Bacillus thuringiensis CryET29 compositions toxic to coleopteran insects and ctenocephalides SPP

Disclosed is a novel .delta.-endotoxin, designated CryET29, that exhibits insecticidal activity against siphonapteran insects, including larvae of the cat flea (Ctenocephalides felis), as well as against colcopteran insects, including the southern corn rootworm (Diabrotica undecimpunctata), western corn rootworm (D. virgifera), Colorado potato beetle (Leptinotarsa decemlineata), Japanese beetle (Popillia japonica), and red flour beetle (Tribolium castaneur). Also disclosed are nucleic acid segments encoding CryET29, recombinant vectors, host cells, and transgenic plants comprising a cryET29 DNA segment. Methods for making and using the disclosed protein and nucleic acid segments are disclosed as well as assays and diagnostic kits for detecting cryET29 and CryET29 sequences in vivo and in vitro.

1. BACKGROUND OF THE INVENTION 
1.1 Field of the Invention 
The present invention relates generally to the fields of molecular biology. 
More particularly, certain embodiments concern methods and compositions 
comprising DNA segments, and proteins derived from bacterial species. More 
particularly, it concerns a novel cryET29 gene from Bacillus thuringiensis 
encoding a coleopteran- and cat flea-toxic crystal protein. Various 
methods for making and using these DNA segments, DNA segments encoding 
synthetically-modified CryET29 proteins, and native and synthetic crystal 
proteins are disclosed, such as, for example, the use of DNA segments as 
diagnostic probes and templates for protein production, and the use of 
proteins, fusion protein carriers and peptides in various immunological 
and diagnostic applications. Also disclosed are methods of making and 
using nucleic acid segments in the development of transgenic plant cells 
containing the DNA segments disclosed herein. 
1.2 Description of the Related Art 
1.2.1 Bacillus thuringiensis Crystal Proteins 
Bacillus thuringiensis is a Gram-positive bacterium that produces 
.delta.-endotoxins known as crystal proteins which are specifically toxic 
to certain orders and species of insects. Many different strains of B. 
thuringiensis have been shown to produce insecticidal crystal proteins. 
Compositions including B. thuringiensis strains which produce insecticidal 
proteins have been commercially available and used as environmentally 
acceptable insecticides because they are quite toxic to the specific 
target insect, but are harmless to plants and other non-targeted 
organisms. 
The B. thuringiensis crystal protein is toxic in the insect only after 
ingestion when the alkaline pH and proteolytic enzymes in the insect 
mid-gut solubilize the crystal protein and release the toxic components. 
These components disrupt the mid-gut cells causing the insect to cease 
feeding and, eventually to die. In fact, B. thuringiensis has proven to be 
an effective and environmentally safe insecticide in dealing with various 
insect pests. 
As noted by Hofte et al., (1989) the majority of insecticidal B. 
thuringiensis strains are active against insect of the order Lepidoptera, 
i.e., caterpillar insects. Other B. thuringiensis strains are 
insecticidally active against insects of the order Diptera, i.e., flies 
and mosquitoes, or against both lepidopteran and dipteran insects. In 
recent years, a few B. thuringiensis strains have been reported as 
producing crystal proteins that are toxic to insects of the order 
Coleoptera, i.e., beetles. To date, there have been no reports of B. 
thuringiensis strains active on fleas of the Genus, Ctenocephalides, in 
the order Siphonaptera. 
The dipteran-active Cyt toxins differ from most of the other B. 
thuringiensis insecticidal crystal proteins in that they are smaller and 
do not share conserved blocks of sequence homology. These proteins 
demonstrate broad cytolytic activity in vitro, yet are specifically toxic 
to larvae of dipteran insects in vivo. These properties have been 
described elsewhere (Chilcott and Ellar, 1988). 
1.2.2 Genetics of Crystal Proteins 
A number of genes encoding crystal proteins have been cloned from several 
strains of B. thuringiensis. The review by Hofte et al. (1989) discusses 
the genes and proteins that were identified in B. thuringiensis prior to 
1990, and sets forth the nomenclature and classification scheme which has 
traditionally been applied to B. thuritigiensis genes and proteins. cryI 
genes encode lepidopteran-toxic CryI proteins. cryII genes encode CryII 
proteins that are toxic to both lepidopterans and dipterans. cryIII genes 
encode coleopteran-toxic CryII proteins, while cryIV genes encode 
dipterantoxic CryIV proteins. 
Recently a new nomenclature has been proposed which systematically 
classifies the cry genes based upon DNA sequence homology rather than upon 
insect specificities. This classification scheme is shown in Table 1. 
TABLE 1 
______________________________________ 
Revised B. thuringiensis .delta.-Endotoxin Gene Nomenclature.sup.a 
New Old GenBank Accession # 
______________________________________ 
Cry1Aa CryIA(a) M11250 
Cry1Ab CryIA(b) M13898 
Cry1Ac CryIA(c) M11068 
Cry1Ad CryIA(d) M73250 
Cry1Ae CryIA(e) M65252 
Cry1Ba CryIB X06711 
Cry1Bb ET5 L32020 
Cry1Bc PEG5 Z46442 
Cry1Ca CryIC X07518 
Cry1Cb CryIC(b) M97880 
Cry1Da CryID X54160 
Cry1Db PrtB Z22511 
Cry1Ea CryIE X53985 
Cry1Eb CryIE(b) M73253 
Cry1Fa CryIF M63897 
Cry1Fb PrtD Z22512 
Cry1G PrtA Z22510 
Cry1H PrtC Z22513 
Cry1Hb U35780 
Cry1Ia CryV X62821 
Cry1Ib CryV U07642 
Cry1Ja ET4 L32019 
Cry1Jb ET1 U31527 
Cry1K U28801 
Cry2Aa CryIIA M31738 
Cry2Ab CryIIB M23724 
Cry2Ac CryIIC X57252 
Cry3A CryIIIA M22472 
Cry3Ba CryIIIB X17123 
Cry3Bb CryIIIB2 M89794 
Cry3C CryIIID X59797 
Cry4A CryIVA Y00423 
Cry4B CryIVB X07423 
Cry5Aa CryVA(a) L07025 
Cry5Ab CryVA(b) L07026 
Cry5B U19725 
Cry6A CryVIA L07022 
Cry6B CryVIB L07024 
Cry7Aa CryIIIC M64478 
Cry7Ab CryIIICb U04367 
Cry8A CryIIIE U04364 
Cry8B CryIIIG U04365 
Cry8C CryIIIF U04366 
Cry9A CryIG X58120 
Cry9B CryIX X75019 
Cry9C CryIH Z37527 
Cry10A CryIVC M12662 
Cry11A CryIVD M31737 
Cry11B Jeg80 X86902 
Cry12A CryVB L07027 
Cry13A CryVC L07023 
Cry14A CryVD U13955 
Cry15A 34kDa M76442 
Cry16A cbm71 X94146 
Cyt1A CytA X03182 
Cyt2A CytB Z14147 
To Be Assigned 
CryET29, Present Invention 
To Be Assigned 
______________________________________ 
.sup.a Adapted from: http://www.susx.ac.uk:80/users/bafn6/bt/index.html 
1.2.3 Identification of Crystal Proteins Toxic To Coleopteran Insects 
The cloning and expression of a gene encoding a 26-kDa mosquitocidal toxin 
from the dipteran-active B. thuringiensis var. israelensis has been 
described (Ward et al., 1984), and the nucleotide sequence of this gene 
was reported (Ward and Ellar, 1986). The molecular mass of the toxin 
protein, CytA, calculated from the deduced amino acid sequence was 
determined to be 27,340 Da. 
The nucleotide sequence of the gene for a 27-kDa mosquitocidal Cyt protein 
isolated from B. thuringiensis var. morrisoni strain PG14 has been 
disclosed (Earp and Ellar, 1987). The sequence of this toxin protein was 
found to differ by only one amino acid residue from the CytIA protein of 
B. thuringiensis var. israelensis. 
The identification of a 25-kDa protein that exhibits cytolytic activity in 
vitro when activated by proteolysis from the mosquitocidal B. 
thuringiensis var. kyushuensis was described earlier (Knowles et al., 
1992), and the nucleotide sequence of the gene for this protein, CytB, was 
reported (Koni and Ellar, 1993). The predicted molecular mass of the CytB 
protein is 29,236 Da and the deduced amino acid sequence is quite 
distinct, although it does share significant sequence similarity with the 
CytA protein of B. thuringiensis var. israelensis. 
The cloning and characterization of the gene for a 30-kDa toxin protein 
with activity on coleopteran and dipteran insects has been described 
(Intl. Pat. Appl. Pub. No. WO 95/02693, 1995). This gene, isolated from B. 
thuringiensis PS201T6, encodes a protein of 29,906 Da which exhibits a 64% 
sequence identity with the CytA toxin of B. thuringiensis var. 
israelensis. 
2. SUMMARY OF THE INVENTION 
The present invention provides a novel B. thuringiensis insecticidal 
crystal protein (designated CryET29) and the gene which encodes it 
(designated cryET29) which contain amino acid and nucleic acid sequences, 
respectively, showing little homology to the .delta.-endotoxin proteins 
and genes of the prior art. Suprisingly, the CryET29 protein of the 
present invention demonstrates remarkable insecticidal activity against 
not only insects of the order Coleoptera, but also against fleas, and in 
particular larvae of the cat flea, Ctenocephalides felis. 
In one important embodiment, the invention provides an isolated and 
purified amino acid segment comprising a B. thuringiensis CryET29 
insecticidal crystal protein (SEQ ID NO:2) comprising the amino acid 
sequence illustrated in FIG. 1A and FIG. 1B. The coding region for the 
CryET29 protein is from nucleotide 29 to 721 of SEQ ID NO:1. The CryET29 
protein exhibits insecticidal activity against Coleopterans such as the 
southern corn rootworm, western corn rootworm, Colorado potato beetle, 
Japanese beetle, and red flour beetle. In related embodiments, methods for 
making and using this protein, derivatives and mutants thereof, and 
antibodies directed against these proteins are also disclosed. 
In another important embodiment, the invention provides an isolated and 
purified nucleic acid segment comprising the cryET29 gene which encodes 
the CryET29 crystal protein disclosed herein. The nucleotide sequence of 
the cryET29 gene is given in SEQ ID NO:1 and illustrated in FIG. 1A and 
FIG. 1B. In related embodiments, methods for making, using, altering, 
mutagenizing, assaying, and quantitating these nucleic acid segments are 
also disclosed. Also disclosed are diagnostic methods and assay kits for 
the identification and detection of related cry gene sequences in a 
variety of in vitro and in vivo methodologies. 
Another aspect of the present invention is a Bacillus thuringiensis cell 
that produces a CryET29 crystal protein. In a preferred embodiment, the 
cell is a Bacillus thuringiensis bacterial strain designated B. 
thuringiensis EG4096 which has been deposited with the Agricultural 
Research Culture Collection, Northern Regional Research Laboratory (NRRL), 
on May 30, 1996 and assigned the Accession No. NRRL B-21582. B. 
thuringiensis EG4096, further described in Examples 1, 2, and 3, is a 
naturally-occurring bacterium that comprises a cryET29 gene (SEQ ID NO: 1) 
of the present invention. EG4096 produces a novel insecticidal crystal 
protein of approximately 26-kDa, which the inventors have designated 
CryET29 (SEQ ID NO:2). Most preferrably, the Bacillus thuringiensis cell 
has the NRRL accession number NRRL B-21582. 
A further aspect of the present invention is a plasmid, cosmid, or vector 
that comprises the nucleic acid sequence of a whole or a portion of the 
cryET729 gene (SEQ NO ID:1), a transformed host cell comprising a native 
or recombinant (cryET29 gene, a culture of a recombinant bacterium 
transformed with such plasmid, the bacterium preferably being B. 
thuringiensis such as the recombinant strains EG11494 and EG11502, 
described in Example 7, and most preferably a biologically-pure culture of 
such a bacterial strain. EG11494 was deposited on May 30, 1996 under the 
terms of the Budapest Treaty with the NRRL and given the Accession number 
NQRRL B-21583. Alternatively, the E. coli recombinant strains EG1 1513 and 
EG11514 comprising the novel cryET29 gene, are also preferred hosts for 
expression of the CryET29 protein. 
2.1 cryET29 DNA Segments 
The present invention also concerns DNA segments, that can be isolated from 
virtually any source, that are free from total genomic DNA and that encode 
the whole or a portion of the novel peptides disclosed herein. The cryET29 
gene (SEQ ID NO:1; FIG. 1A and FIG. 1B) encodes the 26-kDa CryET29 protein 
having an amino acid sequence shown in FIG. 1A and FIG. 1B (SEQ ID NO:2). 
DNA segments encoding these peptide species may prove to encode proteins, 
polypeptides, subunits, functional domains, and the like of crystal 
protein-related or other non-related gene products. In addition these DNA 
segments may be synthesized entirely in vitro using methods that are 
well-known to those of skill in the art. 
As used herein, the term "DNA segment" refers to a DNA molecule that has 
been isolated free of total genomic DNA of a particular species. 
Therefore, a DNA segment encoding a crystal protein or peptide refers to a 
DNA segment that contains crystal protein coding sequences yet is isolated 
away from, or purified free from, total genomic DNA of the species from 
which the DNA segment is obtained, which in the instant case is the genome 
of the Gram-positive bacterial genus, Bacillus, and in particular, the 
species known as B. thuringiensis. Included within the term "DNA segment", 
are DNA segments and smaller fragments of such segments, and also 
recombinant vectors, including, for example, plasmids, cosmids, phagemids, 
phage, viruses, and the like. 
Similarly, a DNA segment comprising an isolated or purified crystal 
protein-encoding gene refers to a DNA segment which may include in 
addition to peptide encoding sequences, certain other elements such as, 
regulatory sequences, isolated substantially away from other naturally 
occurring genes or protein-encoding sequences. In this respect, the term 
"gene" is used for simplicity to refer to a functional protein-, 
polypeptide- or peptide-encoding unit. As will be understood by those in 
the art, this functional term includes not only genomic sequences, 
including extrachromosomal DNA sequences, but also operon sequences and/or 
engineered gene segments that express, or may be adapted to express, 
proteins, polypeptides or peptides. 
"Isolated substantially away from other coding sequences" means that the 
gene of interest, in this case, a gene encoding a bacterial crystal 
protein, forms the significant part of the coding region of the DNA 
segment, and that the DNA segment does not contain large portions of 
naturally-occurring coding DNA, such as large chromosomal fragments or 
other functional genes or operon coding regions. Of course, this refers to 
the DNA segment as originally isolated, and does not exclude genes, 
recombinant genes, synthetic linkers, or coding regions later added to the 
segment by the hand of man. 
In particular embodiments, the invention concerns isolated DNA segments and 
recombinant vectors incorporating DNA sequences that encode a Cry protein 
or peptide species that includes within its amino acid sequence an amino 
acid sequence essentially as set forth in SEQ ID NO:2. More preferably, 
the DNA sequence comprises a nucleic acid sequence that encodes a Cry 
protein or peptide species that includes within its amino acid sequence an 
at least ten amino acid contiguous sequence of SEQ ID NO:2. 
The term "a sequence essentially as set forth in SEQ ID NO:2," means that 
the sequence substantially corresponds to a portion of the sequence of SEQ 
ID NO:2 and has relatively few amino acids that are not identical to, or a 
biologically functional equivalent of, the amino acids of any of these 
sequences. The term "biologically functional equivalent" is well 
understood in the art and is further defined in detail herein (e.g., see 
Illustrative Embodiments). Accordingly, sequences that have between about 
70% and about 80%, or more preferably between about 81% and about 90%, or 
even more preferably between about 91 % and about 99% amino acid sequence 
identity or functional equivalence to the amino acids of SEQ ID NO:2 will 
be sequences that are "essentially as set forth in SEQ ID NO:2." 
It will also be understood that amino acid and nucleic acid sequences may 
include additional residues, such as additional N- or C-terminal amino 
acids or 5' or 3' sequences, and yet still be essentially as set forth in 
one of the sequences disclosed herein, so long as the sequence meets the 
criteria set forth above, including the maintenance of biological protein 
activity where protein expression is concerned. The addition of terminal 
sequences particularly applies to nucleic acid sequences that may, for 
example, include various non-coding sequences flanking either of the 5' or 
3' portions of the coding region or may include various internal 
sequences, i.e., introns, which are known to occur within genes. 
The nucleic acid segments of the present invention, regardless of the 
length of the coding sequence itself, may be combined with other DNA 
sequences, such as promoters, polyadenylation signals, additional 
restriction enzyme sites, multiple cloning sites, other coding segments, 
and the like, such that their overall length may vary considerably. It is 
therefore contemplated that a nucleic acid fragment of almost any length 
may be employed, with the total length preferably being limited by the 
ease of preparation and use in the intended recombinant DNA protocol. For 
example, nucleic acid fragments may be prepared that include a short 
contiguous stretch encoding the whole or a portion of the peptide sequence 
disclosed in SEQ ID NO:2, or that are identical to or complementary to DNA 
sequences which encode the peptidc disclosed in SEQ ID NO:2, and 
particularly the DNA segment disclosed in SEQ ID NO:1. For example, DNA 
sequences such as about 14 nucleotides, and that are up to about 10,000, 
about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, 
about 100, about 50, and about 14 base pairs in length (including all 
intermediate lengths) are also contemplated to be useful. 
It will be readily understood that "intermediate lengths", in these 
contexts, means any length between the quoted ranges, such as 14, 15, 16, 
17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, 
etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all 
integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 
3,000-5,000; and up to and including sequences of about 10,000 nucleotides 
and the like. 
It will also be understood that this invention is not limited to the 
particular nucleic acid sequences which encode peptides of the present 
invention, or which encode the amino acid sequence of SEQ ID NO:2, 
including the DNA sequence which is particularly disclosed in SEQ ID NO:1. 
Recombinant vectors and isolated DNA segments may therefore variously 
include the peptide-coding regions themselves, coding regions bearing 
selected alterations or modifications in the basic coding region, or they 
may encode larger polypeptides that nevertheless include these 
peptide-coding regions or may encode biologically functional equivalent 
proteins or peptides that have variant amino acids sequences. 
The DNA segments of the present invention encompass 
biologically-functional, equivalent peptides. Such sequences may arise as 
a consequence of codon redundancy and functional equivalency that are 
known to occur naturally within nucleic acid sequences and the proteins 
thus encoded. Alternatively, functionally-equivalent proteins or peptides 
may be created via the application of recombinant DNA technology, in which 
changes in the protein structure may be engineered, based on 
considerations of the properties of the amino acids being exchanged. 
Changes designed by man may be introduced through the application of 
site-directed mutagenesis techniques, e.g., to introduce improvements to 
the antigenicity of the protein or to test mutants in order to examine 
activity at the molecular level. 
If desired, one may also prepare fusion proteins and peptides, e.g., where 
the peptide-coding regions are aligned within the same expression unit 
with other proteins or peptides having desired functions, such as for 
purification or immunodetection purposes (e.g., proteins that may be 
purified by affinity chromatography and enzyme label coding regions, 
respectively). 
