Note: Descriptions are shown in the official language in which they were submitted.
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CASE 50I61
The present invention relates to a new DNA sequence of an
expression cassette in which the regulating regions for
the wound-inducible transcriptional regulation in the stem
and in leaves as well as for the constitutive
transcriptional regulation in the potato tubers are
localised, as well as the transference of these in vectors
containing the DNA sequence in the plant genome, by using
Agrobacteria as transfer microorganisms. The transfer DNA
sequence is concerned both for the regulation of
endogenous products as well as for the production of
heterologous products. Heterologous products can be for
example toxic proteins that can be used for combating
plant pests.
Because of the continual increasing need for food and raw
materials due to the growth in world population, and
because of the long-term reduction in areas of land
su~table for growing crops, it is becoming increasingly
the task for biological research to to increase the yields
of crops and their food content. An increase of yields can
be achieved amongst other methods by increasing the
resistance of crops against plant pests and plant diseases
and/or poor soils. An increase of the resistance could
achieved for example in such a way in that the plants
induce and give rise to an increased formation of
protective substances. For this, the metabolism of the
plants must be manipulated. This can be achieved amongst
other ways by changing the DNA contained in the cell
nuclei. It would be desirable to act on in those DNA areas
which are responsible for transcription in one or more of
the parts of the plant or during a specified period in the
plant growth cycle. For this there is a great interest in
,identifying the DNA sequence in the plant genome
,.. . .. .
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responsible for the transcription or expression of
endogenous plant products. In order to find such DNA
sequences, products first have to be sought which appear
at a specific time in the cell growth cycle or in a
specific part of the plant. If the gene belonging to this
is to be identified and isolated, a careful investigation
of the sequence, and above all the identification and
isolation of the desired transcriptional regulatory
regions, is necessary. Suitable models must then be
provided whose ~unctions must established through
experiments. Identifying such DNA sequences is a
challenging project which is subject to substantial
pitfalls and uncertainty. There is however substantial
interest in the possibility of genetically modifying
plants, which justifies the substantial expenditure and
efforts necessary in identifying transcriptional sequences
and manipulating them to determine their utility.
Processes for genetic modification of dicotyledonous and
monocotyledonous plants are known (EP 267159), as well as
th~ following publications of Crouch et al., in: Molecular
Form and Function of the Plant Genome, eds. van Vloten-
Doting, Groots and Hall, Plenum Publishing Corp, 1985,
pp 555-566; Crouch and Sussex, Planta (1981) 153:64-741
Crouch et al., J. Mol. Appl. Genet (1983) 2:273-283; and
Simon et al., Plant Molecular Biology ~1985) 5: 191-201,
in which various forms of storage proteins in Brassica
napus are described and by Beachy et al., EMBO. J. (1985)
4:3047-3053; Sengupta-~opalan et al., Proc. Natl. Acad.
Sci. USA (1985) 82:3320-3324; Greenwood and Chrispeels,
Plant Physiol. (1985) 79:65-71 and Chen et al., Proc.
Natl. Acad. Sci. USA (1986) 83:8560-8564, in which studies
concerned with seed storage proteins and genetic
manipulation are described and by Eckes et al., Mol. Gen.
Genet. (1986) 205:14 - 22 and Fluhr et al., Science (1986)
35 , 232:1106-1112, in which genetic manipulation of light
,.........
:,
.
200709~
inducible plant genes are described.
There is now provided a new DNA sequence of an expression
cassette in which the regulating regions for the wound-
inducible transcriptional regulation in the stem and in
leaves as well as for the constitutive transcriptional
regulation in the potato tubers are localised, as well as
the transference of these in vectors containing the DNA
sequence in the plant genome, by using Agrobacteria as
transfer microorganisms. The DNA sequence contains the
sequence of the regulatory transcriptional starter region
for the wound induction and the potato tuber specificity.
Downstream from the starter region can be a sequence which
contains the information for the modification of the
phenotype of the cell tissues concerned and the formation,
as well as quantitative distribution, of endogenous
products or the formation of heterogenous expression
products for a new function. Conveniently, the
transcription and termination regions in the direction of
transcription should be provided by a linker or polylinker
which contains one or more restriction positions for the
insertion of this sequence. As a rule, the linker has
1-10, usually 1-8, preferably 2-6 reaction positions. In
general the linker has a size of less than 100 bp, usually
less than 60 bp, but is however at least 5 bp. The
transcriptional starter region can be native or homologous
to the host or foreign or heterologous to the host plants.
Of special interest are the transcriptional starter
regions which are associated with potatoes (Solanum
tuberosum) proteinase-inhibitor II-gene, that during the
total potato tuber development from the formation of the
stolon up to the ripe tuber, is expressed. The expression
of the proteinase-inhibitor II-gene cannot be shown in
other plant parts before injury (for example by biting
insects) which induce the expression of the
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proteinase-inhibitor II-gene in leaves and stems. This
wound-inducing expression is not separated only on the
injured parts of the plants. A systemic induction leads to
an accumulation of proteinase-inhibitor II, both in
wounded and also in intact parts of the wounded plants.
