BACILLUS THURINGIENSIS CRYIF AND CRYIX GENES AND PROTEINS TOXIC TO LEPIDOPTERAN INSECTS

GENES ET PROTEINES DE TYPE CRYIF ET CRYIX DU BACILLUS THURINGIENSIS TOXIQUE POUR LES LEPIDOPTERES

English Abstract

Two purified and isolated cryI-type genes
were obtained from a novel B.t. strain. One gene,
designated cryIF, has a nucleotide base sequence
coding for the amino acid sequence illustrated in
Figure 1. The 134 kDa crystal protein, designated
CryIF, produced by this gene is toxic to European
corn borer larvae and other lepidopteran insects.
The second gene, designated cryIX, produces a
crystal protein of about 81 kDa, designated CryIX,
that is also toxic to lepidopteran insects.

Claims

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The embodiments of the invention in which an
exclusive property or privilege is claimed are as follows:
1. A purified and isolated cryIF gene

characterized by a nucleotide base sequence as illustrated
in Figure 1 (SEQ ID NO:1).

2. A purified and isolated cryIF gene
according to claim 1 further characterized in that the
gene has a coding region extending from nucleotide bases
478 to 3999 in the nucleotide base sequence illustrated in
Figure 1 (SEQ ID N0:1).

3. A biologically pure culture of a bacterium
characterized in that the bacterium has been transformed
to express a lepidopteran-toxic protein from the gene of
claim 1 or claim 2 contained within a recombinant plasmid.

4. The bacterium of claim 3 further
characterized in that the bacterium is E. coli.

5. The E. coli bacterium of claim 4 further
characterized in that the bacterium is deposited with the
NRRL with accession number NRRL B-18634.

6. The bacterium of claim 3 further
characterized in that the bacterium is Bacillus
thuringiensis.

7. The Bacillus thuringiensis bacterium of
claim 6 further characterized in that the bacterium is
deposited with the NRRL with accession number NRRL B-
18635.




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8. An insecticide composition characterized in
that the composition contains the bacterium of claim 3
expressing said lepidopteran-toxic protein and an
agriculturally acceptable carrier.

9. A genetically engineered polynucleotide
encoding a lepidopteran-toxic protein that is a C-terminal
truncated derivative of the protein coded by the cryIF
gene of claim 1, wherein said C-terminal truncated
derivative comprises amino acids 1 to 618 as set forth in
Figure 1 (SEQ ID NO: 2).

10. A biologically pure culture of a bacterium
characterized in that the bacterium has been transformed
to express said lepidopteran-toxic protein from the

polynucleotide of claim 9.

11. An insecticide composition comprising the
bacterium of claim 10 expressing said lepidopteran-toxic
protein and an agriculturally-acceptable carrier.

12. A lepidopteran-toxic protein encoded by the
polynucleotide of claim 9.

13. A plant cell that has been transformed to
express a lepidopteran-toxic protein comprising amino
acids 1 to 618 as set forth in Figure 1 (SEQ ID NO: 2)
from a genetically engineered polynucleotide.

14. A plant cell that has been transformed to
express a lepidopteran-toxic protein from the genetically
engineered polynucleotide of claim 9.

Description

CA 02080684 2000-10-11
-1-

BACILLUS THURINGIENSIS cryIF AND cryIX GENES AND
PROTEINS TOXIC TO LEPIDOPTERAN INSECTS


Field of the Invention

The present invention relates to two genes
isolated from Bacillus thuringiensis (hereinafter "B.t.")
encoding insecticidal crystal proteins designated CryIF and
CryIX, respectively, as well as insecticidal compositions
containing the proteins and plants transformed with the
genes. The insecticidal compositions and transformed plants
are toxic to insects of the order Lepidoptera.


Background of the Invention

B.t. is a gram-positive soil bacterium that
produces crystal proteins during sporulation which are
specifically toxic to certain orders and species of insects.
Many different strains of B.t. have been shown to produce
insecticidal crystal proteins. Compositions including B.t.
strains which produce insecticidal proteins have been
commercially available and used as environmentally


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acceptable insecticides because they are quite
toxic to the specific target insect, but are
harmless to plants and other non-targeted
organisms.
A number of genes er-coding crystal
proteins have been cloned froin several strains of
B.t. A good overview is set forth in H. H;fte et
al., Microbiol'. Rev., 53, pp. 242-255 (1989),
hereinafter "Hofte and Whiteley (1989)." While
this reference is not prior art with respect to the
present invention, it provides a good overview of
the genes and proteins obtained from B.t. and their
uses, adopts a nomenclature and classification
scheme for B.t. genes and proteins, and has an
extensive bibliography.
The B.t. crystal protein is active in the
insect only after ingestion. After ingestion by an
insect, the alkaline pH and proteolytic enzymes in
the mid-gut solubilize the crystal allowing the
release of the toxic components. These toxic
components disrupt the mid-gut cells causing the
insect to cease feeding and, eventually, to die.
In fact, B.t. has proven to be an effective and
environmentally safe insecticide in dealing with
various insect pests.
As noted by H fte and Whiteley (1989),
the majority of insecticidal B.t. strains are
active against insects of the order T.apidoptera,
i.e., caterpillar insects. These B.t. strains

characteristically contain cryI-type genes that
make Cryl crystal proteins. Other B.t. strains
produce different classes of crystal proteins,
e.g.,, Crylv protein, active against insects of the


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3 8

order Diptera, i.e., flies and mosquitoes, or CryII
= protein active against both lepidopteran and
dipteran insects. in recent years, a few B.t.
strains have been reported as producing a new class
of crystal protein, CryIII protein, that is
insecticidal to insects of the order Coleoptera,
i.e., beetles.
Lereclus et al., in Chapter 13 of
Regulation of Procaryotic Development, I. Smith et
al. (eds.), American Society for Microbiology,
Washington, D.C., pp. 255-276 (1989), review the
role, structure and molecular organization of
crystal protein genes. A summary of toxin genes
identified in B.t. is provided in Table 2 (p. 260)
and amino acid sequence comparisons of the various
crystal proteins reported in the literature are
diagrammed in Figures 1 and 2. This reference is
not prior art with respect to the present
invention.
Schnepf et al., J. Biol. Chem., 260,
pp. 6264-6272 (1985), report the complete
nucleotide sequence for a toxin gene from B.t.
kurstaki HD-1 (Figure 2, pp. 6266-6267); this gene
was subsequently classified as the crStlAa) gene by -
H fte and Whiteley (1989). The published open
reading frame extends 1176 amino acids and encodes
a protein with a calculated M r of 133,500 daltons
(Da).
= Wabiko et al., DNA, 5, pp. 305-314
(1986),*describe the DNA sequence of an
= insecticidal toxin gene from B.t. subsp. berliner
1715, subsequently classified as crylA(b) by H6fte
and Whiteley (1989). The molecular mass of the


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protean encoded is 130,615 Da and sequential
deletions indicate that the NH2-terminal 612 amino
acid polypeptide is toxic.
Hofte et al., Eur. J. Biochem., 161,
pp. 273-280 (1986), describe the cloning and
nucleotide sequencing of a crystal protein gene
from B.t. subsp. berliner 1715, subsequently
classified as cryl~,(b) by Hofte and Whiteley
(1989). The cloned gene produces a 130 kilodalton
(kDa) protein which coincides with the size of the
major protein observed in the strain. A
restriction map of the cloned toxin gene is
presented (p. 276, Figure 3A). Toxicity data for
the cloned gene is shown in Table 1(p. 275). The
complete nucleotide sequence for this gene is shown
in Figure 3B (p. 276). It has an open reading
frame of 3466 bases which would encode a
protein 1155 amino acids in length having a
molecular weight of 130,533 Da. Similarities of
this sequence to the previously reported sequences
for the cloned crystal genes from B.t. kurstaki
HD-1, B.t. kurstaki HD-73 and B.t. sotto are
discussed on p. 278 and summarized in Figures 6 and
7 (pp. 279 and 280, respectively). Data
identifying a minimal toxic fragment required for
insecticidal activity is also presented (pp. 277
and 278, Figure 4, Table 2).
Adang et al., Gene, 36, pp. 289-300
(1985), report the cloning and complete nucleotide
sequence of a crystal protein gene harbored on the
75 kb plasa-id of strain B.t subsp. kurstaki HD-73.
The restriction map (Figure 2, p. 292) identifies
this gene as cryIA(c) under the current


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classification system of Hofte and Whiteley (1989).
The complete sequence of the gene, spanning 3537
nucleotide base pairs (bp), coding for 1178 amino
acids and potentially encoding a protein of
Mr 133,330 Da, is shown in Figure 3 (p. 294).
Sequence comparisons of this gene and the published
N-terminal sequence for the B<.t. kurstaki HD-1
Dipel gene reveal only 41 base pair differences,
concentrated within the last 376 base pairs of the
HD-1 sequence (p. 293). The 5' regulatory
sequences are identical in both clones. A
schematic of this comparison is shown in Figure 4
(p. 295). Toxicity data against Manduca sexta for
the protein made by the full length HD-73 gene are
also presented (Table II, p. 296).
Brizzard et al., Nucleic Acids Res., 16
(6), pp. 2723-2724 (1988), describe the nucleotide
sequence of crystal protein gene cryA4
(subsequently classified as crylH by Hofte and
Whiteley (1989)) isolated from B.t. strain HD2.
This report makes a cursory statement
distinguishing the amino-terminal region of the
cryIB gene from those of the cryIA(a), cryTA(b) and
cryIA(c) genes. The sequence of this gene is
further differentiated from the three cryIA genes
by virtue of its TTG translational start codon.
Honee et al., Nucleic Acids Res., 16
(13), p. 6240 (1988), describe the complete DNA
sequence for the BTVI crystal protein gene isolated
from B.t. subsp. entomocidus 60.5 (termed cryIC by
H fte and Whiteley (1989)). Extensive homology to
the cryIA(a) crystal protein gene was evident
downstream from the proteolytic cleavage site.


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2 0 8 0 611
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Visser et al., Mol. Gen. Genet., 212,
pp. 219-224 (1988), report the isolation and analysis of five toxin genes
belonging to four

different gene families from B.t. entomocidus 60.5
(see Figure 2, p. 221). Two of these, BTIV and
BTVIII, are crylA(a)-type genes according to the
Hofte and Whiteley (1989) claassification scheme.
Two additional genes, BTVI and BTV, reside in
opposite orientations on the same recombinant
plasmid and are separated by approximately 3
kilobase (kb) of intervening DNA. BTVI is the
crylC gene according to the Hofte and Whiteley
(1989) classification scheme. The authors state
that the restriction map for BTV closely resembles
that identified for a gene isolated from B.t.
strain HD68 subsp. aizawai, now termed the cr ID
gene (p. 222). A fifth gene, BTVII, is also
identified and its restriction map differs
significantly from the other four genes described.
Toxicity data against S. exigua, S. littoralis, H.
virescens and P. brassicae are presented for each
of the isolates (see Table 1, p. 223). BTVI is
highly active against Spodoptera larvae. BTVII is
toxic to P. brasicae.- The BTV gene product was
inactive against all insects teste.d.
Sanchis et al., Mol. Microbiol., 2,
pp. 393-404 (1988), describe the isolation of
recombinant clones containing two novel B.t. toxin
genes from strains aizawai 7.29 (plasmids pHTA4 and
pHTA6) and entomocidus 601 (pHTE4 and pHTE6).
Toxin genes on pHTA6 and pHTE6 have been
subsequently classified as crYIC according to the
Hofte and bdhiteley (1989) classification scheme.


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Restriction map data (see Figure 5, p. 398)
indicate that the novel genes are in close
proximity to each other (3 kb apart). The
restriction maps and molecular organization for the
novel aizawai genes are identical to the
entomocidus genes, except foa: a 1.4 kb insert in
the upstream entomocidus gene. Toxicity data are
presented for Escherichia coli (E. coli) expression
constructs of these novel geries (see Table 2,
p. 400 and discussion on p. 401). The proteins
produced by these cryiC genes are highly toxic to
S. littoralis and P. brassicae. No significant
toxicity was demonstrated for the protein of the
novel gene on plasmid pHTA4.
Sanchis et al., Mol. Microbiol., 3,
pp. 229-238 (1989), report the nucleotide sequence
for the N-terminal coding region (2470 nucleotides)
and 5' flanking region of a gene from B.t. subsp.
aizawai 7.29 now classified as the cryIC gene under
the classification system of Hofte and Whiteley
(1989). The open reading frame encodes a
polypeptide 824 amino acids long with a calculated
molecular weight of 92,906 Da (see sequence,
Figure 1, p. 231). Comparative analysis of this
sequence to other known B.t. toxin genes indicates
that for the N-terminal DNA sequence (amino acids
1-281), the CryIC prot$in has only 58% identity to
other Pi proteins> The more homologous C-terminal
region (amino acids 619-824) has less than 10%
variability. The authors report the identification
of five N-terminal conserved domains present in
lepidopterais-active, dipteran-active and
coleopteran-active endotoxins.