Recombinant vectors form further aspects of the present invention. 
Particularly useful vectors are contemplated to be those vectors in which 
the coding portion of the DNA segment, whether encoding a full length 
protein or smaller peptide, is positioned under the control of a promoter. 
The promoter may be in the form of the promoter that is naturally 
associated with a gene encoding peptides of the present invention, as may 
be obtained by isolating the 5' non-coding sequences located upstream of 
the coding segment or exon, for example, using recombinant cloning and/or 
PCR.TM. technology, in connection with the compositions disclosed herein. 
2.2 DNA Segments as Hybridization Probes and Primers 
In addition to their use in directing the expression of crystal proteins or 
peptides of the present invention, the nucleic acid sequences contemplated 
herein also have a variety of other uses. For example, they also have 
utility as probes or primers in nucleic acid hybridization embodiments. As 
such, it is contemplated that nucleic acid segments that comprise a 
sequence region that consists of at least a 14 nucleotide long contiguous 
sequence that has the same sequence as, or is complementary to, a 14 
nucleotide long contiguous DNA segment of SEQ ID NO:1 will find particular 
utility. Longer contiguous identical or complementary sequences, e.g., 
those of about 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, 10000 etc. 
(including all intermediate lengths and up to and including full-length 
sequences) will also be of use in certain embodiments. 
The ability of such nucleic acid probes to specifically hybridize to 
crystal protein-encoding sequences will enable them to be of use in 
detecting the presence of complementary sequences in a given sample. 
However, other uses are envisioned, including the use of the sequence 
information for the preparation of mutant species primers, or primers for 
use in preparing other genetic constructions. 
Nucleic acid molecules having sequence regions consisting of contiguous 
nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 
nucleotides or so, identical or complementary to the DNA sequence of SEQ 
ID NO:1, are particularly contemplated as hybridization probes for use in, 
e.g., Southern and Northern blotting. Smaller fragments will generally 
find use in hybridization embodiments, wherein the length of the 
contiguous complementary region may be varied, such as between about 10-14 
and about 100 or 200 nucleotides, but larger contiguous complementarity 
stretches may be used, according to the length complementary sequences one 
wishes to detect. 
The use of a hybridization probe of about 14 nucleotides in length allows 
the formation of a duplex molecule that is both stable and selective. 
Molecules having contiguous complementary sequences over stretches greater 
than 14 bases in length are generally preferred, though, in order to 
increase stability and selectivity of the hybrid, and thereby improve the 
quality and degree of specific hybrid molecules obtained. One will 
generally prefer to design nucleic acid molecules having 
gene-complementary stretches of 15 to 20 contiguous nucleotides, or even 
longer where desired. 
Of course, fragments may also be obtained by other techniques such as, 
e.g., by mechanical shearing or by restriction enzyme digestion. Small 
nucleic acid segments or fragments may be readily prepared by, for 
example, directly synthesizing the fragment by chemical means, as is 
commonly practiced using an automated oligonucleotide synthesizer. Also, 
fragments may be obtained by application of nucleic acid reproduction 
technology, such as the PCR.TM. technology of U.S. Pat. Nos. 4,683,195 and 
4,683,202 (each incorporated herein by reference), by introducing selected 
sequences into recombinant vectors for recombinant production, and by 
other recombinant DNA techniques generally known to those of skill in the 
art of molecular biology. 
Accordingly, the nucleotide sequences of the invention may be used for 
their ability to selectively form duplex molecules with complementary 
stretches of DNA fragments. Depending on the application envisioned, one 
will desire to employ varying conditions of hybridization to achieve 
varying degrees of selectivity of probe towards target sequence. For 
applications requiring high selectivity, one will typically desire to 
employ relatively stringent conditions to form the hybrids, e.g., one will 
select relatively low salt and/or high temperature conditions, such as 
provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 
50.degree. C. to about 70.degree. C. Such selective conditions tolerate 
little, if any, mismatch between the probe and the template or target 
strand, and would be particularly suitable for isolating crystal 
protein-encoding DNA segments. Detection of DNA segments via hybridization 
is well-known to those of skill in the art, and the teachings of U.S. Pat. 
Nos. 4,965,188 and 5,176,995 (each incorporated herein by reference) are 
exemplary of the methods of hybridization analyses. Teachings such as 
those found in the texts of Maloy et al., 1994; Segal 1976; Prokop, 1991; 
and Kuby, 1991, are particularly relevant. 
Of course, for some applications, for example, where one desires to prepare 
mutants employing a mutant primer strand hybridized to an underlying 
template or where one seeks to isolate crystal protein-encoding sequences 
from related species, functional equivalents, or the like, less stringent 
hybridization conditions will typically be needed in order to allow 
formation of the heteroduplex. In these circumstances, one may desire to 
employ conditions such as about 0.15 M to about 0.9 M salt, at 
temperatures ranging from about 20.degree. C. to about 55.degree. C. 
Cross-hybridizing species can thereby be readily identified as positively 
hybridizing signals with respect to control hybridizations. In any case, 
it is generally appreciated that conditions can be rendered more stringent 
by the addition of increasing amounts of formamide, which serves to 
destabilize the hybrid duplex in the same manner as increased temperature. 
Thus, hybridization conditions can be readily manipulated, and thus will 
generally be a method of choice depending on the desired results. 
In certain embodiments, it will be advantageous to employ nucleic acid 
sequences of the present invention in combination with an appropriate 
means, such as a label, for determining hybridization. A wide variety of 
appropriate indicator means are known in the art, including fluorescent, 
radioactive, enzymatic or other ligands, such as avidin/biotin, which are 
capable of giving a detectable signal. In preferred embodiments, one will 
likely desire to employ a fluorescent label or an enzyme tag, such as 
urease, alkaline phosphatase or peroxidase, instead of radioactive or 
other environmental undesirable reagents. In the case of enzyme tags, 
calorimetric indicator substrates are known that can be employed to 
provide a means visible to the human eye or spectrophotometrically, to 
identify specific hybridization with complementary nucleic acid-containing 
samples. 
In general, it is envisioned that the hybridization probes described herein 
will be useful both as reagents in solution hybridization as well as in 
embodiments employing a solid phase. In embodiments involving a solid 
phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a 
selected matrix or surface. This fixed, single-stranded nucleic acid is 
then subjected to specific hybridization with selected probes under 
desired conditions. The selected conditions will depend on the particular 
circumstances based on the particular criteria required (depending, for 
example, on the G+C. content, type of target nucleic acid, source of 
nucleic acid, size of hybridization probe, etc.). Following washing of the 
hybridized surface so as to remove nonspecifically bound probe molecules, 
specific hybridization is detected, or even quantitated, by means of the 
label. 
2.3 Recombinant Vectors and Protein Expression 
The invention also discloses and claims a composition comprising a CryET29 
crystal protein. The composition may comprises bacterial host cells which 
express a CryET29 crystal protein, inclusion bodies or crystals containing 
the CryET29 protein, culture supernatant, disrupted cells, cell extracts, 
lysates, homogenates, and the like. The compositions may be in aqueous 
form, or alternatively, in dry, semi-wet, or similar forms such as cell 
paste, cell pellets, or alternatively freeze dried, powdered, lyophilized, 
evaporated, or otherwise similarly prepared in dry form. Such means for 
preparing crystal proteins are well-known to those of skill in the art of 
bacterial protein isolation and purification. In certain embodiments, the 
crystal proteins may be purified, concentrated, admixed with other 
reagents, or processed to a desired final form. Preferably, the 
composition will comprise from about 1% to about 90% by weight of the 
crystal protein, and more preferably from about 5% to about 50% by weight. 
In a preferred embodiment, the crystal protein compositions of the 
invention may be prepared by a process which comprises the steps of 
culturing a Bacillus thuringiensis cell which expresses a CryET29 crystal 
protein under conditions effective to produce such a protein, and then 
obtaining the protein from the cell. The obtaining of such a crystal 
protein may further include purifying, concentrating, processing, or 
admixing the protein with one or more reagents. Preferably, the CryET29 
crystal protein is obtained in an amount of from between about 1% to about 
5% toy weight and more preferably from about 5% to about 50% by weight. 
The invention also relates to a method of preparing a CryET29 crystal 
protein composition. Such a method generally involves the steps of 
culturing a Bacillus thuringiensis cell which expresses a CryET29 crystal 
protein under conditions effective to produce the protein, and then 
obtaining the protein so produced. In a preferred embodiment the Bacillus 
thuringiensis cell is an NRRL B-21582 cell, or any Bacillus thuringiensis 
cell which contains a cryET29gene segment. Alternatively, the recombinant 
plasmid vectors of the invention may be used to transform other suitable 
bacterial or eukaryotic cells to produce the crystal protein of the 
invention. Prokaryotic host cells including Gram-negative cells such as E. 
coli, Pseudomonas spp. and related Enterobacteraceae, or Gram-postive 
cells such as Bacillus spp. (including B. meegateriuni, B. subtilis, and 
B. thuringiensis) and the like are all contemplated to be useful in the 
preparation of the crystal proteins of the invention. Particularly 
preferred E. coli strains include NRRL B-21624, deposited with the NRRL 
under the terms of the Budapest Treaty. 
In such embodiments, it is contemplated that certain advantages will be 
gained by positioning the coding DNA segment under the control of a 
recombinant, or heterologous, promoter. As used herein, a recombinant or 
heterologous promoter is intended to refer to a promoter that is not 
normally associated with a DNA segment encoding a crystal protein or 
peptide in its natural environment. Such promoters may include promoters 
normally associated with other genes, and/or promoters isolated from any 
bacterial, viral, eukaryotic, or plant cell. Naturally, it will be 
important to employ a promoter that effectively directs the expression of 
the DNA segment in the cell type, organism, or even animal, chosen for 
expression. The use of promoter and cell type combinations for protein 
expression is generally known to those of skill in the art of molecular 
biology, for example, see Sambrook et al., 1989. The promoters employed 
may be constitutive, or inducible, and can be used under the appropriate 
conditions to direct high level expression of the introduced DNA segment, 
such as is advantageous in the large-scale production of recombinant 
proteins or peptides. Appropriate promoter systems contemplated for use in 
high-level expression include, but are not limited to, the Pichia 
expression vector system (Pharmacia LKB Biotechnology). 
In connection with expression embodiments to prepare recombinant proteins 
and peptides, it is contemplated that longer DNA segments will most often 
be used, with DNA segments encoding the entire peptide sequence being most 
preferred. However, it will be appreciated that the use of shorter DNA 
segments to direct the expression of crystal pcptides or epitopic core 
regions, such as may be used to generate anti-crystal protein antibodies, 
also falls within the scope of the invention. DNA segments that encode 
peptide antigens from about 8 to about 50 amino acids in length, or more 
preferably, from about 8 to about 30 amino acids in length, or even more 
preferably, from about 8 to about 20 amino acids in length are 
contemplated to be particularly useful. Such peptide epitopes may be amino 
acid sequences which comprise contiguous amino acid sequences from SEQ ID 
NO:2. 
2.4 Crystal Protein Transgenes and Transgenic Host Cells 
In yet another aspect, the present invention provides methods for producing 
a transgenic cell, and in particular a plant or animal cell which 
expresses a nucleic acid segment encoding the novel CryET29 crystal 
protein of the present invention. The process of producing transgenic 
cells is well-known in the art. In general, the method comprises 
transforming a suitable host cell with a DNA segment which contains a 
promoter operatively linked to a coding region that encodes a B. 
thuringiensis CryET29 crystal protein. Such a coding region is generally 
operatively linked to a transcription-terminating region, whereby the 
promoter is capable of driving the transcription of the coding region in 
the cell, and hence providing the cell the ability to produce the 
recombinant protein in vivo. Alternatively, in instances where it is 
desirable to control, regulate, or decrease the amount of a particular 
recombinant crystal protein expressed in a particular transgenic cell, the 
invention also provides for the expression of crystal protein antisense 
mRNA. The use of antisense mRNA as a means of controlling or decreasing 
the amount of a given protein of interest in a cell is well-known in the 
art. 
In a preferred embodiment, the invention encompasses a plant cell which has 
been transformed with a nucleic acid segment of the invention, and which 
expresses a gene or gene segment encoding one or more of the novel 
polypeptide compositions disclosed herein. As used herein, the term 
"transgenic plant cell" is intended to refer to a plant cell that has 
incorporated DNA sequences, including but not limited to genes which are 
perhaps not normally present, DNA sequences not normally transcribed into 
RNA or translated into a protein ("expressed"), or any other genes or DNA 
sequences which one desires to introduce into the non-transformed plant, 
such as genes which may normally be present in the non-transformed plant 
but which one desires to either genetically engineer or to have altered 
expression. 
It is contemplated that in some instances the genome of a transgenic plant 
of the present invention will have been augmented through the stable 
introduction of a cryET29 transgene, either native cryET29, or 
synthetically modified or mutated cryET29. In some instances, more than 
one transgene will be incorporated into the genome of the transformed host 
plant cell. Such is the case when more than one crystal protein-encoding 
DNA segment is incorporated into the genome of such a plant. In certain 
situations, it may be desirable to have one, two, three, four, or even 
more B. thuringiensis crystal proteins (either native or 
recombinantly-engineered) incorporated and stably expressed in the 
transformed transgenic plant. In preferred embodiments, the introduction 
of the transgene into the genome of the plant cell results in a stable 
integration wherein the offspring of such plants also contain a copy of 
the transgene in their genome. The inheritibility of this genetic element 
by the progeny of the plant into which the gene was originally introduced 
is a preferred aspect of this invention. 
A preferred gene which may be introduced includes, for example, a crystal 
protein-encoding a DNA sequence from bacterial origin, and particularly 
one or more of those described herein which are obtained from Bacillus 
spp. Highly preferred nucleic acid sequences are those obtained from B. 
thuringiensis, or any of those sequences which have been genetically 
engineered to decrease or increase the insecticidal activity of the 
crystal protein in such a transformed host cell. 
Means for transforming a plant cell and the preparation of a transgenic 
cell line are well-known in the art (as exemplified in U.S. Pat. Nos. 
5,550,318; 5,508,468; 5,482,852; 5,384,253; 5,276,269; and 5,225,341, all 
specifically incorporated herein by reference), and are briefly discussed 
herein. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) 
and DNA segments for use in transforming such cells will, of course, 
generally comprise either the operons, genes, or gene-derived sequences of 
the present invention, either native, or synthetically-derived, and 
particularly those encoding the disclosed crystal proteins. These DNA 
constructs can further include structures such as promoters, enhancers, 
polylinkers, or even gene sequences which have positively- or 
negatively-regulating activity upon the particular genes of interest as 
desired. The DNA segment or gene may encode either a native or modified 
crystal protein, which will be expressed in the resultant recombinant 
cells, and/or which will impart an improved phenotype to the regenerated 
plant. 
Such transgenic plants may be desirable for increasing the insecticidal 
resistance of a monocotyledonous or dicotyledonous plant, by incorporating 
into such a plant, a transgenic DNA segment encoding a CryET29 crystal 
protein which is toxic to coleopteran insects. Particularly preferred 
plants include corn, wheat, soybeans, turf grasses, ornamental plants, 
fruit trees, shrubs, vegetables, grains, legumes, and the like, or any 
plant into which introduction of a crystal protein transgene is desired. 
In a related aspect, the present invention also encompasses a seed produced 
by the transformed plant, a progeny from such seed, and a seed produced by 
the progeny of the original transgenic plant, produced in accordance with 
the above process. Such progeny and seeds will have a crystal protein 
transgene stably incorporated into its genome, and such progeny plants 
will inherit the traits afforded by the introduction of a stable transgenc 
in Mendelian fashion. All such transgenic plants having incorporated into 
their genome transgenic DNA segments encoding a CryET29 crystal protein or 
polypeptide are aspects of this invention. 
2.5 Site-Speciric Mutagenesis 
Site-specific mutagenesis is a technique useful in the preparation of 
individual peptides, or biologically functional equivalent proteins or 
peptides, through specific mutagenesis of the underlying DNA. The 
technique further provides a ready ability to prepare and test sequence 
variants, for example, incorporating one or more of the foregoing 
considerations, by introducing one or more nucleotide sequence changes 
into the DNA. Site-specific mutagenesis allows the production of mutants 
through the use of specific oligonucleotide sequences which encode the DNA 
sequence of the desired mutation, as well as a sufficient number of 
adjacent nucleotides, to provide a primer sequence of sufficient size and 
sequence complexity to form a stable duplex on both sides of the deletion 
junction being traversed. Typically, a primer of about 17 to 25 
nucleotides in length is preferred, with about 5 to 10 residues on both 
sides of the junction of the sequence being altered. 
In general, the technique of site-specific mutagenesis is well known in the 
art, as exemplified by various publications. As will be appreciated, the 
technique typically employs a phage vector which exists in both a single 
stranded and double stranded form. Typical vectors useful in site-directed 
mutagenesis include vectors such as the M13 phage. These phage are readily 
commercially available and their use is generally well known to those 
skilled in the art. Double stranded plasmids are also routinely employed 
in site directed mutagenesis which eliminates the step of transferring the 
gene of interest from a plasmid to a phage. 
In general, site-directed mutagenesis in accordance herewith is performed 
by first obtaining a single-stranded vector or melting apart of two 
strands of a double stranded vector which includes within its sequence a 
DNA sequence which encodes the desired peptide. An oligonucleotide primer 
bearing the desired mutated sequence is prepared, generally synthetically. 
This primer is then annealed with the single-stranded vector, and 
subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow 
fragment, in order to complete the synthesis of the mutation-bearing 
strand. Thus, a heteroduplex is formed wherein one strand encodes the 
original non-mutated sequence and the second strand bears the desired 
mutation. This heteroduplex vector is then used to transform appropriate 
cells, such as E. coli cells, and clones are selected which include 
recombinant vectors bearing the mutated sequence arrangement. 
The preparation of sequence variants of the selected peptide-encoding DNA 
segments using site-directed mutagenesis is provided as a means of 
producing potentially useful species and is not meant to be limiting as 
there are other ways in which sequence variants of peptides and the DNA 
sequences encoding them may be obtained. For example, recombinant vectors 
encoding the desired peptide sequence may be treated with mutagenic 
agents, such as hydroxylamine, to obtain sequence variants. 
2.6 Antibody Compositions and Methods of Producing 
In particular embodiments, the inventors contemplate the use of antibodies, 
either monoclonal or polyclonal which bind to the crystal proteins 
disclosed herein. Means for preparing and characterizing antibodies are 
well known in the art (See, e.g, Harlow and Lane, 1988; incorporated 
herein by reference). The methods for generating monoclonal antibodies 
(mAbs) generally begin along the same lines as those for preparing 
polyclonal antibodies. Briefly, a polyclonal antibody is prepared by 
immunizing an animal with an immunogenic composition in accordance with 
the present invention and collecting antisera from that immunized animal. 
A wide range of animal species can be used for the production of antisera. 
Typically the animal used for production of anti-antisera is a rabbit, a 
mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively 
large blood volume of rabbits, a rabbit is a preferred choice for 
production of polyclonal antibodies. 
