Note: Descriptions are shown in the official language in which they were submitted.
CA 0229684042000-O1-13
1
DNA SEQUENCE CODING FOR A HYDROXYPHENYLPYRUVATE DIOXYGENASE
AND OVERPRODUCTION THEREOF IN PLANTS
20
The present invention relates to a method of generating plants
with an elevated vitamin E content by expressing an exogenous or
endogenous HPPD gene in plants or plant organs. The invention
furthermore relates to the use of the corresponding nucleic acids
encoding an HPPD gene in transgenic plants to make the latter
resistant to HPPD inhibitors, and to the use of the DNA sequence
encoding an HPPD for generating a test system for identifying
HPPD inhibitors.
An important aim in plant molecular genetics is the generation of
plants with an elevated content of sugars, enzymes and amino
acids. It would also be economically interesting to develop
plants with an elevated vitamin content, eg. an elevated vitamin
E content.
The eight naturally occurring compounds with vitamin E activity
are derivatives of 6-chromanol (Ullmann's Encyclopedia of
Industrial Chemistry, Vol. A 27 (1996), VCH Verlagsgesellschaft,
Chapter 4., 478-488, Vitamin E). The first group (la - d) is
derived from tocol, while the second group is composed of
tocotrienol derivatives (2a - d):
R1
HO
~5I 3
\ 1
R O 4, 8,
R3
la, a-tocopherol: R1 = Rz = R3 = CH3
lb, (i-tocopherol [148-03-8]: R1 = R3 = CH3, R2 = H
lc, y-tocopherol [ 54-2 8-4 ] : R1 = H, R2 = R3 = CH3
ld, b-tocopherol [119-13-lj: R1 = RZ = H, R3 = CH3
3 0 R1
HO
\ ~ 0 _ _/ _ / /
R T 3' 7' 11,
R3
0050/48141 CA 02296840 2000-oi-i3
., ,
2
2a, a-tocotrienol [ 1721-51-3 j : R1 = RZ = R3 = CH3
2b, (3-tocotrienol [ 490-23-3 j : Rl = R3 = CH3, R2 = H
2c, y-tocotrienol [ 14101-61-2 ] : R1 = H, R2 = R3 = CH3
2d, 8-tocotrienol [25612-59-3j: R1 = R2 = H, R3 = CH3
a-Tocopherol is of great economic importance.
The development of crop plants with an elevated vitamin E content
by means of tissue culture or seed mutagenesis and natural
selection has its limits. On the one hand, the vitamin E content
must be detectable as early as at the tissue culture level and,
on the other hand, only those plants can be manipulated via
tissue culture techniques which can successfully be regenerated
into entire plants, starting from cell cultures. Moreover,
following mutagenesis and selection, crop plants may show
undesirable characteristics which have to be eliminated by
back-crossing, in some cases repeated back-crossing. Also,
elevation of the vitamin E content by means of crossing would be
limited to plants of the same species.
Those are the reasons why the genetic engineering approach, viz.
isolating an essential biosynthesis gene which encodes the
vitamin E synthesis performance and transferring it specifically
into crop plants, is superior to the traditional breeding method.
The conditions for this method are that the biosynthesis and its
regulation are known and that genes which affect biosynthesis
performance are identified.
Tocopherol biosynthesis in plants and algae proceeds in a known
! 30 manner and is as follows:
40
0050/48141 CA 02296840 2000-oi-i3
.,
- 3
COOH
=0
HZ OH
HPPD ~ CH2- COOH
OH COZ OH
4-hydroxyphenylpyruvate homogentisic acid
(3) (4)
i 15
OH
H H
4
(5) (6)
b -tocotrienol (2d) b -tocopherol (ld)
~ .
(~- or Y- tocotrienol ---~ ~- or Y-tocopherol
(2b or 2c) (lb or lc)
a -tocotrienol (2a) ~ a -tocopherol (la)
0050/48141 CA 02296840 2000-O1-13
~ i
4
The precursor of the aromatic ring of the tocopherols is
p-hydroxyphenylpyruvate (3), which is converted enzymatically into
homogentisic acid (4) with the aid of the enzyme
hydroxyphenylpyruvate dioxygenase (HPPD), and the homogentisic
acid reacts with phytyl pyrophosphate with elimination of C02 to
give the precursor (6). The tocotrienol biosynthesis route starts
with a condensation reaction between homogentisic acid (4) and
geranylgeranyl pyrophosphate to give the precursor (5). Enzymatic
cyclization of the precursors 5 or 6 gives 8 - tocotrienol or b -
tocopherol, respectively. Some of these biosynthesis enzymes have
been isolated.
While searching for Arabidopsis mutants with defects in the
carotinoid biosynthesis, a white phenotype mutant was identified
which is not capable of producing active HPPD. If this mutant,
termed pds2, is raised in the presence of homogentisic acid, it
produces carotinoids, like the wild type, and greens (Norris et
al., Plant Cell (1995) 7: 2139 - 2149). This work shows that HPPD
activity is a prerequisite for the formation of
photosynthetically active chloroplasts. Without this enzyme, no
plastoquinones are formed, which are required as acceptors for
liberated reduction equivalents during carotinoid biosynthesis
(phytoene desaturation). The fact that HPPD has a key role in the
plastid metabolism makes it an interesting target for herbicides.
Sulcotriones efficiently inhibit the activity of the enzyme
(Schultz et al., FEBS Lett. (1993) 318: 162 - 166).
Sequences of HPPD-specific genes are already known from the
organisms mentioned below:
Access number
Organism Sequence database
nee
Humans HPPD HUMAN X72389
Pig HPPD PIG D13390
Rat HPPD RAT M18405
Mouse HPPD MOUSE D29987
Streptomyces SA11864 U11864
avermitilis
Pseudomonas
sp. strain HPPD PSESP P80064
P.J. 874
Arabidopsis HPPD_ARAB1 AF900228
HPPD ARAB2 U89267
0050/48141 CA 02296840 2000-O1-13
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Furthermore, the following sequences, which show a marked
homology with HPPD sequences, can be found in the databases:
PEA3 MOUSE: Mus muscula (mouse) PEA3 polypeptide, AC X63190;
MELA SHECO: Shewanella colwelliana, melA protein, AC M59289.
WO 96/38567 describes the HPPD DNA sequence from Arabidopsis
thaliana and Daucus carota.
A knowledge of the HPPD DNA sequences is an absolute prerequisite
both for the use in crop protection for the generation of
herbicide-resistant plants and for increasing the vitamin E
synthesis in plants, for example for producing animal feeds with
elevated vitamin E content.
It is an object of the present invention to develop a transgenic
plant with elevated vitamin E content.
20 It is a further object of the present invention to develop a
transgenic plant which is resistant to HPPD inhibitors.
We have found that these objects are achieved, surprisingly, by
overexpressing an HPPD gene in the plants.
It is an additional object of the present invention to develop a
test system for identifying HPPD inhibitors.
We have found that this object is achieved by expressing a barley
HPPD gene in a plant or in a microorganism and subsequently
testing chemicals for inhibition of HPPD enzyme activity.
A first aspect of the present invention relates to the cloning of
the complete barley HPPD gene via isolating the
HPPD-gene-specific cDNA (HvSD36).
During leaf senescence, the vitamin E content in the leaves is
markedly increased (Rise et al., Plant Physiol. (1989) 89:
1028 - 1030).'The monocotyledonous leaf of barley represents a
gradient of cells of different ages since the leaf has a basal
meristem, from which new cells are formed by successive division.
Thus, the oldest cells are located at the leaf tip and the
youngest at the base. Fig. 1 shows a diagram of the primary leaf
of barley on various days after sowing. The total leaf length
measured can be seen from the scale on the left-hand side. Shown,
and termed I - IV, are the leaf sections of the primary leaf which
are differentiated to various degrees and which have been
~
0050/48141 CA 02296840 2000-O1-13
6
selected for gene expression analysis. The plants were raised in
a daily light/dark photoperiod (L/D) and, for inducing
senescence, were excised after 6 days and incubated for 2 days in
the dark (2 nD). A "Northern blot" analysis of RNA from the barley
primary leaf from sections which had differentiated to various
degrees (see Fig. 2) suggest that HPPD expression in barley is
controlled in a development-dependent manner. Thus, copious
accumulation of the approx. 1600 nt long transcript takes place in
the meristematic region on the primary leaf base (I). The content
of this transcript decreases with increasing age of the tissue
(IIa and IIb) and increases again in the fully differentiated
cells with mature chloroplasts (III). Finally, the content of the
1600 nt long transcript is highest in the senescing sections of
the primary leaf (IV). In addition, an approx. 3100 nt long
transcript can be detected only in the meristematic cells on the
base of the primary leaf. Again, this transcript can no longer be
detected with increasing tissue maturation.
