Note: Descriptions are shown in the official language in which they were submitted.
DESCRIPTION ~ ~ 4 1 4 7 2
This application is a division of Application No. 520,375 filed
on October 14, 1986.
PROCESS FOR ALTERING THE HOST RANGE OF
BACILLUS THURINGIENSIS TOXINS, AND NOVEL
TOXINS PRODUCED THEREBY
Background of the Invention
15 The most widely used microbial pesticides are de-
rived from the bacterium Bacillus thuringiensis. This
bacterial agent is used to control a wide range of
leaf-eating caterpillars, Japanese beetles and mos-
quitos. Bacillus thuringiensis produces a proteina-
20 ceous paraspore or crystal which is toxic upon ingestion
by a susceptible insect host. For example, B. thur-
ingiensis var. kurstaki HD-1 produces a crystal called
a delta toxin which is toxic to the larvae of a number
of lepido~teran insects. The cloning and expression
25 of this B.t. crystal protein gene in Escherichia coli
has been described in the published literature
(Schnepf, H.E. and Whiteley, H.R. [1981] Proc. Natl.
Acad. Sci. USA 78:2893-2897). U.S. Patent 4,448,885
and U.S. Patent 4,467,036 both disclose the expression
30 of B.t. crystal protein in E. coli. In U.S. 4,467,036
B. thuringiensis var. kurstaki HD-1 is disclosed as
being available from the well-known NRRL culture reposi-
tory at Peoria, Illinois. Its accession number there is
NRRL B-3792. B, thuringiensis var. kurstaki HD-73
35 is also available from NRRL. Its accession number is
NRRL B-4488.
1~4~47~
Brief Summarv of the Invention
The subject invention concerns a novel process
for altering the insect host range of Bacillus
thurinQiensis toxins, and novel toxins produced as
exemplification of this useful process. This altera-
tion can result in expansion of the insect host
range of the toxin, and/or, amplification of host
toxicity. The process comprises recombining in vitro
the variable regions) of two or more d-endotoxin genes.
Specifically exemplified is the recombining of portions
of two Bacillus _thuringiensis var. kurstaki DNA
sequences, i.e., referred to herein as k-1 and k-73,
to produce chimeric B.t. toxins with expanded host
ranges as compared to the toxins produced by the
parent DNA's.
"Variable regions," as used herein, refers to
the non-homologous regions of two or more DNA
sequences. As shown by the examples presented herein,
the recombining of such variable regions from two
different _B._t. DNA sequences yields, unexpectedly,
a DNA sequence encoding a 8-endotoxin with an expanded
insect host range. In a related example, the recom-
bining of two variable regions of two different
B.t. toxin genes results in the creation of a chimeric
toxin molecule with increased toxicity toward the
target insect. The utility of this discovery by
the inventorsis clearly broader than the examples
disclosed herein. From this discovery, it can be
expected that a large number of new and useful toxins
will be produced. Thus, though the subject process is
exemplified by construction of chimeric toxin-producing
DNA sequences from two well-known B.t. kurstaki DNA
sequences, it should be understood that the process
-3- 13~ 1~~~
is not limited to these starting DNA sequences. The
invention process also can be used to construct chimeric
toxins from any B. th uringiensis toxin-producing
DNA sequence.
Description of the Drawines
FIGURE 1: A schematic diagram plasmid pEWl which
of
contains the DNA seque nce encoding Bacillus
thuringiensi s toxin 1.
k-
FIGURE 2: A schematic diagram plasmid pEW2 which
of
contains the DNA seque nce encoding Bacillus
thuringiensis 73.
toxin k-
FIGURE 3: A schematic diagram plasmid pEW3 which
of
contains the DNA seque nce encoding Bacillus
thuringiensi s chimerictoxin k-73/k -1 (pllY).
FIGURE 4: A schematic diagram plasmid pEW4 which
of
contains the DNA seque nce encoding Bacillus
thuringiensi s chimerictoxin k-1/k- 73 (pYH).
Detailed Disclosure of the Invention
Upon recombining in vitro the variable regions)
of two or more d-endotoxin genes, there is obtained
a genes) encoding a chimeric toxins) which has an
expanded and/or amplified host toxicity as compared
to the toxin produced by the starting genes. This
recombination is done using standard well-known genetic
engineering techniques.
The restriction enzymes disclosed herein can be
purchased from Bethesda Research Laboratories, Gai-
thersburg, MD, or New England Biolabs, Beverly, MA.
The enzymes are used accordi.np to the instructions
provided by the supplier.
-4- ~3~~412
The various methods employed in the preparation
of the plasmids and transformation of host organisms
are well known in the art. These procedures are all
described in Maniatis, ~I., Fritsch, E.F., and Sambrook,
J.(1982) Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, New York. Thus, it
is within the skill of those in the genetic engineering
art to extract DNA from microbial cells, perform
restriction enzyme digestions, electrophorese DPdA
fragments, tail and anneal plasmid and insert DNA,
ligate DNA, transform cells, prepare plasmid DNA,
electrophorese proteins, and sequence DNA.
Plasmids pEWl , pEW2 , pEW3 , and pEW4 , constructed
as described infra, have been deposited in E. coli
hosts in the permanent collection (to be maintained
for at least 30 years) of the Northern Regional Research
Laboratory (NRRL), U.S. Department of Agriculture,
Peoria, Illinois, USA. Their accession numbers and
dates of deposit are as follows:
pEWl--NRRL B-18032; deposited on Nov. 29, 1985
pEW2--NRRL B-18033; deposited on Nov. 29, 1985
pEW3--NRRL B-18034; deposited on Nov. 29, 1985
pEW4--NRRL B-18035; deposited on Nov. 29, 1985
B. thuringiensis strain MTX-36, NRRL B-18101 was deposited
on August 25, 1986.
Plasmid pBR322 is a well-known and available
plasmid. It is maintained in the E. coli host ATCC
37017. Purified pBR322 DNA can be obtained as
described in Bolivar, F., P,odriguez, R.L., Greene, P.J.,
Betlach, M.C., Heynecker, H.1., Boyer, H.W., Crosa,
J.H. and Falkow, S. (1977) Gene 2:95-113; and Sutcliffe,
J.G. (1978)Nucleic Acids Res. 5:2721-2728.
NRRL B-18032, NRRL B-18033, NRRI, B-18034 , NRRL B-
180.35, and NRRL B-18101 are available to the public upon
the grant of a patent which discloses these accession
-5- 1341472
numbers in conjunction with the invention described
herein. It should be understood that the availability
of these deposits dies not constitute a license to
practice the subject invention in derogation of patent
rights granted for the subject invention by govern-
mental action.
As disclosed above, a~ B. thuringiensis toxin-
producing DNA sequence can be used as starting material
for the subject invention. Examples of B. thuringiensis
organisms, other than those previously given, are as
follows:
Bacillus thuringiensis var. israelensis--ATCC 35646
Bacillus thuringiensis M-7--NRRT B-15939
Bacillus thuringiensis var. tenebrionis--DSM 2803
The following B. thuringiensis cultures are avail-
able from the United States Department of Agriculture
(USDA) at Brownsville, Texas. Requests should be made
to Joe Garcia, USDA, ARS, Cotton Insects Research Unit,
P.O. Box 1033, Brownsville, Texas 78520 USA.
B. thuringiensis HD2
B. thuringiensis var. finitimus HD3
B. thurin~iensis var. alesti HD4
B. thuringiensis var. kurstaki HD73
B. thuringiensis var. sotto HD770
B. thuringiensis var. dendrolimus HD7
B. thuringiensis var. kenyae HD5
B. thurinpiensis var. galleriae HD29
B. thuringiensis var. canadensis HD224
B. thuringiensis var. entomocidus HD9
B~ thurin~iensis var. subtoxicus HD109
B. thuringiensis var. aizawai HD11
B. thuringiensis var. morrisoni HD12
B. thuringiensis var. ostriniae HD501
B. thuringiensis var. tolworthi HD537
6 134147
B. thuringiensis var. darmstadiensis HD146
B. thuringiensis var. toumanoffi HD201
B. thuringiensis var. kyushuensis HD541
B. thuringiensis var. thomosoni HD542
B. thurinniensis var. Pakistani HD395
B. thuringiensis var. israelensis HD567
B. thuringiensis var. Indiana HD521
B. thuringiensis var. dakota
B. thuringiensis var. tohokuensis HD866
B. thuringiensis var. kumanotoensis HD867
B. thuringiensis var. tochigiensis HD868
B. thuringiensis var. colmeri HD847
B. thuringiensis var. wuhanensis HD525
Though the main thrust of the subject invention
is direc=ed toward a process for altering the host
range of B. thuringiensis toxins, the process is
also applicable in the same sense to other Bacillus
toxin-producing microbes. Examples of such Bacillus
organisms which can be used as starting material
are as follows:
Bacillus cereus--,'~'~'CC 21281
Bacillus moritai--ATCC 21282
Bacillus popilliae--ATCC 14706
Bacillus lentimorbus--ATCC 14707
Bacillus sphaericus--ATCC 33203
Bacillus thuringiensis M-7, exemplified herein,
is a Bacillus thuringiensis isolate which, surprisingly,
has activity against beetles of the order Coleoptera
but not against Trichoplusia ni, Spodoptera exipua
or Aedes aegypti. Included in the Coleoptera are
134147
various Diabrotica species (family Chrysomelidae) that
are responsible for large agricultural losses, for
example, D. undecimpunctata (western spotted cucumber
beetle), D. longicornis (northern corn_ - rootworm), D.
virgitera (western corn rootworm), and D. undecimpunctata
howardi (southern corn rootworm).
B. thurinaiensis M-7 is unusual in having a unique
parasporal body (crystal) which under phase contrast
microscopy is dark in appearance with a flat, square
configuration.
The pesticide encoded by the DNA sequence used as
starting material for the invention process can be any
toxin produced by a microbe. For example, it can be a
polypeptide which has toxic activity toward a eukaryotic
multicellular pest, such as insects, e.g., coleoptera,
lepidoptera, diptera, hemiptera, dermaptera, and
orthoptera; or arachnids; gastropods; or worms, such as
nematodes and platyhelminths. Various susceptible
insects include beetles, moths, flies, grasshoppers,
lice, and earwigs.
Further, it can be a polypeptide produced in active
form or a precursor or proform requiring further
processing for toxin activity, e.g., the novel crystal
toxin of B. thuringiensis var. kurstaki, which requires
processing by the pest.
The constructs produced by the process of the
invention, containing chimeric toxin-producing DNA
sequences, can be transformed into suitable hosts by
using standard procedures. Illustrative host cells may
include either prokaryotes or eukaryotes,
JJ:in
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normally being limited to those cells which do not
produce substances toxic to higher organisms, such as
mammals. However, organisms which produce substances
toxic to higher organisms could be used, where the toxin
is unstable or the level of application sufficiently low
as to avoid any possibility of toxicity to a mammalian
host. As hosts, of particular interest will be the
prokaryotes and lower eukaryotes, such as fungi.
