Note: Descriptions are shown in the official language in which they were submitted.
WO 91/16432 PCT/1;P91/00733
2080584
MODIFIED BACILLUS THURINGIENSIS
INSECTICIDAL-CRYSTAL PROTEIN GENES AND THEIR
EXPRESSION IN PLANT CELLS
This invention provides a modified Bacillus
thurinctiensis ("Bt") gene (the "modified BtICP gene")
encoding all or an insecticidally-effective portion of
a Bt insecticidal crystal protein ("ICP"). A plant,
transformed with the modified Bt ICP gene can show
higher expression levels of the encoded ICP and
improved insect-resistance.
Background of the Invention
Plant genetic engineering technology has made
significant progress during the last 10 years. It has
become possible to introduce stably foreign genes into
plants. This has provided exciting opportunities for
modern agriculture. Derivatives of the Ti-plasmid of
the plant pathogen, AQrobacterium tumefaciens, have
Proven to be efficient and highly versatile vehicles
for the introduction of foreign genes into plants and
plant cells. In addition, a variety of free DNA
delivery methods, such as electroporation,
microinjection, pollen-mediated gene transfer and
Particle gun technology, have been developed for the
same purpose.
The major aim of plant transformations by genetic
engineering has been crop improvement. In an initial
phase, research has been focused on the engineering
into plants of useful traits such as insect-resistance.
In this respect, progress in engineering insect
resistance in transgenic plants has been obtained
through the use of genes, encoding ICPs, from Bt
strains (Vaeck et al., 1987). A Bt strain is a spore
forming gram-positive bacterium that produces a
WO 91/16432 z o s a 5 ~ ~ PCT/EP91/00733
2
parasporal crystal which is composed of crystal
proteins which are specifically toxic against insect
larvae. Bt ICPs possess a specific insecticidal
spectrum and display no toxicity towards other animals
and humans (Gasser and Fraley, 1989). Therefore, the Bt
ICP genes are highly suited for plant engineering
purposes.
For more than 20 years, Bt crystal spore
preparations have been used as biological insecticides.
The commercial use of Bt sprays has however been
limited by high production costs and the instability of
crystal proteins when exposed in the field (Vaeck et
al., 1987). The heterogeneity of Bt strains has been
well documented. Strains active against Lepidoptera
(Dulmage et al., 1981), Diptera (Goldberg and Margalit,
1977) and Coleoptera (Krieg et al., 1983) have been
described.
Bt strains produce endogenous crystals upon
sporulation. Upon ingestion by insect larvae, the
crystals are solubilized in the alkaline environment of
the insect midgut giving rise to a protoxin which is
subsequently proteolytically converted into a toxic
core fragment or toxin of 60-70 kDa. The toxin causes
cytolysis of the epithelial midgut cells. The
specificity of Bt ICPs can be determined by their
interaction with high-affinity binding sites present on
insects' midgut epithelia.
The identification of Bt ICPs and the cloning and
sequencing of Bt ICP genes has been reviewed by Hofte
and Whiteley (1989). The Bt ICP genes share a number of
common properties. They generally encode insecticidal
proteins of 130 kDa to 140 kDa or of about 70 kDa,
which contain toxic fragments of 60 ~ 10 kDa (Hofte and
Whiteley, 1989). The Bt ICP genes have been classified
into four major groups according to both their
r t
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W091/16432 PCT/EP91/00733
3
structural similarities and insecticidal spectra (Hofte
and Whiteley, 1989): Lepidoptera-specific (CryI),
Lepidoptera- and Diptera-specific (CryII), Coleoptera-
specific (CryIII) and Diptera-specific (Cry IV) genes.
The Zepidoptera-specific genes (CryI) all encode 130-140
kDa proteins. These proteins are generally synthesized
as protoxins. The toxic domain is localized in the N-
terminal half of the protoxin. Deletion analysis of
several CryI genes confirm that 3' portions of the
protoxins are not absolutely required for toxic activity
(Schnepf et al., 1989). Cry II genes encode 65 kDa
proteins (Widner and Whiteley, 1985). The Cry II A
proteins are toxic against both Lepidoptera and Diptera
while the Cry II B proteins are toxic only to
Zepidopteran insects. The Coleoptera-specific genes (Cry
III) generally encode proteins with a molecular weight of
about 70 kDa. (Hofte and Whiteley, 1989). The
corresponding gene (cry III A) expressed in E. coli
directs the synthesis of a 72 kDa protein which is toxic
for the Colorado potato beetle. This 72 kDa protein is
processed to a 66 kDa protein by spore-associated
bacterial proteases which remove the first 57 N-terminal
amino acids (Mc Pherson et al., 1988). Deletion analysis
demonstrated that this type of gene cannot be truncated
at its 3'-end without the loss of toxic activity (Hofte
and Whiteley, 1989). Recently, an anti-coleopteran
strain, which produces a 130 kDa, protein has also been
described (European patent publication 0,382,990). The
cry IV class of crystal protein genes is composed of a
heterogeneous group of Diptera-specific crystal protein
genes (Hofte and Whiteley,1989).
The feasibility of generating insect-resistant
transgenic crops by using Bt ICPs has been
demonstrated. Vaeck et al., 1987; Fischof et al.,
WO 91/16432 PCT/EP91/0073z
X080584
1987 and Barton et al., 1987). Transgenic plants offer
an attractive alternative and provide an entirely new
approach to insect control in agriculture which is at
the same time safe, environmentally attractive and
cost-effective. (Meeusen and Warren, 1989). Successful
insect control has been observed under field conditions
(Delannay et al., 1989 : Meeusen and Warren, 1989).
In all cases, Agrobacterium-mediated gene transfer
has been used to express chimaeric Bt ICP genes in
plants (Vaeck et al., 1987; Barton et al., 1987;
Fischoff et al., 1987). Bt ICP genes were placed under
the control of a strong promoter capable of directing
gene expression in plant cells. It is however
remarkable that expression levels in plant cells were
high enough only to obtain insect-killing levels of Bt
ICP genes when truncated genes were used (Vaeck et al.,
1987 Barton et al., 1987). None of the transgenic
plants containing a full-length Bt ICP gene produced
insect-killing activity. Moreover, Barton et al. (1987)
showed that tobacco calli transformed with the entire
Bt ICP coding sequence became necrotic and died. These
results indicate that the Bt ICP gene presents unusual
problems that must be overcome to obtain significant
levels of expression in plants. Even, when using a
truncated Bt ICP gene for plant transformation, the
steady state levels of Bt ICP mRNA obtained in
transgenic plants are very low relative to levels
produced by both an adjacent NPT II-gene, used as a
marker, and by other chimeric genes (Barton et al.,
1987; Vaeck et al., 1987). Moreover, the Bt ICP mRNA
cannot be detected by northern blot analysis. Similar
observations were made by Fischoff et al. (1987); they
reported that the level of Bt ICP mRNA was much lower
than expected for a chimeric gene expressed from the
CaMV35S promoter. In other words, the cytoplasmic
CA 02080584 2000-05-30
W091/16432 PCT/EP91/00733
5 accumulation of the bt mRNA, and consequently the
synthesis, the accumulation and thereby the expression of
the Bt ICP protein in plant cells, are extremely
inefficient. By contrast, in microorganisms, it has been
shoran that truncated Bt ICP genes are less favorable than
full-length genes (Adang et al., 1985), indicating that
the inefficient expression is solely related to the
heterologous expression of Bt ICP genes in plants.
The problem of obtaining significant Bt ICP
expression levels in plant cells seems to be inherent and
intrinsic to the Bt ICP genes. Furthermore, the
relatively love and poor expression levels obtained in
plants appears to be a common phenomenon for all Bt ICP
genes.
