Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-02-17
2001-01-23
Cochrane Carlson, Karen (Department: 1653)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S069100, C435S252310, C435S320100, C435S410000, C536S023710, C536S024100, C530S350000, 57, 57
Reexamination Certificate
active
06177615
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology. Certain embodiments concern methods and compositions comprising novel nucleic acid segments and their encoded
Bacillus thuringiensis
-derived &dgr;-endotoxins. More particularly, it concerns methods of altering the structure of Cry1 crystal proteins by mutagenesis of the loop regions between the &agr;-helices of the protein's domain 1 or of the loop region between &agr;-helix 7 of domain 1 and &bgr;-strand 1 of domain 2 to give rise to modified Cry1 proteins (Cry1*). Exemplary mutagenized Cry1C* proteins are disclosed which have modified amino acid sequences in loop regions &agr;3,4; &agr;4,5; and &agr;5,6. The resulting novel Cry1C* gene products encode crystal proteins which have improved activity against members of the Order Lepidoptera. Various methods for making and using these recombinantly-engineered proteins, methods for making and using the nucleic acid segments which encode them, and methods for preparing recombinant host cells and transgenic plants comprising the novel synthetically-modified Cry1C* proteins are disclosed. Also disclosed are compositions comprising transgenic plant cells, their progeny, and seeds derived therefrom.
2. Description of the Related Art
The most widely used microbial pesticides are derived from the bacterium
Bacillus thuringiensis. B. thuringiensis
is a Gram-positive bacterium that produces 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.
&dgr;-endotoxins are used to control a wide range of leaf-eating caterpillars and beetles, as well as mosquitoes.
B. thuringiensis
produces a proteinaceous parasporal body or crystal which is toxic upon ingestion by a susceptible insect host. For example,
B. thuringiensis
subsp.
kurstaki
HD-1 produces a crystal inclusion comprising &dgr;-endotoxins which are toxic to the larvae of a number of insects in the order Lepidoptera (Schnepf and Whiteley, 1981).
&dgr;-Endotoxins
&dgr;-endotoxins are a large collection of insecticidal proteins produced by
B. thuringiensis
. Over the past decade research on the structure and function of
B. thuringiensis
toxins has covered all of the major toxin categories, and while these toxins differ in specific structure and function, general similarities in the structure and function are assumed. Based on the accumulated knowledge of
B. thuringiensis
toxins, a generalized mode of action for
B. thuringiensis
toxins has been created and includes: ingestion by the insect, solubilization in the insect midgut (a combination stomach and small intestine), resistance to digestive enzymes sometimes with partial digestion actually “activating” the toxin, binding to the midgut cells, formation of a pore in the insect cells and the disruption of cellular homeostasis (English and Slatin, 1992).
Genes Encoding Crystal Proteins
Many of the &dgr;-endotoxins are related to various degrees by similarities in their amino acid sequences. Historically, the proteins and the genes which encode them were classified based largely upon their spectrum of insecticidal activity. The review by Höfte and Whiteley (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.
Based on the degree of sequence similarity, the proteins were further classified into subfamilies; more highly related proteins within each family were assigned divisional letters such as CryIA, CryIB, CryIC, etc. Even more closely related proteins within each division were given names such as CryIC1, CryIC2, etc.
Recently a new nomenclature has been proposed which systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. This classification scheme is summarized in TABLE 1.
TABLE 1
Revised
B. thuringiensis
&dgr;-Endotoxin 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
a
Adapted from: http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html
Crystal Proteins Find Utility as Bioinsecticides
The utility of bacterial crystal proteins as insecticides was extended when the first isolation of a coleopteran-toxic
B. thuringiensis
strain was reported (Krieg 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. 5,024,837 also describes hybrid
B. thuringiensis
var.
kurstaki
strains which showed activity against lepidopteran insects. U.S. Pat. No. 4,797,279 (corresponding to EP 0221024) discloses a hybrid
B. thuringiensis
containing a plasmid from
B. thuringiensis
var.
kurstaki
encoding a lepidopteran-toxic crystal protein-encoding gene and a plasmid from
B. thuringiensis tenebrionis
encoding a coleopteran-toxic crystal protein-encoding gene. The hybrid
B. thuringiensis
strain produces crystal proteins characteristic of those made by both
B. thuringiensis kurstaki
and
B. thuringiensis tenebrionis.
U.S. Pat. No. 4,910,016 (corresponding to EP 0303379) discloses a
B. thuringiensis
isolate identified as
B. thuringiensis
MT 104 which has insecticidal activity against coleopterans and lepidopterans.
Cry1 Crystal Proteins
The characterization of the lepidopteran-toxic
B. thuringiensis
Cry1Aa crystal protein, and the cloning, DNA sequencing, and expression of the gene which encodes it have been described (Schnepf and Whitely, 1981; Schnepf et al., 1985). In related publications, U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 (specifically incorporated herein by reference), the expression of the native
B. thuringiensis
Cry1Aa crystal protein in
E. coli
is disclosed.
Several cry1C genes have been described in the prior art. A cry1C gene truncated at the 3′ end was isolated from
B. thuringiensis
subsp.
aizawai
7.29 by Sanchis et al. (1988). The truncated protein exhibited toxicity towards Spodoptera species. The sequence of the truncated cry1C gene and its e
Ball, Esq. Timothy K.
Carlson Karen Cochrane
Howrey Simon Arnold & White , LLP
Monsanto Company
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