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Sanchis et al., in European Patent
Application Publication No. 0 295 156, published
December 14, 1988, disclose the DNA and amino acid
sequences of a truncated gene, the crylC gene, and
encoded crystal protein isolated from B.t. subsp.
aizawai 7.29. The sequence revealed is the same as
that in Sanchis et al., Molecular Microbiol., 3,
pp. 229-238 (1989), described above.
Schnepf et al., in U.S. Patent 4,467,036,
issued August 21, 1984, disclose a hybrid
recombinant plasmid capable of replication in an E.
coli strain, and capable of expressing a
polypeptide with the immunological properties of
B.t. crystal protein, and being identifiable with a
PvuII C DNA fragment probe derived from a gene, now
known as crylA(a), of B.t. var. kurstaki strain
HD-i. The following B.t. subspecies and strains
are disclosed as sources of expressible
heterologous DNA: tolworthi, darmstadiensis,
sotto, thuringiensis, kurstaki, HD-290, HD-120,
HD-2, HD-244, HD-73, HD-l, HD-4, HD-8, F-6, F-5 and
F-9.
Generally, the sequences of B.t. genes
encoding delta-endotoxin proteins active against
different orders of insects are not well-conserved.
Rather, the sequences of genes responsible for a
given crystal phenotype and active against the same
insect order are significantly inore related. The
homology of delta-endotoxin amino acid sequences,
as well as similarities in insecticidal activity,
have been used to define an ordered classification
of genes encoding B.t. delta-endotoxin proteins
(Hofte and Whiteley (1989)). The genes encoding


WO 91/16434 PCF/bJS91/02560

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g

the 130-138 kDa, lepidopteran.-active delta-
endotoxin proteins comprise the largest of these
families, the cr I genes.
Within the cryl gene classification
described by Hofte and Whiteley (1989), a
subranking has been established based upon further
refinement of sequence relationship. Thus, the
crylA(a), crYIA(b) and cryIA c) gene sub-families
embrace the previously designated 4.5 kb, 5.3 kb
and 6.6 kb P1 genes, respectively. These genes
originally were differentiated according to the
size of a characteristic HindiIl fragment
associated with the presence of the gene. The
amino acid sequences of CryIA proteins are highly
related (greater than 80% amino acid identity),
with most of the sequence dissimilarity relegated
to a short internal variable region (Whiteley et
al., Ann. Rev. Microbiol., 40, pp. 549-576 (1986)).
It is believed that differences within this
variable region account for the different
insecticidal specificities exhibited by the
proteins encoded by the crylA(a), crylA(b) and
crylA(c) genes.
Recently, additional genes within the
cryl family have been discovered, such as the cryIB
gene found in B.t. subsp. thuringiensis (Brizzard
et al., (1988), supra), and the crylC and crylo
genes found in subsp. aizawai (Sanchis et al.,
(1988), supra, Visser et a1. (1988), M.ra, Hofte
and Whiteley (1989), European Patent Application
Publication No. 0 358 557, published March 14,
1990, of Plant Genetic Systems, N.V.), and the
crylE gene found in B.t. subsp. darmstadiensis


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2 f) C13 13 bi ;111
(EP 0 358 557 (1990), supra) and in B.t._ subsp.
kenyae (Visser et al., J. Bacteriol., 172,
pp. 6783-6788 (1990)). Other cryI-type genes are
disclosed in European Patent Application
Publication No. 0 367 474, published May 9, 1991,
of Mycogen Corporation, in European Patent
Application Publication No. 0 401 979, published
December 12, 1990 of Mycogen Corporation, and in
PCT International Publication No. WO 90/13651,
published November 15, 1990, of Imperial Chemical
Industries PLC. Comparisons of the sequences for
these cryz-type genes to the cryIA genes reveal
significant sequence dissimilarities, particularly
in the N-terminal protein domain.
The present invention is based on the
discovery of at least one additional subgroup of
crVl genes. The prototype of this subgroup, which
the inventors have designated cryIF, was also
isolated from a B.t. subsp. aizawai strain.
However, the sequence of the crylP gene and the
insecticidal specificity of the CryIF protein it
encodes, is distinctly different from the other
cryI genes and their encoded Cryl proteins. This
distinction is also true with respect to the crylC
gene and its encoded protein, even though a
spontaneously cured derivative of a B.t. strain
containing the cr
XIC gene was used in the isolation
of the crylF gene.
In addition, the present invention
includes the identification and isolation of a
second gene, designated cryz, which is located
downstream from the novel cr IF gene. Of the
cryI-type genes discussed above, the gryIX gene


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s 4Y Ci r"
)
appears most closely related to the B.t. toxin gene
disclosed in PCT International Publication
No. WO 90/13651, published November 15, 1990, of
Imperial Chemical Industries PLC.
Data are presented hereinafter concerning
the identification, cloning, sec[uencing and
expression of these novel cr IF and crylX toxin
genes, as well as the insecticidlal activities of
the CryIF and CryIX proteins against lepidopteran
larvae.

Summary of the Invention
One aspect of the present invention
relates to a purified and isolated lepidopteran-
active toxin gene having a nucleotide base sequence
coding for the amino acid sequence illustrated in
Figure 1 and hereinafter designated as the crylF
gene (SEQ ID NO:1). The cryIF gene (SEQ ID NO:1)
has a coding region, i.e., open reading frame,
extending from nucleotide bases 478 to 3999 shown
in Figure 1.
Another aspect of the present invention
relates to the insecticidal protein produced by the
cryIF gene (SEQ ID NO:1). The CryIF protein (SEQ
ID NO:2) has the amino acid sequence, as deduced
from the nucleotide sequence of the crylF gene from
bases 478 to 3999, that is shown in Figure 1. The
protein exhibits insecticidal activity against
insects of the order Lepidoptera, in particular,
Ostrinia nubilalis (European corn borer) and
Spodootera exigua (beet armyworm).
Another aspect of the present invention
relates to a purified and isolated insecticidal
toxin gene hereinafter designated as the cryIX


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~~~~~?~
gene, a portion (SEQ ID No:3) of whose nucleotide 20base sequence is shown in
Figure 2. The present

invention also relates to the insecticidal protein
produced by the cryIX gene and called the CryIX
protein, which protein has a molecular mass of
about 81 kDa. A portion (SEQ ID N0:4) of the amino
acid sequence for the 81 kDa CryIX protein, as
deduced from the truncated portion (SEQ ID No:3) of
the cryIX gene, is also shown in Figure 2. The 81
kDa CryIX protein exhibits insecticidal activity
against lepidopteran insects, such as Plutella
xylostella (diamondback moth) and Ostrinia
nubilalis (European corn borer).
Still another aspect of the present
invention relates to biologically pure cultures of
B.t. and E. coli bacteria deposited with the NRRL
having Accession Nos. NRRL B-18633, B-18635, and
B-18805 and being designated as B.t. strains EG6345
and EG1945, and E. coli strain EG1083,
respectively. B.t. strains EG6345 and EG1945 carry
the cryIF gene and produce the insecticidal CryIF
protein. E. coli strain EG1083 carries the crylX
gene and produces the CryIX protein. Biologically
pure cultures of other B.t. bacteria carrying the
crylF gene or of B.t. strains carrying the crylX
gene are also within the scope of this invention.
Yet another aspect of this invention
relates to insecticidal compositions containing, in
combination with an agriculturally acceptable
carrier, either the CryIF or CryIX protein or
fermentation cultures of a B.t. strain which has
produced the CryIF protein or the CryIX protein.


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The invention also includes a method of
controlling lepidopteran insects by applying to a
host plant for such insects an insecticidally
effective amount of the CryIF protein or the CryIX
protein or of a fermentation culture of a B.t.
strain that has made the CryIF protein or the CryIX
protein.
Still other aspects of the present
invention relate to recombinazzt plasmids containing
the cryIF gene and/or the crylX gene; biologically
pure cultures of a bacterium transformed with such
recombinant plasmids, the bacterium preferably
being B.t., such as the aforementioned B.t. strain
EG1945; as well as plants transformed with the
cryiF gene and/or the cryIX gene.

Brief Description of the Drawings
Figure 1 comprises Figures 1-A through
1-E and shows the partial nucleotide base sequence
of DNA from a 5.7 kb fragment that contains the
crylF gene inserted into plasmid pEG640. The DNA
sequence begins with the 51 Sau3A cloning site and
extends 4020 bp in length. The open reading frame
for the cryIF gene and the deduced amino acid
sequence of the CryIF protein are indicated. The
putative ribosome binding site (RBS) for the crylF
gene is indicated on Figure 1-A. Sites for the
restriction enzymes Sau3A, BamHI and YRnI are also
indicated.
Figure 2 comprises Figures 2-A and 2-B
and shows the partial nucleotide base sequence of
DNA from the 5.7 kb fragment inserted into plasmid
pEG640 that contains a portion of the cryIX gene.


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The DNA sequence begins with nucleotide base
position 4021 in Figure 2-A, which is immediately
adjacent to and downstream from position 4020 in
Figure 1-E, and extends to nucleotide base position
5649 in Figure 2-B, ending at the 31 Sau3A cloning
site. The open reading frame for the truncated
crylX gene and the deduced amino acid sequence of
the CryIX protein encoded by the cr-yIX gene
fragment are indicated. The putative ribosome
binding site (RBS) for the crylX gene is indicated.
Sites for the restriction enzymes KgnI and Sau3A
are indicated.
Figure 3 comprises a photocopy of a
portion of an ethidium bromide stained agarose
electrophoresis gel containing size fractionated
native plasmids of B.t. subsp. aizawai strains
EG6346 in the left lane and EG6345 in the right
lane. The number to the right of Figure 3
indicates the approximately 45 MDa plasmid of B.t.
strain EG6345 which is absent in the cured B.t.
strain EG6346.
Figure 4 comprises Figures 4-A, 4-B and
4-C, which are photocopies of autoradiograms made
by transferring total HindIII-digested DNA from
B.t. strains EG6346 (lane 1 of each autoradiogram),
EG6345 (lane 2 of each autoradiogram) and HD-1
(lane 3 of each autoradiogram) to nitrocellulose
filters, hybridizing the filters with radioactively
labeled probes and exposing the filter to X-ray
film. The DNA in the autoradiogram labeled
Figure 4-A follows hybridization of the DNA to a
32P-labeled 0.7 kb EcoRI probe from the cryIA(a)
gene of B.t. strain HD-1. The autoradiogram


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labeled Figure 4-B follows hybridization of the
DNA to a 32P-labeled intragenic 2.2 kb PvuII probe
from the crylA(a) gene of B.t. strain HD-1. The
autoradiogram labeled Figure 4-C follows
hybridization of the DNA to a 32P-labeled plasmid
pEG640 probe. The numbers to the left of Figure 4
indicate the sizes, in kb, of standard DNA
fragments of phage lambda.
Figure 5 shows a restriction map of
plasmid pEG640. The locations and orientations of
the cryIF gene and a gene designated the cryIX gene
are indicated by arrows. The solid black line
indicates the E. coli cloning vector pGEM"-3Z. The
following letters designate the indicated
restriction enzymes: B BamHI; B2 = BstEII;
C = Clal; E= EcoRI; H Hindill; K = KPnI,=
Pt1 = Pstl; Pv2 $ PvuII; S= SacI; X = XbaI.
Figure 6, based on the same scale as
Figure 5, shows a restriction map of plasmid pEG642
which was created by inserting plasmid pEG640 into
a HindIIl site on the Bacillus vector pEG434. The
abbreviations and other indicators referred to with
respect to Figure 5 apply with respect to Figure 6.
In addition, the crosshatched area of Figure 6
indicates vector pEG434 and the arrow labeled "tet"
indicates the direction of transcription of the
tetracycline resistance gene encoded on plasmid
vector pEG434.
Figure 7 is a photocopy of a Coomassie
stained SDS-polyacrylamide gel showing gradient
purified crystal protein from B.t. strain EG6345
(lane 1), B.t. strain EG6346 (lane 2) and
recombinant B.t. strain EG1945 harboring the crylF


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gene (lane 3). The unnumbered, extreme left lane
adjacent to lane 1 contains molecular weight
standards having the indicated sizes, in kDa.
Figure 8 comprises Figures 8-A, 8-B and
8-C. Figure 8-A is a photocopy of an ethidium
bromide stained agarose electrophoresis gel
containing size-fractionated plasmids of B.t.
strains HD-1 (lane 1), EG6345 (lane 2) and EG6346
(lane 3). Figure 8-B is a photocopy of an
autoradiogram made by transferring the plasmids
resolved by the gel shown in Figure 8-A to a
nitrocellulose filter, hybridizing the filter with
a 32P-labeled 2.2 kb PvuII intragenic fragment
obtained from the crylA(a) gene of HD-1, where
lanes 1 through 3 in Figure 8-B correspond to
lanes 1 through 3 of Figure 8-A. Figure 8-C is a
photocopy of an autoradiogram made by transferring
the plasmids resolved by the gel shown in
Figure 8-A to a nitrocellulose filter, hybridizing
the filter with a 32 P-labeled 0.4 kb PstI-SacI
intragenic N-terminal fragment obtained from the
crylF gene, where lanes I through 3 of Figure 8-C
correspond to lanes 1 through 3 of Figure 8-A. The
numbers to the left of Figure 8-A indicate the
sizes, in MDa, of various plasmids. The letter "L"
to the left of Figure 8-A indicates the linear
degeneration fragments from the breakdown of the
larger plasmids.
Figure 9 comprises a photocopy of an
autoradiogram made by transferring total DNA from
B.t. strain EG6346 digested with restriction
enzymes (as described below) to a nitrocellulose
filter, hybridizing the filter with a 32 P-labeled


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0.6 kb K.nI-BamHI restrictiori fragment containing a
portion of the cryrX gene, and exposing the filter
to X-ray film. The digestiori of total DNA from
B.t. strain EG6346 was carrie:d out with several
restriction enzymes, as follows: As~718 (an
isoschizomer of KpnI) in lane: 1, Clal in lane 2,
Sphl in lane 3, Asp718 (KpnI) + S.hI in lane 4,
Clal +SphI in lane 5, Sstl in lane 6, AsR718
(KpnI) + SstI in lane 7, Clal + SstI in lane 8.
The numbers to the left of Figure 9 indicate the
sizes, in kb, of standard DNA fragments of phage
lambda.
Figure 10 shows a circular restriction
map of the 7.2 kb B.t.-E. coli cloning vector
pEG854, originally described by Baum et al., ARpl.
Environ. Microbiol. 56, pp. 3420-3428 (1990). The
open box represents the pT219u segment of the
vector that contains an ampicillin resistance gene
and a replication origin functional in E. coli.
The shaded box, designated ori 43, contains a
2.8 kb replication origin region derived from a
native B.t. plasmid that is function in B.t.. The
solid black arrow corresponds to a chloramphenicol
acetyltransferase (cat) gene that confers
chloramphenicol resistance on B.t.' strains
transformed with pEG854 or its derivatives.
Cloning vector pEG854 contains a unique C1aI
restriction site within the multiple cloning site,
designated MCS in the Figure. Restriction sites
for Xbal, SfiI, and Notl restriction endonucleases
are also shown.


WO 91/16434 PCT/US91/02560

`cf 6
- 18 8 0 -

Figure 11 shows a circular restriction
map of the 11.8 kb recombinar-t plasmid pEG313
consisting of a 4.6 kb Clal restriction fragment
isolated from total DNA of B.t. strain EG6346
inserted in the Clal site of cloning vector pEG854
(see Figure 10). The Clal sites flanking the 4.6
kb crylX-encoding fragment are indicated in bold
type. An SstI restriction site, located downstream
from the cryIX gene, is contained within the 4.6 kb
Clal restriction fragment. The orientation and
approximate length of the crylX coding region is
indicated by the open boxed arrow. Other
annotations are as described for Figure 10.
Figure 12 shows a circular restriction
map of the 2.86 kb E. coli cloning vector pTZl9u,
used to obtain expression of the cryIX gene in
E. coli. A multiple cloning site region,
containing unique restriction sites for Accl and
Sstl (in bold type), is demarcated by unique
HindIII and EcoRI restriction sites within the
lacZ' gene. vector pTZ19u contains a beta-
lactamase gene (bla) that confers resistance to
ampicillin and also contains the replication region
from an fl filamentous phage (1) used for the
synthesis of single-stranded DNA. The lac promoter
(Plac in bold type) is positioned upstream from the
multiple cloning site region. Restriction sites
for Nae2, Scal and g
MI restriction endonucleases
are also shown.
Figure 13 shows a circular restriction
map of the 7.3 kb recombinant plasmid pEG314
consisting of a 4.4 kb Clal-SstI restriction
fragment derived from pEG313 (see Figure 11)


WO 91/16434 P('T/US91/02560

- 19 - `J~~0 'p) C~4
inserted into the AccI and SstI restriction sites
of vector pTZ19u (see Figure.12). The orientation
and approximate relative length of the cr IX coding
region is indicated by the open arrow. Other
annotations are as described for Figure 12.
Figure 14 is a photocopy of a Coomassie
stained 10% SDS-polyacrylamide gel showing crude
(in lane 1) and gradient purified (in lane 2) CryiX
crystal protein from E. coli strain EG1083. The
numbers to the left of Figure 14 indicate the
sizes, in kDa, of protein molecular weight (MW)
standards displayed in the leftmost (unnumbered)
lane.

