Note: Descriptions are shown in the official language in which they were submitted.
1 ~~'~~~~''~' MA53.C1
DESCRIPTION
NOVEL COLEOPTERAN-ACTIVE BACILLUS T~IURINGIENSIS ISOLATE
AND A NOVEL GENE ENCODING A COLEOPTERAN-ACTIVE T'OHI~T
10 Background of the Invention
Bacillus thuringiensis (B-t.) produces an insect toxin designated as ~
endotoxin. It is synthesized by the B-t. sporulating cell. The toxin, upon
being
ingested in its crystalline form by susceptible insect larvae, is transformed
into
biologically active moieties by the insect gut juice proteases. The primary
target
is insect cells of the gut epithelium, which are rapidly destroyed:
The reported activity spectrum of Ba. covers insect species within the
order Lepidoptera, many of which are major pests in agriculture and forestry.
The
activity spectrum also includes the insect order Diptera, which includes
mosquitos
and black flies. See Couch, T.L. (1980) "Mosquito Pathogenicity of Bacillus
thuringiensis var. israelensis;' Developments in Industrial Microbiology 22:61-
76;
Beegle, C.C., (1978) "Use of Entomogenous Bacteria in Agroecosystems,"
Developments in Industrial Microbiology 20:97-104. Krieg, et al., Z. ang. Ent.
(1983) 96:500-508, describe a B~t. isolate named Bacillus thurin '~ensis var.
tenebrionis, which is reportedly active against two beetles in the order
Coleoptera.
These are the Colorado potato beetle, Leptinotarsa decemlineata, and elastics
alni.
In European Patent Application 0 202 739 there is disclosed a novel B~t.
isolate active against Coleoptera. It is known as B. thurin~iensis var. son
die~o
(B.t.sd.). U.S. Patent No. 4,966,765 discloses the coleopteran-active Bacillus
thurin~iensis isolate B~t. PS86B1. European Patent Application 0 337 604 also
discloses a novel B~t. isolate active against C.oleoptera. This isolate is B-
t. PS43F.
v4 P.~H W~/
2 MA53.C1
Coleopteran-active strains, such as B.t.sd.. B-t. PS86B1, and B~t. PS43F,
can be used to control foliar-feeding beetles. The Colorado potato beetle
(Lentinotarsa decemlineata), for example, is susceptible to the delta-
endotoxin of
B.t.sd. and larvae are killed upon ingesting a sufficient dose of
spore/crystal
preparation on treated foliage.
A number of crops are attacked by flea beetles. These beetles belong
to the family Chrysomelidae, the deeemlineata. The adults can cause extensive
damage by feeding on the foliage.
Brief Summary of the Invention
The subject invention concerns a novel Bacillus thurin '~nsis (B-t.) isolate
and a cloned gene therefrom which encodes a novel coleopteran-active protein.
The novel B-t. isolate, known herein as Bacillus thuring;iensis PS50C (B~t.
PS50C),
has thus far been shown to be active against the Colorado potato beetle
(Leptinotarsa decemlineata). The novel d-endotoxin gene of the invention
encodes an =130 kDa protein. The nucleotide sequence of the gene (open reading
frame only) is shown in Sequence ID No. 1. The predicted peptide sequence of
the toxin is shown in Sequence )D No. 2.
The subject invention also includes mutants of B-t. PS50C which have
substantially the same pesticidal properties as B-t. PS50C. Procedures for
malting
mutants are well )mown in the microbiological art. Ultraviolet light and
nitrosoguanidine are used extensively toward this end.
Further, the invention also includes the treatment of substantially intact
B-t. PS50C cells, and recombinant cells containing the gene of the invention,
to
prolong the pesticidal activity when the substantially intact cells are
applied to the
environment of a target pest. Such treatment can be by chemical or physical
means, or a combination of chemical or physical means, so long as the
technique
does not deleteriously affect the properties of the pesticide, nor diminish
the
cellular capability in protecting the pesticide. The treated cell acts as a
protective
coating for the pesticidal toxin. The toxin becomes available to act as such
upon
ingestion by a target insect.
3
~3.C1
Brief Description of the Sequences
Sequence ID No. l is the nucleotide sequence (open reading frame only)
of the novel gene of the invention.
Sequence ID No. Z is the predicted peptide sequence of the toxin.
Brief Description of the Drawings
Figure 1 - Photograph of a Standard SDS Polyacrylamide Gel of B~t.
PS50C, B.t.sd.. and B~t. PS86B1.
Figure 2 - Restriction map of pMYC1638.
Deta~ed Disclosure of the Invention
The novel Bacillus thu '~~ iensis isolate of the subject invention has the
following characteristics in its biologically pure form:
Characteristics of t~3.t. PS50C
Colony morphology--Large colony, dull surface, typical B~t.
Vegetative cell morphology--typical B-t. .
Culture methods--typical for B-t.
Flagellar serotyping--PS50C belongs to serotype 18, kumamotoensis.
Crystal morphology--a sphere.
RFLP analysis--Southern hybridization of total DNA distinguishes B~t.
PSSOC from B.t.sd. and other B.t. isolates.
Alkali-soluble proteins-SDS polyacrylamide gel electrophoresis (SDS-
PAGE) shows a 130 kDa doublet protein.
A comparison of the characteristics of B. thurin~iensis PS50C (B-tt.
PS50C) to the characteristics of the known B_t. strains B. thuringiensis var.
san
die~o (B.t.sd.), B. thurin 'ensis PS86B1 (NRRL B-18299), and B. thuringiensis
var.
kurstaki (HD-1) is shown in Table 1.
CA 02059242 2001-05-23
4
Table 1. Comparison of B-t. PSSOC, B-t. PS86B1, B.t.sd., and B-t. HD-1
B-t. PSSOC B.t.sd. B_t. PS86B1 B-t. HD-1
Serovar kumamotoensis morrisoni tolworthi kurstaki
Type of inclusionsphere square flat, pointedBipyramid
wafer ellipse, plus
sm. inclusions
Size of alkali-130 kDa 72,000 75,000 130,000
soluble proteinsdoublet 64,000 68,000 68,000
by SDS-PAGE 61,000
Host range Coleoptera Coleoptera Coleoptera Lepidoptera
The cultures disclosed in this application have been deposited in the
Agricultural Research Service Patent Culture Collection (NRRL), Northern
Regional Research Center, 1815 North University Street, Peoria, Illinois
61604,
USA.