With the aid of the so-called "Differential Display" method, a
207 by cDNA fragment was first isolated whose corresponding
transcript accumulates in the primary leaf of barley in the case
of dark-induced senescence. This fragment (sequence protocol:
sequence ID NO:1: nucleotide position 1342 - 1549) was
subsequently used as a probe to isolate a cDNA clone with a
larger insert in a cDNA library (in ~,-ZAP-II) from senescing
barley flag leaves:
Diagram of the cDNA subclone HvSD 36 from the ~,-ZAP-II library:
,! 3 0
T~ -~XhoI SaII HindIII EcoRI NcoI PstI EcoRI PstI BamHI XbaI ~- T3
3 5 14 by 759 by 14 by
The cDNA fragment (sequence protocol: Sequence ID NO:1:
nucleotide position 771 - 1529) was cloned into the EcoRl
40 cleavage site of pBluescript(SK-). In addition, both ends of the
cDNA are equipped with a 14 by adaptor sequence which was required
for ligation into ~,-ZAP-II. Selected restriction sites of the
vector and of the cDNA itself are shown.
45 The 759 by long cDNA fragment was used as probe in a further
experiment to obtain a complete sequence of HvSD 36. To this end,
a cDNA library from RNA of the meristematic section of 5-day-old
. ~ 0050/48141
CA 02296840 2000-O1-13
7
barley seedlings was available. The lambda phage ExCeli Eco RICIP
from Pharmacia (Freiburg) (product number: 27-5011, 45.5kb) was
used for this cDNA library.
A 1565 by long cDNA clone was isolated, see sequence protocol:
sequence ID NO:1: and 2.
Amongst the sequences in the databases, the 434 amino acids long
protein sequence has a homology of 58%, which is the highest
homology with the HPPD sequence from Arabidopsis tha3iana.
To find a genomic clone which contains the complete HPPD gene
sequence, a lambda FIXII library of barley was obtained from
Stratagene (Heidelberg, product number 946104). The library was
prepared using DNA from etiolated leaves of winter barley cv.
Igri. The DNA was subjected to partial digestion with Sau3AI.
Prior to cloning into the Xhol cleavage site of the vector, the
fragment ends and the phage arms were filled up with nucleotides.
Screening of the library with 200,000 pfu in the first round gave
only one clone which hybridized with cDNA HvSD36. After
subjecting this recombinant phage to restriction digestion with
PstI and SacI, fragments of a size of 5400, 3800 and 1800 bp,
respectively, were isolated which can be detected in a "Southern"
blot hybridization with the HvSD36 probe. These sub-fragments
exist in cloned form in the Bluescript vector. Figure 3 shows the
construction of the barley HPPD gene in the form of a diagram.
The invention relates in particular to expression cassettes whose
sequence encodes an HPPD or a functional equivalent thereof, and
," 30 to the use of these expression cassettes for generating a plant
with an elevated vitamin E content. The nucleic acid sequence may
be, for example, a DNA or a cDNA sequence. Encoding sequences
which are suitable for insertion into an expression cassette
according to the invention are, for example, those which encode
an HPPD and which impart, to the host, the ability to overproduce
vitamin E.
In addition, the expression cassettes according to the invention
comprise regulatory nucleic acid sequences which govern
expression of the encoding sequence in the host cell. In
accordance with a preferred embodiment, an expression cassette
according to the invention comprises upstream, ie. on the 5' end
of the encoding sequence, a promoter and downstream, ie. on the
3' end, a polyadenylation signal and, if appropriate, other
regulatory elements which are operatively linked with the
encoding sequence for the HPPD gene which is located in-between.
Operative linkage is to be understood as meaning the sequential
0050/48141 CA 02296840 2000-O1-13
8
arrangement of promoter, encoding sequence, terminator and, if
appropriate, other regulatory elements in such a way that each of
the regulatory elements can fulfill its function as intended when
the encoding sequence is expressed. The sequences preferred for
operative linkage, but not limited thereto, are targeting
sequences for guaranteeing subcellular localization in the
apoplast, in the vacuole, in plastids, in the mitochondrium, in
the endoplasmatic reticulum (ER), in the nucleus, in liposomes or
in other compartments and translation enhancers such as the
5' leader sequence from the tobacco mosaic virus (Gallie et al.,
Nucl. Acids Res. 15 (1987) 8693 - 8711).
For example, the plant expression cassette can be incorporated
into the tobacco transformation vector pBinAR-Hyg. Fig. 4 shows
the tobacco transformation vectors pBinAR-Hyg with 35S
promoter (A) and pBinAR-Hyg with seed-specific promoter phaseolin
796 (B):
- HPT: hygromycin phosphotransferase
- OCS: octopine synthase terminator
- PNOS: nopaline synthase promoter
- those restriction sites which cleave the vector only once are
also shown.
Suitable as promoters of the expression cassette according to the
invention are, in principle, all promoters which can control the
expression of foreign genes in plants. In particular a plant
promoter or a promoter derived from a plant virus is preferably
used. Particularly preferred is the CaMV 35S promoter from
;- 30 cauliflower mosaic viruss (Franck et al., Cell 21 (1980)
285 - 294). It is known that this promoter contains various
recognition sequences for transcriptional effectors which in
their entirety lead to permanent and constitutive expression of
the gene introduced (Benfey et al., EMBO J. 8 (1989) 2195 - 2202).
The expression cassette according to the invention may
additionally comprise a chemically inducible promoter by means of
which expression of the exogenous HPPD gene in the plant can be
controlled at a specific point in time. Such promoters which can
be used are, inter alia, for example the PRP1 promoter (Ward et
al., Plant. Mol. Biol. 22 (1993), 36I-366), a promoter which can
be induced by salicylic acid (WO 95/19443), a promoter which can
be induced by benzenesulfonamide (EP-A 388186), a promoter which
can be induced by tetracyclin (Gatz et al., (1992) Plant J. 2,
397-404), a promoter which can be induced by abscisic acid
~
. , 0050/48141
CA 02296840 2000-O1-13
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(EP-A 335528) or a promoter which can be induced by ethanol or
cyclohexanone (WO 93/21334).
Furthermore, particularly preferred promoters are those which
ensure expression in tissues or plant organs in which the
biosynthesis of vitamin E, or its precursors, takes place.
Promoters which must be mentioned in particular are those which
guarantee leaf-specific expression. Promoters which may be
mentioned are the potato cytosolic FBPase or the potato ST-LSI
promoter (Stockhaus et al., EMBO J. 8 (1989) 2445 - 245).
With the aid of a seed-specific promoter, it was possible stably
to express a foreign protein in the seeds of transgenic tobacco
plants in an amount of up to 0.67% of the total soluble seed
protein (Fiedler and Conrad, Bio/Technology 10 (1995),
1090-1094). The expression cassette according to the invention
can therefore contain, for example, a seed-specific promoter
(preferably the phaseolin promoter (US 5504200), the USP
(Baumlein, H. et al. Mol. Gen. Genet. (1991) 225 (3), 459 - 467)
or LEB4 promoter (Fiedler and Conrad, 1995)), the LEB4 signal
peptide, the gene to be expressed and an ER retention signal. The
construction of such a cassette is shown in the form of a diagram
in Figure 4 by way of example.
An expression cassette according to the invention is prepared by
fusing a suitable promoter with a suitable HPPD DNA sequence and
preferably a DNA which is inserted between promoter and HPPD DNA
sequence and which encodes a chloroplast-specific transit
peptide, and a polyadenylation signal, using customary
;-- 30 recombination and cloning techniques as they are described, for
example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and
L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et
al., Current Protocols in Molecular Biology, Greene Publishing
Assoc. and Wiley-Interscience (1987).
Particularly preferred sequences are those which guarantee
targeting into the apoplast, into plastids, into the vacuole, the
mitochondrium, the endoplasmatic reticulum (ER), or, by means of
the absence of suitable operative sequences, the remaining in the
compartment of formation, the cytosol (Kermode, Crit. Rev. Plant
Sci. 15, 4 (1996), 285 - 423). Localization in the ER has proved
to be especially advantageous for the amount of protein
0050/48141
CA 02296840 2000-O1-13
accumulation in transgenic plants (Schouten et al. , Plant Mol.
Biol. 30 (1996), 781 - 792).
The invention also relates to expression cassettes whose DNA
5 sequence encodes an HPPD fusion protein, a moiety of the fusion
protein being a transit peptide which governs translocation of
the polypeptide. Especially preferred are chloroplast-specific
transit peptides which are cleaved enzymatically from the HPPD
moiety after the HPPD gene product has been translocated into the
10 chloroplasts. Particularly preferred is the transit peptide which
is derived from plastid transketolase (TK) or a functional
equivalent of this transit peptide (eg. the transit peptide of
the small subunit of rubisco or of Ferredoxin NADP
oxidoreductase).
t 15
The HPPD-encoding nucleotide sequence inserted can be prepared
synthetically or obtained naturally or comprise a mixture of
synthetic and natural DNA components. In general, there are
prepared synthetic nucleotide sequences with codons which are
preferred by plants. These codons which are preferred by plants
can be determined from amongst codons with the highest protein
frequency which are expressed in most interesting plant species.
When preparing an expression cassette, various DNA fragments may
be manipulated in order to obtain a nucleotide sequence which
expediently reads in the correct direction and which is provided
with a correct reading frame. To connect the DNA fragments to
each other, adaptors or linkers may be joined onto the fragments.
