Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-06
2003-03-25
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S023710, C435S320100, C435S252300, C435S252330, C435S252310, C435S252340, C435S325000, C435S419000, C435S069100, C436S063000
Reexamination Certificate
active
06537756
ABSTRACT:
1. BACKGROUND OF 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 &dgr;-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. thuringiensis
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 CryIII proteins, while cryIV genes encode dipteran-toxic 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
&dgr;-Endotoxin Gene Nomenclature
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
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 &dgr;-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.
1
A and FIG.
1
B. 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
Donovan William P.
Rupar Mark J.
Slaney Annette C.
Tan Yuping
Ball, Esq. Timothy K.
Howrey Simon Arnold & White , LLP
Monsanto Technology LLC
Prouty Rebecca E.
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