U.S. patent application number 09/138873 was filed with the patent office on 2001-09-06 for transgenic pathogen-resistant organism.
Invention is credited to CHET, ILAN, ECKES, PETER, GORNHARDT, BIRGIT, JACH, GUIDO, LOGEMANN, JURGEN, MUNDY, JOHN, SCHELL, JEFF.
Application Number | 20010020300 09/138873 |
Document ID | / |
Family ID | 6470118 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010020300 |
Kind Code |
A1 |
LOGEMANN, JURGEN ; et
al. |
September 6, 2001 |
TRANSGENIC PATHOGEN-RESISTANT ORGANISM
Abstract
Transgenic pathogen-resistant organism whose genome contains at
least two different genes under the control of active promoters
with pathogen-inhibiting action. This organism is distinguished by
a synergistic pathogen-inhibiting action. This action is evident
particularly when the genes code for the gene products chitinase
(ChiS, ChiG), glucanase (GluG), protein synthesis inhibitor (PSI)
and antifungal protein (AFP).
Inventors: |
LOGEMANN, JURGEN; (NB
LEIDEN, NL) ; JACH, GUIDO; (KOLN, DE) ;
GORNHARDT, BIRGIT; (KOLN, DE) ; MUNDY, JOHN;
(V COPENHAGEN, DK) ; SCHELL, JEFF; (KOLN, DE)
; ECKES, PETER; (KELKHEIM(TAUNUS), DE) ; CHET,
ILAN; (NES ZIONA, IL) |
Correspondence
Address: |
BRUMBAUGH GRAVES DONOHUE & RAYMOND
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
|
Family ID: |
6470118 |
Appl. No.: |
09/138873 |
Filed: |
August 24, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09138873 |
Aug 24, 1998 |
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08812025 |
Mar 6, 1997 |
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5804184 |
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08812025 |
Mar 6, 1997 |
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08457797 |
Jun 1, 1995 |
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5689045 |
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08457797 |
Jun 1, 1995 |
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08134416 |
Oct 8, 1993 |
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Current U.S.
Class: |
800/279 |
Current CPC
Class: |
C12N 9/2402 20130101;
Y10S 47/01 20130101; C12N 15/8282 20130101; C12N 9/2442 20130101;
C07K 14/415 20130101; C12Y 302/01014 20130101; C07K 14/37
20130101 |
Class at
Publication: |
800/279 |
International
Class: |
A61K 038/47 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 1992 |
DE |
P 42 34 131.0 |
Claims
1. Transgenic pathogen-resistant organism characterized in that its
genome contains at least two different genes under the control of
active promoters with pathogen-inhibiting action.
2. Transgenic pathogen-resistant organism according to claim 1,
characterized in that the genes code for gene products which reduce
the vitality of fungi.
3. Transgenic pathogen-resistant organism according to claim 1 or
2, characterized in that the genes are of fungal, bacterial, plant,
animal or viral origin.
4. Transgenic pathogen-resistant organism according to claim 2 or
3, characterized in that the gene products have properties
promoting resistance to fungi.
5. Transgenic pathogen-resistant organism according to claim 4,
characterized in that the gene products are chitinase (ChiS, ChiG),
glucanase (GluG), protein synthesis inhibitor (PSI) and antifungal
protein (AFP).
6. Transgenic pathogen-resistant organism according to any of
claims 1 to 5, characterized in that the latter is a plant.
7. Transgenic pathogen-resistant organism according to claim 6,
characterized in that it is a tobacco, potato, strawberry, corn,
rape or tomato plant.
8. DNA-transfer vectors with inserted DNA sequences according to
one or more of the preceding claims.
9. Process for the generation of pathogen-resistant organisms
according to any of claims 1-7, characterized in that at least one
gene with pathogen-inhibiting action is transferred into the genome
of an organism, and the pathogen-resistant organism is obtained (a)
by crossing the organism with another, optionally transgenic,
organism which contains at least one other gene with
pathogen-inhibiting action, and subsequently selecting, and/or (b)
by transformation of at least one other gene with
pathogen-inhibiting action into the organism.
10. Process according to claim 9, characterized in that
DNA-transfer vectors with inserted DNA sequences corresponding to a
gene with pathogen-inhibiting action as described in any of claims
1 to 5 are used.
11. Process for the generation of pathogen-resistant organisms
according to any of claims 1-7, characterized in that vectors which
comprises more than one gene with pathogen-inhibiting action are
used for the transformation into the genome of an organism.
12. Process for ensuring the resistance of organisms to pathogens,
characterized in that the organism used is a transgenic
pathogen-resistant organism according to any of claims 1 to 7 or an
organism whose genome contains at least one gene complying with the
definitions of claims 1 to 7, and at least one substance which is
not expressed by the organism but corresponds to any other one of
the gene products complying with claims 1 to 7 is applied to the
organism.
Description
[0001] Transgenic pathogen-resistant organism
[0002] The invention relates to a pathogen-resistant organism and
to a process for generating it.
[0003] It is known in the state of the art that infestation of a
plant by pathogens are caused a series of different reactions.
These include, for example, changes in the cell wall structure, the
synthesis of phytoalexins which have antimicrobial activity, the
accumulation of so-called PR proteins (pathogenesis-related),
protease inhibitors and enzymes with hydrolytic functions
(Hahlbrock and Grisebach in Ann. Rev. Plant. Physiol., 30 (1979),
105-130).
[0004] Many pathogens (fungi and insects) have chitin as a
constituent of their cell wall. By contrast, plants possess no
chitin. It has now been demonstrated in some cases that there is
enhanced production of chitinases in plants after infestation by
pathogens. Chitinases are among the enzymes with hydrolytic
functions and they catalyze chitin breakdown. It has now been
possible to show that plants acquire an increased resistance to
pathogens by the production of chitinases.
[0005] It is furthermore known to use a gene from barley plants
whose gene product codes for an inhibitor of fungal protein
synthesis. The incorporation of a corresponding inhibitor gene in
transgenic plants led to improved resistance to fungi.
[0006] Finally, it has also been disclosed that the use of a
polypeptide from Aspergillus giganteus is able to protect, by
virtue of its antifungal activity, plants from infestation by
fungi.
[0007] However, given this state of the art there is a need to
provide further transgenic pathogen-resistant organisms. Moreover,
the organisms which are particularly desired are those whose
resistance is increased overall by comparison with the known
organisms or is extended with respect to the number of possible
pathogens.
[0008] This problem is solved by a transgenic pathogen-resistant
organism having the features of claim 1.
