at 27 C in
the dark.
Embryogenic sections of the developing callus were isolated, placed on callus
maintenance medium W2 (W1 medium but without acetylsalicylic acid and with
~ WO 97/06267 27 - 2200496 PCT/EP96/03366
-
only 0.5 mg/I CuSO4.5H20 and 1 mg/I 2,4-D), and incubated for 3 weeks at 24-
25 C in the light (approx. 20 mEinsteins/s/m2 (with a photoperiod of 16 hours
light and 8 hours dark).
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.
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
lo of a 9 cm Petridish containing W2 medium in a circle with a diameter of
approx.
0.5 cm. When required niacinamide (250 mg/I) was added to the W2 medium
and the tissue pieces were maintained under these conditions for 4 days after
they were bombarded.
15 Bombardment was carried out using the Biolistic PDS-1000/He apparatus (Bio-
Rad). Preparation of the microcarriers (0.4-1.2m) 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.
For the transformation experiments the following plasmid DNA was used.
- plasmid pVE136, the sequence of which is given in SEQ ID No 4. This
plasmid contains the following chimeric genes:
- P35S-bar-3'nos
- PCA55-bamase-3'nos
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' untransiated end of the Agrobacterium T-DNA nopaline synthase
gene, PCA55 is a stamen-specific promoter from corn gene CA55 (WO
WO 97/06267 22" O4Q" PCT/EP96/03366
- 28 _
9213957), and bamase is a DNA encoding barnase (Hartley, 1988,
J. MoI. Bio1.202:913-915)
- Ip asmid pTS172 the sequence of which is given in SEQ ID No 2. This
plasmid contains the following chimeric genes:
- P35S-bar-3'g7
- PE 1-bamase-3'nos
in whiich 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 Agrobacterium T-DNA
gene 7, PE1 is a stamen-specific promoter from rice gene El (WO
9213956), barnase is a DNA encoding barnase (Hartley, 1988,
J.MoI.BioI.202:913-915), and 3'nos is the 3' untransiated end of the
Agrobacterium T-DNA nopaline synthase gene,
- plasrnid 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:
- P35S-bar-3'g7
- P'1772-barnase-3'nos
in which PT72 is a stamen-specific promoter from rice gene T72 (WO
9213956)
The bombarded calli were transferred to selective medium W2 containing 2.5
mg/I phosphinothricin (PPT) and, if neccesary, 100 mg/I niacinamide. The calli
that were placed on medium containing niacinamide were transferred after 4
days to niacinamide-free W2 medium containing 2.5 mg/i PPT. The cells were
cultured at 24-25 C.
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/I
2200496
WO 97/06267 PCT/EP96/03366
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CuSO4.5H20 and 0.5 mg/I 2,4-D). Calli were subcultivated every two weeks at
which time the nonmorphogenic parts of the calli were removed. When the calli
started to form shoots they were transferred to W5 medium (WI medium with
half concentrated MS medium and only 0.5 mg/i CuSO4.5H20 and without
acetylsalicylic acid and 2,4-D, but supplemented with 50 mg/I myo-inositol,
0.25
mg/I pyridoxine.HCI and 0.25 mg/i nicotinic acid) containing 2.5 mg/I PPT. For
the rest of the procedure temperature was maintained at a maximum of 24 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
so shoots) were transferred to 1 liter vessels with W6 medium (half-
concentrated
MS medium supplemented with 1.5% sucrose, 50 mg/i myo-inositol, 0.25 mg/I
pyridoxine.HCI, 0.25 mg/I nicotinic acid, 0.5 mg/I thiamine.HCI, 0.7% agar
(Difco) pH 5.8 and 0.5 mg/I CuSO4.5H20) containing 2.5 mg/I PPT. Once the
shoots and roots had grown out, individual shoots were separated from each
other and transferred to 1 1 vessels containing W6 medium with 2.5 mg/I 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 aI, 1995, Planta 197:
619-626). Transformed shoots were finally transferred to the greenhouse into
soil.
