Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THE INVENTION
A NOVEL BACILLUS THURINGIENSIS STRAIN, CRYSTAL GENE AND
CRYSTAL PROTEIN AND USES THEREOF
FIELD OF THE INVENTION
[0001] The present invention relates to a novel Bacillus
thuringiensis strain, crystal gene and crystal protein and uses thereof. More
specifically, the present invention is concerned with a novel Bacillus
thuringiensis, novel Cry3l protoxin and toxin, nucleotide sequences encoding
same and anti-cancer therapeutic applications for the toxin.
BACKGROUND OF THE INVENTION
[0002] Bacillus thuringiensis has been known for years for coding for
6-endotoxin crystal proteins. A large variety of endotoxins have been
described and characterized, many of them having reported insecticidal
activities. Most of these have molecular weights in the range of 130-140KDa
and 65-80KDa (Schnepf et al., 1998). Recently, a novel endotoxin protein has
been identified and designated Cry3l Aal (also called parasporin) (Mizuki et
al.,
2000). It is an 81 KDa protein encoded by a 2169 bp gene that has been
characterized as having a selective activity as a human Leukemic Cell-
Recognizing Protein (Mizuki et al., (1999) and (2000)). No other member of
this
novel family of endotoxin has yet been reported.
[0003] It is therefore an object of the present invention to provide a
new bacillus thuringiensis strain expressing a new member of this novel family
of b-endoxins displaying advantageous cytotoxicity against human cancer cells.
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SUMMARY OF THE INVENTION
[0004] More specifically, in accordance with the present invention,
there is provided a novel Bacillus thuringiensis strain, named M15, a novel 83-
kDa crystal protein 6-endotoxin assigned the designation Cry31Aa2 by the
Bacillus thuringiensis Pesticide Crystal Protein Nomenclature Committee and
displaying cytotoxicity against certain human cancer cells.
[0005] According to a first aspect of the present invention, there is
also provided a biologically pure culture of a microorganism strain comprising
all of the identifying characteristics of a Bacillus thuringiensis strain
deposited
at the International Depository Authority of Health Canada in Winnipeg under
accession number IDAC010201-5, or a mutant thereof isolated from said strain,
said mutant comprising all of said biochemical characteristics and producing a
crystal protein having cytotoxicity against human cancer cells..
[0006] According to a second aspect of the present invention, there
is also provided an isolated nucleic acid molecule comprising:
(a) a nucleotide sequence encoding a polypeptide comprising
the complete amino acid sequence of SEQ ID NO:2;
(b) a nucleotide sequence encoding a polypeptide comprising
the complete amino acid sequence of SEQ ID NO:8;
(c) a nucleotide sequence encoding a polypeptide comprising
the complete amino acid sequence of SEQ ID NO:14;
(d) a nucleotide sequence encoding a polypeptide comprising
the complete amino acid sequence of SEQ ID NO:13, with the proviso that said
nucleotide sequence does not encode the amino acid sequence at positions
232 to 723 of SEQ ID NO:18;
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(e) a nucleotide sequence encoding a polypeptide comprising
the complete amino acid sequence of SEQ ID NO:15, with the proviso that said
nucleotide sequence does not encode the amino acid sequence at positions
232 to 723 of SEQ ID NO:18;
(f) a nucleotide sequence comprising the nucleotide
sequence set forth in SEQ ID NO:1 or 9;
(g) a nucleotide sequence encoding a crystal protein cytotoxic
against at least one human cancer cell, the crystal protein having at least
94%
identity with the complete amino acid sequence in SEQ ID NO:2;
(h) a nucleotide sequence encoding a crystal protein cytotoxic
against at least one human cancer cell, the crystal protein having at least
97%
identity with the complete amino acid sequence in SEQ ID NO:8;
(i) a nucleotide sequence completely complementary to any
of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g) and (h); or
Q) a nucleotide sequence encoding a crystal protein cytotoxic
against at least one cancer cell, which hybridizes under high stringency
conditions to the nucleotide sequence in (i), wherein the high stringency
conditions comprise hybridization in 5X SSC, 5X Denhardt's solution, 1% SDS
and 100 pg/ml of carrier DNA at 65 C followed by washing in 0.2X SSC, 0.1%
SDS at 65 C.
[0007] According to another aspect of the present invention, there is
also provided an isolated polypeptide comprising:
(a) an amino acid sequence as set forth in SEQ ID NO:2;
(b) an amino acid sequence set forth in SEQ ID NO:8;
(c) an amino acid sequence as set forth in SEQ ID NO:14;
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(d) an amino acid sequence as set forth in SEQ ID NO:13,
with the proviso that said amino acid sequence is not the sequence at
positions
232 to 723 of SEQ ID NO:18;
(e) an amino acid sequence as set forth in SEQ ID NO:15,
with the proviso that said amino acid sequence is not the sequence at
positions
232 to 723 of SEQ ID NO:18
(f) a crystal protein having at least 94% identity with the
complete amino acid sequence of SEQ ID NO:2, wherein the protein becomes
cytotoxic upon treatment with trypsin; or
(g) a crystal protein cytotoxic against at least one cancer cell
and having at least 97% identity with the complete amino acid sequence of
SEQ ID NO:8.
[0008] According to another aspect of the present invention, there is
also provided a recombinant vector comprising an isolated nucleic acid
molecule of the present invention, a recombinant host cell same, a method for
making same comprising inserting such isolated nucleic acid molecule in a
vector.
[0009] According to another aspect of the present invention, there is
also provided a recombinant method for producing a cytotoxic polypeptide,
comprising culturing the host cell under conditions such that the polypeptide
is
expressed and recovering the polypeptide.
[0010] According to another aspect of the present invention, there is
also provided an isolated antibody that binds specifically to a polypeptide of
the
present invention.
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[0010a] In an embodiment, the antibody specifically binds to:
(a) an amino acid sequence as set forth in SEQ ID NO:2;
(b) an amino acid sequence as set forth in SEQ ID NO:8;
(c) a crystal protein having at least 94% identity with the
complete amino acid sequence of SEQ ID NO:2, wherein said protein becomes
cytotoxic upon treatment with trypsin; or
(d) a crystal protein cytotoxic against at least one human
cancer cell and having at least 97% identity with the complete amino acid
sequence of SEQ ID NO:8.
[0011] According to another aspect of the present invention, there is
also provided a method of modulating the level of cry31Aa2 active protein in a
cell comprising a modulation of the level or activity of the sequence SEQ ID
NO:8.
[0012] According to another aspect of the present invention, there is
also provided an in vitro method of using a polypeptide of the present
invention
for lysing a human cancer cell which according a specific embodiment of the
present invention is a HELA, TCS, HL-60, Jurkat, or Hep-G2 cell.
[0013] According to another aspect of the present invention, there is
also provided an in vitro method of testing the cytotoxicity of a polypeptide
of
the present invention against a candidate cancer cell comprising determining
the EC50 of the polypeptide on the candidate cell, wherein the polypeptide is
characterized as possessing cytotoxicity against the candidate cell if the
EC50
of the polypeptide against the candidate cell is measurably lower than that
against a normal T cell.
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[0014] According to another aspect of the present invention, there is
also provided an in vitro method for lysing a human cancer cell comprising
applying a cytotoxic amount of a polypeptide of the present invention on a
human cancer cell.
[0015] According to another aspect of the present invention, there is
also provided a method for obtaining a cytotoxic polypeptide comprising
cleaving a polypeptide of the present invention with a protease able to cleave
between a residue R and a residue I. In an embodiment the residue R and the
Residue I is an arginine at position 250 and an isoleucine at position 251 of
SEQ ID NO:2. In a specific embodiment, the protease is trypsin.
[0016] In order to provide a clear and consistent understanding of
terms used in the present description, a number of definitions are provided
hereinbelow.
[0017] Unless defined otherwise, the scientific and technological
terms and nomenclature used herein have the same meaning as commonly
understood by a person of ordinary skill to which this invention pertains.
Generally, the procedures for molecular biology methods and the like are
common methods used in the art. Such standard techniques can be found in
reference manuals such as for example Sambrook et al. (1989, Molecular
Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.) and Ausubel et al. (1994, Current Protocols in Molecular
Biology, Wiley, New York).
[0018] The terminology "human cancer cell" as used herein refers to
cells associated with at least one type of cancer. Without limiting the
generality
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of this definition, this terminology includes the following cells and
corresponding
tissues, namely acetabulum: HT-1080; amnion: WISH; B-cells: NAGL-1;
blood: J-111, IM-9, jurkat; bone: HOS, MG-63, MEG-01; bone marrow: A549;
MEG-01; FS-1; brain: SF126, U-251, MG, Becker, Marcus, T98G, SK-MG-1,
ONS-76, KNS, B2-17, no. 10, no. 11, KALS-1, KINGS-1, KS-1, KNS-81-FD,
NMC-G1, GB-1, AM-38, YH-13; colon: WiDr, LoVo, CCD 841, CCD-33, Caco-
2; embryonic limb: Miz-1; epidermoid: A-431; whole fetus: HE-1; foreskin:
FLOW7000, Hs68, liver: Chang Liver, Alexander cells, HC, Hep-G2; lung:
MRC-5, MRC-9, HFL1, WI-38, Flow 2000, KNS-62; lymph node: GAK;
lymphoblastoid: Namalwa; maxilla: Raji; melanoma: G-361, A2058;
neuroblastoma: KP-N; ovary: RMG, RKN, RTSG, RMUG; peripheral blast:
MTA; peripheral blood: RPMI 8226, HL-60, CCRF-SB, EB-3, RPMI 788, NC-37,
MOLT-4, KU812, CCRF-CEM, CMK, NOMO, NKM-1, MEG-A2, TMD5, KA13;
pleural effusion: U-937; prostate: DU145, CEACAM-1; sympatho-adrenal cell:
IMR-32, NB-1; umbilical cord: HUV-EC-C; uterine cervix: Ca Ski, HeLa, SKG,
BOKU; uterine endometrium : SNG; uterus: SKN, NJG, SAWANO, TCS,
UtSMC. The terminology "cancer cell" also refers herein to cells associated
with
non-human forms of cancer including Vero, COS-7 and NIH3T3 cells.
[0019] As used herein, a "biologically pure" strain is intended to
mean the strain separated from materials with which it is normally associated
in
nature. Note that a strain associated with other strains, or with compounds or
materials that it is not normally found with in nature, is still defined as
"biologically pure." A monoculture of a particular strain is, of course,
"biologically pure."
Nucleotides
[0020] Nucleotide sequences of the present invention are presented
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herein by single strand, in the 5' to 3' direction, from left to right, using
the one
letter nucleotide symbols as commonly used in the art and in accordance with
the recommendations of the IUPAC-IUB Biochemical Nomenclature
Commission.
[0021] The present description refers to a number of routinely used
recombinant DNA (rDNA) technology terms. Nevertheless, definitions of
selected examples of such rDNA terms are provided for clarity and consistency.
[0022] As used herein, "nucleic acid molecule", refers to a polymer
of nucleotides. Non-limiting examples thereof include DNA (e.g. genomic DNA,
cDNA), RNA molecules (e.g. mRNA) and chimeras thereof. The nucleic acid
molecule can be obtained by cloning techniques or synthesized. DNA can be
double-stranded or single-stranded (coding strand or non-coding strand
[antisense]).
Protein expression
[0023] Prokaryotic expressions are useful for the preparation of
large quantities of the Cry31Aa2 protoxine and toxine encoded by the cry3lAa2
DNA sequence. These proteins can be purified according to standard
protocols that take advantage of the intrinsic properties thereof, such as
size
and charge (e.g. SDS gel electrophoresis, gel filtration, centrifugation, ion
exchange chromatography...). In addition, the protein of interest can be
purified via affinity chromatography using polyclonal or monoclonal
antibodies.
The purified protein can be used for therapeutic applications in accordance
with
the methods and uses of the present invention.
Mutations, mutants and variants
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[0024] As commonly known, a "mutation" is a detectable change in
the genetic material which can be transmitted to a daughter cell. As well
known,
a mutation can be, for example, a detectable change in one or more
deoxyribonucleotides. For example, nucleotides can be added, deleted,
substituted for, inverted, or transposed to a new position. Spontaneous
mutations and experimentally induced mutations exist. A mutant polypeptide
can be encoded from this mutant nucleic acid molecule.
[0025] As used herein, a "mutant" of the novel strain of Bacillus
thuringiensis of the present invention namely the M15 strain deposited under
access no, IDAC010201-5 may or may not have the same identifying biological
characteristics of the M15 strain, as long as the mutant produces a crystal
protein that is cytotoxic against human cancer cells.
Illustrative examples of suitable methods for preparing mutants and variants
of
the inventive microorganism strain include, but are not limited to:
mutagenesis
by irradiation with ultraviolet light or X-rays, or by treatment with a
chemical
mutagen such as nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine),
methylmethanesulfonate, nitrogen mustard and the like; gene integration
techniques, such as those mediated by insertional elements or transposons or
by homologous recombination of transforming linear or circular DNA molecules;
and transduction mediated by bacteriophages such as P1. These methods are
well known in the art and are described, for example, in J. H. Miller,
Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1972); J. H. Miller, A Short Course in Bacterial
Genetics,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); M.
Singer and P. Berg, Genes & Genomes, University Science Books, Mill Valley,
CA (1991); J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A
Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989); P. B. Kaufman et al., Handbook of Molecular and Cellular
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Methods in Biology and Medicine, CRC Press, Boca Raton, FL (1995);
Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J. E.
Thompson, eds., CRC Press, Boca Raton, FL (1993); and P. F. Smith-Keary,
Molecular Genetics of Escherichia coli, The Guilford Press, New York, N.Y.
(1989).
[0026] Mutated strains derived from the M15 strain using known
methods are then preferably selected or screened for improved cytotoxic
crystal proteins production potential or for other desirable properties
related to
their utility in expressing crystal proteins that are cytotoxic to human
cancer
cells. In a specific embodiment of the mutagenesis and screening approach to
strain improvement, mutagenized cells are selected on the basis of their
cytopathic effects or cytocidal activity on target cells and their spectrum of
action.
[0027] The term "variant" refers herein to a protein or nucleic acid
molecule which is substantially similar in structure and biological activity
to the
protein or nucleic acid of the present invention and includes a cry3lAa2
nucleic
sequence or the protein encoded by same having one or more mutations that
does not affect its cytotoxic activity. In particular, a variant of the
nucleotide or
polypeptide sequence of the active portion of Cry31Aa2 possesses the ability
to
lyse human cancer cells including HeLa, TCS, HL-60, Jurkat and Hep-G2. The
methods for determining whether a nucleotide or polypeptide sequence
constitutes a variant of Cry31Aa2 include conducting an EC50 assay on a
cancer cell against which the Cry31Aa2 active toxin displays cytotoxicity.
[0028] The functional derivatives of the present invention can be
synthesized chemically or produced through recombinant DNA technology. All
these methods are well known in the art.
Hybridization
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[0029] "Nucleic acid hybridization" refers generally to the
hybridization of two single-stranded nucleic acid molecules having
complementary base sequences, which under appropriate conditions will form
a thermodynamically favored double-stranded structure. Examples of
hybridization conditions can be found in the two laboratory manuals referred
above (Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra) and are
commonly known in the art. In the case of an hybridization to a nitrocellulose
filter, as for example in the well known Southern blotting procedure, a
nitrocellulose filter can be incubated overnight at 65 C with a labeled probe
in a
solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x
Denhardt's solution, 1% SDS, and 100 pg/mI denatured carrier DNA (e.g.
salmon sperm DNA). The non-specifically binding probe can then be washed
off the filter by several washes in 0.2 x SSC/0.1 % SDS at a temperature which
is selected in view of the desired stringency: room temperature (low
stringency), 42 C (moderate stringency) or 65 C (high stringency). The
selected temperature is based on the melting temperature (Tm) of the DNA
hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such
cases, the conditions of hybridization and washing can be adapted according to
well known methods by the person of ordinary skill. Stringent conditions will
be
preferably used (Sambrook et a!.,1989, supra). In most hybridizations, a 1%
mismatching of bases will lower the melting temperature by 1-1.5 C (Sambrook
et a!.,1989, supra). Consequently, nucleotide sequences sharing 98%
nucleotide identities with the cry3lAa2 gene encoding the trypsin-activated
portion of Cry31Aa2 will still hybridize with the cry3lAa2 gene when the
melting
temperature is lowered by 3 C, respective to the most stringent conditions for
hybridization between two identical cry3lAa2 sequences. The term "high
stringency" conditions refer herein to the conditions required for the
hybridization of nucleotide sequences sharing at least 97% nucleotide
identities
with the cry3lAa2 gene encoding the trypsin-activated portion of Cry31Aa2.
