AU6129000A - Compositions and method for mudulation of gene expression in plants - Google Patents

Description

S&F Ref: 405696D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicants: Ribozyme Pharmaceuticals, Incorporated 2950 Wilderness Place Boulder Colorado 80301 United States of America r r Actual Inventor(s): Address for Service: Dow AgroSciences LLC 9330 Zionsville Road Indianapolis Indiana United States of America Michael G Zwick Brent E Edington James A McSwiggen Patricia Ann Owens Merlo Lining Guo Thomas A Skokut Scott A Young Otto Folkerts Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Invention Title: The following statement is a full performing it known to me/us:- Compositions and Method for Modulation of Gene Expression in Plants description of this invention, including the best method of 5845c Compositions and Method for Modulation of Gene Expression in Plants 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 description 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 cosuppression (reviewed in Jorgensen, 1995 Science 268, 686-691) approaches Tiave 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 ribozyme technology offers an attractive new means to alter gene expression in plants.

Naturally occurring antisence 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- 20 597; Simons 1988 Gene 72: 35-44). The first demonstration of antisense-mediated inhibition of gene expression was reported in mammalian cells (Izant and Weintraub 1984 Cell 36: 1007-1015). There are many examples in the literature for the use of antisense RNA to modulate gene expression in plants. Following are a few examples: *c [n:\libc]03175:MEF 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 doninant suppressor gene. Transgenic potato plants have been produced hich exprs a

RNA

antisense to potato or cassava granule bound starch syntiasc (cG SS) In b ex pr es R N cases, transgenic plants have been constructed which hav r l dued or o (IISS c ty or protein. These transgenic plants give rise to potatoes containing starch with dramatically reduced amylose levels (Visser et al. 1991, Mol. Gen. Genet w 225 2889-296;y Salehuzzaman et al. 1993, Plant Mol. Biol. 23: 947-962).

Kull et al., 1995, J. Genet. Breed 49, 69-76 reported inhibition of amylose biosynthesis in tubers from transgenic potato lines rndiated 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 ribozymes targeted against th GBSS RNA.es rgeted against the Antisense RNA constructs targeted against A-9 desaturase enzyme in canola have been shown to increase the level ofstearic acid (C8:0) from to 40% (Knuton et. al., 1992 Proc. Natl. Acad Sci. 89, 2624). There was no decrease in total oil content or germination efficiency in one of the high stearate lines. Several recent reviews are available which illustrate the utility of plants with modified oil composition (lroe, B. 1994 Plant Pksiol. 104, 821; Kinney, A. J. 1994 Curr. 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 Sthe 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 transcriptional 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 nterest (Ronchi et al 1995 EMBO J 14: 5318-5328). The other mechanism of co-suppressio n is posttranscriptional, 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 degradation of both messages (van Bokland et al., 1994 Plant 1 6: 861-877) The exact molecular basis for co-suppression is unknown.

Unfortunately, both antisense and co-suppression technologies are subject to problems in heritability of the desired trait (Finnegan and McEroy 994 ioges are subec thnoov 1: 883-888). Currently,. there is no esyainnesancand c E r o y 1994 Bio/Tchnology S 12: 883-888). Currently, there is no easy way to specifically inactivate a gene of intcrcst at the DNA level in plants (Pazkowski et al., 1988 EMBO J 7: 4021-4026). Transposon mutagenesis is inefficient and not a stable event, while chemical mutagenesis is highly non-specific.

Applicant believes.that ribozymes 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 ribozymes in modulating gene expression in plant systems Mazzolini et al., 1992 Plant o. Biol 715-731; Kull et al., 1995 .1 Genet. Breed. 49: 69-76). Although there are reports in the literature of ribozyme activity in plants cells, almost allof them involve down regulation of exogenously introduced genes, such as reporter genes in transient assays (Steinecke et al., 1992 EMBOJ. 11:1525-1530; Perriman et al., 1993 Antisense Res. Dev.

3: 253-263; Perriman et al., 1995, Proc. Nal. Acad. Sci. USA, 92, 6165).

There are also several publications, Lamb and Hay, 1990, J. Gen. irol. 71, 2257-2264; Gerlach e al., International PCT Publication No. WO 9/13994; Xu e al., 1992, Science in China (Ser. B) 35, 1434-1443; Edington and Nelson, 1992, in Gene Regulation: Biology ofantisense RNA and DNA, eds. R. P. Erickson and J. G. Izant, pp 209-221, Raven Press, NY.; Atkins et 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 at, 1994, Cell.