The transcription cassette contains in the 5'-3'
transcription direction, a region representative for the
plants for the transcription and the translation, a
desired sequence and a region for the transcriptional and
translational termination. The termination region used is
a homologue of the participating proteinase-inhibitor
II-gene. If a subfragment of the proteinase-inhibitor
II-gene regulator region is fused to a heterologous
promoter, termination area seems to be exchangeable. The
~NA sequence could contain all possible open reading
frames for a desired peptide as well as also one or more
introns. Examples include sequences for enzymes; sequences
that are complementary (a) to a genome sequence whereby
the genome sequence can be an open reading frame; (b) to
an intron; (c) to a non-coded leading sequence; (d) to
ea~h sequence, which inhibits through complementarity, the
transcription mRNA processing (for example splicing) or
the translation. The desired DNA sequence can be
synthetically produced or extracted naturally, or can
contain a mixture of synthetic or natural DNA content. In
general, a synthetic DNA sequence with codons is produced,
which is preferred by the plants. This preferred codon
from the plants can be specified from the codons with the
highest protein frequency which can be expressed in the
most interesting plant species. In the preparation of the
transcription cassettes, the different DNA fragments can
be manipulated in order to contain a DNA sequence, which
leads generally in the correct direction and which is
equipped with the correct reading frame. For the
connections of the DNA fragments to each other, adaptors
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or linkers can be introduced on the fragment ends. Further
manipulations can be introduced which provide the suitable
restriction positions or separate the excess DNA or
restrictio~ positions. Where insertions, deletions or
substitutions, such as for example transitions and
transversions, are concerned, in vitro mutaganese, primer
repair, restriction or ligation can be used.
In suitable manipulations, such as for example
restriction, "chewing-back" or filling up of overhangs for
"blunt-ends", complementary ends of the fragments for the
fusing and ligation could be used. For carrying out the
various steps which serve to ensure the expected success
of the intervention, a cloning is necessary for th~
increase of the DNA amounts and for the DNA analysis.
A large amount of cloning vectors are available which
contain a replication system in E. coli and a marker which
allows a selection of the transformed cells. The vectors
contain for example pBR 332, pUC series, M13 mp series,
pACYC 184 etc. In such a way, the sequence can be
introduced into a suitable restriction position in the
vector. The contained plasmid is used for the
transformation in E. coli. The E. coli cells are
cultivated in a suitable nutrient medium and then
harvested and lysed. The plasmid is then recovered. As a
method of analysis there is generally used a sequence
analysis, a reætriction analysis, electrophor~sis and
further biochemical-molecular biological methods. After
each manipulation, the used DNA sequence can be restricted
and connected with the next DNA sequence. Each plasmid
sequence can be cloned in the same or different plasm~d.
After each introduction method of the desired gene in the
plants further DNA sequences may be necessary. If for
example for the transformation, the Ti- or Ri-plasmid of
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the plant cells is used, at least the right boundary andoften however the right and the left boundary of the Ti-
and Ri-plasmid T-DNA, as flanking areas of the introduced
gene, can be connected. The use of T-DNA for the
transformation of plant cells is being intensi~ely studied
and is well described in EP 120 516; Hoekema, in: The
Binary Plant Vector System Offset-drukkerij Kanters B.8.,
Alblasserdam, 1985, Chapter V; Fraley, at al., Crit. Rev.
Plant Sci., ~:1-46 und An et al., EMBO J. (1985) 4:277-
284.
When the introduced DNA is first integrated once in thegenome, it is then also relatively stable and as a rule no
more comes out. It normally contains a selection marker
which passes on to the transformed plant cells, resistance
against a biocide or an antibiotic such as kanamycin,
G 418, bleomycin, hygromycin or chloramphenicol, amongst
others. The particular marker employed should be one which
will allow for selection of transformed cells compared to
cells lacking the DNA which has been introduced.
A variety of techniques are available for introduction of
DNA into a plant host cell. These techniques include
transformation with T-DNA using Aqrobacterium tumefaciens
or A~robacterium rhizogenes as transformation agent, the
fusion, the injection or the electroporation as well as
further possibilities. If Agrobacteria are used for the
transformation, the introduced DNA must be cloned in
special plasmid and either in an intermediary vector or a
binary vector. The intermediary vectors which are based on
sequences which are homologous with sequences in the T-DNA
can be integrated through homologous re-combination in the
Ti- or Ri- plasmid. These contain also the necessary -
Vir-region for the transfer of the T-DNA. Intermediary
vectors cannot be replicated in Agrobacteria. By means of
Z 0 07 09
helper-plasmid, the intermediary vector of Agrobacterium
tumefaciens can be transferred (conjugation). Binary
vectors can be replicated in E. coli as well as in
Agrobacteria. They contain a selection marker gene and a
linker or polylinker, which are framed from the right and
left T-DNA border regions. They can be transformed
directly in the agrobacteria (Holsters et al., Mol. Gen.