It is known that there are six steps at which gene
expression can be controlled in eucaryotes (Darnell,
1982 )
1) Transcriptional control
2) RNA processing control
3) RNA transport control
4) mRNA degradation control
5) translational control
6) protein activity control
For all genes, transcriptional control is
considered to be of paramount importance (The Molecular
Biology of the Cell; Eds. Alberta, B., Bray, D., hewis,
J., Raff, M., Roberts, K. and Watson , J.D. Garland
Publishing Inc., New York(1989).
In European patent publications ("EP") 385,962 and
359,472, efforts to modify the codon usage of Bt ICP
genes to improve their expression in plant cells have
been reported. However, wholesale (i.e., non-selective)
changes in codon usage can introduce cryptic regulatory
signals in a gene, thereby causing problems in one or
WO 91/16432 ~ ~ ~ ~ ~ PCT/EP91/0073'
6
more of the six steps mentioned above for gene
expression, and thus inhibiting or interfering with
transcription and/or translation of the modified
foreign gene in plant cells. For example, changes in
codon usage can cause differential rates of mRNA
production, producing instability in the mRNA, so
produced (e. g., by exposure of regions of the mRNA,
unprotected by ribosomes, to attack and degradation by
cytoplasmic enzymes). Changes in codon usage also can
inadvertantly cause inhibition or termination of RNA
polymerase II elongation on the so-modified gene.
Summary of the Invention
In accordance with this invention is provided a
process for modifying a foreign gene, particularly a Bt
ICP gene, whose level and/or rate of expression in
plant cells, transformed with the gene, is limited by
the rate and/or level of nuclear production of an mRNA
encoded by the gene: the process comprises the step of
changing adenine and thymine sequences to corresponding
guanine and cytosine sequences encoding the same amino
acids in a plurality of translational codons of the
gene that would otherwise directly or indirectly cause
a nuclear event which would negatively control (i.e.,
inhibit or interfere with) transcription, nuclear
accumulation and/or nuclear export of the mRNA,
particularly transcription, quite particularly
elongation of transcription by RNA polymerase II of the
plant cells. Preferably, the adenine and thymine
sequences are changed to cytosine and guanine sequences
in translational codons of at least one region of the
gene which, during transcription, would otherwise have
thereon a relatively low percentage of RNA polymerase
II as compared to another adjacent upstream (i.e., 5')
region of the gene.
r I
WO 91 / 16432 . - PCT/EP91 /00733
2080584
Also in accordance with this..invention is provided
the modified Bt ICP gene resulting from the process.
Further in accordance with this invention, a
process is provided for improving the resistance of a
plant against insect pests by transforming the plant
cell genome with at least one modified Bt ICP gene.
This invention also relates to a chimaeric gene
that can be used to transform plant cells and that
contains the following operably-linked DNA fragments in
the same transcriptional unit:
1) the modified Bt ICP gene:
2) a promoter suitable for directing transcription
of the modified Bt ICP gene in the plant cells
and
3) suitable transcript 3' end formation and
polyadenylation signals for expressing the
modified Bt ICP gene in the plant cells.
This invention further relates to:
- a cell of a plant, the nuclear genome of which
has been transformed to contain, preferably stably
integrated therein, the modified Bt ICP gene,
particularly the chimaeric gene;
- cell cultures consisting of the plant cell;
- a plant which is regenerated from the
transformed plant cell or is produced from the
so-regenerated plant, the genome of which contains
the modified Bt ICP gene, particularly the
chimaeric gene, and which shows improved
resistance to insect pests:
- seeds of the plant: and
- a vector for stably transforming the nuclear
genome of plant cells with the modified Bt ICP
gene, particularly the chimaeric gene.
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W091/16432 PCT/EP91/00733
8
Detailed description of the Invention
As used herein, "Bt ICP" should be understood as
an intact protein or a part thereof which has
insecticidal activity and which can be produced in
nature by B. thuringiensis. A Bt ICP can be a protoxin,
as well as an active toxin or other insecticidal
truncated part of a protoxin which need not be
crystalline and which need no be a naturally occurring
protein. An example of a Bt ICP is a Bt2 insecticidal
crystal protein (Hofte et al., 1986), as well as its
insecticidally effective parts which are truncated at
its C- and/or N-terminal ends towards its tryspsin
cleavage sites) and preferably having a molecular
weight of 60-80 kDa. Other examples of Bt ICPs are . Bt2,
Bt3, Bt4, Btl3, Btl4, Btl5, Btl8, Bt2l, Bt22, Bt73,
Bt208, Bt245, BtI260 and BtI109P as disclosed in
PCT publications W090/15139 and W090/09445, in Hofte
and Whiteley (1989) and in Canadian Patent Application
2,079,877.
As used herein, "protoxin" should be understood as
the primary translation product of a full-length gene
encoding a Bt ICP.
As used herein, "toxin" or "active toxin" or
"toxic core" should all be understood as a part of a
protoxin which can be obtained by protease (e.g., by
trypsin) cleavage and has insecticidal activity.
As used herein, "truncated Bt gene" should be
understood as a fragment of a full-length Bt gene which
still encodes at least the toxic part of the Bt ICP,
preferentially the toxin.
As used herein, "modified Bt ICP gene" should be
understood as a DNA sequence which encodes a Bt ICP, and
in which the content of adenine ("A") and thymine
("T") has been changed to guanine ("G") and cytosine
( "C" ) in codons , preferably at least 3 , in at least one
region of the DNA sequence without affecting the
WO 91/16432 PCT/EP91/00733
9
2oeo5e4
original amino acid sequence of the Bt ICP. Preferably
in at least two regions, especially in at least three
regions, of the DNA sequence, the A and T content is
changed to G and C in at least 3 codons. For regions
downstream of the translation initiation site of the
DNA sequence, it is preferred that the A-T content of
at least about 10 codons, particularly at least about
33 codons, be changed to G-C.
By "region" of a modified Bt ICP gene is meant any
sequence encoding at least three translational codons
which affect expression of the gene in plants.
In accordance with this invention, it has been
shown by means of mRNA turn-over studies that the
expression pathway of a Bt ICP gene, such as bt2, btl4,
btl5 and btl8, is specifically inhibited at the nuclear
level in plant cells. In a further analysis, nuclei of
transgenic tobacco plants, i.e., N28 - 220 (Vaeck et
al., 1987), were used in a nuclear run-on assay to
determine the distribution and the relative efficiency
of RNA polymerise II complexes to initiate
transcription of chimaeric Bt ICP plant genes. In this
regard, the run-on assay has been used to determine
initially the relative efficiency of RNA polymerise II
complexes to initiate transcription of Bt ICP genes and
thereafter to determine the relative distribution and
migration efficiency of the RNA polymerise II complexes
on the Bt ICP genes.
N28 - 220 contains the bt884 fragment under
control of the TR 2' promoter as a chimaeric gene.
gtgg4 is a 5' fragment of the bt2 gene (Hofte et al . ,
1986) up to codon 610 (Vaeck et al., 1987). Using
nuclear run-on analysis, isolated nuclei of N28 - 220
were incubated with highly labeled radioactive RNA
precursors, so that the RNA transcripts being
synthesized at the time became radioactively labeled.
WO 91/16432 PCT/EP91/007:~"
1~ 2oeo5e~
The RNA polymerise II molecules caught in the act of
transcription in the cell continue elongating the same
RNA molecules in vitro.
The nuclear run-on assays of nuclei of N28 - 220
culture (non-induced cells and induced cells, TRl'-neo,
TR2'-bt884) revealed that transcription from the TRl'
and TR2' promoters is about equally efficient. This
implies that the low Bt ICP (i.e., Bt884) expression
levels are not due to a specifically reduced
transcriptional activity of the TR2' promoter. However,
nuclear run-on analysis with N28 - 220 nuclei indicated
that transcription elongation of the nascent Bt ICP
mRNA is impaired somewhere between 700 to 1000
nucleotides downstream of the start of transcription.
