[1317
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`Intematlonal Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 6 :
`C1fl99m0
`
`(11) International Publication Number:
`
`WO 99/53037
`
`A2
`(43) International Publication Date:
`
`21 October 1999 (21.10.99)
`
`(21) International Application Number:
`
`PCT/USQ9/08202
`
`(22) International Filing Date:
`
`14 April 1999 (14.04.99)
`
`(30) Priority Data:
`60/081,846
`
`15 April 1998 (15.04.98)
`
`US
`
`(71) Applicant (for all designated States except US): PRODIGENE,
`INC. [US/US]; Suite 220, 1500 Research Parkway, College
`Station, TX 77845 (US).
`
`(72) Inventor; and
`Joseph, M.
`JILKA,
`(for US only):
`(75) Inventor/Applicant
`[US/US]; Suite 220, 1500 Research Parkway, College
`Station, TX 77845 (US).
`
`(74) Agent: NEBEL, Heidi, S.; Zarley, McKee, Thomte, Voorhees
`& Sease, Suite 3200, 801 Grand Avenue, Des Moines, IA
`50309—2721 (US).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB,
`GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG,
`KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
`MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI,
`SK, SL, TJ, TM, TR, 'I'I‘, UA, UG, US, UZ, VN, YU, ZA,
`ZW, ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ,
`UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD,
`RU, TJ, TM), European patent (AT, BE, CH, CY, DE, DK,
`ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI
`patent (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR,
`NE, SN, TD, TG).
`
`Published
`
`Without international search report and to be republished
`upon receipt of that report.
`
`
`
`(54) Title: OPTIMIZED NUCLEOTIDE SEQUENCE ENCODING ORGANOPHOSPHOROUS HYDROLASE AND METHODS OF
`USE FOR SAME
`
`EagI
`--- ______ + ____________ +----
`
`HpaI
`BsteII XhoI
`———---+------—+—— ----- +
`
`273bp
`
`193bp
`
`313bp
`
`(57) Abstract
`
`A nucleotide sequence is disclosed which is optimized for plant expression and which encodes organophosphorous hydrolase.
`Expression constructs,
`transformation vectors as well as transformed cells are also disclosed which achieve expression levels superior
`to those demonstrated by expression in bacteria such as E. coli for high throughput production of the enzyme. Transgenic plants may
`themselves be used or the protein may be harvested from said plants for in situ or other environmental detoxification of organophosphorous
`neurotoxiu contaminated areas.
`
`
`
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Zimbabwe
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`Albania
`ES
`LS
`Lesotho
`SI
`Slovenia
`FI
`Armenia
`LT
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`SK
`Slovakia
`Austria
`FR
`LU
`SN
`Luxembourg
`Senegal
`Australia
`GA
`LV
`Latvia
`SZ
`Swaziland
`GB
`TD
`MC
`Monaco
`Chad
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`GE
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`Madagascar
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`GN
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`TM
`Turkmenistan
`Belgium
`The former Yugoslav
`Burkina Faso
`GR
`TR
`Republic of Macedonia
`Turkey
`HU
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`Bulgaria
`Trinidad and Tobago
`Benin
`IE
`UA
`Ukraine
`Mongolia
`Brazil
`[L
`Mauritania
`UG
`Uganda
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`US
`United States of America
`Canada
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`Viet Nam
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`Netherlands
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`Norway
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`KP
`New Zealand
`Cameroon
`Poland
`China
`Portugal
`Cuba
`Romania
`Russian Federation
`Czech Republic
`Sudan
`Germany
`Denmark
`Sweden
`Estonia
`Singapore
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
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`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`
`RO
`RU
`SD
`SE
`SG
`
`VN
`YU
`ZW
`
`