25 Biochem. Suppl. 18A, 110 (X1-406) and Feyter et al., 1996, Mol. Gen. Genet. 250, 329- 338], that propose using hammerhead ribozymes to modulate: virus replication, expression of viral genes and/or reporter genes. None of these publications report the use ofribozymes to modulate the expression of plant genes.

Mazzolini et al., 1992, Plant. Mol. Bio. 20, 715-731; Steinecke ce al., 1992, EMBO.

J. 11, 1525-1530; Perriman et al, 1995, Proc. Na. Aad. Sci. USA., 9 92, 6175-6179; Wegener et 1994, Mol. Gen. Genet. 245, 465-470; and Steinecke et a, 1994, Gene, 149, 47-54, describe the use of hammerhead ribozymes to inhibit expression of reporter genes in plant cells.

Bennett and Cullimore. 1992 Nucleic Acids Res. 20, 831-837 deonstrate hammerhead ribozyme-mediated in vitro cleavage of glna, glnb, ging and gnd RNA, coding for glutamine synthetase enzyme in Phaseolus vulgaris.

Hitz et al., (WO 91/18985) describe a method for usg te soyb -9 dsatura enzyme to modify plant oil composication dcscribcs the use ol soybean A-9 desaturase sequence to isolate 6-9 desaturasc genes ftomn otcr specics.

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. Furthnerore, Applicant believes that the references do not disclose and/or enable the us of ribozymcs to down regulate genes in plant cells, 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 15 not limited to, a hammerhead, hairpin, hepatitis delta virus, group I intron, group II intron, RNaseP RNA, Neurospora VS RNA and-the like. The enzymatic nucleic acid :inon, RNasP RNA, 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 S 20 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 Agrobacteriun, bombarding with DNA coated microprojectiles, whiskers, or electroporation. Any target gene may be modified with the nucleic acids ecoding the enzymatic nucleic acidmolecules with RNA cleaving activity.

Two targets wich 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 andor inhibit gene expression in plants such as monocot plants corn). Riboz d yes 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 j' umayoe esiabl, in some instances, to 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 Stechniques were developed herein to allow directed modulation of gene exprcssio to generate plant cells, plant tissues or plants with altered phenotype.

Ribozymes enzymatic nucleic acids) are nuclcic acid molcctils having an enzymatic activity which is able to repeatedly cleave other scpanItc RNA molecl s in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved n vitro and in vivo (Zaug et al., 1986, Nature 324, 429; Kim et al, 1987, Proc. Nal d Acad Sci USA 84, 8788; Dreyfus, 1988, Einstein Quarterly J. Bio. M 1 92 ro Hascloff and Gerlach, 19 8 8 Nature 334 585; Cech, 1988, JAMA 260, 3030; Murphy and Cech, 1989, Proc Acd Sci. USA., 86, 9218; Jefferies et al., 1989, Nucleic Acids Research 17, 1371).

Because of their sequence-specificity, trans-cleaving 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 RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and 20 abrogates protein expression from that RNA. In this manner, synthesis ofa protein associated with a particular phenotype and/or disease state can be selectively inhibited.

Other features and advantages of thebe apparent from the following description of the preferred embodiments thereof, and from the claims.

Brief Description of the Figures 25 Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem can be 2 base-pairs long. Each N is any nucleotide and each represents a base pair.

Figure 2a is a diagrammatic representation of the hammerhead ribozyme domain known in 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 6 Gerlach (1988, Nature, 334, 585-591) into two portions: and Fiur d s a siilar diagram showing the hammerhead divided by Jeffries and Sv

F

ons (1989, ni a cids Res., 17, 1371-1371) into two portions. c.

Figure 3 is a diagrammatic representation of the gcncral structure of hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs 1 2, 3 or 4) and helix 5 can be optionally provided of length 2 or morc bases (prclcrably 3 20 bas n s, m is from I 20 or more). Helix 2 and helix 5 may be covalently linked b.y one or mor bases r is 2 1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs 4 20 base pairs) to stabilize the ribozyme tructure, and preferably is a protein binding site. In each instance, each N and N' independently is any nonral 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 o an qui d is each independently from 0 to any number, 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 andior phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to 20 its base, sugar or phosphate. 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.

e tacv nbd SFigure 4 is a representation of the general structure of ribozyme domain known in the art.

e o f t e h p s Figure 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.