[0009] The invention is based on the surprising finding that the
incorporation of at least two different genes with
pathogen-inhibiting action into the genome of an organism assists
the latter to resist pathogens to an extent going far beyond an
additive effect of each of the genes on its own.
[0010] The dependent claims indicate further embodiments of the
invention.
[0011] The genes can code for gene products which reduce the
vitality of fungi. In particular, the genes can be of fungal,
bacterial and plant, animal or viral origin. In particular, the
gene products have properties which promote resistance to fungi.
The gene products are chitinase (ChiS, ChiG), glucanase (GluG),
protein synthesis inhibitor (PST) and antifungal protein (AFP).
[0012] The transgenic pathogen-resistant organism can be a plant,
and tobacco, potato, strawberry, corn, rape or tomato plants are
preferred.
[0013] The invention also relates to DNA-transfer vectors with
inserted DNA sequences as are indicated in detail in this
description.
[0014] The invention Furthermore relates to a process for the
generation of pathogen-resistant organisms as are described herein,
wherein at least 1 gene with pathogen-inhibiting action is
transferred into the genome of an organism, and the
pathogen-resistant organism is obtained (a) by crossing the
organism with another, optionally transgenic, organism which
contains at least one other gene with pathogen-inhibiting action,
and subsequently selecting, and/or (b) by transformation of this
other gene with pathogen-inhibiting action into the organism. The
process can be used with DNA-transfer vectors with inserted DNA
sequences corresponding to a gene with pathogen-inhibiting action
as described herein.
[0015] Finally, the invention relates to a process for the
generation of pathogen-resistant organisms, wherein vectors which
comprise more than one gene with pathogen-inhibiting action are
used for the transformation into the genome of an organism.
[0016] The invention also relates to a process for ensuring the
resistance of organisms to pathogens, characterized in that the
organism used is a transgenic pathogen-resistant organism according
to any or claims 1 to 7 or an organism whose genome contains at
least one gene complying with the definitions used herein (see
claims 1 to 7, and at least one substance which is not expressed by
the organism but corresponds to any other one of the gene products
complying with the definitions given in this application (claims 1
to 7) is applied to the organism.
[0017] It was possible to achieve the synergistic effects very
particularly with transgenic pathogen-resistant organisms to which
the gene sequences which coded for proteins of the attached
sequence listings A to E, or corresponded to the latter, were
transferred or transfected.
[0018] ChiS
[0019] A DNA fragment which is 1.8 Kb in size, that codes for a
chitinase called ChiS was isolated from the soil bacterium Serratia
marcescens. In vitro investigations with purified ChiS protein
showed that it is able effectively to inhibit the growth of fungi,
even in low concentrations. The reason for the inhibition is that
the ChiS protein has a chitinase activity which is able to damage
the tips of the fungal hyphae. In this way the fungus is unable to
grow further and is inhibited.
[0020] PSI
[0021] The PSI gene originates from barley and codes for a protein
which inhibits protein synthesis by fungi. In vitro tests show that
even low concentrations of PSI are sufficient to inhibit various
fungi such as, for example, Rhizoctonia solani.
[0022] AFP
[0023] It is possible for a polypeptide which has antifungal
activity to be isolated from the fermentation broth of Aspergillus
giganteus and to be sequenced. This polypeptide is suitable as
antifungal agent, for example as spraying agent and as preservative
for industrial products and human and animal foods. It can
furthermore be combined with other substances which have pesticidal
activity, fertilizers or growth regulators. Inhibitory activities
against fungi were detectable inter alia against various
Aspergillus, Fusaria, Phytophthora and Trichophyton species.
[0024] ChiG and GluG
[0025] Two genes which code, respectively, for a chitinase (ChiG)
and glucanase (GluG) can be isolated from certain types of barley.
Purified ChiG protein or GluG protein inhibits various
phytopathogenic fungi in vitro (inter alia Rhizoctonia solani) (see
R. Leah et al., Journal of Biological Chemistry, Vol. 266, No. 3
(1991), pages 1564-1573).
[0026] The inventors have now found, completely surprisingly, that
an at least binary combination of expression of PSI, AFP, ChiS,
ChiG or GluG leads to synergistic effects in respect of the
acquired resistance to fungi in transgenic plants. In particular,
the effects of the individual substances in the combination are
markedly exceeded. These include resistance to the fungus
Rhizoctonia solani, Sclerotinia infestation, Botrytis infestation,
etc.
[0027] Combinations according to the invention are (DNA and/or
polypeptides):
[0028] binary combinations
[0029] ChiS, GluG; ChiS, PSI; ChiS, ChiG; ChiS, AFP; GluG, PSI;
GluG, ChiG; GluG, AFP; PSI, ChiG; PSI, AFP;
[0030] ternary combinations
[0031] ChiS, GluG, PSI; ChiS, GluG, ChiG; ChiS, GluG, AFP; GluG,
PSI, ChiG; GluG, PSI, AFP; PSI, ChiG, AFP; ChiG, AFP, GluG
[0032] quaternary combinations
[0033] ChiS, GluG, PSI, AFP; ChiS, GluG, PSI, ChiG;
[0034] quinary combination
[0035] ChiS, GluG, PSI, AFP, ChiG
[0036] The invention furthermore relates to the combined use of the
proteins with pathogen-inhibiting action, preferably ChiS, PSI,
AFP, ChiG and GluG, against pathogens. Combined use also means in
this context that at least a first pathogen-inhibiting substance is
expressed by the organism and at least a second substance which has
pathogen-inhibiting action is applied to the organism from
outside.
[0037] The agents according to the invention also include those
which contain the abovementioned proteins in at least binary
combination. The agents according to the invention can contain
other active substances besides the proteins. The other active
substances can be pesticides, fertilizers and/or growth regulators,
and the agents according to the invention can be prepared in
various formulations such as concentrates, emulsions, powders,
formulations on carriers, mixtures with other active substances,
etc. The ChiS/PSI and AFP/PSI combination is particularly
preferred. These proteins can be used particularly effectively to
inhibit the growth of Rhizoctonia solani, especially in tobacco
crops.
[0038] The invention also relates to the use in a process according
to the invention of a DNA sequence which codes at least for a
polypeptide of sequences A to E, or to a pathogen-resistant
organism, where its genome contains at least two different genes
under the control of active promoters with pathogen-inhibiting
action, where the genes are in each case selected from the group of
sequences A to E. The invention furthermore includes DNA sequences
which hybridizes with a DNA sequence which codes for polypeptides
of amino-acid sequences A to E; where these DNA sequences can be of
natural synthetic [sic] or semisynthetic origin and can be related
to the abovementioned DNA sequence by mutations, nucleotide
substitutions, nucleotide deletions, nucleotide insertions and
inversions of nucleotide sequences, and codes for a polypeptide
with pathogenic activity. The invention furthermore relates to a
recombinant DNA molecule which contains at least one DNA sequence
which accords with the preceding statements, where this DNA
molecule can be in the form of a cloning or expression vector.