Genet.(1978) 163: 181-187). The Agrobacterium serving as
host cells should contain a plasmid that carries the
Vir-region, which is necessary for the transfer of the
T-DNA in the plant cells whereby additional T-DNA can be
contained. The bacterium so transformed is used for the
transformation of plant cells. For the transfer of DNA in
the plant cells, plant explanates can be cultivated in
suitable manner with Agrobacterium tumefaciens or
Agrobacterium rhizogenes. From the infected plant material
(for example leaf bits, stem segments, roots as well as
protoplasts or suspensions of cultivated cells), whole
plants can then be regenerated in a suitable medium which
can contain antibiotics or biocides for the selection,
wh~ch then can be tested for the presence of introduced
DNA. In the injection and e~ectroporation, no special
requirements on the plasmid are needed and a simple
plasmid, for example pUC derivative can be used. For the
introduction of foreign genes into the plants a variety of
possibilities can be used. Especially interesting,
however, is the expression of genes which introduce
pesticidal resistance, which after wounding of the plant,
give to the plant a reduced food quality of the plant to
the pests or a toxicity to the pest. Therefore excess
insect eating is avoided which leads to far higher yields
of crop. The corresponding genes can be toxin-genes that
code, for example for B. thurinqiensis ~-endotoxin, or can
be genes which code for insect hormones which lead to a
change in growth of insect larvae (for axample ecdysone).
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Z 0 0 7 09
Alternatively, genes for the most different of starting
materials can be used, including mammalian products, such
as for example blood factors; lymphokines; colony
stimulation factors; interferons; plasminogen activators;
enzymes, such as for example superoxide-dismutase or
chymosin; hormones; thioesterase-2 from rats milk;
phospholipid-acyl-desaturase which takes part in the
synthesis of cicosapentaenoic acid; or human serum
albumin. A further possibility is increasing the amounts
of tuber proteins, especially mutated tuber proteins,
which show an optimised amino acid composition (essential
amino acids) and in this way the nutritive value of the
tubers can be increased. Should the amounts of specified
endogenous products b~ reduced, the expression of the gene
or parts of this gene in the wrong orientation to the
promoter is also conceivable, which leads to synthesis of
an RNA, which is complementary to a total or to parts of
an endogenous gene and thus the transcription of this gene
or the processing and/or translation of the endogenous
mRNA can be inhibited.
The transformed cells grow within the plants in the usual
way tsee also McCormick et al., Plant Cell Reports t1986)
5, 81-84). These plants can be grown normally and crossed
with plants, that possess the same transformed gene or
Z5 other genes. The resulting hybridised individuals have the
corresponding phenotypic properties. Two or more
generations should be grown, in order to secure that the
phenotypic state remains stable and will be passed on,
especially if seeds are to be harvested, in order to
ensure that the corresponding phenotype or other
individual characteristics are included. As host plants
for the wound inducible expression, any plant type can be
used that is of economic interest.
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For tuber specific expression Solanum tuberosum is
suitable. The identification of necessary transcriptional
starter regions can be achieved in a number of ways. As a
rule the mRNAs can be used, which are isolated from
specified parts of the plants ~tubers) or during certain
conditions of the plants (non-wounded/wounded). For the
additional increase in concentration of the mRNA specific
to the cells or associated with plant conditions, cDNA can
be prepared whereby non-specific cDNA from the mRNA or the
cDNA from other tissues or plant conditions (for example
wounded/non-wounded) can be drawn off. The remaining cDNA
can then be used for probing the genome for complementary
sequences using a suitable plant DNA library. Where the
protein is to be isolated, it can be partially sequenced
so that a probe for direct identification of the
corresponding sequences in a plant ~NA library can be
produced. The sequences that are hybridised with the probe
can then be isolated and manipulated. Further, the non-
translated 5'-region, that is associated with the coded
area, can be isolated and used in expression cassettes for
the identification of the transcriptional activity of the
non-translated 5'-regions. The expression cassettes
obtained which use the non-translated 5'-regions can be
transformed in plants (see above), in order to prove their
functionability with a heterologous structure gene ~other
than the open reading frame of the wild types which are
associated with the non-translated 5- region) as well as
the tuber and wound spacificity. In this way, specific
sequences can be identified which are necessary for the
tuber and wound specific transcription. Expression
cassettes that are of special interest contain
transcriptional initiation positions of the
protein-inhibitor II-gene.
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identification of necessary transcriptional starter
regions can be achieved in a number of ways. As a rule the
mRNAs can be used, which are isolated from specified parts
of the plants (tubers) or duri~g certain condi~ions of the
plants (non-wounded/wounded).~For the additional increase
in concentration of the mRNA specific to the cells or
associated with plant conditions, cDNA can be prepared
whereby non-specific cDNA from the mRNA or the cDNA from
other tissues or plant conditions (for example
wounded/non-wounded) can be drawn off. The remaining cDNA
can then be used for probing the genome for complementary
sequences using a suitable plant DNA library. Where the
protein is to be isolated, it can be partially sequenced
so that a probe for direct identification of the
corresponding sequences in a plant DNA library can be
produced. The sequences that are hybridised with the probe
can then be isolated and manipulated. Further, the non-
translated 5'-region, that is associated wi~h the coded
area, can be isolated and used in expression cassettes for
the identification of the transcriptional activity of the
non-translated 5'-regions. The expression cassettes
obtained which use the non-translated 5'-regions can be
transformed in plants (see above), in order to prove their
functionability with a heterologous structure gene (other
than the open reading frame of the wild types which are
associated with the non-translated 5- region) as well as
the tuber and wound specificity. In this way, specific
sequences can be identified which are necessary for the
tuber and wound specific transcription. Expression
cassettes that are of special interest contain
transcriptional initiation positions of the
protein-inhibitor II-gene.