S
Culture Repository No. Deposit date
Bacillus thurin '~ensis NRRL B-18746 January 9, 1991
PSSOC
Escherichia coli NM522 NRRL B-18751 January 11, 1991
[pMYC1638J
The subject cultures have been deposited under conditions that assure that
access to the cultures will be available during the pendency of this patent
application
to one determined by the Commissioner of Patents to be entitled thereto. The
deposits
are available as required by foreign patent laws in countries wherein
counterparts of
the subject application, or its progeny, are filed. However, it should be
understood that
the availability of a deposit does not constitute a license to practice the
subject
invention in derogation of patent rights granted by governmental action.
MA53.C1
Further, the subject culture deposits will be stored and made available
to the public in accord with the provisions of the Budapest 'Treaty for the
Deposit
of Microorganisms, i.e., they will be stored with all the care necessary to
keep
them viable and uncontaminated for a period of at least five years after the
mast
S recent request for the furnishing of a sample of a deposit, and in any case,
for a
period of at least thirty (30) years after the date of deposit or for the
enforceable
life of any patent which may issue disclosing the cultures. The depositor
acknowledges the duty to replace the deposits should the depository be unable
to
furnish a sample when requested, due to the condition of the deposits. All
restrictions on the availability to the public of the subject culture deposits
will be
irrevocably removed upon the granting of a patent disclosing them.
B-t. PSSOC, NRRL B-18746, can be cultured using standard art media
and fermentation techniques. Upon completion of the fermentation cycle, the
bacteria can be harvested by first separating the B-,t. spores and crystals
from the
IS fermentation broth by means well known in the art. The recovered B-t.
spares and
crystals can be formulated into a wettable powder, liquid concentrate,
granules, or
other formulations by the addition of surfactants, dispersants, inert carriers
and
other components to facilitate handling and application for particular target
pests.
These formulation and application procedures are all well known in the art.
Plasmid DNA (pMYC1638) containing the toxin gene from B-t. PS50C
can be purified from E. coli NM522(pMYC1638] by standard procedures well
known in the art. The toxin gene can be excised from the plasmid DNA by
restriction enzyme digestion, as indicated in Figure 2.
Formulated products can be sprayed or applied onto foliage to control
phytophagous beetles or caterpillars.
Another approach that can be taken is to incorporate the spores and
crystals of BB~t. PSSOC into bait granules containing an attractant and
applying
these granules to the soil for control of soil-inhabiting Coleoptera.
Formulated
B-t. PSSOC can also be applied as a seed-coating ar root treatment or total
plant
treatment.
6 MA53.C1
The B-t. PSSOC cells can be treated prior to formulation to prolong the
pesticidal activity when the cells are applied to the environment of a target
pest.
Such treatment can be by chemical or physical means, or by a combination of
chemical and/or physical means, so long as the technique does not
deleteriously
S affect the properties of the pesticide, :nor diminish the cellular
capability in
protecting the pesticide. Examples of chemical reagents are halogenating
agents,
particularly halogens of atomic no. 17-80. More particularly, iodine can be
used
under mild conditions and for sufficient time to achieve the desired results.
Other
suitable techniques include treatment with aldehydes, such as formaldehyde and
glutaraldehyde; anti-infectives, such as zephiran chloride; alcohols, such as
isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative
and
Helly's fixative (See: Humason, Gretchen. L., Animal Tissue Techniques, W.FI.
Freeman and Company, 1967); or a combination of physical (heat) and chemical
agents that prolong the activity of the toxin produced in the cell when the
cell is
applied to the environment of the target pest(s). Examples of physical means
are
short wavelength radiation such as gamma-radiation and X-radiation, freezing,
UV
irradiation, lyophilization, and the like.
The novel toxin gene of the subject invention was obtained from a navel
coleopteran-active B. thurin 'ensis (B-t.) isolate designated B-t. PSSOC. The
gene
was isolated as disclosed in the Examples.
The toxin gene of the subject invention can be introduced into a wide
variety of microbial hosts. Expression of the toxin gene results, directly or
indirectly, in the intracellular production and maintenance of the pesticide.
With
suitable hosts, e.g., Pseudomonas, the microbes can be applied to the situs of
coleopteran insects where they will proliferate and be ingested by the
insects. The
result is a control of the unwanted insects. Alternatively, the microbe
hosting the
toxin gene can be treated under conditions that prolong the activity of the
toxin
produced in the cell. The treated cell then can be applied to the environment
of
target pest(s). The resulting product retains the toxicity of the B-t. toxin.
r
s~
7 MA53.C1
Where the B-t, toxin gene is introduced via a suitable vector into a
microbial host, and said host is applied to the environment in a living state,
it is
essential that certain host microbes be used. Microorganism hosts are selected
which are known to occupy the "phytosphere" (phylloplane, phyllosphere,
rhizosphere, and/or rhizoplane) of one or more crops of interest. These
microorganisms are selected so as to be capable of successfully competing in
the
particular environment (crop and other insect habitats) with the wild-type
microorganisms, provide for stable maintenance and expression of the gene
expressing the polypeptide pesticide, and, desirably, provide for improved
protection of the pesticide from environmental degradation and inactivation.
A large number of microorganisms are known to inhabit the phylloplane
(the surface of the plant leaves) and/or the rhizosphere (the soil surrounding
plant
roots) of a wide variety of important crops. These microorganisms include
bacteria, algae, and fungi. Of particular interest are microorganisms, such as
bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella.
Xanthomonas,
Streptom~, Rhizobium, lZhodopseudomonas. Methvlophilius, Agrobacterium,
Acetobacter, Lactobacillus, Arthrobacter, Azotobacter. Leuconostoc, and
Alcali es~nes; fungi, particularly yeast, e.g., genera Saccharomyces,
Cryptococcus,
Kluyveromyces, Sporobolomvces, Rhodotorula, and Aureobasidium. Of particular
interest are such phytosphere bacterial species as Pseudomonas syrin age,
Pseudomonas fluorescens, Serratia marcescens, Acetobacter linum
A~robacterium tumefaciens. IZhodopseudomonas spheroides, Xanthomonas
cam ep StTIS, Rhizobium melioti, Alcafi enes entrophus, and Azotobacter
vinlandii;
and phytosphere yeast species such as Rhodotorula rubra, R, utinis R, marina,
R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei,
S. pretoriensis, S. cerevisiae, Sporobolomvces roseus, S. odorus, HIuweromvces
veronae, and Aureobasidium pollulans. Of particular interest are the pigmented
microorganisms.