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[0030] As used herein, "chemical derivatives" is meant to cover
additional chemical moieties not normally part of the subject matter of the
invention. Such moieties could affect the physico-chemical characteristic of
the
derivative (e.g. solubility, absorption, half life, decrease of toxicity and
the like).
Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980).
Methods of coupling these chemical-physical moieties to a polypeptide or
nucleic acid sequence are well known in the art.
Recombinant vectors
[00311 The term "recombinant DNA" as known in the art refers to a
DNA molecule resulting from the joining of DNA segments. This is often
referred to as genetic engineering. The same is true for "recombinant nucleic
acid".
[0032] The term "vector" is commonly known in the art and defines a
plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA
vehicle into which DNA of the present invention can be cloned. Numerous
types of vectors exist and are well known in the art.
[0033] The term "expression" defines the process by which a gene is
transcribed into mRNA (transcription), the mRNA is then being translated
(translation) into one polypeptide (or protein) or more.
[0034] The terminology "expression vector" defines a vector or
vehicle as described above but designed to enable the expression of an
inserted sequence following transformation into a host. The cloned gene
(inserted sequence) is usually placed under the control of control element
sequences such as promoter sequences. The placing of a cloned gene under
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such control sequences is often referred to as being operably linked to
control
elements or sequences.
[0035] Operably linked sequences may also include two segments
that are transcribed onto the same RNA transcript. Thus, two sequences, such
as a promoter and a "reporter sequence" are operably linked if transcription
commencing in the promoter will produce an RNA transcript of the reporter
sequence. In order to be "operably linked" it is not necessary that two
sequences be immediately adjacent to one another.
[0036] Expression control sequences will vary depending on
whether the vector is designed to express the operably linked gene in a
prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally
contain transcriptional elements such as enhancer elements, termination
sequences, tissue-specificity elements, and/or translational initiation and
termination sites.
[0037] The DNA construct can be a vector comprising a promoter
that is operably linked to an oligonucleotide sequence of the present
invention,
which is in turn, operably linked to a heterologous gene, such as the gene for
the luciferase reporter molecule. "Promoter" refers to a DNA regulatory region
capable of binding directly or indirectly to RNA polymerase in a cell and
initiating transcription of a downstream (3' direction) coding sequence. For
purposes of the present invention, the promoter is bound at its 3' terminus by
the transcription initiation site and extends upstream (5' direction) to
include the
minimum number of bases or elements necessary to initiate transcription at
levels detectable above background. Within the promoter will be found a
transcription initiation site (conveniently defined by mapping with S1
nuclease),
as well as protein binding domains (consensus sequences) responsible for the
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binding of RNA polymerase. Eukaryotic promoters will often, but not always,
contain "TATA" boxes and "CCAT" boxes. Prokaryotic promoters contain -10
and -35 consensus sequences, which serve to initiate transcription and the
transcript products contain Shine-Dalgarno sequences, which serve as
ribosome binding sequences during translation initiation.
Recombinant host cell
[0038] A host cell or indicator cell has been "transfected" by
exogenous or heterologous DNA (e.g. a DNA construct) when such DNA has
been introduced inside the cell. The transfecting DNA may or may not be
integrated (covalently linked) into chromosomal DNA making up the genome of
the cell. In prokaryotes, yeast, and mammalian cells for example, the
transfecting DNA may be maintained on an episomal element such as a
plasmid. With respect to eukaryotic cells, a stably transfected cell is one in
which the transfecting DNA has become integrated into a chromosome so that
it is inherited by daughter cells through chromosome replication. This
stability
is demonstrated by the ability of the eukaryotic cell to establish cell lines
or
clones comprised of a population of daughter cells containing the transfecting
DNA. Transfection methods are well known in the art (Sambrook et al., 1989,
supra; Ausubel et aL, 1994 supra).
Method for identifying other cancer cells against which Cry3l Aa2 is
c otoxic
[0039] In addition to the EC50 assay described herein, other assays
may be used to determine the effects of Cry31 Aa2 or other proteins
encompassed by the present invention on human cancer cells. In particular,
these effects may be observed by photonic microscopy. Furthermore, assays
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for detecting cytopathic effects can also be used for this purpose.
[0040] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following non-
restrictive description of preferred embodiments thereof, given by way of
example only with reference to the accompanying drawings.
[0041] The present invention seeks to meet these needs and other
needs.
[0042] The present description refers to a number of documents, the
content of which is herein incorporated by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
[0043] Figure 1 illustrates in panel A) a phase-contrast micrograph of a
lysed culture of Bacillus thuringiensis strain M15; in panel B, a transmission
electron micrograph of Bacillus thuringiensis strain M15 containing a spore
and
a tightly bound parasporal inclusion;
[0044] Figure 2 shows a SDS-PAGE analysis of the parasporal inclusion
protein(s) of B. thuringiensis strain M15;
[0045] Figure 3 is the nucleotide sequence of the translated portion of
the Cry3lAa2 gene (SEQ ID NO: 1);
[0046] Figure 4 is the deduced amino acid sequence of the Cry39Aa2
gene (SEQ ID NO: 2);
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[0047] Figure 5 shows a comparison of the deduced amino acid
sequences of Cry3lAa2 (SEQ ID NO:2) and Cry3lAal (SEQ ID NO:18). The
capital letters and dotted lines under the amino acid sequence of Cry31Aa2
(SEQ ID NO:2) correspond to the difference and alignment gaps between the
Cry3lAa2 (SEQ ID NO:2) and Cry3lAal (SEQ ID NO:18). The asterisks under
the Cry3lAa2 sequence indicate the identities between Cry3lAa2 (SEQ ID
NO:2) and Cry31Aal (SEQ ID NO:18);
[0048] Figure 6 shows a restriction map of the recombinant plasmid
pYCP31A2 containing the Cry31Aa2 gene;
[0049] Figure 7 shows a transmission electron micrograph of a B.
thuringiensis Cr{ B transformant expressing the Cry3lAa2 gene. S: spore; P:
parasporal inclusion; Magnification : 20,000 X;
[0050] Figure 8 shows a SDS-PAGE analysis of the parasporal inclusion
protein from a B. thuringiensis transformant expressing the crystal protein
gene
Cry31 Aa2;
[0051] Figure 9 shows the nucleotide sequence (SEQ ID NO:16) and
deduced amino acid sequence (SEQ ID NO:2) of the Cry31Aa2 gene along
with features thereof; and
[0052] Figure 10 shows the nucleotide sequence of the translated
portion of the Cry31Aal gene (SEQ ID NO:17).
DESCRIPTION OF THE PREFERRED EMBODIMENT
Isolation of strain, morphological and biochemical characteristics
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[0053] A Bacillus thuringiensis strain was isolated from dead two-spotted
spider mites (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae)
and named M15. The mites, parasitic on apple leaves, were collected in an
apple orchard located in Frelighsburgh, Quebec, Canada. They were
homogenized in 3 ml of phosphate-buffered saline (PBS) (NaCl 8 g, KCI 0.2 g,
Na2HPO4 1.44 g, KH2PO4 0.24 g I-1). The homogenized solution was
incubated for 16 hr at room temp and heated at 78 C for 15 min. Afterwards,
the homogenate was plated on 2YT agar medium (Bacto Tryptone 16 g, Bacto
Yeast Extract 10 g, NaCl 5 g, Agar 18 g I-1), and incubated for 24 hr at 30 C.
All colonies with a morphology similar to B. thuringiensis were streaked on T3
agar medium (Bacto Tryptone 3 g, Bacto Tryptose 2 g, Bacto Yeast Extract 1.5
g, MnC12 0.005 g, 0.05M Sodium phosphate, pH6.7, Agar 18 g I-1) and
incubated at 30 C for 48 hr. The cultures were examined by phase-contrast
microscopy (Carl Zeiss Canada Ltd., Toronto, Ontario, Canada) for the
presence of spores and crystals. B. thuringiensis M15 was deposited on 29
January 2001 in the International Depository Authority of Health Canada in
Winnipeg under the Budapest Treaty (Bureau of Microbiology, Health Canada,
1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2) under accession no.
I DAC010201-5.
[0054] The M15 strain was characterized for its ability to ferment specific
carbon sources, and for the production, utilization and reduction of specific
compounds (see Table 1 below). The biochemical characteristics of B.
thuringiensis strain M15, obtained using the API 50CH and API 20E kits as
recommended by the manufacturer (bioMerieux, St-Laurent, Quebec, Canada),
were different from those of three controls, B. thuringiensis var. kurstaki HD-
1, -
var. israelensis HD-500 and -var. higo BT205. B. thuringiensis var. kurstaki
HD-
1 and -var. israelensis HD-500 were obtained from "Laboratoire des bacteries
entomopathogenes", Institut Pasteur (Paris, France). B. thuringiensis var.
higo
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BT205 was in the Agriculture Canada collection (Jung et at., 1998). The M15
strain is further characterized in Jung, 2001.
Table 1. The biochemical profile of B. thuringiensis M15 and selected control
strains
Tests B. thuringiensis B. thuringiensis B. thuringiensis B. thuringiensis
var. kurstaki HD-1 var. israelensis HD-500 var. higo BT M 15
205
Fermentatio
n of
Glycerol + + +
D-Arabinose - - - -
L-Arabinose - - - -
Ribose + + + +
D-Xylose - - - -
L-Xylose - - - -
D-Galactose - - - -
D-Glucose + + + +
D-Fructose + + + +
D-Mannose - - - -
L-Sorbose - - - -
Inositol - - - -
D-Mannitol - - - -
D-Sorbitol - - - -
N- + + + +
Acetylglucosa
mine
Arbutin + + + -
Esculin + +
Salicin + - + +
D- + + + -
Cellobiose
D-Maltose + + + +
Lactose - - - -
Melibiose - - - -
Sucrose - - - -
Trehalose + + + +
Starch - + + -
Glycogen + + + -
Gluconate + + -
Production
of
Galactosida
se
Arginine + + + -
dihydrolase
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Ornithine - - - -
decarboxyla
se
Urease + - + +
Tryptophane - - - -
deaminase
Gelatinase + + + +
Oxidase + + + +
Catalase + + + +
H2S - - - -
Indole - - - -
Acetoin + + + +
Citrate + - - -
utilization
Nitrate + - - -
reduction
+, -, and indicate positive, negative, and weak reactions, respectively.
Microscopic characterization of Cry31 Aa2 parasporal inclusion bodies
[0055] The parasporal inclusion bodies produced by a sporulated culture
of B. thuringiensis strain M15 appear roughly spherical when observed under
phase-contrast microscopy (Fig. 1 A) and are tightly coupled to the spores
even
in lysed cultures. Further analysis under the transmission electron microscope
(TEM), however, reveals that the parasporal inclusion body has a polygonal
shape (Fig. 1B). The TEM observation was conducted after the B.
thuringiensis strain M15 was incubated for 5 days at 309C in T3 medium and
the samples ultra-thinly sectioned according to Beveridge et al. (1994).
Arrows
show the roughly spherical parasporal inclusions tightly bound to the white
ovoid spores. In this figure, "S" and "P" denote spore and parasporal
inclusion,
respectively. Magnification used is of 25,000 X.
SDS-PAGE analysis and N-terminal sequencing of the native parasporal
inclusion protein
[0056] The B. thuringiensis strain M15 was grown in T3 medium for 5
CA 02410153 2002-12-05
19
days at 30 C on a rotary shaker to allow crystal protein production. Spores
and
crystals were separated from each other in the tightly bound parasporal
duplexes using an ultrasonic processor model VC 130 (Sonics & Materials, Inc.,
Newtown, CT, USA) and purified by sucrose density gradient centrifugation as
described elsewhere (Thomas and Ellar, 1983). Twenty microliters of the
crystal suspension were added to 3 volumes of gel loading buffer (4% SDS,
20% glycerol, 125 mM Tris-HCI, 10% 2-mercaptoethanol, pH 6.8) in a 1.5-ml
microtube, incubated at 90 C for 7 min and centrifuged for 2 min to remove
unsolubilized materials. Thirty microliters of the supernatant were loaded on
top of 10% SDS-polyacrylamide gels. Discontinuous sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to
Laemmli and Favre (1973).
[0057] Figure 2 shows the B. thuringiensis strain M15 parasporal
inclusion purified by sucrose density gradient centrifugation as subjected to
a
10% SDS-PAGE electrophoresis (lane 4); the crude extracts of the fully lysed
B. thuringiensis var. kurstaki HD-1 subjected to electrophoresis on the same
gel (lane 3) as a control; and high molecular (lane 1) and low molecular
masses
(lane 2) of standard protein markers on the left. At least two major bands of
approximately 86- and 79-KDa in size were revealed. They were transferred to
a polyvinylidene difluoride (PVDF) membrane (Bio-RadTM), excised and
subjected to a pulsed liquid phase sequencer for determination of N-terminal
amino acid sequence.
[0058] The N-terminal amino acid sequence of the crystal protein from B.
thuringiensis strain M15 was determined as follows. The purified parasporal
crystal was added into O.1 N NaOH-3M HEPES solution and solubilized in 10
volumes of gel loading buffer by incubating in boiling water for 5 min. The
crystal protein was separated on 10 % SDS-PAGE and transferred to a
CA 02410153 2002-12-05
polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Mississauga, Ontario,
Canada). The crystal protein band stained with Coomassie brilliant blueTM R-
250 (Bio-Rad) was excised and subjected to a pulsed liquid phase sequencer
model 473A (Applied Biosystems, Foster City, CA, USA) at the Regional
Sequencing Facility (Centre de recherche du Centre hospitalier de I'Universite
Laval, Quebec, Canada).
[0059] The N-terminal sequence analysis revealed that both
polypeptides (86- and 79-KDa) shared identical 20-amino acids residues.
These were Met, Asp, Pro, Phe, Ser, Asn, Tyr, Ser, Glu, Gin, Lys, Tyr, Pro,
Asp, Ser, Asn, Asn, Asn, Gin and Glu (SEQ ID NO: 3).
Southern hybridization and gene cloning
[0060] An 18-mer oligonucleotide sequence, referred to as M15-M, was
deduced from a middle portion of the N-terminal amino acid sequence (Glu,
Gin, Lys, Tyr, Pro, Asp (SEQ ID NO: 4)) of the 86-kDa crystal protein. The
M15-M oligonucleotide was labeled by the Digoxigenin (DIG) oligonucleotide 3'-
end labeling kit containing DIG-11-ddUTP (Roche, Laval, Quebec, Canada) as
recommended by the manufacturer. The labeled oligonucleotide was
precipitated with 0.1 volume of 4M LiCI and 2.5 volumes of ice-cold ethanol,
and transferred at -70 C for 30 min. The reaction was centrifuged at 16,000g
for 15 min at 4 C. The washed pellet was resuspended in nuclease-free water,
and stored at -20 C until use.
[0061] The M 15-M generated had the following sequence: 5'-
GARCARAARTAYCCNGAY-3' (SEQ ID NO: 5).
CA 02410153 2002-12-05
21
[0062] B. thuringiensis strain M15 was grown in Luria-Bertani (LB)
medium (Bacto Tryptone 10 g, Bacto Yeast Extract 5 g, NaCl 5 g I-1) at 30 C
for 16 hr on a rotary shaker. Plasmid DNA was isolated using the alkaline
extraction method as described elsewhere (Birnboim and Doly, 1979) with the
following modifications. LysozymeTM (Sigma-Aldrich Canada Ltd., Oakville,
Ontario, Canada) was added at a concentration of 2 mg.ml-1 and the cell
suspension was incubated at 37 C for 1 hr.