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:
Group Niacinamide treatment
None No treatment
Before 100 100 mg/I niacinamide for four days prior to bombardment
Before 250 250 mg/I niacinamide for four days prior to bombardment
WO 97/06267 2 2 0 0 4 9 5 PCT/EP96/03366,o
- 30 -
Before/After 250 mg/I niacinamide for four days prior to bombardment
pfus 100 -mg/I niacinamide for four days after
bombardment
Results of the experiments are presented in Tables 1, 2 and 3. Plants could be
obtained orily from bombarded calli that were treated with niacinamide.
For the plants that were transformed with plasmid pTS 172 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 male-sterile plants that
otherwise looked completely healthy could be obtained only from bombarded
calli treateci 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 bamase 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-steriie wheat plants
from
control calli a much larger number of calli would have to be bombarded.
WO 97/06267 2200496 PCT/EP96/03366
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Results of wheat transformation experiments
Table 1 :
Plasmid pTS172
Treatment Nr of Nr of PPT- Nr of PPT. Nr of MS
bombarded resistant resistant plants
calli calli plants recovered
recovered recovered
None 60 30 1 a) 0
Before 250 125 30 3 3b)
a) This plant proved to be fertile and to be transformed only with the
chimeric
bar gene
b) The obtained plants looked healthy and tillered vigorously
WO 97/06267 22ry O49G PCT/EP96/03366i*
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Table 2:
Plasmid pTS772
Treatment Nr of Nr of PPT- Nr of PPT Nr of MS
bombarded resistant resistant plants
calli calli plants recovered
recovered recovered
None 250 22 0 0
Before 210 75 7 3 a)b)
250
Before/ 210 45 6 3a
After
a) The obtained plants looked healthy and tillered vigorously
b) Only six plants could be analyzed for MS phenotype since one of the plants
s died prematurely.
WO 97/06267 2200496 PCT/EP96/03366
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Table 3:
Plasmid pVE136
Treatment Nr of Nr of PPT Nr of MS
bombarded resistant plants
calli plants recovered
recovered
None 200 1 0
Before 800 8a) 8
100
a) The obtained plants looked healthy and tillered vigorously
WO 97/06267 2~ ~ 0496 PCT/EP96/0336*
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Example 3: Transformation of oilseed rape with a barnase gene under the
control of a stamen-specific promoter using Agrobacterium mediated
transformation.
Hypocotyl explants of Brassica napus were obtained , cultured and
transformed essentially as described by De Block et aI, 1989, Plant Physiol.
914:694-701 except for the following modifications:
- hypocotyl explants were precultured for 3 days on A2 medium (MS, 0.5
g/I Mes (pH 5.7), 1.2% glucose, 0.5% agarose, 1 mg/I 2,4-D, 0.25 mg/i
naphthalene acetic acid (NAA), 1 mg/1 6-benzylaminopurine (BAP)), and then
transferred to the A2 medium with or without niacinamide for another 4 days.
- infection medium A3 was MS, 0.5 g/I Mes (pH 5.7), 1.2% glucose, 0.1
mg/I NAA, 0.75 mg/I BAP, 0.01 mg/I giberellinic acid (GA3)
- selection medium A5 was 0.5 g/I Mes (pH 5.7), 1.2 % glucose, 40 mg/I
adenine.S04, 0.5 g/I polyvinyl-polypyrrolidone (PVP), 0.5% agarose, 0.1 mg/I
ls NAA, 0.75 mg/I BAP, 0.01 mg/I GA3, 250 mg/I carbenicillin, 250 mg/I
triacillin, 5
mg/I AgN03.
- regeneration medium A6 was MS, 0.5 g/I Mes (pH 5.7), 2% sucrose, 40
mg/I adenine.S04, 0.5 g/I PVP, 0.5% agarose, 0.0025 mg/I BAP, 250 mg/I
triacillin.
- 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/I isobutyric acid (IBA), 100 mg/I
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%)
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).
WO 97/06267 2 2 0 0 4 9 6 PCT/EP96/03366
- 35 -
Plasmid pTHW107 is a vector carrying a T-DNA comprising the following
chimeric genes :
- PTA29-barnase-3'g7
- PSSU-bar-3'nos
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.
io Where required niacinamide (250 mg/I) 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 Southem 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.