S Figure 6 is a schematic representation of an RNaseH accessibility assay.

Specifically, the left side of Figure 6 is a diagram of complenentary

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 represent uncleaved target RNA; the bands unique to each lane represet the cleaved products. the cleaved Figure 7 is a graphical representation of RNaseH accessibility of GBSS

RNA.

Figure 8 is a graphical representation of GBSS RNA clc vi-g by ribh.zyicS a different temperatures.

Figure 9 is a graphical representation of GBSS RNA cleavag ribozymes.

A cleavage by ultipl mays. Figure 10 lists the nucleotide sequence of A-9 desaturase cDNA isolated from Zea Figures 11 and 12 are diagrammatic representations of faty cid biosynthis in plants. Figure 11 has been adapted from Gibson et al., 1994, Plant Cell Envir. 17, 627.

Figures 13 and 14 are graphical representations of RNaseH accessibility of A-9 desaturase

RNA.

Figure 15 shows cleavage of A-9 desaturase RNA by ribozymes in vitro. 10/10 represents the length of the binding arms of a hammerhead ribozymes 10/in 0 means helix 1 and helix 3 each form 10 base-pairs with the target RNA (Fig. 4/6 and 6/6, represent helix2/helixl interaction between a hairpin ribozyme and its target. 4/6 means the hairpin (HP) ribozyme forms four base-paired helix 2 and a six base-paired helix I complex with the target (see Fig. 6/6 means, the hairpin ribozyme forms a 6 basepaired-helix 2 and a six base-paired helix I complex with the target. The cleavage reactions were carried out for 120 min at 26 0

C.

"..o"Figure 16 shows the effect of arm-length variation on the activity of HH and HP ribozymes in vitro. 7/7, 10/10 and 12/12 are essentially as described above for the HH ribozyme. 6/6, 6/8, 6/12 represents varying helix I length and a constant (6 bp) helix 2 for S a hairpin ribozyme. The cleavage reactions were carried out essentially as described 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 A-9 desaturase

RNA.

Figures 20 and 21 show in vitro cleavage ofA-9 desaurase RNA b ribozyes that are transcribed from DNA templates using bacteriophage T7 RNA polynerase enzyme.

Figure 22 diagrammatic representation of a non-limiting strategy to onst transcript comprising multiple ribozyme motifs that are iti s t ae y td co nstructi various sites within GBSS RNA. same or d i f f er c t targting Figure 24 shows cleavage of A-9 desaturase RNA by ribozymes 453 Multiler, represents a multimer ribozyme construct taeted against hammerhead ribozyme sites 453, 464, 475 and 484. 252 Multimer, represents a multimer i ead yme construct targeted against hammerhead ribozyme sites 252, 271, 313 and 326. 23R Multimr, reprcscents multimer ribozyme construct targeted against three hammerhead ribozyme sites 252, 259 and 271 and one hairpin ribozyme site 238 259 Multimer, represents a multimer ribozyme construct targeted against two hammerhead ribozme sites 271 and 313 and one hairpin ribozyme site 259 (HP).tes 271 and 313 and one Figure 25 illustrates GBSS mRNA levels in Ribozyme minus Controls F, I, J, N, P, Q) and Active Ribozyme RPA63 Transformants (AA, DD, EE, FF, GG, HH, Ji,

KK).

Figure 26 illustrates A9 desaturase mRNA levels in Non-transformed plants (NT), 85-06 High Stearate Plants 3, 5, 8, 12, 14), and Transformed (irrelevant ribozyme) Control Plants (RPA63-33, RPA63-51, RPA63-65).

20 Figure 27 illustrates A9 desaturase mRNA levels Nontransfored 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 A9 desaturase mRNA levels in Non-transformed plants (NTY), 113-06 Inactive Ribozyme Plants (02, 04, 07, 10,11).- 25 Figures 29a and 29b illustrate A9 desaturase protein levelsin maize leaves (a) Line Hill, plants a-e nontransformed and ribozyme inactive line RPAi13-17, plants 1-6.

Ribozyme active line RPA85-15, plants 1-15.

Figure 30 illustrates stearic acid in leaves of RPA85-06 plants.

Figure 31 illustrates stearic acid in leaves of RPA85-15 plants, results of three assays. plants, results of three Figure 32 illustrates stearic acid in leaves of RPAl 13-06 plants.