`

`WO 99/53037
`
`PCT/US99/08202
`
`TITLE:
`
`OPTIMIZED NUCLEOTIDE SEQUENCE ENCODING
`
`ORGANOPHOSPHOROUS HYDROLASE AND METHODS OF
`
`USE FOR SAME
`
`CROSS REFERENCE TO RELATED APPLICATION
`
`This is a continuation application of co-pending provisional application,
`
`Serial No. 60/081,846 filed April 15, 1998.
`
`BACKGROUND OF THE INVENTION
`
`Synthetic organophosphorous neurotoxins are used extensively as
`
`agricultural and domestic pesticides, including insecticides, fungicides, and
`
`herbicides. Naturally occurring bacterial isolates capable of metabolizing this
`
`class of compounds have received considerable attention since they provide the
`
`possibility of both environmental and in situ detoxification. Pseudomonas
`
`putida MG, Pseudomonas diminuta, and Flavobacterium, s.pp. have been
`
`shown to possess the ability to degrade an extremely broad spectrum of
`
`organophosphorous phosphotriesters as well as thiol esters.
`
`(McDaniel, et al.,
`
`"Cloning and Sequencing of a Plasmid-Borne Gene (opd) Encoding a
`
`Phosphotriesterase," Journal of Bacteriology, Vol. 170:5, 2306-11 (lVIay 1988).)
`
`Organophosphorous hydrolase (OPH) is a broad spectrum OP hydrolase
`
`that is capable of detoxifying organophosphorous neurotoxins by creating
`
`various phosphoryl bonds (P-O, P-F, P-CN, and P-S) between the phosphorous
`
`center and varying electrophilic leaving groups. (Dave et al.,
`
`"Characterization of Organophosphorous Hydrolases and the Genetic
`
`Manipulation of the Phosphotriesterase from Pseudomonas diminuta",
`
`Chemical-Biological Interactions 87, 55-68 (1993)). This enzyme is often
`
`identified by more limited descriptors such as phosphotriesterase, DFPase,
`
`parathion hydrolase, parathion aryl esterase, or paraoxonase as well as being
`
`called a somanase or sarinase. The hydrolytic reaction rates with several
`
`phosphotriesterases appears to be limited by diffusion to the active center of
`
`the enzyme.
`
`(Caldwell et al., "Limits of Diffusion in the Hydrolysis of
`
`l
`
`

`

`WO 99/53037
`
`PCT/US99/08202
`
`Substrates of the Phosphotriesterase From Pseudomonas diminuta",
`
`Biochemistry 80, 7488-7444 (1991)).
`
`This broad spectrum hydrolase (OPH) is the only enzyme which has
`
`been shown to be able to hydrolyze the P-S bond of various phosphorothioate
`
`pesticides. The toxicity of hydrolyzed products has been shown to be
`
`significantly reduced as indicated by the loss of inhibition of acetyl
`
`cholinesterase activity and by decreased neurotoxic response in animals.
`
`(Kolakowski, et al., Biocatolisis and Biotransformation, Vol. 15, 297-312
`
`(1997)). OP-thioate insecticides (acephate, azinophos-ethyl, demeton-S,
`
`malathion and phosalone) have been shown to be hydrolyzed by OPH. The
`
`hydrolysis of these pesticides has a first order dependency on the amount of
`
`enzyme used and the reaction time. The enzyme hydrolyzed acephate,
`
`azinophos-ethyl, demeton-S and phosalone at relatively fast velocities with
`
`reaction rates which are thousands of times greater than that which occurs
`
`during strong alkaline hydrolysis. In contrast, the enzyme possessed poor
`
`capability for a malathion hydrolysis, although still significantly better than
`
`non-enzymatic hydrolysis under similar conditions. When compared to the
`
`hydrolysis of P-O bond phosphotriester substrates and P-F bond
`
`phosphofluoridate substrates, the thioesters (P-S bond esters) hydrolysis was
`
`much slower in general.
`
`(See Kolakowski, et al., Biokatolisis and
`
`Biotransformation, Vol., 297-312 (1997)).
`
`The genetic expression systems in the purification procedures of
`
`recombinant OPH from E. coli have been described previously in Lai et al.,
`
`1994. However, the expression in E. coli is approximately 5-10
`
`milligrams/liter. This level is inadequate for the intended uses such as
`
`detoxification of pesticides on a commercial level. OPH has also been
`
`expressed in baculovirus systems With similar expression levels as those in E.
`
`coli. As can be seen a need exists in the art for a reliable high expression
`
`system for production of OPH.
`
`The present invention relates to the plant expression of OPH as well as
`
`the creation of a synthetic OPH nucleotide sequence which can be expressed in
`
`2
`
`