[0039] The invention relates to appropriate host organisms and
intermediate hosts which are transformed with a recombinant DNA
molecule which accords with the preceding statements. Preferred as
intermediate host in the generation of a pathogen-resistant
transgenic organism are strains of bakeria, in particular so-called
Agrobakeria strains.
[0040] The invention furthermore relates to the transgenic
pathogen-resistant organisms obtained by the process according to
the invention, in particular tobacco, potato, corn, pea, rape and
tomato plants.
[0041] The DNA sequences according to the invention are, as a rule,
transferred together with a promoter. Promoter sequences are
recognized by the plant transcription apparatus and thus lead to
constitutive expression of the gene associated with them in plants.
The promoter can, however, also be pathogen-inducible and/or
wound-inducible (WUN1) and/or tissue-specific and/or
development-specific.
[0042] The genetic manipulation operations necessary for carrying
out the invention, especially for expression of the gene in plants,
are generally known. See for example the publication by Maniatis et
al. in "Molecular cloning: A laboratory manual", Cold Spring Harbor
(1982).
[0043] The invention is explained in detail in the following
examples.
[0044] All the standard methods of molecular biology were carried
out, unless otherwise indicated, as described by Maniatis et al.
"Molecular cloning: a laboratory manual", Cold Spring Harbor
(1982).
[0045] The DNA coding for amino-acid sequences A to E was initially
cloned in a manner known per se and then transferred by conjugation
into A. Tumefaciens LBA 4404 (A. Hoekema et al., Nature 303,
179-180). This took place by the method described by Van Haute et
al. in EMBO J. 2, 411-418 (1983).
[0046] The transfer of DNA into that Agrobacterium was checked by
isolating Agrobacterium DNA by the method described by Ebert et al.
in Proc. Natl. Acad. Sci. USA 84 5745-5749 (1987). Restriction
cleavage of the DNA, transfer to Hybond-N membrane (Amersham) and
hybridization with a radioactively labeled DNA probe provided
information about successful DNA transfer into the
Agrobacterium.
[0047] The transformed Agrobacterium was then used to transform
tobaco, rape, strawberry, tomato and potato plants.
[0048] The LBA4404 Agrobacteria required for the infection were
initially cultivated in selective antibiotic medium (P. Zambrisky
et al. in EMBO J., 1, 147-152 (1983)), sedimented by centrifugation
and washed in YEB medium without antibiotics (YEB=0.5% meat
extract; 0.2% yeast extract; 0.5% peptone; 0.5% sucrose; 2 mM
MgSO.sub.4). After renewed sedimentation and taking up in
MgSO.sub.4 it was possible to use the bacteria for the
infection.
[0049] The so-called leaf disk method was used for the
infection.
[0050] Sterile leaves were used for the leaf disk infection. Leaf
pieces about 1 cm in size are dipped in the previously described
Agrobacteria suspension and subsequently transferred to 3MS medium
(medium described by T. Murashige and F. Skoog in Physiol. Plant.,
15, 473-497 (1962); 3MS=MS-3% sucrose). After incubation at
25.degree. C. to 27.degree. C. with 16 hours of light for two days,
the leaf pieces were transferred to MSC16 medium (according to T.
Murashige (see above); MSC16=MS+0.5 .mu.g/ml BAP+0.1 .mu.g/ml
NAA+100 .mu.g/ml kanamycin sulfate+500 .mu.g/ml Claforan). Shoots
appearing after 4-6 weeks were cut off and transplanted to MSC15
medium (according to Murashige (see above); MSC15=MS+2% sucrose,
500 .mu.g/ml Claforan+100 .mu.g/ml kanamycin sulfate). Shoots with
root formation were analyzed further.
[0051] Monocotyledonous plants (including corn), but some
dicotyledonous plants too, were transformed by direct gene transfer
into protoplasts. These protoplasts were subsequently regenerated
to intact plants (Example: J. Potrykus in Biotechnology 8 (1990),
535).
[0052] The resulting transgenic plants were infected with the
fungus Rhizoctonia solani for testing purposes. For this purpose,
fungal cultures were grown and thoroughly mixed in standard soil.
This soil was then distributed in a dish and planted with the
plants to be tested.
[0053] For the evaluation, each plant on a dish was assigned a
value from 0 to 3. It was possible to calculate from this for each
plant line an index which resulted from the sum of the values. The
classification is as follows:
[0054] 0=no symptoms (healthy)
[0055] 1=slightly reduced size (compared with a non-infected
control); no or very slight visible infestation
[0056] 2=severe reduction in growth; severe symptoms of
infestation
[0057] 3=dead
[0058] The rating is carried out in each case 14 days after the
start of the series of tests.
EXAMPLE 1
[0059] Fungus inhibition test with combined proteins
[0060] The intention initially was to show that the proteins used
here have synergistic effects in their combination. Fungal growth
tests in vitro were carried out for this purpose.
[0061] These entailed a defined amount of Rhizoctonia solani fungal
mycelium being mixed with 100 .mu.l of potato dextrose solution and
incubated in microtiter plates at 25.degree. C. In this test there
is a linear correlation between the growth of the fungus and the
increase in the optical density at 405 nanometers. The inhibitory
effect of proteins can be detected from a smaller increase in the
optical density.
[0062] 2-3 mycelium balls were taken from a liquid culture of R.
Solani, mixed with 100 .mu.l of KGB medium in an Eppendorf vessel
and carefully homogenized with a glass mortar. This suspension was
then mixed with 10 ml of KGB medium and passed through a sterile
100 .mu.m screen. The optical density of this mycelium fragment
suspension (100 .mu.l aliquot) was adjusted to a value of 0.06-0.07
at 405 nanometers by adding medium. 100 .mu.l samples were placed
on a microtiter plate and mixed with the proteins to be tested. 7
parallels were measured per mixture. Mixtures which were mixed with
the corresponding amounts of buffer served as controls. The plates
were incubated in the dark at 25.degree. C. for 48 hours, and the
optical density of the cultures was measured at regular
intervals.
[0063] Calculation of whether two proteins act together in an
additive synergistic or antagonistic manner in the inhibition of
fungal growth is possible from the measured data with the aid of
the Colby formula which is described hereinafter and generally used
(S. R. Colby in Wheeds, 15 (1967), 20-22).