A wide variety of ways are available for introducing the B~t. gene
expressing the toxin into the microorganism host under conditions which allow
for
~~i~u'~~~c
8 MA53.C1
stable maintenance and expression of the gene. One can provide for DNA
constructs which include the transcriptional and translational regulatory
signals for
expression of the toxin gene, the toxin gene under their regulatory control
and a
DNA sequence homologous with a sequence in the host organism, whereby
integration will occur, and/or a replication system which is functional in the
host,
whereby integration or stable maintenance will occur.
The transcriptional initiation signals will include a promoter and a
transcriptional initiation start site. In same instances, it may be desirable
to
provide for regulative expression of the toxin, where expression of the toxin
will
only occur after release into the environment. This can be achieved with
operators or a region binding to an activator or enhancers, which are capable
of
induction upon a change in the physical or chemical environment of the
microorganisms. For example, a temperature sensitive regulatory region may be
employed, where the organisms may be grown up in the laboratory without
expression of a toxin, but upon release into the environment, expression would
begin. Other techniques may employ a specific nutrient medium in the
laboratory,
which inhibits the expression of the toxin, where the nutrient medium in the
environment would allow for expression of the toxin. Far translational
initiation,
a n'bosomal binding site and an initiation codon will be present.
Various manipulations may be employed for enhancing the expression
of the messenger, particularly by using an active promoter, as well as by
employing
sequences, which enhance the stability of the messenger RNA. The initiation
and
translational termination region will involve stop codon(s), a terminator
region,
and optionally, a polyadenylation signal.
In the direction of transcription, namely in the 5' to 3' direction of the
coding or sense sequence, the construct will involve the transcriptional
regulatory
region, if any, and the promoter, where the regulatory region may be either 5'
or
3' of the promoter, the ribosomal binding site, the initiation codon, the
structural
gene having an open reading frame in phase with the initiation codon, the stop
codon(s), the polyadenylation signal sequence, if any, and the terminator
region.
~(?~~
9 MA53.C1
This sequence as a double strand may be used by itself far transformation of a
microorganism host, but will usually be included with a DNA sequence involving
a marker, where the second DNA sequence may be joined to the toxin expression
construct during introduction of the DNA into the host.
S By a marker is intended a structural gene which provides for selection
of those hosts which have been modified or transformed. The marker will
normally provide for selective advantage, for example, providing far biocide
resistance, e.g., resistance to antibiotics or heavy metals; complementation,
so as
to provide prototropy to an auxotro;phic host, or the like. Preferably,
complementation is employed, so that the modified host may not only be
selected,
but may also be competitive in the field. Cne or more markers may be employed
in the development of the constructs, as well as for modifying the host. The
organisms may be further modified by providing for a competitive advantage
against other wild-type microorganisms in the field. For example, genes
expressing
metal chelating agents, e.g., siderophores, may be introduced into the host
along
with the structural gene expressing the toxin. In this manner, the enhanced
expression of a siderophore may provide for a competitive advantage for the
toxin-
producing host, so that it may effectively compete with the wild-type
microorganisms and stably occupy a niche in the environment.
Where no functional replication system is present, the construct will also
include a sequence of at least 50 basepairs (bp), preferably at least about
100 bp,
and usually not more than about 1000 by of a sequence homologous with a
sequence in the host. In this way, the probability of legitimate recombination
is
enhanced, so that the gene will be integrated into the host and stably
maintained
by the host. Desirably, the toxin gene will be in close proximity to the gene
providing for complementation as well as the gene providing for the
competitive
advantage. Therefore, in the event that a toxin gene is lost, the resulting
organism
will be likely to also lose the complementing gene and/or the gene providing
for
the competitive advantage, so that it will be unable to compete in the
environment
with the gene retaining the intact construct.
~'~'~'[ ~v (,~J_
/V9 'r ~iJ l~~l~
MA53.C1
A large number of transcriptional regulatory regions are available from
a wide variety of microorganism hosts, such as bacteria, bacteriophage,
cyanobacteria, algae, fungi, and 'the like. Various transcriptional regulatory
regions
include the regions associated with the trp gene, lac gene, gal gene, the
lambda left
5 and right promoters, the tac promoter, the naturally-occurring promoters
associated with the toxin gene, where functional in the host. See for example,
U.S.
Patent Nos. 4,332,898, 4,342,832 and 4,3:16,270. The termination region may be
the termination region normally associated with the transcriptional initiation
region
or a different transcriptional initiation region, so long as the two regions
are
10 compatible and functional in the host.
Where stable episomal maintenance or integration is desired, a plasmid
will be employed which has a replication system which is functional in the
host.
The replication system may 'be derived from the chromosome, an episomal
element normally present in the host or a different host, or a replication
system
from a virus which is stable in the host. A large number of plasmids are
available,
such as pBR322, pACYC184, RSF1010, pR01614, and the like. See for example,
Olson et al., (1982) J. Bacteriol. 150:6069, and Bagdasarian et al., (1982)
Gene
16:237, and U.S. Patent Nos. 4,356,270, 4,362,817, and 4,371,625.
The B-t. gene can be introduced between the transcriptional and
translational initiation region and the transcriptional and translational
termination
region, so as to be under the regulatory cantrol of the initiation region.
This
construct will be included in a plasmid, which will include at least one
replication
system, but may include more than one, where one replication system is
employed
for cloning during the development of the plasmid and the second replication
system is necessary for functioning in the ultimate host. In addition, one or
more
markers may be present, which have been described previously. Where
integration
is desired, the plasmid will desirably include a sequence homologous with the
host
genome.
The transformants can be isolated in accordance with conventional ways,
usually employing a selection technique, which allows for selection of the
desired
~~~...a~v~.° o~Cw
11 MA53.C1
organism as against unmodified organisms or transfernng organisms, when
present.
The transformants then can be tested for pesticidal activity.
Suitable host cells, where the pesticide-containing cells will be treated to
prolong the activity of the toxin in the cell when the then treated cell is
applied
S to the environment of target pest(s), may include either prokaryotes or
eukaryotes,
normally being limited to those cells which do not produce substances toxic to
higher organisms, such as mammals. However, organisms which produce
substances toxic to higher organisms could be used, where the toxin is
unstable or
the level of application sufficiently low a:> to avoid any possibility of
toxicity to a
mammalian host. As hosts, of particular interest will be the prokaryotes and
the
lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative
and -
positive, include Enterobacteriaceae, such as Escherichia. Erwinia, Shigella,
Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium:
Spirillaceae, such as photobacterium, Zymomonas, Serratia. Aeromonas. Vibrio,
Desulfovibrio, Spirillum; L.actobacillaceae; Pseudomonadaceae, such as
Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among
eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes
yeast,
such as Saccharomvces and Schizosaccharomyces; and Basidiomycetes yeast, such
as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for purposes
of production include ease of introducing the B-t, gene into the host,
availability
of expression systems, efficiency of expression, stability of the pesticide in
the host,
and the presence of auxiliary genetic capabilities. Characteristics of
interest for
use as a pesticide microcapsule include protective qualities for the
pesticide, such
as thick cell walls, pigmentation, and intracellular packaging or formation of
inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to
pests
for ingestion; ease of killing and fixing without damage to the toxin; and the
like.
