Note: Descriptions are shown in the official language in which they were submitted.
CA 02103248 2002-08-29
1
DESCRIPTION
NOVEL, BACILLUS TRZWNGMM ISOLATES ACr1VE AGAINST
HYMENOPTERANPF= AND GENES ENCODING
HYMENOPTERAN-ACTIVE TONES
Background of the Invention
The development of biological control agents as alternatives to chemical
insecticides for
the control of important pest species is a subject of increasing interest.
Concerns for the
environment and exposure of man to harmful substances in air, food and water
have stimulated
legislation and restrictions regarding the use of chemical pesticides,
particularly for pests found
in the urban environment. Control of insect pests in urban areas is highly
desirable but exposure
to chemical pesticides in the household and from lawns and gardens is of great
concern to the
public. If given a choice, most people would use a non-toxic biological
control rather than a toxic
chemical to control insects in the urban environment. The problem is that very
few biological
alternatives to chemical insecticides are available for purchase and use by
the public.
Bacillus thuringiensic (B.t) produces an insect toxin designated as b-
endotoxln. It is
synthesized by the B.t sporulating cell. The toxin, upon being ingested in its
crystalline form by
susceptible insects, is transformed into biologically active moieties by the
insect gut juice
proteases. Tice primary target is insect cells of the gut epithelium, which
are rapidly destroyed.
The reported activity spectrum ofB.t, covers insect species within the order
Lepidoptera,
many of which are major pests in agriculture and forestry. The activity
spectrum also includes the
insect order Diptera, which includes mosquitos and black flies. See Couch, T.L
(1980)
"Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,"
Developments in Ltdustria!
Microbiology 22:61-76; Beegle, C.C., (1978) "Use of Entomogenous Bacteria in
Agroecosystems,"
Developments in Industrial Microbiology 20:97-104. Krieg, et al (1983) Z ang.
Ent 96:500-508,
describe a at isolate named Bacillus thwingiensis var. tenebrionis, which is
reportedly active
against two beetles in the order Coleoptera. These are the Colorado potato
beetle, Leptinotarsa
decemlineara, and ABelasrica ab-i. In European Patent Application No. 0 202
739 there is
disclosed a novel B.t isolate active against Coleoptera. It is known as A
thwirtgiensis var. san
diego (BJ sd). U.S. Patent No. 4,966,765 discloses the eoleoptesan-active
Bacillus t usingiensis
isolate At PS86BL
Ants comprise a large group of insects (family Formic lddae) from the
taxonomic order,
Hymenoptera. They are among the most common house pests. In many situations,
ants are a
WO 92/20802 PCF/US92/04316
2103248 2
nuisance pest. Foraging ants create problems with hygiene in hospitals and the
food industry.
Ants also create problems in agriculture. Damage can be caused by direct
feeding on plants.
Harvester and fire ants are commonly associated with this type of damage
(Holldobler, B., E.O.
Wilson [1990] The Ants, Belkap Press, Cambridge, Mass. 732 pp.) Some ants
cause indirect
damage by nurturing and protecting sap feeding insects such as mealybugs and
aphids. Ants,
particularly in the genus Solenopsis are capable of producing extremely
painful stings to humans.
It has been estimated that approximately 10,000 stings occur each year
(Habermehl, G.G. [1981]
Venomous Animals and Their Taxies, Springer-Verlag, NY, 195 pp.). The pharaoh
ant
(Monomoriusm pharaonis) is primarily an urban pest. However, this species can
also be an
agricultural pest and damage to corn has been noted (Ebeling, W. [1978] Urban
Entomology, UC
Press, Berkeley, Calif, 695 pp.).
Carpenter ants, Camponotus spp., are distributed throughout North America.
Some of
the more common and/or studied species include C modoc in the Pacific
northwest, C clarithorax
in southern California, and the black, red, and Florida carpenter ants, C.
pennsylvanicus, C.
noveboracensis and C abdominalis, respectively, in the east (Ebeling, W.
[1978] Urban Entomology,
Univ. Calif: Berkeley p. 209-213). Public concern over carpenter ants has been
increasing due
to the greater probability of structural infestations as suburban developments
extend into the
forest habitats of the ants.
Pestiferous species of carpenter ants may be considered nuisance pests because
of their
foraging activity inside homes More significant damage occurs when carpenter
ants extend their
nests into sound wood. Nesting sites may be located in live and dead trees,
sometimes resulting
in damage to shade trees. Nests may also be established in walls and support
beams of structures,
or in voids within doors, walls, and furniture. Preference for moist or
decaying wood has been
reported, but nesting sites are not restricted to such areas. Carpenter ant
populations develop
relatively slowly with colonies of 300-2,000 workers being produced over a 2 -
year or longer period
for various species. Thepresence of reproductives follows this slow
development since their
production has been reported only from well established colonies (Hansen, LD.,
R.D. Akre [1985]
Biology of carpenter ants in Washington state (Hymenoptera: Formicidae:
Camponotus).
Melanderia 43. 62 p.; Pricer, J.I.. [19081 Biol. Bull. 14:177-218). Despite
the slow colony growth,
large colonies with satellite colonies have been found. Worker movement occurs
between the
main colony and the satellites, which serve as areas for further brood
development and colony
expansion (Hansen and Akre [1985], supra).
Current methods for controlling structural infestations of carpenter ants
include sanitation
of potential and current nest sites, minimizing access to structures (eg.
preventing the contact of
tree branches with a structure), and the application of insecticides to repel
(perimeter spray
barriers) and/or eliminate carpenter ants. The use of boric acid dust in dry,
wall voids is reported
to be effective for up to 20 years (Hansen and Akre, supra).
Recommendations for the chemical control of established structural
infestations in the
home are often accompanied with warnings of possible hazards to the applicator
as well as
.~'Y!:l l./:e:, ..; f.l...rr.... ..d'. -. ..`S~'J k.T.' f f .. .. .. .. .. ~
., .:~t. .= ..i. .5. t _...... .._. -. ~... .. - ..
WO 92/20802 PCI /US92/04316
2103248
3
children and pets. Alternative control methods such as effective biological
control agents have
not been found (Akre, R.D., L.D. Hansen, A.L. Antonelli [1989] Fat. Bull.
Washington State
Univ. Coop. Fat. Serv.1989 rev no. EB 0818, 6 pp.).
A need clearly exists for a safe, effective biological control agent for
carpenter ants-
Pharaoh ants, Monomorium pharaonic, have been described as "... the most
persistent
and difficult of all our house-infesting ants to control or eradicate" (Smith,
M.R. [1965] USDA-
ARS Tech. Bull. No. 1326, 105 pp.). It is a tropical species which has
extended its range to more
temperate regions by establishing colonies in heated buildings. Pharaoh ants
frequently infests
buildings where food is prepared, and have been found to carry pathogenic
organisms (Beatson,
SH. [1972] Lancet 1425-427).
attributed to their inaccessible nesting
The difficulty in controlling pharaoh ants may be and
sites, rapid population growth, and dispersion of colonies. Their small size
allows establishment
of colonies in any suitable location, including unusual places such as between
books and in stored
clothing. With multiple queen colonies, and the warm (30 C), humid (63-80%
RH) conditions
that favor pharaoh ants, large colonies can develop rapidly. Portions of these
large colonies may
disperse to form new colonies at any time, probably in response to
overcrowding and unfavorable
mic roenvironmental conditions. Unlike other ant species, pharaoh ants do not
exhibit intercolony
aggression. This permits the adoption of ants from other colonies and may
further enhance the
establishment of new colonies and reinfestations. Pharaoh ants also forage for
food more than
35 m from the nest without distinct trail following, and thus make nests
difficult to find and
eradicate.
Control methods for pharaoh ants emphasize the use of insect growth regulators
(IOR)
or toxicants incorporated into baits. Properly implemented bait programs are
effective, however
it may take over a month to achieve control. Insecticide applications, while
fast acting, usually
do not eliminate colonies, and may be unacceptable in certain areas where
toxic residues are a
concern. In addition, insecticide applications are generally not compatible
with bait programs.
A need exists for safe and effective biological control agents for pharaoh
ants.
Brief Summary of the Im-ention
The subject invention concerns novel Bacillus thunngiensis (Ba.) isolates and
genes
therefrom which encode novel hymenopteran-active proteins. The novel Rt.
isolates, known
herein as Bacillus t/uaingiensis PS14OE2 (Bt PS14OE2), Bacillus duaingiensis
PS86Q3 (Bt.
PS86Q3) and Bacillus duautgiens&c PS211B2 (Rt. PS211B2) have been shown to be
active against,
for example, the pharaoh ant (Monomorium pharaonic). Toxins of the subject
invention control,
for example, fire ants, carpenter ants, argentine ants, and pharaoh ants.
The subject invention also includes mutants of the above isolates which have
substantially
the same pesticidal properties as the parent isolate. Procedures for making
mutants are well
known in the microbiological art. Ultraviolet light and nitrosoguanidine are
used extensively
toward this end.
i1/L6IF.frr5.r.f rh!!,.~.t.....j. 9?r .,.ifs It ;.i~'=!",~. 1'. _. 1. 1.. ._
~A=! ...L ..... ?~. E?f:rr. -......... .... ,,_,. },.... .. r'. u. ,.. .y-.,.
.., ... .
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2103248 4
The subject invention also concerns novel toxins active against ants. A
further aspect of
the invention concerns genes coding for these formicidal toxins. The subject
invention provides
the person skilled in this art with a vast array of formicidal toxins, methods
for using these toxins,
and genes that code for the toxins. The genes or gene fragments of the
invention encode Bacillus
thuringiensis d-endotoxins which have formicidal activity. The genes or gene
fragments can be
transferred to suitable hosts via a recombinant DNA vector.
One aspect of the invention is the discovery of a generalized chemical formula
common
to a wide range of formicidal toxins. This formula can be used by those
skilled in this art to
obtain and identify a wide variety of toxins having the desired formicidal
activity. The subject
invention concerns other teachings which enable the skilled practitioner to
identify and isolate
ant-active toxins and the genes which code therefor. For example,
characteristic features of ant-
active toxin crystals are disclosed herein. Furthermore, characteristic levels
of ammo acid
homology can be used to characterize the toxins of the subject invention. Yet
another
characterizing feature pertains to immunoreactivity with certain antibodies.
Also, nucleotide
probes specific for genes encoding toxins with formicidal activity are
described. Thus, the
identification of toxins of the subject invention can be accomplished by
sequence-specific motifs,
overall sequence similarity, immunoreactivity, and ability to hybridize with
specific probes.
In addition to the teachings of the subject invention which broadly define At.
toxins with
advantageous formicidal activity, a further aspect of the subject invention is
the provision of
specific formicidal toxins and the nucleotide sequences which code for these
toxins. One such
toxin- is the gene expression product of isolate PS86Q3.
Brief Description of the Drawings
Figure 1 is a photograph of a standard SDS polyacrylamide gel of At PS140E2,
and8 t.
PS86Q3.
Figure 2 is a photograph of a standard SDS polyacrylamide gel showing alkali-
soluble
proteins of Bt PS211132 compared to a protein standard.
Figures 3-5 are transmission electron micrographs of ultrathin sections of the
ant-active
At. strains (Figure 3 is At. PS14E2, Figure 4 is At. PS86Q3; and Figure 5 is
At. PS211B2). Cells
were embedded in an epoxy resin and stained with uranyl acetate and lead
citrate.
Brief Description of the Sequences
SEQ ID NO.1 is the nucleotide sequence of gene 17a.
SEQ ID NO.2 is the amino acid sequence of protein 17a.
SEQ ED NO.3 is the nucleotide sequence of gene 17b.
SEQ ID NO.4 is the amino acid sequence of protein 17b.
SEQ ID NO.5 is the nucleotide sequence of gene 33F2.
SEQ ID NO.6 is the amino acid sequence of protein 33F2.
SEQ ID NO.7 is the nucleotide sequence of gene 8603(a).
CA 02103248 2002-08-29
SEQ ID NO.8 is the amino add sequence of protein 86Q3(a).
SEQ ID NO.9 is the nucleotide sequence of gene 63B.
SEQ ID NO. 10 is the amino acid sequence of protein 63B.
SEQ ID NO.11 is the amino acid sequence of a probe which can be used according
to
5 the subject invention.
SEQ ID NO. 12 is DNA coding for the amino acid sequence of SEQ ID NO. 11.
SEQ ID NO. 13 is DNA coding for the amino add sequence of SEQ ID NO. 1L
SEQ ID NO.14 is the amino add sequence of a probe which can be used according
to
the subject invention.
SEQ ID NO. 15 is DNA coding for the amino add sequence of SEQ ID NO. 14.
SEQ ID NO. 16 is DNA coding for the amino add sequence of SEQ ID NO. 14.
SEQ ID NO. 17 is the N-terminal amino add sequence of 17a.
SEQ ID NO. 18 is the N-terminal amino add sequence of 17b.
SEQ ID NO. 19 is the N-terminal amino add sequence of 86Q3(a).
SEQ ID NO.20 is the N-terminal amino acid sequence of 63B.
SEQ ID NO.21 is the N-terminal amino acid sequence of 33F2.
SEQ ID NO.22 is an internal amino acid sequence for 63B.
SEQ ID NO.23 is a synthetic oligonudcotide derived from 17.
SEQ ID NO.24 is the forward oligonucleotide primer from 63B.
SEQ ID NO.25 is the reverse oligonucleotide primer from 63B.
SEQ ID NO.26 is oligonucleotide probe 33F2A.
SEQ ID NO.27 is oligonudeotide probe 33F2B.
SEQ ID NO. 29 is a reverse primer used according to the subject invention.
SEQ ID NO.29 is an oligonudeotide derived from the N-terminal amino acid
sequence
of 86Q3(a) (SEQ ID NO. 19).
SEQ ID NO. 30 is the amino acid sequence coded for by an oligonucleotide used
according to the subject invention (SEQ ID NO. 31).
SEQ ID NO.31 is an oligonucleotidc which codes for the amino acid sequence of
SEQ
ID NO. 30.
SEQ ID NO.32 is the amino acid sequence coded for by the oligonucleotide of
SEQ ID
NO. 33.
SEQ ID NO.33 is a DNA sequence coding for the peptide of SEQ ID NO. 32.
SEQ ID NO.34 is the reverse complement primer to SEQ ID NO. 38, used according
to
the subject invention.
SEQ ID NO.35 is a forward primer according to the subject invention.
SEQ ID NO.36 is an amino acid sequence according to the subject invention.
SEQ ID No. 37 is a reverse primer according to the subject invention.
SEQ ID NO. 38 is the nematode (NEMI) variant of'region 5 of HSfte and
Whiteley,
Microbiological Reviews, (1989) Vol. 53, p. 242-255.
WO 92/20802 PCr/US92/04316
42 10312.18 6
Detailed Disclosure of the Invention
One aspect of the subject invention is the discovery of Bacillus thuringiensis
isolates having
activity against ants. The novel Bacillus thuringiensis isolates of the
subject invention have the
following characteristics in their biologically pure form:
Characteristics of At PS14OE2
Colony morphology-large colony, dull surface, typical At.
Vegetative cell morphology typical At.
Culture methods-typical for At.
Inclusions-an elliptical coated inclusion outside the exosporium, and a long
inclusion inside the exosporium
Approximate molecular weight of alkaliVSDS-soluble polypeptides (kDa)-78, 70,
Serotype-6, entomocidus.
15 Characteristics of At. PS86Q3
Colony morphology-large colony, dull surface, typical At.
Vegetative cell morphology-typical AL
Culture methods-typical for B.L
Inclusions-long amorphic inclusion and a small inclusion, both of which remain
with the
20 spore after lysis
Approximate molecular weight of alkali SDS-soluble polypeptides (kDa)-155,
135, 98,
62,58
Serotype-new serotype (not H-1 through H-27).
25 Characteristics of At. PS211132
Colony morphology: large colony, dull surface, typical R L
Vegetative cell morphology-typical BL
Culture methods-typical for At
Inclusions-large round amorphic inclusion with coat, and elliptical inclusion
30 Approximate molecular weight of alkali/SDS-soluble polypeptides (kDa)--
175,130, 100,
83, 69, 43, 40, 36, 35, 34 and 27
Serotype-6, entomocidus.
A comparison of the characteristics of B thuringiensis PS140E2 (BL PS140E2),
B.
35 thuringiensis PS86Q3 (At. PS86Q3), B thuringiensis PS211B2 (Bt. PS211I32),
B. thuringiensis var.
san diego (BL.sd), and B. thuringiensis var. Iwrstaki (HD-1) is shown in Table
1.
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Table 1. Comparison of B.t PS14OE2, B.t. PS86Q3, Rt PS211B2, Bt.sd, and B.L HD-
1
B.t. PS14OE2 Bt. PS86Q3 llt. PS211B2 B.L HD-1 B.ts d
Inclusions: Ellipse and 2 1 long and 1 Large Bipyramid Flat square
small or 2 small amorphic
inclusions inclusions
Approximate 78,000 155,000 175,000 130,000 72,000
molecular wt. of 70,000 135,000 130,000 68,000 64,000
proteins by 35,000 98,000 100,000
SDS-PAGE 62,000 83,000
58,000 69,000
43,000
40,000
36,000
35,000
34,000
27,000
Host range Hymenoptera Hymenoptera J Hymenoptera Lepidopteran Coleoptera
(Colorado
and Coleopteran Potato Beetle
In addition to the ant-active B.t isolates described herein, the subject
invention concerns
a vast array of B.t b-endotoxins having formicidal activity. In addition to
having formicidal
activity, the toxins of the subject invention will have one or more of the
following characteristics:
1. An amino acid sequence according to the generic formula disclosed herein.
2. A high degree of amino acid homology with specific toxins disclosed herein.
3. A DNA sequence encoding the toxin wherein said sequence hybridizes with
probes or genes disclosed herein.
4. A nucleotide sequence which can be amplified using primers disclosed
herein.
5. A crystal toxin presentation as described herein.
6. Immunoreactivity to an antibody raised to a toxin disclosed herein.
One aspect of the subject invention concerns the discovery of a generic
chemical formula
(hereinafter referred to as the Generic Formula) which can be used to identify
toxins having
activity against ants. This formula describes toxin proteins having molecular
weights in excess of
130,000kDa. The Generic Formula below covers those amino acids in the N-
terminal region
extending two amino acids past the invariant proline residue encountered at
amino acid number
695 in the sequence of 86Q3(a). The organization of the toxins within this
class is delineated by
the following generic sequence motif that is the ultimate determinant of
structure and function.
1 MOXLUEBYPx BXYUBLXxxx xxxXXXXXXX XXXXXBXXxX EXXXKXXXKX
XxxxxxXJXX XXBXXXXXXX XXI-XXXXXXX XXLZBLZBxB PXXXXXXXXX
101 XXBBXXBXXX XXXXXXXXKX xxLBXXBXXXBXXBBXXXBX XXXXXXXUXX
BXZLUXXXXX XXXOBXXXX* XX*xxxxxxx
201 xxxxxXXUZX XOXXLXXBxx xxxxxxxXXE XXXXXxxxXL PXYOXBOXXH
LBLXJXXLxx xxxxxXKXXB XXJXXxBXXXK XXLXXXLXXX XLOBXXXBXX
301 XLXXXxXXXJ xXZXXXXXXY BJXBOXX*LEBXXXXPOBEX XXYXXxxxxx
XLXXOKXLXZ XxxxxxXXXX BXXXXXZXXXX ZXXXXXXxXX XXXBXXXXXX
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8
401 XXXXBxxxxx xxxxXXXXXX LXXXXXXXXX XXX*xxXXXX Xxxxxxxxxx
XXXXXXXXXX XXXXX*XXXX XXPLXXX*XJ XxXXXXXXXX XXXXXBXXXX
501 XXZXXXXXXX xx*x*XXXXX XXXXXXXxxx XXXXXXXLXX LYXXXXXXXJ
XXXUXBXBXB ZXXXXXEXXX XXBXZXXXXX XXBXXXXBXx xxXXLtxxxxx
601 XxxxxxxxxE XLUZXUXBXL XXXUXBXBXB XXXXXXXYXL K*KUPZXXXX
XXXBXBEXXX xUXBXXXXXX XZXXXXXXZx XXXXXXYXBX ZXOxxxxxxX
701 xXLXxxxxxx xxxXUXXXXB BLEKLEBBPX X
Numbering is for convenience and approximate location only.
Symbols used:
A = ala G = gly M = met S = ser
C=cys H=his N=asn T=thr
D=asp I=ile P=pro V=val
E=glu K=lys Q=gin W=trp
F = phe L = leu R = arg Y =tyr
K=KorR
E=EorD
L=Lori
B=M,L,1,V,orF
J=K,R,E,orD
O=AorT
U=Nor Q
Z=GorS
X any naturally occurring amino acid, except C.
= any naturally occurring amino acid.
x = any naturally occurring amino acid, except C (or complete omission of any
amino
acids).
Where a stretch of wild-card amino acids are encountered (X(n) or x(n) where
n>2),
repetition of a given amino acid should be avoided. Similarly, P, C, E, D, K,
or R utilization
should be minimized.
Formicidal toxins according to the - Generic Formula of the subject invention
are
specifically exemplified herein by the toxin encoded by the gene designated
86Q3(a). Since this
toxin is merely exemplary of the toxins represented by the Generic Formula
presented herein, it
should be readily apparent that the subject invention further comprises
equivalent toxins (and
nucleotide sequences coding for equivalent toxins) having the same or similar
biological activity
of 86Q3(a). These equivalent toxins will have amino acid homology with
86Q3(a). This amino
acid homology will typically be greater than 50%, preferably be greater than
75%, and most
preferably be greater than 90%. The amino acid homology will be highest in
certain critical
WO 92/20802 2103248 PCT/US92/04316
9
regions of the toxin which account for biological activity or are involved in
the determination of
three-dimensional configuration which ultimately is responsible for the
biological activity. In this
regard, certain amino acid substitutions are acceptable and can be expected if
these substitutions
are in regions which are not critical to activity or are conservative amino
acid substitutions which
do not affect the three-dimensional configuration of the molecule. For
example, amino acids may
be placed in the following classes: non-polar, uncharged polar, basic, and
acidic. Conservative
substitutions whereby an amino acid of one class is replaced with another
amino acid of the same
type fall within the scope of the subject invention so long as the
substitution does not materially
alter the biological activity of the compound. Table 2 provides a listing of
examples of amino
acids belonging to each class.
Table 2
Class of Amino Acid Examples of Amino Acids
Nonpolar Ala, Val, Leu, De, Pro, Met, Phe, Trp
Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, On
Acidic Asp, Glu
Basic Lys, Arg, His
In some instances, non-conservative substitutions can also be made. The
critical factor
is that these substitutions must not significantly detract from the biological
activity of the toxin.
The information presented in the generic formulae of the subject invention
provides clear
guidance to the person skilled in this art in malting various amino acid
substitutions.
Further guidance for characterizing the formicidal toxins of the subject
invention is
provided in Tables 4 and 5, which demonstrate the relatedness among toxins
within the formicidal
toxins. Time tables show a numeric score for the best matching alignment
between two proteins
that reflects: (1) positive scores for exact matches, (2) positive or negative
scores reflecting the
likelihood (or not) of one amino acid substituting for another in a related
protein, and (3)
negative scores for the introduction of gaps. A protein sequence aligned to
itself will have the
highest possible score-i.e., all exact matches and no gaps. However, an
unrelated protein or a
randomly generated sequence will typically have a low positive score. Related
sequences have
scores between the random background score and the perfect match score.
The sequence comparisons were made using the local homology algorithm of Smith
and
Waterman ([1981] Advances in Applied Mathematics 2:482-489), implemented as
the program
"Bestfit" in the GCG Sequence Analysis Software Package Version ? April 1991.
The sequences
were compared with default parameter values (comparison table: Swgappep.Cmp,
Gap weight:3.0,
Length weight-0.1) except that gap limits of 250 residues were applied to each
sequence compared.
The program output value compared is referred to as the Quality score.
WO 92/20802 PCT/US92/04316
Tables 4 and 5 show the pairwise alignments between the indicated amino acids
of the
ant-active proteins and representatives of dipteran (CryIV; ISRH3 of Sen, K.
et aL [1988] Agric.
BioL Chem. 52:873-878), lepidopteran and dipteran (CryIIA; CryBi of Widner and
Whiteley [1989]
J. Bacteriol 171:965-974), and lepidopteran (CryIA(c); Adang et aL [1981] Gene
36:289-300)
5 proteins.
Table 3 shows which amino acids were compared from the proteins of interest.
Table 3
10 Protein Amino acids compared
86Q3(a) 1-697
63B 1-692
33F2 1-618
17a 1-677
17b 1-678
CryIV 1-633
CryIIA 1-633
CryIA(c) 1-609
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11
Table 4 shows the scores prior to adjustment for random sequence scores.
Table 4
` , _ 86Q3(a) 63B 33F2 17b 17a jCryIVA IQYIJA CryIA(c)
86Q3(a) 1046 389 310 342 340 236 237 238
63B 1038 274 339 338 235 228 232
33F2 927 323 322 251 232 251
17b 1017 1007 238 240 236
17a 1016 240 240 237
CiyIVA 950 245 325
CryII.A 950 244
CryIA(c) 914
Note that ant-active protein 86Q3(a) is more closely related to 63B, 17a, 17b,
and 33F2
than it is to the CryIVA, CryILA, and CrylA(c) toxins.
5 Table 5 shows the same analysis after subtraction of the average score of 50
alignments
of random shuffles of the column sequences with the row sequences.
Table 5
86Q3(a) 63B 1331z2 17b 17a CryNA CryIIA CryIA(c)
86Q3(a) 841 184 118 136 135 41 40 50
10 63B 831 81 133 130 40 33 4v
33F2 740 130 128 65 50 71
17b 811 798 42 44 47
17a 808 43 44 44
CryIVA 761 54 141
CryIIA 755 55
( ryIA(c) 729
Note that in Table 5 the same relationships hold as in Table 4, i.e.,
86Q3(a)'s highest
score, aside from itself, is with 63B.
This degree of relatedness provides the basis for using common or similar
sequence
5 elements from the previously-described known genes to obtain related, but
non-identical genes
from an ant-active isolate.