`

`WO 99/53037
`
`PCT/U 599/08202
`
`plant systems or yeast systems utilizing appropriate leader sequences whereby
`
`the expression level of OPH is greatly increased over that of E. coli or
`
`baculovirus systems. The present invention relates not only to the creation of
`
`the optimized OPH coding sequence, but also to transformation, expression of
`
`the same, and useof the recombinant protein in the hydrolysis of a variety of
`
`organophosphorous neurotoxins such as those in many widely used
`
`agricultural and domestic pesticides.
`
`SUMMARY OF THE INVENTION
`
`The present invention is directed to a nucleotide sequence which
`
`encodes OPH and its expression in plant systems. In addition, the present
`
`invention is directed to a nucleotide sequence which encodes an optimized
`
`OPH gene preferrably with a host specific leader sequence. The present
`
`invention is further directed to expression constructs, vectors and transgenic
`
`plants which contain these OPH nucleotide sequences. In yet another
`
`embodiment the invention is directed to the expression of OPH in plant or
`
`yeast cells. The present invention is also directed to methods for using
`
`transgenic plants which express this OPH to detoxify environments which are
`
`contaminated with insecticides and other neurotoxins. More specifically, the
`
`present invention is directed to transgenic plants which are capable of
`
`expressing OPH at levels greater than that of the typical 10—15 mg/l of E. coli
`
`or baculovirus.
`
`It is to be understood that the OPH nucleotide sequence of the present
`
`invention can be utilized in conjunction with a wide variety of host organisms
`
`including plants, bacteria and yeast. It should also be understood that trivial
`
`modifications to the OPH gene of this invention are encompassed in this
`
`invention. Further, while it is to be understood that the expression of the OPH
`
`of the present invention can be utilized either as present in the transgenic
`
`plants or harvested therefrom to hydrolyze neurotoxins such as pesticides, this
`
`expressed OPH gene can further be utilized to hydrolyze other chemical agents
`
`or compounds containing P-O, P-S, P-CN, and P-S bonds.
`
`3
`
`

`

`WO 99/53037
`
`PCT/US99/08202
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`Fig. 1 is a schematic of optimized OPH nucleotide sequence showing the
`
`restriction sites used in the synthesis of the gene.
`
`Fig. 2 is an optimized nucleotide sequence encoding OPH gene (SEQ ID
`
`NO:3).
`
`Fig. 3A and 3B are a nucleotide sequence which encodes plant leader
`
`sequence (SEQ ID NO:7).
`
`Fig. 4A and 4B are a nucleotide sequence which encodes OPH and which
`
`also contains a plant leader sequence (SEQ ID NO:5).
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The present invention includes novel nucleotide sequences which are
`
`optimized to encode an organophosphorous hydrolase protein. An expression
`
`construct comprising this sequence and regulatory elements for OPH
`
`expression in a host organism is also provided. The present invention also
`
`includes plants, seeds, and plant tissue capable of expressing the novel
`
`nucleotide sequence.
`
`In order to provide a clear and consistent understanding of the
`
`specification and the claims, including the scope given to such terms, the
`
`following definitions are provided:
`
`Coding DNA Sequence: A DNA sequence from which the information
`
`for making a peptide molecule, mRNA, or tRNA are transcribed. A DNA
`
`sequence may be a gene, combination of genes, or a gene fragment.
`
`Gene: A chromosomal region which is responsible for a cellular product.
`
`Microorganism: A member of one of the following classes; bacteria,
`
`fungi, protozoa or viruses.
`
`Plant Tissue: Any tissue of a plant in plant or in culture. This term
`
`includes, but is not limited to, Whole plants, plant cells, plant organs, plant
`
`seeds, embryos, pollen, silk, tassel, leaf, root, protoplasts, callus, cell cultures
`
`and any other group of plant cells organized into structural and/0r functional
`
`4
`
`