Other considerations include ease of formulation and handling, economics,
storage
stability, and the like.
12 MA53.C1
PIost organisms of particular interest include yeast, such as Rhodotorula
sp., Aureobasidium sp., Saccharornvces sp., and Sporobolornyces sp.;
phylloplane
organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such
other organisms as Escherichia, Lactobacillus sp., Bacillus sp., Streptomyces
sp.,
and the like. Specific organisms include Pseudomonas ae~nosa, Pseudomonas
fluorescens, Saccharom,~ces cerevisiae, Bacillus thurin 'ensis, Escherichia
colic
Bacillus subtilis, Strentomyces lividans, and the like.
The cell will usually be intact and be substantially in the proliferative
form when treated, rather than in a spore form, although in some instances
spores
may be employed.
Treatment of the recombinant microbial cell can be done as disclosed
infra. The treated cells generally will have enhanced structural stability
which will
enhance resistance to environmental conditions. ~llrhere the pesticide is in a
proform, the method of inactivation should be selected so as not to inhibit
processing of the proform to the mature form of the pesticide by the target
pest
pathogen. For example, formaldehyde will crosslink proteins and could inlu'bit
processing of the proform of a polypeptide pesticide. The method of
inactivation
or killing retains at least a substantial portion of the bio-availability or
bioactivity
of the toxin.
The cellular host containing the B-t. insecticidal gene may be grown in
any convenient nutrient medium, where the DNA construct provides a selective
advantage, providing for a selective medium so that substantially all or all
of the
cells retain the B-t, gene. These cells may then be harvested in accordance
with
conventional ways. Alternatively, the cells can be treated prior to
harvesting.
The B-t. cells may be formulated in a variety of ways. They may be
employed as wettable powders, granules or dusts, by mixing with various inert
materials, such as inorganic minerals (phyllasilicates, carbonates, sulfates,
phosphates, and the like) or botanical materials (powdered corncobs, rice
hulls,
walnut shells, and the like). The formulations may include spreader-sticker
adjuvants, stabilizing agents, other pesticidal additives, or surfactants.
Liquid
13 IVIA53.C1
formulations may be aqueous-based or non-aqueous and employed as foams, gels,
suspensions, emulsifiable concentrates, or the like. The ingredients may
include
rheological agents, surfactants, emulsifiers, dispersants, or polymers.
The pesticidal concentration will vary widely depending upon the nature
of the particular formulation, particularly whether it is a concentrate or to
be used
directly. The pesticide will be present in at least 1% by weight and may be
100%
by weight. The dry formulations will have from about 1-95% by weight of the
pesticide while the liquid formulations will generally be from about 1-60% by
weight of the solids in the liquid phase. The formulations will generally have
from
about 102 to about 104 cells/mg. These formulations will be administered at
about
50 mg (liquid or dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the coleopteran
pest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling, or the
like.
Following are examples which illustrate procedures, including the best
mode, for practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture proportions
are by
volume unless otherwise noted.
Example 1 - Culturing B.t PS50C NRRL B-18746
A subculture of B-t. PS50C, NRRL B-18746 can be used to inoculate the
following medium, a peptone, glucose, salts medium.
Bacto Peptone 7.5 g/(
Glucose 1.0 gIl
KHZP04 3.4 g/!
KZHPO4 4.35 g/I
Salt Solution 5.0 m1/1
CaCl2 Solution 5.0 ml/I
Salts Solution (100 ml)
llrlgS0~.7H20 2.46 g
fit; '~~:~~s~
14 MA53.C1
MnSO~.H20 0.04 g
ZnS04.7Hz0 0.28 g
FeS04.7H20 0.40 g
CaCl2 Solution (100 ml)
CaC12.2H20 3.66 g
pH 7.2
The salts solution and CaCl2 solution are filter-sterilized and added to
the autoclaved and cooked broth at the time of inoculation. ?Masks are
incubated
at 30 ° C on a rotary shaker at 200 rpm for 64 hr.
The above procedure can be readily scaled up to large fermentors by
procedures well known in the art.
The B-t. spores and crystals, obtained in the above fermentation, can be
isolated by procedures well known in the art. A frequently-used procedure is
to
subject the harvested fermentation broth to separation techniques, e.g.,
centrifugation.
Example 2 - Testing of B.t. PSSOC NRRL B-18746 Spores and C , sr~tals
B-t. PS50C, NRRL B-18746 spores and crystals are toxic to the Colorado
potato beetle (CPB). The assay for the Colorado potato beetle was conducted as
follows:
CPB Biaassav - Early second instar larvae of Lentinotarsa decemlineata
are placed on potato leaves which have been dipped in suspensions containing
Bacillus thurin 'ensis preparations. The larvae are incubated at 25 ° C
for 4 days,
and larval mortality is recorded and analyzed using probit analysis.
Example 3 - Cloning of a Novel Toxin Gene from B t Isolate PSSOC
Total cellular DNA was prepared from Bacillus thurin '~ensis (B-t.) cells
grown to an optical density, at 600 nm, of 1Ø The cells were recovered by
CA 02059242 2001-05-23
centrifugation and protoplasts were prepared in TES buffer (30 mM Tris-HC1, 10
mM EDTA, 50 mM NaCI, pH = 8.0) containing 20% sucrose and 50 mg/ml
lysozyme. The protoplasts were lysed by addition of SDS to a final
concentration
of 4%. The cellular material was precipitated overnight at 4°C in 100
mM (final
5 concentration) neutral potassium chloride. The supernate was extracted twice
with
phenol/chloroform (1:1). rJucleic acids were precipitated with ethanol and DNA
was purified by isopycnic banding on cesium chloride-ethidium bromide
gradients.
