Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-14
2001-06-19
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C436S094000, C536S023710, C514S002600
Reexamination Certificate
active
06248536
ABSTRACT:
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 novel cryET33 and cryET34 genes from
Bacillus thuringiensis
encoding coleopteran-toxic crystal proteins. Various methods for making and using these DNA segments, DNA segments encoding synthetically-modified Cry 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
One of the unique features of
B. thuringiensis
is its production of crystal proteins during sporulation 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 proteins having insecticidal activity against lepidopteran and dipteran insects 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 mechanism of insecticidal activity of the
B. thuringiensis
crystal proteins has been studied extensively in the past decade. It has been shown that the crystal proteins are toxic to the insect only after ingestion of the protein by the insect. The alkaline pH and proteolytic enzymes in the insect mid-gut solubilize the proteins, thereby allowing the release of components which are toxic to the insect. These toxic components disrupt the mid-gut cells, cause the insect to cease feeding, and, eventually, bring about insect death. For this reason,
B. thuringiensis
has proven to be an effective and environmentally safe insecticide in dealing with various insect pests.
As noted by Höfte et al., (1989) the majority of insecticidal
B. thuringiensis
strains are active against insects 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 (Krieg et al., 1983; Sick et al., 1990; Lambert et al., 1992).
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 Höfte et al. (1989) discusses the genes and proteins that were identified in
B. thuringiensis
prior to 1990, and sets forth the nomen-clature 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 I
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
Cry1Be
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
Cry11a
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
CryET33, Present Invention
To Be Assigned
To Be Assigned
CryET34, 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 utility of bacterial crystal proteins as insecticides was extended when the first isolation of a coleopteran-toxic
B. thuringiensis
strain was reported (Kreig et al., 1983; 1984). This strain (described in U.S. Pat. No. 4,766,203, specifically incorporated herein by reference), designated
B. thuringiensis
var. tenebrionis, is reported to be toxic to larvae of the coleopteran insects
Agelastica alni
(blue alder leaf beetle) and
Leptinotarsa decemlineata
(Colorado potato beetle).
U.S. Pat. No. 4,766,203 (specifically incorporated herein by reference) relates to a 65-70 kilodalton (kDa) insecticidal crystal protein identified in B. thuringiensis tenebrionis (see also Berhnard, 1986). Sekar et al., (1987) report the cloning and characterization of a gene for a coleopteran-toxic crystal protein from B. thuringiensis tenebrionis. The predicted size of the polypeptide (as deduced from the gene sequence) is 73 kDa, however, the isolated protein consists primarily of a 65-kDa component. Hofte et al. (1987) also reports the DNA sequence for the cloned gene from B. thuringiensis tenebrionis, with the sequence of the gene being identical to that reported by Sekar et al. (1987).
McPherson et al. (1988) discloses a DNA sequence for the cloned insect control gene from B. thuringiensis tenebrionis; the sequence was identical to that reported by Sekar et al. (1987).
E. coli
cells and
Pseudomonas fluorescens
cells harboring the cloned gene were found to be toxic to Colorado potato beetle larvae.
Intl. Pat. Appl. Publ. No. WO 91/07481 dated May 30, 1991, describes
B. thuringiensis
mutants that produce high yields of the same insecticidal proteins originally made by the parent strains at lesser yields. Mutants of the coleopteran-toxic B. thuringiensis tenebrionis strain are disclosed.
A coleopteran-toxic strain, designated
B. thuringiensis
var. san diego, was reported by Herrnstadt et al. (1986) to produce a 64-kDa crystal protein toxic to some coleopterans, including
Pyrrhalta luteola
(elm leaf beetle);
Anthonomus gradis
(boll weevil),
Leptinotarsa decemlineata
(Colorado potato beetle),
Osiorhynchus sulcatus
(black vine weevil),
Tenebrio molitor
(yellow mealworm),
Haltica zombacina;
and Diabrotica undecimpunctata undecimpunctata (western spotted cucumber beetle).
The DNA sequence of a coleopteran toxin gene from B. thuringiensis san diego was reported by Herrnstadt et al. (1987); and was disclosed in U.S. Pat. No. 4,771,131. The sequence of the toxin gene of B. thuringiensis san diego is identical to that reported by Sekar et al. (1987) for the cloned coleopteran toxin gene of B. thuringiensis tenebrionis. Krieg et al., (1987) demonstrated that B. thuringiensis san diego was identical to B. thuringiensis tenebrionis, based on various diagnostic tests.
Another
B. thuringiensis
strain, EG2158
Donovan Judith C.
Donovan William P.
Slaney Annette C.
Ball Timothy K.
Carlson Karen Cochrane
Howrey Simon Arnold & White , LLP
Monsanto Company
Tu Stephen
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