Thus, certain toxins according to the subject invention can be defined as
those which have
formicidal activity and have an alignment value (according to the procedures
of Table 5) greater
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12
than 100 with 86Q3(a). As used herein, the term "alignment value" refers to
the scores obtained
using the methods described above which were used to create the scores
reported in Table 5.
The toxins of the subject invention can also be characterized in terms of the
shape and
location of toxin inclusions.
Inclusion We
PS86Q3-Long amorphic inclusion and a small inclusion, both of which remain
with the spore after lysis. See Figure 3.
PS14OE2-An elliptical coated inclusion situated outside the exosporium, and a
long inclusion inside the exosporium. See Figure 4.
PS211B2--Large round amorphic inclusion with coat, and an elliptical
inclusion.
See Figure 5.
The genes and toxins according to the subject invention include not only the
full length
sequences disclosed herein but also fragments of these sequences, or fusion
proteins, which retain
the characteristic formicidal activity of the sequences specifically
exemplified herein.
It should be apparent to a person skilled in this art that genes coding for
ant-active toxins
can be identified and obtained through several means. The specific genes may
be obtained from
a culture depository as described below. These genes, or portions thereof, may
be constructed
synthetically, for example, by use of a gene machine. Variations of these
genes may be readily
constructed using standard techniques for making point mutations. Also,
fragments of these genes
cann be made using commercially available exonucleases or endonucleases
according to standard
procedures. For example, enzymes such as Ba131 or site-directed mutagenesis
can be used to
systematically cut off nucleotides from the ends of these genes. Also, genes
which code for active
fragments may be obtained using a variety of other restriction enzymes.
Proteases may be..thtd
to directly obtain active fragments of these toxins.
Equivalent toxins and/or genes encoding these equivalent toxins can also be
located from
B a isolates and/or DNA libraries using the teachings provided herein. There
are a number of
methods for obtaining the ant-active toxins of the instant invention which
occur in nature. For
example, antibodies to the ant-active toxins disclosed and claimed herein can
be used to identify
and isolate other toxins from a mixture of proteins. Specifically, antibodies
may be raised to the
portions of the ant-active toxins which are most constant and most distinct
from other At. toxins.
These antibodies can then be used to specifically identify equivalent toxins
with the characteristic
formicidal activity by immunoprecipitation, enzyme linked immunoassay (ELISA),
or Western
blotting. Antibodies to the toxins disclosed herein, or to equivalent toxins,
or fragments of these
toxins, can readily be prepared using standard procedures in this art. The
genes coding for these
toxins can then be obtained from the microorganism.
A further method for identifying the toxins and genes of the subject invention
is through
the use of oligonucleotide probes. These probes are nucleotide sequences
having a detectable
label. As is well known in the art, if the probe molecule and nucleic acid
sample hybridize by
WO 92/20802 2.0324 PCT/US92/04316
13
forming a strong bond between the two molecules, it can be reasonably assumed
that the probe
and sample are essentially identical. The probe's detectable label provides a
means for
determining in a known manner whether hybridization has occurred. Such a probe
analysis
provides a rapid method for identifying formicidal endotoxin genes of the
subject invention.
The nucleotide segments which are used as probes according to the invention
can be
synthesized by use of DNA synthesizers using standard procedures. In the use
of the nucleotide
segments as probes, the particular probe is labeled with any suitable label
known to those skilled
in the art, including radioactive and non-radioactive labels. Typical
radioactive labels include 32P,
125j 3S, or the like. A probe labeled with a radioactive isotope can be
constructed from a
nucleotide sequence complementary to the DNA sample by a conventional nick
translation
reaction, using a DNase and DNA polymerase. The probe and sample can then be
combined in
a hybridization buffer solution and held at an appropriate temperature until
annealing occurs.
Thereafter, the membrane is washed free of extraneous materials, leaving the
sample and bound
probe molecules typically detected and quantified by autoradiography and/or
liquid scintillation
counting.
Non-radioactive labels include, for example, ligands such as biotin or
thyroxine, as well
as enzymes such as hydrolases or perixodases, or the various chemiluminescers
such as luciferin,
or fluorescent compounds like fluorescein and its derivatives. The probe may
also be labeled at
both ends with different types of labels for ease of separation, as, for
example, by using an isotopic
label at the end mentioned above and a biotin label at the other end.
Duplex formation and stability depend on substantial complementarity between
the two
strands of a hybrid, and, as noted above, a certain degree of mismatch can be
tolerated.
Therefore, the probes of the subject invention include mutations (both single
and multiple),
deletions, insertions of the described sequences, and combinations thereof,
wherein said mutations,
insertions and deletions permit formation of stable hybrids with the target
polynucleotide of
interest. Mutations, insertions, and deletions can be produced in a given
polynucleotide sequence
in many ways, and these methods are known to an ordinarily skilled artisan.
Other methods may
become known in the future.
The known methods include, but are not limited to:
(1) synthesizing chemically or otherwise an artificial sequence which is a
mutation,
insertion or deletion of the known sequence;
(2) using a probe of the present invention to obtain via hybridization a new
sequence
or a mutation, insertion or deletion of the probe sequence, and
(3) mutating, inserting or deleting a test sequence in vitro or in vivo.
It is important to note that the mutational, insertional, and deletional
variants generated
from a given probe may be more or less efficient than the original probe.
Notwithstanding such
differences in efficiency, these variants are within the scope of the present
invention.
Thus, mutational, insertional, and deletional variants of the disclosed test
sequences can
be readily prepared by methods which are well known to those skilled in the
are. These variants
WO 92/20802 PCT/US92/04316
2103245'. :. ;.:
14
can be used in the same manner as the instant probes so long as the variants
have substantial
sequence homology with the probes. As used herein, substantial sequence
homology refers to
homology which is sufficient to enable the variant to function in the same
capacity as the original
probe. Preferably, this homology is greater than 50%; more preferably, this
homology is greater
than 75%; and most preferably, this homology is greater than 90%. The degree
of homology
needed for the variant to function in its intended capacity will depend upon
the intended use of
the sequence. It is well within the skill of a person trained in this art to
make mutational,
insertional, and deletional mutations which are designed to improve the
function of the sequence
or otherwise provide a methodological advantage.
Specific nucleotide probes useful, according to the subject invention, in the
rapid
identification of ant-active genes are
(i) DNA coding for a peptide sequence whose single letter amino acid
designation
is "REWINGAN" (SEQ II) NO. 11) or variations thereof which embody point
mutations according to the following: position 1, R or K, position 3, W or Y,
position 4, I or L; position 7, A or N; position 8, N or Q; a specific example
of
such a probe is "AGA(A or G)T(G or A)(G or T)(A or T)T(A or T)AATGG(A
or T)GC(G or T)(A or C)A" (SEQ ID NO. 12); another example of such a
probe is "GA(A or G)TGG(A or T)TAAATGGT(A or G)(A or C)(G or
C)AA-" (SEQ 1D NO. 13);
(ii) DNA coding for a peptide sequence whose single letter amino acid
designation
is "PTFDPDLY" (SEQ ID NO. 14) or variations thereof which embody point
mutations according to the following: position 3, f or L, position 4, I2 or Y;
position 5. P or T, position 6, D or It position 7, L or H or D or N; a
specific
example of such a probe is "CC(A or T)AC(C or T)TTT(T Tor
G)ATCCAGAT(C or G)(T or A)(T or C)TAT" (SEQ ID NO. 15); another
example of such a probe is "CC(T or A)AC(T or A)TT(T or C)GAT(C or
A)CA(G or C)AT(C or A)(T or A)TTAT" (SEQ ID NO. 16);
(iii) additional useful probes for detecting ant-active At. genes include
"GCAATTI'I'AA ATGAATTATA TCC" (SEQ ID NO. 23), "CAAYTACAAG
CWCAACC" (SEQ ID NO. 24), "AATGAAGTWT ATCCWGTWAA T"
(SEQ ID NO. 27), "GCAAGCGGCC GCTTATGGAA TAAATTCAAT
TYKRTCWA" (SEQ ID NO. 28), "AGACFGGATC CATGGCWACW
ATWAATGAAT TATAYCC" (SEQ ID NO. 29), "TAACGTGTAT
WCGST1TTAA TITWGAYTC' (SEQ ID NO. 31), "TGGAATAAAT
TCAATTYKRT CWA" (SEQ IDNO.33), "AGGAACAAAY TCAAKWCGRT
CTA" (SEQ ID NO. 34), and "TCTCCATCFT CTGARGWAAT" (SEQ ID
NO. 37).
The potential variations in the probes listed is due, in part, to the
redundancy of the
genetic code. Because of the redundancy of the genetic code, ie., more than
one coding
CA 02103248 2002-08-29
nucleotide triplet (codon) can be used for most of the amino adds used to make
proteins.
Therefore different nucleotide sequences can code for a particular amino acid.
Thus, the amino
acid sequences of the at toxins and peptides can be prepared by equivalent
nucleotide sequences
encoding the same amino acid sequence of the protein or peptide. Accordingly,
the subject
5 invention includes such equivalent nucleotide sequences. Also, inverse or
complement sequences
are an aspect of the subject invention and can be readily used by a person
skilled in this art. In
addition it has been shown that proteins of identified structure and function
may be constructed
by changing the amino acid sequence if such changes do not alter the protein
secondary structure
(Kaiser, E.T., Kezdy, F.J. [1984] Science 223:249-255). Thus, the subject
invention includes
10 mutants of the amino acid sequence depicted herein which do not alter the
protein secondary
structure, or if the structure is altered, the biological activity is
substantially retained. Further,
the invention also includes mutants of organisms hosting all or part of a
toxin encoding a gene
of the invention. Such microbial mutants can be made by techniques well known
to persons
skilled in the art. For example, UV irradiation can be used to prepare mutants
of host organisms.
15 Likewise, such mutants may include asporogenous host cells which also can
be prepared by
procedures well known in the art.
The toxin genes or gene fragments exemplified according to the subject
invention can be
obtained from R thuringienth (Re) isolates designated PS17, PS33F2, PS63B, and
PS86Q3.
Subcultures of the E colt host harboring the toxin genes of the invention were
deposited in the
permanent collection of the Northern Research Laboratory, U.S. Department of
Agriculture,
Peoria, Illinois, USA. The accession numbers are as follows:
Culture Repository No. posit Date
At PS140E2 NRRL B-18812 April 23, 1991
Rt. PS96Q3 NRRL B-18765 February 6, 1991
At. PS211B2 NRRL B-18921 November 15, 1991
Bt PS17 NRRL B-18243 July 28, 1987
At. PS33F2 NRRL B-18244 July 28, 1987
At PS63B NRRL B-18246 July 28, 1987
E cols NM522(pMYC2316)(33F2) NRRL B-18785 March 15, 1991
E coil NM522(pMYC2321) NRRL B-18770 February 14, 1991
B. tali NM522(pMYC2317) NRRL B-18816 April 24, 1991
B. coil NM522(pMYCI627)(17a) NRRL B-18651 May 11, 1990
E coil NM522(pMYC1628)(17b) NRRL B-18652 May 11, 1990
E coil NM522(pMYC1642)(63B) NRRL 13-18961 April 10, IM
E cols MR618(pMYC1647)(86Q3) NRRL B-18970 April 29, 1992
The subject cultures have been deposited under conditions that assure that
access to the
cultures will be available during the pendency of this patent application to
one determined by the
Commissioner of Patents and Trademarks to be entitled thereto.
CA 02103248 2002-08-29
16
The deposits are available as required by foreign patent laws in countries
wherein
counterparts of the subject application, or its progeny, are filed. However,
it should be
understood that the availability of a deposit does not constitute a license to
practice the subject
invention in derogation of patent rights granted by governmental action.
S Further, the subject culture deposits will be stored and made available to
the public in
accord with the provisions of the Budapest Treaty for the Deposit of
Microorganisms, ic., they
will be stored with all the care necessary to keep them viable and
uncontaminated for a period
of at least five years after the most recent request for the furnishing of a
sample of the deposit,
and in any case, for a period of at least 30 (thirty) years after the date of
deposit or for the
enforceable life of any patent which may issue disclosing the cultures. The
depositor
acknowledges the duty to replace the deposits should the depository be unable
to furnish a sample
when requested, due to the condition of the deposit(s). All restrictions on
the availability to the
public of the subject culture deposits will be irrevocably removed upon the
granting of a patent
disclosing them.
1S The At isolates of the invention can be cultured using standard art media
and
fermentation techniques. Upon completion of the fermentation cycle, the
bacteria can be
harvested by first separating theB.t spores and crystals from the fermentation
broth by means well
known in the att. The recovered Bar. spores and crystals can be formulated
into a wettable
powder, liquid concentrate, granules, or other formulations by the addition of
surfactants,
dispersants, inert carriers and other components to facilitate handling and
application for
particular target pests. These formulation and application procedures are all
well known in the
art
Formulated products can be sprayed or applied as baits to control hymenopteran
pests.
When applied with a bait, theB.x itself maybe used, or another suitable host,
as described herein,
may be transformed with a At. gene and used to express toxins. A vegetable off
or other liquid
substance can be added to a bait to make it more attractive to the pests.
Various attractants,
including pheromone compounds, are well known to those skilled in the art and
can be used as
a component of the bait. The bait and toxin or toxin-producing microbe can be
used as part of
a trap.
The At cells of the invention can be treated prior to formulation to prolong
the
pesticidal activity when the cells are applied to the environment of a target
pest. Such treatment
can be by chemical or physical means, or by a combination of chemical and/or
physical means, so
long as the technique does not deleteriously affect the properties of the
pesticide, nor diminish
the cellular capability in protecting the pesticide. Examples of chemical
reagents are halogenating
agents, particularly halogens of atomic no. 17-80. More particularly, iodine
can be used under
mild conditions and for sufficient time to achieve the desired results. Other
suitable techniques
include treatment with aldehydes, such as formaldehyde and glutaraldehyde;
anti-infectives, such
as tephiran chloride; alcohols, such as isopropyl and ethanol; various
histologic fixatives, such as
Bouln's f xative and Helly's fixative (See: Humason, Gretchen. L,Animal T
Fssue Techniques, W.H.
WO 92/20802 Q PCr/US92/04316
210324
17 O
Freeman and Company, 1967); or a combination of physical (heat) and chemical
agents that
prolong the activity of the toxin produced in the cell when the cell is
applied to the environment
of the target pest(s). Examples of physical means are short wavelength
radiation such as gamma-
radiation and X-radiation, freezing, UV irradiation, lyophilization, and the
like.
Genes encoding toxins having activity against the target susceptible pests can
be isolated
from the At. isolate of the invention by use of well known procedures.
The toxin genes of the subject invention can be introduced into a wide variety
of
microbial hosts. Expression of the toxin gene results, directly or indirectly,
in the intracellular
production and maintenance of the pesticide. With suitable hosts, e.g.,
Pseudomonas, the
microbes can be applied to the situs of hymenopteran insects where they will
proliferate and be
ingested by the insects. The result is a control of the unwanted insects.
Alternatively, the microbe
hosting the toxin gene can be treated under conditions that prolong the
activity of the toxin
produced in the cell. The treated cell then can be applied to the environment
of target pest(s).
The resulting product retains the toxicity of the At toxin.
Where the At. toxin gene is introduced via a suitable vector into a microbial
host, and
said host is applied to the environment in a living state, it is essential
that certain host microbes
be used. Microorganism hosts are selected which are known to occ'ipy the
"phytosphere"
(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more
crops of interest.
These microorganisms are selected so as to be capable of successfully
competing in the particular
environment (crop and other insect habitats) with the wild-type
microorganisms, provide for stable
maintenance and expression of the gene expressing the polypeptide pesticide,
and, desirably,
provide for improved protection of the pesticide from environmental
degradation and inactivation.
A large number of microorganisms are known to inhabit the phylloplane (the
surface of
the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of
a wide varidy of
important crops. These microorganisms include bacteria, algae, and fungi. Of
particular interest
are microorganisms, such as bacteria, e g., genera Pseudomonas, Erwinia,
Serratia, Kiebsiella,
Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophzhus,
Agrobacterium,
Acetobacter, Lactobacillus, Arthrobacter, Azetobacter, d euconostoc, and
Alcabgenes; fungi,
particularly yeast, e.g., genera Saccharomyces, Coptococcus, Kfuyveromyces,
Sporobolomyces,
P.haodotorula, and Aureobas dawn Of particular interest are such phytosphere
bacterial species as
Pseudomonas syringae, Pseudomonas fluorescens, Serrana marcescens, Acetobacter
xylinum,
Agrobacteririm tumefaciens, Rhodopseudomonas spherofdes, Xanthomonas
campestris, Rhizobium
melon, Alcafigenes entrophus, and Acetobacter vinlandu, and phytosphere yeast
species such as
Ri xdotorula rubra, R gluwus, R marina, R auranhaca, Cryptococcus albidus, C.
difuens, C.
laumuu, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces
roseus, S. odorus,
Khqveromyces veronae, and Aureobasidium poflulans. Of particular interest are
the pigmented
microorganisms.
A wide variety of ways are available for introducing the At gene expressing
the toxin into
the microorganism host under conditions which allow for stable maintenance and
expression of
.1..S1:e.....Ltx.:J'e!a..r..~. ". rf,1:Yd~=eu.:r~ie~W:.... ?!t i !
'fii...n..... r..r r.lr. .I~I .uSõ l:J{:.1 {fi.M , A`..,, . .., jo ~.."~.~....
.. ...... ...
WO 92/20802 PCT/US92/04316
2103248
18
the gene. One can provide for DNA constructs which include the transcriptional
and translational
regulatory signals for expression of the toxin gene, the toxin gene under
their regulatory control
and a DNA sequence homologous with a sequence in the host organism, whereby
integration will
occur, and/or a replication system which is functional in the host, whereby
integration or stable
maintenance will occur.
The transcriptional initiation signals will include a promoter and a
transcriptional
initiation start site. In some instances, it may be desirable to provide for
regulative expression
of the toxin, where expression of the toxin will only occur after release into
the environment. This
can be achieved with operators or a region binding to an activator or
enhancers, which are capable
of induction upon a change in the physical or chemical environment of the
microorganisms. For
example, a temperature sensitive regulatory region may be employed, where the
organisms may
be grown up in the laboratory without expression of a toxin, but upon release
into the
environment, expression would begin. Other techniques may employ a specific
nutrient medium
in the laboratory, which inhibits the expression of the toxin, where the
nutrient medium in the
environment would allow for expression of the toxin. For translational
initiation, a ribosomal
binding site and an initiation codon will be present.
Various manipulations may be employed for enhancing the expression of the
messenger,
particularly by using an active promoter, as well as by employing sequences,
which enhance the
stability of, the messenger RNA. The initiation and translational termination
region will involve
stop codon(s), a terminator region, and optionally, a polyadenylation signal.
In the direction of transcription, namely in the 5' to 31 direction of the
coding or sense
sequence, the construct will involve the transcriptional regulatory region, if
any, and the promoter,
where the regulatory region may be either 5' or 3' of the promoter, the
ribosomal binding site,
the initiation codon, the structural gene having an open reading frame in
phase with the initiation
codon, the stop codon(s),'the polyadenylation signal sequence, if any, and the
terminator region.
This sequence as a double strand may be used by itself for transformation of a
microorganism
host, but will usually be included with a DNA sequence involving a marker,
where the second
DNA sequence may be joined to the toxin expression construct during
introduction of the DNA
into the host.
By a marker is intended a structural gene which provides for selection of
those hosts
which have been modified or transformed. The marker will normally provide for
selective
advantage, for example, providing for biocide resistance, e.g., resistance to
antibiotics or heavy
metals; complementation, so as to provide prototropy to an auxotrophic host,
or the like.
Preferably, complementation is employed, so that the modified host may not
only be selected, but
may also be competitive in the field. One or more markers may be employed in
the development
of the constructs, as well as for modifying the host. The organisms may be
further modified by
providing for a competitive advantage against other wild-type microorganisms
in the field. For
example, genes expressing metal chelating agents, e.g., siderophores, may be
introduced into the
host along with the structural gene expressing the toxin. In this manner, the
enhanced expression
r ~.;. ~~ . 11 ..,l Sr .. .. .. ,..,.,
./. t.rr {., ,. . f1,.A y7~ ,. r.. . ., ' ~y .. ~. . . ~~. may. ..+. x. r , ~
WO 92/20802 PCT/US92/04316
1248
of a siderophore may provide for a competitive advantage for the toxin-
producing host, so that
it may effectively compete with the wild-type microorganisms and stably occupy
a niche in the
environment.
Where no functional replication system is present, the construct will also
include a
5 sequence of at least 50 basepairs (bp), preferably at least about 100 bp,
and usually not more than
about 1000 bp of a sequence homologous with a sequence in the host. In this
way, the probability
of legitimate recombination is enhanced, so that the gene will be integrated
into the host and
stably maintained by the host. Desirably, the toxin gene will be in close
proximity to the gene
providing for complementation as well as the gene providing for the
competitive advantage.
10 Therefore, in the event that a toxin gene is lost, the resulting organism
will be likely to also lose
the complementing gene and/or the gene providing for the competitive
advantage, so that it will
be unable to compete in the environment with the gene retaining the intact
construct.
A large number of transcriptional regulatory regions are available from a wide
variety of
microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae,
fungi, and the like.
Various transcriptional regulatory regions include the regions associated with
the trp gene, lac
gene, gal gene, the lambda left and right promoters, the tac promoter, the
naturally-occurring
promoters associated with the toxin gene, where functional in the host. See
for example, U.S.
Patent Nos. 4,332,898, 4,342,832 and 4,356,270. The termination region may be
the termination
region normally associated with the transcriptional initiation region or a
different transcriptional
initiation region, so long as the two regions are compatible and functional in
the host.
Where stable episomal maintenance or integration is desired, a plasmid will be
employed
which has a replication system which is functional in the host. The
replication system may be
derived from the chromosome, an episomal element normally present in the host
or a different
host, or, a replication system from a virus which is stable in the host. A
large number of plasthills
are available, such as pBR322, pACYC184, RSF1010, pRO1614, and the lace. See
for example,
Olson et aL (1982) J BacterioL 150:6069; Bagdasarian et aL (1981) Gene 16:237,
and US. Patent
Nos. 4,356,270, 4,362,817, and 4,371,625.
The At. gene can be introduced between the transcriptional and translational
initiation
region and the transcriptional and translational termination region, so as to
be under the
regulatory control of the initiation region. This construct will be included
in a plasmid, which will
include at least one replication system, but may include more than one, where
one replication
system is employed for cloning during the development of the plasmid and the
second replication
system is necessary for functioning in the ultimate host. In addition, one or
more markers may
be present, which have been described previously. Where integration is
desired, the plasmid will
desirably include a sequence homologous with the host genome.
The transformants can be isolated in accordance with conventional ways,
usually
employing a selection technique, which allows for selection of the desired
organism as against
unmodified organisms or transferring organisms, when present. The
transformants then can be
tested for pesticidal activity.
WO 92/20802 PCT/US92/04316
2103248
Suitable host cells, where the pesticide-containing cells will be treated to
prolong the
activity of the toxin in the cell when the then treated cell is applied to the
environment of target
pest(s), may include either prokaryotes or eukaryotes, normally being limited
to those cells which
do not produce substances toxic to higher organisms, such as mammals. However,
organisms
5 which produce substances toxic to higher organisms could be used, where the
toxin is unstable or
the level of application sufficiently low as to avoid any possibility of
toxicity to a mammalian host.
As hosts, of particular interest will be the prokaryotes and the lower
eukaryotes, such as fungi.
Illustrative prokaryotes, both Gram-negative and -positive, include
Enterobacteriaceae, such as
Escherichia, Erwinia, Shigella, Salmonella, and Proteus; BadIlaceae;
Rhizobiceae, such as
10 Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia,
Aeromonas, Vibno,
Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as
Pseudomonas and
Acetobacter, Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are
fungi, such as
Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and
Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,
Aureobasidium,
15 Sporobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for purposes
of production
include ease of introducing the Bt. gene into the host, availability of
expression systems, efficie icy
of expression, stability of the pesticide in the host, and the presence of
auxiliary genetic
capabilities. Characteristics of interest for use as a pesticide microcapsule
include protective
20 qualities for the pesticide, such as thick cell walls, pigmentation, and
intracellular packaging or
formation of inclusion bodies; leaf affinity, lack of mammalian toxicity;
attractiveness to pests for
ingestion; ease of killing and fixing without damage to the toxin; and the
like. Other
considerations include ease of formulation and handling, economics, storage
stability, and the like.
Host organisms of particular interest include yeast, such as Rhodotorulkl'sp.,
Aureobasid um sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane
organisms such as
Pseudomonas sp., Erwura sp. and Flavobacterium sp.; or such other organisms
'as Escherichia,
Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like. Specific
organisms include
Pseudomonas aeruginosa, Pseudomonas fkeorescens, Saccharomyces cerevisiae,
Bacilkts thunngtensis,
Escherichia cob, Bacillus subtilis, Streptomyces lividans, and the like.
The cell will usually be intact and be substantially in the proliferative form
when treated,
rather than in a spore form, although in some instances spores may be
employed.
Treatment of the recombinant microbial cell can be done as disclosed infra.
The treated
cells generally will have enhanced structural stability which will enhance
resistance to
environmental conditions. Where the pesticide is in a proform, the method of
inactivation should
be selected so as not to inhibit processing of the proform to the mature form
of the pesticide by
the target pest pathogen. For example, formaldehyde will crosslink proteins
and could inhibit
processing of the proform of a polypeptide pesticide. The method of
inactivation or killing retains
at least a substantial portion of the bio-availability or bioactivity of the
toxin.
WO 92/20802 2103248 PCT/US92/04316
21
The cellular host containing the B.L insecticidal gene may be grown in any
convenient
nutrient medium, where the DNA construct provides a selective advantage,
providing for a
selective medium so that substantially all or all of the cells retain the Rt.
gene. These cells may
then be harvested in accordance with conventional ways. Alternatively, the
cells can be treated
prior to harvesting.
The Rt. cells may be formulated in a variety of ways. They may be employed as
wettable
powders, baits, granules or dusts, by mixing with various inert materials,
such as inorganic minerals
(phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical
materials (powdered
corncobs, rice hulls, walnut shells, and the like). The formulations may
include spreader-sticker
adjuvants, stabilizing agents, other pesticidal additives, or surfactants.