`

`WO 99/53037
`
`PCT/US99/08202
`
`units. The use of this term in conjunction with, or in the absence of, any
`
`specific type of plant tissue as listed above or otherwise embraced by this
`
`definition is not intended to be exclusive of any other type of plant tissue.
`
`Plant Transformation Vector: A plant, bacterial or viral vector that is
`
`capable of transforming plant tissue such that following transformation the
`
`plant tissue contains the DNA to be introduced by the vector in the plant
`
`tissues.
`
`Substantial Sequence Homology: Substantial functional and/or
`
`structural equivalents between sequences of nucleotides or amino acids.
`
`Functional and/or Structural Differences between sequences having
`
`substantial sequence homology will be diminimous.
`
`Synthetic Gene: A DNA sequence that exists in total or in part through
`
`manufacture by manufacture in vitro.
`
`Transgenic Plant: a plant which contains and expresses DNA that was
`
`not pre-existing in the plant, either at all or in the post-transformation
`
`quantity, prior to the introduction of the DNA into the plant.
`
`Organophosphorous hydrolase protein: as used herein this term shall
`
`mean any protein or protein fragment which maintains the functional
`
`properties of OPH as determined by the assays described in Daie et al.,
`
`"Characterization of Organophosphorous Hydrolases and the Genetic
`
`Manipulation of the Phosphotriesterase from Pseudomonas diminuta",
`
`Chemical-Biological Interactions, 87, 55-68 (1993), and Caldwell et al., "Limits
`
`of Diffusion in the Hydrolysis of Substrates of the Phosphotriesterase From
`
`Pseudomonas diminuta", Biochemistry 30, 7438-7444 (1991). Typically this
`
`will be an amino acid with at least 30%-50% homology with at least one form
`
`of the protein as disclosed herein. 80% homology is preferred and 90%
`
`homology is most preferred especially including conservative substitutions.
`
`Homology is calculated by standard methods which involve aligning two
`
`sequences to be compared so that the maximum matching occurs, and
`
`calculating the percentage of matches. Substantially equivalent substances to
`
`these include those wherein one or more of the residues of the native sequence
`
`5
`
`

`

`WO 99/53037
`
`PCT/US99/08202
`
`is deleted, substituted for, or inserted by a different amino acid or acids.
`
`Preferred substitutions are those which are conservative, i.e., wherein a
`
`residue is replaced by another of the same general type. As is well understood,
`
`naturally occurring amino acids can be sub classified as acidic, basic, neutral
`
`and polar, or neutral and nonpolar. Furthermore, three of the encoded amino
`
`acids are aromatic. It is generally preferred that peptides differing from the
`
`native OPH sequence contain substitutions which are from the same group as
`
`that of the amio acid replaced. Thus, in general, the basic amino acids Lys and
`
`Arg are interchangeable; the acidic amino acids aspartic and glutamic are
`
`interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, and Asn are
`
`interchangeable;
`
`the nonpolar aliphatic acids Gly, Ala, Val, Ile, and Leu are
`
`conservative with respect to each other (but because of size, Gly and Ala are
`
`more closely related and Val, Ile and Leu are more closely related), and the
`
`aromatic amino acids Phe, Trp, and Tyr are interchangeable. While proline is
`
`a non-olar neutral amino acid, it represents difficulties because of its effects on
`
`conformation, and substitutions by or for proline are not preferred, except
`
`when the same or similar conformational results can be obtained. Polar amino
`
`acids which represent conservative changes include Ser, Thr, Gln, Asn; and to
`
`a lesser extent, Met. In addition, although classified in different categories,
`
`Ala, Gly, and Ser seem to be interchangeable, and Cys additionally fits into
`
`this group, or may be classified with the polar neutral amino acids. In general,
`
`whatever substitutions are made are such that the functional properties of the
`
`intact proteinaceous molecule is retained and ancillary properties, such as
`
`non-toxicity are not substantially disturbed as described earlier.
`
`Conditions of high stringency: as used herein this term shall mean
`
`nucleotide hybridization conditions equivalent to the following: 42°C in a
`
`buffer containing 50% formamide, 1M sodium chloride, 1% SDS, 10% dextran
`
`sulfate, 100 ug/ml denatured salmon sperm, or those described in Reppert,
`
`S.M. et a1. (1994) Neuron 13:1177-1185.
`
`