Detailed Description of the Preferred Embodiments
The isolation and purification of the
cryIF gene (SEQ ID NO:1) and the lepidopteran-toxic
CryIF crystal protein (SEQ ID NO:2), and the
characterization of the native B.t. strain EG6345,
the cured B.t. strain EG6346 derived from B.t.
strain EG6345, and the recombinant B.t. strain
EG1945, both of which produce the CryIF protein,
are described in the Examples. The utility of
recombinant B.t. strain EG1945 and of the CryIF
crystal protein in insecticidal compositions and
methods is also illustrated in the Examples.
Similarly, the isolation and purification
of the cryIX gene and the characterization of its
lepidopteran-toxic CryIX crystal protein are also
illustrated in the Examples. The methods and
procedures described in the Examples for the cr IF


W091 / 16434 PC'T/L'S91 /02560
-20-
and its Cr IF
gene y protein are also generally
applicable to the cryIX gene and its insecticidal
CryIX protein.
The cr I-type gene of this invention, the
crylF gene (SEQ ID NO:1), has the nucleotide base
sequence shown in Figure 1. The coding region of
the crylF gene extends from nucleotide base
position 478 to position 3999 shown in Figure 1.
A comparison of the nucleotide base pairs
of the crylF gene coding region with the
corresponding coding region of the prior art cr I
genes indicates significant differences between the
new cryIF gene and the prior art cr I genes. The
crylF gene is only about 67% to about 78%
homologous (positionally identical) with the
cryIA(a), cryIA(b) and cryIA(c) genes and the crylB
and cryIC genes. There is even less homology with
the cr II, crylli and cryIV genes, described in
Hofte and Whiteley (1989). The homology is
discussed in more detail hereinafter.
The CryI-type protein of this invention,
the CryIF protein (SEQ ID NO:2) that is encoded by
the cryIF gene, has the amino acid sequence shown
in Figure 1. In this disclosure, references to the
CryIF "protein" (and to the CryIX "protein") are
synonymous with its description as a "crystal
protein," "protein toxin," "insecticidal protein,"
"delta endotoxin" or the like, unless the context
indicates otherwise.
30. The deduced size of the CryIF protein is
133,635 Da. The prior art Cryl-type proteins,
encoded by the respective cryI genes, have similar
deduced sizes. Despite the apparent size


WO 91/16434 PCT/U591/02960

- 21 - ~~U~~ ~rJC}
similarity, comparison of the amino acid sequence
of the CryIF protein with published sequences of
the other prior art CryI-type proteins shows
significant differences betweeri them and the CryIF
protein. The CryIF protein is only about 58% to
about 72% identical with the other prior art Cryl-
type proteins, even when considering the C-terminal
regions which are more related than the N-terminal
regions.
The cryIX gene of this invention contains
approximately 2100-2200 basepairs in its coding
region, of which approximately 1140 basepairs are
shown for the truncated upstream portion (SEQ ID
NO:3) of the cryIX gene in Figure 2. The crylX
gene of this invention is contained in isolated
form on a DNA fragment carried on a recombinant
plasmid, in E. coli strain EG1083 which has been
deposited in the NRRL under accession No. NRRL B-
18805. The CryIX protein of this invention,
produced by the cryIX gene, is about 81 kDa in size
and exhibits insecticidal activity against insects
of the order Lepidoptera. The amino acid sequence
(SEQ ID NO:4) -for a portion of the CryIX protein,
deduced from the truncated portion of the crylX
gene shown in Figure 2, is shown in Figure 2. The
380 amino acids of this initial portion (SEQ ID
NO:4) of the CryIX protein shown in Figure 2
represent approximately one-half of the CryIX
protein encoded by the cr IX gene.
The CryIF and CryIX proteins have been
shown to be insecticidal against insects of the
order Lepidoptera, as set forth in more detail in
Examples 6 and 11, respectively.


WO 91/16434 PCT/US91/02560
- 22 -

The present invention is intended to
cover mutants and recombinant or genetically
engineered derivatives of the: crylF gene and crylX
gene that yield lepidopteran-toxic proteins with
essentially the same properties as the respective
CryIF and CryIX proteins.
The cryIF gene and cryIX gene are also
useful as DNA hybridization probes, for discovering
similar or closely related cryi-type genes in other
B.t. strains. The crylF or cryzX gene, or portions
or derivatives thereof, can be labeled for use as a
hybridization probe, e.g., with a radioactive
label, using conventional procedures. The labeled
DNA hybridization probe may then be used in the
manner described in the Examples.
The cryIF or cryIX gene may be introduced
into a variety of microorganism hosts, using
procedures well known to those skilled in the art
for transforming suitable hosts under conditions
which allow for stable maintenance and expression
of the cloned crylF or crylX gene, as the case may
be. Suitable hosts that allow the er IF and cryIX
genes to be expressed and the respective CryIF and
CryIX proteins to be produced include Bacillus
thuringiensis and other Bacillus species such as B.
subtilis or B. megaterium. E. coli or Pseudomonas
fluorescens are also suitable hosts for these
genes. It should be evident that genetically
altered or engineered microorganisms containing the
crylF gene or crylX gene can also contain other
toxin genes present in the same microorganism and


WO 91/16434 FCI'/U591/02560
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that these genes could concurrently produce
insecticidal crystal proteins different from the
cryIF and CryIX proteins.
The Bacillus and E. coli strains
described in this disclosure may be cultured using
conventional growth media and standard fermentation
techniques. The B.t. strains harboring the cr IF
gene (or the cryIX gene) may be fermented, as
described in the Examples, until the cultured B.t.
cells reach the stage of their growth cycle when
CryIF crystal protein (or CryIX crystal protein) is
formed. For sporogenous B.t. strains, fermentation
is typically continued through the sporulation
stage, when crystal protein is formed along with
spores. The B.t. fermentation culture is then
typically harvested by centrifugation, filtration
or the like to separate fermentation culture
solids, containing the crystal protein, from the
aqueous broth portion of the culture.
The B.t. strains exemplified in this
disclosure are sporulating varieties (spore forming
or sporogenous strains), but the crylF gene and the
crylX gene also have utility in asporogenous
Bacillus strains, i.e., strains that produce the
crystal protein without production of spores. It
should be understood that references to
"fermentation cultures" of B.t. strains (containing
the ,c1e IF or crylX gene) in this disclosure are
intended to cover sporulated B.t. cultures, i.e.,
B.t. cultures containing the CryIF or CryIX crystal
protein and spores, and sporogenous Bacillus strain
cultures that have produced crystal protein during
the vegetative stage, as well as asporogenous


WO 9l/15434 P("T/US91/02560

24 - 208063d
-

Bacillus strains containing the cr IF or cr IX gene
in which the culture has reached the growth stage
where crystal protein is actually produced.
The separated fermentation solids are
primarily CryIF or CryIX crystal protein, as the
case may be, and B.t. spores, along with some cell
debris, some intact cells, and residual
fermentation medium solids. If desired, the
crystal protein may be separa'ted from the other
recovered solids via conventional methods, e.g.,
sucrose density gradient fractionation. Highly
purified CryIF or CryIX protein may be obtained by
solubilizing the recovered crystal protein and then
reprecipitating the protein from solution.
The CryIF protein is an effective
insecticidal compound against lepidopteran insects
like the European cornborer, the beet armyworm, and
the tobacco budworm, for example. Likewise, the
CryIX protein is insecticidal to lepidopteran
insect species. The CryIF protein or CryIX protein
may be utilized as the active ingredient in
insecticidal formulations useful for the control of
lepidopteran insects. Such insecticidal
formulations or compositions typically contain
agriculturally acceptable carriers or adjuvants in
addition to the active ingredient.
The CryZF protein or CryIX protein may be
employed in insecticidal formulations in isolated
or purified form, e.g., as the crystal protein
itself. Alternatively, the CryIF protein or CryIX
protein may be present in the recovered
fermentation solids, obtained from culturing of a
Bacillus strain, e.g., Bacillus thuringiensis, or


"'0 91/16434 4'CI'/US91/02560
- 25 -

other microorganism host carrying the crylF or
cryIX gene and capable of producing the
corresponding CryIF or CryIX protein. Preferred
Bacillus hosts for the crylF gene include B.t.
strain EG6345 and genetically improved B.t. strains
derived from B.t. strain EG6345, such as B.t.
strain EG6346. The derivative B.t. strains may be
obtained via plasmid curing arad/or conjugation
techniques and contain the native cryIF gene-
containing plasmid from B.t. strain EG6345.
Genetically engineered or transformed B.t. strains
or other host microorganisms, containing a
recombinant plasmid that expresses the cloned cryIF
gene and obtained by recombinant DNA procedures,
may also be used.
Examples of such transformants include
B.t. strain EG1945 which contains the cloned cryIF
gene on a recombinant plasmid.
The recovered fermentation solids contain
primarily the crystal protein and (if a sporulating
B.t. host is employed) spores; cell debris and
residual fermentation medium solids may also be
present. The recovered fermentation solids
containing the CryIF or CryIX protein may be dried,
if desired, prior to incorporation into the
insecticidal formulation.
The formulations or compositions of this
invention containing the insecticidal CryIF or
CryIX protein as the active component are applied
at an insect:tcidally effective amount which will
vary depending on such factors as, for example, the
specific lepidopteran insects to be controlled, the
specific plarit or crop to be treated and the method


WO 91/16434 PC3'/1J591/02560

- 26 208063d
-

of applying the insecticidally active compositions.
An insecticidally effective amount of the
insecticide formulation is employed in the insect
control method of this invention.
The insecticide compositions are made by
formulating the insecticidally active component
with the desired agriculturally acceptable carrier.
The formulated compositions may be in the form of a
dust or granular material, or a suspension in oil
(vegetable or mineral) or water or oil/water
emulsions, or as a wettable powder, or in
combination with any other carrier material
suitable for agricultural application. Suitable
agricultural carriers can be solid or liquid and
are well known in the art. The term
"agriculturally acceptable carrier" covers all
adjuvants, e.g., inert components, dispersants,
surfactants, tackifiers, binders, etc. that are
ordinarily used in insecticide formulation
technology; these are well known to those skilled
in insecticide formulation.
The formulations containing the CryIF or
CryIX protein and one or more solid or liquid
adjuvants are prepared in known manners, e.g., by
homogeneously mixing, blending and/or grinding the
insecticidally active CryIF or CryIX protein
component with suitable adjuvants using
conventional formulation techniques.
The insecticidal compositions of this
invention are applied to the environment of the
target lepidopteran insect, typically onto the
foliage of the plant or crop to be protected by
conventional methods, preferably by spraying.


WO 91/16434 JPCi'/US91/02560

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Other application techniques, e.g., dusting,
sprinkling, soaking, soil injection, seed coating,
seedling coating or spraying, or the like, are also
feasible. These application procedures are well
known in the art.
The crylF or crylX gene or its functional
equivalent, hereinafter sometimes referred to as
the "toxin gene," can be introduced into a wide
variety of microorganism hosts. Expression of the
cr IF gene results in the production of
insecticidal CryIF crystal protein. Likewise,
expression of the cryzx gene results in production
of the insecticidal CryIX protein. Suitable hosts
include B.t. and other species of Bacillus, such as
B. subtilis or B. megaterium, for example. Other
bacterial hosts such as E. coli and Pseudomonas
fluorescens may also be used. Various procedures
well known to those skilled in the art are
available for introducing the crylF or crylX gene
into the microorganism host under conditions which
allow for stable maintenance and expression of the
gene in the resulting transformants.
The transformants, i.e., host
microorganisms that harbor a cloned gene in a
recombinant plasmid, can be isolated in accordance
with conventional ways, usually employing a
selection technique, which allows growth of only
those host microorganisms that contain a
recombinant plasmid. The transformants then can be
tested for insecticidal activity. Again, these
techniques are standard procedures.


WO 91/16434 PG?/U591/02560
- 28 2

Characteristics of particular interest in
selecting a host cell for purposes of production
include ease of introducing the gene into the host,
availability of expression systems, efficiency of
expression, stability of the CryIF or CryIX
insecticidal protein in the host, and the presence
of auxiliary genetic capabilities. The cellular
host containing the insecticidal crylF or cryTX
gene may be grown in any convenient nutrient
medium, where expression of the crZIF or crylX gene
is obtained and corresponding CryIF or CryIX
protein produced, typically upon sporulation. The
sporulated cells containing the crystal protein may
then be harvested in accordance with conventional
ways, e.g., centrifugation or filtration.
The cryaF and cryIX genes may also be
incorporated into a plant which is capable of
expressing the gene and producing CryIF or CryIX
protein, as the case may be, rendering the plant
more resistant to insect attack. Genetic
engineering of plants with the cryIF or cryIX gene
may be accomplished by introducing the desired DNA
containing the gene into plant tissues or cells,
using DNA molecules of a variety of forms and
origins that are well know to those skilled in
plant genetic engineering. An example of a
technique for introducing DNA into plant tissue is
disclosed in European Patent Application
Publication No. 0 289 479, published November 2,
1988, of Monsanto Company.
DNA containing the crylE or crylX gene or
a modified crylF or crylX gene capable of producing
the corresponding CryIF or CryIX protein may be


'/0 91/16434 PCT/YJ591/02560
_ 29

delivered into the plant cells or tissues directly
by infectious plasmids, such +as Ti, the plasmid
from Agrobacterium tumefaciens, viruses or
microorganisms like A. tumefaciens, by the use of
lysosomes or liposomes, by microinjection by
mechanical methods and by other techniques familiar
to those skilled in plant genetic engineering.
Slight variations may be made in the
crXIF or crYIX gene nucleotide base sequences,
since the various amino acids forming the proteins
encoded by the respective genes usually may be
determined by more than one codon, as is well known
to those skilled in the art. Moreover, there may
be some variations or truncation in the coding
region of the cryIF and cryIX nucleotide base
sequences which allow expression of the gene and
production of functionally equivalent forms of the
corresponding CryIF and CryIX insecticidal
proteins. These variations which can be determined
without undue experimentation by those of ordinary
skill in the art with reference to the present
specification are to be considered within the scope
of the appended claims, since they are fully
equivalent to the specifically claimed subject
matter.
The.present invention will now be
described in more detail with reference to the
following specific, non-limiting examples. The
examples relate to work which was actually done
based on techniques generally known in the art and
using commercially available equipment.