As is well known in the art, a given composition may vary in its 
immunogenicity. It is often necessary therefore to boost the host immune 
system, as may be achieved by coupling a peptide or polypeptide immunogen 
to a carrier. Exemplary and preferred carriers are keyhole limpet 
hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as 
ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as 
carriers. Means for conjugating a polypeptide to a carrier protein are 
well known in the art and include glutaraldehyde, 
m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and 
bis-biazotized benzidine. 
As is also well known in the art, the immunogenicity of a particular 
immunogen composition can be enhanced by the use of non-specific 
stimulators of the immune response, known as adjuvants. Exemplary and 
preferred adjuvants include complete Freund's adjuvant (a non-specific 
stimulator of the immune response containing killed Mycobacterium 
tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide 
adjuvant. 
The amount of immunogen composition used in the production of polyclonal 
antibodies varies upon the nature of the immunogen as well as the animal 
used for immunization. A variety of routes can be used to administer the 
immunogen (subcutaneous, intramuscular, intradermal, intravenous and 
intraperitoneal). The production of polyclonal antibodies may be monitored 
by sampling blood of the immunized animal at various points following 
immunization. A second, booster, injection may also be given. The process 
of boosting and titering is repeated until a suitable titer is achieved. 
When a desired level of immunogenicity is obtained, the immunized animal 
can be bled and the serum isolated and stored, and/or the animal can be 
used to generate mAbs. 
mAbs may be readily prepared through use of well-known techniques, such as 
those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by 
reference. Typically, this technique involves immunizing a suitable animal 
with a selected immunogen composition, e.g., a purified or partially 
purified crystal protein, polypeptide or peptide. The immunizing 
composition is administered in a manner effective to stimulate antibody 
producing cells. Rodents such as mice and rats are preferred animals, 
however, the use of rabbit, sheep frog cells is also possible. The use of 
rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice 
are preferred, with the BALB/c mouse being most preferred as this is most 
routinely used and generally gives a higher percentage of stable fusions. 
Following immunization, somatic cells with the potential for producing 
antibodies, specifically B lymphocytes (B cells), are selected for use in 
the mAb generating protocol. These cells may be obtained from biopsied 
spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen 
cells and peripheral blood cells are preferred, the former because they 
are a rich source of antibody-producing cells that are in the dividing 
plasmablast stage, and the latter because peripheral blood is easily 
accessible. Often, a panel of animals will have been immunized and the 
spleen of animal with the highest antibody titer will be removed and the 
spleen lymphocytes obtained by homogenizing the spleen with a syringe. 
Typically, a spleen from an immunized mouse contains approximately 
5.times.10.sup.7 to 2.times.10.sup.8 lymphocytes. 
The antibody-producing B lymphocytes from the immunized animal are then 
fused with cells of an immortal myeloma cell, generally one of the same 
species as the animal that was immunized. Myeloma cell lines suited for 
use in hybridoma-producing fusion procedures preferably are 
non-antibody-producing, have high fusion efficiency, and enzyme 
deficiencies that render then incapable of growing in certain selective 
media which support the growth of only the desired fused cells 
(hybridomas). 
Any one of a number of myeloma cells may be used, as are known to those of 
skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For 
example, where the immunized animal is a mouse, one may use P3-X63/Ag8, 
X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag 14, FO, NSOAU, MPC-11, MPC11-X45-GTG 
1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, 
IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are 
all useful in connection with human cell fusions. 
One preferred murine myeloma cell is the NS-1 myeloma cell line (also 
termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human 
Genetic Mutant Cell Repository by requesting cell line repository number 
GM3573. Another mouse myeloma cell line that may be used is the 
8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line. 
Methods for generating hybrids of antibody-producing spleen or lymph node 
cells and myeloma cells usually comprise mixing somatic cells with myeloma 
cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 
1:1, respectively, in the presence of an agent or agents (chemical or 
electrical) that promote the fusion of cell membranes. Fusion methods 
using Sendai virus have been described (Kohler and Milstein, 1975; 1976), 
and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter 
et al., 1977). The use of electrically induced fusion methods is also 
appropriate (Goding, 1986, pp. 71-74). 
Fusion procedures usually produce viable hybrids at low frequencies, about 
1.times.10.sup.-6 to 1.times.10.sup.31 8. However, this does not pose a 
problem, as the viable, fused hybrids are differentiated from the 
parental, unfused cells (particularly the unfused myeloma cells that would 
normally continue to divide indefinitely) by culturing in a selective 
medium. The selective medium is generally one that contains an agent that 
blocks the de novo synthesis of nucleotides in the tissue culture media. 
Exemplary and preferred agents are aminopterin, methotrexate, and 
azaserine. Aminopterin and methotrexate block de novo synthesis of both 
purines and pyrimidines, whereas azaserine blocks only purine synthesis. 
Where aminopterin or methotrexate is used, the media is supplemented with 
hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where 
azaserine is used, the media is supplemented with hypoxanthine. 
The preferred selection medium is HAT. Only cells capable of operating 
nucleotide salvage pathways are able to survive in HAT medium. The myeloma 
cells are defective in key enzymes of the salvage pathway, e.g., 
hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. 
The B-cells can operate this pathway, but they have a limited life span in 
culture and generally die within about two weeks. Therefore, the only 
cells that can survive in the selective media are those hybrids formed 
from myeloma and B-cells. 
This culturing provides a population of hybridomas from which specific 
hybridomas are selected. Typically, selection of hybridomas is performed 
by culturing the cells by single-clone dilution in microtiter plates, 
followed by testing the individual clonal supernatants (after about two to 
three weeks) for the desired reactivity. The assay should be sensitive, 
simple and rapid, such as radioimmunoassays, enzyme immunoassays, 
cytotoxicity assays, plaque assays, dot immunobinding assays, and the 
like. 
The selected hybridomas would then be serially diluted and cloned into 
individual antibody-producing cell lines, which clones can then be 
propagated indefinitely to provide mAbs. The cell lines may be exploited 
for mAb production in two basic ways. A sample of the hybridoma can be 
injected (often into the peritoneal cavity) into a histocompatible animal 
of the type that was used to provide the somatic and mycloma cells for the 
original fusion. The injected animal develops tumors secreting the 
specific monoclonal antibody produced by the fused cell hybrid. The body 
fluids of the animal, such as serum or ascites fluid, can then be tapped 
to provide mAbs in high concentration. The individual cell lines could 
also be cultured in vitro, where the mAbs are naturally secreted into the 
culture medium from which they can be readily obtained in high 
concentrations. mAbs produced by either means may be further purified, if 
desired, using filtration, centrifugation and various chromatographic 
methods such as HPLC or affinity chromatography. 
2.7 Crystal Protein Screening and Immunodetection Kits 
The present invention also provides compositions, methods and kits for 
screening samples suspected of containing a CryET29 .delta.-endotoxin or a 
gene encoding such a crystal protein. Such screening may be performed on 
samples such as transformed host cells, transgenic plants, progeny or seed 
thereof, or laboratory samples suspected of containing or producing such a 
polypeptide or nucleic acid segment. A kit can contain a novel nucleic 
acid segment or an antibody of the present invention. The kit can contain 
reagents for detecting an interaction between a sample and a nucleic acid 
or an antibody of the present invention. The provided reagent can be 
radio-, fluorescently- or enzymatically-labeled. The kit can contain a 
known radiolabeled agent capable of binding or interacting with a nucleic 
acid or antibody of the present invention. 
The reagent of the kit can be provided as a liquid solution, attached to a 
solid support or as a dried powder. Preferably, when the reagent is 
provided in a liquid solution, the liquid solution is an aqueous solution. 
Preferably, when the reagent provided is attached to a solid support, the 
solid support can be chromatograph media, a test plate having a plurality 
of wells, or a microscope slide. When the reagent provided is a dry 
powder, the powder can be reconstituted by the addition of a suitable 
solvent, that may be provided. 
In still further embodiments, the present invention concerns 
immunodetection methods and associated kits. It is proposed that the 
crystal proteins or peptides of the present invention may be employed to 
detect antibodies having reactivity therewith, or, alternatively, 
antibodies prepared in accordance with the present invention, may be 
employed to detect crystal proteins or crystal protein-related 
epitope-containing peptides. In general, these methods will include first 
obtaining a sample suspected of containing such a protein, peptide or 
antibody, contacting the sample with an antibody or peptide in accordance 
with the present invention, as the case may be, under conditions effective 
to allow the formation of an immunocomplex, and then detecting the 
presence of the immunocomplex. 
In general, the detection of immunocomplex formation is quite well known in 
the art and may be achieved through the application of numerous 
approaches. For example, the present invention contemplates the 
application of ELISA, RIA, immunoblot (e.g., dot blot), indirect 
immunofluorescence techniques and the like. Generally, immunocomplex 
formation will be detected through the use of a label, such as a 
radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish 
peroxidase, or the like). Of course, one may find additional advantages 
through the use of a secondary binding ligand such as a second antibody or 
a biotin/avidin ligand binding arrangement, as is known in the art. 
For assaying purposes, it is proposed that virtually any sample suspected 
of comprising either a crystal protein or peptide or a crystal 
protein-related peptide or antibody sought to be detected, as the case may 
be, may be employed. It is contemplated that such embodiments may have 
application in the titering of antigen or antibody samples, in the 
selection of hybridomas, and the like. In related embodiments, the present 
invention contemplates the preparation of kits that may be employed to 
detect the presence of crystal proteins or related peptides and/or 
antibodies in a sample. Samples may include cells, cell supernatants, cell 
suspensions, cell extracts, enzyme fractions, protein extracts, or other 
cell-free compositions suspected of containing crystal proteins or 
peptides. Generally speaking, kits in accordance with the present 
invention will include a suitable crystal protein, peptide or an antibody 
directed against such a protein or peptide, together with an 
immunodetection reagent and a means for containing the antibody or antigen 
and reagent. The immunodetection reagent will typically comprise a label 
associated with the antibody or antigen, or associated with a secondary 
binding ligand. Exemplary ligands might include a secondary antibody 
directed against the first antibody or antigen or a biotin or avidin (or 
streptavidin) ligand having an associated label. Of course, as noted 
above, a number of exemplary labels are known in the art and all such 
labels may be employed in connection with the present invention. 
The container will generally include a vial into which the antibody, 
antigen or detection reagent may be placed, and preferably suitably 
aliquotted. The kits of the present invention will also typically include 
a means for containing the antibody, antigen, and reagent containers in 
close confinement for commercial sale. Such containers may include 
injection or blow-molded plastic containers into which the desired vials 
are retained. 
2.8 ELISAs and Immunoprecipitation 
ELISAs may be used in conjunction with the invention. In an ELISA assay, 
proteins or peptides incorporating crystal protein antigen sequences are 
immobilized onto a selected surface, preferably a surface exhibiting a 
protein affinity such as the wells of a polystyrene microtiter plate. 
After washing to remove incompletely adsorbed material, it is desirable to 
bind or coat the assay plate wells with a nonspecific protein that is 
known to be antigenically neutral with regard to the test antisera such as 
bovine serum albumin (BSA), casein or solutions of milk powder. This 
allows for blocking of nonspecific adsorption sites on the immobilizing 
surface and thus reduces the background caused by nonspecific binding of 
antisera onto the surface. 
After binding of antigenic material to the well, coating with a 
non-reactive material to reduce background, and washing to remove unbound 
material, the immobilizing surface is contacted with the antisera or 
clinical or biological extract to be tested in a manner conducive to 
immune complex (antigen/antibody) formation. Such conditions preferably 
include diluting the antisera with diluents such as BSA, bovine gamma 
globulin (BGG) and phosphate buffered saline (PBS)/Tween.RTM.. These added 
agents also tend to assist in the reduction of nonspecific background. The 
layered antisera is then allowed to incubate for from about 2 to about 4 
hours, at temperatures preferably on the order of about 25.degree. to 
about 27.degree. C. Following incubation, the antiscra-contacted surface 
is washed so as to remove non-immunocomplexed material. A preferred 
washing procedure includes washing with a solution such as PBS/Tween.RTM., 
or borate buffer. 
Following formation of specific immunocomplexes between the test sample and 
the bound antigen, and subsequent washing, the occurrence and even amount 
of immunocomplex formation may be determined by subjecting same to a 
second antibody having specificity for the first. To provide a detecting 
means, the second antibody will preferably have an associated enzyme that 
will generate a color development upon incubating with an appropriate 
chromogenic substrate. Thus, for example, one will desire to contact and 
incubate the antisera-bound surface with a urease or peroxidase-conjugated 
anti-human IgG for a period of time and under conditions which favor the 
development of immunocomplex formation (e.g., incubation for 2 hours at 
room temperature in a PBS-containing solution such as PBS Tween.RTM.). 
After incubation with the second enzyme-tagged antibody, and subsequent to 
washing to remove unbound material, the amount of label is quantified by 
incubation with a chromogenic substrate such as urea and bromocresol 
purple or 2, 2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) 
and H.sub.2 O.sub.2, in the case of peroxidase as the enzyme label. 
Quantitation is then achieved by measuring the degree of color generation, 
e.g., using a visible spectra spectrophotometer. 
The anti-crystal protein antibodies of the present invention are 
particularly useful for the isolation of other crystal protein antigens by 
immunoprecipitation. Immunoprecipitation involves the separation of the 
target antigen component from a complex mixture, and is used to 
discriminate or isolate minute amounts of protein. For the isolation of 
membrane proteins cells must be solubilized into detergent micelles. 
Nonionic salts are preferred, since other agents such as bile salts, 
precipitate at acid pH or in the presence of bivalent cations. 
In an alternative embodiment the antibodies of the present invention are 
useful for the close juxtaposition of two antigens. This is particularly 
useful for increasing the localized concentration of antigens, e.g. 
enzyme-substrate pairs. 
2.9 Western Blots 
The compositions of the present invention will find great use in immunoblot 
or western blot analysis. The anti-peptide antibodies may be used as 
high-affinity primary reagents for the identification of proteins 
immobilized onto a solid support matrix, such as nitrocellulose, nylon or 
combinations thereof. In conjunction with immunoprecipitation, followed by 
gel electrophoresis, these may be used as a single step reagent for use in 
detecting antigens against which secondary reagents used in the detection 
of the antigen cause an adverse background. This is especially useful when 
the antigens studied are immunoglobulins (precluding the use of 
immunoglobulins binding bacterial cell wall components), the antigens 
studied cross-react with the detecting agent, or they migrate at the same 
relative molecular weight as a cross-reacting signal. 
Immunologically-based detection methods for use in conjunction with Western 
blotting include enzymatically-, radiolabel-, or fluorescently-tagged 
secondary antibodies against the toxin moiety are considered to be of 
particular use in this regard. 
2.10 Epitopic Core Sequences 
The present invention is also directed to protein or peptide compositions, 
free from total cells and other peptides, which comprise a purified 
protein or peptide which incorporates an epitope that is immunologically 
cross-reactive with one or more anti-crystal protein antibodies. In 
particular, the invention concerns epitopic core sequences derived from 
Cry proteins or peptides. 
As used herein, the term "incorporating an epitope(s) that is 
immunologically cross-reactive with one or more anti-crystal protein 
antibodies" is intended to refer to a peptide or protein antigen which 
includes a primary, secondary or tertiary structure similar to an epitope 
located within a crystal protein or polypeptide. The level of similarity 
will generally be to such a degree that monoclonal or polyclonal 
antibodies directed against the crystal protein or polypeptide will also 
bind to, react with, or otherwise recognize, the cross-reactive peptide or 
protein antigen. Various immunoassay methods may be employed in 
conjunction with such antibodies, such as, for example, Western blotting, 
ELISA, RIA, and the like, all of which are known to those of skill in the 
art. 
The identification of Cry immunodominant epitopes, and/or their functional 
equivalents, suitable for use in vaccines is a relatively straightforward 
matter. For example, one may employ the methods of Hopp, as taught in U.S. 
Pat. No. 4,554,101, incorporated herein by reference, which teaches the 
identification and preparation of epitopes from amino acid sequences on 
the basis of hydrophilicity. The methods described in several other 
papers, and software programs based thereon, can also be used to identify 
epitopic core sequences (see, e.g., Jameson and Wolf, 1988; Wolf et al., 
1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these "epitopic 
core sequences" may then be readily incorporated into peptides, either 
through the application of peptide synthesis or recombinant technology. 
Preferred peptides for use in accordance with the present invention will 
generally be on the order of about 8 to about 20 amino acids in length, 
and more preferably about 8 to about 15 amino acids in length. It is 
proposed that shorter antigenic crystal protein-derived peptides will 
provide advantages in certain circumstances, for example, in the 
preparation of immunologic detection assays. Exemplary advantages include 
the ease of preparation and purification, the relatively low cost and 
improved reproducibility of production, and advantageous biodistribution. 
It is proposed that particular advantages of the present invention may be 
realized through the preparation of synthetic peptides which include 
modified and/or extended epitopic/immunogenic core sequences which result 
in a "universal" epitopic peptide directed to crystal proteins, and in 
particular Cry and Cry-related sequences. These epitopic core sequences 
are identified herein in particular aspects as hydrophilic regions of the 
particular polypeptide antigen. It is proposed that these regions 
represent those which are most likely to promote T-cell or B-cell 
stimulation, and, hence, elicit specific antibody production. 
An epitopic core sequence, as used herein, is a relatively short stretch of 
amino acids that is "complementary" to, and therefore will bind, antigen 
binding sites on the crystal protein-directed antibodies disclosed herein. 
Additionally or alternatively, an epitopic core sequence is one that will 
elicit antibodies that are cross-reactive with antibodies directed against 
the peptide compositions of the present invention. It will be understood 
that in the context of the present disclosure, the term "complementary" 
refers to amino acids or peptides that exhibit an attractive force towards 
each other. Thus, certain epitope core sequences of the present invention 
may be operationally defined in terms of their ability to compete with or 
perhaps displace the binding of the desired protein antigen with the 
corresponding protein-directed antisera. 
In general, the size of the polypeptide antigen is not believed to be 
particularly crucial, so long as it is at least large enough to carry the 
identified core sequence or sequences. The smallest useful core sequence 
anticipated by the present disclosure would generally be on the order of 
about 8 amino acids in length, with sequences on the order of 10 to 20 
being more preferred. Thus, this size will generally correspond to the 
smallest peptide antigens prepared in accordance with the invention. 
However, the size of the antigen may be larger where desired, so long as 
it contains a basic epitopic core sequence. 
The identification of epitopic core sequences is known to those of skill in 
the art, for example, as described in U.S. Pat. No. 4,554,101, 
incorporated herein by reference, which teaches the identification and 
preparation of epitopes from amino acid sequences on the basis of 
hydrophilicity. Moreover, numerous computer programs are available for use 
in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 
1988; Wolf et al., 1988). Computerized peptide sequence analysis programs 
(e.g., DNAStar.RTM. software, DNAStar, Inc., Madison, Wis.) may also be 
useful in designing synthetic peptides in accordance with the present 
disclosure. 
Syntheses of epitopic sequences, or peptides which include an antigenic 
epitope within their sequence, are readily achieved using conventional 
synthetic techniques such as the solid phase method (e.g., through the use 
of commercially available peptide synthesizer such as an Applied 
Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized 
in this manner may then be aliquotted in predetermined amounts and stored 
in conventional manners, such as in aqueous solutions or, even more 
preferably, in a powder or lyophilized state pending use. 