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lo
Expressions & Abbreviations
Abbreviations:
bp = Base pairs
cDNA = A copy of a mRNA produced by reverse
S transcriptase.
mRNA = Messenger ribonucleic acid.
T-DNA = Transfer-DNA (localised on the Ti-plasmid from
Agrobacterium tumefaciens)
Terms:0 Blunt ends = DNA ends in which both DNA strands are
exactly the same length.
Chewing-back = Enzymatic removal of nucleotides of a
DNA strand which is longer than the
complementary strand of a DNA
molecule.
Electrophoresis = A biochemical process of separation
for separating nucleic acids fro~
proteins according to size and charge.
En~onuclease = An enzyme that splits independently of
the chain length.
Exonuclease = An enzyme that can attack only at the
end of a polynucleotide chain.
Expression = Activity of a gene.
Gene = Genetic factor; a unit of inheritance,
carrier of part information for a
particular specified characteristic.
Genes consist of nucleic acids (eg
DNA, RNA).
Genome = Totality of the gene localised in the
chromosomes of the cell.
20070~3~
Genome-sequence = The DNA sequence of the genome whereby
three nucleotide bases lying within it
form a codon which code again for a
specific amino acid.
RNA splicing = A gene does not always show up as a
colinear unity but can contain non-
coded sequences ~introns) which must
be spliced from the mRNA (splicing).
Heterologous gene(s) or ~NA = Foreign genes or foreign
DNA.
Homologous gene(s) or DNA = Gene or DNA derived from the
same species.
Klenow enzyme = Fragments of DNA polymerase I of a
size 76,000 D ob~ained by splitting
with a subtilisin. Possess 5' - 3'
polymerase and 3' - 5' exonuclease
activity but not the 5' - 3'
exonuclease activity of the
holoenzyme.
20 Clone = Cell population that is derived from
one of its own mother cells.
Descendants are genotypically the
same. By cloning, the homogeneity of
cell lines can be increased further.
25 Ligation = Enzymatic formation of a
phosphodiester bond between
5'-phosphate groups and 3'-hydroxy
groups of the DNA.
Linker, Polylinker = Synthetic DNA sequence that contains
one or more (polylinker) restriction
cutting regions in direct sequence.
Northern blots, = Transfer and fixing of
Southern blots, electrophoretically separate RNA or DNA
on a nitrocellulose or nylon membrane.
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Phenotype = A sum of characteristics which
expressed in an organism as opposed to
its genotype.
Plasmid = Additional extrachromosomal DNA gene
carrier in bacteria cells (possibly
also in eukaryons) which reduplicate
themselves independently of the
bacterial chromosomes. The plasmid can
be integrated in other DNA hosts.
10 Primer = Starting piece; polynucleotide strand
on which further nucleotides can be
attached.
Promoter = Control sequence of the DNA expression
which realises the transcription of
homologous or heterologous DNA gene
sequences.
Regulator protein = Proteins that stand with DNA in
exchange activity and steer the gene
expression.
Re~lication = Doubling of the DNA sequence.
Restriction enzymes = Restriction endonucleases that
are in sub-units of the endo
DNA's (for example EcoRI
(specificity G AATTC and
EcoRII CC (AT) GG, from E.coli)
show themselves through a high
specificity of the substrate
knowledge ( = splitting
position3.
Restriction positions = A splitting position which is
produced specifically by
restriction enzymes.
Termination = A last stage of the protein and/or the
RNA synthesis.
., .
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Transformation = Introduction of exogenous DNA of a
bacterial species which is in a
receiver cell.
Transcription = Overwriting on an RNA the genetic
information contained in the DNA.
Translation = Translation of the genetic information
which is memorised in the form of a
linear sequence of bases in nucleic
acids. The product of the translation
is a polypeptide that comprises a
sequence of amino acids.
Transition = Base pair exchange: purine-pyrimidine
to purine-pyrimidine e.g. A-T
exchanging G-C.5 Transversion = Base pair exchange: purine-pyrimidine
to pyrimidine-purine e.g. A-T
replacing T-A.
Deletion = Removal of one or more base pairs;
Insertion = Introduction of one or more base
pairs;
Transition, Transversion, Deletion and
Insertion are point mutations.
Transposo~ = A unity comprising resistance gene (S) and
two IS elements (IS = integrated segments).
IS elements or IS sequences are DNA
sequences that can influence the expression
of adjacent genes. Deletions can be induced
and possess the feature of being able to
extend themselves in the bacteria genome in
various positions singly and also
repeatedly.
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14
Vectors = Host specific replicatable structures, that
take up genes and carry these into other
cells. Plasmid can also be used as vertors.