This means that RNA polymerise II is not able to
transcribe the Bt ICP coding sequence with 100 %
efficiency. Filter binding assays using labeled Bt DNA
fragments spanning this region and protein extract
prepared from tobacco nuclei reveal that this DNA
region undergoes specific interactions with proteins
present in nuclei. These interactions are the prime
candidates that cause or affect the impaired elongation
of transcription by RNA polymerise II through this
region. By modification of this region to abolish
specific protein binding, Bt ICP expression levels will
increase. However, other mechanisms responsible for
impaired elongation in this region cannot be excluded.
Further in accordance with this invention,
sequences within the coding region involved in negative
control of cytoplasmic Bt ICP mRNA levels have been
identified by deletion analysis. To this end, 24
deletion derivatives of pVE36 have been constructed.
Three main types of deletion mutants have been
constructed (see fig. 3):
WO 91/16432 PCT/EP91/00733
11 2080584
- 5' end deletions
- 3' end deletions
- internal deletions.
The expression of a mutant hybrid bt2-neo gene
(encoding a fusion protein of Bt2 (Hofte et al., 1986)
and NPTII) has been studied by means of transient
expression experiments using the cat gene as a
reference. To this end, the neo mRNA levels were
measured in relation to cat mRNA levels in RNA extracts
of SR1 protoplasts. The ratio between the neo and cat
mRNA level was used to quantify on a relative basis the
nptII transcript (i.e., mRNA) levels produced by the
different constructions. These experiments show that
progressive deletions of the carboxy-terminal (i. e.,
3' ) part or the amino-terminal (i. e. , 5' ) part of the
Bt ICP coding sequence result in a gradual increase of
the nptII transcript level. Furthermore, since the
changes in transcript levels are not very abrupt, these
results suggest that the low transcript levels produced
by Bt ICP genes are not controlled by a single factor.
Nevertheless, individual modifications of bt2 coding
sequence can significantly reduce the interference
and/or inhibition of the expression of the mRNA encoded
by Bt ICP genes in plant cells at the level of
transcript elongation, nuclear accumulation and nuclear
export. The modifications) may also affect cytoplasmic
regulation and metabolism of such mRNAs and their
translation.
Deletion analysis clearly indicates that several
internal sequences, located within the Bt ICP coding
region, might be involved in the negative regulation of
the Bt ICP expression. By way of example, a 326 by
region (fig. 6b) was identified in the bt2 gene that is
involved in the negative control of BT ICP expression
CA 02080584 2000-05-30
W091/16432 PCT/EP91/00733
12
and that is located between nucleotide position 674 and
nucleotide position 1000, particularly a 268 by region
between nucleotide positions 733 and 1000, quite
particularly a 29 by region between nucleotide positions
765 and 794 which carries two perfect CCAAT boxes which
are known to be able to cause a reduction in elongation
efficiency and termination of transcription by RNA
polynerase II in animal systems (Connelly and Manley,
1989). This internal gene fragment or inhibitory zone
may itself comprise a plurality of inhibitory zones which
reduce Bt ICP expression levels or which interact
directly or indirectly with other zones to inhibit or
interfere with expression. Codon usage of this
inhibitory zone has been modified in a second step by
substituting A - T with G - C without affecting the amino
acid sequence. In this regard, this internal 326 by
fragment (fig.6b) has been replaced with a modified Bt
ICP fragment of this invention containing 63 moclified
codons. The effect of such modification of this
inhibitory zone on Bt ICP expression has been analyzed
both in transient and stable plant transformants. The
results show that such modification of codon usage causes
a significant increase of Bt ICP expression levels and
hence improved insect-resistance.
In addition, N-terms.nal deletion mutants of the bt2 gene
have been made by deleting the first N-terminal 28 amino
acids (Hofte et al., 1986). It is known for the bt2
gene that the first 28 codons can be deleted without
loss of toxicity (Hofte et al., 1986; Vaeck et al.,
1987). Also, codon usage for three codons, 29 to 31,
has been changed in accordance with this invention by
replacing A - T with G - C without affecting the amino
acid sequence. E~rthermore, an optimal translation
initiation (ATG) site was created
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W091/16432 PCT/EP91/00733
13
based on the consensus sequence of Joshi (1987) as shown
in fig. 6a. Plants transformed with this modified Bt ICP
gene show significantly higher Bt ICP expression levels.
In accordance with this invention, all or part of a
modified Bt ICP gene of the invention can be stably
inserted in a conventional manner into the nuclear genome
of a plant cell, and the so-transformed plant cell can be
used to produce a transgenic plant showing improved
expression of the Bt ICP gene. In this regard, a
disarmed Ti-plasmid, containing the modified Bt ICP gene,
in Agrobacterium (e.g., A. tumefaciens) can be used to
transform a plant cell using the procedures described,
for example, in EP 116,718 and EP 270,822, PCT
publication 84/02913, EPA 0,242,246 and Gould et al.
(1991) .
Preferred Ti-plasmid vectors contain the foreign
DNA sequence between the border sequence, 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 233,247), pollen mediated
transformation (as described, for example, in EP 270,356,
PCT publication WO 85/01856, and US patent 4,684,611),
plant RNA virus-mediated transformation (as described,
for example, in EP 67,553 and US patent 4,407,956),
liposome-mediated transformation (as described, for
example, in US patent 4,536,475) and other methods such
as the recently described methods for transforming
certain lines of corn (Fromm et al., 1990; Gordon-Kamm et
al., 1990).
Preferably, the modified Bt ICP gene is inserted in a
plant genome downstream of, and under the control of, a
promoter which can direct the expression of the
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W091/16432 PCT/EP91/00733
14
gene in the plant cells. Preferred promoters include,
but are not limited to, the strong constitutive 35S
promoter (Odell et al., 1985) of cauliflower mosaic
virus; 35S promoters have been obtained from different
isolates (Hull and Howell, Virology 86, 482-493 (1987)).
Other preferred promoters include the TRl' promoter and
the TR2' promoter (Velten et al., 1984). Alternatively,
a promoter can be utilized which is not constitutive but
rather is specific for one or more tissues or organs.
For example, the modified Bt ICP gene can be selectively
expressed in the green tissues of a plant by placing the
gene under the control of a light-inducible promoter such
as the promoter of the ribulose - 1,5 - phosphate -
carboxylase small subunit gene as described in EPA
0,193,259. Another alternative is to use a promoter
whose expression is inducible by temperature or chemical
factors .
It is also preferred that the modified Bt ICP gene
be inserted upstream of suitable 3' transcription
regulation signals (i.e., transcript 3' end formation and
polyadenylation signals) such as the 3' untranslated end
of the octopine synthase gene (Gielen et al., 1984) or T-
DNA gene 7 (Velten and Schell, 1985).
The resulting transformed plant of this invention
shows improved expression of the modified Bt ICP gene
and hence is characterized by the production of high
levels of Bt ICP. Such a plant can be used in a
conventional breeding scheme to produce more
transformed plants with the same improved insect-
resistance characteristics or to introduce the moclified
Bt ICP gene into other varieties of the same or related
plant species. Seeds, which are obtained from the
transformed plants, contain the modified BtICP gene as
a stable genomic insert.
CA 02080584 2000-05-30
W091/16432 PCT/EP91/00733
5 Furthermore, at least two modified BtICP genes,
coding for two non-competitively binding anti-
Lepidopteran or anti-Coleopteran Bt ICPs, can be cloned
into a plant expression vector (EPA 0,408,403). Plants
transformed with such a vector are characterized by the
10 simultaneous expression of at least two modified BtICP
genes. The resulting transgenic plant is particularly
useful to prevent or delay development of resistance to
Bt ICP of insects feeding on the plant.
The following Examples illustrate the invention and
15 are not intended to limit its scope. The Figures,
referred to in the Examples, are as follows:
Fig.1 -- Comparison of the transcription initiation
frequency of RNA polymerase II complexes in nuclei of
N28-220. Hybridisation efficiencies of labelled nptII
mRNA and Bt ICP mRNA with their complementary DNA
counterparts present on a Southern blot were compared.