Total cellular DrTA from B-t. subsp. kumamotoensis (B.t.kum.), isolate
PSSOC, was digested with I~indIII and fractionated by electrophoresis on a
0.8%
10 (w/v) agarose-TAE (50 mIvl Tris-HCI, 20 mM NaOAc, 2.5 mM EDTA, pH = 8.0)
buffered gel. A Southern blot of the gel was hybridized with a [32P]-
radiolabeled
oligonucleotide probe. Results showed that the hybridizing fragments of PSSOC
are approximately 12 Kb and 1.7 Kb in size.
A library was constructed from PSSOC total cellular DNA partially
15 digested with Sau3A and size fractionated by gel electrophoresis. The 9-23
Kb
region of the gel was excised and the DNA was electroeluted and then
concentrated using an Eluvtip-d~ ion exchange column (Schleicher and Schuel,
Keene, NH). The isolated Sau3A fragments were ligated into BamHI-digested
LambdaGEM-11~ (PROrrfEGA). The packaged phage were plated on E. coli
KW251 cells (PROMEGA) at a high titer and screened using the radiolabeled
oligonucleotide probe. Hybridizing plaques were purified and rescreened at a
lower plaque density. Sinhle isolated, purified plaques that hybridized with
the
probe were used to infect F- coli KW251 cells in liquid culture for
preparation of
phage for DNA isolation. I>NA was isolated by standard procedures. Preparative
amounts of DNA were dif;ested with XhoI (to release the inserted DNA from
lambda sequences) and separated by electrophoresis on a 0.6% agarose-TAE gel.
The large fragments were purified by ion exchange chromatography as above and
ligated to XhoI-digested, de,phosphorylated pHTBIueII (an E. co ' - thurin
'ensis
shuttle vector comprised of pBluescript s/k [Stratagene] and the replication
origin
from a resident B-t. plasmicl [D. Lereclus et al. 1989. FEMS Microbiology
Letters
*Trade-mark
16 MA53.C1
60:211-218]). The ligation mix was introduced by transformation into competent
E. coli NM522 cells (ATCC 47000) and plated on LB agar containing ampicillin,
isopropyl-(,B)-D-thiogalactoside (IPTG) and 5-bromo-4-chloro-4-indolyl-(~)-D-
galactoside (XGAL). White colonies, with putative restriction fragment
insertions
in the (~)-galactosidase gene of pHTBIueII, were subjected to standard rapid
plasmid purification procedures. Plasmids were analyzed by XhoI digestion and
agarose gel electrophoresis. The desired plasmid construct, pMYC1638, contains
an approximately 12 Kb XhoI insert. A partial restriction map (Figure 2) of
the
cloned insert indicates that the toxin gene is novel compared to the maps of
other
toxin genes encoding insecticidal proteins. The nucleotide sequence (open
reading
frame only) is shown in Sequence >D No. 1. The predicted peptide sequence of
the toxin is shown in Sequence 1D No. 2.
Plasmid pMYC1638 was introduced into an acrystalliferous (Cry ) B-t.
host (HD-1 cryB obtained from A. Aronson, Purdue University) by
electroporation. Expression of an approximately 130 kDa protein was verified
by
SDS-PAGE. Broth containing spores and crystals was used for the determination
of toxicity to Leptinotarsa decemlineata.
Plasmid pMYC1638 containing the B_t, toxin gene, can be removed from
the transformed host microbe by use of standard well-known procedures. For
example, E. coli NM522[pMYC1638] NRRL B-18751 can be subjected to cleared
lysate isopycnic density gradient procedures, and the like, to recover
pMYC1638.
Example 4 - Insertion of Toxin Gene Into Plants
The novel gene coding for the novel insecticidal toxin, as disclosed
herein, can be inserted into plant cells using the Ti plasmid from A rg
obacter
tumefaciens. Plant cells can then be caused to regenerate into plants
(~ambryski,
P., Joos, H., Gentello, C., Leemans, J., Van Montague, M. and Schell, J [1983]
Cell
32:1033-1043). A particularly useful vector in this regard is pEND4K (Klee,
H.J.,
Yanofsky, M.F. and Nester, E.W. [1985] BiofTechnology 3:637-642). This plasmid
can replicate both in plant cells and in bacteria and has multiple cloning
sites for
aC'n~..~.~"'~ ~~c d
17 MA53.C1
passenger genes. The toxin gene, for Pxample, can be inserted into the Baml-II
site of pEND4I~, propagated in E. coli, and transformed into appropriate plant
cells.
Example 5 - Cloning of Novel B. thuri~~nsis Gene Into Baculoviruses
The novel gene of the invention can be cloned into baculoviruses such
as Autographa californica nuclear polyhedrosis virus (AcNPV). Plasmids can be
constructed that contain the AcNPV genome cloned into a commercial cloning
vector such as pUCB. The AcNPV genome is modified so that the coding region
of the polyhedrin gene is removed and a unique cloning site for a passenger
gene
is placed directly behind the polyhedrin promoter. Examples of such vectors
are
pGP-B6874, descnbed by Pennock et al. (Pennock, G.D., Shoemaker, C. and
Miller, L.K. [1984] Mol. Cell. Biol. 4:399-406), and pAC380, descn'bed by
Smith
et al. (Smith, G.E., Summers, M.D. and Eraser, M.J. [1983] Mol Cell. Biol.
3:2156-
2165). The gene coding for the novel protein toxin of the invention can be
modified with BamPII linkers at appropriate regions both upstream and
downstream from the coding region and inserted into the passenger site of one
of
the AcNPV vectors.
