Note: Descriptions are shown in the official language in which they were submitted.
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RECEPTOR FOR A BACILLUS THTJRINGIENSIS TOXIN
Acknowledgment of Government Support
Work resulting in the present invention was supported in part by Research
Agreement 58-3198-3-011 from the Office of International Cooperation and
Development, U.S.D.A. and by Cooperative Agreement 58-5410-1-135 from the
Arthropod-Borne Animal Disease Laboratory, Agricultural Research Service,
U.S.D.A. and by Grant HD-18702 from the National Institutes of Health. The
U.S.
government has certain rights in this invention.
Technical Field
The invention relates to receptors that bind toxins from Bacillus
thuringiensis
and thus to pesticides and pest resistance. More particularly, the invention
concerns
recombinantly produced receptors that bind BT toxin and to their use in assays
for
improved pesticides, as well as in mediation of cell and tissue destruction,
dissociation, dispersion, cell-to-cell association, and changes in morphology.
Background Art
It has long been recognized that the bacterium Bacillus thuringiensis (BT)
2 o produces bactericidal proteins that are toxic to a limited range of
insects, mostly in the
orders Lepidoptera, Coleoptera and Diptera. Advantage has been taken of these
toxins in controlling pests, mostly by applying bacteria to plants or
transforming
plants themselves so that they generate the toxins by virtue of their
transgenic
character. The toxins themselves are glycoprotein products of the cry gene as
2 5 described by Hofte, H. et al. Microbiol Rev ( 1989) 53:242. It has been
established
that the toxins function in the brush border of the insect midgut epithelial
cells as
described by Gill, S.S. et al. Annu Rev Entomol (1992) 37:615. Specific
binding of
BT toxins to midgut brush border membrane vesicles has been reported by
Hofmann,
C. et al. Proc Natl Acad Sci USA (1988) 85:7844; Van Rie, 3. et al. Eur
JBiochem
3 0 (1989) 186:239; and Van Rie, J. et al. Appl Environ Microbiol ( 1990)
56:1378.
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Presumably, the toxins generated by BT exert their effects by some kind of
interaction with receptors in the midgut. The purification of a particular
receptor from
Manduca sexta was reported by the present inventors in an article by
Vadlamudi, R.K.
et al. J Biol Chem ( 1993) 268:12334. In this report, the receptor protein was
isolated
by immunoprecipitating toxin-binding protein complexes with toxin-specific
antisera
and separating the complexes by SDS-PAGE followed by electroelution. However,
to
date, there has been no structural information concerning any insect receptor
which
binds BT toxin, nor have, to applicants' knowledge, any genes encoding these
receptors been recovered.
Disclosure of the Invention
The present invention is based, in part, on the isolation and characterization
of
a receptor that is bound by members of the BT-toxin family of insecticidal
proteins,
hereinafter the BT-R, protein. The present invention is further based on the
isolation
and characterization of a nucleic acid molecule that encodes the BT-toxin
receptor,
hereinafter BT Rl gene. Based on these observations, the present invention
provides
compositions and methods for use in identifying agents that bind to the BT-R,
protein
as a means for identifying insecticidal agent and fox identifying other
members of the
BT-R, family of proteins.
Brief Description of the Drawings
Figure 1 show the nucleotide sequence and deduced amino acid sequence of
cDNA encoding the BT-R, protein from M. sexta.
Figure 2 (panels a and b) shows a comparison of amino acid sequences of
2 5 cadherin motifs (BTRcad-1 to 11 ) in BT-R, to those of other cadherins.
Figure 3 shows a block diagram of the cadherin-like structure of BT-R,.
Figure 4 shows the clone characterization of the BamHI-SacI fragment of
BT-R,. LM is HindIII cut Lambda marker; UP is the uncut plasmid clone; NP is
NsiI
cut plasmid; XP is XhoI cut plasmid; BSP is BamHI and SacI cut plasmid showing
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the cloned fragment from BT-R,; RM is mRNA size marker; and RT1 and RT2 are
transcribed mRNAs from the cloned BT-R, fragment.
Figure 5 illustrates the detection of protein expression from the plasmid
containing the Bam-Sac fragment of BT-R, using 35S-methionine as a tag. LCR is
a
luciferase control mRNA to show that the rabbit reticulocyte lysates are
functional;
RR1 and RR2 are expression products of the Bam-Sac fragment of BT-R, produced
in
rabbit reticulocytes from mRNA; LCT is a luciferase control plasmid to show
that the
transcription/translation kit is functional; and TT1 and TT2 are expression
products of
the Bam-Sac fragment of BT-R, produced in a transcription/translation kit.
Figure 6 shows a radio-blot of the Bam-Sac fragment of BT-R, with
~zsl_labeled CrylAb. BBMV is the brush border membrane vesicles from the
midgut
of M. sexta containing the wild-type BT-R, receptor protein; RBK is a rabbit
reticulocyte blank; RR1 and RR2 are the expression products of the Bam-Sac
fragment of BT-R, produced in rabbit reticulocytes from mRNA; TBK is a
transcription/translation kit blank; TT1 and TT2 are expression products of
the Bam-
Sac fragment of BT-R, produced in a transcription/translation kit. The arrows
point to
two of the bands.
Figure 7 shows the presence of a BT-R, homologue in Pink Bollworm and
European Corn Borer identified using toxin binding similar to that used to
identify the
2 0 original BT-R, clone.
Figure 8 shows the binding of CrylAb to fragments of the BT-R, protein.
Modes of Carrying Out the Invention
I. General Description
2 5 The present invention is based, in part, on the isolation and
characterization of
a novel protein expressed in the midgut of Manduca sexta that binds to members
of
the BT-toxin family of proteins, hereinafter the BT-R, protein. The present
invention
specifically provides purified BT-R" the amino acid sequence of BT-R" as well
as
nucleotide sequences that encode BT-R,. The BT-R, protein and nucleic acid
3 0 molecules can serve as targets in identifying insecticidal agents.
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II. Specific Embodiments
A. BT-R, Protein
Prior to the present invention, although members of the BT-toxin family of
protein were known, no one had identified the receptor that is bound by these
toxin
proteins. The present invention provides, in part, the amino acid sequences of
a
BT-toxin receptor that is expressed in the midgut of Maduca sexta.
In one embodiment, the present invention provides the ability to isolate or
produce a previously unknown protein by using known purification methods, the
cloned nucleic acid molecules herein described or by synthesizing a protein
having the
amino acid sequence herein disclosed.
As used herein, BT-R, refers to a protein that has the amino acid sequence of
BT-R, provided in Figure l, as well as allelic variants of the BT-R, sequence,
and
conservative substitutions mutants of the BT-R, sequence that have BT-R,
activity.
BT-R, is comprised of a single subunit, has a molecular weight of 210 kD, and
has the
amino acid sequence provided in Figure 1. A prediction of the structure of BT-
R, is
provided in Figure 3.
The BT-R, protein of the present invention includes the specifically
identified
and characterized variant herein described, as well as allelic variants,
conservative
2 o substitution variants and homologues (Figure 7) that can be
isolated/generated and
characterized without undue experimentation following the methods outlined
below.
For the sake of convenience, all BT-R, proteins will be collectively referred
to as the
BT-R, proteins, the BT-R, proteins of the present invention or BT-R,.
The term "BT-R," includes all naturally occurring allelic variants of the
2 5 Manduca sexta BT-R, protein provided in Figure 1. In general, naturally
occurring
allelic variants of Manduca sexta BT-R, will share significant homology, at
least 75
%, and generally at least 90%, to the BT-R, amino acid sequence provided in
Seq. ID
No:2. Allelic variants, though possessing a slightly different amino acid
sequence
than Seq. ID No:2, will be expressed as a transmembrane protein in the
digestive tract
3 0 of an insect or other organism. Typically, allelic variants of the BT-R,
protein will
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contain conservative amino acid substitutions from the BT-R, sequence herein
described or will contain a substitution of an amino acid from a corresponding
position in a BT-R, homologue (a BT-R, protein isolated from an organism other
than
Manduca sexta).
One class of BT-R, allelic variants will be proteins that share a high degree
of
homology with at least a small region of the amino acid sequence provided in
Seq. ID
No:-, but may further contain a radical departure from the sequence, such as a
non-
conservative substitution, truncation, insertion or frame shift. Such alleles
are termed
mutant alleles of BT-R, and represent proteins that typically do not perform
the same
1 o biological functions as does the BT-R, variant of Seq. ID No:2.
The BT-R, proteins of the present invention are preferably in isolated form.