Figure 33 illustrates stearic acid in leaves of RPA 13-17 plants.

Figure 34 illustrates stearic acid in leaves of control plants.

Figure 35 illustrates leaf stearate in RI plants from a high stcarate plant cross (RPA85-15.07 self).

Figure 36 illustrates A9 desaturase levels in next generation maize leaves (R *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 A9 desaturase.

Figure 38 illustrates stearic acid in individual somatic embryos from a culture (308/430-015) transformed with antisense A9 desaturase.

Figure 39 illustrates stearic acid in individual leaves from plants regenerated from a culture (308/430-012) transformed with antisense A9 desaturase.

Figure 40 illustrates amylose content in a single kernel of untransformed control line 15 (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 A-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 sie on the RNA, but O unable to cleave that RNA.NA but By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which ha complementarity in a substrate binding region to a specified genc target, and also has an enzymatic activity which is active to specifically cleave that target. That is, tle enzymatic nucleic acid molecule is able to intermolcularly cleave INA (or DNA) and thereby inactivate a target RNA molecule. This complcmcntarity 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, polyribozymes, molecular scissors, self-splicing RNA, self-cleaving RNA, cis-cleaving RNA, autolytic RNA, endoribonuclease, minizyme, leadzyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The term encompasses enzymatic RNA molecule which include one or more ribonucleoides and may include a majority of other types of nucleotides or abasic moieties, as described o. below.

20 By "complementarity" 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.

*25 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 containin a phosphate and either a pure O 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 DNA that is complementary to and derived from a mRNA.

By "dsDNA" is meant a double stranded cDNA.

By "sense" RNA is meant RNA 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 comnplementarity may exist with any part of the target RNA, at the 5' non-coding sequence, 3' non-coding sequence, introns, or 15 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 nopaline synthase promoter, the manopine synthetase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S); plant romoters, such as the ribulose 1, 6 -biphosphate (RUBP) carboxylase small subunit (ssu), the bera-conglycin promoter, the phaseolin promoter, the ADH promoter, heat-shock promoters and tissue specific promoters. Promoter may also contain certain enhancer sequence ele d ents that may improve the transcription efficiency.

By "enhancer" is meant nucleotide sequence element which can stimulaic promotcr activity (Adh). c an st m l By "constitutive promoter" is meant promoter element that directs continuous gene expression in all cells types and at all times (actin, ubiquitin, CaMV 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, napin, ACP).

eeds (ein oleosin, By "development-specific" promoter is meant promoter element responsible for 15 gene expression at specific plant developmental stage, such as in early or late embryogenesis. n o r late C* C y "inducible promoter" is meant promoter element which is rsponsbl for expression of genes in response to a specific signal,.such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone metabolites; and stress.

pr20 By a "plant" is meant a photosynthetic organism, either eukaryotic and prokaryotic.

ba By "angiosperm" is meant a plant having its seed enclosed in an ovary coffee, tobacco, bean, pea).

S By "gymnosperm" is meant a plant having its seed exposed and not enclosed in an 25 ovary 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.

By "transgenic plant" is meant a plant expressing a foreign gene.

By "open reading frame" is meant a nucleotide sequence, without introns, encoding an amino acid sequence, with a defined translation nitation and termination region.

The invention provides a method for producing a class of en atic g agents which exhibit a high degree of specificity for the RNA of a desired target. The c av zyn at nucleic acid molecule may be targeted to a highly specific sequence region ofa 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 hav bn 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 ofribozyme 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.

20 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 25 molecule that acts to cleave the target RNA. Thus, t a enzymatic nuic aid firt recognizes and then binds a target RNA through complementary base-pairing, and once 9 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 RNA target, it is rcleascd 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 hairpin motif, but may also be formed in the motif of a hepatitis A virus, group I intron, group I 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 1992, ADS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel e al., EP03602 57 Hampe and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989 a n 82, E53, l 2 5 lol 7 a Gerlach, 1989, Gene, 82, 43, and Hampel e al., 1990 Nu Act"j' /v 18, 299 Ih l l e hepatitis A virus motif is described by Perrotta and Been, 1992 Biochnemi.ry 3 1 16; of the RNaseP motif by Guerrier-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 described by Collins (Saville and Collins, 1990 Ccl 6 685-696: 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 14, 363); Group 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., U.S. Patent 4,987,071.

These specific motifs aree al., U.S. Patent 4 9 87071.