`

`WO 99/53037
`
`PCT/US99/08202
`
`Optimized OPH Gene
`
`The amino acid and nucleic acid sequence for the native plasmid-borne
`
`organophosphorus degrading from Pseudomonas diminuta has been
`
`determined. McDaniel et al., "Cloning and Sequencing of a Plasmid-Born
`
`Gene (opd) Encoding a Phosphotriesterase," Journal of Bacteriology, 170
`
`(5)2280-11, (May 1988). The amino acid sequence was back—translated into a
`
`DNA coding sequence using a maize codon preference table
`
`(http://www.gcg.com/techsupport/data/maize-high.cod). This sequence, which
`
`was optimized for corn, was searched for putative deleterious sequences
`
`compiled in Table 1 of Example 1. Codons were chosen so that deleterious
`
`mRNA signals would be eliminated in the DNA coding sequence. In addition,
`
`convenient restriction enzyme cites were added to facilitate downstream
`
`cloning. The optimized DNA coding sequence was divided into convenient
`
`cloning lengths and then each of these lengths were further subdivided into
`
`oligonucleotides of approximately 50 bases in length with 10 base overhangs.
`
`Oligonucleotides corresponding to both the sense and antisense strand were
`
`synthesized and 5' phosphorylated. The oligonucleotides were then annealed
`
`and ligated. Following ligation, varying dilutions were subjected to PCR using
`
`primers specific to the 5' and 3' ends. PCR product was analyzed using
`
`polyacrylamide gels, and DNA of the proper expected lengths was extracted
`
`from the gel and the DNA eluted from the extracted gel material. The DNA
`
`was cloned into a pCR 2.1 TOPO(Invitrogen) plasmid as per the
`
`manufacturer's instructions. Transformed colonies containing the PCR insert
`
`were grown overnight and the plasmid DNA purified. The plasmid DNA was
`
`then analyzed for the correct PCR insert as per standard protocols known to
`
`those of skill in the art. The inserts were sequenced to verify that the correct
`
`sequence was created and the optimized gene was assembled properly using
`
`standard protocols known to those of skill in the art.
`
`Appropriate Leader Seguence
`
`To enable the OPH gene to be expressed in a host organism, an
`
`appropriate leader sequence or subcellular localization signal is preferably
`
`7
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`WO 99/53037
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`PCT/US99/08202
`
`integrated onto the OPH encoding nucleotide sequence gene. For example, a
`
`plant leader sequence will ensure high expression in plants while a yeast
`
`leader sequence will express high levels of OPH expression in yeast. Upon
`
`assembly of the OPH gene with a 5‘ leader sequence specific for the host
`
`organism of choice, the OPH gene containing the appropriate leader can be
`
`cloned into the standard expression cassette for that host to ensure high
`
`expression in that organism. For example, a 5' fragment can be assembled to
`
`contain a plant leader sequence to ensure high expression in plants. The OPH
`
`gene containing the plant leader may be cloned into a standard plant upon
`
`expression cassette to ensure high expression in plants.
`
`Transformation technigues
`
`In the early 1980s, it became clear that plants were amenable to genetic
`
`engineering because foreign genes can be stably introduced into plant
`
`chromosomal DNA by a variety of techniques. (Gasser, C. S. & Fraley, R. T.,
`
`"Genetically Engineering Plants for Crop Improvement," Science, 224, 1293
`
`(1989); Potrykus, 1., "Gene Transfer to Plants; Assessment of Published
`
`Approaches and Results," Annu. Rev. Plant Physiol. Plant Mol. Biol., 42, 205
`
`(1991).) If a given foreign gene contains the appropriate regulatory sequences,
`
`the gene product will be synthesized by the transformed plant. Furthermore,
`
`most plant cells are totipotent, thereby allowing regeneration of a fertile
`
`"transgenic" plant from a single transformed cell. If the transgenic plant
`
`flowers and produces viable seed, the acquired trait will be preserved in the
`
`progeny. (Greenberg & Glick, "The Use of Recombinet DNA Technology to
`
`Produce Genetically Modified Plants," Methods in Plant Molecular Biology and
`
`Biotechnology (Glick & Thompson, eds.) (CRC Press 1993).
`
`Once a nucleotide acid sequence of interest is isolated or synthesized, it
`
`is included into an expression cassette which typically comprises a
`
`transcription unit of a promoter operably linked to the nucleotide sequence
`
`which is functional in a plant cell and a termination or polyadenylation signal.
`
`This expression cassette typically forms part of a plant transformation vector.
`
`This plant transformation vector will also contain the appropriate regulatory
`
`8
`
`