e~ ~...~9~~.b tame~9
18 MA53.C1
SEQUENCE LISTTNG
(1) GENERAL INFORMATION:
(i) APPLICANT:
Foncerrada,
Luis
R
Payne,
Jewel
M
Sick,
August
J
(ii) TITLE
OF
INVENTION:
Novel
Coleopteran-Active
Bacillus
th
i
i
i
ur
nc1
ens
s
Isolate
and
a
Novel
Gene
Encoding
a
Coleoperan-Active
Toxin
(iiij NUMBER
OF
SEQUENCES:
2
(iv )
CORRESPONDENCE
ADDRESS:
A ADDRESSEE: Roman Saliwanchik
B STREET: 2421 N.W. 41st Street, Ste A-1
C CITY: Gainesville
D STATE: FL
E COUNTRY: USA
F ZIP: 32606
(v) COMPUTER
READABLE
FORM:
(A) MEDIUM TYPE: Floppy disk
(B COMPUTER: IBM PC compatible
)
C OPERATING SYSTEM: PC-DOS/M5-DOS
(
)
D SOFTWARE: PatentIn Release #1.0, Version
( #1.25
)
(vi) CURRENT
APPLICATION
DATA:
A APPLICATION NUMBER:
B FILING DATE:
~
~
C CLASSIFICATION:
(viii)ATTORNEY/AGENT
INFORMATION:
(A)
NAME:
Saliwanchik,
Roman
(ix) TELECOMMUNICATION
INFORMATION:
(A)
TELEPHONE:
904-375-8100
(B)
TELEFAX:
904-372-5800
(2)
INFORMATTON
FOR
SEQ
ID
NO:1:
(i) SEQUENCE
CHARACTERISTICS:
A LENGTH: 3471 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: double
~
~
D TOPOLOGY: linear
(ii) MOLECULE
TYPE:
DNA
(genomic)
(iii) HYPOTHETICAL:
NO
(iv) ANTI-SENSE:
NO
(vi) ORIGINAL
SOURCE:
A ORGANISM: Bacillus thuringiensis
B STRAIN: kumamotoensia
~
~
C INDIVIDUAL ISOLATE: PS50C
(vii) IMMEDIATE
SOURCE:
(A)
LIBRARY:
LAMBDAGEM
(TM)
-
11
LIBRARY
OF
LUTS
FONCERRADA
(B)
CLONE:
50C
(xi) SEQUENCE
DESCRIPTION:
SEQ
ID
N0:1:
ATGAGTCCAA 60
ATAATCAAAA
TGAATATGAA
ATTATAGATG
CGACACCTTC
TACATCTGTA
TCCAGTGATT 120
CTAACAGATA
CCCTTTTGCG
AATGAGCCAA
CAGATGCGTT
ACAAAATATG
AATTATAAAG 180
ATTATCTGAA
AATGTCTGGG
GGAGAGAATC
CTGAATTATT
TGGAAATCCG
GAGACGTTTA 240
TTAGTTCATC
CACGATTCAA
ACTGGAATTG
GCATTGTTGG
TCGAATACTA
GGAGCTTTAG 300
GGGTTCCATT
TGCTAGTCAG
ATAGCTAGTT
TCTATAGTTT
CATTGTTGGT
CAATTATGGC 360
CGTCAAAGAG
CGTAGATATA
TGGGGAGAAA
TTATGGAACG
AGTGGAAGAA
~1L'fr~.~IvP,~ r...8
R.r ~.. ....~ .~,m ~,s ~KVd
19 MAS3.C1
CTCGTTGATC CTCTTGCTGA ATTAAAAGGG420
AAAAAATAGA
AAAATATGTA
AAAGATAAGG
CTAGGAAATGCTTTGGATGTATATCAGCAGTCACTTGAAGATTGGCTGGA AAATCGCAAT480
GATGCAAGAACTAGAAGTGTTGTTTCTAATCAATTTATAGCTTTAGATCT TAACTTTGTT540
AGTTCAATTCCATCTTTTGCAGTATCCGGACACGAAGTACTATTATTAGC AGTATATGCA600
CAGGCTGTGAACCTACATTTATTGTTATTAAGAGATGCTTCTATTTTTGG AGAAGAGTGG660
GGATTTACACCAGGTGAAATTTCTAGATTTTATAATCGTCAAGTGCAACT TACCGCTGAA720
TATTCAGACTATTGTGTAAAGTGGTATAAAATC:GGCTTAGATAAATTGAA AGGTACCACT780
TCTAAAAGTTGGCTGAATTATCATCAGTTCCG7'AGAGAGATGACATTACT GGTATTAGAT840
TTGGTGGCGTTATTTCCAAACTATGACACACATATGTATCCAATCGAAAC AACAGCTCAA900
CTTACACGGGATGTGTATACAGATCCGATAGCATTTAACATAGTGACAAG TACTGGATTC960
TGCAACCCTTGGTCAACCCACAGTGGTATTCTTTTTTATGAAGTTGAAAA CAACGTAATT1020
CGTCCGCCACACTTGTTTGATATACTCAGCTCAGTAGAAATTAATACAAG TAGAGGGGGT1080
ATTACGTTAAATAATGATGCATATATAAACTACTGGTCAGGACATACCCT AAAATATCGT1140
AGAACAGCTGATTCGACCGTAACATACACAGCTAATTACGGTCGAATCAC TTCAGAAAAG1200
AATTCATTTGCACTTGAGGATAGGGATATTTTTGAAATTAATTCAACTGT GGCAAACCTA1260
GCTAATTACTACCAAAAGGCATATGGTGTGCCGGGATCTTGGTTCCATAT GGTAAAAAGG1320
GGAACCTCATCAACAACAGCGTATTTATATTCAAAAACACATACAGCTCT CCAAGGGTGT1380
ACACAGGTTTATGAATCAAGTGATGAAATACCTCTAGATAGAACTGTACC GGTAGCTGAA1440
AGCTATAGTCATAGATTATCTCATATTACCTCCCATTCTTTCTCTAAAAA TGGGAGTGCA1500
TACTATGGGAGTTTCCCTGTATTTGTTTGGACACATACTAGTGCGGATTT AAATAATACA1560
ATATATTCAGATAAAATCACTCAAATTCCAGCGGTAAAGGGAGACATGTT ATATCTAGGG1620
GGTTCCGTAGTACAGGGTCCTGGATTTACAGGAGGAGATATATTAAAAAG AACCAATCCT1680
AGCATATTAGGGACCTTTGCGGTTACAGTAAATGGGTCGTTATCACAAAG ATATCGTGTA1740
AGAATTCGCTATGCCTCTACAACAGATTTTGAATTTACTCTATACCTTGG CGACACAATA1800
GAAAAAAATAGATTTAACAAAACTATGGATAATGGGGCATCTTTAACGTA TGAAACATTT1860
AAATTCGCAAGTTTCATTACTGATTTCCAATTCAGAGAAACACAAGATAA AATACTCCTA1920
TCCATGGGTGATTTTAGCTCCGGTCAAGAAGTTTATATAGACCGAATCGA ATTCATCCCA1980
GTAGATGAGACATATGAGGCGGAACAAGATTTAGAAGCGGCGAAGAAAGC AGTGAATGCC2040