As used herein, a protein is said to be isolated when physical, mechanical or
chemical
methods are employed to remove the BT-R, protein from cellular constituents
that are
normally associated with the protein. A skilled artisan can readily employ
standard
purification methods to obtain an isolated BT-R, protein. The nature and
degree of
isolation will depend on the intended use.
The cloning of the BT-R, encoding nucleic acid molecule makes it possible to
generate defined fragments of the BT-R, proteins of the present invention. As
discussed below, fragments of BT-R, are particularly useful in: generating
domain
2 0 specific antibodies; identifying agents that bind to toxin binding domain
on BT-R,;
identifying toxin-binding structures; identifying cellular factors that bind
to BT-R,;
isolating homologues or other allelic forms of BT-R,; and studying the mode of
action
of BT-toxins.
Fragments of the BT-R, proteins can be generated using standard peptide
2 5 synthesis technology and the amino acid sequence of Manduca sexta BT-R,
disclosed
herein. Alternatively, as illustrated in Example 5, recombinant methods can be
used
to generate nucleic acid molecules that encode a fragment of the BT-R,
protein.
Fragments of the BT-R, protein subunits that contain particularly interesting
structures can be identified using art-known methods such as by using an
3 0 immunogenicity plot, Chou-Fasman plot, Gamier-Robson plot, Kyte-Doolittle
plot,
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Eisenberg plot, Karplus-Schultz plot or Jameson-Wolf plot of the BT-R,
protein.
Fragments containing such residues are particularly useful in generating
domain
specific anti-BT-R, antibodies or in identifying cellular factors that bind to
BT-R,.
One particular fragment that is preferred for use in identifying insecticidal
agents is a
soluble fragment of BT-R, that can bind to a member of the BT family of
toxins. In
Example 5, a fragment of BT-R, that binds to a BT-toxin is disclosed.
As described below, members of the BT-R, family of proteins can be used for,
but are not limited to: 1 ) a target to identify agents that bind to BT-R" 2)
a target or
bait to identify and isolate binding partners and cellular factors that bind
to BT-R"
3) an assay target to identify BT-R, and other receptor-mediated activity, and
4) a
marker of cells that express a member of the BT-R, family of proteins.
B. Anti-BT-R, Antibodies
The present invention further provides antibodies that bind BT-R,. The most
preferred antibodies will selectively bind to BT-R, and will not bind (or will
only bind
weakly) to non-BT-R, proteins. Anti- BT-R, antibodies that are especially
contemplated include monoclonal and polyclonal antibodies as well as fragments
containing the antigen binding domain and/or one or more complement
determining
regions (CDRs) of these antibodies.
2 0 Antibodies are generally prepared by immunizing a suitable mammalian host
using a BT-R, protein (synthetic or isolated), or fragment, in isolated or
immunoconjugated form (Harlow, Antibodies, Cold Spring Harbor Press, NY (
1989)).
Regions of the BT-R, protein that show immunogenic structure can readily be
identified using art-known methods. Other important regions and domains can
readily
2 5 be identified using protein analytical and comparative methods known in
the art, such
as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or
Jameson-Wolf analysis. Fragments containing these residues are particularly
suited in
generating specific classes of anti-BT-R, antibodies. Particularly useful
fragments
include, but are not limited to, the BT-toxin binding domain of BT-R,
identified in
3 0 Example S.
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Methods for preparing a protein for use as an immunogen and for preparing
immunogenic conjugates of a protein with a Garner such as BSA, KLH, or other
Garner proteins are well known in the art. In some circumstances, direct
conjugation
with reagents such as carbodiimide may be used; in other instances linking
reagents
like those supplied by Pierce Chemical Co., Rockford, IL, may be effective.
Administration of a BT-R, immunogen is conducted generally by injection
over a suitable time period in combination with a suitable adjuvant, as is
generally
understood in the art. During the immunization schedule, titers of antibodies
can be
taken to determine adequacy of antibody formation.
1 o Although the polyclonal antisera produced in this way may be satisfactory
for
some applications, for many other applications, monoclonal antibody
preparations are
preferred. Immortalized cell lines which secrete a desired monoclonal antibody
may
be prepared using the standard method of Kohler and Milstein or modifications
which
effect immortalization of lymphocytes or spleen cells, as is generally known.
The
immortalized cell lines secreting the desired antibodies are screened by
immunoassay
in which the antigen is the BT-R, protein or BT-R, fragment. When the
appropriate
immortalized cell culture secreting the desired antibody is identified, the
cells can be
cultured either in vitro or by production in ascites fluid.
The desired monoclonal antibodies are then recovered from the culture
2 o supernatant or from the ascites supernatant. Fragments of the monoclonals
or the
polyclonal antisera which contain the immunologically significant portion can
be used
as antagonists, as well as the intact antibodies. Use of immunologically
reactive
fragments, such as the Fab, Fab', of F(ab'), fragments is often preferable,
especially in
a therapeutic context, as these fragments are generally less immunogenic than
the
2 5 whole immunoglobulin.
The antibodies or fragments may also be produced, using current technology,
by recombinant means. Regions that bind specifically to the desired regions of
the
BT-R, protein can also be produced in the context of chimeric or CDR grafted
antibodies of multiple species origin.
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As described below, anti-BT-R, antibodies are useful as modulators of BT-R,
activity, are useful in in vitro and in vivo antibody based assays methods for
detecting
BT-R, expression/activity, in generating toxin conjugates, for purifying
homologues
of Manduca sexta BT-R" in generating anti-ideotypic antibodies that mimic the
BT-R, protein and in identifying competitive inhibitors of BT-toxin/BT-R,
interactions.
C. BT-R, Encoding Nucleic Acid Molecules
As described above, the present invention is based, in part, on isolating
nucleic
acid molecules from Manduca sexta that encode BT-R,. Accordingly, the present
invention further provides nucleic acid molecules that encode the BT-R,
protein, as
herein defined, preferably in isolated form. For convenience, all BT-R,
encoding
nucleic acid molecules will be referred to as BT-R, encoding nucleic acid
molecules,
the BT RI genes, or BT Rl. The nucleotide sequence of the Manduca sexta
nucleic
acid molecule that encodes one allelic form of BT-R, is provided in Figure 1.
As used herein, a "nucleic acid molecule" is defined as an RNA or DNA
molecule that encodes a peptide as defined above, or is complementary to a
nucleic
acid sequence encoding such peptides. Particularly preferred nucleic acid
molecules
will have a nucleotide sequence identical to or complementary to the Manduca
sexta
2 o DNA sequences herein disclosed. Specifically contemplated are genomic DNA,
cDNAs, synthetically prepared DNAs, and antisense molecules, as well as
nucleic
acids based on an alternative backbone or including alternative bases, whether
derived
from natural sources or synthesized. A skilled artisan can readily obtain
these classes
of nucleic acid molecules using the herein described BT RI sequences. However,
2 5 such nucleic acid molecules, are defined fixrther as being novel and
unobvious over
any prior art nucleic acid molecules encoding non-BT-R, proteins. For example,
the
BT Rl sequences of the present invention specifically excludes previously
identified
nucleic acid molecules that share only partial homology to BT RI. Such
excluded
sequences include identified members of the cadhedrin family of proteins.
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As used herein, a nucleic acid molecule is said to be "isolated" when the
nucleic acid molecule is substantially separated from contaminant nucleic acid
molecules that encode polypeptides other than BT-R,. A skilled artisan can
readily
employ nucleic acid isolation procedures to obtain an isolated BT-R, encoding
nucleic
acid molecule.
The present invention further provides fragments of the BT-R, encoding nucleic
acid molecules of the present invention. As used herein, a fragment of a BT-R,
encoding nucleic acid molecule refers to a small portion of the entire BT Rl
sequence.
The size of the fragment will be determined by its intended use. For example,
if the
fragment is chosen so as to encode the toxin binding domain of BT-R,
identified in
Example S, then the fragment will need to be large enough to encode the toxin
binding
domain of the BT-R, protein. If the fragment is to be used as a nucleic acid
probe or
PCR primer, then the fragment length is chosen so as to obtain a relatively
small number
of false positives during probing/priming. Fragments of the Manduca sexta BT
R~
gene that are particularly useful as selective hybridization probes or PCR
primers can be
readily identified from the entire BT RI sequence using art-known methods.