The promoter and terminator regions according to the invention
may advantageously be provided, in the direction of
transcription, with a linker or polylinker which comprises one or
more restriction sites for insertion of this sequence. As a rule,
the linker has 1 to 10, in most cases 1 to 8, preferably 2 to 6,
restriction sites. In general, the linker within the regulatory
regions has a size of less than 100 bp, frequently less than
60 bp, but at least 5 bp. The promoter according to the invention
may be both native, or homologous, but also foreign, or
heterologous, to the host plant. The expression cassette
according to the invention comprises, in the 5'-3' transcription
direction, the promoter according to the invention, any desired
DNA sequence and a region for transcriptional termination.
Various termination regions can be exchanged for each other as
desired.
It is furthermore possible to employ manipulations which provide
suitable restriction sites or which remove excess DNA or
restriction sites. Where insertions, deletions or substitutions,
. ~ 0050/48141
CA 02296840 2000-O1-13
11
eg. transitions and transversions, are suitable, it is possible
to use in vitro mutagenesis, primer repair, restriction or
ligation. In the case of suitable manipulations, eg. restriction,
chewing back or filling up overlaps for blunt ends, complementary
ends of the fragments may be provided for ligation.
What may be of importance for the success according to the
invention is, inter alia, attaching the specific ER retention
signal SEKDEL (Schouten, A. et al. Plant Mol. Biol. 30 (1996),
781 - 792), which results in a three to four times higher than ,
average expression level. Other retention signals which occur
naturally in plant and animal proteins which are localized in the
ER may also be used for constructing the cassette.
Preferred polyadenylation signals are plant polyadenylation
signals, preferably those which correspond essentially to T-DNA
polyadenylation signals from Agrobacterium tumefaciens, in
particular of gene 3 of the T-DNA (octopine synthase) of the Ti
plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 et seq.), or
functional equivalents.
An expression cassette according to the invention may comprise,
for example, a constitutive promoter (preferably the CaMV 35S
promoter), the LeB4 signal peptide, the gene to be expressed and
the ER retention signal. The preferred ER retention signal used
is the amino acid sequence KDEL (lysine, aspartic acid, glutamic
acid, leucine).
The fused expression cassette which encodes an HPPD gene is
preferably cloned into a vector, for example pBinl9, which is
suitable for transforming Agrobacterium tumefaciens. Agrobacteria
which are transformed with such a vector can then be used in the
known manner for transforming plants, in particular crop plants,
eg. tobacco plants, for example by immersing scarified leaves or
leaf sections in an agrobacteria solution and subsequently
growing them in suitable media. The transformation of plants by
means of agrobacteria is known, inter alia, from F.F. White,
Vectors for Gene Transfer in Higher Plants; in Transgenic Plants,
Vol. 1, Engineering and Utilization, Eds. S.D. Kung and R. Wu,
Academic Press, 1993, pp. 15 - 38. The transformed cells of the
scarified leaves or leaf sections can be used for regenerating,
in the known manner, transgenic plants which contain a gene for
expression of an HPPD gene integrated into the expression
cassette according to the invention.
0050/48141
CA 02296840 2000-O1-13
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To transform a host plant with an HPPD-encoding DNA, an
expression cassette according to the invention is incorporated
into a recombinant vector in the form of an insertion, and the
vector DNA of this recombinant vector additionally comprises
functional regulation signals, for example sequences for
replication or integration. Suitable vectors are described, inter
alia, in "Methods in Plant Molecular Biology and Biotechnology"
(CRC Press), Chapter. 6/7, pp. 71 - 119 (1993).
Using the above-cited recombination and cloning techniques, the
expression cassettes according to the invention can be cloned
into suitable vectors which allow their multiplication, for
example in E. coli. Suitable cloning vectors are, inter alia,
pBR332, pUC series, Ml3mp series and pACYC184. Especially
suitable are binary vectors which are capable of replicating not
only in E. coli, but also in agrobacteria.
The invention furthermore relates to the use of an expression
cassette according to the invention for transforming plants,
plant cells, plant tissues or plant organs. The preferred purpose
of the use is to raise the vitamin E content of the plant.
Depending on the choice of the promoter, expression may take
place specifically in the leaves, in the seeds or in other plant
organs. The present invention also relates to such transgenic
plants, their propagation material and their plant cells, plant
tissues or plant organs.
In addition, the expression cassette according to the invention
may also be employed for transforming bacteria, cyanobacteria,
yeasts, filarnentous fungi and algae with the purpose of raising
the vitamin E production.
The transfer of foreign genes into the genome of a plant is
termed transformation. This process exploits the previously
described methods of transforming and regenerating plants from
plant tissues or plant cells to obtain transient or stable
transformation. Suitable methods are protoplast transformation by
polyethylene-glycol induced DNA uptake, the ballistic method with
the gene gun - the so-called particle bombardment method,
electroporation, incubation of dry embryos in DNA-containing
solution, microinjection and Agrobacterium-mediated gene
transfer. The abovementioned methods are described, for example,
in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic
Plants, Vol. 1, Engineering and Utilization, Eds. S.D. Kung and R.
Wu, Academic Press (1993) 128 - 143, and in Potrykus Annu. Rev.
Plant Physiol. Plant Molec. Biol. 42 (1991) 205 - 225). The
0050/48141
CA 02296840 2000-O1-13
13
construct to be expressed is preferably cloned into a vector
which is suitable for transforming Agrobacterium tumefaciens, for
example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
Agrobacteria transformed with an expression cassette according to
the invention can also be used, in a known manner, for
transforming of plants, in particular crop plants such as
cereals, maize, oats, soya, rice, cotton, sugar beet, canola,
sunflowers, flax, hemp, potatoes, tobacco, tomatoes, oilseed .
rape, alfalfa, lettuce and the various tree, nut and grapevine
species, for example by immersing scarified leaves or leaf
sections in an agrobacteria solution and subsequently growing
them in suitable media.
Functionally equivalent sequences which encode an HPPD gene are,
in accordance with the invention, those sequences which still
have the desired functions despite a different nucleotide
sequence. Thus, functional equivalents embrace naturally
occurring variants of the sequences described herein and also
artificial nucleotide sequences, eg. artificial nucleotide
sequences which have been obtained by chemical synthesis and
which are adapted to the codon usage of a plant.
A functional equivalent is also to be understood as meaning, in
particular, natural or artificial mutations of an originally
isolated HPPD-encoding sequence which continues to show the
desired function. Mutations encompass substitutions, additions,
deletions, inversions or insertions of one or more nucleotide
residues. Thus, the present invention also encompasses those
nucleotide sequences which are obtained by modifying the present
nucleotide sequence. The purpose of such a modification may be,
for example, a further limitation of the encoding sequence
contained therein, or else, for example, the insertion of further
cleavage sites for restriction enzymes.
Functional equivalents are also those variants whose function is
less or more pronounced in comparison with the starting gene or
gene fragment.
Also suitable are artificial DNA sequences as long as they, as
described above, mediate the desired characteristic of raising
the vitamin E content in the plant by overexpressing the HPPD
gene in crop plants. Such artificial DNA sequences can be
determined for example by back-translation of proteins
constructed with the aid of molecular modeling and which have
HPPD activity, or by in vitro selection. Especially suitable are
encoding DNA sequences which were obtained by back-translating a
- ~ 005048141 CA 02296840 2000-O1-13
14
polypeptide sequence in accordance with the codon usage specific
to the host plant. The specific codon usage can be determined
readily by an expert familiar with plant genetic methods using
computer evaluations of other, known genes of the plant to be
transformed.
Further suitable equivalent nucleic acid sequences according to
the invention which must be mentioned are sequences which encode
fusion proteins, a component of the fusion protein being a plant
HPPD polypeptide or a functionally equivalent moiety thereof. The
second moiety of the fusion protein can be, for example, a
further polypeptide with enzymatic activity or an antigenic
polypeptide sequence with the aid of which the detection of HPPD
expression is possible (eg. myc-tag or his-tag). However, this is
preferably a regulatory protein sequence, eg. a signal or transit
peptide, which leads the HPPD protein to the desired site of
action.
However, the invention also relates to the expression products
and fusion products, of a transit peptide and a polypeptide with
HPPD activity, which have been produced in accordance with the
invention.
Raising the vitamin E content means, for the purposes of the
present invention, the artificially acquired ability of an
elevated vitamin E biosynthesis performance by means of
functional overexpression of the HPPD gene in the plant in
contrast to the non-genetically-engineered plant for the duration
of at least one plant generation.
The vitamin E biosynthesis site is generally the leaf tissue, so
that leaf-specific expression of the HPPD gene is expedient.
However, it will be understood readily that vitamin E
biosynthesis is not necessarily restricted to the leaf tissue,
but may also take place tissue-specifically in all other organs
of the plant, for example in fatty seeds.
In addition, constitutive expression of the exogenous HPPD gene
is advantageous. On the other hand, inducible expression may also
appear desirable.
The efficacy of expression of the transgenically expressed HPPD
gene can be determined for example in vitro by shoot meristem
propagation. In addition, changes in the nature and level of HPPD
gene expression, and its effect on the vitamin E biosynthesis
' 0050/48141
CA 02296840 2000-O1-13
performance on test plants, can be tested in greenhouse
experiments.