On 16.12.1988 the following microorganism was deposited at
the German Collection for Microorganisms (DSM) in
Braunschweig, Germany (deposit number):
Agrobacterium tumefaciens A. tum. M 14, containing the
vector pM 14 (DSM 5088)
Description of the Fiqures
10 Figure 1 shows the vector pM 14 on which the 3.4 kb long
EcoRI/HindIII DNA sequence is localised. This DNA sequence
contains the 1.3 kb long ScaI/HindIII fragment of the
proteinase-inhibitor II-promoter, the 1.8 kb long
BamHI/Sst~ fragment of the ~-glucuronidase and the a . 26 kb
long SphI/SphI fragment of the proteinase-inhibitor II.
There are further, records of the cutting positions as
we~1 as positions of the transcription starts.
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For a better understanding of this invention the following
examples are given. An explanation for these experiments
is given as follows:
1. Cloning Vectors
For cloning, the vectors pUC18/19 (Yanisch-Perron et
al Gene (1985), 33, 103-119) were used.
For plant transformations, the gene structures were
cloned either in the intermediate vector pMPK110
(Eckes, Doktorarbeit (1985) - Standort der Arbeit -
Universitatsbibliothek Koln) or the binary vector
BIN19 (Bevan, Nucl Acids Research (1984), 12, 8711-
8720).
2. Bacterial Species
For the pUC-and M13 vectors the E. coli species
BMH71-18 (Nessing et al, Proc. Nat. Acad. Sci. USA
(1977), 24, 6342-6346) or TB1 was used. For the
vectors pMPK110 and BIN19, the species TBl was
exclusively used. TB1 is a recombinant, negative,
tetracyclines resistant derivative of the species
JM101 (Yanisch-Perron et al., Gene (1985), 33, 103-
119). The genotype of the TB1 species is (Bart
Barrel, personal communication): F'(traD36, proAB,
lacl, lacZ~M15), ~(lac, pro), SupE, thiS, recA,
Srl::TnlO( TCR ) .
As helper species for the conjugative transfer of the
pMPK plasmid from the TB1 cells in Agrobacterium
tumefaciens, the E. coli species GJ23 (Van Haute et
al., EMBO J. (1983), 2, 411-417) can be used.
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The plant transformation was carried out with the
help of the Aqrobacterium tumefaciens species
GV3850kan (Jones et al., EMBO J. (1985) 4, 2411-2418,
pMPK plasmid or GV2260 (Deblaere et al Nucl. Acids
Res. (1985), 13, 4777-4788; Binl9-derivative).
Medium
YT-Medium: 0.5% Yeast extract, 0.5% NaCl;. 0.8%
bacto-trypton, if necessary in 1.5%
agar.
YEB-Medium: 0.5% beef extract, 0.1% yeast extract,
0.5~ peptone, 0.5% saccharose, 2 mM
MgS04, if necessary in 1.5% agar.
MS-Medium: According to Murashige and Skoog
(Physiologia Plantarum (1962), 15,
473-497).
3. Conjugation and/or Transformation of Aqrobacterium
tumefaciens
For the transfer of the recombinant pMPKllO plasmid
there were mixed together 20 yl each of an overnight
culture, washed under selection, of the pMPXllO
containing TBl cells (5U ug/ml spectinomycin and 25
~g/ml streptomycin in YT-medium), of the helper
species GJ23 (10 ~g/ml tetracyclines and 25 ~g/ml
kanamycin in YT-medium) and of the Agrobacterium
tumefaciens species GV3850Kan (50 ~g/ml erythromycin
and 50 ~g/ml chloramphenicol in YEB-medium~ and
incubated overnight on a YEB agar plate, without
selection, at 28aC. The resulting bacterial growth
was plated out on YEB plates that contained 50 ~g/ml
chloramphenicol, 50 ~g/ml erythromycin, 100 ~g/ml
spectinomycin and 300 ~g/ml streptomycin. The
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20070!9~
colonies were washed after three stage incubation at
28 C and combined by the same selection on YEB
plates. From the resulting single colony the total
DNA was isolated, and this was tested after suitable
restriction cleavage with help of southern blots for
the integration of the recombinant DNA.
For Binl9 derivatives, the introduction of the DNA in
Agrobacteria was carried out by direct transformation
by the method of Holsters et al. (Mol. Gen. Genet.
(1978), 163, 181-187). The plasmid DNA transformed
Agrobacteria were isolated by the method of Birnboim
and Doly) Nucl. Acids Res. (1979), 7, 1513-1523) and
opened up gel electrophore~ically by a suitable
restriction cleavage.
4. Plant Transformation
A) Tobacco: 10 ml of an overnight culture of
Agrobacterium tumefaciens, washed under
selection was centrifuged, the supernatant
discarded and the bacteria resuspended in the
same volume of anabiotic-free medium. In a
sterile petri dish, leaf discs of sterile
plants, (ca 1 cm2), from which the middle vein
had been removed, were bathed in this bacterial
suspension. The leaf discs were then compactly
laid down in petri dishes which contained
MS-medium with 2% saccharose and 0.8% bacto-
agar. After two d~ys incubation at 25 C in the
dark, they were transferred to MS-medium which
contained 100 mg/l kanamycin, 500 mg/l claforan,
1 mg~l benzylaminopurine (BAP), 0.2 mgJ1
naphthylacetic acid (NAA) and 0.8% bacto-agar.