Another class of fragments of BT-R, encoding nucleic acid molecules are the
expression control sequence found upstream and downstream from the BT-R,
encoding
region found in genomic clones of the BT RI gene. Specifically, tissue and
2 o developmental specific expression control elements can be identified as
being 5' to the
BT-R, encoding region found in genomic clones of the BT Rl gene. Such
expression
control sequence are useful in generating expression vectors for expressing
genes in the
digestive tract of a transgenic organism. As described in more detail below, a
skilled
artisan can readily use the BT Rl cDNA sequence herein described to isolate
and
2 5 identify genomic BT R~ sequences and the expression control elements found
in the
BT R I gene.
Fragments of the BT-R, encoding nucleic acid molecules of the present
invention (i.e., synthetic oligonucleotides) that are used as probes or
specific primers for
the polymerase chain reaction (PCR), or to synthesize gene sequences encoding
BT-R,
3 o proteins, can easily be synthesized by chemical techniques, for example,
the
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phosphotriester method of Matteucci, et al., JAm Chem Soc (1981) 103:3185-
3191, or
using automated synthesis methods. In addition, larger DNA segments can
readily be
prepared by well known methods, such as synthesis of a group of
oligonucleotides that
define various modular segments of the BT RI gene, followed by ligation of
oligonucleotides to build the complete modified BT RI gene.
The BT-R, encoding nucleic acid molecules of the present invention may
further be modified so as to contain a detectable label for diagnostic and
probe
purposes. As described above, such probes can be used to identify nucleic acid
molecules encoding other allelic variants or homologues of the BT-R, proteins
and as
1 o described below, such probes can be used to identify the presence of a BT-
R, protein
as a means for identifying cells that express a BT-R, protein. A variety of
such labels
are known in the art and can readily be employed with the BT-R, encoding
molecules
herein described. Suitable labels include, but are not limited to, biotin,
radiolabeled
nucleotides, biotin, and the like. A skilled artisan can employ any of the art-
known
labels to obtain a labeled BT-R, encoding nucleic acid molecule.
D. Isolation of Other BT-R, Encoding Nucleic Acid Molecules
The identification of the BT-R, protein from Manduca sexta and the
corresponding encoding nucleic acid molecules, has made possible the
identification of
2 0 and isolation of: 1 ) BT-R, proteins from organisms other than Manduca
sexta,
hereinafter referred to collectively as BT-R, homologues, 2) other allelic and
mutant
forms of the Manduca sexta BT-R, protein (described above), and 3) the
corresponding
genomic DNA that contains the BT RI gene. The most preferred source of BT-R,
homologues are insects, the most preferred being members of the Lepidopteran,
2 5 Coleopteran and Dipteran orders of insects. Evidence of the existence of
BT-R,
homologues is provided in Figure 7.
Essentially, a skilled artisan can readily use the amino acid sequence of the
Manduca sexta BT-R, protein to generate antibody probes to screen expression
libraries prepared from cells and organisms. Typically, polyclonal antiserum
from
3 0 mammals such as rabbits immunized with the purified protein (as described
above) or
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monoclonal antibodies can be used to probe an expression library, prepared
from a
target organism, to obtain the appropriate coding sequence for a BT-R,
homologue. The
cloned cDNA sequence can be expressed as a fusion protein, expressed directly
using its
own control sequences, or expressed by constructing an expression cassette
using
control sequences appropriate to the particular host used for expression of
the enzyme.
Alternatively, a portion of the BT-R, encoding sequence herein described can
be
synthesized and used as a probe to retrieve DNA encoding a member of the BT-R,
family of proteins from organisms other than Manduca sexta, allelic variants
of the
Manduca sexta BT-R, protein herein described, and genomic sequence containing
the
1 o BT RI gene. Oligomers containing approximately 18-20 nucleotides {encoding
about a
6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA
libraries to obtain hybridization under stringent conditions or conditions of
sufficient
stringency to eliminate an undue level of false positives.
Additionally, pairs of oligonucleotide primers can be prepared for use in a
polymerase chain reaction (PCR) to selectively amplify/clone a BT-R,-encoding
nucleic
acid molecule, or fragment thereof. A PCR denature/anneal/extend cycle for
using
such PCR primers is well known in the art and can readily be adapted for use
in
isolating other BT-R, encoding nucleic acid molecules. Regions of the Manduca
sexta
BT RI gene that are particularly well suited for use as a probe or as primers
can be
2 0 readily identified by one skilled in the art.
Non-Manduca sexta homologues of BT Rl, naturally occurring allelic variants
of the Manduca sexta BT RI gene and genomic BT RI sequences will share a high
degree of homology to the Manduca sexta BT Rl sequence herein described. In
general, such nucleic acid molecules will hybridize to the Manduca sexta BT RI
2 5 sequence under high stringency. Such sequences will typically contain at
least 70%
homology, preferably at least 80%, most preferably at least 90% homology to
the
Manduca sexta BT RI sequence of Seq. ID No:l.
In general, nucleic acid molecules that encode homologues of the Manduca
sexta BT-R, protein will hybridize to the Manduca sexta BT Rl sequence under
3 0 stringent conditions. "Stringent conditions" are those that ( 1 ) employ
low ionic
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strength and high temperature for washing, for example, 0.01 SM NaCI/0.001 SM
sodium titrate/0.1 % SDS at SO°C., or (2) employ during hybridization a
denaturing
agent such as formamide, for example, SO% (vol/vol) formamide with 0.1% bovine
serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/SO mM sodium phosphate
buffer at pH 6.S with 7S0 mM NaCI, 7S mM sodium citrate at 42°C.
Another example
is use of SO% formamide, S x SSC (0.7SM NaCI, 0.075 M sodium citrate), SO mM
sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, S x Denhardt's
solution,
sonicated salmon sperm DNA (S0 pg/ml), 0.1 % SDS, and 10% dextran sulfate at
42°C., with washes at 42°C. in 0.2 x SSC and 0.1% SDS. A skilled
artisan can
readily determine and vary the stringency conditions appropriately to obtain a
clear
and detectable hybridization signal.
The presence of similar receptors in noninsect organisms as well as other
insects besides those harboring BT-R, is supported by the sequence similarity
of the
BT-R, protein to that of the various members of the cadherin superfamily of
proteins,
which are membrane giycoproteins believed to mediate calcium-dependent cell
aggregation and sorting. See, for example, Takeichi, M. Science ( 1991 ) 2S
1:1451;
and Takeichi, M. NRev Biochem (1990) 59:237.
Included in this superfamily are desmoglien, desmocollins, the Drosophila fat
tumor suppressor, Manduca sexta intestinal peptide transport protein and T-
cadherin.
2 0 All of these proteins share common extracellular motifs although their
cytoplasmic
domains differ. Goodwin, L. et al. Biochem Biophys Res Commun (1990) 173:1224;
Holton, J.L. et al. J Cell Sci (1990) 97:239; Bestal, D.3. J Cell Biol (1992)
119:451;
Mahoney, P.A. et al. Cell (1991) 853; Dantzig, A.H. et al. Science (1994)
264:430;
and Sano, K. et al. EMBO J (1993) 12:2249. Inclusion of BT-R, in the cadherin
2 5 superfamily is further supported by the report that EDTA decreases the
binding of
CrylAb toxin of BT to the 210 kD receptor of M. sexta (Martinez-Ramirez, A.C.
et al.
Biochm Biophys Res Commun (1994) 201:782).
It is noted below that the amino acid sequence of BT-R, reveals that a calcium-
binding motif is present. This is consistent with the possibility that cells
having
3 0 receptors to bind toxin may themselves survive although they render the
tissues in
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which they are included permeable to solutes and thus effect disintegration of
the
tissue. Such a mechanism is proposed for the death of insects that ingest the
toxin via
the epithelial cells in their midgut by Knowles, B.H. et al. Biochim Biophys
Acta
( 1987) 924:509. Such a mechanism is also supported in part by the results set
forth in
Example 4 hereinbelow which indicate that the effect of the toxin on embryonic
293
cells modified to express the receptor at their surface is reversible.
E. rDNA Molecules Containing a BT-R, Encoding Nucleic Acid Molecule
The present invention further provides recombinant DNA molecules (rDNAs)
1 o that contain a BT-R, encoding sequences as herein described, or a fragment
thereof,
such as a soluble fragment of BT-R, that contains the BT-toxin binding site.
As used
herein, a rDNA molecule is a DNA molecule that has been subjected to molecular
manipulation in vitro. Methods for generating rDNA molecules are well known in
the
art, for example, see Sambrook et al., Molecular Cloning (1989). In the
preferred rDNA
molecules of the present invention, a BT-R, encoding DNA sequence that encodes
a
BT-R, protein or a fragment of BT-R" is operably linked to one or more
expression
control sequences and/or vector sequences.