DNA fragments were obtained from a cligest of plasmid
pGSH163. A schematic view of the region is given. The
lengths of the fragments blotted on Hybond-NT"' filter
(1), the homologous genes on plasmid pGSHl63 (2), and the
densitometric values (3) are as follows:
Digest: 1 2 3
BamHI/HindIII 2358 neo 12386
1695 bt2 6565
154 bt2 -
6250 vector -
Fig. 2a -- Determination of the distribution of the RNA
polymerase II complexes on the Bt ICP coding sequence in
nuclei of N28-220. The hybridisation of labelled RNA
prepared by nuclear run on with DNA fragments of the Bt
ICP coding sequence was quantitated. The restriction
WO 91/16432 PCT/EP91/00733
16 2080584
fragments and scanning values are given in the table
and figure. The scanning value is proportional to "X",
the size of the DNA fragment and the ~ UTP per RNA
fragment hybridising. "X" is directly proportional to
the number of RNA polymerises passing through the DNA
fragment. "X" is proportional to the scanning value
divided by the number of UTPs. The X values of the
different restriction fragments are shown in the
figure. In this regard, conversion of the different
densitometric values into relative hybridisation
efficiencies by normalising the values of the number of
dATPs present in the DNA fragment, complementary to the
hybridising RNA, generates the value "X". "X" is a
relative measure of the number and the length of the
extension of the transcripts. "X" thus reflects the
number of RNA polymerises transcribing a specific DNA
sequence and their elongation rate. DNA fragments
present on the Southern digests of plasmid DNA of plant
vector pGSH163 each have the following lengths of
fragments blotted on Hybond-N filter (1), homologous
genes on plasmid pGSH163 (2) and densitometric values
(3)
Digest: 1 2 3
BamHI/EcoRI 8877 neo 15333
726 bt2(2) 2926
583 bt2(3) 635
271 bt2 (1) -
Ba~I/EcoRV 8887 neo 15182
84 bt2 2466
729 bt2 1102
BamHI/HindIII 6250 - -
2358 neo 12386
WO 91/16432 PCT/EP91/00733
17 208084
1695 bt2 6565
154 - -
BamHI/SacI 8053 neo 14194
1353 bt2(1) 4572
1051 bt2(2) 615
XmnI 4973 neo 13219
2107 - -
1401 - -
729 bt2(3) 736
628 bt2(2) 1817
305 bt2(4) -
188 bt2(5)
120 bt2(1) -
Fig. 2b -- Schematic view of nine bt884 DNA fragments
that were inserted into the polylinker of M13 vectors,
MP18 and MP19 (Yanisch-Perron et al., 1985). The Bt ICP
coding sequence is shown from AUG to 1600 nucleotides
downstream. The relevant restriction sites and sizes of
the DNA fragments are indicated. The nucleotide
numbering is relative to the AUG. The subclones were
named pJD7l, pJD72, pJD73, etc. (to pJD79), as
indicated. The inserts were oriented into the M13
sector such that single standed M13 carried the
fragments of the Bt ICP coding sequence in an anti-
sense orientation.
Fig. 2c -- Schematic representation of three nuclear
run-on analyses with N28-220 nuclei as described by Cox
and Goldberg (1988). Assays were performed for periods
of 5, 10 and 30 minutes. The labeled nuclear RNA was
allowed to hybridize with 5 ~g of single stranded
pJD71-pJD79 and MP18 DNA, which were immobilised on
nylon membranes. The membranes were autoradiographed,
WO 91/16432 PCT/EP91/00733
la 2080584
and densitometric values were obtained by scanning the
autoradiographs. The abscissa shows the nucleotide
position relative to the AUG of the Bt (i.e., bt2)
coding sequence. The center of each of the single
stranded Bt DNA fragments is indicated in the graph.
The ordinate gives the relative hybridisation signal
for each fragment corrected for the number of dATPs in
the fragment and adjusted to 100% for the value of
pJD71 for each of the three incubation periods. All
values are corrected for non-specific hybridisation to
single stranded MP18 DNA. The relative values are a
measure for the reactivation of bt mRNA synthesis by
RNA polymerise II. The assay does not distinguish
between the number of mRNA extensions and the length of
~A extensions.
Fig. 3 - - Construction of deletion mutants of the
bt860-neo gene to measure the effect on cytoplasmic
Bt
ICP mRNA levels. The parental vector pVE36 is shown.
The following
deletion mutants
were generated:
1. PJD50: pJD50 was derived from pVE36 by digesting
with BamHI and SphI. The 5'and 3'
protruding ends were filled in with Klenow
DNA polymerise I enzyme. The treated DNA
was ligated and then used to transform
MC1061 cells. Transformants were selected
for amps phenotype.
2. PJD51: pJD51 was derived from pVE36 by digesting
with SpeI and SphI. The 5'and 3'
protruding ends were filled in with Klenow
DNA polymerise I enzyme. The treated DNA
was ligated and then used to transform
MC1061 cells. Transformants were selected
for amps phenotype.
WO 91 / 16432 PCT/ E P91 /00733
i9 ~08058~
3. PJD52: pJD52 was derived from pVE36 by digesting
with EcoRV and SphI. The 5'and 3'
protruding ends were filled in with Klenow
DNA polymerase I enzyme. The treated DNA
was ligated and then used to transform
MC1061 cells. Transformants were selected
for amps phenotype.
4. PJD53: pJD53 was derived from pVE36 by digesting
with XcaI and SphI. The 3' protruding ends
were f il led in with Klenow DNA polymerase
I enzyme. The treated DNA was ligated and
then used to transform MC1061 cells.
Transformants were selected for amps
phenotype.
5. PJD54: pJD54 was derived from pVE36 by digesting
with AflII and SphI. The 5' and 3'
protruding ends were filled in with Klenow
DNA polymerase I enzyme. The treated DNA
was ligated and then used to transform
MC1061 cells. Transformants were selected
for amps phenotype.
6. PJD55: pJD55 was derived from pVE36 by digesting
with ClaI and SphI. The 5' and 3'
protruding ends were filled in with Klenow
DNA polymerase I enzyme. The treated DNA
was ligated and then used to transform
MC1061 cells. Transformants were selected
for amps phenotype.
7. PJD56: pJD56 was derived from pVE36 by digesting
with XhoI and SphI. The 5' and 3'
protruding ends were filled in with Klenow
DNA polymerase I enzyme. The treated DNA
was ligated and then used to transform
MC1061 cells. Transformants were selected
for amps phenotype.
WO 91/16432 PCT/EP91/00733
2~ 200584
8. PJD57: pJD57 was derived from pVE36 by digesting
with AflII and BamHI. The 5' and 3'
protruding ends were filled in with Klenow
DNA polymerise I enzyme. The treated DNA
was ligated and then used to transform
MC1061 cells. Transformants were selected
for amps phenotype.
9. PJD58: pJD58 was derived from pVE36 by digesting
with XcaI and BamHI. The 5' protruding
ends were filled in with Klenow DNA
polymerise I enzyme. The treated DNA was
ligated and then used to transform MC1061
cells. Transformants were selected for
amps phenotype.
lO.PJD59: pJD59 was derived from pVE36 by digesting
with EcoRV and BamHI. The 5' protruding
ends were filled in with Klenow DNA
polymerise I enzyme. The treated DNA was
ligated and then used to transform MC1061
cells. Transformants were selected for
amps phenotype .
11. PJD60: pJD60 was derived from pVE36 by digesting
with SpeI and BamHI. The 5' protruding
ends were filled in with Klenow DNA
polymerise I enzyme. The treated DNA was
ligated and then used to transform MC1061
cells. Transformants were selected for
amp'' phenotype .
12. PJD61: PJD61 was derived from PJD50. PVE36 was
digested with XbaI and filled in with
Klenow polymerise I. PJD50 was linearized
with BamHI and filled in with Klenow
polymerise I. The 375bp XbaI fragment of
PVE36 was ligated in the filled in BamHI
of pJD50. The ligation mixture was used
to
WO 91 / 16432 PCT/EP91 /00733
21 2o8o5e4
transform MC1061 cells. Transformants were
selected for amps phenotype.
