Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
1999-02-19
2001-08-28
Navarro, Mark (Department: 1645)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C435S410000, C435S412000, C435S415000, C435S424000, C435S426000, C435S427000, C435S430000, C800S278000, C800S279000, C800S295000, C800S300100, C800S301000, C800S314000, C800S320000, C800S320100, C800S320300, C800S322000
Reexamination Certificate
active
06281016
ABSTRACT:
1. BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention provides new proteins for combatting insects, and particularly, coleopteran, dipteran, and lepidopteran insects sensitive to the disclosed &dgr;-endotoxins derived from
Bacillus thuringiensis
. The invention provides novel chimeric crystal proteins and the chimeric cry gene segments which encode them, as well as methods for making and usmg these DNA segments, methods of producing the encoded proteins, methods for making synthetically-modified chimeric crystal proteins, and methods of making and using the synthetic crystal proteins.
1.2 Description of Related Art
1.2.1
B. Thuringiensis
Crystal Proteins
The Gram-positive soil bacteriumn
B. thuringiensis
is well known for its production of proteinaceous parasporal crystals, or &dgr;-endotoxins, that are toxic to a variety of lepidopteran, coleopteran, and dipteran larvae.
B. thuringiensis
produces crystal proteins during sporulation which are specifically toxic to certain species of insects. Many different strains of
B. thuringiensis
have been shown to produce insecticidal crystal proteins, and compositions comprising
B. thuringiensis
strains which produce proteins having insecticidal activity have been used commercially as environmentally-acceptable insecticides because of their toxicity to the specific target insect, and nontoxicity to plants and other non-targeted organisms.
Commercial formulations of naturally occurring
B. thuringiensis
isolates have long been used for the biological control of agricultural insect pests. In commercial production, the spores and crystals obtained from the fermentation process are concentrated and formulated for foliar application according to conventional agricultural practices.
1.2.2 Nomenclature of Crystal Proteins
A review by Höfte et al., (1989) describes the general state of the art with respect to the majority of insecticidal
B. thuringiensis
strains that have been identified which are active against insects of the Order Lepidoptera, i.e., caterpillar insects. This treatise also describes
B. thuringiensis
strains having insecticidal activity against insects of the Orders Diptera (ie. flies and mosquitoes) and Coleoptera (ie. beetles). A number of genes encoding crystal proteins have been cloned from several strains of
B. thuringiensis
. Höfte 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. cry1 genes encode lepidopteran-toxic Cry1 proteins. cry2 genes encode Cry2 proteins that are toxic to both lepidopterans and dipterans. cry3 genes encode coleopteran-toxic Cry3 proteins, while cry4 genes encode dipteran-toxic Cry4 proteins, 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
Cry1Bd
CryE1
U70726
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
Cry1Ga
PrtA
Z22510
Cry1Gb
CryH2
U70725
Cry1Ha
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
cbm7l
X94146
Cry17A
cbm71
X99478
Cry18A
CryBP1
X99049
Cry19A
Jeg65
Y08920
Cyt1Aa
CytA
X03182
Cyt1Ab
CytM
X98793
Cyt1B
U37196
Cyt2A
CytB
Z14147
Cyt2B
CytB
U52043
A
Adapted from: http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html
1.23 Mode of Crystal Protein Toxicity
All &dgr;-endotoxin crystals are toxic to insect larvae by ingestion. Solubilization of the crystal in the midgut of the insect releases the protoxin form of the &dgr;-endotoxin which, in most instances, is subsequently processed to an active toxin by midgut protease. The activated toxins recognize and bind to the brush-border of the insect midgut epithelium through receptor proteins. Several putative crystal protein receptors have been isolated from certain insect larvae (Knight et al., 1995; Gill et al., 1995; Masson et al., 1995). The binding of active toxins is followed by intercalation and aggregation of toxin molecules to form pores within the midgut epithelium. This process leads to osmotic imbalance, swelling, lysis of the cells lining the midgut epithelium, and eventual larvae mortality.
1.2.4 Molecular Biology of &dgr;-Endotoxins
With the advent of molecular genetic techniques, various &dgr;-endotoxin genes have been isolated and their DNA sequences determined. These genes have been used to construct certain genetically engineered
B. thuringiensis
products that have been approved for commercial use. Recent developments have seen new &dgr;-endotoxin delivery systems developed, including plants that contain and express genetically engineered &dgr;-endotoxin genes.
The cloning and sequencing of a number of &dgr;-endotoxin genes from a variety of
Bacillus thuringiensis
strains have been described and are summarized by Höfte and Whiteley, 1989. Plasmid shuttle vectors designed for the cloning and expression of &dgr;-endotoxin genes in
E. coli
or
B. thuringiensis
are described by Gawron-Burke and Baum (1991). U.S. Pat. No. 5,441,884 discloses a site-specific recombination system for constructing recombinant
B. thuringiensis
strans containing &dgr;-endotoxin genes that are free of DNA not native to
B. thuringiensis.
The Cry1 family of crystal proteins, which are primarily active against lepidopteran pests, are the best studied class of &dgr;-endotoxins. The pro-toxin form of Cry1 &dgr;-endotoxins consist of two approximately equal sized segments. The carboxyl-half, or pro-toxin segment, is not toxic and is thought to be important for crystal formation (Arvidson et al., 1989). The amino-half of the protoxin comprises the active-toxin segment of the Cry1 molecule and may be further divided into three structural domains as determined by the recently described crystallographic structure for the active toxin segment of the Cry1Aa &dgr;-endotoxin (Grochulski et al., 1995). Domain 1 occupies the first third of the active toxin and is essential for channel formation (Thompson et al., 1995). Domain 2 and domain 3 occupy the middle and last third of the active toxin, respectively. Both domains 2 and 3 have been implicated in receptor binding and insect specificity, depending on the insect and &dgr;-endotoxin being examined (Thompson et al., 1995).
1.2.5 Chumeric Crystal Proteins
In recent years, researchers have focused effort on the construction of hybrid &dgr;-endotoxins with the hope of producing proteins with enhanced activity or improved properties. Advances in the art of molecular genetics over the past decade have facilitated a logical and orderly approach to engineering proteins with improved properties. Site-specific and random mutagenesis methods, the advent of polymerase chain reaction (PCR™) methodologies, and the development of recombinant methods for generating gene fusions and constructing chimeric proteins have facilitated an assortment of methods for chan
Gilmer Amy Jelen
Malvar Thomas
Ball T. K.
Howrey Simon Arnold & White , LLP
Monsanto Company
Navarro Mark
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