The choice of vector and/or expression control sequences to which the BT-R,
encoding sequence is operably linked depends directly, as is well known in the
art, on
2 o the functional properties desired, e.g., protein expression, and the host
cell to be
transformed. A vector contemplated by the present invention is at least
capable of
directing the replication or insertion into the host chromosome, and
preferably also
expression, of the BT-R, encoding sequence included in the rDNA molecule.
Expression control elements that are used for regulating the expression of an
2 5 operably linked protein encoding sequence are known in the art and
include, but are not
limited to, inducible promoters, constitutive promoters, secretion signals,
enhancers,
transcription terminators and other regulatory elements. Preferably, an
inducible
promoter that is readily controlled, such as being responsive to a nutrient in
the host
cell's medium, is used. Further, for soluble fragments, it may be desirable to
use
3 o secretion signals to direct the secretion of the BT-R, protein, or
fragment, out of the cell.
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In one embodiment, the vector containing a BT-R, encoding nucleic acid
molecule will include a prokaryotic replicon, i.e., a DNA sequence having the
ability to
direct autonomous replication and maintenance of the recombinant DNA molecule
intrachromosomally in a prokaryotic host cell, such as a bacterial host cell,
transformed
therewith. Such replicons are well known in the art. In addition, vectors that
include a
prokaryotic replicon may also include a gene whose expression confers a
detectable
marker such as a drug resistance. Typical bacterial drug resistance genes are
those that
confer resistance to ampicillin or tetracycline.
Vectors that include a prokaryotic replicon can further include a prokaryotic
or
viral promoter capable of directing the expression (transcription and
translation) of the
BT-R, encoding sequence in a bacterial host cell, such as E. toll. A promoter
is an
expression control element formed by a DNA sequence that permits binding of
RNA
polymerase and transcription to occur. Promoter sequences compatible with
bacterial
hosts are typically provided in plasmid vectors containing convenient
restriction sites for
insertion of a DNA segment of the present invention. Typical of such vector
plasmids
are pUCB, pUC9, pBR322 and pBR329 available from Biorad Laboratories
(Richmond,
CA), pPL and pKK223 available from Pharmacia, Piscataway, NJ.
Expression vectors compatible with eukaryotic cells, preferably those
compatible with vertebrate cells, can also be used to variant rDNA molecules
that
2 0 contain a BT-R, encoding sequence. Eukaryotic cell expression vectors are
well known
in the art and are available from several commercial sources. Typically, such
vectors are
provided containing convenient restriction sites for insertion of the desired
DNA
segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-
1/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the
vector
2 5 pCDM8 described herein, and the like eukaryotic expression vectors.
Eukaryotic cell expression vectors used to construct the rDNA molecules of the
present invention may further include a selectable marker that is effective in
an
eukaryotic cell, preferably a drug resistance selection marker. A preferred
drug
resistance marker is the gene whose expression results in neomycin resistance,
i.e., the
3 0 neomycin phosphotransferase (neo) gene. Southern et al., JMoI Anal Genet
(1982)
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1:327-341. Alternatively, the selectable marker can be present on a separate
plasmid,
and the two vectors are introduced by cotransfection of the host cell, and
selected by
culturing in the presence of the appropriate drug for the selectable marker.
F. Host Cells Containing an Exogenously Supplied BT-R, Encoding
Nucleic Acid Molecule
The present invention further provides host cells transformed with a nucleic
acid
molecule that encodes a BT-R, protein of the present invention, either the
entire BT-R,
protein or a fragment thereof. The host cell can be either prokaryotic or
eukaryotic.
Eukaryotic cells useful for expression of a BT-R, protein are not limited, so
long as the
cell line is compatible with cell culture methods and compatible with the
propagation of
the expression vector and expression of a BT RI gene. Preferred eukaryotic
host cells
include, but are not limited to, yeast, insect and mammalian cells, the most
preferred
being cells that do not naturally express a BT-R, protein.
Any prokaryotic host can be used to express a BT-R,-encoding rDNA molecule.
The preferred prokaryotic host is E. coli.
Transformation of appropriate cell hosts with an rDNA molecule of the present
invention is accomplished by well known methods that typically depend on the
type of
vector used and host system employed. With regard to transformation of
prokaryotic
2 0 host cells, electroporation and salt treatment methods are typically
employed, see, for
example, Cohen et al., Proc Acad Sci USA (1972) 69:2110; and Maniatis et al.,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring
Harbor, NY (1982). With regard to transformation of vertebrate cells with
vectors
containing rDNAs, electroporation, cationic lipid or salt treatment methods
are typically
2 5 employed, see, for example, Graham et al., Virol (1973) 52:456; Wigler et
al., Proc Natl
Acad Sci USA (1979) 76:1373-76.
Successfully transformed cells, i.e., cells that contain an rDNA molecule of
the
present invention, can be identified by well known techniques. For example,
cells
resulting from the introduction of an rDNA of the present invention can be
cloned to
3 0 produce single colonies. Cells from those colonies can be harvested, lysed
and their
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DNA content examined for the presence of the rDNA using a method such as that
described by Southern, JMoI Biol (1975) 98:503, or Berent et al., Biotech
(1985) 3:208
or the proteins produced from the cell assayed via an immunological method.
G. Production of a BT-R, Protein Using an rDNA Molecule
The present invention further provides methods for producing a BT-R, protein
that uses one of the BT-R, encoding nucleic acid molecules herein described.
In general
terms, the production of a recombinant BT-R, protein typically involves the
following
steps.
First, a nucleic acid molecule is obtained that encodes a BT-R, protein or a
fragment thereof, such as the nucleic acid molecule depicted in Figure 1. The
BT-R,
encoding nucleic acid molecule is then preferably placed in an operable
linkage with
suitable control sequences, as described above, to generate an expression unit
containing
the BT-R, encoding sequence. The expression unit is used to transform a
suitable host
and the transformed host is cultured under conditions that allow the
production of the
BT-R, protein. Optionally the BT-R, protein is isolated from the medium or
from the
cells; recovery and purification of the protein may not be necessary in some
instances
where some impurities may be tolerated.
Each of the foregoing steps can be done in a variety of ways. For example, the
2 0 desired coding sequences may be obtained from genomic fragments and used
directly in
an appropriate host. The construction of expression vectors that are operable
in a variety
of hosts is accomplished using an appropriate combination of replicons and
control
sequences. The control sequences, expression vectors, and transformation
methods are
dependent on the type of host cell used to express the gene and were discussed
in detail
2 5 earlier. Suitable restriction sites can, if not normally available, be
added to the ends of
the coding sequence so as to provide an excisable gene to insert into these
vectors. A
skilled artisan can readily adapt any hosdexpression system known in the art
for use
with BT-R, encoding sequences to produce a BT-R, protein.
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H. Identification of Agents and Cellular Constituents that Bind to a
BT-R, Protein
Another embodiment of the present invention provides methods for identifying
agents and cellular constituents that bind to BT-R,. Specifically, agents and
cellular
constituents that bind to BT-R, can be identified by: 1) the ability of the
agent/constituent to bind to BT-R" 2) the ability to block BT-toxin binding to
BT-R, ,
and/or 3) the ability to kill BT-R, expressing cells. Activity assays for BT-
R, activity
and binding and competitive assays using a BT-R, protein are suitable for use
in high
through-put screening methods, particularly using a soluble fragment of BT-R,
that
contains the BT-toxin binding domain, such as that disclosed in Example 5.
In detail, in one embodiment, BT-R, is mixed with an agent or cellular
extract.
After mixing under conditions that allow association of BT-R, with the agent
or
component of the extract, the mixture is analyzed to determine if the
agent/component
bound to the BT-R,. Binding agents/components are identified as being able to
bind
to BT-R,. Alternatively or consecutively, BT-R, activity can be directly
assessed as a
means for identifying agonists and antagonists of BT-R, activity.
Alternatively, targets that are bound by a BT-R, protein can be identified
using
a yeast two-hybrid system or using a binding-capture assay. In the yeast two
hybrid
system, an expression unit encoding a fusion protein made up of one subunit of
a two
2 o subunit transcription factor and the BT-R, protein is introduced and
expressed in a
yeast cell. The cell is further modified to contain 1 ) an expression unit
encoding a
detectable marker whose expression requires the two subunit transcription
factor for
expression and 2) an expression unit that encodes a fusion protein made up of
the
second subunit of the transcription factor and a cloned segment of DNA. If the
cloned
2 5 segment of DNA encodes a protein that binds to the BT-R, protein, the
expression
results in the interaction of the BT-R, and the encoded protein. This brings
the two
subunits of the transcription factor into binding proximity, allowing
reconstitution of
the transcription factor. This results in the expression of the detectable
marker. The
yeast two hybrid system is particularly useful in screening a library of cDNA
3 o encoding segments for cellular binding partners of BT-R,.