The invention furthermore relates to transgenic plants
5 transformed with an expression cassette according to the
invention, and to transgenic cells, tissues, organs and
propagation material of such plants. Especially preferred in this
context are transgenic crop plants, eg. barley, wheat, rye,
maize, oats, soya, rice, cotton, sugar beet, canola, sunflowers,
10 flax, hemp, potatoes, tobacco, tomatoes, oilseed rape, alfalfa,
lettuce and the various tree, nut and grapevine species.
Plants for the purposes of the invention are mono- and
dicotyledonous plants or algae.
As already mentioned, HPPD is a suitable target for
sulcotrione-type herbicides. To allow even more efficient HPPD
inhibitors, it is necessary to provide suitable test systems with
which inhibitor/enzyme binding studies can be carried out. To
this end, for example, the complete barley HPPD cDNA sequence is
cloned into an expression vector (pQE, Qiagen) and overexpressed
in E. coli.
The HPPD protein expressed with the aid of the expression
cassette according to the invention is particularly suitable for
finding HPPD-specific inhibitors.
To this end, the HPPD can be employed, for example, in an enzyme
assay in which the HPPD activity is determined in the presence
and absence of the active substance to be tested. A comparison of
the two activity determinations allows qualitative and
quantitative findings on the inhibitory behavior of the active
substance to be tested to be obtained.
The test system according to the invention allows a large number
of chemical compounds to be screened rapidly and simply for
herbicidal properties. The method allows the targeted and
reproducible selection, amongst a large number of substances, of
those with great potency in order to subject these substances
subsequently to further in-depth tests with which the expert is
familiar.
The invention furthermore relates to herbicides which can be
identified with the above-described test system.
0050/48141
CA 02296840 2000-O1-13
16
Overexpression in a plant of the gene sequence Seq ID NO: l, which
encodes an HPPD, results in an elevated resistance to HPPD
inhibitors. The invention also relates to the transgenic plants
thus generated.
The invention furthermore relates to:
- A method of transforming a plant, which comprises introducing
an expression cassette according to the invention into a plant
cell, into callus tissue, into an entire plant or into plant
protoplasts.
- The use of a plant for generating plant HPPD.
;;;;v 15 - The use of the expression cassette according to the invention
for generating plants with elevated resistance to HPPD
inhibitors by means of higher expression of a DNA sequence
according to the invention.
- The use of the expression cassette according to the invention
for generating plants with an elevated vitamin E content by
means of expressing, in plants, a DNA sequence according to the
invention.
- The use of the expression cassette according to the invention
for generating a test system for identifying HPPD inhibitors.
The invention is illustrated by the examples which follow, but
not limited thereto:
35
45
0050/48141
CA 02296840 2000-O1-13
17
General cloning methods
The cloning steps carried out within the scope of the present
invention, eg. restriction cleavages, agarose gel
electrophoresis, purification of DNA fragments, transfer of
nucleic acids onto nitrocellulose and nylon membranes, linking
DNA fragments, transformation of E. coli cells, growing bacteria,
multiplying phages and sequence analysis of recombinant DNA, were
carried out as described by Sambrook et al. (1989) Cold Spring
Harbor Laboratory Press; ISBN 0-87969-309-6).
The bacterial strains used hereinbelow (E. coli, XL-I Blue) were
obtained from Stratagene and, in the case of NP66, Pharmacia. The
i. 15 agrobacterial strain used for the transformation of plants
(Agrobacterium tumefaciens, C58C1 with plasmid pGV2260 or
pGV3850kann) was described by Deblaere et al. in (Nucl. Acids
Res. 13 (1985) 4777). Alternatively, the agrobacterial strain
LBA4404 (Clontech) or other suitable strains may also be
employed. Vectors which can be used for cloning are the vectors
pUCl9 (Vanish-Perron, Gene 33 (1985), 103 - 119) pBluescript SK-
(Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan
et al., Nucl. Acids Res. 12 (1984), 8711 - 8720) and pBinAR
(Hofgen and Willmitzer, Plant Science 66 (1990) 221 - 230).
Sequence analysis of recombinant DNA
Recombinant DNA molecules were sequenced using a laser
fluorescence DNA sequencer by Licor (available from MWG Biotech,
Ebersbach) following the method of Sanger (Sanger et al., Proc.
Natl. Acad. Sci. USA 74 (1977), 5463 - 5467).
Generation of plant expression cassettes
Into plasmid pBinl9 (Bevan et al., Nucl. Acids Res. (1984) 12,
8711) there was inserted a 35S CaMV promoter in the form of an
EcoRI-KpnI.fragment corresponding to nucleotides 6909 - 7437 of
cauliflower mosaic virus (Franck et a1. Cell 21 (1980) 285). The
polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid
pTiACHS (Gielen et al., EMBO J. 3 (1984) 835), nucleotides
11749 - 11939, was isolated as a PvuII-HindIII fragment and, after
addition of SphI linkers, cloned into the PvuII cleavage site
between the SpHI-HindIII cleavage site of the vector pBmAR-Hyg.
This gave the plasmid pBinAR (Hofgen and Willmitzer, Plant
Science 66 (1990) 221 - 230).
005048141 CA 02296840 2000-O1-13
18
Use Examples
Example 1
Isolation of HPPD-specific cDNA sequences
The composition of the mRNA population from primary leaves of
nine-day-old barley plants which had been grown in an L/D
photoperiod (16 hours light/8 hours dark) was compared with that
of primary leaves of 11-day-old barley plants in which, after a
raising period of nine days, senescence was subsequently induced
by a two-day dark treatment (Humbeck and Krupinska, J. Photochem.
Photobiol. 36 (1996), 321 - 326) with the aid of the DDRT-PCR
method published by Liang and Pardee (Science (1992) 257, 967 -
972). In each case 0.2 p,g of the total RNA was converted into cDNA
using the enzyme "Superskript RT" (Gibco BRL, Eggenstein). In
addition to the RNA, the reaction batches (20 ~1) also contained
~tM dNTPs, 10 ~M DTT, lxRT buffer and in each case 1 ~M
(dT)12VN primer. The anchor "primers" required for these
20 reactions were synthesized on the basis of the data of Liang and
Pardee:
1. 5'-TTTTTTTTTTTTAG-3'
2. 5'-TTTTTTTTTTTTCA-3'
3, 5'-TTTTTTTTTTTTAC-3'
4. 5'-TTTTTTTTTTTTGT-3'
After the cDNAs were synthesized, amplification of the relevant
sequences was effected in each case in ten batches, which differ
by the use of the random "primers" given hereinbelow:
1. 5 '-TACAACGAGG-3' 2. 5'-GGAACCAATC-3'
3. 5 '-AAACTCCGTC-3' 4. 5'-TGGTAAAGGG-3'
5. 5 '-CTGCTTGATG-3' 6. 5'-GTTTTCGCAG-3'
5 '-GATCTCAGAC-3' 8. 5'-GATCTAACCG-3'
7.
9. 5 '-GATCATGGTC-3' 10. 5'-GATCTAAGGC-3'
In a volume of in each case 20 ~,1, the PCR reaction batches
contained lxPCR buffer, 2 ~M dNTPs, 2.5 ~.Ci (a 33P)-dATP, 1 [~M
(dT)12VN-..primer", 1/10 vol. RT mix (Sambrook et al. Molecular
Cloning - A Laboratory Manual, 1989), 1 U Taq DNA polymerase
(Boehringer, Mannheim) and 1 p,M 10-mer random "primers". The
PCR-reactions proceeded in a Uno block (Biometra) following the
program below:
1. 94°C 2 min
2. 94°C 30 s
0050/48141
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v 19
3. 40°C 2 min
4. 72°C 30 s
5. 72°C 5 min
6. 4°C storage until further processing
Steps 2, 3 and 4 were carried out 40 times in succession. This
gave approximately 100 cDNA bands per reaction and "primer"
combination.
In contrast to the protocol of Liang and Pardee, the amplified
cDNA fragments were separated in non-denaturing polyacrylamide
gels of the following composition: 6% (w/v) acrylamide (Long
Ranger, AT Biochem), 1.2 x TBE buffer, 0.005% (v/v) TEMED and
0.005% (w/v) APS (Bauer et al, Nucl. Ac. Res. (1993) 21,
4272 - 4280 ) .
In each case 3.5 [~1 of each PCR batch were treated with 2
~.1 of
loading buffer (dye II, Sambrook et al., 1989) and then loaded
onto the gel. To determine the reproducibility of the cDNA
band
patterns (Fig. 5), in each case two independent RNA preparations
(9 and 9', 11 and 11') were prepared from the barley primary
leaves harvested on days 9 and 11 and used in parallel in
the
analysis below. What is shown is the result of two different
primer combinations (A and B); by way of example, two differences
in the band pattern between the sample of days 9 and 11 were
emphasized by arrows. Only those bands which occurred equally
in
the two samples from senescing plants and which did not occur
in
the two comparison samples were taken into consideration when
analyzing the gels at a later point in time. Electrophoresis
was
carried out over a period of 2.5 hours at 40 watt (0.8 w/cm3)
in
1 x TBE buffer. After separation of the cDNA fragments had
occurred, the gel was transferred onto filter paper (Schleicher
&
Schiill, Dassel). After the gel had been dried at 50C, an
X-ray
film was placed on top of it. cDNA bands which were only found
in
the case of samples 11 and 11' in the autoradiograph were
excised
from the dry gel using a surgical blade, and the DNA was eluted
by boiling in 100 ~1 1 x TE buffer. The ethanol-precipitated
DNA
was resuspended in 10 ~.1 of water for further tests. After
reamplification with the "primers" previously used for this
batch, the DNA was cloned and sequenced and also employed
as a
probe for Northern blot hybridizations.