In general, due to the relative stability of peptides, they may be readily 
stored in aqueous solutions for fairly long periods of time if desired, 
e.g., up to six months or more, in virtually any aqueous solution without 
appreciable degradation or loss of antigenic activity. However, where 
extended aqueous storage is contemplated it will generally be desirable to 
include agents including buffers such as Tris or phosphate buffers to 
maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to 
include agents which will inhibit microbial growth, such as sodium azide 
or Merthiolate. For extended storage in an aqueous state it will be 
desirable to store the solutions at about 4.degree. C., or more 
preferably, frozen. Of course, where the peptides are stored in a 
lyophilized or powdered state, they may be stored virtually indefinitely, 
e.g., in metered aliquots that may be rehydrated with a predetermined 
amount of water (preferably distilled) or buffer prior to use. 
2.11 Crystal Protein Compositions As Insecticides and Methods of Use 
The inventors contemplate that the crystal protein compositions disclosed 
herein will find particular utility as insecticides for topical and/or 
systemic application to field crops, grasses, fruits and vegetables, and 
ornamental plants. In a preferred embodiment, the bioinsecticide 
composition comprises an oil flowable suspension of bacterial cells which 
expresses a novel crystal protein disclosed herein. Preferably the cells 
are B. thuringiensis EG4096, EG11494, or EG11502 cells, however, any such 
bacterial host cell expressing the novel nucleic acid segments disclosed 
herein and producing a crystal protein is contemplated to be useful, such 
as B. megaterium, B. subtilis, E. coli, or Pseudomonas spp. 
In another important embodiment, the bioinsecticide composition comprises a 
water dispersible granule. This granule comprises bacterial cells which 
expresses a novel crystal protein disclosed herein. Preferred bacterial 
cells are B. thuringiensis EG4096, EG11494, or EG11502 cells, however, 
bacteria such as B. megateriuni, B. subtilis, E. coli, or Pseudomonas spp. 
cells transformed with a DNA segment disclosed herein and expressing the 
crystal protein are also contemplated to be useful. 
In a third important embodiment, the bioinsecticide composition comprises a 
wettable powder, dust, pellet, or collodial concentrate. This powder 
comprises bacterial cells which expresses a novel crystal protein 
disclosed herein. Preferred bacterial cells are B. thuringiensis EG4096, 
EG11494, or EG11502 cells, however, bacteria such as B. megaterium, B. 
subtilis, E. coli, or Pseudomonas spp. cells transformed with a DNA 
segment disclosed herein and expressing the crystal protein are also 
contemplated to be useful. Such dry forms of the insecticidal compositions 
may be formulated to dissolve immediately upon wetting, or alternatively, 
dissolve in a controlled-release, sustained-release, or other 
time-dependent manner. 
In a fourth important embodiment, the bioinsecticide composition comprises 
an aqueous suspension of bacterial cells such as those described above 
which express the crystal protein. Such aqueous suspensions may be 
provided as a concentrated stock solution which is diluted prior to 
application, or alternatively, as a diluted solution ready-to-apply. 
For these methods involving application of bacterial cells, the cellular 
host containing the crystal protein gene(s) 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 B. thuringiensis gene. These cells may then be harvested 
in accordance with conventional ways. Alternatively, the cells can be 
treated prior to harvesting. 
When the insecticidal compositions comprise intact B. thuringiensis cells 
expressing the protein of interest, such bacteria may be formulated in a 
variety of ways. They may be employed as wettable powders, granules or 
dusts, by mixing with various inert materials, such as inorganic minerals 
(phyllosilicates, carbonates, sulfates, phosphates, and the like) or 
botanical materials (powdered corncobs, rice hulls, walnut shells, and the 
like). The formulations may include spreader-sticker adjuvants, 
stabilizing agents, other pesticidal additives, or surfactants. Liquid 
formulations may be aqueous-based or non-aqueous and employed as foams, 
suspensions, emulsifiable concentrates, or the like. The ingredients may 
include Theological agents, surfactants, emulsifiers, dispersants, or 
polymers. 
Alternatively, the novel CryET29 or CryET29-derived protein may be prepared 
by native or recombinant bacterial expression systems in vitro and 
isolated for subsequent field application. Such protein may be either in 
crude cell lysates, suspensions, colloids, etc., or alternatively may be 
purified, refined, buffered, and/or further processed, before formulating 
in an active biocidal formulation. Likewise, under certain circumstances, 
it may be desirable to isolate crystals and/or spores from bacterial 
cultures expressing the crystal protein and apply solutions, suspensions, 
or collodial preparations of such crystals and/or spores as the active 
bioinsecticidal composition. 
Regardless of the method of application, the amount of the active 
component(s) are applied at an insecticidally-effective amount, which will 
vary depending on such factors as, for example, the specific coleopteran 
insects to be controlled, the specific plant or crop to be treated, the 
environmental conditions, and the method, rate, and quantity of 
application of the insecticidally-active composition. 
The insecticide compositions described may be made by formulating either 
the bacterial cell, crystal and/or spore suspension, or isolated protein 
component with the desired agriculturally-acceptable carrier. The 
compositions may be formulated prior to administration in an appropriate 
means such as lyophilized, freeze-dried, cdessicated, or in an aqueous 
carrier, medium or suitable diluent, such as saline or other buffer. The 
formulated compositions may be in the form of a dust or granular material, 
or a suspension in oil (vegetable or mineral), or water or oil/water 
emulsions, or as a wettable powder, or in combination with any other 
carrier material suitable for agricultural application. Suitable 
agricultural carriers can be solid or liquid and are well known in the 
art. The term "agriculturally-acceptable carrier" covers all adjuvants, 
e.g., inert components, dispersants, surfactants, tackifiers, binders, 
etc. that are ordinarily used in insecticide formulation technology; these 
are well known to those skilled in insecticide formulation. The 
formulations may be mixed with one or more solid or liquid adjuvants and 
prepared by various means, e.g., by homogeneously mixing, blending and/or 
grinding the insecticidal composition with suitable adjuvants using 
conventional formulation techniques. 
The insecticidal compositions of this invention are applied to the 
environment of the target coleopteran insect, typically onto the foliage 
of the plant or crop to be protected, by conventional methods, preferably 
by spraying. The strength and duration of insecticidal application will be 
set with regard to conditions specific to the particular pest(s), crop(s) 
to be treated and particular environmental conditions. The proportional 
ratio of active ingredient to carrier will naturally depend on the 
chemical nature, solubility, and stability of the insecticidal 
composition, as well as the particular formulation contemplated. 
Other application techniques, e.g., dusting, sprinkling, soaking, soil 
injection, seed coating, seedling coating, spraying, aerating, misting, 
atomizing, and the like, are also feasible and may be required under 
certain circumstances such as e.g., insects that cause root or stalk 
infestation, or for application to delicate vegetation or ornamental 
plants. These application procedures are also well-known to those of skill 
in the art. 
The insecticidal composition of the invention may be employed in the method 
of the invention singly or in combination with other compounds, including 
and not limited to other pesticides. The method of the invention may also 
be used in conjunction with other treatments such as surfactants, 
detergents, polymers or time-release formulations. The insecticidal 
compositions of the present invention may be formulated for either 
systemic or topical use. 
The concentration of insecticidal composition which is used for 
environmental, systemic, or foliar application will vary widely depending 
upon the nature of the particular formulation, means of application, 
environmental conditions, and degree of biocidal activity. Typically, the 
bioinsecticidal composition will be present in the applied formulation at 
a concentration of at least about 1% by weight and may be up to and 
including about 99% by weight. Dry formulations of the compositions may be 
from about 1% to about 99% or more by weight of the composition, while 
liquid formulations may generally comprise from about 1% to about 99% or 
more of the active ingredient by weight. Formulations which comprise 
intact bacterial cells will generally contain from about 10.sup.4 to about 
10.sup.12 cells/mg. 
The insecticidal formulation may be administered to a particular plant or 
target area in one or more applications as needed, with a typical field 
application rate per hectare ranging on the order of from about 50 g to 
about 500 g of active ingredient, or of from about 500 g to about 1000 g, 
or of from about 1000 g to about 5000 g or more of active ingredient. 
2.12 Pharmaceutical Compositions and Methods for the Treatment of Fleas 
Since the novel crystal protein of the present invention is the first such 
B. thunrizgiensis .delta.-endotoxin identified which has insecticidal 
activity against fleas; the inventors also contemplate the formulation of 
pharmaceutical compositions which may be given to animals as prophylaxis 
and/or treatment of infestation by fleas, and in particular by infestation 
of members of the Genus Ctenocephalides, such as Ctenocephalides felis 
(common name, cat flea) and C. canis (common name, dog flea). While these 
are only two members of the Order Siphonaptera for which the present 
invention's compositions demonstrate insecticidal activity, it is 
contemplated that the compositions may be useful in treating other related 
insects which commonly attack animals may also be controlled by the novel 
compositions disclosed herein. Such insects are described in detail in 
U.S. Pat. No. 5,449,681, incorporated herein by reference, and include 
members of the Genera Culex, Culiseta, Bovicola, Callitroga, Chrysops, 
Cimes, Ctenocephalis, Dermatophilus, Dermatobia, and Damalinia among 
others. 
As such, one aspect of the invention comprises a pharmaceutical composition 
comprising a crystal protein composition disclosed herein for 
administration to an animal to prevent or reduce flea or related insect 
infestation. A method of reducing such flea infestation in an animal is 
also disclosed and claimed herein. The method generally comprises 
administering to an animal an insecticidally-effective amount of a CryET29 
composition. Means for administering such insecticidal compositions to an 
animal are well-known in the art. U.S. Pat. No. 5,416,102 (specifically 
incorporated herein by reference) provides teaching for methods and 
formulations for preventing flea infestation using an insecticidal 
composition. 
Such anti-siphonapteran veterinary compositions may be delivered in a 
variety of methods depending upon the particular application. Examples of 
means for administering insecticidal compositions to an animal are 
well-known to those of skill in the art, and include, e.g., flea collars, 
flea sprays, dips, powders and the like. Methods for providing such 
formulations to an animal are also well-known to those of skill in the 
art, and include direct application or passive application such as the 
device described in U.S. Pat. No. 4,008,688 for the application of 
insecticides by a pet bed assembly. The animal to be treated may be any 
animal which is sensitive to or susceptible to attack or infestation by a 
flea which is killed or inhibited by a CryET29 composition as disclosed 
herein. Such animals may be feline, canine, equine, porcine, lupine, 
bovine, murine, etc. and the like, although the inventors contemplate that 
feline and canine animals will be particularly preferred as animals to be 
treated by the novel compositions disclosed herein. 
It is further contemplated that in addition to topical administration of 
the pharmaceutical compositions disclosed, systemic administration may in 
some cases be preferable or desirable. For oral administration, the 
compositions may be formulated with an inert diluent or with an 
assimilable edible carrier, or they may be enclosed in hard- or soft-shell 
gelatin capsule, or they may be compressed into tablets, or they may be 
incorporated directly with the food of the diet. For oral therapeutic 
administration, the active compounds may be incorporated with excipients 
and used in the form of ingestible tablets, buccal tables, troches, 
capsules, elixirs, suspensions, syrups, wafers, and the like. Such 
compositions and preparations should contain at least 0.1% of active 
compound. The percentage of the compositions and preparations may, of 
course, be varied and may conveniently be between about 2 to about 60% of 
the weight of the unit. The amount of active compounds in such 
therapeutically useful compositions is such that a suitable dosage will be 
obtained. 
For oral prophylaxis of fleas, the crystal protein may be incorporated with 
excipients and used in the form of a gel, paste, powder, pill, tablet, 
capsule, or slurry which may be given to the animal for ingestion. 
Alternatively the compositions may be formulated as an additive to pet 
foods, treats, or other edible formulations. When formulated as a tablet 
or capsule, or the like, the composition may also contain the following: a 
binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, 
such as dicalcium phosphate; a disintegrating agent, such as corn starch, 
potato starch, alginic acid and the like; a lubricant, such as magnesium 
stearate; and a sweetening agent, such as sucrose, lactose or saccharin 
may be added or a flavoring agent to make the composition more palatable 
to the animal being treated. One such means for delivering flea 
prophylactics to an animal is a sauce as described in U.S. Pat. No. 
4,702,914, specifically incorporated herein by reference. 
When the dosage unit form is a capsule, it may contain, in addition to 
materials of the above type, a liquid carrier. Various other materials may 
be present as coatings or to otherwise modify the physical form of the 
dosage unit. For instance, tablets, pills, or capsules may be coated with 
shellac, sugar or both. Of course, any material used in preparing any 
dosage unit form should be pharmaceutically pure and substantially 
non-toxic in the amounts employed. In addition, the active compounds may 
be incorporated into sustained-release preparation and formulations. 
Alternatively, the pharmaceutical compositions disclosed herein may be 
administered parenterally, intramuscularly, or even intraperitoneally. 
Solutions of the active compounds as free base or pharmacologically 
acceptable salts may be prepared in water suitably mixed with a 
surfactant, such as hydroxypropylcellulose. Dispersions may also be 
prepared in glycerol, liquid polyethylene glycols, and mixtures thereof 
and in oils. Under ordinary conditions of storage and use, these 
preparations contain a preservative to prevent the growth of 
microorganisms. The pharmaceutical forms suitable for injectable use 
include sterile aqueous solutions or dispersions and sterile powders for 
the extemporaneous preparation of sterile injectable solutions or 
dispersions. In all cases the form must be sterile and must be fluid to 
the extent that easy syringability exists. It must be stable under the 
conditions of manufacture and storage and must be preserved against the 
contaminating action of microorganisms, such as bacteria and fungi. The 
carrier can be a solvent or dispersion medium containing, for example, 
water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid 
polyethylene glycol, and the like), suitable mixtures thereof, and/or 
vegetable oils. Proper fluidity may be maintained, for example, by the use 
of a coating, such as lecithin, by the maintenance of the required 
particle size in the case of dispersion and by the use of surfactants. The 
prevention of the action of microorganisms can be brought about by various 
antibacterial ad antifungal agents, for example, parabens, chlorobutanol, 
phenol, sorbic acid, thimerosal, and the like. In many cases, it will be 
preferable to include isotonic agents, for example, sugars or sodium 
chloride. Prolonged absorption of the injectable compositions can be 
brought about by the use in the compositions of agents delaying 
absorption, for example, aluminum monostearate and gelatin. 
When systemic administration is desired, e.g., parenteral administration in 
an aqueous solution, the solution should be suitably buffered if necessary 
and the liquid diluent first rendered isotonic with sufficient saline or 
glucose. These particular aqueous solutions are especially suitable for 
intramuscular, subcutaneous and intraperitoneal administration. In this 
connection, sterile aqueous media which can be employed will be known to 
those of skill in the art in light of the present disclosure. Some 
variation in dosage will necessarily occur depending on the condition, 
size, and type of animal being treated. The person responsible for 
administration will, in any event, determine the appropriate dose for the 
individual subject. Moreover, for human administration, preparations 
should meet sterility, pyrogenicity, general safety and purity standards 
as required by FDA Office of Biologics standards. 
Sterile injectable solutions are prepared by incorporating the active 
compounds in the required amount in the appropriate solvent with various 
of the other ingredients enumerated above, as required, followed by 
filtered sterilization. Generally, dispersions are prepared by 
incorporating the various sterilized active ingredients into a sterile 
vehicle which contains the basic dispersion medium and the required other 
ingredients from those enumerated above. In the case of sterile powders 
for the preparation of sterile injectable solutions, the preferred methods 
of preparation are vacuum-drying and freeze-drying techniques which yield 
a powder of the active ingredient plus any additional desired ingredient 
from a previously sterile-filtered solution thereof. 
The compositions disclosed herein may be formulated in a neutral or salt 
form. Pharmaceutically-acceptable salts, include the acid addition salts 
(formed with the free amino groups of the protein) and which are formed 
with inorganic acids such as, for example, hydrochloric or phosphoric 
acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and 
the like. Salts formed with the free carboxyl groups can also be derived 
from inorganic bases such as, for example, sodium, potassium, ammonium, 
calcium, or ferric hydroxides, and such organic bases as isopropylamine, 
trimethylamine, histidine, procaine and the like. Upon formulation, 
solutions will be administered in a manner compatible with the dosage 
formulation and in such amount as is therapeutically effective. The 
formulations are easily administered in a variety of dosage forms such as 
creams, lotions, sprays, dips, emulsions, colloids, or alternatively, when 
systemic administration is desirable, injectable solutions, drug release 
capsules and the like. 
As used herein, "carrier" includes any and all solvents, dispersion media, 
vehicles, coatings, diluents, antibacterial and antifungal agents, 
isotonic and absorption delaying agents, buffers, carrier solutions, 
suspensions, colloids, and the like. The use of such media and agents for 
pharmaceutical active substances is well known in the art. Except insofar 
as any conventional media or agent is incompatible with the active 
ingredient, its use in the therapeutic compositions is contemplated. 
Supplementary active ingredients can also be incorporated into the 
compositions. 
The phrase "pharmaceutically-acceptable" refers to molecular entities and 
compositions that do not produce an allergic or similar untoward reaction 
when administered to a animal. The preparation of an aqueous composition 
that contains a protein as an active ingredient is well understood in the 
art. Typically, such compositions are prepared as injectables, either as 
liquid solutions or suspensions; solid forms suitable for solution in, or 
suspension in, liquid prior to injection can also be prepared. The 
preparation can also be emulsified. 
Another aspect of the invention encompasses methods and compositions for 
use in the control and eradication of siphonapteran insects from 
environmental areas where infestation by such insects is suspected. The 
method generally involves applying to an area suspected of containing such 
insects an insecticidally-effective amount of a CryET29 composition as 
dislosed herein. The inventors further contemplate the use of the protein 
of the present invention as an active ingredient in a pharmaceutical 
composition for administration to body or to the living areas and environs 
of an animal to prevent, lessen, or reduce the infestation of fleas and 
related insects in such areas. The crystal protein composition may be 
formulated in a powder, spray, fog, granule, rinse, shampoo, flea collar, 
dip, etc. suitable for administration to the body of the animal or to the 
living quarters, bedding materials, houses, yards, kennels, pet boarding 
facilities etc. of such an animal using techniques which are known to 
those of skill in the art of veterinary insecticide formulations. An 
example of oral formulation of veterinary insecticides is found in the 
teachings of U.S. Pat. No. 5,416,102. The inventors contemplate that the 
use of such compositions in the prevention or eradication of fleas on pets 
such as dogs, cats, and other fur-bearing animals may represent a 
significant advance in the state of the art considering the novel 
compositions disclosed herein are the first crystal proteins identified 
which have such desirable anti-siphonapteran insecticidal activity. 