WO 91/16434 F'GT/US91/02560
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The novel B.t. strain EG6345 and a cured
derivative B.t. strain EG6346 were isolated
following the procedures described in Example 1.

Examlale 1
Isolation of B.t. Strainst EG634S and EG6346
Crop dust samples were obtained from
various sources throughout the U.S. and abroad,
typically grain storage facilities. The crop dust
samples were treated by suspending the crop dust in
an aqueous buffer and heating the suspension at
60'C for 30 min. to enrich for heat resistant spore
forming Bacillus-type bacteria such as B.t. The
treated dust suspensions were diluted in aqueous
buffer, and the dilutions were spread on agar
plates to allow each individual bacterium from the
crop dust to grow into a colony on the surface of
the agar plate.
After extensive screening of crop dust
samples, a B.t. subsp. aizawai strain, designated
B.t. strain EG6345, was isolated from a maize grain
dust sample. A sporulated culture of B.t. strain
EG6345 was spread for the growth of individual
colonies on a nutrient salts agar plate and
incubated for 3 days at 306C. After incubation,
one colony was noted on this plate which displayed
a different colony morphology (i.e., shinier) than
the parent B.t. strain EG6345. The colony,
designated B.t. strain EG6346, was isolated as an
individual colony.
A sample of the isolated B~t. strain
EG6346 was further purified by streaking on an agar
plate containing Spizizen's glucose peptone beef


'0 91/16434 PCT/US91/02560

9 ON "ul
- 31 -

extract (SGPB). A sample of this SGPB agar plate
culture was used for agarose gel electrophoresis
analysis of plasmid DNA using the standard Gonzalez
technique (Gonzalez et al., Proc. Natl. Acad. Sci.
U.S.A., 79, pp. 6951-6955 (1982)). The agarose gel
electrophoretic analysis was coupled with standard
plasmid curing (ie., plasmid loss) and conjugation
(ie., plasmid transfer) studies. The plasmid array
of the new isolate of B.t. strain EG6346 was
compared to that of B.t. strain EG6345 using
agarose gel electrophoresis of plasmid DNA.
The agarose gel electrophoresis analyses
of plasmid DNA, coupled with the plasmid curing and
conjugation studies, indicated that B.t. strain
EG6345 contained two plasmids of approximately 115
MDa and 45 MDa that encoded crystal protein. B.t.
strain EG6346 was identified as a spontaneously
cured derivative of B.t. strain EG6345 which
contained the plasmid of approximately 115 MDa, but
which lacked the approximately 45 MDa plasmid.
Figure 3 is a photograph of a portion of an
ethidium bromide stained agarose gel containing
size-fractionated plasmids of B.t. strains EG6346
(left lane) and EG6345 (right lane). As
illustrated in Figure 3, B.t. strain EG6346 does
not contain the approximately 45 MDa plasmid
contained in B.t. strain EG6345. Both B.t. strain
EG6345 and the cured derivative B.t. strain EG6346
produced large bipyramidal inclusions during
sporulation, as detected by phase contrast
microscopy of sporulated cultures.


",0 91/16434 PC,T/U591/02560
- 32 -

Following the Southern blot technique
(E.M. Southern, J. Mol. Biol., 98, pp. 503-517
(1975)), total DNA, prepared from both B.t. strains
EG6345 and EG6346, was digested with Fiindill,
electrophoresed through a 0.7% agarose gel,
transferred to a nitrocellulose filter and
hybridized at 50'C overnight to either a 32P-
labeled 0.7 kb EcoRT N-terminal probe isolated from
the B.t. strain HD-l crYIA(a) gene or a similarly
labeled 2.2 kb intragenic PvuII probe also isolated
from the HD-1 cryIA(a) gene. Digested DNA from
B.t. subsp. kurstaki HD-1, which harbors the
crylA(a), cryIA(b) and crylA(c) genes, was included
as a control. The results of the Southern blot
analyses are illustrated in Figure 4-A and 4-B.
Figure 4-A is the Southern blot of the agarose gel
containing the total HindIiI-digested DNA from B.t.
strains EG6346 (lane 1), EG6345 (lane 2) and HD-1
(lane 3), following hybridization to the
radiolabeled EcoRI probe. Figure 4-B shows the
Southern blot of total Hindill-digested DNA from
the B.t. strains indicated with respect to
Figure 4-A, and in the same order, following
hybridization to the radiolabeled PvuIS probe.
As shown in Figure 4-A, the 0.7 kb EcoRI
probe detected the expected 4.5, 5.3 and 6.6 kb
fragments in HD-1 DNA (lane 3) corresponding to the
previously described characteristic HindIIl
fragments for the crylA(a), cUIA(b) and cryIA(c)
genes, respectively. This probe also detected a
prominent 5.3 kb band in B.t. strain EG6345
(lane 2) which was absent in the cured derivative
B.t. strain EG6346 (lane 1). This result indicated


WO 97/16434 P+L .T'/US91/02560
- 33 -

that the 45 MDa plasmid of EG6345 harbored at least
one crylA(b) gene. The N-terminal 0.7 kb EcoRI
probe also hybridized to a 1.4 kb Hindlll fragment
of unknown origin in both B.t. strains EG6345 and
5. EG6346.
The hybridization pattern obtained with
the radiolabeled intragenic PvuII probe was more
complex as can be seen in Figure 4-B. This probe,
as expected, also hybridized to the 4.5, 5.3 and
6.6 kb fragments in HD-1 (lane 3) confirming the
presence of the respective crylA(a), crylA(b~ and
crylA(c) genes in this strain. Internal HindIIl
fragments of 1.1 kb and 0.9 kb, derived from the
resident crylA(a) and cryIA(b) genes in HD-1,
respectively, were also detected with the PvuII
probe.
The 5.3, 2.8 and 0.9 kb fragments were
also-detected by the PvuII probe in the DNA of B.t.
strain EG6345, indicating the presence of the
crylA(b) gene in this strain (lane 2). These bands
were not detected in B.t. strain EG6346 (lane 1).
However, the 1.4 kb Hindlll fragment, detected in
both B,t. strains EG6345 and EG6346 by the EcoRI
probe, was similarly detected in both strains by
the PvuII probe. A faintly hybridizing 2.5 kb
Hindlll fragment was also detected with the PvuII
probe in both S.t. strains EG6345 and EG6346. This
band corresponds in size to the characteristic
HindIIl fragment of the cgYIC gene detected in
other B.t. subsp. aizawai strains.
The Pvuil probe also hybridized to two
large Hindlll fragments present in both B.t.
strains EG63465 and EG6346. These fragments,


WO 91/16434 PCI'/IUS91/02560

- 34 - vI]
approximating 8.2 and 10.4 kb in size, were not
detected by the EcoRI probe in either of B.t.
strains EG6345 or EG6346, nor were they observed
with either probe in HD-i DNA. The unusual size of
the hybridizing fragments, along with the
production of large, bi-)yrami.dal crystal protein
inclusions by B.t. strain EG6346, indicated the
presence of one or more novel. toxin genes in B.t.
strains EG6345 and EG6346.

Ex!jmpls 2
Isolation of the crylP Gane in E. coli
A genomic library was constructed for
B.t. strain EG6346 and was screened at low
stringency conditions with the intragenic 2.2 kb
PvuII probe obtained from the cryIA(a) toxin gene.
DNA from B.t. strain EG6346 was chosen as the
substrate DNA due to its apparent lack of cryIA-
type toxin genes, whose presence could potentially
increase the difficulty in screening the library at
low stringency with the PvuII probe.
More specifically, high molecular weight
DNA, obtained from B.t. strain EG6346, was
partially digested with Sau3A and size-fractionated
on a 10% to 40% sucrose gradient in 100 mM NaCl-
10mM Tris hydrochloride (pH 7.4)-imM EDTA (TE).
Gradient fractions, containing DNA ranging in size
from 5 to 10 kb, were pooled, dialyzed against TE
10:1 (pH 7.4), extracted with 2-butanol to reduce
the volume and ethanol precipitated. The purified
insert DNA was ligated to E. coli plasmid vector
pGEM'"-32 digested with BamHI at a 1:2 molar ratio


'10 91/16434 P'CT/US91/02560

- 35 - ( QU 0634~
of vector to insert and at a final DNA
concentration of 20 ,ug/m1, using T4 DNA ligase
available from Promega Corp. Transformation of
E. coli DH5al cells was based on the Hanahan
procedure (Hanahan, J. Aiol. Biol., 166, pp. 557-580
(1983)) and transformed colonies were plated on
agar plates of standard LB medium containing 100
jug/ml ampicillin and 50 )ag/ml X-gal (5-bromo-4-
chloro-3-indolyl-beta-D-galactoside).
Approximately 3.3 x 106 colonies were
screened for the presence of cryI-related toxin
gene sequences under low stringency conditions,
using as a probe the 32P-labeled 2.2 kb PvuII
intragenic fragment obtained from a crylA(a) gene
present within B.t. strain HD-1. The low
stringency conditions include hybridization
conducted at 50-55'C overnight in 3X SSC (1X SSC
comprises 0.15 M NaC1, 0.015M sodium citrate), 10X
Denhardt's solution (1X Denhardt's solution
comprises 0.02% bovine serum albumin, 0.02% Ficoll,
0.02% polyvinylpyrrolidone), 200 pg/ml heparin and
0.1% SDS. The probe hybridized strongly to one
E. coli recombinant colony, designated E. coli
strain EG1943, which contained an 8.4 kb
recombinant plasmid, designated pE.G640, that
consisted of plasmid pGEM%-3Z ligated to a 5.7 kb
Sau3A insert of DNA from B.t. strain EG6346.
A restriction map for the pEG640 plasmid
was generated as shown in Figure 5 using those
restriction enzymes indicated above in the Brief
Description of the Drawings and methods well known
to those skilled in the art. The relative
positions of restriction sites and localization of


WO 91/16434 PCT/US91/02560

- 36 (yd
toxin gene sequences within the map were initially
accomplished by low stringency hybridization of
Southern blots containing digested pEG640 DNA to
the radiolabeled EcoRI and PvuII toxin gene probes
as set forth above in Example 1.
Initial mapping data identified two
regions on the pEG640 insert which reacted with
varying intensity to the toxin gene probes. The
larger region, spanning over 3 kb in length,
hybridized strongly to the PvuII probe at low and
high stringency hybridization conditions. The high
stringency conditions are the same as the above-
identified low stringency conditions, except that
the temperature is increased to 65'C. The larger
3 kb region on the 5.7 kb insert of the pEG640
plasmid also reacted well with the EcoRI probe at
low stringency hybridization conditions. A smaller
region, positioned in close proximity to the
vector, weakly hybridized to the EcoRI probe at low
stringency conditions only. These data indicated
the presence of two different toxin genes on the
5.7 kb insert of the pEG640 plasmid.
Pthen purified 32P-labeled pEG640 DNA was
used to probe HindliI genomic digests, a single
10.4 kb hybridizing band was detected in B.t.
strains EG6345.and EG6346, as illustrated in
lanes 1 and 2 of Figure 4-C, respectively. This
10.4 kb fragment was also detected in both B.t.
strains EG6345 and EG6346 with the PvuII probe as
can be seen in Figure 4-B, lanes 1 and 2,
respectively. No hybridizable bands were detected
in the DNA from B.t. strain HD-i, as evidenced by


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lane 3 in Figure 4-C, which is consistent with the 2
absence of these novel gene sequences in this
strain.

Example 3
Sequence Analyses of crylF and crylX Genes
Standard dideoxy sequencing procedures
(Sanger et al., Proc. Natl. Acaci. Sci. U.S.A., 74,
pp. 5463-5467 (1977), with Sequenase'", available
from United States Biochemical C:orp., were used to
determine the DNA sequence of the 5.7 kb pEG640
insert from the recombinant E. coli strain EG1943.
Sequencing of the insert was initiated in both
directions and on both strands from the SP6 and T7
promoters present on vector pGEM"'-3Z and utilized
the specific primers supplied by Promega Corp.
Preparation and denaturation of the double stranded
template was also according to manufacturers'
directions (Promega Corp. and United States
Biochemical Corp.). Subsequent 17mer
oligonucleotide primers were synthesized on an
Applied Biosystems, Inc. DNA synthesizer,
Model 380B.
The DNA sequence, which is flanked by
Sau3A cloning sites (GATC) extends 5649 nucleotide
bases in length and is shown in Figures 1 and 2.
Translation of the sequence revealed the presence
of two open reading frames which are separated by
approximately 500 bases of non-coding DNA sequence
and which are out of frame with respect to one
another. The genes potentially encoded by these
open reading frames have been designated cr'yIF (SEQ
ID NO:l) and crylX (SEQ ID NO:3). Justification
for this designation derives from sequence