[0063] The plasmid DNA was then purified with Wizard TM Plus SV
minipreps DNA purification system following the manufacturer's
recommendation (Promega, Nepean, Ontario, Canada). Three samples of the
plasmid were then digested with Hindlll, HindlIl/EcoRl and EcoRl (Gibco BRL),
respectively, electrophoresed on a 0.7% agarose gel and transferred onto a
NytranTM nylon membrane (Schleicher & Schuell, Keene, New Hampshire,
USA) by the method of Southern (1975). They were then probed with the DIG-
labeled 18-mer M15-M oligonucleotide.
[0064] This Southern blot hybridization was performed using the DIG-
labeled oligonucleotides with the standard hybridization solution (5X SSC, 1%
blocking reagent (Roche), 0.1% N-lauroylsarcosine, 0.02% SDS) for 13 hr at
39 C. After hybridization, the membrane was washed twice for 15 min each in
4X wash solution (4X SSC, 0.1% SDS) at 39 C. Following the washes,
detection of signals on the membrane was performed with the color-substrate
solution containing NBT (4-Nitroblue tetrazolium chloride, Roche) and BCIP (5-
Bromo-4-chloro-3-indolyl-phosphate, Roche) as recommended by the
manufacturer. After hybridization and post-hybridization washes at 39 C, the
M15-M probe strongly hybridized to an 8-kb Hindlll, a 2.6-kb Hindlll/EcoRl,
and
a 2.6-kb EcoRl fragment.
CA 02410153 2002-12-05
22
[0065] The purified B. thuringiensis M15 plasmid DNA was digested with
Hindlll and ligated with the Hind lll-digested SAP (Shrimp Alkaline
Phosphatase, Roche)-treated pBluescriptTM II KS(+) (Stratagene, La Jolla,
California, USA). After ligation, the recombinant DNA was transformed into E.
coli DH5a (Gibco BRL, Burlington, Ontario, Canada). Preparation of E. coli
DH5a competent cells and transformation were done as described (Sambrook
et al., 1989).
[0066] The transformants were grown on LB agar plates containing 100
g ml-1 ampicillin (Sigma-Aldrich Canada Ltd.) and 40 g ml-1 X-Gal (5'-
Bromo-4-chloro-3-indolyl-p-D-galactopyranoside, Sigma-Aldrich Canada Ltd.)
at 37 C. White colonies were toothpicks-transferred to 1 ml of fresh LB media
supplemented with 100 .tg ml-1 ampicillin, and incubated overnight at 37 C.
[0067] The recombinant DNA were then isolated by the cracking
procedure (Sambrook et al., 1989) and electrophoresed on 0.7% agarose gel to
assess the size of the undigested recombinant plasmids.
[0068] The three recombinant plasmids with the highest molecular
weight were selected and digested with Hindlll. They were designated
pYCH27, pYCH40 and pYCH217, respectively. All three plasmids contained
an 8-kb Hindlll insert. In addition, pYCH27 and pYCH40 also contained a 0.75-
kb and a 1.9-kb Hindlll fragment, respectively. They were then electrophoresed
on a 0.7% agarose gel, transferred onto a NytranTM nylon membrane by the
method of Southern (1975) and probed with the M15-M oligonucleotide. The
M15-M probe hybridized to the 8-kb Hindlll fragments in pYCH27, pYCH40 and
pYCH217 as revealed by Southern blot hybridization.
CA 02410153 2002-12-05
23
[0069] The 8-kb Hindlll fragments from pYCH27, pYCH40 and pYCH217
were doubly digested with Hindlll/EcoRl, electrophoresed on agarose gel,
Southern transferred, and hybridized with the M15-M probe. For each of the
three recombinant plasmids, a single 2.6-kb fragment was detected (data not
shown). This confirms that this 2.6-kb fragment is the same as the one in the
EcoRl-digested plasmid DNA of strain M15.
[0070] The 8-kb Hindlll insert was excised from recombinant plasmid
pYCH217, digested with various restriction enzymes [EcoRl, Bglll (Gibco BRL),
Dral, Sphl (Amersham Pharmacia Biotech)], and a restriction map constructed.
The 8-kb Hindlll fragment contains a 3.4-kb HindlII/EcoRl, a 2.6-kb
EcoRl/EcoRl, a 1.4-kb EcoRl/EcoRl and a 0.6-kb EcoRl/Hindlll fragment. The
open reading frame of a crystal protein gene, cry3lAa2, is indicated by a gray
arrow. The hatched box indicates the region homologous to the DIG-labeled
18-mer M15-M oligonucleotide probe. Selected subfragment sizes are
indicated below the structural map.
[0071] To identify the region homologous to the M15-M probe, the
recombinant plasmid pYCH217 was doubly digested with Hindlll/EcoRl, and
the resulting fragments were subcloned into EcoRl-digested pBluescriptTM II
KS(+). After ligation, four subclones were obtained to give the recombinant
plasmids pYC12S, pYC22S, pYC30S, and pYC31S. Plasmids pYC12S and
pYC30S contained a 1.4-kb and a 2.6-kb insert, respectively, while pYC22S
and pYC31 S both harbored a 2.6-kb insert along with a 0.6-kb and a 1.4-kb
fragment, respectively. Only the 2.6-kb EcoRl/EcoRl fragment from subclones,
pYC22S, pYC30S and pYC31S hybridized with the M15-M probe. To further
localize the region of hybridization of the M15-M probe in the 2.6-kb
EcoRl/EcoRl fragment, the recombinant plasmid pYC30S was digested with
EcoRl, EcoRl/Dral, EcoRI/Sphl, and EcoRl/BgllI, respectively, and then
CA 02410153 2002-12-05
24
hybridized with the M15-M probe. The M15-M probe detected a 2.6-kb EcoRl,
a 0.6-kb Dral, a 1.6-kb EcoRl/Sphl, and a 0.85-kb EcoRl/BgIII fragment,
respectively. It was thus determined that the region of hybridization of the
M15-
M probe lied between the BgIII and Dral sites within the 2.6-kb EcoRl
fragment.
Characterization of a new crystal protein gene, cry31Aa2
[0072] The nucleotide sequences of the 2.6-kb EcoRI/EcoRl, 1.4-kb
EcoRl/EcoRl and 0.6-kb EcoRl/Hindlll fragments were determined. An open
reading frame (ORF) of 2,226-bp in length that codes for a polypeptide of 742
amino acids with a predicted molecular mass of 83,068Da (Figures 3 and 4)
was found. The start codon is not ATG but GTG. One potential promoter-like
sequence in the 5' non-coding region (Lereclus et al., 1989; Baum and Malvar,
1995) shows a 13-bp spacing between the putative -10 and -35 sequences
located 138-bp upstream from the start codon (GTG). The potential ribosome
binding site (RBS) (GAAAGGTGG (SEQ ID NO: 6)) is located 7-bp upstream of
the start codon (GTG) and is partially complementary to the 3' end
(UCUUUCCUCC (SEQ ID NO: 7)) of B. subtilis 16S rRNA (McLaughlin et al.,
1981; Moran et al., 1982). Both potential -35 and -10 boxes and a putative
ribosome-binding site are underlined in figure 9. The calculated free energy
of
interaction (AG, 25 C) between the B. subtilis 16S rRNA and the putative
ribosome binding site is -14.8 kcal-mol-1 (Tinoco et al., 1973). A terminal
inverted repeat that could form a stem-and-loop secondary structure with a
calculated energy (AG, 25 C) of -12.2 kcal=mol-1 (Tinoco et al., 1973) is
located 112-bp downstream from the stop codon (TAA), which is marked with
asterisks in figure 9, and may function as a transcription terminator
(indicated
by arrows). The 18-mer M15-M oligonucleotide sequence based on the N-
terminal amino acid sequence (Glu, Gin, Lys, Tyr, Pro, Asp (SEQ ID NO: 4)) of
the crystal protein is homologous to a region located 24-bp downstream from
CA 02410153 2002-12-05
the start codon (GTG). The sequence of the DIG-labeled 18-mer
oligonucleotide (M15-M) probe is indicated in bold capital letters in figure
9.
The crv3lAa2 gene expression in B. thuringgiensis Cry- B strain
[0073] The 3.6-kb Hindlll/Sphl fragment containing the entire crystal
protein gene was excised from the recombinant plasmid pYCH217, and then
cloned into the E. coli-B. thuringiensis shuttle vector pHPS9 doubly digested
with Hindlll/Sphl to yield recombinant plasmid pYCP31A2 (Figure 6). The E.
coli-B. thuringiensis shuttle vector pHPS9 (Haima, et al., 1990) was purchased
from American Type Culture Collection (Manassas, VA, USA). To express the
cloned cry3lAa2 crystal protein gene in the acrystalliferous B. thuringiensis
strain Cry- B, the 3.6-kb Hindlll/Sphl fragment was cloned into the
Hindlll/Sphl
doubly-digested E. coli-B. thuringiensis shuttle vector pHPS9 to yield
recombinant plasmid pYCP31A2 (Fig. 6).
[0074] The B. thuringiensis var. kurstaki HD-1 acrystalliferous Cry-B
strain ((Stahly et al., 1978) provided by the Bacillus Genetic Stock Center,
The
Ohio State University (Columbus, OH, USA)), was transformed with the cloned
B. thuringiensis M15 crystal protein gene by electroporation as described by
Vehmaanpera (1989) with the following modifications. Bacterial cells cultured
in 200 ml of LB supplemented with 0.25 M sucrose and 0.05 M potassium
phosphate, pH7.0 (LBSP) to an optical density of 1.0 at 600 nm were
centrifuged, washed three times with ice-cold SHMG buffer (250 mM sucrose, 1
mM HEPES, 1 mM MgC12, 10% (v/v) glycerol, pH 7.0), and then resuspended
in 1 ml of ice-cold SHMG buffer. The cell suspension was mixed with plasmid
DNA at a final DNA concentration of 10 g ml-1 in a 0.2-cm electroporation
cuvette (Bio-Rad), kept on ice for 30 min, and then electroporated by a Gene
PulserTM model 1652076 (Bio-Rad) at 25 F, 2.5kV and 400Q with the pulse
CA 02410153 2002-12-05
26
once. After electroporation, 3ml of LBSP supplemented with 10% (v/v) glycerol
(LBSPG) were immediately added into the cuvette and incubated at 37 C for 2
hr with shaking.
[0075] The selected B. thuringiensis transformant was cultured in 250 ml
of nutrient broth supplemented with 5 gg m1-1 of erythromycin (Sigma-Aldrich
Canada Ltd.) and 5 g ml-1 of chloramphenicol (Sigma-Aldrich Canada Ltd.) at
37 C until cell autolysis was observed. The lysate was harvested and then
washed twice with 10 mM EDTA (pH 8.0)-1 M NaCI-1 mM phenylmethylsulfonyl
fluoride (Sigma-Aldrich Canada Ltd.).
[0076] The B. thuringiensis Cry- B transformant containing the B.
thuringiensis M15 parasporal crystal protein gene was incubated in nutrient
broth (Bacto Beef Extract 3 g, Bacto Peptone 5 g I-1) at 30 C for 3 days to
allow expression of the toxin gene and crystal formation. The presence of
parasporal inclusions was examined by phase-contrast microscopy. When
observed under a phase-contrast microscope, the B. thuringiensis
transformants expressing the cry31 Aa2 gene contained, in addition to the
spore, a roughly spherical inclusion, whereas no inclusions were found in the
B.
thuringiensis transformant harboring the non-recombinant shuttle vector pHPS9
alone (data not shown). Under the transmission electron microscope (TEM),
however, the parasporal inclusion body has a nearly perfect hexagonal shape
(Fig. 7). Both inclusions in the transformant, spore and crystal, are
separated
from each other as opposed to what is found in B. thuringiensis strain M15
where they are tightly bound to each other.
[0077] The parasporal inclusion from a B. thuringiensis transformant was
purified by sucrose density gradient centrifugation as described previously
(Thomas and Ellar, 1983). It was then subjected to a 10% discontinuous
CA 02410153 2002-12-05
27
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SIDS-PAGE) (lane
3) as reported previously (Laemmli and Favre, 1973). High molecular (lane 1)
and low molecular masses (lane 2) of standard protein markers are indicated
on the left. The parasporal inclusion protein in the B. thuringiensis
transformant
is composed of a single major polypeptide of 83-kDa (Fig. 8).
Preparation of inclusion proteins, proteolytic processing, and toxin
activation
[0078] The spore-inclusion mixture was harvested from sporulated
cultures and the inclusions were partially purified by a biphasic separation
method described in Goodman (1967) using polyethylene glycol 6000 (Wako
Pure Chemical, Osaka, Japan) and sodium dextran sulfate 500 (Sigma, St.
Louis, Mo.). Inclusions were further purified by sucrose density gradient
centrifugation as described in Saitoh et al., (1998a). The purified inclusions
were stored at 20 C until use.
[0079] Solubilization of purified inclusions was done in 50 mM Na2CO3
(pH 10.0) containing 1 mM EDTA and 10 mM dithiothreitol for 1 h at 37 C. After
centrifugation at 20,000 x g for 5 min at 4 C to remove unsolubilized
materials,
the pH of the solution was adjusted to 8Ø
[0080] The native 83KDa protoxine displayed no cytocidal activity
against cancer cells. This protein was therefore cleaved with three enzymes,
namely trypsin, chymotrypsin and proteinase K to identify an active toxine.
The
solubilized proteins (1.3 mg m11) were therefore treated with proteinase K
(final
concentrations, 0.0003, 0.003, 0.03, and 0.3 mg m11), trypsin (0.03, 0.3, 3,
and
30 mg m11), and chymotrypsin (0.03, 0.3, 3, and 30 mg m11) in 50 mM Na2CO3
(pH 10.0) for 1.5 h at 37 C. After protease treatment, phenylmethylsulfonyl
CA 02410153 2002-12-05
28
fluoride (Wako Pure Chemical) was added to the solution to stop the
proteolytic
reaction, and the mixture was examined for both sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) profiles and cytopathic effect
(CPE) on certain cancer cells including MOLT-4 and Hela. The CPE was
monitored under a phase-contrast microscope for 24 h, and the degree of
cytopathy was graded on the basis of the ratio of damaged cells as described
in Mizuki et al., (1999).
One-dose assay, hemolytic assay, and dose-response study
[0081] One-dose assays for cytotoxicity and hemolytic activity were
carried out as described in Mizuki et al., (1999). Each well of a MicroTest
plate
received 90 pl of cell suspension containing 2 x 104 cells. After
preincubation
for 16 h at 37 C, 10 pI of the trypsin-activated sample solution (1.3 mg ml)
was
added to the well.
[0082] Thirteen human cells, two monkey cells and one mouse cell were
used for dose-response studies. A hemolytic assay was done using human
erythrocytes according to the method described in Saitoh et al., (1998b). Each
well containing 90 1u1 of cell suspension (2 x 104 cells) received 10 N1 of
trypsin-
activated inclusion proteins which had been prepared in 10-fold serial
dilutions
in 50 mM Na2CO3 (pH 10.0) containing 10 mM DTT and 1 mM EDTA. Five
wells were used for each dilution, and the test was repeated at least three
times. The CPE was monitored under a phase-contrast microscope at
appropriate intervals for 24 h postinoculation.
[0083] For assessment of the level of cytotoxicity, a cell proliferation test
using an MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium
bromide] assay as described in Behl (1992) and Heiss (1997) was conducted
CA 02410153 2002-12-05
29
24 h postinoculation by using a PremixTM WST-1 kit (Takara Co.). The average
of absorbance in mock-inoculated negative controls was used as a blank value.
The arbitrary unit was defined on the basis of the relative value of
absorbance
at 450 nm to the blank (1.0). The 50% effective concentrations (EC50s) were
deduced from the dose-response curves using a log-probit program. Table 2
below presents the EC50s.