Liquid formulations may
be aqueous-based or non-aqueous and employed as foams, gels, suspensions,
emulsifiable
concentrates, or the like. The ingredients may include rheological agents,
surfactants, emulsifiers,
dispersants, or polymers.
The pesticidal concentration will vary widely depending upon the nature of the
particular
formulation, particularly whether it is a concentrate or to be used directly.
The pesticide will be
present in at least 1% by weight and may be. 100% by weight. The dry
formulations will have
from about 1-95% by weight of the pesticide while the liquid formulations will
generally be from
about 1-60% by weight of the solids in the liquid phase. The formulations will
generally have
from about 102 to about 104 cells/mg. These formulations will be administered
at about 50 mg
(liquid or dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the hymenopteran
pest(s), e.g.,
plants, soil or water, by spraying, dusting, sprinkling, baits or the like.
Following are examples which illustrate procedures, including the best mode,
or
practicIng the invention. These examples should not be construed as limiting.
All percentages
are by weight and all solvent mixture proportions are by volume unless
otherwise noted.
Example 1 Culturing B.t Isolates of the Invention
A subculture of a Bt. isolate can be used to inoculate the following medium, a
peptone,
glucose, salts medium.
Bacto Peptone 7.5 g/l
Glucose 1.0 gIl
KH2PO4 3.4 g/l
K2HPO4 435 g/l
Salts Solution 5.0 ml/1
Ca%72 Solution 5.0 ml/1
Salts Solution (100 ml)
MgSO4.7H20 2.46 g
MnSO4.1-120 0.04 g
WO 92/20802 PCT/US92/04316
22
ZnSO4.7H20 0.28 g
FeSO4=7H20 0.40 g
CaC12 Solution (100 ml)
CaC12.2H20 3.66 g
pH 7.2
The salts solution and CaC12 solution are filter-sterilized and added to the
autoclaved and
cooked broth at the time of inoculation. Flasks are incubated at 30 C on a
rotary shaker at 200
rpm for 64 hr.
E nple 2 - Purification of Protein and Amino Acid Sequencing
The At. isolates PS86Q3, PS17, PS63B, and PS33F2 were cultured as described in
Example 1. The parasporal inclusion bodies were partially purified by sodium
bromide (28-38%)
isopycnic gradient centrifugation (Pfannenstiel, M.A, E.J. Ross, V.C. Kramer,
K.W. Nickerson
[1984] FEMS MicrobioL Lett. 21:39). The proteins were bound to PVDF membranes
(Millipore,
Bedford, MA) by western blotting techniques (Towbin, H., T. Staehlelin, K.
Gordon [1979] Prot.
NatL Acad. Sci. USA 76:4350) and the N-terminal amino acid sequences were
determined by the
standard Edman reaction with an automated gas-phase sequenator (Hunkapiller,
LW., R.M.
Hewick, W.L. Dreyer, and L.E. Hood [1983] Meth. Enrymol 91:399). The sequences
obtained
were:
17a: AILNELYPSVPYNV(SEQIDNO.17)
17b. AILNELYPSVPYN V (SEQIDNO.18)
86Q3(a): MATINELYPNVPYNVL(SEQIDNO.19)
63B: QLQAQPLIPYNVLA(SEQIDNO.20)
33F2: A TL N E V Y P V N (SEQ ID NO. 21)
In addition, internal amino acid sequence data were derived for 63B. The toxin
protein
was partially digested with Staphylococcus aureus V8 protease (Sigma Chem.
Co., St. Louis, MO)
essentially as described (Cleveland, D.W., S.G. Fischer, M.W. Kirschner, U.K.
Laemmli [1977] J
BioL Chem. 252:1102). The digested material was blotted onto PVDF membrane and
a ca. 28 kDa
limit peptide was selected for N-terminal sequencing as described above. The
sequence obtained
was:
63B(2) VQRILDEKLSFQLIK(SEQIDNO.22)
From these sequence data oligonucleotide probes were designed by utilizing a
codon
frequency table assembled from available sequence data of other Bt. toxin
genes. The probes were
synthesized on an Applied Biosystems, Inc. DNA synthesis machine.
Protein purification and subsequent amino acid analysis of the N-terminal
peptides listed
above has led to the deduction of several oligonucleotide probes for the
isolation of toxin genes
from formicidal B.L isolates. RFLP analysis of restricted total cellular DNA
using radiolabeled
oligonucleotide probes has elucidated different genes or gene fragments.
WO 92/20802 2103248 PCI /US92/04316
23
Example 3 - Cloning of Novel Toxin Genes and Transformation into Escherichia
coli
Total cellular DNA was prepared by growing the cells At. PS17 to a low optical
density
(ODD = 1.0) and recovering the cells by centrifugation. The cells were
protoplasted in TES
buffer (30 mM Tris-Cl, 10 mM EDTA, 50 mM NaCl, pH = &0) containing 20 %
sucrose and 50
mg/ml lysozyme. The protoplasts were lysed by addition of SDS to a final
concentration of 4%.
The cellular material was precipitated overnight at 4 C in 100 mM (final
concentration) neutral
potassium chloride. The supernate was extracted twice with phenol/chloroform
(1:1). The DNA
was precipitated with ethanol and purified by isopycnic banding on a cesium
chloride-ethidium
bromide gradient.
Total cellular DNA from PS17 was digested with EcoRI and separated by
electrophoresis
on a 0.8% (w/v) Agarose-TAE (50 mM Tris-HCI, 20 mM NaOAc22.5 mM EDTA, pH=8.0)
buffered gel. A Southern blot of the gel was hybridized with a [32P]-
radiolabeled oligonucleotide
probe derived from the N-terminal amino acid sequence of purified 130 kDa
protein from PS17.
The sequence of the oligonucleotide synthesized is (GCAATTTTAAATGAATTATATCC)
(SEQ
ID NO. 23). Results showed that the hybridizing EcoRl fragments of PS17 are
5.0 kb, 4.5 kb, 2.7
kb and 1.8 kb in size, presumptively identifying at least four new ant-active
toxin genes, 17d, 17b,
17a and 17e, respectively.
A library was constructed from PS17 total cellular DNA partially digested with
Sau3A and
size fractionated by electrophoresis. The 9 to 23 kb region of the gel was
excised and the DNA
was electroeluted and then concentrated using an Elutip7m ion exchange column
(Schleicher and
Schuel, Keene NH). The isolated Sau3A fragments were ligated into LambdaGEM-
11Tt''I
(PROMEGA). The packaged phage were plated on KW251 E. cold cells (PROMEGA) at
a high
titer and screened using the above radiolabeled synthetic oligonucleotide as a
nucleic acid
hybridization probe. Hybridizing plaques were purified and rescreened at a
lower plaque denary.
" Single isolated purified plaques that hybridized with the probe were used to
infect KW251 E coli
cells in liquid culture for preparation of phage for DNA isolation. DNA was
isolated by standard
procedures.
Recovered recombinant phage DNA was digested with EcoRI and separated by
electrophoresis on a 0.8% agarose-TAE gel. The gel was Southern blotted and
hybridized with
the oligonucleotide probe to characterize the toxin genes isolated from the
lambda library. Two
patterns were present, clones containing the 4.5 kb (17b) or the 2.7 kb (17a)
EcoRI fragments.
Preparative amounts of phage DNA were digested with Sall (to release the
inserted DNA from
lambda arms) and separated by electrophoresis on a 0.6% agarose-TAE gel. The
large fragments,
electroeluted and concentrated as described above, were ligated to SaII-
digested and
dephosphorylated pBClac, an coli/Rz shuttle vector comprised of replication
origins from
pBC16 and pUC19. The ligation mix was introduced by transformation into NM522
competent
E. coli cells and plated on LB agar containing ampicillin, isopropyl-(Beta)-D-
thiogalactoside
(IPTG) and 5-Bromo-4-Chloro-3-indolyl-(Beta)-D-galactoside (XGAL).. White
colonies, with
putative insertions in the (Beta)-galactosidase gene of pBClac, were subjected
to standard rapid
WWO 92/20802 PCT/US92/04316
2103248 24
plasmid purification procedures to isolate the desired plasmids. The selected
plasmid containing
the 2.7 kb EcoRI fragment was named pMYC1627 and the plasmid containing the
4.5 kb EcoRI
fragment was called pMYCI628.
The toxin genes were sequenced by the standard Sanger dideoxy chain
termination method
using the synthetic oligonucleotide probe, disclosed above, and by "walking"
with primers made
to the sequence of the new toxin genes.
The PS17 toxin genes were subcloned into the shuttle vector pHT3101 (Lereclus,
D. et
al [1989] FMS Microbiol Lett 60:211-218) using standard methods for expression
in B.t. Briefly,
Salt fragments containing the 17a and 17b toxin genes were isolated from
pMYC1629 and
pMYC1627, respectively, by preparative agarose gel electrophoresis,
electroelution, and
concentrated, as described above. These concentrated fragments were ligated
into Sail-cleaved
and dephosphorylated pI TT3101. The ligation mixtures were used separately to
transform frozen,
competent E cob NM522. Plasmids from each respective recombinant E cob strain
were
prepared by alkaline lysis and analyzed by agarose gel electrophoresis. The
resulting subclones,
pMYC2311 and pMYC23O9, harbored the 17a and 17b toxin genes, respectively.
These plasmids
were transformed into the acrystalliferous B.t strain, HD-1 cryB (Aronson, A.,
Purdue University,
West Lafayette, IN), by standard electroporation techniques (Instruction
Manual, Biorad,
Richmond, CA).
Recombinant At strains HD-1 cryB [pMYC2311] and [pMYC2309] were grown to
sporulation and the proteins purified by NaBr gradient centrifugation as
described above for the
wild-type Bt. proteins.
Example 4 -- Molecular Cloning of a Gene Encoding a Novel Toxin from Bacillus
thunngiensis
Strain PS63B
Example 2 shows the aminotermiml and internal polypeptide sequences of the 63B
toxin
protein as determined by standard Edman protein sequencing. From these
sequences, two
oligonucleotide primers were designed using a colon frequency table assembled
from B.tw genes
encoding 6-endotoxins. The sequence of the forward primer (63B-A) was
complementary to the
predicted DNA sequence at the 5' end of the gene:
63B-A - 5' CAA T/CTA CAA GCA/T CAA CC 3' -(SEQ ID NO. 24)
The sequence of the reverse primer (63B-IN) was complementary to the inverse
of the internal
predicted DNA sequence:
63B-INT - 5' TTC ATC TAA AAT TCT TTG ATFAC 3' (SEQ ID NO. 25)
These primers were used in standard polymerase chain reactions (Cetus
Corporation) to amplify
an approximately 460 bp fragment of the 63B toxin gene for use as a DNA
cloning probe.
Standard Southern blots of total cellular DNA from 63B were hybridized with
the radiolabeled
PCR probe. Hybridizing bands included an approximately 4.4 kbp XbaI fragment,
an
approximately 2.0 kbp HindiII fragment, and an approximately 6.4 kbp Spel
fragment.
CA 02103248 2002-08-29
Total cellular DNA was prepared from Bau7bsi lava ginsir (8t) calls grown to
an
optical density of L0 at 600 nm. The cells were recovered by centrifugation
and protoplasts were
prepared in lysis mix (300 mM sucrose, 25 mM Tris-HCI, 25 mM EDTA, pH = &0)
and lysozyme
at a concentration of 20 mg/mL The protoplasts were ruptured by addition of
ten volumes of 0.1
5 M NaCl, 0.1 M Tris-HCl pH 8.0, and 0.1% SDS. The cellular material was
quickly frozen at
70 C and thawed to 37 C twice. The supernatant was extracted twice with
phenol/chloroform
(1:1). The nucleic acids were precipitated with ethanol. To remove as much RNA
as possible
from the DNA preparation, RNase at final concentration of 200 ug/ml was added.
After
incubation at 37 C for 1 hour, the solution was extracted once with
pbenollchloroform and
10 precipitated with ethanol
A gene library was constructed from 63D total cellular DNA partially digested
with NdeII
and size fractioned by gel electrophoresis. The 9-23 kb region of the gel was
excised and the DNA
was electroeluted and then concentrated using an Elutip-d ion exchange column
(Schleicher and
Schuel, Keene, NH). The isolated NdeII fragments were ligated into BamHl-
digested
15 LambdaGEM-11 (PROMEGA). The packaged phage were plated on 8 coli KW251
cells
(PROMEGA) at a high titer and screened using the radiolabeled approximately
430 bp fragment
probe amplified with the 63B-A and 63B internal primers (SEQ ID NOS. 27 and
28, respectively)
by polymerase chain reaction. Hybridizing plaques were purified and rescreened
at a lower plaque
density . Single isolated, purified plaques that hybridized with the probe
were used to infect
20 KW251 cells in liquid culture for preparation of phage for DNA isolation.
DNA was isolated by
standard procedures (Maniatis, T., E.F. Fritsch, J. Sambrook [1982] Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York). Preparative
amounts of DNA
were digested with Sall (to release the inserted DNA from lambda sequences)
and separated by
electrophoresis on a 0.6% agarose-TAE gel. The large fragments were purified
by Ion exchange
*
25 chromatography as above and ligated to Sall-digested, dephosphorylated
pHTBlueII (anE. coWB.t
*
shuttle vector comprised of pBlueScript S/K [Stratagene, San Diego, CA] and
the replication
origin from a resident AL plasmid [Lereclus, D. et aL (1989) FEMS MicrobioL
Lett. 60:211-218]).
The ligation mix was introduced by transformation into competent F. coil NM522
cells (ATCC
47000) and plated on LB agar containing ampiclllin (100 rughnl), IPTG (2%),
and XGAL (2%).
White colonies, with putative restriction fragment insertions in the (Beta)-
galactosidase gene of
pHlBludIU, were subjected to standard rapid plasmid purification procedures
(Maniatis er aL,
supra). Plasmids ere analyzed by Sall digestion and agarose gel
electrophoresis. The desired
plasmid construct, pMYC1641, contains an approximately 14 kb Sail insert.
For subcioning, preparative amounts of DNA were digested with Xbal and
electrophoresed on an agarose gel. The approximately 4.4 kbp band containing
the toxin gene was
e :ised from the gel, electroeluted from the gel slice, and purified by ion
exchange
chromatography as above. This fragment was ligated into Xbal cut pHTBlueII and
the resultant
plasmid was designated pMYC1642.
*Trade-mark
WO 92/20802 PCT/US92/04316
2103248 :: .
26
Example 5 - Cloning of a Novel Toxin Gene From At PS33F2 and Transformation
into
Escherichia coli
Total cellular DNA was prepared from B.t PS33F2 cells grown to an optical
density, at
600 nm, of 1Ø Cells were pelleted by centrifugation and resuspended in
protoplast buffer (20
mg/ml lysozyme in 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA). After
incubation at
37 C for 1 hour, protoplasts were lysed by the addition of nine volumes of a
solution of 0.1 M
NaCI, 0.1% SDS, 0.1 M Tris-Cl followed by two cycles of freezing and thawing.
The cleared lysate
was extracted twice with phenol:chloroform (1:1). Nucleic acids were
precipitated with two
volumes of ethanol and pelleted by centrifugation. The pellet was resuspended
in 10 mM Tris-Cl,
1 mM EDTA ('TE) and RNase was added to a final concentration of 50 ug/mL After
incubation
at 37 C for 1 hour, the solution was extracted once each with phenol:
chloroform (1:1) and TE-
saturated chloroform. DNA was precipitated from the aqueous phase by the
addition of one-tenth
volume of 3 M NaOAc and two volumes of ethanol DNA was pelleted by
centrifugation, washed
with 70% ethanol, dried, and resuspended in TE.
Plasmid DNA was extracted from protoplasts prepared as described above.
Protoplasts
were lysed by the addition of nine volumes of a solution of 10 mM Tris-Cl, 1
mM EDTA, 0.085
N NaOH, 01% SDS, PH=&O. SDS was added to 1% final concentration to complete
lysis. One-
half volume of 3 M KOAc was then added and the cellular material was
precipitated overnight
at 4 C. After centrifugation, the DNA was precipitated with ethanol and
plasmids were purified
by isopycnic centrifugation on cesium chloride-ethidium bromide gradients.
Restriction Fragment Length Polymorphism (RFLP) analyses were performed by
standard
hybridization of Southern blots of PS33F2 plasmid and total cellular DNA with
32P-labelled
oligonucleotideprobes designed to the N-terminal amino acid sequence disclosed
in Example 2.
Probe 33F2A: 5' GCA/T ACA/T TTA AAT GAA GTA/T TAT 3' (SEQ ID NO 261
Probe 33F2B 5' AAT GAA GTA/T TAT CCAIT GTA/T AAT 3' (SEQ ID NO 27)
Hybridizing bands included an approximately 5.85 kbp EcoRI fragment. Probe
33F2A and a
reverse PCR primer were used to amplify a DNA fragment of approximately 1.8
kbp for use as
a hybridization probe for cloning the 33F2 toxin gene. The sequence of the
reverse primer was:
5' GCAAGCGGCCGCTI'ATGGAATAAATTCAATT C/T T/G A/G TC T/A A 3' (SEQ ID
NO. 28).
A gene library was constructed from 33F2 plasmid DNA digested with EcoRL
Restriction
digests were fractionated by agarose gel electrophoresis. DNA fragments 4.3-
6.6 kbp were excised
from the gel, electroeluted from the gel slice, and recovered by ethanol
precipitation after
purification on an Elutip-D ion exchange column (Schleicher and Schuel, Keene
NH). The EcoRI
inserts were ligated into EcoRl-digested pHTBluell (an E. cob/E thuringiensis
shuttle vector
comprised of pBluescript S/K [Stratagene] and the replication origin from a
resident Rt plasmid
(Lereclus, A et aL [1989] FEMS Microbial Lett 60:211-218]). The ligation
mixture was
transformed into frozen, competent NM522 cells (ATCC 47000). Transformants
were plated on
LB agar containing ampicillin, isopropyl-(Beta)-D-thiogalactoside (IPTG), and
5-bromo-4-chloro-
WO 92/20802 PCT/US92/04316
d
27
3-indolyl-(Beta)-D-galactoside (XGAL). Colonies were screened by hybridization
with the
radiolabeled PCR amplified probe described above. Plasmids were purified from
putative toxin
gene clones by alkaline lysis and analyzed by agarose gel electrophoresis of
restriction digests. The
desired plasmid construct, pMYC2316, contains an approximately 5.85 kbp Eco4RI
insert; the
toxin gene residing on this DNA fragment (33F2a) is novel compared to the DNA
sequences of
other toxin genes encoding formicidal proteins.
Plasmid pMYC2316 was introduced into the acrystalliferous (Cry-) Bt host, HD-1
CryB
(A. Aronson, Purdue University, West Lafayette, IN) by electroporation.
Expression of an
approximately 120-140 kDa crystal protein was verified by SDS-PAGE analysis.
Crystals were
purified on NaBr gradients N.A. Pfannenstiel et aL [1984] FEMS MicrobioL Lett
21:39) for
determination of toxicity of the cloned gene product to Pratylenchus spp.
Example 6 - Cloning of a Novel Toxin Gene from at Isolate PS86Q3
Total cellular DNA was prepared from Bacillus th gienszs (B t.) cells grown to
an
optical density of 1.0 at 600 nm. The cells were recovered by centrifugation
and protoplasts were
prepared in lysis mix (300 mM sucrose, 25 mM Tris-HCI, 25 mM EDTA, pH = &0)
containing
lysozyme at a concentration of 20 mglml. The protoplasts were ruptured by
addition of ten
volumes of 0.1 M NaCl, 0.1% SDS, 0.1 M Tris-CI, pH = 8Ø The cleared lysate
was quickly
frozen at --70 C and thawed to 37 C twice The supernate was extracted twice
with
phenoichloroform (1:1). The pellet was resuspended in 10 mM Tris-CI, 1 mM
EDTA,pH = 8.0
(TB), and RNase was added to a final concentration of 50 4uglml. After
incubation at 37 C for
one hour, the solution was extracted once with phenol:chloroform (1:1) and
then with TE-
saturated chloroform. DNA was precipitated from the aqueous phase by the
addition of one-tenth
volume of 3M NaOAc and two volumes of ethanol. DNA was pelleted by
centrifugation, was'h$d
with 70% ethanol, dried, and resuspended in TB.
Total cellular DNA from isolate PS86Q3 was used as template for polymerase
chain
reaction (PCR) analysis. according to protocols furnished by Perkin Elmer
Cetus. An
oligonucleotide derived from the N-terminal amino acid sequence of the toxin
protein was used
as a 5' primer. The sequence of this oligonucleotide is:
5'- AGACTGGATCCATGGC(A or T)AC(A or T)AT(A or T)AATGAATTATA (T or C)CC-3'
(SEQ ID NO. 29).
An oligonucleotide coding for the amino acid sequence "ESKLKPNTRY" (SEQ ID NO.
30) can be used as the reverse 3' primer. The sequence of this oligonucleotide
can be: "5'-
TAACGTGTAT(A or T)CG(C or G)TITTAATIT(T or A)GA(C or T)TC-3"(SEQ ID NO.
31).
The reverse "YIDKIEFIP" (SEQ ID NO. 32) oligonucleotide was also used as a
reverse
3' primer in conjunction with the above mentioned 5' primer. The sequence of
the reverse
primer can be: "5'-TGGAATAAATTCAATT(C or T)(T or G)(A or G)TC(T or A)A-3"'
(SEQ
ID NO. 33).
w_.,." '-..-.. ._,_,.,... '__ -.. .. --ramõ ..... . ,...-_.. :`r. .. ..,. .:.
r:,..... :-._.. -. .. ..r..,....,... ... ..., r;,.... _ ...
WO 92/20802 PCT/US92/04316
21x3248
28
Amplification with the 5' primer and SEQ ID NO. 31 generates an approximately
2.3
kbp DNA fragment and an approximately 4.3 kbp DNA fragment. Amplification with
the 5'
primer and SEQ ID NO. 33 generates an approximate 1.8 kbp DNA fragment and an
approximately 3.7 kbp DNA fragment. The approximately 2.3 kbp fragment was
radiolabeled with
32P and used as a hybridization probe to generate restriction fragment
polymorphism (RFLP)
patterns and to screen recombinant phage libraries.
A Southern blot of total cellular DNA digested with EcoRV was probed with the
radiolabeled 2.3 kbp probe described above. The resultant RFLP includes 9.5
kbp, 6.4 kbp, and
4.5 kbp hybridizing fragments.
A gene library was constructed from PS86Q3 total cellular DNA partially
digested with
Ndell and size fractioned by gel electrophoresis. The 9-23 kb region of the
gel was excised and
the DNA was electroeluted and then concentrated using an Elutip-d ion exchange
column
(Schleicher and Schuel, Keene, NH). The isolated Ndel fragments were ligated
into BamHI-
digested LambdaGEM-11 (PROMEGA). The packaged phage were plated on R coil
KW251 cells
(PROMEGA) at a high titer and screened using the radiolabeled probe described
above.
Hybridizing plaques were purified and rescreened at a lower plaque density
Single isolated,
purified plaques that hybridized with the probe were used to infect KW251
cells in liquid culture
for preparation of phage for DNA isolation. DNA was isolated by standard
procedures (Maniatis
et aL, supra). Preparative amounts of DNA were digested with Sall (to release
the inserted DNA
from lambda sequences) and separated by electrophoresis on a 0.6% agarose-TAE
gel. The large
fragments were purified by ion exchange chromatography as above and ligated to
Sall-digested,
dephosphorylated pHTBlueU (an E. cok/8.L shuttle vector comprised of
pBluescript S/K
[Stratagene, San Diego, CA]) and the replication origin from a resident At.
plasmid (Lereclus et
aL [19891, supra). The ligation mix was introduced by transformation into
competent E-`coli
NM522 cells (ATCC 47000) and plated on LB agar containing ampicillin, IPM, and
KGAL-
White colonies, with putative restriction fragment insertions in the (Beta)-
galactosidase gene of
pHTBluell, were subjected to standard rapid plasmid purification procedures
(Maniatis et aL,
supra). Plasnud DNA was analyzed by Sall digestion and agarose gel
electrophoresis. The desired
plasmid construct, pMYC1647, contains an approximately 12 kb Sall insert.
Plasmid pMYC1647 was introduced by electroporation into an acrystalliferous
(Cry) At.,
HD-1 CryB (AL Aronson, Purdue University) host to yield MR515, a recombinant
At. clone of
86Q3(a). Expression of an approximately 155 kDa protein was verified by SDS-
PAGE. Spores
and crystals were removed from broth cultures and were used for determination
of toxicity to
pharaoh ants.
Example 7 - Activity of the Rt. Toxin Protein and Gene Product Aeainst Ants
Broths were tested for the presence of fi-exotoxin by a larval house fly
'bioassay
(Campbell, D.P., Dieball, D.E., Bracket, J.M. = [1987] "Rapid HPLC assay for
the fi-exotoxin of
WO 92/20802 2103248 PCT/US92/04316
29
Bacillus thuringiensi c, ' J. Agric. Food Chem. 35:156-158). Only isolates
which tested free of 9-
exotoxin were used in the assays against ants.
A bait was made consisting of 10% Bacillus thuringiensis isolates of the
invention and
Crosse and Blackwell mint apple jelly. Approximately 100 ants were placed in
each plastic test
chamber replicate with the baits. Control experiments were performed with
untreated mint apple
jelly. Each test was replicated a minimum of 10 times. Mortality was assessed
at 7, 14 and 21
days after introduction of the bait to the ants. Results are shown below:
Table 6. Toxicity of B. thunngiensis Isolates to the Pharaoh Ant (Monomoruum
Pharaonic)
At. Isolate Percent Mortality
PS14OE2 91
PS 86Q3 84
Control 11
PS211B2 90.0
Control 3.8
Exjrle 8 Activity Against Pharaoh Ants
Mint apple jelly containing 10% AL (100,000 ppm) was fed to 5 replicates of
approximately 100 worker ants for 21 days. Total mortality (in %) over the
test period is
compared to control.
Table 7. Three week mortality on pharaoh ant workers.
Sample Rate ppm Percent Mortality
MR315 100000 40.1
86Q3 100000 29.2
211B2 100000; 58.5
MAJ Blank 25.0
Control Blank 14.4
MR515 a recombinant At. clone of 86Q3(a) gene, 10% in MAT (Example 6)
86Q3 = spray dried powder of At. PS86Q3, 10% in MAJ
211B2 = spray dried power of At. PS211B2,10% in MAT
MAJ = Mint apple jelly, Crosse & Blackwell
Control = rearing diet of water, frozen flies, mealworms/honey agar
WO 92/20802 PCF/US92/04316
21U3248 30
Table & Three week mortality (%) on pharaoh ant workers.