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2 0
comparisons to other toxin genes and is discussed
below. The partial DNA sequence for the portion of
the 5.7 kb insert of pEG640 including the cr IF
gene (SEQ ID NO:l) and the deduced amino acid
sequence of the crystal protein encoded by the
cryIF gene, designated the CryIF protein (SEQ ID
NO:2), are illustrated in Figure 1. The partial,
truncated DNA sequence (SEQ ID NO:3) for the
portion of the 5.7 kb insert of pEG640 including
the truncated crylX gene, and the deduced,
truncated amino acid sequence (SEQ ID NO:4) of the
crystal protein encoded by the crylX gene,
designated the CryIX protein are illustrated in
Figure 2. The beginning of the sequences in
Figure 2 follow immediately after the end of the
sequences illustrated in Figure 1, and two figures
are used merely for the sake of convenience.
The crylF open reading frame, which is
the larger of the two, encodes a CryIF protein
consisting of 1174 amino acids and having a deduced
size of 133,635 Da. The position of the cryIF gene
within pEG640 and its relationship to the position
of the crylX gene is schematically represented in
Figure 5. An NH2-terminal methionine translational
start site was identified for the crylF gene at
nucleotide base position 478 of the sequence. It
was immediately preceded by a putative ribosome
binding site (RES). The cryIF gene open reading
frame terminates at nucleotide base position 3999.
A putative promoter sequence was identified for the
cryIF gene 53 nucleotide bases upstream of the
ribosome binding site. The nucleotide base
sequence as counted from both base pairs 10 and 35
positions upstream of the methionine start is


wO 91/16434 P4'T/[JS91/02560
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exactly homologous to that identified for the HD-1
cryIA(a) gene promoter (Wong et al., J. Biol.
Chem., 258, pp. 1960-1967 (1983)).
As indicated in Figure 2, an NH2-terminal
methionine codon, signifying the translational
start site of the cr IX open reading frame, was
identified at nucleotide base position 4508. The
crylX open reading frame continued an additional
1141 nucleotides, encoding 380 amino acids, and
terminated with the GATC cloning site delimiting
the insert DNA. The sequence presented here for
the crylX gene represents an artificially truncated
version of the native gene present within B.t.
strain EG6346. Although a putative ribosome
binding site has been identified upstream of the
cryI7t sequence, it was not possible to identify
promoter regions located 10 and 35 base pairs
upstream from the methionine start for the cryzX
gene within the intervening DNA sequence between
the cryIF and crylX open reading frames by sequence
inspection. Tnspection of the intervening DNA
sequence between the cr IF and cryzl{ genes has
identified a stem-loop termination structure at
positions 4090-4132 (see Figure 2) that is nearly
identical to that described downstream of the HD-
1-Dipel crylA(a) gene (Wong et al., J. Biol. Chem.,
258, pp. 1960-1967 (1983)).
The sequence analysis program of Queen
and Korn was used to compare the sequences of the
crylF and crYIX genes to the published sequences of
other B.t. toxin genes (Queen et al., Nucleic Acids
Res., 12, pp. 581-599 (1984)). The nucleotide base
sequences anei deduced amino acid sequences of the


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- 40 - 2 0 0

cryIF and cr IX genes were aligned with the
published sequences of various delta-endotoxin
genes and the results of the comparisons are
tabulated in Table 1. As shoi,rn in Table 1, the
amino acid sequence of the N-terminal region (amino
acids 1-618) of the cr yIF-encoded protein differs
significantly from the N-terminal region of other
CryI-type encoded proteins (about 40%-50%
identity). These sequence differences are likely
responsible for the unique insecticidal activity
spectrum of the CryIF protein (see Example 5
below), since previous studies of truncated cryI
genes indicate that it is the N-terminal region of
the protein that determines insecticidal activity
(Schnepf et al., J. Biol. Chem., 260, pp. 6273-6278
(1985); Hofte et al., Eur. J. Biochem., 161, pp.
273-280 (1986).


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- 41

TABLE 1

Amino Acid (aa) Comparisons of the
N-terminal Region of CryIF Proteina
Protein Class (Bd-terYainal region) ~ Similarity
CryIA(a) (aa 1-608) 51Ø
CryIA(b) (aa 1-609) 52.0
cryIA(c) (aa 1-610) 49.0
CryIB (aa 1-637) 40.1
CryIC (aa 1-617) 48.8
CryID (aa 1-593) 52.0
CrylE (aa 1-602) 48.1
a Amino acids 1-602 of the CryIF protein were
compared to the N-terminal regions of CryIA(a)
(Schnepf et al., (1985), su ra), CryIA(b) (Hofte
et al., (1986), supra), CryIA(c) (Adang et al.,
(1985), supra), CryIB (Brizzard et al. (1988),
supra), CryIC (Ho6ee et al., (1988), supra),
CryID and CryIE (both in EP 0 358 557 (1990),
supra).

The nucleotide base sequence of the
entire crYIF gene and the amino acid sequences of
the CryIF protein were also compared to other
crystal protein genes and their respectively
encoded proteins. The comparisons were tabulated
in Table 2.


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TABLE 2

8equence Comparisons of cryYF and crylX
Ganes and Encoded Proteins
With Other B.t. Genes and Proteins

crylF CryIF crylX CryX
DNA aaa DNA aa
Gene tXpe
crylA(a) 77.6b 71.7b 52.5 35.2
crylA(b) 75.8 70.4 53.4 36.4
crylA(c) 75.8 69.9 53.4 36.7
crylB 66.6 58.3 70.4 62.9
cryiC 75.3 70.0 51.9 36.7
crylD 75.6 71.5 43.1 31.8
cryIE 77.2 69.8 51.2 34.8
cryllA 43.9 24.6 47.9 26.2
cryIIIA 53.0 35.6 55.3 38.8
cryIVD 44.5 20.8 45.0 22.9
a aa means amino acid.
b g Identity, i.e., positional identity.
Comparisons of the complete DNA sequence
indicate the crylF gene was related to, but
distinct from, the crYxA class og toxin genes
(about 76-78% identity) (Table 2). Of the three
crylA genes compared to the cryIF gene, cryIF was
most related to the HD-i crylA(a) nucleotide
sequence with about 78% of the nucleotides
conserved between the two genes.
Table 2 indicates that the CryIF protein
is significantly more related to other Cryl
proteins than to the cryllA, CryIIiA or CryIVD


WO 91/16434 PCT/US91/02560
- 43 - 0
proteins. Amino acid identity ranged from about
70-72% for the CryIF protein and the CryIA, CryIC,
CryID and CryIE proteins.
The crylF gene sequence was less related
to crylB (about 67%) and, as expected, much less
related to dipteran and coleopteran toxin genes
(cryll, cryIII and cr IV genes).
The crystal protein genes thus far
disclosed in the previously cited references have
been divided into four major classes and several
subclasses characterized by both structural
similarities and the insecticidal spectrum of the
encoded crystal proteins (Hofte and Whiteley (1989)
p. 242). The four major classes, I, II, 111 and
IV, encode lepidopteran-specific, lepidopteran- and
dipteran-specific, coleopteran-specific and
dipteran-specific proteins, respectively. Table 1
of Hofte and Whiteley (1989) at p. 243 lists the
genes presently assigned to these four major
classes.
The cryl genes can be distinguished from
the other crystal protein genes by sequence
homolocgy. The amino acid sequences encoded by the
cryl genes exhibit greater than 50% identity
(Table 3, H fte and Whiteley (1989) at p. 245).
The amino acid sequences of three Cryl-encoded
proteins (CryIA(a), CryIA(b) and CryYA(c)) show
greater than 80% identity, and thus they are
considered members of the same subgroup (CryIA).
There is ample justification for
designating the novel toxin gene identified in B.t.
strain EG6345 as cryIF. The CryIF protein exhibits
greater than 50% amino acid identity to the other


1*/091/16434 PC'V'/US91/02560
- 44 - rl~~~06U4

Cryl proteins. More specifically, the CryIF
protein is about 70-72% identical to the CrYIA
subgroup proteins, about 58% identical to the CryIB
protein and about 70% identical to the CryIC and
CryID proteins. The CryIF protein is less related
to the crystal proteins encoded by the other
crystal protein gene classes c:r II, cryIll and
cryIV (see Table 2).
However, the CryIF protein is not greater
than about 80% identical to the crystal proteins
encoded by the crylA subgroup of genes, and thus
the cryIF gene does not belong to the crylA
subgroup. The CryIF protein is only somewhat
related to the CryIB, CryIC, CryID and CrylE
proteins, and thus, the cr IF gene is not a member
of a new subgroup including any of the cr IB,
cryIC, cryID or cryIE genes.
Further substantiation of the crylF
designation, i.e., its categorization as a cryl-
type gene, is that the CryIB protein is about 55-
56% identical to the proteins encoded by the crylA
subgroup of genes and the CryID protein is about
70-71% identical to the CryIA subgroup and CryIC
proteins (see Hofte and Whiteley (1989) Table 3).
The cryIX truncated nucleotide base
sequence (SEQ ID NO:3) and the deduced amino acid
sequence (SEQ ID NO:4) were similarly compared to
other toxin gene sequences, as shown in Table 2.
The cryIX nucleotide base sequence is also distinct
from, but related to, the other cryl genes in
Table 2, such as that of the cr IB gene (about 70%
identical).


~ '!O 91/1643d PCr/IJS91/02560
2080t~84
- 45 -

EXample 4
Expression of the Cloned arylP Gene
Studies were conducted to demonstrate the
production of the CryIF protein by the crylF gene.
Table 3 summarizes the relevant
characteristics of the B.t. and E. coli strains and
plasmids used during these procedures. A plus (+)
indicates the presence of the designated element,
activity or function and a minus (-) indicates the
absence of the same. The designations S and r
indicate sensitivity and resistance, respectively,
to the antibiotic with which each is used. The
abbreviations used in the table have the following
meanings: Amp (ampicillin); Cry (crystalliferous);
Tc (tetracycline).


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Table 3

Strain or Plasmid Relevant Characteristics
B. thuringiensis

HD73-26 Cry

EG1078 HD73-26 harboring pEG310 (cr IF- c
yIX
+
EG1945 HD73-26 harboring pEG642 (crylF+ cr IX+
EG6345 crylF+ cryIX+

EG6346 cr IF+ crySX+, derivative of EG6345
cured of 45 MDa plasmid

E. coli

DH5 cl, Cry , Amps
GM2163 Cry-, ,Amps

EG1943 DH50~. harboring pEG640(crylF+ cryIX+)
Plasmids

pEG310 crylF cryIX+ deletion mutant plasmid
of pEG642

pEG434 Tcr Bacillus vector
pGEM"-3Z Ampr E. coli vector

pEG640 Fmpr pGEIV-3Z with 5.7 kb insert (c IF+ cryIX+)
pEG642 Tcr, E. coli-Bacillus shuttle vector consisting of
pEG640 lTgateT into the Hindll% site of pEG434

It has been reported that E. coli cells
harboring cloned B.t. toxin genes fail to produce
significant amounts of the toxin protein required
for critical evaluations of insecticidal activity
(Donovan et al., Mol. Gen. Genet., 214, pp. 365-372
(1988)). Returning the cloned B.t. toxin gene to a
Bacillus species, and ideally to a B.t. host,


EV 91/16434 PC'f/U591/02560
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maximizes toxin gene expression from its native
promoter. Accordingly, the cloned crylF gene was
introduced into the Cry recipient B.t. strain
HD73-26, as described below.
The pEG640 plasmid construct was ligated
to the vector pEG434 (Mettus et al., Applied and
Environ. Microbiol., 192, pp. 288-289 (1990)) at
the unique Hindill site present on both pEG640 and
pEG434 and the ligation mixture used to transform
E. coli strain GM2163, which is defective for both
adenine and cytosine methylation (Marinus et al.,
Mol. Gen. Genet., 56, pp. 1128-1134 (1983)). The
resulting 11.4 kb recombinant plasmid, designated
pEG642 and having a restriction map as shown in
Figure 6, possessed both E. coli and Bacillus
replication origins and a selectable marker for
tetracycline resistance (Tcr) that could function
in a B~t. host. Plasmid pEG642 DNA was isolated
from E. coli strain GM2163 by alkaline/SDS lysis
followed by ethanol precipitation using standard
procedures. Plasmid DNA was then used to transform
the B.t. Cry- recipient strain HD73-26 by
electroporation. A single Tcr HD73-26 trafasformant
containing pEG642, designated B.t. strain EG1945,
was chosen for further study. Microscopic
examination of sporulated cultures of B.t. strain
EG1945 revealed the presence of crystalline
inclusions (large, irregularly shaped rods and
bipyramidal shapes).
Renografin density gradient purified
crystal protein from B.t. strain EG1945 was used
for SDS-PAGE analyses of the cr1rIF gene product.
The purified CryIF protein from the recombinant


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B.t. strain EG1945 was compared to similarly
purified proteins obtained from the native B.t.
strains EG6345 and EG6346 harboring the cryIF gene.
Crystal protein preparations (2.8 j.tg of EG6345,
0.7 pg of EG6346 and 0.70 )ig of EG1945) were loaded
onto a 5-20% gradient SD5-polyacrylamide gel and
electrophoresed. Figure 7 is a photograph of the
resulting Coomassie stained SDS-polyacrylamide gel,
in which lanes 1, 2 and 3 contain proteins from
native B.t. strains EG6345 and EG6346 and
recombinant B.t. strain EG1945, respectively.
As indicated in lane 3, a single high
molecular weight protein, approximating 135 kDa in
size, was observed in recombinant B.t. strain
EG1945, consistent with expression of the single
crylF gene. The size of the observed protein
correlates well with the predicted molecular weight
of 133,635 Da deduced from the amino acid sequence.
At least three distinct protein species
were observed in lane 1 of Figure 7, from B.t.
strain EG6345, which confirms the DNA hybridization
result shown in Figure 4, verifying the presence of
the crylA(b), cryIC and cryIF genes in this strain.
It is possible, however, that other similarly sized
proteins encoded by additional toxin genes are also
present in B.t. strain EG6345, e.g., the crylX
gene.
Similarly, B.t. strain EG6346, which was
used to construct the library from which crylF was
cloned, contains at least two crystal proteins, the
largest of which appears to co-migrate with the
approximately 135 kDa recombinant czylF protein in
B.t. strain EG1945. The smaller protein present in


WO 91 / 16434 FCT/US91 /02560

2 'S`~'~ 8 ~y~
- 49 -

B.t. strain EG6346, also evident in B.t. strain
EG6345, is believed to represent the protein
encoded by the crylC gene which has been identified
in each of these strains by DNA hybridization
analysis with a cr IC specific oligonucleotide
probe.