[0084] The protein exhibited cytotoxicity against HeLa, TCS, HL-60,
Jurkat and Hep-G2 cells when treated with trypsin. No cytocidal activity was
induced after treatment with chymotrypsin or proteinase K on HeLa, MOLT-4
and Sawano cells. Without protease digestion, inclusion proteins showed no
cytocidal toxicity. Trypsin cleaves the cry31 Aa2 protein after the arginine
at
position 250 of the full native protein sequence (SEQ ID NO: 2). The sequence
of the trypsin-activated protein is designated SEQ ID NO: 8 and the
corresponding nucleotide sequence is designated SEQ ID NO: 9.
[0085] Table 2 shows the results of one-dose assays of trypsin-activated
Cry31 Aa2 as compared to those of trypsin-activated Cry3lAal against several
species of cultured cells. The toxicity spectrum of the protein from the
recombinant Cry-B was similar to that of the protein of the wild strain M15.
Both cloned proteins were highly or moderately cytocidal against HeLa, TCS,
HL-60, Jurkat and Hep-G2 but were slightly toxic or nontoxic for normal T
cells
and for Sawano, UtSMC, MOLT-4, A549, MRC-5, HC, Caco-2 and the non
human cells tested.
Table 2 Effective concentration 50 of trypsin-activated cry31Aa2 as compared
to that of activated cry31Aa 1 on various cells
EC50(ug/ml) EC50(ug/ml)
Organ Cell Cell type Cry3lAa2 Cry3lAal
CA 02410153 2009-09-30
1 Human Uterus HeLa Cervix cancer 0.30 0.23
2 Sawano Uterus cancer, adenocarcinoma >10
3 TCS Cervix cancer, keratinizing squamous 0.32
4 UtSMC Uterus normal smooth muscle >10
5 Blood MOLT-4 T cell leukaemia >10 1.06
6 HL-60 T cell leukaemia 0.05
7 Jurkat T cell leukaemia 0.02
8 T cell Normal T cell >10
9 Lung A549 Lung cancer >10
10 MRC-5 Lung normal fibroblast >10
11 Liver HC Normal hepatocyte cell >10
12 Hep-G2 Liver carcinoma hepatocellular 0.02
13 Colon Caco-2 fColon cancer, adenocarcinoma >10
14 Monkey Kidney Vero Monkey, kidney epithelial cell >10
15 COS-7 Monkey, kidney SV40 transformed cell >10
16 Mouse Embryo NIH3T3 Mouse, embryo fibroblast cell >10
Comparison of Cry3lAa2 and Cry31Aal
[0001] As may be seen in Figure 5, the Cry3lAa2 amino acid sequence
shares extensive homology with Cry31Aal except for a substitution of 26
amino acid residues and an addition of 19 contiguous codons in Cry31Aa2
(Figure 5). This 19 amino acid sequence is as follows
SYQNMKTEIVNTDLPYNTN and is designated SEQ ID NO: 10 while the
corresponding nucleotide sequence is designated SEQ ID NO: 11. The
asterisks under the Cry3lAa2 sequence indicate the identities between
Cry31Aa2 and Cry3lAal. The capital letters and dotted lines under the amino
acid sequence of Cry3lAa2 in Figure 5, correspond to the difference and
alignment gaps between the Cry31Aa2 and Cry3lAal proteins. The 83-KDa
Cry3lAa2 protein exhibits 93.9% amino acid sequence identities with the
Cry3lAal protein. The five conserved amino acid blocks of the Cry3lAa2
CA 02410153 2009-09-30
31
protein were especially identical to those of the Cry3lAal protein except for
the
substitution of a single lysine residue in the second conserved block of
Cry3lAa2. The bold lines above the Cry3lAa2 sequence correspond to the five
conserved amino acid blocks found in the amino acid sequence of Cry3lAal.
Both Cry3lAa2 and Cry3lAal show very low amino acid sequence homology
to the known B. thuringiensis Cry and Cyt proteins (Mizuki et al., 2000). The
trypsin-activated protein shares a 96.9% identity with the corresponding
sequence of Cry3lAal.
[0002] Table 2 above shows that both Cry3lAa2 and Cry3lAal proteins
display cytotoxicity against a number of human cancer cells. The cytocidal
activity of the Cry3lAal was due to the cleavage by proteinase K and trypsin
(Mizuki et al., 2000) while that of Cry3lAa2 was due to trypsin. The
comparison
of the amino acid sequence of Cry3lAal with that of Cry3lAa2 indicates which
amino acids of the amino acid sequence of members of the Cry3l family can
be substituted without abrogating this cytoxicity against human cancer cells.
Although certain of these substitutions surely provide for the specificity of
each
of Cry3lAa2 and Cry3lAal against specific human cancer cells, they each
display a significant toxicity against a number of human cancer cells. Hence,
it
is submitted that the amino acids at positions 24, 37, 39, 51, 56, 59, 87, 97,
138, 158, 170, 251, 389, 444-446, 466, 481, 507, 510, 518, 551, 582, 637, 725
and 742 can be replaced by any other amino acid without abrogating the
cytotoxicity of the protein that it constitutes against at least some cancer
cells.
Sequences encompassing substitutions at these positions in the complete
Cry31Aa2 protein sequence (SEQ ID NO:2) and in the trypsin-activated
Cry3lAa2 protein sequence (SEQ ID NO:8) starting after the arginine at
position 250 are within the scope of the present invention and are designated
herein as SEQ ID NOs:12 and 13, respectively.
CA 02410153 2002-12-05
32
[0088] Certain substitutions are preferred however and correspond to
either a substitution by an amino acid having similar chemical properties or,
even more preferred, a substitution by the amino acid found at the
corresponding position in Cry3lAal. Amino acids are categorized herein into 5
groups of amino acids according to their chemical properties, namely small
nonpolar (i.e. C, P, A and T), small polar (i.e. S, G, D and N), large polar
(i.e. E,
0, K and R), intermediate polarity (i.e. Y, H and W), aand large nonpolar
(i.e. F,
M, L, I and V) . Hence, the amino acid at position 24 of the Cry31 Aa2, is
preferably a polar amino acid, most preferably a large polar amino acid and
even more preferably glutamate or lysine. The amino acid at position 37 is
preferably methionine or alanine. The amino acid at position 39 is preferably
threonine or asparagine. The amino acid at position 51, is preferably a
nonpolar
amino acid, most preferably a small nonpolar amino acid and even more
preferably alanine or threonine. The amino acid at position 56 is preferably
proline or serine. The amino acid at position 59 is preferably an amino acid
of
intermediate polarity and most preferably tyrosine or tryptophan. The amino
acid at position 87, is preferably a polar amino acid, most preferably a small
polar amino acid and even more preferably asparagine or aspartate. The amino
acid at position 97, is preferably a polar amino acid, most preferably a large
polar amino acid and even most preferably arginine or lysine. The amino acid
at position 138 is preferably a polar amino acid, most preferably a large
polar
amino acid and even more preferably glutamate or lysine. The amino acid at
position 158 is preferably alanine or asparagine. The amino acid at position
170
is preferably a polar amino acid, most preferably a small polar amino acid and
even more preferably glycine or serine.
[0089] The amino acid at position 251 is preferably a nonpolar amino
acid, most preferably a large nonpolar amino acid and even more preferably
isoleucine or methionine. The amino acid at position 389 is preferably a polar
CA 02410153 2009-09-30
33
amino acid, most preferably a large polar amino acid and even more preferably
lysine or arginine. The amino acid at position 444 is preferably serine or
histidine. The amino acid at position 445 is preferably a polar amino acid,
most
preferably a small polar amino acid and even more preferably glycine or
serine.
The amino acid at position 446 is preferably glycine or proline. The amino
acid
at position 466 is preferably a polar amino acid, most preferably a large
polar
amino acid and even more preferably glutamine or arginine. The amino acid at
position 481 is preferably an amino acid of intermediate polarity and most
preferably tyrosine or tryptophan. The amino acid at position 507 is
preferably
alanine or leucine. The amino acid at position 510 is preferably glycine or
histidine. The amino acid at position 518 is preferably a nonpolar amino acid
and is preferably alanine or valine. The amino acid at position 551 is
preferably
a nonpolar amino acid, most preferably a small nonpolar amino acid and even
more preferably alanine or proline. The amino acid at position 582 is
preferably
a nonpolar amino acid, most preferably a small nonpolar amino acid and even
more preferably alanine or threonine. The amino acid at position 637 is
preferably arginine or isoleucine. The amino acid at position 725 is
preferably
glycine or arginine. Finally, the amino acid at position 742 is preferably
valine or
serine. Sequences encompassing the most preferred substitutions listed above
at these positions in the complete Cry3lAa2 protein sequence (SEQ ID NO:2)
and in the trypsin-activated Cry3lAa2 protein sequence (SEQ ID NO:8) starting
after the arginine at position 250 are within the scope of the present
invention
and are designated herein as SEQ ID NOs:14 and 15, respectively.
[0090] Sequences encompassing all the possible substitutions to the
cry3lAa2 gene nucleotide sequence, the crystal protein and the trypsin-
activated crystal protein derived from the crystal protein of the Bacillus
thuringiensis M15 deposited under no. IDAC010201-5 as described above are
within the scope of the present invention.
CA 02410153 2009-09-30
34
[0091] Although the present invention has been described hereinabove
by way of preferred embodiments thereof, it can be modified, without departing
from the spirit and nature of the subject invention as defined in the appended
claims.
CA 02410153 2002-12-05
REFERENCES
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13. Hofte, H., and Whiteley, H. R. (1989). Insecticidal crystal proteins of B.
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14. Jung, Y. C., Kim, S. U., Cote, J. -C., Lecadet, M. -M., and Chung, Y. S.,
and Bok, S. H. (1998). Characterization of a new Bacillus thuringiensis
subsp. higo strain isolated from rice bran in Korea. J. lnvertebr. Pathol.
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15. Kim, H. S. (2000). Comparative study of the frequency, flagellar serotype,
crystal shape, toxicity, and cry gene contents of Bacillus thuringiensis from
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16.Laemmli, U. K., and Favre, M. (1973). Maturation of the head of
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17.Lecadet, M. -M., Frachon, E., Cosmao Dumanoir, V. et at. (1999).
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18. Lee, D. W., Akao, T., Yamashita, S., Katayama, H., Maeda, M., Saitoh, H.,
Mizuki, E., and Ohba, M. (2000). Noninsecticidal parasporal proteins of a
Bacillus thuringiensis serovar shandongiensis isolate exhibit a preferential
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19. Lereclus, D., Bourgouin, C., Lecadet, M. -M., Klier, A., and Rapoport, G.
(1989). Role, structure, and molecular organization of the genes coding for
the parasporal 6-endotoxins of B. thuringiensis. In "Regulation of
Procaryotic Development: Structural and Functional Analysis of Bacterial
Sporulation and Germination" (Smith, I., R. A. Slepecky, and P. Setlow,
Eds.), pp. 255-276, Washington, D.C. Am. Soc. for Microbiol.
20. McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981). Unique
features in the ribosome binding site sequence of the Gram-positive
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21.Mizuki, E., Ohba, M., Akao, T., Yamashita, S., Saitoh, H., and Park, Y. S.
(1999). Unique activity associated with non-insecticidal Bacillus
thuringiensis parasporal inclusions: in vitro cell-killing action on human
CA 02410153 2002-12-05
37
cancer cells. J. App!. Micro biol. 86, 477-486.
22. Mizuki, E., Park, Y. S., Saitoh, H., Yamashita, S., Akao, T., Higuchi, K.,
and
Ohba, M. (2000). Parasporin, a human leukemic cell-recognizing
parasporal protein of Bacillus thuringiensis. Clin. Diag. Lab. Immunol. 7,
625-634.
23. Moran, C. P., Lang, N., Jr., LeGrice, S. F. J., Lee, G., Stephens, M.,
Sonenshein, A. L., Pero, J., and Losick, R. (1982). Nucleotide sequences
that signal the initiation of transcription and translation in Bacillus
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Mol Gen. Gene. 186, 339-346.
24.Ohba, M. (1996). Bacillus thuringiensis populations naturally occuring on
mulberry leaves: a possible source of the populations associated with
silkworm-rearing insectaries. J. Appl. Bacteriol. 80, 56-64.
25.Ohba, M., and Aizawa, K. (1986). Insect toxicity of Bacillus thuringiensis
isolated from soils of Japan. J. Invertebr. Pathol. 47, 12-20.
26. Roh, J. Y., Park, H. W., Jin, B. R., Kim, H. S., Yu, Y. M., and Kang, S.
K.
(1996). Characterization of novel non-toxic Bacillus thuringiensis isolates
from Korea. Lett. Appl. Microbiol. 23, 249-252.
27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). "Molecular cloning.
A Laboratory manual," 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, New York.
28.Saitoh, H., K. Higuchi, E. Mizuki, and M. Ohba. (1998a). Larvicidal
toxicity of
Japanese Bacillus thuringiensis against the mosquito Anopheles stephensi.
Med. Vet. Entomol. 12:98-102.
29.Saitoh, H., K. Higuchi, E. Mizuki, S.-H. Hwang, and M. Ohba. (1998b).
Characterization of mosquito larvicidal parasporal inclusions of a Bacillus
thuringiensis serovar higo strain. J. Appl. Microbiol. 84:883-888
30.Schnepf, H. E., Wong, H. C., and Whiteley, H. R. (1985). The amino acid
sequence of a crystal protein from Bacillus thuringiensis deduced from the
DNA base sequence. J. Biol. Chem. 260, 6264-6272.
31. Southern, E. (1975). Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517.
32.Stahly, D. P., Dingman, D. W., Bulla, L. A., Jr., and Aronson, A. I.
(1978).
Possible origin and function of the parasporal crystal in Bacillus
thuringiensis. Biochem. Biophys. Res. Commun. 84, 581-588.
CA 02410153 2002-12-05
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33. Thomas, W. E., and Ellar, D. J. (1983). B. thuringiensis var. israelensis
crystal S-endotoxin: effects on insect and mammalian cells in vitro and in
vivo. J. Cell. Sci. 60, 181-197.
34.Tinoco, I., Jr., Borer, P. N., Dengler, B., Levine, M. D., Uhlenbeck, 0.
C.,
Crothers, D. M., and Gralla, J. (1973). Improved estimation of secondary
structure in ribonucleic acids. Nature New Biol. 246, 40-41.
35.Vehmaanpera, J. (1989). Transformation of Bacillus amyloliquefaciens by
electroporation. FEMS Microbiol. Lett. 61, 165-170.
36. Jung, Y.-C. (2001) Bacillus thuringiensis Strain M15, a Novel
Autoagglutinable, Non-Serotypeable Strain-Cloning and Characterization of
a Novel cry31A-type Crystal Protein Gene, cry3lAa2, and Two New
Insertion Sequences, IS231M and -N. Departement de sciences
biologiques, Faculte des arts et des sciences, Universite de Montreal.
