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WO1997010328A2 - Compositions and method for modulation of gene expression in plants - Google Patents

Compositions and method for modulation of gene expression in plants Download PDF

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Publication number
WO1997010328A2
WO1997010328A2 PCT/US1996/011689 US9611689W WO9710328A2 WO 1997010328 A2 WO1997010328 A2 WO 1997010328A2 US 9611689 W US9611689 W US 9611689W WO 9710328 A2 WO9710328 A2 WO 9710328A2
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Prior art keywords
nucleic acid
plant
gene
gaa
cugauga
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PCT/US1996/011689
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French (fr)
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WO1997010328A3 (en
Inventor
Michael G. Zwick
Brent E. Edington
James A. Mcswiggen
Patricia Ann Owens Merlo
Lining Guo
Thomas A. Skokut
Scott A. Young
Otto Folkerts
Donald J. Merlo
Original Assignee
Ribozyme Pharmaceuticals, Inc.
Dowelanco
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Publication date
Application filed by Ribozyme Pharmaceuticals, Inc., Dowelanco filed Critical Ribozyme Pharmaceuticals, Inc.
Priority to JP9506989A priority Critical patent/JPH11509733A/en
Priority to EP96927999A priority patent/EP0842286A2/en
Priority to MX9800454A priority patent/MX9800454A/en
Priority to BR9610402-3A priority patent/BR9610402A/en
Priority to AU67617/96A priority patent/AU6761796A/en
Publication of WO1997010328A2 publication Critical patent/WO1997010328A2/en
Publication of WO1997010328A3 publication Critical patent/WO1997010328A3/en

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    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
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    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

  • the present invention concerns compositions and methods for the modulation of gene expression in plants, specifically using enzymatic nucleic acid molecules
  • Naturally occurring antisense RNA was first discovered in bacteria over a decade ago (Simons and Kleckner, 1983 Cell 34, 683-691). It is thought to be one way in which bacteria can regulate their gene expression (Green et al., 1986 Ann Rev Biochem 55: 567- 597; Simons 1988 Gene 72: 35-44) The first demonstration of antisense-mediated inhibition of gene expression was repo ⁇ ed m mammalian cells (Izant and Wemtraub 1984 Cell 36: 1007-1015). There are many examples m the literature for the use of antisense RNA to modulate gene expression in plants. Following are a few examples Shewmaker et al , U.S. Patent Nos. 5, 107.065 and 5. 453.566 disclose methods for regulating gene expression in plants using antisense RNA
  • Transgenic potato plants have been produced which express RN ⁇ antisense to potato or cassava granule bound starch syntha.sc (GBSS)
  • GBSS cassava granule bound starch syntha.sc
  • transgenic plants have been constructed which have reduced oi no GBSS activity or protein. These transgenic plants give rise to potatoes containing starch with dramatically reduced amylose levels (Visser et al. 1991 , Mol. Gen Genet. 225. 2889-296, Salehuzzaman et al. 1993, Plant Mol. Biol. 23: 947-962)
  • Antisense RNA constructs targeted against ⁇ -9 desaturase enzyme in canola have been shown to increase the level of stearic acid (C18:0) from 2% to 40% (Knutzon et. al., 1992 Proc. Natl. Acad. Sci. 89, 2624). There was no decrease in total oil content or germinanon efficiency in one of the high stearate lines.
  • Several recent reviews are available which illustrate the utility of plants with modified oil composition (Ohlrogge, J. B. 1994 Plant Physiol. 104, 821; Kmney, A. J. 1994 Cwrr. Opin. Cell Biol. 5, 144; Gibson et al. 1994 Plant Cell Envir. 17, 627).
  • Hitz et al, (WO 91/18985) describe a method for using the soybean ⁇ -9 dcsat-irasc enzyme to modify plant oil composition.
  • the application describes the use of soybean ⁇ -9 desaturase sequence to isolate ⁇ -9 desaturasc genes from other species.
  • references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the use of ribozymes in maize. Furthermore, Applicant believes that the references do not disclose and/or enable the use of ribozymes to down regulate genes in plant ceils, let alone plants.
  • the invention features modulation of gene expression in plants specifically using enzymatic nucleic acid molecules.
  • the gene is an endogenous gene.
  • the enzymatic nucleic acid molecule with RNA cleaving activity may be in the form of, but not limited to, a hammerhead, hai ⁇ in, hepatitis delta virus, group I intron, group II intron, RNaseP RNA, Neurospora VS RNA and the like.
  • the enzymatic nucleic acid molecule with RNA cleaving activity may be encoded as a monomer or a multimcr, preferably a multimer.
  • the nucleic acids encoding for the enzymatic nucleic acid molecule with RNA cleaving activity may be operably linked to an open reading frame.
  • Gene expression in any plant species may be modified by transformation of the plant with the nucleic acid encoding the enzymatic nucleic acid molecules with RNA cleaving activity.
  • technologies for transforming a plant include but are not limited to transformation with Agrobacterium, bombarding with DNA coated microprojectiles, whiskers, or electroporation.
  • Any target gene may be modified with the nucleic acids encoding the enzymatic nucleic acid molecules with RNA cleaving activity.
  • Two targets which are exemplified herein are delta 9 desaturase and granule bound starch synthase (GBSS).
  • nucleic acid-based reagents such as enzymatic nucleic acids (ribozymes)
  • Ribozymes can be used to modulate a specific trait of a plant cell, for example, by modulating the activity of an enzyme involved in a biochemical pathway. It may be desirable, in some instances, to Robinson,,93 alone,,. > PCT US96/11689 97/10328
  • ribozymes can be used to achieve this Enzymatic nucleic acid-based techniques were developed herein to allow directed modulation of gene expression to generate plant cells, plant tissues or plants with altered phenotype.
  • Ribozymes i.e., enzymatic nucleic acids
  • enzymatic nucleic acids arc nucleic acid molecules having an enzymatic activity which is able to repeatedly cleave other separate RN ⁇ molecules in a nucleotide base sequence-specific manner.
  • Such enzymatic RN ⁇ molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro and in vivo (Zaug et ⁇ l, 1986, Nature 324, 429; Kim et al., 1987, Proc. Natl Acad. Set USA 84, 8788; Dreyfus, 1988, Einstein Quarterly J.
  • tr ⁇ /w-cleavmg ribozymes may be used as efficient tools to modulate gene expression in a variety of organisms including plants, animals and humans (Bennett et al, supra; Edington et al, supra; Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med Chem. 38, 2023-2037).
  • Ribozymes can be designed to cleave specific R ⁇ A targets within the background of cellular R ⁇ A. Such a cleavage event renders the mR ⁇ A non-functional and abrogates protein expression from that R ⁇ A. In this manner, synthesis of a protein associated with a particular phenotype and/or disease state can be selectively inhibited.
  • Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art.
  • Stem II can be > 2 base-pairs long.
  • Each ⁇ is any nucleotide and each • represents a base pair.
  • Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known the art
  • Figure 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion
  • Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions
  • Figure 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids Res., 17, 1371-1371) into two portions.
  • FIG 3 is a diagrammatic representation of the general structure of a hairpin ribozyme.
  • Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is I , 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 - 20 bases, i. ., m is from 1 - 20 or more).
  • Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is > 1 base).
  • Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site.
  • each N and N' independently is any no ⁇ nal or modified base and each dash represents a potential base-pairing interaction.
  • These nucleotides may be modified at the sugar, base or phosphate.
  • Complete base-pairing is not required in the helices, but is preferred.
  • Helix 1 and 4 can be of any size (i.e.. o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained.
  • Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect.
  • Helix 4 can be formed from two separate molecules, i.e., without a connecting loop.
  • the connecting loop when present may be a ribonucleotidc with or without modifications to its base, sugar or phosphate, "q" is > 2 bases.
  • the connecting loop can also be replaced with a non-nucleotide linker molecule.
  • H refers to bases A, U, or C.
  • Y refers to pyrimidine bases.
  • " " refers to a covalent bond.
  • Figure 4 is a representation of the general structure of the hepatitis ⁇ virus ribozyme domain known in the art.
  • Figure 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.
  • Figure 6 is a schematic representation of an RNaseH accessibility assay. Specifically, the left side of Figure 6 is a diagram of complementary DNA oligonucleotides bound to accessible sites on the target RNA. Complementary DNA oligonucleotides are represented by broad lines labeled A, B, and C. Target RNA is represented by the thin, twisted line. The right side of Figure 6 is a schematic of a gel separation of uncut target RNA from a cleaved target RNA. Detection of target RNA is by autoradiography of body-labeled, T7 transcript. The bands common to each lane 97/10328
  • Figure 7 is a graphical representation of RNaseH accessibility of GBSS RNA
  • Figure 8 is a graphical representation of GBSS RN ⁇ cleavage by nbo/ymes at different temperatures.
  • Figure 9 is a graphical representation of GBSS RNA cleavage by multiple ribozymes.
  • Figure 10 lists the nucleotide sequence of ⁇ -9 desaturase cDN ⁇ isolated from 7.eu mays.
  • Figures 1 1 and 12 are diagrammanc representations of fatty acid biosynthesis in plants.
  • Figure 1 1 has been adapted from Gibson et al, 1994, Plant Cell Envir. 17, 627.
  • Figures 13 and 14 are graphical representations of RNaseH accessibility of ⁇ -9 desaturase RNA.
  • Figure 15 shows cleavage of ⁇ -9 desaturase RNA by ribozymes in vitro.
  • 10/10 represents the length of the binding arms of a hammerhead (HH) ribozyme.
  • 10/10 means helix 1 and helix 3 each form 10 base-pairs with the target RNA (Fig. 1).
  • 4/6 and 6/6 represent helix2/helixl interaction between a hai ⁇ in ribozyme and its target.
  • 4/6 means the hai ⁇ in (HP) ribozyme forms four base-paired helix 2 and a six base-paired helix 1 complex with the target (see Fig. 3).
  • 6/6 means, the hai ⁇ in ribozyme forms a 6 base- paired helix 2 and a six base-paired helix 1 complex with the target.
  • the cleavage reactions were earned out for 120 mm at 26°C.
  • Figure 16 shows the effect of arm-length variation on the activity of HH and HP ribozymes in vitro. Ill, 10/10 and 12/12 are essentially as described above for the HH ribozyme. 6/6, 6/8, 6/12 represents varying helix 1 length and a constant (6 bp) helix 2 for a hai ⁇ in ribozyme. The cleavage reactions were earned out essentially as desc ⁇ bed above.
  • Figures 17, 18, 19 and 23 are diagrammatic representations of non-limiting strategies to construct a transcript comprising multiple ribozyme motifs that are the same or different, targeting various sites within ⁇ -9 desaturase RNA.
  • Figures 20 and 21 show in vitro cleavage of ⁇ -9 desaturase RNA by ribozymes that are transcribed from DNA templates using bacte ⁇ ophage T7 RNA polymerase enzyme
  • Figure 22 diagrammatic representation of a non-limiting strategy to construct a transcript compnsing multiple ribozyme motifs that arc the same or different targeting various sites within GBSS RNA
  • Figure 24 shows cleavage of ⁇ -9 desaturase RN ⁇ by ribozymes 453 Multimer, represents a multimer ribozyme construct targeted against hammerhead ⁇ bozyme sites 453, 464, 475 and 484.
  • 252 Multimer represents a multimer ribozyme construct targeted against hammerhead ribozyme sites 252, 271 , 313 and 326 238 Multimer, represents a multimer ribozyme construct targeted against three hammerhead ⁇ bozyme sites 252, 259 and 271 and one hai ⁇ in ribozyme site 238 (HP).
  • 259 Multimer represents a multimer ⁇ bozyme construct targeted against two hammerhead ribozyme sites 271 and 313 and one hai ⁇ in ⁇ bozyme site 259 (HP)
  • Figure 25 illustrates GBSS mRNA levels in Ribozyme minus Controls (C, F, I, J, N, P, Q) and Active Ribozyme RPA63 Transformants (AA, DD, EE, FF, GG, HH, JJ, KK).
  • Figure 26 illustrates ⁇ 9 desaturase mRNA levels in Non-transformed plants (NT), 85-06 High Stearate Plants (1, 3, 5, 8, 12, 14), and Transformed (irrelevant ribozyme) Control Plants (RPA63-33, RPA63-51, RPA63-65).
  • Figure 27 illustrates ⁇ 9 desaturase mRNA levels in Non-transformed plants
  • NTO 85-15 High Stearate Plants
  • 02, 05, 09, 14 85-15 Normal Stearate Plants
  • Figure 28 illustrates ⁇ 9 desaturase mRNA levels in Non-transformed plants (NTY), 113-06 Inactive Ribozyme Plants (02, 04, 07, 10,11).
  • Figures 29a and 29b illustrate ⁇ 9 desaturase protein levels in maize leaves (R0) (a)
  • Figure 30 illustrates stea ⁇ c acid in leaves of RPA85-06 plants
  • Figure 31 illustrates stea ⁇ c acid in leaves of RPA85-15 plants, results of three assays.
  • Figure 32 illustrates stearic acid in leaves of RPA 1 13-06 plants.
  • Figure 33 illustrates stearic acid in leaves of RPA1 13-17 plants.
  • Figure 34 illustrates stearic acid in leaves of control plants.
  • Figure 35 illustrates leaf stearate in RI plants from a high stearate plant cross (RPA85- 15.07 self).
  • Figure 36 illustrates ⁇ 9 desaturase levels in next generation maize leaves (RI ). * indicates those plants that showed a high stearate content.
  • Figure 37 illustrates stearic acid in individual somatic embryos from a culture (308/430-012) transformed with antisense ⁇ 9 desaturase.
  • Figure 38 illustrates stearic acid in individual somatic embryos from a culture
  • Figure 39 illustrates stearic acid in individual leaves from plants regenerated from a culture (308/430-012) transformed with antisense ⁇ 9 desaturase.
  • Figure 40 illustrates amylose content in a single kernel of untransformed control line (Q806 and antisense line 308/425- 12.2.1.
  • Figure 41 illustrates GBSS activity in single kernels of a southern negative line (RPA63-0306) and Southern positive line RPA63-0218.
  • Figure 42 illustrates a transformation vector that can be used to express the enzymatic nucleic acid of the present invention.
  • the present invention concerns compositions and methods for the modulation of gene expression in plants specifically using enzymatic nucleic acid molecules.
  • inhibitor or “modulate” is meant that the activity of enzymes such as GBSS and ⁇ -9 desaturase or level of mRNAs encoded by these genes is reduced below that observed in the absence of an enzymatic nucleic acid and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.
  • enzymatic nucleic acid molecule it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave that target That is, the enzymatic nucleic acid molecule is able to mtcrmolccularly cleave RN ⁇ (or DN ⁇ ) and thereby inactivate a target RNA molecule.
  • nucleic acids may be modified at the base, sugar, and/or phosphate groups.
  • enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, RNAzyme, poly ⁇ bozymes, molecular scissors, self-splicing RNA, self-cleaving RNA, cis-cleaving RNA, autolytic RNA, endoribonuclease, minizyme, leadzyme or DNA enzyme. All of these terminologies desc ⁇ be nucleic acid molecules with enzymatic activity.
  • the term encompasses enzymatic RNA molecule which include one or more ribonucleotides and may include a majority of other types of nucleotides or abasic moieties, as desc ⁇ bed below.
  • complementa ⁇ ty is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequences by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
  • vectors any nucleic acid- and/or viral-based technique used to deliver and/or express a desired nucleic acid.
  • RNA RNA
  • plant gene is meant a gene encoded by a plant.
  • endogenous gene is meant a gene normally found in a plant cell in its natural location in the genome.
  • nucleic acid is meant a molecule which can be single-stranded or double- stranded, composed of nucleotides containing a sugar, a phosphate and either a purine or pyrimidine base which may be same or different, and may be modified or unmodified.
  • genomic is meant genetic material contained in each cell of an organism and/or a virus.
  • RNA that can be translated into protein by a cell.
  • cDNA is meant DN ⁇ that is complementary to and derived from a mRNA.
  • dsDNA is meant a double stranded cDNA.
  • RNA By “sense” RNA is meant RN ⁇ transcript that comprises the mRNA sequence.
  • antisense RNA an RNA transcript that comprises sequences complementary to all or part of a target RNA and/or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript and/or mRNA.
  • the complementarity may exist with any part of the target RNA, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • Antisense RNA is normally a mirror image of the sense RNA.
  • expression is meant the transcription and stable accumulation of the enzymatic nucleic acid molecules, mRNA and/or the antisense RNA inside a plant cell. Expression of genes involves transcription of the gene and translation of the mRNA into precursor or mature proteins.
  • cosuppression is meant the expression of a foreign gene, which has substantial homology to an gene, and in a plant cell causes the reduction in activity of the foreign and/or the endogenous protein product.
  • altered levels is meant the level of production of a gene product in a transgenic organism is different from that of a normal or non-transgenic organism.
  • promoter nucleotide sequence element within a gene which controls the expression of that gene. Promoter sequence provides the recognition for RNA polymerase and other transcription factors required for efficient transcription. Promoters from a variety of sources can be used efficiently in plant cells to express ribozymes. For example, promoters of bacterial origin, such as the octopine synthetase promoter, the walnut, chalk, chalk, chalk, chalk, chalk, chalk, chalk, chalk, the like.
  • nopaline synthase promoter the manopme synthetase promoter, promoters of viral o ⁇ gin, such as the cauliflower mosaic virus (35S); plant promoters, such as the ⁇ bulose- 1,6-biphosphate (RUBP) carboxylase small subunit (ssu), the beta-conglycinin promoter, the phaseolm promoter, the ADH promoter, heat-shock promoters, and tissue specific promoters.
  • Promoter may also contain certain enhancer sequence elements that may improve the transcription efficiency.
  • enhancer nucleotide sequence element which can stimulate promoter activity (Adh).
  • constitutive promoter is meant promoter element that directs continuous gene expression in all cells types and at all times (actin, ubiquitin, CaMV 35S).
  • tissue-specific promoter is meant promoter element responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (zein, oleosin, napm, ACP).
  • development-specific promoter is meant promoter element responsible for gene expression at specific plant developmental stage, such as in early or late embryogenesis.
  • inducible promoter is meant promoter element which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; and stress.
  • a “plant” is meant a photosynthetic organism, either eukaryotic and prokaryotic.
  • angiosperm is meant a plant having its seed enclosed in an ovary (e.g., coffee, tobacco, bean, pea).
  • gymnosperm is meant a plant having its seed exposed and not enclosed in an ovary (e.g., pine, spruce).
  • seed leaf By “monocotyledon” is meant a plant characterized by the presence of only one seed leaf (primary leaf of the embryo). For example, maize, wheat, rice and others.
  • cotyledon is meant a plant producing seeds with two cotyledons (primary leaf of the embryo). For example, coffee, canola, peas and others. -. thorough speaking,, revision PCT/US96/11689 97/10328
  • transgenic plant is meant a plant expressing a foreign gene.
  • open reading frame is meant a nucleotide sequence, without introns, encoding an ammo acid sequence, with a defined translation initiation and termination region.
  • the invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RN ⁇ of a desired target.
  • the cn/.ymaiic nucleic acid molecule may be targeted to a highly specific sequence region of a target such that specific gene inhibition can be achieved.
  • enzymatic nucleic acid can be targeted to a highly conserved region of a gene family to inhibit gene expression of a family of related enzymes.
  • the ribozymes can be expressed in plants that have been transformed with vectors which express the nucleic acid of the present invention.
  • ribozyme The enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.
  • enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RN ⁇ target, it is released from that RNA to search for another target and can repeatedly .bind and cleave new targets.
  • the enzymatic nucleic acid molecule is formed in a hammerhead or hai ⁇ in motif, but may also be formed in the 97/10328
  • Group II introns are described by Griffin et al, 1995, Chem. Biol. 2, 761 ; Michels and Pyle, 1995, Biochemistry 34, 2965; and of the Group I intron by Cech et al, U.S. Patent 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
  • the enzymatic nucleic acid molecules of the instant invention will be expressed within cells from eukaryotic promoters [e.g., Gerlach et al. International PCT Publication No. WO 91/13994; Edington and Nelson, 1992, in Gene Regulation: Biology of Antisense RNA and DNA, eds. R. P. Erickson and J. G. Izant, pp 209-221, Raven Press, NY.; Atkins et al, International PCT Publication No. WO 94/00012; Lenee et al, International PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins et al, 1 95, J. Gen. Virol.
  • eukaryotic promoters e.g., Gerlach et al. International PCT Publication No. WO 91/13994; Edington and Nelson, 1992, in Gene Regulation: Biology of Antisense RNA and DNA, eds. R. P. Erickson and
  • any ribozyme can be expressed in eukaryotic plant cells from an appropriate promoter.
  • the ribozymes expression is under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter.
  • the ⁇ bozyme RNA is introduced into the plant.
  • plants may be transformed using Agrobacterium technology, sec US Patent 5, 177.010 to University of Toledo, 5,104,310 to Texas A&M, European Patent Application 0I 3 I 624B I , European Patent Applications 120516, 159418B1 and 176,1 12 to Schilperoot, US Patents 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 1 16718, 290799, 320500 all to MaxPlanck, European Patent Applications 604662 and 627752 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and US Patent 5,231,019 all to Ciba Geigy, US Patents 5,463,174 and 4,762,785 both to Calgene, and US Patents 5,004,863 and 5,159,135 both to Agracetus; whiskers technology, see
  • tissue which is contacted with the foreign material (typically plasmids containing RNA or DNA) may vary as well.
  • tissue would include but would not be limited to embryogenic tissue, callus tissue type I and II, and any tissue which is receptive to transformation and subsequent regeneration into a transgenic plant.
  • Another variable is die choice of a selectable marker. The preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker.
  • selectable markers include but are not limited to chlorosulfuron, hygromyacin, PAT and/or bar, bromoxynil, kanamycin and the like.
  • the bar gene may be isolated from Strptomuces, particularly from the hygroscopicus or viridochromogenes species.
  • the bar gene codes for phosphinothricin acetyl transferase (PAT) that inactivates the active ingradient in the herbicide bialaphos phosphinothricin (PPT).
  • PAT phosphinothricin acetyl transferase
  • PPT herbicide bialaphos phosphinothricin
  • the ribozymes may be expressed individually as monomers, i.e., one ribozyme targeted against one site is expressed per transcript. Alternatively, two or more ribozymes targeted against more than one target site are expressed as part of a single RNA transcript. A single RNA transcript comprising more than one ribozyme targeted against more than one cleavage site are readily generated to achieve efficient modulation of gene expression. Ribozymes within these multimer constructs are the same or different.
  • the multimer construct may comprise a plurality of hammerhead ribozymes or hai ⁇ in ribozymes or other ⁇ bozyme motifs.
  • the multimer construct may be designed to include a plurality of different ⁇ bozyme motifs, such as hammerhead and hai ⁇ in ribozymes. More specifically, multimer ribozyme constructs arc designed, wherein a series of ribozyme motifs are linked together in tandem in a single RN ⁇ transcript. The ribozymes are linked to each other by nucleotide linker sequence, wherein the linker sequence may or may not be complementary to the target RNA. Multimer ribozyme constructs (polyribozymes) are likely to improve the effectiveness of ribozyme-mediated modulation of gene expression.
  • ribozymes can also be augmented by their release from the primary transcript by a second ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595, both hereby inco ⁇ orated in their totality by reference herein; Ohkawa, J., et al., 1992, Nucleic Acids Symp. Ser. , 27, 15-6; Taira, K., et ⁇ ., 1991 , Nucleic Acids Res., 19, 5125-30; Ventura, M., et al, 1993, Nucleic Acids Res., 21 , 3249-55; Chowrira et c/., 1994 J. Biol. Chem. 269, 25856).
  • Ribozyme-mediated modulation of gene expression can be practiced in a wide variety of plants including angiosperms, gymnosperms, monocotyledons, and dicotyledons.
  • Plants of interest include but are not limited to: cereals, such as rice, wheat, barley, maize; oil-producing crops, such as soybean, canola, sunflower, cotton, maize, cocoa, saf lower, oil palm, coconut palm, flax, castor, peanut; plantation crops, such as coffee and tea; fruits, such as pineapple, papaya, mango, banana, grapes, oranges, apples; vegetables, such as cauliflower, cabbage, melon, green pepper, tomatoes, carrots, lettuce, celery, potatoes, broccoli; legumes, such as soybean, beans, peas; flowers, such as carnations, chrysanthemum, daisy, tulip, gypsophila, alstromeria, marigold, petunia, rose; trees such as olive, cork, poplar
  • Ribozyme-mediated down regulation of the expression of genes involved in caffeine synthesis can be used to significantly change caffeine concentration in coffee beans.
  • Expression of genes, such as 7-methylxanthos ⁇ ne and/or 3-methyl transferase in coffee plants can be readily modulated using ribozymes to decrease caffeine synthesis (Adams and Zarowitz, US Patent No. 5,334,529; inco ⁇ orated by reference herein).
  • Transgenic plants expressing ribozymes targeted against genes involved in ripening of fruits such as ethylene-form g enzyme, pectin mcthyltransfcrasc, pectin cstcrasc, polygalacturonase, 1 -am ⁇ nocyclopropane carboxylic acid ( ⁇ CC) syntha.sc, ⁇ CC oxidase genes (Smith et al, 1988, Nature, 334, 724; Gray et al, 1992, Pl. Mol. Biol, 19, 69; Tieman et al, 1992, Plant Cell, 4, 667; Picton et al, 1993, The Plant J. 3, 469; Shewmaker et al, supra; James et al, 1996, Bio/Technology, 14, 56), would delay the ripening of fruits, such as tomato and apple.
  • ethylene-form g enzyme pectin mcthyltransfcrasc, pectin cstcrasc,
  • Transgenic plants expressing ribozymes targeted against genes involved in flower pigmentation such as chalcone synthase (CHS), chalcone flavanone isomerase (CHI), phenylalanme ammonia lyase, or dehydroflavonol (DF) hydroxylases, DF reductase (Krol van der, et al, 1988, Nature, 333, 866; Krol van der, et al. 1990, Pl. Mol Biol. 14, 457; Shewmaker et al, supra; Jorgensen, 1996, Science, 268, 686), would produce flowers, such as roses, petunia, with altered colors.
  • CHS chalcone synthase
  • CHI chalcone flavanone isomerase
  • DF dehydroflavonol
  • DF reductase Krol van der, et al, 1988, Nature, 333, 866; Krol van der, et al. 1990, Pl. Mol Biol. 14, 4
  • Lignins are organic compounds essential for maintaining mechanical strength of cell walls in plants. Although essential, lignins have some disadvantages. They cause indigestibility of sillage crops and are undesirable to paper production from wood pulp and others. Transgenic plants expressing ribozymes targeted against genes involved in lignin production such as, O-methyltransferase, cinnamoyI-CoA:NADPH reductase or cinnamoyl alcohol dehydrogenase (Doorsselaere et al, 1995, The Plant J. 8, 855; Atanassova et al, 1995, The Plant J. 8, 465; Shewmaker et al, supra; Dwivedi et al, 1994, Pl. Mol. Biol, 26, 61 ), would have altered levels of lignin.
  • starch biosynthesis occurs in both chloroplasts (short te ⁇ n starch storage) and in the amyloplast (long term starch storage).
  • Starch granules normally consist of a linear chain of ⁇ ( l -4)-l ⁇ nked ⁇ -D-glucose units (amylose) and a branched form of amylose cross-linked by ⁇ ( l-6) bonds (amylopectin)
  • An enzyme involved in the synthesis of starch in plants is starch synthase which produces linear chains of ⁇ (l - 4)-glucose using ADP-glucose.
  • starch synthase Two mam forms of starch synthase are found in plants: granule bound starch synthase (GBSS) and a soluble form located in the stroma of chloroplasts and in amyloplasts (soluble starch syntha.sc) Both lorms ol this cii/yme utilize ADP-D-glucose while the granular bound form also utilizes UD -D-glucosc, with a preference for the former.
  • GBSS granule bound starch synthase
  • soluble form located in the stroma of chloroplasts and in amyloplasts
  • the GBSS known as waxy protein
  • the Wx (waxy) locus encodes a granule bound glucosyl transferase involved in starch biosynthesis. Expression of this enzyme is limited to endosperm, pollen and the embryo sac in maize. Mutations in this locus have been termed waxy due to the appearance of mutant kernels, which is the phenotype resulting from an reduction in amylose composition m the kernels. In maize, this enzyme is transported into the amyloplast of the developing endosperm where it catalyses production of amylose. Com kernels are about 70% starch, of which 27% is linear amylose and 73% is amylopectin.
  • Waxy is a recessive mutation in the gene encoding granule bound starch synthase (GBSS). Plants homozygous for this recessive mutation produce kernels that contain 100% of their starch in the form of amylopectin.
  • GBSS granule bound starch synthase
  • Ribozymes with their catalytic activity and increased site specificity (as desc ⁇ bed below), represent more potent and perhaps more specific inhibitory molecules than antisense oligonucleondes. Moreover, these ribozymes are able to inhibit GBSS activity and the catalytic activity of the ⁇ bozymes is required for their inhibitory effect. For those of ordinary skill in the art, it is clear from the examples that other ribozymes may ⁇ ⁇ -,,. n ,- o PCT/US96/11689 O 97/10328
  • the invention features ⁇ bozymes that inhibit enzymes involved in amylose production, e.g., by reducing GBSS activity
  • RNA molecules contain substrate binding domains that bind to accessible regions of the target mRNA.
  • the RN ⁇ molecules also contain domains that catalyze the cleavage of RNA.
  • the RNA molecules are preferably ribozymes of the hammerhead or hai ⁇ in motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, amylose production is reduced or inhibited. Specific examples arc provided below infra.
  • Preferred embodiments include the ribozymes having binding arms which are complementary to the binding sequences in Tables IIIA, VA and VB. Examples of such ribozymes are shown in Tables IIIB - V. Those in the art will recognize that while such examples are designed to one plant's (e.g.. maize) mRNA, similar ribozymes can be made complementary to other plant species' mRNA. By complementary is thus meant that the binding arms enable ribozymes to interact with the target RNA in a sequence-specific manner to cause cleavage of a plant mRNA target. Examples of such ribozymes consist essentially of sequences shown in Tables IIIB - V.
  • Preferred embodiments are the ribozymes and methods for their use in the inhibition of starch granule bound ADP (UDP)-glucose: ⁇ -l,4-Z)-glucan 4- ⁇ -glucosyl transferase i.e.. granule bound starch synthase (GBSS) activity in plants.
  • UDP starch granule bound ADP
  • GBSS granule bound starch synthase
  • ribozymes that cleave target molecules and inhibit amylose production are expressed from transcription units inserted into the plant genome.
  • the recombinant vectors capable of stable integration into the plant genome and selection of transformed plant lines expressing the ribozymes are expressed either by constitutive or inducible promoters in the plant cells. Once expressed, the ribozymes cleave their target mRNAs and reduce amylose production of their host cells.
  • the ribozymes expressed in plant cells are under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter. Modification of com starch is an important application of ribozyme technology which is capable of reducing specific gene expression.
  • a high level of amylopectin is desirable for the wet milling process of corn and there is also some evidence that high amylopectin corn leads to increased digestibility and therefore energy availability in feed.
  • Nearly 10% of wet-milled com has the waxy phenotype, but because of its recessive nature the traditional waxy varieties are very difficult for the grower lo handle Ribozymes targeted to cleave the GBSS mRNA and thus reduce GBSS activity in plants, and in particular, corn endosperm will act as a dominant trait and produce corn plants with the waxy phenotype that will be easier for the grower to handle.
  • Fatty acid biosynthesis in plant tissues is initiated in the chloroplast.
  • Fatty acids are synthesized as thioesters of acyl carrier protein (ACP) by the fatty acid synthase complex (FAS).
  • ACP acyl carrier protein
  • FAS fatty acid synthase complex
  • Fatty acids with chain lengths of 16 carbons follow one of three paths: 1) they are released, immediately after synthesis, and transferred to glycerol 3-phosphate (G3P) by a chloroplast acyl transferase for further modification within the chloroplast; 2) they are released and transferred to Co-enzyme A (CoA) upon export from the plastid by thioesterases; or 3) they are further elongated to C18 chain lengths.
  • G3P glycerol 3-phosphate
  • CoA Co-enzyme A
  • the C18 chains are rapidly desaturated at the C9 position by stearoyi-ACP desaturase. This is followed by immediate transfer of the oleic acid (18:1) group to G3P within the chloroplast, or by export from the chloroplast and conversion to oleoyl-CoA by thioesterases (Somerville and Browse, 1991 Science 252: 80-87). The majority of C16 fatty acids follow the third pathway.
  • Di- and tri-unsaturated chains are then released into the acyl-CoA pool and transfe ed to the C3 position of the glycerol backbone in diacyl glycerol in the production of triglycerides (Frentzen, 1993 in Lipid Metabolism in Plants., p.195-230, (ed. Moore.T.S.) CRC Press, Boca Raton, FA.).
  • a schematic of the plant fatty acid biosynthesis pathway is shown in Figures 1 1 and 12.
  • the three predominant fatty acids in co seed oil are linoleic acid (18:2, ⁇ 59%), oleic acid (18:1 , ⁇ 26%), and palmitic acid (16:0, -11%).
  • This predominance of C18 chain lengths may reflect the abundance and activity of several key enzymes, such as the fatty acid synthase responsible for production of CI 8 carbon chains, the stearoyl-ACP desaturase ( ⁇ -9 desaturase) for production of 18: 1 and a microsomal ⁇ - 12 desaturase for conversion of 18: 1 to 18:2.
  • the fatty acid synthase responsible for production of CI 8 carbon chains the stearoyl-ACP desaturase ( ⁇ -9 desaturase) for production of 18: 1
  • ⁇ - 12 desaturase for conversion of 18: 1 to 18:2.
  • ⁇ -9 desaturase also called stearoyl-ACP desaturase
  • ACP acyl carrier protein
  • Rat and yeast ⁇ -9 desaturases are membrane bound microsomal enzymes using acyl-CoA chains as substrates, whereas cyanobacterial ⁇ -9 desaturase uses acyl chains on diacyl glycerol as substrate.
  • Com (maize) has been used minimally for the production of margarine products because it has traditionally not been utilized as an oil crop and because of the relatively low seed oil content when compared with soybean and canola.
  • com oil has low levels of linolenic acid (18:3) and relatively high levels of palmitic (16:0) acid (desirable in margarine).
  • Applicant believes that reduction in oleic and linoleic acid levels by down- regulation of ⁇ -9 desaturase activity will make com a viable alternative to soybean and canola in the saturated oil market.
  • Margarine and confectionary fats are produced by chemical hydrogenation of oil from plants such as soybean. This process adds cost to the production of the margarine and also causes both cis and trans isomers of the fatty acids. Trans isomers are not naturally found in plant de ⁇ ved oils and have raised a concern for potential health risks. Applicant believes that one way to eliminate the need for chemical hydrogenation is to genetically engineer the plants so that desaturation enzymes are down-regulated. ⁇ -9 desaturase introduces the first double bond into 18 carbon fatty acids and is the key step effecting the extent of desaturation of fatty acids.
  • the invention concerns compositions (and methods for their use) for the modification of fatty acid composition in plants. This is accomplished through the inhibition of genetic expression, with ribozymes, antisense nucleic acid, cosuppression or triplex DN ⁇ , which results in the reduction or elimination of certain enzyme activities in plants, such as ⁇ -9 desaturase Such activity is reduced in monocotyledon plants, such as maize, wheat, rice, palm, coconut and others. ⁇ -9 desaturase activity may also be reduced in dicotyledon plants such as sunflower, saffiower, cotton, peanut, olive, sesame, cuphea, flax, jojoba, grape and others.
  • the invention features ribozymes that inhibit enzymes involved in fatty acid unsaturation, e.g., by reducing ⁇ -9 desaturase activity.
  • RNA molecules contain substrate binding domains that bind to accessible regions of the target mRNA.
  • the RNA molecules also contain domains that catalyze the cleavage of RNA.
  • the RNA molecules are preferably ribozymes of the hammerhead or hai ⁇ in motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, stearate levels are increased and unsaturated fatty acid production is reduced or inhibited. Specific examples are provided below in the Tables listed directly below.
  • the ribozymes have binding arms which are complementary to the sequences in the Tables VI and VIII.
  • binding arms of the ribozymes are able to interact with the target RNA in a sequence-specific manner and enable the ribozyme to cause cleavage of a plant mRNA target.
  • Examples of such ribozymes are typically sequences defined in Tables VII and VIII.
  • the active ribozyme typically contains an enzymatic center equivalent to those in the examples, and binding arms able to bind plant mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such binding and/or cleavage.
  • ⁇ bozymes that cleave target molecules and reduce unsaturated fatty acid content in plants are expressed from transcription units inserted into the plant genome.
  • the recombinant vectors capable of stable integration into the plant genome and selection of transformed plant lines expressing the ribozymes are expressed either by constitutive or inducible promoters in the plant cells. Once expressed, the ribozymes cleave their target mRN ⁇ s and reduce unsaturated fatty acid production of their host cells.
  • the ribozymes expressed in plant cells are under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter.
  • Modification of fatty acid profile is an important application of nucleic acid-based technologies which are capable of reducing specific gene expression.
  • a high level of saturated fatty acid is desirable in plants that produce oils of commercial importance.
  • this invention features the isolation of the cDNA sequence encoding ⁇ -9 desaturase in maize.
  • hai ⁇ in and hammerhead ribozymes that cleave ⁇ -9 desaturase mRNA are also described.
  • Those of ordinary skill in the art will understand from the examples described below that other ⁇ bozymes that cleave target mRNAs required for ⁇ -9 desaturase activity may now be readily designed and are within the scope of the invention.
  • com RNA While specific examples to com RNA are provided, those in the art will recognize that the teachings are not limited to com. Furthermore, the same target may be used in other plant species.
  • RNA sequences are utilized in the ribozyme targeted to that specific RNA.
  • Example 1 Isolation of ⁇ .9 desaturase cDNA from Zea mays
  • PCR primers were designed and synthesized to two conserved peptides involved in diiron-oxo group binding of plant ⁇ -9 desaturases.
  • a 276 bp DNA fragment was PCR amplified from maize embryo cDNA and was cloned in to a vector. The predicted amino acid sequence of this fragment was similar to the sequence of the region separated by the two conserved peptides of dicot ⁇ -9 desaturase proteins. This was used to screen a maize embryo cDNA library. A total of 16 clones were isolated; further restriction mapping and hybridization identified one clone which was sequenced.
  • cDNA insert is: a 1621 nt cDNA; 145 nt 5' and 294 nt 3' untranslated regions including a 18 nt poly A tail; a 394 amino acid open reading frame encoding a 44.7 kD polypeptide; and 85% amino acid identity with castor bean ⁇ -9 desaturase gene for the predicted mature protein.
  • the complete sequence is presented in Figure 10.
  • HH ribozyme sites Approximately two hundred and fifty HH ribozyme sites and approximately forty three HP sites were identified in the com ⁇ -9 desaturase mRNA.
  • a HH site consists of a uridine and any nucleotide except guanosine (UH).
  • UH guanosine
  • Tables VI and VIII have a list of HH and HP ribozyme cleavage sites. The numbering system starts with 1 at the 5' end of a ⁇ -
  • Ribozymes such as those listed in Tables VII and VIII, can be readily designed and synthesized to such cleavage sites with between 5 and 100 or more bases as substrate binding arms (see Figs. 1 - 5). These substrate binding arms within a ribozyme allow the ribozyme to interact with their target in a sequence-specific manner.
  • Example 3 Selection of Ribozvme Cleavage Sites for 9 desaturase
  • RNase H assays These oligonucleotides covered 108 sites within ⁇ -9 desaturase RNA.
  • RNase H assays (Fig. 6) were performed using a full length transcript of the ⁇ -9 desaturase cDNA.