Sample Rate ppm Percent Mortality
50000 100.0
140E2
86Q3 50000 99.6.
211B2 50000 100.0
MAJ Blank 753
Control Blank 39.0
140E2 = 5% 140E2 purified protein in MAJ
86Q3 = 5% 86Q3 purified protein in MAJ
211B2 5% 211B2 purified protein in MAJ
MAJ = Mint apple jelly, Crosse & Blackwell
Control = rearing diet of water, frozen flies, mealworms/honey agar
Example 9 - Cloning of Novel Ant-Active Genes Using Generic Oligonucleotide
Primers
The formicidal gene of a new formicidal At. can be obtained from DNA of the
strain by
performing the standard polymerase chain reaction procedure as in Example 6
using the
ohgonucleotides of SEQ ID NO. 33 or AGGAACAAAYTCAAKWCGRTCTA (SEQ ID NO. 34)
as reverse primers and SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.
16, SEQ
ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 29, or SEQ ID NO. 24 as forward primers.
The
expected PCR fragments would be approximately 330 to 600 bp with either
reverse primer and
SEQ ID NO. 12 or SEQ ID NO.13,1000 to 1400 bp with either reverse primer and
SEQ ID NO.
15, or SEQ ID NO. 16, and 1800 to 2100 bp with either reverse primer and any
of the thr N
terminal primers, SEQ ID NO. 27, SEQ ID NO. 23, SEQ ID NO. 29, and-SEQ ID NO.
24.
Alternatively, a complement from the primer family described by SEQ ID NO. 12
and SEQ ID
NO. 13 can be used as reverse primer with SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID
NO. 23,
SEQ ID NO. 27, SEQ ID NO. 29, or SEQ ID NO. 24 as forward primers. The
expected PCR
fragments would be approximately 650 to 1000 bp with SEQ ID NO. 15 or SEQ ID
NO. 16, and
1400 to 1800 bp for the four N-terminal primers (SEQ ID NO. 27, SEQ ID NO. 23,
SEQ ID NO.
29, and SEQ ID NO. 24).
As another alternative, the reverse primer SEQ ID NO. 31 can be used with any
of the
four N-terminal forward primers to yield fragments of approximately 2550-3100
bp; 1750-2150 bp
with the forward primers SEQ ID NOS. 15 or 16; 850-1400 bp with SEQ ID NOS. 12
or 13; and
550-1050 bp with the forward primer TITAGATCGT(A or C)TTGA(G or A)TTT(A or
G)T(A
or T)CC (SEQ ID NO. 35).
As yet another alternative, the ITSED (SEQ ID NO 37) reverse primer
(TCfCCATCTTCTGA(G or A)G(T or A)AAT) (SEQ ID NO. 37) can be used with the N-
terminal forward primers (SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 27, and SEQ
ID NO.
SUBSTITUTE SHEET
WO 92/20802 PCT/US92/04316
2103248
31
ID NOS. 15 or 16;1800-2400 bp with forward primers SEQ ID NOS. 12 or 13; and
1500-2050 bp
with forward primer SEQ ID NO. 35.
Amplified DNA fragments of the indicated sizes can be radiolabeled and used as
probes
to clone the entire gene as in Example 6.
Example 10 - Insertion of Toxin Gene Into Plants
One aspect of the subject invention is the transformation of plants with genes
coding for
a formicidal toxin. The transformed plants are resistant to attack by ants.
Genes coding for formicidal toxins, as disclosed herein, can be inserted into
plant cells
using a variety of techniques which are well known in the art. For example, a
large number of
cloning vectors comprising a replication system in E cob and a marker that
permits selection of
the transformed cells are available for preparation for the insertion of
foreign genes into higher
plants. The vectors comprise, for example, pBR322, pUC series, M13mp series,
pACYC184, etc.
Accordingly, the sequence coding for the At. toxin can be inserted into the
vector at a suitable
restriction site. The resulting plasmid is used for transformation into E
cold. The E coli cells
are cultivated in a suitable nutrient medium, then harvested and lysed. The
plasmid is recovered.
Sequence analysis, restriction analysis, electrophoresis, and other
biochemical-molecular biological
methods are generally carried out as methods of analysis. After each
manipulation, the DNA
sequence used can be cleaved and joined to the next DNA sequence. Each plasmid
sequence can
be cloned in the same or other plasmids. Depending on the method of inserting
desired genes
into the plant, other DNA sequences may be necessary. If, for example, the Ti
or Ri plasmid is
used for the transformation of the plant cell, then at least the right border,
but often the right and
the left border of the Ti or Ri plasmid T -DNA, has to be joined as the
flanking region of the
genes to be inserted. -c4
The use of T -DNA for the transformation of plant cells has been intensively
researched
and sufficiently described in EP 120 516; Hoekema (1985) In: The Binary Plant
Vector System,
Offset-durkkerij Kanters B. T., Alblasserdam, Chapter 5; Fraley et aL, Cut.
Rev. Plant Sci. 4:1-46;
and An et aL (1985) EMBO 14:277-287.
Once the inserted DNA has been integrated in the genome, it is relatively
stable there
and, as a rule, does not come out again. It normally contains a selection
marker that confers on
the transformed plant cells resistance to a biocide or an antibiotic, such as
kanamycin, G 418,
bieomycin, hygromycin, or chloramphemcol, inter aba. The individually employed
marker should
accordingly permit the selection of transformed cells rather than cells that
do not contain the
inserted DNA.
A large number of techniques are available for inserting DNA into a plant host
cell.
Those techniques include transformation with T -DNA using Agrobacterium
tumefaciew or
Agrobacterium rhrzogenes as transformation agent, fusion, injection, or
electroporation as well as
other possible methods. If agrobacteria are used for the transformation, the
DNA to be inserted
has to be cloned into special plasmids, namely either into an intermediate
vector or into a binary
WO 92/20802 PCT/US92/04316
2103248 32
vector. The intermediate vectors can be integrated into the Ti or Ri plasmid
by homologous
recombination owing to sequences that are homologous to sequences in the T -
DNA. The Ti or
Ri plasmid also comprises the vir region necessary for the transfer of the T -
DNA. Intermediate
vectors cannot replicate themselves in agrobacteria. The intermediate vector
can be transferred
into Agrobacterium tumefaciens by means of a helper plasmid (conjugation).
Binary vectors can
replicate themselves both in E. cold and in agrobacteria. They comprise a
selection marker gene
and a linker or polylinker which are framed by the right and left T -DNA
border regions. They
can be transformed directly into agrobacteria (Holsters et aL [19781 MoL Gen.
Genet. 163:181-187).
The agrobacterium used as host cell is to comprise a plasmid carrying a vir
region. The vir region
Is necessary for the transfer of the 'I -DNA into the plant cell. Additional T-
DNA may be
contained. The bacterium so transformed is used for the transformation of
plant cells. Plant
explants can advantageously be cultivated with Agrobacterdum tumefaciens or
Agrobacterium
rhdzogmes for the transfer of the DNA into the plant cell. Whole plants can
then be regenerated
from the infected plant material (for example, pieces of leaf, segments of
stalk, roots, but also
protoplasts or suspension-cultivated cells) in a suitable medium, which may
contain antibiotics or
biocides for selection. The plants so obtained can then be tested for the
presence of the inserted
DNA. No special demands are made of the plasmids in the case of injection and
electroporation.
It is possible to use ordinary plasnuds, such as, for example, pUC
derivatives.
The transformed cells grow inside the plants in the usual manner They can form
germ
cells and transmit the transformed trait(s) to progeny plants. Such plants can
be grown in the
normal manner and crossed with plants that have the same transformed
hereditary factors or other
hereditary factors. The resulting hybrid individuals have the corresponding
phenotypic properties.
P. xample 11- Cloning of Novel B. thunnsiensis Genes Into Insect Viruses , d 4
A number of viruses are known to infect insects. These viruses include, for
example,
baculoviruses and entomopoxviruses. In one embodiment of the subject
invention, ant-active
genes, as described herein, can be placed with the genome of the insect virus,
thus enhancing the
pathogenicity of the virus. Methods for constructing insect viruses which
comprise at. toxin genes
are well known and readily practiced by those skilled in the art. These
procedures are described,
for c zample, in Meriyweather et aL (Merryweather, A.T., U. Weyer, M.F.G.
Harris, M. Hirst, T.
Booth, R.D. Possee (1990) J. Gen. YtroL 71:1535-1544) and Martens et aL
(Martens, J.W.M., G.
Honee, D Zuidema, JWM. van Lent, B. Visser, J.M. Vlak (1990) AppL
Environmental MurobwL
56(9):2764-2770)-
It should be understood that the examples and embodiments described herein are
for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of this
application and the scope of the appended claims.
WO 92/20802 Pcr/US92/04316
2103248
32-1
BUDAPEST VdZATZ ON TYPE INTRPHATIONAL
RECOGIlTION OF TUC DWOSI? OF 1NICROORCAUISMS
FOR THE PURPOSES OIF PATENT PROCEDURZ
INTZRN TIONAL 1'OPl1
rT0 RECEIPT IN THE CASE OP AN ORIGINAL DEPOSIT
Dr. Jewel Payne issued pursuant to Rule 7.1 by the
Mycogen Corporation INTERNATIONAL DEPOSITARY AUTHORITY
5451 Cberlin Drive Identified at the bottom of this page
San Diego, CA 92121
NAME AND ADDRESS
OP DEPOSITOR
I I. IDENTIFICATION Or THR MXCROORCANISN
Identification reference given by the Accession number given by the
DEPOSITORS IRtZKNATIONAL DEPOSITARY AUTHORITY1
Bacillus thuri><S iensis PS86Q3 NRRL B-19765
Xi. SCXX=XPIC OESCI%XX TION AND/OR PROPOSED TAXONOMIC DZSZCASATION
The main. uorgnni>vae Identified under a above vas aeco.pe*da4 bys
a scientific description
a proposed taxonomic designation
(Mark with a er'oen whore applicable)
Ill. RECEIPT AND ACCEPTANCE
This International Depositary Authority accepts the faicroorganiss identified
under I above,
which was received by it on Feb.6,1991' (date of the original deposit)),
IV. RECEIPT Or R }UZST FOR CONVERSION
The microorganism identified under I above was received by this International
Depositary Authority on (date of the original deposit) and
a request to convert the original deposit to a deposit under the Budapest
Treaty
was received by it on (ants of receipt of request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: AOricu3.tural Research Culture Signature(s) of person(s) having the
power
Collection (NRRL) to represent the International Depositary
International Depositary Authorit Authors of uthoriaed official(s):
LAddress: 1615 N. University Street Date,
Peoria, Illinois 61604 U.S.A. 1J / y
Where Rule 6.4(d) applies, such date Is the date on which the status of
international 1eponitary
authority was acquired.
SUBSTITUTE SHED''
WO 92/20802 PCT/US92/04316
2103248
2suwu'isS'l 'A'f+a..l va ana lava+a....ea i. ..
RECOGNITION OF THE DEPOSIT OF MICR.OOi3CAMISi4S
FOR Tat PURPOSES or PATEN? PROCCOURE
INNTERHATIOHAL FORM
FT-' RECEIPT IN THE CASE Or AN ORIGINAL DEPOSIT
Dr. Jecl Payne issued pursuant to Rule 7.1 by the
Mycogen Corporation INTERNNATIONA DEPOSITARY AUTHORITY
5451 Oberlin Drive identified at the bottom of this page
San Diego, CA 92121
NAME AND ADDRESS
OF DEPOSITOR
1. ID0fZFICATION Of THE MICROORGANISM
Identification teterence given by the Accession number given by the
DEPOSITORa INTSMATIONNAL DEPOSITARY AUTHORITY!
Bacillus illus thuurin i~ s PS140E2 NRRL 8-18812
II. SCIZTYIPIC D&SCRIPTION AND/OR PROPOSED TAXOSOHIC DESIGNATYON
The microorganism identified under I above was accompanied bys
EJ a scientific description
t.. .~ 4 proposed tauonoeic designation
(Mark with a cross where applicable)
Ill. Ractipt. ATE ACCEPTANCB
This International Depositary Authority accepts the Microorganism identified
under I above,
which was received by it onflpr.23,199(date of the original deposit)
2V. RECEI or REQUEST FOR CONVERSION
The microorganism identified under I above vas received by this International
Depositary Authority on (date of the original deposit) and
a request to convert the original deposit to a deposit under the Budapest
Treaty
was received by it on (date of receipt of request for conversion)
V. IirrERNATIOHAL DEPOSITARY 'AUTHORITY
Names Agricultural Research Culture Signature(s) of person(s) having the power
Collection (NRRL) to represent the International Depositary
International Depositary Authorit Authority Or Of author204 official(s):
Addreass 1815 N. University Street Datei
Peoria, Illinois 61604 U.S.A.
Where Rule 6.4(d) applies, such data is the data on which the statue
ofinternationaL depositary
authority was acquired.
SUBSTITUTE SHE
.- .... :-.,, :, ,-..-:. ,.:-:: -.. _., ..,.-.rs: -, r, zr.. .::r .m....
........,. ., F.....:,.,, i:. z, .r .'..'r. ..., o...ii ,
WO 92/20802 PCT/US92/04316
2(03248
32-3
BUDAPEST TBATY ON THE INTERNATIONAL
RECOCNITIOH Of Tans DEPOSIT or NICROOR ZSN.S
FOR THE pwpoSz$ or PATS" PROCIMU
INTLPR3rtATIOWAL FORM
' ~ r~ Jewel Payne RECEIPT IN THE CASE OF AN ORXGINAL DEPOSIT
Corporation issued pur'uant to Rule 7.1 by the
Mycogeri po SPPP ATIONJd7. DEPOSITARY AUTHORITY
5451 Oberlin Drive identified at the bottom of this page
San Diego, CA 92121
NME AND ADDRESS
OF DEPOSITOR
1. IDOMITICATION OF THE NICROORGA)USH
saentification reference given by the Accession number given by the
DaDFOSI?ORz IN7@ -TIGt1AL DQOSITARY AUTHORITY:
Bacillus thuringiensis PS211B2 NRRL B-16921
II. SCIENTIPIC DESCRIPTION AND/OR PROPOSED TA39o1fof{IC DESIe iATION
The microorganism identified under I above was accompanied bys
a scientific description
m a proposed taxonomic designation
(Mark with a cross where applicable) -
III. RECEIPT AND ACCtpTANCZ
This International Depositary Authority accepts the microorganism identified
under I above,
which was received by it onNOV. 15,1991 (date of the original deposit)l
2'V. RECEIPT OF REQUEST ?OR CONY SXOH
The microorganism Identified under I above was received by this International
Depositary Authority on (date of the original deposit) and
a request to convert the original deposit to a deposit under the Budapest
Treaty
was received by it on (date of receipt of request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Names Agricultural Research Culture Signature(s) of parson(s) having the power
Collection (NRRL) to represent the International Depositary
International Depositary Authority Authority or Of authorizg4 otiieial(s)e
Addsesas 1815 N. University Street Data ~ `-
Peoria, Illinois 61604 U.S.A. e~y
Where Rule 6.4(d) applies, such date is the data on which the status of
international depositary
authority was acquired.
SUBSTITUTE SHE
WO 92/20802 PC r/US92/04316
2103248 32-4
BP/A/II/12
page 14
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORH
TO
Dr. Jewel Payne RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Entomology issued pursuant to Rule 7.1 by the
Pal Corporation INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
5457 Oberlin Dr.
San Diego, CA 92121
HAMS AND ADDRESS
L = OF DEPOSITOR
1. IDENTIFICATION OF THE MICWORG)WISM
Identification reference given by the Accession number given by the
DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Bacillus thuriiensis PS17 NRRL B-18243
3g. s IC oN AND/OR F 'i71I DMIC DESIGNATION
The microorganism identified under I above was accaosvpanied by:
a scientific description
.~4
a proposed taxonomic designation -
.~(l4ar& with a cross where applicable)
III. RECEIPT AM ACCEPT NCE
This International Depositary Authority accepts the microorganism identified
under I above,
which was received by it on July 28 ,1987(date of the original deposit)1
IV. IONAL DEPOSITARY AUTHORITY
tee: Agricultural Research Culture Signature(s) of person(s) having the power
Collection (Mn) to represent the T!!15021 ational Depositary
International Depositary Authority Authority o~roff 4iorize d official (s) :
Address. 1815 N. University Street 1 ~1+4, _ _ a--
Peoria, Illinois 61604 U.S.A. Date: ~(
f iU ds7
Where Rule 6.4(d) applies, such date is the date on which the status of
international depositary
authority was acquired; where a deposit made outside the Budapest Treaty after
the acquisition
of the status of international depositary authority is converted into a
deposit under the
Budapest Treaty, such date is the date on which the microorganism was received
by the
international depositary authority.
Form BP/4 (sole page) SUBSTITUTE SHE
WO 92/20802 PCF/US92/04316 2 10 3248 32-5
HP/A/II/11
page 14
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
FTO
Dr. Jewel Payne RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
tolocgy issued pursuant to Rule 7.1 by the
INTERNATIONAL DEPOSITARY AUTHORITY
Mycogen Corporation identified at the bottom of this page
5457 Oberlin Dr.
.can Diego, CA 92121
NAME AND ADDRESS
t OF DEPOSITOR
1. IDENTIFICATION OF THE MXCRCORGANISM
Identification reference given by the Accession number given by the
DEPOSMTOR: INTERNATIONAL DEPOSIU AUTHORITY:
Bacillus thuringiensis ps33F2 NRRL B-18244
II. s C DESCRIPTION AND/OR PROPOSED TAXON02ZC DESIMFATION
The microorganism identified under I above was accompanied by:
~- a scientific description
-.a
a proposed taxonomic designation
(21axk with a cross where applicable)
IYI.' '1 PT ANA ACC'EPTANcz
This International Depositary authority accepts the microorganism identified
under I above,
1.
which was received by it on July 28,1987(date of the original deposit)
TV. ' ZNTEFWATIOUAL DEPOSITARY AUTHORITY
e: Agricultural Research Culture Signature(s) of person(s) having the power
Collection (NRRL) to represent the International Depositary
International Depositary Authority Authority or of uthorized official(s):
Address: 1815 N. University Street
Peoria, Illinois 61604 U.S.A. Date:
r~)
Y7
l Where Rule 6.4(d) applies, such data is the date an which the status of
international depositary
authority was acquired; where a deposit made outside the Budapest Treaty after
the acquisition
of the status of international depositary authority is converted into a
deposit under the
Budapest Treaty, such date is the date on which the microorganism was received
by the
international depositary authority.
`Fora BP/4 (sole page) SUBSTITUTE SHMET
WO 92/20802 PCT/US92/04316
32-6
2103248
BP/A/II/12
page 14
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
TO
'Dr. Jewel Payne RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Entomology issued pursuant to Rule 7.1 by the
INTERNATIONAL DEPOSITARY AUTHORITY
Mycogen Corporation identified at the bottom of this page
5457 Oberlin Dr.
San Diego, CA 92121
j NAME AND ADDRESS
OF DEPOSITOR
1. IDENTIFICATION OF TEE MICROORGANISM
Identification reference given by the Accession number given by the
DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Bacillus thurinSiensis PS63B NRRL B-18246
11. SCI IFIC DESCRIPTION AM/oR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied by:
a scientific description
a proposed taxonomic designation
f1ara with a cross where applicable)
xxl. RECt.IIPT AND ACCEPTANCE
This International Depositary authority accepts the microorganism identified
under I above,
which was received by it on July 28 ,1987(date of the original deposit)1
IV. INTERNATIONAL DEPOSITARY AUTHORITY
..ame; Agricultural Research Culture Signature(s) of person (a) having the
power
Collection (NRRL) to represent the ternational Depositary
International Depositary Authority Authority or of au riled official (s)
radr..a: 1815 N. University Street
Peoria, Illinois 61604 U.S.A. Date:
1 Where Rule 6.4 (d) applies, such date is the date on which the status of
international depositary
authority was acquired; where a deposit made outside the Budapest Treaty after
the acquisition
of the status of international depositary authority is converted into a
deposit under the
Budapest Treaty, such date is the date on which the microorganism was received
by the
international depositary authority.
q
Form BP/4 (sole page) SUBSTITUTE SHEET
r r = T r f 5
ryt vl w
r ~ i ! i f i'' y <4
WO 92/20802 2:103248 PCT/US92/04316
32-7
BUDAPEST TREATY ON THE INTERNATIONAL
ECOGNITION OF THE DEPOSIT OF MICAC ;ANISMS
FOR THE PURPOSES OF PATENT PRO(. IRE
INTERNATIONAL FORM
` s. L?nore Linda R. N d RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7.1 by the
Mycogen Corporation INTERNATIONAL DEPOSITARY AUTHORITY
5451 Oberlin Dr. identified at the bottom of this page
San Diego, CA 92121
NAME AND ADDRESS
L OF DEPOSITOR
I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the Accession number given by the
DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Escherichia cols NM522/pMYC2316 MR608 NRRL B-18785
It. SCIENTIFIC DESCRIPTION AaO/Oft PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied byt
a scientific description
a proposed taxonomic designation
(Mark with a cross where applicable)
III RECEIPT AM ACCEPTANCE
This international Depositary_ Auth r4laccepts the microorganism identiified
under I above,
which was received by it an i~`ilar.ll~Ss (date of the original deposit)
IV. RECEIPT OF REOUZST FOR CONVERSION
The microorganism identified under I above was received by this International
Depositary Authority on (date of the original deposit) and
a request to convert the original deposit to a deposit under the Budapest
Treaty
was received by it on (date of receipt of request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: Agricultural Research Culture Signature(s) of rson(s) having the power
Collection (NRRL) to represent the ternational Depositary
International Depositary Authorit Authors to au ized attic al(s):
Address: 1815 N. University Street Date:
Peoria, Illinois 61604 frJU.S.A.
Where Rule 6.4(d) applies, such date is the date on which the status of
international depositar
authority was acquired. SUBSTITUTE SUBSTITUTE SHEET
WO 92/20802
PCF/US92/04316
32-8
,BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICR-"M JISXS
FOR THE PURPOSES OF PATENT PRE JURE
INTERNATIONAL FORM
Linda R. Nyga d RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
i' I~ issued pursuant to Rule 7.1 by the
My en enOr Corporation R. INTERNATIONAL DEPOSITARY AUTHORITY
5451 Oberlin Dr. identified at the bottom of this page
San Diego, CA 92121
NAME AND ADDRESS
OF DEPOSITOR
1. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the Accession number given by the
DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Escherichia coil NM522/PM"1C 2321 MR607 NRRL B-18770
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAEONONIC DESIGNATION
The nieroorganisn identified under I above, was accompanied byt
a scientific description
a proposed taxonomic designation
(Mark with a cross where applicable)
III. RECEIPT AND ACCEPTANCE
This International Depositary Authority accepts the microorganism identified
under I above,
which was received by it on Feb.14,1991 (date of the original deposit)1
IV. RECEIPT OF REOUEST FOR CONVERSION
The microorganism identified under I above was received by this International
-Depositary Authority on (date of the original deposit) and
a request to convert the original deposit to a deposit under the Budapest
Treaty
was received by it on (date of receipt of request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Names Agricultural Research Culture Signature(s) of person(s) having the power
Collection (NRRL) to represent the International Depositary
International Depositary Authority Authori o of a horized official(s):
Address: 1815 N. University Street Date:
Peoria, Illinois 61604 U.S.A. ~ycfr
Wbere Rule 6.4(d) applies. such date is the data on which the status of
international depositary
authority was acquired.
Form ~/4 (sole page) SUBSTITUTE SHEET
N-Y
err:~Ar eta rh.~ rI fr ~<<i ~~ A`rt. ~f ` . , .,
WO 92/2M2 PCT/US92/04316
2103248
32-9
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORa"ISMS
POR 72EC PURPOSES OF PAT3WT PROCEDdm..
INTERNATIONAL FORM
RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7.1 by the
i4s Lenore Linda R. Nygaard INTERtiATIONAL DEPOSITARY AUTHORITZ
Mycogen Corporation identified at the bottom of this page
5451 Oberlin Dr.
San Diego, CA 92121
NAHE AND ADDRESS
OF DEPOSITOR
1. IDE33TZrICATION Or T3lE MICROORGANISM
Identification reference given by the Accession number given by the
DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Eseherichia coli
19-1522/pMYC2317 MR609 NRRL B-18816
II. SCIglITZFZC DESCR ION AN/OR PROPOSED TAXONOMIC DESIC ATION
The microorganism identified under I above was accompanied bys
a scientific description
fl : 1, a proposed taxonomic designation
(Mark with a cress where applicable)
111. RECZXPT AND AOCETAaaCL
This International Depositary Authority accepts the microorganism identified
under I above,
which was received by it on Apr.24,1991 (date of the original depokit)1
W. RECEIPT or REQUEST FOR CONVSSIOM
The microorganism identified under I above was received by this International
I Depositary Authority on (date of.the original deposit) and
a requestt to convert the original deposit to a deposit under the Budapest
Treaty
was received by it on (date of receipt of request for conversion)
V. INTERUATIONAL DEPOSITARY AUTHORITY
Names Agricultural Research Culture Signature(s) of person(s) having the power
Collection (NRRL) to represent the International Depositary
International Depositary Authorit Authority or of authrariz d officiatts):
( Address: 1815 N. University Street Dac9: Peoria, Illinois G-1604 U.S.A.
Where Rule 6.4(d) applies. such dat',is the date on which the status of
international. depositar=
authority was acquired. SUBSTITUTE SHE
WO 92/20802 PACT/US92/04316
2103248 32- 10
BP/A/II/12
page 14
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
14s[ enure Linda R. Nygaard 1 RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Mycogen corporation issued pursuant to Rule 7.1 by the
5451 Oberlin Dr. INTERNATIONAL DEPOSITARY AUTHORITY
San Diego, CA 92121 identified at the bottom of this page.