Exaatple 3
Plasmid Localization of the cryZE' Gene
To determine the location of the cryIF
gene in B.t. strains EG6345 and EG6346 and to
compare its location to that of the crylA(b) gene
present in B.t. strain EG6345, plasmid DNAs of B.t.
strains EG6345 and EG6346 were resolved by agarose
gel electrophoresis. The resulting ethidium
bromide stained gel is illustrated in Figure 8-A.
Plasmids from strain HD-1 (lane 1) were included as
controls and were used as size standards. Lane 2
shows the plasmids from B.t. strain EG6345, while
lane 3 shows the plasmids from B.t. strain EG6346.
Plasmid DNAs resolved by the gel of
Figure 8-A were transferred to nitrocellulose and
hybridized to either the intragenic radiolabeled
2.2 kb PvuII cryIA(a) probe or to a cryIF gene-
specific probe consisting of a radiolabeled gel-
purified 0.4 kb PstI-SacI fragment isolated from
the N-terminal region of the cryIF gene on pEG640.
Hybridizations were conducted at 65"C overnight to
assure specificity of the reaction with each probe.
As shown in the autoradiogram of Figure 8-B, the
PvuII intragenic cryA(a) probe hybridized strongly
to the 44 AiDa plasmid present in HD-1 (lane 1)
which harbors a c IA b gene. Hybridization of


WO 91 / 16434 1'Cf/1JS91 /02560

- 5o - 2 0 ~'10 `'18
the PvuII probe to this plasmid was expected, since
the nucleotide base sequence of the probe is highly
conserved among all three crYIA genes. Similarly,
the PvuII probe also hybridized to the large 110
MDa plasmid in strain HD-1 containing the cryIA (a)
and crYIA(c) toxin genes.
The PvuII probe also hybridized to the
45 MDa plasmid containing the cr IA b gene present
within B.t. strain EG6345 (lane 2). Differences in
the hybridization signal intensity of the PvuII
probe in detecting the cryIA(b) gene in B.t.
strains HD-1 and EG6345 may be attributed to
different amounts of DNA loaded onto the gel shown
in Figure 8-A. Lack of a hybridization band from
the PvuII probe in strain EG6346 (lane 3) was
entirely consistent with the classification of this
strain as a cured derivative of B.t. strain EG6345
not containing the 45 MDa plasmid. The 115 MDa
plasmid present within B.t. strains EG6345 and
EG6346 was weakly detected by the PvuII probe. The
reduced hybridization signal observed in each of
these strains, as compared to strain HD-1, may be
attributed to quantitative differences in the
amounts of DNA loaded, as we11 as to the reduced
sequence homology between the PvuII probe and the
novel toxin genes present on this large plasmid.
Hybridization of the crylF PstI-SacI
intragenic probe to plasmid DNAs from B.t. strains
HD-1 (lane 1), EG6345 (lane 2) and EG6346 (lane 3)
is shown in the autoradiogram of Figure 8-C. The
specificity of this probe for the crylF gene is
confirmed by the lack of hybridization to plasmids
harboring cry.IA genes in B.t. strains FiD-1 or


'VO 91/16434 ACI'/US91/02560
- 51 - 2 EG6345, and by its hybridization to the 115 MDa
plasmid present in B.t. strains EG6345 and EG6346.
Both the PvuII and the PstI-SacI probes hybridized
to a low molecular weight smear, identified as "L"
in Figure 8-A, which represents linear fragments of
sheared larger toxin plasmids.

le ~
Insect Toxicity of the cry,IF Protein
The insecticidal activity of CryIF
protein was determined against several lepidopteran
larvae including Ostrinia nubilalis (European
cornborer), Spodoptera exi a(beet armyworm),
Heliothis virescens (tobacco budworm), Heliothis
zea (bollworm) and Lymantria dispar (gypsy moth),
using Renografin' density gradient purified CryIF
crystal protein from recombinant B.t. strain
EG1945, which harbors the crylF gene on plasmid
pEG642.
Activity was measured using a diet-
surface overlay technique where the surface of an
agar-based artificial diet was covered with an
aliquot suspension containing CryIF protein
crystals. After delivery of the aliquot to the
diet surface, the diluent was allowed to evaporate,
at which time one larva of the test species was
placed in each cup. Each 2 ml well (cup) contained
1 ml diet having a surface area of 175 mm2
Bioassays were held at 280C for 7 days, at which
time mortality was scored. Bioassays were first
conducted at three doses with 1 to 10 dilutions.
If the CryIF protein demonstrated sufficient
activity, eight dose assays (1 to 2 dilutions) were


WO 91/16434 PCT/US91/02560

2
52

conducted to determine LC50 values via the well-
known technique of probit analysis (Daum, Bull.
Entomol. Soc. Am., 16, pp. 10-15 (1970)). Each
dose was tested against 32 insects. The diluent,
0.005% Triton' X-100, served as a control
treatment. All insects were tested as newly
hatched first-stage larvae. The results of
effective insecticidal activity are set forth in
Table 4 in comparison with the results of
insecticidal bioassays using other CrylA crystal
proteins.

Table 4

Insecticidal Activitp of CryI Crystal Proteins
LC50 in ng protein/mm2

Heliothis Spodoptera Ostrinia
Protein virescens exiqua nubilalis
CryIA(a) 2.2 >57 0.27
CryIA(b) 0.7 33 0.17
CryIA(c) 0.03 >57 0.08

CryiF 0.31 26 0.17
The CryIF protein exhibited the greatest
toxicity to Ostrinia nubilalis larvae as indicated
in Table 4. The LC50 value obtained is similar to
LC50 values obtained for the purified CryIA(b)
crystal protein which is highly toxic to Ostrinia
nubilalis larvae. In addition, the CryIF protein
was toxic to Spodoptera exiqua larvae. CryIF
protein was considerably more toxic to Spodo tera


VO 91/16434 Pt;T/US91/02560
53

exigua than purified CryIA(a) and CryIA(c) crystal
proteins and slightly more toxic than purified
CryIA(b) crystal protein. Purified CryIF crystal
protein was also toxic to Heliothis virescens, with
a toxicity between that of purified CryIA(c) and
CryIA(b) crystal protein. CryIF crystal protein
exhibited little toxicity to Iieliothis zea or
Lymantria dispar at the doses tested.

Examp l e 7
Analysis of Insecticidal Activity of CryZX Fragment
The sequence of the cryIX gene present on
plasmid pEG642 (and likewise present on plasmid
pEG640) does not encode a sufficient number of
amino acids to constitute a "minimum toxic
fragment" as defined by deletion analyses of cryTA
genes (Schnepf et al., J. Biol. Chem,, 260,
pp. 6273-6278 (1985)), H fte et al., (1986) su ra).
Nonetheless, to assess the contribution of crylX,
if any, to the overall toxicity of the pEG642
construct, the following study was performed.
Plasmid pEG310, containing a deletion in
the cryIF gene, was constructed by restriction
enzyme deletion from plasmid pEG642 of an N-
terminal region of the cry%F gene which is flanked
by BstEII sites (Figure 6). Following religation,
plasmid pEG310 was introduced into the Cry B.t.
HD73-26 recipient via electroporation, resulting in
a recombinant strain designated B.t. strain EG1078.
Fully sporulated cultures, containing the intact
crylX gene sequence from plasmid pEG642, but not
the cryTF gene which had been deleted, were assayed
by the insect bioassay procedure previously


WO 91/16434 FCI'/U591/02560

54 - 203O~~~,~~~
described in Example 6 for toxicity against
Ostrinia nubilalis. B.t. strain EG1945, containing
the intact crylF gene, was the positive control.
Thirty insect larvae were assayed, at a
protein dose of 4.00 ng/550mm2. At this dose, the
B.t. strain EG1945 was 100% toxic to larvae of
Ostrinia nubilalis. However, B.t. strain EG1078,
containing the cryIF deletion mutant, exhibited 0t
mortality for Ostrinia nubilalis larvae. Thus, it
was concluded that the sequence of the cr IX gene
present on plasmid pEG642 does not contribute to
the observed toxicity of B.t. strain EG1945 and
that the cryIF gene product is the active
insecticide in the strain.

Examplt 8
Southern Blot Analysis
of the crvTX G ne in B.t. Strain
EG6346
Following the Southern blot technique
cited in Example 1, total DNA was obtained from
B.t. strain EG6346, digested to completion with the
restriction endonucleases As~718 (an isoschizomer
of Ksnl), C1aI, Sstl, and SshI both individually
and in combination, electrophoresed through a 0.8%
agarose gel, transferred to a nitrocellulose
filter, and hybridized at 65 C overnight to a 32p
labgled 0.6 kb EpnIaBamHl probe that was isolated
from pEG640 (previously described in Example 2) and
that contained a portion of the cr IX coding
region. The positions of the gi and BamFil sites
flanking the crylX probe are shown in Figure 5.


WO 91/16434 PCT/L1S91/02560
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The results of the Southern blot analysis
are shown in Figure 9. Total DNA from B.t. strain
EG6346 digested with restriction endonucleases
exhibited, in each instance, a single DNA fragment
hybridizing to the cr IX probe. Most importantly,
B.t. strain EG6346 DNA digested with Clal (lane 2)
yielded a 4.6 kb restriction jEragment that
hybridized to the probe. In addition, B.t. strain
EG6346 DNA digested with both ClaI and SstI
(lane 8) yielded a 4.4 kb restriction fragment that
was detected by the cr IX probe. Since a ClaI
restriction site was present only 309 bp upstream
from the cryIX open reading frame shown in
Figure 2, these results indicated that the entire
cryiX gene was likely to be contained on the 4.6 kb
Clal restriction fragment. This assumption was
shown to be correct by the fact that the CryIX
protein is only 81 kDa, which corresponds to a gene
of about 2.1-2.2 kb in length. In addition, the
absence of an SstI site immediately upstream to or
within the sequenced portion of the cryIX gene
displayed in Figure 2-A indicated that the SstI
restriction site detected by Southern blot analysis
was located downstream from crylX.
Exam2l !
gaolatioa of the Entire cs~gt$X ene in B. coli
A genomic library was constructed from
Clag-digested DNA of B.t. strain EG6346 and
screened under moderate stringency conditions with
the 0.6 kb nDnI-BaaaFiI cryIX probe derived from
pEG640 to ida:ntify recombinant E. coli colonies
containing cr.ylX gene sequences.


WO 91/16434 PCT/US91/02560
- 56 -

More specifically, total DNA obtained
from B.t. strain EG6346 was digested to completion
with Cla2, electrophoresed th:rough a 0.8% agarose
gel, and DNA fragments in the 4.3-5.0 kb range
excised from the gel with a clean razor blade. DNA
fragments within the agarose gel slice were
purified using the GeneClean II kit and procedure
available from Bio 101, Inc. of La Jolla, CA.
The E. coli-B.t. cloning vector pEG854,
depicted as a circular restriction map in Figure 10
and described by Baum et al., Appl. Environ.
Microbiol., 56, pp. 3420-3428 (1990), was used to
clone the crylX gene on the Clal restriction
fragments. The C1aI restriction fragments were
ligated to C1aI-digested pEG854 vector DNA
pretreated with calf intestinal alkaline
phosphatase to prevent self-ligation.
Transformation of E. coli HB101 cells with the
ligation mixture was achieved by electroporation
using the high-efficiency transformation procedure
of Dower et al., Nucleic Acids Res., 16, pp. 6127-
6145. Transformed cells were plated on agar plates
of standard LB rmedium containing 50 pg/mi
ampiciliin. Colonies were screened under moderaie
stringency conditions for the presence of the cryIX
gene sequence using the colony blot hybridization
procedure outlined in Example 2. The hybridization
step was performed at 65'C, rather than at 50-55 C
as in Example 2, using the 0.6 kb KpnI-BamHI cryIX
probe described in Example 9. Filter washes were
performed at 65=C in 3X SSC, 0.1% SDS. The crylX
probe hybridized strongly to one E. coli
recombinant colony, designated E. coli strain


'0 91/16434 PCT/U591/02560
- 57 -

EG1082, that contained an 11.8 kb recombinant
plasmid, designated pEG313, that consisted of a 4.6
kb C1aI restriction fragment from B.t. strain
EG6346 inserted into the C1aI restriction site of
cloning vector pEG854.
A circular restriction map of recombinant
plasmid pEG313 is depicted in F'igure 11. The
orientation of the 4.6 kb ClaI restriction fragment
was determined by restriction endonuclease mapping
using methods well known to those skilled in the
art.

Example 10
Expression of the cryIR G ne in E. coli
and Production of Cry%8 ProtaTn
To achieve expression of the crylX gene
in E. coli and to characterize its encoded crystal
protein, a 4.4. kb DNA fragment containing the
crylX gene was inserted into the E. coli cloning
vector pTZ19u, obtained from U.S. Biochemical
Corporation. A circular restriction map of cloning
vector pTZ19u, designated Plac in Figure 12, can be
used to direct the transcription of cloned genes
inserted into the multiple cloning site region
demarcated by the unique HindIIl and EcoRI
restriction sites within the lacZ' gene.
Accordingly, a 4.4 kb ClaI-SstI restriction
fragment containing the entire cryIX gene, as
indicated by the Southern blot analysis in
Example 8, was isolated from the recombinant
plasmid pEG313 (see Figure 11) and ligated to
pTZ19u DNA digested with Accl and SstI, two
restriction endonucleases with cleavage sites
within the multiple cloning site region of the


WO 91/16434 1'CI'/tJS91/02560
- 58

cloning vector. Note that the Accl restriction
site is compatible with that of C1aI, thereby
allowing for efficient ligaticin of the cryIX gene
fragment and orienting the cryIX gene in the same
direction as the lac promoter. The ligation
mixture was used to transform E. coli DH5;yL cells
as described in Example 2. Using the X-gal
screening procedure, a recombinant E. coli colony,
designated EG1083, was recovered that contained a
7.3 kb recombinant plasmid, designated pEG314, that
consisted of a 4.4 kb C1ai-Sstl restriction
fragment derived from pEG313 inserted into the AccI
and Sstl sites of vector pTZ19u. A circular
restriction map of recombinant plasmid pEG314,
containing the cryIX gene inserted downstream from
the lac promoter of pTZ19u, is depicted in
Figure 13.
E. coli strain EG1083, containing pEG314
which carried the crylX gene, was grown in Luria
broth containing 50 pg/ml ampicillin and 1mM
isopropyl-beta-D-thiogalactopyranoside (IPTG) at
376C overnight. IPTG is an inducer of the lac
promoter and is commonly used to optimize
transcription from that promoter in E. coli. After
overnight growth, cells were examined by phase-
contrast microscopy. E. coli strain EG1083 cells,
but not E. coli strain DR5& cells containing
pTZ19u, contained multiple phase-bright inclusions.
Subsequent lysis of the recombinant cells with
lysozyme released the large inclusions, some of
which appeared rhomboid in shape. The inclusions
were purifieci from E. coli strain EG1083 by
Renografin" density .gradient centrifugation and


WO 91/16434 1'C"T/US91/02560
- 59 -

examined by SDS-polyacrylamide gel electrophoresis.
Figure 14 is.a photocopy of the resulting
Coomassie-stained 10% SDS-polyacrylamide gel, in
which lanes 1 and 2 contain CryIX crystal protein
from E. coli strain EG1083 before and after
Renografin' density gradient centrifugation,
respectively. Protein molecular weight standards
are displayed in the leftmost lane. Based on these
standards, the CryIX crystal protein migrates with
an apparent molecular mass of 81 kDa.
Example 11
Insect Toxicity of the CryIR Protein
The insecticidal activity of the 81 kDa
CryIX protein was determined against lepidopteran
species, using Renografin'o density gradient
purified CryIX crystal protein from recombinant E.
coli strain EG1083, which harbors the cr.xX gene on
plasmid pEG314.
Activity was measured using a diet-
surface overlay technique where the surface of an
agar-based artificial diet was covered with an
aqueous suspension containing CryIX protein
crystals. Insect larvae were placed on the diet.
surface after the diluent had evaporated and held
at 28'C for seven days, at which time mortality was
scored.
In this bioassay screening.procedure, the
purified CryIX protein exhibited insecticidal
activity against larvae of Plutella arylostella
(diamondback moth).