  • RNA was screened for accessible cleavage sites by the method described generally in Draper et al, supra. Briefly, DNA oligonucleotides representing ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to generate a substrate for T7 RNA polymerase transcription from co cDNA clones. Labeled RNA transcripts were synthesized in vitro from these templates.
  • RNAseH was added and the mixtures were incubated for 10 minutes at 37°C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a Molecular Dynamics phosphor imaging system (Figs. 13 and 14).
  • HH and hai ⁇ in (HP) ribozymes were designed to the sites covered by the oligos which cleaved best in the RNase H assays. These ribozymes were then subjected to analysis by computer folding and the ribozymes that had significant secondary structure were rejected.
  • RNA synthesis was chemically synthesized.
  • the general procedures for RNA synthesis have been desc ⁇ bed previously (Usman et al, 1987, J. Am. Chem. Soc, 109, 7845-7854 and in Scaringe et al., 1990, Nucl Acids Res., 18, 5433-5341 ; Wincott et al ,
  • oligonucleotide synthesis reagents for the 394 Det ⁇ tylation solution was 2% TCA in methylene chloride (ABI); capping was performed with 16% N- Methyl imidazole in THF (ABI) and 10% acetic anhydride/ 10% 2,6-lut ⁇ d ⁇ ne in THF (ABI); oxidation solution was 16.9 mM l2, 49 mM pyridine, 9% water in THF
  • Deprotection of the R ⁇ A was performed as follows.
  • TEA and 1.0 mL TEA*3HF to provide a 1.4 M HF concentration were heated to 65°C for 1.5 h.
  • the resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.
  • the TEAB solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB (10 mL), the R ⁇ A was eluted with 2 M TEAB (10 mL) and dried down to a white powder.
  • Qiagen 500® anion exchange cartridge Qiagen Inc.
  • Inactive hammerhead ribozymes were synthesized by substituting a U for G5 and a U for A 14 (numbering from (Hertel, K. J., et al, 1992, Nucleic Acids Res., 20, 3252) The hai ⁇ in ribozymes were synthesized as described above for the hammerhead RNAs.
  • Ribozymes were also synthesized from DNA templates using bacte ⁇ ophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol 180, 51 ). Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; Sec Wincott et al, 1996, supra, the totality of which is hereby inco ⁇ orated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study arc shown below in Tables VII and VIII.
  • Target RNA used in this study was 1621 nt long and contained cleavage sites for all the HH and HP ribozymes targeted against ⁇ -9 desaturase RNA.
  • a template containing T7 RNA polymerase promoter upstream of ⁇ -9 desaturase target sequence was PCR amplified from a cDNA clone.
  • Target RNA was transcribed from this PCR amplified template using T7 RNA polymerase.
  • the transcript was internally labeled during transcription by including [ ⁇ - 32 P] CTP as one of the four ribonucleotide triphosphates.
  • the transcription mixture was treated with DNase-I, following transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription.
  • the transcription mixture was resolved on a denaturing polyacrylamide gel. Bands corresponding to full- length RNA was isolated from a gel slice and the RNA was precipitated with isopropanol and the pellet was stored at 4°C.
  • Ribozyme cleavage reactions were carried out under ribozyme excess (k at/KM) conditions (Herschlag and Cech, 1990, Biochemistry 29, 10159-10171). Briefly, 1 mM ribozyme and ⁇ 10 nM internally labeled target RNA were denatured separately by heating to 65°C for 2 min in the presence of 50 mM Tris.HCl, pH 7.5 and 10 mM
  • RNAs were renatured by cooling to the reaction temperature (37°C, 26°C or
  • Cleavage reaction was initiated by mixing the ribozyme and target RNA at appropriate reaction temperatures. Aliquots were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on 4 % sequencing gel.
  • Example 7 Cleavage of the target RNA using multiple ribozvme combinations for ⁇ 9 desaturase
  • ribozymes were incorporated into a multimer ribozyme construct which contains two or more ribozymes embedded in a contiguous stretch of complementary RNA sequence.
  • Non-limiting examples of multimer ribozymes are shown in Figures 17, 18, 19 and 23.
  • the ribozymes were made by annealling complementary oligonucleotides and cloning into an expression vector containing the Cauliflower Mosaic Virus 35S enhanced promoter (Franck et al. 1985 Cell 21, 285), the maize Adh 1 intron (Dennis et al, 1984 Nucl Acids Res.
  • Ribozymes targeted to cleave ⁇ -9 desaturase mRNA are endogenously expressed in plants, either from genes inserted into the plant genome (stable transformation) or from episomal transcription units (transient expression) which are part of plasmid vectors or viral sequences. These ribozymes can be expressed via RNA polymerase I, II, or III plant or plant virus promoters (such as CaMV). Promoters can be either constitutive, tissue specific, or developmentally expressed.
  • the ribozymes were designed with 3 bp long stem II and 20 bp (total) long substrate binding arms targeted against site 259.
  • the active version is RPA 114, the inactive is RPA 1 15.
  • the parent plasmid, pDAB367 was digested with Not I and filled in with Klenow to make a blunt acceptor site.
  • the vector was then digested with Hind III restriction enzyme.
  • the ribozyme containing plasmids were cut with Eco RI, filled-in with Klenow and recut with Hind III.
  • the insert containing the entire ribozyme transcription unit was 97/10328 3Q
  • the ⁇ bozymes were designed with 3 bp long stem fl regions Total length ol the substrate binding arms of the multimer construct was 42 bp.
  • the active version is RPA 118, the inactive is 119
  • the constructs were made as described above for the 259 monomer.
  • the multimer construct was designed with four hammerhead ribozymes targeted against sites 453, 464, 475 and 484 within ⁇ -9 desaturase RNA
  • transgenic plants can be identified by standard assays.
  • the transgenic plants can be evaluated for reduction tn ⁇ -9 desaturase expression and ⁇ -9 desaturase activity as discussed in the examples infra.
  • GBSS mRNA polypeptide coding region (see table IIIA).
  • a hammer-head site consists of a undine and any nucleotide except guanine (UH).
  • UH guanine
  • sequence of GBSS coding region for com SEQ. I.D. No.25.
  • the numbe ⁇ ng system starts with I at the 5' end of a GBSS cDNA clone having the following sequence (5' to 3')
  • Ribozymes can be readily designed and synthesized to such sites with between 5 and 100 or more bases as substrate binding arms (see Figs. 1 - 5) as described above.
  • the secondary structure of GBSS mRNA was assessed by computer analysis using folding algorithms, such as the ones developed by M. Zuker ( Zuker, M., 1989 Science,
  • RNA RNA stems of over eight nucleotides and contained potential hammerhead ribozyme cleavage sites were identified. These sites which were then assessed for oligonucleotide accessibility with RNase H assays (see Fig. 6). Fifty-eight DNA oligonucleotides, each twenty one nucleotides long were used in these assays.
  • oligonucleotides covered 85 sites The position and designation of these oligonucleotides were 195, 205, 240, 307, 390, 424, 472, 481 , 539, 592, 625, 636, 678, 725, 741 , 81 1 , 859, 891 , 897, 912, 918, 928, 951 , 958, 969 , 993, 999, 1015, 1027, 1032, 1056, 1084, 1 105, 1 156, 1 168, 1 186, 1 195, 1204, 1213.
  • Example 1 RNaseH Assays for GBSS
  • RNase H assays were performed using a full length transcript of the GBSS coding region, 3' noncoding region, and part of the 5' noncoding region.
  • the GBSS RNA was screened for accessible cleavage sites by the method desc ⁇ bed generally in Draper et al, supra, hereby inco ⁇ orated by reference herein. Briefly, DNA oligonucleotides representing hammerhead ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to generate a substrate for T7 RNA polymerase transcription from com cDNA clones. Labeled RNA transcripts were synthesized in vitro from these templates.
  • RNAseH was added and the mixtures were incubated for 10 minutes at 37°C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a phosphor imaging system (Fig. 7)*
  • the ⁇ bozymes were chemically synthesized.
  • the method of synthesis used follows the procedure for normal RNA synthesis as described above (and in Usman et al , supra, Sca ⁇ nge et al, and Wincott et al, supra) and are incorporated by reference herein, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • the average stepwise coupling yields were >98%.
  • Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from (Hertel et al, supra).
  • Hairpin ⁇ bozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chow ⁇ ra and Burke, 1992, Nucleic Acids Res., 20, 2835-). All ribozymes were modified to enhance stability by modification of five ribonucleotides at both the 5' and 3' ends with 2'-0- methyl groups. Ribozymes were purified by gel electrophoresis using general methods. (Ausubel et al, 1990 Current Protocols in Molecular Biology Wiley & Sons, NY) or were purified by high pressure liquid chromatography, as described above and were resuspended in water.
  • Target RNA used in this study was 900 nt long and contained cleavage sites for all the 23 HH ribozymes targeted against GBSS RNA.
  • a template containing T7 RNA polymerase promoter upstream of GBSS target sequence was PCR amplified from a cDNA clone.
  • Target RNA was transcribed from this PCR amplified template using T7 RNA polymerase.
  • the transcript was internally labeled during transcription by including [ ⁇ -32p] CTP as one of the four ribonucleotide triphosphates.
  • the transcription mixture was treated with DNase-1 , following transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription.
  • the transcription mixture was resolved on a denaturing polyacrylamide gel. Bands corresponding to full-length RNA was isolated from a gel slice and the RNA was precipitated with isopropanol and the pellet was stored at 4°C.
  • Ribozyme cleavage reactions were carried out under ribozyme excess (kcat/K ) conditions (Herschlag and Cech, supra). Briefly, 1000 nM ribozyme and ⁇ 10 nM internally labeled target RNA were denatured separately by heating to 90°C for 2 min. in the presence of 50 mM Tris.HCl, pH 7.5 and 10 mM MgCl2- The RNAs were renarured by cooling to the reaction temperature (37°C, 26°C and 20°C) for 10-20 min. Cleavage reaction was initiated by mbcing the ribozyme and target RNA at appropriate reaction temperatures.
  • RNA cleavage reaction Four of the lead ⁇ bozymes (892, 919, 959, 1241 ) were incubated with internally labeled target RNA in the following combinations: 892 alone; 892 + 919; 892 + 919 + 959; 892 + 919 + 959 + 1241.
  • the fraction of RNA cleavage increased in an additive manner with an increase in the number of ribozymes used in the cleavage reaction (Fig. 9) Ribozyme cleavage reactions were earned out at 20°C as described above.
  • a multimer ribozyme was constructed which contained four hammerhead ribozymes targeting sites 892, 919, 959 and 968 of the GBSS mRNA.
  • Two DNA oligonucleotides (Macromolecular Resourses, Fort Collins, CO) were ordered which overlap by 18 nucleotides. The sequences were as follows:
  • Oligo 1 CGCGGA TCC TGG TAG GAC TGA TGA GGC CGA AAG GCC GAA ATGTTGTGCTGATGAGGCCGAAAG GCCGAAATGCAGAAAGCG GTC TTTGCGTCCCTGTAGATG CCGTGGC
  • Oligo2 CGCGAGCTCGGCCCTCTCTTTCGGCCTTTCGGCCTC ATC AGG TGCTAC CTC AAG AGC AAC TAC CAG TTT CGG CCTTTC GGC CTC ATC AGCCACGGCATCTACAGGG
  • the DNA fragments were ligated into BamVWS.st I digested pD ⁇ B 353.
  • the gaiion was transformed into competent DH5 ⁇ F' bacteria (Gibco/BRL). Potential clones were screened by digestion with Bam UEco RI. Clones were confirmed by sequencing. The total length of homology with the target sequence is 96 nucleotides.
  • a single ribozyme to site 919 of the GBSS mRNA was constructed with 10/10 a ⁇ ns as follows. Two DNA oligos were ordered:
  • Oligo 1 GAT CCG ATG CCGTGG CTG ATG AGG CCG AAA GGC CGA AAC TGG TAG TT
  • Oligo2 AACTACCAGTTTCGGCCTTTCGGCCTC ⁇ TC ⁇ GC CACGGC ⁇ TC G
  • the oligos are phosphorylated individually in IX kinase buffer (Gibco/BRL) and heat denatured and annealed by combining at 90°C for 10 min, then slow cooled to room temperature (22°C).
  • the vector was prepared by digestion of pDAB 353 with Sst I and blunting the ends with T4 DNA polymerase. The vector was redigested with Bam HI and gel purified as above.
  • the annealed oligos are ligated to the vector in IX ligation buffer (Gibco/BRL) at 16°C overnight. Potential clones were digested with Bam HVEco RI and confirmed by sequencing.
  • Example 16 Plant Transformation Plasmids pDAB 367. Used in the ⁇ .9 Ribozyme Experiments, and pDAB353 used in the GBSS Ribozvme Experiments
  • Plasmid pDAB367 has the following DNA structure: beginning with the base after the final C residue of d e Sph I site of pUC 19 (base 441 ; Ref. 1 ), and reading on the strand contiguous to the LacZ gene coding strand, the linker sequence CTGCAGGCCGGCC TTAATTAAGCGGCCGCGTTTAAACGCCCGGGCATTTAAATGGCGCGCCGC GATCGCTTGCAGATCTGCATGGGTG, nucleotides 7093 to 7344 of CaMV DNA (2), the linker sequence CATCGATG, nucleotides 7093 to 7439 of CaMV, the linker sequence GGGGACTCTAGAGGATCCAG, nucleotides 167 to 186 of MSV (3), nucleotides 188 to 277 of MSV (3), a C residue followed by nucleotides 1 19 to 209 of maize Adh IS containing parts of exon 1 and intron I (4), nucleotides 555 to 672 containing
  • Plasmid pDAB353 has the following DNA structure: beginning with the base after the final C residue of the Sph I site of pUC 19 (base 441; Ref. 1), and reading on the strand contiguous to the LacZ gene coding strand, the linker sequence CTGCAGATCTGCATGGGTG, nucleotides 7093 to 7344 of CaMV DNA (2), the linker sequence CATCGATG, nucleotides 7093 to 7439 of CaMV, the linker sequence GGGGACTCTAGAG, nucleotides 119 to 209 of maize Adh IS containing parts of exon 1 and intron 1 (4), nucleotides 555 to 672 containing parts of Adh IS intron 1 and exon 2 (4), and the linker sequence GACGGATCCGTCGACC, where GGATCC represents the recognition sequence for BamH I restriction enzyme.
  • GUS beta- giucuronidase
  • Example 17 Plasmid pDAB359 a Plant Transformation Plasmid which Contains the Gamma-Zein Promoter, the Antisense GBSS. and a the Nos Polvadenvlation Sequence
  • Plasmid pDAB359 is a 6702 bp double-stranded, circular DNA that contains the following sequence elements: nucleotides 1-404 from pUC18 which include lac operon sequence from base 238 to base 404 and ends with the Hindlll site of the M 13mpl 8 polylinker (1,2); the Nos polyadenylation sequence from nucleotides 412 to 668 (3); a synthetic adapter sequence from nucleotides 679-690 which converts a Sac I site to an Xho I site by changing GAGCTC to GAGCTT and adding CTCGAG: maize granule bound starch synthase cDNA from bases 691 to 2953, corresponding to nucleotides 1- 2255 of SEQ.
  • the GBSS sequence in plasmid pDAB359 was modified from the original cDNA by the addition of a 5' Xho I and a 3' Nco I site as well as the deletion of internal Nco I and Xho I sites using Klenow to fill in the enzyme recognition sequences.
  • Bases 2971 to 4453 are 5' untranslated sequence of the maize 27 kD gamma- zein gene corresponding to nucleotides 1078 to 2565 of the published sequence (4).
  • the gamma-zein sequence was modified to contain a 5' Kpn I site and 3' BamH/Sall/Nco I sites.
  • nucleotides 4454 to 6720 of pDAB359 are identical to pucl 8 bases 456 to 2686 including the Kpn I/EcoRI/Sac I sites of the M13/mpI 8 polylinker from 4454 to 4471 , a lac operon fragment from 4471 to 4697, and the ⁇ -lacatmase gene from 5642 to 6433 (1 , 2).
  • Example 18 Construction of Plasmid pDAB430. containing Antisense ⁇ 9 Desaturase, Expressed bv the Ubiquitin Promoter/intron (Ubi 1 )
  • Plasmid pDAB421 contains a unique blunt-end Srfl cloning site flanked by the maize Ubiquitin promoter/intron and the nopaline synthase polyadenylation sequences.
  • pDAB421 was prepared as follows: digestion of pDAB355 with restriction enzymes Kpnl and BamHI drops out the R coding region on a 2.2 kB fragment. Following gel purification, the vector was ligated to an adapter composed of two annealed oligonucleotides OF235 and OF236.
  • OF235 has the sequence 5' - GAT CCG CCC GGG GCC CGG GCG GTA C - 3' and OF236 has the sequence 5' - CGC CCG GGC CCC GGG CG - 3'.
  • Clones containing this adapter were identified by digestion and linearization of plasmid DNA with the enzymes Srfl and Smal which cut in the adapter, but not elsewhere in the plasmid.
  • One representative clone was sequenced to verify that only one adapter was inserted into the plasmid.
  • the resulting plasmid pDAB421 was used in subsequent construction of the ⁇ 9 desaturase antisense plasmid pDAB430.
  • plasmid pDAB430 (antisense ⁇ 9 desaturase)
  • the antisense ⁇ 9 desaturase construct present in plasmid pDAB430 was produced by cloning of an amplification product in the blunt-end cloning site of plasmid pDAB421. Two constructs were produced simultaneously from the same experiment.
  • the first construct contains the ⁇ 9 desaturase gene in the sense orientation behind the ubiquitin promoter, and the c-myc tag fused to the C-terminus of the ⁇ 9 desaturase open reading frame for immunological detection of ove ⁇ roduced protein in transgenic lines This construct was intended for testing of ribozymes in a system which did not express maize ⁇ 9 desaturase.
  • the ⁇ 9 desaturase cl)N ⁇ sequence described herein was amplified with two primers
  • the N-tcrmmal primer OF279 has the sequence 5'- GTG CCC ACA ATG GCG CTC CGC CTC AAC GAC - 3'.
  • the underlined bases correspond to nucleotides 146- 166 of the cDNA sequence
  • C- terminal primer OF280 has the sequence 5' - TCA TCA CAG GTC CTC CTC GCT GAT CAG CTT CTC CTC CAG TTG GAC CTG CCT ACC GTA - 3' and is the reverse complement of the sequence 5' - TAC GGT AGG GAC GTC CAA CTG GAG GAG AAG CTG ATC AGC GAG GAG GAC CTG TGA TGA - 3'.
  • the underlined bases correspond to nucleotides 1304-1324 of the cDNA sequence, the bases in italics correspond to the sequence of the c-myc epitope.
  • the 1179 bp of amplification product was purified through a 1.0% agarose gel, and ligated into plasmid pDAB421 which was linearized with the restriction enzyme Srfl. Colony hybridization was used to select clones containing the pDAB421 plasmid with the insert.
  • the orientation of the insert was determined by restriction digestion of plasmid DNA with diagnostic enzymes Kpnl and BamHl. A clone containing the ⁇ 9 desaturase coding sequence in the sense orientation relative to the Ubiquitin promoter/intron was recovered and was named pDAB429.
  • Plasmid pDAB430 was subjected to sequence analysis and it was determined that the sequence contained three PCR induced errors compared to the expected sequence. One error was found in the sequence corresponding to primer OF280 and two nucleotide changes were observed internal to the coding sequence. These errors were not corrected, because antisense downregulation does not require 100% sequence identity between the antisense transcript and the downregulation target.
  • Example 19 Helium Blasting of Embryogenic Maize Cultures and the Subsequent Regeneration of Transgenic Progeny
  • Hi-II is a hybrid made by lntermating 2 R3 lines derived from a B73xA 188 cross (Armstrong et al. 1990). When cultured, this genotype produces callus tissue known as Type II. Type II callus is friable, grows quickly, and exhibits the ability to maintain a high level of embryogenic activity over an extended time period.
  • Type II cultures were initiated from 1.5-3.0 mm immature embryos resulting from controlled pollinations of greenhouse grown Hi-II plants.
  • the initiation medium used was N6 (Chu, 1978) which contained I .Omg/L 2,4-D, 25 mM L-prolinc, 1 0 mg L casein hydrolysate, 10 mg L AgNO3, 2.5 g L gel ⁇ te and 2% sucrose adjusted to pH 5.8.
  • N6 Cho, 1978
  • L-prolinc 1 0 mg L casein hydrolysate
  • 10 mg L AgNO3, 2.5 g L gel ⁇ te and 2% sucrose adjusted to pH 5.8.
  • selection occurred for Type II callus and against non- embryogenic and or Type I callus.
  • Type II callus was transferred to a maintenance medium in which AgNO3 was omitted and L-prolinc reduced to 6mM.
  • Plasmid DNA was adsorbed onto the surface of gold particles prior to use in transformation experiments.
  • the experiments for the GBSS target used gold particles which were spherical with diameters ranging from 1.5-3.0 microns (Aldrich Chemical Co., Milwaukee, WI).
  • Transfomation experiments for the ⁇ 9 desaturase target used 1.0 micron spherical gold particles (Bio-Rad, Hercules, CA). All gold particles were surface-sterilized with ethanol prior to use. Adso ⁇ tion was accomplished by adding 74 ⁇ l of 2.5 M calcium chloride and 30 ⁇ l of 0.1 M spermidine to 300 ⁇ l of plasmid DNA and sterile H2O.
  • the concentration of plasmid DNA was 140 ⁇ g.
  • the DNA-coated gold particles were immediately vortexed and allowed to settle out of suspension. The resulting clear supematent was removed and the particles were resuspended in 1 ml of 100% ethanol. The final dilution of the suspension ready for use in helium blasting was 7.5 mg DNA gold per ml of ethanol.
  • Helium blasting involved accelerating the suspended DNA-coated gold particles towards and into prepared tissue targets.
  • the device used was an earlier prototype to the one described in a DowElanco U.S. Patent (#5,141 ,131) which is inco ⁇ orated herein by reference, although both function in a similar manner.
  • the device consisted of a high pressure helium source, a syringe containing the DN ⁇ /gold suspension, and a pneumatically-operated multipurpose valve which provided controlled linkage between the helium source and a loop of pre-loaded DNA/gold suspension.
  • tissue targets Prior to blasting, tissue targets were covered with a sterile 104 micron stainless steel screen, which held the tissue in place du ⁇ ng impact. Next, targets were placed under vacuum in the main chamber of the device. The DNA-coated gold particles were accelerated at the target 4 times using a helium pressure of 1500 psi. Each blast delivered 20 ⁇ l of DNA/gold suspension. Immediately post-blasting, the targets were placed back on maintenance medium plus osmoticum for a 16 to 24 hour recovery period.
  • BastaTM resistant callus was established as a line, plant regeneration was initiated by transferring callus tissue to petri plate containing cytokinin-based induction medium which were then placed in low light (125 ft-candles) for one week followed by one week in high light (325 ft-candles).
  • the induction medium was composed of MS salts and vitamins (Murashige and Skoog, 1962), 30 g L sucrose, 100 mg/L myo-inositol, 5 mg/L 6- benzylaminopurine, 0.025 mg L 2,4-D, 2.5 g/L gelrite adjusted to pH 5.7.
  • the tissue was non-selectively transfe ⁇ ed to hormone-free regeneration medium and kept in high light.
  • the regeneration medium was composed of MS salts and vitamins, 30 g/L sucrose and 2.5 g/L gelrite adjusted to pH 5.7. Both induction and regeneration media contained 30 mg/L BastaTM.
  • Tissue began differentiating shoots and roots in 2-4 weeks. Small (1.5-3 cm) plantlets were removed and placed in tubes containing SH medium.
  • SH medium is composed of SH salts and vitamins (Schenkillon worth endeavour_._ PCT/US96/11689 97/10328
  • Ro plants were sclf-pollinatcd and/or cross-pollinated with non-iransgenic inbrcds to obtain transgenic progeny.
  • Ri seed produced from Ro pollinations was replanted.
  • the R] plants were grown to maturity and pollinated to produce R2 seed in the quantities needed for the analyses.
  • Example 20 Production and Regeneration of ⁇ .9 Transgenic Material.
  • BMS does not produce a GBSS mRNA which is homologous to that found endogenously in maize. Therefore, a double transformation system was developed to produce transformants which expressed both target and ribozymes.
  • "ZM" BMS suspensions obtained from Jack Widholm, University of Illinois, also see W. F. Sheridan, "Black Mexican Sweet Com: Its Use for Tissue Cultures” in Maize for Biological Research, W. F. Sheridan, editor. University Press. University of North Dakokta, Grand Forks, ND, 1982, pp.
  • 385-388 were prepared for helium blasting four days after subculture by transfer to a 100 x 20 mm Petri plate (Fisher Scientific, Pittsburgh, PA) and partial removal of liquid medium, forming a thin paste of cells.
  • Targets consisted of 100- 125 mg fresh weight of cells on a 1/2" antibiotic disc (Schleicher and Schuell, Keene, NH) placed on blasting medium, DN6 [N6 salts and vitamins (Chu ei al, 1978), 20 g ' L sucrose, 1.5 mg L 2,4-dichlorophenoxyacet ⁇ c acid (2,4-D).
  • the antibiotic discs were transferred to DN6 medium made with 0.8% TC agar for one week of target tissue recovery. After recovery, each target was spread onto a 5.5 cm Whatman #4 filter placed on DN6 medium minus proline with 3 mg/L Basta® (Hoechst, Frankfort, Germany). Two weeks later, the filters were transferred to fresh selection medium with 6 mg/L Basta®. Subsequent transfers were done at two week intervals.
  • Isolates were maintained by subculture to fresh medium every two weeks.
  • Basta®-resistant isolates which expressed GBSS were subjected to a second transformation.
  • targets of transgenic callus were prepared 4 days after subculture by spreading tissue onto 1/2" filters.
  • AMCF-ARM with 2% TC agar was used for blasting, due to maintenance of transformants on AMCF-ARM selection media.
  • Each sample was covered with a sterile 104 ⁇ m mesh screen and blasting was done at 1500 psi.
  • Target tissue was co-bombarded with pDAB 319 (35S-ALS; 35T- GUS) and RPA63 (active ribozyme multimer) or pDAB319 and RPA64 (inactive ribozyme multimer), or shot with pDAB 319 alone.
  • AMCF-ARM nonselective medium
  • CSN chlorsulfuron
  • GBSS functional target gene
  • RNase protection assay RPA
  • Northern blot analysis were performed on ribozyme-expressing and vector control tissues to compare levels of GBSS transcript in the presence or absence of active ribozyme.
  • GBSS values were normalized to an internal control ( ⁇ 9 desaturase); Northern blot data is shown in Figure (25).
  • Northern blot results revealed a significantly lower level of GBSS message in the presence of ribozyme, as compared to vector controls.
  • RPA data showed that some of the individual samples expressing active ribozyme ("L" and "O") were significantly different from vector controls and similar to a nontransformed control.
  • Plant material co-transformed with the pDAB308 and one of the following ribozyme containing vectors, pRPA63, pRPA64, pRPA85, pRPA1 13, ⁇ RPA1 14, pRPAl 15, pRPAl 18 or pRPAl 19 were analyzed at the callus level, Ro level and select lines analyzed at the Fl level.
  • Leaf material was harvested when the plantlets reached the 6-8 leaf stage.
  • DNA from the plant and callus material was prepared from lyophilized tissue as described by Saghai-Maroof et al(supra).
  • Probes specific for the ribozyme coding region were hybridized to the membranes. Probe DNA was prepared by boiling 50 ng of probe DNA for 10 minutes then quick cooling on ice before being added to the Ready-To-Go DNA labeling beads (Pharmacia 7/10328
  • the DNA from the RPA63 and RPA64 was digested with the restriction enzymes Hindlll and EcoRI and the blots containing these samples were hybridized to the RPA63 probe.
  • the RPA63 probe consists of the RPA63 ribozyme multimer coding region and should produce a single 1.3 kb hybridization product when hybridized to the RP ⁇ 63 or RPA64 materials.
  • the 1.3 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the ribozyme coding region and the nopaline synthase poly A 3' end.
  • RPA 122 is the 252 multimer ribozyme in pDAB 353 replacing the GUS reporter.
  • the RPA 122 probe consists of the RPA 122 ribozyme multimer coding region and the nopaline synthase 3' end and should produce a single 2.1 kb hybridization product when hybridized to die RPA85 or RPA113 materials.
  • the 2.1 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the bar gene, the ribozyme coding region and the nopaline synthase poly A 3' end.
  • the DNA from the RPA114 and RPA115 was digested with the restriction enzymes Hindlll and Smal and the blots containing these samples were hybridized to the RPA 115 probe.
  • the RPA 115 probe consist of the RPA115 ribozyme coding region and should produce a single 1.2 kb hybridization product when hybridized to the RPA114 or RPA115 materials.
  • the 1.2 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the ribozyme coding region and the nopaline synthase poly A 3' end.
  • the DNA from the RPA118 and RPA119 was digested with the restriction enzymes Hindlll and Smal and the blots containing these samples were hybridized to the RPA1 18 probe.
  • the RPA1 18 probe consist of the RPA118 ribozyme coding region and should produce a single 1.3 kb hybridization product when hybridized to the RPA1 18 or RPA119 materials.
  • the 1.3 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the ribozyme coding region and the nopaline synthase poly A 3' end.
  • PCR Polymerase Chain Reaction
  • PCR Polymerase Chain Reaction
  • RPA1 14/RPA1 15 258 ribozyme monomer
  • This primer is used to amplify across the Eco RV site in the 35S promoter. Primers were prepared using standard oligo synthesis protocols on an Applied Biosystems Model 394 DNA/RNA synthesizer.
  • RNA Extraction Buffer 50 mM Tris-HCl pH 8.0, 4% para-amino salicylic acid, 1% Tri-iso-propylnapthalenesulfonic acid, 10 mM dithiothreitol, and 10 M Sodium meta-bisulfite
  • RNA pellet was washed with 70% ethanol and dried under vacuum. RNA was resuspended in sterile H2O and stored at -80°C.
  • RT-PCR thermostable rTth DNA Polymerase
  • the PCR reaction was performed for 35 cycles using the following parameters; incubation at 96°C for I minute, dcnaiuraiion ai 94"C for 0 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 3 mins. ⁇ n aliquot of 0.2x vol. of each RT-PCR reaction was electrophoresed on a 2% 3: 1 Agarose (FMC) gel using standard TAE agarose gel conditions.
  • FMC Agarose
  • This primer covers the 10 base pair ribozyme arm and the first 6 bases of the ribozyme catalytic domain.
  • GBSS ribozyme expression in transgenic callus and plants was confirmed by RT-PCR.
  • GBSS multimer ribozyme expression in stably transformed callus was also determined by Ribonuc lease Protection Assay.
  • This primer spans the junction of the BAR gene and the RPA85/113 ribozyme.
  • This primer covers the 10 base pair ribozyme arm and the first 6 bases of the ribozyme catalytic domain.
  • RPA118/RPA1 19 453 ribozyme multimer
  • This primer covers die Adh I intron footprint upstream of the first ribozyme arm.
  • Expression of ⁇ 9 desaturase ribozymes in transgenic plant lines 85-06, 1 13-06 and 85-15 were confirmed by RT-PCR.
  • P ⁇ mers were prepared using standard oligo synthesis protocols on an Applied Biosystems Model 394 DNA/RNA synthesizer.
  • RNA was dried under vacuum, resuspended in loading buffer (20mM phosphate buffer pH 6.8, 5mM EDTA; 50% formamide: 16% formaldehyde: 10% glycerol) and denatured for 10 minutes at 65°C. Electrophoresis was at 50 volts through 1 % agarose gel in 20 mM phosphate buffer (pH 6.8) with buffer recirculation. BRL 0.24-9.5 Kb RNA ladder (Gibco/BRL, Gaithersburg, MD) were stained in gels with ethiduim bromide.
  • the probes were made using the Ambion Maxiscript kit and were typically 10 s cpm/ microgram or higher. The probes were made the same day they were used. They were gel purified, resuspended in RNase-freelOmM Tris (pH 8) and kept on ice. Probes were diluted to 5xl0 5 cpm/ul immediately before use. 5 ⁇ g of RNA derived from callus or 20 ⁇ g of RNA derived from protoplasts was incubated with 5 x 10 5 cpm of probe in 4M Guanidine Buffer.
  • RNA Running Buffer 95% Formamide/20mM EDTA/0.1% Bromophenol Blue/0.1% Xylene Cyanol ). The sample was heated to 95° C for 3 minutes, and loaded onto an 8% denaturing acrylamide gel. The gel was vacuum dried and exposed to a phosphorimager screens for 4 to 12 hours.
  • Part B Results demonstrating reductions in GBSS mRNA levels in nongenerable callus expressing both a GBSS and GBSS targeted ribozyme RNA.
  • the production of nonregenerable callus expressing RNAs for the GBSS target gene and an active multimer ribozyme targeted to GBSS mRNA was performed. Also produced were transgenics expressing GBSS and a ribozyme (-) control RNA. Total RNA was prepared from the transgenic lines. Northern analysis was performed on 7 ribozyme (-) control transformants and 8 active RPA63 lines. Probes for this analysis were a full length maize GBSS cDNA and a maize ⁇ 9 cDNA fragment.
  • GBSS mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions
  • level of GBSS mRNA was compared to the level of ⁇ 9 mRNA within that sample.
  • the level of full length GBSS transcript was compared between ribozyme expressing and ribozyme minus calli to identify lines with ribozyme mediated target RNA reductions. Blot to blot variation was controlled by performing duplicate analyses.
  • a range in GBSS/ ⁇ 9 ratio was observed between ribozyme (-) transgenics.
  • the target mRNA is produced by a transgene and may be subject to more variation in expression men the endogenous ⁇ 9 mRNA.
  • Active lines (RPA 63) AA, EE, KK, and JJ were shown to reduce the level of GBSS/ ⁇ 9 most significantly, as much as 10 fold as compared to ribozyme (-) control transgenics this is graphed in Figure 25. Those active lines were shown to be expressing GBSS targeted ⁇ bozyme by RT-PCR as desc ⁇ bed herein.
  • the probes for this analysis were cDNA fragments from a maize ⁇ 9 desaturase cDNA and a maize actin cDNA.
  • the level of ⁇ 9 mRNA was compared to the level of actin mRNA within that sample.
  • a ratio was calculated for each sample.
  • ⁇ 9/actin ratio values ranging from 0.55 to 0.88 were calculated for the 85-06 plants.
  • the average ⁇ 9/actm value for non- transformed controls was 2.7. There is an apparent 4 fold reduction in ⁇ 9/actm ratios between 85-06 and NT leaves.
  • Plants were produced which were transformed with inactive versions of the ⁇ 9 desaturase ribozyme genes. Data was presented demonstrating control levels of leaf stearate in the inactive ⁇ 9 ribozyme transgenic lines RPA 1 13-06 and 1 13- 17. Ribozyme expression and northern analysis was performed for the RP ⁇ 1 13-06 line. ⁇ 9 dcsaiurasc protein levels were dete ⁇ nined in plants of the RP ⁇ I 13- 17 line Kibo/ymc expression was measured as described herein. Plants 1 13-06-04, -07, and - 10 expressed detectable levels of RPA 1 13 inactive ⁇ 9 ribozyme.
  • the supernatant was assayed for total protein concentration by Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). One hundred micrograms of total protein was brought up to a final volume of 500 ⁇ l in Buffer A, added to 50 ⁇ l of mixed SP-sepharose beads (Pharmacia Biotech Inc., Piscataway, NJ), and resuspended by vortexing briefly. Proteins were allowed to bind to sepharose beads for 10 minutes while on ice.
  • the ⁇ 9 desaturase-sepharose material was centrifuged (10,000 x g) for 10 seconds, decanted, washed three times with Buffer A (500 ⁇ l), and washed one time with 200 mM sodium chloride (500 ⁇ l). Proteins were eluted by boiling in 50 ⁇ l of Treatment buffer (125 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol. and 10% 2-mercaptoethanol) for 5 mintues. Samples were centrifuged (10,000 x g) for 5 minutes. The supernatant was saved for Western anaylsis and the pellet consisting of sepharose beads was discarded.
  • Treatment buffer 125 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol. and 10% 2-mercaptoethanol
  • Part B Western analysis method which was used to demonstrate reductions in stearoyl- ACP ⁇ 9 desaturase.
  • Partially purified proteins were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels (10% PAGE) as described by Lacmmli, U. K. ( 1 70) Cleavage of structural proteins during assembly of the head of phage T4, Nature 227, 660-685.
  • SDS sodium dodecyl sulfate
  • Proteins were electrophoretically transferred to ECL 1M nitrocellulose membranes (Amersham Life Sciences, Arlington Heights, Illinois) using a Pharmacia Semi-Dry Blotter (Pharmacia Biotech Inc., Piscataway, NJ), using Towbin buffer (Towbin et al. 1979). The nonspecific binding sites were blocked with 10% dry milk in phosphate buffer saline for 1 h. Immunoreactive polypeptides were detected using the ECLTM Western Blotting Detection Reagent (Amersham Life Sciences, Arlington Heights, Illinois) with rabbit antiserum raised against E. coli expressed maize ⁇ 9 desaturase. The antibody was produced according to standard protocols by Berkeley Antibody Co.
  • the secondary antibody was goat antirabbit serum conjugated to horseradish peroxidase (BioRad). Autoradiograms were scanned with a densitometer and quantified based on the relative amount of purified E. coli ⁇ 9 desaturase. These experiments were duplicated and the mean reduction was recorded.
  • RPA85-15 contains an intact copy of the fused multimer gene. ⁇ 9 desaturase was partially purified from R0 maize leaves, using the protocol described herein. Western analysis was performed on ribozyme active (RPA85-15) and ribozyme inactive
  • Example 31 E. coli Expression and Purification of Maize ⁇ .-9 desaturase enzyme
  • the mature protein encoding portion of the maize ⁇ -9 desaturase cDNA was inserted into the bacterial T7 expression vector pET9D (Novagen Inc., Madison, WI).
  • the mature protein encoding region was deduced from the mature castor bean polypeptide sequence.
  • the alanine at position 32 (nts 239-241 of cDN ⁇ ) was designated as die first residue. This is found within the sequence Ala.Val.Ala.Ser.Met.Thr. Restriction endonuciease Nhe I site was engineered into the maize sequence by PCR, modifying GCCTCC to GCTAGC and a BamHl site was added at the 3' end. This does not change the amino acid sequence of the protein.
  • the cDNA sequence was cloned into pET9d vector using die Nhe I and Bam HI sites.
  • the recombinant plasmid is designated as pDAB428.
  • the maize ⁇ -9 desaturase protein expressed in bacteria has an additional methionine residue at the 5' end.
  • This pDAB428 plasmid was transformed into the bacterial strain BL21 (Novagen, Inc., Madison, WI) and plated on LB/kanamycin plates (25 mg/ml). Colonies were resuspended in 10 ml LB with kanamycin (25 mg/ml) and IPTG (ImM) and were grown in a shaker for 3 hours at 37°C.
  • the cells were harvested by centrifugation at lOOOxg at 4°C for 10 minutes.
  • the cells were lysed by freezing and thawing the cell pellet 2X, followed by the addition of 1 ml lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.1 % Triton XI 00, 100 ug/ml DNAse I, 100 ug ml RNAse A, and 1 mg/ml lysozyme).
  • the mixture was incubated for 15 minutes at 37°C and then centrifuged at 1000 Xg for 10 minutes at 4°C. The supernatant is used as the soluble protein fraction.
  • the supernatant adjusted to 25 mM sodium phosphate buffer (pH 6.0), was chilled on ice for 1 hr. Afterwards, d e resulting flocculant precipitant was removed by centrifugation. The ice incubation step was repeated twice more after which the solution remained clear.
  • the clarified solution was loaded onto a Mono S HR 10/10 column (Pharmacia) that had been equilibrated in 25 mM sodium phosphate buffer (pH 6.0). Basic proteins bound to the column matrix were eluted using a 0-500 mM NaCl gradient over 1 hr (2 ml min; 2 ml fractions).