13. PJD62: PJD62 was derived from PJD50. PVE36 was
digested with XcaI and EcoRV. PJD50 was
linearized with BamHI and filled in with
Klenow polymerise I. The 367bp XcaI-EcoRV
fragment of PVE36 was ligated in the
filled in BamHI of pJD50. The ligation
mixture was used to transform MC1061
cells. Transformants were selected for
amps phenotype .
14. PJD63: PJD63 was derived from PJD50. PVE36 was
digested with Xcal and EcoRV. PJD50 was
linearized with BamHI and filled in with
Klenow polymerise I. The 474bp XcaI-EcoRV
fragment of PVE36 was ligated in the
filled in BamHI of pJD50. The ligation
mixture was used to transform MC1061
cells. Transformants were selected for
amps phenotype.
15. PJD64: PJD64 was derived from PJD50. PVE36 was
digested with EcoRI and EcoRV and filled
in with Klenow polymerise I. PJD50 was
linearized with BamHI and filled in with
Klenow polymerise I. The 458bp EcoRI-EcoRV
fragment of PVE36 was ligated in the
filled in BamAI of pJD50. The ligation
mixture was used to transform MC1061
cells. Transformants were selected for
amps phenotype.
16. PJD65: PJD65 was derived from PJD50. PVE36 was
digested with EcoRI and XbaI and filled in
with Klenow polymerise I. PJD50 was
linearized with BamHI and filled in with
Klenow polymerise I. The 327bp EcoRI-XbaI
WO 91 / 16432 PCT/EP91 /0073.'
22 2 o s o 5 84
fragment of PVE36 was ligated in the
filled in BamHI of pJD50. The ligation
mixture was used to transform MC1061
cells. Transformants were selected for
amps phenotype .
17. PJD66: PJD66 was derived from PJD50. PVE36 was
digested with SpeI and XcaI and filled in
with Klenow polymerise I. PJD50 was
linearized with BamHI and filled in with
l0 Klenow polymerise I. The 1021bp SpeI-XcaI
fragment of PVE36 was ligated in the
filled in BamHI of pJD50. The ligation
mixture was used to transform MC1061
cells. Transformants were selected for
amp'' phenotype.
18.PPS56D1: PPS56D1 was derived from PJD56 by
digesting with EcoRV. The treated DNA was
ligated and then used to transform MC1061
cells. Transformants were selected for
amps phenotype.
19. PPS56D2: PPS56D2 was derived from PJD56 by
digesting with XcaI and AflII. The 5'
protruding ends were filled in with Klenow
polymerise I. The treated DNA was ligated
and then used to transform MC1061 cells.
Transformants were selected for amps
phenotype.
20. PPS56D3: PPS56D3 was derived from PJD56 by
digesting with SpeI and EcoRV. The 5'
protruding ends were filled in with Klenow
polymerise I. The treated DNA was ligated
and then used to transform MC1061 cells.
Transformants were selected for amps
phenotype.
WO 91/16432 PCT/EP91/00733
23
21. PPS56D4: PPS56D4 was derived from PJD56 by
digesting with XcaI and partially with
EcoRV. The treated DNA was ligated and
then used to transform MC1061 cells.
Transformants were selected for amps
phenotype.
22. PPS56D6: PPS56D6 was derived from PJD56 by
digesting with SpeI and partially with
EcoRV. The 5' protruding ends were filled
in with Klenow polymerise I. The treated
DNA was ligated and then used to transform
MC1061 cells. Transformants were selected
for amps phenotype.
23. PPS56D7: PPS56D7 was derived from PJD56 by
digesting with SpeI and XcaI. The 5'
protruding ends were filled in with Klenow
polymerise I. The treated DNA was ligated
and then used to transform MC1061 cells.
Transformants were selected for amps
phenotype.
24. PPS56D8: PPS56D8 was derived from PPS56D2 by
digesting with SpeI and partially with
EcoRV. The 5' protruding ends were filled
in with Klenow polymerise I. The treated
DNA was ligated and then used to transform
MC1061 cells. Transformants were selected
for amp'' phenotype.
Fig. 4 -- Effect of deletions in the Bt ICP coding
sequence on cytoplasmic Bt ICP mRNA levels. The
cytoplasmic mRNA levels specified by the invariable cat
reference gene and the different Bt ICP deletion
mutants described in fig. 3 are listed in the table.
The measurements were converted into relative Bt ICP
mRNA abundances. Bt ICP and cat mRNA quantizations were
WO 91/16432 PCT/EP91/0073?
24 2 ~ g 0 5 8 4
done as described by Cornelissen (1989). Total RNA was
slot blotted and hybridised with radioactively labeled
RNA complementary to the neo and cat coding sequences.
Values were quantitated with the aid of calibration
curves of cold cat and Bt ICP riboprobe transcripts.
Fig. 5 -- Relative transcript levels produced by the
deletion derivatives of pVE36.
Fig. 6a -- Schematic presentation of the synthetic DNA
sequences used to introduce a N-terminal deletion and a
change of the codons 29, 30 and 31 of the bt2 coding
sequence. The oligo nucleotides were annealed according
to Engler et al. (1988) and cloned into the BstXI
restriction site of plasmid pVE36, yielding pPS027. The
7360 by fragment of pPS027 was ligated to the the 1177
by ClaI restriction fragment of pVE36, yielding plasmid
pPS028. pPS028 is identical to pVE36 apart for the N-
terminal modification.
Fig. 6b -- Schematic presentation of the synthetic DNA
sequences used to introduce an internal modification
into the bt2 coding sequence. The oligonucleotides were
annealed and ligated as described by Engler et al.
(1988) and the resulting concatemeric DNA fragment was
cut with the restriction enzymes XbaI and EcoRI to
release the modified 327 by XbaI-EcoRI restriction
fragment. This fragment was ligated into the 3530 by
EcoRI-XbaI fragment of pPS023 which is a pUCl9
derivative (Yanisch-Perron et al., 1985) that carries
the 1533 by AflII (filled in) BamHI fragment of pVE36
in the HindIII (filled in) BamHI site of pUCl9,
resulting in plasmid pPS024. Plasmid pPS024 was
linearised by digestion with restriction enzyme XbaI
and the 375 by XbaI restriction fragment of pPS023 was
introduced resulting in pPS025. The 1177 by Clal
WO 91/16432 PCT/EP91/00733
25 2 0 8 0 5 g~
fragment of pPS025 was introduced in the 7360 by ClaI
restriction fragment of pPS027 yielding pPS029. pPS029
is identical to pVE36 but carries both the amino
terminal modification and the internal modification of
the Bt ICP coding sequence.
Fig. 6c -- Nucleotide sequences 800 to 4000 of the
plasmids pVE36 and pPS029. "x" refers to not known
nucleotides.
l0
Fig. 7 -- Schematic presentation of the effect of the
mutations on the AT content of the Bt ICP plant gene.
The modified regions are indicated.
Fig. 8a -- Schematic presentation of the plasmid
constructions used in the transient expression assay.
The relevant genes are indicated.
Fig. 8b -- Accumulation profiles of CAT (Neumann et
al., 1987) and the modified BtICP (Engvall and Pesce,
1978) in a typical transient expression assay.
Unless otherwise stated in the Examples, all
procedures for making and manipulating recombinant DNA
are carried out by the standardized procedures
described in Sambrook et al., Molecular Cloning - A
laboratory Manual, Cold Spring Harbor Laboratory
(1989) .
Buample i. Determination o! the $fficiencv of
Transcription Initiation
The relative efficiency of RNA polymerase II
complexes to initiate transcription at chimaeric BtICP
plant genes was studied, using transgenic plant N28-220
which is described by Vaeck et al. (1987) and contains
copies of the T-DNA of plasmid pGSH163 This T-DNA
carries the chimaeric plant genes PTetbt8843'g7 and
WO 91/16432 PCT/EP91/0073z
26 208058
PT~~,neo3'ocs. Nuclei of 25 g of induced leaves of
N28-220 were prepared according to Cox and Goldberg
(1988) and stored the nuclei at a temperature of -70°C.