WO 91/16434 PCI'/L1S91/02560
- 60 - 2

In another bioassay screening procedure,
using a cell paste of E. coli strain EG1083 that
had produced CryIX protein instead of the purified
CryIX protein, insecticidal activity was exhibited
against larvae of ostrinia nubilalis (European corn
borer).
To assure the availability of materials
to those interested members of the public upon
issuance of a patent on the present application
deposits of the following microorganisms were made
prior to the filing of present application with the
ARS Patent Collection, Agricultural Research
Culture Collection, Northern Regional Research
Laboratory (NRRL), 1815 North University Street,
Peoria, Illinois 61064, as indicated in the
following Table 5:
Table 5

Bacterial Strain NRRL Accession No. Date of Deposit
B.thuringiensis HD73-26 B-18508 June 12, 1989
B.thuringiensis EG6345 B-18633 March 27, 1990

B.thurinqiensis EG1945 B-18635 March 27, 1990
E. coli EG1943 B-18634 March 27, 1990
E. coli EG1083 B-18805 March 29, 1991
These microorganism deposits were made
under the provisions of the "Budapest Treaty on the
International Recognition of the Deposit of
Microorganisms for the Purposes of Patent
Procedure".


WO 91/16434 I'CI'/US91/02560
61 -

The present invention may be embodied in
other specific forms without d+eparting from the
spirit or essential attributes thereof and,
accordingly, reference should be made to the
appended claims, rather than to the foregoing
specification as indicating the scope of the
invention.


WO 91/16434 PCI/IJS91/02560
- 62 -

SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT: Gawron-Burke, Cynthia
Chambers, Judith A.
Gonzalez Jr., Jose M.

(ii) TITLE OF INVENTION: BACILLUS TF3I:JRINGIENSIS crylF and crylX
GENES AND PROTEINS TOXIC TO LEPIDOPTERAN INSECTS

(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Panitch Schwarze et al. c/o A.S. Nadel
(B) STREET: 1601 Market Street, 36th Floor
(C) CITY: Philadelphia
(D) STATE: PA
(E) COUNTRY: U.S.A.
(F) ZIP: 19103

(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/510327
(B) FILING DATE: 16-APR-1990

(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME:- Eqol, Christopher
(B) REGISTRATION NAMBER: 27633
(C) REFERENCE/DOCKET NUMBER: 7205-27 U1
(2) INFORMATION FOR SEQ ID NO:1:'

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4020 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 478..4002

(xi) SEQUENCE DESCRIPTION: SEQ I?) NO:l:


WO 91/16434 PCT/US91/02560

- 63 - 2 0 Pior, S, d
GATCTTCAAA TGAGAAAATA AGGGTATTCC GTATGGGATG CCTTTA'TTT GGTTGGGAAG 60
AAGGATTAAA AATCAAAAAT GTAAATCAGA TATAGTCCAG ATAATTTTTT AAAGAGTGTA 120

.GTATATTAAA AATAATGTTC TTATAACATA TATGTTGATT TTAAGAAAAT ATTTTGTTTA 180
AGAATTCAAT CCATATGAGT ATAAAAAGTT AAAAGGCCCA AAAATAAGTT AAGGGAAATC 240
AACTCTTTAA TACAAAAGTT TATCTCAGGA ATTCTCAACT ATGGATAGCA GGAAGAGAAG 300
TAAGCACACT ATTAACATAT TAGGTCTATT TAAATTAAGG: GCATATAGTG ATATTTTATA 360
AGATTGGTTG CACTTTGTGC ATTTTTTCAT AAGATGAGTC ATATGTTTTA CATTGTAATA 420
CAGTAAGAGG TTTTAGTTTT AAAGAACAAC TATTATGATG AAATGTGGAG GGAACAT 477
ATG GAG AAT AAT ATT CAA AAT CAA TGC GTA CCT TAC AAT TGT TTA AAT 525
Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn
1 5 10 15
AAT CCT GAA GTA GAA ATA TTA AAT GAA GAA AGA AGT ACT GGC AGA TTA 573
Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu
20 25 30
CCG TTA GAT ATA TCC TTA TCG CTT ACA CGT TTC CTT TTG AGT GAA TTT 621
Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe
35 40 45

GTT CCA GGT GTG GGA GTT GCG TTT GGA TTA TTT GAT TTA ATA TGG GGT 669
Va1 Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly
50 55 60

TTT ATA ACT CCT TCT GAT TGG AGC TTA TTT CTT TTA CAG ATT GAA CAA 717
Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln
65 70 75 80
TTG ATT GAG CAA AGA ATA GAA ACA TTG GAA AGG AAC CGG GCA ATT ACT 765
Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95
ACA TTA CGA GGG TTA GCA GAT AGC TAT GAA ATT TAT ATT GAA GCA CTA 813
Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu I1e Tyr Ile Glu Ala Leu
100 105 110
=AGA GAG TGG GAA GCA AAT CCT AAT AAT GCA CAA TTA AGG GAA GAT GTG 861
Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val
115 120 125

CGT ATT CGA TTT GCT AAT ACA GAC GAC GC'T TTA ATA ACA GCA ATA AAT 909
Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asrn
130 135 140

AAT TTT ACA CTT ACA AGT TTT GAA ATC CCT CTT TTA TCG GTC TAT GTT 957
Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val
145 150 155 160
CAA GCG GCG AAT TTA CAT TTA TCA CTA TTA AGA GAC GCT GTA TCG TTT 1005
Gln Ala Ala Asn Leu His Lau Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175


WO 91/16434 PC'T/U591/02560

- 64 - 2, 0 89634
GGG CAG GGT TGG GGA CTG GAT ATA GCT ACT GTT AAT AAT CAT TAT AAT 1053
Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asri His Tyr Asn
180 185 190
AGA TTA ATA AAT CTT ATT CAT AGA TAT ACG AA.A, CAT TGT TTG GAC ACA 1101
Arg Leu I1e Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr
195 200 205

TAC AAT CAA GGA TTA GAA AAC TTA AGA GGT ACT AAT ACT CGA CAA TGG 1149
Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp
210 215 220

GCA AGA TTC AAT CAG TTT AGG AGA GAT TTA ACA CTT ACT GTA TTA GAT 1197
Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Va1 Leu Asp
225 230 235 240
ATC GTT GCT CTT TTT CCG AAC TAC GAT GTT AGA ACA TAT CCA ATT CAA 1245
Ile Va1 Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro 21e Gln
245 250 255
ACG TCA TCC CAA TTA ACA AGG GAA ATT TAT ACA AGT TCA GTA ATT GAG 1293
Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile G1u
260 265 270
GAT TCT CCA GTT TCT GCT AAT ATA CCT AAT GGT TTT AAT AGG GCG GAA 1341
Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu
275 280 285

TTT GGA GTT AGA CCG CCC CAT CTT ATG GAC TTT ATG AAT TCT TTG TTT 1389
Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe
290 295 300

GTA ACT GCA GAG ACT GTT AGA AGT CAA ACT GTG TGG GGA GGA CAC TTA 1437
Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu
305 310 315 320
GTT AGT TCA CGA AAT ACG GCT GGT AAC CGT ATA AAT TTC CCT AGT TAC 1485
Va1 Ser Ser Arg Asn Thr Ala G1y Asn Arg Ile Asn Phe Pro Ser Tyr
325 330 335
GGG GTC TTC AAT CCT GGT GGC GCC ATT TGG ATT GCA GAT GAG GAT CCA 1533
Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro
340 345 350
CGT CCT TTT TAT CGG ACA TTA TCA GAT CCT GTT TTT GTC CGA GGA GGA 1581
Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly
355 360 365

TTT GGG AAT CCT CAT TAT GTA CTG GGG CTT AGG GGA GTA GCA TTT CAA 1629
Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln
370 375 380

CAA ACT GGT ACG AAC CAC ACC CGA ACA TTT AGA AAT AGT GGG ACC ATA 1677
Gln Thr Gly Thr Asn His Tllr Arg Thr Phe Arg Asn Ser Gly Thr Ile
385 390 395 400
GAT TCT CTA GAT GAA ATC CCA CCT CAG GAT AAT AGT GGG GCA CCT TGG 1725
Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp
405 410 415


WO 91/16434 POff'/US91/02560

- 65 - 2 ~3 ~i ~j~~'j ~~~
'U~(1 rc
AAT GAT TAT AGT CAT GTA TTA AAT CAT GTT ACA TTT GTA CGA TGG CCA 1773
Asn Asp Tyr Ser His Val Leu Asn His Va]. Thr Phe Val Arg Trp Pro
420 425 430
GGT GAG ATT TCA GGA AGT GAT TCA TGG AGA GCT CCA ATG TTT TCT TGG 1821
Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp
435 440 445

ACG CAC CGT AGT GCA ACC CCT ACA AAT ACA ATT GAT CCG GAG AGG ATT 1869
Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile
450 455 460

ACT CAA ATA CCA TTG GTA AAA GCA CAT ACA CTT CAG TCA GGT ACT ACT 1917
Thr Gin Ile Pro Leu Val Lys Ala His Thr Lelu Gln Ser Gly Thr Thr
465 470 475 480
GTT GTA AGA GGG CCC GGG TTT ACG GGA GGA GAT ATT CTT CGA CGA ACA 1965
Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr
485 490 495
AGT GGA GGA CCA TTT GCT TAT ACT ATT GTT AAT ATA AAT GGG CAA TTA 2013
Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gin Leu
500 505 510
CCC CAA AGG TAT CGT GCA AGA ATA CGC TAT GCC TCT ACT ACA AAT CTA 2061
Pro Gin Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
515 520 525

AGA ATT TAC GTA ACG GTT GCA GGT GAA CGG ATT TTT GCT GGT CAA TTT 2109
Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe
530 535 540

AAC AAA ACA ATG GAT ACC GGT GAC CCA TTA ACA TTC CAA TCT TTT AGT 2157
Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser
545 550 555 560
TAC GCA ACT ATT AAT ACA GCT TTT ACA TTC CCA ATG AGC CAG AGT AGT 2205
Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser
565 570 575
TTC ACA GTA GGT GCT GAT ACT TTT AGT TCA GGG AAT GAA GTT TAT ATA 2253
Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile
580 585 590
.GAC AGA TTT GAA TTG ATT CCA GTT ACT GCA ACA TTT GAA GCA GPdA TAT 2301
Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala G1u Tyr
595 600 605

GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GCG CTG TTT ACT TCT ATA 2349
Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile
610 615 620

AAC CAA ATA GGG ATA AAA ACA GAT GTG ACG GAT TAT CAT ATT GAT CAA 2397
Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln
625 630 635 640
GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA,TTT TGT CTG GAT GAA 2445
Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu
645 650 655


WO 91/16434 POt'/lJ591 /02560
-66-

AAG CGA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA CTC AGT GAT 2493
Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp
660 665 670
GAG CGG AAT TTA CTT CAA GAT CCA AAC TTC AAA GGC ATC AAT AGG CAA 2541
Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln
675 680 685

CTA GAC CGT GGT TGG AGA GGA AGT ACG GAT ATT ACC ATC CAA AGA GGA 2589
Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg G1y
690 695 700

GAT GAC GTA TTC AAA GAA AAT TAT GTC ACA CTA CCA GGT ACC TTT GAT 2637
Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp
705 710 71:5 720
GAG TGC TAT CCA ACG TAT TTA TAT CAA AAA ATA GAT GAG TCG AAA TTA 2685
Giu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Giu Ser Lys Leu
725 730 735
AAA CCC TAT ACT CGT TAT CAA TTA AGA GGG TAT ATC GAG GAT AGT CAA 2733
Lys Pro Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln
740 745 750
GAC TTA GAA ATC TAT TTG ATC CGC TAT AAT GCA AAA CAC GAA ACA GTA 2781
Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val
755 760 765

AAT GTG CTA GGT ACG GGT TCT TTA TGG CCG CTT TCA GTC CAA AGT CCA 2829
Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gin Ser Pro
770 775 780

ATC AGA A.AG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC CTT GAA TGG 2877
Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp
785 790 795 800
A.AT CCT GAT CTA GAT TGT TCC TGC AGA GAC'GGG GAA AAA TGT GCA CAT 2925
Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His
805 810 815
CAT TCG CAT CAT TTC TCC TTG GAC ATT GAT GTT GGA TGT ACA GAC TTA 2973
His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu
820 825 830
AAT GAG GAC TTA GAT GTA TGG GTG ATA TTC AAG ATT AAG ACG CAA GAT 3021
Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gin Asp
835 840 845

GGC CAT GCA AGA CTA GGA AAT CTA GAG TTT CTC GAA GAG AAA CCA TTA 3069
Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu
850 855 860

GTC GGG GAA GCA CTA GCT CGT GTG AAA AGA GCA GAG AAA AAA TGG AGA 3117
Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg
865 870 875 880
GAT AAA CGT GAA AAA TTG GAA TTG GAA ACA AAT ATT GTT TAT AAA GAG 3165
Asp Lys Arg Glu Lys Leu Giu Leu Giu Thr Asn Ile Val Tyr Lys Glu
885 890 895


WO 91/16434 PCT/US91/02560
-67-
~~090684
GCA AAA GAA TCT GTA GAT GCT TTA TTT GTA AAC TCT CAA TAT GAT CAA 3213
Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln
900 905 910
TTA CAA GCG GAT ACG AAT ATT GCC ATG ATT CAT GCG GCA GAT AAA CGT 3261
Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg
915 920 925

GTT CAT AGA ATT CGG GAA GCG TAT CTT CCA GAG TTA TCT GTG ATT CCG 3309
Val His Arg Ile Arg Glu A1. 'yr Leu Pro Glu Leu Ser Val Ili:~ Pro
930 935 940

GGT GTA AAT GTA GAC ATT TTC GAA GAA TTA AAA GGG CGT ATT TTC ACT 3357
Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr
945 950 955 960
GCA TTC TTC CTA TAT GAT GCG AGA AAT GTC ATT AAA AAC GGT GAT TTC 3405
Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe
965 970 975
AAT AAT GGC TTA TCA TGC TGG AAC GTG AAA GGG CAT GTA GAT GTA GAA 3453
Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu
980 985 990
GAA CAA AAC AAC CAC CGT TCG GTC CTT GTT GTT CCG GAA TGG GAA GCA 3501
Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala
995 1000 1005

GAA GTG TCA CAA GAA GTT CGT GTC TGT CCG GGT CGT GGC TAT ATC CTT 3549
Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu
1010 1015 1020