[0064] To do this it was initially necessary to calculate the
growth inhibition E to be expected theoretically with an additive
behavior (the expected efficacy). This is given by:
E=W1+W2-((W1.times.W2)/100)
[0065] where W1 and W2 indicate the efficacies of the individual
proteins, which is defined as that percentage deviation of the
growth plot (in the presence of the protein) from the untreated
control. The efficacy for a protein (at a defined time in the
growth plot) is given by:
W1=(OD(K)-OD(P))/OD(K).times.100 (percent)
[0066] In this, OD(K) is the optical density of the untreated
control and OD(P) is the optical density of the culture treated
with the protein.
[0067] Thus, on combined use of two proteins, the following
statements were possible: if the efficacy G measured in the
experiment is identical to the expected value E, the behavior is
additive. If, on the other hand, G is greater than E, the behavior
is synergistic.
[0068] Using this test model, it emerged that the proteins ChiS,
PSI, AFP, ChiG and GluG used in the Example surprisingly have
synergistic inhibitory effects on various fungi, and these effects
were achieved both by the combination of two types of protein and
by multiple combination of the abovementioned proteins.
[0069] For example, the following values were determined from the
combination of ChiS and PSI protein and from the combination of AFP
protein and PSI protein on the fungus Rhizoctonia solani (in each
case two different ChiS and AFP concentrations with a constant RIP
concentration):
[0070] ChiS+PSI
[0071] The expected values were: E1=29.9% and E2=44.5% The measured
values were: G1=60.4% and G2=64.1% The proteins ChiS and PSI
therefore act together in a synergistic manner in the inhibition of
the growth of R. Solani.
[0072] FIG. 1 shows the results obtained with the combination of
the proteins and with the individual substances. According to the
Figure, various ChiS concentrations (0.5 .mu.g/ml and 0.05
.mu.g/ml) are combined with PSi protein (1.0 .mu.g/ml).
[0073] AFP+PSI
[0074] The expected values were: E1=39.9% and E2=41.9% The measured
values were: G1=57.7% and G2=65.4% The AFP and PSI combination also
according to this shows a synergistic Inhibition of growth of the
fungus R. Solani. FIG. 2 indicates the test results with various
AFP concentrations (0.4 .mu.g/ml and 0.04 .mu.g/ml) combined with
PSI protein (1.0 .mu.g/ml).
EXAMPLE 2
[0075] Transgenic plants
[0076] In order to obtain the organisms according to the invention
with DNA sequences which act together synergistically, initially
transgenic plants which contained at least one of the genes which
act together synergistically were generated.
[0077] ChiS in transgenic plants
[0078] Initially a ChiS gene was fused to plant regulatory
sequences.
[0079] A ChiS gene 1.8 Kb in size was sequenced by using synthetic
oligonucleotides in the dideoxy sequencing method of Sanger et al.
in Proc. Natl. Acad. Sci. USA, 74 (1977), 5463-5467.
[0080] The 35S promoter originating from cauliflower mosaic virus
(CamV) (400 bp (according to Topfer et al. in Nucl. Acid. Res., 15
(1987), 5890)) underwent transcriptional fusion to the ChiS gene.
The termination signal, which is 0.2 Kb in size, of the 35S gene of
CamV, whose functionality in dicotyledonous plants is known, was
used 3' from the ChiS gene. The chimeric gene 35S-ChiS was cloned
into the pLS034 vector by means of the Agrobacterium tumefaciens
transformation system in tobacco and potato plants, and
kanamycin-resistant plants were regenerated.
[0081] It was possible to detect both the ChiS gene and the
corresponding mRNA as well as the gene product protein in the
resulting plants.
[0082] PSI in transgenic plants
[0083] PolyA RNA was initially isolated from ripe barley seeds
(Horaeum vulgare L. cv. Piggy) and deposited in a cDNA gene bank in
.lambda.-gt-11-phages. The details of the process are to be found
in R. Lea in Plant. Biol., 12 (1989), 673-682. Monospecific PSI
antibodies were then used to identify cDNA clones.
[0084] Subsequently, the PSI-positive .lambda.-gt-11-phages were
isolated, cloned further and sequenced by the dideoxy sequencing
method of Sanger et al. indicated above. The DNA cloned into E.
coli was then transferred in the manner described above by
conjugation into Agrobacterium LBA4404.
[0085] Both the transferred gene and mRNA and gene product were
detectable in corresponding transgenic tobacco, potato, rape,
strawberry and tomato plants.
[0086] AFP in transgenic plants
[0087] For the cloning in the vector, the cDNA sequence of the
antifungal peptide is provided with ends which can be ligated into
BamH1 and Sall restriction cleavage sites. The cloning vector used
was pDH51 (Pietrzak et al. in Nucl. Acids Res. 14 (1986), 5857).
The vector pDH51 was opened with the restriction enzymes BamH1 and
Sall between promoter and terminator. The vector pDH51 is a pUC18
derivative which contains promoter and terminator sequences of the
35S transcript from cauliflower mosaic virus. These sequences are
recognized by the plant's transcription apparatus and lead to
strong constitutive expression of the gene associated with them in
plants. The DNA of the antifungal peptide is then cloned via the
BamH1 and Sall cleavage site into the vector. Finally, the
transcription unit--promoter, gene and terminator--is cut out of
the vector using the restriction enzyme EcoRI and cloned into a
plant transformation vector. The following vectors and their
derivatives can, for example, be used as plant transformation
vector:
[0088] pOCA18 (Olszewski et al. in Nucl. Acids Res., 16 (1988),
10765) pPCV310 (Koncz and Shell in MGG 204 (1986), 383) and pBin19
(Bevan et al. Nucl. Acids. Res. 12 (1984), 8711)
[0089] After the transcription unit and the vector had been ligated
via the EcoRI cleavage site, the construct was conjugated into the
Agrobacterium strain MP90RK (Koncz and Shell (see above)) or IHA101
(Hood et al. in J. Bacteriol. 168 (1986), 1291).
[0090] Transgenic tobacco, potato, strawberry, rape and tomato
plants were then transformed by the method described above.
Transformed shoots are selected on the basis of the cotransferred
resistance to the antibiotic kanamycin. Expression of the
antifungal protein in the transformed crop plants was checked and
confirmed by DNA analysis (Southern blotting), RNA analysis
(Northern blotting) and protein analysis with specific antibodies
(Western blotting).
[0091] ChiG and GluG in transgenic plants
[0092] ChiG- and GluG-transgenic plants which were both Southern-,
Northern- and Western-positive were obtainable in analogy to the
plants described above.
[0093] ChiS, PSI, AFP, ChiG, GluG in transgenic monocotyledonous
plants
[0094] It was possible by means of direct gene transfer to
integrate the abovementioned genes into the genome of
monocotyledonous plants such as, for example, corn. This resulted
in transgenic plants which were Southern- and Northern- and
Western-positive.