CA 02410153 2004-04-01
1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: AGRICULTURE AGROALIMENTAIRE CANADA
(ii) TITLE OF INVENTION: A Novel Bacillus Thuringiensis Strain,
Crystal Gene and Crystal Protein and Uses Thereof
(iii) NUMBER OF SEQUENCES: 18
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Goudreau Gage Dubuc
(B) STREET: Stock Exchange Tower suite 3400, 800 Place
Victoria
(C) CITY: Montreal
(D) STATE: Quebec
(E) COUNTRY: Canada
(F) ZIP: H4Z 1E9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,410,153
(B) FILING DATE: 05-DEC-2002
(C) CLASSIFICATION: C12N-15/31
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gauvreau, Julie
(B) REGISTRATION NUMBER: 11,127
(C) REFERENCE/DOCKET NUMBER: 12292.5
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 514 397-7602
(B) TELEFAX: 514 397-4382
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2229 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GTGGACCCAT TTTCTAATTA TTCTGAACAA AAATACCCAG ATTCAAATAA TAACCAAGAA 60
CA 02410153 2004-04-01
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CTAATTACAG AATCCTCTTC ATTTTATTCG GATACTACTA ATGAAAATAT GAAAACTTAC 120
CATCCAATTG AACAAGATAT TCTCAAATTT GCAAATCAAG AATTTCCCGA TAATTATTAT 180
CAACATTCCG ATGTTTCTAA TTCATATCAA AATATGAAAA CAGAAATCGT AAATACAGAT 240
TTACCCTATA ATACAAATAA TATAAATAGT ATGCGAAATA CTCTATGCAG AGATTTACCT 300
CCCGAGACTA ACATGAGCAT TTATGATAAT TTACGATCTA CTGTTACTGT TCCTTCATTT 360
TCTAATCAAT TTGATCCTAT AAAATTTCTT CACGATATTG AAATTGCTAT AGAAACTGGA 420
TCATTTTCTG CATTAACGCA ATCTAACATG AATCAAGGTG GTACTGATAT TGCTCCAATG 480
TTAATCTCTA CATTTTTTAA AGTTGCAGGT AGTTTACTTC CATTTCCTCT ATCATCATTA 540
GGTGCTTTGG CTTCCTTTTA TGTTACAGAT TCACAAACAG GCGCTATGGC AAATTTATGG 600
AGACAAATGG TAGATTATGT TGAAAGAAGA ATTGATTCTA AAATATTAGA TTATCATAAT 660
TTTATTATGG GAGCAGAACT CGCAGCATTA AATGCAAGTT TAAAAGAATA CGCACGAGTA 720
GTTAAAATTT TTGAAAATGA TATGAACAGA ATAGCTGAAC CACCTTCAAC TGGAGTTATC 780
ACTCAATTCA GAATTCTTAA TGATAATTTC ATTAAATATA TTGCAAAATT ACAATTCTCA 840
ACAAATCAAT CAGATTTACA ATATCCTGTC CTAACTTTAC CATTACGTGC ACAAGCATGT 900
GTAATGCATT TAATGTTATT AAAAGATGCA ACGACTTCTG TGTGGGGACA ACAAATAGAC 960
TCGCAACAAT TAAATGGGTA TAAAGCAGAA TTAATACGTT TAATAAAAGT ATATACTAAT 1020
GATGTAAACA CAACGTATAA TCAAGGGCTA GAGCTAGAAA AAGCTAAACC ACTAAATTAT 1080
TCTGATCCTG AAGAATATTT ACAAGCAGGA CGTCCAGATA TATCTGTATT ACGCAGTAAC 1140
TTTAAAGAGG TTATGAAGTG GAATAAAGTA GCGAAATATA AACGTGGAAT GGCTATGAGT 1200
GCTTTATCAT TAGCTGCATT ATTTCCAACT TTCGGACCAA ATTATCCAAA ACAAGCATTA 1260
AAAGTTGTGC AATCTAGACA AATTTTTGCA CCTGTAATTG GAATACCAGG CGGTATAACA 1320
AGTCAAGATA GTGGTCCCAC TTTTGGTAGT ATGAGATTTG ATGTAAAAAC TTATGATCAA 1380
ATTGATGCGT TACGACAACT AATGGAATTA TATATTCAAC CTTTAAAATC TGCTTACTTT 1440
TGGATATATG AATCGGATTG GAAAGTTCGT GCAACTTATG TCAATGATTA TATTGGTAAA 1500
AGAGGGTCAA ATACAGGTGC TGCTTGGCAC ATGTGGTCAA GTGATCCTTC AGCCATATAC 1560
ACTTCTGCAC TAGGAGCAGC AGGATACGCT CCTAACGTTG TTGGTGTAAG ATATTCACAT 1620
GGGGGTAGTT ACACAAAAGG TATGGCACCC GCAAATACTA ATGCGTATGC TCCATTTGAA 1680
TTTAAATATC CTGGTTATAA ACTACACAGT GTTAGTGCTT ATGGATTAAG TAAAGCACCT 1740
GATGCAGCTG ATTCTGTTAT GTTTGGATTT AGACCTGTAT TGTTAGAAAA TGAAGCAAAT 1800
CAATTATTAA CAGATACAGC ATTGCAAATT CCAGCAGAAA TAGGAATAAC AGATGTCGTA 1860
CCTGCATTTG GTAGAACAGA AGAACCTATT AATGGTCAAG ATGCAATAAG AATATGGGAA 1920
CA 02410153 2004-04-01
3
AGTTTTACAA GTGGATTTGG CTTTACTTAT ACTGTTGATT CTCCACAAAA ACAAAAATAT 1980
AAAATCATTT ATAGAATTGC AAATAACTTA AGCGCTTCTA CAGTTTCTTT AACCTATAAT 2040
AATCAAACAT TTTTCACTGA TATTTTAAAT ACTTCATTAG ATCCAAATGG AGTAAGAGGA 2100
AATTATGGTT CTTATACACT TGTAGAAGGT CCTATTATTG AATTTTCTCA AGGAACTAAT 2160
ATCTTTAAAC TAGGATCACA AAAAGGAGAA TTCGCTATAG ATTCCATTAT TTTTAGTCCT 2220
GTTGTTTAA 2229
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Asp Pro Phe Ser Asn Tyr Ser Glu Gln Lys Tyr Pro Asp Ser Asn
1 5 10 15
Asn Asn Gln Glu Leu Ile Thr Glu Ser Ser Ser Phe Tyr Ser Asp Thr
20 25 30
Thr Asn Glu Asn Met Lys Thr Tyr His Pro Ile Glu Gln Asp Ile Leu
35 40 45
Lys Phe Ala Asn Gln Glu Phe Pro Asp Asn Tyr Tyr Gln His Ser Asp
50 55 60
Val Ser Asn Ser Tyr Gln Asn Met Lys Thr Glu Ile Val Asn Thr Asp
65 70 75 80
Leu Pro Tyr Asn Thr Asn Asn Ile Asn Ser Met Arg Asn Thr Leu Cys
85 90 95
Arg Asp Leu Pro Pro Glu Thr Asn Met Ser Ile Tyr Asp Asn Leu Arg
100 105 110
Ser Thr Val Thr Val Pro Ser Phe Ser Asn Gln Phe Asp Pro Ile Lys
115 120 125
Phe Leu His Asp Ile Glu Ile Ala Ile Glu Thr Gly Ser Phe Ser Ala
130 135 140
Leu Thr Gln Ser Asn Met Asn Gln Gly Gly Thr Asp Ile Ala Pro Met
145 150 155 160
Leu Ile Ser Thr Phe Phe Lys Val Ala Gly Ser Leu Leu Pro Phe Pro
CA 02410153 2004-04-01
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165 170 175
Leu Ser Ser Leu Gly Ala Leu Ala Ser Phe Tyr Val Thr Asp Ser Gln
180 185 190
Thr Gly Ala Met Ala Asn Leu Trp Arg Gln Met Val Asp Tyr Val Glu
195 200 205
Lys Arg Ile Asp Ser Lys Ile Leu Asp Tyr His Asn Phe Ile Met Gly
210 215 220
Ala Glu Leu Ala Ala Leu Asn Ala Ser Leu Lys Glu Tyr Ala Arg Val
225 230 235 240
Val Lys Ile Phe Glu Asn Asp Met Asn Arg Ile Ala Glu Pro Pro Ser
245 250 255
Thr Gly Val Ile Thr Gln Phe Arg Ile Leu Asn Asp Asn Phe Ile Lys
260 265 270
Tyr Ile Ala Lys Leu Gln Phe Ser Thr Asn Gln Ser Asp Leu Gln Tyr
275 280 285
Pro Val Leu Thr Leu Pro Leu Arg Ala Gln Ala Cys Val Met His Leu
290 295 300
Met Leu Leu Lys Asp Ala Thr Thr Ser Val Trp Gly Gln Gln Ile Asp
305 310 315 320
Ser Gln Gln Leu Asn Gly Tyr Lys Ala Glu Leu Ile Arg Leu Ile Lys
325 330 335
Val Tyr Thr Asn Asp Val Asn Thr Thr Tyr Asn Gln Gly Leu Glu Leu
340 345 350
Glu Lys Ala Lys Pro Leu Asn Tyr Ser Asp Pro Glu Glu Tyr Leu Gln
355 360 365
Ala Gly Arg Pro Asp Ile Ser Val Leu Arg Ser Asn Phe Lys Glu Val
370 375 380
Met Lys Trp Asn Lys Val Ala Lys Tyr Lys Arg Gly Met Ala Met Ser
385 390 395 400
Ala Leu Ser Leu Ala Ala Leu Phe Pro Thr Phe Gly Pro Asn Tyr Pro
405 410 415
Lys Gln Ala Leu Lys Val Val Gln Ser Arg Gln Ile Phe Ala Pro Val
420 425 430
Ile Gly Ile Pro Gly Gly Ile Thr Ser Gln Asp Ser Gly Pro Thr Phe
435 440 445
Gly Ser Met Arg Phe Asp Val Lys Thr Tyr Asp Gln Ile Asp Ala Leu
450 455 460
Arg Gln Leu Met Glu Leu Tyr Ile Gln Pro Leu Lys Ser Ala Tyr Phe
465 470 475 480
Trp Ile Tyr Glu Ser Asp Trp Lys Val Arg Ala Thr Tyr Val Asn Asp
485 490 495
CA 02410153 2004-04-01
Tyr Ile Gly Lys Arg Gly Ser Asn Thr Gly Ala Ala Trp His Met Trp
500 505 510
Ser Ser Asp Pro Ser Ala Ile Tyr Thr Ser Ala Leu Gly Ala Ala Gly
515 520 525
Tyr Ala Pro Asn Val Val Gly Val Arg Tyr Ser His Gly Gly Ser Tyr
530 535 540
Thr Lys Gly Met Ala Pro Ala Asn Thr Asn Ala Tyr Ala Pro Phe Glu
545 550 555 560
Phe Lys Tyr Pro Gly Tyr Lys Leu His Ser Val Ser Ala Tyr Gly Leu
565 570 575
Ser Lys Ala Pro Asp Ala Ala Asp Ser Val Met Phe Gly Phe Arg Pro
580 585 590
Val Leu Leu Glu Asn Glu Ala Asn Gln Leu Leu Thr Asp Thr Ala Leu
595 600 605
Gln Ile Pro Ala Glu Ile Gly Ile Thr Asp Val Val Pro Ala Phe Gly
610 615 620
Arg Thr Glu Glu Pro Ile Asn Gly Gln Asp Ala Ile Arg Ile Trp Glu
625 630 635 640
Ser Phe Thr Ser Gly Phe Gly Phe Thr Tyr Thr Val Asp Ser Pro Gln
645 650 655
Lys Gln Lys Tyr Lys Ile Ile Tyr Arg Ile Ala Asn Asn Leu Ser Ala
660 665 670
Ser Thr Val Ser Leu Thr Tyr Asn Asn Gln Thr Phe Phe Thr Asp Ile
675 680 685
Leu Asn Thr Ser Leu Asp Pro Asn Gly Val Arg Gly Asn Tyr Gly Ser
690 695 700
Tyr Thr Leu Val Glu Gly Pro Ile Ile Glu Phe Ser Gln Gly Thr Asn
705 710 715 720
Ile Phe Lys Leu Gly Ser Gln Lys Gly Glu Phe Ala Ile Asp Ser Ile
725 730 735
Ile Phe Ser Pro Val Val
740
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
CA 02410153 2004-04-01
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Asp Pro Phe Ser Asn Tyr Ser Glu Gln Lys Tyr Pro Asp Ser Asn
1 5 10 15
Asn Asn Gln Glu
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Glu Gln Lys Tyr Pro Asp
1 5
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 15
(D) OTHER INFORMATION: /note= "N IS A, C, G, OR T"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GARCARAART AYCCNGAY 18
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02410153 2004-04-01
7
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GAAAGGTGG 9
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus subtilis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
UCUUUCCUCC 10
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 492 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ile Ala Glu Pro Pro Ser Thr Gly Val Ile Thr Gln Phe Arg Ile Leu
1 5 10 15
Asn Asp Asn Phe Ile Lys Tyr Ile Ala Lys Leu Gln Phe Ser Thr Asn
20 25 30
Gln Ser Asp Leu Gln Tyr Pro Val Leu Thr Leu Pro Leu Arg Ala Gln
35 40 45
Ala Cys Val Met His Leu Met Leu Leu Lys Asp Ala Thr Thr Ser Val
50 55 60
Trp Gly Gln Gln Ile Asp Ser Gln Gln Leu Asn Gly Tyr Lys Ala Glu
CA 02410153 2004-04-01
8
65 70 75 80
Leu Ile Arg Leu Ile Lys Val Tyr Thr Asn Asp Val Asn Thr Thr Tyr
85 90 95
Asn Gln Gly Leu Glu Leu Glu Lys Ala Lys Pro Leu Asn Tyr Ser Asp
100 105 110
Pro Glu Glu Tyr Leu Gln Ala Gly Arg Pro Asp Ile Ser Val Leu Arg
115 120 125
Ser Asn Phe Lys Glu Val Met Lys Trp Asn Lys Val Ala Lys Tyr Lys
130 135 140
Arg Gly Met Ala Met Ser Ala Leu Ser Leu Ala Ala Leu Phe Pro Thr
145 150 155 160
Phe Gly Pro Asn Tyr Pro Lys Gln Ala Leu Lys Val Val Gln Ser Arg
165 170 175
Gln Ile Phe Ala Pro Val Ile Gly Ile Pro Gly Gly Ile Thr Ser Gln
180 185 190
Asp Ser Gly Pro Thr Phe Gly Ser Met Arg Phe Asp Val Lys Thr Tyr
195 200 205
Asp Gln Ile Asp Ala Leu Arg Gln Leu Met Glu Leu Tyr Ile Gln Pro
210 215 220
Leu Lys Ser Ala Tyr Phe Trp Ile Tyr Glu Ser Asp Trp Lys Val Arg
225 230 235 240
Ala Thr Tyr Val Asn Asp Tyr Ile Gly Lys Arg Gly Ser Asn Thr Gly
245 250 255
Ala Ala Trp His Met Trp Ser Ser Asp Pro Ser Ala Ile Tyr Thr Ser
260 265 270
Ala Leu Gly Ala Ala Gly Tyr Ala Pro Asn Val Val Gly Val Arg Tyr
275 280 285
Ser His Gly Gly Ser Tyr Thr Lys Gly Met Ala Pro Ala Asn Thr Asn
290 295 300
Ala Tyr Ala Pro Phe Glu Phe Lys Tyr Pro Gly Tyr Lys Leu His Ser
305 310 315 320
Val Ser Ala Tyr Gly Leu Ser Lys Ala Pro Asp Ala Ala Asp Ser Val
325 330 335
Met Phe Gly Phe Arg Pro Val Leu Leu Glu Asn Glu Ala Asn Gln Leu
340 345 350
Leu Thr Asp Thr Ala Leu Gln Ile Pro Ala Glu Ile Gly Ile Thr Asp
355 360 365
Val Val Pro Ala Phe Gly Arg Thr Glu Glu Pro Ile Asn Gly Gln Asp
370 375 380
Ala Ile Arg Ile Trp Glu Ser Phe Thr Ser Gly Phe Gly Phe Thr Tyr
385 390 395 400
CA 02410153 2004-04-01
9
Thr Val Asp Ser Pro Gln Lys Gln Lys Tyr Lys Ile Ile Tyr Arg Ile
405 410 415
Ala Asn Asn Leu Ser Ala Ser Thr Val Ser Leu Thr Tyr Asn Asn Gln
420 425 430
Thr Phe Phe Thr Asp Ile Leu Asn Thr Ser Leu Asp Pro Asn Gly Val
435 440 445
Arg Gly Asn Tyr Gly Ser Tyr Thr Leu Val Glu Gly Pro Ile Ile Glu
450 455 460
Phe Ser Gln Gly Thr Asn Ile Phe Lys Leu Gly Ser Gln Lys Gly Glu
465 470 475 480
Phe Ala Ile Asp Ser Ile Ile Phe Ser Pro Val Val
485 490
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1479 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATAGCTGAAC CACCTTCAAC TGGAGTTATC ACTCAATTCA GAATTCTTAA TGATAATTTC 60
ATTAAATATA TTGCAAAATT ACAATTCTCA ACAAATCAAT CAGATTTACA ATATCCTGTC 120