CGT GTC ACA GCG TAC AAG GAG GGA TAT GGA GAA GGT TGC GTA ACC ATT 3597
Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile
1025 1030 1035 1040
CAT GAG ATC GAG AAC AAT ACA GAC GAA CTG AAG TTT AGC AAC TGC GTA 3645
His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Va1
1045 1050 1055
GAA GAG GAA GTC TAT CCA AAC AAC ACG GTA ACG TGT AAT GAT TAT ACT 3693
Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr
1060 1065 1070
GCA AAT CAA GAA GAA TAC GGG GGT GCG TAC ACT TCC CGT AAT CGT GGA 3741
Ala Asn G1n Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly
1075 1080 1085

TAT GAC GAA ACT TAT GGA AGC AAT TCT TCT GTA CCAi GCT GAT TAT GCG 3789
Tyr Asp Glu Thr Tyr Gly f3er Asn Ser Ser Val Pro Ala Asp Tyr Ala
1090 1095 1100

TCA GTC TAT GAA GAA AAA TCG TAT ACA GAT GGA CGA AGA GAC AAT CCT 3837
Ser Val Tyr Glu Glu Lys .~er Tyr Thr Asp Gly Arg Arg Asp Asn Pro
1105 1110 1115 1120
TGT GAA TCT AAC AGA GGA TAT GGG GAT TAC ACA CCA CTA CCA GCT GGC 3885
Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly
1125 1130 1135


WO 91/16434 PC'I'/US91/02560

- 68 - 2 008 ~~
TAT GTG ACA AAA GAA TTA GAG TAC TTC CCA GAA ACC GAT AAG GTA TGG 3933
Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lyn Val T:p
1140 1145 1150
ATT GAG ATC GGA GAA ACG GAA GGA ACA TTC ATC GTG GAC AGC GTG GAA 3981
Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu
1155 1160 1165

TTA CTC CTT ATG GAG GAA TAGTCTCATA CAAAATTAGT T 4020
Leu Leu Leu Met Glu Glu
1170 117
(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1174 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn
1 5 10 15
Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu
20 25 30
Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe
35 40 45

Va1 Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly
50 55 60
Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu G1n Ile Glu Gln
65 70 75 80
Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95

Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110
Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val
115 120 125
Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn
130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val
145 150 155 160
Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175

Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn
180 185 190


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06{.~d">':~

Arg Leu Ile Asn Leu I1e His Arg Tyr Thr Lys His Cys Leu Asp Thr
195 200 205
Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp
210 215 220
Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp
225 230 235 240
Ile Va1 Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln
245 250 255

Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val I1e Glu
260 265 270
Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu
275 280 285
Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe
290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu
305 310 315 320
Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr
325 330 335

Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro
340 345 350
Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly
355 360 365'
Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe G1n
370 375 380

Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg.Asn,Ser Gly Thr Ile
385 390 395 400
Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp
405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro
420 425 430
Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp
435 440 445
Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile
450 455 460

Thr Gln Ile Pro Leu Vai Lys Ala His Thr Leu Gin Ser Gly Thr Thr
465 470 475 480
Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr
485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr I1e Val Asn Ile Asn Gly Gln Leu
500 505 510


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- 70 - ~~ ~ ~ a n l~c~~~
~~y~ t~
Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
515 520 525
Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe
530 535 540
Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser
545 550 555 560
Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phie Pro Met Ser Gln Ser Ser
565 570 575

Phe Thr Va1 Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr I1e
580 585 590
Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr
595 600 605
Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile
610 615 620

Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln
625 630 635 640
Val Ser Asn Leu Va1 Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu
645 650 655

Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp
660 665 670
Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly I1e Asn Arg Gln
675 680 685
Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly
690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp
705 710 715 720
Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glu Ser Lys Leu
725 730 735

Lys Pro Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr Ile Glu Asp Ser G1n
740 745 750
Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val
755 760 765
Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gln Ser Pro
770 775 780

Yle Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp
785 790 795 800
Asn Pro Asp Leu Asp Gys Ser Cys Arg Asp Gly Glu Lys Cys Ala His
805 810 815

His Ser His His Phe Ser Leu Asp Ile AspVal Gly Cys Thr Asp Leu
820 825 830


WO 91/16434 PfT/US91/02560
s ~~f,r ~ i'~ ~ a {
- ~
Asn Glu Asp Leu Asp Val Trp Val I1e Phe Lys I].e Lys Thr G1n Asp
835 840 845

Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu
850 855 860
Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg
865 870 875 880
Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu
885 890 895
Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln
900 905 910

Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg
915 920 925
Val His Arg 21e Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro
930 935 940
Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Tie Phe Thr
945 950 955 960
Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe
965 970 975

Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu
980 985 990
Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala
995 1000 1005
Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu
1010 1015 1020

Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile
1025 1030 1035 1040
His Glu I1e Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val
1045 1050 1055
Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr
1060 1065 1070

Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly
1075 1080 1085
Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala
1090 1095 1100
Ser Val Tyr Glu Glu I,ys Ser Tyr Thr Asp Gly Arg Arg Asp Asn Pro
1105 1110 1115 1120
Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly
1125 1130 1135
Tyr Va1 Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp
1140 1145 1150


WO 91/16434 PCT/U591/02560
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~~~~0 ('3
Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe I1e Val Asp Ser Val Glu
1155 1160 1165
Leu Leu Leu Met Glu Glu
1170
(2) INFORMATION FOR SEQ ID NO:3:

( i ) SEQUENCE CFiARACTERISTICS :
(A) LENGTH: 1629 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 488..1629

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TTAAATATCG TTTTCAAATC AATTGCCTAA GAGCATCATT ACAAATAGAT AAGTAATTTG 60
=. TTGTAATGAA AAACGGACAT CACCTCCATT GAAACGGTGA GATGTCCGTT TTACTATGTT 120
ATTTTCTAGT AATACATATG TACAGAGCAA CTTAATTAAG CAGAGATATT TTCCCCTATC 180
GATGAAAATA TCTCTGCTTT TTCTTTCTTT ATTCGGTATA TGCTTTACTT GTAATTGAAA 240
ATAAAGCACT AATAAGAGTA TTTATAGGTG TTTGAAGTTA TTTCAGTTTA TTTTTAAAGG 300
AGGTTTAAAA ACGTTAGAAA GTTATTAAGG AATAATACTT ATTAGTAAAT TCCACATATA 360
TTTTATAATT AATTATGAAA TATATGTATA AATTGAAAAT GCTTTATTTG ACATTACAGC 420
TAAGTATAAT TTTGTATGAA TAAAATTATA TCTGAAAATT AAATAATATT ACAGTGGAGG 480
GATTAAT ATG AAA CTA AAG AAT CCA GAT AAG CAT CAA AGT TTT TCT AGC 529
Met Lys Leu Lys Asn Pro Asp Lys His G1n Ser Phe Ser Ser
1 5 10

AAT GCG AAA GTA GAT AAA ATC TCT ACG GAT TCA CTA AAA AP.T GAA ACA 577
Asn Ala Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr
15 20 25 30
GAT ATA GAA TTA CAA AAC ATT AAT CAT GAA GAT TGT TTG AAA ATA TCT 625
Asp Ile Glu Leu Gin Asn I].e Asn His Glu Asp Cys Leu Lys Ile Ser
35 40 45
GAG TAT GAA AAT GTA GAG CC:G TTT GTT AGT GCA TCA ACA ATT CAA AC.11. 673
Glu Tyr Giu Asn Val Glu Pro Phe Val Ser Ala Ser Thr Ile Gin Thr
50 55 60
GGT ATT AGT ATT GCG GGT AAA ATA C.TT GGC ACC CTA GGC GTT CCT TTT 721
Gly Ile Ser Ile Ala Gly Lys Ile Leu Gly Thr Leu Gly Va1 Pro Phe


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65 70 75 cp080639~~
GCA GGA yA GTA GCT AGT CTT TAT AGT TTT ATC TTA GGT GAG CTA TGG( 769
Ala Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly Glu Leu Trp
80 85 90

CCT AAG GGG AAA AAT CAA TGG GAA ATC TTT ATG GAA CAT GTA GAA GAG 817
Pro Lys Gly Lys Asn Gln Trp Glu Ile Phe Met Glu His Val Glu Glu
95 100 105 110
ATT ATT AAT CAA AAA ATA TCA ACT TAT GCA AGA AAT AAA GCA CTT ACA 865
Ile Ile Asn Gln Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr
115 120 125
GAC TTG AAA GGA TTA GGA GAT G C C TTA GCT GTC TAC CAT GAA TCG CTT 913
Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val Tyr His Glu Ser Leu
130 135 140
GAA AGT TGG GTT GGA AAT CGT AAG AAC ACA AGG GCT AGG AGT GTT GTC 961
Glu Ser Trp Val Gly Asn Arg Lys Asn Thr Arg Ala Arg Ser Val Val
145 150 155

AAG AGC CAA TAT ATC GCA TTA GAA TTG ATG TTC GTT CAG AAA CTA CCT 1009
Lys Ser Gln Tyr Ile Ala Leu Glu Leu Met Phe Val Gln Lys Leu Pro
160 165 170

TCT TTT GCA GTG TCT GGA GAG GAG GTA CCA TTA TTA CCG ATA TAT GCC 1057
Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala
175 180 185 190
CAA GCT GCA AAT TTA CAT TTG TTG CTA TTA AGA GAT GCA TCT ATT TTT 1105
Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe
195 200 205
GGA AAA GAG TGG GGA TTA TCA TCT TCA GAA ATT TCA ACA TTT TAT AAC 1153
Gly Lys Glu Trp Gly Leu Ser Ser Ser Glu Ile Ser Thr Phe Tyr Asn
210 215 220
CGT CAA GTC GAA CGA GCA GGA GAT TAT TCC GAC CAT TGT GTG AAA TGG 1201
Arg G1n Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp
225 230 235

TAT AGT ACA GGT CTA AAT AAC TTG AGG GGT ACA AAT GCC GAA AGC TGG 1249
Tyr Ser Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp
240 245 250

GTT CGT TAT AAT CAA TTT CGT AAA GAT ATG ACA TTA ATG GTA CTT GAT 1297
Val Arg Tyr Asn Gln Phe Arg Lys Asp Met Thr Leu Met Val Leu Asp
255 260 265 270
TTA GTC GCA CTA TTC CCA AGC TAT GAT ACA CTT GTA TAT CCA ATT AAA 1345
Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Leu Val Tyr Pro Ile Lys
275 280 285
ACT ACT TCT CAA CTT ACA AGA GAA GTA TAT ACA GAC GCA ATT GGG ACA 1393
Thr Thr Ser Gin Leu Thr ls,rg Glu Val Tyr Thr Asp Ala Ile Gly Thr
290 295 300
GTA CAT CCG AAT GCA AGT TTT GCA AGT ACG ACT TGG TAT AAT AAT AAT 1441
Val His Pro Asn Ala Ser Phe Ala Ser Thr Thr Trp Tyr Asn Asn Asn


WO 91/16434 PCf/US91/02560
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305 310 315

GCC CCT TCG TTC TCT ACC ATA GAG TCT GCT GTT GTT CGA AAC CCG CAT 1489
Ala Pro Ser Phe Ser Thr 21e Glu Ser Ala Val Val Arg Asn Pro His
320 325 330

CTA CTC GAT TTT CTA GAA CAA GTT ACA ATT TAC AGC TTA TTA AGT AGG 1537
Leu Leu Asp Phe Leu Glu Gln Val Thr Ile ",Cyr Ser Leu Leu Ser Arg
335 340 :345 350
TGG AGT AAC ACT CAG TAT ATG AAT ATG TGG GGA GGA CAT AGA CTT GAA 1585
Trp Ser Asn Thr Gln Tyr Met Asn Met Trp Gly Gly His Arg Leu Glu
355 360 365
TTC CCA ACA ATC GGA GGA ATG TTA AAT ACC TCA ACA CAA GGA TC 1629
Phe Arg Thr I1e Gly Gly Met Leu Asn Thr Ser Thr Gln Gly
370 375 380
(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 380 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Lys Leu Lys Asn Pro Asp Lys His Gin Ser Phe Ser Ser Asn Ala
1 5 10 15
Lys Val Asp Lys Ile Ser Thr Asp Ser Leu Lys Asn Glu Thr Asp Ile
20 25 30
Glu Leu Gln Asn Ile Asn His Giu Asp Cys Leu Lys 21e Ser Glu Tyr
35 40 45

Glu Asn Val Glu Pro Phe Val Ser Ala Ser Thr I1e Gin Thr Gly Ile
50 55 60
Ser Ile Ala Gly Lys Ile Leu Gly Thr Leu Gly Val Pro Phe Ala Gly
65 70 75 80
Gln Val Ala Ser Leu Tyr Ser Phe Ile Leu Gly Glu Leu Trp Pro Lys
85 90 95

Gly Lys Asn Gln Trp Glu I1e Phe Met Glu His Val Glu Glu Ile Ile
100 105 110
Asn Gin Lys Ile Ser Thr Tyr Ala Arg Asn Lys Ala Leu Thr Asp Leu
115 120 125
Lys Gly Leu Gly Asp Ala Leu Ala Val Tyr His Glu Ser Leu Glu Ser
130 135 140

Trp Val Gly Asn Arg Lys Asn Thr Arg Ala Arg Ser Val Val Lys Ser
145 150 155 160


WO 91/16434 PCT/[!S91/02560
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~+J ~ ~Jd
-75-

Gln Tyr Ile Ala Leu Glu Leu Met Phe Val Gln Lys Leu Pro Ser Phe
165 170 175
Ala Val Ser Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala Gln Ala
180 185 190
Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe Gly Lys
195 200 205

Glu Trp Gly Leu Ser Ser Ser Glu I1e Ser Thr Phe Tyr Asn Arg Gln
210 215 220
Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp Tyr Ser
225 230 235 240
Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Val Arg
245 250 255
Tyr Asn Gln Phe Arg Lys Asp Met Thr Leu Met Va1 Leu Asp Leu Val
260 265 270

Ala Leu Phe Pro Ser Tyr Asp Thr Leu Val Tyr Pro Ile Lys Thr Thr
275 280 285
Ser Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His
290 295 300
Pro Asn Ala Ser Phe Ala Ser Thr Thr Trp Tyr Asn Asn Asn Ala Pro
305 310 315 320
Ser Phe Ser Thr Ile Glu Ser Ala Val Val Arg Asn Pro His Leu Leu
325 330 335

Asp Phe Leu Glu Gin Val Thr Ile Tyr Ser Leu Leu Ser Arg Trp Ser
340 345 350
Asn Thr Gln 'Tyr Met Asn Met Trp Gly Gly His Arg Leu Glu Phe Arg
355 360 365
Thr Ile Gly Gly Met Leu Asn Thr Ser Thr Gln Gly
370 375 380