Illustrative pro;~caryotes, both Gram-negative and -
positive, include Entereobacteriaceae, such as
Escherichia, Erwinia, Shigella, Salmonella, and Proteus;
Baceillaceae; Rhizobiaceae, such as Rhizobium;
Spirillaceae, such as photobacterium, Zymomonas,
Serratia, Aeromonas, Vibrio, 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 Aureaobasidium, ~orobolomyces, and the
like.
Characteristics of particular interest in selecting
a host cell for purposes of production include ease of
introducing the chimeric toxin-producing gene into the
host, availability of expression systems, efficiency of
expression, stability of the pesticide in the host, and
the presence of auxiliary genetic capabilities.
Characteristics of interest for use as a pesticide micro-
capsule include protective qualities for the pesticide,
JJ:in
i
- 1~414~2
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 Rhodotorula sp., Aureobasidium sp.,
Saccharomvces sp., and ~orobolomyces sp.; phylloplane
organisms such Pseudomonas sp., Erwinia sp. and
Flavobacterium sp.; or such other organisms as
Escherichia, Lactobacillus sp., Bacillus sp., and the
like. Specific organisms include Pseudomonas aerucrinosa,
Pseudomonas fluorescens, Saccharomyces cerevisiae,
Bacillus thurinc~iensis, Escherichia coli, Bacillus
subtilis, and the like.
The chimeric toxin-producing genes) can be
introduced into the host in any convenient manner, either
providing for extrachromosomal maintenance or integration
into the host genome.
Various constructs may be used, which include
replication systems from plasmids, viruses, or
centromeres in combination with an autonomous replicating
segment (ars) for stable maintenance. Where only
integration is desired, constructs can be used which may
provide for replication, and are either transposons or
have transposon-like insertion activity or provide for
homology with the genome of the host. DNA sequences can
be employed having the chimeric toxin-producing gene
between sequences which are homologous with sequences in
the genome of the host, either chromosomal or plasmid.
Desirably, the chimeric toxin-producing genes) will be
present in multiple copies. See for example, U.S. Patent
JJ:in
-lo- ~~4~4~'~
No. 4,399,216. Thus, conjugation, transduction,
transfection and transformation may be employed for
introduction of the gene.
A large number of vectors are presently available
which depend upon eukaryotic and prokaryotic replication
systems, such as ColEl, P-1 incompatibility plasmids,
e.g., pRK290, yeast 2m ~ plasmid, lambda, and the like.
Where an extrachromosomal element is employed, the
DNA construct will desirably include a marker which
allows for a selection of those host cells containing the
construct. The marker is commonly one which provides for
biocide resistance, e.g., antibiotic resistance or heavy
metal resistance, complementation providing prototrophy
to an auxotrophic host, or the like. The replication
systems can provide special properties, such as runaway
replication, can involve cos cells, or other special
feature.
Where the chimeric toxin-producing genes) has
transcriptional and translational initiation and
termination regulatory signals recognized by the host
cell, it will frequently be satisfactory to employ those
regulatory features in conjunction with the gene.
However, in those situations where the chimeric toxin-
producing gene is modified, as for example, removing a
leader sequence or providing a sequence which codes for
the mature form of the pesticide, where the entire gene
encodes for a precursor, it will frequently be necessary
to manipulate the DNA sequence, so that a transcriptional
initiation regulatory sequence may be provided which is
different from the natural one.
A wide variety of transcriptional initiation
sequences exist for a wide variety of hosts. The
sequence can provide for constitutive expression of the
pesticide or regulated expression, where the regulation
JJ:in
_11- 7 2
1 ~ ~ ~ ~
may be inducible by a chemical, e.g., a metabolite, by
temperature, or by a regulatable repressor. See for
example, U.S. Patent No. 4,374,927. The particular
choice of the promoter will depend on a number of
factors, the strength of the promoter, the interference
of the promoter with the viability of the cells, the
effect o.f regulatory mechanisms endogenous to the cell on
the promoter, and the like. A large number of promoters
are available from a variety of sources, including
commercial sources.
The cellular host containing the chimeric toxin-
producing pesticidal 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
chimeric toxin-producing gene. These cells may then be
harvested in accordance with conventional ways and
modified in the various manners described above.
Alternatively, the cells can be fixed prior to
harvesting.
Host cells transformed to contain chimeric toxin-
producing DNA sequences can be treated to prolong
pesticidal activity when the cells are applied to the
environment of a target pest. This treatment can involve
the killing of the host cells under protease deactivating
or cell wall strengthening conditions, while retaining
pesticidal activity.
The cells may be inhibited from proliferation in a
variety of ways, so long as the technique does not
deleteriously affect the properties of the pesticide, nor
diminish the cellular capability in protecting the
pesticide. The techniques ;nay involve physical
treatment, chemical treatment, changing the physical
JJ:in
-12- 1 3 4 1 4 7 2
character of the cell or leaving the physical character
of the cell substantially intact, or the like.
Various techniques for inactivating the host cells
include heat, usually 50°C to 70°C; freezing; W
irradiation; lyophilization; toxins, e.g., antibiotics;
phenols; anilides, e.g., carbanilide and salicylanilide;
hydroxyurea; quaternaries; alcohols; antibacterial dyes;
EDTA and amidines; non-specific organic and inorganic
chemicals, such as halogenating agents, e.g.,
chlorinating, brominating or iodinating agents;
aldehydes, e.g., glutaraldehyde or formaldehyde; toxic
gases, such as ozone and ethylene oxide; peroxide;
psoralens; desiccating agents; or the like, which may be
used individually or in combination. The choice of agent
will depend upon the particular pesticide, the nature of
the host cell, the nature of the modification of the
cellular structure, such as fixing and preserving the
cell wall with cross-linking agents, or the like.
The cells generally will have enhanced structural
stability which will enhance resistance to environmental
degradation in the field. 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 ~~ polypeptide
pesticide. The method of inactivation or killing retains
at least a substantial portion of the bioavailability or
bioacLivity of the toxin.
JJ:in
-13- '~34~472
The method of treating the organism can fulfill
a number of functions. First, it may enhance structural
integrity. Second, it may provide for enhanced proteo-
lytic stability of the toxin, by modifying the toxin so
as to reduce its susceptibility to proteolytic degrada-
tion and/or by reducing the proteolytic activity of
proteases naturally present in the cell. The cells
are preferably modified at an intact stage and when
there has been a substantial build-up of the toxin
protein. These modifications can be achieved in a
variety of ways, such as by using chemical reagents
having a broad spectrum of chemical reactivity. The
intact cells can be combined with a liquid reagent
medium containing the chemical reagents, with or without
agitation at temperatures in the range of about -10 to
60°C. The reaction time may be determined empirically
and 4ii11 vary widely with the reagents and reaction
conditions. Cell concentrations will vary from about
10E2 to 10E10 per ml.
Of particular interest as chemical reagents are
halogenating agents, particularly halogens of atomic
no. 17-80. More particularly, iodine can be used under
mild conditions and for sufficient time to achieve the
desired results. Other suitable techniques include
treatment with aldehydes, such as formaldehyde and
glutaraldehyde; anti-infectives, such as zephiran
chloride and cetylpyridinium chloride; alcohols, such
as isopropyl and ethanol; various histologic fixatives,
such as Bouin's fixative and Helly's fixative (See:
Humason, Gretchen L., Animal Tissue Techniques, W.H.
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).
For halogenation with iodine, temperatures will
generally range from about 0 to 50°C, but the reaction
can be conveniently carried out at room temperature.
-~4- 141472
Conveniently, the iodination may be performed using
triiodide or iodine at 0.5 to 5% in an acidic aqueous
medium, particularly an aqueous carboxylic acid solution
that may vary from about 0.5-5M. Conveniently, acetic
acid may be used, although other carboxylic acids,
generally of from about 1 to 4 carbon atoms, may also be
employed. The time for the reaction will generally
range from less than a minute to about 24 hrs, usually
from about 1 to 6 hrs. Any residual iodine may
be removed by reaction with a reducing agent, such
as dithionite, sodium thiosulfate, or other reducing
agent compatible with ultimate usage in the field.
In addition, the modified cells may be subjected to
further treatment, such as washing to remove all of the
reaction medium, isolation in dry form, and formulation
with typical stickers, spreaders, and adjuvants generally
utilized in agricultural applications, as is well known
to those skilled in the art.
Of particular interest are reagents capable of
crosslinking the cell wall. A number of reagents are
known in the art for this purpose. The treatment
should result in enhanced stability of the pesticide.
That is, there should be enhanced persistence or residual
activity of the pesticide under field conditions.
Thus, under conditions where the pesticidal activity
of untreated cells diminishes, the activity of treated
cells remains for periods of from 1 to 3 times longer.
The cells can be formulated for use in the environ-
ment in a variety of ways. They can be employed as
wettable powders, granules, or dusts, by mixing with
various inert materials, such as inorganic minerals
(phyllosilicates, carbonates, sulfates, or phosphates)
-15- 1341472
or botanical materials (powdered corncobs, rice hulls,
or walnut shells). The formulations can include spreader/
sticker adjuvants, stabilizing agents, other pesticidal
additives, or surfactants. Liquid formulations can be
aqueous-based or non-aqueous and employed as foams, gels,
suspensions, emulsifiable concentrates, and the like. The
ingredients can include rheological agents, surfactants,
emulsifiers, dispersants, polymers, and the like.
The pesticidal concentration will vary depending
upon the nature of the particular formulation, e.g.,
whether it is a concentrate or to be used undiluted.
The pesticide will generally be present at a concentra-
tion of at least about 1% by weight, but can be up to
100% by weight. The dry formulations will have from
about 1 to 95% by weight of the pesticide, while the
liquid formulations will generally be from about 1 to
607 by weight of the solids in the liquid phase. The
formulations will generally have from about lE2 to lE8
cells /mg.
The formulations can be applied to the environment
of the pest(s), e.g., plants, soil or water, by spray-
ing, dusting, sprinkling, or the like. These formula-
tions can be administered at about 2 oz (liquid or dry)
to 2 or more pounds per hectare, as required.
Following are examples which illustrate oroce-
dures, including the best mode, for practicing the
invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent
mixture proportions are by volume unless otherwise
noted.
Example 1--Construction of plasmid pEWl
The k-1 gene is the hd-1 gene described by
Schnepf et al. (J. Biol. Chem. 260:6264-6272 1985).