CTAACTTTAC CATTACGTGC ACAAGCATGT GTAATGCATT TAATGTTATT AAAAGATGCA 180
ACGACTTCTG TGTGGGGACA ACAAATAGAC TCGCAACAAT TAAATGGGTA TAAAGCAGAA 240
TTAATACGTT TAATAAAAGT ATATACTAAT GATGTAAACA CAACGTATAA TCAAGGGCTA 300
GAGCTAGAAA AAGCTAAACC ACTAAATTAT TCTGATCCTG AAGAATATTT ACAAGCAGGA 360
CGTCCAGATA TATCTGTATT ACGCAGTAAC TTTAAAGAGG TTATGAAGTG GAATAAAGTA 420
GCGAAATATA AACGTGGAAT GGCTATGAGT GCTTTATCAT TAGCTGCATT ATTTCCAACT 480
TTCGGACCAA ATTATCCAAA ACAAGCATTA AAAGTTGTGC AATCTAGACA AATTTTTGCA 540
CCTGTAATTG GAATACCAGG CGGTATAACA AGTCAAGATA GTGGTCCCAC TTTTGGTAGT 600
ATGAGATTTG ATGTAAAAAC TTATGATCAA ATTGATGCGT TACGACAACT AATGGAATTA 660
TATATTCAAC CTTTAAAATC TGCTTACTTT TGGATATATG AATCGGATTG GAAAGTTCGT 720
GCAACTTATG TCAATGATTA TATTGGTAAA AGAGGGTCAA ATACAGGTGC TGCTTGGCAC 780
CA 02410153 2004-04-01
ATGTGGTCAA GTGATCCTTC AGCCATATAC ACTTCTGCAC TAGGAGCAGC AGGATACGCT 840
CCTAACGTTG TTGGTGTAAG ATATTCACAT GGGGGTAGTT ACACAAAAGG TATGGCACCC 900
GCAAATACTA ATGCGTATGC TCCATTTGAA TTTAAATATC CTGGTTATAA ACTACACAGT 960
GTTAGTGCTT ATGGATTAAG TAAAGCACCT GATGCAGCTG ATTCTGTTAT GTTTGGATTT 1020
AGACCTGTAT TGTTAGAAAA TGAAGCAAAT CAATTATTAA CAGATACAGC ATTGCAAATT 1080
CCAGCAGAAA TAGGAATAAC AGATGTCGTA CCTGCATTTG GTAGAACAGA AGAACCTATT 1140
AATGGTCAAG ATGCAATAAG AATATGGGAA AGTTTTACAA GTGGATTTGG CTTTACTTAT 1200
ACTGTTGATT CTCCACAAAA ACAAAAATAT AAAATCATTT ATAGAATTGC AAATAACTTA 1260
AGCGCTTCTA CAGTTTCTTT AACCTATAAT AATCAAACAT TTTTCACTGA TATTTTAAAT 1320
ACTTCATTAG ATCCAAATGG AGTAAGAGGA AATTATGGTT CTTATACACT TGTAGAAGGT 1380
CCTATTATTG AATTTTCTCA AGGAACTAAT ATCTTTAAAC TAGGATCACA AAAAGGAGAA 1440
TTCGCTATAG ATTCCATTAT TTTTAGTCCT GTTGTTTAA 1479
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ser Tyr Gln Asn Met Lys Thr Glu Ile Val Asn Thr Asp Leu Pro Tyr
1 5 10 15
Asn Thr Asn
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
CA 02410153 2004-04-01
11
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TCATATCAAA ATATGAAAAC AGAAATCGTA AATACAGATT TACCCTATAA TACAAAT 57
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 24
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 37
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 39
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 51
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 56
(D) OTHER INFORMATION: /note= "Xaa=anyt amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 59
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 87
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 97
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
CA 02410153 2004-04-01
12
(B) LOCATION: 138
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 158
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 170
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 251
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 389
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 444
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 445
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 446
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 466
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE :
(A) NAME/KEY: Modified-site
(B) LOCATION: 481
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 507
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 510
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 518
CA 02410153 2004-04-01
13
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 551
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 582
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 637
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 725
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 742
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Asp Pro Phe Ser Asn Tyr Ser Glu Gln Lys Tyr Pro Asp Ser Asn
1 5 10 15
Asn Asn Gln Glu Leu Ile Thr Xaa Ser Ser Ser Phe Tyr Ser Asp Thr
20 25 30
Thr Asn Glu Asn Xaa Lys Xaa Tyr His Pro Ile Glu Gln Asp Ile Leu
35 40 45
Lys Phe Xaa Asn Gln Glu Phe Xaa Asp Asn Xaa Tyr Gln His Ser Asp
50 55 60
Val Ser Asn Ser Tyr Gln Asn Met Lys Thr Glu Ile Val Asn Thr Asp
65 70 75 80
Leu Pro Tyr Asn Thr Asn Xaa Ile Asn Ser Met Arg Asn Thr Leu Cys
85 90 95
Xaa Asp Leu Pro Pro Glu Thr Asn Met Ser Ile Tyr Asp Asn Leu Arg
100 105 110
Ser Thr Val Thr Val Pro Ser Phe Ser Asn Gln Phe Asp Pro Ile Lys
115 120 125
Phe Leu His Asp Ile Glu Ile Ala Ile Xaa Thr Gly Ser Phe Ser Ala
130 135 140
Leu Thr Gln Ser Asn Met Asn Gln Gly Gly Thr Asp Ile Xaa Pro Met
145 150 155 160
Leu Ile Ser Thr Phe Phe Lys Val Ala Xaa Ser Leu Leu Pro Phe Pro
CA 02410153 2004-04-01
14
165 170 175
Leu Ser Ser Leu Gly Ala Leu Ala Ser Phe Tyr Val Thr Asp Ser Gln
180 185 190
Thr Gly Ala Met Ala Asn Leu Trp Arg Gln Met Val Asp Tyr Val Glu
195 200 205
Lys Arg Ile Asp Ser Lys Ile Leu Asp Tyr His Asn Phe Ile Met Gly
210 215 220
Ala Glu Leu Ala Ala Leu Asn Ala Ser Leu Lys Glu Tyr Ala Arg Val
225 230 235 240
Val Lys Ile Phe Glu Asn Asp Met Asn Arg Xaa Ala Glu Pro Pro Ser
245 250 255
Thr Gly Val Ile Thr Gln Phe Arg Ile Leu Asn Asp Asn Phe Ile Lys
260 265 270
Tyr Ile Ala Lys Leu Gln Phe Ser Thr Asn Gln Ser Asp Leu Gln Tyr
275 280 285
Pro Val Leu Thr Leu Pro Leu Arg Ala Gln Ala Cys Val Met His Leu
290 295 300
Met Leu Leu Lys Asp Ala Thr Thr Ser Val Trp Gly Gln Gln Ile Asp
305 310 315 320
Ser Gln Gln Leu Asn Gly Tyr Lys Ala Glu Leu Ile Arg Leu Ile Lys
325 330 335
Val Tyr Thr Asn Asp Val Asn Thr Thr Tyr Asn Gln Gly Leu Glu Leu
340 345 350
Glu Lys Ala Lys Pro Leu Asn Tyr Ser Asp Pro Glu Glu Tyr Leu Gln
355 360 365
Ala Gly Arg Pro Asp Ile Ser Val Leu Arg Ser Asn Phe Lys Glu Val
370 375 380
Met Lys Trp Asn Xaa Val Ala Lys Tyr Lys Arg Gly Met Ala Met Ser
385 390 395 400
Ala Leu Ser Leu Ala Ala Leu Phe Pro Thr Phe Gly Pro Asn Tyr Pro
405 410 415
Lys Gln Ala Leu Lys Val Val Gln Ser Arg Gln Ile Phe Ala Pro Val
420 425 430
Ile Gly Ile Pro Gly Gly Ile Thr Ser Gln Asp Xaa Xaa Xaa Thr Phe
435 440 445
Gly Ser Met Arg Phe Asp Val Lys Thr Tyr Asp Gln Ile Asp Ala Leu
450 455 460
Arg Xaa Leu Met Glu Leu Tyr Ile Gln Pro Leu Lys Ser Ala Tyr Phe
465 470 475 480
Xaa Ile Tyr Glu Ser Asp Trp Lys Val Arg Ala Thr Tyr Val Asn Asp
485 490 495
CA 02410153 2004-04-01
Tyr Ile Gly Lys Arg Gly Ser Asn Thr Gly Xaa Ala Trp Xaa Met Trp
500 505 510
Ser Ser Asp Pro Ser Xaa Ile Tyr Thr Ser Ala Leu Gly Ala Ala Gly
515 520 525
Tyr Ala Pro Asn Val Val Gly Val Arg Tyr Ser His Gly Gly Ser Tyr
530 535 540
Thr Lys Gly Met Ala Pro Xaa Asn Thr Asn Ala Tyr Ala Pro Phe Glu
545 550 555 560
Phe Lys Tyr Pro Gly Tyr Lys Leu His Ser Val Ser Ala Tyr Gly Leu
565 570 575
Ser Lys Ala Pro Asp Xaa Ala Asp Ser Val Met Phe Gly Phe Arg Pro
580 585 590
Val Leu Leu Glu Asn Glu Ala Asn Gln Leu Leu Thr Asp Thr Ala Leu
595 600 605
Gln Ile Pro Ala Glu Ile Gly Ile Thr Asp Val Val Pro Ala Phe Gly
610 615 620
Arg Thr Glu Glu Pro Ile Asn Gly Gln Asp Ala Ile Xaa Ile Trp Glu
625 630 635 640
Ser Phe Thr Ser Gly Phe Gly Phe Thr Tyr Thr Val Asp Ser Pro Gln
645 650 655
Lys Gln Lys Tyr Lys Ile Ile Tyr Arg Ile Ala Asn Asn Leu Ser Ala
660 665 670
Ser Thr Val Ser Leu Thr Tyr Asn Asn Gln Thr Phe Phe Thr Asp Ile
675 680 685
Leu Asn Thr Ser Leu Asp Pro Asn Gly Val Arg Gly Asn Tyr Gly Ser
690 695 700
Tyr Thr Leu Val Glu Gly Pro Ile Ile Glu Phe Ser Gln Gly Thr Asn
705 710 715 720
Ile Phe Lys Leu Xaa Ser Gln Lys Gly Glu Phe Ala Ile Asp Ser Ile
725 730 735
Ile Phe Ser Pro Val Xaa
740
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 492 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
CA 02410153 2004-04-01
16
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 139
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 194
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 195..196
(D) OTHER INFORMATION: /note= "Xaa can be any naturally
occurring amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 216
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 231
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 257
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 260
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 268
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 301
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 332
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 387
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
CA 02410153 2004-04-01
17
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 475
(D) OTHER INFORMATION: /note= "Xaa= any amino acid"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 492
(D) OTHER INFORMATION: /note= "Xaa=any amino acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Xaa Ala Glu Pro Pro Ser Thr Gly Val Ile Thr Gln Phe Arg Ile Leu
1 5 10 15
Asn Asp Asn Phe Ile Lys Tyr Ile Ala Lys Leu Gln Phe Ser Thr Asn
20 25 30
Gln Ser Asp Leu Gln Tyr Pro Val Leu Thr Leu Pro Leu Arg Ala Gln
35 40 45
Ala Cys Val Met His Leu Met Leu Leu Lys Asp Ala Thr Thr Ser Val
50 55 60
Trp Gly Gln Gln Ile Asp Ser Gln Gln Leu Asn Gly Tyr Lys Ala Glu
65 70 75 80
Leu Ile Arg Leu Ile Lys Val Tyr Thr Asn Asp Val Asn Thr Thr Tyr
85 90 95
Asn Gln Gly Leu Glu Leu Glu Lys Ala Lys Pro Leu Asn Tyr Ser Asp
100 105 110
Pro Glu Glu Tyr Leu Gln Ala Gly Arg Pro Asp Ile Ser Val Leu Arg
115 120 125
Ser Asn Phe Lys Glu Val Met Lys Trp Asn Xaa Val Ala Lys Tyr Lys
130 135 140
Arg Gly Met Ala Met Ser Ala Leu Ser Leu Ala Ala Leu Phe Pro Thr
145 150 155 160
Phe Gly Pro Asn Tyr Pro Lys Gln Ala Leu Lys Val Val Gln Ser Arg
165 170 175
Gln Ile Phe Ala Pro Val Ile Gly Ile Pro Gly Gly Ile Thr Ser Gln
180 185 190
Asp Xaa Xaa Xaa Thr Phe Gly Ser Met Arg Phe Asp Val Lys Thr Tyr
195 200 205
Asp Gln Ile Asp Ala Leu Arg Xaa Leu Met Glu Leu Tyr Ile Gln Pro
210 215 220
Leu Lys Ser Ala Tyr Phe Xaa Ile Tyr Glu Ser Asp Trp Lys Val Arg
225 230 235 240
Ala Thr Tyr Val Asn Asp Tyr Ile Gly Lys Arg Gly Ser Asn Thr Gly
245 250 255
CA 02410153 2004-04-01
18
Xaa Ala Trp Xaa Met Trp Ser Ser Asp Pro Ser Xaa Ile Tyr Thr Ser
260 265 270
Ala Leu Gly Ala Ala Gly Tyr Ala Pro Asn Val Val Gly Val Arg Tyr
275 280 285
Ser His Gly Gly Ser Tyr Thr Lys Gly Met Ala Pro Xaa Asn Thr Asn
290 295 300
Ala Tyr Ala Pro Phe Glu Phe Lys Tyr Pro Gly Tyr Lys Leu His Ser
305 310 315 320
Val Ser Ala Tyr Gly Leu Ser Lys Ala Pro Asp Xaa Ala Asp Ser Val
325 330 335
Met Phe Gly Phe Arg Pro Val Leu Leu Glu Asn Glu Ala Asn Gln Leu
340 345 350
Leu Thr Asp Thr Ala Leu Gln Ile Pro Ala Glu Ile Gly Ile Thr Asp
355 360 365
Val Val Pro Ala Phe Gly Arg Thr Glu Glu Pro Ile Asn Gly Gln Asp
370 375 380
Ala Ile Xaa Ile Trp Glu Ser Phe Thr Ser Gly Phe Gly Phe Thr Tyr
385 390 395 400
Thr Val Asp Ser Pro Gln Lys Gln Lys Tyr Lys Ile Ile Tyr Arg Ile
405 410 415
Ala Asn Asn Leu Ser Ala Ser Thr Val Ser Leu Thr Tyr Asn Asn Gln
420 425 430
Thr Phe Phe Thr Asp Ile Leu Asn Thr Ser Leu Asp Pro Asn Gly Val
435 440 445
Arg Gly Asn Tyr Gly Ser Tyr Thr Leu Val Glu Gly Pro Ile Ile Glu
450 455 460
Phe Ser Gln Gly Thr Asn Ile Phe Lys Leu Xaa Ser Gln Lys Gly Glu
465 470 475 480
Phe Ala Ile Asp Ser Ile Ile Phe Ser Pro Val Xaa