WO 97/06267 220^ 496 PCT/EP96/0336*
- 36 -
Table 4:
Treatment Id. Vegetative Reproductive Copy No. of Phenotype of
No. phenotypea phenotypeb the the F1-progenyd
transgenesc
no 1 stressed sterile 3 stressed/sterile
treatment 2 stressed sterile 4-6 ND
3 stressed sterile 3 stressed/sterile
4 normal sterile 1 normal/sterile
stressed (bud fall) ND ND
Before 1 normal sterile 1 normal/sterile
250 2 normal sterile 3 normal/sterile
3 normal sterile 1 ND
4 normal sterile 3 ND
5 normal sterile 2 ND
a. Vegetatively stressed plants have a small size and flower early, leaves
5 are oblong and dark green.
b. Reproductive phenotype regards male sterility; in flowers where the
buds fell off prematurely this phenotype was not scored, except where
some buds resulted in flowers.
c. Copy number of the transgenes was estimated by comparative
Southern. ND: not determined.
d. Fl-progeny was obtained by pollinating the transformed plants with
pollen obtained from an untransformed N90-740 line. Fl-Progeny
resistant to phosphinotricin was scored for vegetative and reproductive
phenotype.
= WO 97/06267 22004 9 b PCT/EP96/03366
- 37 -
Example 4: Aarobacterium-mediated transformation of oilseed rape using
niacinamide in the culture medium.
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/I niacinamide
for 1 day prior to infection with Agrobacterium (BEFORE), treated for 2 days
during the infection with 250 mg/I niacinamide (DURING), or treated for 1 day
after the Agrobacterium infection with 250 mg/I niacinamide (AFTER).
All hypocotyl explants were infected with Agrobacterium tumefaciens strain
C58C1 Rif carrying T-DNA vector pTHW142 and a helper Ti-plasmid pMP90
(Koncz and Shell, 1986 su ra (or a derivative thereof).
Piasmid pTHW142 is a vector carrying a T-DNA comprising the following
chimeric genes:
- PSSU-bar-3'g7
- p35S-uidA-3'35S
In which uidA is a DNA encoding b-glucuronidase (Jefferson et al., 1986, Proc.
Nati. Acad. Sci. USA 83, 8447-8451) and 3' 35S is the 3' untransiated end of
the cauliflower mosaic virus 35S transcript.
The complete sequence of the T-DNA of pTHW142 is presented in SEQ ID No
5.
After the Agrobacterium infection, hypocotyl explants were transferred to
selection medium A5, and if appropriate to A5 medium containing 250 mg/I
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
CA 02200496 2008-10-08
-38-
Table 5. Niacinamide treatment either before or after the Agrobacterium
infection significantly increase the transformation efficiency.
Table 5:
Treatment Transformation Remarksb
frequency81
NONE 16% small, green calli
BEFORE 32% large, green calli
DURING 16% very small, light green calli
large, green calli
AFTER 29% developing shoots
a. Determined as the number of transformed calli (PPT-resitant and GUS-
positive) developing per 100 hypocotyl explants
b. Size 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
2200496
= WO 97/06267 PCT/EP96/03366
- 39 -
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: PLANT GENETIC SYSTEMS N.V.
(B) STREET: Plateaustraat 22
(C) CITY: Ghent
(E) COUNTRY: Belgium
(F) POSTAL.CODE (ZIP): 9000
(G) TELEPHONE: 32 9 235 84 58
(H) TELEFAX: 32 9 223 19 23
(I) TELEX: 11.361 Pgsgen
(ii) TITLE OF INVENTION: Genetic Transformation using a PARP inhibitor
(iii) NUMBER OF SEQUENCES: 5
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4946 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: T-DNA of plasmid pTHW107
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (1..25)
(D) OTHER INFORMATION:/label= RB
/note= "T-DNA right border"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (97..330)
(D) OTHER INFORMATION:/label= 3'g7
/note= "3' untranslated region containing the
polyadenylation signal of gene 7 of Agrobacterium T-DNA "
2200496
WO 97/06267 PCT/EP96/033610
- 40 -
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (331..882)
(D) OTHER INFORMATION:/label= bar
/note= "region coding for phosphinothricin acetyl
transferase"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (883..2608)
(D) OTHER INFORMATION:/label= PSSU
/note= "promoter region of Rubisco small subunit gene of
Arabidopsis thali..."