  • the putative protein of interest was subjected to SDS-PAGE, blotted onto PVDF membrane, visualized with coomassie blue, excised, and sent to Harvard Microchem for amino-terminal sequence analysis. Comparison of the protein's amino terminal sequence to that encoded by the cDNA clone revealed that the protein was indeed ⁇ 9.
  • Spectrophotomet ⁇ c analysis of the duron-oxo component associated with the expressed protein (Fox et al., 1993 Proc. Natl. Acad. Sci USA. 90. 2486-2490), as well as identification using a specific ⁇ onheme iron stain (Lcong et al., 1992 Anal. Biochem. 207, 317-320) confirmed that the purified protein was ⁇ -9
  • Protein Precipitation ⁇ 9 was purified from com kernels following homogenization using a Warring blender in 25 mM sodium phosphate buffer (pH 7.0) containing 25 mM sodium bisulfite and a 2.5% polyvinylpolypyrrolidone. The crude homogenate was filtered through cheesecloth, centrifuged (10,000xg) for 0.25 h and the resulting supernatant was filtered once more through cheesecloth. In some cases, the supernatant was fractionated via saturated ammonium sulfate precipitation by precipitation at 20% v/v followed by 80% v/v.
  • Acyl Carrier Protein-Sepharose Chromatography AC? was purchased from Sigma Chemical Company and purified via precipitation at pH 4.1 (Rock and Cronan. 1981 J. Biol. Chem. 254, 71 16-7122) before linkage to the beads.
  • ACP-sepharose was prepared by covalently binding 100 mg of ACP to cyanogen bromide activated sepharose 4B beads, essentially as desc ⁇ bed by Pharmacia, Inc., in the package insert After linkage and blocking of the remaining sites with glycine, the ⁇ CP-scpharose material was packed into a HR 5/5 column (Pharmacia, Inc.) and equilibrated in 25 mM sodium phosphate buffer (pH 7.0).
  • the ⁇ -9 protein purified from com was determined to have a molecular size of approximately 38 kDa by SDS-PAGE analysis (Hames, 1981 in Gel Electrophoresis of Proteins: A Practical Approach , eds Hames BD and Rickwood, D. , IRL Press, Oxford).
  • Phenyl Sepharose Chromatography The fractions containing ⁇ 9 obtained from the ACP- Sepharose column were adjusted to 0.4 M ammonium sulfate (25 mM sodium phosphate, pH 7.0) and loaded onto a Pharmacia Phenyl Superose column (HR 10/10). Proteins were eluted by running a gradient (0.4 - 0.0 M ammonium sulfate) at 2 ml min for 1 hour. The ⁇ 9 protein typically eluted between 60- and 30 mM ammonium sulfate as detennmed by enzymatic and western analysis.
  • Example 32 Evidence for the Increase in Stearic Acid in Leaves as a Result of Transformation of Plants with ⁇ 9 Desaturase Ribozymes
  • the fatty acid methyl esters were removed from the reaction mixture by extraction with hexane.
  • One ml of hexane aid 1 ml of 0.9% (w/v) NaCl was added followed by vigorous shaking of the test tubes. After centrifugation of the tubes at 2000 ⁇ m for 5 minutes the top hexane layer was removed and used for fatty acid methyl ester analysis.
  • Gas chromatograph analysis was performed by injection of 1 ⁇ l of the sample on a Hewlett Packard (Wilmington, DE) Series II model 5890 gas chromatograph equipped with a flame lonization detector and a J&W Scientific (Folsom, CA) DB-23 column.
  • the oven temperature was 150°C throughout the run and the flow of the carrier gas (helium) was 80 cm/see The run time was 20 minutes
  • the conditions allowed for the separation of the 5 fatty acid methyl esters of interest C I 0, palmityl methyl ester; C18:0, stearyl methyl ester; C18.1 , oleoyl methyl ester; C I :2, linolcoyl methyl ester; and C18:3, linolenyl methyl ester.
  • Data collection and analysis was performed with a Hewlett Packard Se ⁇ es II Model 3396 integrator and a PE Nelson (Pcrkm Elmer, Norwalk, CT) data collection system.
  • the percentage of each fatty acid in the sample was taken directly from the readouts of the data collection system. Quantitative amounts of each fatty acid were calculated using the peak areas of a standard (Matreya, pleasant Gap, PA) which consisted of a known amount of the five fatty acid methyl esters. The amount calculated was used to estimate the percentage, of total fresh weight, represented by the five fatty acids in the sample. An adjustment was not made for loss of fatty acids during the extraction and esterification procedure. Recovery of the standard sample, after subjecting it to the extraction and esterification procedure (with no tissue present), ranged from 90 to 100% depending on the original amount of the sample. The presence of plant tissue in the extraction mixture had no effect on the recovery of the known amount of standard.
  • Part B Demonstration of an increase in steanc acid in leaves due to introduction of ⁇ 9 desaturase ribozymes.
  • Leaf tissue from individual plants was assayed for stearic acid as described in Part A.
  • a total of 428 plants were assayed from 35 lines transformed with active ⁇ 9 desaturase nbozymes (RPA85, RPA 1 14, RPA 1 18) and 406 plants from 31 lines transformed with ⁇ 9 desaturase inactive ribozymes (RPA1 13, RPA1 15, RPA1 19).
  • Table XI summarizes the results obtained for stearic acid levels in these plants. Seven percent of the plants from the active lines had stearic acid levels greater than 3%, and 2% had levels greater than 5%.
  • Part B Results demonstrating reductions m ⁇ 9 desaturase levels in next generation (RI ) maize leaves expressing ribozymes targeted to ⁇ 9 desaturase mRNA
  • RI next generation
  • ⁇ 9 desaturase was partially purified from RI maize leaves, using the protocol described herein. Western analysis was performed on several of the high stearate plants. In leaves of next generation plants, a 40-50% reduction of ⁇ 9 desaturase was observed in those plants that had high stearate content ( Figure 36). The reduction was comparable to RO maize leaves. This reduction was observed in either OQ414 plants crossed with RP ⁇ 85- I 5 pollen or RPA85-15 plants crossed with self or siblings. Therefore, this suggests that the gene encoding the ribozyme is heritable.
  • Somatic embryos make up a large part of this embryogenic callus.
  • the somatic embryos continued to form in callus because e callus was transferred every two weeks.
  • the somatic embryos in embryogenic callus continued to proliferate but usually remained in an early stage of embryo development because of the 2,4-D in the culture medium.
  • the somatic embryos regenerated into plantlets because the callus was subjected to a regeneration procedure described herein. During regeneration the somatic embryo formed a root and a shoot, and ceases development as an embryo.
  • Somatic embryos were made to develop as seed embryos, i.e., beyond the early stage of development found in embryogenic callus and no regeneration, by a specific medium treatment.
  • This medium treatment involved transfer of the embryogenic callus to a Murashige and Skoog medium (MS; described by Murashige and Skoog in 1962) which contains 6% (w/v) sucrose and no plant hormones.
  • MS Murashige and Skoog medium
  • ABA abscisic acid
  • the somatic embryos were assayed for fatty acid composition as described herein after 3 to 7 days of growth on the ABA medium.
  • the fatty acid composition of somatic embryos grown on the above media was compared to the fatty acid composition of embryogenic callus and maize zygotic embryos 12 days after pollination (Table XIII).
  • the fatty acid composition of the somatic embryos was different than that of the embryogenic callus.
  • the embryogenic callus had a higher percentage of C16.0 and C18:3, and a lower percentage of Cl 8: 1 and Cl 8:2.
  • the percentage of lipid represented by the fresh weight was different for the embryogenic callus when compared to the somatic embryos; 0.4% versus 4.0%.
  • the fatty acid composition of the zygotic embryos and somatic embryos were very similar and their percentage of lipid represented by the fresh weight were nearly identical. It was 97/10328
  • somatic embryo culture system desc ⁇ bed above would be an useful in vitro system for testing the effect of certain genes on lipid synthesis in developing embryos of maize.
  • Somatic embryos were produced using th method described herein from embryogenic callus transformed with pD ⁇ B308/pD ⁇ B430
  • the somatic embryos from 16 different lines were assayed for fatty acid composition.
  • the stearic acid content of somatic embryos from these two lines is compared to the stearic acid content of somatic embryos from their control lines in Figures 37 and 38.
  • the control lines were from the same culture that the transformed lines came from except that they were not transformed.
  • stearic acid in somatic embryos ranged from 1 to 23% while the controls ranged from 0.5 to 3%.
  • steanc acid in somatic embryos ranged from 2 to 15% while the controls ranged from 0.5 to 3%. More than 50% of the somatic embryos had stearic acid levels which were above the range of the controls m both the transformed lines. The above results indicate that an antisense- ⁇ 9 desaturase gene can be used to raise the stearic acid levels in somatic embryos of maize.
  • Part C Demonstration of an increase in stearic acid in leaves due to introduction of an antisense- ⁇ 9 desaturase gene.
  • Embryogenic cultures from lines 308/430-12 and 308/430- 15 were used to regenerate plants. Leaves from these plants were analyzed for fatty acid composition using the method previously described. Only 4 plants were obtained from the 308/430-15 culture and the stearic acid level in the leaves of these plants were normal, 1-2%).
  • the stearic acid levels in leaves from plants of line 308/430-12 are shown in Figure 39. The stearic acid levels in leaves ranged from 1 to 13% in plants from line 308/430-12.
  • antisense is meant a non-enzymatic nucleic acid molecule that binds to a RNA (target RNA) by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004).
  • target RNA RNA
  • RNA-RNA or RNA-DNA or RNA-PNA protein nucleic acid
  • amylose content was assayed by the method of Hovenkamp-Hermelink et al.
  • the matenal was then centnfuged at 5000 g for 2 mm and the supernatant was discarded The pellet was washed three times by resuspending in water and removing supernatant by centrifugation After washing, die starch was filtrated through 20 ⁇ m nylon membrane and centnfuged. Pellet was then lyophilized and stored in - 20 °C until used for activity assay.
  • a standard GBSS reaction mixture contained 0.2 M T ⁇ cine, pH 8.5, 25 mM Glutathione, 5 mM EDTA, 1 mM ⁇ 4 C ADPG (6 nci/ ⁇ mol), and 10 mg starch in a total volume of 200 ⁇ l. Reactions were conducted at 37 °C for 5 min and terminated by adding 200 ⁇ l of 70% ethanol (v/v) m 0.1 M KCI.
  • CQ806 Twenty five individual kernels of CQ806, a conventional maize inbred line, were analyzed. The amylose content of CQ806 ranged from 24.4% to 32.2%, averaging 29.1%). The single kernel distribution of amylose content is skewed slightly towards lower amylose contents. Forty nine single kernels of 308/425-12.2.1.1 were analyzed. Given that 308/425-12.2.1.1 resulted from self pollination of a hemizygous individual, the expected distribution would consist of 4 distinct genetic classes present in equal frequencies since endosperm is a triploid tissue. The 4 genetic classes consist of individuals carrying 0, 1, 2, and 3 copies of the antisense construct.
  • the dist ⁇ bution of amylose contents would be tetramodal.
  • One of the modes of the resulting distribution should be indistinguishable from the non-transgenic parent. If there is no dosage effect for the transgene (individuals carrying 1, 2 or 3 copies of the transgene are phenotypically equivalent), then the distribution should be bimodal with one of the modes identical to the parent The number of individuals included in the modes should be 3:1 of transgenieparental The distribution for 308/425-12.2.1.1 is distinctly trimodal. The central mode is approximately twice the size of either other mode. The two distal modes are of approximately equal size. Goodness of fit to a 1 :2: 1 ratio was tested and the fit was excellent. 97/10328
  • the central mode includes two generic classes: individuals with 1 or 2 copies of the antisense construct.
  • the mode with the lowest amylose content thus represents those individuals which are fully homozygous (3 copies) for the antisense construct.
  • the 2: 1 ratio was tested and could not be rejected on the basis of the data.
  • Example 37 The same two-step screening strategy as in the antisense study (Example 37) was used to analyze ribozyme-GBSS plants. 160 lines representing 1 1 transformation events were examined in the pooled starch level. Among the control lines (both untransformed line and Southem negative line), the amylose content varied from 28% to 19%. No significant reduction was observed among all lines carrying ribozyme gene (Southem positive line). More than 20 selected lines were further analyzed in the single kernel level, no significant amylose reduction as well as segregation pattern were found. It was apparent that ribozyme did not cause any alternation in the phenotypic level.
  • Transformed lines were further examined by their GBSS activity (as described in Example 36). For each line, 30 kernels were taken from the frozen ear and starch was purified. Table XIV shows the results of 9 plants representing one transformation event of the GBSS activity in the pooled starch samples, amylose content in the pooled starch samples, and Southem analysis results. Three southem negative lines: RPA63.0283, RPA63.0236, and RPA63.0219 were used as control. The GBSS activities of control lines RPA63.0283, RPA63.0236, and RPA63.0219 were around 300 units/mg starch.
  • GBSS activities at the single kernel level of line RP ⁇ 63.021 was further examined, using RP ⁇ 63.0306 (Southern negative and GBSS activity normal in pooled starch) as control. About 30 kernels from each line were taken, and starch samples were purified from each kernel individually. Figure 41 clearly indicated reduced GBSS activity in line RPA63.0218 compared to RPA63.0306.
  • the small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" b-gaiactosidase message by the ligation of new b-galactosidase sequences onto the defective message ["].
  • RNAse P RNA M1 RNA
  • RNA portion of a ubiquitous nbonucleoprotein enzyme • RNA portion of a ubiquitous nbonucleoprotein enzyme.
  • Reaction mechanism possible anack by M* * -OH to generate cleavage products with 3'-OH and 5 " -phosphate.
  • RNAse P is found throughout the prokaryotes and eukaryotes.
  • the RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.
  • Reaction mechanism attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2 ⁇ 3'-cyclic phosphate and 5'-OH ends.
  • Reaction mechanism attack by 2 -OH 5' to the scissile bond to generate cleavage products with 2', 3 '-cyclic phosphate and 5' -OH ends.
  • RNA RNA as the infectious agent.
  • Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [ 3I ]
  • HDV Hepatitis Delta Virus
  • Circular form of HDV is active and shows increased nuclease stability [ 37 ]
  • a group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge. Mass.) (1995), 83(4). 529-38.
  • X represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252).
  • the length of stem II may be ⁇ 2 base-pairs.
  • X represents stem II region of a HH ribozyme (Hertel et al.. 1992 Nucleic Acids Res. 20 3252).
  • the length of stem II may be ⁇ 2 base-pairs.

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Abstract

An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a gene in a plant. A transgenic plant comprising nucleic acids encoding for an enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a gene in said plant.

Description

97/10328
1
DESCRIPTION
COMPOSITIONS AND METHOD FOR MODULATION OF GENE EXPRESSION TN PLANTS
This application is a continuation-in-part of I ) a Non-Provisional application by Edington, entitled "Method for the production of transgenic plants deficient in starch granule bound glucose starch glycosyl transfcrasc activity" filed on September 2, 1 94 as U.S.S.N 08/300,726; and 2) a Provisional application by Zwick et al , entitled "Composition and method for modification of fatty acid saturation profile m plants" filed on July 13, 1995, as U.S.S.N 60/001,135. Both of these applications in their entirety, including drawings, are hereby incoφorated by reference herein
Background of the Invention
The present invention concerns compositions and methods for the modulation of gene expression in plants, specifically using enzymatic nucleic acid molecules
The following is a brief descπption of regulation of gene expression in plants. The discussion is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention.
There are a variety of strategies for modulating gene expression in plants. Traditionally, antisense RNA (reviewed in Bourque, 1995 Plant Sci 105, 125-149) and co- suppression (reviewed in Jorgensen, 1995 Science 268, 686-691) approaches have been used to modulate gene expression. Insertion mutagenesis of genes have also been used to silence gene expression. This approach, however, cannot be designed to specifically inactivate the gene of interest. Applicant believes that πbozyme technology offers an attractive new means to alter gene expression in plants.
Naturally occurring antisense RNA was first discovered in bacteria over a decade ago (Simons and Kleckner, 1983 Cell 34, 683-691). It is thought to be one way in which bacteria can regulate their gene expression (Green et al., 1986 Ann Rev Biochem 55: 567- 597; Simons 1988 Gene 72: 35-44) The first demonstration of antisense-mediated inhibition of gene expression was repoπed m mammalian cells (Izant and Wemtraub 1984 Cell 36: 1007-1015). There are many examples m the literature for the use of antisense RNA to modulate gene expression in plants. Following are a few examples Shewmaker et al , U.S. Patent Nos. 5, 107.065 and 5. 453.566 disclose methods for regulating gene expression in plants using antisense RNA
It has been shown that an antisense gene expressed in plants can act as a dominant suppressor gene Transgenic potato plants have been produced which express RNΛ antisense to potato or cassava granule bound starch syntha.sc (GBSS) In both of these cases, transgenic plants have been constructed which have reduced oi no GBSS activity or protein. These transgenic plants give rise to potatoes containing starch with dramatically reduced amylose levels (Visser et al. 1991 , Mol. Gen Genet. 225. 2889-296, Salehuzzaman et al. 1993, Plant Mol. Biol. 23: 947-962)
Kull et ai, 1995, J. Genet. & Breed. 49, 69-76 reported inhibition of amylose biosynthesis in tubers from transgenic potato lines mediated by the expression of antisense sequences of the gene for granule-bound starch synthase (GBSS). The authors, however, indicated a failure to see any in vivo activity of πbozymes targeted against the GBSS RNA.
Antisense RNA constructs targeted against Δ-9 desaturase enzyme in canola have been shown to increase the level of stearic acid (C18:0) from 2% to 40% (Knutzon et. al., 1992 Proc. Natl. Acad. Sci. 89, 2624). There was no decrease in total oil content or germinanon efficiency in one of the high stearate lines. Several recent reviews are available which illustrate the utility of plants with modified oil composition (Ohlrogge, J. B. 1994 Plant Physiol. 104, 821; Kmney, A. J. 1994 Cwrr. Opin. Cell Biol. 5, 144; Gibson et al. 1994 Plant Cell Envir. 17, 627).
Homologous transgene inactivation was first documented in plants as an unexpected result of inserting a transgene in the sense orientation and finding that both the gene and the transgene were down-regulated (Napoli et al., 1990 Plant Cell 2: 279-289). There appears to be at least two mechanisms for inactivation of homologous genetic sequences. One appears to be transcπptional inactivation via methylation, where duplicated DNA regions signal endogenous mechanisms for gene silencing. This approach of gene modulation involves either the introduction of multiple copies of transgenes or transformation of plants with transgenes with homology to the gene of interest (Ronchi et al 1995 EMBO J. 14: 5318-5328) The other mechanism of co-suppression is post- transcπptional, where the combined levels of expression from both the gene and the transgene is thought to produce high levels of transcript which triggers threshold-induced ~ „„« n,,o PCT US96/11689 O 97/10328
3 degradation of both messages (van Bokland et al , 1994 Plant J 6 861 -877) The exact molecular basis for co-suppression is unknown
Unfortunately, both antisense and co-suppression technologies are subject to problems in heπtability of the desired trait (Fmnegan and McElroy 1994 Bin/Technology 12: 883-888). Currently, there is no easy way to specifically inactivate a gene of interest at the DNA level in plants (Pazkowski et al , 1988 EMBO J 7 4021 -4026) 'I ransposon mutagenesis is inefficient and not a stable event, while chemical mutagenesis is highly non-specific
Applicant believes that πbozymes present an attractive alternative and because of their catalytic mechanism of action, have advantages over competing technologies. However, there have been difficulties in demonstrating the effectiveness of πbozymes m modulating gene expression in plant systems ( Mazzolmi et al., 1992 Plant Mol Biol. 20- 715-731; Kull et al., 1995 J Genet. & Breed 49: 69-76) Although there are reports in the literature of ribozyme activity in plants cells, almost all of them involve down regulation of exogenously introduced genes, such as reporter genes in transient assays (Stemecke et al., 1992 EMBO J. 11:1525-1530; Pemman et al., 1993 Antisense Res. Dev 3: 253-263; Pemman et al, 1995, Proc. Natl. Acad. Sci. USA, 92, 6165).
There are also several publications, [e.g., Lamb and Hay, 1990, J. Gen. Virol. 71 , 2257-2264; Gerlach et al, International PCT Publication No. WO 91/13994; Xu et al, 1992, Science in China (Ser. B) 35, 1434-1443; Edington and Nelson, 1992, in Gene Regulation: Biology of antisense RNA and DNA, eds. R. P. Enckson and J. G Izant, pp 209-221, Raven Press, NY.; Atkins et al, International PCT Publication No WO 94/00012; Lenee et al, International PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins et al, 1995, J. Gen. Virol 76, 1781-1790; Gruber et al, 1994, J Cell Biochem. Suppl. 18A, 110 (XI -406) and Feyter et al, 1996, Mol Gen. Genet. 250, 329- 338], that propose using hammerhead πbozymes to modulate: virus replication, expression of viral genes and/or reporter genes. None of these publications report the use of ribozymes to modulate the expression of plant genes.
Mazzolini et al, 1992, Plant. Mol. Bio. 20, 715-731 , Stemecke et al, 1992, EMBO J. 11, 1525-1530; Pemman et al, 1995, Proc. Natl. Aead. Sci USA., 92, 6175-6179, Wegener et al, 1994, Mol. Gen. Genet. 245, 465-470; and Stemecke et al , 1994, Gene, 149, 47-54, descπbe the use of hammerhead ribozymes to inhibit expression of reporter genes in plant cells. Bennett and Cullimore, 1992 Nucleic Acids Res. 20, 831-837 demonstrate hammerhead ribozyme-mediated in vitro cleavage of glna, glnb, glng and glnd RNA, coding for glutamine synthetase enzyme in Phaseolus vulgar is.
Hitz et al, (WO 91/18985) describe a method for using the soybean Δ-9 dcsat-irasc enzyme to modify plant oil composition. The application describes the use of soybean Δ-9 desaturase sequence to isolate Δ-9 desaturasc genes from other species.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the use of ribozymes in maize. Furthermore, Applicant believes that the references do not disclose and/or enable the use of ribozymes to down regulate genes in plant ceils, let alone plants.
Summary Of The Invention
The invention features modulation of gene expression in plants specifically using enzymatic nucleic acid molecules. Preferably, the gene is an endogenous gene. The enzymatic nucleic acid molecule with RNA cleaving activity may be in the form of, but not limited to, a hammerhead, haiφin, hepatitis delta virus, group I intron, group II intron, RNaseP RNA, Neurospora VS RNA and the like. The enzymatic nucleic acid molecule with RNA cleaving activity may be encoded as a monomer or a multimcr, preferably a multimer. The nucleic acids encoding for the enzymatic nucleic acid molecule with RNA cleaving activity may be operably linked to an open reading frame. Gene expression in any plant species may be modified by transformation of the plant with the nucleic acid encoding the enzymatic nucleic acid molecules with RNA cleaving activity. There are also numerous technologies for transforming a plant: such technologies include but are not limited to transformation with Agrobacterium, bombarding with DNA coated microprojectiles, whiskers, or electroporation. Any target gene may be modified with the nucleic acids encoding the enzymatic nucleic acid molecules with RNA cleaving activity. Two targets which are exemplified herein are delta 9 desaturase and granule bound starch synthase (GBSS).
Until the discovery of the inventions herein, nucleic acid-based reagents, such as enzymatic nucleic acids (ribozymes), had yet to be demonstrated to modulate and or inhibit gene expression in plants such as monocot plants (e.g., corn). Ribozymes can be used to modulate a specific trait of a plant cell, for example, by modulating the activity of an enzyme involved in a biochemical pathway. It may be desirable, in some instances, to „,,„„,,.> PCT US96/11689 97/10328
5 decrease the level of expression of a particular gene, rather than shutting down expression completely: ribozymes can be used to achieve this Enzymatic nucleic acid-based techniques were developed herein to allow directed modulation of gene expression to generate plant cells, plant tissues or plants with altered phenotype.
Ribozymes (i.e., enzymatic nucleic acids) arc nucleic acid molecules having an enzymatic activity which is able to repeatedly cleave other separate RNΛ molecules in a nucleotide base sequence-specific manner. Such enzymatic RNΛ molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro and in vivo (Zaug et αl, 1986, Nature 324, 429; Kim et al., 1987, Proc. Natl Acad. Set USA 84, 8788; Dreyfus, 1988, Einstein Quarterly J. Bio Med, 6, 92; Hascloff and Gerlach, 1988, Nature 334 585; Cech, 1988, JAMA 260, 3030; Murphy and Cech, 1989, Proc. Natl. Acad. Sci. USA., 86, 9218; Jeffeπes et al., 1989, Nucleic Acids Research 17, 1371).
Because of their sequence-specificity, trα/w-cleavmg ribozymes may be used as efficient tools to modulate gene expression in a variety of organisms including plants, animals and humans (Bennett et al, supra; Edington et al, supra; Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RΝA targets within the background of cellular RΝA. Such a cleavage event renders the mRΝA non-functional and abrogates protein expression from that RΝA. In this manner, synthesis of a protein associated with a particular phenotype and/or disease state can be selectively inhibited.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Brief Description of the Figures
Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be > 2 base-pairs long. Each Ν is any nucleotide and each • represents a base pair.
Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known the art; Figure 2b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; Figure 2c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and Figure 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids Res., 17, 1371-1371) into two portions.
Figure 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is I , 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 - 20 bases, i. ., m is from 1 - 20 or more). Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is > 1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any noπnal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base-pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e.. o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotidc with or without modifications to its base, sugar or phosphate, "q" is > 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases. " " refers to a covalent bond.
Figure 4 is a representation of the general structure of the hepatitis Δ virus ribozyme domain known in the art.
Figure 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.
Figure 6 is a schematic representation of an RNaseH accessibility assay. Specifically, the left side of Figure 6 is a diagram of complementary DNA oligonucleotides bound to accessible sites on the target RNA. Complementary DNA oligonucleotides are represented by broad lines labeled A, B, and C. Target RNA is represented by the thin, twisted line. The right side of Figure 6 is a schematic of a gel separation of uncut target RNA from a cleaved target RNA. Detection of target RNA is by autoradiography of body-labeled, T7 transcript. The bands common to each lane 97/10328
represent uncleaved target RNA; the bands unique to each lane represent the cleaved • products.
Figure 7 is a graphical representation of RNaseH accessibility of GBSS RNA
Figure 8 is a graphical representation of GBSS RNΛ cleavage by nbo/ymes at different temperatures.
Figure 9 is a graphical representation of GBSS RNA cleavage by multiple ribozymes.
Figure 10 lists the nucleotide sequence of Δ-9 desaturase cDNΛ isolated from 7.eu mays.
Figures 1 1 and 12 are diagrammanc representations of fatty acid biosynthesis in plants. Figure 1 1 has been adapted from Gibson et al, 1994, Plant Cell Envir. 17, 627.
Figures 13 and 14 are graphical representations of RNaseH accessibility of Δ-9 desaturase RNA.
Figure 15 shows cleavage of Δ-9 desaturase RNA by ribozymes in vitro. 10/10 represents the length of the binding arms of a hammerhead (HH) ribozyme. 10/10 means helix 1 and helix 3 each form 10 base-pairs with the target RNA (Fig. 1). 4/6 and 6/6, represent helix2/helixl interaction between a haiφin ribozyme and its target. 4/6 means the haiφin (HP) ribozyme forms four base-paired helix 2 and a six base-paired helix 1 complex with the target (see Fig. 3). 6/6 means, the haiφin ribozyme forms a 6 base- paired helix 2 and a six base-paired helix 1 complex with the target. The cleavage reactions were earned out for 120 mm at 26°C.
Figure 16 shows the effect of arm-length variation on the activity of HH and HP ribozymes in vitro. Ill, 10/10 and 12/12 are essentially as described above for the HH ribozyme. 6/6, 6/8, 6/12 represents varying helix 1 length and a constant (6 bp) helix 2 for a haiφin ribozyme. The cleavage reactions were earned out essentially as descπbed above.
Figures 17, 18, 19 and 23 are diagrammatic representations of non-limiting strategies to construct a transcript comprising multiple ribozyme motifs that are the same or different, targeting various sites within Δ-9 desaturase RNA. Figures 20 and 21 show in vitro cleavage of Δ-9 desaturase RNA by ribozymes that are transcribed from DNA templates using bacteπophage T7 RNA polymerase enzyme
Figure 22 diagrammatic representation of a non-limiting strategy to construct a transcript compnsing multiple ribozyme motifs that arc the same or different targeting various sites within GBSS RNA
Figure 24 shows cleavage of Δ-9 desaturase RNΛ by ribozymes 453 Multimer, represents a multimer ribozyme construct targeted against hammerhead πbozyme sites 453, 464, 475 and 484. 252 Multimer, represents a multimer ribozyme construct targeted against hammerhead ribozyme sites 252, 271 , 313 and 326 238 Multimer, represents a multimer ribozyme construct targeted against three hammerhead πbozyme sites 252, 259 and 271 and one haiφin ribozyme site 238 (HP). 259 Multimer, represents a multimer πbozyme construct targeted against two hammerhead ribozyme sites 271 and 313 and one haiφin πbozyme site 259 (HP)
Figure 25 illustrates GBSS mRNA levels in Ribozyme minus Controls (C, F, I, J, N, P, Q) and Active Ribozyme RPA63 Transformants (AA, DD, EE, FF, GG, HH, JJ, KK).
Figure 26 illustrates Δ9 desaturase mRNA levels in Non-transformed plants (NT), 85-06 High Stearate Plants (1, 3, 5, 8, 12, 14), and Transformed (irrelevant ribozyme) Control Plants (RPA63-33, RPA63-51, RPA63-65).
Figure 27 illustrates Δ9 desaturase mRNA levels in Non-transformed plants
(NTO), 85-15 High Stearate Plants (01, 06, 07, 10, 11, 12), and 85-15 Normal Stearate Plants (02, 05, 09, 14).
Figure 28 illustrates Δ9 desaturase mRNA levels in Non-transformed plants (NTY), 113-06 Inactive Ribozyme Plants (02, 04, 07, 10,11).
Figures 29a and 29b illustrate Δ9 desaturase protein levels in maize leaves (R0) (a)
Line Hill, plants a-e nontransformed and ribozyme inactive line RPA1 13-17, plants 1 -6 (b) Ribozyme active line RPA85-I5, plants 1- 15
Figure 30 illustrates steaπc acid in leaves of RPA85-06 plants
Figure 31 illustrates steaπc acid in leaves of RPA85-15 plants, results of three assays. Figure 32 illustrates stearic acid in leaves of RPA 1 13-06 plants.
Figure 33 illustrates stearic acid in leaves of RPA1 13-17 plants.
Figure 34 illustrates stearic acid in leaves of control plants.
Figure 35 illustrates leaf stearate in RI plants from a high stearate plant cross (RPA85- 15.07 self).
Figure 36 illustrates Δ9 desaturase levels in next generation maize leaves (RI ). * indicates those plants that showed a high stearate content.
Figure 37 illustrates stearic acid in individual somatic embryos from a culture (308/430-012) transformed with antisense Δ9 desaturase.
Figure 38 illustrates stearic acid in individual somatic embryos from a culture
(308/430-015) transformed with antisense Δ9 desaturase.
Figure 39 illustrates stearic acid in individual leaves from plants regenerated from a culture (308/430-012) transformed with antisense Δ9 desaturase.
Figure 40 illustrates amylose content in a single kernel of untransformed control line (Q806 and antisense line 308/425- 12.2.1.
Figure 41 illustrates GBSS activity in single kernels of a southern negative line (RPA63-0306) and Southern positive line RPA63-0218.
Figure 42 illustrates a transformation vector that can be used to express the enzymatic nucleic acid of the present invention.
Detailed Description Of The Invention
The present invention concerns compositions and methods for the modulation of gene expression in plants specifically using enzymatic nucleic acid molecules.
The following phrases and terms are defined below:
By "inhibit" or "modulate" is meant that the activity of enzymes such as GBSS and Δ-9 desaturase or level of mRNAs encoded by these genes is reduced below that observed in the absence of an enzymatic nucleic acid and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.
By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave that target That is, the enzymatic nucleic acid molecule is able to mtcrmolccularly cleave RNΛ (or DNΛ) and thereby inactivate a target RNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA to allow the cleavage to occur One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, RNAzyme, polyπbozymes, molecular scissors, self-splicing RNA, self-cleaving RNA, cis-cleaving RNA, autolytic RNA, endoribonuclease, minizyme, leadzyme or DNA enzyme. All of these terminologies descπbe nucleic acid molecules with enzymatic activity. The term encompasses enzymatic RNA molecule which include one or more ribonucleotides and may include a majority of other types of nucleotides or abasic moieties, as descπbed below.
By "complementaπty" is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequences by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver and/or express a desired nucleic acid.
By "gene" is meant a nucleic acid that encodes an RNA.
By "plant gene" is meant a gene encoded by a plant.
By "endogenous" gene is meant a gene normally found in a plant cell in its natural location in the genome.
By "foreign" or "heterologous" gene is meant a gene not normally found in the host plant cell, but that is introduced by standard gene transfer techniques. By "nucleic acid" is meant a molecule which can be single-stranded or double- stranded, composed of nucleotides containing a sugar, a phosphate and either a purine or pyrimidine base which may be same or different, and may be modified or unmodified.
By "genome" is meant genetic material contained in each cell of an organism and/or a virus.
By "mRNA" is meant RNA that can be translated into protein by a cell.
By "cDNA" is meant DNΛ that is complementary to and derived from a mRNA.
By "dsDNA" is meant a double stranded cDNA.
By "sense" RNA is meant RNΛ transcript that comprises the mRNA sequence.
By "antisense RNA" is meant an RNA transcript that comprises sequences complementary to all or part of a target RNA and/or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript and/or mRNA. The complementarity may exist with any part of the target RNA, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. Antisense RNA is normally a mirror image of the sense RNA.
By "expression", as used herein, is meant the transcription and stable accumulation of the enzymatic nucleic acid molecules, mRNA and/or the antisense RNA inside a plant cell. Expression of genes involves transcription of the gene and translation of the mRNA into precursor or mature proteins.
By "cosuppression" is meant the expression of a foreign gene, which has substantial homology to an gene, and in a plant cell causes the reduction in activity of the foreign and/or the endogenous protein product.
By "altered levels" is meant the level of production of a gene product in a transgenic organism is different from that of a normal or non-transgenic organism.
By "promoter" is meant nucleotide sequence element within a gene which controls the expression of that gene. Promoter sequence provides the recognition for RNA polymerase and other transcription factors required for efficient transcription. Promoters from a variety of sources can be used efficiently in plant cells to express ribozymes. For example, promoters of bacterial origin, such as the octopine synthetase promoter, the „,„„,,«- 97/10328 1 2
nopaline synthase promoter, the manopme synthetase promoter, promoters of viral oπgin, such as the cauliflower mosaic virus (35S); plant promoters, such as the πbulose- 1,6-biphosphate (RUBP) carboxylase small subunit (ssu), the beta-conglycinin promoter, the phaseolm promoter, the ADH promoter, heat-shock promoters, and tissue specific promoters. Promoter may also contain certain enhancer sequence elements that may improve the transcription efficiency.
By "enhancer" is meant nucleotide sequence element which can stimulate promoter activity (Adh).
By "constitutive promoter" is meant promoter element that directs continuous gene expression in all cells types and at all times (actin, ubiquitin, CaMV 35S).
By "tissue-specific" promoter is meant promoter element responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (zein, oleosin, napm, ACP).
By "development-specific" promoter is meant promoter element responsible for gene expression at specific plant developmental stage, such as in early or late embryogenesis.
By "inducible promoter" is meant promoter element which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; and stress.
By a "plant" is meant a photosynthetic organism, either eukaryotic and prokaryotic.
By "angiosperm" is meant a plant having its seed enclosed in an ovary (e.g., coffee, tobacco, bean, pea).
By "gymnosperm" is meant a plant having its seed exposed and not enclosed in an ovary (e.g., pine, spruce).
By "monocotyledon" is meant a plant characterized by the presence of only one seed leaf (primary leaf of the embryo). For example, maize, wheat, rice and others.
By "dicotyledon" is meant a plant producing seeds with two cotyledons (primary leaf of the embryo). For example, coffee, canola, peas and others. -.„„,,„ PCT/US96/11689 97/10328
By "transgenic plant" is meant a plant expressing a foreign gene.
By "open reading frame" is meant a nucleotide sequence, without introns, encoding an ammo acid sequence, with a defined translation initiation and termination region.
The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNΛ of a desired target. The cn/.ymaiic nucleic acid molecule may be targeted to a highly specific sequence region of a target such that specific gene inhibition can be achieved. Alternatively, enzymatic nucleic acid can be targeted to a highly conserved region of a gene family to inhibit gene expression of a family of related enzymes. The ribozymes can be expressed in plants that have been transformed with vectors which express the nucleic acid of the present invention.
The enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNΛ target, it is released from that RNA to search for another target and can repeatedly .bind and cleave new targets.
In one of the preferred embodiments of the inventions herein, the enzymatic nucleic acid molecule is formed in a hammerhead or haiφin motif, but may also be formed in the 97/10328
1 4 motif of a hepatitis Δ virus, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al , 1992, AIDS Research and Human Retroviruses 8, 183, of haiφin motifs by Hampel et al, EP0360257, Hampel and Tπtz, 1989 Biochemistry 28, 4929, Feldstem <?t al , 1 89, Gene 82, 53, Hascloff and Gerlach, 1989, Gene, 82, 43, and Hampel el al , 1990 Nucleic Λt ul Res I X, 299. of i c hepatitis Δ virus motif is described by Perrotta and Been, 1992 Biochemistry 31 , 1 ; ol the RNaseP motif by Guemer-Takada et al, 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res 24, 835, Neurospora VS RNA ribozyme motif is descπbed by Collins (Saville and Collins, 1990 Cell 61 , 6X5-696, Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830, Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II introns are described by Griffin et al, 1995, Chem. Biol. 2, 761 ; Michels and Pyle, 1995, Biochemistry 34, 2965; and of the Group I intron by Cech et al, U.S. Patent 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
The enzymatic nucleic acid molecules of the instant invention will be expressed within cells from eukaryotic promoters [e.g., Gerlach et al. International PCT Publication No. WO 91/13994; Edington and Nelson, 1992, in Gene Regulation: Biology of Antisense RNA and DNA, eds. R. P. Erickson and J. G. Izant, pp 209-221, Raven Press, NY.; Atkins et al, International PCT Publication No. WO 94/00012; Lenee et al, International PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins et al, 1 95, J. Gen. Virol. 76, 1781-1790; McElroy and Brettell, 1994, T1BTECH 12, 62; Gruber et al, 1994, J. Cell Biochem. Suppl. 18A, 1 10 (Xl-406)and Feyter et al, 1996, Mol. Gen. Genet. 250, 329- 338; all of these are incoφorated by reference herein]. Those skilled in the art will realize from the teachings herein that any ribozyme can be expressed in eukaryotic plant cells from an appropriate promoter. The ribozymes expression is under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter.