-16- 1 3 4 1 4 ~ 2
The k-1 gene was resected from the 5' end with Ba131
up to position 504. To this position was added a
Sall linker (5'GTCGACC3'). The 3' end of the gene was
cleaved at position 4211 with the enzyme Ndel and
blunt ended with the Klenow fragment of DNA polymerase.
The cloning vector pUC8 (Messing, J. and Vieira,
J. [1982] Gene 19:269-276) which can be purchased from
Pharmacia, Piscataway, NJ, was cleaved with Sall and
EcoRI and cloned into plasmid pBR322 which had been
cut with the same enzymes. The trp promoter (Genblock,
available from Pharmacia) was blunt ended at the S'
end with Klenow and inserted into this hybrid vector
by blunt end ligation of the 5' end to the Smal site of
the vector, and by insertion of the 3' end at the Sall
site of the vector. The k-1 gene was then inserted
using the Sall site at the 5' end and by blunt end
ligation of the 3' end to the PvuII site of the vector.
A schematic drawing of this construct, called pEWl, is
shown in Fig. 1 of the drawings.
Plasmid pEWl contains the DNA sequence encoding
Bacillus thurin~iensis toxin k-1.
Example 2--Construction of plasmid pEW2
The k-73 gene is the HD-73 gene described by
Adang et al. (Gene 36:289-300 1985). The k-73 gene
was cleaved at position 176 with Nsil. The sequence
was then cleaved at position 3212 with HindIII and the
3036 base fragment consisting of residues 176-3212 was
isolated by agarose gel electrophoresis.
Plasmid pEWl, prepared as described in Example 1,
was also cleaved with HindIII (position 3345 in
Table 1) and partially digested with NsiI (position
556 in Table 1). The 3036 base fragment from k-73,
-17- X341472
disclosed above, was inserted into the Nsil to HindIII
region of pEWl replacing the comparable fragment of
the k-1 gene, and creating plasmid pEW2. A schematic
diagram of pEW2 is shown in Fig. 2 of the drawings.
Plasmid pEW2 contains the DNA sequence encoding
Bacillus thurinQiensis toxin k-73.
Example 3--Construction of plasmid pEW3
The k-1 gene was cut with Sacl at position 1873.
The gene was then submitted to partial digestion with
HindIII and the 1427 base fragment consisting of
residues 1873 to 3345 was isolated by agarose gel
electrophoresis. Plasmid pEW2 was cut with SacI
and HindIII and the large fragment representing the
entire plasmid minus the SacI to HindIII fragment of
the k-2 gene was isolated by agarose gel electrophore-
sis. The 1427 base fragment from the k-1 gene was then
ligated into the Sacl to HindIII region of pEW2, creat-
ing plasmid pEW3. A schematic diagram of pEW3 is
shown in Fig. 3 of the drawings.
Plasmid pEW3 contains the DNA sequence encoding
Bacillus thuringiensis chimeric toxin k-73/k-1 (pHy),
The nucleotide sequence encoding the chimeric
toxin is shown in Table 1. The deduced amino acid
sequence is shown in Table lA.
Example 4--Construction of Dlasmid pEW4
The k-1 gene was cut at position 556 with NsiI.
The gene was then cut with Sacl at position 1873 and
the 1317 base fragment from Nsil to Sacl was isolated
by agarose gel electrophoresis. Plasmid pEW2 was cut
with Sacl and then submitted to partial digestion with
Nsil. The large fragment representing the entire
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1~~+1472
plasmid, minus the Nsil to Sacl region of the k-73
gene, was isolated by agarose gel electrophoresis.
The 1317 base NsiI to Sacl fragment of gene k-1 was
then ligated into Nsil to SacI region of pEW2 to
S create plasmid pEW4. A schematic diagram of pEW4
is shown in Fig. 4 of the drawings.
The nucleotide sequence encoding the chimeric
toxin is shown in Table 2. The deduced amino acid
sequence is shown in Table 2A.
Plasmid pEW4 contains the DNA sequence encoding
Bacillus thuringiensis chimeric toxin k-1/k-73 (PYH).
Example 5--Insertion of Chimeric Toxin Genes Into Plants
Genes coding for chimeric insecticidal toxins,
as disclosed herein, can be inserted into plant cells
using the Ti plasmid from Agrobacter tumefaciens.
Plant cells can then be caused to regenerate into
plants (Zambryski, P., Joos, H., Gentello, C., Leemans,
J., Van Montague, M. and Schell, J. [1983] EMBO J.
2:2143-2150; Bartok, K., Binns, A., Matzke, A. and
Chilton, M-D. [1983] Cell 32:1033-1043). A particu-
larly useful vector in this regard is pEND4K (Klee,
H.J., Yanofsky, M.F. and Nester, E.W. [1985] Bio/
Technology 3:637-642). This plasmid can replicate
both in plant cells and in bacteria and has multiple
cloning sites for passenger genes. Toxin genes,
for example, can be inserted into the BamHI site of
pEND4K, propagated in E, coli, and transformed into
appropriate plant cells.
Example 6--Cloning of B. thuringiensis genes into
baculoviruses
Genes coding for Bacillus thuringiensis
chimeric toxins, as disclosed herein, can be cloned
a
-19- 1341472
into baculoviruses such as Autographa californica
nuclear polyhedrosis virus (AcNPV). Plasmids can
be constructed that contain the AcNPV genome cloned
into a commercial cloning vector such as pUC8. The
AcNPV genome is modified so that the coding region of
the polyhedrin gene is removed and a unique cloning
site for a passenger gene is placed directly behind the
polyhedrin promoter. Examples of such vectors are nGP-
B6874,described by Pennock et al. (Pennock, G.D.,
Shoemaker, C. and Miller, L.K. [1984] Mol. Cell. Biol.
4:399-406), and pAC380,described by Smith et al. (Smith,
G.E., Summers, M.D. and Fraser, M.J. [1983] Mol. Cell.
Biol. 3:2156-2165). The genes coding for k-1, k-73,
k-73/k-1, k-1/k-73, or other B.t. genes can be modified
with BamHI linkers at appropriate regions both up-
stream and downstream from the coding regions and
inserted into the passenger site of one of the AcNPV
vectors.
Example 7--Chimeric Toxin Denoted ACB-1
Enhanced toxicity against all three insects
tested was shown by a toxin denoted ACB-1. The toxin
ACB-1 (Table 3A) is encoded by plasmid pACB-1 (Table 3).
The insecticidal activity encoded by pACB-1, in com-
parison with pEW3 (Example 3), is as follows:
LC50 (O.D.575/ml)
Clone T. ni H. zea S. exigua
pEW3 4.3 23.0 12.3
pACB-1 1.2 3.9 1.2
-20- ~~4'~4~~
The above test was conducted using the conditions
described previously.
The above results show that the ACB-1 toxin has
the best composite activity as compared to the other
S toxins tested herein against all three insects.
Plasmid pACB-1 was constructed between the variable
region of MTX-36, a wild B. thuringiensis strain,
having the deposit accession number NRRL B-18101, and
the variable region of HD-73 as follows: MTX-36;
N-terminal to Sacl site. HD-73; Sacl site to C-terminal.
Total plasmid DNA was prepared from strain MTX-36
by standard procedures. The DNA was submitted to
complete digestion by restriction enzymes SpeI and Dral.
The digest was separated according to size by agarose
gel electrophoresis and a 1962 by fragment was
purified by electroelution using standard procedures.
Plasmid pEW2 was purified and digested completely
with Spel and then submitted to partial digestion with
Dral. The digest was submitted to agarose gel electro-
phoresis and a 4,138 by fragment was purified by
electroelution as above.
The two fragments (1962 by from MTX-36 and 4138 by
from pEW2 were ligated together to form construct
pACB.
Plasmid DNA was prepared from pACB, digested
completely with Sacl and Ndel and a 3760 by fragment
was isolated by electroelution following agarose gel
electrophoresis.
Plasmid oEWl was digested completely with Sacl and
NdeI and a 2340 b~ fragment was isolated by electroelution
following agarose gel electrophoresis.
The two fragments (3760 bn from pACB and 2340 from
pEWl) were ligated together to form construct pACB-1.
-21- 1 3 4 1 4 7 2
The complete nucleotide sequence of the ACB-1
gene was determined and the deduced amino acid sequence
of the toxin was compared with that determined for the
toxin encoded by pEW3 (EW3). The result was that the
deduced amino acid sequence of the ACB-1 toxin was
identical to that of EW3 with two exceptions: (1)
Aspartic acid residue 411 in EW3 was changed to
asparagine in ACB-1 and (2) glycine residue 425 in
EW3 was changed to glutamic acid in ACB-1. These two
amino acid changes account for all of the changes in
insect toxicity between these strains. The amino
acid sequence of the EW3 toxin is as reported in
Table 1. A schematic representation of these two toxins
is as follows:
20
NH2 NH2
411 - Asp X11 - Asn
425 - Gly 425 - Glu
COON COOH
EW3 ACB-1
-22- ~ ~ 4 1 4 7 2
The above disclosure is further exemplification
of the subject invention process for altering the host
range of Bacillus toxins which comprises recombining
in vitro the variable region of two or more toxin genes.
Once a chimeric toxin is produced, the gene encoding
the same can be sequenced by standard procedures,
as disclosed above. The sequencing data can be used
to alter other DNA by known molecular biology procedures
to obtain the desired novel toxin. For example, the
above-noted changes in the ACB-1 gene from HD-73, makes
it possible to construct the ACB-1 gene as follows:
Plasmid pEW3, NRRL B-18034, was modified by
altering the coding sequence for the toxin. The 151 by
DNA fragment bounded by the Accl restriction site at
nucleotide residue 1199 in the coding sequence, and
the Sacl restriction site at residue 1350 were removed
by digestion with the indicated restriction endonu-
cleases using standard procedures. The removed
151 by DNA fragment was replaced with the following
synthetic DNA oligomer by standard procedures:
A TAC AGA AAA AGC GGA ACG GTA GAT TCG CTG AAT GAA
ATA CCG CCA CAG AAT AAC AAC GTG CCC CCG AGG CAA
GAA TTT AGT CAT CGA TTA AGC CAT GTT TCA ATG TTT
AGA TCT GGC TTT AGT AAT AGT AGT GTA AGT ATA ATA
AGA GCT
The net result of this change is that the aspartic
residue at position 411 in the toxin encoded by pEW3
(Table lA) is converted to asparagine, and the glycine
residue at position 425 is converted to a glutamic
residue. All other amino acids encoded by these genes
are identical.
-23-
1341472
The changes made at positions 411 and 425, dis-
cussed above, clearly illustrate the sensitivity of
these two positions in toxin EW3. Accordingly, the
scope of the invention is not limited to the particular
amino acids depicted as participating in the changes.