`

`WO 99/53037
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`PCT/US99/08202
`
`sequences in addition to the gene of interest. The vector DNA facilitates
`
`manipulation of the gene in the host cell, such as E. coli. or yeast, prior to
`
`plant transformation. In addition, the host vector-cell can act as a vehicle to
`
`transfer the gene to the host plant such as the case of the plant pathogenic
`
`bacterium, Agrobacterium tumefaciens. An idealized vector would contain a
`
`multiple cloning site, an antibiotic resistance or other selective gene, a broad-
`
`host bacterial origin of replication, and a selection marker or gene for selection
`
`of the foreign DNA in transformed plants.
`
`PROMOTERS
`
`The constructs, promoters or control systems used in the methods of the
`
`invention may include a tissue specific promoter, an inducible promoter or a
`
`constitutive promoter.
`
`A large number of suitable promoter systems are available. For
`
`example one constitutive promoter useful for the invention is the cauliflower
`
`mosaic virus (CaMV) 358. It has been shown to be highly active in many plant
`
`organs and during many stages of development when integrated into the
`
`genome of transgenic plants including tobacco and petunia, and has been
`
`shown to confer expression in protoplasts of both dicots and monocots.
`
`Organ-specific promoters are also well known. For example, the E8
`
`promoter is only transcriptionally activated during tomato fruit ripening, and
`
`can be used to target gene expression in ripening tomato fruit (Deikman and
`
`Fischer, EMBO J. (1988) 723315; Giovannoni et al., The Plant Cell (1989)
`
`1:53). The activity of the E8 promoter is not limited to tomato fruit, but is
`
`thought to be compatible with any system wherein ethylene activates
`
`biological processes. Similarly the Lipoxegenase ("the LOX gene") is a fruit
`
`specific promoter.
`
`Seed specific promoters include the Napin promoter described in united
`
`States Patent 5,110,728 to Calgene, which describes and discloses the use of
`
`the napin promoter in directing the expression to seed tissue of an acyl carrier
`
`protein to enhance seed oil production; the D03 promoter from carrots which is
`
`early to mid embryo specific and is disclosed at Plant Physiology, Oct. 1992
`
`9
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`WO 99/53037
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`PCT/U 599/08202
`
`100(2) p. 576-581, "Hormonal and Environmental Regulation of the Carrot
`
`Lea-class Gene Dc 3, and Plant Mol. Biol., April 1992, 18(6) p. 1049-1063,
`
`"Transcriptional Regulation of a Seed Specific Carrot Gene, DC 8": the
`
`phaseolin promoter described in United States Patent 5,504,200 to Mycogen
`
`which discloses the gene sequence and regulatory regions for phaseolin, a
`
`protein isolated from P. vulgaris which is expressed only while the seed is
`
`developing within the pod, and only in tissues involved in seed generation.
`
`Other organ-specific promoters appropriate for a desired target organ
`
`can be isolated using known procedures. These control sequences are
`
`generally associated with genes uniquely expressed in the desired organ. In a
`
`typical higher plant, each organ has thousands of mRNAs that are absent from
`
`other organ systems (reviewed in Goldberg, Phil, Trans. R. Soc. London (1986)
`
`B314—348. mRNAs are first isolated to obtain suitable probes for retrieval of
`
`the appropriate genomic sequence which retains the presence of the natively
`
`associated control sequences. An example of the use of techniques to obtain
`
`the cDNA associated with mRNA specific to avocado fruit is found in
`
`Christoffersen et al., Plant Molecular Biology (1984) 3:385. Briefly, mRNA
`
`was isolated from ripening avocado fruit and used to make a cDNA library.
`
`Clones in the library were identified that hybridized with labeled RNA isolated
`
`from ripening avocado fruit, but that did not hybridize with labeled RNAs
`
`isolated from unripe avocado fruit. Many of these clones represent mRNAs
`
`encoded by genes that are transcriptionally activated at the onset of avocado
`
`fruit ripening.
`
`Another very important method that can be used to identify cell type
`
`specific promoters that allow even to identification of genes expressed in a
`
`single cell is enhancer detection (O'Kane, C., and Gehring, W.J. (1987),
`
`"Detection in situ of genomic regulatory elements in Drosophila", Proc. Natl.
`
`Acad. Sci. USA, 84, 9123-9127). This method was first developed in
`
`Drosophila and rapidly adapted to mice and plants (Wilson, C., Pearson, R.K.,
`
`Bellen, H.J., O'Kane, C.J., Grossniklaus, U., and Gehring, W.J. (1989), "P-
`
`element-mediated enhancer detection: an efficient method for isolating and
`
`10
`
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`