[0095] Combination of various fungus-resistance genes in transgenic
plants
[0096] The previously obtained tobacco, corn, rape, strawberry,
potato and tomato plants were crossed together and selected for
plants containing in each case the fungus-resistant genes of both
parents. In addition, transgenic plants were obtained by
transforming them initially with one and then with one or more
other gene. Finally, plants were also transformed with vectors
which contained various resistance genes. Fungus-resistance tests
were done with this plant material. Surprisingly, in all cases
synergistic effects, not just additive effects, in respect of
fungus resistance are observed.
[0097] For example, a tobacco plant which expresses ChiS and PSI
shows a considerably greater resistance to Rhizoctonia infestation
than the plants which expressed only ChiS or PSI or which would
result from the additive resistance.
[0098] A synergistic inhibitory effect on infestation with
Rhizoctonia solani also results from combined expression of PSI-
and AFP-transgenic tobacco. Combination of two or more different
genes (ChiS, RIP, AFP, ChiG and GluG) in a wide variety of
transgenic plants also led to synergistic inhibitory effects on
various fungi.
[0099] Whereas wild-type plants have index values from 38 to 46 in
tests on 20 seedlings, it emerges with transgenic tobacco according
to the invention that the latter grows as well in the presence of
the fungus Rhizoctonia solani as do control plants (index value
10-12) cultivated on Rhizoctonia-free soil.
[0100] Sequence listing A and A' (AFP)
[0101] Seq IDNo.: 1 (A)
[0102] Sequence type: complete nucleotide sequence with
corresponding protein to the extent that it is encoded by an open
reading frame, active protein (A')
[0103] Sequence length: 51 amino acids (A')
[0104] Strandedness: single strand
[0105] Topology: linear
[0106] Molecule type: cDNA
[0107] Original source: Aspergillus giganteus fermentation
broth
[0108] Name: antifungal peptide (AFP)
[0109] Features (A): open reading frame of 177 nucleotides, the
N-terminal amino acid of the active protein is marked by *.
[0110] Properties: antifungal agent, especially on Rhizoctonia
solani, various Aspergillus, Fusaria and Trichophyton species.
1 TTGCGACCCCCGTTGAAGCCGATTCTCTCACCGCTGGTGGTCTGGATGCAAGAGATGAGA 1
------------------------------------------------------------ 60
AACGCTGGGGGCAACTTCGGCTAAGAGAGTGGCGACCACCAGACCTACGTTCTCTACTCT M Q E
M 4 - GCGCGGCTTTTGGCCACATACAATGGCAAATGCTACAAGAAGGATAATATC-
TGCAAGTAC 61 -----------------------------------------------------
-------- 120 CGCGCCCAAAACCGGRGRATGTTACCGTTTACGATGTTCTTCCTATTATAGA-
CGTTCATG A R V L A T Y N G K C Y K K D N I C K Y -
AAGGCACAGAGCGGCAAGACTGCCATTTGCAAGTGCTATGTCAAAAA- GTGCCCCCCGGAC 121
------------------------------------------------- ------------ 180
TTCCGTGTCTCGCCCTTCTGACGGTAAACGTTCACGATACAGTTTTTC- AGGGGGCGGGCTG K A
Q S G K T A I C K C Y V K X C P R D -
GGCGGCGAAATGCGAGTTTGACAGCTACAAGGGGAA- GTGCTACTGCTAGACGGTGAGCGAA 181
------------------------------------- ------------------------ 240
CCGCGCTTTACGCTCAAACTGTCGATGTTCCCCTTC- ACGATGACGATCTGCCACTCGCGTT G A
K C E F D S Y K G K C Y C * GGGACGAATAGGCTGGGGGTTATTTTACTCTGCT 241
----------------------------------- 275
CCCTGCTTCATCCGACCCCCAATAAAATGAGACGA A'
Ala-Thr-Tyr-Asn-Gly-Lys-Cys-Tyr-Lys-Lys-Asp-Asn-Ile-Cys-
Lys-Tyr-Lys-Ala-Gln-Ser-Gly-Lys-Thr-Ala-Ile-Cys-Lys-Cys-
Tyr-Val-Lys-Lys-Cys-Pro-Arg-Asp-Gly-Ala-Lys-Cys-Glu-Phe-
Asp-Ser-Tyr-Lys-Gly-Lys-Cys-Tyr-Cys.
[0111]
Ala-Thr-Tyr-Asn-Gly-Lys-Cys-Tyr-Lys-Lys-Asp-Asn-Ile-Cys-Lys-Tyr-Lys-
-Ala-Gln-Ser-Gly-Lys-Thr-Ala-Ile-Cys-Lys-Cys-Tyr-Val-Lys
-Lys-Cys-Pro-Arg-Asp-Gly-Ala-Lys-Cys-Glu-Phe-Asp-Ser-Tyr-Lys-Gly-Lys-Cys--
Tyr-Cys.
[0112] Sequence listing B and B' (PSI)
[0113] Seq IDNo.: 2
[0114] Sequence type: nucleotide with corresponding protein
[0115] Sequence length: 1078 base pairs (B'=incomplete PSI-cDNA
clone)
[0116] Strandedness: single strand
[0117] Topology: linear
[0118] Molecule type: complementary DNA
[0119] Original source: barley seeds (Hordeum vulgare L. cv.
Piggy)
[0120] Immediate experimental source: cDNA gene bank in
.lambda.-gt-11 phages
[0121] Name: protein synthesis inhibitor
[0122] Features: 42 bp-lona 5'-non-translating region open reading
frame of 843 base pairs (the stop codon is marked by an asterisk)
193 base pair-long 3'-non-translated end, possible polyadenylation
signals are underlined
[0123] Properties: antifungal activity, especially on spores of
Trichoderma reesii and fusarium sporotrichoides and on Rhizoctonia
solani.