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 ne or more of the target gene RNA regions, and that it have nucleotide sequences within or urrounding 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 Gerlach et a. International PCT Publication No. WO 91/13994; Edington and Nelson, 1992, in Gene Regulation Binology of ntisense 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 e al, International 25 PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins etal., 1995, I Gen. Virol.

76, 1781-1790; McElroy and Brettell, 1994, TIBTECH 12, 62 Gruber et al., 1994, Cell.

Biochem. Suppl. 18A, 110 (XI-406)and Feyter et al. 1996, Mo. Gen. Genet. 250, 329- 338; all of these are incorporated 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 o m e in e ukaryo ti c p l an t c el l s 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 ribozye RNA is introduced into the plant. Although examples are provided below for the construction of the plasmids used in the transformation experiments illustrated herein, it is well within the skill of an artisan to design numerous different types of plasmids which can be used in the transformation of plants._see Bevan, 1984 Nucl. Acids Res. 12. 8711-8721, which is incorporated by reference. There are also numerous ways to transfor plants In the examples below embryogenic maize cultures were helium blasted. In addition to using the gene gun (US Patents 4,945,050 to Comell and 5,141, 31 to DowElanco) plants may he transformed using Agrobacterium technology, see US Patent 5,177.010 to 1 inMvcr'Sy o Toledo, 5,104,310 to Texas A&M, European Patent Application 0131624 1 European Patent Applications 120516, 159418B1 and 176,112 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 116718, 290799, 320500 all to MaxPlanck, Europan 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, W0920969 6 and W09321 3 3 5 both to PGS; all of which are incorporated 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 20 include but would not be limited to embryogenic tissue, callus tissue type I and and any tissue which is receptive to transformation and subse uent regeneration into a transgenic plant. Another variable is the choice of a selectable marker. The preference for a particular marker is at the discretion of the artisan, but any of the following selectable m: arkers may be usedalong ith 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 hygroscopcus or rdochromogenes species The ar g f h ygroscopicus or viridochromogenes species. The bar gene codes for phosphinothricin acetyl transferase (PAT) that inactivates the active ingradient in the herbicide bialaphos phosphinothricin (PPT). Thus, 30 numerous combinations of technologies may be used in employing ribozyme mediated modulation. n bozyme mediated 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 f a sngle 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 ae the same or different. For example, the multimer construct may comprise a lurality arf hammerhead ribozyFnes or hairpin ribozymes or other ribozyme motifs. Alternatively the multimer construct may be designed to include a plurality of different ribozymne notif, such as ha erh c c ad and hairpin ribozymes. More specifically, multimer ribozye construs h tarc a lsigne d wherein a series of ribozyme motifs are linked togcther i c l tand r c 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. Multicr ribozyme constructs (polyribozymes) are likely to iprove the cffcctivctcss of ribozyme-mediated modulation of gene expression.

The activity ofribozymes 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 a., PCT WO 94/02595, both hereby incorporated i their totality by reference herein; Ohkawa, et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira, elity byl. 1991, Nucleic Acids Res., 19, 5125-30; Ventura, e al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et 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, S barley, maize; oil-producing crops, such as soybean, canola, sunflower, cotton, maize, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut; plantation crops, such as coffee and tea; fruits, such as pineapple, papaya, mango, banana, grapes, oranges, apples; Svegetables, such as cauliflwer, cabbage, melon, gre pepper, tomatoes, carrots, lettuce celery, potatoes, broccoli; legumes, such as soybean, beans, peas; flowers, such as carnations, chrysanthemum, daisy, tulip, gypsophila, alsromeria, gold, petunia, rose; t.:rees 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 ofgenes involved in caffeine synthesis can be used to significantly change caffeine concentration in coffee beans.

Expression of genes, such as 7 -methylxanthosine 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; incorporated by reference herein).

C.

C

C C

CCC.

C

C..

C

C C

C

C C 17 Transgenic tobacco plants expressing ribozymes targeted against genes inlvolv~ed i nicotine Production, such as N-methylputrescine ox-ds optrcneN inty transferase (Shewmaker et supra), wxoul prdelaes wot teredn N-iry concentration.udpouelae wihatrdncie Transgenic plants e~r ing ibozymes tarete aainst genes Involved in ripening Of fruits, such as ethylene-form-n eympctin rncthyltrallsfcrasc, PctinI cstcrasc, polygalacturolse, Il-aminocyclopropane carboxylic acid (ACC) syntlizsc, ACC oxidasc genes (Smith el al., 1988, Nature, 334, 724; Gray et 1992, P1. Mol. Biol. 1 9, 69; Tieman et 1992, Plant Cell, 4, 667; Picton el 1993, The Plant 1. 3, 469; Shewmnaker et al. supra; James et al, 1996, BiolTechnojog, 14, 56), would dclay thc ripcill ig of fruits, such as tomato and apple.