The scope of the invention includes substitution of all
19 other amino acids at these positions. This can
be shown by the following schematic:
NH2 NH2
411 - Asp ~ 411 - X
425 - Gly ~ 425 - Y
COON COOH
EW3
wherein X is one of the 20 common amino acids except
Asp when the amino acid at position 425 is Gly; Y is
one of the 20 common amino acids except Gly when the
amino acid at position 411 is Asp. The 20 common amino
acids are as follows: alanine, arginine, asparagine,
aspartate, cysteine, glutamine, glutamate, glycine,
histidine, isoleucine, leucine, lysine, methionine,
phertylalanine, proline, serine, threonine, tryotophan,
tyrosine, and valine.
-24- 1 3 4 1 ~+ 7 2
Example 8--Chimeric Toxin Denoted SYW1
Enhanced toxicity against tested insects was
shown by a toxin denoted SYW1. The toxin SYW1 (Table
4A) is encoded by plasmid pSYWl (Table 4). The
insecticidal activity encoded by pSYWl, in comparison
with pEWl (Example 1) and pEW2 (Example 2), is as
follows:
LC50 (O.D.575~m1)
Clone T. ni H. zea S, exigua
pEWl 3.5 12.3 18.8
pEW2 1.4 52.3 5.9
pSYWl 0.7 1.9 12.0
The above test was conducted using the conditions
described previously.
Plasmid pSYW1 was constructed as follows:
Plasmid DNA from pEW2 was prepared by standard
procedures and submitted to complete digestion with
restriction enzyme AsuII followed by partial digestion
with EcoRI. A 5878 by fragment was purified by
electroelution following agarose gel electrophoresis
of the digest by standard procedures.
Plasmid DNA from strain HD-1 was prepared and
submitted to complete digestion with restriction
enzymes AsuII and EcoRI. A 222 by fragment was
purified by electroelution following agarose gel
electrophoresis of the digest.
The two fragments (5878 by from pEW2 and 222 by
from HD-1) were ligated together, by standard proce-
dures, to form construct pSYWI.
The amino acid changes (3) in toxin SYWl from EW3
are as follows: (1) Arginine residue 289 in EW3 was
_25_ ~ ~ 4 ~ ~t
changed to glycine in SYWl, (2) arginine residue 311
in EW3 was changed to lysine in SYW1, and (3) the
tyrosine residue 313 was changed to glycine in SYW1.
A schematic representation of these two toxins is
as follows:
NH2 NH2
289 - Arg 289 - Gly
311 - Arg 311 - Lys
313 - Tyr 313 - G1u
COON COON
EW3 SYW1
The changes made at positions 289, 311, and 313,
discussed above, clearly illustrate the sensitivity
of these three positions in toxin EW3. Accordingly,
the scope of the invention is not limited to the parti-
cular amino acids depicted as participating in the
changes. The scope of the invention includes substitution
of all the common amino acids at these positions. This
can be shown by the following schematic:
X341472
-26-
NH2 NH2
2 89 - Arg '~ 289
-
X
311 - Arg ~ 11
-
Y
313 - Tyr '~ 313
- Z
COOH COON
EW3
wherein X is one of the 20 common amino acids except
Arg when the amino acid at position 311 is Arg and the
amino acid at position 313 is Tyr; Y is one of the 20
common amino acids except Arg when the amino acid at
position 289 is Arg and the amino acid at position 313
is Tyr; and Z is one of the 20 common amino acids
except Tyr when the amino acid at position 289 is
Arg and the amino acid at position 311 is Arg.
Construction of the SYW1 gene can be carried out
by procedures disclosed above for the construction of
the ACB-1 gene from plasmid pEW3 with appropriate
changes in the synthetic DNA oligomer.
35
~~41472
As is well known in the art, the amino acid
sequence of a protein is determined by the nucleotide
sequence of the D;IA. Because of the redundancy of
the genetic code, i.e., more than one coding nucleotide
triplet (codon) can be used for most of the amino acids
used to make proteins, different nucleotide sequences
can code for a particular amino acid. Thus, the
genetic code can be depicted as follows:
Phenylalanine (Phe) TTK Histidine (His) CAK
Leucine (Leu) XTY Glutamine (Gln) CAJ
Isoleucine (Ile) ATM Asparagine (Asn) AAK
Methionine (I~fet ) ATG Lys ine (Lys ) AAJ
Valine (Val) GTL Aspartic acid (Asp) GAK
Serine (Ser) QRS Glutamic acid (Glu) GAJ
Proline (Pro) CCL Cysteine (Cys) TGK
Threonine (Thr) ACL Tryptophan (Trp) TGG
Alanine (Ala) GCL Arginine (Arg) WGZ
Tyrosine (Tyr) TAB Glycine (Gly) GGL
Termination signal TAJ
Key: Each 3-letter deoxynucleotide trivlet corresponds
to a trinucleotide of mRNA, having a 5'-end on the
left and a 3'-end on the right. All DNA sequences
given herein are those of the strand whose sequence
corresponds to the mRNA sequence, with thymine substi-
tuted for uracil. The letters stand for the purine or.
pyrimidine bases forming the deoxynucleotide sequence.
A = adenine
G - guanine
C = cytosine
T = thymine
X = T or C if Y is A or G
X = C if Y is C or T
Y = A, G, C or T if X is C
Y = A or G if X is T
-28- 141472
W = C or A if Z is A or G
W = C if Z is C or T
Z = A, G, C or T if W is C
Z - A or G if W is A
QR = TC if S is A, G, C or T; alternatively QR =
AG if S is T or C
J = A or G
K = T or C
L = A, T, C ar G
M = A, C or T
The above shows that the novel amino acid sequence
of the chimeric toxins, and other useful proteins,
can be prepared by equivalent nucleotide sequences
encoding the same amino acid sequence of the proteins.
Accordingly, the subject invention includes such
equivalent nucleotide sequences. 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. and Kezdy, F.J.
11984] Science 223:249-255). Thus, the subject inven-
tion includes muteins of the amino acid sequences
depicted herein which do not alter the protein
secondary structure.
The one-letter symbol for the amino acids used
in Tables lA and 2A is well known in the art. For
convenience, the relationship of the three-letter
abbreviation and the one-letter symbol for amino acids
is as follows:
Ala A
Arg R
Asn N
Asp D
-29- 1 3 4 1 4 7 2
Cys C
Gln Q
Glu E
Gly G
His H
Ile I
Leu L
Lys K
Met M
Phe F
Pro P
Ser S
Thr T
Trp W
Tyr Y
Val V
The work described herein was all done in
conformity with physical and biological containment
requirements specified in the NIH Guidelines.
30
- t341472
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-31- 1341472
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-33-
~34fi~~2
Table 1
Nucleotide Sequence of Plasmid pEW3 Encoding
Chimeric Toxin
Numbering of the nucleotide bases is the same as
Schnepf et al. (J. Biol. Chem. 260:6264-6272 [1985])
for HD-1 and Adang et al. (Gene 36:289-300 [1985])
for HD-73. Only protein coding sequences are shown.
( st art HO-73 ) ATG GATAACAATC 4CiC~
CGAACATCAA TGAATGCATT CCTTATAATT GTTTAAGTAA CCCTGAAGTA
GAAGTATTAG GTGGAGAAAG AATAGAAACT GGTTACACCC CAATCGATAT 50C~
TTCCTTGTCG CTAACGCAAT TTCTTTTGAG TGAATTTGTT CCCGGTGCTG
GATTTGTGTT AGGACTAGTT GATATAATAT GGGGAATTTT TGGTCCCTCT 6C~C~
CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA ACCAAAGAAT
AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTAGAA GGACTAAGCA 7C~C~
ATCTTTATCA AATTTACGCA GAATCTTTTA GAGAGTGGGA AGCAGATCCT
ACTAATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 8G0
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG
TTCCTCTTTT ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 9C~C~
TTGAGAGATG TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC
TATCAATAGT CGTTATAATG ATTTAACTAG GCTTATTGGC AACTATACAG iCrUCr
ATTATGCTGT ACGCTGGTAC AATACGGGAT TAGAACGTGT ATGGGGACCG
GATTCTAGAG ATTGGGTAAG GTATAATCAA TTTAGAAGAG AATTAACACT IloC~
AACTGTATTA GATATCGTTG CTCTGTTCCC GAATTATGAT AGTAGAAGAT
ATCCAATTCG AACAGTTTCC CAATTAACAA GAGAAATTTA TACAAACCCA 1200
GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC AGGGCATAGA
AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTAAC AGTATAACCA l3CrCr
TCTATACGGA TGCTCATAGG GGTTATTATT ATTGGTCAGG GCATCAAATA
ATGGCTTCTC CTGTAGGGTT TTCGGGGCCA GAATTCACTT TTCCGCTATA 14C~~
TGGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GGTGAACTAG
GTCAGGGCGT GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500
AATATAGGGA TAAATAATCA ACAACTATCT GTTCTTGACG GGACAGAATT
TGCTTATGGA ACCTCCTCAA ATTTGCCATC CGCTGTATAC AGAAAAAGCG l6Crp
GARCGGTAGA TTCGCTGGAT GAAATACCGC CACAGAATAA CAACGTGCCA
CCTAGGCAAG GATTTAGTCA TCGATTAAGC CATGTTTCAA TGTTTCGTTC 1700
AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT (end hd-73)
(start HD-1) CCAACGT TTTCTTGGCA GCATCGCAGT l9Un
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT
AACAAAATCT ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG 2UUCr
GATTTACAGG AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGRTTTCA
ACCTTAAGAG TAAATATTAC TGCACCATTA TCACAAAGAT ATCGGGTAAG 2lCrCr
AATTCGCTAC GCTTCTACTA CAAATTTACA ATTCCATACA TCAATTGACG
GAAGACCTAT TAATCAGGGT AATTTTTCAG CAACTATGAG TAGTGGGAGT 220Cr
AATTTACAGT CCGGAAGCTT TAGGACTGTA GGTTTTACTA CTCCGTTTAA
CTTTTCAAAT GGATCAAGTG TATTTACGTT AAGTGCTCAT GTCTTCAATT 23«C~
CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC GGCAGAAGTA
ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATGA 2400
~CTGTTTACT TCTTCCAATC AAATC~GGTT AAAAACAGAT GTGACGGATT
-34- 1 3 4 1 4 7 2
Table 1 (cont.)