To obtain the ribozyme mediated modulation, the πbozyme RNA is introduced into the plant. Although examples are provided below for the construction of the plasmids used in the transformation expeπments illustrated herein, it is well within the skill of an 97/10328
aπisan to design numerous different types of plasmids which can be used in the transformation of plants._see Bevan, 1984 , Nucl Acids Res. 12, 871 1 -8721 , which is incoφorated by reference. There are also numerous ways to transform plants. In the examples below embryogenic maize cultures were helium blasted. In addition to using the gene gun (US Patents 4,945,050 to Cornell and 5, 141 , 131 to DowElanco), plants may be transformed using Agrobacterium technology, sec US Patent 5, 177.010 to University of Toledo, 5,104,310 to Texas A&M, European Patent Application 0I 3 I 624B I , European Patent Applications 120516, 159418B1 and 176,1 12 to Schilperoot, US Patents 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 1 16718, 290799, 320500 all to MaxPlanck, European Patent Applications 604662 and 627752 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and US Patent 5,231,019 all to Ciba Geigy, US Patents 5,463,174 and 4,762,785 both to Calgene, and US Patents 5,004,863 and 5,159,135 both to Agracetus; whiskers technology, see US Patents 5,302,523 and 5,464,765 both to Zeneca; electroporation technology, see WO 87/06614 to Boyce Thompson Institute, 5,472,869 and 5,384,253 both to Dekalb, WO9209696 and WO9321335 both to PGS; all of which are incoφorated by reference herein in totality. In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign material (typically plasmids containing RNA or DNA) may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue type I and II, and any tissue which is receptive to transformation and subsequent regeneration into a transgenic plant. Another variable is die choice of a selectable marker. The preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to chlorosulfuron, hygromyacin, PAT and/or bar, bromoxynil, kanamycin and the like. The bar gene may be isolated from Strptomuces, particularly from the hygroscopicus or viridochromogenes species. The bar gene codes for phosphinothricin acetyl transferase (PAT) that inactivates the active ingradient in the herbicide bialaphos phosphinothricin (PPT). Thus, numerous combinations of technologies may be used in employing ribozyme mediated modulation.
The ribozymes may be expressed individually as monomers, i.e., one ribozyme targeted against one site is expressed per transcript. Alternatively, two or more ribozymes targeted against more than one target site are expressed as part of a single RNA transcript. A single RNA transcript comprising more than one ribozyme targeted against more than one cleavage site are readily generated to achieve efficient modulation of gene expression. Ribozymes within these multimer constructs are the same or different. For example, the multimer construct may comprise a plurality of hammerhead ribozymes or haiφin ribozymes or other πbozyme motifs. Alternatively, the multimer construct may be designed to include a plurality of different πbozyme motifs, such as hammerhead and haiφin ribozymes. More specifically, multimer ribozyme constructs arc designed, wherein a series of ribozyme motifs are linked together in tandem in a single RNΛ transcript. The ribozymes are linked to each other by nucleotide linker sequence, wherein the linker sequence may or may not be complementary to the target RNA. Multimer ribozyme constructs (polyribozymes) are likely to improve the effectiveness of ribozyme-mediated modulation of gene expression.
The activity of ribozymes can also be augmented by their release from the primary transcript by a second ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595, both hereby incoφorated in their totality by reference herein; Ohkawa, J., et al., 1992, Nucleic Acids Symp. Ser. , 27, 15-6; Taira, K., et α ., 1991 , Nucleic Acids Res., 19, 5125-30; Ventura, M., et al, 1993, Nucleic Acids Res., 21 , 3249-55; Chowrira et c/., 1994 J. Biol. Chem. 269, 25856).
Ribozyme-mediated modulation of gene expression can be practiced in a wide variety of plants including angiosperms, gymnosperms, monocotyledons, and dicotyledons. Plants of interest include but are not limited to: cereals, such as rice, wheat, barley, maize; oil-producing crops, such as soybean, canola, sunflower, cotton, maize, cocoa, saf lower, oil palm, coconut palm, flax, castor, peanut; plantation crops, such as coffee and tea; fruits, such as pineapple, papaya, mango, banana, grapes, oranges, apples; vegetables, such as cauliflower, cabbage, melon, green pepper, tomatoes, carrots, lettuce, celery, potatoes, broccoli; legumes, such as soybean, beans, peas; flowers, such as carnations, chrysanthemum, daisy, tulip, gypsophila, alstromeria, marigold, petunia, rose; trees such as olive, cork, poplar, pine; nuts, such as walnut, pistachio, and others. Following are a few non-limiting examples that describe the general utility of ribozymes in modulation of gene expression.
Ribozyme-mediated down regulation of the expression of genes involved in caffeine synthesis can be used to significantly change caffeine concentration in coffee beans. Expression of genes, such as 7-methylxanthosιne and/or 3-methyl transferase in coffee plants can be readily modulated using ribozymes to decrease caffeine synthesis (Adams and Zarowitz, US Patent No. 5,334,529; incoφorated by reference herein). Transgenic tobacco plants expressing ribozymes targeted against genes involved in nicotine production, such as N-methylputrescine oxidase or putrescine N-methyl transferase (Shewmaker et al, supra), would produce leaves with altered nicotine concentration.
Transgenic plants expressing ribozymes targeted against genes involved in ripening of fruits, such as ethylene-form g enzyme, pectin mcthyltransfcrasc, pectin cstcrasc, polygalacturonase, 1 -amιnocyclopropane carboxylic acid (ΛCC) syntha.sc, ΛCC oxidase genes (Smith et al, 1988, Nature, 334, 724; Gray et al, 1992, Pl. Mol. Biol, 19, 69; Tieman et al, 1992, Plant Cell, 4, 667; Picton et al, 1993, The Plant J. 3, 469; Shewmaker et al, supra; James et al, 1996, Bio/Technology, 14, 56), would delay the ripening of fruits, such as tomato and apple.
Transgenic plants expressing ribozymes targeted against genes involved in flower pigmentation, such as chalcone synthase (CHS), chalcone flavanone isomerase (CHI), phenylalanme ammonia lyase, or dehydroflavonol (DF) hydroxylases, DF reductase (Krol van der, et al, 1988, Nature, 333, 866; Krol van der, et al. 1990, Pl. Mol Biol. 14, 457; Shewmaker et al, supra; Jorgensen, 1996, Science, 268, 686), would produce flowers, such as roses, petunia, with altered colors.
Lignins are organic compounds essential for maintaining mechanical strength of cell walls in plants. Although essential, lignins have some disadvantages. They cause indigestibility of sillage crops and are undesirable to paper production from wood pulp and others. Transgenic plants expressing ribozymes targeted against genes involved in lignin production such as, O-methyltransferase, cinnamoyI-CoA:NADPH reductase or cinnamoyl alcohol dehydrogenase (Doorsselaere et al, 1995, The Plant J. 8, 855; Atanassova et al, 1995, The Plant J. 8, 465; Shewmaker et al, supra; Dwivedi et al, 1994, Pl. Mol. Biol, 26, 61 ), would have altered levels of lignin.
Other useful targets for useful ribozymes are disclosed in Draper et al, Intemational PCT Publication No. WO 93/23569, Sullivan et al, International PCT Publication No. WO 94/02595, as well as by Stinchcomb et al. International PCT Publication No. WO 95/31541, and hereby incoφorated by reference herein in totality
Modulation of granule bound starch svnthase gene expression in plants:
In plants, starch biosynthesis occurs in both chloroplasts (short teπn starch storage) and in the amyloplast (long term starch storage). Starch granules normally consist of a linear chain of α( l -4)-lιnked α-D-glucose units (amylose) and a branched form of amylose cross-linked by α( l-6) bonds (amylopectin) An enzyme involved in the synthesis of starch in plants is starch synthase which produces linear chains of α (l - 4)-glucose using ADP-glucose. Two mam forms of starch synthase are found in plants: granule bound starch synthase (GBSS) and a soluble form located in the stroma of chloroplasts and in amyloplasts (soluble starch syntha.sc) Both lorms ol this cii/yme utilize ADP-D-glucose while the granular bound form also utilizes UD -D-glucosc, with a preference for the former. The GBSS, known as waxy protein, has a molecular mass of between 55 to about 70 kDa in a variety of plants in which it has been characterized Mutations affecting the GBSS gene in several plant species has resulted in the loss of amylose, while the total amount of starch has remained relatively unchanged In addition to a loss of GBSS activity, these mutants also contain altered, reduced levels, or no GBSS protein (Mac Donald and Preiss, Plant Physiol. 78: 849-852 (1985), Sano, Theor Appl. Genet. 68: 467-473 (1984), Hovenkamp-Hermelink et al Theor. Appl. Genet 75. 217- 221 91987), Shure et al. Cell 35, 225-233 (1983), Echt and Schwartz Genetics 99: 275- 284 (1981) ). The presence of a branching enzyme as well as a soluble ADP-glucose starch glycosyl transferase in both GBSS mutants and wild type plants indicates the existence of independent pathways for the formation of the branched chain polymer amylopectin and the straight chain polymer amylose.
The Wx (waxy) locus encodes a granule bound glucosyl transferase involved in starch biosynthesis. Expression of this enzyme is limited to endosperm, pollen and the embryo sac in maize. Mutations in this locus have been termed waxy due to the appearance of mutant kernels, which is the phenotype resulting from an reduction in amylose composition m the kernels. In maize, this enzyme is transported into the amyloplast of the developing endosperm where it catalyses production of amylose. Com kernels are about 70% starch, of which 27% is linear amylose and 73% is amylopectin. Waxy is a recessive mutation in the gene encoding granule bound starch synthase (GBSS). Plants homozygous for this recessive mutation produce kernels that contain 100% of their starch in the form of amylopectin.
Ribozymes, with their catalytic activity and increased site specificity (as descπbed below), represent more potent and perhaps more specific inhibitory molecules than antisense oligonucleondes. Moreover, these ribozymes are able to inhibit GBSS activity and the catalytic activity of the πbozymes is required for their inhibitory effect. For those of ordinary skill in the art, it is clear from the examples that other ribozymes may Λ Λ-,,.n,-o PCT/US96/11689 O 97/10328
be designed that cleave target mRNAs required for GBSS activity in plant species other than maize.
Thus, in a preferred embodiment, the invention features πbozymes that inhibit enzymes involved in amylose production, e.g., by reducing GBSS activity These endogenously expressed RNA molecules contain substrate binding domains that bind to accessible regions of the target mRNA. The RNΛ molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or haiφin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, amylose production is reduced or inhibited. Specific examples arc provided below infra.
Preferred embodiments include the ribozymes having binding arms which are complementary to the binding sequences in Tables IIIA, VA and VB. Examples of such ribozymes are shown in Tables IIIB - V. Those in the art will recognize that while such examples are designed to one plant's (e.g.. maize) mRNA, similar ribozymes can be made complementary to other plant species' mRNA. By complementary is thus meant that the binding arms enable ribozymes to interact with the target RNA in a sequence-specific manner to cause cleavage of a plant mRNA target. Examples of such ribozymes consist essentially of sequences shown in Tables IIIB - V.
Preferred embodiments are the ribozymes and methods for their use in the inhibition of starch granule bound ADP (UDP)-glucose: α-l,4-Z)-glucan 4-α-glucosyl transferase i.e.. granule bound starch synthase (GBSS) activity in plants. This is accomplished through the inhibition of genetic expression, with ribozymes, which results in the reduction or elimination of GBSS activity in plants.
In another aspect of the invention, ribozymes that cleave target molecules and inhibit amylose production are expressed from transcription units inserted into the plant genome. Preferably, the recombinant vectors capable of stable integration into the plant genome and selection of transformed plant lines expressing the ribozymes are expressed either by constitutive or inducible promoters in the plant cells. Once expressed, the ribozymes cleave their target mRNAs and reduce amylose production of their host cells. The ribozymes expressed in plant cells are under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter. Modification of com starch is an important application of ribozyme technology which is capable of reducing specific gene expression. A high level of amylopectin is desirable for the wet milling process of corn and there is also some evidence that high amylopectin corn leads to increased digestibility and therefore energy availability in feed. Nearly 10% of wet-milled com has the waxy phenotype, but because of its recessive nature the traditional waxy varieties are very difficult for the grower lo handle Ribozymes targeted to cleave the GBSS mRNA and thus reduce GBSS activity in plants, and in particular, corn endosperm will act as a dominant trait and produce corn plants with the waxy phenotype that will be easier for the grower to handle.
Modification of fattv acid saturation profile in plants1
Fatty acid biosynthesis in plant tissues is initiated in the chloroplast. Fatty acids are synthesized as thioesters of acyl carrier protein (ACP) by the fatty acid synthase complex (FAS). Fatty acids with chain lengths of 16 carbons follow one of three paths: 1) they are released, immediately after synthesis, and transferred to glycerol 3-phosphate (G3P) by a chloroplast acyl transferase for further modification within the chloroplast; 2) they are released and transferred to Co-enzyme A (CoA) upon export from the plastid by thioesterases; or 3) they are further elongated to C18 chain lengths. The C18 chains are rapidly desaturated at the C9 position by stearoyi-ACP desaturase. This is followed by immediate transfer of the oleic acid (18:1) group to G3P within the chloroplast, or by export from the chloroplast and conversion to oleoyl-CoA by thioesterases (Somerville and Browse, 1991 Science 252: 80-87). The majority of C16 fatty acids follow the third pathway.
In com seed oil the predominant triglycerides are produced in the endoplasmic reticulum. Most oleic acids (18:1) and some palmitic acids (16:0) are transferred to G3P from phosphatidic acids, which are then converted to diacyl glycerides and phosphatidyl choline. Further desaturation of the acyl chains on phosphatidyl choline by membrane bound desaturases takes place in the endoplasmic reticulum. Di- and tri-unsaturated chains are then released into the acyl-CoA pool and transfe ed to the C3 position of the glycerol backbone in diacyl glycerol in the production of triglycerides (Frentzen, 1993 in Lipid Metabolism in Plants., p.195-230, (ed. Moore.T.S.) CRC Press, Boca Raton, FA.). A schematic of the plant fatty acid biosynthesis pathway is shown in Figures 1 1 and 12. The three predominant fatty acids in co seed oil are linoleic acid (18:2, ~59%), oleic acid (18:1 , ~26%), and palmitic acid (16:0, -11%). These are average values and may be somewhat different depending on the genotype; however, composite samples of US Com Belt produced oil analyzed over the past ten years have consistently had this composition (Glover and Mertz, 1987 in: Nutritional Quality of Cereal Grains: genetic and agronomic improvement., p.183-336, (eds. Olson, R.A. and Frey, K.J.) Am. Soc. Agronomy. Inc. Madison, WI.; Fitch-Haumann, 1985 J. Am. Oil Chem. Soc. 62: 1524- 1531; Strecker et al., 1990 in Edible fats and oils processing: basic principles and modem practices (ed. Erickson, D.R.) Am. Oil Chemists Soc. Champaign, II.) This predominance of C18 chain lengths may reflect the abundance and activity of several key enzymes, such as the fatty acid synthase responsible for production of CI 8 carbon chains, the stearoyl-ACP desaturase (Δ-9 desaturase) for production of 18: 1 and a microsomal Δ- 12 desaturase for conversion of 18: 1 to 18:2.
Δ-9 desaturase (also called stearoyl-ACP desaturase) of plants is a soluble chloroplast enzyme which uses C18 and occasionally C16-acyl chains linked to acyl carrier protein (ACP) as a substrate (McKeon, T.A. and Stumpf, P.K., 1982 J. Biol Chem. 257: 12141-12147). This contrasts to the mammalian, lower eukaryotic and cyanobacterial Δ-9 desaturases. Rat and yeast Δ-9 desaturases are membrane bound microsomal enzymes using acyl-CoA chains as substrates, whereas cyanobacterial Δ-9 desaturase uses acyl chains on diacyl glycerol as substrate. To date several Δ-9 desaturase cDNA clones from dicotelydenous plants have been isolated and characterized (Shanklin and Somerville, 1991 Proc. Natl Acad. Sci. USA 88: 2510-2514; Knutzon et al., 1991 Plant Physiol 96: 344-345; Sato et al., 1992 Plant Physiol 99: 362-363; Shanklin et al., 1991 Plant Physiol. 97: 467-468; Slocombe et al., 1992 Plant. Mol. Biol 20: 151-155; Taylor et al., 1992 Plant Physiol 100: 533-534; Thompson et al., 1991 Proc. Natl Acad. Sci. USA 88: 2578-2582). Comparison of the different plant Δ-9 desaturase sequences suggests that this is a highly conserved enzyme, widi high levels of identity both at the amino acid level (-90%) and at the nucleotide level (-80%). However, as might be expected from its very different physical and enzymological properties, no sequence similarity exists between plant and other Δ-9 desaturases (Shanklin and Somerville, supra).
Purification and characterization of the castor bean desaturase (and others) indicates that the Δ-9 desaturase is active as a homodimer; the subunit molecular weight is - 41 kDa. The enzyme requires molecular oxygen, NADPH, NADPH ferredoxin oxidoreductase and ferredoxin for activity in vitro. Fox et al. , 1993 (Proc. Natl. Acad. Sci. USA 90: 2486-2490) showed that upon expression in E. coli the castor bean enzyme contains four catalytically active ferrous atoms per homodimer. The oxidized enzyme contains two identical diferπc clusters, which in the presence of dithionite are reduced to the diferrous state. In the presence of stearoyl-CoA and O2 the clusters return to the diferπc state. This suggests that the desaturase belongs to a group of O2 activating proteins containing dnron-oxo clusters. Other members of this group are nbonucleotide reductase and methane monooxygenase hydroxylase Comparison of the predicted primary structure for these catalytically diverse proteins shows that all contain a conserved pair of ammo acid sequences (Asp/Glu)-Glu-Xaa-Λrg-l lιs separated by -80- 100 amino acids.
Traditional plant breeding programs have shown that increased stearate levels can be achieved without deleterious consequences to the plant In saffiower (Ladd and Knowles, 1970 Crop Sci. 10: 525-527) and in soybean (Hammond and Fehr, 1984 J. Amer. Oil Chem. Soc. 61 : 1713-1716; Graef et al, 1985 Crop Sci. 25: 1076- 1079) stearate levels have been increased significantly This demonstrates the flexibility in fatty acid composition of seed oil.
Increases in Δ-9 desaturase activity have been achieved by the transformation of tobacco with the Δ-9 desaturase genes from yeast (Polashock et al., 1992 Plant Physiol 100, 894) or rat (Graybum et. al., 1992 BioTechnology 10, 675). Both sets of transgenic plants had significant changes in fatty acid composition, yet were phenotypically identical to control plants.
Com (maize) has been used minimally for the production of margarine products because it has traditionally not been utilized as an oil crop and because of the relatively low seed oil content when compared with soybean and canola. However, com oil has low levels of linolenic acid (18:3) and relatively high levels of palmitic (16:0) acid (desirable in margarine). Applicant believes that reduction in oleic and linoleic acid levels by down- regulation of Δ-9 desaturase activity will make com a viable alternative to soybean and canola in the saturated oil market.
Margarine and confectionary fats are produced by chemical hydrogenation of oil from plants such as soybean. This process adds cost to the production of the margarine and also causes both cis and trans isomers of the fatty acids. Trans isomers are not naturally found in plant deπved oils and have raised a concern for potential health risks. Applicant believes that one way to eliminate the need for chemical hydrogenation is to genetically engineer the plants so that desaturation enzymes are down-regulated. Δ-9 desaturase introduces the first double bond into 18 carbon fatty acids and is the key step effecting the extent of desaturation of fatty acids.
Thus, in a preferred embodiment, the invention concerns compositions (and methods for their use) for the modification of fatty acid composition in plants. This is accomplished through the inhibition of genetic expression, with ribozymes, antisense nucleic acid, cosuppression or triplex DNΛ, which results in the reduction or elimination of certain enzyme activities in plants, such as Δ-9 desaturase Such activity is reduced in monocotyledon plants, such as maize, wheat, rice, palm, coconut and others. Δ-9 desaturase activity may also be reduced in dicotyledon plants such as sunflower, saffiower, cotton, peanut, olive, sesame, cuphea, flax, jojoba, grape and others.
Thus, in one aspect, the invention features ribozymes that inhibit enzymes involved in fatty acid unsaturation, e.g., by reducing Δ-9 desaturase activity. These endogenously expressed RNA molecules contain substrate binding domains that bind to accessible regions of the target mRNA. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or haiφin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, stearate levels are increased and unsaturated fatty acid production is reduced or inhibited. Specific examples are provided below in the Tables listed directly below.
In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the Tables VI and VIII. Those in the an will recognize that while such examples are designed to one plant's (e.g., com) mRNA, similar ribozymes can be made complementary to other plant's mRNA. By complementary is thus meant that the binding arms of the ribozymes are able to interact with the target RNA in a sequence-specific manner and enable the ribozyme to cause cleavage of a plant mRNA target. Examples of such ribozymes are typically sequences defined in Tables VII and VIII. The active ribozyme typically contains an enzymatic center equivalent to those in the examples, and binding arms able to bind plant mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such binding and/or cleavage.
The sequences of the ribozymes that are particularly useful in this study, are shown in Tables VII and VIII. Those in the art will recognize that ribozyme sequences listed in the Tables are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity For example, stem-loop II sequence of hammerhead ribozymes listed in Table IV (5'-GGCGΛAΛGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequences, pielei bly provided that a minimum of a two base-paired stem structure can form Similarly, stem- loop IV sequence of haiφin ribozymes listed in Tables V and VIII (5'-CΛCGUUGUG-.V) can be altered (substitution, deletion, and/or insertion) to contain any sequence, preferably provided that a minimum of a two base-paired stem structure can form Such πbozymes are equivalent to the ribozymes descπbed specifically in the Tables
In another aspect of the invention, πbozymes that cleave target molecules and reduce unsaturated fatty acid content in plants are expressed from transcription units inserted into the plant genome. Preferably, the recombinant vectors capable of stable integration into the plant genome and selection of transformed plant lines expressing the ribozymes are expressed either by constitutive or inducible promoters in the plant cells. Once expressed, the ribozymes cleave their target mRNΛs and reduce unsaturated fatty acid production of their host cells. The ribozymes expressed in plant cells are under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter.
Modification of fatty acid profile is an important application of nucleic acid-based technologies which are capable of reducing specific gene expression. A high level of saturated fatty acid is desirable in plants that produce oils of commercial importance.
In a related aspect, this invention features the isolation of the cDNA sequence encoding Δ-9 desaturase in maize.
In preferred embodiments, haiφin and hammerhead ribozymes that cleave Δ-9 desaturase mRNA are also described. Those of ordinary skill in the art will understand from the examples described below that other πbozymes that cleave target mRNAs required for Δ-9 desaturase activity may now be readily designed and are within the scope of the invention.
While specific examples to com RNA are provided, those in the art will recognize that the teachings are not limited to com. Furthermore, the same target may be used in other plant species. The complementary arms suitable for targeting the specific plant
RNA sequences are utilized in the ribozyme targeted to that specific RNA. The examples 97/10328 25
and teachings herein are meant to be non-limiting, and those skilled in the art will recognize that similar embodiments can be readily generated in a variety of different plants to modulate expression of a variety of different genes, using the teachings herein, and are within the scope of the inventions.
Standard molecular biology techniques were followed in the examples herein.
Additional information may be found in Sambrook, J., l-'ritsch, L. I'., and Mainatis, T. (1989), Molecular Cloning a Laboratory Manual, second edition, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, which is incoφorated herein by reference.
Examples Example 1 : Isolation of Δ.9 desaturase cDNA from Zea mays
Degenerate PCR primers were designed and synthesized to two conserved peptides involved in diiron-oxo group binding of plant Δ-9 desaturases. A 276 bp DNA fragment was PCR amplified from maize embryo cDNA and was cloned in to a vector. The predicted amino acid sequence of this fragment was similar to the sequence of the region separated by the two conserved peptides of dicot Δ-9 desaturase proteins. This was used to screen a maize embryo cDNA library. A total of 16 clones were isolated; further restriction mapping and hybridization identified one clone which was sequenced. Features of the cDNA insert are: a 1621 nt cDNA; 145 nt 5' and 294 nt 3' untranslated regions including a 18 nt poly A tail; a 394 amino acid open reading frame encoding a 44.7 kD polypeptide; and 85% amino acid identity with castor bean Δ-9 desaturase gene for the predicted mature protein. The complete sequence is presented in Figure 10.
Example 2: Identification of Potential Ribozyme Cleavage Sites for Δ9 desaturase
Approximately two hundred and fifty HH ribozyme sites and approximately forty three HP sites were identified in the com Δ-9 desaturase mRNA. A HH site consists of a uridine and any nucleotide except guanosine (UH). Tables VI and VIII have a list of HH and HP ribozyme cleavage sites. The numbering system starts with 1 at the 5' end of a Δ-
9 desaturase cDNA clone having the sequence shown in Fig. 10.
Ribozymes, such as those listed in Tables VII and VIII, can be readily designed and synthesized to such cleavage sites with between 5 and 100 or more bases as substrate binding arms (see Figs. 1 - 5). These substrate binding arms within a ribozyme allow the ribozyme to interact with their target in a sequence-specific manner. Example 3: Selection of Ribozvme Cleavage Sites for 9 desaturase
The secondary structure of Δ-9 desaturase mRNA was assessed by computer analysis using algoπthms, such as those developed by M. Zuker ( Zuker, M , 1989
Science, 244, 48-52). Regions of the mRNA that did not form secondary folding structures with RNA/RNA stems of over eight nucleotides and coniaincd potential hammerhead ribozyme cleavage sues were identified
These sites were assessed for oligonucleotide accessibility by RNase H assays (see Example 4 infra).
Example 4: RNaseH Assays for Δ9 desaturase
Forty nine DNA oligonucleotides, each twenty one nucleotides long were used in
RNase H assays. These oligonucleotides covered 108 sites within Δ-9 desaturase RNA. RNase H assays (Fig. 6) were performed using a full length transcript of the Δ-9 desaturase cDNA. RNA was screened for accessible cleavage sites by the method described generally in Draper et al, supra. Briefly, DNA oligonucleotides representing ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to generate a substrate for T7 RNA polymerase transcription from co cDNA clones. Labeled RNA transcripts were synthesized in vitro from these templates. The oligonucleotides and the labeled transcripts were annealed, RNAseH was added and the mixtures were incubated for 10 minutes at 37°C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a Molecular Dynamics phosphor imaging system (Figs. 13 and 14).
Example 5: Hammerhead and Hairpin Ribozvmes for Δ9 desaturase
Hammerhead (HH) and haiφin (HP) ribozymes were designed to the sites covered by the oligos which cleaved best in the RNase H assays. These ribozymes were then subjected to analysis by computer folding and the ribozymes that had significant secondary structure were rejected.
The πbozymes were chemically synthesized. The general procedures for RNA synthesis have been descπbed previously (Usman et al, 1987, J. Am. Chem. Soc, 109, 7845-7854 and in Scaringe et al., 1990, Nucl Acids Res., 18, 5433-5341 ; Wincott et al,
1995, Nucleic Acids Res. 23, 2677). Small scale syntheses were conducted on a 394 7/10328
Applied Biosystems, Inc. synthesizer using a modified 2.5 μmol scale protocol with a 5 min coupling step for alkylsilyl protected nucleotides and 2.5 mm coupling step for -O- methylated nucleotides. Table II outlines the amounts, and the contact times, of the reagents used m the synthesis cycle. A 6.5-foId excess (163 μL of 0.1 M = 16.3 μmol) of phosphoramidite and a 24-fold excess of 5-ethyl tetrazolc (238 μL of 0.25 M = 59.5 μmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle Λvcrage coupling yields on the 394, determined by coloπmetπc quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394: Detπtylation solution was 2% TCA in methylene chloride (ABI); capping was performed with 16% N- Methyl imidazole in THF (ABI) and 10% acetic anhydride/ 10% 2,6-lutιdιne in THF (ABI); oxidation solution was 16.9 mM l2, 49 mM pyridine, 9% water in THF
(Millipore). B & J Synthesis Grade acetonitrile was used directly from the reagent bottle. 5-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from Ameπcan Intemational Chemical, Inc.
Deprotection of the RΝA was performed as follows. The polymer-bound oligoribonucleotide, tπtyl-off, was transferred from the synthesis column to a 4 mL glass screw top vial and suspended in a solution of methylamine (MA) at 65°C for 10 min. After cooling to -20°C, the supernatant was removed from the polymer support. The support was washed three times with 1.0 mL of EtOH:MeCΝ:H2θ/3:l : l , vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder.
The base-deprotected oligoribonucleotide was resuspended in anhydrous
TEA«HF/NMP solution (250 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL
TEA and 1.0 mL TEA*3HF to provide a 1.4 M HF concentration) and heated to 65°C for 1.5 h. The resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.
For anion exchange desalting of the deprotected oligomer, the TEAB solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB (10 mL), the RΝA was eluted with 2 M TEAB (10 mL) and dried down to a white powder.
Inactive hammerhead ribozymes were synthesized by substituting a U for G5 and a U for A 14 (numbering from (Hertel, K. J., et al, 1992, Nucleic Acids Res., 20, 3252) The haiφin ribozymes were synthesized as described above for the hammerhead RNAs.
Ribozymes were also synthesized from DNA templates using bacteπophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol 180, 51 ). Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; Sec Wincott et al, 1996, supra, the totality of which is hereby incoφorated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study arc shown below in Tables VII and VIII.
Example 6: Long substrate tests for Δ.9 desaturase ribozymes
Target RNA used in this study was 1621 nt long and contained cleavage sites for all the HH and HP ribozymes targeted against Δ-9 desaturase RNA. A template containing T7 RNA polymerase promoter upstream of Δ-9 desaturase target sequence, was PCR amplified from a cDNA clone. Target RNA was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [α-32P] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase-I, following transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription. The transcription mixture was resolved on a denaturing polyacrylamide gel. Bands corresponding to full- length RNA was isolated from a gel slice and the RNA was precipitated with isopropanol and the pellet was stored at 4°C.
Ribozyme cleavage reactions were carried out under ribozyme excess (k at/KM) conditions (Herschlag and Cech, 1990, Biochemistry 29, 10159-10171). Briefly, 1 mM ribozyme and < 10 nM internally labeled target RNA were denatured separately by heating to 65°C for 2 min in the presence of 50 mM Tris.HCl, pH 7.5 and 10 mM
MgCl2- The RNAs were renatured by cooling to the reaction temperature (37°C, 26°C or
20°C) for 10-20 min. Cleavage reaction was initiated by mixing the ribozyme and target RNA at appropriate reaction temperatures. Aliquots were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on 4 % sequencing gel.
The results from ribozyme cleavage reactions, at 26°C or 20°C, are summarized in Table IX and Figures 15 and 16. Of the ribozymes tested, seven hammerheads and two 7/10328 2g
haiφins showed significant cleavage of Δ-9 desaturase RNA (Figures 15 and 16). Ribozymes to other sites showed varied levels of activity.
Example 7: Cleavage of the target RNA using multiple ribozvme combinations for Δ9 desaturase Several of the above ribozymes were incorporated into a multimer ribozyme construct which contains two or more ribozymes embedded in a contiguous stretch of complementary RNA sequence. Non-limiting examples of multimer ribozymes are shown in Figures 17, 18, 19 and 23. The ribozymes were made by annealling complementary oligonucleotides and cloning into an expression vector containing the Cauliflower Mosaic Virus 35S enhanced promoter (Franck et al. 1985 Cell 21, 285), the maize Adh 1 intron (Dennis et al, 1984 Nucl Acids Res. 12, 3983) and the Nos polyadenylation signal (DePicker β/ α/., 1982 J. Molec. Appl. Genet. 1, 561). Cleavage assays with T7 transcripts made from these multimer-containing transcription units are shown in Figures 20 and 21. These are non-limiting examples; those skilled in the art will recognize that similar embodiments, consisting of other ribozyme combinations, introns and promoter elements, can be readily generated using techniques known in the art and are within the scope of this invention.
Example 8: Construction of Ribozyme expressing transcription units for Δ9 desaturase
Ribozymes targeted to cleave Δ-9 desaturase mRNA are endogenously expressed in plants, either from genes inserted into the plant genome (stable transformation) or from episomal transcription units (transient expression) which are part of plasmid vectors or viral sequences. These ribozymes can be expressed via RNA polymerase I, II, or III plant or plant virus promoters (such as CaMV). Promoters can be either constitutive, tissue specific, or developmentally expressed.
Δ9 259 Monomer Ribozvme Constructs (RPA 114, 115)
These are the Δ-9 desaturase 259 monomer hammerhead ribozyme clones. The ribozymes were designed with 3 bp long stem II and 20 bp (total) long substrate binding arms targeted against site 259. The active version is RPA 114, the inactive is RPA 1 15. The parent plasmid, pDAB367, was digested with Not I and filled in with Klenow to make a blunt acceptor site. The vector was then digested with Hind III restriction enzyme. The ribozyme containing plasmids were cut with Eco RI, filled-in with Klenow and recut with Hind III. The insert containing the entire ribozyme transcription unit was 97/10328 3Q
gel-punfied and ligated into the pDAB 367 vector The constructs are checked by digestion with SgfllHind III and Xba l/Sst I and confirmed by sequencing
Δ9 453 Multimer Ribozvme Constructs (RPA 1 18, 1 19)
These are the Δ-9 desaturase 453 Multimer hammerhead ribozyme clones (sec Figure 17). The πbozymes were designed with 3 bp long stem fl regions Total length ol the substrate binding arms of the multimer construct was 42 bp. The active version is RPA 118, the inactive is 119 The constructs were made as described above for the 259 monomer. The multimer construct was designed with four hammerhead ribozymes targeted against sites 453, 464, 475 and 484 within Δ-9 desaturase RNA
Δ9 252 Multimer Ribozvme Constructs (RPA 85, 113)
These are the Δ-9 desaturase 252 multimer ribozyme clones placed at the 3 'end of bar (phosphoinothπcm acetyl transferase, Thompson et al., 1987 EMBO J 6. 2519-2523) open reading frame. The multimer contracts were designed with 3 bp long stem II regions. Total length of the substrate binding arms of the multimer construct was 91 bp. RPA 85 is the active ribozyme, RPA 113 is the inactive. The vector was constructed as follows: The parent plasmid pDAB 367 was partially digested with Bgl II and the single cut plasmid was gel-punfied. This was recut with Eco RI and again gel-punfied to isolate the correct Bgl YUEco RI cut fragment. The Bam HI/ Eco RI inserts from the ribozyme constructs were gel-isolated (this contains the ribozyme and the NOS poly A region) and ligated into the 367 vector. The identitiy of positive plasmids were confirmed by performing a Nco I / Sst I digest and sequencing.
Useful transgenic plants can be identified by standard assays. The transgenic plants can be evaluated for reduction tn Δ-9 desaturase expression and Δ-9 desaturase activity as discussed in the examples infra.
Example 9: Identification of Potential Ribozvme Cleavage Sites in GBSS RNA
Two hundred and forty one hammer-head ribozyme sites were identified in the com
GBSS mRNA polypeptide coding region (see table IIIA). A hammer-head site consists of a undine and any nucleotide except guanine (UH). Following is the sequence of GBSS coding region for com (SEQ. I.D. No.25). The numbeπng system starts with I at the 5' end of a GBSS cDNA clone having the following sequence (5' to 3')
72 GACCGATCGATCGCCACAGCCAACACCACCCGCCGAGGCGACGCGACAGCCGCCΛ GGAGGAAGGAATAAACT
73 144
CACTGCCAGCCAGTGAAGGGGGAGAAGTGTACTGCTCCGTCCACCAGTGCGCGCA CCGCCCGGCAGGGCTGC
145 216
TCATCTCGTCGACGACCAGTGGATTΛATCGGCΛTGGCGGCTCTΛGCCΛCGTCGCΛ GCTCGTCGCAACGCGCG
217 288 CCGGCCTGGGCGTCCCGGACGCGTCCACGTTCCGCCGCGGCGCCGCGCΛGGGCCT GAGGGGGGGCCGGACGG
289 360
CGTCGGCGGCGGACACGCTCAGCATTCGGACCAGCGCGCGCGCGGCGCCCAGGCT CCAGCACCAGCAGCAGC 361 432
AGCAGGCGCGCCGCGGGGCCAGGTTCCCGTCGCTCGTCGTGTGCGCCAGCGCCGG CATGAACGTCGTCTTCG
433 504
TCGGCGCCGAGATGGCGCCGTGGAGCAAGACCGGCGGCCTCGGCGACGTCCTCGG CGGCCTGCCGCCGGCCA
505 576
TGGCCGCGAATGGGCACCGTGTCATGGTCGTCTCTCCCCGCTACGACCAGTACAA GGACGCCTGGGACACCA
577 648 GCGTCGTGTCCGAGATCAAGATGGGAGACAGGTACGAGACGGTCAGGTTCTTCCA CTGCTACAAGCGCGGAG 649 720
TGGACCGCGTGTTCGTTGACCACCCACTGTTCCTGGAGAGGGTTTGGGGAAAGAC CGAGGAGAAGATCTACG 721 792
GGCCTGACGCTGGAACGGACTACAGGGACAACCAGCTGCGGTTCAGCCTGCTATG CCAGGCAGCACTTGAAG
793 864
CTCCAAGGATCCTGAGCCTCAACAACAACCCATACTTCTCCGGACCATACGGGGA GGACGTCGTGTTCGTCT
865 936 GCAACGACTGGCACACCGGCCCTCTCTCGTGCTACCTCAAGAGCAACTACCAGTCC CACGGCATCTACAGGG
937 1008
ACGCAAAGACCGCTTTCTGCATCCACAACATCTCCTACCAGGGCCGGTTCGCCTTC TCCGACTACCCGGAGC
1009 10X0
TGAACCTCCCGGAGAGATTCΛAGTCGTCCTTCGΛΓΓTCΛTCGΛCGGC ΎΛCGΛ ΛΛ GCCCGTGGAAGGCCGGA
1081 1 152 AGATCAACTGGATGAAGGCCGGGATCCTCGAGGCCGACAGGGTCCTCACCGTCAG CCCCTACTACGCCGAGG
1153 1224
AGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAACATCATGCGCCTCAC CGGCATCACCGGCATCG 1225 1296
TCAACGGCATGGACGTCAGCGAGTGGGACCCCAGCAGGGACAAGTACATCGCCGT
GAAGTACGACGTGTCGA
1297 1368
CGGCCGTGGAGGCCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGC TCCCGGTGGACCGGAACA
1369 1440
TCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGACCCGACGTCAT
GGCGGCCGCCATCCCGC
1441 1512 AGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAAGA AGAAGTTCGAGCGCATGC
1513 1584
TCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAAGTTCAA CGCGGCGCTGGCGCACC 1585 1656
ACATCATGGCCGGCGCCGACGTGCTCGCCGTCACCAGCCGCTTCGAGCCCTGCGGC CTCATCCAGCTGCAGG
1657 1728
GGATGCGATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGACAC CATCATCGAAGGCAAGA
1729 1800 CCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGΛGCCGGCGGA CGTCAAGAAGGTGGCCA
1801 1872
CCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGATGGT GAGGAACTGCATGATCC
1873 1944
AGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGGΛGΛΛCGTGCΪGCTCΛGCCT
CGGGGTCGCCGGCGGCG
1945 2016 AGCCAGGGGTCGAAGGCGAGGAGATCGCGCCGCTCGCCAAGGAGΛΛCGTGGCCG CGCCCTGAAGAGTTCGGC
2017 2088
CTGCAGGCCCCCTGATCTCGCGCGTGGTGCAAACATGTTGGGACATCTTCTTATAT ATGCTGTTTCGTTTAT 2089 2160
GTGATATGGACAAGTATGTGTAGCTGCTTGCTTGTGCTAGTGTAATATAGTGTAG TGGTGGCCAGTGGCACA
2161 2232
ACCTAATAAGCGCATGAACTAATTGCTTGCGTGTGTAGTTAAGTACCGATCGGTA ATTTTATATTGCGAGTA
2233
AATAAATGGACCTGTAGTGGTGGAAAAAAAAAAAA (SEQ I.D. NO. 25).
There are approximately 53 potential haiφin ribozyme sites in the GBSS mRNA. The ribozyme and target sequences are listed in Table V.
Ribozymes can be readily designed and synthesized to such sites with between 5 and 100 or more bases as substrate binding arms (see Figs. 1 - 5) as described above.