L NAME AND ADDRESS
OF DEPOSITOR
a. IDENTIFICATION OF THE MICROORGANISM
identification reference given by the Accession number given by the
DPOSZ=Rt INTERNATIONAL DEPOSITARY AUTHORITY:
Escherichia coli NM522/pMYC1627 MR398 NRRL B-18651
I2. S C DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied by.
ED a scientific description
a proposed . taxonomic designation
(Mark with a cross where applicable)
Z=- R CEIPT AND ACCEPTANCE
This International Depositary Authority accepts the microorganism identified
under I above,
which was received by it on May 11,1990 (date of the original deposit)
Iv. X TZRNATZONAL DEPOSITARY AUTHORITY
mez Agricultural Research Culture Signature(s) of person(s) having the power
Collect on (NRRL) to represent the I ornational Depositary
International Depositary Authority Authority or f au riaed official(s):
Address:.1815 N. University, Street.''
Peoria, Illinois 61604 U.S.A. Date:
1 Where Rule 6.4(d) applies, such date is the date on which the status of
international depositary
authority was acquired; where a deposit made outside the Budapest Treaty after
the acquisition
of the status of international depositary authority is converted into a
deposit under the
Budapest Treaty, such date is the date on which the microorganism was received
by the
international depositary-authority.
Form BP/4 (sole page) SUBSTITUTE SH
=..., rr~' ,,,,,. .., fxr+ ~;~:.,. ..,<., ..=,..,:~a=.:~+r:.;.a=~ ........,
,.........., ............::.Y*"r.r=,.;,--t+f rr;. ..,...... ~rs,S+..;2,,
F......... .........F....... .3r... ... . .. ...,
WO 92/20802 PCT/US92/04316
2103248
32-11
BP/A/II/12
page 14
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Ms1 -Lenore Linda R. Nygaard RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Mycogen Corporation issued pursuant to Rule 7.1 by the
5451 Oberlin Dr. INTERNATIONAL DEPOSITARY AUTHORITY
San Diego, CA 92121 identified at the bottom of this page.
NAME AND ADDRESS
L OF DEPOSITOR
1. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the Accession number given by the
DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
Eseberichia coli NM522/pMYC1628 MR399 NRRL B-18652
XI. S C DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was aeeowpanied by:
a scientific description
X(~ a proposed taxonomic designation
(Mark with a cross where applicable)
III. RECEIPT ANDACCEpTANCE
This International Depositary Authority accepts the microorganism identified
under I above,
which was received by it ca May 11,1990 (date of the original deposit)1
IV. INTERNATIONAL DE'POSITVM AUTHORITY
boa; Agricultural Research Culture Signature(s) of person(s) having the power
Collection (NRR,L) to represent the ternational Depositary
International Depositary Authority Authority or f au prized offi (s) :
Address: 1815 N. University Street ~/~''
Peoria, Illinois 61604 U.S.A. Date:
t. Ill'/yv
l Where Rule 6.4(d) aspplles, such date is the date on which the status of
international depositary
authority was acquired; where a deposit made outside the Budapest Treaty after
the acquisition
of the status of international depositary authority is converted into a
deposit under the
Budapest Treaty, such date is the date on which the microorganism was received
by the
International depositary =authority.
Form BP/4 (sole page) SUBSTITUTE SHEET
WO 92/20802 PCT/US92/04316
21O 2~0 32-12
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF NICR006IGA (zsMa
FOR THE PURPOSES OF PATEN? FROC=URE
INTERNATIONAL FORM
1=" RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Ms. Lenore Linda R. Nygaard Issued pursuant to Rule 7.1 by the
Mycogen Corporation INTERNATIONAL DEPOSITARY AUTHORITY
5451 Oberlin Drive Identified at the bottom of this page
San Diego, CA 92121
HAMS AND ADDRESS
OF DEPOSITOR
I. IDENTI?ICATION OF THE MICROORGANISM
Identification reference given by the Accession number given by the
DEPOSITORa INTERNATIONAL DEPOSITARY AUTHORITY:
Escherichia coli
NM 522/pMYC 1642 MR626 NRRL B-18961
EI. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism Identified under I above was accompanied byt
a, scientific description
a. a proposed taxonomic designation
dfark with a cross where applicable)
1 X I . RECEIPT AND ACCEPTANCE
This International Depositary authority accepts the microorganism identified
under I above,
which was received by it on 4-10-92 (date of the original deposit)l
IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International
Depositary Authority on (date of the original deposit) and
a request to convert the original deposit to a deposit under the Budapest
Treaty
was received by It on (date of receipt of request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
names Agricultural Research Culture Signature(s) of person(s) having the power
Collection (NRRL) to represent the International Depositary
International Depositary Authorit Authority or of A thosizoo official(s)a
Addressi 1815 N. University Street Date: 4 -%
Peoria, Illinois 61604 U.S.A. ((,-y')-
Where Rule 6.4(d) applies, such date is the date on which the status of
International depositary
authority was acquired. -~^ SHEET
Forte BP/4 (sole page) SUBSTI i UTE
WO 92/20802 2103248 PCT/US92/04316
33
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Payne, Jewel M.
Kennedy, M. Keith
Randall, John Brooks
Meier, Henry
Uick, Heidi Jane
Foncerrada, Luis
Schnepf, Harry E.
Schwab, George E.
(ii) TITLE OF INVENTION: Novel Bacillus thuringiensis isolates
Active Against Hymenopteran Pests and Genes Encoding
Hymenopteran Active Toxins
(iii) NUMBER OF SEQUENCES: 38
(iv) CORRESPONDENCE ADDRESS:
A) ADDRESSEE: David R. Saliwanchik
B) STREET: 2421 N.W. 41st Street, Suite A-1
C CITY: Gainesville
D STATE: FL
E COUNTRY: USA
F ZIP: 32606
(v) COMPUTER ) MDIUMATYYPPE Floppy disk
B) COMPUTER: IBM PC compatible
C OPERATING SYSTEM: PC-DOS/MS-DOS
D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
JA APPLICATION NUMBER: US
B FILING DATE:
C CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
JA NAME: Saliwanchik, David R.
B REGISTRATION NUMBER: 31,794
C REFERENCE/DOCKET NUMBER: M/SCJ 104
(ix) TELECOMMUNICATION INFORMATION:
M TELEPHONE: 904-375-8100
TELEFAX 904-372-5800
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS
JA LENGTH: 4155 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: double
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
JA~ ORGANISM: Bacillus thuringiensis
BSTRAIN: PS17
C) INDIVIDUAL ISOLATE: PS17a
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. soli NM522(pMYC1627) NRRL B-18651
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATGGCAATTT TAAATGAATT ATATCCATCT GTACCTTATA ATGTATTGGC GTATACGCCA 60
CCCTCTTTTT TACCTGATGC GGGTACACAA GCTACACCTG CTGACTTAAC AGCTTATGAA 120
CAATTGTTGA AAAATTTAGA AAAAGGGATA AATGCTGGAA CTTATTCGAA AGCAATAGCT 180
GATGTACTTA AAGGTATTTT TATAGATGAT ACAATAAATT ATCAAACATA TGTAAATATT 240
GGTTTAAGTT TAATTACATT AGCTGTACCG GAAATTGGTA TTTTTACACC TTTCATCGGT .300
TTGTTTTTTG CTGCATTGAA TATACATGAT GCTCCACCTC CTCCTAATGC AAAAGATATA 360
TTTGAGGCTA TGAAACCAGC GATTCAAGAG ATGATTGATA GAACTTTAAC TGCGGATGAG 420
CAAACATTTT TAAATGGGGA AATAAGTGGT TTACAAAATT TAGCAGCAAG ATATCAATCT 480
SUBSTITUTE SHEET
WO 92/20802
FCT/US92/04316
2103248 34
ACAATGGATG ATATTCAAAG CCATGGAGGA TTTAATAAGG TAGATTCTGG ATTAATTAAA 540
AAGTTTACAG ATGAGGTACT ATCGTTAAAT AGTTTTTATA CAGATCGTTT ACCTGTATTT 600
ATTACAGATA ATACAGCGGA TCGAACTTTG TTAGGTCTTC CTTATTATGC TATACTTGCG 660
AGCATGCATC TTATGTTATT AAGAGATATC ATTACTAAGG GTCCGACATG GGATTCTAAA 720
ATTAATTTCA CACCAGATGC AATTGATTCC TTTAAAACCG ATATTAAAAA TAATATAAAG 780
CTTAACTCTA AAACTATTTA TGACGTATTT CAGAAGGGAC TTGCTTCATA CGGAACGCCT 840
TCTGATTTAG AGTCCTTTGC AAAAAAACAA AAATATATTG AAATTATGAC AACACATTGT 900
TTAGATTTTG CAAGATTGTT TCCTACTTTT GATCCAGATC TTTATCCAAC AGGATCAGGT 960
GATATAAGTT TACAAAAAAC ACGTAGAATT CTTTCTCCTT TTATCCCTAT ACGTACTGCA 1020
GATGGGTTAA CATTAAATAA TACTTCAATT GATACTTCAA ATTGGCCTAA TTATGAAAAT 1080
GGGAATGGCG CGTTTCCAAA CCCAAAAGAA AGAATATTAA AACAATTCAA ACTGTATCCT 1140
AGTTGGAGAG CGGGACAGTA CGGTGGGCTT TTACAACCTT ATTTATGGGC AATAGAAGTC 1200
CAAGATTCTG TAGAGACTCG TTTGTATGGG CAGCTTCCAG CTGTAGATCC ACAGGCAGGG 1260
CCTAATTATG TTTCCATAGA TACTTCTAAT CCAATCATAC AAATAAATAT GGATACTTGG 1320
AAAACACCAC CACAAGGTGC GAGTGGGTGG AATACAAATT TAATGAGAGG AAGTGTAAGC 1380
GGGTTAAGTT TTTTACAACG AGATGGTACGAGACTTAGTG CTGGTATGGG TGGTGGTTTT 1440
GCTGATACAA TATATAGTCT CCCTGCAACT CATTATCTTT CTTATCTCTA TGGAACTCCT 1500
TATCAAACTT CTGATAACTA TTCTGGTCAC,GTTGGTGCAT TGGTAGGTGT GAGTACGCCT 1560
CAAGAGGCTA CTCTTCCTAA TATTATAGGTCAACCAGATG AACAGGGAAA TGTATCTACA 1620
ATGGGATTTC CGTTTGAAAA AGCTTCTTAT GGAGGTACAG TTGTTAAAGA ATGGTTAAAT 1680
GGTGCGAATG CGATGAAGCT TTCTCCTGGG CAATCTATAG GTATTCCTAT TACAAATGTA 1740
ACAAGTGGAGAATATCAAAT TCGTTGTCGT TATGCAAGTA ATGATAATAC TAACGTTTTC 1800
TTTAATGTAG ATACTGGTGG AGCAAATCCA ATTTTCCAAC AGATAAACTT TGCATCTACT 1860
GTAGATAATA ATACGGGAGT ACAAGGAGCA AATGGTGTCT ATGTAGTCAA ATCTATTGCT .1920
ACAACTGATA ATTCTTTTAC AGAAATTCCT GCGAAGACGA TTAATGTTCA TTTAACCAAC 1980
CAAGGTTCTT CTGATGTCTT TTTAGACCGT ATTGATTTCATACCTTTTTC TCTACCTCTT 2040
ATATATCATG GAAGTTATAA TACTTCATCA GGTGCAGATG ATGTTTTATG GTCTTCTTCA 210D.--t
AATATGAATT ACTACGATAT AATAGTAAAT GGTCAGGCCA ATAGTAGTAG TATCGCTAGT 2160
TCTATGCATT TTGTTAATGA AGGAAAAGTG ATAAAAACAA TTGATATTCC AGGGCATTCG .2220
GAAACCTTCT TTGCTACGTT CCCAGTTCCA GAAGGATTTA ATGAAGTTAG AATTCTTGCT 2280
GGCCTTCCAG AAGTTAGTGG AAATAATACC GTACAATCTA ATAATCCGCC TCAACCTAGT 2340
AATAATGGTGGTGGTGATGG TGGTGGTAAT GGTGGTGGTG ATGGTGGTCA ATACAATTTT 2400
TC'1"ZTAAGCG GATCTGATCA TACGACTATT TATCATGGAA AACTTGAAAC TGGGATTCAT 2460
GTACAAGGTA ATTATACCTA TACAGGTACT CCCGTATTAA TACTGAATGC TTACAGAAAT 2520
AATACTGTAG TATCAAACAT TCCAGTATAT TCTCCTTTTG ATATAACTAT ACAGACAGAA 2580
GCTGATAGCC TTGAGCTTGA ACTACAACCT AGATATGGTT TTGCCACAGT GAATGGTACT 2640
GCAACAGTAA AAAGTCCTAA TGTAAATTAC GATAGATCAT TTAAACTCCC AATAGACTTA 2700
CAAAATATCA CAACACJMT AAATGCATTA TTCGCATCTG GAACACAAAA TATGCTTGCT 2760
CTTAATGAAA GTGATCATGA TATTGAAGAA GTTGTATTAA AAGTGGATGC CTTATCAGAT 2820
GAAGTATTTG GAGATGA AA GAAGGCTTTA CGTAAATTGG TGAATCAAGC AAAACGTTTG 2880
AGTAGAGCAA GAAATCTTCT GATAGGTGGG AGTTTTGAAA ATTGGGATGC ATGGTATAAA 2940
GGAAGAAATG TAGTAACTGT ATCTGATCAT GAACTATTTA AGAGTGATCA TGTATTAACA 3000
CCACCACCAG GATTGTCTCC ATCTTATATT TTCCAAAAAG TGGAGGAATC TAAATTAAAA 3060
CCAAATACAC GTTATATTGT TTCTGGATTC ATCGCACATG GAAAAGACCT AGAAATTGTT 3120
WO 92/20802 PCT/US92/04316
2103248
GTTTCACGTT ATGGGCAAGA AGTGCAAAAG GTCGTGCAAG TTCCTTATGG AGAAGCATTC 3180
CCGTTAACAT CAAATGGACC AGTTTGTTGT CCCCCACGTT CTACAAGTAA TGGAACCTTA 3240
GGAGATCCAC ATTTCTTTAG TTACAGTATC GATGTAGGTG CACTAGATTT ACAAGCAAAC 3300
CCTGGTATTG AATTTGGTCT TCGTATTGTA AATCCAACTG GAATGGCACG CGTAAGCAAT 3360
TTGGAAATTC GTGAAGATCG TCCATTAGCA GCAAATGAAA TACGACAAGT ACAACGTGTC 3420
GCAAGAAATT GGAGAACCGA GTATGAGAAA GAACGTGCGG AAGTAACAAG TTTAATTCAA 3480
CCTGTTATCA ATOGAATCAA CGGATTGTAT GAAAATGGAA ATTGGAACGG TTCTATTCGT 3540
TCAGATATTT CGTATCAGAA TATAGACGCG ATTGTATTAC CAACGTTACC AAAGTTACGC 3600
CATTGGTTTA TGTCAGATAG ATTCAGTGAA CAAGGAGATA TAATGGCTAA ATTCCAAGGT 3660
GATTTAAATC GTGCGTATGC ACAACTGGAA CAAAGTACGC TTCTGCATAA TGGTCATTTT 3720
ACAAAAGATG CAGCTAATTG GACAATAGAA GGCGATGCAC ATCAGATAAC ACTAGAAGAT 3780
GGTAGACGTG TATTGOGACT TCCAGATTGG TCTTCGAGTG TATCTCAAAT GATTGAAATC 3840
GAGAATTTTA ATCCAGATAA AGAATACAAC TTAGTATTCC ATGGGCAAGG AGAAGGCACG 3900
GTTACGTTGG AGCATGGAGA AGAAACAAAATATATAGAAA CGCATACACA TCATTTTGCG 3960
AATTTTACAA CTTCTCAACG TCAAGGACTC ACGTTTGAAT CAAATAAAGT GACAGTGACC 4020
ATTTCTTCAG AAGATGGAGA ATTCTTAGTG GATAATATTG CGCTTGTGGA AGCTCCTCTT 4080
CCTACAGATG ACCAAAATTC TGAGGGAAAT ACGGCTTCCA GTACGAATAG CGATACAAGT 4140
ATGAACAACA ATCAA 4155
(2) INFORMATION FOR SEQ ID AO:2
(i) S CE CHARACTERISTICS:
JjUZ
LENGTH: 1385 amino acids
8 TYPE: amino acid
C STRANDEDNESS single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
IAI ORGANISM: BACILLUS THURINGIENSIS .-rt
BSTRAIN: PS17
C INDIVIDUAL' ISOLATE: PS17a
(vii) IMMEDIATE SOURCE:
(8)CLONE: E. cols NM522(pMYC1627) NRRL B-18651
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Not Ala Ile Lou Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val Leu
1 5 10 15
Ala Tyr Thr Pro Pro Ser Phe Leu Pro Asp Ala Gly Thr Gin Ala Thr 25
Pro Ala Asp Leu Thr Ala Tyr Glu Gln Leu Leu Lys Asn Leu Glu Lys
35 40 45
Gly Ille Asn Ala Gly Thr, Tyr Ser Lys Ala Ile Ala Asp Val Leu Lys
s 55
Gly Ile Phe Ile Asp Asp Thr Ile Asn Tyr Gln Thr Tyr Val Asn Ile
65 70 75 80
Gly Len Ser Leu 8ie Thr Leu Ala Val PO Glu Ile Gly Ile Phe Thr
Pro Phe Ile Gl09 Leu Phe Phe Ala Ala Leu Asn Lys His Asp Ala Pro 1
Pro Pro Pro Asn Ala Lys Asp 1iee Phe Glu Ala Met LYS Pro Ala Ile
Gin Glu Net Ile Asp Arg Thr Leu Thr Ala Asp Glu Gin Thr Phe Leu
130 135 140
~... '',, .....A _7,f ~1r........ ...;!-a.L. .. ..., 4'T
:r
. C._.i, ..,`~ t,._ . ..
WO 92/20802 PCT/US92/04316
2103248 36
Asn Gly Glu Ile Ser Gly Lau Gin Asn Leu Ala Ala Arg Tyr Gln Ser
145 150 155 160
Thr Met Asp Asp lie Gln Ser His Gly lly Phe Asn Lys Val Asp Ser
16 15
Gly Leu Ile Lys Lye Pile Thr Asp Glu Valll Leu Ser Lou Ann Ser Pile
180 185 190
Tyr Thr Asp Arg Leu Pro Val Phe Ile Thr Asp Asn Thr Ala Asp Arg
195 200 205
Thr Lau Leu Gly Leu Pro Tyr Tyr Ala Ile Lau Ala Ser Met His Lau
210 215 220
Met Lau Lau Arg Asp Ile Ile Thr Lys Gly Pro Thr Trp Asp Ser Lys
225 230 235 240
Ile Asn Phe Thr Pro Asp Ala Ile Asp Ser Phe Lys Thr Asp Ile Lys
245 250 255
Asn Asn Ile Lys Lou s Tyr Ser Lys 265 Thr Ile Tyr Asp Val 27e Gin Lys
Gly Lou Ala Ser Tyr Gly Thr PPrro Ser Asp Leu Glu SSerr Phe Ala Lys
27 25
Lys Gln Lye Tyr Ile Gin Ile Met Thr Thr His s Lau Asp Phe Ala
290 295 300
Arg Lau The Pro Thr The Asp Pro Asp Leu Tyr Pro Thr Gly Ser Giy
30310 315 320
Asp Ile Ser Leu G32n Lys Thr Arg Arg lie e Lau Ser Pro Phe 335 Ile
Ile Arg Thr 3laa Asp Gly Leu Thr Len Ann Asn Thr'Ser lie e Asp Thr 345
Ser Asn Tr
Pro Ann Tyr Gin A36n Giy Asn Gly Ala 36e Pro Asn Pro
Lys Glu Arg Ile Lau Lys G35 in The Lys Leu Tyr Prroo Ser Trp Arg Ala
G1 Gin Tyr Gly Gly Lau Lou Gin Pro Tyr Lau Trp Ala-Ile Gin Val
385 390 395 400
Gin Asp Ser Val GG01u5 Thr Arg Lou Tyr G1 Gin Lau Pro Ala VVal Asp 45
Pro Gin Ala Glyy Pro Ann Tyr Val Ser Ileee Asp Ser Ser Ann Pro Ile
420 425 430
Ile Gin lie Asn Met Asp Thr Trp Lys Thr Pro Pro 44n Gly Ala-Ser
43; 4ib
Gly Trp Asn Thr Asn Lau 4ett Arg Giy Ser Val Seer Gly Lou Ser Pile
450
46
Lau Gin Arg Asp Gly Thr Arg Lau Ser Ala Glq Met Gly Gly Gly Phe
465 470 475 480
Ala Asp Thr Ile Tyrr Ser Lou Pro Ala 4Thr His Tyr Lau Ser Tyr Lou
48 49
Tyr Gly Thr PPro ro Tyr Gin Thr SerA0p Ann Tyr Ser G1y His Val Gly
Ala Lau Val
1l Gly Val Ser Thr Proo Gin Glu Ala Thr 52eu Pro Ann Ile
Ile Glyy Gin Pro Asp Glu Gin Gly Asn Val Ser Thr Met Gly Phe Pro
53D 535 540
Phe Glu Lys Ala Ser Tyr Gly Gly Thr Val Val Lys Glu Trp Leu Asn
545 550 555 560
Gly Ala Asn Ala Met Lys Lau Ser Pro 5G ly Gin Ser Ile G1y lie e Pro
Ile Thr Asn Val Thr Ser Gly Glu Tyr Gin Ile Arg Cys Sg Tyr Ala
Ser Asn 59p Asn Thr Asn Val Pile Phe Asn Val Asp Thr Gly Gly Ala
6 605
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37
Asn PProo Ile Phe Gln Gin 6le Asn Phe Ala Ser Thr Val Asp Asn Asn
1 620
Thr Gly Val Gln Gly Ala Asn Gly Val Tyr Val Val Lys Ser Ile Ala
625 630 635 640
Thr Thr Asp Asn Ser Phe Thr Glu Ile Pro Ala Lys Thr Ile Asn Val
645 650 655
His Lau Thr AAenn Gin Gly Ser Ser Asp Val Phe Leu Asp 6ra Ile Glu
660 6 Phe Ile Pro Phe Ser Leu Pro Lau Ile Tyr His Gly Ser Tyrr Asn Thr
675 680 685
Ser Ser Gly Ala Asp Asp Val Leu Trp Ser Ser Ser Asn Met Asn Tyr
690 695 700
Tyr Asp Ile Ile Val Asn Gly Gin Ala Asn Ser Ser Ser Ile Ala Ser
705 710 715 720
Ser Met His Lau Len Asn Lys Gly Lys Val Ile Lys Thr Ile Asp Ile
725 73
Pro Gly His Ser Glu Thr Phe Phe Ala Thr Phe Pro Val Pro Glu Giy
740 745 750
The Asn Glu Val Arg Ile Leu Ala Giy Lau Pro Glu Val Ser Gly Asn
755 760 765
Ile Thr Val Gin Ser Asn Asn Pro Pro Gin Pro Ser Asn Asn Gly Gly
770 775 780
G1y Asp Gly Gly Gly Asn Gly Gly Gly Asp Gly Gly Gin Tyr Asn Phe
785 790 795 800
Ser Lau Ser Gly Ser Asp His Thr Thr lie Tyr His Gly Lys L81u Gin
805 810
Thr Gly Ile HHis Val GinGly AsnTyr Thr Tyr Thr Gly Thr Pro Val
825 830
Lau Ile Leu Asn Ala Tyr Arg Asn Asn Thr Val Val Ser Ser Ile Pro
835 840 845
Val Tyr Ser Pro Phe Asp I815e Thr Ile Gln Thr Gin Ala Asp Ser Lau
850
Gin Lau Glu Lau Gin Pro Arg TyrGly Phe Ala Thr Val Asn Gly Thr
865 870 875 880
Ala Thr Val Lys Ser Pro Asn Val Asn Tyr Asp Arg Ser Phe Lps Lau
885 890 895 ,e=~
Pro Ile Asp Leu Gin Aan Ile Thr Thr Gln Val Asn Ala Lau Phe-Ala
900 905 910
Ser Gly TThr5 Gln Asn Met Lau 92aa His Asn Val Ser Asp His Asp Ile
Glu GG310 Val Val Leu Lys Val Asp Ala Lau Ser Asp Glu Val Phe.Gly 935 0