ATCATATTGA TCAAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT 25C>U
TGTCTGGATG AAAAACAAGA ATTGTCCGAG AAAGTCAAAC ATGCGAAGCG
ACTTAGTGAT GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA ~b~rir
ATAGACAACT AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA
GGAGGCGATG ACGTATTCAA AGAGAATTAC GTTACGCTAT TGGGTACCTT ~7C>0
TGATGAGTGC TATCCAACGT ATTTATATCA AAAAATAGAT GAGTCGAAAT
TAAAAGCCTA TACCCGTTAT CAATTAAGAG GGTATATCGA AGATAGTCAA 28r=r«
GACTTAGAAA TCTATTTAAT TCGCTACAAT GCAAAACATG AAACAGTAAA
TGTGCCAGGT ACGGGTTCCT TATGGCCGCT TTCAGCCCAA AGTCCAATCG 2900
GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA ATGGAATCCT
GACTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA 3Cr00
TCATTTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAGATGG GCACGCAAGA 3100
CTAGGGAATC TAGAGTTTCT CGAAGAGAAA CCATTAGTAG GAGRAGCGCT
AGCTCGTGTG AAAAGAGCGG AGAAAAAATG GAGAGACAAA CGTGAAAAAT 3200
TGGAATGGGA AACAAATATC GTTTATAAAG AGGCAAAAGA ATCTGTAGAT
GCTTTATTTG TAAACTCTCA ATATGATCAA TTACAAGCGG ATACGAATAT 3300
TGCCATGATT CATGCGGCAG ATAAACGTGT TCATAGCATT CGAGAAGCTT
ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG TCAATGCGGC TATTTTTGAA 34C>Cr
GAATTAGAAG GGCGTATTTT CACTGCATTC TCCCTATATG ATGCGAGAAA
TGTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC TGGAACGTGA 350?
AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGTTC GGTCCTTGTT
CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG 3600
TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
GTTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 3700
AGCAACTGCG TAGAAGAGGA AATCTATCCA AATRACACGG TAACGTGTAA
TGATTATACT GTAAATCAAG AAGAATACGG AGGTGCGTAC ACTTCTCGTA 3800
ATCGAGGATA TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC
TATGAAGAAA AATCGTATAC AGATGGACGA AGAGAGAATC CTTGTGAATT 3900
TAACAGAGGG TATAGGGATT ACACGCCACT ACCAGTTGGT TATGTGACAA
ARGAATTAGA ATACTTCCCA GAAACCGATA AGGTATGGAT TGAGATTGGA 4~~C~0
GAAACGGAAG GAACATTTAT CGTGGACAGC GTGGAATTAC TCCTTATGGA
GGAA (end HD-1>
-35-
~34~47~
Table lA
Deduced Amino Acid Sequence of Chimeric Toxin Produced
by Plasmid pEW3
M D t~! N F N I N E C I F Y N C L S N F E V E V L G r, E R I E
T r, Y T P I L~ I S L S L T C, F L L S E F V F G A G F V L G L
V Li I I W r, I F G F' S ~! W Li A F L V i i I E 6t L I N n F I E E
F A R ~J t:! A I S R L E G L S t~J L 'r G! I 'f A E S F F: E 4J E A L~
F' T t~l F' A L Fs E E M R I G! F tJ D M N S A L T T A I F' L F A V
i! N Y n' V F' L L S V Y V i=! A A N L H L. S V L F D V S V F G i!
F; bJ r, F Lt A A T I N S F~ Y N L~ L T R L I r, tJ Y T L~ Y A V R l~J
Y N T G L E F; V W G F Ci S R L~ W V R Y N ~:! F F F E L T L T V
L D I V A L F F' N Y C~ S R R Y F' I R T' V S G: L T R E I Y T N
F' V L E N F D G S F R G S A ~! G I E R S I R S F' H L M L~ I L
N S I T I Y T Li A H F; G Y Y 'i bJ S G H G! T M A S F V G F S G
F'EFTt= F'LYGTM13NAAFilaR I VAG!LGn!GVYF
T L S S T L Y R R F F N I G I N N i' ~t L S V L Li G T E F A Y
G T S S N L F S A V Y F. K S r, T V L~ S L D E i F F n! N N N V
F F F f! r, F S H R L S ti V 5 M F fi S G F S N 5 S V S I I R A
F' T F S lJ l' H R S A E F N N I I F' S S G! I T C! I F L T K S T
N L r, S r, T S V V K G F G F T r, G D I L F R T S F G r! I S T
L R V N I T A F L S r. F Y F V R I F Y A S T T N L n! F H T S
I L~ r, fi F' I N ~; G N F S A T M S S G S N L ~' S G S F R T V r,
F T T F' F N F S N G S S V F T L S A H V F PJ S G N E V Y I D
R I E F V F' A E V T F E A E Y Lt L E R A i! K A V N E L F T S
S N n:; I G L K T Li V T G Y H I to n' V S N L V E C L S Li E F C
L L~ E K n~ E L 5 E K V K H A K F L S C~ E R N L L G! Li F' N F R
G I ~J R ~? L D R G W R G S T D I T I n! r, r, L~ L~ V F K E N Y V
T L L G T F D E C Y F' T Y L Y G' K I Lt E S K L K A Y T F; Y i-!
L R G '~( I E D S [! L~ L E I Y L I F; Y N A K H E T V N V F' G T
S L W F' L S A i'! S F I G K C G E F ~d R C A F H L E 4J N F I;i
L D C S ~ F; I) r, E K C A H H 5 H H F S L C~ I L~ V ~ C T L~ L N
E Li L G ~' W V I F K I K T G! Li G H A R I_ 6 N L E F L E E K F'
L V r E A L A R V K fi A E K K W R D K R E K L E W E T N I V
Y K E A K E S V Li A L F V N S t! Y D l! L n! A D T ~d i A M I H
A A D K R V H S I R E A Y L F' E L S V I F' G V N A A I F E E
L E ~ R I F T A F S L Y D A Ft ~J V I K N r, D F N N r, L S C W
N V K G H V L~ V E E n! ~d N i! F, S V L V L F' E W E A E V S u! E
V R V C F' r, F G Y I L R V T A Y K E G Y G E G C V T I H E I
E N N T Lt E L K F S N C V E E E I Y F' N N T V T C N Li Y T V
N a E E Y r, r, A Y T S F N R G Y N E A P 5 V F' A P Y A S V Y
E E K S Y T L~ G R R E N F C E F N R G Y R L~ Y T F L F' V ~ Y
V T K E L E Y F F E T D K V W I E I G E T E ~ T F I V Lt S V
E L L L M E E
-36-
~~4~~72
Table 2
Nucleotide Sequence of Plasmid pEW4 Encoding
Chimeric Toxin
Numbering of nucleotide bases is the same as Schnepf
et al. (J. Biol. Chem. 260:6264-6272 [1985)) for
HD-1 and Adang et al. (Gene 36:289-300 [1985]) for
HD-73. Only protein coding sequences are shown.
tstart HD-1) ATGG ATAACAATCC GAACATCAAT
GAATGCATTC CTTATAATTG TTTAAGTAAC CCTGAAGTAG AAGTATTAGG 6C~0
TGGAGAAAGA ATAGAAACTG GTTACACCCC AATCGATATT TCCTTGTCGC
TAACGCAATT TCTTTTGAGT GAATTTGTTC CCGGTGCTGG ATTTGTGTTA 700
GGACTAGTTG ATATAATATG GGGAATTTTT GGTCCCTCTC AATGGGACGC
ATTTCCTGTA CAAATTGAAC AGTTAATTAA CCAAAGAATA GAAGAATTCG B00
CTAGGAACCA AGCGATTTCT AGATTAGAAG GACTAAGCAA TCTTTATCAA
ATTTACGCAG AATCTTTTAG AGAGTGGGAA GCAGATCCTA CTAATCCAGC 900
ATTAAGAGAA GAGATGCGTA TTCAATTCAA TGACATGAAC AGTGCCCTTA
CAACCGCTAT TCCTCTTTTG GCAGTTCAAA ATTATCAAGT TCCTCTTTTA 100Cr
TCAGTATATG TTCAAGCTGC AAATTTACAT TTATCAGTTT TGAGAGATGT
TTCAGTGTTT GGACAAAGGT GGGGATTTGA TGCCGCGACT ATCAATA6TC 1100
GTTATAATGA TTTAACTAGG CTTATTGGCA ACTATACAGA TTATGCTGTG
CGCTGGTACA ATACGGGATT AGAGCGTGTA TGGGGACCGG ATTCTAGAGA 1200
TTGGGTAAGG TATAATCAAT TTAGAAGAGA GCTAACACTT ACTGTATTAG
ATATCGTTGC TCTATTCTCA AATTATGATA GTCGAAGGTA TCCAATTCGA 1300
ACAGTTTCCC AATTAACAAG AGAAATTTAT ACGAACCCAG TATTAGAAAA
TTTTGAT~GT AGTTTTCGTG GRATGGCTCA GAGAATAGAA CA~AATATTA 1400
GGCAACCACA TCTTATGGAT ATCCTTAATA GTATAACCAT TTATACTGAT
GTGCATAGAG GCTTTAATTA TTGGTCAGGG CATCAAATAA CAGCTTCTCC l5ui~
TGTAGGGTTT TCAGGACCAG AATTCGCATT CCCTTTATTT GGGAATGCGG
GGAATGCArC TCCACCCGTA CTTGTCTCAT TAACTGGTTT GGGGATTTTT ibC>C~
AGAACATTAT CTTCACCTTT ATATAGAAGA ATTATACTTG GTTCAGGCCC
AAATAATCAG GAACTGTTTG TCCTTGATGG AACGGAGTTT TCTTTTGCCT 17C»~
CCCTAACGAC CAACTTGCCT TCCACTATAT ATAGACAAA~ GGGTACAGTC
GATTCACTA6 ATGTAATACC GCCACAGGAT AATAGTGTAC CAGCTCGTGC 180Cr
GGGATTTAGC CATCGATTGA GTCATGTTAC AATGCTGAGC CAAGCAGCTG
GAGCAGTTTA CACCTTGAGA GCTCAACGT (stop HD-11
(start HLv-73) CCT ATGTTCTCTT
GGATACATCG TAGTGCTGAA TTTAATAATA TAATTGCATC GGATAGTATT 1800
ACTCAAATCC CTGCAGTGAA GGGAAACTTT CTTTTTAATG GTTCTGTAAT
TTCAGGACCA GGATTTACTG GTGGGGACTT AGTTAGATTA AATAGTAGTG l9UCa
GAAATAACAT TCAGAATAGA GGGTATATTG AAGTTCCAAT TCACTTCCCA
TCGACATCTA CCAGATATCG AGTTCGTGTA CGGTATGCTT. CTGTAACCCC 2000
GATTCACCTC AACGTTAATT GGGGTAATTC ATCCATTTTT TCCAATACAG
TACCAGCTAC AGCTACGTCA TTAGATAATC TACAATCAAG TGATTTTGGT ~~10~?
TATTTTGAAA GTGCCAATGC TTTTACATCT TCATTAGGTA ATATAGTAGG
TGTTAGAAAT TTTAGTGGGA CTGCAGGAGT GATAATAGAC AGATTTGAAT 2200
TTATTCCAGT TACTGCAACA CTCGAGGCTG AATATAATCT GGRAAGAGCG
-37-
d
Table 2 (cont.)