485 490
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 742 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(ix) FEATURE:
(A) NAME/KEY: Modified-site
CA 02410153 2004-04-01
19
(B) LOCATION: 24
(D) OTHER INFORMATION: /note= "Xaa=glutamate or lysine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 37
(D) OTHER INFORMATION: /note= "Xaa= methionine or alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 39
(D) OTHER INFORMATION: /note= "Xaa=threonine or
asparagine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 51
(D) OTHER INFORMATION: /note= "Xaa=alanine or threonine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 56
(D) OTHER INFORMATION: /note= "Xaa= proline or serine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 59
(D) OTHER INFORMATION: /note= "Xaa=tyrosine or tryptophan"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 87
(D) OTHER INFORMATION: /note= "Xaa= asparagine or
aspartate"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 97
(D) OTHER INFORMATION: /note= "Xaa=arginine or lysine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 138
(D) OTHER INFORMATION: /note= "Xaa= glutamate or lysine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 158
(D) OTHER INFORMATION: /note= "Xaa= alanine or asparagine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 170
(D) OTHER INFORMATION: /note= "Xaa=glycine or serine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 251
(D) OTHER INFORMATION: /note= "Xaa= isoleucine or
methionine"
CA 02410153 2004-04-01
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 389
(D) OTHER INFORMATION: /note= "Xaa= lysine or arginine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 444
(D) OTHER INFORMATION: /note= "Xaa= serine or histidine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 445
(D) OTHER INFORMATION: /note= "Xaa= glycine or serine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 446
(D) OTHER INFORMATION: /note= "Xaa= glycine or proline"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 466
(D) OTHER INFORMATION: /note= "Xaa= glutamine or arginine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 481
(D) OTHER INFORMATION: /note= "Xaa= tyrosine or
tryptophan"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 507
(D) OTHER INFORMATION: /note= "Xaa= alanine or leucine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 510
(D) OTHER INFORMATION: /note= "Xaa= glycine or histidine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 518
(D) OTHER INFORMATION: /note= "Xaa= alanine or valine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 551
(D) OTHER INFORMATION: /note= "Xaa= alanine or proline"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 582
(D) OTHER INFORMATION: /note= "Xaa= alanine or threonine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 637
(D) OTHER INFORMATION: /note= "Xaa= arginine or
isoleucine"
CA 02410153 2004-04-01
21
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 725
(D) OTHER INFORMATION: /note= "Xaa= glycine or arginine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 742
(D) OTHER INFORMATION: /note= "Xaa= valine or serine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Asp Pro Phe Ser Asn Tyr Ser Glu Gln Lys Tyr Pro Asp Ser Asn
1 5 10 15
Asn Asn Gln Glu Leu Ile Thr Xaa Ser Ser Ser Phe Tyr Ser Asp Thr
20 25 30
Thr Asn Glu Asn Xaa Lys Xaa Tyr His Pro Ile Glu Gln Asp Ile Leu
35 40 45
Lys Phe Xaa Asn Gln Glu Phe Xaa Asp Asn Xaa Tyr Gln His Ser Asp
50 55 60
Val Ser Asn Ser Tyr Gln Asn Met Lys Thr Glu Ile Val Asn Thr Asp
65 70 75 80
Leu Pro Tyr Asn Thr Asn Xaa Ile Asn Ser Met Arg Asn Thr Leu Cys
85 90 95
Xaa Asp Leu Pro Pro Glu Thr Asn Met Ser Ile Tyr Asp Asn Leu Arg
100 105 110
Ser Thr Val Thr Val Pro Ser Phe Ser Asn Gln Phe Asp Pro Ile Lys
115 120 125
Phe Leu His Asp Ile Glu Ile Ala Ile Xaa Thr Gly Ser Phe Ser Ala
130 135 140
Leu Thr Gln Ser Asn Met Asn Gln Gly Gly Thr Asp Ile Xaa Pro Met
145 150 155 160
Leu Ile Ser Thr Phe Phe Lys Val Ala Xaa Ser Leu Leu Pro Phe Pro
165 170 175
Leu Ser Ser Leu Gly Ala Leu Ala Ser Phe Tyr Val Thr Asp Ser Gln
180 185 190
Thr Gly Ala Met Ala Asn Leu Trp Arg Gln Met Val Asp Tyr Val Glu
195 200 205
Lys Arg Ile Asp Ser Lys Ile Leu Asp Tyr His Asn Phe Ile Met Gly
210 215 220
Ala Glu Leu Ala Ala Leu Asn Ala Ser Leu Lys Glu Tyr Ala Arg Val
225 230 235 240
Val Lys Ile Phe Glu Asn Asp Met Asn Arg Xaa Ala Glu Pro Pro Ser
245 250 255
CA 02410153 2004-04-01
22
Thr Gly Val Ile Thr Gln Phe Arg Ile Leu Asn Asp Asn Phe Ile Lys
260 265 270
Tyr Ile Ala Lys Leu Gln Phe Ser Thr Asn Gln Ser Asp Leu Gln Tyr
275 280 285
Pro Val Leu Thr Leu Pro Leu Arg Ala Gln Ala Cys Val Met His Leu
290 295 300
Met Leu Leu Lys Asp Ala Thr Thr Ser Val Trp Gly Gln Gln Ile Asp
305 310 315 320
Ser Gln Gln Leu Asn Gly Tyr Lys Ala Glu Leu Ile Arg Leu Ile Lys
325 330 335
Val Tyr Thr Asn Asp Val Asn Thr Thr Tyr Asn Gln Gly Leu Glu Leu
340 345 350
Glu Lys Ala Lys Pro Leu Asn Tyr Ser Asp Pro Glu Glu Tyr Leu Gln
355 360 365
Ala Gly Arg Pro Asp Ile Ser Val Leu Arg Ser Asn Phe Lys Glu Val
370 375 380
Met Lys Trp Asn Xaa Val Ala Lys Tyr Lys Arg Gly Met Ala Met Ser
385 390 395 400
Ala Leu Ser Leu Ala Ala Leu Phe Pro Thr Phe Gly Pro Asn Tyr Pro
405 410 415
Lys Gln Ala Leu Lys Val Val Gln Ser Arg Gln Ile Phe Ala Pro Val
420 425 430
Ile Gly Ile Pro Gly Gly Ile Thr Ser Gln Asp Xaa Xaa Xaa Thr Phe
435 440 445
Gly Ser Met Arg Phe Asp Val Lys Thr Tyr Asp Gln Ile Asp Ala Leu
450 455 460
Arg Xaa Leu Met Glu Leu Tyr Ile Gln Pro Leu Lys Ser Ala Tyr Phe
465 470 475 480
Xaa Ile Tyr Glu Ser Asp Trp Lys Val Arg Ala Thr Tyr Val Asn Asp
485 490 495
Tyr Ile Gly Lys Arg Gly Ser Asn Thr Gly Xaa Ala Trp Xaa Met Trp
500 505 510
Ser Ser Asp Pro Ser Xaa Ile Tyr Thr Ser Ala Leu Gly Ala Ala Gly
515 520 525
Tyr Ala Pro Asn Val Val Gly Val Arg Tyr Ser His Gly Gly Ser Tyr
530 535 540
Thr Lys Gly Met Ala Pro Xaa Asn Thr Asn Ala Tyr Ala Pro Phe Glu
545 550 555 560
Phe Lys Tyr Pro Gly Tyr Lys Leu His Ser Val Ser Ala Tyr Gly Leu
565 570 575
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Ser Lys Ala Pro Asp Xaa Ala Asp Ser Val Met Phe Gly Phe Arg Pro
580 585 590
Val Leu Leu Glu Asn Glu Ala Asn Gln Leu Leu Thr Asp Thr Ala Leu
595 600 605
Gln Ile Pro Ala Glu Ile Gly Ile Thr Asp Val Val Pro Ala Phe Gly
610 615 620
Arg Thr Glu Glu Pro Ile Asn Gly Gln Asp Ala Ile Xaa Ile Trp Glu
625 630 635 640
Ser Phe Thr Ser Gly Phe Gly Phe Thr Tyr Thr Val Asp Ser Pro Gln
645 650 655
Lys Gln Lys Tyr Lys Ile Ile Tyr Arg Ile Ala Asn Asn Leu Ser Ala
660 665 670
Ser Thr Val Ser Leu Thr Tyr Asn Asn Gln Thr Phe Phe Thr Asp Ile
675 680 685
Leu Asn Thr Ser Leu Asp Pro Asn Gly Val Arg Gly Asn Tyr Gly Ser
690 695 700
Tyr Thr Leu Val Glu Gly Pro Ile Ile Glu Phe Ser Gln Gly Thr Asn
705 710 715 720
Ile Phe Lys Leu Xaa Ser Gln Lys Gly Glu Phe Ala Ile Asp Ser Ile
725 730 735
Ile Phe Ser Pro Val Xaa
740
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 492 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /note= "Xaa= isoleucine or
methionine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 139
(D) OTHER INFORMATION: /note= "Xaa= lysine or arginine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 194
(D) OTHER INFORMATION: /note= "Xaa= serine or histidine"
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(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 195
(D) OTHER INFORMATION: /note= "Xaa= glycine or serine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 196
(D) OTHER INFORMATION: /note= "Xaa= glycine or proline"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 216
(D) OTHER INFORMATION: /note= "Xaa= glutamine or arginine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 231
(D) OTHER INFORMATION: /note= "Xaa= tyrosine or
tryptophan"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 257
(D) OTHER INFORMATION: /note= "Xaa= alanine or leucine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 260
(D) OTHER INFORMATION: /note= "Xaa= glycine or histidine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 268
(D) OTHER INFORMATION: /note= "xaa= alanine or valine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 301
(D) OTHER INFORMATION: /note= "Xaa= alanine or proline"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 332
(D) OTHER INFORMATION: /note= "Xaa= alanine or threonine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 387
(D) OTHER INFORMATION: /note= "Xaa= arginine or
isoleucine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 475
(D) OTHER INFORMATION: /note= "Xaa= glycine or arginine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 492
CA 02410153 2004-04-01
(D) OTHER INFORMATION: /note= "Xaa= valine or serine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Xaa Ala Glu Pro Pro Ser Thr Gly Val Ile Thr Gln Phe Arg Ile Leu
1 5 10 15
Asn Asp Asn Phe Ile Lys Tyr Ile Ala Lys Leu Gln Phe Ser Thr Asn
20 25 30
Gln Ser Asp Leu Gln Tyr Pro Val Leu Thr Leu Pro Leu Arg Ala Gln
40 45
Ala Cys Val Met His Leu Met Leu Leu Lys Asp Ala Thr Thr Ser Val
50 55 60
Trp Gly Gln Gln Ile Asp Ser Gln Gln Leu Asn Gly Tyr Lys Ala Glu
65 70 75 80
Leu Ile Arg Leu Ile Lys Val Tyr Thr Asn Asp Val Asn Thr Thr Tyr
85 90 95
Asn Gln Gly Leu Glu Leu Glu Lys Ala Lys Pro Leu Asn Tyr Ser Asp
100 105 110
Pro Glu Glu Tyr Leu Gln Ala Gly Arg Pro Asp Ile Ser Val Leu Arg
115 120 125
Ser Asn Phe Lys Glu Val Met Lys Trp Asn Xaa Val Ala Lys Tyr Lys
130 135 140
Arg Gly Met Ala Met Ser Ala Leu Ser Leu Ala Ala Leu Phe Pro Thr
145 150 155 160
Phe Gly Pro Asn Tyr Pro Lys Gln Ala Leu Lys Val Val Gln Ser Arg
165 170 175
Gln Ile Phe Ala Pro Val Ile Gly Ile Pro Gly Gly Ile Thr Ser Gln
180 185 190
Asp Xaa Xaa Xaa Thr Phe Gly Ser Met Arg Phe Asp Val Lys Thr Tyr
195 200 205
Asp Gln Ile Asp Ala Leu Arg Xaa Leu Met Glu Leu Tyr Ile Gln Pro
210 215 220
Leu Lys Ser Ala Tyr Phe Xaa Ile Tyr Glu Ser Asp Trp Lys Val Arg
225 230 235 240
Ala Thr Tyr Val Asn Asp Tyr Ile Gly Lys Arg Gly Ser Asn Thr Gly
245 250 255
Xaa Ala Trp Xaa Met Trp Ser Ser Asp Pro Ser Xaa Ile Tyr Thr Ser
260 265 270
Ala Leu Gly Ala Ala Gly Tyr Ala Pro Asn Val Val Gly Val Arg Tyr
275 280 285
Ser His Gly Gly Ser Tyr Thr Lys Gly Met Ala Pro Xaa Asn Thr Asn
290 295 300
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Ala Tyr Ala Pro Phe Glu Phe Lys Tyr Pro Gly Tyr Lys Leu His Ser
305 310 315 320
Val Ser Ala Tyr Gly Leu Ser Lys Ala Pro Asp Xaa Ala Asp Ser Val
325 330 335
Met Phe Gly Phe Arg Pro Val Leu Leu Glu Asn Glu Ala Asn Gln Leu
340 345 350
Leu Thr Asp Thr Ala Leu Gln Ile Pro Ala Glu Ile Gly Ile Thr Asp
355 360 365
Val Val Pro Ala Phe Gly Arg Thr Glu Glu Pro Ile Asn Gly Gln Asp
370 375 380
Ala Ile Xaa Ile Trp Glu Ser Phe Thr Ser Gly Phe Gly Phe Thr Tyr
385 390 395 400
Thr Val Asp Ser Pro Gln Lys Gln Lys Tyr Lys Ile Ile Tyr Arg Ile
405 410 415
Ala Asn Asn Leu Ser Ala Ser Thr Val Ser Leu Thr Tyr Asn Asn Gln
420 425 430