Example 10: Selection of Ribozvme Cleavage Sites for GBSS
The secondary structure of GBSS mRNA was assessed by computer analysis using folding algorithms, such as the ones developed by M. Zuker ( Zuker, M., 1989 Science,
244, 48-52. Regions of the mRNA that did not form secondary folding structures with
RNA RNA stems of over eight nucleotides and contained potential hammerhead ribozyme cleavage sites were identified. These sites which were then assessed for oligonucleotide accessibility with RNase H assays (see Fig. 6). Fifty-eight DNA oligonucleotides, each twenty one nucleotides long were used in these assays. These oligonucleotides covered 85 sites The position and designation of these oligonucleotides were 195, 205, 240, 307, 390, 424, 472, 481 , 539, 592, 625, 636, 678, 725, 741 , 81 1 , 859, 891 , 897, 912, 918, 928, 951 , 958, 969, 993, 999, 1015, 1027, 1032, 1056, 1084, 1 105, 1 156, 1 168, 1 186, 1 195, 1204, 1213. 1222, 1240, 1269, 1284, 1293, 1345, 1351, 1420, 1471 , 1533, 1563, 1714, 1750, 1786, 1806, 1819, 1921 , 1954, and 1978. Secondary sites were also covered and included 202, 394, 384, 385, 484, 624, 627, 628, 679, 862, 901 , 930, 950, 952, 967, 990, 991 , 1026, 1035, 1 108, 1159, 1225,1273, 1534, 1564, 1558, and 1717.
Example 1 1 : RNaseH Assays for GBSS
RNase H assays (Fig. 7) were performed using a full length transcript of the GBSS coding region, 3' noncoding region, and part of the 5' noncoding region. The GBSS RNA was screened for accessible cleavage sites by the method descπbed generally in Draper et al, supra, hereby incoφorated by reference herein. Briefly, DNA oligonucleotides representing hammerhead ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to generate a substrate for T7 RNA polymerase transcription from com cDNA clones. Labeled RNA transcripts were synthesized in vitro from these templates. The oligonucleotides and the labeled transcripts were annealed, RNAseH was added and the mixtures were incubated for 10 minutes at 37°C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a phosphor imaging system (Fig. 7)*
Example 12: Hammerhead Ribozymes for GBSS
Hammerhead ribozymes with 10/10 (i.e., able to form 10 base pairs on each arm of the ribozyme) nucleotide binding arms were designed to the sites covered by the oligos which cleaved best in the RNase H assays. These ribozymes were then subjected to analysis by computer folding and the ribozymes that had significant secondary structure were rejected. As a result of this screening procedure 23 ribozymes were designed to the most open regions in the GBSS mRNA, the sequences of these ribozymes are shown in Table IV.
The πbozymes were chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described above (and in Usman et al , supra, Scaπnge et al, and Wincott et al, supra) and are incorporated by reference herein, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise coupling yields were >98%. Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from (Hertel et al, supra). Hairpin πbozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowπra and Burke, 1992, Nucleic Acids Res., 20, 2835-). All ribozymes were modified to enhance stability by modification of five ribonucleotides at both the 5' and 3' ends with 2'-0- methyl groups. Ribozymes were purified by gel electrophoresis using general methods. (Ausubel et al, 1990 Current Protocols in Molecular Biology Wiley & Sons, NY) or were purified by high pressure liquid chromatography, as described above and were resuspended in water.
Example 13: Long Substrate Tests for GBSS
Target RNA used in this study was 900 nt long and contained cleavage sites for all the 23 HH ribozymes targeted against GBSS RNA. A template containing T7 RNA polymerase promoter upstream of GBSS target sequence, was PCR amplified from a cDNA clone. Target RNA was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [α-32p] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase-1 , following transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription. The transcription mixture was resolved on a denaturing polyacrylamide gel. Bands corresponding to full-length RNA was isolated from a gel slice and the RNA was precipitated with isopropanol and the pellet was stored at 4°C.
Ribozyme cleavage reactions were carried out under ribozyme excess (kcat/K ) conditions (Herschlag and Cech, supra). Briefly, 1000 nM ribozyme and < 10 nM internally labeled target RNA were denatured separately by heating to 90°C for 2 min. in the presence of 50 mM Tris.HCl, pH 7.5 and 10 mM MgCl2- The RNAs were renarured by cooling to the reaction temperature (37°C, 26°C and 20°C) for 10-20 min. Cleavage reaction was initiated by mbcing the ribozyme and target RNA at appropriate reaction temperatures. Alquots were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on 4% sequencing gel. The results from ribozyme cleavage reactions, at the three different temperatures, are summarized in Figure 8. Seven lead ribozymes were chosen (425, 892, 919, 959, 968, 1241, and 1787). One of the active ribozymes (81 1 ) produced a strange pattern of cleavage products; as a result, it was not chosen as one of our lead ribozymes
Example 14: Cleavage of the GBSS RNA Using Multiple Ribozvme Combinations
Four of the lead πbozymes (892, 919, 959, 1241 ) were incubated with internally labeled target RNA in the following combinations: 892 alone; 892 + 919; 892 + 919 + 959; 892 + 919 + 959 + 1241. The fraction of RNA cleavage increased in an additive manner with an increase in the number of ribozymes used in the cleavage reaction (Fig. 9) Ribozyme cleavage reactions were earned out at 20°C as described above. These data suggest that multiple ribozymes targeted to different sites on the same mRNA will increase the reduction of target RNA in an additive manner.
Example 15: Construction of Ribozvme Expressing Transcription Units for GBSS Cloning of GBSS Multimer Ribozymes RPA 63 (active) and RPA64 (inactive)
A multimer ribozyme was constructed which contained four hammerhead ribozymes targeting sites 892, 919, 959 and 968 of the GBSS mRNA. Two DNA oligonucleotides (Macromolecular Resourses, Fort Collins, CO) were ordered which overlap by 18 nucleotides. The sequences were as follows:
Oligo 1: CGCGGA TCC TGG TAG GAC TGA TGA GGC CGA AAG GCC GAA ATGTTGTGCTGATGAGGCCGAAAG GCCGAAATGCAGAAAGCG GTC TTTGCGTCCCTGTAGATG CCGTGGC
Oligo2: CGCGAGCTCGGCCCTCTCTTTCGGCCTTTCGGCCTC ATC AGG TGCTAC CTC AAG AGC AAC TAC CAG TTT CGG CCTTTC GGC CTC ATC AGCCACGGCATCTACAGGG
Inactive versions of the above were made by substituting T for G5 and T for A 14 within the catalytic core of each ribozyme motif.
These were annealed in 1 X Klenow Buffer (Gibco/BRL) at 90°C for 5 minutes and slow cooled to room temperature (22°C). NTPs were added to 0.2 mM and the oligos extended with Klenow enzyme at l unit ul for one hour at 37°C. This was phenol/chloroform extracted and ethanol precipitated, then resuspended in I X React 3 buffer (Gibco/BRL) and digested with Bam HI and Sst I for 1 hour at 37°C. The DNA was gel purified on a 2% agarose gel using the Qiagen gel extraction kit.
The DNA fragments were ligated into BamVWS.st I digested pDΛB 353. The gaiion was transformed into competent DH5α F' bacteria (Gibco/BRL). Potential clones were screened by digestion with Bam UEco RI. Clones were confirmed by sequencing. The total length of homology with the target sequence is 96 nucleotides.
919 Monomer Ribozvme (RPA66)
A single ribozyme to site 919 of the GBSS mRNA was constructed with 10/10 aπns as follows. Two DNA oligos were ordered:
Oligo 1: GAT CCG ATG CCGTGG CTG ATG AGG CCG AAA GGC CGA AAC TGG TAG TT
Oligo2: AACTACCAGTTTCGGCCTTTCGGCCTCΛTCΛGC CACGGC ΛTC G
The oligos are phosphorylated individually in IX kinase buffer (Gibco/BRL) and heat denatured and annealed by combining at 90°C for 10 min, then slow cooled to room temperature (22°C). The vector was prepared by digestion of pDAB 353 with Sst I and blunting the ends with T4 DNA polymerase. The vector was redigested with Bam HI and gel purified as above. The annealed oligos are ligated to the vector in IX ligation buffer (Gibco/BRL) at 16°C overnight. Potential clones were digested with Bam HVEco RI and confirmed by sequencing.
Example 16: Plant Transformation Plasmids pDAB 367. Used in the Δ.9 Ribozyme Experiments, and pDAB353 used in the GBSS Ribozvme Experiments
Part A PDAB367
Plasmid pDAB367 has the following DNA structure: beginning with the base after the final C residue of d e Sph I site of pUC 19 (base 441 ; Ref. 1 ), and reading on the strand contiguous to the LacZ gene coding strand, the linker sequence CTGCAGGCCGGCC TTAATTAAGCGGCCGCGTTTAAACGCCCGGGCATTTAAATGGCGCGCCGC GATCGCTTGCAGATCTGCATGGGTG, nucleotides 7093 to 7344 of CaMV DNA (2), the linker sequence CATCGATG, nucleotides 7093 to 7439 of CaMV, the linker sequence GGGGACTCTAGAGGATCCAG, nucleotides 167 to 186 of MSV (3), nucleotides 188 to 277 of MSV (3), a C residue followed by nucleotides 1 19 to 209 of maize Adh IS containing parts of exon 1 and intron I (4), nucleotides 555 to 672 containing parts of Adh IS intron 1 and exon 2 (4), the linker sequence GACGGΛTC G, and nucleotides 278 to 317 of MSV. This is followed by a modified BAR coding region from pIJ4104 (5) having the AGC seπne codon in the second position replaced by a GCC alanine codon, and nucleotide 546 of the coding region changed from G to A to eliminate a Bgl II site. Next, the linker sequence TGAGATCTGAGCTCGΛΛTTTCCCC, nucleotides 1298 to 1554 of Nos (6), and a G residue followed by the rest of the pUC 19 sequence (including the Eco RI site).
Part B pDAB353
Plasmid pDAB353 has the following DNA structure: beginning with the base after the final C residue of the Sph I site of pUC 19 (base 441; Ref. 1), and reading on the strand contiguous to the LacZ gene coding strand, the linker sequence CTGCAGATCTGCATGGGTG, nucleotides 7093 to 7344 of CaMV DNA (2), the linker sequence CATCGATG, nucleotides 7093 to 7439 of CaMV, the linker sequence GGGGACTCTAGAG, nucleotides 119 to 209 of maize Adh IS containing parts of exon 1 and intron 1 (4), nucleotides 555 to 672 containing parts of Adh IS intron 1 and exon 2 (4), and the linker sequence GACGGATCCGTCGACC, where GGATCC represents the recognition sequence for BamH I restriction enzyme. This is followed by the beta- giucuronidase (GUS) gene from pRAJ275 (7), cloned as an Nco I/Sac I fragment, the linker sequence GAATTTCCCC, the poly A region in nucleotides 1298 to 1554 of Nos (6), and a G residue followed by the rest of the pUC 19 sequence (including the Eco RI site).
The following are herein incoφorated by reference:
1. Messing, J. (1983) in "Methods in Enzymology" (Wu, R. et al, Eds) 101 :20-78.
2. Franck, A., H. Guilley, G. Jonard, K. Richards, and L. Hirth (1980) Nucleotide sequence of Cauliflower Mosaic Virus DNA. Cell 21 :285-294. Λ „., „ «„«, PCT/US96/11689 O 97/10328
39
3. Mullineaux, P. M., J. Donson, B. A. M. Morπs-Krsinich. M. I. Boulton, and J. W. Davιes (1984) The nucleotide sequence of Maize Streak Virus DNΛ. EMBO J. 3:3063- 3068.
4. Dennis, E. S., W. L. Gerlach, A. J. Pryor, J. L. Bennetzen. A. Ing s, D. Llewellyn, M . M. Sachs, R. J. Ferl, and W. J. Peacock (1984) Molecular analysis of the alcohol dehydrogenase (Adhl) gene of maize. Nucl. Acids Res. 12:3983-4000
5. White, J., S-Y Chang, M. J. Bibb, and M. J. Bibb (1990) Λ cassette containing the bar gene of Streptomyces hygroscopicus: a selectable marker for plant transformation. Nucl. Acids. Res. 18: 1062. 6. DePicker, A., S. Stachel, P. Dhaese, P. Zambryski, and I I. M. Goodman ( 1982) Nopaline Synthase: Transcript mapping and DNA sequence J. Molcc. Λppl. Genet. 1 :561-573.
7. Jefferson, R. A. (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Molec. Biol. Reporter 5:387-405.
Example 17: Plasmid pDAB359 a Plant Transformation Plasmid which Contains the Gamma-Zein Promoter, the Antisense GBSS. and a the Nos Polvadenvlation Sequence
Plasmid pDAB359 is a 6702 bp double-stranded, circular DNA that contains the following sequence elements: nucleotides 1-404 from pUC18 which include lac operon sequence from base 238 to base 404 and ends with the Hindlll site of the M 13mpl 8 polylinker (1,2); the Nos polyadenylation sequence from nucleotides 412 to 668 (3); a synthetic adapter sequence from nucleotides 679-690 which converts a Sac I site to an Xho I site by changing GAGCTC to GAGCTT and adding CTCGAG: maize granule bound starch synthase cDNA from bases 691 to 2953, corresponding to nucleotides 1- 2255 of SEQ. I.D. No. 25. The GBSS sequence in plasmid pDAB359 was modified from the original cDNA by the addition of a 5' Xho I and a 3' Nco I site as well as the deletion of internal Nco I and Xho I sites using Klenow to fill in the enzyme recognition sequences. Bases 2971 to 4453 are 5' untranslated sequence of the maize 27 kD gamma- zein gene corresponding to nucleotides 1078 to 2565 of the published sequence (4). The gamma-zein sequence was modified to contain a 5' Kpn I site and 3' BamH/Sall/Nco I sites. Additional changes in the gamma-zein sequence relative to the published sequence include a T deletion at nucleotide 104, a TACA deletion at nucleotide 613, a C to T conversion at nucleotide 812, an A deletion at nucleotide 1165 and an A insertion at nucleotide 1353. Finally, nucleotides 4454 to 6720 of pDAB359 are identical to pucl 8 bases 456 to 2686 including the Kpn I/EcoRI/Sac I sites of the M13/mpI 8 polylinker from 4454 to 4471 , a lac operon fragment from 4471 to 4697, and the β-lacatmase gene from 5642 to 6433 (1 , 2).
The following are incoφorated by reference herein:
pUC18- Norrander, J., Kempe, T., Messing, J. Gene ( 1983) 26: 101 - 106; Pouwcls. P.I I., Enger-Valk, B.E., Brammar, W. J. Cloning Vectors, Elsevier 1985 and supplements
NosA - DePicker, A., Stachel, S., Dhaese, P., Zambryski, P., and Goodman, H.M. (1982) Nopaline Synthase: Transcript Mapping and DNA Sequence J. Molec. Appl. Genet. 1 :561-573.
Maize 27kD gamma-zein - Das, O.P., Poliak, E.L., Ward, K., Messing, J. Nucleic Acids Research 19, 3325 - 3330 (1991 ).
Example 18: Construction of Plasmid pDAB430. containing Antisense Δ9 Desaturase, Expressed bv the Ubiquitin Promoter/intron (Ubi 1 )
Part A Construction of plasmid pDAB421 Plasmid pDAB421 contains a unique blunt-end Srfl cloning site flanked by the maize Ubiquitin promoter/intron and the nopaline synthase polyadenylation sequences. pDAB421 was prepared as follows: digestion of pDAB355 with restriction enzymes Kpnl and BamHI drops out the R coding region on a 2.2 kB fragment. Following gel purification, the vector was ligated to an adapter composed of two annealed oligonucleotides OF235 and OF236. OF235 has the sequence 5' - GAT CCG CCC GGG GCC CGG GCG GTA C - 3' and OF236 has the sequence 5' - CGC CCG GGC CCC GGG CG - 3'. Clones containing this adapter were identified by digestion and linearization of plasmid DNA with the enzymes Srfl and Smal which cut in the adapter, but not elsewhere in the plasmid. One representative clone was sequenced to verify that only one adapter was inserted into the plasmid. The resulting plasmid pDAB421 was used in subsequent construction of the Δ9 desaturase antisense plasmid pDAB430.
Part B Construction of plasmid pDAB430 (antisense Δ9 desaturase) The antisense Δ9 desaturase construct present in plasmid pDAB430 was produced by cloning of an amplification product in the blunt-end cloning site of plasmid pDAB421. Two constructs were produced simultaneously from the same experiment. The first construct contains the Δ9 desaturase gene in the sense orientation behind the ubiquitin promoter, and the c-myc tag fused to the C-terminus of the Δ9 desaturase open reading frame for immunological detection of oveφroduced protein in transgenic lines This construct was intended for testing of ribozymes in a system which did not express maize Δ9 desaturase. This construct was never used, but the primers used to amplify and construct the Δ9 desaturase antisense gene were the same The Δ9 desaturase cl)NΛ sequence described herein was amplified with two primers The N-tcrmmal primer OF279 has the sequence 5'- GTG CCC ACA ATG GCG CTC CGC CTC AAC GAC - 3'. The underlined bases correspond to nucleotides 146- 166 of the cDNA sequence C- terminal primer OF280 has the sequence 5' - TCA TCA CAG GTC CTC CTC GCT GAT CAG CTT CTC CTC CAG TTG GAC CTG CCT ACC GTA - 3' and is the reverse complement of the sequence 5' - TAC GGT AGG GAC GTC CAA CTG GAG GAG AAG CTG ATC AGC GAG GAG GAC CTG TGA TGA - 3'. In this sequence the underlined bases correspond to nucleotides 1304-1324 of the cDNA sequence, the bases in italics correspond to the sequence of the c-myc epitope. The 1179 bp of amplification product was purified through a 1.0% agarose gel, and ligated into plasmid pDAB421 which was linearized with the restriction enzyme Srfl. Colony hybridization was used to select clones containing the pDAB421 plasmid with the insert. The orientation of the insert was determined by restriction digestion of plasmid DNA with diagnostic enzymes Kpnl and BamHl. A clone containing the Δ9 desaturase coding sequence in the sense orientation relative to the Ubiquitin promoter/intron was recovered and was named pDAB429. An additional clone containing the Δ9 desaturase coding sequence in the anitsense orientation relative to the promoter was named pDAB430. Plasmid pDAB430 was subjected to sequence analysis and it was determined that the sequence contained three PCR induced errors compared to the expected sequence. One error was found in the sequence corresponding to primer OF280 and two nucleotide changes were observed internal to the coding sequence. These errors were not corrected, because antisense downregulation does not require 100% sequence identity between the antisense transcript and the downregulation target.
Example 19: Helium Blasting of Embryogenic Maize Cultures and the Subsequent Regeneration of Transgenic Progeny
Part A Establishment of embryogenic maize cultures. The tissue cultures employed in transformation experiments were initiated from immature zygotic embryos of the genotype "Hi-II". Hi-II is a hybrid made by lntermating 2 R3 lines derived from a B73xA 188 cross (Armstrong et al. 1990). When cultured, this genotype produces callus tissue known as Type II. Type II callus is friable, grows quickly, and exhibits the ability to maintain a high level of embryogenic activity over an extended time period.
Type II cultures were initiated from 1.5-3.0 mm immature embryos resulting from controlled pollinations of greenhouse grown Hi-II plants. The initiation medium used was N6 (Chu, 1978) which contained I .Omg/L 2,4-D, 25 mM L-prolinc, 1 0 mg L casein hydrolysate, 10 mg L AgNO3, 2.5 g L gelπte and 2% sucrose adjusted to pH 5.8. For approximately 2-8 weeks, selection occurred for Type II callus and against non- embryogenic and or Type I callus. Once Type II callus was selected, it was transferred to a maintenance medium in which AgNO3 was omitted and L-prolinc reduced to 6mM.
After approximately 3 months of subculture in which the quantity and quality of embryogenic cultures was increased, the cultures were deemed acceptable for use in transformation experiments.
Part B Preparation of plasmid DNA. Plasmid DNA was adsorbed onto the surface of gold particles prior to use in transformation experiments. The experiments for the GBSS target used gold particles which were spherical with diameters ranging from 1.5-3.0 microns (Aldrich Chemical Co., Milwaukee, WI). Transfomation experiments for the Δ9 desaturase target used 1.0 micron spherical gold particles (Bio-Rad, Hercules, CA). All gold particles were surface-sterilized with ethanol prior to use. Adsoφtion was accomplished by adding 74 μl of 2.5 M calcium chloride and 30 μl of 0.1 M spermidine to 300 μl of plasmid DNA and sterile H2O. The concentration of plasmid DNA was 140 μg. The DNA-coated gold particles were immediately vortexed and allowed to settle out of suspension. The resulting clear supematent was removed and the particles were resuspended in 1 ml of 100% ethanol. The final dilution of the suspension ready for use in helium blasting was 7.5 mg DNA gold per ml of ethanol.
Part C Preparation and helium blasting of tissue targets. Approximately 600 mg of embryogenic callus tissue per target was spread over the surface of petri plates containing Type II callus maintenance medium plus 0.2 M sorbitol and 0.2 M mannitol as an osmoticum. After an approximately 4 hour pretreatment, all tissue was transferred to petri plates containing 2% agar blasting medium (maintenance medium plus osmoticum plus 2% agar). 7/10328 43
Helium blasting involved accelerating the suspended DNA-coated gold particles towards and into prepared tissue targets. The device used was an earlier prototype to the one described in a DowElanco U.S. Patent (#5,141 ,131) which is incoφorated herein by reference, although both function in a similar manner. The device consisted of a high pressure helium source, a syringe containing the DNΛ/gold suspension, and a pneumatically-operated multipurpose valve which provided controlled linkage between the helium source and a loop of pre-loaded DNA/gold suspension.
Prior to blasting, tissue targets were covered with a sterile 104 micron stainless steel screen, which held the tissue in place duπng impact. Next, targets were placed under vacuum in the main chamber of the device. The DNA-coated gold particles were accelerated at the target 4 times using a helium pressure of 1500 psi. Each blast delivered 20 μl of DNA/gold suspension. Immediately post-blasting, the targets were placed back on maintenance medium plus osmoticum for a 16 to 24 hour recovery period.
Part D Selection of transformed tissue and the regeneration of plants from transgenic cultures. After 16 to 24 hours post-blasting, the tissue was divided into small pieces and transferred to selection medium (maintenance medium plus 30 mg/L Basta™). Every 4 weeks for 3 months, the tissue pieces were non-selectively transferred to fresh selection medium. After 8 weeks and up to 24 weeks, any sectors found proliferating against a background of growth inhibited tissue were removed and isolated. Putatively transformed tissue was subcultured onto fresh selection medium. Transgenic cultures were established after 1 to 3 additional subcultures.
Once Basta™ resistant callus was established as a line, plant regeneration was initiated by transferring callus tissue to petri plate containing cytokinin-based induction medium which were then placed in low light (125 ft-candles) for one week followed by one week in high light (325 ft-candles). The induction medium was composed of MS salts and vitamins (Murashige and Skoog, 1962), 30 g L sucrose, 100 mg/L myo-inositol, 5 mg/L 6- benzylaminopurine, 0.025 mg L 2,4-D, 2.5 g/L gelrite adjusted to pH 5.7. Following the two week induction period, the tissue was non-selectively transfeσed to hormone-free regeneration medium and kept in high light. The regeneration medium was composed of MS salts and vitamins, 30 g/L sucrose and 2.5 g/L gelrite adjusted to pH 5.7. Both induction and regeneration media contained 30 mg/L Basta™. Tissue began differentiating shoots and roots in 2-4 weeks. Small (1.5-3 cm) plantlets were removed and placed in tubes containing SH medium. SH medium is composed of SH salts and vitamins (Schenk „„_._ PCT/US96/11689 97/10328
44 and Hildebrandt, 1972). 10 g/L sucrose, 100 mg/L myo-inositol, 5 mL L FeEDTA, and either 7 g/L Agar or 2.5 g/L Gelrite adjusted to pH 5.8. Plantlets were transferred to 10 cm pots containing approximately 0.1 kg of Metro-Mix® 360 (The Scotts Co., Marysville, OH) in the greenhouse as soon as they exhibited growth and developed a sufficient root system ( 1 -2 weeks). At the 3-5 leaf stage, plants were transferred to 5 gallon pots containing approximately 4 kg Mctro-Mix υ 360 and grown to mammy. These Ro plants were sclf-pollinatcd and/or cross-pollinated with non-iransgenic inbrcds to obtain transgenic progeny. In the case of transgenic plants produced for the GBSS target, Ri seed produced from Ro pollinations was replanted. The R] plants were grown to maturity and pollinated to produce R2 seed in the quantities needed for the analyses.
Example 20: Production and Regeneration of Δ.9 Transgenic Material.
Part A Transformation and isolation of embryogenic callus. Six ribozyme constructs, described previously, targeted to Δ9 desaturase were transformed into regenerable Type II callus cultures as described herein. These 6 constructs consisted of 3 active/inactive pairs; namely, RPA85/RPA113, RPA1 14/RPA115, and RPA1 18/RPA1 19. A total of 1621 tissue targets were prepared, blasted, and placed into selection. From these blasting experiments 334 independent Basta®-resistant transformation events ("lines") were isolated from selection. Approximately 50% of these lines were analyzed via DNA PCR or GC/FAME as a means of determining which ones to move forward to regeneration and which ones to discard. The remaining 50% were not analyzed either because they had become non-embryogenic or contaminated.
Part B Regeneration of Δ9 plants from transgenic callus. Following analyses of the transgenic callus, twelve lines were chosen per ribozyme construct for regeneration, with 15 Ro plants to be produced per line. These lines generally consisted of 10 analysis- positive lines plus 2 negative controls, however, due to the poor regencrability of some of the cultures, plants were produced from less than 12 lines for constructs RPA 113, RPA 115, RPA 118, and RPA 119. An overall total of 854 Ro plants were regenerated from 66 individual lines (see Table X). When the plants reached maturity, self- or sib- pollinations were given the highest priority, however, when this was not possible, cross- pollinations were made using the inbreds CQ806, CS716, OQ414, or HOi as pollen donors, and occasionally as pollen recipients. Over 715 controlled pollinations have been made, with the majority (55%) being comprised of self- or sib-pollinations and the 97/10328
45 minority (45%) being comprised of Fl crosses. Ri seed was collected approximately 45 days post-pollination.
Example 21 : Production and Regeneration of Transgenic Maize for the GBSS
Part A Transformation of embryogenic maize callus and the subsequent selection and establishment of transgenic cultures. RPA63 and RPΛ64, an active/inactive pair of ribozyme multimers targeted to GBSS, were inserted along with bar selection plasmid pDAB308 into Type II callus as described herein. A total of 1 15 Basta™-rcsιstant independent transformation events were recovered from the selection of 590 blasted tissue targets. Southern analysis was performed on callus samples from established cultures of all events to determine the status of the gene of interest.
Part B Regeneration of plants from cultures transformed with ribozymes targeted to GBSS as well as the advancement to the R2 generation. Plants were regenerated from
Southern "positive" transgenic cultures and grown to maturity in a greenhouse. The primary regenerates were pollinated to produce Ri seed. From 30 to 45 days after pollination, seed was harvested, dried to the correct moisture content, and replanted. A total of 752 R] plants, representing 16 original lines, were grown to sexual maturity and pollinated. Approximately 19 to 22 days after pollination, ears were harvested and 30 kernels were randomly excised per ear and frozen for later analyses.
Example 22: Testing of GBSS-Targeted Ribozymes in Maize Black Mexican Sweet (BMS) Stably Transformed Callus
Part A Production of BMS callus stably transformed with GBSS and GBSS-targeted ribozymes. BMS does not produce a GBSS mRNA which is homologous to that found endogenously in maize. Therefore, a double transformation system was developed to produce transformants which expressed both target and ribozymes. "ZM" BMS suspensions (obtained from Jack Widholm, University of Illinois, also see W. F. Sheridan, "Black Mexican Sweet Com: Its Use for Tissue Cultures" in Maize for Biological Research, W. F. Sheridan, editor. University Press. University of North Dakokta, Grand Forks, ND, 1982, pp. 385-388) were prepared for helium blasting four days after subculture by transfer to a 100 x 20 mm Petri plate (Fisher Scientific, Pittsburgh, PA) and partial removal of liquid medium, forming a thin paste of cells. Targets consisted of 100- 125 mg fresh weight of cells on a 1/2" antibiotic disc (Schleicher and Schuell, Keene, NH) placed on blasting medium, DN6 [N6 salts and vitamins (Chu ei al, 1978), 20 g'L sucrose, 1.5 mg L 2,4-dichlorophenoxyacetιc acid (2,4-D). 25 mM L-prolme; pH= 5.8 before autoclaving 20 minutes at 121 °C] solidified with 2% TC agar (JRH Bioscicnces, Lenexa, Kansas) in 60 x 20 mm plates. DNA was precipitated onto gold particles. For the first transformation, pDAB 426 (Ubi/GBSS) and pDAB 308 (35T/Bar) were used. Targets were individually shot using DowElanco Helium Blasting Device I. With a vacuum pressure of 650 mm Hg and at a distance of 15.5 cm from target to device nozzle, each sample was blasted once with DNA/gold mixture at 500 psi. Immediately after blasting, the antibiotic discs were transferred to DN6 medium made with 0.8% TC agar for one week of target tissue recovery. After recovery, each target was spread onto a 5.5 cm Whatman #4 filter placed on DN6 medium minus proline with 3 mg/L Basta® (Hoechst, Frankfort, Germany). Two weeks later, the filters were transferred to fresh selection medium with 6 mg/L Basta®. Subsequent transfers were done at two week intervals. Isolates were picked from the filters and placed on AMCF-ARM medium (N6 salts and vitamins, 20 g/L sucrose, 30 g L mannitol, 100 mg/L acid casein hydrolysate, and 1 mg/L 2,4-D, 24 mM L-proline; pH*= 5.8 before autoclaving 20 minutes at 121 °C) solidified with 0.8% TC agar containing 6 mg L Basta®. Isolates were maintained by subculture to fresh medium every two weeks.
Basta®-resistant isolates which expressed GBSS were subjected to a second transformation. As with BMS suspensions, targets of transgenic callus were prepared 4 days after subculture by spreading tissue onto 1/2" filters. However, AMCF-ARM with 2% TC agar was used for blasting, due to maintenance of transformants on AMCF-ARM selection media. Each sample was covered with a sterile 104 μm mesh screen and blasting was done at 1500 psi. Target tissue was co-bombarded with pDAB 319 (35S-ALS; 35T- GUS) and RPA63 (active ribozyme multimer) or pDAB319 and RPA64 (inactive ribozyme multimer), or shot with pDAB 319 alone. Immediately after blasting, all targets were transferred to nonselective medium (AMCF-ARM) for one week of recovery. Subsequently, the targets were placed on AMCF-ARM medium containing two selection agents, 6 mg/L Basta® and 2 μg/L chlorsulfuron (CSN). The level of CSN was increased to 4 ug/L after 2 weeks. Continued transfer of the filters and generation of isolates was done as described in the first transformation, with isolates being maintained on AMCF- ARM medium containing 6 mg/L Basta and 4 μg/L CSN.
Part B Analysis of BMS stable transformants expressing GBSS and GBSS-targeted ribozymes. Isolates from the first transformation were evaluated by Northern blot 7/10328
47 analysis for detection of a functional target gene (GBSS) and to deteπnine relative levels of expression. In 12 of 25 isolates analyzed, GBSS transcript was detected. A range of expression was observed, indicating an independence of transformation events. Isolates generated from the second transformation were evaluated by Northern blot analysis for detection of continued GBSS expression and by RT-PCR to screen for the presence of ribozyme transcript. Of 19 isolates tested from one previously transformed line, 18 expressed the active ribozyme, RPA63, and all expressed GBSS. GBSS was detected in each of 6 vector controls; ribozyme was not expressed in these samples. As described herein, RNase protection assay (RPA) and Northern blot analysis were performed on ribozyme-expressing and vector control tissues to compare levels of GBSS transcript in the presence or absence of active ribozyme. GBSS values were normalized to an internal control (Δ9 desaturase); Northern blot data is shown in Figure (25). Northern blot results revealed a significantly lower level of GBSS message in the presence of ribozyme, as compared to vector controls. RPA data showed that some of the individual samples expressing active ribozyme ("L" and "O") were significantly different from vector controls and similar to a nontransformed control.
Example 23: Analysis of Plant and Callus Materials
Plant material co-transformed with the pDAB308 and one of the following ribozyme containing vectors, pRPA63, pRPA64, pRPA85, pRPA1 13, ρRPA1 14, pRPAl 15, pRPAl 18 or pRPAl 19 were analyzed at the callus level, Ro level and select lines analyzed at the Fl level. Leaf material was harvested when the plantlets reached the 6-8 leaf stage. DNA from the plant and callus material was prepared from lyophilized tissue as described by Saghai-Maroof et al(supra). Eight micrograms of each DNA was digested with the restriction enzymes specific for each construct using conditions suggested by the manufacturer (Bethesda Research Laboratory, Gaithersburg, MD) and separated by agarose gel electrophoresis. The DNA was blotted onto nylon membrane as described by Southern, E. 1975 "Detection of specific sequences among DNA fragments separated by gel electrophoresis," J Mol. Biol. 98:503 and Southern, E. 1980 "Gel electrophoresis of restriction fragments" Methods Enzmol. 69:152, which are incoφorated by reference herein.
Probes specific for the ribozyme coding region were hybridized to the membranes. Probe DNA was prepared by boiling 50 ng of probe DNA for 10 minutes then quick cooling on ice before being added to the Ready-To-Go DNA labeling beads (Pharmacia 7/10328
48
LKB, Piscataway, NJ) with 50 microcuπes of α32P-dCTP (Amersham Life Science. Arlington Heights, IL). Probes were hybridized to the genomic DNA on the nylon membranes. The membranes were washed at 60°C in 0.25X SSC and 0.2% SDS for 45 minutes, blotted dry and exposed to XAR-5 film overnight with two intensifying screens
The DNA from the RPA63 and RPA64 was digested with the restriction enzymes Hindlll and EcoRI and the blots containing these samples were hybridized to the RPA63 probe. The RPA63 probe consists of the RPA63 ribozyme multimer coding region and should produce a single 1.3 kb hybridization product when hybridized to the RPΛ63 or RPA64 materials. The 1.3 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the ribozyme coding region and the nopaline synthase poly A 3' end. The DNA from the RPA85 and RPA113 was digested with the restriction enzymes Hindlll and EcoRI and the blots containing these samples were hybridized to the RPA122 probe. RPA 122 is the 252 multimer ribozyme in pDAB 353 replacing the GUS reporter. The RPA 122 probe consists of the RPA 122 ribozyme multimer coding region and the nopaline synthase 3' end and should produce a single 2.1 kb hybridization product when hybridized to die RPA85 or RPA113 materials. The 2.1 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the bar gene, the ribozyme coding region and the nopaline synthase poly A 3' end. The DNA from the RPA114 and RPA115 was digested with the restriction enzymes Hindlll and Smal and the blots containing these samples were hybridized to the RPA 115 probe. The RPA 115 probe consist of the RPA115 ribozyme coding region and should produce a single 1.2 kb hybridization product when hybridized to the RPA114 or RPA115 materials. The 1.2 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the ribozyme coding region and the nopaline synthase poly A 3' end. The DNA from the RPA118 and RPA119 was digested with the restriction enzymes Hindlll and Smal and the blots containing these samples were hybridized to the RPA1 18 probe. The RPA1 18 probe consist of the RPA118 ribozyme coding region and should produce a single 1.3 kb hybridization product when hybridized to the RPA1 18 or RPA119 materials. The 1.3 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the ribozyme coding region and the nopaline synthase poly A 3' end.
Example 24: Extraction of Genomic DNA from Transgenic Callus
Three hundred mg of actively growing callus were quick frozen on dry ice. It was ground to a fine powder with a chilled Bessman Tissue Pulverizer (Spectrum, Houston, TX) and extracted with 400μl of 2x CTAB buffer (2% Hexadecyltrimethylammonium Bromide, 100 mM Tris pH 8.0, 20 mM EDTA, 1.4 M NaCl, 1 % polyvinylpyrrolidone). The suspension was lysed at 65°C for 25 minutes, then extracted with an equal volume of chloroform: isoamyl alcohol. To the aqueous phase was added 0.1 volumes of 10% CTAB buffer (10% Hexadecyltrimethylammonium Bromide, 0.7 M NaCl). Following extraction with an equal volume of chloroforrmisoamyl alcohol, 0.6 volumes of cold isopropyl alcohol was added to the aqueous phase, and placed at -20°C for 30 minutes. After a 5 minute centrifugation at 14,000 φm, the resulting precipitant was dried for 10 minutes under vacuum. It was resuspended in 200 μl TE ( lOmM Tris, I mMEDTΛ, pH 8.0) at 65°C for 20 minutes. 20% Chelex (Biorad, ) was added to the DNΛ to a final concentration of 5% and incubated at 56°C for 15-30 minutes to remove impurities. The DNA concentration was measured on a Hoefer Fluorimeter (Hoefer, San Francisco).
Example 25: PCR Analysis of Genomic Callus DNA
Use of Polymerase Chain Reaction (PCR) to demonstrate the stable insertion of ribozyme genes into the chromosome of transgenic maize calli.
Part A Method used to detect ribozyme DNA The Polymerase Chain Reaction (PCR) was performed as described in the suppliers protocol using AmpliTaq DNA Polymerase (GeneAmp PCR kit, Perkin Elmer, Cetus). Aliquots of 300 ng of genomic callus DNA, 1 μl of a 50 μM downstream primer (5* CGC AAG ACC GGC AAC AGG 3' ), lμl of an upstream primer and Iμl of Perfect Match (Stratagene, Ca) PCR enhancer were mixed with the components of the kit. The PCR reaction was performed for 40 cycles using the following parameters; denaturation at 94°C for 1 minute, annealing at 55°C for 2 minutes, and extension at 72βC for 3 mins. An aliquot of 0.2x vol. of each PCR reaction was electrophoresised on a 2% 3:1 Agarose (FMC) gel using standard TAE agarose gel conditions.
Part B Upstream primer used for detection of Δ.9 desaturase ribozyme genes
RPA85 RPA1 13 251 multimer fused to BAR 3* ORF
RPA1 14/RPA1 15 258 ribozyme monomer
RPA 118/RPA 119 452 ribozyme multimer
5* TGG ATT GAT GTG ATA TCT CCA C 3' This primer is used to amplify across the Eco RV site in the 35S promoter. Primers were prepared using standard oligo synthesis protocols on an Applied Biosystems Model 394 DNA/RNA synthesizer.
Example 26: Preparation of Total RNA from Transgenic Maize Calli and Plant
Part A Preparation of total RNA from transgenic non-regcncrablc and regenerable callus tissue. Three hundred milligrams of actively growing callus was quick frozen on dry ice The tissue was ground to a fine powder with a chilled Bessman Tissue Pulverizer (Spectrum, Houston, TX) and extracted with RNA Extraction Buffer (50 mM Tris-HCl pH 8.0, 4% para-amino salicylic acid, 1% Tri-iso-propylnapthalenesulfonic acid, 10 mM dithiothreitol, and 10 M Sodium meta-bisulfite) by vigorous vortcx g. The homogcnatc was then extracted with an equal volume of phenol containing 0.1% 8-hydroxyquinoline. After centrifugation, the aqueous layer was extracted with an equal volume of phenol containing chlorofomrisoamyl alcohol (24:1), followed by extraction with chloroform:octanol (24: 1). Subsequently, 7.5 M Ammonium acetate was added to a final concentration of 2.5 M, the RNA was precipitated for 1 to 3 hours at 4°C. Following 4°C centrifugation at 14,000 φm, RNA was resuspended in sterile water, precipitated with 2.5 M NH4OAC and 2 volumes of 100% ethanol and incubated ovemite at -20°C.
The harvested RNA pellet was washed with 70% ethanol and dried under vacuum. RNA was resuspended in sterile H2O and stored at -80°C.