CAGAAGGCGG TGAATGCGCT GTTTACGTCT ACAAACCAAC TAGGGCTAAA 23U«
AACAAATGTA ACGGATTATC ATATTGATCA AGTGTCCAAT TTAGTTACGT
ATTTATCGGA TGAATTTTGT CTGr,ATGAAA AGCGAGAATT GTCCGAGAAA 'r4C>0
GTCAAACATG CGAAGCGACT CAGTGATGAA CGCAATTTAC TCCAAGATTC
AAATTTCAAA GACATTAATA GGCAACCAGA ACGTGGGTGG GGCGGAAGTA 2500
CAGGGATTAC CATCCAAGGA GGGGATGACG TATTTAAAGA AAATTACGTC
ACACTATCAG GTACCTTTGA TGAGTGCTAT CCAACATATT TGTATCAAAA ~6C~C>
AATCGATGAA TCAAAATTAA AAGCCTTTAC CCGTTATCAA TTAAGAGGGT
ATATCGAAGA TAGTCAAGAC TTAGAAATCT ATTTAATTCG CTACAATGCA 2700
AAACATGAAA CAGTAAATGT GCCAGGTACG GGTTCCTTAT GGCCGCTTTC
AGCCCAAAGT CCAATCGGAA AGTGTGGAGA GCCGAATCGA TGCGCGCCAC 2800
ACCTTGAATG GAATCCTGAC TTAGATTGTT CGTGTAGGGA TGGAGAAAAG
TGTGCCCATC ATTCGCATCA TTTCTCCTTA GACATTGATG TAGGATGTAC 2900
AGACTTAAAT GAGGACCTAG GTGTATGGGT GATCTTTAAG ATTAAGACGC
AAGATGGGCA CGCAAGACTA GGGAATCTAG AGTTTCTCGA AGAGAAACCA 3000
TTAGTAGGAG AAGCGCTAGC TCGTGTGAAA AGAGCGGAGA AAAAATGGAG
AGACAAACGT GAAAAATTGG AATGGGAAAC AAATATCGTT TATAAAGAGG 3100
CAAAAGAATC TGTAGATGCT TTATTTGTAA ACTCTCAATA TGATCAATTA
CAAGCGGATA CGAATATTGC CATGATTCAT GCGGCAGATA AACGTGTTCA 3200 _
TAGCATTCGA GAAGCTTATC TGCCTGAGCT GTCTGTGATT CCGGGTGTCA
ATGCGGCTAT TTTTGAAGAA TTAGAAGGGC GTATTTTCAC TGCATTCTCC 3300
CTATATGATG CGAGAAATGT CATTAAAAAT GGTGATTTTA ATAATGGCTT
ATCCTGCTGG AACGTGAAAG GGCATGTAGA TGTAGAAGAA CAAAACAACC 3400
AACGTTCGGT CCTTGTTGTT CCGr,AATGrr, AAr,~AGAArT GTCACAAGAA
GTTCGTGTCT GTCCGGGTCG TGGCTATATC CTTCGTGTCA CAGCGTACAA 3500
GGAGGGATAT GGAGAAGGTT GCGTAACCAT TCATGAGATC GAGAACAATA
CAGACGAACT GAAGTTTAGC AACTGCr,TAG AAGAGGAAAT CTATCCAAAT 360?
AACACGGTAA CGTGTAATGA TTATACTGTA AATCAAGAAG AATACGGAGG
TGCGTACACT TCTCGTAATC GAGGATATAA C~AAGCTCCT TCCGTACGAG 370C>
CTGATTATGC GTCAGTCTAT GAAGAAAAAT CGTATACAGA TGGACGAAGA
GAGAATCCTT GTGAATTTAA CAGAGGGTAT AGGGATTACA CGCCACTACC 38i~i~
AGTTGGTTAT GTGACAAAAG AATTAGAATA CTTCCCAGAA ACCGATAAGG
TATGGATTGA GATTGGAGAA ACGGAAGGAA CATTTATCGT GGACAGCGTG 3900
GAAT'('ACTCC TTATGGAGGA A (end HD-73)
i
-3s- 1 3 4 1 4 7 2
Table 2A
Deduced Amino Acid Sequence of Chimeric Toxin Produced
by Plasmid pEW4
M G tJ N F N T N E C i F Y N C L S N F E V E V L r, r, E R I E
TT3YT~F I G I SLSLTr_.r.FLLSEFVF'G~AGFVLGL
V D I I ld G I F G F S n W D A F F' V n I E G! L I N r! R I E E
F A R N r! A I S R L E ~ L 5 tJ L Y >>! I Y A E S F R E 4J E A D
F' T N F' A L R E E M R I r. F N D M N 5 A L T T A I F L L A V
r~ N Y G! V F' L L S V Y V G! A A N L H L S V L R G V S V F G G!
R W r, F G A A T I N S R Y N D L T R L. I G N Y T Lt Y A V R W
Y N T G L E R V W r, F' G S R D W V iY N L; F R R E L T L T V
L D I V A L F S N Y D S R R Y F I F T V S ('~ L T R E I Y T N
F V L E N F D G S F F i3 M A i! R I E ~~! N I R ~? F' H L M D I L
N S I T I Y T D V H F ~3 F N Y W S G H r. I T A S F V G F S G
F E F A F F' L F G N A r; to A A F' F' v L V S L T G L r, I F F T
L S S F L Y F: F; I I L G S G F' N N n! E L F V L D r, T E F S F
A S L T T PJ L F' S T I Y F; i>! F; G T V D S L D V I ~ F' G! D N S
V F F R A G F S H R L '= H 'J T P1 l_ S n! A A G A V Y T L R A n!
RF'~iFSW I HRSAEF NfJ I I ASDS I Ty! I F'AVK G
l'J F L F N ~ S V I S G P ~ F T G G D L. V R L N S S G N PJ I n!
N R r, Y I E V F' I H F P S T S T F; Y f; V R V R Y A S V T F' I
H L tJ V N W G PJ S S I F S N T V F' A T A T S L Lt PJ L G! S S D
F G Y F E S A N A F T S S L G N I V r-, V F; N F S G T A G V I
I G R F E F I F' V T A T L E A E Y N L E R A l.! K A V N A L F
T S T N ~7 L G L K T N V T G Y H I D n V S N L v T Y L S D E
F C L D E K R E L S E K V K H A K R L S D E F N L L G D S N
F K G I N R G! F E R G W r, G S T G I T I ~: G G G G V F K E N
Y V T L S G T F Lt E C Y F' T Y L Y i=! K I D E S K L K A F T F~
Y a L R G Y I E D S G G L E I Y L i R Y N A K H E T V N V F
G T G S L W F L S A G S F' I G K C G E F N R C A F H L E W,N
P L~ L D C S C R D r, E K C A H H S H H F S L D I D V G C T G
L N E D L G V W V I F K I K T G! G r, H A R L G N L E F L E E
K F L V r, E A L A R V K R A E K K W R G K R E K L E W E T N
I V Y K E A K E S V D A L F V N S i! Y G ~:? L l~ A D T N I A M
I H A A D K R V H S I R E A Y L F' E L 5 V I F' G V N A A I F
E E L E G R I F T A F S L Y D A R N V I K N G G F N N G L S
C ld N V K r, H V G V E E G! N N G! R S V L V V F E W E A E V S
t:! E V R V C F ~ R G Y I L R ',i T A Y K E 6 Y G E ~ C V T I H
E I E N N T G E L K F S N C V E E E I Y F' N N T V T C N D Y
T V N n! E E Y r, r, A Y T S R t'J R ~ Y N E A F' S V F A G Y A S
V Y E E K S Y T D G R F.' E N F C E F N R G Y R G Y T F L F V
G Y V T K E L E Y F F F_ T L~ K V W I E I G E T E G T F I V G
S V E L L_ L M E E
-39-
1341472
Table 3
Nucleotide Sequence of Plasmid pACB-1 Encoding
Chimeric Toxin ACB-1
The nucleotide differences as compared to the sequence
shown in Table 1 are underlined at Dositions 1618
and 1661 and code for amino acid changes at positions
411 and 425 as shown in Table 3A.
' (start HD-73) ATG GATAACAATC ~i00
CGAACATCAA TGAATGCATT CCTTATAATT GTTTAAGTAA CCCTGAAGTA
GAAGTATTAG GTGGAGAAAG AATAGAAACT GGTTACACCC CAATCGATAT SC~C~
TTCCTTGTCG CTAACGCAAT TTCTTTTGAG TGAATTTGTT CCCGGTGCTG
GATTTGTGTT AGGACTAGTT GRTATAATAT GGGGARTTTT TGGTCCCTCT 6««
CAATGGGACG CATTTCTTGT ACAAATTGAA CAGTTAATTA ACCAAAGART
AGAAGAATTC GCTAGGAACC AAGCCATTTC TAGATTRGAA GGACTAAGCA 7C~C~
ATCTTTATCA AATTTRCGCA GRATCTTTTA GRGAGTGGGA RGCAGRTCCT
ACTRATCCAG CATTAAGAGA AGAGATGCGT ATTCAATTCA ATGACATGAA 800
CAGTGCCCTT ACAACCGCTA TTCCTCTTTT TGCAGTTCAA AATTATCAAG
TTCCTCTTTT ATCAGTATAT GTTCAAGCTG CAAATTTACA TTTATCAGTT 900
TTGAGAGRTG TTTCAGTGTT TGGACAAAGG TGGGGATTTG ATGCCGCGAC
TATCAATAGT CGTTATAATG ATTTAACTAG GCTTATTGGC AACTATACAG 1000
ATTATGCTGT ACGCTGGTAC AATACGGGAT TAGAACGTGT ATGGGGACCG
GATTCTRGAG ATTGGGTAAG GTATRATCAA TTTAGAAGAG AATTAACACT 1100
AACTGTATTA GATATCGTTG CTCTGTTCCC GRATTATGAT AGTAGAAGAT
ATCCAATTCG~AACAGTTTCC CAATTAACAA GAGAAATTTA TACAAACCCA 1200
GTATTAGAAA ATTTTGATGG TAGTTTTCGA GGCTCGGCTC AGGGCATAGA
AAGAAGTATT AGGAGTCCAC ATTTGATGGA TATACTTARC AGTATAACCA 130«
TCTATACGGA TGCTCATAGG GGTTATTATT:ATTGGTCAGG GCATCAAATA
ATGGCTTCTC.CTGTAGGGTT TTCGrrr,CCA GAATTCACTT TTCCGCTATA 1400
TCiGAACTATG GGAAATGCAG CTCCACAACA ACGTATTGTT GCTCAACTAG ,
GTCAGGGCGT GTATAGAACA TTATCGTCCA CTTTATATAG AAGACCTTTT 1500
AATATAGGGA TAAATRATCA ACAACTATCT GTTCTTGACG GGACAGAATT
TGCTTATGGA ACCTCCTCAA ATTTGCCATC CGCTGTATAC AGAAAAAGCG 1600
GAACGGTAGA TTCGCTG_AAT GAAATACCGC CACAGAATAA CAACGTGCCA
CCTAGGCAAG _AATTTAGTCA TCGATTARGC CATGTTTCAA TGTTTCGTTC 1700
AGGCTTTAGT AATAGTAGTG TAAGTATAAT AAGAGCT (end hd-73)
(start Hti-1) CCARCGT TTTCTTGGCA GCATCGCRGT 1900
GCTGAATTTA ATAATATAAT TCCTTCATCA CAAATTACAC AAATACCTTT
AACRAAATCT ACTAATCTTG GCTCTGGAAC TTCTGTCGTT AAAGGACCAG ~~OC>0
GATTTACAGG AGGAGATATT CTTCGAAGAA CTTCACCTGG CCAGATTTCA
ACCTTAAGAG TAAATATTAC TGCACCATTA TCACAAAGAT ATCGGGTAAG ~1c'>~~
AATTCGCTAC GCTTCTACTA CAAATTTACA ATTCCATACA TCAATTGACG
GAAGACCTAT TAATCAGGGT AATTTTTCAG CAACTATGAG TAGTGGGAGT ~~00
AATTTACAGT~CCGGAAGCTT TAGGACTGTA GGTTTTACTA CTCCGTTTAA
CTTTTCAAAT GGATCARGTG TATTTACGTT AAGTGCTCAT GTCTTCAATT 2300
CAGGCAATGA AGTTTATATA GATCGAATTG AATTTGTTCC GGCAGAAGTA
ACCTTTGAGG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGRATGA 2900
GCTGTTTACT TCTTCCAATC AAATCGGGTT AAAAACAGAT GTGACGGATT
ATCATATTGA TCRAGTATCC AATTTAGTTG AGTGTTTATC AGATGAATTT L5V'J
TGTCTGGaTG AAAAACAAGA ATTGTCCGAG AAAGTCRAAC ATGCGAAGCG
-40-
134~~~~
Table 3 (cont.)