2.13 Biological Functional Equivalents 
Modification and changes may be made in the structure of the peptides of 
the present invention and DNA segments which encode them and still obtain 
a functional molecule that encodes a protein or peptide with desirable 
characteristics. The following is a discussion based upon changing the 
amino acids of a protein to create an equivalent, or even an improved, 
second-generation molecule. In particular embodiments of the invention, 
mutated crystal proteins are contemplated to be useful for increasing the 
insecticidal activity of the protein, and consequently increasing the 
insecticidal activity and/or expression of the recombinant transgene in a 
plant cell. The amino acid changes may be achieved by changing the codons 
of the DNA sequence, according to the codons given in Table 2. 
TABLE 2 
______________________________________ 
Amino Acids Codons 
______________________________________ 
Alanine Ala A GCA GCC GCG GCU 
Cysteine Cys UGC UGU 
Aspartic acid 
Asp 
D GAC GAU 
Glutamic acid 
Glu 
E GAA GAG 
Phenylalanine 
Phe 
F UUC UUU 
Glycine GGly 
GGA GGC GGG GGU 
Histidine Hs CAC CAU 
Isoleucine Ile 
I AUA AUC AUU 
Lysine K Lys 
AAA AAG 
Leucine LLeu 
UUA UUG CUA CUC CUG CUU 
Methionine Met 
M AUG 
Asparagine Asn 
N AAC AAU 
Proline PPro 
CCA CCC CCG CCU 
Glutamine Qn CAA CAG 
Arginine Rrg AGA AGG CGA CGC CGG CGU 
Serine S Ser 
AGC AGU UCA UCC UCG UCU 
Threonine Tr ACA ACC ACG ACU 
Valine V Val 
GUA GUC GUG GUU 
Tryptophan Trp 
W UGG 
Tyrosine Yyr UAC UAU 
______________________________________ 
For example, certain amino acids may be substituted for other amino acids 
in a protein structure without appreciable loss of interactive binding 
capacity with structures such as, for example, antigen-binding regions of 
antibodies or binding sites on substrate molecules. Since it is the 
interactive capacity and nature of a protein that defines that protein's 
biological functional activity, certain amino acid sequence substitutions 
can be made in a protein sequence, and, of course, its underlying DNA 
coding sequence, and nevertheless obtain a protein with like properties. 
It is thus contemplated by the inventors that various changes may be made 
in the peptide sequences of the disclosed compositions, or corresponding 
DNA sequences which encode said peptides without appreciable loss of their 
biological utility or activity. 
In making such changes, the hydropathic index of amino acids may be 
considered. The importance of the hydropathic amino acid index in 
conferring interactive biologic function on a protein is generally 
understood in the art (Kyte and Doolittle, 1982, incorporate herein by 
reference). It is accepted that the relative hydropathic character of the 
amino acid contributes to the secondary structure of the resultant 
protein, which in turn defines the interaction of the protein with other 
molecules, for example, enzymes, substrates, receptors, DNA, antibodies, 
antigens, and the like. 
Each amino acid has been assigned a hydropathic index on the basis of their 
hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), 
these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine 
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); 
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); 
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); 
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and 
arginine (-4.5). 
It is known in the art that certain amino acids may be substituted by other 
amino acids having a similar hydropathic index or score and still result 
in a protein with similar biological activity, i.e., still obtain a 
biological functionally equivalent protein. In making such changes, the 
substitution of amino acids whose hydropathic indices are within .+-.2 is 
preferred, those which are within .+-.1 are particularly preferred, and 
those within .+-.0.5 are even more particularly preferred. 
It is also understood in the art that the substitution of like amino acids 
can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 
4,554,101, incorporated herein by reference, states that the greatest 
local average hydrophilicity of a protein, as governed by the 
hydrophilicity of its adjacent amino acids, correlates with a biological 
property of the protein. 
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values 
have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); 
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine 
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline 
(-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine 
(-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); 
phenylalanine (-2.5); tryptophan (-3.4). 
It is understood that an amino acid can be substituted for another having a 
similar hydrophilicity value and still obtain a biologically equivalent, 
and in particular, an immunologically equivalent protein. In such changes, 
the substitution of amino acids whose hydrophilicity values are within 
.+-.2 is preferred, those which are within .+-.1 are particularly 
preferred, and those within .+-.0.5 are even more particularly preferred. 
As outlined above, amino acid substitutions are generally therefore based 
on the relative similarity of the amino acid side-chain substituents, for 
example, their hydrophobicity, hydrophilicity, charge, size, and the like. 
Exemplary substitutions which take various of the foregoing 
characteristics into consideration are well known to those of skill in the 
art and include: arginine and lysine; glutamate and aspartate; serine and 
threonine; glutamine and asparagine; and valine, leucine and isoleucine.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 
The present invention provides a novel .delta.-endotoxin, designated 
CryET29, which is toxic to larvae of the cat flea, Ctenocephalides felis, 
as well as against coleopteran insects such as the southern and western 
corn rootworm, Colorado potato beetle, Japanese beetle, and the red flour 
beetle. It is important to note that the trivial name for Ctenocephalides 
felis is somewhat misleading in that the organism parasitizes not only 
felines, but is the major parasitic flea for canines as well (see e.g., 
U.S. Pat. No. 4,547,360, specifically incorporated herein by reference). 
4.1 cryET29 DNA Probes and Primers 
In another aspect, DNA sequence information provided by the invention 
allows for the preparation of relatively short DNA (or RNA) sequences 
having the ability to specifically hybridize to gene sequences of the 
selected polynucleotides disclosed herein. In these aspects, nucleic acid 
probes of an appropriate length are prepared based on a consideration of a 
selected crystal protein gene sequence, e.g., a sequence such as that 
shown in SEQ ID NO: 1. The ability of such nucleic acid probes to 
specifically hybridize to a crystal protein-encoding gene sequence lends 
them particular utility in a variety of embodiments. Most importantly, the 
probes may be used in a variety of assays for detecting the presence of 
complementary sequences in a given sample. 
In certain embodiments, it is advantageous to use oligonucleotide primers. 
The sequence of such primers is designed using a polynucleotide of the 
present invention for use in detecting, amplifying or mutating a defined 
segment of a crystal protein gene from B. thuringiensis using PCR.TM. 
technology. Segments of related crystal protein genes from other species 
may also be amplified by PCR.TM. using such primers. 
4.2 Expression Vectors 
The present invention contemplates an expression vector comprising a 
polynucleotide of the present invention. Thus, in one embodiment an 
expression vector is an isolated and purified DNA molecule comprising a 
promoter operatively linked to an coding region that encodes a polypeptide 
of the present invention, which coding region is operatively linked to a 
transcription-terminating region, whereby the promoter drives the 
transcription of the coding region. 
As used herein, the term "operatively linked" means that a promoter is 
connected to an coding region in such a way that the transcription of that 
coding region is controlled and regulated by that promoter. Means for 
operatively linking a promoter to a coding region are well known in the 
art. 
In a preferred embodiment, the recombinant expression of DNAs encoding the 
crystal proteins of the present invention is preferable in a Bacillus host 
cell. Preferred host cells include B. thuringiensis, B. megaterium, B. 
subtilis, and related bacilli, with B. thuringiensis host cells being 
highly preferred. Promoters that function in bacteria are well-known in 
the art. An exemplary and preferred promoter for the Bacillus crystal 
proteins include any of the known crystal protein gene promoters, 
including the cryET29 gene promoter, and promoters specific for B. 
thuringiensis sigma factors, such as .sigma..sup.E and .sigma..sup.K (for 
a review see Baum and Malvar, 1995) Alternatively, mutagenized or 
recombinant crystal protein-encoding gene promoters may be engineered by 
the hand of man and used to promote expression of the novel gene segments 
disclosed herein. 
In an alternate embodiment, the recombinant expression of DNAs encoding the 
crystal proteins of the present invention is performed using a transformed 
Gram-negative bacterium such as an E. coli or Pseudomnonas spp. host cell. 
Promoters which function in high-level expression of target polypeptides 
in E. Coli and other Gram-negative host cells are also well-known in the 
art. 
Where an expression vector of the present invention is to be used to 
transform a plant, a promoter is selected that has the ability to drive 
expression in plants. Promoters that function in plants are also well 
known in the art. Useful in expressing the polypeptide in plants are 
promoters that are inducible, viral, synthetic, constitutive as described 
(Poszkowski et al., 1989; Odell et al., 1985), and temporally regulated, 
spatially regulated, and spatio-temporally regulated (Chau et al., 1989). 
A promoter is also selected for its ability to direct the transformed plant 
cell's or transgenic plant's transcriptional activity to the coding 
region. Structural genes can be driven by a variety of promoters in plant 
tissues. Promoters can be near-constitutive, such as the CaMV 35S 
promoter, or tissue-specific or developmentally specific promoters 
affecting dicots or monocots. 
Where the promoter is a near-constitutive promoter such as CaMV 35S, 
increases in polypeptide expression are found in a variety of transformed 
plant tissues (e.g., callus, leaf, seed and root). Alternatively, the 
effects of transformation can be directed to specific plant tissues by 
using plant integrating vectors containing a tissue-specific promoter. 
An exemplary tissue-specific promoter is the lectin promoter, which is 
specific for seed tissue. The Lectin protein in soybean seeds is encoded 
by a single gene (Le1) that is only expressed during seed maturation and 
accounts for about 2 to about 5% of total seed mRNA. The lectin gene and 
seed-specific promoter have been fully characterized and used to direct 
seed specific expression in transgenic tobacco plants (Vodkin et al., 
1983; Lindstrom et al., 1990.) 
An expression vector containing a coding region that encodes a polypeptide 
of interest is engineered to be under control of the lectin promoter and 
that vector is introduced into plants using, for example, a protoplast 
transformation method (Dhir et al., 1991). The expression of the 
polypeptide is directed specifically to the seeds of the transgenic plant. 
A transgenic plant of the present invention produced from a plant cell 
transformed with a tissue specific promoter can be crossed with a second 
transgenic plant developed from a plant cell transformed with a different 
tissue specific promoter to produce a hybrid transgenic plant that shows 
the effects of transformation in more than one specific tissue. 
Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et 
al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), corn light 
harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al., 
1985), pea small subunit RuBP Carboxylase (Poulsen et al., 1986; Cashmore 
et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti 
plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone 
isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et 
al., 1989), CaMV 35s transcript (Odell et al., 1985) and Potato patatin 
(Wenzler et al., 1989). Preferred promoters are the cauliflower mosaic 
virus (CaMV 35S) promoter and the S-E9 small subunit RuBP carboxylase 
promoter. 
The choice of which expression vector and ultimately to which promoter a 
polypeptide coding region is operatively linked depends directly on the 
functional properties desired, e.g., the location and timing of protein 
expression, and the host cell to be transformed. These are well known 
limitations inherent in the art of constructing recombinant DNA molecules. 
However, a vector useful in practicing the present invention is capable of 
directing the expression of the polypeptide coding region to which it is 
operatively linked. 
Typical vectors useful for expression of genes in higher plants are well 
known in the art and include vectors derived from the tumor-inducing (Ti) 
plasmid of Agrobacterium tumefaciens described (Rogers et al., 1987). 
However, several other plant integrating vector systems are known to 
function in plants including pCaMVCN transfer control vector described 
(Fromm et al., 1985). Plasmid pCaMVCN (available from Pharmacia, 
Piscataway, N.J.) includes the cauliflower mosaic virus CaMV 35S promoter. 
In preferred embodiments, the vector used to express the polypeptide 
includes a selection marker that is effective in a plant cell, preferably 
a drug resistance selection marker. One preferred drug resistance marker 
is the gene whose expression results in kanamycin resistance; i.e., the 
chimeric gene containing the nopaline synthase promoter, Tn5 neomycin 
phosphotransferase II (nptII) and nopaline synthase 3' nontranslated 
region described (Rogers etal, 1988). 
RNA polymerase transcribes a coding DNA sequence through a site where 
polyadenylation occurs. Typically, DNA sequences located a few hundred 
base pairs downstream of the polyadenylation site serve to terminate 
transcription. Those DNA sequences are referred to herein as 
transcription-termination regions. Those regions are required for 
efficient polyadenylation of transcribed messenger RNA (mRNA). 
Means for preparing expression vectors are well known in the art. 
Expression (transformation vectors) used to transform plants and methods 
of making those vectors are described in U.S. Pat. Nos. 4,971,908, 
4,940,835, 4,769,061 and 4,757,011, the disclosures of which arc 
incorporated herein by reference. Those vectors can be modified to include 
a coding sequence in accordance with the present invention. 
A variety of methods has been developed to operatively link DNA to vectors 
via complementary cohesive termini or blunt ends. For instance, 
complementary homopolymer tracts can be added to the DNA segment to be 
inserted and to the vector DNA. The vector and DNA segment are then joined 
by hydrogen bonding between the complementary homopolymeric tails to form 
recombinant DNA molecules. 
A coding region that encodes a polypeptide having the ability to confer 
insecticidal activity to a cell is preferably a CryET29 B. thuringiensis 
crystal protein-encoding gene. In preferred embodiments, such a 
polypeptide has the amino acid residue sequence of SEQ ID NO:2, or a 
functional equivalent of this sequence. In accordance with such 
embodiments, a coding region comprising the DNA sequence of SEQ ID NO: 1 
is also preferred. 
4.3 Characteristics of the CryET29 Crystal Protein 
The present invention provides novel polypeptides that define a whole or a 
portion of a B. thuringiensis CryET29 crystal protein. 
In a preferred embodiment, the invention discloses and claims an isolated 
and purified CryET29 protein. The CryET29 protein comprises an amino acid 
sequence as disclosed in SEQ ID NO:2. CryET29 has a calculated isoelectric 
constant (pI) equal to 5.88. The amino acid composition of the CryET29 
protein is given in Table 3. 
TABLE 3 
______________________________________ 
Amino Acid Composition of CryET29 
# 
Amino Acid 
# Residues 
% Total Amino Acid 
Residues 
% Total 
______________________________________ 
Ala 18 7.7 Leu 13 5.6 
Arg 7 3.0 Lys 16 6.9 
Asn 15 6.4 Met 4 1.7 
Asp 15 6.4 Phe 12 5.1 
Cys 1 0.4 Pro 6 2.5 
Gln 15 6.4 Ser 16 6.9 
Glu 10 4.3 Thr 17 7.3 
Gly 5 2.1 Trp 2 0.8 
His 3 1.2 Tyr 10 4.3 
Ile 20 8.6 Val 26 11.2 
Acidic (Asp + Glu) 25 10.7 
Basic (Arg + Lys) 23 9.9 
Aromatic 
(Phe + Trp + Tyr) 24 10.2 
Hydrophobic 
(Aromatic + Ile + Leu + Met + Val) 
87 37.3 
______________________________________ 
4.4 Transformed or Transgenic Plant Cells 
A bacterium, a yeast cell, or a plant cell or a plant transformed with an 
expression vector of the present invention is also contemplated. A 
transgenic bacterium, yeast cell, plant cell or plant derived from such a 
transformed or transgenic cell is also contemplated. Means for 
transforming bacteria and yeast cells are well known in the art. 
Typically, means of transformation are similar to those well known means 
used to transform other bacteria or yeast such as E. coli or 
Saccharoinyces cerevisiae. 
Methods for DNA transformation of plant cells include 
Agrobacterium-mediated plant transformation, protoplast transformation, 
gene transfer into pollen, injection into reproductive organs, injection 
into immature embryos and particle bombardment. Each of these methods has 
distinct advantages and disadvantages. Thus, one particular method of 
introducing genes into a particular plant strain may not necessarily be 
the most effective for another plant strain, but it is well known which 
methods are useful for a particular plant strain. 
There are many methods for introducing transforming DNA segments into 
cells, but not all are suitable for delivering DNA to plant cells. 
Suitable methods are believed to include virtually any method by which DNA 
can be introduced into a cell, such as by Agrobacterium infection, direct 
delivery of DNA such as, for example, by PEG-mediated transformation of 
protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated 
DNA uptake, by electroporation, by agitation with silicon carbide fibers, 
by acceleration of DNA coated particles, etc. In certain embodiments, 
acceleration methods are preferred and include, for example, 
microprojectile bombardment and the like. 
Technology for introduction of DNA into cells is well-known to those of 
skill in the art. Four general methods for delivering a gene into cells 
have been described: (1) chemical methods (Graham and van der Eb, 1973; 
Zatloukal et al., 1992); (2) physical methods such as microinjection 
(Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm etal., 
1985) and the gene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) 
viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a; 
1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992; 
Wagner et al., 1992). 
4.5.1 Electroporation 
The application of brief, high-voltage electric pulses to a variety of 
animal and plant cells leads to the formation of nanometer-sized pores in 
the plasma membrane. DNA is taken directly into the cell cytoplasm either 
through these pores or as a consequence of the redistribution of membrane 
components that accompanies closure of the pores. Electroporation can be 
extremely efficient and can be used both for transient expression of 
clones genes and for establishment of cell lines that carry integrated 
copies of the gene of interest. Electroporation, in contrast to calcium 
phosphate-mediated transfection and protoplast fusion, frequently gives 
rise to cell lines that carry one, or at most a few, integrated copies of 
the foreign DNA. 
The introduction of DNA by means of electroporation, is well-known to those 
of skill in the art. In this method, certain cell wall-degrading enzymes, 
such as pectin-degrading enzymes, are employed to render the target 
recipient cells more susceptible to transformation by electroporation than 
untreated cells. Alternatively, recipient cells are made more susceptible 
to transformation, by mechanical wounding. To effect transformation by 
electroporation one may employ either friable tissues such as a suspension 
culture of cells, or embryogenic callus, or alternatively, one may 
transform immature embryos or other organized tissues directly. One would 
partially degrade the cell walls of the chosen cells by exposing them to 
pectin-degrading enzymes (pectolyases) or mechanically wounding in a 
controlled manner. Such cells would then be recipient to DNA transfer by 
electroporation, which may be carried out at this stage, and transformed 
cells then identified by a suitable selection or screening protocol 
dependent on the nature of the newly incorporated DNA. 
4.5.2 Microprojectile Bombardment 
A further advantageous method for delivering transforming DNA segments to 
plant cells is microprojectile bombardment. In this method, particles may 
be coated with nucleic acids and delivered into cells by a propelling 
force. Exemplary particles include those comprised of tungsten, gold, 
platinum, and the like. 
An advantage of microprojectile bombardment, in addition to it being an 
effective means of reproducibly stably transforming monocots, is that 
neither the isolation of protoplasts (Cristou et al., 1988) nor the 
susceptibility to Agrobacteritim infection is required. An illustrative 
embodiment of a method for delivering DNA into a monocot cell by 
acceleration is a Biolistics Particle Delivery System, which can be used 
to propel particles coated with DNA or cells through a screen, such as a 
stainless steel or Nytex screen, onto a filter surface covered with corn 
cells cultured in suspension. The screen disperses the particles so that 
they are not delivered to the recipient cells in large aggregates. It is 
believed that a screen intervening between the projectile apparatus and 
the cells to be bombarded reduces the size of projectiles aggregate and 
may contribute to a higher frequency of transformation by reducing damage 
inflicted on the recipient cells by projectiles that are too large. 