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (2658..3031)
(D) OTHER INFORMATION:/label= 3'nos
/note= '3' untranslated region containing the
polyadenylation signal of the nopaline synthase gene of Agrobacterium
T-DNA"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (3032..3367)
(D) OTHER INFORMATION:/label= barnase
/note= "region coding for barnase"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (3368..4876)
(D) OTHER INFORMATION:/label= PTA29
/note= "promoter region of TA29 gene of Nicotiana tabacum"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (4922..4946)
(D) OTHER INFORMATION:/label= LB
/note= "T-DNA left border"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 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
WO 97/06267 22('~ O 496 PCT/EP96/03366
- 41 -
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
WO 97/06267 22004C) 6 PCT/EP96/033660
- 42 -
CACCAATTAG GTTTCTTATT ATGTGCCAAA TTCAATATAA TTATAGAGGA TAT.TTCAAAT 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
WO 97/06267 2200496 PCT/EP96/03366
- 43 -
ATGTTTATTC TAGTCCAGCC ACCCACCTTA TGCAAGTCTG CTTTTAGCTTGATTCAAAAA 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) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6548 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
W 97/0626' 2 2 0 0 4 7 6 PCT/EP96/03366*
- 44 -
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: plasmid pTS172
(ix) IFEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (2019..2288)
(D) OTHER INFORMATION:/label= 3'nos
/note= "3' untranslated region containing the
polyadenylation signal of the nopaline synthase gene of Agrobacterium
T-DNA"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (2289..2624)
(D) OTHER INFORMATION:/label= barnase
/note= "region coding for barnase"
(ix) =EATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (2625..4313)
(D) OTHER INFORMATION:/label= PE1
/note= "promoter region of El gene of rice"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:4336..5710
(D) OTHER INFORMATION:/label= P35S
/note= "35S promoter region of Cauliflower mosaic virus"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:5711..6262
(D) OTHER INFORMATION:/label= bar
/note= "region coding for phosphinothricin acetyl
transferase"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:6263..6496
(D) OTHER INFORMATION:/label= 3'g7
/note= "3' untranslated region containing the
polyadenylation signal of gene 7 of Agrobacterium T-DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
AATTCAAGCT TGACGTCAGG TGGCACTTTT CGGGGAAATG TGCGCGGAAC CCCTATTTGT 60
TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA GACAATAACC CTGATAAATG 120
CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC ATTTCCGTGT CGCCCTTATT 180
WO 97/06267 2~ ~ 0496 PCT/EP96/03366
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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
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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
iC
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
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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
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- 48 -
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
(2) INFORKATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1601 base pairs
(B) TYPE: nucleic acid
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(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: N0
(vi) ORIGINAL SOURCE:
(A) ORGANISM: T72 promoter region
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (1..1601)
(D) OTHER INFORMATION:/label= PT72
/note= "promoter region of T72 gene of rice"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 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
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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
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6291 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: plasmid pVE136
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (425..687)
(D) OTHER INFORMATION:/label= 3'nos
/note= "3'untranslated region containing the
polyadenylation signal of the nopaline synthase gene of Agrobacterium
T-DNA"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (803..1138)
(D) OTHER INFORMATION:/label= barnase
/note= "region coding for barnase"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (1138..2317)
(D) OTHER INFORMATION:/label= PCa55
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/note= "stamen-specific promoter from corn gene CA55"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:2355..3187
(D) OTHER INFORMATION:/label= P35S