This method causes the nascent precursor mRNA chains
and the RNA polymerise II complexes to halt while the
complexes remain associated at the DNA. A batch of
these nuclei was assayed for the ability to incorporate
radioactively labeled UTP as a measure for the
transcriptional viability of the nuclei (Cox and
Goldberg (1988). This incorporation could be
successfully repressed by addition of a-amanitin to a
final concentration of 2 ~Cg/ml. This shows that the UTP
incorporation was due to transcript elongation by RNA
polymerise II and that RNA synthesis on the protein
coding genes which are occupied by RNA polymerise II
can be reactivated under the appropriate experimental
conditions.
Batches of the nuclei of N28-220 were used to
synthesize radioactively labeled RNA as described by
Cox and Goldberg (1988). The radioactive RNA
synthesized is a direct representation of the
distribution of the RNA polymerises II complexes on the
DNA in the nuclei. As the DNA of N28-220 carries two
genes which can be assayed, namely the chimaeric neo
gene and the chimaeric Bt ICP gene, it is possible to
compare the distribution of RNA polymerise II complexes
on these two genes. To this end, the radioactive RNA
was extracted from the nuclei according to Cox and
Goldberg (1988) and used as a probe in a conventional
Southern hybridisation. The Southern blot contained DNA
fragments carrying the Bt ICP and neo coding sequences
in a molar excess relative to the neo and Bt ICP RNA
species present in the radioactive probe. A detailed
description of the Southern blot is given in fig. 1.
The hybridisation experiment resulted in hybridisation
WO 91 / 16432 PCT/E P91 /00733
27 2o8o5e4
signals to both the neo and Bt ICP coding sequences
(fig. 1). Densitometric scanning showed that the
intensity of the hybridisation signal to the neo and Bt
ICP coding regions was nearly identical. This result
implies that the number of transcripts initiating from
the TR dual promoter is about similar in both
directions. As in plant N28-220 the cytoplasmic neo
mRNA level is several magnitudes higher than that of Bt
ICP~ this shows that the Bt ICP coding sequence indeed
negatively controls accumulation of cytoplasmic Bt ICP
mRNA, but that this phenomenon is not due to a dominant
negative effect on transcription initiation of the
chimaeric Bt ICP plant gene.
$$~pl. 2. Transcription Blonqatioa
The relative distribution of RNA polymerase II
complexes on the Bt ICP plant genes present in
transgenic plant N28-220 which is described by Vaeck et
al. (1987) was investigated. To this end, a second
experiment was carried out with batches of the nuclei
of N28-220 described in Example 1.
The nuclei were incubated as described by Cox and
Goldberg (1988) to synthesize radioactively labeled
RNA. The radioactive RNA was extracted as described
previously to provide a probe for a Southern
hybridisation. The Southern blot prepared for this
experiment contained several fragments of the Bt ICP
coding sequence in molar excess relative to the
complementary RNA present in the probe. The rationale
of the experiment was that if the RNA polymerase II
complexes were equally distributed over the Bt ICP
coding region, the hybridisation with the different Bt
ICP DNA fragments present on the Southern blot would be
proportional to the size and dATP content of the
different fragments. A detailed description of the DNA
WO 91 / 16432 PCT/EP91 /0073?
28 zoso5~~
fragments present on the Southern is given in fig 2a.
The hybridisation of the radioactive RNA extracted from
the nuclei of N28-220 with the Southern revealed that
the complete Bt ICP coding sequence as present in
N28-220 is transcribed by RNA polymerise II.
Quantification of the hybridisation signals by
densitometric scanning of the autoradiogram showed that
more radioactively labeled RNA was hybridising with DNA
fragments representing Bt ICP sequences located 5' on
the Bt ICP coding sequence than with Bt ICP sequences
located 3' on the Bt ICP coding sequence. The actual
values are given in fig 2a. This in vitro experiment
demonstrates that in vivo the RNA polymerises are not
evenly distributed over the Bt ICP coding sequence.
The sites) involved in reducing the RNA
polymerise II elongation were then determined more
accurately. Nine M13 derivatives were made that carry
overlapping fragments of the Bt2 coding sequence
spanning the region from the AUG to 1584 nucleotides
downstream. The inserts were oriented into the vector
such that, in single stranded M13 derivatives, the Bt
sequences were complementary to the Bt transcript. A
schematic view of the M13 clones is given in fig. 2b.
A molar excess of each single stranded anti-Bt DNA
was bound to nylon filters to serve as a DNA target for
hybridisation with labeled RNA prepared from nuclear
run-on assays with N28-220 nuclei as described by Cox
and Goldberg (1988). Three nuclear run-ons that
differed only in their time period of incubation were
carried out simultaneously. The incubation time
determines the length of extension of the nascent mRNA
chain. Shorter incubation periods give a more accurate
view of the position of the RNA polymerise II complexes
relative to the substrate DNA and their ability to
elongate at the moment of the start of incubation.
WO 91 / 16432 PCT/EP91 /00733
29 _ 2 0 8 0 5 84
Hence, the shorter the in vitro incubation period, the
more accurate the view in predicting the in vivo
situation.
The results are shown in fig. 2c. The data for the
5 minute incubation show that, in vivo, at a very
discrete inhibitory zone along the bt2 coding sequence,
one or more factors interfere with transcript
elongation and that such factor(s~ remain present in
such inhibitory zone during the course of the _in vitro
~A extension reaction. Increased incubation periods
show that, on a subset of DNA templates, RNA synthesis
resumed downstream of such inhibitory zone in this
assay without significantly removing the inhibition in
the inhibitory zone itself. In this regard, the data
indicate that:
1. The inhibitory zone causes the RNA polymerases to
pause and not to terminate.
2. This pause is only transitory for a small fraction
of the Bt DNA templates which were used.
3. The continued RNA polymerase elongation,
downstream of the inhibitory zone, is done by a
large number of polymerases on the relatively
small fraction of the Bt DNA templates.
It is believed, therefore, that low cytoplasmic Bt
mRNA levels are due at least in part to inefficient
production of precursor mRNA caused by inefficient
elongation of a nascent transcript and/or stalling of
~A Polymerase II complexes from transcribing at an
inhibitory zone.
The inhibitory zone was assayed for its ability to
interact with proteins present in nuclei of tobacco
protoplasts. A crude nuclear extract was prepared from
tobacco SR1 leaf protoplasts according to Luthe and
WO 91/16432 PCT/EP91/0073?
3~ 2080584
Quatrano (1980) and used for filter binding assay
essentially as described by Diffley and Stillman
(1986). 100 ng samples of protein extract were mixed
with different amounts of radioactively labeled 532 by
XbaI-AccI bt884 DNA fragment, ranging from 0 to 1670
picomolar, in a final volume of 0.150 ml binding buffer
(10 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA and
5% glycerol). After 45 minutes incubation at room
temperature, the samples were filtered through an
alkali-washed nitrocellulose membrane and washed twice
with 0.150 ml of an ice-cold solution containing 10 mM
Tris pH 7.5, 50 mM NaCl and 1 mM EDTA. The retention of
DNA-protein complex was quantified by scintillation
counting and revealed that the binding had a
dissociation constant in the 100 picomolar range. The
binding was not affected by preincubation of the
nuclear extract with a molecular excess of a specific
competitor DNA.