Asp Glu Lys Lys Ala Leu Arg Lys Lau Val Asn Gln Ala Lys Arg Lau
945 950 955 960
Ser Arg Ala Arg A65 Lau Lau Ile Gly 9G ly Ser Phe Glu Asn Trp Asp
Ala Trp Tyr Lyas Gly Avg Asn Val Val Thr Val Ser Asp Hiss Gin Lau 95 Phe Lys
Ser Asp His Val Leu Leu Pro Pro Pro Giy Lau Ser Pro Ser
995 1000 1005
Tyr Ile Phe Gln Lys Val Glu Glu Ser Lys Lau Lyys Pro Asn Thr Arg
1010 1015 1020
Tyr Ile Val Ser Gly Phe Ile Ala His Gly Lys Asp Lau Gin Ile Val
1025 1030 1035 1040
Val Ser Arg Tyr G1y Gin Glu Val Gin Lys Val Val Gin Val Pro Tyr
1045 1050 1055
Gly Glu Ala Phe Pro Lau Thr Ser Asn Gly Pro Val Cys Cvs Pro Pro
1060 1065 1070
WO 92/20802 PCT/US92/04316
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Arg Ser Thr Ser Asn Gly Thr Lou Gly Asp Pro His Phe Phe Ser Tyr
1075 1080 1085
Ser Ile Asp Val Gly Ala Lou Asp Lou Gln Ala Asn Pro Gly Ile Glu
1090 1095 1100
Phe Gly Lou Arg Ile Val Asn Pro Thr Gly Met Ala Arg Val Ser Asn
1105 1110 1115 1120
Lou Glu Ile Arg Glu Asp Arg Pro Lou Ala Ala Asn Glu Ile Ar Gln
1125 1130 1135
Val Gln Arg V140 Ala Arg Asn Trp AArrg5Thr Glu Tyr Glu L150G1u Arg
Ala Glu Val Thr Ser Lou Ile Gin Pro Val Ile Asn Ara Ile Asn Gly
1155 1160 1165
Lou TTyyr Glu Asn Gly Asn Trp Asa Gly Ser Ile Arg Ser Asp Ile Ser
1970 1175 1180
Tyr Gln Asn Ile Asp Ala Ile Val Lou Pro Thr Lou Pro Lys Lou Arg
1185 1190 1195 1200
His Trp Phe Met Ser Asp Arg Phe Ser Glu Gln Gly Asp Ile Met Ala
1205 1210 1215
Lys Phe Gln Gly Ala Lou Asn Arg Ala Tyr Ala Gin, Leu Glu Gin Ser
1220 1225 1230
Thr Lou Lou His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr
1235 1240 1245
Ile Glu Gly Asp Ala His Gin Ile Thr Lou Glu Asp Gly Arg Arg Val
1250 1255 1260
Lou Arg Lou Pro Asp Trrpp Ser Ser Ser Val Ser Gin Met lie Gin Ile
1265 2270 1275 1280
Glu Asn Phe Asn Pro Asp Lys Glu Tyr Asn Lou Val Phe His G1y Gin
1285 1290 1295
Gly Glu Gly Thr Val Thr Lou Glu His Gly Glu Glu Thr Lys Tyr Ile
1300 1305 1310
Glu Thr His Thr His His Phe Ala Asn Phe Thr Thr Sex Gln Arg Gln
1315 1320 1325
Gly Leu Thr Phe Glu Ser Asn Lys Val Thr Val Thr Ile Ser Ser Glu
1330 1335 1340
Asp Gly Glu Phe Lou Val Asp,Asn Ile Ala Lou Val Glu Ala Pro Lou
1345 1350 1355 1360
Pro Thr Asp Asp G1n-Asn Ser Glu Gly Asn Thr Ala Ser Ser Thr Asn
1365 1370 1375
Ser Asp Thr S380 Met Asn Asn Asn Gins
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 3867 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: double
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
M A ORGANISM: Bacillus thuringiensis
B STRAIN: PS17
C INDIVIDUAL ISOLATE: PS17b
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC1628) NRRL B-18652
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
ATGGCAATTT TAAATGAATT ATATCCATCT GTACCTTATA ATGTATTGGC GTATACGCCA 60
WO 92/20802 PCT/US92/04316
2103248
39
CCCTCTTTTT TACCTGATGC GGGTACACAA GCTACACCTG CTGACTTAAC AGCTTATGAA 120
CAATTGTTGA AAAATTTAGA AAAAGGGATA AATGCTGGAA CTTATTCGAA AGCAATAGCT 180
GATGTACTTA AAGGTATTTT TATAGATGAT ACAATAAATT ATCAAACATA TGTAAATATT 240
GGTTTAAGTT TAATTACGAT AGCTGTACCG GAAATTGGTA TTTTTACACC TTTCACCGGT 300
TTGTTTTTTG CTGCATTGAA TAAACATGAT GCTCCACCTC CTCCTAATGC AAAAGATATA 360
TTTGAGGCTA TGAAACCAGC GATTCAAGAG ATGATTGATA GAACTTTAAC TGCGGATGAG 420
CAAACATTGT TAAATGGGGA AATAAGTGGT TTACAAAATT TAGCAGCAAG ATACCAGTCT 480
ACAATGGATG ATATTCAAAG CCATGGAGGA TTTAATAAGG TAGATTCTGG ATTAATTAAA 540
AAGTTTACAG ATGAGGTACT ATCGTTAAAT AGTTTTTATA CAGATCGTTT ACCTGTATTT 600
ATTACAGATA ATACAGCGGA TCGAACTTTG TTAGGTCTTC CTTATTATGC TATACTTGCG 660
AGCATGCATC TTATGTTATT AAGAGATATC ATTACTAAGG GTCCGACATG GGATTCTAAA 720
ATTAATTAAA CACCAGATGC AATTGATTCC TTTAAAACCG ATATTAAAAA TAATATAAAG 780
CTTTACTCTA AAACTATTTA TGACGTATTT CAGAAGGGAC TTGCTTCATA CGGAACGCCT 840
TCTGATTTAG AGTCCTTTGC AAAAAAACAA AAATATATTG AAATTATGAC AACACATTGT 900
TTAGATTTTG CAAGATTGTT TCCTACTTTT GATCCAGATC TTTATCCAAC AGGATCAGGT 960
GATATAAGTT TACAAAAAAC ACGTAGAATT CTTTCTCCTT TTATCCCTAT ACGTACTGCA 1020
GATGGGTTAA CATTAAATAA TACTTCAATT GATACTTCAA ATTGGCCTAA TTATGAAAAT 1080
GGGAATGGCG CGTTTCCAAA CCCAAAAGAA AGAATATTAA AACAATTCAA ACTGTATCCT 1140
AGTTGGAGAG CGGCACAGTA CGGTGGGCTT TTACAACCTT ATTTATGGGC AATAGAAGTC 1200
CAAGATTCTG TAGAGACTCG TTTGTATGGG CAGCTTCCAG CTGTAGATCC ACAGGCAGGG 1260
CCTAATTATG TTTCCATAGA TTCTTCTAAT CCAATCATAC AAATAAATAT GGATACTTGG 1320
AAAACACCAC CACAAGGTGC GAGTGGGTGG AAAACAAATT TAATGAGAGG AAGTGTAAGC 1380
GGGTTAAGTT TTTTACAACG AGATGGTACG AGACTTAGTG CTGGTATGGG TGGTGGTTTT 1440
GCTGATACAATATATAGTCT CCCTGCAACT CATTATCTTT CTTATCTCTA TGGAACTCCT 1500
TATCAAACTT CTGATAACTA TTCTGGTCAC GTTGGTGCAT TGGTAGGTGT GAGTACGCCT 1560
CAAGAGGCTA CTCTTCCTAA TATTATAGGT CAACCAGATG AACAGGGAAA TGTATCTACA 1620
ATGGGATTTC CGTTTGAAAA AGCTTCTTAT GGAGGTACAG TTGTTAAAGA ATGGTTAAAT 1680
GGTGCGAATGCGATGAAGCT TTCTCCTGGG CAATCTATAG GTATTCCTAT TACAAATGAA 1740
ACAAGTGGAG ACAATAAAAT TCGTTGTCGT TATGCAAGTA ATGATAATAC TAACGTTTTC 1800
TTTAATGTAG ATACTGGTGG AGCAAATCCA ATTTTCCAAC AGAAAAAATT TGCATCTACT 1860
GTAGATAATA ATACGGGAGT ACAAGGAGCA AATGGTGTCT ATGTAGTCAA ATCTATTGCT 1920
ACAACTGATA ATTCTTTTAC AGTAAAAATT CCAGGGAAGA CGATTAATGT TCATTTAACC 1980
AACCAAGGTT CTTCTGATGT CTTTTTAGAT CGTATTGAGT TTGTTCCAAT TCTAGAATCA 2040
AATACTGTAA CTATATTCAA CAATCTATAT ACTACAGGTT CAGCAAATCT TATACCAGCA 2100
ATAGCTCCTC TTTGGAGTAC TAGTTCAGAT AAAGCCCTTA CAGGTTCTAT GTCAATAACA 2160
GGTCGAACTA CCCCTAACAG TGATGATGCT TTGCTTCGAT TTTTTAAAAC TAATTATGAT 2220
ACACAAACCA TTCCTATTCC GGGTTCCGGA AAAGATTTTA CAAATACTCT AGAATAATAA 2280
GACATAGTTT CTATTGATAT TTTTGTCGGA TCTGGTCTAC ATGGATCCGA TGGATCTATA 2340
AAATTAGATT TTACCAATAA TAATAGTGGT AGTGGTGGCT CTCCAAAGAG TTTCACCGAG 2400
CAAAATGATT TAGAGAATAT CACAACACAA GTGAATGCTC TATTCACATC TAATACACAA 2460
GATGCACTTG CAACAGATGT GAGTGATCAT GATATTGAAG AAGTGGTTCT AAAAGTAGAT 2520
GCATTATCTG ATGAAGTGTT TGGAAAAGAG AAAAAAACAT TGCGTAAATT TGTAAATCAA 2580
GCGAAGCGCT TAAGCAAGGC GCGTAATCTC CTGGTAGGAG GCAATTTTGA TAACCATGAT. 2640
GCTTGGTATA GAGGAAGAAA TGTAGTAAAC GTATCTAATC ACGAACTTTT GAAGAGTGAT 2700
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CATGTATTAT TACCACCACC AGGATTGTCT CCATCTTATA TTTTCCAAAA AGTGGAGGAA 2760
TCTAAATTAA AACGAAATAC ACGTTATACG GTTTCTGGAT TTATTGCGCA TGCAACAGAT 2820
TTAGAAATTG TGGTTTCTCG TTATGGGCAA GAAATAAAGA AAGTGGTGCA AGTTCCTTAT 2880
GGAGAAGCAT TCCCATTAAC ATCAAGTGGA CCAGTTTGTT GTATCCCACA TTCTACAAGT 2940
AATGGAACTT TAGGCAATCC ACATTTCTTT AGTTACAGTA TTGATGTAGG TGCATTAGAT 3000
GTAGACACAA ACCCTGGTAT TGAATTCGGT CTTCGTATTG TAAATCCAAC TGGAATGGCA 3060
CGCGTAAGCA ATTTGGAAAT TCGTGAAGAT CGTCCATTAG CAGCAAATGA AATACGACAA 3120
GTACAACGTG TCGCAAGAAA TTGGAGAACC GAGTATGAGA AAGAACGTGC GGAAGTAACA 3180
AGTTTAATTC AACCTGTTAT CAATCGAATC AATGGATTGT ATGACAATGG AAATTGGAAC 3240
GGTTCTATTC GTTCAGATAT TTCGTATCAG AATATAGACG CGATTGTATT ACCAACGTTA 3300
CCAAAGTTAC GCCATTGGTT TATGTCAGAT AGATTTAGTG AACAAGGAGA TATCATGGCT 3360
AAATTCCAAGGTGCATTAAA TCGTGCGTAT GCACAACTGG AACAAAATAC GCTTCTGCAT 3420
AATGGTCATT TTACAAAAGA TGCAGCCAAT TGGACGGTAG AAGGCGATGC ACATCAGGTA 3480
GTATTAGAAG ATGGTAAACG TGTATTACGA TTGCCAGATT GGTCTTCGAG TGTGTCTCAA 3540
ACGATTGAAA TCGAGAATTT TGATCCAGAT AAAGAATATC AATTAGTATT TCATGGGCAA 3600
GGAGAAGGAA CGGTTACGTT GGAGCATGGA GAAGAAACAA AATATATAGA AACGCATACA 3660
CATCATTTTG CGAATTTTAC AACTTCTCAA CGTCAAGGAC TCACGTLTGA ATCAAATAAA 3720
GTGACAGTGA CCATTTCTTC AGAAGATGGA GAATTCTTAG TGGATAATAT TGCGCTTGTG 3780
GAAGCTCCTC TTCCTACAGA TGACCAAAAT TCTGAGGGAA ATACGGCTTC CAGTACGAAT 3840
AGCGATACAA GTATGAACAA CAATCAA 3867
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
JA LENGTH: 1289 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE-
JAI ORGANISM: BACILLUS THURINGIENSIS
B STRAIN: PS17
CINDIVIDUAL ISOLATE: PSI7b
(Vii) IMMEDIATE SOURCE:
(B)-CLONE: E. coli NM522(pMYC1628) NRRL B-18652
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ala Ile Leu Aan Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val Leu
1 5 10 15
Ala Tyr Thr Prro Pro Ser Phe Leu 2ro Asp Ala Gly Thr Gan Ala Thr
Pro Ala 3sp Leu Thr Ala Tyr l40 U GGin Leu Len Lys 45n Leu Gin Lys
Gly Sae Aen Ala Gly Thr Tyr Ser Lys Ala Ile Ala Asp Val Len Lye
Ely Ile Phe Ile ASP ASP Thr Ile Asn Tyr 75n Thr Tyr Val Asn Sae
Gly Leu Ser Leu Ile Thr Leu Ala Val Pro Glu Ile Gly Ile Phe Thr
85 90 95
Pro Phe Ile Gly Leu Phe Phe Ala Ala Len Asn Lys His sp Ala Pro 105 Pro Pro Pro
Asn Ala Lys Asp 1iee Phe Glu Ala Met Lye Pro Ala Ile
115 SUBSTITUTE SHEET
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4 8
41
Gln Glu Met Ile Asp Arg Thr Leu Thr Ala Asp Giu Gln Thr Phe Leu
130 135 140
Aen Gly Glu Ile Ser Gly Len Gin Asn Leu Ala Ala Arg Tyr Gin Ser
145 150 155 160
Thr Met Asp Asp lie Gin Ser His Gly lly Phe Asn Lys Val AAsp Ser
16
Gly Leu Ile lye Lys Phe Thr Asp G1 85 Val Leu Ser Len lean Ser Phe
Tyr Thr 1sp Arg Leu Pro Val Phe Ile Thr Asp Asn 205Thr Ala Asp Arg
200
Thr Lelu0 Leu Gly Leu Pro T
22yr Tyr Ala Ile Leu Alaa Ser Met His Leu
2
Met Len Leu Arg Asp Ile 1111e Thr Lys Gly Pro Thr Trp Asp Ser Lys
225 230 235 240
Ile Asn Phe Thr Pro Asp Ala Ile Asp Ser Phe Lys Thr Asp lie Lys
250 25
Asn Asn Ile Lye Leu TyrSer Lys 26 Thr Ile Tyr Asp Val 2Phe 7e Gin Lys
Gly Leu Ala Ser Tyr ply Thr Pro Ser Asp Leu Glu Ser Phe Ala Lys
25 285
Lys Gin Lys Tyr Ile Giu lie e Met Thr Thr His 300 Leu Asp Phe Ala
2
Arqq Len Phe Pro Thr Phe Asp Pro Asp Len Tyr Pro Thr Gly Ser Giy
305 310 315 320
Asp Ile Ser Leu Gin Lye Thr Arg Arg lie Len Ser Pro Phe lie Pro 33 335
Ile Arg Thr Ala Asp Gly Len Thr LLeu5 Asn Asn Thr Ser lie Asp Thr 3 35
Ser ken 3rp Pro Asn Tyr Glu sn Gly Asn Gly Ala P65 Pro Asn Pro
Lys Glu Arg Ile Leu Lys Gin Phe Lys Leu Tyr Prroo Ser Trp Arg Ala
370 5 Ala Gln Tyr Gly Gly Leu Leu Gin Pro Tyr Leu Trp Ala Ile Glu Val
385 390 395 400
Gln Asp Ser Val 405 Thr Arg Len Tyr 4ly Gin Len Pro Ala 41V 5 Asp
Pro Gin Ala 42y Pro'Asn Tyr Val Ser Ile Asp Ser Ser Asn Pro Ile 425 430
Ile Gln 4iee Asn Met Asp Thr 4Trp Lys Thr Pro Pro Gin G1y Ala Ser
445
Gly Trp ken Thr Asn Leu M4 t Arg Gly Ser Val 460 Gly Leu Ser Phe
4~b 55
Leu Gln Arg Asp Gly Thr Arg Leu Ser Ala Giy Met Gly Gly Gly Phe
465 470 475 480
Ala Asp Thr Ile Tyr Ser Leu Pro Ala Thr His Tyr Len Ser Tyr Len
485 495
Tyr Gly Thr Pro Tyr Gin Thr Ser Asp Asn Tyr Ser Gly His Val Gly
5 505
Ala Leu Val Gly Val Ser Thr 5Pro 20 Gln Glu Ala Thr L25 Pro Asn Ile
515 5
Ile Gay Gin Pro Asp Glu Gin Gly Asn Val Ser Thr Met Gly Phe Pro 55 540
Phe Gin Lys Ala Ser Tyr Giy G1y Thr Val Val Lys Glu Trp Leu Asn
545 550 555 560
Gly Ala Asn Ala Met Lys Len Ser Pro Giy Gin Ser Ile Gly lie Pro
Ile Thr Asn Vaal Thr Ser Gly Glu Tyr Gin Ile Arg Cys Ar0 Tyr Ala
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Ser Asn Assp Asn Thr Asn Val Phe Phe Asn Val Asp T605 Gly Gly Ala
600 Asn Pro Ile Phe Gln Gln lie Asn Phe Ala Ser T6hrr Val Asp Asn Asn
6 615
Thr Gly Val Gln Gly Ala Asn Gly Val Tyr Val Val Lys Ser Ile Ala
625 630 635 640
Thr Thr Asp Asn Ser Phe Thr Val Lys Glee Pro Ala Lys Thr lie Asn
645 Val His Lau Thr Asn Gln Gly Ser Ser Asp Val Phe Lau AAsp Arg Ile
660 665 6
Glu Phe Val Pro Ile Lau Gin Ser Asn Thr Val Thr Ile Phe Asn Asn
675 680 685
Ser Tyr Thr Thr Gly Ser Ala Asn Leu Ile Pro Ala Ile Ala Pro Leu
690 695 700
Trp Ser Thr Ser Ser 710 Lys Ala Lau Thr GGly Ser Met Ser Ile 720
705 Gly Arg Thr Thr P
Lys
725 73 ro Asn Ser Asp Asp AAla Lau Lau Arg Phe 7Phe 35
Thr Asn Tyr AAssp Thr Gin Thr Ile Pro Ile Pro Gly Ser 7Gly 50 Lys Asp
Phe Thr Asn T Dhr Lau Glu Ile Gin Asp Ile Val Ser Ile Asp Ile Phe
755 760 765
Val 77y Ser Gly Lau His yly Ser Asp Gly Ser 1810 Lys Lau Asp Phe
Thr Asn Asn Asn Ser Gly Ser Gly Gly Ser Pro Lys Ser Phe Thr Gin
785 790 795 800
Gin Asn Asp Lau G05 Asn Ile Thr Thr G10n Val Asn Ala Leu Phe Thr
8 8 815
Ser Asn Thr Gin Asp Ala Lau Ala Thr Asp Val Ser Asp His Asp Ile
820 825 830
Gin Gin VVaallVal Lau Lys Val Asp Ala Lau Ser Asp Gin Val Phe Gly
83 84 85
Lys GG5u0 Lys Lys Thr Leu 8 g Lys Phe Val Asn G16nn Ala Lys Arg Leu
Ser Lys Ala Arg Asn Lau Leu Val Gly Gly Asn Phe Asp Asn Leu Asp
865 870 875 880 ..- '
Ala Trp Tyr Arg G11 Arg Asn Val Val ken Val Ser Asn His G] Lau
Sal 895
Lau Lye Ser 9 p His Val Lau Lau PPrro Pro Pro Gly Lau Ser Pro Ser
Tyr Ile PPhe Gin Lys ValGlu Gin Ser Lys Lau Lys 9 g Asn Thr Arg
91 920 Tyr TT3 Val Ser Gly Phe IIle Ala His Ala Thr As Lau Glu Ile Val
Val Ser ArgTyr Gly Gin Gin Ile Lys Lys Val VaD0l Gin Val Pro Tyr
945 950 955 960
Gly Glu Ala Phe Pro Lau Thr Ser Ser G7lg Pro Val Cys Cys lie Pro 975
Asn Pro His Phe PPhhe Ser Tyr
His Ser Thr SSerr Asn Gly Thr Leu Gly
Ser Ile Asp Val Gly Ala Lau Asp Val Asp Thr Asn Pro Gly Ile Glu
995 1000 1005
Phe Gly Lau Arg Ile Val Asn Pro Thr Gly Met Ala Arg Val Ser Asn
1010 1015 1020
Lau Glu Ile Arg Gin Asp Arg Pro Lau Ala Ala Asn Glu Ile Arg Gln
1025 1030 1035 1040
Val Gin Arg Val Ala Arg Asn Trp Arg Thr Glu Tyr Glu Lys Gin Arg
1045 1050 1055
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Ala Glu Val Thr Ser Leu Ile Gln Pro Val Ile Asn Arg Ile Asn Gly
1060 1065 1070
Leu Tyr Asp Asn Gly Asn Trp Asn Gly Ser Ile Arg Ser Asp Ile Ser
1075 1080 1085
Tyr Gln Asn Ile Asp Ala Ile Val Leu Pro Thr Leu Pro Lys Leu Arg
1090 1095 1100
His Trp Phe Met Ser Asp Arg Phe Ser Glu Gln Gly Asp Ile Met Ala
1105 1110 1115 1120
Lys Phe Gln Gly Ala Leu Asn Arg Ala Tyr Ala Gln Leu Glu Gln Asn
1125 1130 1135
Thr Leu Leu His Asn Gly His Phe Thr Lys Asp Ala Ala Asn Trp Thr
1140 1145 1150
Val Glu Gly Asp Ala His Gln Val Val Leu Glu Asp Gly Lys Arg Val
1155 1160 1165
Leu Arg Leu Pro Asp Trp Ser Ser Ser Val Ser Gin Thr Ile Glu Ile
1170 1175 1180
Glu Asn Phe Asp Pro Asp Lys Glu Tyr Gin Leu Val Phe His Gly Gin
1185 1190 1195 1200
Gly Glu Gly Thr Val Thr Leu Glu His G1 Glu Glu Thr Lys Tyr Ile
1205 1210 1215
Glu Thr His Thr His His Phe Ala Asn Phe Thr Thr Ser Gln Arg Gin
1220 1225 1230
Gly Lou Thr Phe Glu Ser Asn Lys Val Thr Val Thr Ile Ser Ser Giu
1235 1240 1245
Asp Glyy Glu Phe Leu Val Asp Asn Ile Ala Leu Val Glu Ala Pro Leu
1250 1255 1260
Pro Thr Asp Asp Gln Asn Ser Glu Gly Asn Thr Ala Ser Ser Thr Asn
1265 1270 1275 1280
Ser Asp Thr Ser Met Asn Asn Asn Gln
1285
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS
LENGTH: 37?1 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: double
D TOPOLOGY: linear . f~
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
SA ORGANISM: Bacillus thuringiensis
!C) INDIVIDUAL ISOLATE: 33F2
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC2316) B-18785
(ix) FEATURE:
JA) NAME/KEY: misc feature
Bj)}) LOCATION: 4..24
D OTHER INFORMATION: /function= "oligonucleotide
hybridization probe-
/,Product= "GCA/T ACA/T TTA AAT GAA GTA/T TAT"
/standard name= "probe an
/note= "P?obe A"
(ix) FEATURE :
A NAME/KEY: misc feature
B LOCATION: 13..33
D OTHER INFORMATION: /function= "oligonucleotide
hybridization probe"
/product= "AAT GAA GTA/T TAT CCA/T GTA/T AAT"
/standard name= "Probe B"
/label= probe-b
/note= "Probe b"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
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ATGGCTACAC TTAATGAAGT ATATCCTGTG AATTATAATG TATTATCTTC TGATGCTTTT 60
CAACAATTAG ATACAACAGG TTTTAAAAGT AAATATGATG AAATGATAAA AGCATTCGAA 120
AAAAAATGGA AAAAAGGGGC AAAAGGAAAA GACCTTTTAG ATGTTGCATG GACTTATATA 180
ACTACAGGAG AAATTGACCC TTTAAATGTA ATTAAAGGTG TTTTATCTGTATTAACTTTA 240
ATTCCTGAAG TTGGTACTGT GGCCTCTGCA GCAAGTACTA TTGTAAGTTT TATTGTACCT 300
AAAATATTTG GAGATAAACC AAATGCAAAA AATATATTTG AAGAGCTCAA GCCTCAAATT 360
GAAGCATTAA TTCAACAAGA TATAACAAAC TATCAAGATG CAATTAATCA AAAAAAATTT 420
GACAGTCTTC AGAAAACAAT TAATCTATAT ACAGTAGCTA TAGATAACAA TGATTACGTA 480
ACAGCAAAAA CGCAACTCGA AAATCTAAAT TCTATACTTA CCTCAGATAT CTCCATATTT 540
ATTCCAGAAG GATATGAAAC TGGAGGTTTA CCTTATTATG CTATGGTTGC TAATGCTCAT 600
ATATTATTGT TAAGAGACGC TATAGTTAAT GCAGAGAAAT TAGGCTTTAG TGATAAAGAA 660
GTAGACACAC ATAAAAAATA TATCAAAATG ACAATACACA ATCATACTGA AGCAGTAATA 720
AAAGCATTCT TAAATGGACT TGACAAATTT AAGAGTTTAG ATGTAAATAG CTATAATAAA 780
AAAGCAAATT ATATTAAAGG TATGACAGAA ATGGTTCTTG ATCTAGTTGC TCTATGGCCA 840
ACTTTCGATC CAGATCATTA TCAAAAAGAA GTAGAAATTG AATTTACAAG AACTATTTCT 900
TCTCCAATTT ACCAACCTGT ACCTAAAAAC ATGCAAAATA CCTCTAGCTC TATTGTACCT 960
AGCGATCTAT TTCACTATCA AGGAGATCTT GTAAAATTAG AATTTTCTAC AAGAACGGAC 1020
AACGATGGTC TTGCAAAAAT TTTTACTGGT ATTCGAAACA CATTCTACAA ATCGCCTAAT 1080
ACTCATGAAA CATACCATGT ACATTTTAGT TATAATACCC AATCTAGTGG TAATATTTCA 1140
AGAGGCTCTT CAAATCCGAT TCCAATTGAT CTTAATAATC CCATTATTTC AACTTGTATT 1200
AGAAATTCAT TTTATAAGGC AATAGCGGGA TCTTCTGTTT TAGTTAATTT TAAAGATGGC 1260
ACTCAAGGGTATGCATTTGC CCAAGCACCA ACAGGAGGTG CCTGGGACCA TTCATTTATT 1320
GAATCTGATGGTGCCCCAGA AGGGCATAAA TTAAACTATA TTTATACTTC TCCAGGTGAT 1380
ACATTAAGAG ATTTCATCAA TGTATATACT CTTATAAGTACTCCAACTAT AAATGAACTA 1440
TCAACAGAAA AAATCAAAGG CTTTCCTGCG GAAAAAGGAT ATATCAAAAA TCAAGGGATC 1500
ATGAAATATT ACGGTAAACC AGAATATATT AATGGAGCTC AACCAGTTAA TCTGGAAAAC 1560