2 CTTAATAGCACATCTTGTCCGTCTTAGCTTTGCATTACATCCATGGCGGCAAAGATGGCG * M
A A K M A -1 1
AAGAACGTGGACAAGCCGCTCTTCACCGCGACGTTCAACGTCCAGGCCAGCTCCGCCGAC K N V
D K F L F T A T F N V Q A S S A D 10 20 TACGCCACCTTCATCGCCGGCATC-
CGCAACAAGCTCCGCAACCCGGCGCACTTCTCCCAC Y A T F I A G I R N K L R N P
A H F S H 30 40 AACCGCCCCGTGCTGCCGCCGGTCGAGCCCAACGTCCCGCCGAGCAGGT-
GGTTCCACGTG N R P V L P P V E P F V P P S R W F H V 50 60
GTGCTCAAGGCCTCGCCGACCAGCGCCGGGCTCACGCTGGCCATTCGGGCGGACAACATC V L K
A S P T S A G L T L A I R A D N I 70 80 TACCTGGAGGGCTTCAAGAGCAGC-
GACGGCACCTGGTGGGAGCTCACCCCGGGCCTCATC Y L E G F K S S D G T W W E L
T P G L I 90 100 CCCGGCGCCACCTACGTGGGGTTCGGCGGCACCTACCGCGACCTCCTCG-
GCGACACCGAC P G A T Y V G F G G T Y R D L L G D T D 110 120
AAGCTGACCAACGTCGCTCTCGGCCGGCAGCAGCTCCCGGACGCGGTGACCGCCCTCCAC K L T
N V A L G R Q Q L A D A V T A L B 130 140
GGGCGCACCAAGGCCGACAAGCCGTCCGGCCCGAAGCAGCAGCAGGCGAGGGAGGCGGTG G R T
K A D K P S G P K Q Q Q A R E A V 150 160 CCGACGCTGCTGGTCATGCTGAA-
CGAGGCCACGCGGTTCCAGACGGTGTCTGGGTTCGTG T T L L L M V N I A T R T Q T
V S G E V 170 180 GCCGGGTTGCTGCACCCCAAGGCGGTGGAGAAGAAGAGCGGGAAGATC-
GGCAATGAGATG A G L L M P K A V E K K S G K I G N E M 190 200
AAGGCCCAGGTGAACGGGTGGCAGGACCTGTCCGCGGCGCTGCTGAAGACGGACGTGAAG K A Q
V N G W Q D L S A A L L K T D V K 210 220 CCTCCGCCGGGAAAGTCGCCAGG-
GAAGTTCGCGCCGATCGAGAAGATGGGCGTGAGGACG P P P G K S P A K F A P I E K
M G V R T 230 240 GCTGTACAGGCCGCCAACACCCTGGGGATCCTGCTGTTCGTGGAGGTG-
CCGGGTGGGTTG A V Q A A N T L G I L L F V E V P G G L 250 260
ACGGTGGCCAAGGCGCTGGACGTGTTCCATGCGAGTGGTGGGAAATAGGTAGTTTTCCAG T V A
K A L E L F H A S G G K * GTATACGTGCATGGGTAGTGTAAAAGTCG+E,uns
AATAAACATGTCACAGAGTGACGGACTGATA TA+E,uns
AATAAATAAATAAACGTGTCACAGAGTTACATATAAACA+E,uns AATAAATAAATAATTAAAA
ATGTCCAGTTTA.sub.47
TCGGTGACGACGCTGCTCCTCATGGTGAACGAGGCCACGCGGTTCCAGACGGTGTCGGGG A V T
T L L L M V N E A T R F Q T V S G 170 180
TTCGTGGCCGGGCTGCTGCACCCCAAGGCGGTGGAGAAGAAGAGCGGGAAGATCGGCAAT F V A
G L L H P K A V E K K S G K I G N 190 200
GAGATGAAGGCCCAGGTGAACGGGTGGCAGGACCTGTCCGCGCGGCTGCTGAAGACGGAC E M K
A Q V N G N Q D L S A A L L K T D 210 220 GTGAAGCCCCCGCCGGGAAAGTC-
GCCAGCGAAGTTCACGCCGATCGAGAAGATGGGCGTG V K P P P G K S P A K F T P I
E K M G V 230 240 AGGACTGCTGAGCAGGCTGCGGCTACTTTGGGGATCCTGCTG-
TTCGTTGAGGTGCCGGGT R T A E Q A A A T L G I L L F V E V P G 250 260
GGGTTGACGGTGGCCAAGGCGCTGGAGCTGTTTCATGCGAGTGGTGGGAAATAGGTAGTT G L T
V A K A L E L F H A S G G K * 270 280
TTGCAGGTATACCTGCATGGGTAAATGTAAAAGTCG+E,uns AATAAAAATGTCACAGAGTGACGG
ACTGATATA+E,uns AATAAATT+E,uns AATAAACATGTCATCATGAGTGACAGACTGAT-
ATAAATAAATA
[0124] Sequence listing C (ChiS)
[0125] Seq IDNo.: 3
[0126] Sequence type: nucleotide
[0127] Strandedness: single strand (the activated strand is double
strand)
[0128] Topology: linear
[0129] Molecule type: cDNA
[0130] Immediate experimental source: plasmid pLChiS from E. Coli
strain A 5187
[0131] Original source: Cosmid bank from Serratia Marcescens
[0132] Name: ChiS protein (chitinase)
[0133] Properties: exo-chitinase
3 1 CAGGGCGTTG TCAATAATGA CAACACCCTG GCTGAAGAGT GTGGTGCAAT 51
ACTGATAAAT ATTTATCTTT GGTTAATAGA AAATTCACTA TCCTTATTTG 101
TCATGTTTTG ATTTATCTTT GCTTAATAGA ATTCACGCTT GCTGAATAAA 151
ACGCAGTTGA TAGCGCTGTT GTTTTTGCGC CTTTTTTATT TATAGTACTG 201
AATGTACGCG GTGGGAATGA TTATTTCGCC ACGTGGAAAG ACGCTGTTGT 251
TATTTATTGA TTTTAACGTT CGCGGATTAT TGCGGAATTT TTTCGCTTCG 301
GCAATGCATC GCGACGATTA ACTCTTTTAT GTTTATCCTC TCGGAATAAA 351
GGAATCAGTT ATGCGCAAAT TTAATAAACC GCTGTTGGCG CTGTTGATCG 401
GCAGCACGGT GTGTTCCGCG GCGCAGGCCG CCGCGCCGGG CAAGCCGACG 451
ATCGCCTGGG GCAACACCAA GTTCGCCATC GTTGAAGTTG ACCAGGCGGC 501
TACCGCTTAT AATAATTTGG TGAAGGTAAA AAATGCCGCC GATGTTTCCG 551
TCTCCTGGAA TTTATGGAAT GGCGACACCG GCACGACGGC AAAAGTTTTA 601
TTAAATGGCA AAGAGGCGTG GAGTGGTCCT TCAACCGGAT CTTCCGGTAC 651
GGCGAATTTT AAAGTGAATA AAGGCGGCGG TTATCAAATG CAGGTGGCAC 701
TGTGCAATGC GGACGGCTGC ACCGCCAGTG ACGCCACCGA AATTGTGGTA 751
GCCGACACCG ACGGCAGCGA TTTGGCGCCG TTGAAAGAGC CGCTGCTGGA 801
AAAGAATAAA CGGTATAAAC AGAACTCCGG CAAAGTGGTC GGTTCTTATT 851
TCGTCGAGTG GGGCGTTTAC GGGCGCAATT TCACCGTCGA CAAGATCCCG 901
GCGCAAAACC TGACCCACCT GCTGTACGGC TTTATCCCGA TCTGCGGCGG 951
CAATGGCATC AACGACAGCC TGAAAGAGAT TGAAGGCAGC TTCCAGGCGT 1001
TGCAGCGCTC CTGCCAGGGC CGCGAGGACT TCAAAGTCTC GATCCACGAT 1051
CCGTTCGCCC CGCTGCAAAA AGCGCAGAAG GGCGTGACCG CCTGGGATGA 1101
CCGGTACAAG GGCAACTTCG GCCAGCTGAT GGCGCTGAAG CAGGCGCATC 1151