ERamPle 3. Construction of Deletion Mutants
The previous two examples demonstrate that the Bt
ICP coding sequence in a chimaeric plant gene
negatively affects the cytoplasmic Bt ICP mRNA level
directed by the chimaeric plant gene. It is shown that
this negative control is not at the level of
transcription initiation but at least in part due to a
reduced ability of RNA polymerase II to generate
precursor Bt ICP mRNA. A deletion analysis of the
chimaeric Bt ICP plant gene was performed to identify
whether impaired transcription elongation is the
exclusive mechanism by which the Bt ICP sequence
interferes with gene expression. The rationale of the
experiment is that the introduction of specific
deletions in the Bt ICP coding region could remove or
inactivate the sequence elements) responsible for the
WO 91/16432 PCT/EP91/00733
31 2080584
negative control. As a result such mutant gene would
direct an increased level of cytoplasaic mRNA. This
method can therefore be used to map and identify the
sequences) involved fn the negative control.
To perform this analysis, a deletion series of the
bt860-neo gene (Vaeck et al., 1987) was made. Fig. 3
gives a schematic representation. The resultant
deletion derivatives do not specify a Bt ICP and
therefore are assayed at RNA level only. In order to
obtain accurate Bt ICP mRNA concentration values, the
deletion mutants were compared in a transient
expression system using tobacco leaf protoplasts of SR1
(Cornelissen and Vandewiele, 1989). The relative mRNA
abundances were calculated using a correction factor
provided by the mRNA level specified by the cat
reference gene present on the same plasmid as the
mutant Bt ICP gene. Four hours after introduction of
the genes the tobacco leaf protoplasts were harvested,
and total RNA was prepared and analysed (fig. 4).
The mutants nos. 50-60 (fig. 3) show that
progressive deletions of the carboxy-terminal part or
the amino-terminal part of the Bt ICP coding sequence
result in a gradually increasing neo transcript level.
As there are not very abrupt changes in transcript
levels, these results suggest that the low transcript
level produced by full length Bt ICP genes is
controlled by a number of signals. Deletions within the
Bt ICP coding sequence indeed did not localise a
specific sequence element which, by itself, is
responsible for the low Bt ICP mRNA level. Similarly,
cloning of fragments of the Bt ICP coding sequence in
pJD50 (fig. 3) did not allow identification of such a
region.
The relative transcript levels were plotted
against the length of the Bt ICP sequence present in
WO 91/16432 PCT/EP91/0073'
32 2 0 8 0 5 8 4
the different deletion derivatives. Fig. 5 suggests
that hybrid Bt ICP-neo transcript levels drop with
increasing length of the Bt ICP sequence. In this
respect, the mutants nos. 61-66 (fig. 3) form an
exception as they show in average a low transcript
level relative to the length of the Bt ICP sequence.
These results show that the low transcript levels
of Bt ICP plant genes in tobacco are not exclusively
due to an impaired elongation of the nascent transcript
but that a number of signals operate to cause a reduced
expression capacity of the chimaeric Bt ICP gene.
Example 4.
To determine whether cytoplasmic events are
important in causing inefficient expression of the bt2
gene in plants, the following test was carried out.
Cytoplasmic bt2 mRNA steady state levels in transgenic
leaf protoplasts of N28-220 are normally found to be
below 1 transcript per cell. The steady state level is
determined by, and is proportional to, the number of
bt2 transcripts entering per time unit the cytoplasm
and the cytoplasmic half-life of the transcript. When
steady state levels are achieved, the absolute numbers
of transcripts entering and leaving the cytoplasmic bt2
mRNA pool are equal. Therefore, the cytoplasmic half-
life and cytoplasmic steady state level of the bt2
transcript will reveal whether its cytoplasmic steady
state level is due to a relatively low import of bt2
transcript, a relatively high turnover (i.e.,
conversion to a protein) rate, or a combination of
both.
The cytoplasmic turnover of bt884 transcripts was
determined according to Gallie et al. (1989). A capped
and polyadenylated synthetic bt884 mRNA was produced in
vitro according to protocols of Promega Corporation
WO 91 / 16432 PCT/EP91 /00733
33 - 2 0 8 0 5 84
(Madison, Wisconsin, USA) and introduced into tobacco
leaf protoplasts simultaneously with a synthetic bar
(De Block et al., 1987) mRNA. The two synthetic
transcripts differed only in their coding sequences. At
various times after RNA delivery, samples were taken,
and total RNA was isolated. Northern analyses revealed
that the half-lives (T 1/2) of the synthetic bt884 and
bar transcripts were about 8 ~ 3 hours and 5 + 2 hours,
respectively. See Table 1, below. These data show that
the bt884 coding sequence, more particularly the bt884
codon usage and the AU-rich motifs in the bt884 coding
sequence, do not render the bt884 mRNA more unstable
than the bar mRNA which is known to accumulate in the
cytoplasm to about 1000 transcripts per tobacco leaf
protoplast (calculated from Cornelissen, 1989). The low
cytoplasmic steady state level of the bt884 transcripts
is, therefore, caused by a lack of import of
transcripts into the cytoplasm. Thus, the expression
defect of the bt884 gene has to be restored by
introduction of modifications in the bt884 coding
sequence that improve the expression pathway in the
nucleus.
Expression of the btl4, btl5 and btl8 genes in
tobacco revealed that these genes also direct low
cytoplasmic mRNA steady state levels. Therefore, a
similar analysis was carried out with synthetic btl4,
btl5 and btl8 transcripts to identify whether the
expression defect had a cytoplasmic or nuclear
character. Table 1, below, shows that all three
transcripts behave as stable mRNAs in the cytoplasm of
tobacco leaf protoplasts. Therefore, btl4, btl5 and
btl8 genes, like the bt884 gene, must be deficient in
exporting high levels of bt transcript to the
cytoplasm, and to improve the expression of such genes,
it is necessary to modify their coding sequences so
WO 91/16432 ' PCT/EP91/0073~
34
2oao5a4
that nuclear events, which interfere with efficient
gene expression, are avoided or ameliorated.
Table 1
Half-life determination of synthetic bt and bar mRNAs
in Nicotiana tabacum cv. Petite Havanna SR1 leaf
protoplasts
Example 1'LmRNA T1/2 2"dmRNA T1/2
(Hours) (Hours)
A bt884 8+/-3 bar 5+/-2
B btl4 7+/-2 bar 6+/-3
C btl5 12+/-5 bar 21+/-12
p btl8 10+/-5 bar 12+/-5
Legend
The synthetic bar transcripts had a length of 783 bases
and included a cap, the TMV leader (77 bases, Danthinne
and Van Emmelo, 1990), the bar coding sequence (552
bases: De Block et al., 1987), a trailer of 52
nucleotides consisting of the bases GAUCA CGCGA AUU and
39 bases from the pGEM-3Z (Promega) polylinker (KpnI
(T4 DNA pol.)-HindIII (T4 DNA pol.), and a poly(A) of
the composition (A)33G(A)32G(A)3z, followed by the
nucleotides GCU.
The synthetic bt884 transcripts had a length of 2066
bases and included a cap, the TMV leader (77 bases),
the bt884 coding sequence followed by the trailer until
the Klenow treated PstI site (1843 nucleotides), the
trailer continued with AAUUC CGGGG AUCAA UU, 39 bases
of the pGEM-3Z polylinker and the (A)~G(A)3ZG(A)2~
poly(A), followed by the nucleotides CG.
WO 91 / 16432 PCT/EP91 /00733
35 2 0 8 0 5 84
The synthetic btl4 transcripts had a length of 2289
bases and included a cap, the TMV leader (77 bases),
the btl4 coding sequence till the Rlenow treated BclI
site (2023 bases), plus 26 supplementary nucleotides CG
UCG ACC UGC AGC CAA GCU UGC UGA, a trailer starting
with UUGAU UGACC GGAUC CGGCU CUAGA AUU, followed by 39
bases of the pGEM-3Z polylinker, and the
(A)33G(A)32G(A)Z~ poly(A), followed by the nucleotides
CGGUA CCC.