Part B Preparation of total RNA from transgenic maize plants. A five cm section (-150 mg) of actively growing maize leaf tissue was excised and quick frozen in dry ice. The leaf was ground to a fine powder in a chilled mortar. Following manufacturers instructions, total RNA was purified from the powder using a Qaigen RNeasy Plant Total RNA kit (Qiagen Inc., Chatsworth, CA). Total RNA was released from the RNeasy columns by two sequential elution spins of prewarmcd (50°C) sterile water (30 μl each) and stored at - 80°C.
Example 27: Use of RT-PCR Analysis to Demonstrate Expression of Ribozvme RNA in Transgenic Maize Calli and Plants
Part A Method used to detect ribozyme RNA. The Reverse Transcription-Polymerase
Chain Reaction (RT-PCR) was performed as described in the suppliers protocol using a thermostable rTth DNA Polymerase (rTth DNA Polymerase RNA PCR kit, Perkin
Elmer Cetus). Aliquots of 300 ng of total RNA (leaf or callus) and 1 μl of a 15 μM downstream primer (5' CGC AAG ACC GGC AAC AGG 3' ) were mixed with the RT components of the kit. The reverse transcription reaction was performed in a 3 step ramp up with 5 minute incubations at 60°C, 65°C, and 70°C. For the PCR reaction, l μl of upstream primer specific for the ribozyme RNA being analyzed was added to the RT reaction with the PCR components. The PCR reaction was performed for 35 cycles using the following parameters; incubation at 96°C for I minute, dcnaiuraiion ai 94"C for 0 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 3 mins. Λn aliquot of 0.2x vol. of each RT-PCR reaction was electrophoresed on a 2% 3: 1 Agarose (FMC) gel using standard TAE agarose gel conditions.
Part B Specific upstream primers used for detection of GBSS ribozymes.
GBSS Active and Inactive Multimer
5' CAG ATC AAG TGC AAA GCT GCG GAC GGA TCT G 3"
This primer covers the Adh I intron footprint upstream of the first ribozyme aπn. GBSS 918 Intron (-) Monomer:
5' ATC CGA TGC CGT GGC TGA TG 3'
This primer covers the 10 base pair ribozyme arm and the first 6 bases of the ribozyme catalytic domain.
GBSS ribozyme expression in transgenic callus and plants was confirmed by RT-PCR.
GBSS multimer ribozyme expression in stably transformed callus was also determined by Ribonuc lease Protection Assay.
Part C Specific upstream primers used for detection of Δ9 desaturase ribozymes. RPA85/RPA113 252 multimer fused to BAR 3* ORF
5' GAT GAG ATC CGG TGG CAT TG 3'
This primer spans the junction of the BAR gene and the RPA85/113 ribozyme.
RPA114/RPA115 259 ribozyme monomer
5' ATC CCC TTG GTG GAC TGA TG 3' This primer covers the 10 base pair ribozyme arm and the first 6 bases of the ribozyme catalytic domain.
RPA118/RPA1 19 453 ribozyme multimer
5* CAG ATC AAG TGC AAA GCT GCG GAC GGA TCT G 3'
This primer covers die Adh I intron footprint upstream of the first ribozyme arm. Expression of Δ9 desaturase ribozymes in transgenic plant lines 85-06, 1 13-06 and 85-15 were confirmed by RT-PCR. Pπmers were prepared using standard oligo synthesis protocols on an Applied Biosystems Model 394 DNA/RNA synthesizer.
Example 28: Demonstration of Ribozvme Mediated Reduction in Target mRNA Levels in Transgenic Maize Callus and Plants
Part A Northern analysis method which was used to demonstrated reductions in target mRNA levels. Five μg of total RNA was dried under vacuum, resuspended in loading buffer (20mM phosphate buffer pH 6.8, 5mM EDTA; 50% formamide: 16% formaldehyde: 10% glycerol) and denatured for 10 minutes at 65°C. Electrophoresis was at 50 volts through 1 % agarose gel in 20 mM phosphate buffer (pH 6.8) with buffer recirculation. BRL 0.24-9.5 Kb RNA ladder (Gibco/BRL, Gaithersburg, MD) were stained in gels with ethiduim bromide. RNA was transferred to GeneScreen membrane filter ( DuPont NEN, Boston MA) by capillary transfer with sterile water. Hybridization was performed as described by DeLeon et al. (1983) at 42 °C, the filters were washed at 55 °C to remove non-hybridized probe. The blot was probed sequentially with cDNA fragments from the target gene and an internal RNA control gene. The internal RNA standard was utilized to distinguish variation in target mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions. For each sample the level of target mRNA was compared to the level of control mRNA within that sample. Fragments were purified by Qiaex resin (Qaigen Inc. Chatsworth, CA) from Ix TAE agarose gels. They were nick-translated using an Amersham Nick Translation Kit (Amersham Coφoration, Arlington Heights , 111.) with alpha 32p dCTP. Autoradiography was at -70° C with intensifying screens (DuPont, Wilmington DE) for one to three days. Autoradiogram signals for each probe were measured after a 24 hour exposure by densitometer and a ratio of target/internal control mRNA levels was calculated.
Ribonuclease protection assays were performed as follows: RNA was prepared using the Qiagen RNeasy Plant Total RNA Kit from either BMS protoplasts or callus material. The probes were made using the Ambion Maxiscript kit and were typically 10s cpm/ microgram or higher. The probes were made the same day they were used. They were gel purified, resuspended in RNase-freelOmM Tris (pH 8) and kept on ice. Probes were diluted to 5xl05cpm/ul immediately before use. 5 μg of RNA derived from callus or 20 μg of RNA derived from protoplasts was incubated with 5 x 105 cpm of probe in 4M Guanidine Buffer. [4M Guanidine Buffer: 4M Guanidine Thiocyanate/0.5% Sarcosyl/25mM Sodium Citrate (pH 7.4)]. 40 ul of PCR mineral oil was added to each tube to prevent evaporation. The samples were heated to 95° for 3 minutes and placed immediately into a 45° water bath. Incubation continued overnight. 600 μl of RNase Treatment Mix was added per sample and incubated for 30 minutes at 37°C. (RNase Treatment Mix: 400 mM NaCl, 40 units/ml RNase Λ and Tl ). 12 μl of 20% SlλS were added per tube, immediately followed by addition of 12 ul (20 mμ/ml) Proicma.se K to each tube. The tubes were vortexed gently and incubated for 30 minutes at 37ϋC 750 ul of room temperature RNase-free isopropanol was added to each tube, and mixed by inverting repeatedly to get the SDS into solution. The samples were then microfugcd at top speed at room temperature for 20 minutes. The pellets were air dried for 45 minutes. 15 ul of RNA Running Buffer was added to each tube, and vortexed hard for 30 seconds. (RNA Running Buffer: 95% Formamide/20mM EDTA/0.1% Bromophenol Blue/0.1% Xylene Cyanol ). The sample was heated to 95° C for 3 minutes, and loaded onto an 8% denaturing acrylamide gel. The gel was vacuum dried and exposed to a phosphorimager screens for 4 to 12 hours.
Part B Results demonstrating reductions in GBSS mRNA levels in nongenerable callus expressing both a GBSS and GBSS targeted ribozyme RNA. The production of nonregenerable callus expressing RNAs for the GBSS target gene and an active multimer ribozyme targeted to GBSS mRNA was performed. Also produced were transgenics expressing GBSS and a ribozyme (-) control RNA. Total RNA was prepared from the transgenic lines. Northern analysis was performed on 7 ribozyme (-) control transformants and 8 active RPA63 lines. Probes for this analysis were a full length maize GBSS cDNA and a maize Δ9 cDNA fragment. To distinguish variation in GBSS mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions, the level of GBSS mRNA was compared to the level of Δ9 mRNA within that sample. The level of full length GBSS transcript was compared between ribozyme expressing and ribozyme minus calli to identify lines with ribozyme mediated target RNA reductions. Blot to blot variation was controlled by performing duplicate analyses.
A range in GBSS/ Δ9 ratio was observed between ribozyme (-) transgenics. The target mRNA is produced by a transgene and may be subject to more variation in expression men the endogenous Δ9 mRNA. Active lines (RPA 63) AA, EE, KK, and JJ were shown to reduce the level of GBSS/Δ9 most significantly, as much as 10 fold as compared to ribozyme (-) control transgenics this is graphed in Figure 25. Those active lines were shown to be expressing GBSS targeted πbozyme by RT-PCR as descπbed herein.
Reductions in GBSS mRNA compared to Δ9 mRNA were also seen by RNAse protection assay.
Part C Demonstration of reductions in Δ9 desaturase levels in transgenic plants expressing ribozymes targeted to Δ9 desaturase mRNA. The high stearate transgenics, RPA85-06 and RPA85-15, each contained an intact copy of the fused ribozyme multimer gene. Within each line, plants were screened by RT-PCR for the presence of ribozyme RNA. Using the protocol described in Example 27. RPΛ85 ribozyme expression was demonstrated in plants of the 85-06 and 85-15 lines which contained high steaπc acid in their leaves. Northern analysis was performed on the six high stearate plants from each line as well as non-transformed (NT) and transformed control (TC) plants. The probes for this analysis were cDNA fragments from a maize Δ9 desaturase cDNA and a maize actin cDNA. To distinguish vaπation in Δ9 mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions, the level of Δ9 mRNA was compared to the level of actin mRNA within that sample. Using densitometer readings described above a ratio was calculated for each sample. Δ9/actin ratio values ranging from 0.55 to 0.88 were calculated for the 85-06 plants. The average Δ9/actm value for non- transformed controls was 2.7. There is an apparent 4 fold reduction in Δ9/actm ratios between 85-06 and NT leaves. Comparing Δ9/actin values between 85-06 high stearate and TC plants, on average a 3 fold reduction in Δ9/actm was observed for the 85-06 plants. This data is graphed in Figure 26. Ranges in Δ9/actιn ratios from 0.35 to 0.53, with an average of 0.43 were calculated for the RPA85-15 high stearate transgenics. In this experiment the average Δ9/actin ratio for the NT plants was 1.7. Comparing the average Δ9/actin ratio between NT controls and 85-15 high stearate plants, a 3.9 fold reduction in 85-15 Δ9 mRNA was demonstrated. An apparent 3 fold reduction in Δ9 mRNA level was observed for RPA85-15 high stearate transgenics when Δ9/actm ratios were compared between 85-15 high stearate and normal stearate (TC) plants. These data are graphed in Figure 27. These data indicate πbozyme-mediated reduction of Δ9- desaturase mRNA in transgenic plants expressing RPA85 ribozyme, and producing increased levels of steaπc acid in the leaves.
Example 29: Evidence of Δ9 Desaturase Down Regulation in Maize Leaves as a Result of Active Ribozyme Activity 97/10328
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Plants were produced which were transformed with inactive versions of the Δ9 desaturase ribozyme genes. Data was presented demonstrating control levels of leaf stearate in the inactive Δ9 ribozyme transgenic lines RPA 1 13-06 and 1 13- 17. Ribozyme expression and northern analysis was performed for the RPΛ 1 13-06 line. Δ9 dcsaiurasc protein levels were deteπnined in plants of the RPΛ I 13- 17 line Kibo/ymc expression was measured as described herein. Plants 1 13-06-04, -07, and - 10 expressed detectable levels of RPA 1 13 inactive Δ9 ribozyme. Northern analysis was performed on 5 plants of the 113-06 line with leaf stearate ranging from 1.8 - 3.9 %, all of which fall within the range of controls. No reduction in Δ9 desaturase mRNA correlating with ribozyme expression or elevations in leaf stearate were found in the RPA 1 13-06 plants as compared to controls, graphed in Figure 28. Protein analysis did not indicate any reduction in Δ9 desaturase protein levels correlating with elevated leaf stearate in the RPA 113-17 plants. This data is graphed in Figure 29(a). Taken together, the data from the two RPA 1 13 inactive transgenic lines indicate ribozyme activity is responsible for the high strearate phenotype observed in the RPA85 lines. The RPA85 ribozyme is the active version of the RPA113 ribozyme.
Example 30: Demonstration of Ribozyme Mediated Reduction in Stearoyl-ACP Δ9 Desaturase levels in Maize Leaves (RO) Δ.9 Desaturase Levels in Maize Leaves (R0)
Part A Partial purification of stearoyl-ACP Δ9-desaturase from maize leaves. All procedures were performed at 4°C unless stated otherwise. Maize leaves (50 mg) were harvested and ground to a fine powder in liquid N2 with a mortar and pestle. Proteins were extracted in one equal volume of Buffer A consisting of 25 mM sodium-phosphate pH 6.5, 1 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, 10 mM phenylmethylsulfonyl fluoride, 5 mM leupeptin, and 5 mM antipapin. The crude homogenate was centrifuged for 5 minutes at 10,000 x g. The supernatant was assayed for total protein concentration by Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). One hundred micrograms of total protein was brought up to a final volume of 500 μl in Buffer A, added to 50 μl of mixed SP-sepharose beads (Pharmacia Biotech Inc., Piscataway, NJ), and resuspended by vortexing briefly. Proteins were allowed to bind to sepharose beads for 10 minutes while on ice. After binding, the Δ9 desaturase-sepharose material was centrifuged (10,000 x g) for 10 seconds, decanted, washed three times with Buffer A (500 μl), and washed one time with 200 mM sodium chloride (500 μl). Proteins were eluted by boiling in 50 μl of Treatment buffer (125 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol. and 10% 2-mercaptoethanol) for 5 mintues. Samples were centrifuged (10,000 x g) for 5 minutes. The supernatant was saved for Western anaylsis and the pellet consisting of sepharose beads was discarded.
Part B Western analysis method which was used to demonstrate reductions in stearoyl- ACP Δ9 desaturase. Partially purified proteins were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels (10% PAGE) as described by Lacmmli, U. K. ( 1 70) Cleavage of structural proteins during assembly of the head of phage T4, Nature 227, 660-685. To distinguish variation in Δ9 desaturase levels, included on each blot as a reference was purified and quantified overexpressed Δ9 desaturase from E. coli as described hercforth. Proteins were electrophoretically transferred to ECL1M nitrocellulose membranes (Amersham Life Sciences, Arlington Heights, Illinois) using a Pharmacia Semi-Dry Blotter (Pharmacia Biotech Inc., Piscataway, NJ), using Towbin buffer (Towbin et al. 1979). The nonspecific binding sites were blocked with 10% dry milk in phosphate buffer saline for 1 h. Immunoreactive polypeptides were detected using the ECL™ Western Blotting Detection Reagent (Amersham Life Sciences, Arlington Heights, Illinois) with rabbit antiserum raised against E. coli expressed maize Δ9 desaturase. The antibody was produced according to standard protocols by Berkeley Antibody Co. The secondary antibody was goat antirabbit serum conjugated to horseradish peroxidase (BioRad). Autoradiograms were scanned with a densitometer and quantified based on the relative amount of purified E. coli Δ9 desaturase. These experiments were duplicated and the mean reduction was recorded.
Part C Demonstration of Reductions in Δ9 desaturase levels in R0 maize leaves expressing ribozymes targeted to Δ9 desaturase mRNA. The high stearate transgenic line,
RPA85-15, contains an intact copy of the fused multimer gene. Δ9 desaturase was partially purified from R0 maize leaves, using the protocol described herein. Western analysis was performed on ribozyme active (RPA85-15) and ribozyme inactive
(RPA 113-17) plants and nontransformed (Hill) plants as described above in part B. The natural variation of Δ9 desaturase was determined for the nontransformed line (Hill) by
Western analysis see Figure 29 A. No reduction in Δ9 desaturase was observed with the ribozyme inactive line RPA 113-17, all of which fell within the range as compared to the nontransformed line (Hill). An apparent 50% reduction of Δ9 desaturase was observed in six plants of line RPA85-15 (Figure 29 B) as compared with the controls. Concurrent with this, these same six plants also had increased stearate and reduced Δ9 desaturase mRNA (As described in Examples 28 and 32). However, nine active ribozyme plants from line RPA85-15 did not have any significant reduction as compared with nontransformed line (Hill) and inactive ribozyme line (RPA 1 13- 17) (Figures 29 A and B). Collectively, these results suggest that the ribozyme activity m the six plants from line RPA85-15 is responsible for the reduced Δ9 desaturase.
Example 31 : E. coli Expression and Purification of Maize Δ.-9 desaturase enzyme
Part A The mature protein encoding portion of the maize Δ-9 desaturase cDNA was inserted into the bacterial T7 expression vector pET9D (Novagen Inc., Madison, WI). The mature protein encoding region was deduced from the mature castor bean polypeptide sequence. The alanine at position 32 (nts 239-241 of cDNΛ) was designated as die first residue. This is found within the sequence Ala.Val.Ala.Ser.Met.Thr. Restriction endonuciease Nhe I site was engineered into the maize sequence by PCR, modifying GCCTCC to GCTAGC and a BamHl site was added at the 3' end. This does not change the amino acid sequence of the protein. The cDNA sequence was cloned into pET9d vector using die Nhe I and Bam HI sites. The recombinant plasmid is designated as pDAB428. The maize Δ-9 desaturase protein expressed in bacteria has an additional methionine residue at the 5' end. This pDAB428 plasmid was transformed into the bacterial strain BL21 (Novagen, Inc., Madison, WI) and plated on LB/kanamycin plates (25 mg/ml). Colonies were resuspended in 10 ml LB with kanamycin (25 mg/ml) and IPTG (ImM) and were grown in a shaker for 3 hours at 37°C. The cells were harvested by centrifugation at lOOOxg at 4°C for 10 minutes. The cells were lysed by freezing and thawing the cell pellet 2X, followed by the addition of 1 ml lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.1 % Triton XI 00, 100 ug/ml DNAse I, 100 ug ml RNAse A, and 1 mg/ml lysozyme). The mixture was incubated for 15 minutes at 37°C and then centrifuged at 1000 Xg for 10 minutes at 4°C. The supernatant is used as the soluble protein fraction.
The supernatant, adjusted to 25 mM sodium phosphate buffer (pH 6.0), was chilled on ice for 1 hr. Afterwards, d e resulting flocculant precipitant was removed by centrifugation. The ice incubation step was repeated twice more after which the solution remained clear. The clarified solution was loaded onto a Mono S HR 10/10 column (Pharmacia) that had been equilibrated in 25 mM sodium phosphate buffer (pH 6.0). Basic proteins bound to the column matrix were eluted using a 0-500 mM NaCl gradient over 1 hr (2 ml min; 2 ml fractions). The putative protein of interest was subjected to SDS-PAGE, blotted onto PVDF membrane, visualized with coomassie blue, excised, and sent to Harvard Microchem for amino-terminal sequence analysis. Comparison of the protein's amino terminal sequence to that encoded by the cDNA clone revealed that the protein was indeed Δ 9. Spectrophotometπc analysis of the duron-oxo component associated with the expressed protein (Fox et al., 1993 Proc. Natl. Acad. Sci USA. 90. 2486-2490), as well as identification using a specific πonheme iron stain (Lcong et al., 1992 Anal. Biochem. 207, 317-320) confirmed that the purified protein was Δ-9
Part B Production of polyclonal antiserum
The E. coli produced Δ-9 protein, as determined by amino terminal sequencing, was gel purified via SDS-PAGE, excised, and sent in the gel matrix to Berkeley Antibody Co.,
Richmond, CA, for production of polyclonal sera in rabbits. Titcrs of the antibodies against Δ-9 were performed via western analysis using the ECL Detection system
(Amersham, Inc.)
Part C Purification of Δ9 desaturase from co kernels
Protein Precipitation: Δ9 was purified from com kernels following homogenization using a Warring blender in 25 mM sodium phosphate buffer (pH 7.0) containing 25 mM sodium bisulfite and a 2.5% polyvinylpolypyrrolidone. The crude homogenate was filtered through cheesecloth, centrifuged (10,000xg) for 0.25 h and the resulting supernatant was filtered once more through cheesecloth. In some cases, the supernatant was fractionated via saturated ammonium sulfate precipitation by precipitation at 20% v/v followed by 80% v/v. Extracts obtained from high oil germplasm were fractionated by adding a 50% polyethylene glycol solution (mw=8000) at final concentrations of 5- and 25% v/v. In all cases, the Δ9 protein precipitated at either 80% ammonium sulfate or 25% polyethylene glycol. The resulting pellets were then dialyzed extensively in 25mM sodium phosphate buffer (pH 6.0).
Cation Exchange Chromotography: The solubilized pellet material described above was clarified via centrifugation and applied to Mono S HR 10/10 column equilibrated in 25 mM sodium phosphate buffer (pH 6.0). After extensive column washing, basic proteins bound to the column matrix were eluted using a 0-500 mM NaCl gradient over 1 hr (2 ml/min: 2 ml fractions). Typically, the Δ9 protein eluted between 260-and 350 mM NaCl., as determined by enzymatic and western analysis. After dialysis, this material was further fracionated by acyl earner protein (ACP)- sepharose and phenyl superose chromatography. . „„„«, PCT/US96/11689 97/10328
59
Acyl Carrier Protein-Sepharose Chromatography: AC? was purchased from Sigma Chemical Company and purified via precipitation at pH 4.1 (Rock and Cronan. 1981 J. Biol. Chem. 254, 71 16-7122) before linkage to the beads. ACP-sepharose was prepared by covalently binding 100 mg of ACP to cyanogen bromide activated sepharose 4B beads, essentially as descπbed by Pharmacia, Inc., in the package insert After linkage and blocking of the remaining sites with glycine, the ΛCP-scpharose material was packed into a HR 5/5 column (Pharmacia, Inc.) and equilibrated in 25 mM sodium phosphate buffer (pH 7.0). The dialyzed fractions identified above were then loaded onto the column (McKeon and Stumpf, 1982 J. Biol. Chem. 257, 12141- 12147; Thompson et al, 1991 Proc. Natl. Acad. Sci. USA 88, 2578-2582). After extensive column washing, ΛCP- binding proteins were eluted using 1 M NaCl. Enzymatic and western analysis, followed by amino terminal sequencing, indicated that the eluent contained Δ-9 protein. The Δ-9 protein purified from com was determined to have a molecular size of approximately 38 kDa by SDS-PAGE analysis (Hames, 1981 in Gel Electrophoresis of Proteins: A Practical Approach , eds Hames BD and Rickwood, D. , IRL Press, Oxford).
Phenyl Sepharose Chromatography: The fractions containing Δ9 obtained from the ACP- Sepharose column were adjusted to 0.4 M ammonium sulfate (25 mM sodium phosphate, pH 7.0) and loaded onto a Pharmacia Phenyl Superose column (HR 10/10). Proteins were eluted by running a gradient (0.4 - 0.0 M ammonium sulfate) at 2 ml min for 1 hour. The Δ9 protein typically eluted between 60- and 30 mM ammonium sulfate as detennmed by enzymatic and western analysis.
Example 32: Evidence for the Increase in Stearic Acid in Leaves as a Result of Transformation of Plants with Δ9 Desaturase Ribozymes
Part A Method used to determine the stearic acid levels in plant tissues. The procedure for extraction and esterification of fatty acids from plant tissue was modified from a described procedure (Browse et. al, 1986, Anal. Biochem. 152, 141-145). One to 20 mg of plant tissue was placed in Pyrex 13 mm screw top test tubes. After addition of 1 ml of methanolic HCL (Supelco, Bellefonte, PA), the tubes were purged with nitrogen gas and sealed. The tubes were heated at 80°C for 1 hour and allowed to cool. The heating in the presence of the methanolic HCL results in the extraction as well as the esterification of die fatty acids. The fatty acid methyl esters were removed from the reaction mixture by extraction with hexane. One ml of hexane aid 1 ml of 0.9% (w/v) NaCl was added followed by vigorous shaking of the test tubes. After centrifugation of the tubes at 2000 φm for 5 minutes the top hexane layer was removed and used for fatty acid methyl ester analysis. Gas chromatograph analysis was performed by injection of 1 μl of the sample on a Hewlett Packard (Wilmington, DE) Series II model 5890 gas chromatograph equipped with a flame lonization detector and a J&W Scientific (Folsom, CA) DB-23 column. The oven temperature was 150°C throughout the run and the flow of the carrier gas (helium) was 80 cm/see The run time was 20 minutes The conditions allowed for the separation of the 5 fatty acid methyl esters of interest. C I 0, palmityl methyl ester; C18:0, stearyl methyl ester; C18.1 , oleoyl methyl ester; C I :2, linolcoyl methyl ester; and C18:3, linolenyl methyl ester. Data collection and analysis was performed with a Hewlett Packard Seπes II Model 3396 integrator and a PE Nelson (Pcrkm Elmer, Norwalk, CT) data collection system. The percentage of each fatty acid in the sample was taken directly from the readouts of the data collection system. Quantitative amounts of each fatty acid were calculated using the peak areas of a standard (Matreya, Pleasant Gap, PA) which consisted of a known amount of the five fatty acid methyl esters. The amount calculated was used to estimate the percentage, of total fresh weight, represented by the five fatty acids in the sample. An adjustment was not made for loss of fatty acids during the extraction and esterification procedure. Recovery of the standard sample, after subjecting it to the extraction and esterification procedure (with no tissue present), ranged from 90 to 100% depending on the original amount of the sample. The presence of plant tissue in the extraction mixture had no effect on the recovery of the known amount of standard.
Part B Demonstration of an increase in steanc acid in leaves due to introduction of Δ9 desaturase ribozymes. Leaf tissue from individual plants was assayed for stearic acid as described in Part A. A total of 428 plants were assayed from 35 lines transformed with active Δ9 desaturase nbozymes (RPA85, RPA 1 14, RPA 1 18) and 406 plants from 31 lines transformed with Δ9 desaturase inactive ribozymes (RPA1 13, RPA1 15, RPA1 19). Table XI summarizes the results obtained for stearic acid levels in these plants. Seven percent of the plants from the active lines had stearic acid levels greater than 3%, and 2% had levels greater than 5%. Only 3% of the plants from the inactive lines had stearic acid levels greater than 3%. Two percent of the control plants had leaves with stearate greater than 3%. The controls included 49 non-transformed plants and 73 plants transformed with a gene not related to Δ9 desaturase. There were no plants from the inactive lines or controls that had leaf stearate greater than 4%. Two of the lines transformed with the active Δ9 desaturase ribozyme RPA85 produced many plants which exhibited increased stearate in their leaves. Line RPA85-06 had 6 out of the 15 plants assayed with stearic acid levels which were between 3 and 4 %, about 2-fold greater than the average of the 97/10328
61 controls (Figure 30) The average steaπc acid content of the control plants ( 122 plants) was 1 69% (SD+/-049%) The average stearic acid content of leaves from line RPA85-06 was 2.86% (+/-0 57%) Line RPA85- 15 had 6 out of 15 plants assayed with stearic acid levels which were approximately 4-fold greater than the average of the controls (Figure 31) The average leaf stearic acid content of line RPΛ85- 15 was "! 83% ( i /-2 53%) When the leaf analysis was repeated for RPA85-15 plants, the stcaπc acid level in leaves from plants previously shown to have normal stearic acid levels remained normal and leaves from plants with high stearic acid were again found to be high (Figure 31) The steaπc acid levels in leaves of plants from two lines which were transformed with an inactive Δ9 desaturase ribozyme, RPAl 13, is shown in Figures 32 and 33 RPΛ I 13-06 had three plants with a stearic acid content of 3% or higher The average stcaπc acid content of leaves from line RPAl 13-06 was 2.26% (+/-0 65%) RPA 1 13-17 had no plants with leaf steanc acid content greater than 3% The average stearic acid content of leaves from line RPAl 13-17 was 1 76% (+/-029%) The stearic acid content of leaves from 15 control plants is shown in Figure 34 The average stearic acid content for these 15 control plants was 1 70% (+/-0 6%) When compared to the control and inactive Δ9 desaturase ribozyme data, the results obtained for stearic acid content in RPA85-06 and RPA85-15 demonstrate an increase in steaπc acid content due to the introduction of the Δ9 desaturase ribozyme
Example 33 Inhentance of the High Steaπc Acid Trait in Leaves
Part A Results obtained with steanc acid levels leaves from offspπng of high steaπc acid plants. Plants from line RPA85-15 were pollinated as described herein Twenty days after pollination zygotic embryos were excised from immature kernels from these RPA85-15 plants and placed in a tube on media as described herein for growth of regenerated plantlets. After the plants were transferred to the greenhouse, fatty acid analysis was performed on the leaf tissue. Figure 35 shows the stearic acid levels of leaves from 10 different plants for one of the crosses, RPA85-15 07 selfed Fifty percent of the plants had high leaf steanc acid and 50% had normal leaf steaπc acid. Table XII shows the results from 5 different crosses of RPA85-15 plants The number of plants with high stearic acid ranged from 20 to 50%
Part B Results demonstrating reductions m Δ9 desaturase levels in next generation (RI ) maize leaves expressing ribozymes targeted to Δ9 desaturase mRNA In next generation maize plants that showed a high stearate content (see above Part A), Δ9 desaturase was partially purified from RI maize leaves, using the protocol described herein. Western analysis was performed on several of the high stearate plants. In leaves of next generation plants, a 40-50% reduction of Δ9 desaturase was observed in those plants that had high stearate content (Figure 36). The reduction was comparable to RO maize leaves. This reduction was observed in either OQ414 plants crossed with RPΛ85- I 5 pollen or RPA85-15 plants crossed with self or siblings. Therefore, this suggests that the gene encoding the ribozyme is heritable.
Example 34: Increase in Stearic Acid in Plant Tissues Using Antisense- Δ.9 Desaturase
Part A Method for culturing somatic embryos of maize. The production and regeneration of maize embryogenic callus has been described herein. Somatic embryos make up a large part of this embryogenic callus. The somatic embryos continued to form in callus because e callus was transferred every two weeks. The somatic embryos in embryogenic callus continued to proliferate but usually remained in an early stage of embryo development because of the 2,4-D in the culture medium. The somatic embryos regenerated into plantlets because the callus was subjected to a regeneration procedure described herein. During regeneration the somatic embryo formed a root and a shoot, and ceases development as an embryo. Somatic embryos were made to develop as seed embryos, i.e., beyond the early stage of development found in embryogenic callus and no regeneration, by a specific medium treatment. This medium treatment involved transfer of the embryogenic callus to a Murashige and Skoog medium (MS; described by Murashige and Skoog in 1962) which contains 6% (w/v) sucrose and no plant hormones. The callus was grown on the MS medium with 6% sucrose for 7 days and then the somatic embryos were individually transferred to MS medium with 6% sucrose and 10 μM abscisic acid (ABA). The somatic embryos were assayed for fatty acid composition as described herein after 3 to 7 days of growth on the ABA medium. The fatty acid composition of somatic embryos grown on the above media was compared to the fatty acid composition of embryogenic callus and maize zygotic embryos 12 days after pollination (Table XIII). The fatty acid composition of the somatic embryos was different than that of the embryogenic callus. The embryogenic callus had a higher percentage of C16.0 and C18:3, and a lower percentage of Cl 8: 1 and Cl 8:2. The percentage of lipid represented by the fresh weight was different for the embryogenic callus when compared to the somatic embryos; 0.4% versus 4.0%. The fatty acid composition of the zygotic embryos and somatic embryos were very similar and their percentage of lipid represented by the fresh weight were nearly identical. It was 97/10328
63 concluded that the somatic embryo culture system descπbed above would be an useful in vitro system for testing the effect of certain genes on lipid synthesis in developing embryos of maize.
Part B Increase in steaπc acid in somatic embryos of maize as a result of the introduction of an antisense- Δ9 desaturase gene Somatic embryos were produced using th method described herein from embryogenic callus transformed with pDΛB308/pDΛB430 The somatic embryos from 16 different lines were assayed for fatty acid composition. Two lines, 308/430-12 and 308/430-15, were found to produce somatic embryos with high levels of steaπc acid. The stearic acid content of somatic embryos from these two lines is compared to the stearic acid content of somatic embryos from their control lines in Figures 37 and 38. The control lines were from the same culture that the transformed lines came from except that they were not transformed. For line 308/430-12, stearic acid in somatic embryos ranged from 1 to 23% while the controls ranged from 0.5 to 3%. For line 308/430-15, steanc acid in somatic embryos ranged from 2 to 15% while the controls ranged from 0.5 to 3%. More than 50% of the somatic embryos had stearic acid levels which were above the range of the controls m both the transformed lines. The above results indicate that an antisense- Δ9 desaturase gene can be used to raise the stearic acid levels in somatic embryos of maize.
Part C Demonstration of an increase in stearic acid in leaves due to introduction of an antisense- Δ9 desaturase gene. Embryogenic cultures from lines 308/430-12 and 308/430- 15 were used to regenerate plants. Leaves from these plants were analyzed for fatty acid composition using the method previously described. Only 4 plants were obtained from the 308/430-15 culture and the stearic acid level in the leaves of these plants were normal, 1-2%). The stearic acid levels in leaves from plants of line 308/430-12 are shown in Figure 39. The stearic acid levels in leaves ranged from 1 to 13% in plants from line 308/430-12. About 30% of the plants from line 308/430-12 had stearic acid levels above the range observed in the controls, 1-2%. These results indicate that the stearic acid levels can be raised in leaves of maize by introduction of an antisense- Δ9 desaturase gene.
By "antisense" is meant a non-enzymatic nucleic acid molecule that binds to a RNA (target RNA) by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004). Example 35' Amylose Content Assav of Maize Pooled Starch Sample and Sinnle Kernel
The amylose content was assayed by the method of Hovenkamp-Hermelink et al
(Potato Research 31 241-246) with modifications For pooled starch sample, 10 mg to 100 mg starch was dissolved in 5 ml 45% perchloric acid in plastic culture tube The solution was mixed occasionally by vortexing After one hour, 0 2 l of the starch solution was diluted to 10 ml by H2O 04 ml of the diluted solution was then mixed with 0 5 ml diluted Lugol's solution (Sigma) in 1 ml cuvet Readings at 618 nm and 550 nm were immediately taken and the R ratio (618 nm/550 nm) was calculated Using standard equation P (percentage of amylose) = (4 5R-2 6)/(7 3-3 R) generated from potato amylose and maize amylopectin (Sigma, St Louis), amylose content was determined For frozen single kernel sample, same procedure as above was used except it was extracted in 45% perchloπc acid for 20 mm instead for one hour
Example 36- Starch Puπfication and Granular Bound Starch Svnthase (GBSS) Assay
The puπfication of starch and following GBSS activity assay were modified from the methods of Shure et al. (Cell, 35:225-233, 1983) and Nelson et al. (Plant Physiology, 62:383-386, 1978). Maize kernel was homogenized in 2 volume (v/w) of 50 mM Tns- HCl, pH 8.0, 10 mM EDTA and filtrated through 120 μm nylon membrane. The matenal was then centnfuged at 5000 g for 2 mm and the supernatant was discarded The pellet was washed three times by resuspending in water and removing supernatant by centrifugation After washing, die starch was filtrated through 20 μm nylon membrane and centnfuged. Pellet was then lyophilized and stored in - 20 °C until used for activity assay.
A standard GBSS reaction mixture contained 0.2 M Tπcine, pH 8.5, 25 mM Glutathione, 5 mM EDTA, 1 mM ^4C ADPG (6 nci/μmol), and 10 mg starch in a total volume of 200 μl. Reactions were conducted at 37 °C for 5 min and terminated by adding 200 μl of 70% ethanol (v/v) m 0.1 M KCI. The matenal was centnfuged and unmcoφorated ADPG in the supernatant is removed The pellet was then washed four time with 1ml water each in the same fashion After washing, pellet was suspended in 500 μl water, placed into scintillation vial, and the incoφorated ADPG was counted by a Beckman (Fullerton, CA) scintillation counter Specific activity was given as pmoles of ADPG incoφorated into starch per mm per mg starch 97/10328
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Example 37 Analysis of Antisense-GBSS Plants
Because of the segregation of R2 seeds, single kernels should therefore be analyzed for amylose content to identify phenotype. Because of the large amount of samples generated in this study, a two-step screening strategy was used In the first step, 30 kernels were taken randomly from the same ear, freezc-dπcd and homogenized into starch flour. Amylose assays on the starch flours were carried out. Lines with reduced amylose content were identified by statistical analysis. In the second step, amylose content of the single kernels m the lines with reduced amylose content was further analyzed (25 to 50 kernels per ear). Two sets of controls were used in the screening, one of the sets were untransformed lines with the same genetic background and the other were transformed lines which did not carry transgene due to segregation (Southern analysis negative line)
81 lines representing 16 transformation events were examined at the pooled starch level. Among those lines, six with significant reduction of amylose content by statistical analysis were identified for further single kernel analysis One line, 308/425-12.2.1 , showed significant reduction of amylose content (Figure 40).
Twenty five individual kernels of CQ806, a conventional maize inbred line, were analyzed. The amylose content of CQ806 ranged from 24.4% to 32.2%, averaging 29.1%). The single kernel distribution of amylose content is skewed slightly towards lower amylose contents. Forty nine single kernels of 308/425-12.2.1.1 were analyzed. Given that 308/425-12.2.1.1 resulted from self pollination of a hemizygous individual, the expected distribution would consist of 4 distinct genetic classes present in equal frequencies since endosperm is a triploid tissue. The 4 genetic classes consist of individuals carrying 0, 1, 2, and 3 copies of the antisense construct. If there is a large dosage effect for the transgene, then the distπbution of amylose contents would be tetramodal. One of the modes of the resulting distribution should be indistinguishable from the non-transgenic parent. If there is no dosage effect for the transgene (individuals carrying 1, 2 or 3 copies of the transgene are phenotypically equivalent), then the distribution should be bimodal with one of the modes identical to the parent The number of individuals included in the modes should be 3:1 of transgenieparental The distribution for 308/425-12.2.1.1 is distinctly trimodal. The central mode is approximately twice the size of either other mode. The two distal modes are of approximately equal size. Goodness of fit to a 1 :2: 1 ratio was tested and the fit was excellent. 97/10328
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Further evidence was available demonstrating that the mode with the highest amylose content was identical to the non-transgenic parent. This was done using discriminant analysis. The CQ806 and 308/425-12.2.1.1 data sets were combined for this analysis. The distance metrics used in the analysis were calculated using amylose contents only. The estimates of variance from the individual analyses were used in all tests. No pooled estimate of variance was employed. The original data was lesicd for reclassification. Based on the discriminant analysis, the entire mode of the 308/425- 12.2.1.1 distribution with the highest amylose content would be more appropriately classified as parental. This is strong confirmation that this mode of the distribution is parental. Of the remaining two modes, the central mode is approximately twice the size of the lowest amylose content mode. This would be expected if the central mode includes two generic classes: individuals with 1 or 2 copies of the antisense construct. The mode with the lowest amylose content thus represents those individuals which are fully homozygous (3 copies) for the antisense construct. The 2: 1 ratio was tested and could not be rejected on the basis of the data.
This analysis indicates that the antisense GBSS gene as functioning in 308/425- 12.2.1.1 demonstrates a dosage dependent reduction in amylose content of maize kernels.
Example 38: Analysis of Ribozyme-GBSS Plants
The same two-step screening strategy as in the antisense study (Example 37) was used to analyze ribozyme-GBSS plants. 160 lines representing 1 1 transformation events were examined in the pooled starch level. Among the control lines (both untransformed line and Southem negative line), the amylose content varied from 28% to 19%. No significant reduction was observed among all lines carrying ribozyme gene (Southem positive line). More than 20 selected lines were further analyzed in the single kernel level, no significant amylose reduction as well as segregation pattern were found. It was apparent that ribozyme did not cause any alternation in the phenotypic level.
Transformed lines were further examined by their GBSS activity (as described in Example 36). For each line, 30 kernels were taken from the frozen ear and starch was purified. Table XIV shows the results of 9 plants representing one transformation event of the GBSS activity in the pooled starch samples, amylose content in the pooled starch samples, and Southem analysis results. Three southem negative lines: RPA63.0283, RPA63.0236, and RPA63.0219 were used as control. The GBSS activities of control lines RPA63.0283, RPA63.0236, and RPA63.0219 were around 300 units/mg starch. In lines RPA63.021 1 , RPA63.0218, RPA63.0209, and RPA63.0210, a reduction of GBSS activity to more than 30% was observed. The correlation of varied GBSS activity to the Southern analysis in this group (from RPA63.0314 to RPA63.0210 of Table XIV) indicated that the reduced GBSS activity was caused by the expression of ribozyme gene incorporated into the maize genome
GBSS activities at the single kernel level of line RPΛ 63.021 (Southern positive and reduced GBSS activity in pooled starch) was further examined, using RPΛ63.0306 (Southern negative and GBSS activity normal in pooled starch) as control. About 30 kernels from each line were taken, and starch samples were purified from each kernel individually. Figure 41 clearly indicated reduced GBSS activity in line RPA63.0218 compared to RPA63.0306.