CAGCAAACAT TAATATTCGA ATTTCATGCT TCAAAAACAG CTCAATATAC CATTCTAAAA 1620;4
CGTTATGCCA GTACCCAAGG AAAAAAAGGT TATTATCTTT TAGATAATCA GGAACTGCAA 1680
ACGCTTAATA TATCAACTTC ACACAACGGT TATGTAACCG GTAATATTGG TGAAAATTAT 1740
GATTTATATA CAATAGGTTC ATATACAATT ACAGAAGGTA ACCATACTCT TCAAATCCAA 1800
CATAATGATA AAAATGGAAT GGTTTTAGAT CGTATTGAAT TTGTTCCTAA AGATTCACTT 1860
CAAGATTCAC CTCAAGATTC ACCTCCAGAAGTTCACGAAT CAACAATTAT TTTTGATAAA 1920
TCATCTCCAA CTATATGGTC TTCTAACAAA CACTCATATA GCCATATACA TTTAGAAGGA 1980
TCATATACAA GTCAGGGAAG TTATCCACAC AATTTATTAA TTAATTTATT TCATCCTACA 2040
GACCCTAACA GAAATCATAC TATTCATGTT AACAATGGTG ATATGAATGT TGATTATGGA 2100
AAAGATTCTG TAGCCGATGG GTTAAATTTTAATAAAATAA CTGCTACGAT ACCAAGTGAT 2160
GCTTGGTATA GCGGTACTAT TACTTCTATG CACTTATTTA ATGATAATAA TTTTAAAACA 2220
ATAACTCCTA AATTTGAACT TTCTAATGAA TTAGAAAACA TCACAACTCA AGTAAATGCT 2280
TTATTCGCAT CTAGTGCACA AGATACTCTC GCAAGTAATG TAAGTGATTA CTGGATTGAA 2340
CAGGTCGTTA TGAAAGTCGA TGCCTTATCA GATGAAGTAT TTGAACAAGA GAAAAAAGCA 2400
TTACGTAAAT TGGTAAATCA AGCAAAACGT CTCAGTAAAA TACGAAATCT TCTCATAGGT 2460
GGTAATTTTG ACAATTTAGT CGCTTGGTAT ATGGGAAAAG ATGTAGTAAA AGAATCGGAT 2520
CATGAATTAT TTAAAAGTGA TCATGTCTTA CTACCTCCCC CAACATTCCA TCCTTCTTAT 2580
ATTTTCCAAA AGGTGGAAGA ATCAAAACTA AAACCAAATA CACGTTATAC TATTTCTGGT 2640
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TTTATCGCAC ATGGAGAAGA TGTAGAGCTT GTTGTCTCTC GTTATGGGCA AGAAATACAA 2700
AAAGTGATGC AAGTGCCATA TGAAGAAGCA CTTCCTCTTA CATCTGAATC TAATTCTAGT 2760
TGTTGTGTTC CAAATTTAAA TATAAATGAA ACACTAGCTG ATCCACATTT CTTTAGTTAT 2820
AGCATCGATG TTGGTTCTCT GGAAATGGAA GCGAATCCTG GTATTGAATT TGGTCTCCGT 2880
ATTGTCAAAC CAACAGGTAT GGCACGTGTA AGTAATTTAG AAATTCGAGA AGACCGTCCA 2940
TTAACAGCAA AAGAAATTCG TCAAGTACAA CGTGCAGCAA GAGATTGGAA ACAAAACTAT 3000
GAACAAGAAC GAACAGAGAT CACAGCTATA ATTCAACCTG TTCTTAATCA AATTAATGCG 3060
TTATACGAAA ATGAAGATTG GAATGGTTCT ATTCGTTCAA ATGTTTCCTA TCATGATCTA 3120
GAGCAAATTA TGCTTCCTAC TTTATTAAAA ACTGAGGAAA TAAATTGTAA TTATGATCAT 3180
CCAGCTTTTT TATTAAAAGT ATATCATTGG TTTATGACAG ATCGTATAGG AGAACATGGT 3240
ACTATTTTAG CACGTTTCCA AGAAGCATTA GATCGTGCAT ATACACAATT AGAAAATGGT 3300
AATCTCCTGC ATAACGGTCA TTTTACAACT GATACAGCGA ATTGGACAAT AGAAGGAGAT 3360
GCCCATCATA CAATCTTAGA AGATGGTAGA CGTGTGTTAC GTTTACCAGA TTGGTTTTCT 3420
AATGCAACTC AAACAATTGA AATTGAAGAT TTTGACTTAG ATCAAGAATA CCAATTGCTC 3480
ATTCATGCAA AAGGAAAAGG TTCCATTACT TTACAACATG GAGAAGAAAA CGAATATGTG 3540
GAAACACATA CTCATCATAC AAATGATTTT ATAACATCCC AAAATATTCC TTTCACTTTT 3600
AAAGGAAATC AAATTGAAGT CCATATTACT TCAGAAGATG GAGAGTTTTT AATCGATCAC 3660
ATTACAGTAA TAGAAGTTTC TAAAACAGAC ACAAATACAA ATATTATTGA AAATTCACCA 3720
ATCAATACAA GTATGAATAG TAATGTAAGA GTAGATATAC CAAGAAGTCT C 3771
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
I LENGTH: 1257 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
V
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thurin iensis
(C)INDIVIDUALISOLATE: PS33F2
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC2316) B-18785
(ix) FEATURE:
M NAME/KEY: Protein
LOCATION: 1..1257
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ala Thr Leu Asn Glu Val Tyr Pro Val Asn Tyr Asn Val Leu Ser
1 5 10 15
Ser Asp Ala Phhe Gin Gin Leu Asp Thr Thr Gly Phe Lys Ser Lys Tyr
2 25 30
Asp Glu Net Ile Lys Ala Phe Glu Lys Lys Trp Lys Lys Gly Ala Lys
35 40 45
Gly Lps Asp Leu Leu Asp Val Ala Trp Thr Tyr sole Thr Thr Gly Glu
lie Asp Pro Leu Asn 7aal Ile Lys Gly Val 7eeu Ser Val Leu Thr Leu
65 Ile Pro Glu Val Gly Thr Val Ala Ser Ala Ala Ser Thr Ile Val Ser
85 90 95
Phe Ile Trp Pro Lys Ile Phe Gly Asp Lys Pro Asn Ala Lye Asn Ile
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Phe Glu Glu Leu Lys Pro Gln Ile Glu Ala Leu Ile Gln Gln Asp Ile
115 120 125
Thr Asn Tyr Gin Asp Ala Ile Asn Gin Lys Lys Phe Asp Ser Leu Gln
130 135 140
LLye Thr Ile Asn Len Tyrr Thr Val Ala Ile Asp Asn Asn Asp Tyr Val
Thr Ala Lys Thr Gin Leu Glu Asn Leu Asn Ser Ile Leu Thr Ser Asp
165 170 175
Ile Ser Ile Phe Ile Pro Glu Gly Tyr Glu Thr Gly Gly Leu Pro Tyr
180 185 190
Tyr Ala Met Val Ala Asn Ala Hiss Ile Leu Leu Leu 205 Asp Ala Ile
Val Asn Ala Glu Lys Len G1y Phe Ser Asp Lys Gin Val Asp Thr His
Lys Lys Tyr Ile Lys Met Thrr Ile His Asn His Thr Glu Ala Val Ile
225 230 235 240
Lys Ala Phe Leu Aen Gly Leu Asp Lys Phe Lys Ser Leu Asp Val Asn
245 250 255
Ser Tyr Asn Lye Lys Ala Asn Tyr lie e Lys Gly Met Thr GG in Met Val
Leu Asp Leu Val Ala Leu TrpPro Thr Phe Asp Pro Asp His Tyr Gin
275 280 285
Lys Gin Val Glu Ile Glu PPhhe Thr Arg Thr Ile Seer Ser Pro Ile Tyr
29
Gln Pro Val Pro Lys Asn Met Gin Asn Thr Ser Ser Ser Ile Val Pro
'305 310 315 320
Ser Asp Leu Phe His Tyr Gin Gly Asp Leeuu Val Lys Leu Glu Phe Ser 3 335
Thr Arg Thr 3sp Asn Asp GlyLeu 3A4aa Lys Ile Phe Thr 359 Ile Arg
Asn Thr 35e Tyr Lys Ser Pro Aen Thr His Glu Thr Tyr His Val Asp 360 365
Phe 3err Tyr Asn Thr Gin Ser Ser Gly Asn Ile Seer Arg Gly Ser Ser
70 375
Asn Pro Ile Pro Ile Asp Lou Asn Asn Pro Ile Ile"Ser Thr Cys Ile
385 390 395 400
Arg Asn Ser Phe Tyr Lye Ala Ile AlaG41y Ser Ser Val Leu 4aall Asn
405, Phe Lys Asp 4ly Thr Gin Gly Tyr Ala Phe Ala Gin Ala PPrroo Thr Gly
Gly Ala 4Trp Asp His Ser Phelie Glu Ser Asp Gly Alaa Pro Glu Gly
44 45
His Lyee Len Asn Tyr Ile Tyr Thr Ser Pro Gly AAssp Thr Leu Arg Asp
4
Phe Ile Asn; Val Tyr; Thr Len Ile Ser Thr Pro Thar Ile Asn Glu Leu
465 470 475 480
Ser Thr Glu Lys I18e e Lys Gly Phe Pro Ala Glu Lys Gly Tyr lie Lys
495
Asn Gln Gly I0e e Met Lys Tyr Tyr 5G ly Lys Pro Glu Tyr lie e Asn Gly
Ala Gin Pro Val Asn Leu Glu Asn Gin Gin Thr Leu Ile Phe Gin Phe
515 520 525
His Ala Ser Lys Thr Ala Gln Tyr Thr Ile Arg Ile Arg Tyr Ala Ser
530 535 540
Thr Gln Gly Thr Lys Gl Tyr Phe Arg Leu Asp Asn Gin Gin Len Gin
545 550 555 560
Thr Leu, Asn Ile Pro Thr Ser His Asn sly Tyr Val Thr Gly A5e5 Ile
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Gly Glu Asn Tyr Asp Lau Tyr Thr Ile Gly Ser Tyr Thr Ile Thr Gin
580 585 590
Gly Asn His Thr Lau Gln Ile Gln His Asn Asp Lys Asn Gly Met Val
595 600 605
Lau AAsp Arg Ile Glu Phe Val Pro Lys Asp Ser LLauu Gln Asp Ser Pro
60 5
Gln Asp Ser Pro Pro Glu Val His Glu Ser Thr Ile Ile Phe Asp Lys
625 630 635 640
Ser Ser Pro Thr IIllee Trp Ser Ser Asn L6ye His Ser Tyr Ser His s Ile
6O 6
His Lou Glu GG16y Ser Tyr Thr Ser Gin Gly Ser Tyr Pro His Asn Leu
65 6
Lau Ile Asn Leu Phe His Pro Thr Asp Pro Asn Arg Asn His Thr Ile
675 680 685
His Vaal Asn Asn Gly Asp MMeett Asn Val Asp Tyr Goy Lys Asp Ser Val
Ala Asp Gly Lau Asn Phe Asn Lys Ile Thr Ala Thrrr Ile Pro Ser Asp
705 710 715 720
Ala Trp Tyr Ser GGly Thr Ile Thr Ser Met His Lou Phe Asn Asp Asn
72 7 Asn Phe Lys Thr Ile Thr Pro Lys Phe Glu Leu Ser Asn Gin Lau Glu
740 745 750
Asn Ile Thr Thr Gin Val Asn.Allaa Leu Phe Ala Ser S65 Ala Gln Asp
755 7 7
Thr Lou Ala Ser Asn Val Ser Asp Tyr Trp Ile Glu Gln Val Val Met
770 775 780
L s Val Asp Ala Lau Ser Asp Glu Val Phe Gly Lys Glu Lys Lys Ala
795 790 795 800
Lou Arg Lys Lau Val Asn Gin Ala Lys $1g Lau Ser Lys Ile A Asn 819
Lau Lou Ile Gly Gly Asn Phe Asp 825 Lau Val Ala Trp Tyr Met Gly
8210 83
Lys Asp VVall Val Lys Giu Ser Asp His Glu Lou Phe Lye Ser Asp His
83 Val Lou oLen Pro Pro Pro ass Thr Phe His Pro Ser Tyr Ile Phe Gin Lys 86
Val Gin Glu Ser Lys Leu Lys Pro Asn Thr Arg Tyr Thr Ile Ser Giy
865 870 875 880
Phe Ile Ala His GGlly Glu Asp Val Glu Lau Val Val Ser Arg Tvr Gly
S 89
Gin Glu Ile GGinn Lys Val Met Gin Val Pro Tyr Gin Gin 91aa Lau Pro
9 Lau Thr Ser Glu Ser Asn Ser SSerr Cys Cys Val Pro As 925n Lau Asn Ile
915
Asn Glu ThrLau Ala Asp Pro His Phe Phe Ser Tyr Ser Ile Asp Val
930 935 940
Glp Ser Leu Gin Met Glu Ala Asn Pro Gly Ile Glu Phe Giy Leu Arg
945 950 955 960
Ile Val Lys Pro T965 Gly Met Ala Arg 9al Ser Asn Lau Glu lie Arg
Glu Asp Arg Prro Leu Thr Ala Lys 9Glu 85 Ile Arg Gin Val Ginn Arg Ala
9
9
Ala Arg Asp Trp Lys Gin Asn Ty00Glu Gin Gin Arg l r Glu Ile Thr
Ala Ile Ile Gin Pro Val Lau Assn Gin Ile Asn Ala Lau Tyr Giu Asn
1010 1015 1020
Gin Asp Trp Asn Gly Ser Ile Arg Ser Asn Val Ser Tyr His Asp Lau
1025 1030 1035 1040
WO 92/20802 PCl'/US92/04316
2103248 48
Glu Gln Ile Met Leu Pro Thr Len Leu Lys Thr Glu Gin Ile Asn Cys
1045 1D50 1055
Asn Tyr Asp His Pro Ala Phe Leu Leu Lys Val Tyr His Trp Phe Net
1060 1065 1070
Thr Asp 1075I1e Gly Glu His GGly0Thr Ile Leu Ala OgSPhe Gin Glu 10 Ala Leu Asp
Arg Ala Tyr Thr Ginn Leu Glu Ser Arg Asn Leu Len His
1090 1095 1100
Asn Gly His Phe Thr Thr Asp Thr Ala Asn Trp Thr Ile Glu Gly Asp
1105 1110 1115 1120
Ala His His Thr Ile Leu Glu Asp Gly Arg Arg Val Leu Arg Leu Pro
1125 1130 1135
Asp Trp Ser Ser Asn Ala Thr Gin Thr Ile Gin Ile Glu Asp Phe Asp
1140 1145 1150
Leu Asp Gin Gin Tyr Gin Leu Leu Ile His Ala Lys G1y Lys Gly Ser
1155 1160 1155
Ile Thr Leu Gln His Gly Glu Glu Asn Gin Tyr Val Glu Thr His Thr
1170 1175 1180
His His Thr Asn Asp Phe Ile Thr Ser Gln Asn Ile Pro Phe Thr Phe
1185 1190 1195 1200
Lys Gly Asn Gin Ile Gin Val His Ile Thr Ser Glu Asp Gly Glu Phe
1205 1210 1215
Leu Ile Asp His Ile Thr Val Ile Gin Val Ser Lys Thr Asp Thr Asn
1220 1225 1230
Thr Asn Ile Ile Glu Asn Ser Pro lie Asn Thr Ser Met Asn Ser Asn
1235 1240 1245
Val rgoVal Asp Ile Pro AArrg5Ser Leu
22
(2) INFORMATION FOR SEQ ID NO:7:
(1) SEQUENCE CHARACTERISTICS:
A) LENGTH: 3738 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: double
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL : NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
1 A ORGANISM: Bacillus thuringiensis
C) INDIVIDUAL ISOLATE: PS86Q3
(Vii) IMMEDIATE SOURCE:
(A) LIBRRARY: Lambdagem (TM) - 11 LIBRARY
(E: 86Q3a
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATGGCAACAA TTAATGAGTT GTATCCAGTT CCTTATAATG TGCTAGCTCA TCCAATTAAA 60
GAAGTCGATG ATCCTTATTC TTGGTCAAAT TTATTAAAGG GTATACAAGA AGGTTGGGAA 120
GAATGGGGAA AAACAGGACA AAAAAAACTT TTTGAAGACC ATCTTACGAT TGCATGGAAT 180
CTTTATAAAA CAGGAAAATT AGATTATTTC GCTTTGACAA AAGCATCAAT ATCATTGATT 240
GGATTTATTC CAGGGGCAGA AGCAGCAGTT CCCTTTATTA ATATGTTTGT AGACTTTGTT 300
TGGCCTAAAT TATTTGGTGC GAATACAGAA GGAAAAGATC AACAGTTGTT TAATGCTATC 360
ATGGATGCAG TTAATAAAAT GGTAGATAAT AAGTTCTTAA GTTATAATCT TAGTACACTT 420
AATAAAACAA TTGAAGGACT TCAAGGTAAT TTAGGCCTAT TTCAAAATGC TATACAAGTA 480
GCCATTTGTC AAGGCAGTAC ACCAGAAAGA GTAAATTTTG ATCAAAATTG TACACCATGT 540
AATCCAAATC AACCTTGTAA AGATGATTTG GATAGAGTTG CTTCACGTTT TGATACGGCT 600
AATTCTCAAT TCACACAGCA TTTACCAGAA TTTAAAAATC CTTGGTCGGA TGAAAACTCT 660
SUBSTITUTE SHEET
WO 92/20802 21 0 3 2 4 8 PCT/US92/04316
49
ACTCAGGAAT TTAAAAGAAC ATCTGTTGAA TTAACTTTAC CAATGTATAC AACAGTAGCT 720
ACGTTACATC TTTTATTATA TGAAGGATAT ATAGAATTTA TGACAAAATG GAATTTTCAC 780
AATGAACAAT ATTTAAATAA TTTAAAGGTA GAATTACAAC AATTGATACA CTCATATTCA 840
GAAACTGTTC GTACAAGTTT CCTTCAATTT TTACCTACCT TGAATAATCG TTCAAAATCA 900
TCCGTAAATG CTTATAACCG TTATGTCCGC AATATGACTG TTAACTGTTT AGATATTGCT 960
GCTACATGGC CTACATTTGA TACACATAAT TATCATCAAG GTGGTAAATT AGATTTAACT 1020
CGTATTATTC TTTCAGATAC AGCAGGACCA ATAGAAGAAT ATACTACTGG CGACAAAACT 1080
TCAGGACCTG AACATAGTAA CATTACACCA AATAATATTC TAGATACACC ATCTCCAACA 1140
TATCAGCACT CATTTGTATC TGTTGATTCT ATTGTATATT CTAGAAAAGA ATTACAACAA 1200
TTAGACATAG CTACTTATAG TACAAATAAT AGTAATAATT GTCACCCTTA TGGATTACGA 1260
CTTTCATATA CAGATGGAAG CAGATATGAT TATGGAGATAATCAACCTGA TTTTACTACT 1320
TCCAATAACA ATTATTGTCA TAATAGCTAT ACTGCCCCTA TTACACTTGT GAATGCACGA 1380
CATTAATATA ATGCAAAAGG CTCTTTACAA AATGTAGAAT CTTTAGTGGT TAGTACTGTA 1440
AATGGTGGAA GTGGTTCATG CATTTGTGATGCATGGATTA ATTATTTACG TCCTCATCAA 1500
ACAAGTAAAA ATGAATCACG TCCTGATCAA AAAATTAATG TTTTGTATCC AATAACAGAA 1560
ACTGTAAATA AGGGGACTGG AGGAAATTTA GGAGTTATTT CTGCCTATGT TCCAATGGAA 1620
CTTGTACCAG AAAACGTTAT TGGAGATGTT AATGCTGATA CTAAATTGCC ACTTACACAA 1680
TTAAAGGGCT`TTCCATTTGA AAAATATGGT TCTGAGTATA ATAATCGGGG TATCTCTCTP 1740
GTTCGCGAAT GGATAAATGG TAACAATGCA GTTAAACTTT CTAATAGTCA ATCTGTTGGC 1800
ATACAAATTA CGAATCAAAC CAAACAAAAA TATGAAA".CAC GTTGCCGTTA TGCGAGTAAA 1860
GGAGATAATA ATGTTTATTT TAATGTGGAT TTAAGTGAAA ATCCATTTAG AAATTCCATT 1920
TCTTTTGGAT CTACTGAAAG TTCTGTTGTA GGAGTACAAG GTGAAAATGG AAAGTATATA 1980
TTGAAATCAA TCACAACGGT AGAAATACCT GCTGGAAGTT TCTATGTTCA TATAACAAAC 2040
CAAGGTTCTT CAGATCTCTT TTTAGATCGT ATTGAGTTTG TTCCAAAAAT CCAATTCCAA 2100
TTCTGTGATA ATAATAATCT TCACTGTGAT TGTAATAACC CTGTTGACAC CGATTGTACA 2160
TTTTGTTGCG TTTGCPICTAG TCTTACTACT TGTGATTGTA ATAACCCTCG TGGCCTAGAT 2220
TGTACGCTAT GTTGTCAGGT AGAAAATCAG'CTACCTTCTT TTGTGACACT TACAGATTTA 22W'
CAAAATATTA CGACACAAGT AAATGCATTA GTTGCATCGA GCGAACATGA TACACTTGCA 2340
ACAGACGTGA GTGATTATGA GATTGAAGAA GTTGTACTGA AAGTAGATGC ATTATCTGGT 2400
GAAGTGTTTG AAAAGCATTG CGTAAATTGG TAAATCACAC AAAACGTTTA 2460
AGCAAAGCGC GTAACCTCTT GATAGGAGGA AATTTTGATA ACTTGGATGC TTGGTACAGA 2520
GGCCGAAATG.TAGTAAACGT ATCTGATCAT GAACTATTTA AGAGTGATCATGTATTATTG 2580
CCACCACCAA CACTGTACTC ATCTTATATG TTCCAAAAAG TAGAGGAATC GAAATTAAAA 2640
GCGAATACAC GTTATACTGT GTCTGGTTTT ATTGCACATG CAGAAGATTT AGAAATTGTT 2700
GTGTCTCGTT ATGGGCAAGAAGTGAAGAAA GGGGTACAAGTTCCATATGG AGAAGCATTC 2760
CCATTGACAT CGAGGGGAGC GATTTGTTGC CCTCCACGTT CTACAAGTAA TGGAAAACCT 2820
GCTGATCCAC ATTTCTTTAG TTACAGTATT GATGTGGGAA CATTAGATGT AGAAGCAAAC 2880
CCTGGTATCGAATTGGGTCT TCGTATTGTA GAACGAACTG GAATGGCACG TGTAAGTAAT 2940
TTAGAAATTCGTGAAGATCG TCCATTAAAG AAAAATGAAC TCCGCAATGT ACCACGTGCA 3000
GCAAGAAATT GGAGAACAGC ATATGACCAA GAACGTGCAG AAGTAACGGC CTTGATTCAA 3060
CCTGTATTAAATCAAATCAA TGCGTTGTAT GAAAATGAAG ATTGGAATGG AGCAATTCGT 3120
TCTGGAGTTT CTTATCATGA CTTAGAAGCA ATTGTTTTAC CAACATTACC AAAATTAAAT 3180
CATTGGTTTA TGTCTGATAT GTTAGGGGAA CAAGGTTCCA TTTTAGCTCA ATTTCAAGAA 3240
GCATTAGATC GTGCGTATAC GCAACTCGAA GAAAGTACAA TTCTGCATAA TGGTCATTTC 3300
WO 92/20802 PCT/US92/04316
213248 so
ACAACAGATG CAGCAAATTG GACGATAGAA GGCGATGCAC ATCATGCGAT ATTAGAAGAT 3360
GGTAGACGCG TATTACGTCT TCCAGATTGG TCTTCTAGCG TTTCACAAAC CATTGAAATA 3420
GAAAATTTTG ATCCAGATAA AGAATATCAG TTAGTTTTCC ATGCACAAGG AGAAGGAACG 3480
GTCTCCCTTC AACATGGTGA AGAAGGAGAA TATGTGGAAA CACACCCGCA TAAGTCTGCG 3540
AATTTTACAA CTTCACACCG TCAAGGAGTC ACATTTGAAA CAAATAAAGT AACAGTTGAA 3600
ATTACCTCAG AAGATGGAGA ATTCCTAGTC GATCATATTG CTCTTGTGGA AGCTCCTCTT 3660
CCTACAGATG ACCAAAGTTC AGATGGAAAT ACGACTTCCA ATACGAATAG CAATACAAGT 3720
ATGAATAATA ATCAATAA 3738
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 1245 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(Vi) ORIGINAL SOURCE:
(A) ORGANISM: BACILLUS THURINGIENSIS
(C) INDIVIDUAL ISOLATE: PS86Q3
(vii) IMMEDIATE SOURCE:
((A LIBRARY: LAMBDAGEM (tm) - 11 library
B~ CLONE: 86Q3A
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Ala Thr Ile Assn Glu Lou Tyr Pro Val Pro Tyr Asn Val Lou Ala
His Pro Ile LLys Gin Val Asp Asp Pro Tyr Ser Trp Ser Assn Leu Lou
Lys Gly 15e Gln Gin Gly Trp G40 lu Glu Trp Gly Lys 4Thr 5 Gly Gin Lys
Lys Lou Phe Glu Asp His Len Thr Ile Ala Trp Asn Lou Tyr Lys Thr
50 55 60
Gly Lys Lou Asp Tyr Phe Ala Lou Thr Lys Ala Ser Ile Ser Lou Ile =~'~
65 .70 75 80
Gly Phe Ile Pro Gly Ala Gin Ala Ala Val Pro Phe Ile Asn Met Phe
85 90 95
Val Asp Phe ~aall Trp Pro Lys Lou Pile Gly Ala Asn Thr Glluu Gly Lys 10
Asp Gln Gln Lou Phe Asn Ala Ile Met Asp Ala Val Asn Lys Met Val
115 120 125
Asp Asn Lys Phe Lou Ser Tyr Asn Lou Ser Thr Len Asn Lys Thr Ile
130 135 140 '
Gin Gly Lou Gln Gly'Asn Len Gly Lou Phe Gln Asn Ala Ile Gln Val
145 150 155 160
Ala Ile Cys Gln Zly Ser Thr Pro Glu A1rrg Val Asn Phe Asp Ginn Asn 175
Cys Thr Pro Cys Asn Pro Asn Gin Pro Cysss Lys Asp Asp Lou Asp Arg
180 185 190
Val Ala Ser Arg Phe Asp Thr Ala Asn Ser Gln Phe Thr Gln His Lou
195 200 205
Pro Gin Phe Lys Asn Pro Trp Ser Asp Glu Asn Ser Thr Gin Glu Pile
210 Lys Arg Thr Ser Val Glu Lou Thr Lou Pro Met Tyr Thr Thr Val Ala
225 230 235 240
Thr Lou His Lou Lou Lou Tyr Glu Gly Tyr Ile Gin Phe Met Thr Lys
245 250 255
SUBSTITUTE SHEET
WO 92/20802 2103248 PCF/US92/04316
51
Trp Asn Phe His Asn Gin Gin Tyr Leu ken Asn Leu Lys Val Glu Leu
260 265 270
Gin Gln Leu Ile His Ser Tyr Ser Glu Thr Val Arg Thr Ser Phe Leu
275 280 285
Gln The Leu Pro Thr Leu Asn Asn Arg Ser Lys Ser Ser Val Asn Ala
290 295 300
Tyr Asn Arg Tyr Val Ar Asn Met Thr Val Asn Cys Leu Asp Ile Ala
Ala Thr Trp Pro Thr Phe Asp Thr His 33nn Tyr His Gin Gly G35 Lys
Leu Asp Leu Thr Arg Ile Ile Leu Ser Asp Thr Ala G1y Pro Ile Glu
340 345 350
Glu Tyr Thr Thr Gly Asp Lys Thr Ser Gly Pro Glu His Ser Asn Ile
355 360 365
Thr 3ro Asn Asn Ile Len AAsp Thr Pro Ser Pro Thr Tyr Gin His Ser
365 Val Ser Val Asp Seerr Ile Val Tyr Ser 39g Lys Glu Leu Gin Gin
Leu Asp Ile Ala Thr Tyr Ser Thr Asn Asn Ser Asn Asn Cys His Pro
405 410 415
Tyr Gly Leu 4 g LeuSer Tyr Thr AAsp Gly Ser Arg Tyr 4Asp Tyr Sly
Asp ken 4 5 Pro Asp Phe Thr 440 Ser Asn Asn Asn Tyr Cys His Asn
Ser Tyrr Thr Ala Pro Ile 4Thr Leu Val Asn Ala 46g His Len Tyr Asn