TTGTTTACGAATACAAAAGATGGCTTACGACCAGGTGTAACGGATTATGA AGTAAATCAA2100
GCGGCAAACTTAGTGGAATGCCTATCGGATGATTTATATCCAAATGAAAA ACGATTGTTA2160
TTTGATGCGGTGAGAGAGGCAAAACGCCTCAGTGGGGCACGTAACTTACT ACAAGATCCA2220
GATTTCCAAGAGATAAACGGAGAAAATGGATGGGCGGCAAGTACGGGAAT TGAGATTGTA2280
GAAGGGGATGCTGTATTTAAAGGACGTTATCTACGCCTACCAGGTGCACG AGAAATTGAT2340
ACGGAAACGTATCCAACGTATCTGTATCAAAAAGTAGAGGAAGGTGTATT AAAACCATAC2400
ACAAGATATAGACTGAGAGGGTTTGTGGGAAGTAGTCAAGGATTAGAAAT TTATACGATA2460
CGTCACCAAACGAATCGAATTGTAAAGAATGTACCAGATGATTTATTGCC AGATGTATCT2520
CCTGTAAACTCTGATGGCAGTATCAATCGATGCAGCGAACAAAAGTATGT GAATAGCCGT2580
TTAGAAGGAGAAAACCGTTCTGGTGATGCACATGAGTTCTCGCTCCCTAT CGATATAGGA2640
GAGCTGGATTACAATGAAAATGCAGGAATATGGGTTGGATTTAAGATTAC GGACCCAGAG2700
~C' a~,~~°~
20 MA53.C1
GGATACGCAA GTCGAAGAGGGACCTTTGTCAGGAGACGCA2760
CACTTGGAAA
TCTTGAATTA
TTAGAGCGCTTGCAAAGACAAGAACAACAGTGGAAGATTCAAATGACAAGAAGACGTGAA2820
GAGACAGATAGAAGATACATGGCATCGAAACAAGCGGTAGATCGTT.'TATATGCCGATTAT2880
CAGGATCAACAACTGAATCCTGATGTAGAGATTACAGATCTTACTGCGGCTCAAGATCTG2940
ATACAGTCCATTCCTTACGTATATAACGAAATGTTCCCAGAAATACCAGGGATGAACTAT3000
ACGAAGTTTACAGAATTAACAGATCGACTCCAACAAGCGTGGAATTTGTATGATCAGCGA3060
AATGCCATACCAAATGGTGATTTTCGAAATGGGTTAAGTAATTGGAATGCAACGCCTGGC3120
GTAGAAGTACAACAAATCAATCATACATCTGTCCTTGTGATTCCAAACTGGGATGAACAA3180
GTTTCACAACAGTTTACAGTTCAACCGAATCAAAGATATGTATTACGAGTTACTGCAAGA3240
AAAGAAGGGGTAGGAAATGGATATGTAAGTATTCGTGATGGTGGAAATCAATCAGAAACG3300
CTTACTTTTAGTGCAAGCGATTATGATACAAATGGTGTGTATAATGACCAAACCGGCTAT3360
ATCACAAAAACAGTGACATTCATCCCGTATACAGATCAAATGTGGATTGAAATAAGTGAA3420
ACAGAAGGTACGTTCTATATAGAAAGTGTAGAATTGATTGTAGACGTAGAG 3471
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 1157 amino acids
B)) TYPE: amino acid
C STRANDEDNESS: single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(ivj ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
A ORGANISM: Bacillus thuringiensis
B STRAIN: kumamotoensis
~C~ INDIVIDUAL ISOLATE: PS50C
(vii) IMMEDIATE SOURCE:
(A) LIBRARY~ Lambdagem (TM) - 11 LIBRARY OF LUIS
FONCERRADA
(B) CLONE: 50C
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Thr Pro
1 5 ZO 15
Ser Thr Ser Val Ser Ser Asp Ser Asn Arg Tyr Pro Phe Ala Asn Glu
20 25 30
Pro Thr 35p Ala Leu Gln Asn 4e0t Asn Tyr Lys Asp 4yr Leu Lys Met
Ser Gly Gly Glu Asn Pro Glu Leu Phe Gly Asn Pro GSlu Thr Phe Ile
50 55 60
Ser Ser Ser Thr Ile Gln Thr Gly Ile Gly Ile Val Gly Arg Ile Leu
65 70 75 80
Gly Ala Leu Gly 851 Pro Phe Ala Ser 910n Ile Ala Ser Phe 9yr Ser
Phe Ile Val G1y Gln Leu Trp Pro Ser Lys Ser Val Asp Ile TSrp Gly
100 105 110
Glu Ile i15 Glu Arg Val Glu i2O Leu Val Asp Gln iys I12 Glu Lys
~~:'.' a~°~~
21 MA53.C1
Tyr 130 Lys Aap Lys Ala i35 Ala Glu Leu Lys i~~ Leu Gly Aan Ala
Leu Asp Val Tyr Gln Gln Ser Leu Glu Asp Trp LeuO Glu Asn Arg Asn
145 150 15b 160
Asp Ala Arg Thr Arq Ser Val Val Ser Asn Gln Phe Ile Ala Leu Asp
165 170 175
Leu Asn Phe 1810 Ser Ser Ile Pro i85 Phe Ala Val Ser i9y His Glu
Val Leu Leu Leu Ala Val Tyr Ala Gln Ala Val Asn Leu HiOs Leu Leu
195 200 205
Leu 210 Arg Aap Ala Ser 215 Phe Gly Glu Glu z2p Gly Phe Thr Pro
G1y Glu Ile Ser Arg Phe Tyr Aan Arg Gln Val GlDn Leu Thr Ala Glu
225 230 235 240
Tyr Ser Asp Tyr 2y5 Val Lys Trp Tyr 2y0 Ile Gly Leu Asp 2y5 Leu
Lys Gly Thr Thr S4er Lys Ser Trp Leu A5an Tyr His Gln Phe ASrg Arg
260 265 270
Glu Met 275 Leu Leu Val Leu 28p Leu Val Ala Leu 285 Pro Asn Tyr
Asp Thr Hia Met Tyr Pro Ile Gluo Thr Thr Ala Gln Leu Thr Arg Aap
290 295 300
Val Tyr Thr Asp Pro Ile Ala Phe Asn Ile Val Thr Ser Thr Gly Phe
305 310 315 320
Cys Asn Pro Trp 325 Thr His Ser Gly 330 Leu Phe Tyr Glu 335 Glu
Asn Asn Val Ile Arg Pro Pro His Leu Phe Asp Ile Leu Ser Ser Val
340 345 350
Glu Ile 355 Thr Ser Arg Gly 36y Ile Thr Leu Asn 365 Asp Ala Tyr