Growing shoots were put into hormone-free
,,
Z00709~
18
MS-medium with 250 mg/l claforan and tested for
nopaline content (Otten et al. Biochimica et
Biophysica Acta (1978), 527, 497-500). Positive
shoots were put into soil after root growth.
B) Potatoes: 10 small leaves of a sterile potato
culture, wounded with a scalpel, were put into
10 ml MS-medium with 2% saccharose which
contained 30 to 50 ul of an overnight culture of
Agrobacterium tumefaciens, washed under
selection. After 3-5 minutes gentle shaking, the
petri dishes were incubated at 25C in the dark.
After two days, the leaves were laid in MS-
medium with 1.6% glucose, 2 mg/l zeatinribose,
0.02 mg/l naphthylacetic acid, 0.02 mg/l
gibberellic acid, 500 mg/l claforan, 50 mg/l
kanamycin and 0.8% bacto-agar. After one week
incubation at 25C and 3000 lux the claforan
concentration in the medium was reduced by half.
5. AnalYsis of the Genomic DNA from Transgenic Plants
The isolation of genomic plant DNA was carried out by
the method of Rogers and Bendich (Plant Mol. Biol
(1985), 5, 69-76).
For DNA analysis 10-20 ~g DNA was tested after
suitable restriction cleavage with the aid of
southern blots by integration of the DNA sequences
being analysed.
' ~
:
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19
6. AnalYsis of the Total RNA from Transqenic Plants
The isolation of the total plant RNA was carried out
by the method of Longemann et al (Analytical Biochem
(1987), 163, 16-2-).
For the analysis, 50 ,ug samples of total RNA were
tested with the use of northern blots to determine
the presence of the sought transcripts.
7. CAT Test
The activity of the chloramphenicol-acetyltransferase
(CAT) in transgenic plants was carried out by the
method of Colot et al (EMBO J (1987) 6, 3559-3564).
The Bradford protein determination was carried out
however before the heat treatment (75C, 10 minutes)
of the extract. For the determination of the CAT
activity, an amount of heat treated extracts was used
that corresponded to a protein content of 500 ~g of
the measured extracts.
8. GUS Test
The activity of the ~-glucuronidase (GUS) in
transgenic plants was determined by the method of
Jefferson (Plant Mol. Biol. Rep. (1987), 5, 387-405).
The protein determination was carried out by the
CAT-Test according to Bradford (Anal. Biochem (1976),
72, 248-254). For the determination of the GUS
activity, 50 ,ug protein was used, in which the
incubation was carried out at 37~C for 30 minutes.
' ~
~00709~
The following examples illustrate the isolation
identification as well as the function and use of the
wound induced and tuber specific proteinase inhibitor to
promoters from potato tubers.
Example 1
Cloning and structural analysis of a proteinase inhibitor
II-gene from Solanum tuberosum.
cDNA clones, that have been coded for the
proteinase-inhibitor II of potato were isolated and
sequenced from the potato variety Berolina (Sanchez-
Serrano et al., Mol. Gen. Genet (1986), 203 15-20). These
cDNA clones were used to isolate a homologous genomic
proteinase-inhibitor II-clone from the monohaploid potato
line AM 80/5793 (Max-Planck Institut fur
Zuchtungsforschung, Koln); by restriction and sequence
analysis the exact structure of the gene was determined.
Further, the transcription start could be established by
es~ablished by RNase-digestion of an SP6-antisense-
RNA/mRNA hybrid (Keil et al., Nucl. Acids Res (1986), 14,
5641-5650). -.
Example 2
Identification of the regulatory regions responsible for
wound inducibility of the proteinase-inhibitor II-gene.
It could be shown th t the isolated proteinase-inhibitor
II-gene (see Example I) in transgenic Wisconsin 38 tobacco
plants which themselves contain no homologous sequences
for the proteinase inhibitor II-gene, were induced by
wounding leaves and stems (Sanchez-Serrano et al., EMBO J
(1987), 6, 303-306). The DNA fragment which was thus
,*~ .
~ ` ~
200709~
introduced into the plants, reached from a EcoRI
restriction cutting position, that was located ca. 3 kb 5'
before the transcription start of the proteinase inhibitor
II-gene, to an EcoRI cutting position, that was located
ca. l.S kb 3' behind the polyadenylation position.
In another experiment, the proteinase-inhibitor II-gene
was introduced after deletion of the single intron by
exchange of the intron-containing genomic sequence through
the corresponding cDNA sequence in tobacco (Wisconsin 38).
In this case a HindII cutting position 1.5 kb 5' in front
of the transcription start was used. The EcoRI cutting
position 1.5 kb 3' behind the gene was retained. Analysis
of the mRNA that resulted from this construction of the
transgenic tobacco plants resulted in a wound inducibility
of the intronless proteinase-inhibitor II-gene which was
comparable with ~he corresponding intron containing gene.
It thus appears that the intron of the proteinase
inhibitor II-gene does not contain any necessary
re~ulatory elements for the wound inducibility..
Example 3
Promoter deletion constructions.