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The BT-R, protein used in the above assays can be: an isolated and fully
characterized protein, a fragment of a BT-R, protein (such as a soluble
fragment
containing the BT-toxin binding site), a cell that has been altered to express
a BT-R,
protein/fragment or a fraction of a cell that has been altered to express a BT-
R,
protein/fragment. Further, the BT-R, protein can be the entire BT-R, protein
or a
defined fragment of the BT-R, protein. It will be apparent to one of ordinary
skill in
the art that so long as the BT-R, protein or fragment can be assayed for agent
binding,
e.g., by a shift in molecular weight or activity, the present assay can be
used.
The method used to identify whether an agent/cellular component binds to a
BT-R, protein will be based primarily on the nature of the BT-R, protein used.
For
example, a gel retardation assay can be used to determine whether an agent
binds to
BT-R, or a fragment thereof. Alternatively, immunodetection and biochip
technologies can be adopted for use with the BT-R, protein. A skilled artisan
can
readily employ numerous art-known techniques for determining whether a
particular
agent binds to a BT-R, protein.
Agents and cellular components can be further, or alternatively, tested for
the
ability to block the binding of a BT-toxin to a BT-R, protein/fragment.
Alternatively,
antibodies to the BT-toxin binding site or other agents that bind to the BT-
toxin
binding site on the BT-R, protein can be used in place of the BT-toxin.
2 0 Agents and cellular components can be further tested for the ability to
modulate the activity of a BT-R, protein using a cell-free assay system or a
cellular
assay system. As the activities of the BT-R, protein become more defined,
functional
assays based on the identified activity can be employed.
As used herein, an agent is said to antagonize BT-R, activity when the agent
2 5 reduces BT-R, activity. The preferred antagonist will selectively
antagonize BT-R"
not affecting any other cellular proteins. Further, the preferred antagonist
will reduce
BT-R, activity by more than 50%, more preferably by more than 90%, most
preferably eliminating all BT-R, activity.
As used herein, an agent is said to agonize BT-R, activity when the agent
3 o increases BT-R, activity. The preferred agonist will selectively agonize
BT-R" not
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affecting any other cellular proteins. Further, the preferred antagonist will
increase
BT-R, activity by more than 50%, more preferably by more than 90%, most
preferably more than doubling BT-R, activity.
Agents that are assayed in the above method can be randomly selected or
rationally selected or designed. As used herein, an agent is said to be
randomly
selected when the agent is chosen randomly without considering the specific
sequences of the BT-R, protein or BT-toxin. An example of randomly selected
agents
is the use of a chemical library or a peptide combinatorial library, or a
growth broth of
an organism or plant extract.
As used herein, an agent is said to be rationally selected or designed when
the
agent is chosen on a nonrandom basis that takes into account the sequence of
the
target site and/or its conformation in connection with the agent's action.
Agents can
be rationally selected or rationally designed by utilizing the peptide
sequences that
make up the BT-R, protein and BT-toxin. For example, a rationally selected
peptide
agent can be a peptide whose amino acid sequence is identical to a fragment of
a
BT-R, protein or BT-toxin.
The agents tested in the methods of the present invention can be, as examples,
peptides, small molecules, and vitamin derivatives, as well as carbohydrates.
A
skilled artisan can readily recognize that there is no limit as to the
structural nature of
2 0 the agents used in the present screening method. One class of agents of
the present
invention are peptide agents whose amino acid sequences are chosen based on
the
amino acid sequence of the BT-R, protein or BT-toxin. Small peptide agents can
serve as competitive inhibitors of BT-R, protein activity.
Peptide agents can be prepared using standard solid phase (or solution phase)
2 5 peptide synthesis methods, as is known in the art. In addition, the DNA
encoding
these peptides may be synthesized using commercially available oligonucleotide
synthesis instrumentation and produced recombinantly using standard
recombinant
production systems. The production using solid phase peptide synthesis is
necessitated if non-gene-encoded amino acids are to be included.
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Another class of agents of the present invention are antibodies immunoreactive
with critical positions of the BT-R, protein. As described above, antibodies
are
obtained by immunization of suitable mammalian subjects with peptides,
containing
as antigenic regions, those portions of the BT-R, protein intended to be
targeted by the
antibodies. Critical regions particularly include the BT-toxin binding domain
identified in Example 5. Such agents can be used in competitive binding
studies to
identify second generation BT-R, binding agents.
The cellular extracts tested in the methods of the present invention can be,
as
examples, aqueous extracts of cells or tissues, organic extracts of cells or
tissues or
partially purified cellular fractions. A skilled artisan can readily recognize
that there
is no limit as to the source of the cellular extract used in the screening
method of the
present invention. The preferred source for isolating cellular binding
partners of
BT-R, are cells that express BT-R, or cells that are in close proximity to BT-
R,
expressing cells.
An outline of one screening method is as follows. Cells are modified by
transfection, retroviral infection, electroporation or other known means, to
express a
BT-R, protein and then cultured under conditions wherein the receptor protein
is
produced and displayed. If desired, the cells are then recovered from the
culture for
use in the assay, or the culture itself can be used per se.
2 0 In the assays, the modified cells are contacted with the candidate toxin
and the
effect on metabolism or morphology is noted in the presence and absence of the
candidate. The effect may be cytotoxic -- i.e., the cells may themselves
exhibit one of
the indices of cell death, such as reduced thymidine uptake, slower increase
in optical
density of the culture, reduced exclusion of vital dyes (e.g., trypan blue),
increased
2 5 release of viability markers such as chromium and rubidium, and the like.
The
differential response between the toxin-treated cells and the cells absent the
toxin is
then noted. The strength of the toxin can be assessed by noting the strength
of the
response.
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These assays may be conducted directly as described above or competitively
with known toxins. For example, one approach might be to measure the
diminution in
binding of labeled BT cry toxin in the presence and absence of the toxin
candidate.
In addition to simply screening candidates, the screen can be used to devise
improved forms of toxins which are more specific or less specific to
particular classes
of insects as desired. The ability to determine binding affinity (Ka and Kd),
dissociation and association rates, and cytotoxic effects of a candidate
allows quick,
accurate and reproducible screening techniques for a large number of toxins
and other
ligands under identical conditions which was not possible heretofore. Such
l0 information will facilitate the selection of the most effective toxins and
ligands for
any given receptor obtained from any desired host cell.
Competition assays may also employ antibodies that are specifically
immunoreactive with the receptor. Such antibodies can be prepared in the
conventional manner by administering the purified receptor to a vertebrate
animal,
monitoring antibody titers and recovering the antisera or the antibody-
producing cells
for immortalization, to obtain immortalized cells capable of secreting
antibodies of the
appropriate specificity. Techniques for obtaining immortalized B cells and for
screening them for secretion of the desired antibody are now conventional in
the art.
The resulting monoclonal antibodies may be more effective than the polyclonal
2 0 antisera as competition reagents;. furthermore, the availability of the
immortalized cell
line secreting the desired antibody assures uniformity of production of the
same
reagent over time. The information and the structural characteristics of
toxins and
ligands tested will permit a rational approach to designing more efficient
toxins and
ligands. Additionally, such assays will lead to a better understanding of the
function
2 5 and the structure/function relationship of both toxin/ligand and BT-R,
analogs. In
turn, this will allow the development of highly effective toxins/ligands.
Ligands
include natural and modified toxins, antibodies (anti-receptor and
antiidiotypic
antibodies which mimic a portion of a toxin that binds to a receptor, and
whatever
small molecules bind the receptors.
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I. Uses of Agents that Bind to a BT-R, Protein
As provided in the Background section, BT-R, is the target for the
insecticidal
activity of BT-toxins. Agents that bind a BT-R, protein can be used: 1 ) to
kill BT-R,
expressing cells, 2) to identify agents that block the interaction of a BT-
toxin with
BT-R, and 3) in methods for identifying cells that express BT-R,.
The methods employed in using the BT-R, binding agents will be based
primarily on the nature of the BT-R, binding agent and its intended use. For
example,
a BT-R, binding agent can be used to: deliver a conjugated toxin to a BT-R,
expressing cell; modulate BT-R, activity; directly kill BT-R, expressing
cells; or
screen for and identify competitive binding agents. An agent that inhibits the
activity
of BT-R, can be used to directly inhibit the growth of BT-R, expressing cells.