To test if the relevant cDNA fragment actually represents a
senescence-specifically occurring transcript, hybridizations were
carried out with RNA from leaves of various developmental stages:
' 0050/48141
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A. 1. RNA from primary leaves from plants raised for 9 days in
an L/D photoperiod
A. 3. RNA from primary leaves from 10-day-old plants raised
5 without a light phase on day 10
A. 4 RNA from primary leaves from 11-day-old plants which
lacked a light phase on days 10 and 11
10 A. 5 RNA from primary leaves from 12-day-old plants which
underwent a further light phase after 2 days in the dark
The samples for RNA analysis were harvested in each case in the
middle of the original night phase.
B. RNA from flag leaves which had been collected in the
field at seven different points in time (Fig. 6). The
leaves were fully grown on 29 May and showed less than
10~ of the original chlorophyll content on 21 June. The
beginning of the senescence processes is shown in
Figure 6 by an arrow (ie. 17 days after reaching the full
length on 15 June). The beginning of senescence was
defined as the day on which photosystem II efficacy
dropped (Humbeck et al., Plant Cell Environment (1996)
19 : 337 - 344 ) .
To hybridize a filter with the above-described RNA samples, a
specific probe for the rbcS gene, which encodes the small
sub-unit of ribulose-1,5-bisphosphate carboxylase, was also
employed in addition to the HPPD probe, for comparison reasons.
Figure 6 shows hybridization of the "Northern blots" A and B with
cDNA HvSD36 and with a probe which is specific for the rbcS gene.
Filter A carries RNA from barley primary leaves after a raising
period of 9 days in an L/D photoperiod (9), after subsequent
incubation in the dark for one and two days, respectively (10,
11) and after subsequent return to light conditions for one day
(12). Filter B contains RNA from flag leaves which had been
harvested in the field in the period from 29.05. to 21.06.1992.
The arrow indicates the beginning of senescence on 15.06. As can
been seen from Figure 6, the amount of rbcS-specific mRNA is high
when the amount of HPPD-specific mRNA is relatively low. In
primary leaves of nine-day-old plants, the HPPD-specific mRNA is
not detectable prior to transfer into the dark and accumulates
markedly during the dark phase. When the plants are returned to
light conditions, the amount of this mRNA drops markedly. In the
case of the flag leaves, small amounts of the HPPD-specific mRNA
can already be detected in fully-grown, non-senescent leaves. As
' 0050/48141
CA 02296840 2000-O1-13
21
early as 4 days prior to the actual beginning of senescence,
expression levels are higher. The highest amount of this mRNA can
be found in senescent leaves. A size comparison with known RNA
species showed that the transcript detected with the cDNA probe
HvSD36 (s: senescence; d: dark, fragment number 36 in the DDRT
gel) has a length of approx. 1.6 kb.
By means of DDRT PCR, three cDNA fragments were obtained
independently of each other which showed this expression pattern
and which, on the basis of sequence analysis, actually represent
the same transcript. The longest fragment had a size of 230 bp.
The 230 by long PCR product was finally cloned into the SmaI
cleavage site of vector pUCl8 using the "Sure CloneTM ligation
kit" (Pharmacia, Freiburg) following the manufacturer's
t 15 instructions. The recombinant plasmid was transformed into
competent cells of E. coli strain DH5 a. Since, for methodology
reasons, the fragment represents the 3' end of the relevant
transcript, the sequence information was first insufficient to
identify an unambiguous homology with a sequence in the
databases. To isolate a longer corresponding cDNA, a lambda ZAPII
library (Stratagene, Heidelberg) of RNA of senescent flag leaves
was screened using the 230 by long fragment as the probe. For this
step, the probe was labeled with Dig-dUTP following the
instructions of the "DNA Labeling and Detection Kit" (Boehringer,
Mannheim). The library was examined following the protocol of the
"ZAP-cDNA Synthesis Kit" (Stratagene, Heidelberg).
In the case of the probe described herein, 150,000 pfu were
examined. Of these, 39 phage plaques gave a positive signal. Of
these, further work was carried out on 12 phage populations.
Following phage preparation, the fragments inserted were enriched
via PCR and separated by electrophoresis. Southern blot
hybridization with the HvSD36 probe allowed those phage
populations which had the largest "inserts" with positive signal
to be selected amongst the 12 phage populations thus treated.
After replating, the phages were subjected to a further
hybridization step. Single phage plaques were excised and, after
elution, subjected to an in vivo excision using a helper phage
and following the protocol from Stratagene (ExassistTM
Interference-Resistant Helper Phage with SOLR TM Strain). The
so-called "phagemids" obtained from this treatment contain the
cDNA cloned in pBLueskript (SK-).
Following a subsequent plasmid preparation, the relevant "insert~
was excised from the Bluescript plasmid using EcoRI. The cDNA
clone obtained in the case of HvSD36 contains an "insert" with a
length of approx. 800 bp. Complete sequencing of the cDNA was
0050/48141 CA 02296840 2000-O1-13
22
carried out using the "SequiTherm Excel Long-Read
DNA-Sequenzierungs-Rit" (Epicentre Technologies, Biozym
Diagnostic, Oldendorf) using IRD41-labeled universal "primers"
which bind to sequence regions in the Bluescript vector.
Detection of the DNA fragments was effected via the infrared
laser of the automatic sequencer 4000L by Licor. After
sequencing, an exactly 759 by long sequence was present whose
sides are flanked by an in each case 14 by long adaptor sequence.
These adaptor sequences were used for ligating the cDNA fragments
with the arms of phage lambda ZAPII (Stratagene, Heidelberg) when
generating the c-DNA library.
Amongst the sequences in the databases, the protein sequence
HvSD36, which has a total of over 180 amino acids, has a homology
of 41% with the sequence of human HPPD which is the highest.
Taking into consideration the length of the transcript detected
in the "Northern blot" (approx. 1600 nt), it can be assumed that
850-900 by are still missing from the cDNA.
To complete the cDNA, a further cDNA library was investigated.
mRNA was isolated from the basal meristematic zone of 5-day-old
barley seedlings with the aid of "Dynabeads" (Dynal, Hamburg) and
transcribed into cDNA using the "Time Saver cDNA SyntheseRit"
(Pharmacia, Freiburg). This was followed by ligation of
EcoRI/NotI adaptors (Pharmacia, Freiburg) to the cDNA with
subsequent ligation into the lambda ExCell vector (Pharmacia,
Freiburg). Finally, the recombinant phage DNA was packaged into
phage proteins with the aid of "Gigapack II Gold Set" (Stratagene,
Heidelberg). Using the 759 by long probe HvSD36, 400,000 pfu were
screened, and 5 phages were detected by the probe. Excision of the
"phagemids" from the phage was effected in vivo with the aid of
bacterial strain NP66 following the instructions of Pharmacia
(Freiburg). The recombinant pExCell plasmids were isolated from
the individual bacterial colonies and transferred into bacterial
strain D115 a for propagation.
The longest cDNA clone HvSD36 isolated in this manner has a
length of 1565 by and was sequenced completely (see sequence
protocol).
Example 2
Characterization of the genomic sequence
To identify a genomic clone which contains the gene sequence of
HPPD, a lambda FIXII library of barley was obtained from
Stratagene (Heidelberg). The library was prepared using DNA from
005048141 CA 02296840 2000-O1-13
23
etiolated leaves of winter barley cv. Igri. The DNA was partially
digested with Sau3AI. Prior to cloning into the XhoI cleavage
site of the vector, the fragment ends and the phage arms were
filled up with nucleotides. Screening of the library with
200,000 pfu in the first round only gave one clone which
hybridized with cDNA HvSD36. After subjecting this recombinant
phage to restriction digestion with PstI and SacI, fragments
5400, 3800 and 1800 by in length were subsequently isolated which
can be detected with the HvSD36 probe when carrying out a
"Southern" blot hybridization. These sub-fragments exist in
cloned form in the Bluescript vector.
The library was screened following the protocol given for the
HybondN membrane. Labeling of the probe for screening the library
(;~ 15 and for the "Southern" blot hybridizations was effected via
"random priming" with 32P-dATP using the Klenow enzyme (Sambrook
et al., (1989) Molecular cloning. A laboratory manual. Cold
Spring Harbor Laboratory, New York).