Thr Phe Phe Thr Asp Ile Leu Asn Thr Ser Leu Asp Pro Asn Gly Val
435 440 445
Arg Gly Asn Tyr Gly Ser Tyr Thr Leu Val Glu Gly Pro Ile Ile Glu
450 455 460
Phe Ser Gln Gly Thr Asn Ile Phe Lys Leu Xaa Ser Gln Lys Gly Glu
465 470 475 480
Phe Ala Ile Asp Ser Ile Ile Phe Ser Pro Val Xaa
485 490
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3313 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
AAGAGAATAT TCCTGTAGAT ATTATATTTA AATATAGTCT CACTCCTCTT GCTTATCATA 60
CCGTTGATAC CAATCATGTA AAACTCAAAC ATATTGGATT AGTCCCTTTT TCTTCTTCCG 120
ATCCTAATCT ATACAGCATA CAAGGTGAAT TTCAATTTTT TTATGAATAA ACAATACTTA 180
TGAAAAAACT ATTTATAAGT ATATTAAAGG ACAACAAAGT GAGCATAATG ATGGTTTTGA 240
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TGGGAAAGAA TAATAGGCTT TAGTCAATAG TGGTTCAGTT AATTATTGAT ATATTTTGAT 300
ATTTATAATA CAAAAATTTC TCAAAAATTC TCCTTGCTTA TGTCCATTTA TACCCAAAAA 360
AGCGAGGACA ATGTATATAT TTCTCTATCT ATCATAGAGT AAATATAGAC TGTATACATT 420
TTTAGTCTTA TCTTTGAGTT TTTATATATT TTAAAGTTTG TTTGATAAAT TTTCAGGAAA 480
AAAAGATCTC AACGACTTTT GTATGTCGGT TGTTTACTAT GTGAAAGGTG GAGATATTGT 540
GGACCCATTT TCTAATTATT CTGAACAAAA ATACCCAGAT TCAAATAATA ACCAAGAACT 600
AATTACAGAA TCCTCTTCAT TTTATTCGGA TACTACTAAT GAAAATATGA AAACTTACCA 660
TCCAATTGAA CAAGATATTC TCAAATTTGC AAATAAAGAA TTTCCCGATA ATTATTATCA 720
ACATTCCGAT GTTTCTAATT CATATCAAAA TATGAAAACA GAAATCGTAA ATACAGATTT 780
ACCCTATAAT ACAAATAATA TAAATAGTAT GCGAAATACT CTATGCAGAG ATTTACCTCC 840
CGAGACTAAC ATGAGCATTT ATGATAATTT ACGATCTACT GTTACTGTTC CTTCATTTTC 900
TAATCAATTT GATCCTATAA AATTTCTTCA CGATATTGAA ATTGCTATAG AAACTGGATC 960
ATTTTCTGCA TTAACGCAAT CTAACATGAA TCAAGGTGGT ACTGATATTG CTCCAATGTT 1020
AATCTCTACA TTTTTTAAAG TTGCAGGTAG TTTACTTCCA TTTCCTCTAT CATCATTAGG 1080
TGCTTTGGCT TCCTTTTATG TTACAGATTC ACAAACAGGC GCTATGGCAA ATTTATGGAG 1140
ACAAATGGTA GATTATGTTG AAAAAAGAAT TGATTCTAAA ATATTAGATT ATCATAATTT 1200
TATTATGGGA GCAGAACTCG CAGCATTAAA TGCAAGTTTA AAAGAATACG CACGAGTAGT 1260
TAAAATTTTT GAAAATGATA TGAACAGAAT AGCTGAACCA CCTTCAACTG GAGTTATCAC 1320
TCAATTCAGA ATTCTTAATG ATAATTTCAT TAAATATATT GCAAAATTAC AATTCTCAAC 1380
AAATCAATCA GATTTACAAT ATCCTGTCCT AACTTTACCA TTACGTGCAC AAGCATGTGT 1440
AATGCATTTA ATGTTATTAA AAGATGCAAC GACTTCTGTG TGGGGACAAC AAATAGACTC 1500
GCAACAATTA AATGGGTATA AAGCAGAATT AATACGTTTA ATAAAAGTAT ATACTAATGA 1560
TGTAAACACA ACGTATAATC AAGGGCTAGA GCTAGAAAAA GCTAAACCAC TAAATTATTC 1620
TGATCCTGAA GAATATTTAC AAGCAGGACG TCCAGATATA TCTGTATTAC GCAGTAACTT 1680
TAAAGAGGTT ATGAAGTGGA ATAAAGTAGC GAAATATAAA CGTGGAATGG CTATGAGTGC 1740
TTTATCATTA GCTGCATTAT TTCCAACTTT CGGACCAAAT TATCCAAAAC AAGCATTAAA 1800
AGTTGTGCAA TCTAGACAAA TTTTTGCACC TGTAATTGGA ATACCAGGCG GTATAACAAG 1860
TCAAGATAGT GGTCCCACTT TTGGTAGTAT GAGATTTGAT GTAAAAACTT ATGATCAAAT 1920
TGATGCGTTA CGACAACTAA TGGAATTATA TATTCAACCT TTAAAATCTG CTTACTTTTG 1980
GATATATGAA TCGGATTGGA AAGTTCGTGC AACTTATGTC AATGATTATA TTGGTAAAAG 2040
CA 02410153 2004-04-01
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AGGGTCAAAT ACAGGTGCTG CTTGGCACAT GTGGTCAAGT GATCCTTCAG CCATATACAC 2100
TTCTGCACTA GGAGCAGCAG GATACGCTCC TAACGTTGTT GGTGTAAGAT ATTCACATGG 2160
GGGTAGTTAC ACAAAAGGTA TGGCACCCGC AAATACTAAT GCGTATGCTC CATTTGAATT 2220
TAAATATCCT GGTTATAAAC TACACAGTGT TAGTGCTTAT GGATTAAGTA AAGCACCTGA 2280
TGCAGCTGAT TCTGTTATGT TTGGATTTAG ACCTGTATTG TTAGAAAATG AAGCAAATCA 2340
ATTATTAACA GATACAGCAT TGCAAATTCC AGCAGAAATA GGAATAACAG ATGTCGTACC 2400
TGCATTTGGT AGAACAGAAG AACCTATTAA TGGTCAAGAT GCAATAAGAA TATGGGAAAG 2460
TTTTACAAGT GGATTTGGCT TTACTTATAC TGTTGATTCT CCACAAAAAC AAAAATATAA 2520
AATCATTTAT AGAATTGCAA ATAACTTAAG CGCTTCTACA GTTTCTTTAA CCTATAATAA 2580
TCAAACATTT TTCACTGATA TTTTAAATAC TTCATTAGAT CCAAATGGAG TAAGAGGAAA 2640
TTATGGTTCT TATACACTTG TAGAAGGTCC TATTATTGAA TTTTCTCAAG GAACTAATAT 2700
CTTTAAACTA GGATCACAAA AAGGAGAATT CGCTATAGAT TCCATTATTT TTAGTCCTGT 2760
TGTTTAATAG TGTAGTACCA TTAGACCCAG ACCCATGGTT TCCAGTCCAG AATATTCCCC 2820
AGATTTCATA GTATGCTTCG ATCCCGCATG TTTTATGTAC AAACACATCC TTTTTAGATA 2880
GCATTCCAAT TATAGGGATG CTCTTTTTTT GATIfTCTGGC CTATCCTTCT CATTTCATAG 2940
ATTTTTAATT AGTACCCTTT ACAAAAAGTA AACCCACCAT CTTCGAACAA ATCTTTGATT 3000
TCTATTTTTA AGAATAATCA ATCTGTTGAA CAATTTATAA TTCTTTTGAA GAGAATTTCA 3060
TTTTATTTGT TCGCTTAAGT TGATAGGCAT GTGGTTCTAC CCCTAATAAG TGTCACAGAA 3120
CACTAATTCT AAGACATTTA TCGTAAAAAA ATAGTAAATT CATACAATAC AGTTAAACTT 3180
TCCTCAGTAG CTCACGTTTT TCGATTTCGG GTGTTTTTAC TCATTTCCCC CTTTGTTTTT 3240
AGGAGAGAGT GCTGGCTGGG GGTTTGGGGG CTAGCCCCCA AGAACTTAAC GTAACTGAAT 3300
ATGGAATAAG CTT 3313
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2172 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
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GTGGACCCGT TTTCTAATTA TTCTGAACAA AAATACCCAG ATTCAAATAA TAACCAAGAA 60
CTAATTACAA AATCCTCTTC ATTTTATTCG GATACTACTA ATGAAAATGC AAAAAATTAC 120
CACCCAATTG AACAAGATAT TCTCAAATTT ACAAATCAAG AATTTTCCGA TAATCATTAT 180
CAACATTCCG ATGTTTCAAA TGATATAAAT AGTATGCGAA ATACTCTATG CAAAGATTTA 240
CCTCCTGAGA CTAACATGAG CATTTATGAT AATTTACGAT CTACTGTTAC TGTTCCTTCA 300
TTTTCTAATC AATTTGATCC TATAAAATTT CTTCACGATA TTGAAATTGC TATACAAACT 360
GGATCATTTT CTGCATTAAC GCAATCTAAC ATGAATCAAG GTGGTACTGA TATTAATCCA 420
ATGTTAATCT CTACATTTTT TAAAGTTGCA AGTAGTTTAC TTCCATTTCC TCTATCATCA 480
TTAGGTGCTT TAGCTTCCTT TTATGTTACA GATTCACAAA CAGGCGCTAT GGCAAATTTA 540
TGGAGACAAA TGGTAGATTA TGTTGAAAAA AGAATTGATT CTAAAATATT AGATTATCAT 600
AATTTTATTA TGGGAGCAGA ACTCGCAGCA TTAAATGCAA GTTTAAAAGA ATACGCACGA 660
GTAGTTAAAA TTTTTGAAAA TGATATGAAC AGAATGGCTG AACCACCTTC AACTGGAGTT 720
ATCACTCAAT TCAGAATTCT TAATGATAAT TTCATTAAAT ACATTGCAAA ATTACAATTC 780
TCAACAAATC AATCAGATTT ACAATATCCT GTCCTAACTT TACCATTACG TGCACAAGCA 840
TGTGTAATGC ATTTAATGTT ATTAAAAGAT GCAACGACTT CTGTGTGGGG ACAACAAATA 900
GACTCGCAAC AATTAAATGG GTATAAAGCA GAATTAATAC GTTTAATAAA AGTATATACT 960
AATGATGTAA ACACAACGTA TAATCAAGGG CTAGAGCTAG AAAAAGCTAA ACCACTAAAT 1020
TATTCTGATC CTGAAGAATA TTTACAAGCA GGGCGTCCAG ATATATCTGT ATTACGCAGT 1080
AACTTTAAAG AGGTTATGAA GTGGAATAGA GTAGCGAAAT ATAAACGTGG AATGGCTATG 1140
AGTGCTTTAT CATTAGCTGC ATTATTTCCA ACTTTCGGAC CAAATTATCC AAAACAAGCA 1200
TTAAAAGTTG TGGAATCTAG ACAAATTTTT GCACCTGTAA TTGGAATACC AGGCGGTATA 1260
ACAAGTCAAG ATCATTCTGG CACTTTTGGT AGTATGAGAT TTGATGTAAA AACTTATGAT 1320
CAAATTGATG CGTTACGACG ACTAATGGAA TTATATATTC AACCTTTAAA ATCTGCCTAC 1380
TTCTATATAT ATGAATCGGA TTGGAAAGTT CGTGCAACTT ATGTCAATGA CTATATTGGT 1440
AAAAGAGGGT CTAATACAGG TCTTGCCTGG GGAATGTGGT CAAGTGATCC TTCAGTCATA 1500
TACACTTCTG CACTAGGAGC AGCAGGATAC GCTCCTAACG TTGTTGGTGT AAGATATTCA 1560
CATGGGGGTA GTTACACAAA AGGTATGGCA CCCCCAAATA CTAATGCGTA TGCTCCATTT 1620
GAATTTAAAT ATCCTGGTTA TAAACTACAC AGTGTTAGTG CTTATGGATT AAGTAAAGCA 1680
CCTGATACAG CTGATTCTGT TATGTTTGGA TTTAGACCTG TATTGTTAGA AAATGAAGCA 1740
AATCAATTAT TAACAGATAC AGCATTGCAA ATTCCAGCAG AAATAGGAAT AACAGATGTC 1800
GTACCTGCAT TTGGTAGAAC AGAAGAACCT ATTAATGGTC AAGATGCAAT AATAATATGG 1860
CA 02410153 2004-04-01
GAAAGTTTTA CAAGTGGATT TGGCTTTACT TATACTGTTG ATTCTCCACA AAAACAAAAA 1920
TATAAAATCA TTTATAGAAT TGCAAATAAC TTAAGCGCTT CTACAGTTTC TTTAACCTAT 1980
AATAATCAAA CATTTTTCAC TGATATTTTA AATACTTCAT TAGATCCAAA TGGAGTAAGA 2040
GGAAATTATG GTTCTTATAC ACTTGTAGAA GGTCCTATTA TTGAATTTTC TCAAGGAACT 2100
AATATCTTTA AACTAAGATC ACAAAAAGGA GAATTCGCTA TAGATTCCAT TATTTTTAGT 2160
CCTGTTTCAT AA 2172
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 723 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Met Asp Pro Phe Ser Asn Tyr Ser Glu Gln Lys Tyr Pro Asp Ser Asn
1 5 10 15
Asn Asn Gln Glu Leu Ile Thr Lys Ser Ser Ser Phe Tyr Ser Asp Thr
20 25 30
Thr Asn Glu Asn Ala Lys Asn Tyr His Pro Ile Glu Gln Asp Ile Leu
40 45
Lys Phe Thr Asn Gln Glu Phe Ser Asp Asn His Tyr Gln His Ser Asp
50 55 60
Val Ser Asn Asp Ile Asn Ser Met Arg Asn Thr Leu Cys Lys Asp Leu
65 70 75 80
Pro Pro Glu Thr Asn Met Ser Ile Tyr Asp Asn Leu Arg Ser Thr Val
85 90 95
Thr Val Pro Ser Phe Ser Asn Gln Phe Asp Pro Ile Lys Phe Leu His
100 105 110
Asp Ile Glu Ile Ala Ile Gln Thr Gly Ser Phe Ser Ala Leu Thr Gln
115 120 125
Ser Asn Met Asn Gin Gly Gly Thr Asp Ile Asn Pro Met Leu Ile Ser
130 135 140
Thr Phe Phe Lys Val Ala Ser Ser Leu Leu Pro Phe Pro Leu Ser Ser
145 150 155 160
Leu Gly Ala Leu Ala Ser Phe Tyr Val Thr Asp Ser Gln Thr Gly Ala
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165 170 175
Met Ala Asn Leu Trp Arg Gln Met Val Asp Tyr Val Glu Lys Arg Ile
180 185 190
Asp Ser Lys Ile Leu Asp Tyr His Asn Phe Ile Met Gly Ala Glu Leu
195 200 205
Ala Ala Leu Asn Ala Ser Leu Lys Glu Tyr Ala Arg Val Val Lys Ile
210 215 220
Phe Glu Asn Asp Met Asn Arg Met Ala Glu Pro Pro Ser Thr Gly Val
225 230 235 240
Ile Thr Gln Phe Arg Ile Leu Asn Asp Asn Phe Ile Lys Tyr Ile Ala
245 250 255
Lys Leu Gln Phe Ser Thr Asn Gln Ser Asp Leu Gln Tyr Pro Val Leu
260 265 270
Thr Leu Pro Leu Arg Ala Gln Ala Cys Val Met His Leu Met Leu Leu
275 280 285
Lys Asp Ala Thr Thr Ser Val Trp Gly Gln Gln Ile Asp Ser Gln Gln
290 295 300
Leu Asn Gly Tyr Lys Ala Glu Leu Ile Arg Leu Ile Lys Val Tyr Thr
305 310 315 320
Asn Asp Val Asn Thr Thr Tyr Asn Gln Gly Leu Glu Leu Glu Lys Ala
325 330 335
Lys Pro Leu Asn Tyr Ser Asp Pro Glu Glu Tyr Leu Gln Ala Gly Arg
340 345 350
Pro Asp Ile Ser Val Leu Arg Ser Asn Phe Lys Glu Val Met Lys Trp
355 360 365
Asn Arg Val Ala Lys Tyr Lys Arg Gly Met Ala Met Ser Ala Leu Ser
370 375 380
Leu Ala Ala Leu Phe Pro Thr Phe Gly Pro Asn Tyr Pro Lys Gln Ala
385 390 395 400
Leu Lys Val Val Gln Ser Arg Gln Ile Phe Ala Pro Val Ile Gly Ile
405 410 415
Pro Gly Gly Ile Thr Ser Gln Asp His Ser Gly Thr Phe Gly Ser Met
420 425 430
Arg Phe Asp Val Lys Thr Tyr Asp Gln Ile Asp Ala Leu Arg Arg Leu
435 440 445
Met Glu Leu Tyr Ile Gln Pro Leu Lys Ser Ala Tyr Phe Tyr Ile Tyr
450 455 460
Glu Ser Asp Trp Lys Val Arg Ala Thr Tyr Val Asn Asp Tyr Ile Gly
465 470 475 480
Lys Arg Gly Ser Asn Thr Gly Leu Ala Trp Gly Met Trp Ser Ser Asp
485 490 495
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Pro Ser Val Ile Tyr Thr Ser Ala Leu Gly Ala Ala Gly Tyr Ala Pro
500 505 510
Asn Val Val Gly Val Arg Tyr Ser His Gly Gly Ser Tyr Thr Lys Gly
515 520 525
Met Ala Pro Pro Asn Thr Asn Ala Tyr Ala Pro Phe Glu Phe Lys Tyr
530 535 540
Pro Gly Tyr Lys Leu His Ser Val Ser Ala Tyr Gly Leu Ser Lys Ala
545 550 555 560
Pro Asp Thr Ala Asp Ser Val Met Phe Gly Phe Arg Pro Val Leu Leu
565 570 575
Glu Asn Glu Ala Asn Gln Leu Leu Thr Asp Thr Ala Leu Gln Ile Pro
580 585 590
Ala Glu Ile Gly Ile Thr Asp Val Val Pro Ala Phe Gly Arg Thr Glu
595 600 605
Glu Pro Ile Asn Gly Gln Asp Ala Ile Ile Ile Trp Glu Ser Phe Thr
610 615 620
Ser Gly Phe Gly Phe Thr Tyr Thr Val Asp Ser Pro Gln Lys Gln Lys
625 630 635 640
Tyr Lys Ile Ile Tyr Arg Ile Ala Asn Asn Leu Ser Ala Ser Thr Val
645 650 655
Ser Leu Thr Tyr Asn Asn Gln Thr Phe Phe Thr Asp Ile Leu Asn Thr
660 665 670
Ser Leu Asp Pro Asn Gly Val Arg Gly Asn Tyr Gly Ser Tyr Thr Leu
675 680 685
Val Glu Gly Pro Ile Ile Glu Phe Ser Gln Gly Thr Asn Ile Phe Lys
690 695 700
Leu Arg Ser Gln Lys Gly Glu Phe Ala Ile Asp Ser Ile Ile Phe Ser
705 710 715 720
Pro Val Ser