`WO 99/53037
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`PCT/U 899/08202
`
`characterizing developmentally regulated genes in Drosophila", Genes & Dev.,
`
`3, 1301—1813; Skarnes, WC. (1990), "Entrapment vectors: a new tool for
`
`mammalian genetics", Biotechnology, 8, 827-831; Topping, J.F., Wei, W., and
`
`Lindsey, K. (1991), "Functional tagging of regulatory elements in the plant
`
`genome", Development, 112, 1009-1019; Sundaresan, V., Springer, P.S., Volpe,
`
`T., Haward, S., Jones, J.D.G., Dean, 0., Ma, H., and Martienssen, RA, (1995),
`
`"Patterns of gene action in plant development revealed by enhancer trap and
`
`gene trap transposable elements", Genes & Dev., 9, 1797—1810).
`
`The promoter used in the method of the invention may also be an
`
`inducible promoter. An inducible promoter is a promoter that is capable of
`
`directly or indirectly activating transcription of a DNA sequence in response to
`
`an inducer. In the absence of an inducer, the DNA sequence will not be
`
`transcribed. Typically, the protein factor that binds specifically to an inducible
`
`promoter to activate transcription is present in an inactive form which is then
`
`directly or indirectly converted to the active form by the inducer. The inducer
`
`may be a chemical agent such as a protein, metabolite (sugar, alcohol etc.), a
`
`growth regulator, herbicide, or a phenolic compound or a physiological stress
`
`imposed directly by heat, salt, toxic elements etc. or indirectly through the
`
`action of a pathogen or disease agent such as a virus. A plant cell containing
`
`an inducible promoter may be exposed to an inducer by externally applying the
`
`inducer to the cell such as by spraying, watering, heating, or similar methods.
`
`Examples of inducible promoters include the inducible 7 O kd heat shock
`
`
`promoter of D. melanogaster (Freeling, M., Bennet, D.C., Maize ADN 1, Ann.
`
`Rev. of Genetics, 19:297-323) and the alcohol dehydrogenase promoter which is
`
`induced by ethanol (Nagao, R.T., et al., Miflin, B.J., Ed. Oxford Surveys of
`
`Plant Molecular and Cell Biology, Vol. 3, p. 384-438, Oxford University Press,
`
`Oxford 1986) or the Lex A promoter which is triggered with chemical
`
`treatment and is available through Ligand pharmaceuticals. The inducible
`
`promoter may be in an induced state throughout seed formation or at least for
`
`a period which corresponds to the transcription of the DNA sequence of the
`
`recombinant DNA molecule(s).
`
`ll
`
`