4555 Ala Lys Giy Ser Len Gin Assn Val Glu Ser Len Val l Val Ser Thr Val
465 470 475 480
Asn Gly Gly Ser 48y Ser Cys Ile Cys 4sp Ala Trp Ile Asn TVr Len 49
Arg Pro Pro Gin Thr Ser Lys ken 5Glu 05 Ser Arg Pro Asp Gin Lys Ile
Asn Val Len Tyr Pro,Ile Thr Gin Thr Val Asn Lys G2y Thr Gly Sly
Asn LLeu0 Gly Val Ile Ser Ala Tyr Val Pro Met GG41uu Leu Vai Pro Glu
535
Asn Val Ile Gly Asp Vai Asn Ala Asp Thr L YS Leu Pro Len Thr-Gin
545 550 555 560
Leu Lys Gly The Pro Phe Glu Lys Tyr GG7y Ser Gin Tyr Asn 57n Arg
56 Gly Ile Ser L80 Val Arg Gin Trp lie Asn Gly Asn Asn laa Val Lys
5 585 59
Leu Ser 5Asn 95 Ser Gln Ser Val G60y Ile Gin Ile Thr 60n Gin Thr Lys
Gin LLyss Tyr Glu Ile Arg Cys Arg Tyr Ala Ser L a Gly Asp Asn Asn
6 6
Val Tyr Phe ken Val Asp Leu Ser Glu Asn Pro Phe Arg Asn Ser Ile
625 630 635 640
Ser Phe Gly Ser T64hr Glu Ser Ser Vai Val50 G1y Val Gin Sly Gin Asn
Gly Lys Tyr lie Leu Lys Ser Ile Thr Thr Vai Glu Ile Pro o Ala Gly
66 665
Ser Phe TVr Val His Ile Thr 6Asn 80 Gln Gly Ser Ser Asp Leu Phe Leu
Asp Ar Ile Glu The Val Pro Lys Ile Gln Phe Gin Phe Cys Asp Asn
699 695 700
Asn Asn Leu His Cys Asp Cys Asn Asn Pro Val Asp Thr Asp Cys Thr
705 710 715 720
WO 92/20802 1 0 3 2 4 8 PCF/US92/04316
52
The Cys Cys Val Cvs Thr Ser Leu Thr 73p Cys Asp Cys Asn 73n Pro
Arg Gly Leu Assp eye Thr Leu Cys Is Gln Val Glu Asn 75 nn Len Pro
74 Ser Phe Val Thr Leu Thr Asp Leu Gin Asn Ile Thr Thr Gin Val Asn
755 760 765
Ala Leu Val Ala Ser Ser Glu His Asp Thr Leu Ala Thr Asp Val Ser
770 775 780
Asp Tyr Glu Ile Glu Glu Val Val Leu Lys Val Asp Ala Leu Ser Gl
785 790 795 80~
Glu Val Phe Gly Lys Glu Lys Lys Ala Leu Arg Lys Leu Val Asn His
805 810 815
Thr Lys Arg LLeen Ser Lys Ala Arg 825 Len Leu Ile Gly Gly Asn The
Asp Asn L3e5 Asp Ala Trp Tyr 8Arrg Giy Arg Asn Val Vaal Asn Val Ser 845
Asp His Glu Len The Lys Ser Asp His Val Len Leon Pro Pro Pro Thr ass 8
Leu Tyr Ser Ser Tyr Met Phe Gin Lys Val Glu Glu Ser Lys Leu Lys
865 870 875 880
Ala Aan Thr Arg Tyr Thr Val Ser Gly The Ile Ala His Ala Glu Asp
885 890 895
Leu Glu Ile Val Val Ser Arg Tyr GGiy Gln Glu Val Lys Lyes Val Val
9
Gln ValP1o Tyr Gly Glu Ala Pao Pro Len Thr Ser 9 g Gly Ala Ile
95 Cys C
ons Pro Pro Arg Ser Thr Ser Asn Gly Lys Pro Ala Asp Pro His
45 935 9
The Phe Ser Tyr Ser Ile Asp Val Gly Thr Lei Asp Val Glu Ala Asn
945 950 955 960
Pro Gly Ile Glu Leu Gly Leu Arg Ile Val Glu Arg Thr Gly Met Ala
965 970 975
Arg Val Ser AA8sn Lou Gin Ile Arg Giu Asp Arg Pro Leu LLye Lys Asn
9 985 9
Glu Leu Ara lien Val Gin Arg Ala Ala Arg Asn Trp Ara Thr Ala Tyr
995 1000 1005
Asp Gin Gin Arg Ala Gin Val Thr Ala Leu Ile Gin Pro Val Leu Asn
1010 1015 1020
Gin Ile Asn Ala Leu Tyr Glu Asn Glu Asp Trp Asn Gly Ala Ile Ara
1025 1030 1035 1040
Ser Gly Val Ser THis Asp Len Glu Ala Ile Val Len Pro Thr Len
1045 1050 1055
Pro Lys Leu Aen His Trp Phe Met Ser Asp Met Leu Gly Glu Gin Gly
1060 1065 1070
Ser Ile Leu Ala Gin Phe Gin Glu Ala Leu Asp Arg Ala Tyr Thr Gin
1075 1080 1085
Leu Glu Glu Ser Thr Ile Leu His Asn Gly His Phe Thr Thr Asp Ala
1090 1095 1100
Ala Asn Trp Thr Ile Glu Giy Asp Ala His His Ala Ile Leu Glu Asp
1105 1110 1115 1120
Gly Arg Arg Val Leu Arg Leu Pro Asp Trp Ser Ser Ser Val Ser Gln
1125 1130 1135
Thr Ile Glu Ile Gin Asn Phe Asp Pro Asp Lys Glu Tyr Gin Leu Val
1140 1145 1150
The His Ala Gln Gly Glu Gly Thr Val Ser Leu Gln His Gly Glu Gin
1155 1160 1165
G1y Gin Tyr Val Giu Thr His Pro His Lys Ser Ala Asn The Thr Thr
1170 1175 1180
WO 92/20802 219 43 2 4 8 PCI'/US92/04316
53
Ser His Arg Gin Gly Val Thr Phe Glu Thr Asn Lys Val Thr Val Glu
1185 1190 1195 1200
Ile Thr Ser Glu Asp Gly Glu Phe Leu Val Asp His Ile Ala Leu Val
1205 1210 1215
Glu Ala Pro Leu Pro Thr Asp Asp Gln Ser Ser Asp Gly Asn Thr Thr
1220 1225 1230
Ser Asn Thr Asn Ser Asn Thr Ser Met Asn Asn Asn Gin
1235 1240 1245
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
JA LENGTH: 2412 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: double
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
1A1 ORGANISM: Bacillus thuringiensis
C; INDIVIDUAL ISOLATE: PS63B
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. soli NM522(pMYCI642) NRRL B-18961
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATGACTTGTC AATTACAAGC GCAACCACTT ATTCCCTATA ACGTACTAGC AGGAGTTCCA 60
ACTAGTAATA CAGGTAGTCC AAT GGCAAT GCAGGTAATC AATTTGATCA GTTTGAGCAA 120
ACCGTTAAAG AGCTCAAGGA AGCATGGGAA GCGTTCCAAA AAAACGGAAG TTTCTCATTA 180
GCAGCTCTTG AAAAGGGATT TGATGCAGCA ATCGGAGGAG GATCCTTTGA TTATTTAGGT 240
TTAGTTCAAG CCGGCCTAGG ATTAGTTGGT ACGCTAGGCG CCGCAATCCC TGGTGTTTCA 300
GTGGCAGTGC CTCTTATTAG CATGCTTGTT GGTGTTTTTT GGCCAAAGGG CACAAACAAC 360
CAAGAAAACC TTATTACAGT TATTGATAAG GAAGTTCAGA GAATACTAGA TGAAAAGCTA 420
TCTGATCAGT TAATAAAGAA ATTGAAAGCA GATTTAAATG CTTTTACGGA CCTAGTAACT 480
CGTTTGGAAG AAGTAATAAT AGATGCAACT TTCGAGAATC ACAAGCCTGT ACTACAAGTA, 540
AGTAAATCAA ATTATATGAA AGTGGATTCA GCATATTTCT CAACAGGAGG TATTCTTACT 6OO
CTTGGCATGA GTGATTTTCT TACTGATACC TATTCAAAGC TTACCTTCCC ATTATATGTA 660
CTAGGCGCAA CTATGAAACT TTCAGCATAT CATAGTTATA TACAATTCGG AAATACATGG 720
CTTAATAAAG TTTATGATTT ATCATCAGAT GAGGGAAAAA CAATGTCGCA GGCTTTAGCA 780
CGAGCTAAAC AGCATATGCG CCAGGACATA GCATTTTATA CAAGCCAAGC TTTAAACATG 840
TTTCAGGGGA ATCTCCCTTC ATTATCATCT AATAAATATG CAATTAATGA CTATAATGTA 900
TACAATCGAG CAATGGTATT GAATGGCTTA GATATAGTAG CAACATGGCC TACCCTATAT 960
C:CAGATGACT ATTCGTCTCA GATAAAAATG GAGAAAACAC GCGTGATCTT TTCAGATATG 1020
GTCGGGCAAA GTGAGAGTAG AGATGGAAGC GTAACGATTA AAAATATTTT TGACAATACA 1080
GATTCACATC AACATGGATC CATAGGTCTC AATTCAATCT CTTATTTCCC AGATGAGTTA 1140
CAGAAAGCAC AACTTCGCAT GTATGATTAT AATCACAAAC CTTATTGTAC GGACTGTTTC 1200
TGCTGGCCGT ATGGAGTGAT TTTAAACTAT AACAAGAATA CCTTTAGATA TGGCGATAAT 1260
GATCCAGGTC TTTCAGGAGA CGTTCAACTC CCAGCACCTA TGAGTGTAGT TAATGCCCAA 1320
ACTCAAACAG CCCAATATAC AGATGGAGAA AACATATGGA CAGATACTGG CCGCAGTTGG 1380
CTTTGTACTC TACGTGGCTA CTGTACTACA AACTGTTTTC CAGGAAGAGG TTGTTATAAT 1440
AATAGTACTG GATATGGAGA AAGTTGCAAT CAATCACTTC CAGGTCAAAA AATACATGCA 1.500
CTATATCCTT TTACACAAAC AAATGTGCTG GGACAATCAG GCAAACTAGG ATTGCTAGCA 1560
AGTCATATTC CATATGACCT AAGTCCGAAC AATACGATTG GTGACAAAGA TACAGATTCT 1620
WO 92/20802 PCT/US92/04316
2103248
54
ACGAATATTG TCGCAAAAGG AATTCCAGTG GAAAAAGGGT ATGCATCCAG TGGACAAAAA 1680
GTTGAAATTA TACGAGAGTG GATAAATGGT GCGAATGTAG TTCAATTATC TCCAGGCCAA 1740
TCTTGGGGAA TGGATTTTAC CAATAGCACA GGTGGTCAAT ATATGGTCCG CTGTCGATAT 1800
GCAAGTACAA ACGATACTCC AATCTTTTTT AATTTAGTGT ATGACGGGGG ATCGAATCCT 1860
ATTTATAACC AGATGACATT CCCTGCTACA AAAGAGACTC CAGCTCACGA TTCAGTAGAT 1920
AACAAGATAC TAGGCATAAA AGGAATAAAT GGAAATTATT CACTCATGAA TGTAAAAGAT 1980
TCTGTCGAAC TTCCATCTGG GAAATTTCAT GTTTTTTTCA CAAATAATGG ATCATCTGCT 2040
ATTTATTTAG ATCGACTTGA GTTTGTTCCT TTAGATCAAC CAGCAGCGCC AACACAGTCA 2100
ACACAACCAA TTAATTATCC TATCACAAGT AGGTTACCTC ATCGTTCCGG AGAACCACCT 2160
GCAATAATAT GGGAGAAATC AGGGAATGTT CGCGGGAATC AACTAACTAT ATCGGCACAA 2220
GGTGTTCCAG AAAATTCCCA AATATATCTT TCGGTGGGTG GCGATCGCCA AATTTTAGAC 2280
CGTAGCAACG GATTTAAATT AGTTAATTAC TCACCTACTT ATTCTTTCAC TAACATTCAG 2340
GCTAGCTCGT CAAATTTAGT AGATATTACA AGTGGTACCA TCACTGGCCA AGTACAAGTA 2400
TCTAATCTAT AA 2412
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 803 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
A ORGANISM: Bacillus thuringiensis
(C) INDIVIDUAL ISOLATE: PS63B
(vii) IMMEDIATE SOURCE:
(B) CLONE: E. coli NM522(pMYC1642) NRRL B-18961
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Net Thr Cys Gln Leu Gln Ala Gln Pro Leu Ile Pro Tyr Asn Val Leu
1 5 10 15
AlaGly Val Pro ThrSer Asn Thr Gly Ser Pro Ile Gly 3A sn Ala Gly 25
Asn Gin Phe Asp Gin Phe Glu Gln Thr Val Lys Glu Leu Lys Glu Ala
35 40 45
Trp Gin Ala Phe Gin Lye Asn Gly Ser Phe Ser Len Ala Ala Leu Glu
50 55 60
LLys G1y Phe Asp Ala Ala Ile Gly Gly Gly 7S 5er Phe Asp Tyr Leu Gly
6 so
Leu Val Gin Ala G85y Leu Gly Len Val Gly Thr Len Gly Ala Ala Ile 90 95
Pro Gly Val Ser Val Ala Val Pro Leu Ile Ser Met Leu Val Gly Val
100 105 110
Phe Trp Pro Lys Gly Thr Asn Asn Gln Glu Asn Leu Ile Thr Val Ile
115 120 125
Asp Lys0 Glu Val Gin Arg 13e Len Asp Glu Lys Len Ser Asp Gin Leu
1
Ile Lys Lys Leu Asn Ala Asp Len Asn Ala Phe Thr Asp Leu Val Thr
145 150 155 160
Arg Leu Glu Glu Vaal Ile Ile Asp Ala T hr Phe Glu Asn His LL ye Pro
15 170
Val Leu Gin Val Ser Lys Ser Asn Tyr Met Lys Val Asp Ser Ala Tyr
180 185 190
WO 92/20802 PCT/US92/04316
210324
Phe Ser Thr Gly Gly Ile Leu Thr Leu Gly Met Ser AsOp Phe Leu Thr 200 2
Asp Thr Tyr Ser Lys Len ZT1r Phe Pro Leu Tyr Val Len Gly Ala Thr
Met Lys Leu Ser Ala Iyr r His Ser Tyr Ile Gin Phe Gly Asn Thr T
225 233 235 2rp
Leu Asn Lys Val Tyr Asp Leu Ser Ser Asp Glu Gly Lys Thr Met Seer
245 250 255
Gin Ala Leu Ala Arg Ala Lys Gin His Met Arg Gin Asp Ile Ala Phe
260 265 270
Tyr Thr Ser Gin Ala Leu Asn MMeett Phe Thr Gly Asn Leu Pro Ser Leu
Ser Ser Asn Lys Tyr Ala Ile Asn Asp Tyr Asn Val Tyr Thr Arg Ala
290 295 300
Met Val Leu Asn Gly Leu Asp Ile Val Ala Thr Trp Pro Thr Leu Tyr
305 310 315 320
Pro Asp Asp Tyr Ser Ser Gin Ile Lys 3eu0 Glu Lys Thr Arg Val Ile
325 335
Phe Ser Asp Met Val Gly Gin Ser Glu Ser Arg Asp Gly Ser Val Thr
3 345 Ile Lys Asn Ile Phe Asp Asn Thr Asp Ser His Gin His Gly Ser Ile
355 360 365
Gly Leu0 Asn Ser Ile Ser Tyr Phe Pro Asp Glu Leu Gin Lys Ala Gin
3 37 380
Leu e5 Arg Met Tyr Asp Tv0 Asn His Lys Pro Tyr Cys Thr Asp Cys Phe
j9 400
3
Cys Trp Pro Tyr GGly Val Ile Leu Asn Tyr Asn Lye Asn Thr Phe Arg
405 4 415
Tyr Gly Asp Asn Asp Pro Giy Leu Ser GlyAsp Val Gin Leu Pro Ala
420 425 430
Pro Met Ser Val Val Asn Ala GGiin Thr Gin Thr Ala G44nn Tyr Thr Asp
5
Gly Glu Asn Ile Trp Thr AAsp Thr Gly Arg Ser Trp Len Cys Thr Leu
Ar Gly Tyr Cys Thr Thr As55n Cys Phe Pro.Gly,Arg Gly Cys Tyr Asn
465 470 475 480 = -r
Asn Ser ThrGly Tyr Gly Gin Ser Cys sn Gin Ser Leu Pro G4I9K -Gin
4B 49
Lys Ile His Ala Leu Tyr Pro Phe T 505 hr Thr Asn Val 510 Giy Gin
Ser Gly LyL e Leu Giy Leu Leu 52Ala 0 Ser His Ile Pro Tyr r Asp Leu Ser
5
Pro,53snn ken Thr Ile Gly Asp Lys Asp Thr Asp Ser Thr Asn Ile Val 5 540
;Ala Lys Gly Ile Pro ValGlu Lys Gly Tyr Ala Ser Ser Gly Gin Lys
545 550 555 560
Val Gin Ile Ile AArra Glu Trp Ile Asn Gly Ala Asn Val Val G7n Leu
5 Ser Pro Gly Gin Ser Trp Gly Met Ass? Phe Thr Asn Ser SThr Gly Giy
Gin Tyr 9Met 5 Val Arg Cys Arg Tyrr Ala Ser Thr Asn Asp Thr Pro Ile
Phe Phe Asn Leu Val Tyr 6A 13 Gly Gly Ser Asn PP2o Ile Tyr Asn Gin
610 6
Met Thr Phe Pro Ala Thr Lys Glu Thr Pro Ala His Asp Ser Val Aspp
625 630 635 64D
Asn Lys Ile Leu 6lly Ile Lys Gly Ile 55n Gly Asn Tyr Ser Leu Met 655
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Asn Val Lys Asp Ser Val Glu Leu Pro Ser Gly Lys Phe 67ss Val Phe
Phe Thr Asn Asn Gly Ser Ser AAl8aa Ile Tyr Leu Asp Arrg Leu Glu Phe
675 Val Pro Leu Asp Gin Pro Ala Ala Pro Thr Gin Ser Thr55 Gln Pro Ile
690 695 700
Asn Tyr Pro Ile Thr Ser Arg Leu Pro-His Arg Ser Gly Glu Pro Pro
705 710 715 720
Ala Ile Ile Trp Glu Lys Ser Gly Asn Val Arg Gly Asn Gln Leu Thr
725 730 735
Ile Ser Ala G41nn Gly Val Pro Glu 745 Ser Gin Ile Tyr Leon Ser Val
7
7
Gly Gly As Arg Gln lie Leu Asp Arg Ser Asn Gly ?65 Lys Len Val
70 Asn Tvr Ser Pro Thr Tyr Ser Phe Thr Asn Ile G8 0n Ala Ser Ser Ser
775 Asn Leu Val Asp Ile Thr Ser Gly Thr Ile Thr Gly Gin Val Gin Val
785 790 795 800
Ser Aen Leu
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 8 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
A1rg Glu Trp Ile. Asn Gly Ala Asn
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE-CHARACTERISTICS:
A LENGTH: 21 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12
AGARTRKWTW AATGGWGCKN A 21
(2)INFORMATION.FOR SEQ ID NO:13
(i.) SEQUENCE CHARACTERISTICS:
A LENGTH: 20 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi.) SEQUENCE DESCRIPTION: SEQ ID NO:13
GARTGGWTAA ATGGTRMSAA 20
(2) INFORMATION FOR SEQ ID NO:14
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 8 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
WO 92/20802 QQ PCT/US92/04316
iO3N4V
Pro Thr Phe Asp Pro Asp Leu Tyr
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
IAI LENGTH: 24 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CCNACYTTTK ATCCAGATSW YTAT 24
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 24 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION SEQ ID NO:16:
CCWACWTTYG ATMCASATMW TTAT 24
(2) INFORMATION FOR SEQ ID NO:17:
(1.) SEQUENCE CHARACTERISTICS:
A) LENGTH: 14 amino acids
B) TYPE: amino acid
C STRANDEDNESS single
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ala Ile Leu Asn Glu Leu Tyr Pro Ser Val Pro Tyr Asn Val
1 5 10
(2) INFORMATION FOR SEQ ID NO 18 :
(i) SEQUENCE CHARACTERISTICS:
Al LENGTH: 14 amino acids = -''~
B) TYPE: amino acid
C j single
D) TOPOLOGY linear
(ii) MOLECULE TYPE: protein
(xi.)" SEQUENCE DESCRIPTION SEQ ID NO:18:
Ala Ile Leu Asn Glu Leu. Tyr Pro Ser Val Pro Tyr Asn Val
l 5 10
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
Al ENGTH: 16 amino acids
B) TYPE: amino acid
C j single
ID ) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE.DESCRIPTION: SEQ ID NO:19:
Met Ala Thr Ile Asn Glu Leu Tyr Pro Asn Val Pro Tyr Asn Val Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:'14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
SUSSTiTUTE SHEET
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Gin Leu Gln Ala Gln Pro Leu Ile Pro Tyr Asn Val Leu Ala
1 5 10
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 10 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Ala Thr Leu Asn Glu Val Tyr Pro Val Asn
1 5 10
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 15 amino acids
B) TYPE: amino acid
C STRANDEDNESS: single
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Val Gin Arg Ile Leu AspGlu Lys Leu Ser Phe Gln Leu Ile Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS
A LENGTH: 23 bases
B TYPE: nucleic acid
C STRANDEDNESS single
D TOPOLOGY: linear
(ii) MOLECULE TYPE DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GCAATTTTAA ATGAATTATA TCC 23
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
JA LENGTH: 17 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CAAYTACAAG CWCAACC 17
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEAUELENGTH: R RISTICS:
s
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
TTCATCTAAA ATTCTTTGWA C 21
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(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 21 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GCWACWTTAA ATGAAGTWTA T 21
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 21 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
AATGAAGTWT ATCCWGTWAA T 21
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 38 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(Li) MOLECULE TYPE DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
GCAAGCGGCC GCTTATGGAA TAAATTCAAT TYKRTCWA 38
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH 37 bases
B TYPE: nucleic acid
C STRANDEDNESS: single .1 ,
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
AGACTGGATC CATGGCWACW ATWAATGAAT TATAYCC 37
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
Al ENGTH: 10 amino acids
B) TYPE: amino acid
C j single
ID ) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Glu Ser Lys Leu Lys Pro Asn Thr Arg Tyr
(,2) INFORMATION FOR SEQ, ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 29 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
WO 92/20802 PCT/US92/04316
210324
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
TAACGTGTAT WCGSTTTTAA TTTWGAYTC 29
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 9 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Tyr Ile Asp Lys lie Glu Phe Ile Pro
(2) INFORMATION FOR SEQ ID NO:33:
(i)SEQUENCE CHARACTERISTICS:
A LENGTH: 23 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
TGGAATAAAT TCAATTYRRT CWA 23
(2) INFORMATION FOR SEQ ID NO:34:
(k) SEQUENCE CHARACTERISTICS:
A LENGTH: 23 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
AGGAACAAAY TCAAICWCGRT CTA 23
(2) INFORMATION FOR SEQ ID NO:35:
(xi) SEQUENCE DESCRIPTION : SEQ ID NO-.35: TTTAGATCGT MTTGARTTTR TWCC 24
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 5 amino acids
B TYPE: amino acid
C STRANDEDNESS single
D TOPOLOGY: linear
(ii} MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Ile Thr Ser Glu Asp.
5
(2) INFORMATION FOR SEQ ID NO:37:
(i) SE - CHARACTERISTICS:
LENGTH: 20 bases
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
TCTCCATCTT CTGARGWAAT 20
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(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 8 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
feu Asp Arg Ile Glu Phe Val Pro
-.4