For the bombardment, cells in suspension are preferably concentrated on 
filters or solid culture medium. Alternatively, immature embryos or other 
target cells may be arranged on solid culture medium. The cells to be 
bombarded are positioned at an appropriate distance below the 
macroprojectile stopping plate. If desired, one or more screens are also 
positioned between the acceleration device and the cells to be bombarded. 
Through the use of techniques set forth herein one may obtain up to 1000 
or more foci of cells transiently expressing a marker gene. The number of 
cells in a focus which express the exogenous gene product 48 hours 
post-bombardment often range from 1 to 10 and average 1 to 3. 
In bombardment transformation, one may optimize the prebombardment 
culturing conditions and the bombardment parameters to yield the maximum 
numbers of stable transformants. Both the physical and biological 
parameters for bombardment are important in this technology. Physical 
factors are those that involve manipulating the DNA/microprojectile 
precipitate or those that affect the flight and velocity of either the 
macro- or microprojectiles. Biological factors include all steps involved 
in manipulation of cells before and immediately after bombardment, the 
osmotic adjustment of target cells to help alleviate the trauma associated 
with bombardment, and also the nature of the transforming DNA, such as 
linearized DNA or intact supercoiled plasmids. It is believed that 
pre-bombardment manipulations are especially important for successful 
transformation of immature embryos. 
Accordingly, it is contemplated that one may wish to adjust various of the 
bombardment parameters in small scale studies to fully optimize the 
conditions. One may particularly wish to adjust physical parameters such 
as gap distance, flight distance, tissue distance, and helium pressure. 
One may also minimize the trauma reduction factors (TRFs) by modifying 
conditions which influence the physiological state of the recipient cells 
and which may therefore influence transformation and integration 
efficiencies. For example, the osmotic state, tissue hydration and the 
subculture stage or cell cycle of the recipient cells may be adjusted for 
optimum transformation. The execution of other routine adjustments will be 
known to those of skill in the art in light of the present disclosure. 
4.5.3 Agrobacterium-Mediated Transfer 
Agrobacterium-mediated transfer is a widely applicable system for 
introducing genes into plant cells because the DNA can be introduced into 
whole plant tissues, thereby bypassing the need for regeneration of an 
intact plant from a protoplast. The use of Agrobacterium-mediated plant 
integrating vectors to introduce DNA into plant cells is well known in the 
art. See, for example, the methods described (Fraley et al., 1985; Rogers 
et al., 1987). Further, the integration of the Ti-DNA is a relatively 
precise process resulting in few rearrangements. The region of DNA to be 
transferred is defined by the border sequences, and intervening DNA is 
usually inserted into the plant genome as described (Spielmann et al., 
1986; Jorgensen et al., 1987). 
Modern Agrobacterium transformation vectors are capable of replication in 
E. coli as well as Agrobacterium, allowing for convenient manipulations as 
described (Klee et al., 1985). Moreover, recent technological advances in 
vectors for Agrobacterium-mediated gene transfer have improved the 
arrangement of genes and restriction sites in the vectors to facilitate 
construction of vectors capable of expressing various polypeptide coding 
genes. The vectors described (Rogers et al., 1987), have convenient 
multi-linker regions flanked by a promoter and a polyadenylation site for 
direct expression of inserted polypeptide coding genes and are suitable 
for present purposes. In addition, Agrobacterium containing both armed and 
disarmed Ti genes can be used for the transformations. In those plant 
strains where Agrobacterium-mediated transformation is efficient, it is 
the method of choice because of the facile and defined nature of the gene 
transfer. 
Agrobacterium-mediated transformation of leaf disks and other tissues such 
as cotyledons and hypocotyls appears to be limited to plants that 
Agrobacterium naturally infects. Agrobacteriuni-mediated transformation is 
most efficient in dicotyledonous plants. Few monocots appear to be natural 
hosts for Agrobacterium, although transgenic plants have been produced in 
asparagus using Agrobacterium vectors as described (Bytebier et al., 
1987). Therefore, commercially important cereal grains such as rice, corn, 
and wheat must usually be transformed using alternative methods. However, 
as mentioned above, the transformation of asparagus using Agrobacteriuni 
can also be achieved (see, for example, Bytebier et al., 1987). 
A transgenic plant formed using Agrobacterium transformation methods 
typically contains a single gene on one chromosome. Such transgenic plants 
can be referred to as being heterozygous for the added gene. However, 
inasmuch as use of the word "heterozygous" usually implies the presence of 
a complementary gene at the same locus of the second chromosome of a pair 
of chromosomes, and there is no such gene in a plant containing one added 
gene as here, it is believed that a more accurate name for such a plant is 
an independent segregant, because the added, exogenous gene segregates 
independently during mitosis and meiosis. 
More preferred is a transgenic plant that is homozygous for the added 
structural gene; i.e., a transgenic plant that contains two added genes, 
one gene at the same locus on each chromosome of a chromosome pair. A 
homozygous transgenic plant can be obtained by sexually mating (selfing) 
an independent segregant transgenic plant that contains a single added 
gene, germinating some of the seed produced and analyzing the resulting 
plants produced for enhanced insecticidal activity relative to a control 
(native, non-transgenic) or an independent segregant transgenic plant. 
It is to be understood that two different transgenic plants can also be 
mated to produce offspring that contain two independently segregating 
added, exogenous genes. Selfing of appropriate progeny can produce plants 
that are homozygous for both added, exogenous genes that encode a 
polypeptide of interest. Back-crossing to a parental plant and 
out-crossing with a non-transgenic plant are also contemplated. 
Transformation of plant protoplasts can be achieved using methods based on 
calcium phosphate precipitation, polyethylene glycol treatment, 
electroporation, and combinations of these treatments (see, e.g., Potrykus 
et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiya et al., 
1986; Callis et al., 1987; Marcotte et al., 1988). 
Application of these systems to different plant strains depends upon the 
ability to regenerate that particular plant strain from protoplasts. 
Illustrative methods for the regeneration of cereals from protoplasts are 
described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada et al., 
1986; Abdullah etal., 1986). 
To transform plant strains that cannot be successfully regenerated from 
protoplasts, other ways to introduce DNA into intact cells or tissues can 
be utilized. For example, regeneration of cereals from immature embryos or 
explants can be effected as described (Vasil, 1988). In addition, 
"particle gun" or high-velocity microprojectile technology can be utilized 
(Vasil, 1992). 
Using that latter technology, DNA is carried through the cell wall and into 
the cytoplasm on the surface of small metal particles as described (Klein 
et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metal 
particles penetrate through several layers of cells and thus allow the 
transformation of cells within tissue explants. 
4.6 Methods for Producing Insect-Resistant Transgenic Plants 
By transforming a suitable host cell, such as a plant cell, with a 
recombinant cryET29 gene-containing segment, the expression of the encoded 
crystal protein (i.e., a bacterial crystal protein or polypeptide having 
insecticidal activity against coleopterans) can result in the formation of 
insect-resistant plants. 
By way of example, one may utilize an expression vector containing a coding 
region for a B. thuringiensis crystal protein and an appropriate 
selectable marker to transform a suspension of embryonic plant cells, such 
as wheat or corn cells using a method such as particle bombardment 
(Maddock et al., 1991; Vasil et al., 1992) to deliver the DNA coated on 
microprojectiles into the recipient cells. Transgenic plants are then 
regenerated from transformed embryonic calli that express the insecticidal 
proteins. 
The formation of transgenic plants may also be accomplished using other 
methods of cell transformation which are known in the art such as 
Agrobacterium-mediated DNA transfer (Fraley et al., 1983). Alternatively, 
DNA can be introduced into plants by direct DNA transfer into pollen (Zhou 
et al., 1983; Hess, 1987; Luo et al., 1988), by injection of the DNA into 
reproductive organs of a plant (Pena et al., 1987), or by direct injection 
of DNA into the cells of immature embryos followed by the rehydration of 
desiccated embryos (Neuhaus et al., 1987; Benbrook et al., 1986). 
The regeneration, development, and cultivation of plants from single plant 
protoplast transformants or from various transformed explants is well 
known in the art (Weissbach and Weissbach, 1988). This regeneration and 
growth process typically includes the steps of selection of transformed 
cells, culturing those individualized cells through the usual stages of 
embryonic development through the rooted plantlet stage. Transgenic 
embryos and seeds are similarly regenerated. The resulting transgenic 
rooted shoots are thereafter planted in an appropriate plant growth medium 
such as soil. 
The development or regeneration of plants containing the foreign, exogenous 
gene that encodes a polypeptide of interest introduced by Agrobacterium 
from leaf explants can be achieved by methods well known in the art such 
as described (Horsch et al., 1985). In this procedure, transformants are 
cultured in the presence of a selection agent and in a medium that induces 
the regeneration of shoots in the plant strain being transformed as 
described (Fraley et al., 1983). 
This procedure typically produces shoots within two to four months and 
those shoots are then transferred to an appropriate root-inducing medium 
containing the selective agent and an antibiotic to prevent bacterial 
growth. Shoots that rooted in the presence of the selective agent to form 
plantlets are then transplanted to soil or other media to allow the 
production of roots. These procedures vary depending upon the particular 
plant strain employed, such variations being well known in the art. 
Preferably, the regenerated plants are self-pollinated to provide 
homozygous transgenic plants, as discussed before. Otherwise, pollen 
obtained from the regenerated plants is crossed to seed-grown plants of 
agronomically important, preferably inbred lines. Conversely, pollen from 
plants of those important lines is used to pollinate regenerated plants. A 
transgenic plant of the present invention containing a desired polypeptide 
is cultivated using methods well known to one skilled in the art. 
A transgenic plant of this invention thus has an increased amount of a 
coding region (e.g., a cry gene) that encodes the Cry polypeptide of 
interest. A preferred transgenic plant is an independent segregant and can 
transmit that gene and its activity to its progeny. A more preferred 
transgenic plant is homozygous for that gene, and transmits that gene to 
all of its offspring on sexual mating. Seed from a transgenic plant may be 
grown in the field or greenhouse, and resulting sexually mature transgenic 
plants are self-pollinated to generate true breeding plants. The progeny 
from these plants become true breeding lines that are evaluated for, by 
way of example, increased insecticidal capacity against coleopteran 
insects and cat flea larvae, preferably in the field, under a range of 
environmental conditions. The inventors contemplate that the present 
invention will find particular utility in the creation of transgenic 
plants of commercial interest including various turf grasses, wheat, corn, 
rice, barley, oats, a variety of ornamental plants and vegetables, as well 
as a number of nut- and fruit-bearing trees and plants. 
4.7 Nomenclature of the Novel Proteins 
The inventors have arbitrarily assigned the designation CryET29 to the 
novel protein of the invention. Likewise, the arbitrary designation of 
cryET29 has been assigned to the novel nucleic acid sequence which encodes 
this polypeptide. Formal assignment of the gene and protein designations 
based on the revised nomenclature of crystal protein endotoxins (Table 1) 
will be assigned by a committee on the nomenclature of B. thuringiensis, 
formed to systematically classify B. thuringiensis crystal proteins. The 
inventors contemplate that the arbitrarily assigned designations of the 
present invention will be superseded by the official nomenclature assigned 
to these sequences. 
4.8 Definitions 
The following words and phrases have the meanings set forth below. 
Expression: The combination of intracellular processes, including 
transcription and translation undergone by a coding DNA molecule such as a 
structural gene to produce a polypeptide. 
Promoter: A recognition site on a DNA sequence or group of DNA sequences 
that provide an expression control element for a structural gene and to 
which RNA polymerase specifically binds and initiates RNA synthesis 
(transcription) of that gene. 
Regeneration: The process of growing a plant from a plant cell (e.g., plant 
protoplast or explant). 
Structural gene: A gene that is expressed to produce a polypeptide. 
Transformation: A process of introducing an exogenous DNA sequence (e.g., a 
vector, a recombinant DNA molecule) into a cell or protoplast in which 
that exogenous DNA is incorporated into a chromosome or is capable of 
autonomous replication. 
Transformed cell: A cell whose DNA has been altered by the introduction of 
an exogenous DNA molecule into that cell. 
Transgenic cell: Any cell derived or regenerated from a transformed cell or 
derived from a transgenic cell. Exemplary transgenic cells include plant 
calli derived from a transformed plant cell and particular cells such as 
leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells 
obtained from a transgenic plant. 
Transgenic plant: A plant or progeny thereof derived from a transformed 
plant cell or protoplast, wherein the plant DNA contains an introduced 
exogenous DNA molecule not originally present in a native, non-transgenic 
plant of the same strain. The terms "transgenic plant" and "transformed 
plant" have sometimes been used in the art as synonymous terms to define a 
plant whose DNA contains an exogenous DNA molecule. However, it is thought 
more scientifically correct to refer to a regenerated plant or callus 
obtained from a transformed plant cell or protoplast as being a transgenic 
plant, and that usage will be followed herein. 
Vector: A DNA molecule capable of replication in a host cell and/or to 
which another DNA segment can be operatively linked so as to bring about 
replication of the attached segment. A plasmid is an exemplary vector. 
5. EXAMPLES 
The following examples are included to demonstrate preferred embodiments of 
the invention. It should be appreciated by those of skill in the art that 
the techniques disclosed in the examples which follow represent techniques 
discovered by the inventors to function well in the practice of the 
invention, and thus can be considered to constitute preferred modes for 
its practice. However, those of skill in the art should, in light of the 
present disclosure, appreciate that many changes can be made in the 
specific embodiments which are disclosed and still obtain a like or 
similar result without departing from the spirit and scope of the 
invention. 
5.1 Example 1 
Isolation of B. thuringiensis EG4096 
Crop dust samples were obtained from various sources throughout the U.S. 
and abroad, typically grain storage facilities. The crop dust samples were 
treated and spread on agar plates to isolate individual Bacillus-type 
colonies as described (Donovan et al., 1993). EG4096 is a wild-type B. 
thuringiensis strain isolated from a crop dust sample from Thailand. Phase 
contrast microscopy was used to visually examine the crystal morphology of 
the bacterial colonies from this crop dust. The colony designated EG4096 
contained endospores and crystalline inclusions of a unique morphology 
resembling short needles. The array of plasmids native to strain EG4096 is 
also unique. 
Insect bioassay of this wild-type B. thuringiensis strain determined that 
it had insecticidal activity against larvae of coleopteran insects, 
including Southern corn rootworm, western corn rootworm, Colorado potato 
beetle, red flour beetle, and Japanese beetle. EG4096 also exhibits 
insecticidal activity against larva of the cat flea. 
Characterization of EG4096 included the analysis of crystal protein 
produced by the strain during sporulation and the cloning and expression 
of the gene encoding the crystal protein, which has been designated 
cryET29. The insecticidal activity of both the wild-type strain and of a 
recombinant B. thuringiensis expressing the cloned cryET29 toxin gene was 
determined. 
5.2 Example 2 
Evaluation of the Native Plasmids of B. thuringienisis Strain EG4096 
The complement of native plasmids contained within isolated B. 
thuringiensis EG4096 was determined by modified Eckhardt agarose gel 
electrophoresis as described by Gonzalez et at., (1982). The pattern of 
native plasmids did not correspond to patterns of typical known serovars 
(Carlton and Gonzalez, 1985). The plasmid sizes are 5.0, 7.2, 6.0 (open 
circular), 39, 80 and 100 MDa. 
5.3 Example 3 
Evaluation of the Crystal Protein of B. thuringiensis EG4096 
EG4096 was grown in DSM+glucose sporulation medium [0.8% (wt/vol) Difco 
nutrient broth, 0.5% (wt/vol) glucose, 10 mM K.sub.2 HPO.sub.4, 10 mM 
KH.sub.2 PO.sub.4, 1 mM Ca(NO.sub.3).sub.2, 0.5 mM MgSO.sub.4, 10 .mu.M 
MnCl.sub.2, 10 .mu.M FeSO.sub.4 ] for three days at 30.degree. C. during 
which the culture grew to stationary phase, sporulated and lysed, thus 
releasing the protein inclusions into the medium. The cultures were 
harvested by centrifugation which pelleted the spores and crystals. The 
pellet was washed in a solution of 0.005% Triton X-100.RTM., 2 mM EDTA and 
recentrifuged. The washed pellet was resuspended at one-tenth the original 
volume of 0.005% Triton X-100.RTM., 2 mM EDTA. 
Crystal protein was solubilized from the spores-crystals suspension by 
incubating the suspension in solubilization buffer [0.14 M Tris-HCl pH 
8.0, 2% (wt/vol) sodium dodecyl sulfate (SDS), 5% (vol/vol) 
2-mercaptoethanol, 10% (vol/vol) glycerol, and 0.1% bromphenol blue] at 
100.degree. C. for 5 min. The solubilized crystal protein was size 
fractionated by SDS-PAGE. After size fractionation the proteins were 
visualized by Coomassie Brilliant Blue R-250 staining. This analysis 
showed that the major crystal protein present in sporulated cultures of 
EG4096 is approximately 25-kDa in size. This novel protein was designated 
CryET29. 
To further characterize CryET29, the NH.sub.2 -terminal amino acid sequence 
of the protein was determined. A sporulated culture of EG4096 was washed 
and resuspended. The suspension was solubilized and run on an acrylamide 
gel following the procedures for SDS-PAGE analysis. After electrophoresis 
the proteins were transferred to a BioRad PVDF membrane following standard 
western blotting procedures. After transfer, the membrane was rinsed 
3.times. in dH.sub.2 O and washed in Amido Black 1013 stain for 1 min 
(Sigma Chemical Co., St. Louis, Mo.). The filter was destained 1 min in 5% 
acetic acid and then rinsed in 3 changes of dH.sub.2 O. The portion of the 
filter containing the approximately 25-kDa protein band was excised with a 
razor blade. This procedure resulted in a pure form of CryET29 being 
obtained as a protein blotted onto a PVDF membrane (BioRad, Hercules, 
Calif.). 
The determination of the NH.sub.2 -terminal amino acid sequence of the 
purified CryET29 protein immobilized on the membrane was performed in the 
Department of Physiology at the Tufts Medical School, Boston, Mass. using 
standard automated Edman degradation procedures The NH.sub.2 -terminal 
sequence was determined to be: 
(SEQ ID NO:3) 
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 
MetPhePheAsnArgValIleTheLeuThrValProSerSerAsp 
Computer algorithms (Korn and Queen, 1984) were used to compare the 
N-terminal sequence of the CryET29 protein with amino acid sequences of 
all B. thuringiensis crystal proteins of which the inventors are aware 
including the sequences of all B. thuringiensis crystal proteins which 
have been published in scientific literature, international patent 
applications, or issued patents. A list of the crystal proteins whose 
sequences have been published along with the source of publication is 
shown in Table 4. 