Further, identified cellular factors that bind to BT-R, can, themselves, be
used in
binding/competitive assays to identify agonist and antagonists of BT-R,.
J. Methods for Identifying the Presence of a BT-R, protein or gene
The present invention further provides methods for identifying cells, tissues
or
organisms expressing a BT-R, protein or a BT Rl gene. Such methods can be used
to
diagnose the presence of cells or an organism that expresses a BT-R, protein
in vivo or
in vitro. The methods of the present invention are particularly useful in the
2 0 determining the presence of cells that are a target for BT-toxin activity
or for
identifying susceptibility of an organism to a BT-toxin or BT-toxin-like
agent.
Specifically, the presence of a BT-R, protein can be identified by determining
whether
a BT-R, protein, or nucleic acid encoding a BT-R, protein, is expressed in a
cell,
tissue or organism.
2 5 A variety of immunological and molecular genetic techniques can be used to
determine if a BT-R, protein is expressed/produced in a particular cell or
sample. In
general, an extract containing nucleic acid molecules or an extract containing
proteins
is prepared. The extract is then assayed to determine whether a BT-R, protein,
or a
BT-R, encoding nucleic acid molecule, is produced in the cell.
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For example, to perform a diagnostic test based on nucleic acid molecules, a
suitable nucleic acid sample is obtained and prepared using conventional
techniques.
DNA can be prepared, for example, simply by boiling a sample in SDS. The
extracted nucleic acid can then be subjected to amplification, for example by
using the
polymerase chain reaction (PCR) according to standard procedures, such as a RT-
PCR
method, to selectively amplify a BT-R, encoding nucleic acid molecule or
fragment
thereof. The size or presence of a specific amplified fragment (typically
following
restriction endonuclease digestion) is then determined using gel
electrophoresis or the
nucleotide sequence of the fragment is determined (for example, see Weber and
May
Am JHum Genet (1989) 44:388-339; Davies, J. et al. Nature (1994) 371:130-
136)).
The resulting size of the fragment or sequence is then compared to the known
BT-R,
proteins encoding sequences, for example via hybridization probe. Using this
method,
the presence of a BT-R, protein can be identified.
To perform a diagnostic test based on proteins, a suitable protein sample is
obtained and prepared using conventional techniques. Protein samples can be
prepared, for example, simply by mixing a sample with SDS followed by salt
precipitation of a protein fraction. The extracted protein can then be
analyzed to
determine the presence of a BT-R, protein using known methods. For example,
the
presence of specific sized or charged variants of a protein can be identified
using
mobility in an electric filed. Alternatively, antibodies can be used for
detection
purposes. A skilled artisan can readily adapt known protein analytical methods
to
determine if a sample contains a BT-R, protein.
Alternatively, BT-R, protein or gene expression can also be used in methods to
identify agents that decrease the level of expression of a BT Rl gene. For
example,
2 5 cells or tissues expressing a BT-R, protein can be contacted with a test
agent to
determine the effects of the agent on BT-R, protein/gene expression. Agents
that
activate BT-R, protein/gene expression can be used as an agonist of BT-R,
activity
whereas agents that decrease BT-R, protein/gene expression can be used as an
antagonist of BT-R, activity.
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K. Methods to Sensitize Cells
The present invention further provides methods of sensitizing cells such that
they become susceptible to killing with a BT-toxin, or a BT-toxin analog.
Specifically, host cells transformed to express BT-R, receptor, or a homolog
of the
BT-R, receptor, become sensitive to the mode of action of BT-toxins. The
binding of
a BT-toxin to a BT-R, receptor expressed on the surface of the transformed
cells
results in induction of a cellular death and apoptosis of the cell expressing
the BT-R,
receptor. Accordingly, the BT-R, receptor is an appropriate candidate for use
in
transforming cells in which it is desirable to induce cell death.
There are numerous situations in which it is desirable to introduce the
selected
gene into a selected population of cells, thus bringing about cell death. One
such
example is in the therapeutic treatment of cancer cells. In using specifically
targeted
vectors for delivery of BT-R,-encoding DNA molecules into a tumor cell, tumor
cells
within a patient can be engineered to express a BT-R, protein. Such cells then
become susceptible to death upon treatment with a BT-toxin. Since BT-toxin is
not
normally toxic to mammalian cells, this method is particularly applicable to
inducing
cell death in particular cells in a mammalian host. Other situations where it
may be
desirable to stimulate cell death in particular cells or cell lines are in the
treatment of
autoimmune disorders and in the treatment of cells harboring pathogens, such
as
2 0 malaria or HIV agents.
The choice of the actual steps employed to introduce a BT-R,-encoding DNA
molecule into a cell to render the cells susceptible to treatment with BT-
toxin is based
primarily on the cell type that is to be altered, the conditions under which
the cell type
will be altered, and the overall use envisioned. A skilled artisan can readily
adapt art-
2 5 known methods for use with the BT-R,-encoding DNA molecule of the present
invention.
L. Animal Models and Gene Therapy
The BT RI gene and the BT-R, protein can also serve as a target for generating
3 0 transgenic organisms in which the pattern of BT-R, expression has been
altered. For
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example, in one application, BT-R, deficient insects or insect cells can be
generated
using standard knock-out procedures to inactivate a BT RI gene, or, if such
animals
are non-viable, inducible BT RI antisense molecules can be used to regulate BT
Rl
activity/expression. Alternatively, cells or an organism can be altered so as
to contain
a Manduca sexta BT-R, encoding nucleic acid molecule or an antisense-BT-R,
expression unit that directs the expression of a BT-R, protein or an antisense
molecule
in a tissue specific fashion. In such uses, an organism or cells, for example
insects or
insect cells, is generated in which the expression of a BT Rl gene is altered
by
inactivation or activation and/or replaced by a Manduca sexta BT R~ gene. This
can
be accomplished using a variety of art-known procedures such as targeted
recombination. Once generated, the BT RI expression altered cells or organisms
can
be used to 1 ) identify biological and pathological processes mediated by the
BT-R,
protein, 2) identify proteins and other genes that interact with the BT-R,
protein,
3) identify agents that can be exogenously supplied to overcome a BT-R,
protein
deficiency and 4) serve as an appropriate screen for identifying mutations
within the
BT RI gene that increases or decreases activity.
For example, it is possible to generate transgenic insects, such as members of
the dipteran order, expressing the Manduca sexta minigene encoding BT-R, in a
tissue
specific-fashion and test the effect of over-expression of the protein in
tissues and
2 0 cells that normally do not contain the BT-R, protein.
M. Use of Expression Control Elements of the BT-R, Gene
The present invention further provides the expression control sequences found
5' of the of the newly identified BT RI gene in a form that can be used in
generating
2 5 expression vectors. Specifically, the BT RI expression control elements,
such as the
BT-R, promoter, that can readily be identified as being 5' from the ATG start
codon
in the BT RI gene, can be used to direct the expression of an operably linked
protein
encoding DNA sequence. Since BT-R, expression is mostly tissue-specific, the
expression control elements are particularly useful in directing the
expression of an
3 0 introduced transgene in a tissue specific fashion. A skilled artisan can
readily use the
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BT-R, gene promoter and other regulatory elements to generate expression
vectors
using methods known in the art.
Without further description, it is believed that one of ordinary skill in the
art
can, using the preceding description and the following illustrative examples,
make and
utilize the compounds of the present invention and practice the claimed
methods. The
following working examples therefore, specifically point out preferred
embodiments
of the present invention, and are not to be construed as limiting in any way
the
remainder of the disclosure.
Example 1
Purification and Sequence Determination of BT-R, Protein
Midguts of M. sexta were extracted and the BT-R, protein purified according
to the method of Vadlamudi, R.K. et al. JBiol Chem (1993) 268:1233, referenced
above and incorporated herein by reference. The electroeiuted band was
confirmed to
contain BT-R, protein by binding to'ZSI-crylAb toxin. In gel electrophoresis,
the
protein bound to toxin had an apparent weight of approximately 210 kD under
reducing and nonreducing conditions.
The purified electroeluted BT-R, was subjected to cyanogen bromide digestion
and the cyanogen bromide fragments separated on a 17% high-resolution tricine
SDS-
2 0 polyacrylamide gel as described by Schagger, H. et al. Anal Biochem (
1987) 166:368.