ACTTAGTGAT GAGCGGAATT TACTTCAAGA TCCAAACTTC AGAGGGATCA ~bGC~
ATAGACAACT AGACCGTGGC TGGAGAGGAA GTACGGATAT TACCATCCAA
GGAGGCGATG ACGTATTCAA AGAGAATTAC GTTACGCTAT TGGGTACCTT i7U~
TGATGAGTGC TATCCAACGT ATTTATATCA AAAAATAGAT GAGTCGAAAT
TAAAAGCCTA TACCCGTTAT CAATTAAGAG GGTATATCGA AGATAGTCAA ~8C~o
GACTTAGAAA TCTATTTAAT TCGCTACAAT GCAAAACATG AAACAGTAAA
TGTr,CCAr,~;T ACGGGTTCCT TATGGCCGCT TTCAGCCCfIA AGTCCAATCG ~9CW
GAAAGTGTGG AGAGCCGAAT CGATGCGCGC CACACCTTGA ATGGAATCCT
~3r=CTTAGATT GTTCGTGTAG GGATGGAGAA AAGTGTGCCC ATCATTCGCA 3C~C~i>
~TCAT~fTCTCC TTAGACATTG ATGTAGGATG TACAGACTTA AATGAGGACC
TAGGTGTATG GGTGATCTTT AAGATTAAGA CGCAAr"~Trr GCACGCAAGA 3100
CTAGGr~AATC TAGAGTTTCT CGAAGAGAAA CCAT1A~TIaG GHGAAGCGCT
AGCTC~TGT~ AAAAGAGCGG AGAAAAAATG GAGAGHCAAA CG-fGAAAAAT 3~oio
TGGAATGGGA AACAAATATC GTTTATAAAG AGGCAA~aAr,A ATCTGTAGAT
GCTTTATTTG TAAACTCTCA A-fATGATCAA TTACAAr;CGr, ATACGAATAT 33~:>i>
IGCCATGATT CATGCGrCAG ATAAACGTGT TCATAGCA1-T CGAGAAGCTT
ATCTGCCTGA GCTGTCTGTG ATTCCGGGTG TCAATGCGGC TATTTTTGAA 34C»~
GAATTAGAAG GGCGTATTTT CACTGCATTC TCCCTATATG ATGCGAGAAA
1'GTCATTAAA AATGGTGATT TTAATAATGG CTTATCCTGC TG~;Ar=aCGTGA 35C~«
AAGGGCATGT AGATGTAGAA GAACAAAACA ACCAACGT'I'C GGTCCTTGTT
CTTCCGGAAT GGGAAGCAGA AGTGTCACAA GAAGTTCGTG TCTGTCCGGG 36C~C~
TCGTGGCTAT ATCCTTCGTG TCACAGCGTA CAAGGAGGGA TATGGAGAAG
~TTGCGTAAC CATTCATGAG ATCGAGAACA ATACAGACGA ACTGAAGTTT 37«c>
~1~~CAACT~Cr, TAGAAGAGGA AATCTATCCA AATAACACGG TAACGTGTAA
TGATTATACT GTAAATCAAG AAGAATACGG AGGTGC,3TAC ACTTCTCGTA 38oC~
ATCGAG;aT;'~ TAACGAAGCT CCTTCCGTAC CAGCTGATTA TGCGTCAGTC
TATGAAGAAA AATCGTATAC F'GATGGACGA AGAGAGAATC CTTGTGAATT 39C~C>
TAACAGAGGG TATAGGGATT ACACGCCACT ACCAGTTGGT TATGTGACAA
l-;AGAATTAGA ATACTTCCCA GAAACCGATA AGGTATGGAT TGAGATTGGA 4C~CeC~
~AAACGr,AAG GAACATTTAT CGTGGACAGC GTGGAATTAC TCCTTATGGA
GGAA tend HG-1>
1 3
Table 3A
Deduced Amino Acid Sequence of Chimeric Toxin
ACB-1
M D I'J N P N I N E C I P Y N C L S N F' E V E V L G G E R I E
T G Y~~~T P t D I S L S L T CI F L L S E F V P G A G F V L G L
V Lv I I W G I F G P S C~ W D A F L V et I E Ct L I N CI R I E E
F .A R N ft A I S .fi L E G L S N L Y Gt I Y A E S F R E W E A D
P T N P A L R E E M R I G? F N D M N S A L T T A I P L.F A V
C: N Y G! V P L L S V Y V G A A 1J L H L S V L Ft D V S V F G C?
fi~4Jr,F C~AAT I NSF:YNDLTfiL I GNYTDYAVfiW
Y N,T G L E fi V 4J G P D S fi D W V fi Y N Gt F R R E L T L T V
L D I V A L F F' N Y D S fi fi Y F I R T V S G L T R E I Y T N
P V L E N F D G S F R G S A C: G I E fi S I fi S F H L M D I L
N 5 I T I Y T D A H R G Y Y Y W S r, H r,! I M A S P V G F S G
P E F~ T F F L Y: G .T M G N A A F 6t a R I V A ~~ L G Ct G V Y fi
T L S S T L Y R F; F' F N I G I N N G Ct L S V L Lt G T E F A Y
G T S S N L F' S A V Y R K S G T V D S L N E I P P r~ PJ N N V
P P F: t? E F S H fi L S H V S M F R S G F s N S 5 V S I I fi A
F' T F S l~l f! H R S A E F N N I I F' S S r! I T Ct I F L T K S T
N L G S G T S V V K G P G F T r, r D I L R fi T S F G n; I S T
L R V N I T A F L S G! Fs Y F; V R I R Y A S T T N L i=! F H T S
I D G R P I N It G N F S A T M S S G S N L G! S G S F R T V G
F T T F' F N F S N G S S V F T L S A H 'J F N S G N E V Y I L~
R I E F V F' A E V T F E A E Y Li L E Ft A t; K A V N E L F T S
S N rt I G L K T D V T f.~ Y H I D ~; V S N L V E C L S D E F C
L D E K ~:~ E L S E K V K H A K R L S L~ E F~ N L L C? L~ F' N F R
G I N R ~! L L~ fi G 4J R G S T C~ I T I n! r, r, L~ L V F K E N Y V
T- L L r, T F D E C Y F' T Y L Y f! K I C~ E S K L K A Y T R Y
L fi G Y I E D S ft Li L E I Y L I R Y N A K H E T V N V F' G T
r, S L DJ F' L S A G! S F' I r, K C G E F ~J F: C A F H L E ~J N P D
L D C S C fi D G E K C A H H S H H F S L Ci I D V G C T L~ L N
E D L G V 4J V I F K I K T r. L~ r, H A R L G N L E F L E E K P
L V G E A L A R V K fi A E K K 4J F; D K R E K L E W E T N I V
Y K E A K E 5 V L~ A L F V N 5 L; Y L~ G! L _n.., A D T .i'J I A M I H
A A D K R V H S I R E A Y L F' E L 5 V I F G V N A A I F E E
'L.E G R I F T A.F S L Y D A R N V I K N G Li F N N.G L S C W
N V K r, .H V D V E E Ct N N it R S V L V L F E W E 'A E V S G! E
V fi.V C.P G R G Y I L R V T A Y K E G Y G E G C V T I H E I
E N N T L~ E L K F S N C V E E E I Y F N N T V T C N Ll Y T V
N C? E E Y G G A Y T S fi N fi G Y N E A P S V F' A D Y A S V Y
E E K S Y'vT D G R R E N P C E F N fi G Y R L~ Y T P L P ~V G Y
V T K E L E Y F P E T~D K V W i E I G E T E G T F I V Li S V
E L L L N E E
~i
Table 4
Nucleotide Sequence of Plasmid pSYWI Encoding
Chimeric Toxin SYW1
The nucleotide differences as compared to the sequence
shown in Table 1 are underlined at positions 1252, 1319,
1320, 1323, 1324, and 1326; and code for amino acid
changes at positions 289, 311, and 313, as shown
in Table 4A.