A genomic "Southern blot" was carried out with total DNA from
barley (farina) (Fig. 7). In each case 15 ~tg of DNA were digested
with BamHI (B), EcoRI (E), HindIII (H) or XBAI (X) and separated
in a 0.75 agarose gel. After transfer to a Hybond N+ membrane
(Amersham, Braunschweig), hybridization was effected with the
incomplete, 759 by long cDNA probe from HvSD36 following
instructions of the membrane manufacturer. The following
fragments were detected:
BamHI: 6.0, 3.9 and 3.0 kbp
~' 30 EcoRI: >10 kbp
~HindIII: 8.3, 2.6, 1.1 and 1.0 kbp
XbaI: 9.0, 5.2 and 4.2 kbp
The fragment lengths were estimated by comparison with a DNA size
standard (Kb-Leiter, GibcoBRL, Eggenstein).
Example 3
Homology comparison of the HvSD36 protein sequence
A comparison of the HvSD36 protein sequence with protein
sequences in the database revealed homologies to the following
protein sequences known to date:
~
~ 0050/48141 CA 02296840 2000-O1-13
24
20 30 40 50
5 HPPD Hv .......... .......... .......... ........MP PTPTTPAATG
HPPD_Ath .......... .........~ ..~~~~~~~~ ~-.MGHQNAA VSENQNHDDG
HPPD_HUMAN .......... .......... .......... .......... ..........
HPPD_RAT .......... .......... .......... .......... ..........
HPPD_PIG .......... .......... .......... .......... ..........
HPPD_MOUSE .......... ........~. .......... .......... ..........
HPPD_PSESP .......... .......... .......... .......... ..........
10 MELA_SHECO .......... .......... .......... .......... ..........
PEA3 MOUSE MTKSSNHNCL LRPENKPGLW GPGAQAASLR PSPATLWSS PGHAEHPPAA
60 70 80 90 100
HPPDHv AAAAVTPEHA RPHRMVRFNPRSDRFHTLSFHHVEFWCADAASAAGRFAFA
_ Ath AASSPGFKLV GFSKFVRKNPKSDKFKVKRFHHIEFWCGDATNVARRFSWG
HPPD
t;',' 15 HPPDHUMAN _ M TTYSDKGAKPERGRFLH--FHSVTFWVGNAKQAASFYCSK
_ RAT YWDKGPKP ERGRFLH--FHSVTFWVGNAKQAASFYCNK
HPPD
_ PIG M TSYSDKGEKPERGRFLH--FHSVTFWGNARQAASYYCSK
HPPD
_ MOUSE M TTYNNKGPKPERGRFLH--FHSVTFWVGNAKQAASFYCNK
HPPD
_ PSESP ADLYENP MGLMGFEFIELASPTPNTLE
HPPD
_ SHECO MASEQNP LGLLGIEFTEFATPDLDFMH
MELA
_ MOUSE PAQTPGPQVS ASARGPGPVAGGSGRMERRMKGGYL---DQRVPYTFCSKS
PEA3
110 120 130 140 150
HPPDHv LGAPLAARSD LSTGNSAHASQLLRSGSLAFLFT--APYANG-CDAA----
_ Ath LGMRFSAKSD LSTGNMVHASYLLTSGDLRFLFT--APYSPS-LSAGEIKP
HPPD
_ HUMAN MGFEPLAYRG LETGSREWSHVIKQGKIVFVLS--SA----------LNP
HPPD
_ RAT MGFEPLAYKG LETGSREWSHVIKQGKIVFVLC--SA----------LNP
HppD
HPPDPIG IGFEPLAYKG LETGSREWSHVVKQDKIVFVFS--SA----------LNP
_ MOUSE MGFEPLAYRG LETGSREWSHVIKRGKIVFVLC--SA----------LNP
HPPD
_ PSESP PIFEIMGFTK VATHRSKDV-HLYRQGAINLILN--NE-------------
HPPD
_ SHECO KVFIDFGFSK LKKHKQKDI-WYKQNDINF LLN--NE-------------
MELA
_ MOUSE PGNGSLGEAL MVPQGKLMDPGSLPPSDSEDLFQDLSHFQETWLAEAQVPD
PEA3_
y- 30
160 170 180 190 200
HPPDHv --TASLPSFS ADAARRFSADHGIAVRSVALRVADAAEAFRASRRRGARPA
_ Ath TTTASIPSFD HGSCRSFFSSHGLGVRAVAIEVEDAESAFSISVANGAIPS
HPPD
_ HUMAN --------WN KEMGDHL-VKHGDGVKDIAFEVEDCDYIVQKARERGAKIM
HPPD
_ RAT --------WN KEMGDHL-VKHGDGVKDIAFEVEDCEHIVQKARERGAKIV
HPPD
_ PIG --------WN KEMGDHL-VKHGDGVKDIAFEVEDCDYIVQKARERGAIIV
HppD
HPPDMOUSE --------WN KEMGDHL-VKHGDGVKDIAFEVEDCDHIVQKARERGAKIV
_ PSESP ---------P HSVASYFAAEHGPSVCGMAFRVKDSQKAYKRALELGAQPI
HPPD
_ SHECO ---------K QGFSAQFAKTHGPAISSMGWRVEDANFAFEGAVARGAKPA
MELA
_ MOUSE SDEQFVPDFH ---SENLAFHSPTTRIKKEPQSPRTDPALSCSRKPPLPYH
PEA3
210 220 230 240 250
HPPDHv FAPV------ -----DLGRGFAFAEVELYG--DWLRFVSHP--DG--TD
_ Ath SPPI------ -----VLNEAVTIAEV'KLYG--DVVLRYVSYKAEDT--EK
HPPD
HPPD_ REP------- -WVEQDKFGKVKFAVLQTYG--DTTHTLVEKMN-----YI
HUMAN
HPPD_ REP------- -WVEEDKFGKVKFAVLQTYG--DTTHTLVEKIN-----YT
RAT
HPPD_ REEVC-CAAD VRGHHTPLDRAR----QVWE--GT---LVEKMT-----FC
PIG
HppD_ REP------- -WVEQDKFGKVKFAVLQTYG--DTTHTLVEKIN-----YT
MOUSE
HPPDPSESP HI-------- ----ETGPMELNLPAIKGIG--GAPLYLIDRFGEGSSIYD
MELA_ AD-------- ----EV--KDLPYPAIYGIG--DSLIYFIDTFGDDNNIYT
SHECO
PEA3_ HGEQCLYSRQ IAIKSPAPGAPGQSPLQPFSRAEQQQSLLRASSSSQSHPG
MOUSE
' 0050/48141
CA 02296840 2000-O1-13
260 270 280 29D 300
HPPDHv VPFLPGFEGV TNPDA-----VDYGLTRFDH -APAAAYIAG
WGNVP--EL
_ Ath SEFLPGFERV EDASSF---PLDYGIRRLDH -GPALTYVAG
HPPD AVGNVP--EL
_ HUMAN GQFLPGYEAP AFMDPLLPKLPKCSLEMIDHIVGNQPDQEM-VSASEW---
HPPD
_ RAT GRFLPGFEAP TYKDTLLPKLPSCNLEIIDHIVGNQPDQEM-ESASEW---
HPPD
5 _ PIG LDSRPQPSQT LLHRLLLSKLPKCGLEIIDHIVGNQPDQEM-ESASQW---
HPPD
HPPDMOUSE GRFLPGFEAP TYKDTLLPKLPRCNLEIIDHIVGNQPDQEM-QSASEW---
_ PSESP IDFV--FLEG VDRHPVGA-----GLKIIDHLTHNWRGRM-A---YWANF
HPPD
_ SHECO SDF-----EA LDEPIITQ---EKGFIEVDHLTNNVHKGTM-E---YWSNF
MELA
_ MOUSE HGYLGEHSSV FQQPVDMCHSFTSPQGGGREPLPAPYQHQLSEPCPPYPQQ
PEA3
10 310 320 330 340 350
HPPDHv FT---GFHEF AEFTAEDVGTTESGLNSWL ANNSEGVLLPLNEPVHGTKR
_ Ath FT---GFHQF AEFTADDVGTAESGLNSAVLASNDEMVLLPINEPVHGTKR
HPPD
_ HUMAN YLKNLQFHRF WSVDDTQVHTEYSSLRSIW ANYEESIKMPINEPAPG-KR
HPPD
_ RAT YLKNLQFHRF WSVDDTQVHTEYSSLRSIW ANYEESIKMPINEPAPG-RR
HPPD
_ PIG YMRNLQFHRF WSVDDTQIHTEYSALRSWM ANYEESIKMPINEPAPG-KR
HPPD
_ MOUSE YLKNLQFHRF WSVDDTQVHTEYSSLRSIW TNYEESIKMPINEPAPG-RK
HPPD
;: 15 HPPDPSESP YEKLFNFREI RYF---DIKGEYTGLTSKAMTAPDGMIRIPLNE--ESSKG
'-
_ SHECO YKDIFGFTEV RYF---DIKGSQTALISYAI.RSPDGSFCIPINE--GKGDD
MELA
_ MOUSE NFKQ-EYHDP LYEQAGQPASSQGGVSGHRYPGAGWIKQERTDFAYDSDV
PEA3
360 370 380 390 400
20 HPPDHv RSQIQTFLEH HGGPGVQH-IAVASSDVLRTLRKMRARSAMGGFDFLPPPL