From the results of Example 2, chimeric constructions of
the deletions, as well as the restriction fragments of the
proteinase inhibitor II-gene with the bacterial
chloramphenicol-acetyltransferase-gene (CAT) resulting
from the exonuclease III digestion and of the 3' region of
the proteinase inhibitor II-gene, were produced.
The deletion fragments resulted from the sequence analysis
of the proteinase-inhibitor II-gene. They were cloned with
the help of an EcoRI cutting position that was located 5'
- . .
-:
zoo~o~
before the corresponding promoter deletion in the
polylinker of the phage vector M13mpl9, and a ScaI cutting
position 32 bp 3' of the transcription start and 18 bp 5'
of the ATG start codon of the proteinase-inhibitor II-gene
S in the EcoRI/SmaI splitting intermediary vector pMPK110.
The promoter fragment resulting from this had a length of
700, 514, 210 and 150 bp 5' of the transcription start of
the proteinase-inhibitor II-gene.
On the one hand, a fragment served as a restriction
fragment that spread from a HindIII cutting position
1.3 kb 5' in front of the transcription start up to the
above mentioned ScaI cutting position (pM 11) but also a
441 bp long SspI/ScaI fragment of the promoter region that
respectively were cloned in the Smai cutting position of
pMPK 110. The HindIII cutting position of the first
fragment first had to be filled with T4-DNA polymerase.
The CAT gene was cloned as 800 bp long BamHi fragments
tVelten et al., Nucl Acids Res (1985). 13, 6981-699~), .. -
re~pectively 3' behind the promoter fragment of the
proteinase-inhibitor II-gene in the BamHI cutting position
of the polylinker of pMPKllO.
The 260 bp long RsaI/SspI fragment of the
proteinase-inhibitor II-gene that was supplied with SphI
link, was cloned in the corresponding restriction cutting
position of the polylinker of pMPKllO, as polyadenylation
signal. The RsaI cutting position was located 11 bp in
front of the TGA stop codon and the SspI cutting position
was located 74 bp 3' behind the polyadenylation position
of the proteinase inhibitor II-gene.
The construction was introduced using the Aqrobacterium
tumefaciens transformation system in the tobacco variety
..
20070~
Wisconsin 38. From the resulting transgenic tobacco plants
were unwounded and wounded leaves were tested in the
presence of CAT-mRNA and/or for the activity of the CAT
enzymes. The results showed that for maximal wound
inducibility, the area of 700 - 1300 bp 5' before the
transcription start is necessary (pM 11). After deletion
of this region only a very small wound inducibility was
observed, if at all. Interestingly no CAT activity was
observed with a construction that contained the total
promoter of the proteinase inhibitor I~-gene up to 1500 bp
5' before the transcription start, but not the 3'-end of
the proteinase-inhibitor II-gene. In this construction, 3'
behind the promoter a 1000 bp long SalI eye fragment was
cloned in the corresponding cutting position of the
pMPK110 polylinXer which contained, 3' behind the CAT
gene, the polyadenylation signal of the gene 7 of the
T-DNA of Aqrobacterium tumefaciens (Velten et al., Nucl
Acids Res (1985), 13, 6981-6998). This polyadenylation
signal is functional in plant cells.
To summarise: the regulatory region of the proteinase
inhibitor II-gene functions in a specific manner also in
combination with a heterologous bacterial gene, in this
case the chloramphenicol acetyltransferase. For maximal
inducibility of the chimeric gene an enhancer is necessary
that is located in the region of 700 - 1300 bp 5' in front
of the transcription start and also ~ 260 bp long fragment
of the 3'-region of the proteinase-inhibitor II-gene which
stretches up to 74 bp 3' behind the polyadenylation
position.
. :-
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24
Example 4
Fusion of a proteinase inhibitor II promoter fragment with
a heterologous promoter.
Should the proteinase inhibitor II promoter actually
contain an enhancer, this must be able to activate an
inactive promoter that contains only the TATA and CAAT
steering element.
For this, first, a CAT construction with an inactive
promoter was prepared. The 35 S-promoter of the
cauliflower mosaic virus constitutively and very strongly
expressed in plant cells was cloned as 550 bp long
EcoRI/RpnI fragments in the corresponding restriction
cutting positions in front of the 1000 bp long
SalI-fragment of the CAT gene with the gene
7-polyadenylation signal in pUC18 (pDHCAT1). The KpnI
cutting position was located on position +10 in relation
to the transcription start of the 35 S-promoter. The EcoRV
cu~ting position on position -90 in relation to the
transcription starts of the 35 S-promoter and the HincII
cutting position 3' behind the gene 7-polyadenylation
signal was usecl, in order to clone the resulting
(-90)35S/CAT/g7pA-fragment in the HincII cutting position
of pMPK110 (pMP35SCAT1). For this a partial digestion of
pDHCAT1 with HincII was necessary since a second HincII
cutting position was present in front of the CAT gene that
must remain uncleaved. In order to be able to use the SmaI
cutting position located 5' in front of the (-90)35 S-
promoter, for the insertion of DNA fragments, the SmaI
cutting position was eliminated immediately 5' in front of
the CAT-gene. For this pMP(-90)35SCAT1 was cleaved with
SalI, the cutting position was filled up with Rlenow
enzyme and then cleaved with PstI. The ligation with
. .