CTGACCTGAA AATCCTGCCG TCGATCGGCG GCTGGACGCT GTCCGACCCG 1201
TTCTTCTTCA TGGGCGACAA GGTGAAGCGC GATCGCTTGG TCGGTTCGGT 1251
GAAAGAGTTC CTGCAGACCT GGAAGTTCTT CGACGGCGTG GATATCGACT 1301
GGGAGTTGGG GGGCGGCAAA GGCGCCAACC CTAACCTGGG CAGCCCGCAA 1351
GACGGGGAAA CGTATGTGCT GCTGATGAAG GAGCTGCGGG CGATGCTGGA 1401
TCAGCTGTCG GTGGAAACGG GCCGCAAGTA TGAGCTGACC TCCGCCATCA 1451
GCGCCGGTAA GGACAAGATC GACAAGGTGG CTTACAACGT TGCGCAGAAC 1501
TCGATGGATC ACATGTTGCT GATGAGCTAC GACTTCTATG GCGCCTTCGA 1551
TCTGAAGAAC GTGGGGCATC AGACCGCGCT GAATGCGCCG GCCTGGAAAC 1601
CGGACACCGC CTACACCACG GTGAACGGCG TCAATGCGCT GCTGGCGCAG 1651
GGCGTCAAGC CGGGCAAAAT CGTCGTCGGC ACCGCCATGT ATGGCCGCGG 1701
CTGGACCGGG GTGAACGGCT ACCAGAACAA TATTCCGTTC ACCGGCACCG 1751
CCACCGGGCC GGTTAAAGGC ACCTGGGAGA ACGGTATCGT GGACTACCGC 1801
CAAATCGCCG GCCAGTTCAT GAGCGGCGAG TGGCAGTATA CCTACGACGC 1851
CACGGCGGAA GCGCCTTACG TGTTCAAACC TTCCACCGGC GATCTGATCA 1901
CCTTCGACGA TGCCCGCTCG GTGCAGGCTA AAGGCAAGTA CGTGTTGGAT 1951
AAGCAGCTGG GCGGCCTGTT CTGGTGGGAG ATCGACGCGG ATAACGGCGA 2001
TATTCTCAAC AGCATGAACG CCAGCCTGGG CAACAGCGCC GGCGTTCAAT 2051
AATCGGTTGC AGTGGTTGCC GGGGGATATC CTTTCGCCCC CGGCTTTTTC 2101
GCCGACGAAA GTTTTTTTAC GCCGCACAGA TTGTGGCTCT GCCCCGAGCA 2151
AAACGCGCTC ATCGGACTCA CCCTTTTGGG TAATCCTTCA GCATTTCCTC 2201
CTGTCTTTAA CGGCGATCAC AAAAATAACC GTTCAGATAT TCATCATTCA 2251
GCAACAAAGT TTTGGCGTTT TTTAACGGAG TTAAAAACCA GTAAGTTTGT
[0134] Sequence listing D (ChiG)
[0135] Seq IDNo.: 4
[0136] Sequence type: nucleotide
[0137] Sequence length: 1013 nucleotides
[0138] Molecule type: cDNA
[0139] Original source: barley seeds (Hordeum vulgare L.)
[0140] Name: ChiG (chitinase G)
[0141] Feature: 63 pb-long 5'-non-translating initial region, 798
pb open reading frame, 152 pb-long 3'-non-translated end, reading
stop codons are marked by an asterisk, the probable signal peptide
sequences are underlined, the amino-acid sequence of a 26 kD
chitinase preprotein with 266 amino acids is indicated below the
nucleotide sequence, the underlined AT-rich sequence in position
905 is probably a polyadenylation signal.
[0142] Properties antifungal activity, especially on Trichoderma
reesii and Fusarium sporotrichoides as well as Rhizoctonia solani
and Botrytis cinerea. D
4 CCTACGACAGTAGCGTAACGGTAAACACCGAGTACGGTACTCTGTGCTTTGTTGGCTCGC 60 *
* ACAATGAGATCGCTCGCGGTGGTGGT- GGCCGTGGTAGCCACGGTGGCCATGGCCATCGGC
120 +E,uns M R S L A V V V A V V A T V A M A I G -20 -10
ACGGCGCGCGGCAGCGTGTCCTCCATCGTCTCGCGCGC- ACAGTTTGACCGCATGCTTCTC 180
+E,uns T A R G S V S S I V S R A Q F D R M L L -1 1 10
CACCGCAACGACGGCGCCTGCCAGGCCAAGGGCTTCTACACCTACGACGCCT- TCGTCGCC 240
+E,uns R R N D G A C Q A K G F Y T Y D A F V A 20 30
GCCGCAGCCGCCTTCCCGGGCTTCGGCACCACCGGCAGCGCCGACGCCCAGAAGCGCGAG 300
+E,uns A A A A F P G F G T T G S A D A Q K R +E,uns E 40 50
GTGGCCGCCTTCCTAGCACAGACCTCCCACGAGACCACCGGCGGGTGGGCGACTGCACCG 360
+E,uns V A A F L A Q T S H E T T G G W +E,uns A T A P 60 70
GACGGGGCCTTCGCCTGGGGCTACTGCTTCAAGCAGGAACGTGGCGCCTCCTCCGACTAC 420
+E,uns D G A F W W G Y C F K Q E R G A S S D Y 80 90
TGCACCCCGAGCGCACAATGGCCGTGCGCCCCCGGGAAGCGCTACTACGGCCGCGGGCCA 480 C
T P S A Q W P C A P G K R Y +E,uns Y G R G P 100 110
ATCCAGCTCTCCCACAACTACAA- CTATGGACCTGCCGGCCGGGCCATCGGGGTCGATCTG 540
+E,uns I Q L S H N Y N Y G P A G R A I G V D L 120 130
CTGGCCAACCCGGACCTGGTGGCCACGGACGCCACTGT- GGGCTTTAAGACGGCCATCTGG 600
+E,uns L A N P D L V A T D A T V G F K T A I W 140 150
TTCTGGATGACGGCGCAGCCGCCCAAGCCATCGAGCCATGCTGTGATCGCCGG- CCAGTGG 660
F W +E,uns M T A Q P P K P S S H A V I A G Q W 160 170
AGCCCGTCAGGGGCTGACCGGGCCGCAGGCCGGGTGCCCGGGTTTGGTGTGATCACCAAC 720
+E,uns S P S G A D R A A G R V P G F G V I T N 180 190
ATCATCAACGGCGGGATCGAGTGCGGTCACGGGCAGGACAGCCGCGTCGCCGATCGAATC 780
+E,uns I I N G G I E C G H G Q D S R V A D R I 200 210
GGGTTTTACAAGCGCTACTGTGACATCCTCGGCGTTGGCTACGGCAACAACCTCGATTGC 840
+E,uns G F Y K R Y C D I L G V G Y G N N L D C 220 230
TACAGCCAGAGACCCTTCGCCTAATTAATTAGTCATGTATTAATCTTGGCCCTCCATAAA 900
+E,uns Y S Q R P F A * 240 ATAC+E,uns
AATAAGAGCATCGTCTCCTATCTACATGCTGTAAGATGTAACTATGGTAACCTTTT 960
ATGGGGAACATAACAAAGGCATCTCGTATAGATGCTTTGCTA.sub.12 1013
[0143] Sequence listing E (GluG)
[0144] Seq IDNo.: 5
[0145] Sequence type: nucleotide with corresponding protein
[0146] Sequence length: 1249 nucleotides
[0147] Molecule type: cDNA
[0148] Original source: barley seed (Hordeum vulgare L.)