TABLE 4 
______________________________________ 
B. thuringiensis Crystal Proteins Described in the Literature 
Crystal Protein 
Source or Reference 
______________________________________ 
Cry1A(a) J. Biol. Chem., 260:6264-6272 
Cry1A(b) DNA, 5:305-314 
Cry1A(c) Gene, 36:289-300 
Cry1B Nucl. Acids Res., 16:4168-4169 
Cry1C Nucl. Acids Res., 16:6240 
Cry1Cb Appl. Environ. Micro., 59: 1131-1137 
Cry1C(b) Nucl. Acids Res., 18:7443 
Cry1D Nucl. Acids Res., 18:5545 
Cry1E EPO 358 557 A2 
Cry1F J. Bacteriol., 173:3966-3976 
Cry1G FEBS, 293:25-28 
CryV WO 90/13651 
Cry2A J. Biol. Chem., 263:561-567 
Cry2B J. Bacteriol., 171:965-974 
Cry2C FEMS Microbiol. Lett., 81:31-36 
Cry3A Proc. Natl. Acad. Sci. USA, 84:7036-7040 
Cry3B Nucl. Acids Res., 18:1305 
Cry3B2 Appl. Environ. Microbiol., 58:3921-3927 
Cry3B3 U.S. 5,378,625 
Cry3C Appl. Environ. Microbiol., 58:2536-2542 
Cry3D Gene, 110:131-132 
Cry4A Nucl. Acids Res., 15:7195 
Cry4B EPO 308,199 
Cry4C J. Bacteriol., 166:801-811 
Cry4D J. Bacteriol., 170:4732, 1988 
Cry5 Molec. Micro., 6:1211-1217 
Cry33AkD WO 94/13785 
Cry33BkD WO 94/13785 
Cry34kD J. Bacteriol., 174:549-557 
Cry40kD J. Bacteriol., 174:549-557 
Cry201T635 WO 95/02693 
Cry517 J. Gen. Micro., 138:55-62 
Crya7A021 EPO 256,553 B1 
CryAB780RF1 WO 94/21795 
CryAB780RF2 WO 94/21795 
CryAB78100kD WO 94/21795 
Crybtpgs 1208 EPO 382 990 
Crybtpgs 1245 EPO 382 990 
Crybts02618A WO 94/05771 
CryBuibui WO 93/03154 
CryET4 U.S.5,322,687 
CryET5 U.S.5,322,687 
CryGei87 EPO 238,441 
CryHD511 U.S.5,286,486 
CryHD867 U.S.6,286,486 
CryIPL U.S.5,231,008 
CryMITS JP 6000084 
CryPS17A WO 92/19739 
CryPS17B U.S.5,350,576 and 5,424,410 
CryP16 WO 95/00639 
CryP18 WO 95/00639 
CryP66 WO 95/00639 
CryPS33F2 WO 92/19739 and U.S.5,424,410 
CryPS40D1 U.S.5,273,746 
CryPS43F WO 93/04587 
CryPS 50Ca WO 93/04587 and EPO 498,537 A2 
CryPS 50Cb WO 93/15206 
Cryps52A1 U.S.4,849,217 
CryPS63B WO 92119739 
CryPS69D1 U S. 5,424,410 
Cryps71M3 WO 95/02694 
CryPS80JJ1 WO 94/16079 
CryPS81IA U.S.5,273,746 
CryPS81IA2 EPO 405 810 
Cryps81A2 EPO 401 979 
CryPS81IB WO 93/14641 
CryPS81IB2 U.S.5,273,746 
Cryps81f U.S.5,045,469 
Cryps81gg U.S.5,273,746 
Cryps81rr1 EPO 401 979 
Cryps86A1 U S. 5,468,636 
CryX FEBS Lett., 336:79-82 
CryXenA24 WO 95/00647 
CrycytA Nucl. Acids Res., 13:8207-8217 
______________________________________ 
The N-terminal sequence of the CryET29 protein was not found to be 
homologous to any of the known B. thuringiensis crystal proteins 
identified in Table 4. 
5.4 Example 4 
Isolation of a DNA Fragment Comprising the B. thuringiensis EG4096 cryET29 
Gene 
In order to identify the gene encoding the CryET29 protein, an 
oligonucleotide probe specific for the NH.sub.2 -terminal amino acid 
sequence of the protein was designed. Using codons typically found in B. 
thuringiensis toxin genes, an oligo of 35 nucleotides was synthesized by 
Integrated DNA Technologies, Inc. (Coralville, Iowa) and designated wd270. 
The sequence of wd270 is: 
5'-ATGTTTTTTAATAGAGTAATTACATTAACAGTACC-3' (SEQ ID NO: 4) 
Radioactively-labeled wd270 was used as a probe in Southern blot studies as 
described below to identify a DNA restriction fragment containing the 
cryET29 gene. Total DNA was extracted from EG4096 by the following 
procedure. Vegetative cells were resuspended in a lysis buffer containing 
50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 4 mg/ml lysozyme. 
The suspension was incubated at 37.degree. C. for one hr. Following 
incubation, the suspension was extracted with an equal volume of phenol, 
one time with an equal volume of phenol:chloroform:isoamyl alcohol 
(50:48:2), and once with an equal volume of chloroform:isoamyl alcohol 
(24:1). DNA was precipitated from the aqueous phase by the addition of 
one-tenth volume 3 M sodium acetate then two volumes of 100% ethanol. The 
precipitated DNA was collected by centrifugation, washed with 70% ethanol 
and resuspended in dH.sub.2 O. 
The extracted DNA was then digested, in separate reactions, with various 
restriction endonucleases, including EcoRI. The digested DNA was size 
fractionated by electrophoresis through an 0.8% agarose gel in 1.times. 
TBE overnight at 2 V/cm. The fractionated DNA fragments were transferred 
to an Immobilon-NC nitrocellulose filter (Millipore Corp., Bedford, Mass.) 
according to the method of Southern (1975). DNA was fixed to the filter by 
baking at 80.degree. C. in a vacuum oven. 
To identify the DNA fragment(s) containing the sequence encoding the 
NH.sub.2 -terminus of the CryET29 protein (see Example 3) the 
oligonucleotide wd270 was radioactively labeled at the 5' ends and used as 
a hybridization probe. To radioactively label the probe, 1 to 5 pmoles 
wd270 was added to a reaction containing [.gamma.-.sup.32 p] ATP (3 .mu.l 
of 3,000 Ci/mmole at 10 mCi/mi in a 20 .mu.l reaction volume), a 10 
.times. reaction buffer (700 mM Tris-HCl, pH 7.8, 100 mM MgCl.sub.2, 50 mM 
DTT), and 10 units T4 polynucleotide kinase (Promega Corporation, Madison, 
Wis.). The reaction was incubated 20 minutes at 37.degree. C. to allow the 
transfer of the radioactive phosphate to the 5' end of the 
oligonucleotide, thus making it useful as a hybridization probe. 
The labeled probe was then incubated with the nitrocellulose filter 
overnight at 45.degree. C. in 3.times. SSC, 0.1% SDS, 10.times. Denhardt's 
reagent (0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% ficoll), 0.2 mg/ml 
heparin. Following incubation the filter was washed in several changes of 
3.times. SSC, 0.1 % SDS at 45.degree. C. The filter was blotted dry and 
exposed to Kodak X-OMAT AR X-ray film (Eastman Kodak Company, Rochester, 
N.Y.) overnight at -70.degree. C. with a DuPont Cronex Lightning Plus 
screen. 
The labeled probe was then incubated with the nitrocellulose filter which 
was then washed and exposed to X-ray film to obtain an autoradiogram. 
Examination of the autoradiogram identified two distinct EcoRi restriction 
fragments, of approximately 5.0 kb and 7.0 kb, that specifically 
hybridized to the labeled wd270 probe. This result indicated that strain 
EG4096 either contained two closely related, or identical, copies of the 
cryET29 gene, both of which hybridize to the wd270 oligonucleotide. 
5.5 Example 5 
Cloning of the cryET29 Gene of B. thuringiensis EG4096 
To isolate the 5.0 and 7.0 kilobase (kb) EcoRI restriction fragments 
containing the cryET29 gene, total genomic DNA was isolated from strain 
EG4096 as described in Example 4. The DNA was digested with EcoRI and 
electrophoresecl through a 0.8% agarose, 1.times. TBE gel, overnight at 2 
V/cm of gel length. The gel was stained with Ethidium bromide so that the 
digested DNA could be visualized when exposed to long-wave UV light. Gel 
slices containing DNA fragments of approximately 5.0 and 7.0 kb were 
excised from the gel with a razor blade and placed in separate dialysis 
bags containing a small volume (1 ml) of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA 
(TE). The DNA fragments were eluted from the gel slices into the TE buffer 
by placing the dialysis bags in a horizontal electrophoresis apparatus 
filled with 1.times. TBE and applying 100 V for 2 hr. This results in the 
DNA fragments migrating out of the gel slice into the TE buffer. The TE 
buffer containing the eluted fragments was then phenol:chloroform 
extracted and ethanol precipitated. 
To create a library in E. coli of the two sets of size selected EcoRI 
restriction fragments (approximately 5.0 and 7.0 kb), the fragments were 
ligated into the cloning vector pUC 18 (Yanisch-Perron, et al., 1985). The 
plasmid DNA vector pUC 18 can replicate at a high copy number in E. coli 
and carries the gene for resistance to the antibiotic ampicillin, which 
may be used as a selectable marker. The two sets of fragments were mixed, 
in separate reactions, with EcoRI-digested pUC18 that had been treated 
with bacterial alkaline phosphatase (GibcoBRL, Gaithersburg, Md.) to 
remove the 5' phosphates from the digested plasmid to prevent re-ligation 
of the vector to itself. T4 ligase and a ligation buffer (Promega 
Corporation, Madison, Wis.) were added to the reaction containing the 
digested pUC18 and the size-selected EcoRI fragments. These were incubated 
at room temperature for I hour to allow the insertion and ligation of the 
EcoRI fragments into the pUC 18 vector DNA. 
The ligation mixtures described above were introduced, separately, into 
transformation-competent E. coli DH5.alpha..TM. cells (purchased from 
GibcoBRL, Gaithersburg, Md.) following procedures described by the 
manufacturer. The transformed E. coli cells were plated on LB agar plates 
containing 50 .mu.g/ml ampicillin and incubated overnight at 37.degree. C. 
Both transformations yielded approximately 300 ampicillin-resistant 
colonies indicating the presence of a recombinant plasmid in the cells of 
each colony. 
To isolate the colonies harboring the cloned 5.0 and 7.0 kb EcoRl fragments 
that contain the crvET29 gene sequences the transformed E. coli colonies 
were first transferred to nitrocellulose filters. This was accomplished by 
simply placing a circular filter (Millipore HATF 085 25, Millipore Corp., 
Bedford, Mass.) directly on top of the LB-ampicillin agar plates 
containing the transformed colonies. When the filter is slowly peeled off 
of the plate the colonies stick to the filter giving an exact replica of 
the pattern of colonies from the original plate. Enough cells from each 
colony are left on the plate that 5 to 6 hr of growth at 37.degree. C. 
will restore the colonies. The plates are then stored at 4.degree. C. 
until needed. The nitrocellulose filters with the transferred colonies 
were then placed, colony-side up, on fresh LB-ampicillin agar plates and 
allowed to grow at 37.degree. C. until they reached a size of 
approximately 1 mm in diameter. 
To release the DNA from the recombinant E. coli cells onto the 
nitrocellulose filter the filters were placed, colony-side up, on 2 sheets 
of Whatman 3 MM Chr paper (Whatman International LTD., Maidstone, England) 
soaked with 0.5 N NaOH, 1.5 M NaCl for 15 min. This treatment lyses the 
cells and denatures the released DNA allowing it to stick to the 
nitrocellulose filter. The filters were then neutralized by placing the 
filters, colony-side up, on 2 sheets of Whatman paper soaked with 1 M 
NH.sub.4 -acetate, 0.02 M NaOH for 10 min. The filters were then rinsed in 
3.times. SSC, air dried, and baked for 1 hr at 80.degree. C. in a vacuum 
oven to prepare them for hybridization. 
The NH.sub.2 -terminal oligonucleotide specific for the cryET29 gene, 
wd270, was labeled at the 5' end with .gamma.-.sup.32 P and T4 
polynucleotide kinase as described above. The labeled probe was added to 
the filters in 3.times. SSC, 0.1% SDS, 10.times. Denhardt's reagent (0.2% 
BSA, 0.2% polyvinylpyrrolidone, 0.2% ficoll), 0.2 mg/ml heparin and 
incubated overnight at 45.degree. C. These conditions were chosen to allow 
hybridization of the labeled oligonucleotide to related sequences present 
on the nitrocellulose blots of the transformed E. coli colonies. Following 
incubation the filters were washed in several changes of 3.times. SSC, 0.1 
% SDS at 45.degree. C. The filters were blotted dry and exposed to Kodak 
X-OMAT AR x-ray film (Eastman Kodak Company, Rochester, N.Y.) overnight at 
-70.degree. C. with a DuPont Cronex Lightning Plus screen. 
Several colonies from each transformation (the 5.0 and 7.0 kb ligation 
mixes described above) hybridized to wd270. These colonies were identified 
by lining up the signals on the autoradiogram with the colonies on the 
original transformation plates. The isolated colonies were then grown in 
LB-ampicillin liquid medium from which the cells could be harvested and 
recombinant plasmid prepared by the standard alkaline-lysis miniprep 
procedure (described in Maniatis et al., 1982). The isolated plasmids were 
digested with the restriction enzyme EcoRI to determine if the cloned 
fragments of EG4096 DNA were of the expected size. All of the hybridizing 
plasmids from both the 5.0 kb and 7.0 kb constructions had the expected 
size insert fragment. The DNA from these plasmid digests were 
electrophoresed through an agarose gel and transferred to nitrocellulose 
as described above. The blot was then hybridized with the oligonucleotide, 
wd270, that had been radioactively labeled at the 5' end with 
.gamma.-.sup.32 P and T4 polynucleotide kinase. EcoRI fragments from two 
of the five plasmids containing 5.0 kb inserts hybridized to the probe 
confirming the presence of the cryET29 gene on those fragments. One of the 
plasmids with the 5.0 insert containing the cryET29 gene was designated 
pEG1298. EcoRI fragments from five of the six plasmids containing 7.0 kb 
inserts hybridized to the probe confirming the presence of the cryET29 
gene on those fragments. One of the plasmids with the 7.0 kb insert 
containing the crvET29 gene was designated pEG 1299, 
The E. coli strain containing pEG1298 has been designated EG11513. EG11513 
has been deposited with the Agricultural Research Culture Collection, 
Northern Regional Research Laboratory (NRRL) having Accession No. NRRL 
B-21624. The E. coli strain containing pEG 1299 has been designated EG 
11514. 
5.6 Example 6 
Determination of the DNA Sequence of the cryET29 gene 
A partial DNA sequence of the genes cloned on pEG1298 and pEG1299 was 
determined following established dideoxy chain-termination DNA sequencing 
procedures (Sanger et al., 1977). Preparation of the double stranded 
plasmid template DNA was accomplished using a Qiagen Plasmid Kit (Qiagen 
Inc., Chatsworth, Calif.) following manufacturer's procedures. The 
sequencing reactions were performed using the Sequenase.TM. Version 2.0 
DNA Sequencing Kit (United States Biochemical/Amersham Life Science Inc., 
Cleveland, Ohio) following manufacturer's procedures and using .sup.35 
S-dATP as the labeling isotope (obtained from Du Pont NEN.RTM. Research 
Products, Boston, Mass.). Denaturing gel electrophoresis of the reactions 
was done on a 6% (w/v) acrylamide, 42% (w/v) urea sequencing gel. The 
dried gel was exposed to Kodak X-OMAT AR X-ray film (Eastman Kodak 
Company, Rochester, N.Y.) overnight at room temperature. 
The NH.sub.2 -terminal specific oligonucleotide wd270 was used as the 
initial sequencing primer. The partial DNA sequences indicated that the 
plasmids pEG1298 and pEG1299 contained either identical, or nearly 
identical, copies of the cryET29 gene of B. thuringiensis strain EG4096. 
The entire DNA sequence for the copies of cryET29 on the two plasmids was 
completed using the procedures described above. Successive 
oligonucleotides to be used for priming sequencing reactions were designed 
from the sequencing data of the previous set of reactions. In this way the 
DNA sequencing progressed along both the top and bottom strand of the 
cryET29 gene in a step-wise fashion. 
The DNA sequence of both copies of the crvET29 (SEQ ID NO: 1) gene is 
identical and is shown in FIG. 1. The protein coding portion of the 
cryET29 gene is comprised of 696 nucleotides, including a stop codon. The 
CryET29 protein (SEQ ID NO: 2), as deduced from the DNA sequence, consists 
of 231 amino acids with a predicted molecular mass of 26,194 daltons. 
Database searches were then conducted to determine if the deduced amino 
acid sequence of the CryET29 protein shares identity with other 
characterized proteins, especially other insecticidal toxin proteins. 
Database searches using on-line servers were performed with the BLASTP 
program (Altschul et al., 1990) provided by the National Center for 
Biotechnology Information (Bethesda, Md.). The BLASTP searches were run 
with the BLOSUM62 matrix. The searched database consisted of non-redundant 
GenBank CDS translations +PDB+SwissProt+SPupdate+PIR. 
Only four proteins in these databases were identified with any significant 
identity to CryET29. These included the dipteran toxin CytB (55% identity; 
Koni and Ellar, 1993); the coleopteran/dipteran toxin CytA (44.2% 
identity; Ward et al., 1984); the dipteran toxin PS201T6 (41.1% identity; 
Intl. Pat. Appl. Publ. No. WO 95/02693) and the 27-kDa Bacillus 
thuringiensis morrissoni dipteran toxin (44.2% identity; Earp and Ellar, 
1987). 
5.7 Example 7 
Expression of the Cloned cryET29 Gene 
To characterize the properties of the CryET29 protein it was necessary to 
express the cloned cryET29 gene in B. thuringiensis cells that are 
negative for crystal proteins (Cry.sup.31). The cloned EcoRI fragments on 
pEG1298 and pEG1299 was inserted into a plasmid vector capable of 
replicating in B. thuringiensis, thus allowing the expression of cloned 
genes. pEG 1298 and pEG 1299 were digested with EcoRI to remove the cloned 
5 kb and 7 kb fragments, respectively. The digested plasmids were resolved 
on an agarose gel and the desired fragments were purified from gel slices 
using the GeneClean.RTM. procedure of Bio101, Inc. (Vista, Calif.). The 
fragments were ligated, separately, into a B. thuringiensis/E. coli 
shuttle vector that had been digested with EcoRI and treated with 
bacterial alkaline phosphatase. The shuttle vector pEG1297 had been 
constructed by ligating the 3.1 kb EcoRI fragment of the Bacillus pNN101 
(Norton et al., 1985) into NdeI digested pUC18. pEG1297 is capable of 
replication in both E. coli and B. thuringiensis and confers Amp.sup.R to 
E. coli and tetracycline (Tet) resistance (Tet.sup.R) to B. thuringiensis. 
The two ligation mixtures were first introduced into E. coli 
DH5.alpha..RTM. cells by transformation procedures described by the 
manufacturers (Gibco-BRL, Gaithersburg, Md.). Plasmid DNA was prepared 
from Amp.sup.R transformants and restriction enzyme analysis was performed 
to confirm the proper construction. The plasmid consisting of the 5-kb 
EcoRI fragment of pEG 1298 inserted into pEG 1297 was designated pEG 1302. 