Ile 3~n0 Tyr Trp Ser Gly 3~5 ThDr Leu Lys Tyr $~ Arg Thr Ala Asp
Ser Thr Val Thr Tyr Thr Ala Asn Tyr Gly Ar Ile Thr Ser Glu Lys
385 390 398 4D0
Aan Ser Phe Ala Leu Glu Asp Arg Asp Ile Dhe Glu Ile Asn Ser Thr
405 410 415
Val Ala Asn Leu Ala Aan Tyr Tyr Gln Lys Ala Tyr Gly Val Pro Gly
420 425 430
Ser Trp P435 His Met Val Lys 4g Gly Thr Ser Ser T4~5 Thr Ala Tyr
Leu Tyr Ser Lys Thr His Thr AlOa Leu Gln Gly C~s Thr G1n Val Tyr
4 0 455 4 0
Glu Ser Ser Asp Glu Tle Pro Leu Asp Arg Thr Val Pro Va1 Ala Glu
465 470 475 480
Ser Tyr Ser His Bg Leu Ser His Ile 49r0 Ser His Ser Phe 49~ Lys
Aan Gly Ser 5D0 TyrS Tyr Gly Ser 5h05 Pro Val Phe Val 52p Thr His
Thr Ser 515 Aap Leu Aan Asn 5 O Ile Tyr Ser Asp 5y5 IlDe Thr Gln
Ile 530 Ala Val Lys G1y 5~~ Met Leu Tyr Leu Gly GZly Ser Val Val
5 54
22 MA53.C1
Gln Gly Pro Gly Phe Thr Gly G1y Asp Ile Leu Lys Arg Thr Asn Pro
545 550 555 560
Ser Ile Leu Gly Thr Phe Ala Val Thr Val Asn Gly Ser Leu Ser Gln
565 570 575
Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Phe Glu Phe
580 585 590
Thr Leu 5y5 Leu Gly Asp Thr 600 Glu Lys Asn Arg 605 Asn Lys Thr
Met Asp A9sn Gly Ala Ser Leu Thr Tyr Glu Thr Phe Lys Phe Ala Ser
610 615 620
Phe Ile Thr Asp Phe Gln Phe Arg Glu Thr Gln Asp Lys Ile Leu Leu
625 630 635 640
Ser Met Gly Asp Phe Ser Ser Gly Gln Glu Val Tyr Ile Asp Arg Ile
645 650 655
Glu Phe Ile 660 Val Asp Glu Thr Ty5 Glu Ala Glu Gln 6~~ Leu Glu
Ala Ala 6y5 Lys Ala Val Asn 68a0 L6~eu Phe Thr Asn 685 LyOs Asp Gly
Leu 6 ~ P7ro Gly Val Thr 69p Tyr Glu Val Asn 710Q Ala Ala Asn Leu
Val Glu Cys Leu Ser Asp As5p Leu Tyr Pro Asn Glu Lys Arg Leu Leu
705 71D 715 720
Phe Asp Ala Val A72rg Glu Ala Lys Arg 730 5er Gly Ala Arg '36 Leu
Leu Gln Asp Pro AsSp Phe GIn Glu Ile Asn Gly Glu Asn G1y Trp Ala
740 745 750
Ala Ser 755 Gly Ile Glu Ile 7610 Glu Gly Asp Ala 765 Phe Lys Gly
Arg 7yr0 Leu Arg Leu Pro 7~~ Ala Arg Glu Ile 7~~ Thr Glu Thr Tyr
Pro T7hr Tyr Leu Tyr Gln LySs Val Glu Glu Glp VaDl Leu Lys Pro ~yr
785 790 79b $D0
Thr Arg Tyr Arg 8p5 Arg Gly Phe Val 8iy Ser Ser Gln Gly 8i6 Glu
Ile Tyr Thr Ile Arg His Gln Thr Asn ArOg Ile Val Lys Asn Val Pro
820 825 830
Asp Asp $36 Leu Pro Asp Val 8~r0 Pro Val Asn Ser 8~~ Gly Ser Ile
Asn 8 ~ Cys Ser Glu Gln By5 Tyr Val Asn Ser ~~ Lebu Glu Gly Glu
Asn Arg Ser Gly Asp Ala H5is Glu Phe Sex Leu Pro Ile Asp Ile G1p
865 870 875 880
Glu Leu Asp Tyr 885 Glu Asn Ala Gly 89o Trp Val Gly Phe 8y5 Ile
Thr Asp Pro Glu Gly Tyr Ala Thr Leu Gly Asn Leu Glu Leu V9al Glu
900 905 910
Glu Gly Pro Leu Ser Gly Asp Ala Leu Glu Arg Leu Glri Arg Glu Glu
915 920 925
Gln Gln Trp Lys Ile Gln Met Thr Arg Arg Arg Glu Glu Thr Asp Arg
930 935 940
~~ Tyr Met Ala Ser 9ys0 Gln Ala Val Asp 9 ~ Leu Tyr Ala Asp 9~r0
~~.j...s~~'m ~i~i
23 MA53.C1
Gln Asp Gln Gln Leu Asn Pro Asp Val Glu Ile Thr Asp Leu Thr Ala
965 970 975
Ala Gln Asp Leu Ile Gln Ser Ile Pro Tyr Val Tyr Asn Glu Met Phe
980 985 990
Pro Glu Ile Pro Gly Met Asn Tyr Thr Lys Phe Thr Glu Leu Thr Asp
995 1000 1005
Arg Leu Gln Gln Ala Trp Asn l,eu Tyr Aep Gln Arg Asn Ala Ile Pro
1010 1015 1020
Asn Gly Asp Phe Arg Asn GIy Leu Ser Asn Trp Asn Ala Thr Pro G1p
1025 1030 2035 1040
Val Glu Val Gln Gln Ile Asn FIis Thr Ser Val Leu Val Ile Pro Asn
1045 1050 1055
Trp Asp Glu Gln Val Ser Gln Gln Phe Thr Val Gln Pro Asn Gln Arg
1060 1065 1070
Tyr Val Leu Arg Val Thr Ala Arg Lys Glu Gly Val Gly Asn Gly Tyr
1075 1080 1085
Val Ser IIe Arg Asp Gly G1y Asn Gln Ser Glu Thr Leu Thr Phe Ser
1090 1095 1100
Ala Ser Asp Tyr Asp Thr Asn Gly Val Tyr Asn Asp Gln Thr Gly Tyr
1105 1110 1115 1120
Ile Thr Lys Thr Val Thr Phe Ile Pro Tyr Thr Asp Gln Met Trp Ile
1125 1130 1135
Glu Ile Ser Glu Thr Glu Gly Thr Phe Tyr Ile Glu Ser Val Glu Leu
1140 1145 1150
Ile Val i155Va1 Glu