`

`WO 99/53037
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`
`Another example of an inducible promoter is the chemically inducible
`
`gene promoter sequence isolated from a 27 kd subunit of the maize
`
`glutathione—S-transferase (GST II) gene. Two of the inducers for this promoter
`
`are N,N-diallyl-Z,2-dichloroacetamide (common name: dichloramid) or benzyl-
`
`=2-chloro-4-(trifluoromethy1)-5-thiazolecarboxy1ate (common name: flurazole).
`
`In addition, a number of other potential inducers may be used with this
`
`promoter as described in published PCT Application No. PCT/GB90/00110 by
`
`ICI.
`
`Another example of an inducible promoter is the light inducible
`
`chlorophyll a/b binding protein (CAB) promoter, also described in published
`
`PCT Application No. PCT/GB90/00110 by ICI.
`
`Inducible promoters have also been described in published Application
`
`No. EP89/103888.7 by Ciba-Geigy. In this application, a number of inducible
`
`promoters are identified, including the PR protein genes, especially the tobacco
`
`PR protein genes, such as PR-la, PR-lb, PR-lc, PR-I, PR-A, PR-S, the
`
`cucumber chitinase gene, and the acidic and basic tobacco beta-1,3-glucanase
`
`genes. There are numerous potential inducers for these promoters, as
`
`described in Application No. EP89/103888.7.
`
`The preferred promoters may be used in conjunction with naturally
`
`occurring flanking coding or transcribed sequences of the seed specific
`
`Polycomb genes or with any other coding or transcribed sequence that is
`
`critical to Polycomb formation and/or function.
`
`It may also be desirable to include some intron sequences in the
`
`promoter constructs since the inclusion of intron sequences in the coding
`
`region may result in enhanced expression and specificity. Thus, it may be
`
`advantageous to join the DNA sequences to be expressed to a promoter
`
`sequence that contains the first intron and exon sequences of a polypeptide
`
`Which is unique to cells/tissues of a plant critical to seed specific Polycomb
`
`formation and/or function.
`
`Additionally, regions of one promoter may be joined to regions from a
`
`different promoter in order to obtain the desired promoter activity resulting in
`
`12
`
`

`

`WO 99/53037
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`a chimeric promoter. Synthetic promoters which regulate gene expression may
`
`also be used. The expression system may be further optimized by employing
`
`supplemental elements such as transcription terminators and/or enhancer
`
`elements.
`
`OTHER REGULATORY ELEMENTS
`
`In addition to a promoter sequence, an expression cassette or construct
`
`should also contain a transcription termination region downstream of the
`
`structural gene to provide for efficient termination. The termination region or
`
`polyadenylation signal may be obtained from the same gene as the promoter
`
`sequence or may be obtained from different genes. Polyadenylation sequences
`
`include, but are not limited to the Agrobacterium octopine synthase signal
`
`(Gielen et al., EMBO J. (1984) 3:835-846) or the nopaline synthase signal
`
`(Depicker et al., M01. and Appl. Genet. (1982) 12561-573).
`
`MARKER GENES
`
`Recombinant DNA molecules containing any of the DNA sequences and
`
`promoters described herein may additionally contain selection marker genes
`
`which encode a selection gene product which confer on a plant cell resistance
`
`to a chemical agent or physiological stress, or confers a distinguishable
`
`phenotypic characteristic to the cells such that plant cells transformed with
`
`the recombinant DNA molecule may be easily selected using a selective agent.
`
`One such selection marker gene is neomycin phosphotransferase (NPT II)
`
`which confers resistance to kanamycin and the antibiotic G-418. Cells
`
`transformed with this selection marker gene may be selected for by assaying
`
`for the presence in vitro of phosphorylation of kanamycin using techniques
`
`described in the literature or by testing for the presence of the mRNA coding
`
`for the NPT II gene by Northern blot analysis in RNA from the tissue of the
`
`transformed plant. Polymerase chain reactions are also used to identify the
`
`presence of a transgene or expression using reverse transcriptase PCR
`
`amplification to monitor expression and PCR on genomic DNA. Other
`
`commonly used selection markers include the ampicillin resistance gene, the
`
`tetracycline resistance and the hygromycin resistance gene. Transformed
`
`l3
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`

`

`WO 99/53037
`
`PCT/USQ9/08202
`
`plant cells thus selected can be induced to differentiate into plant structures
`
`which will eventually yield whole plants. It is to be understood that a
`
`selection marker gene may also be native to a plant.
`
`TRANSFORMATION
`
`Various methods are known in the art to accomplish the genetic
`
`transformation of plants and plant tissues. Some of these methods include
`
`transformation, or agroinfection, by Agrobacterium tumefaciens,
`
`electroporation, particle bombardment or biollistic projection, or direct gene
`
`transfer.
`
`Various methods are known in the art to accomplish the genetic
`
`transformation of plants and plant tissues. Among these methods for
`
`introducing foreign DNA into plants is Agrobacterium species transformation
`
`and transformation by direct gene transfer.
`
`Agrobacterium tumefaciens is the etio

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