( st art HLi-73 ATG GATAACAATC4<.~C~
>
CGAACATCAATGAATGCATTCCTTATAATTGTTTAAGTAACCCTGAAGTA
GAAGTATTAGGTGGAGAAAGAATAGAAACTGGTTACACCCCAATCGATAT5C~
TTCCTTGTCGCTAACGCAATTTCTTTTGAGTGAATTTGTTCCCGGTGCTG
GATTTGTGTTAGr,ACTA~TTGATATAATATGrr,GAATTTTTGGTCCCTCT6
CAATGGGACGCATTTCTTGTACAAATTGAACAGTTAATTAACCAAAGAAT
AGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCA7C~
ATCTTTATCA'AATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCT
ACTAATCCAGCATTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAABCKr
CAGTGCCCTTACAACCGCTATTCCTCTTTTTGCAGTTCAAAATTATCAAG
TTCCTCTTTTATCAGTATATGTTCAAGCTGCAAATTTACATTTATCAGTT9C~C~
TTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGATGCCGCGAC
TATCAATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAACTATACAGlr?C
ATTATGCTGTACGCTGGTAGAATACGGGATTAGAACGTGTATGGGGACCG
GATTCTAGAGATTGGGTAAGGTATAATCAATTTAGAAGAGAATTAACACT11<r~r
AACTGTATTAGATATCGTTGCTCTGTTCCCGAATTATGATAGTAGAAGAT
ATCCRATTCGAACAGTTTCCCAATTARCAAGAGAAATTTATACAAACCCA1'Cr
GTATTAGAAAATTTTGATGGTAGTTTTCGAGGCTCGGCTCAGGGCATAGA
GGAAGTATTAGGAGTCCACATTTGATGGATATACTTAACAGTATAACCA130
A
_ ~AA GGGGAATATTATTGGTCAGGGCATCAAATA
TCTATACGGAT~GCTCATA
ATGGCTTCTC_ TTCGGr,GCCAGAAT1'CACTTTTCCGCTATA1~Cr
CTGTAGGGTT
TGGAACTATGGGAAATGCAGCTCCACAACAACGTATTGTTGCTCAACTAG
GTCAGGGCGTGTATAGAACATTATCGTCCACTTTATATAGAAGACCTTTT1~<?
AATATAGGGATAAATAATCAACAACTATCTGTTCTTGACGGGACAGAATT
TGCTTATGGAACCTCCTCAAATTTGCCATCCGCTrTATACAGAAAAAGCG16x,
GAACGGTAGATTCGCTGGATGAAATACCGCCACAGAATAACAACGTGCCA
CCTAGGCAAGGATTTAGTCATCGAT'fAAGCCATGTTTCAATGTTTCGTTC170
AGGCTTTAGTAATAGTAGTGTAAGTATAATAAGAGCT
tend
hd-73)
(sta.rt .HLi-i) CCAACGT TTTCTTGGCAGCATCGCAGT19GC~
GCTGAATTTAATAATATAATTCCTTCATCACAAATTACACAAATACCTTT
AACAAAATCTACTAATCTTGGCTCTGGAACTTCTGTCGTTAAAGGACCAG~~?C~
GATTTACAGG~AGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCA
ACCTTAAGAGTAAATATTACTGCACCATTATCACAAAGATATCGGGTAAG2lU~r
AATTCGCTACGCTTCTACTACAAATTTACAATTCCATACATCAATTGACG
GAAGACCTA'f-TAATCAGGGTAATTTTTCAGCAACTATGAGTAGTGGGAGT~~Cr
AATTTACAGT~CCGGAAGCTTTAGGACTGTAGGTTTTACTACTCCGTTTAA
CTTTTCAAAT TATTTACGTTAAGTGCTCATGTCTTCAATT23~r0
GGATCAAGTG
CAGGCAATGA AATTTGTTCCGGCAGAAGTA
AGTTTATATA
GATCGAATTG
ACCTTTGAGG CGGTGAATGA
CAGAATRTGA X400
TTTA~AAAGA
r,CACAAAA6G
GCTGTTTACT AAAAACAGATGTGACGGATT
TCTTCCAATC
AAATCGGGTT
-43- 1 3 4 ~ 4 7 2
Table 4 (cont.)
ATCATATTGATCAAGTATCCAATTTAGTTGAGTGTTTATCAGATGAATTT~5C7
TGTCTGGATGAAAAACAAGAATTGTCCGAGAAAGTCAAACATGCGAAGCG
ACTTAGTGATGAGCGGAATTTAGTTCAAGATCCAAACTTCAGAGGGATCA~bvn
ATAGACAACTAGACCGTGGCTGGA~A~GAAGTACGGATATTACCATCCAA
r,GAGGCGATGACGTATTCAAAGAGAATTACGTTACGCTATTGGGTACCTT27C>
TGATGAGTGCTATCCAACGTATTTATATCAAAAAATAGATGAGTCGAAAT
TAAAAGCCTATACCCGTTATCAATTAAGAGGGTATATCGAAGATAGTCAA~~8C~
GACTTAGAAATCTATTTAATTCGCTACAATGCAAAACATGAAACAGTAAA
TGTGCCAGGTACGGGTTCCTTATGGCCGCTTTCAGCCCAAAGTCCAATCG29CW
GAAAGTGTGGAGAGCCGAATCGATGCGCGCCACACCTTGAATGGAATCCT
GACTTAGATTGTTCGTGTAGGGATGGAGAAAAGTGTGCCCATCATTCGCA3000
TCATTTCTCCTTAGACATTGATGTAGGATGTACAGACTTAAATGAGGACC
TAGGTGTATGGGTGATCTTTAAGATTAAGACGCAAGATGGGCACGCAAGA31C>ci
CTAGGGAATCTAGAGTTTCTCGAAGAGAAACCATTAGTAGGAGAAGCGCT
AGCTCGTGTGAAAAGAGCGGAGAAAAAATGGAGAGACAAACGTGAAAAAT3200
TGGAATGGGAAACAAATATCGTTTATAAAGAGGCAAAAGAATCTGTAGAT
GCTTTATTTGTAAACTCTCAATATGATCAATTACAAGCGGATACGAATAT3300
TGCCATGATTCATGCGGCAGATAAACGTGTTCATAGCATTCGAGAAGCTT
ATCTGCCTGAGCTGTCTGT.GATTCCGGGTGTCAATGCGGCTATTTTTGAA3400
GAATTAGAAGGGCGTATTTTCACTGCATTCTCCCTATATGATGCGAGAAA
TGTCATTAAAAATGGTGATTTTAATAATGGCTTATCCTGCTGGAACGTGA35C
AAGGGCATGTAGATGTAGAAGAACAAAACAACCAACGTTCGGTCCTTGTT
CTTCCGGAATGGGAAGCAGAAGTGTCACAAGAAGTTCGTGTCTGTCCGGG36C7
TCGTGGCTATATCCTTCGTGTCACAGCGTACAAGGAGGGATATGGAGAAG
GTTGCGTAACCATTCATGAGATCGAGAACAATACAGACGAACTGAAGTTT3700
AGCAACTGCGTAGAAGAGGAAATCTATCCAAATAACACGGTAACGTGTAA
TGATTATACTGTAAATCAAGAAGAATACGGAGGTGCGTACACTTCTCGTA3BC>0
ATCGAGGATATAACGAAGCTCCTTCCGTACCAGCTGATTATGCGTCAGTC
TATGAAGAAAAATCGTATACAGATGGACGAAGAGAGAATCCTTGTGAATT3900
TAACAGAGGGTATAGGGATTACACGCCACTACCAGTTGGTTATGTGACAA
AAGAATTAGAATACTTCCCAGAAACCGATAAGGTATGGATTGAGATTGGA4C~OC~
GAAACGGAAGGAACATTTATCGTGGACAGCGTGGAATTACTCCTTATGGA
GGAA (endHG-1)
44
tified B. t. crystal protein
1 toxicity against at least Table 4A
ny of said crystal protein Amino Acid Sequence of Chimeric Toxin SYW1
hereby the recombinant
protein toxin is identifiedN I N EC i F YN C LS N F EV E VL G G ER I E
~
I D I SL S L Tr!F LL S E FV F'r,A G F VL G L
in having an altered G I F GF S C7WD A FL V itIE r7LI N r,,~RI E E
host
A I S RL E r,LS t~JLY CtI YA E SF R E ~JE A D
L R E EM R I G!F N LiM N S AL T TA I F'LF A V
F' L L SV Y V ~tA A NL H L SV L C;D V S.VF G i=!
A A T IN S R YN D LT R L IG CJYT D Y AV R l~J
E R V WG P D SR D WV R Y NC?F RR E L TL T V
'3. t. crystal proteinL F F NY D S RR Y FI R T VS C!LT R E iY T N
toxin
F D G SF R G SA G GI E G SI R SP H L MD I L
t one target lepidopteranY T D AH K G EY Y WS G _ ~I M AS P V GF S G
H
F L Y GT _ G _A A-Fr?Q R IV A nL G C~GV Y R
rocess of: M N
the
b
p L Y R RP F N Ir,I NN C!C7LS V LD G T EF A Y
y
5t arent DNA se uence L F S AV Y R KS G TV D S LD E IP P G,NN N V
p q F S H RL S H VS M ~'R 5 G FS N 5S V S II R A
~
xin with at least a ~ H R 5A E F NN I IF S S ~!I T ~I P L TK 5 T
part of
T S V VK G P GF T GG D I LR R TS F G GI S T
A sequence encoding T A F LS G7R YR V RI R Y AS T TN L CtFH T S
a
I N C7GN F S AT M SS G S NL G;SG S F RT V G
~ to obtain a recombinantN F 5 NG S S VF T LS A H VF N SG N E VY I D
F A E VT F E AE Y LtL E R.A6!K AV N E LF T S
protein toxin which L K T DV T D YH I DG?V S NL V EC L S L~E F C
is
E L 5 EK V K HA K RL S D ER N LL L!D F'N F R
~ded by said parent L D R GW R G S.T D IT I C,GG D DV F K EN Y V
DNA
F D E CY F T YL Y G!K I D ES K LK A Y TR Y~G!
E D S (?D L E .IY L~IR Y N AK H ET V N VF G T
from said recombinant L S A QS P I GK C GE F N R.CA F.H L E WN F D
F S D V G C TD L N
R D G EK C A HH S HH L I D
4J V I FK I K TG7D GH A R LG N LE F L EE K P
L A R VK R A EK ~KWR D K RE K LE W E TN I V
to verify whether saidE 5 V DA L F VN S C,Y D C;LG A DT N I AM I H
E
V H S IR E A YL P EL S V IF G VN A A IF E
host range or increasedF T A FS L Y DA R NV I K N.GD FN N G LS C W
V D V EE Q N Nn R SV L V LF E WE A E VS C~E
! host as compared G R G YI L R VT A YK E G YG E GC V T IH .EI
to any
E L K'FS .NC VE E EI Y F NN T VT C N CiY T V
it DNA sequences; : G G A YT ~ R :R G YN E A FS V FA D Y AS V Y
S N
T D G RR E N FC E FN R G YR D YT F L F'V r,Y
lifted B. t. crystal ~E Y F PE T D KV W IE I G ET E GT F I VD S '
protein V
E E
i toxicity against
at least
ny of said crystal protein
hereby the recombinant
motein toxin is identified