Other embodiments are within die following claims.
Table I
68
TABLE 1
Characteristics of naturally occurring ribozymes
Group I Introns
• Size: -150 to > 1000 nucleotides.
• Requires a U in the target sequence immediately 5' of the cleavage site
• Binds 4-6 nucleotides at the 5'-side of the cleavage site
• Reaction mechanism: anack by the 3'-OH of guanosine to generate cleavage products with 3'-OH and 5 "-guanosine
• Additional protein cofactors required in some cases to help folding and maintamance of the active structure ['].
• Over 300 known members of this class. Found as an intervening sequence in Terrahvmena thermophila rRNA. fungal mitochondria, chloroplasts. phage T4. blue-green algae, and others.
• Major structural features largely established through phylogenetic comparisons, mutagenesis. and biochemical studies [V]
• Complete kinetic framework established for one ribozyme [VΛ7].
• Studies of ribozyme folding and substrate docking underway [V,10].
• Chemical modification investigation of important residues well established f",12].
• The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" b-gaiactosidase message by the ligation of new b-galactosidase sequences onto the defective message ["].
RNAse P RNA (M1 RNA)
• Size: -290 to 400 nucleotides.
• RNA portion of a ubiquitous nbonucleoprotein enzyme.
• Cleaves tRNA precursors to form mature tRNA [l4].
• Reaction mechanism: possible anack by M**-OH to generate cleavage products with 3'-OH and 5 "-phosphate.
• RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.
• Recruitment of endogenous RNAse P for therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA [15,16]
• Important phosphate and 2' OH contacts recently identified [","]
Group II Introns
• Size: >1000 nucleotides.
• Trans cleavage of target RNAs recently demonstrated f",20].
• Sequence requirements not fully determined.
• Reaction mechanism. 2"-OH of an internal adenosine generates cleavage products with 3'-OH and a "lariat" RNA containing a 3"-5" and a 2'-5" branch point.
• Only natural ribozyme with demonstrated participation in DNA cleavage [2 ~] in addition to RNA cleavage and ligation
• Major structural features largely established through phylogenetic comparisons [: ] Table 1
69
• Important 2* OH contacts beginning to be identified [24]
• Kinetic framework under development [25]
Neurospora VS RNA
• Size: ~ 144 nucleotides.
• Trans cleavage of haiφin target RNAs recently demonstrated [26].
• Sequence requirements not fully determined.
• Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2\3"-cyclic phosphate and 5'-OH ends.
• Binding sites and structural requirements not fully determined.
• Only 1 known member of this class. Found in Neurospora VS RNA.
Hammerhead Ribozyme
(see text for references)
• Size: -13 to 40 nucleotides.
• Requires the target sequence UH immediately 5' of the cleavage site.
• Binds a variable number nucleotides on both sides of the cleavage site.
• Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2\3'-cyclic phosphate and 5'-OH ends.
• 14 known members of this class. Found in a number of plant padiogens (virusoids) that use RNA as the infectious agent.
• Essential structural features largely defined, including 2 crystal structures []
• Minimal ligation activity demonstrated (for engineering through in vitro selection) []
• Complete kinetic framework established for two or more ribozymes [].
• Chemical modification investigation of important residues well established [].
Hairpin Ribozyme
• Size: ~50 nucleotides.
• Requires the target sequence GUC immediately 3' of the cleavage site.
• Binds 4-6 nucleotides at the 5'-side of the cleavage site and a variable number to the 3'-side of the cleavage site.
• Reaction mechanism: attack by 2 -OH 5' to the scissile bond to generate cleavage products with 2', 3 '-cyclic phosphate and 5' -OH ends.
• 3 known members of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent.
• Essential structural features largely defined [21,2t,29,i0]
• Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [3I]
• Complete kinetic framework established for one ribozyme [32].
• Chemical modification investigation of important residues begun [","].
Hepatitis Delta Virus (HDV) Ribozyme
• Size: ~60 nucleotides.
• Trans cleavage of target RNAs demonstrated ["].
• Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Folded ribozyme contains a pseudoknot structure [36]. Table I
70
• Reaction mechanism: attack b\ 2*-OH 5 ' to the scissile bond to generate cleavage products with 2'.3"-cyclιc phosphate and 5"-OH ends.
• Only 2 known members of this class. Found in human HDV
• Circular form of HDV is active and shows increased nuclease stability [37]
1 Mohr, G.. Caprara. M.G.. Guo, Q.. Lambowitz. A M Nature. 370. 147-150 ( 1994)
2 Michel. Francois: Westhof. Eric. Slippery substrates Nat. Struct. Biol (1994), 1( 1), 5-7
3 Lisacek, Fredenque: Diaz. Yolande: Michel. Francois Automatic identification of group I intron coresin genomic DNA sequences. J. Mol. Biol. (1994), 235(4). 1206-17.
4 Herschiag, Daniel; Cech. Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry (1990), 29(44), 10159-71.
5 Herschiag, Daniel, Cech. Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry (1990). 29(44), 10172-80.
6. Knitt, Deborah S.: Herschiag. Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an
Unconventional Origin of an Apparent pKa. Biochemistry ( 1 96). 35(5). 1560-70.
7 Bevilacqua, Philip C: Sugimoto. Naoki: Turner. Douglas H.. A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena πbozyme. Biochemistry (1996), 35(2), 648-58.
8. Li, Yi; Bevilacqua. Philip C: Mathews. David, Turner. Douglas H.. Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is dπven by a favorable entropy change. Biochemistry (1995), 34(44), 14394-9.
9. Banerjee, Alo e Raj, Turner. Douglas H.. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12.
10. Zaninkar, Patrick P.: Williamson. James R.. The P9.1 -P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996). 24(5). 854-8.
1 1. Strobel. Scott A.; Cech. Thomas R.. Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington. D. C.) (1995), 267(5198), 675-9
12. Strobel. Scott A.; Cech, Thomas R.. Exocyciic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splιce Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11.
13. Sullenger, Bruce A.: Cech, Thomas R.. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) ( 1994). 371 (6498), 619-22.
14. Robertson. H.D.: Altman, S.. Smith, J.D. J. Biol. Chem., 2 1. 5243-5251 (1972).
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16. Yuan, Y.; Hwang, E. S.; Altman. S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10.
17 Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 1(2). 210-18.
18. Pan, Tao; Loria, Andrew; Zhong. Kun. Probing of ternary interactions in RNA 2'-hydroxyl-base contacu between the RNase P RNA and pre-tRNA. Proc Natl Acad. Sci. U. S. A. (1995). 92(26). 12510- 14.
19. Pyle, Anna Marie; Green. Justin B.. Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry (1994). 33(9). 2716-25.
20. Michels. William J. Jr.; Pyle, Anna Marie. Conversion of a Group II intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides Elucidation of Reaction Mechanism and Structure/Function Relationships. Biochemistry ( 1995). 34(9), 2965-77
21. Zimmerly, Steven; Guo. Huatao; Eskes. Robert: Yang. Jian: Perl an. Philip S.: Lambowitz. Alan M. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge. Mass.) (1995), 83(4). 529-38.
22. Griffin. Edmund A.. Jr.; Qin. Zhifeng; Michels. Williams J.. Jr.. Pyle. Anna Marie. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2'-hydroxyl groups. Chem. Biol (1995), 2(1 1). 761-70 Table 1
23 Michel. Francois. Ferat. Jean Luc. Structure and activities of group II introns Annu. Rev Biochem ( 1995). 64. 435-61
24 Abramovitz. Dana L.. Friedman. Richard A.; Pyle. Anna Mane Catalytic role of 2 -hydroxyl groups within a group II intron active site. Science (Washington, D. C ) ( 1996). 271 (5254). 1410-13.
25 Daniels. Danette L.. Michels. William J., Jr., Pyle. Anna Mane Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J Mol. Biol ( 1996), 256( 1 ). 31-49
26 Guo. Hans C. T.: Collins, Richard A. Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995). 14(2). 368-76
27 Hampel. Arnold; Tritz. Richard; Hicks. Margaret: Cruz. Phillip 'Hairpin' catalytic RNA model, evidence for helixes and sequence requirement for substrate RNA Nucleic Acids Res (1990). 18(2). 299-304
28 Chowrira, Bharat M.: Berzal-Herranz, Alfredo: Burke. John M. Novel guanosine requirement for catalysis by the haiφin ribozyme. Nature (London) (1991 ), 354(6351 ). 320-2
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Essential nucleotide sequences and secondary structure elements of the haiφin ribozyme EMBO J. (1993), 12(6), 2567-73.
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Table II
72
Table II: 2.5 μmol RNA Synthesis Cycle
Figure imgf000074_0001
Wait time does not include contact time duπng delivery.
Table IIIA
73
Table IIIA: GBSS Hammerhead Substrate Sequence
Substrate Seq. ID No.
GGUCGUCUC UCCCCGCU 27
UCGUCUCUC CCCGCUAC 29
UCCCCGCUA CGACCAGU 31
CGACCAGUA CAAGGACG 33
ACCAGCGUC GUGUCCGA 35
CGUCGUGUC CGAGAUCA 37
UCCGAGAUC AAGAUGGG 39
AGACAGGUA CGAGACGG 41
GAGACGGUC AGGUUCUU 43
GGUCAGGUU CUUCCACU 45
GUCAGGUUC UUCCACUG 47
CAGGUUCUU CCACUGCU 49
AGGUUCUUC CACUGCUA 51
CCACUGCUA CAAGCGCG 53
CCGCGUGUU CGUUGACC 55
CGCGUGUUC GUUGACCA 57
GUGUUCGUU GACCACCC 59
CCCACUGUU CCUGGAGA 61
CCACUGUUC CUGGAGAG 63
GAGAGGGUU UGGGGAAA 65
AGAGGGUUU GGGGAAAG 67
GAGAAGAUC UACGGGCC 69
GAAGAUCUA CGGGCCUG 71
AACGGACUA CAGGGACA 73
GCUGCGGUU CAGCCUGC 75
CUGCGGUUC AGCCUGCU 77
AGCCUGCUA UGCCAGGC 79
GCAGCACUU GAAGCUCC 81
UUGAAGCUC CAAGGAUC 83
CCAAGGAUC CUGAGCCU 85
CUGAGCCUC AACAACAA 87
CAACCCAUA CUUCUCCG 89
CCCAUACUU CUCCGGAC 91
CCAUACUUC UCCGGACC 93
AUACUUCUC CGGACCAU 95
CGGACCAUA CGGGGAGG 97
GAGGACGUC GUGUUCGU 99
CGUCGUGUU CGUCUGCA 101
GUCGUGUUC GUCUGCAA 103
GUGUUCGUC UGCAACGA 105
CCGGCCCUC UCUCGUGC 107
GGCCCUCUC UCGUGCUA 109
CCCUCUCUC GUGCUACC 111
AUGGACGUC AGCGAGUG 113
GGACAAGUA CAUCGCCG 115
AAGUACAUC GCCGUGAA 117
CGUGAAGUA CGACGUGU 119
CGACGUGUC GACGGCCG 121
GCGGAGGUC GGGCUCCC 123
GUCGGGCUC CCGGUGGA 125
CGGAACAUC CCGCUGGU 127
Figure imgf000075_0001
Figure imgf000075_0002
GGUGGCGUU CAUCGGCA 129 Table IIIA
74
Figure imgf000076_0001
Figure imgf000076_0002
Figure imgf000076_0003
Table IIIA
75
nt. Substrate Seq. ID Position No.
1878 CGGUAAUUU UAUAUUGC 211 1880 GGUAAUUUU AUAUUGCG 213 1882 GUAAUUUUA UAUUGCGA 215 1922 AAUUUUAUA UUGCGAGU 217 1928 UUUUAUAUU GCGAGUAA 219 1934 UUGCGAGUA AAUAAAUG 221 1955 GAGUAAAUA AAUGGACC 223 1970 GGACCUGUA GUGGUGGA 225 1979 2012 2013 2033 2035 2055 2063 2065 2066 2068 2069 2071 2073 2080 2081 2082 2085 2086 2087 2094 2104 2110 2117 2121 2127 2132 2135 2137 2142 2165 2168 2181 2184 2188 2197 2200 2201 2205 2211 2215 2218
Figure imgf000077_0001
Table IUB
76
Table III B: Hammerhead Ribozyme Sequence Targeted Against GBSS mRNA
nt. Position HH Ribozyme Sequence Seq. ID
No.
12 UGGCUGUGGC CUGAUGA X GAA AUCGAUCGGU 267
68 GCAGUGAGUU CUGAUGA X GAA AUUCCUUCCU 268
73 GGCUGGCAGU CUGAUGA X GAA AGUUUAUUCC 269
103 GACGGAGCAG CUGAUGA X GAA ACACUUCUCC 270
109 CUGGUGGACG CUGAUGA X GAA AGCAGUACAC 271
113 CGCACUGGUG CUGAUGA X GAA ACGGAGCAGU 272
146 UCGACGAGAU CUGAUGA X GAA AGCAGCCCUG 273
149 UCGUCGACGA CUGAUGA X GAA AUGAGCAGCC 274
151 GGUCGUCGAC CUGAUGA X GAA AGAUGAGCAG 275
154 ACUGGUCGUC CUGAUGA X GAA ACGAGAUGAG 276
169 CAUGCCGAUU CUGAUGA X GAA AUCCACUGGU 277
170 CCAUGCCGAU CUGAUGA X GAA AAUCCACUGG 278 173 CCGCCAUGCC CUGAUGA X GAA AUUAAUCCAC 279 186 GACGUGGCU A CUGAUGA X GAA AGCCGCCAUG 280 188 GCGACGUGGC CUGAUGA X GAA AGAGCCGCCA 281 196 GACGAGCUGC CUGAUGA X GAA ACGUGGCUAG 282 203 GCGUUGCGAC CUGAUGA X GAA AGCUGCGACG 283 206 CGCGCGUUGC CUGAUGA X GAA ACGAGCUGCG 284 230 ACGCGUCCGG CUGAUGA X GAA ACGCCCAGGC 285 241 GCGGAACGUG CUGAUGA X GAA ACGCGUCCGG 286
247 GCCGCGGCGG CUGAUGA X GAA ACGUGGACGC 287
248 CGCCGCGGCG CUGAUGA X GAA AACGUGGACG 288 292 GUCCGCCGCC CUGAUGA X GAA ACGCCGUCCG 289 308 UCCGAAUGCU CUGAUGA X GAA AGCGUGUCCG 290
314 CGCUGGUCCG CUGAUGA X GAA AUGCUGAGCG 291
315 GCGCUGGUCC CUGAUGA X GAA AAUGCUGAGC 292 344 GCUGGUGCUG CUGAUGA X GAA AGCCUGGGCG 293
385 GAGCGACGGG CUGAUGA X GAA ACCUGGCCCC 294
386 CGAGCGACGG CUGAUGA X GAA AACCUGGCCC 295 391 CACGACGAGC CUGAUGA X GAA ACGGGAACCU 296 395 CGCACACGAC CUGAUGA X GAA AGCGACGGGA 297 398 UGGCGCACAC CUGAUGA X GAA ACGAGCGACG 298 425 CGACGAAGAC CUGAUGA X GAA ACGUUCAUGC 299 428 CGCCGACGAA CUGAUGA X GAA ACGACGUUCA 300
430 GGCGCCGACG CUGAUGA X GAA AGACGACGUU 301
431 CGGCGCCGAC CUGAUGA X GAA AAGACGACGU 302 434 UCUCGGCGCC CUGAUGA X GAA ACGAAGACGA 303 473 GGACGUCGCC CUGAUGA X GAA AGGCCGCCGG 304 482 GGCCGCCGAG CUGAUGA X GAA ACGUCGCCGA 305 485 GCAGGCCGCC CUGAUGA X GAA AGGACGUCGC 306 527 AGACGACCAU CUGAUGA X GAA ACACGGUGCC 307 533 GGGGAGAGAC CUGAUGA X GAA ACCAUGACAC 308 536 AGCGGGGAGA CUGAUGA X GAA ACGACCAUGA 309 538 GUAGCGGGG A CUGAUGA X GAA AGACGACCAU 310 540 UCGUAGCGGG CUGAUGA X GAA AGAGACGACC 311 Table IIIB
77
nt. Position HH Ribozyme Sequence Seq. ID No.
547 GUACUGGUCG CUGAUGA X GAA AGCGGGGAGA 312
556 GGCGUCCUUG CUGAUGA X GAA ACUGGUCGUA 313
581 UCUCGGACAC CUGAUGA X GAA ACGCUGGUGU 314
586 CUUGAUCUCG CUGAUGA X GAA ACACGACGCU 315
593 CUCCCAUCUU CUGAUGA X GAA AUCUCGGACA 316
610 GACCGUCUCG CUGAUGA X GAA ACCUGUCUCC 317
620 GGAAGAACCU CUGAUGA X GAA ACCGUCUCGU 318
625 GCAGUGGAAG CUGAUGA X GAA ACCUGACCGU 319
626 AGCAGUGGAA CUGAUGA X GAA AACCUGACCG 320
628 GUAGCAGUGG CUGAUGA X GAA AGAACCUGAC 321
629 UGUAGCAGUG CUGAUGA X GAA AAGAACCUGA 322 637 UCCGCGCUUG CUGAUGA X GAA AGCAGUGGAA 323
661 GUGGUCAACG CUGAUGA X GAA ACACGCGGUC 324
662 GGUGGUCAAC CUGAUGA X GAA AACACGCGGU 325 665 GUGGGUGGUC CUGAUGA X GAA ACGAACACGC 326
679 CCUCUCCAGG CUGAUGA X GAA ACAGUGGGUG 327
680 CCCUCUCCAG CUGAUGA X GAA AACAGUGGGU 328
692 UCUUUCCCCA CUGAUGA X GAA ACCCUCUCCA 329
693 GUCUUUCCCC CUGAUGA X GAA AACCCUCUCC 330 716 CAGGCCCGUA CUGAUGA X GAA AUCUUCUCCU 331 718 GUCAGGCCCG CUGAUGA X GAA AGAUCUUCUC 332 742 GUUGUCCCUG CUGAUGA X GAA AGUCCGUUCC 333
763 UAGCAGGCUG CUGAUGA X GAA ACCGCAGCUG 334
764 AUAGCAGGCU CUGAUGA X GAA AACCGCAGCU 335 773 CUGCCUGGCA CUGAUGA X GAA AGCAGGCUGA 336 788 UUGGAGCUUC CUGAUGA X GAA AGUGCUGCCU 337 795 AGGAUCCUUG CUGAUGA X GAA AGCUUCAAGU 338 803 UGAGGCUCAG CUGAUGA X GAA AUCCUUGGAG 339 812 GGUUGUUGUU CUGAUGA X GAA AGGCUCAGGA 340 826 UCCGGAGAAG CUGAUGA X GAA AUGGGUUGUU 341
829 UGGUCCGGAG CUGAUGA X GAA AGUAUGGGUU 342
830 AUGGUCCGGA CUGAUGA X GAA AAGUAUGGGU 343 832 GUAUGGUCCG CUGAUGA X GAA AGAAGUAUGG 344 841 GUCCUCCCCG CUGAUGA X GAA AUGGUCCGGA 345 854 AGACGAACAC CUGAUGA X GAA ACGUCCUCCC 346
859 GUUGCAGACG CUGAUGA X GAA ACACGACGUC 347
860 CGUUGCAGAC CUGAUGA X GAA AAC AC GAC GU 348 863 AGUCGUUGCA CUGAUGA X GAA ACGAACACGA 349 888 UAGCACGAGA CUGAUGA X GAA AGGGCCGGUG 350 890 GGUAGCACGA CUGAUGA X GAA AGAGGGCCGG 351 892 GAGGUAGCAC CUGAUGA X GAA AGAGAGGGCC 352 898 GCUCUUGAGG CUGAUGA X GAA AGCACGAGAG 353 902 AGUUGCUCUU CUGAUGA X GAA AGGUAGCACG 354 913 GUGGGACUGG CUGAUGA X GAA AGUUGCUCUU 355 919 GAUGCCGUGG CUGAUGA X GAA ACUGGUAGUU 356 929 CGUCCCUGUA CUGAUGA X GAA AUGCCGUGGG 357 931 UGCGUCCCUG CUGAUGA X GAA AGAUGCCGUG 358
951 UGGAUGCAGA CUGAUGA X GAA AGCGGUCUUU 359
952 GUGGAUGCAG CUGAUGA X GAA AAGCGGUCUU 360
953 UGUGGAUGCA CUGAUGA X GAA AAAGCGGUCU 361 959 AGAUGUUGUG CUGAUGA X GAA AUGCAGAAAG 362 968 CCUGGUAGGA CUGAUGA X GAA AUGUUGUGGA 363 Table IIIB
78
nt. Position HH Ribozyme Sequence Seq. ID No.
970 GCCCUGGUAG CUGAUGA X GAA AGAUGUUGUG 364
973 CCGGCCCUGG CUGAUGA X GAA AGGAGAUGUU 365
985 GGAGAAGGCG CUGAUGA X GAA ACCGGCCCUG 366
986 CGGAGAAGGC CUGAUGA X GAA AACCGGCCCU 367
991 GUAGUCGGAG CUGAUGA X GAA AGGCGAACCG 368
992 GGUAGUCGGA CUGAUGA X GAA AAGGCGAACC 369 994 CGGGUAGUCG CUGAUGA X GAA AGAAGGCGAA 370 1000 CAGCUCCGGG CUGAUGA X GAA AGUCGGAGAA 371 1016 AUCUCUCCGG CUGAUGA X GAA AGGUUCAGCU 372
1027 GGACGACUUG CUGAUGA X GAA AUCUCUCCGG 373
1028 AGGACGACUU CUGAUGA X GAA AAUCUCUCCG 374 1033 AUCGAAGGAC CUGAUGA X GAA ACUUGAAUCU 375 1036 GAAAUCGAAG CUGAUGA X GAA ACGACUUGAA 376
1039 GAUGAAAUCG CUGAUGA X GAA AGGACGACUU 377
1040 CGAUGAAAUC CUGAUGA X GAA AAGGACGACU 378
1044 CCGUCGAUGA CUGAUGA X GAA AUCGAAGGAC 379
1045 GCCGUCGAUG CUGAUGA X GAA AAUCGAAGGA 380
1046 AGCCGUCGAU CUGAUGA X GAA AAAUCGAAGG 381 1049 CGUAGCCGUC CUGAUGA X GAA AUGAAAUCGA 382 1057 GGGCUUCUCG CUGAUGA X GAA AGCCGUCGAU 383 1085 UCAUCCAGUU CUGAUGA X GAA AUCUUCCGGC 384 1106 CGGCCUCGAG CUGAUGA X GAA AUCCCGGCCU 385 1109 UGUCGGCCUC CUGAUGA X GAA AGGAUCCCGG 386 1124 UGACGGUGAG CUGAUGA X GAA ACCCUGUCGG 387 1127 GGCUGACGGU CUGAUGA X GAA AGGACCCUGU 388 1133 AGUAGGGGCU CUGAUGA X GAA ACGGUGAGGA 389 1141 CUCGGCGUAG CUGAUGA X GAA AGGGGCUGAC 390 1144 CUCCUCGGCG CUGAUGA X GAA AGUAGGGGCU 391 1157 UGCCGGAGAU CUGAUGA X GAA AGCUCCUCGG 392 1160 CGAUGCCGGA CUGAUGA X GAA AUGAGCUCCU 393 1162 GGCGAUGCCG CUGAUGA X GAA AGAUGAGCUC 394 1169 AGCCCCUGGC CUGAUGA X GAA AUGCCGGAGA 395 1187 UGAUGUUGUC CUGAUGA X GAA AGCUCGCAGC 396 1196 UGAGGCGCAU CUGAUGA X GAA AUGUUGUCGA 397 1205 UGAUGCCGGU CUGAUGA X GAA AGGCGCAUGA 398 1214 CGAUGCCGGU CUGAUGA X GAA AUGCCGGUGA 399 1223 UGCCGUUGAC CUGAUGA X GAA AUGCCGGUGA 400 1226 CCAUGCCGUU CUGAUGA X GAA ACGAUGCCGG 401 1241 CCCACUCGCU CUGAUGA X GAA ACGUCCAUGC 402 1270 CACGGCGAUG CUGAUGA X GAA ACUUGUCCCU 403 1274 ACUUCACGGC CUGAUGA X GAA AUGUACUUGU 404 1285 CGACACGUCG CUGAUGA X GAA ACUUCACGGC 405 1294 CACGGCCGUC CUGAUGA X GAA ACACGUCGUA 406 1346 CCGGGAGCCC CUGAUGA X GAA ACCUCCGCCU 407 1352 GGUCCACCGG CUGAUGA X GAA AGCCCGACCU 408 1370 CCACCAGCGG CUGAUGA X GAA AUGUUCCGGU 409
1384 CCUGCCGAUG CUGAUGA X GAA ACGCCACCAG 410
1385 GCCUGCCGAU CUGAUGA X GAA AACGCCACCA 411 1388 CCAGCCUGCC CUGAUGA X GAA AUGAACGCCA 412 1421 CGGCCGCCAU CUGAUGA X GAA ACGUCGGGUC 413 1436 UGAGCUGCGG CUGAUGA X GAA AUGGCGGCCG 414 1445 CCAUCUCCAU CUGAUGA X GAA AGCUGCGGGA 415 Table IIIB
79
nt. Position HH Ribozyme Sequence Seq. ID
No.
1472 CCAGCAGAAC CUGAUGA X GAA AUCUGCACGU 416
1475 UGCCCAGCAG CUGAUGA X GAA ACGAUCUGCA 417
1476 GUGCCCAGCA CUGAUGA X GAA AACGAUCUGC 418
1501 CAUGCGCUCG CUGAUGA X GAA ACUUCUUCUU 419
1502 GCAUGCGCUC CUGAUGA X GAA AACUUCUUCU 420 1514 CGGCGCUCAU CUGAUGA X GAA AGCAUGCGCU 421
1534 CUUGCCUGGG CUGAUGA X GAA ACUUCUCCUC 422
1535 CCUUGCCUGG CUGAUGA X GAA AACUUCUCCU 423 1559 CGUUGAACUU CUGAUGA X GAA ACCACGGCGC 424
1564 CGCCGCGUUG CUGAUGA X GAA ACUUGACCAC 425
1565 GCGCCGCGUU CUGAUGA X GAA AACUUGACCA 426 1589 CGCCGGCCAU CUGAUGA X GAA AUGUGGUGCG 427 1610 UGGUGACGGC CUGAUGA X GAA AGCACGUCGG 428 1616 AGCGGCUGGU CUGAUGA X GAA ACGGCGAGCA 429
1627 GCAGGGCUCG CUGAUGA X GAA AGCGGCUGGU 430
1628 CGCAGGGCUC CUGAUGA X GAA AAGCGGCUGG 431 1643 GCAGCUGGAU CUGAUGA X GAA AGGCCGCAGG 432 1646 CCUGCAGCUG CUGAUGA X GAA AUGAGGCCGC433
1666 GGGCGUUCCG CUGAUGA X GAA AUCGCAUCCC 434
1690 UCCACCGGUG CUGAUGA X GAA ACGCGCAGGC 435
1703 UGGUGUCGAC CUGAUGA X GAA AGUCCACCGG 436
1706 UGAUGGUGUC CUGAUGA X GAA ACGAGUCCAC 437
1715 UGCCUUCGAU CUGAUGA X GAA AUGGUGUCGA 438
1718 UCUUGCCUUC CUGAUGA X GAA AUGAUGGUGU 439
1735 GCCCAUGUGG CUGAUGA X GAA ACCCGGUCUU 440
1736 GGCCCAUGUG CUGAUGA X GAA AACCCGGUCU 441 1751 AGUCGACGCU CUGAUGA X GAA AGGCGGCCCA 442 1757 CGUUGCAGUC CUGAUGA X GAA ACGCUGAGGC 443 1769 CCGGCUCCAC CUGAUGA X GAA ACGUUGCAGU 444 1787 CCACCUUCUU CUGAUGA X GAA ACGUCCGCCG 445 1807 GGCGCGCUGC CUGAUGA X GAA AGGUGGUGGC 446 1820 CGACCACCUU CUGAUGA X GAA AUGGCGCGCU 447 1829 CCGGCGUGCC CUGAUGA X GAA ACCACCUUGA 448 1843 CAUCUCCUCG CUGAUGA X GAA ACGCCGGCGU 449 1871 AGAGAUCCUG CUGAUGA X GAA AUCAUGCAGU 450 1878 UUCCAGGAGA CUGAUGA X GAA AUCCUGGAUC 451 1880 CCUUCCAGGA CUGAUGA X GAA AGAUCCUGGA 452 1882 GCCCUUCCAG CUGAUGA X GAA AGAGAUCCUG 453 1922 CCCCGAGGCU CUGAUGA X GAA AGCAGCACGU 454 1928 CGGCGACCCC CUGAUGA X GAA AGGCUGAGCA 455 1934 CGCCGCCGGC CUGAUGA X GAA ACCCCGAGGC 456 1955 CCUCGCCUUC CUGAUGA X GAA ACCCCUGGCU 457 1970 CGAGCGGCGC CUGAUGA X GAA AUCUCCUCGC 458 1979 UCUCCUUGGC CUGAUGA X GAA AGCGGCGCGA 459
2012 CUGCAGGCCG CUGAUGA X GAA ACUCUUCAGG 460
2013 CCUGCAGGCC CUGAUGA X GAA AACUCUUCAG 461 2033 CCACGCGCGA CUGAUGA X GAA AUCAGGGGGC 462 2035 CACCACGCGC CUGAUGA X GAA AGAUCAGGGG 463 2055 AAGAUGUCCC CUGAUGA X GAA ACAUGUUUGC 464 2063 UAUAUAAGAA CUGAUGA X GAA AUGUCCCAAC 465
2065 CAUAUAUAAG CUGAUGA X GAA AGAUGUCCCA 466
2066 GCAUAUAUAA CUGAUGA X GAA AAGAUGUCCC 467 Table IIIB
80
nt. Position HH Ribozyme Sequence Seq. ID No.
2068 CAGCAUAUAU CUGAUGA X GAA AGAAGAUGUC 468
2069 ACAGCAUAUA CUGAUGA X GAA AAGAAGAUGU 469 2071 AAAC AGCAU A CUGAUGA X GAA AU AAGAAGAU 470 2073 CGAAACAGCA CUGAUGA X GAA AUAUAAGAAG 471
2080 ACAUAAACGA CUGAUGA X GAA ACAGCAUAUA 472
2081 CACAUAAACG CUGAUGA X GAA AACAGCAUAU 473
2082 UCACAUAAAC CUGAUGA X GAA AAACAGCAUA 474
2085 AUAUCACAUA CUGAUGA X GAA ACGAAACAGC 475
2086 CAUAUCACAU CUGAUGA X GAA AACGAAACAG 476
2087 CCAUAUCACA CUGAUGA X GAA AAACGAAACA 477 2094 UACUUGUCCA CUGAUGA X GAA AUCACAUAAA 478 2104 CAGCUACACA CUGAUGA X GAA ACUUGUCCAU 479 2110 AGCAAGCAGC CUGAUGA X GAA ACACAUACUU 480 2117 UAGCACAAGC CUGAUGA X GAA AGCAGCUACA 481 2121 ACACUAGCAC CUGAUGA X GAA AGCAAGCAGC 482 2127 UAUAUUACAC CUGAUGA X GAA AGCACAAGCA 483 2132 UACACUAU AU CUGAUGA X GAA ACACUAGCAC 484 2135 CACUACACUA CUGAUGA X GAA AUUACACUAG 485 2137 ACCACUACAC CUGAUGA X GAA AUAUUACACU 486 2142 UGGCCACCAC CUGAUGA X GAA ACACUAUAUU 487 2165 AUGCGCUUAU CUGAUGA X GAA AGGUUGUGCC 488 2168 UUCAUGCGCU CUGAUGA X GAA AUUAGGUUGU 489 2181 CGCAAGCAAU CUGAUGA X GAA AGUUCAUGCG 490 2184 ACACGCAAGC CUGAUGA X GAA AUUAGUUCAU 491 2188 CUACACACGC CUGAUGA X GAA AGCAAUUAGU 492 2197 GGUACUUAAC CUGAUGA X GAA ACACACGCAA 493
2200 AUCGGUACUU CUGAUGA X GAA ACUACACACG 494
2201 GAUCGGUACU CUGAUGA X GAA AACUACACAC 495 2205 UACCGAUCGG CUGAUGA X GAA ACUUAACUAC 496 2211 UAAAAUUACC CUGAUGA X GAA AUCGGUACUU 497 2215 AAUAUAAAAU CUGAUGA X GAA ACCGAUCGGU 498
2218 CGCAAUAUAA CUGAUGA X GAA AUUACCGAUC 499
2219 UCGCAAUAUA CUGAUGA X GAA AAUUACCGAU 500
2220 CUCGCAAUAU CUGAUGA X GAA AAAUUACCGA 501
2221 ACUCGCAAUA CUGAUGA X GAA AAAAUUACCG 502 2223 UUACUCGCAA CUGAUGA X GAA AUAAAAUUAC 503 2225 AUUUACUCGC CUGAUGA X GAA AUAUAAAAUU 504 2232 UCCAUUUAUU CUGAUGA X GAA ACUCGCAAUA 505 2236 CAGGUCCAUU CUGAUGA X GAA AUUUACUCGC 506 2248 UUUCCACCAC CUGAUGA X GAA ACAGGUCCAU 507
Where "X" represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252). The length of stem II may be ≥ 2 base-pairs. Table IV
Table TV: HH Ribozyme Sequences Tested against GBSS mRNA nt. HH Ribozyme Sequence Sequenoe Position D.
425 CGACGAAGAC CUGAUGAGGCCGAAAGGCCGAA ACGUUCAUGC 2 593 CUCCCAUCUU CUGAUGAGGCCGAAAGGCCGAA AUCUCGGACA 3 742 GUUGUCCCUG CUGAUGAGGCCGAAAGGCCGAA AGUCCGUUCC 4 812 GGUUGUUGUU CUGAUGAGGCCGAAAGGCCGAA AGGCUCAGGA 5 892 GAGGUAGCAC CUGAUGAGGCCGAAAGGCCGAA AGAGAGGGCC 6 913 GUGGGACUGG CUGAUGAGGCCGAAAGGCCGAA AGUUGCUCUU 7 919 GAUGCCGUGG CUGAUGAGGCCGAAAGGCCGAA ACUGGUAGUU 8 953 UGUGGAUGCA CUGAUGAGGCCGAAAGGCCGAA AAAGCGGUCU 9 959 AGAUGUUGUG CUGAUGAGGCCGAAAGGCCGAA AUGCAGAAAG 10 968 CCUGGUAGGA CUGAUGAGGCCGAAAGGCCGAA AUGUUGUGGA 11 1016 AUCUCUCCGG CUGAUGAGGCCGAAAGGCCGAA AGGUUCAGCU 12 1028 AGGACGACUU CUGAUGAGGCCGAAAGGCCGAA AAUCUCUCCG 13 1085 UCAUCCAGUU CUGAUGAGGCCGAAAGGCCGAA AUCUUCCGGC 14 1187 UGAUGUUGUC CUGAUGAGGCCGAAAGGCCGAA AGCUCGCAGC 15 1196 UGAGGCGCAU CUGAUGAGGCCGAAAGGCCGAA AUGUUGUCG-. 16 1226 CCAUGCCGUU CUGAUGAGGCCGAAAGGCCGAA ACGAUGCCGG 17 1241 CCCACUCGCU CUGAUGAGGCCGAAAGGCCGAA ACGUCCAUGC 18 1270 CACGGCGAUG CUGAUGAGGCCGAAAGGCCGAA ACUUGUCCCU 19 1352 GGUCCACCGG CUGAUGAGGCCGAAAGGCCGAA AGCCCGACCϋ 20 1421 CGGCCGCCAU CUGAUGAGGCCGAAAGGCCGAA ACGUCGGGUC 21 1534 CUUGCCUGGG CUGAUGAGGCCGAAAGGCCGAA ACUUCUCCUC 22 1715 UGCCUUCGAU CUGAUGAGGCCGAAAGGCCGAA AUGGUGUCGA 23 1787 CCACCUUCUU CUGAUGAGGCCGAAAGGCCGAA ACGUCCGCCC- 24 I able VΛ
Table V A: GBSS Hairpin Ribozyme and Substrate Sequences
(0
C CD Hairpin Ribozyme Sequence
AGAAGUCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
C AGAAGUGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA H m AGAAGCCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
CO AGAAGCCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA z AGAAGCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA m AGAAGUGGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
∑u AGAAGUUCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
30 AGAAGCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA c AGAAGUCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA m r AGAAGCCAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGCGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGCCCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAAGCCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
Figure imgf000084_0001
GAAGCAUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
Figure imgf000084_0002
l ablc VU
Table VB: GBSS Hairpin Ribozyme and Substrate Sequences
Ribozyme Sequence Seq. ID Substrate Seq. ID No.
No.
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 538 ACCACCC GCC GAGGCGAC 539
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 540 CGCGACA GCC GCCAGGAG 5 1
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 542 GUGUACU GCU CCGUCCAC 543
(0 ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 544 CUGCUCC GUC CACCAGUG 545
C CD ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 546 GCGCACC GCC CGGCAGGG 547 tn ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 548 CAGGGCU GCU CAUCUCGU 549
H ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 550 CAUGGCG GCU CUAGCCAC 551
H
C ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 552 CGUCGCA GCU CGUCGCAA 553 H ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 554 CGCGCCG GCC UGGGCGUC 555 m ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 556 CGUCCCG GAC GCGUCCAC 557
(0 x ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 558 ACGUUCC GCC GCGGCGCC 559 m ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 560 GGGGCCG GAC GGCGUCGG 561
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 562 GCAUUCG GAC CAGCGCGC 563
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 564 GGUUCCC GUC GCUCGUCG 565 c 3D ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 566 ACCGGCG GCC UCGGCGAC 567
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 568 CUCGGCG GCC UGCCGCCG 569
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 570 GCGGCCU GCC GCCGGCCA 571 at ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 572 GCCUGCC GCC GGCCAUGG 573
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 574 ACCCACU GUU CCUGGAGA 575
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 576 CGGGCCU GAC GCUGGAAC 577 CCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 578 UGGAACG GAC UACAGGGA 579
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 580 ACAACCA GCU GCGGUUCA 581
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 582 AGCUGCG GUU CAGCCUGC 583 CCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 584 CGGUUCA GCC UGCUAUGC 585 CCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 586 UUCUCCG GAC CAUACGGG 587
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 588 CACACCG GCC CUCUCUCG 589
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 590 ACUACCA GUC CCACGGCA 591 CCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 592 AAAGACC GCU UUCUGCAU 593
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 594 AGGGCCG GUU CGCCUUCU 595
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 596 CUUCUCC GAC UACCCGGA 597
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 598 ACCGUCA GCC CCUACUAC 599
Figure imgf000085_0001
CCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 600 GUCGACG GCC GUGGAGGC 601
t able VB
Position Ribozyme Sequence Seq. ID Substrate Seq. ID No.
No. GAAGGAUGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 602 ACAUCCC GCU GGUGGCGU 603 GAAGGUCCCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 604 GGGACCC GAC GUCAUGGC 605
AGAAGCCAUGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 606 CAUGGCG GCC GCCAUCCC 607 GAAGCGGGAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 608 UCCCGCA GCU CAUGGAGA 609
AGAAGCACGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 610 ACGUGCA GAU CGUUCUGC 611
AGAAGAACGAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 612 UCGUUCU GCU GGGCACGG 613
AGAAGCGCCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 614 CGGCGCC GAC GUGCUCGC 615
AGAAGGUGACACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 616 GUCACCA GCC GCUUCGAG 617
AGAAGCUGGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 618 ACCAGCC GCU UCGAGCCC 619
AGAAGCAGGGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 620 CCCUGCG GCC UCAUCCAG 621
AGAAGGAUGAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 622 UCAUCCA GCU GCAGGGGA 623
AGAAGCCCAUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 624 AUGGGCC GCC UCAGCGUC 625 GAAGCCGGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 626 GCCGGCG GAC GUCAAGAA 627
AGAAGCACGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 628 ACGUGCU GCU CAGCCUCG 629
AGAAGAGCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 630 CUGCUCA GCC UCGGGGUC 631
AGAAGCGCGAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 632 UCGCGCC GCU CGCCAAGG 633
AGAAGAACUCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 634 GAGUUCG GCC UGCAGGCC 635 GAAGGGGGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 636 GCCCCCU GAU CUCGCGCG 637 GAAGCAUAUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 638 AUAUGCU GUU UCGUUUAU 639 GAAGCUACAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 640 UGUAGCU GCU UGCUUGUG 641
Figure imgf000086_0001
GAAGUACUUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 642 AAGUACC GAU CGGUAAUU 643
Table VI
85
Table VI: Delta-9 Desaturase HH Ribozyme Target Sequences
Substrate Seq. ID No.
GUCCAGGUUACACAUUC 645
UCCAGGUUACACAUUCA 647
UUACACAUUCAAUGCCA 649
UACACAUUCAAUGCCAC 651
UGCCACCUCACAAGAUU 653
CACAAGAUUGAAAUUUU 655
AUUGAAAUUUUCAAGUC 657
UUGAAAUUUUCAAGUCG 659
UGAAAUUUUCAAGUCGC 661
GAAAUUUUCAAGUCGCU 663
UUUCAAGUCGCUUGAUG 665
AAGUCGCUUGAUGAUUG 667
UUGAUGAUUGGGCUAGA 669
AUUGGGCUAGAGAUAAU 671
CUAGAGAUAAUAUCUUG 673
GAGAUAAUAUCUUGACG 675
GAUAAUAUCUUGACGCA 677
UAAUAUCUUGACGCAUC 679
UGACGCAUCUCAAGCCA 681
ACGCAUCUCAAGCCAGU 683
AAGCCAGUCGAGAAGUG 685
AGAAGUGUUGGCAGCCA 687
CACAGGAUUUCCUCCCG 689
ACAGGAUUUCCUCCCGG 691
CAGGAUUUCCUCCCGGA 693
GAUUUCCUCCCGGACCC 695
CCCAGCAUCUGAAG6AU 697
UGAAGGAUUUCAUGAUG 699
GAAGGAUUUCAUGAUGA 701
AAGGAUUUCAUGAUGAA 703
GAUGAAGUUAAGGAGCU 705
AUGAAGUUAAGGAGCUC 707
AAGGAGCUCAGAGAACG 709
AAGGAAAUCCCUGAUGA 711
CUGAUGAUUAUUUUGUU 713
UGAUGAUUAUUUUGUUU 715
AUGAUUAUUUUGUUUGU 717
UGAUUAUUUUGUUUGUU 719
GAUUAUUUUGUUUGUUU 721
UAUUUUGUUUGUUUGGU 723
AUUUUGUUUGUUUGGUG 725
UUGUUUGUUUGGUGGGA 727
ACACUGCUCGUCACGCC 729
CUGCUCGUCACGCCAAG 731
CAAGGACUU UGGCGACU 733
AAGGACUUUGGCGACUU 735
UGGCGACUUAAAGCUUG 737
GGCGACUUAAAGCUUGC 739
UUAAAGCUUGCACAAAU 741
GCACAAAUCUGCGGCAU 743
UGCGGCAUCAUCGCCUC 745
GGCAUCAUCGCCUCAGA 747
Figure imgf000087_0001
Figure imgf000087_0002
CAUCGCCUCAGAUGAGA 749 Table VI
86
Figure imgf000088_0001
Figure imgf000088_0002
Figure imgf000088_0003
Table VI
87
Figure imgf000089_0001
Table VII
88
Table VII: Delta-9 Desaturase HH Ribozyme Sequences nt. Ribozyme sequence Seq. ID No. Position
13 AAGCGGCA CUGAUGA X GAA AGGGCGCG 894
21 GAACGAAC CUGAUGA X GAA AGCGGCAG 895
24 GAGGAACG CUGAUGA X GAA ACAAGCGG 896
25 CGAGGAAC CUGAUGA X GAA AACAAGCG 897
28 GCGCGAGG CUGAUGA X GAA ACGAACAA 898
29 AGCGCGAG CUGAUGA X GAA AACGAACA 899 32 GCGAGCGC CUGAUGA X GAA AGGAACGA 900 38 CUGGUGGC CUGAUGA X GAA AGCGCGAG 901 63 GAGAUUGG CUGAUGA X GAA AUGUGUGU 902 69 CCUCGCGA CUGAUGA X GAA AUUGGGAU 903 71 GCCCUCGC CUGAUGA X GAA AGAUUGGG 904 92 CCGCCGCA CUGAUGA X GAA ACCCUGCU 905
11 GGAGCCGG CUGAUGA X GAA AGCGCGGC 906
118 GGGAGCCG CUGAUGA X GAA AAGCGCGG 907 124 GGGAAGGG CUGAUGA X GAA AGCCGGAA 908
129 CCAAUGGG CUGAUGA X GAA AGGGGAGC 909
130 GCCAAUGG CUGAUGA X GAA AAGGGGAG 910 135 UGGAGGCC CUGAUGA X GAA AUGGGAAG 911 141 CCAUCGUG CUGAUGA X GAA AGGCCAAU 912 154 UUGAGGCG CUGAUGA X GAA AGCGCCAU 913 160 ACGUCGUU CUGAUGA X GAA AGGCGGAG 914 169 CAGAGCGC CUGAUGA X GAA ACGUCGUU 915 175 G AGAGGCA CUGAUGA X GAA AGCGCGAC 916 181 GGCGGGGA CUGAUGA X GAA AGGCAGAG 917 1 B3 GCGGCGGG CUGAUGA X GAA AGAGGCAG 918 193 CGGGCGGC CUGAUGA X GAA AGCGGCGG 919
228 CGGCGACG CUGAUGA X GAA ACCUGCCG 920
229 ACGGCGAC CUGAUGA X GAA AACCUGCC 921 232 GCGACGGC CUGAUGA X GAA ACGAACCU 922 238 AUGGAGGC CUGAUGA X GAA ACGGCGAC 923 243 ACGUCAUG CUGAUGA X GAA AGGCGACG 924 252 AGACGGCG CUGAUGA X GAA ACGUCAUG 925 259 UUGGUGGA CUGAUGA X GAA ACGGCGGA 926 261 CCUUGGUG CUGAUGA X GAA AGACGGCG 927 271 UUAUUCUC CUGAUGA X GAA ACCUUGGU 928 278 UGGCUUCU CUGAUGA X GAA AUUCUCGA 929
288 GAGGAGCA CUGAUGA X GAA AUGGCUUC 930
289 GGAGGAGC CUGAUGA X GAA AAUGGCUU 931 293 CCUUGGAG CUGAUGA X GAA AGCAAAUG 932 296 CUCCCUUG CUGAUGA X GAA AGGAGCAA 933 307 UGGACAUG CUGAUGA X GAA ACCUCCCU 934 313 GUAACCUG CUGAUGA X GAA ACAUGUAC 935
319 GAAUGUGU CUGAUGA X GAA ACCUGGAC 936
320 UGAAUGUG CUGAUGA X GAA AACCUGGA 937
326 UGGCAUUG CUGAUGA X GAA AUGUGUAA 938
327 GUGGCAUU CUGAUGA X GAA AAUGUGUA 939 338 AAUCUUGU CUGAUGA X GAA AGGUGGCA 940 346 AAAAUUUC CUGAUGA X GAA AUCUUGUG 941
352 GACUUGAA CUGAUGA X GAA AUUUCAAU 942
353 CGACUUGA CUGAUGA X GAA AAUUUCAA 943
354 GCGACUUG CUGAUGA X GAA AAAUUUCA 944
355 AGCGACUU CUGAUGA X GAA AAAAUUUC 945 360 CAUCAAGC CUGAUGA X GAA ACUUGAAA 946 364 CAAUCAUC CUGAUGA X GAA AGCGACUU 947 Table VII
89
nt. Ribozymesequence Seq. ID No. Position
371 UCUAGCCC CUGAUGA X GAA AUCAUCAA 948 377 AUUAUCUC CUGAUGA X GAA AGCCCAAU 949 383 CAAGAUAU CUGAUGA X GAA AUCUCUAG 950 386 CGUCAAGA CUGAUGA X GAA AUUAUCUC 951 388 UGCGUCAA CUGAUGA X GAA AUAUUAUC 952 390 GAUGCGUC CUGAUGA X GAA AGAUAUUA 953 398 UGGCUUGA CUGAUGA X GAA AUGCGUCA 954 400 ACUGGCUU CUGAUGA X GAA AGAUGCGU 955 409 CACUUCUC CUGAUGA X GAA ACUGGCUU 956 419 UGGCUGCC CUGAUGA X GAA ACACUUCU 957 434 CGGGAGGA CUGAUGA X GAA AUCCUGUG 958 435 CCGGGAGG CUGAUGA X GAA AAUCCUGU 959 436 UCCGGGAG CUGAUGA X GAA AAAUCCUG 960 439 GGGUCCGG CUGAUGA X GAA AGGAAAUC 961 453 AUCCUUCA CUGAUGA X GAA AUGCUGGG 962 462 CAUCAUGA CUGAUGA X GAA AUCCUUCA 963 463 UCAUCAUG CUGAUGA X GAA AAUCCUUC 964 464 UUCAUCAU CUGAUGA X GAA AAAUCCUU 965 475 AGCUCCUU CUGAUGA X GAA ACUUCAUC 966 476 GAGCUCCU CUGAUGA X GAA AACUUCAU 967 484 CGUUCUCU CUGAUGA X GAA AGCUCCUU 968 505 UCAUCAGG CUGAUGA X GAA AUUUCCUU 969 515 AACAAAAU CUGAUGA X GAA AUCAUCAG 970 516 AAACAAAA CUGAUGA X GAA AAUCAUCA 971 518 ACAAACAA CUGAUGA X GAA AUAAUCAU 972 519 AACAAACA CUGAUGA X GAA AAUAAUCA 973 520 AAACAAAC CUGAUGA X GAA AAAUAAUC 974 523 ACCAAACA CUGAUGA X GAA ACAAAAUA 975 524 CACCAAAC CUGAUGA X GAA AACAAAAU 976 527 UCCCACCA CUGAUGA X GAA ACAAACAA 977 528 CUCCCACC CUGAUGA X GAA AACAAACA 978 544 UCCUCGGU CUGAUGA X GAA AUCAUGUC 979 545 UUCCUCGG CUGAUGA X GAA AAUCAUGU 980 557 UGUUGGUA CUGAUGA X GAA AGCUUCCU 981 559 UAUGUUGG CUGAUGA X GAA AGAGCUUC 982 567 UAGUCUGG CUGAUGA X GAA AUGUUGGU 983 575 GUUAAGCA CUGAUGA X GAA AGUCUGGU 984 580 AGGGUGUU CUGAUGA X GAA AGCAUAGU 985 581 GAGGGUGU CUGAUGA X GAA AAGCAUAG 986 589 ACACCGUC CUGAUGA X GAA AGGGUGUU 987 598 UCAUCUCU CUGAUGA X GAA ACACCGUC 988 637 CUCGUCCA CUGAUGA X GAA ACAGCCCA 989 638 CCUCGUCC CUGAUGA X GAA AACAGCCC 990 680 GUUGAGCA CUGAUGA X GAA AUCACCAU 991 685 UACUUGUU CUGAUGA X GAA AGCAGAUC 992 693 GGUACAUA CUGAUGA X GAA ACUUGUUG 993 695 GAGGUACA CUGAUGA X GAA AUACUUGU 994 699 CAGUGAGG CUGAUGA X GAA ACAUAUAC 995 703 CUCCCAGU CUGAUGA X GAA AGGUACAU 996 719 CUGCCUCA CUGAUGA X GAA AUCCACCC 997 730 GUCUUCUC CUGAUGA X GAA AUCUGCCU 998 742 AGAUACUG CUGAUGA X GAA AUUGUCUU 999 743 AAGAUACU CUGAUGA X GAA AAUUGUCU 1000 747 CAAUAAGA CUGAUGA X GAA ACUGAAUU 1001 749 GCCAAUAA CUGAUGA X GAA AUACUGAA 1002 751 GAGCCAAU CUGAUGA X GAA AGAUACUG 1003 752 AGAGCCAA CUGAUGA X GAA AAGAUACU 1004 TableVII
90
nt. Ribozymesequence Seq. ID No. Position
754 CCAGAGCC CUGAUGA X GAA AUAAGAUA 1005
759 CCAUUCCA CUGAUGA X GAA AGCCAAUA 1006
770 AGUCCUAG CUGAUGA X GAA AUCCAUUC 1007
773 CUCAGUCC CUGAUGA X GAA AGGAUCCA 1008
785 AUAAGGAU CUGAUGA X GAA AUUCUCAG 1009
788 AAGAUAAG CUGAUGA X GAA AUUAUUCU 1010
791 ACCAAGAU CUGAUGA X GAA AGGAUUAU 1011
792 AACCAAGA CUGAUGA X GAA AAGGAUUA 1012
794 GAAACCAA CUGAUGA X GAA AUAAGGAU 1013
796 AUGAAACC CUGAUGA X GAA AGAUAAGG 1014
800 GUAGAUGA CUGAUGA X GAA ACCAAGAU 1015
801 UGUAGAUG CUGAUGA X GAA AACCAAGA 1016
802 GUGUAGAU CUGAUGA X GAA AAACCAAG 1017
805 GAGGUGUA CUGAUGA X GAA AUGAAACC 1018
807 AGGAGGUG CUGAUGA X GAA AGAUGAAA 1019
813 CUUGGAAG CUGAUGA X GAA AGGUGUAG 1020
816 GCUCUUGG CUGAUGA X GAA AGGAGGUG 1021
817 CGCUCUUG CUGAUGA X GAA AAGGAGGU 1022
834 GUGAGAUG CUGAUGA X GAA AGGUCGCC 1023
835 UGUGAGAU CUGAUGA X GAA AAGGUCGC 1024
838 CCGUGUGA CUGAUGA X GAA AUGAAGGU 1025
840 UCCCGUGU CUGAUGA X GAA AGAUGAAG 1026
857 GGCGUGAC CUGAUGA X GAA AGCAGUGU 1027
860 CUUGGCGU CUGAUGA X GAA ACGAGCAG 1028
873 AGUCGCCA CUGAUGA X GAA AGUCCUUG 1029
874 AAGUCGCC CUGAUGA X GAA AAGUCCUU 1030
882 CAAGCUUU CUGAUGA X GAA AGUCGCCA 1031
883 GCAAGCUU CUGAUGA X GAA AAGUCGCC 1032
889 AUUUGUGC CUGAUGA X GAA AGCUUUAA 1033
898 AUGCCGCA CUGAUGA X GAA AUUUGUGC 1034
907 GAGGCGAU CUGAUGA X GAA AUGCCGCA 1035
910 UCUGAGGC CUGAUGA X GAA AUGAUGCC 1036
915 UCUCAUCU CUGAUGA X GAA AGGCGAUG 1037
942 UCUUGGUG CUGAUGA X GAA ACGCAGUU 1038
952 UUCUCCAC CUGAUGA X GAA AUCUUGGU 1039
966 CGAUCUCA CUGAUGA X GAA ACAGCUUC 1040
967 UCGAUCUC CUGAUGA X GAA AACAGCUU 1041
973 UCAGGGUC CUGAUGA X GAA AUCUCAAA 1042
986 GACCACGG CUGAUGA X GAA ACCAUCAG 1043
994 GCCAGAGC CUGAUGA X GAA ACCACGGU 1044
998 GUCAGCCA CUGAUGA X GAA AGCGACCA 1045
1024 GGCAUUGA CUGAUGA X GAA AUCUUCUU 1046
1026 CAGGCAUU CUGAUGA X GAA AGAUCUUC 1047
1047 GCCCGUCA CUGAUGA X GAA ACAUCAGG 1048
1048 UGCCCGUC CUGAUGA X GAA AACAUCAG 1049
1071 AGUGCUCG CUGAUGA X GAA ACAGCUUG 1050
1072 AAGUGCUC CUGAUGA X GAA AACAGCUU 1051
1080 CCAUGGAG CUGAUGA X GAA AGUGCUCG 1052
1081 ACCAUGGA CUGAUGA X GAA AAGUGCUC 1053
1083 CGACCAUG CUGAUGA X GAA AGAAGUGC 1054
1090 CUCUGCGC CUGAUGA X GAA ACCAUGGA 1055
1102 UAAACGCC CUGAUGA X GAA AGCCUCUG 1056
1108 GCGGUGUA CUGAUGA X GAA ACGCCAAG 1057
1109 GGCGGUGU CUGAUGA X GAA AACGCCAA 1058
1110 UGGCGGUG CUGAUGA X GAA AAACGCCA 1059
1125 UGUCGGCG CUGAUGA X GAA AGUCCCUG 1060
1135 AACUCGAG CUGAUGA X GAA AUGUCGGC 1061 Table VI I
nt. Ribozymesequence Seq. IDNo. Position
1138 AGGAACUC CUGAUGAX GAAAGGAUGUC 1062 1143 CGACGAGGCUGAUGAXGAAACUCGAGG 1063 1144 UCGACGAG CUGAUGAX GAAAACUCGAG 1064 1147 CUGUCGAC CUGAUGAX GAAAGGAACUC 1065 1150 CACCUGUC CUGAUGAX GAAACGAGGAA 1066 1181 ACCCGACACUGAUGAX GAAACCAGUCA 1067 1185 CUUCACCCCUGAUGAXGAAACAGACCA 1068 1212 UGCAAAGG CUGAUGAX GAAAGUCCUGC 1069 1216 AGGGUGCACUGAUGAXGAAAGGUAGUC 1070 1217 AAGGGUGCCUGAUGAXGAAAAGGUAGU 1071 1225 CUUGAAGCCUGAUGAXGAAAGGGUGCA 1072 1229 GAUUCUUGCUGAUGAX GAAAGCAAGGG 1073 1230 UGAUUCUU CUGAUGAX GAAAAGCAAGG 1074 1237 AGCCUCCUCUGAUGAX GAAAUUCUUGA 1075 1292 CCAGCUGACUGAUGAX GAAAGGCAGCG 1076 1293 CCCAGCUGCUGAUGAX GAAAAGGCAGC 1077 1294 ACCCAGCU CUGAUGAX GAAAAAGGCAG 1078 1303 CUACCGUACUGAUGAXGAAACCCAGCU 1079 1305 CCCUACCGCUGAUGAXGAAAUACCCAG 1080 1310 GACGUCCCCUGAUGAXGAAACCGUAUA 1081 1318 CACAGUUGCUGAUGAXGAAACGUCCCU 1082 1331 AGGUUUCC CUGAUGAXGAAAUCUCACA 1083 1348 UCUAAGCACUGAUGAXGAAACCGCAGC 1084 1353 UCUUGUCU CUGAUGAXGAAAGCAGACC 1085 1354 GUCUUGUCCUGAUGAXGAAAAGCAGAC 1086 1372 GUAACGCACUGAUGAXGAAACACAGCA 1087 1378 ACCUAUGUCUGAUGAXGAAACGCAGAC 1088 1379 GACCUAUGCUGAUGAXGAAAACGCAGA 1089 1383 UGGAGACCCUGAUGAXGAAAUGUAACG 1090 1387 AACCUGGACUGAUGAXGAAACCUAUGU 1091 1389 AAAACCUGCUGAUGAXGAAAGACCUAU 1092 1395 UUGAUCAACUGAUGAXGAAACCUGGAG 1093 1396 UUUGAUCACUGAUGAXGAAAACCUGGA 1094 1397 AUUUGAUCCUGAUGAXGAAAAACCUGG 1095 1401 GACCAUUUCUGAUGAXGAAAUCAAAAC 1096 1409 CGACACGGCUGAUGAXGAAACCAUUUG 1097 1416 UAUAAGAC CUGAUGAX GAAACACGGGA 1098 1419 CUCUAUAACUGAUGAXGAAACGACACG 1099 1421 CGCUCUAUCUGAUGAX GAAAGACGACA 1100 1422 UCGCUCUACUGAUGAXGAAAAGACGAC 1101 1424 UAUCGCUCCUGAUGAX GAAAUAAGACG 1102 1432 CGUUCUCC CUGAUGAXGAAAUCGCUCU 1103
CACAGACCCUGAUGAXGAAACACGUUC 1104
1448 ACACCACACUGAUGAX GAAACCAACAC 1105 1457 AACAAAGCCUGAUGAXGAAACACCACA 1106 1461 UAAAAACACUGAUGAXGAAAGCUACAC 1107 1462 AUAAAAAC CUGAUGAXGAAAAGCUACA 1108 1465 AAAAUAAACUGAUGAXGAAACAAAGCU 1109 1466 CAAAAUAACUGAUGAX GAAAACAAAGC 1110 1467 ACAAAAUACUGAUGAX GAAAAACAAAG 1111 1468 UACAAAAU CUGAUGAX GAAAAAACAAA 1112 1469 AUACAAAACUGAUGAXGAAAAAAACAA 1113 1471 AAAUACAACUGAUGAX GAAAUAAAAAC 1114 1472 AAAAUACACUGAUGAXGAAAAUAAAAA 1115 1473 AAAAAUACCUGAUGAX GAAAAAUAAAA 1116 1476 CAGAAAAACUGAUGAX GAAACAAAAUA 1117 1478 AGCAGAAACUGAUGAX GAAAUACAAAA 1118 Table VII
92
nt. Ribozyme sequence Seq. ID No. Position
1479 AAGCAGAA CUGAUGA X GAA AAUACAAA 1119 1480 AAAGCAGA CUGAUGA X GAA AAAUACAA 1120 1481 CAAAGCAG CUGAUGA X GAA AAAAUACA 1121 1482 UCAAAGCA CUGAUGA X GAA AAAAAUAC 1122 1487 GUACAUCA CUGAUGA X GAA AGCAGAAA 1123 1488 UGUACAUC CUGAUGA X GAA AAGCAGAA 1124 1494 ACAGGUUG CUGAUGA X GAA ACAUCAAA 1125 1546 AGACAAAG CUGAUGA X GAA ACGGCAUG 1126 1549 GACAGACA CUGAUGA X GAA AGUACGGC 1127 1550 CGACAGAC CUGAUGA X GAA AAGUACGG 1128 1553 CAGCGACA CUGAUGA X GAA ACAAAGUA 1129 1557 CCGCCAGC CUGAUGA X GAA ACAGACAA 1130 1571 CAUACCGA CUGAUGA X GAA ACACACCG 1131 1572 ACAUACCG CUGAUGA X GAA AACACACC 1132 1573 AACAUACC CUGAUGA X GAA AAACACAC 1133 1577 AAAUAACA CUGAUGA X GAA ACCGAAAC 1134 1581 ACUCAAAU CUGAUGA X GAA ACAUACCG 1135 1582 AACUCAAA CUGAUGA X GAA AACAUACC 1136 1584 GCAACUCA CUGAUGA X GAA AUAACAUA 1137 1585 AGCAACUC CUGAUGA X GAA AAUAACAU 1138 1590 AUCUGAGC CUGAUGA X GAA ACUCAAAU 1139 1594 ACAGAUCU CUGAUGA X GAA AGCAACUC 1140 1599 UUUUAACA CUGAUGA X GAA AUCUGAGC 1141 1603 UUUUUUUU CUGAUGA X GAA ACAGAUCU 1142 1604 UUUUUUUU CUGAUGA X GAA AACAGAUC 1143
Where "X" represents stem II region of a HH ribozyme (Hertel et al.. 1992 Nucleic Acids Res. 20 3252). The length of stem II may be ≥ 2 base-pairs.
Table VIII: Delta-9 Desaturase Hairpin Ribozyme and Substrate Seq
nt. Ribozyme Position
14 GAACAAGCAGAAGAGGGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
17 AACGAACAAGAAGCAGAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
CO 108 GGAAGCGCAGAAGCCGCCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA c GGAAGGGGAGAAGGAAGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA to 120
(0 155 GUCGUUGAAGAAGAGCGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
176 CGGGGAGAAGAAGAGCGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA c 186 CGGCGAGCAGAAGGGAGAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
H m 189 GGGCGGCGAGAAGCGGGGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
196
(0 CGGCGGCGAGAAGCGAGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA z 200 GCGGCGGCAGAAGGCGGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA m 203 GCGGCGGCAGAAGCGGGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
∑ϋ 206 GCUGCGGCAGAAGCGGCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
3 209 GCUGCUGCAGAAGCGGCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA c 235 AUGGAGGCAGAAGCGACGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA f 253 GUGGAGACAGAAGACGUCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
256 UUGGUGGAAGAAGCGGACACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA σ> 406 CACUUCUCAGAAGGCUUGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
442 GAUGCUGGAGAAGGGAGGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
508 AAAUAAUCAGAAGGGAUUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
570 UAAGCAUAAGAAGGUAUGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
625 ACAGCCCAAGAAGUGGGGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
634 CUCGUCCAAGAAGCCCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
655 UUCUCCUCAGAAGUCCAUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
681 ACUUGUUGAGAAGAUCACACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
726 UCUUCUCAAGAAGCCUCAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
853 GCGUGACGAGAAGUGUUCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
916 CGCUUCUCAGAAGAGGCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
Figure imgf000095_0001
Ribozyme
AGAAGCUUCUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAAGGGUCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGGCAUUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAAGGUGGGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGCUUGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAAGGCUCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGACCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAAGCGUGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGAAAGGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGGUUUCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGCAGCAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGACCGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAAGGUCUUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGAAAAAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAAGACAAAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAAGAGCAAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
Figure imgf000096_0002
Figure imgf000096_0001
Table IX
Table EX: Cleavage of D elta-9 Desaturase RNA by HH Ribozymes
Figure imgf000097_0001
TABLE X:
Figure imgf000098_0001
Table XI Stearic acid levels in leaves from plants transformed with active and inactive ribozymes compared to control leaves.
Figure imgf000099_0001
Table XII Inheritance of the high stearic acid trait in leaves from crosses of high stearic acid plants.
Figure imgf000100_0001
Table XIII Comparison of fatty acid composition of embryogenic callus, somatic embryos and zygotic embryos.
Figure imgf000101_0001
Table XIV: GBSS activity, amylose content, and Southern analysis results of selected Ribozym
Figure imgf000102_0001

Claims

/10328101Claims
1. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of.a plant gene
2. The enzymatic nucleic acid molecule of claim I . wherein s;ιιcl phut is ;ι monocotyledon.
3. The enzymatic nucleic acid molecule of claim 1 , wherein said plant is a dicotyledon.
4. The enzymatic nucleic acid molecule of claim I , wherein said plant is a gymnosperm.
5. The enzymatic nucleic acid molecule of claim 1, wherein said plant is an angiosperm.
6. The enzymatic nucleic acid molecule of claim 1 , wherein said nucleic acid is in a hammerhead configuration.
7. The enzymatic nucleic acid molecule of claim 1 , wherein said nucleic acid is in a haiφin configuration.
8. The enzymatic nucleic acid molecule of claim 1 , wherein said nucleic acid is in a hepatitis Δ virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration.
9. The enzymatic nucleic acid of any of claims 1-8, wherein said nucleic acid comprises between 12 and 100 bases complementary to RNA of said gene.
10. The enzymatic nucleic acid of any of claims 1-8, wherein said nucleic acid comprises between 14 and 24 bases complementary to RNA of said gene.
1 1. The enzymatic nucleic acid of claim 6, wherein said hammerhead comprises a stem II region of length greater than on equal to two base-pairs.
12 The enzymatic nucleic acid of claim 7, wherein said haiφin comprises a stem II region of length between three and seven base-pairs. 0328
102
13. The enzymatic nucleic acid of claim 7, wherein said hairpin comprises a stem IV region of length greater than or equal to two base-pairs.
14. The enzymatic nucleic acid of claim 2, wherein said monocotyledon plant is selected from a group consisting of maize, rice, wheat, and barley.
15. The enzymatic nucleic acid of claim 3, wherein said dicotyledon plant is selected from a group consisting of canola, sunflower, saffiower, soybean, cotton, peanut, olive, sesame, cuphea, flax, jojoba, and grape.
16. The enzymatic nucleic acid of claim 1 , wherein said gene is involved in fatty acid biosynthesis in said plant.
17. The enzymatic nucleic acid of claim 16, wherein said gene is Δ-9 desaturase.
18. The enzymatic nucleic acid of any of claims 16 or 17, wherein said plant is selected from a group consisting of maize, canola, flax, sunflower, cotton, peanuts, saffiower, soybean and rice.
19. The enzymatic nucleic acid of claim 1, wherein said gene is involved in starch biosynthesis in said plant.
20. The enzymatic nucleic acid of claim 19, wherein said gene is granule bound starch synthase.
21. The enzymatic nucleic acid of any of claims 19 or 20, wherein said plant is selected from a group consisting of maize, potato, wheat, and cassava.
22. The enzymatic nucleic acid of claim 1 , wherein said gene is involved in caffeine synthesis.
23. The enzymatic nucleic acid of claim 22, wherein said gene is selected from a group consisting of 7-methylguanosine and 3-methyl transferase.
24. The enzymatic nucleic acid of any of claims 22 or 23, wherein said plant is a coffee plant.
25. The enzymatic nucleic acid of claim 1 , wherein said gene is involved in nicotine production in said plant. /10328 103
26 The enzymatic nucleic acid of claim 25, wherein said gene is selected from a group consisting of N-methylputrescme oxidase and putrescine Λ'-methyl transferase.
27 The enzymatic nucleic acid of any of claims 25 or 26, wherein aid plant is a tobacco plant
28. The enzymatic nucleic acid of claim 1 , wherein said gene is involved in fruit ripening process in said plant.
29. The enzymatic nucleic acid of claim 28, wherein said gene is selected from a group consisting of ethylene-forming enzyme, pectin mcthyltransfcrasc, pectin esterase, polygalacturonase, 1-amιnocyclopropane carboxylic acid (ACC) synthase, and ACC oxidase.
30. The enzymatic nucleic acid of any of claims 28 or 29, wherein said plant is selected from a group consisting of apple, tomato, pear, plum and peach.
31. The enzymatic nucleic acid of claim 1, wherein said gene is involved in flower pigmentation in said plant.
32. The enzymatic nucleic acid of claim 31, wherein said gene is selected from a group consisting of chalcone synthase, chalcone flavanone isomerase, phenylalamne ammonia lyase, dehydroflavonol hydroxylases, and dehydroflavonol reductase.
33. The enzymatic nucleic acid of any of claims 31 or 32, wherein said plant is selected from a group consisting of rose, petunia, chrysanthamum, and mangold.
34. The enzymatic nucleic acid of claim 1 , wherein said gene is involved in lignin production in said plant.
35. The enzymatic nucleic acid of claim 34, wherein said gene is selected from a group consisting of O-methyltransferase, cιnnamoyl-CoA:ΝADPH reductase and cinnamoyl alcohol dehydrogenase.
36. The enzymatic nucleic acid of any of claims 34 or 35, wherein said plant is selected from a group consisting of tobacco, aspen, poplar, and pine.
37. A nucleic acid fragment compnsing a cDNA sequence coding for maize Δ-9 desaturase, wherein said sequence is represented by the sequence I.D. No. 1.
38. The enzymatic nucleic acid molecule of claim 17, wherein said nucleic acid specifically cleaves any of sequences defined in Table VI, wherein said nucleic acid is in a hammerhead configuration.
39. The enzymatic nucleic acid molecule of claim 17, wherein said nucleic acid specifically cleaves any of sequences defined in Table VIII, wherein said nucleic acid is in a haiφin configuration.
40. The enzymatic nucleic acid molecule of any of claims 38 or 39, consisting essentially of one or more sequences selected from the group shown in Tables
VII and VIII.
41. The enzymatic nucleic acid molecule of claim 20, wherein said nucleic acid specifically cleaves any of sequences defined in Table IIIA, wherein said nucleic acid is in a hammerhead configuration.
42. The enzymatic nucleic acid molecule of claim 20, wherein said nucleic acid specifically cleaves any of sequences defined in Tables VA and VB, wherein said nucleic acid is in a haiφin configuration.
43. The enzymatic nucleic acid molecule of any of claims 41 or 42, consisting essentially of one or more sequences selected from the group shown in Tables IIIB, IV, VA and VB.
44. The enzymatic nucleic acid molecule of claim 41, consisting essentially of sequences defined as any of SEQ. I.D. NOS. 2-24.
45. A plant cell comprising the enzymatic nucleic acid molecule of any of claims 1-8, 11-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-39, 41-42 or 44.
46. A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid molecule of any of claims 1-8, 11-17, 19-20, 22-23, 25-26, 28-29, 31 -32, 34- 35, 37-39, 41-42 or 44.
47. An expression vector comprising nucleic acid encoding the enzymatic nucleic acid molecule of any of claims 1-8, 11-17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-39, 41-42 or 44, in a manner which allows expression and/or delivery of that enzymatic nucleic acid molecule within a plant cell
48 An expression vector comprising nucleic acid encoding a plurality of enzymatic nucleic acid molecules of any of claims 1 -8, 1 1 - 17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-39, 41 -42 or 44, in a manner which allows expression and/or delivery of said enzymatic nucleic acid molecules within «ι plant cell
49 A plant cell compnsing the expression vector of claim 47
50 A plant cell comprising the expression vector of claim 48
51 A transgenic plant and the progeny thereof, compnsing the expression vector of claim 47.
52. A transgenic plant and the progeny thereof, comprising the expression vector of claim 48.
53 A plant cell compnsing the enzymatic nucleic acid of any of claims 16 or 17
54. The plant cell of claim 53, wherem said cell is a maize cell
55. The plant cell of claim 53, wherein said cell is a canola cell.
56. A transgenic plant and the progeny thereof, compnsing the enzymatic nucleic acid of any of claims 16 or 17.
57. The transgenic plant and the progeny thereof of claim 56, wherein said plant is a maize plant.
58 The transgenic plant and the progeny thereof of claim 56, wherein said plant is a canola plant.
59. A plant cell compnsing the enzymatic nucleic acid of any of claims 19 or 20.
60. The plant cell of claim 59, wherein said cell is a maize cell
61. A transgenic plant and the progeny thereof, compnsing the enzymatic nucleic acid of any of claims 19 or 20.
62. The transgenic plant and progeny thereof of claim 61 , wherein said plant is a maize plant.
63. A method for modulating expression of an gene in a plant by administering to said plant the enzymatic nucleic acid molecule ofany of claims I -8.
64. The method of claim 63, wherein said plant is a monocot plant.
65. The method of claim 63, wherein said plant is a dicot plant.
66. The method of claim 63, wherein said plant is a gymnosperm.
67. The method of claim 63, wherein said plant is an angiosperm.
68. The method of claim 63, wherein said gene is Δ-9 desaturase.
69. The method of claim 68, wherein said plant is a maize plant.
70. The method of claim 68, wherein said plant is a canola plant.
71. The method of claim 63, wherein said gene is granule bound starch synthase.
72. The method of claim 71 , wherein said plant is a maize plant.
73. The expression vector of claim 47, wherein said vector comprises:
a) a transcription initiation region;
b) a transcription termination region;
c) a gene encoding at least one said enzymatic nucleic acid molecule; and,
wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.
74. The expression vector of claim 47, wherein said vector comprises:
a) a transcription initiation region;
b) a transcription termination region; 10328
c) an open reading frame;
d) a gene encoding at least one said enzymatic nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and,
wherein said gene is operably linked to said initiation region, aid open reading frame and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.
75. The expression vector of claim 47, wherein said vector comprises:
a) a transcription initiation region;
b) a transcription termination region;
c) an intron;
d) a gene encoding at least one said enzymatic nucleic acid molecule; and,
wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.
76. The expression vector of claim 47, wherein said vector comprises:
a) a transcription initiation region;
b) a transcription termination region;
c) an intron;
d) an open reading frame;
e) a gene encoding at least one said enzymatic nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and.wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.
77. The enzymatic nucleic acid of Claim 1 , wherein said plant is selected from the group consisting of maize, rice, soybeans, canola, alfalfa, cotton, wheat, barley, sunflower, flax and peanuts.
78. A transgenic plant comprising nucleic acids encoding for an enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic aci molecule modulates the expression of a gene in said plant .
79. The transgenic plant of Claim 78, wherein said Plant is selected from the group consisting of maize, rice, soybeans, canola, alfalfa, cotton, wheat, barley, sunflower, flax and peanuts.
80. The transgenic plant of Claim 78, wherein said gene is granule bound starch synthase (GBSS).
81. The transgenic plant of Claim 78, wherein said gene is delta 9 desaturase.
82. The transgenic plant of Claim 78, wherein the plant is transformed with
Agrobacteriurn, bombarding with DNA coated microprojectiles, whiskers, or electroporation.
83. The transgenic plant of Claim 82, wherein said bombarding with DNA coated microprojectiles is done with the gene gun.
84. The transgenic plant of any of Claims 78 or 82, wherein said plant contains a selectable marker selected from the group consisting of chlorosulfuron, hygromycin, bar gene, bromoxynil, and kanamycin and the like.
85. The transgenic plant of any of Claims 78 or 82, wherein said nucleic acid is operably linked to a promoter selected from the group consisting of octopine synthetase, the nopaline synthase, the manopine synthetase, cauliflower mosaic virus (35S); ribulose-1, 6-biphosphate (RUBP) carboxylase small subunit (ssu), the beta-conglycinin, the phaseolin promoter, napin, gamma zein, globulin, the
ADH promoter, heat-shock, actin, and ubiquitin.
86. The transgenic plant of Claim 78, said enzymatic nucleic acid molecule is in a hammerhead, haiφin, hepatitis Δ virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration
87. The transgenic plant of Claim 86, wherein said enzymatic nucleic acid with RNA cleaving activity encoded as a monomer.
88. The transgenic plant of Claim 86, wherein said enzymatic nucleic acid with RNA cleaving activity encoded as a multimer.
89. The transgenic plant of Claim 78, wherein the nucleic acids encoding for said enzymatic nucleic acid molecule with RNA cleaving activity is operably linked to the 3' end of an open reading frame.
90. The transgenic plant of Claim 78, wherein said gene is an endogenous gene.
91. A transgenic maize plant comprising in the 5' to 3' direction of transcription:
a promoter functional in said plant;
a double strand DNA (dsDNA) sequence encoding for a delta 9 gene of SEQ ID. No. 1, wherein transcribed strand of said dsDNA is complementary to RNA endogenous to said plant; and
a termination region functional in said plant.
92. A transgenic maize plant comprising in the 5' to 3' direction of transcription,
a promoter functional in said plant;
a double strand DNA (dsDNA) sequence encoding for a granule bound starch synthase (GBSS) gene of SEQ ID NO. 25, wherein transcribed strand of said dsDNA is complementary to RNA endogenous to said plant; and
a termination region functional in said plant.
93. The enzymatic nucleic acid molecule of claim 1, wherein said gene is an endogenous gene.
94. The method of modulating expression of a gene of claim 63, wherein siad gene is an endogenous gene.
95. The vector of Figure 42, wherein said vector is employed for transformation of a plant cell.
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EP0842286A2 (en) 1998-05-20
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