The separated fragments were transferred to Problott membranes (Applied
Biosystems) and five bands were extracted and subjected to microsequencing
using
standard instrumentation. The amino acid sequences obtained were:
1. (Met)-Leu-Asp-Tyr-Glu-Val-Pro-Glu-Phe-Gln-Ser-Ire-Thr-Ire-Arg-
Val-Val-Ala-Thr-Asp-Asn-Asn-Asp-Thr-Arg-His-Val-Gly-Val-Ala;
2. (Met)-X-Glu-Thr-Tyr-Glu-Leu-Ire-Ire-His-Pro-Phe-Asn-Tyr-Tyr-Ala;
3. (Met)-X-X-X-His-Gln-Leu-Pro-Leu-Ala-Gln-Asp-Ire-Lys-Asn-His;
4. (Met)-Phe/Pro-Asn/Ire-Val-Arg/Tyr-Val-Asp-Ile/Gly;
S. (Met)-Asn-Phe-Phe/His-Ser-Val-Asn-Arg/Asp-Glu.
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Example 2
Recovery of cDNA
An M. sexta cDNA library was constructed from midgut tissue in ~.gtl0 using
the Superscript Choice System according to the manufacturer's instructions
(Life
Technologies, Inc.). Degenerate oligonucleotide probes were constructed based
on
the peptide sequences determined in Example 1 using the methods and approach
described in Zhang, S. et al. Gene ( 1991 ) 105:61. Synthetic oligonucleotides
corresponding to peptides 1-3 of Example 1 were labeled with a3zP using
polynucleotide kinase and used as probes as described in the standard cloning
manual
of Maniatis, T. et al. Molecular Cloning: A Laboratory Manual (Cold Spring
Harbor
Laboratory, Cold Spring Harbor, New York, 2nd ed. 1989). A clone hybridizing
to all
three probes identified from 40 positive clones as hybridizing to all three of
the probes
was plaque-purified from a screen of 4 X 105 recombinants and subcloned into
pBluescript (Stratagene). It contained an insert of 5571 bp.
Double-stranded cDNA in pBluescript was sequenced in both directions by the
dideoxy termination method with Sequanase (USB) according to the
manufacturer's
instructions. The sequencing showed an open reading frame of 4584 base pairs
or
1528 amino acids along with a polyadenylation signal at position 5561. The
sequence
obtained and the deduced amino acid sequence is shown in Figure 1.
2 0 Thus, the deduced protein has a molecular mass of 172 kD and a pI of
approximately 4.5. The amino acid sequences of the cyanogen bromide fragments
of
native receptor match perfectly within the deduced amino acid sequence. The
open
reading frame begins with an ATG that is flanked by the consensus translation
initiation sequence GAGATGG for eucaryotic mRNAs as described by Kozak, M.
2 5 Nucleic Acids Res ( 1987) 15 :8125.
As shown in Figure 1, the deduced amino acid sequence includes a putative
signal, shown underlined, preceding the mature N-terminus Asn-Glu-Arg-etc.
Eleven
repeats (cadl-cadl l) are shown in the extracellular region upstream of the
membrane
domain, shown with the heavy underline, at positions 1406-1427. The end of the
1 lth
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repeat is shown with an arrowhead. The positions of the five CNBR fragments
are
also shown under the complete sequence.
Figure 2 compares the BT-R, sequence obtained herein with other members of
the cadherin family. Like known cadherins, the external domain of BT-R, is
highly
repetitive and contains 11 repeats {cad!-cad! l; see Figure 2 A). The other
cadherins
compared in Figure 2 B are mouse P cadherin (mP EC 1 ); Drosophila fat EC 18
(fat
EC 18) and protocadherin (PC42 EC2), and Manduca sexta intestinal transporter
(HPT-1-EC-1). The eleven repeats of the cadherin motif in BT-R, (cad!-cad! 1)
are
individually aligned with a single motif sequence from each of the other
members of
the cadherin family. Conserved residues are boxed. The greatest similarity of
BT-R,
to the cadherins is with the extracellular repeats of the cadherin motif of
mouse P-
cadherin, Drosophila fat tumor suppresser and the protocadherins, although
homologies are not high (20-40 homology and 30-60 percent similarity). The
conserved repeats of BT-R, included AXDXD, DXE, DXNDXXP, one glutamic acid
residue and two glycine residues (Figure 2 B). Motifs A/VXDXD, DXNDN are the
consensus sequences for calcium binding and two such regions are present in a
typical
cadherin repeat. In all repeats of BT-R" the sequence DXNDN is preceded by 8
to 14
hydrophobic amino acids. Similar hydrophobic sequences also have been observed
in
the cadherins. The length of the hydrophobic stretches suggests that these
areas are
2 o not transmembrane regions buy that the represent J-sheet structures
commonly present
in cadherin-like repeats. BT-R, contains a putative cytoplasmic domain of 101
amino
acids, smaller than vertebrate cadherin cytoplasmic domains (160 amino acids),
and
shows no homology to any of the cadherin cytoplasmic domains or to cytoplasmic
domains of other proteins to which it has been compared in a current sequence
data
2 5 base.
To confirm that the sequenced clone encoded full-length BT-R, protein, total
mRNA was prepared from midgets of M. sexta subjected to Northern blot by
hybridization with the antisense 4.8 kb SacI fragment of the BT-R, cDNA clone.
The
Northern blot analysis was conducted by hybridizing to the antisense probe at
42°C
3 0 and 50% formamide, 5 X Denhardt's Reagent, 5 X SSCP and 50 p.g/ml salmon
sperm
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DNA. The filter was then washed two times with 1 X SSC + 0.1 % SDS and two
times with 0.15 X SSC + 0.1% SDS at 42°C. Each wash was roughly 20
minutes.
The filter was then exposed to X-ray film for 24 hours. The 4.8 kb probe
hybridized
to a single 5.6 kb band.
The BT-R, clone was translated using rabbit reticulolysate and the resulting
translated products were immunoprecipitated with antisera raised against
native
protein encoded by BT-R,. For the in vitro translation, pBluescript plasmid
containing BT-R, cDNA was linearized and transcribed with T3 polymerase
(Pharmacia). The translation was conducted according to manufacturer's
instructions
l0 with nuclease-treated rabbit reticulolysate (Life Technologies, Inc.).
After one hour
of incubation at 30°C, the reaction mixture was combined with an equal
volume of
SDS buffer or lysed with 50 mM Tris buffer containing 1 % NP40 and 250 mM NaCI
(pH 8.0) for immunoprecipitation. Preimmune serum was used as a control.
Translation and immunoprecipitation products were electrophoresed on a 7.5%
SDS-
polyacrylamide gel fixed, treated with Enhance (Dupont NEN), dried and exposed
to
X-ray film for 12 hours.
Two protein bands of approximately 172 kD and 150 kD as determined by
SDS-PAGE were obtained; it is postulated that the 150 kD translation product
was
due to initiation of translation from an internal methionine at amino acid
242. This is
2 0 consistent with the observations of Kozak, M. Mol Cell Biol ( 1989)
9:5073.
Thus, both results confirm that a full-length clone was obtained.
Example 3
Recombinant Production and Characteristics of the BT-R, Protein
2 5 The BT-R, cDNA clone was subcloned into the mammalian expression vector
pcDNA3 (Invitrogen) and the construct transfected into COS-7 cells. Membranes
isolated from the COS-7 transfectants were solubilized, electrophoresed and
ligand
blotted with'ZSI-CrylAb toxin. The cells were harvested 60 hours after
transfection,
washed with phosphate-buffered saline and lysed by freezing in liquid
nitrogen. Cell
3 0 membranes were prepared by differential centrifugation as described by
Elshourbagy,
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N.A. et al. JBiol Ckem (1993) 266:3873. Control cells were COS-7 cells
transfected
with pcDNA3.
The cell membranes (10 p.g) were separated on 7.5% SDS-PAGE blotted to a
nylon membrane and blocked with Tris-buffered saline containing 5% nonfat dry
milk
powder, 5% glycerol and 1% Tween-20. The nylon membrane was then incubated
with'ZSI-CryIAb toxin (2 X 105 cpm/ml) for two hours with blocking buffer,
dried and
exposed to X-ray film at -70°C. The labeled toxin bound to a 210 ~ 5 kD
protein; the
210 kD band was observed only in lanes containing membranes prepared from
either
M. sexta or COS-7 cells transfected with the BT-R, cDNA construct containing
4810
by of cDNA comprising the open reading frame.