_ Ath KSQIQTYLEH NEGAGLQH-LALMSEDIFRTLREMRKRSSIGGFDFMPSPP
HPPD
_ HUMAN KSQIQEYVDY NGGAGVQH-IALKTEDIITAIRHLRER----GLEFLSVP-
HPPD
_ RAT KSQIQEYVDY NGGAGVQH-IALRTEDIITTIRHLRER----GMEFLAVP-
HPPD
_ PIG KSQIQEYVDY NGGAGVQH-IALKTEDIITAIRSLRER----GVEFLAVP-
HPPD
HPPD_ KSQIQEYVDY NGGAGVQH-IALKTEDIITAIRHLRER----GTEFLAAP-
MOUSE
HPPD_ AGQIEEFLMQ FNGEGIQH-VAFLSDDLIKTWDHLKSI--=-GMRFMTAPP
PSESP
25 MELASHECO RNQIDEYLKE YDGPGVQH-LAFRSRDIVASLDAMEGS---SIQTLDIIP
PEAS_ PGCASMYLHP EGFSGPSPGDGVMGYGYEKSLRPFPDDVCIVPRKFEGDIK
MOUSE
410 420 430 440 450
HPPDHv PKYYEGVRRL AGD--VLSEAQIKECQELGVLVDRDDQG----VLL-----
HPPD_ PTYYQNLKKR VGD--VLSDDQIKECEELGILVDRDDQG----TLL-----
Ath
HPPD_ STYYKQLREK LKTAKIKVKENIDALEELKILVDYDEKG----YLL-----
HUMAN
HPPD_ SSYYRLLREN LKTSKIQVKENMDVLEELKILVDYDEKG----YLL-----
RAT
HPPD_ FTYYRQLQEK LKSAKIRVKESIDVLEELKILVDYDEKG----YLL-----
PIG
HPPD_ SSYYKLLREN LKSAKIQVKESMDVLEELHILVDYDEKG----YLL-----
MOUSE
HPPD_ DTYYEMLEGR LPN----HGEPVGELQARGILLDGSSESGDKRLLL-----
PSESP
MELA_ E-YYDTIFEK LPQ----VTEDRDRIKHHQILVDGDEDG----YLL-----
SHECO
PEA3MOUSE QEGIGAFREG PPYQR------RGALQLWQFLVALLDDPTNAHFIAWTGRG
460 470 480 490 500
HPPDHv QIFTKPVGDR PTLFLEMIQRIGCMEKDERGEE----YQKGGCGGFGKGNF
HPPD_ QIFTKPLGDR PTIFIEIIQRVGCMMKDEEGKA----YQSGGCGGFGKGNF
Ath
HPPD_ QIFTKPVQDR PTLFLEVIQRHNHQ-------------- -GFGAGNF
HUMAN
HPPD_ QIFTKPMQDR PTLFLEVIQRHNHQ-------------------GFGAGNF
RAT
HPPD_ QIFTKPMQDR PTVFLEVIQRNNHQ-------------- ---GFGAGNF
PIG
HPPD_ QIFTKPMQDR PTLFLEVIQRHNHQ-------------------GFGAGNF
MOUSE
HPPD_ QIFSETLMGP --VFFEFIQR-----KGDD------------GFGEGNF
PSESP
MELA_ QIFTKNLFGP --IFIEIIQR-----KNNL--------------GFGEGNF
SHECO
PEAS_ MEFKLIEPEE VARLWGIQKNRPAMNYDKLSRSLRYYYEKG
MOUSE IMQKVAGERY
510 520 530
540 550
HPPDHv --------SE LFK-SIE-DY--EKS--LEA
KQSAAV-QGS
- ~ 0050/48141
CA 02296840 2000-O1-13
26
HPPD Ath --------SE LFK-SIE-EY--EKT--LEAKQLVG
_ HUMAN --------NS LFK-AFEEEQ--NLRGNLTNMETNGVVPGM
HPPD
_ RAT --------NS LFK-AFEEEQ--ALRG
HPPD
_ PIG --------NS LFK-AFEEEQ--ELRGNLTDTDPNGVPFRL
HPPD
_ MOUSE --------NS LFK-AFEEEQ--ALRGNLTDLEPNGVRSGM
HPPD
_ PSESP --------KA LFE-SIERDQ--VRRGVLST-D
HPPD_
MELA SHECO --------KA LFE-SIERDQ--VRRGVL
_ MOUSE VYKFVCEPEA LFSLAFPDNQRPALKAEFDRPVSEEDTVPL SHLDESPAYL
PEA3
560 570
HPPD_Hv
HPPD_Ath
HPPD_HUMAN
HPPD_RAT
HPPD_PIG
HPPD_MOUSE
HPPD PSESP
MELA SHECO
,:.
PEA3 MOUSE PELTGPAPPF GHRGGYSY
Key: HPPD Hv: Hordeum vulgate 4-hydroxyphenylpyruvate
dioxygenase (HvSD36)
HPPD_Ath: Arabidopsis thaliana
4-hydroxyphenylpyruvate dioxygenase
HPPD HUMAN: H.sapiens 4-hydroxyphenylpyruvate
dioxygenase
HPPD_PIG: pig 4-hydroxyphenylpyruvate dioxygenase
HPPD_RAT: rat F alloantigen
HPPD MOUSE: mouse 4-hydroxyphenylpyruvate
dioxygenase
MELA_SHECO: S. colwelliana melA protein
HPPD_PSESP: Pseudomonas sp. (strain P.J.874)
4-hydroxyphenylpyruvate dioxygenase
PEA3 MOUSE: Mus musculus (mouse) PEA3 polypeptide
The greatest homology was with the Arabidopsis sequence,
viz. 58% over the entire sequence (62% over 412 amino
acids), followed by HPPD_RAT with 35% (over 365 amino
acids), HPPD HUMAN 34% (over 365 amino acids), HPPD MOUSE
34% (over 371 amino acids).
Example 4
Raising barley (Hordeum vulgate)
Barley seedlings (Hordeum vulgate L. cv. Carina, Ackermann
Saatzucht, Irbach, Germany) were raised over a period of 15 days
under controlled conditions in a controlled-environment cabinet
in so-called Mitscherlich pots in soil containing 4 g of Osmocote
5M (Urania, Hamburg, Germany) per liter. To ensure uniform
growth, the seeds were germinated on moist filter paper in the
dark for 2 days at 4~C and 1 day at 21~C, and only those seedlings
- ~ 0050/48141 CA 02296840 2000-O1-13
27
were planted which showed the same longitudinal growth of the
primary root. After these seedlings had been transferred onto
soil, they were covered with screened soil to a depth of 1.5 cm.
Thereafter, the plants were incubated for 9 days at 16 hours light
(120 Eumm-2~s'1) and 8 hours darkness in conjunction with a
temperature shift (21~C during the day, 16~C during the night).
After 9 days, the plants were kept for 2 days (days 10 and 11) in
the dark at the abovementioned temperature in order to induce
senescence.
Example 5
Raising tobacco
The tobacco plants were raised following the known method. The
tobacco cultivar used is Nicotiana tabacum cv. Xanthi.
Example 6
Transformation of tobacco
The expression cassette according to the invention comprising the
HPPD gene with Sequence 1 was cloned into vector pBinAR-Hyg
(Fig. 4). Tobacco plants as described in Example 5 were
subsequently transformed with this vector following the known
method.
Example 7
Increasing the tocopherol biosynthesis in tobacco
The HPPD cDNA was provided with a CaMV 35S promoter and
,'- 30 overexpressed in tobacco using the 35S promoter. In parallel, the
seed-specific phaseolin gene promoter was used to increase the
tocopherol content specifically in the tobacco seed. Tobacco
plants which had been transformed with the relevant constructs
were raised in the greenhouse. The a-tocopherol content of the
total plant and of the seeds of the plant was subsequently
determined. In all cases, the a-tocopherol concentration was
increased in comparison with the untransformed plant.