200709~
SmaI/PstI cleaved pDHCAT1 gave pMP (-90)35SCAT10, in which
the SalI and the SmaI cutting positions, which are located
between the (-90)35 S-promoter and the CAT gene, were
fused and could thus not be used again. This construction
was introduced in Wisconsin 38 tobacco plants whereby an
activity of the (-90)35 S-promoter could not be
demonstrated either in unwounded nor in wounded leaves of
these transgenic plants.
In order to be able to answer the question whether the
proteinase-inhibitor II-promoter can activate this
inactive (-90)35 S-promoter, a deletion fragment 5', which
was cloned in M13mpl9, was cloned in front of this
promoter with the resulting sequence analysis of the
proteinase-inhibitor II-gene. The deletion fragment which
stretched from position -195 to -1300 in relation to the
transcription start of the proteinase inhibitor II-gene,
could be split off by a Eco~I/HindIII cleavage from the
M13mpl-vector. After filling the cutting positions with
T4-DNA polymerise this fragment was inserted in the SmaI
cu~Eting position of pMP(-90)35SCAT10. The resulting
vectors pM21 and pM22 contained the promoter fragment of
the proteinase-inhibitor II-gene in both orientations.
The analysis of the transgenic tobacco plants containing
this construction gave both in northern blot and also in
the CAT test a definite wound inducibility of the CAT
genes in leaves.
These results show that the proteinase-inhibitor
II-promoter actually possesses an enhancer, which can
specifically activate in both orientations an inactive
promoter. Further, the region from +32 to -195 of the
proteinase inhibitor II-gene is clearly not necessary for
the wound inducibility. Interestingly, in the combination
-
:
200709~
26
of the (-1300/-195)-promoter fragment of the
proteinase-inhibitor II-gene with the (-90)35 S-promoter,
the 3' region of the proteinase-inhibitor II-gene is not
necessary in order to have wound inducibility of the CAT
gene, as is typical for the total proteinase-inhibitor
II-promoter from position 1300 to +32 in relation to the
transcription start.
ExamPle 5
Fusions of the regulatory regions of the proteinase
inhibitor II-gene with another bacterial gene.
In order to show that the regulatory region of the
proteinase inhibitor II-gene functions also in combination
with another gene, there was constructed not only a fusion
with the gene for the bacterial chloramphenicol-
acetyltransferase but also a fusion with a gene which iscoded for the bacterial ~-glucuronidase (GUS).
The GUS gene was first cut from the vector pBI101
(Jefferson et al., EMBO J (1987), 6, 3901-3907), together
with the polyaclenylation signal of the nopaline synthase
gene (Nos) of Aqrobacterium tumefaciens, by an EcoRI/SmaI
cleavage and after Klenow filling, the restriction cutting
positions in the HincII cutting position of pUC18, was
cloned ~pGUS; Meike Koster, personal communication).
Through a BamHI/PstI cleavage, the CAT gene was cleaved
from pM11. Instead of this, a 1800 bp long BamHI/SstI
fragment from the plasmid pGUS, which contains a gene for
the ~-glucuronidase without the Nos-3' end, was ligated.
For this, the PstI and the SstI cutting positions had to
previously be filled with T4-DNA polymerase. The resulting
vector pM14 (see Figure 1) now contained the GUS gene 3'
,.. ,.............. - ~
Z O 0 7 09
behind the proteinase-inhibitor II-promoter and 5' in
front of the 3' end of the proteinase inhibitor II-gene
(see Figure 1). Since the potato transformation with the
binary vector system functions efficiently as with
intermediary vectors, this chimeric gene was cloned round
as 3.4 kb long Eco~I/HindIII fragments in the
corresponding cutting positions of the binary vector BINlg
(pS9). These constructions were introduced both in tobacco
Wisconsin 38 (pM14) and also in the potato variety
Berolina (pS9).
The analysis of the resulting transgenic tobacco and
potato plants with northern blots showed a definite
activity of the GUS gene in wounded leaves, but in non-
wounded leaves none or only a small activity of the GUS
gene could be shown. The analysis of the mRNA and the GUS
activity in potato tubers showed interestingly around a 10
times greater activity of the gene in comparison to that
in wounded leaves. Therefore the proteinase-inhibitor
II-promoter not only contained the steering element for
the wound induction but also that for expression in potato
tubers.
Example 6
Expression of genes which are a code for toxic proteins
under control of regulatory regions of the protein
inhibitor II-gene.
A use for regulatory regions of the proteinase-inhibitor
II-gene is the wound specific expression of toxic proteins
in leaves for combating plant pests such as insects or
certain microorganisms. Thus a gene which codes for the
toxic thionine was kept under the control of the
proteinase-inhibitor II-promoter. Thionine was normally
. .
200709~
28
expressed in the endosperm of various cereals but also in
the leaves of barley seedlings (Bohlmann und Apel, MGG
(1987), 207, 446-454). Under control of the
proteinase-inhibitor II-promoter on the other hand in
transgenic tobacco plants, a wound specific accumulation
of thionine mRNA in leaves could be demonstrated.
.. .,., ~, ..
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