The discrepancy between the 210 kD protein expressed and the calculated 172
kD molecular weight is due to glycosylation of the protein; in vitro
translation of the
cDNA clone, as described above, which does not result in glycosylation, does
produce
the 172 kD protein. To verify this, the COS-7 produced protein was subjected
to
digestion with N-glycosidase-F by first denaturing the purified protein by
boiling in
1 % SDS for 5 minutes followed by addition of NP-40 to a final concentration
of 1
in the presence of 0.1 % SDS, and then incubating the denatured protein in
sodium
phosphate buffer, pH 8.5 at 37°C with N-glycosidase-F for 10 hours.
Controls were
incubated under the same conditions without enzyme. Digestion products were
2 0 separated on a 7.5% SDS-PAGE and stained with Coomassie brilliant blue.
This
glycosidase treatment reduced the molecular weight of BT-R, protein from 210
to 190
kD; this indicates N-glycosylation at some of the 16 consensus N-glycosylation
sites
in the protein. Treatment of BT-R, with O-glycosidase and neuraminidase did
not
alter the mobility of the protein.
2 5 In addition, embryonic 293 cells were transfected with the BT-R, cDNA
clone
in pcDNA3 and incubated with the labeled toxin (0.32 nM) in the presence of
increasing concentrations (0 to 10-6 M) of unlabeled toxin. Nonspecific
binding was
measured as bound radioactivity in the presence of 1 TM unlabeled toxin. A
value for
the dissociation constant (Kd) of 1015 pM was determined by Scatchard
analysis; this
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is approximately the same value that was obtained for the natural receptor as
described by Vadlamudi, R.K. et al. JBiol Chem (1993) (supra).
Example 4
Physiological Effect of BT Toxin on Modified Embryonic 293 Cells
Both unmodified embryonic 293 cells, and 293 cells which have been
modified to produce the BT-R, receptor as described in Example 3, when
cultured in
vitro form adherent star-shaped clusters. When BT toxin (200 nM) is added to
serum-
free medium, the clusters round up and release from the plastic surfaces of
the culture
dish. This effect is also observed under known conditions of cytotoxicity for
293
cells. The foregoing effect is observed only when the cells are cultured in
serum-free
medium since the toxin binds to serum and would thus be ineffective under
conditions
where serum is present.
However, in the presence of anti-receptor antisera, this effect of BT toxin is
blocked. Also, when serum is added back to a culture of modified E293 cells
which
has been treated in serum-free conditions with the toxin, the cells revert to
their
normal star-shaped adherent cluster shapes. This indicates that the effect of
the toxin
is reversible.
2 0 Example 5
Identification Of A Fragment Of BT-R, That Binds To A BT Toxin
To understand some of the properties of BT-R" research has been undertaken
to define the location of the BT-R,/CrylAb protein-protein interaction. The
full-
length wild-type amino acid sequence of BT-R, is provided in Fig. 1 with a
block
2 5 diagram of a possible cadherin-like structure for BT-R, shown in Fig 3. In
both
figures, restriction digest sites from the cDNA are provided relative to the
positions at
which they would disrupt the amino acid coding sequence.
A small fragment lying between the BamHI and Sacl restriction sites of wild-
type BT-R, was cloned into the vector pCITE (Novagen). This vector contains
3 0 transcription/translation sequences designed for use in a rabbit
reticulocyte lysate
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(RRL) system. The clone has been analyzed by restriction mapping and mRNA
expression (Fig. 4). Lane UP shows the uncut plasmid and lanes NP and XP show
restriction digests using NsiI and XhoI, respectively. NsiI is used because it
has only
one restriction site lying within the Bam-Sac fragment and does not cut
anywhere
within the pCITE vector. The BSP lane shows the restriction digest of the
clone
using BamHI and SacI. The digest releases the cloned fragment which separates
at
about 700 base pairs. The RT 1 and RT2 lanes show mRNA transcription from the
clone after linearization with XhoI. The mRNA separates at the expected 1350
base
pairs.
1 o Protein for analysis has been prepared from this clone in two ways. First,
an
RRL translation kit was employed to produce protein from the mRNA
transcription
reaction described above. Second, the plasmid was added directly to an RRL
based
transcription and translation (TNT) coupled kit. Protein production was
detected
using 35S-methionine as a tag (Fig. 5). The LCR lane shows production of
luciferase
protein from mRNA in an RRL kit and the LCT lane is luciferase protein from a
plasmid containing the luciferase coding sequence translated in the TNT kit.
Both are
positive controls to demonstrate that the two translation kits are
operational. The
major bands for luciferase translation are observed at 66 kDa. The lanes
labeled as
RR, and RR2 show expression of the polypeptide sequence of the Bam-Sac
fragment
2 0 of BT-R, translated from mRNA in the RRL kit. The lanes TT 1 and TT2 are
translations from the pCITE plasmid containing the Bam-Sac fragment from the
TNT
kit. All four lanes possess a major band at 30 kDa which is the expected size
of the
Bam-Sac fragment with the addition of a coded antibody tag called S-tag. S-tag
is
part of the multicloning site of pCITE.
The clone was then tested for its ability to bind the insecticidal toxin
CrylAb.
Polypeptide translation of the Bam-Sac fragment of BT-R, was carned out in
duplicate as described above. The only change is that the 35S-methionine tag
was left
out of the reaction mixtures to produce non-radiolabeled proteins. The
proteins were
separated by SDS-PAGE, blotted to nitrocellulose and hybridized with'ZSI-
labeled
3 0 Cry 1 Ab (Fig. 6). BBMV is wild-type BT-R, prepared from the midgut brush
border
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membrane vesicles (BBMV) of M. sexta, and, is used as a positive control. RBK
and
TBK are RRL and TNT control reactions prepared without mRNA or plasmid present
to determine whether proteins endogenous to either kit bind CrylAb. R, and RR2
are
translations from the RRL kit and TT 1 and TT2 are from the TNT kit. A single
30-
kDa band appears in each of these lanes. Two are marked by arrows. These bands
demonstrate that the Bam-Sac fragment of BT-R, is capable of binding CrylAb
insecticidal toxin.
To further understand the nature of this binding site, a set of truncation
mutants of BT-R, was prepared through the use of restriction digests. The cDNA
was
1 o digested at specific sites to remove increasingly larger portions of the C-
terminus.
The restriction enzymes used were Nsil, BamHI, NruI, CIaI, XhoI and StuI
(Figs. 1
and 3). The procedure involved linearizing the plasmid at each one of these
sites and
transcribing up to the truncation. The shortened mRNAs then were translated in
an
RRL kit blotted to nitrocellulose and hybridized with''SI-labeled CrylAb.
Translation of the wild-type BT-R, from the cDNA showed binding to a 172-kDa
protein band, the expected size of wild-type BT-R,. It also shows smaller
bands that
bind Cry1 Ab although the nature of these bands has not been determined. A
blank
made by preparing an RRL reaction mixture without any mRNA gaves several bands
below 66 kDa that show some type of binding of CrylAb to the reticulocytes.
The
2 0 specificity of this binding has not been determined. The truncation
mutants created by
NsiI, BamHI, NruI, CIa.I, XhoI and StuI restriction digests did not show any
binding
to CrylAb except in the region where the reticulocytes bind CrylAb. This data
demonstrates that the removal of the last 100 amino acids from wild type BT-R,
by
NsiI restriction results in the loss of the ability of BT-R, to bind CrylAb.
This
2 5 localizes the toxin binding site on the BT-R, clone and provides a soluble
fragment of
the receptor that can be used in toxin and other binding studies.
A clone of a fragment of BT-R" called the Bam-Sac fragment, has been
prepared. It was prepared using BamHI and SacI restriction digests (Fig. 1 )
and
cloning of the resulting fragment into a vector called pCITE. The polypeptide
3 0 sequence was translated and tested for binding to the insecticidal toxin
CrylAb
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(Figure 8). The Bam-Sac fragment binds to CrylAb, providing first insight into
the
location of the CrylAb binding site within the BT-R, sequence. It lies in the
last 234
C-terminal amino acids. This evidence is further supported by a set of
truncation
mutants that has been prepared. Removal of the 100 most C-terminal amino acids
from wild type BT-R, results in the loss of CrylAb binding. The C-terminal end
of
BT-R, is the location of the CrylAb binding site.
Example 6
Identification Of Homologue of BT-R, That Binds To A BT Toxin
1 o Western blots of tissue extracts prepared from Pink bollworm and European
com
borer were prepare and probed with labeled Cryl a (Figure 7). The results show
that
homologues of BT-R, are present in these two insects and can be readily
isolated
using the methods described herein.