The plasmid consisting of the 7-kb EcoRI fragment of pEG1299 inserted into 
pEG1297 was designated pEG1 303. 
pEG1302 and pEG1303 were separately introduced into a Cry.sup.31 B. 
thuringiensis strain, EG10368, by electroporation (Macaluso and Mettus, 
1991). Cells transformed to tetracycline resistance were selected by 
incubation overnight on LB agar plates containing 10 .mu.g/ml Tet. One 
Tet.sup.R colony from each transformation was selected for further 
analysis. Recombinant strain EG11494 contains pEG1302 (NRRL B-21583) and 
recombinant strain EG11502 contains pEG1303. 
EG11494 and EG11502 were grown in C2 sporulation medium containing 10 
.mu.g/ml tetracycline for 3 days at 30.degree. C. until sporulation and 
cell lysis had occurred. Microscopic examination of the sporulated 
cultures demonstrated that the recombinant strains were producing small 
crystalline inclusions. These crystals resemble the crystals produced by 
the wild-type strain EG4096, indicating that the cryET29 gene in each 
recombinant was a functional gene capable of directing the expression of 
the CryET29 protein. 
The sporulated cultures of EG11494 and EG11502 were harvested by 
centrifugation, washed, and resuspended in 0.005% Triton X-l100.RTM. in 
one-tenth the original volume. The crystal protein in the suspensions was 
characterized by SDS-PAGE analysis which revealed the production of an 
approximately 25-kDa protein by both EG11494 and EG11502. The 25-kDa 
proteins produced by the recombinant strains are identical in size as 
determined by migration on an SDS gel, to the crystal protein of EG4096. 
The amount of toxin protein contained in a particular sample was quantified 
for insect bioassays by SDS-PAGE. The Coomassie stained SDS-PAGE gel was 
scanned on a densitometer and compared with a standard curve generated by 
loading known amounts of a protein, such as bovine serum albumin, on the 
same gel. 
5.8 Example 8 
Toxicity of CryET29 to Southern Corn Rootwornm Larvae 
The toxicity to southern corn rootworm (SCRW) larvae (Diabrotica 
undecinipunctata howardi) was determined for wild-type B. thuringiensis 
EG4096 and for the two recombinant strains expressing the CryET29 protein, 
EG11494 and EG11502. 
EG4096, EG11494, and EG11502 were grown in C2 medium at 30.degree. C. for 3 
days until sporulation and cell lysis had occurred. The cultures were 
harvested by centrifugation, washed twice in 1.times. original volume 
0.005% Triton X-100.RTM., and resuspended in 1/10 the original culture 
volume on 0.005% Triton X-100.RTM.. For comparison, a recombinant B. 
thuringiensis strain, EG11535, expressing the coleopteran-toxic protein 
CryIIIB2 (Donovan et al., 1992), was grown and harvested in the same 
manner. 
SCRW larvae were bioassayed via surface contamination of an artificial diet 
similar to Marrone et al. (1985), but without formalin. Each bioassay 
consisted of eight serial aqueous dilutions with aliquots applied to the 
surface of the diet. After the diluent (an aqueous 0.005% Triton 
X-100.RTM. solution) had dried, first instar larvae were placed on the 
diet and incubated at 28.degree. C. Thirty-two larvae were tested per 
dose. Mortality was scored after 7 days. Data from replicated bioassays 
were pooled for probit analysis (Daum, 1970) with mortality being 
corrected for control death, the control being diluent only (Abbot, 1925). 
Results are reported as the amount of CryET29 crystal protein per well 
(175 mm.sup.2 of diet surface) resulting in an LC.sub.50, the co 
ncentration killing 50% of the test insects. 95% confidence intervals are 
also reported (Table 5). 
TABLE 5 
______________________________________ 
Insecticidal Activity of the CryET29 Protein to SCRW Larvae 
Sample LC.sub.50 (.mu.g protein/well) 
95% C.I. 
______________________________________ 
EG4096 35.3 29-43 
EG11494 24.3 20-30 
EG11502 26.7 22-32 
EG11535 (CryIIIB2) 
17.8 14-23 
______________________________________ 
The results shown in Table 5 demonstrate that the CryET29 protein has 
significant activity on larvae of the sou thern cor n rootworm. The 
CryET29 produced by the two recombinant strains, EG11494 and EG 11502, 
also exhibit significant toxicity. The SCRW activity of the CryET29 
protein produced in EG11494 and EG11502 is somewhat lower than that seen 
for the CryllIB2 protein, although the 95% confidence intervals do overlap 
slightly, indicating that the difference may not be significant. 
5.9 Example 9 
Toxicity of CryET29 to Western Corn Rootworm Larvae 
The toxicity to western corn rootworm (WCRW) larvae (Diabrotica virgifera 
virgifera) was determined for wild-type B. thuringiensis EG4096 and for 
the two recombinant strains expressing the CryET29 protein, EG11494 and 
EG11502. 
The samples were prepared and the bioassays performed essentially as 
described for the SCRW a ssays in Example 8. The wild-type B. 
thuringiensis strain EG4961, which produces the Coleopteran-active 
CryIIIB2 protein, was included in the assay as a positive control (Table 
6). 
TABLE 6 
______________________________________ 
Insecticidal Activity of the CryET29 Protein to SCRW Larvae 
Sample LC.sub.50 (.mu.g protein/well) 
95% C.I. 
______________________________________ 
EG4961 (CryIIIB2) 
73.8 44-211 
EG4096 12.9 7-110 
EG11494 8.7 4-19 
EG11502 13.9 9-29 
______________________________________ 
The results in Table 6 demonstrate that the CryET29 protein has significant 
activity on larvae of the WCRW. Furthermore, the activity of the CryET29 
produced by the recombinant strains EG11494 and EG11502 have significantly 
higher activity (i.e., lower LC.sub.50 s) than the protein produced by the 
coleopteran-active B. thuringiensis strain EG4096961. 
5.10 Example 10 
of CryET29 to Colorado Potato Beetle Larvae 
The toxicity to Colorado potato beetle (CPB) (Leptinotarsa decemlineata) 
larvae was determined for the wild-type B. thuringiensis strain EG4096 and 
for the recombinant strain expressing the CryET29 protein, EG11494. The 
recombinant strain EG7231, which expresses the CryIIIB2 protein, was grown 
for purposes of comparison. 
The assay on CPB larvae was performed using similar techniques to the SCRW 
assay, except for the substitution of BioServe's #9380 insect diet (with 
potato flakes added) for the artificial diet. Mortality was scored at 
three days instead of seven days. For this assay 16 insects were used per 
dose (Table 7). 
TABLE 7 
______________________________________ 
Percent Mortality of CPB Larvae Treated With 
CryET29-Producing Strains 
Dose in .mu.g/well 
EG4096 EG11494 EG7231 (CryIIIB2) 
______________________________________ 
4.375 100 68.75 
8.75 100 75 
9.375 100 
17.5 100 75 
35 100 93 
______________________________________ 
The results shown in Table 7 demonstrate the insecticidal activity of the 
CryET29 protein on CPB larvae. 
5.11 Example 11 
Toxicity of B. thuringiensis EG4096 to Red Flour Beetle Larvae 
Toxicity of EG4096 to red flour beetle larvae (Triboliurn castaneum) was 
determined by applying a washed and concentrated sporulated culture of 
EG4096 to an artificial diet and allowing the larvae to feed on the diet. 
Sixteen larvae were treated in this manner and the percent mortality was 
scored after two weeks. Larvae treated with the EG4096 suspension 
exhibited 44% mortality compared to 13% for the untreated check. In 
addition the surviving larvae treated with EG4096 exhibited significant 
stunting in their growth which is indicative of a sublethal dose of an 
active toxin. The larvae in the untreated check showed no such stunting. 
These results demonstrate that EG4096, which produces the CryET29 protein, 
is toxic to red flour beetle. 
5.12 Example 12 
Toxicity of B. thuringiensis EG4096 to Japanese Beetle Larvae 
The toxicity to Japanese beetle (JB) larvae (Popillia japonica) was 
determined for B. thuringiensis EG4096, which produces the CryET29 
protein. Freeze-dried powders were prepared from washed and concentrated 
sporulated cultures of EG4096. The amount of CryET29 protein present in 
the sample was determined by SDS-PAGE and quantitative densitometry of the 
Coomassie stained gels. 
The freeze-dried powders were resuspended in a diluent containing 0.005% 
Triton X-100.RTM. and incorporated into 100 ml of hot (50-60.degree. C.) 
liquid artificial diet (based on the insect diet described by Ladd (1986). 
The mixtures were allowed to solidify in Petri dishes, and 19-mm diameter 
plugs of the solidified diet were placed into 5/8 ounce plastic cups. One 
JB larva was introduced per cup which were then covered with a lid and 
held at 25.degree. C. for fourteen days before larval mortality was 
scored. 
Table 8 shows the average of results from two replications of the bioassay 
using 20 larvae per replication. The dosages were based on the amount of 
CryET29 protein in the sample. Percent mortality was corrected according 
to Abbott (1925). 
TABLE 8 
______________________________________ 
Toxicity of EG4096 to Japanese Beetle Larvae 
Amount CryET29 (ppm) 
% Mortality 
______________________________________ 
250 ppm 9 
500 ppm 69 
1000 ppm 92 
2000 ppm 96 
______________________________________ 
The results shown in Table 8 demonstrate that the CryET29 protein produced 
by EG4096 has significant insecticidal activity on JB larvae. 
5.13 Example 13 
Toxicity of B. thuringiensis EG4096 to Cat Flea Larvae 
The toxicity to larvae of the cat flea (Ctenocephalides felis) was 
determined for B. thuringiensis EG4096, which produces the CryET29 
protein. Freeze-dried powders were prepared from washed and concentrated 
sporulated cultures of EG4096. The amount of CryET29 protein present in 
the sample was determined by SDS-PAGE. 
To perform the bioassay an amount of the freeze-dried powder containing 1 
mg of CryET29 protein was mixed with 1 gram of dried bovine blood 
resulting in a concentration of 1000 ppm. The mixture was suspended in 
0.1% Triton X-100.RTM. and poured into a glass Petri dish to dry. The 
dried sample was then ground into a fine powder and evenly distributed 
into 32 bioassay wells. One cat flea larva was added to each well which 
was then covered with a lid and kept at high humidity. The assays were 
then scored after seven days. 
The assay is performed in this manner using a powder of EG4096 as the 
sample and the results are shown in Table 9. Thirty-two larvae were 
assayed at each dose. Percent mortality was scored after 1, 4, and 7 days. 
A B. thuringiensis strain that does not produce a toxin protein, EG2205, 
was used to assess control mortality. 
TABLE 9 
______________________________________ 
Toxicity of EG4096 to First Instar Cat Flea Larvae 
% Mortality 
Strain CryET29 (ppm) 
1 Day 4 Day 7 Day 
______________________________________ 
EG4096 500 6.25 15.60 15.60 
EG4096 1000 9.40 34.40 43.75 
EG4096 5000 46.90 78.10 87.50 
EG4096 10000 84.40 93.75 100.00 
EG2205 No toxin 3.10 15.60 15.60 
______________________________________ 
The results shown in Table 9 demonstrate that the CryET29 protein produced 
by Bacillus thuringiensis strain EG4096 has significant insecticidal 
activity on larvae of the cat flea, Ctenocephalides felis. 
The uniqueness of the activity of the CryET29 toxin on cat fleas larvae was 
demonstrated by assaying other Bacillus thuringiensis insecticidal crystal 
proteins in the manner described above. Samples containing spores and 
crystals were tested from recombinant strains of B. thuringiensis 
expressing the following toxin proteins: Cry1Aa, Cry1Ab, CrylAc, Cry2S, 
Cry3A, Cry3B, Cry3B2, and Cry3B3. The characteristics of these other 
classes of insecticidal crystal protein genes are described by Hofte et 
al., (1989). For a detailed description of the Cry3 toxins, see U.S. Pat. 
No. 5,187,091 and U.S. Pat. No. 5,264,364, specifically incorporated 
herein by reference. None of these toxins showed any toxicity toward the 
larvae of the cat flea indicating that the CryET29 toxin protein is unique 
among B. thuringiensis insecticidal proteins isolated to date with respect 
to its cat flea larvae toxicity. 
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__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 4 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 693 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..693 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- ATG TTC TTT AAT CGC GTT ATT ACA TTA ACA GT - #A CCA TCT TCA GAT GTG 
48 
Met Phe Phe Asn Arg Val Ile Thr Leu Thr Va - #l Pro Ser Ser Asp Val 
# 15 
- GTT AAT TAT AGT GAA ATT TAT CAG GTA GCT CC - #A CAA TAT GTG AAT CAA 
96 
Val Asn Tyr Ser Glu Ile Tyr Gln Val Ala Pr - #o Gln Tyr Val Asn Gln 
# 30 
- GCT CTT ACG CTA GCT AAA TAT TTC CAA GGA GC - #A ATT GAT GGT TCA ACA 
144 
Ala Leu Thr Leu Ala Lys Tyr Phe Gln Gly Al - #a Ile Asp Gly Ser Thr 
# 45 
- TTA CGT TTT GAT TTT GAA AAA GCC TTA CAA AT - #T GCA AAT GAT ATT CCA 
192 
Leu Arg Phe Asp Phe Glu Lys Ala Leu Gln Il - #e Ala Asn Asp Ile Pro 
# 60 
- CAG GCA GCA GTG GTA AAC ACT TTA AAT CAA AC - #T GTG CAG CAA GGT ACA 
240 
Gln Ala Ala Val Val Asn Thr Leu Asn Gln Th - #r Val Gln Gln Gly Thr 
# 80 
- GTC CAA GTA TCA GTG ATG ATA GAC AAG ATT GT - #A GAC ATT ATG AAG AAT 
288 
Val Gln Val Ser Val Met Ile Asp Lys Ile Va - #l Asp Ile Met Lys Asn 
# 95 
- GTA TTA TCT ATT GTA ATT GAT AAC AAA AAG TT - #T TGG GAT CAG GTA ACA 
336 
Val Leu Ser Ile Val Ile Asp Asn Lys Lys Ph - #e Trp Asp Gln Val Thr 
# 110 
- GCT GCT ATT ACA AAT ACA TTC ACA AAT CTA AA - #T TCG CAA GAA AGC GAA 
384 
Ala Ala Ile Thr Asn Thr Phe Thr Asn Leu As - #n Ser Gln Glu Ser Glu 
# 125 
- CGA TGG ATT TTT TAT TAC AAA GAA GAT GCA CA - #T AAA ACT AGT TAC TAT 
432 
Arg Trp Ile Phe Tyr Tyr Lys Glu Asp Ala Hi - #s Lys Thr Ser Tyr Tyr 
# 140 
- TAT AAT ATC TTA TTT GCT ATA CAG GAT GAG GA - #A ACA GGT GGG GTA ATG 
480 
Tyr Asn Ile Leu Phe Ala Ile Gln Asp Glu Gl - #u Thr Gly Gly Val Met 
145 1 - #50 1 - #55 1 - 
#60 
- GCG ACA TTA CCG ATT GCA TTT GAT ATT AGT GT - #A GAT ATT GAA AAA GAA 
528 
Ala Thr Leu Pro Ile Ala Phe Asp Ile Ser Va - #l Asp Ile Glu Lys Glu 
# 175 
- AAG GTT CTA TTT GTT ACT ATC AAG GAT ACT GA - #A AAT TAT GCG GTT ACA 
576 
Lys Val Leu Phe Val Thr Ile Lys Asp Thr Gl - #u Asn Tyr Ala Val Thr 
# 190 
- GTA AAA GCT ATT AAT GTA GTA CAA GCA CTT CA - #A TCT TCC CGA GAT TCA 
624 
Val Lys Ala Ile Asn Val Val Gln Ala Leu Gl - #n Ser Ser Arg Asp Ser 
# 205 
- AAA GTT GTA GAT GCT TTT AAA TCG CCA CGT CA - #C TTA CCT AGA AAA AGA 
672 
Lys Val Val Asp Ala Phe Lys Ser Pro Arg Hi - #s Leu Pro Arg Lys Arg 
# 220 
# 693 AAC TCT 
His Lys Ile Cys Ser Asn Ser 
225 2 - #30 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 231 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- Met Phe Phe Asn Arg Val Ile Thr Leu Thr Va - #l Pro Ser Ser Asp Val 
# 15 
- Val Asn Tyr Ser Glu Ile Tyr Gln Val Ala Pr - #o Gln Tyr Val Asn Gln 
# 30 
- Ala Leu Thr Leu Ala Lys Tyr Phe Gln Gly Al - #a Ile Asp Gly Ser Thr 
# 45 
- Leu Arg Phe Asp Phe Glu Lys Ala Leu Gln Il - #e Ala Asn Asp Ile Pro 
# 60 
- Gln Ala Ala Val Val Asn Thr Leu Asn Gln Th - #r Val Gln Gln Gly Thr 
# 80 
- Val Gln Val Ser Val Met Ile Asp Lys Ile Va - #l Asp Ile Met Lys Asn 
# 95 
- Val Leu Ser Ile Val Ile Asp Asn Lys Lys Ph - #e Trp Asp Gln Val Thr 
# 110 
- Ala Ala Ile Thr Asn Thr Phe Thr Asn Leu As - #n Ser Gln Glu Ser Glu 
# 125 
- Arg Trp Ile Phe Tyr Tyr Lys Glu Asp Ala Hi - #s Lys Thr Ser Tyr Tyr 
# 140 
- Tyr Asn Ile Leu Phe Ala Ile Gln Asp Glu Gl - #u Thr Gly Gly Val Met 
145 1 - #50 1 - #55 1 - 
#60 
- Ala Thr Leu Pro Ile Ala Phe Asp Ile Ser Va - #l Asp Ile Glu Lys Glu 
# 175 
- Lys Val Leu Phe Val Thr Ile Lys Asp Thr Gl - #u Asn Tyr Ala Val Thr 
# 190 
- Val Lys Ala Ile Asn Val Val Gln Ala Leu Gl - #n Ser Ser Arg Asp Ser 
# 205 
- Lys Val Val Asp Ala Phe Lys Ser Pro Arg Hi - #s Leu Pro Arg Lys Arg 
# 220 
- His Lys Ile Cys Ser Asn Ser 
225 2 - #30 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 15 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- Met Phe Phe Asn Arg Val Ile Thr Leu Thr Va - #l Pro Ser Ser Asp 
# 15 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 34 TAAT TACATTAACA GTAC 
__________________________________________________________________________ 
All of the compositions and methods disclosed and claimed herein can be 
made and executed without undue experimentation in light of the present 
disclosure. While the compositions and methods of this invention have been 
described in terms of preferred embodiments, it will be apparent to those 
of skill in the art that variations may be applied to the composition, 
methods and in the steps or in the sequence of steps of the method 
described herein without departing from the concept, spirit and scope of 
the invention. More specifically, it will be apparent that certain agents 
which are both chemically and physiologically related may be substituted 
for the agents described herein while the same or similar results would be 
achieved. All such similar substitutes and modifications apparent to those 
skilled in the art are deemed to be within the spirit, scope and concept 
of the invention as defined by the appended claims. Accordingly, the 
exclusive rights sought to be patented are as described in the claims 
below.