The synthetic btl5 transcripts had a length of 2198
bases and included a cap, the TMV leader (77 bases) the
btl5 coding sequence as in pVE35 (PCT publication
W090/15139) followed by the trailer till the Klenow
treated BamHI site (1989 bases), the trailer then
continued with AAUU, 39 bases of the pGEK-3Z polylinker
and the (A) ~G (A) 3zG (A) 2~ poly (A) , followed by the
nucleotides CG.
The synthetic btl8 transcripts had a length of 2184
bases and included a cap, the TMV leader (77 bases) the
btl8 coding sequence until the Klenow treated BcLI site
(1918 bases), followed by 26 nucleotides until the
translation stop CG UCG ACC UGC AGC CAA GCU UGC UGA, a
trailer starting with UUGAU UGACC GGAUC GAUCC GGCUC
AGAUC AAUU, 39 bases of the pGEM-3Z polylinker and the
(A) 33G (A) 32G (A) 2~ poly (A) , followed by the nucleotides
CG.
Example 5. Construction of Modified Ht ICP t3enes
Examples 1-4 show that the expression in a plant
of a Bt ICP gene is negatively affected by the Bt ICP
coding sequence at both transcriptional and post-
transcriptional levels, but principally by nuclear
events. These examples also show that the control of
expression is not confined to a specific DNA sequence
WO 91 / 16432 PCT/EP91 /0073'
36 2 0 8 0 5 84
within the Bt ICP coding sequence. Instead, the
negative effect on gene expression is an intrinsic
property of the Bt ICP coding sequence. On this basis,
it is believed that, by directed change of the DNA
sequence of the Bt ICP coding region, an improvement of
gene expression will occur. The improvement will be of
a cumulative type as the negative influence of the Bt
ICP coding region is spread over the complete coding
sequence. Similarly, an improvement of gene expression
will be obtained by reduction of the length of the Bt
ICP coding sequence. This improvement will have a
cumulative effect if used in combination with
modifications of the Bt ICP coding region.
Therefore, two types of modifications were
introduced into a Bt ICP (i.e., bt2) coding sequence
which, as will be shown, indeed resulted in a
significant increase in Bt ICP plant gene expression.
First, the DNA sequence was modified in the central
region of the toxic core fragment of the Bt ICP as
transcription elongation is impaired in this region.
Secondly, the length of the Bt ICP coding sequence was
reduced as the negative influence is proportional to
the length of the Ht ICP coding sequence. A detailed
description of the mutations is given in figs. 6a, b
and c. As shown in fig. 7, the modifications change the
AT-content of the chimaeric Bt ICP gene significantly.
The modifications change the primary DNA structure of
the Bt ICP coding sequence without affecting the amino
acid sequence of the encoded protein. It is evident
that, if more DNA mutations were to be introduced into
the Bt ICP coding sequence, a further improvement of
gene expression would be obtained.
To determine the effect of the modifications, the
expression properties of the modified BtICP gene and
the parental bt860-neo gene were compared in a
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37
transient expression system as described by Cornelissen
and Vandewiele (1989) and Denecke et al. (1989).
Basically, the accumulation profiles of the genes under
study were compared by relating their profiles to the
profile of a reference gene present in the same
experiment. Fig. 8a shows the vectors used in the assay,
and fig. 8b shows that the accumulation of the reference
CAT protein is nearly identical in both experiments. It
is not possible to measure the accumulation of Bt ICP
encoded by the parental bt860-neo gene, but the modified
Bt ICP gene clearly directs an increased synthesis of Bt
ICP.
These results demonstrate that mutation of the Bt
ICP coding sequence relieves the negative influence of
the Bt ICP coding sequence on the expression of a Bt ICP
plant gene.
Example 6. Clonincs and Expression of Modified BT ICP
Genes In Tobacco and Potato Plants
Using the procedures described in patent
publications EPA 0,193,259, EPA 0,305,275 and EPA
0,382,990 the modified Bt ICP (i.e., bt2) genes of figs.
6 and 7 are inserted into the intermediate T-DNA vector,
pGSH1160 (Deblaere et al., 1988) between the vector's T
DNA terminal border repeat sequences.
To obtain significant expression in plants, the
modified Bt ICP genes are placed under the control of the
strong TR2' promoter (Velten et al., 1984) and are fused
to the transcript 3' end formation and polyadenylation
signals of the T-DNA gene 7 (Velten and Schell, 1985).
In addition, the translation initiation context or
site are changed in accordance with the Joshi consensus
sequence (Joshi, 1987) in order to optimize the
WO 91 / 16432 PCT/EP91 /0073
38 2080584
translation initiation in plant cells. To this end, an
oligo duplex (figs. 6a and 6b) is introduced to create
the following sequence at translation initiation site:
AAAACCATGGCT. In this way, an additional codon (i.e.,
GCT) coding for alanine is introduced. Additionally,
KpnI and BstXI sites are created upstream of the ATG
translation initiation codon.
Using standard procedures (Deblaere et al., 1985),
the intermediate plant expression vectors, containing
the modified BtICP gene, are transferred into the
Aqrobacterium strain C58C1 RifR (US patent application
821,582; EPA 86300291.1) carrying the disarmed Ti-
plasmid pGV2260 (Vaeck et al., 1987). Selection for
spectinomycin resistance yields cointegrated plasmids,
consisting of pGV2260 and the respective intermediate
plant expression vectors. Each of these recombinant
Aqrobacterium strains is then used to transform
different tobacco plant cells (Nicotiania tabacum) and
potato plant cells (Solanum tuberosum) so that the
modified Bt ICP genes are contained in, and expressed
by, different tobacco and potato plant cells.
The transgenic tobacco plants containing the
modified Bt ICP genes are analyzed with an ELISA assay.
These plants are characterized by a significant
increase in levels of Bt (Ht2) proteins, compared to a
transgenic tobacco plant containing a non-modified Bt
ICP (bt2) gene.
The insecticidal activity of the expression
products of the modified Bt ICP (bt2) genes in leaves
of transformed tobacco and potato plants is evaluated
by recording the growth rate and mortality of larvae of
Tobacco hornworm (Manduca sexta), Tobacco budworm
(Heliotis virescens) and potato tubermoth (Phthorimaea
operculella) fed on leaves of these two types of
plants. These results are compared with the growth rate
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W091/16432 PCT/EP91/00733
39
of larvae fed leaves from tobacco and potato plants
transformed with the unmodified or parental Bt ICP (bt2)
gene and from untransformed potato and tobacco plants.
Toxicity assays are performed as described in
EPA 0,305,275 and EPA 0,193,259.
A significantly higher mortality rate is obtained
among larvae fed on leaves of transformed plants
containing and expressing the modified Bt ICP genes.
Tobacco and potato plants containing the modified Bt ICP
genes show considerably higher expression levels of Bt
ICPs compared to tobacco and potato plants containing the
unmodified Bt ICP gene.
The insecticidal activity of three transgenic
tobacco plants containing the modified Bt ICP genes is
determined against second and third instar larvae of
Heliotis virescens. The control plant was not
transformed. The results are summarized in Table 2,
below.
Table 2
Plant ~ mortality of insects (recorded
after 5 days)
Control 11
No. 1 100
No. 2 88.5
No. 3 100
Needless to say, this invention is not limited to
tobacco and potato plants transformed with the modified
Bt ICP gene. It includes any plant, such as tomato,
alfalfa, sunflowers, corn, cotton, soybean, sugar beets,
rapeseed, brassicas and other vegetables, transformed
with the modified Bt ICP gene.
WO 91/16432 PCT/EP91/007?
208 0584
Nor is the invention limited to the use of
Agrobacterium tumefaciens Ti-plasmids for transforming
plant cells with a modified Bt ICP gene. Other known
techniques for plant transformation, such as by means
5 of liposomes, by electroporation or by vector systems
based on plant viruses or pollen, can be used for
transforming monocotyledonous and dicotyledons with
such a modified Bt ICP gene.
Nor is the invention limited to the bt2 gene, but
10 rather encompasses all Cry I, Cry II, CryIII and Cry IV
Bt ICP genes.
20
30
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41
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