/note= "35S promoter region of Cauliflower mosaic virus"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:3188..3739
(D) OTHER INFOFtMATION:/label= bar
/note= "region coding for phosphinotricin acetyl
transferase"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:3757..4017
(D) OTHER INFORMATION:/label= 3'nos
/note= "3' untranslated region containing the
polyadenylation
signal of the nopaline synthase gene of Agrobacterium
T-DNA"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:699..702
(D) OTHER INFORMATION:/note= "region with unknown
sequence (may contain up to 15 nucleotides)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 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
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- 52 -
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 AFIAAAGTGTA 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
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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
2200496
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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
GCAGAAAAP,A AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT 5100
GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT 5160
4Q
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 TGGTCCTGC~. ACTTTATCCG 5460
CCTCCATCCA GTCTATTAAT TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA 5520
GTTTGCGCAA CGTTGTTGCC ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA 5580
TGGCTTCATT CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT 5640
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=
- 55 -
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
(2) INFORMATION FOR SEQ ID NO: 5:
--
-~1Y SEQUENCE HARA 5:
(A) LENGTH: 5560 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: T-DNA of plasmid pTHW142
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:1..25
(D) OTHER INFORMATION:/label= RB
/note= "right border sequence of octopine TL-DNA from
pTiB6S3"
(ix) FEATURE:
(A) NAME/KEY:.-
(B) LOCATION:complement (84..296)
(D) OTHER INFORMATION:/label= 3'g7
/note= "3' untranslated region containing the
polyadenylation signal of gene 7 of Agrobacterium T-DNA"
(ix) FEATURE:
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(A) NAME/KEY: -
(B) LOCATION:complement (318..869)
(D) OTHER INFORMATION:/label= bar
/note= "region coding for posphinotricin acetyl
transferase"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (830..2760)
(D) OTHER INFORMATION:/label= PSSU
/note= "promoter region of Rubisco small subunit gene of
Arabidopsis thali..."
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (2765..3058)
(D) OTHER INFORMATION:/label= 3'35S
/note= "3' untranslated region of the CaMV 35S transcript
containing polyadenylation signals"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (3059..5056)
(D) OTHER INFORMATION:/label= uidA
/note= "region coding for beta-glucoronidase"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (4483..4671)
(D) OTHER INFORMATION:/label= IV2
/note= "region corresponding to the second intron of the
ST-LS1 gene"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:complement (5067..5502)
(D) OTHER INFORMATION:/label= P35S
/note= "35S promoter region of CaMV"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:5533..5560
(D) OTHER INFORMATION:/label= LB
/note= "left border sequence of octopine TL-DNA from
pTIB6S3"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:5058..5059
(D) OTHER INFORMATION:/note= "region with unknown
sequence (may contain up to 20 nucleotides)"
(ix) FEATURE:
(A) NAME/KEY: -
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(B) LOCATION:5077..5078
(D) OTHER INFORMATION:/note= "region with unknown
sequence (may contain up to 20 nucleotides)"
(ix) FEATURE:
(A) NAME/KEY: -
(B) LOCATION:5476..5479
(D) OTHER INFORMATION:/note= "region with unknown
sequence (may contain up to 20 nucleotides)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AATTACAACG GTATATATCC TGCCAGTACT CGGCCGTCGA GTACATGGTC GATAAGAAAA 60
GGCAATTTGT AGATGTTAAT TCCCATCTTG AAAGAAATAT AGTTTAAATA TTTATTGATA 120
AAATAACAAG TCAGGTATTA TAGTCCAAGC AAAI=1ACATAA 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
WO 97/06267 220O 4 9 b PCT/EP96/03361
- 58 -
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 ,.760
AATTAGCTTG CATGCCTGCA GGTCACTGGA TTTTGGTTTT AGGAATTAGA AATTTTATTG 2820
ATAGAAGTAT TTTACAAATA CAAATACATA CTAAGGGTTT CTTATATGCT CAACACATGA 2880
GCGAAACCCT ATAAGAACCC TAATTCCCTT ATCTGGGAAC TACTCACACA TTATTCTGGA 2940
WO 97/06267 2 2 oo4 9 b PCT/PP96/03366
- 59 -
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
WO 97/06267 220O4{) O PCT/EP96/03366
- 60 -
AATTGCTATA TACTACATTC TTTTATTTTG P.AAAAAATAT 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