45
~
~ 0050/48141 CA 02296840 2000-O1-13
28
SEQUENCE PROTOCOL
(1) GENERAL
INFORMATION
(i) APPLICANT
(A) NAME: BASF AG
(B) STREET: Carl Bosch
(C) TOWN: Ludwigshafen
(D) FEDERAL COUNTRY: Germany
(F) POSTCODE: 67056
(G) TELEPHONE: 0621-60-52698
(ii) TITLE OF APPLICATION: HPPD sequence from
barley
(iii) NUMBER OF SEQUENCES: 2
(iv) COMPUTER-READABLE FORM:
(A) RECORDING MEDIUM: floppy disk
(B) COMPUTER: IBM PC compatible
_ (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn release #1.0, Version
#1.25 (EPA)
(2) INFORMATION
ON SEQ
ID NO:
1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1565 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETIC: NO
(iii) ANTISENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: hppd from barley
(D) DEVELOPMENTAL STAGE: senescence
(vi1) IMMEDIATE SOURCE:
(A) LIBRARY: lambda FIXII library of barley
4
(B) CLONE: pHvSD36.seq
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) POSITION: 9..1313
(x) PUBLICATION DETAILS:
(A) AUTHORS: Krupinska, Karin
(B) TITLE: Overexpression of HPPD
(C) JOURNAL: overexpression of HPPD
(G) DATE: 1998
(K) RELEVANT RESIDUES IN SEQ ID NO: 1 FROM 1
TO 1565
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CGCACACC ATG CCG CCC ACC CCC ACC ACC CCC GCG GCT ACC GGC GCC GCC 50
Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala
1 5 10
- 0050/48141 CA 02296840 2000-O1-13
..' 2 9
GCC GCG GTG ACG CCG GAG CAC GCG CGA CCG CAC CGA ATG GTC CGC TTC 98
Ala Ala Val Thr Pro Glu His Ala Arg Pro His Arg Met Val Arg Phe
15 20 25 30
AAC CCG CGC AGC GAC CGC TTC CAC ACG CTC TCC TTC CAC CAC GTC GAG 146
Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu
35 40 45
TTC TGG TGC GCG GAC GCC GCC TCC GCC GCC GGC CGC TTC GCG TTC GCG 194
Phe Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala
50 55 60
CTC GGC GCG CCG CTC GCC GCC AGG TCC GAC CTC TCC ACG GGG AAC TCC 242
Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser
65 70 75
GCG CAC GCC TCC CAG.CTG CTC CGC TCG GGC TCC CTC GCC TTC CTC TTC 290
Ala His Ala Ser Gln Leu Leu Arg Ser Gly Ser Leu Ala Phe Leu Phe
80 85 90
ACC GCG CCC TAC GCC AAC GGC TGC GAC GCC GCC ACC GCC TCC CTG CCC 338
Thr Ala Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro
95 100 105 110
TCC TTC TCC GCC GAC GCC GCG CGC CGG TTC TCC GCC GAC CAC GGG ATC 386
Ser Phe Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Ile
115 120 125
GCG GTG CGC TCC GTA GCG CTG CGC GTC GCA GAC GCC GCC GAG GCC TTC 434
Ala Val Arg Ser Val Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe
130 135 140
CGC GCC AGT CGT CGA CGG GGC GCG CGC CCG GCC TTC GCC CCC GTG GAC 482
t,: Arg Ala Ser Arg Arg Arg Gly Ala Arg Pro Ala Phe Ala Pro Val Asp
145 150 155
CTC GGC CGC GGC TTC GCG TTC GCG GAG GTC GAG CTC TAC GGC GAC GTC 530
Leu Gly Arg Gly Phe Ala Phe Ala Glu Val Glu Leu Tyr Gly Asp Val
160 165 170
GTG CTC CGC TTC GTC AGC CAC CCG GAC GGC ACG GAC GTG CCC TTC TTG 578
Val Leu Arg Phe Val Ser His Pro Asp Gly Thr Asp Val Pro Phe Leu
175 180 185 190
CCG GGG TTC GAG GGC GTA ACC AAC CCG GAC GCC GTG GAC TAC GGC CTG 626
Pro Gly Phe Glu Gly Val Thr Asn Pro Asp Ala Val Asp Tyr Gly Leu
195 200 205
ACG CGG TTC GAC CAC GTC GTC GGC AAC GTC CCG GAG CTT GCC CCC GCC 674
Thr Arg Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala
210 215 220
005048141 CA 02296840 2000-O1-13
GCA GCC TACATC GCC GGGTTC ACG GGGTTC CAC GAGTTC GCC GAG TTC 722
Ala Ala TyrIle Ala GlyPhe Thr GlyPhe His GluPhe Ala Glu Phe
225 230 235
ACG GCG GAGGAC GTG GGCACG ACC GAGAGC GGG CTCAAC TCG GTG GTG ?70
Thr Ala GluAsp Val GlyThr Thr GluSer Gly LeuAsn Ser Val Val
240 245 250
CTC GCC AACAAC TCG GAGGGC GTG CTGCTG CCG CTCAAC GAG CCG GTG 818
Leu Ala AsnAsn Ser GluGly Val LeuLeu Pro LeuAsn Glu Pro Val
255 260 265 270
CAC GGC ACCAAG CGC CGGAGC CAG ATACAG ACG TTCCTG GAA CAC CAC 866
His Gly ThrLys Arg ArgSer Gln IleGln Thr PheLeu Glu His His
275 280 285
GGC GGC CCGGGC GTG .CAGCAC ATC GCGGTG GCC AGCAGT GAC GTG CTC 914
Gly Gly ProGly Val GlnHis Ile AlaVal Ala SerSer Asp Val Leu
290 295 300
AGG ACG CTCAGG AAG ATGCGT GCG CGCTCC GCC ATGGGC GGC TTC GAC 962
Arg Thr LeuArg Lys MetArg Ala ArgSer Ala MetGly Gly Phe Asp
305 310 315
TTC CTG CCACCC CCG CTGCCG AAG TACTAC GAA GGCGTG CGA CGC CTT 1010
Phe Leu ProPro Pro LeuPro Lys TyrTyr Glu GlyVal Arg Arg Leu
320 325 330
GCC GGG GATGTC CTC TCGGAG GCG CAGATC AAG GAATGC CAG GAG CTG 1058
Ala Gly AspVal Leu SerGlu Ala GlnIle Lys GluCys Gln Glu Leu
335 340 345 350
GGT GTG CTCGTC GAT AGGGAC GAC CAAGGG GTG TTGCTC CAA ATC TTC 1106
Gly Val LeuVal Asp ArgAsp Asp GlnGly Val LeuLeu Gln Ile Phe
355 360 365
ACC AAG CCAGTA GGG GACAGG CCG ACCTTG TTC CTGGAG ATG ATC CAG 1154
Thr Lys ProVal Gly AspArg Pro ThrLeu Phe LeuGlu Met Ile Gln
370 375 380
AGG ATC GGGTGC ATG GAGAAG GAC GAGAGA GGG GAAGAG TAC CAG AAG 1202
Arg Ile GlyCys Met GluLys Asp GluArg Gly GluGlu Tyr Gln Lys
385 390 395
GGT GGC TGCGGC GGG TTCGGC AAA GGCAAC TTC TCCGAG CTG TTC AAG 1250
Gly Gly CysGly Gly PheGly Lys GlyAsn Phe SerGlu Leu Phe Lys
400 405 410
TCC ATT GAAGAT TAC GAGAAG TCC CTTGAA GCC AAGCAA TCT GCT GCA 1298
Ser Ile GluAsp Tyr GluLys Ser LeuGlu Ala LysGln Ser Ala Ala
415 420 425 430
0050/48141
CA 02296840 2000-O1-13
31
GTT CAG GGA TCA TAGGATAGAA GCTGGTCCTT GTATCATGGT CTCATGGAGC 1350
Val Gln Gly Ser
435
AAAAGAAAAC AATGTTGTTT GTAATATGCG TCGCACAATT ATATCAATGT TATAATTGGT 1410
GAAGCTGAAG ACAGATGTAT CCTATGTATG ATGGGTGTAA TGGATGGTAG AGGGGCTCAC 1470
ACATGAAGAA AATGTAGCGT TGACATTGTT GTACAATCTT GCTTGCAAGT AAAATAAAGA 1530
ACAGATTTTG AGTTCTGCAA AAAA.A 1565
(2) INFORMATION ON SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 434 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Ala
1 5 10 15
Val Thr Pro Glu His Ala Arg Pro His Arg Met Val Arg Phe Asn Pro
20 25 30
Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu Phe Trp
35 40 45
Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Leu Gly
50 55 60
Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala His
65 70 75 80
Ala Ser Gln Leu Leu Arg Ser Gly Ser Leu Ala Phe Leu Phe Thr Ala
85 90 95
Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro Ser Phe
100 105 110
Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Ile Ala Val
115 120 125
Arg Ser Val Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe Arg Ala
130 135 140
Ser Arg Arg Arg Gly Ala Arg Pro Ala Phe Ala Pro Val Asp Leu Gly
145 150 155 160
0050/48141 CA 02296840 2000-O1-13
-' 32
Arg Gly Phe Ala Phe Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu
165 170 175
Arg Phe Val Ser His Pro Asp Gly Thr Asp Val Pro Phe Leu Pro Gly
180 185 190
Phe Glu Gly Val Thr Asn Pro Asp Ala Val Asp Tyr Gly Leu Thr Arg
195 200 205
Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala Ala Ala
210 215 220
Tyr Ile Ala Gly Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala
225 230 235 240
Glu Asp Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Val Val Leu Ala
l, 245 250 255
Asn Asn Ser Glu Gly Val Leu Leu Pro Leu Asn Glu Pro Val His Gly
260 265 270
Thr Lys Arg Arg Ser Gln Ile Gln Thr Phe Leu Glu His His Gly Gly
275 280 285
Pro Gly Val Gln His Ile Ala Val Ala Ser Ser Asp Val Leu Arg Thr
29p 295 300
Leu Arg Lys Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp Phe Leu
305 310 315 320
Pro Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Leu Ala Gly
325 330 335
f,- Asp Val Leu Ser Glu Ala Gln Ile Lys Glu Cys Gln Glu Leu Gly Val
340 345 350
Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu Gln Ile Phe Thr Lys
355 360 365
Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gln Arg Ile
370 375 380
Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gln Lys Gly Gly
385 390 395 ~ 400
Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile
405 410 415
Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gln Ser Ala Ala Val Gln
420 425 430
Gly Ser