[0149] Name: GluG (glucanase)
[0150] Feature: 48 bp-long 5'-non-translating initial region open
reading frame of 1002 bp 199 pb-long 3'-non-translated end, the
underlined At-rich sequence at position 1083 and 1210 are probably
polyadenylation signals, the derived amino-acid sequence of the
encoded preprotein of 334 amino acids is indicated below the
nucleotide sequence. E
5 GGCAGCATTGCATAGCATTTGAGCACCAGATACTCCGTGTGTGCACCAATGGCTAGAAAA 60
+E,uns M A R K -28 * *
GATGTTGCCTCCATGTTTGCAGTTGCTCTCTTCATTGGAGCATTCGCTGCTGTTCCTACG 120
+E,uns D V A S M F A V A +E,uns L I G A F A A V P T -20 -10
AGTGTGCAGTCCATCGGCGTATGCTACGGCGTGATCGGCAACAACCTCCCCTCCCGGAGC 180
+E,uns S V Q S I G V C Y G V I G N N L P S R S -1 +1 10
GACGTGGTGCAGCTCTACAGGTCCAAGGGCATCAACGGCATGCGCAT{overscore
(CTACTTCGCCGAC)} 240 D V V Q L Y R S K G I N G M R I Y F A D 20 30
{overscore (GGGCAGGCCCTCTCGGCCGTCCGCAACTCCGGCATCGGCCTCATCCTCGACATC-
GGC)}AAC 300 G Q A L S A V R N S G I G L I L D I G N 40 50
GACCAGCTCGCCAACATCGCCGCCAGCACCTCCAACGCGGCCTCCTGGGTCCAGAACAAC 360 D
Q L A N I A A S T S N A A S W V Q N N 60 70
GTGCGGCCCTACTACCCTGCCGTGAACATCAAGTACATCGCCGCCGGCAACGAGGTGCAG 420 V
R P Y Y P A V N I K Y I A A G N E V Q 80 90
GGCGGCGCCACGCAGAGCATCCTGCCGGCCATGCGCAACCTCAACGCGGCCCTCTCCGCG 480 G
G A T Q S I L P A M R N L N A A L S A 100 110
GCGGGGCTCGGCGCCATCAAGGTGTCCACCTCCATCCGGTTCGACGAGGTGGCCAACTCC 540 A
G L G A I K V S T S I R F D E V A N S 120 130
TTCCCGCCCTCCGCCGGCGTGTTCAAGAACGCCTACATGACGGACGTGGCCCGGCTCCTG 600 F
P P S A G V F K N A Y M T D V A R L L 140 150
GCGAGCACCGGCGCGCCGCTGCTCGCCAACGTCTACCCCTACTTCGCGTACCGTGACAAC 660 A
S T G A P L L A N V Y P Y F A Y R D N 160 170
CCCGGGAGCATCAGCCTGAACTACGCGACGTTCCAGCCGGGCACCACCGTGCGTGACCAG 720 P
G S I S L N Y A T F Q P G T T V R D Q 180 190
AACAACGGGCTGACCTACACGTCCCTGTTCGACGCGATGGTGGACGCCGTGTACGCGGCG 780 N
N G L T Y T S L F D A M V D A V Y A A 200 210
CTGGAGAAGGCCGGCGCGCCGGCGGTGAAGGTGGTGGTGTCGGAGAGCGGGTGGCCGTGG 840 L
E K A G A P A V K V V V S E S G W P S 220 230
GCGGGCGGGTTTGCGGCGTCGGCCGGCAATGCGCGGACGTACAACCAGGGGCTGATCAAC 900 A
G G F A A S A G N A R T Y N Q G L I N 240 250
CACGTCGGCGGGGGCACGCCCAAGAAGCGGGAGGCGCTGGAGACGTACATCTTCGCCATG 960 H
V G G G T P K K R E A L E T Y I F A M 260 270
TTCAAGGAGAACCAGAAGACCGGCCACGCCACGGAGAGGAGCTTCGGGCTCTTCAACCCG 1020 F
N E N Q K Y G D A T E R S F G L F N P 280 290
GACAAGTCGCCGGCATACAACATCCAGTTCTAGTACGTGTAGCTACCTAGCTCACATACC 1080 D
K S P A Y N I Q F * 300 TA+E,uns
AATAAATAAGCTGCACGTACGTACGTAATGCGGCATCCAAGTGTAACGTAGACACGTA 1140
CATTCATCCATGGAAGAGTGCAACCAAGCATGCGTTAACTTCCTGGTGATGATACAT- CAT 1200
CATGGTATGAATAAAAGATATGGAAGATGTTATGA.sub.15 1249
[0151]
Sequence CWU 1
1
* * * * *