Transgenic plants expressing ribozymes targeted against genes involved in flower pigmentation, such as chalcone synthase chalcone flavanone isomerase

(CH-I),

phenylalanine ammonia lyase, or -dehydroflavonol (DF) hydroxylases, DF reductase (Krol van der, et al., 1988, Nature, 333, 866; KroI van der, et 1990, P1. MoL. Biol. 14, 457; Shewmaker et supra; Jorgensen, 1996, Science, 2 .68, 686), would produce flowers, such as roses, petunia, with altered colors.

Lign ins are organic compounds essential for maintaining mechanical strength of cell walls in plants. Although essential, lignins have some disadvantages. They cause 20 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, cinnamoyl..CoA:NADPH reductase or cinnarnoyl alcohol dehydrogenase (Doorsselaere et 1995,' The Plant 8, 855; Atanassova et al., 1995, The Plant .1 8, 465; Shewmaker et supra; Dwivedi et aZ, 25 1994, P. Mfol Biol., .26, 6 would have altered levels of lignin.

Other useful targets for useful ribozymes are disclosed in Draper et a., International PCT Publication No. WO 93/23569, Sullivan et International

PCT

Publication No. WO 94/02595, as well as by Stinchcomb et International

PCT

Publication No. WO 95131541, and hereby incorporated by reference herein in totality.

Modulation o f granule bound starch s nthase gene ee sion in nlants: In plants, starch biosynthesis occurs in both chloroplasts (short term starch storage) and in the amyloplast (long term starch storage). Starch granules normally consist of a linear chain of ct(l-4)-linked a-D-glucose units (amylose) and a branched form of amylose cross-linked by bonds (amylopectin). An enzyme involved in the synthesis of starch in plants is starch synthase which produces linear chains of (lI 4 )-glucose using ADP-glucose. Two main forms of starch synthase are found in plants: granule bound starch synthase (GBSS) and a soluble form located il tile stro la n o chloroplasts and in amyloplasts (soluble starch synthase). Roth rms of thl clzym utilize ADP-D-glucose while the granular bound form also utilizcs UDP)'i)-tgl csc 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 charactrizd.

Mutations affecting the GBSS gene in several plant species has resulted in tie 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 Waxy is a recessive mutation in the gene encoding granule bound starch synthase

(GBSS).

.s homryo sac in maize. Mutations in this locus have been termed waxy due to th appe r starch in the of mutant ke els, which is the in.

3 0 Ribozymes, with th e i r ca a l y ti c activity a nd i n cr eased s i te sp e ci f i city as d esc ribed amylosense composition in the keels. In maize bozy s ear e i tinhiit GBSS antivit amyloplast of the d eveloping endosperm whes is required for their inhbitnory effect. For those ofordnare about 70% starch, of whi i ear fro he exnam yloples that other bozyes may 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 described below), represent more potent and perhaps more specific inhibitory molecules than antisense oligonucleotides. Moreover, these ribozymes are able to inhibit GBSS activity and the catalytic activity of the ribozymes is required for their inhibitory effect. For those of ordinary skill in the art, it is clear from the examples that other ribozymes may be designed that cleave target mRNAs required for GBSS than maize. ac ti vity in plant species other Thus, in a preferred embodiment, the invention features riboz es that inhibit enzymes involved in amylose production, by rcducing G3SS activity T nhi es endogenously expressed RNA molecules contain substrate nding domains thait bi h to accessible regions of the target mRNA. The RNA lmol cul also contain domains that catalyze the cleavage of RNA. The RNA molecules ar rfrably r c bozy n i n of the hammerhead or hairpin motif. Upon binding, the ribozyes cleave the target n m

RNA

s preventing translation and protein accumulation. In the absence of the expreion of th target gene, amylose production is reduced or inhibited. Spccific examples ar on providd below infra.

Preferred embodiments include the ribozymes having binding arms which are complemeny to the binding sequences in Tables lAII, VA and VB. Examples of such ribozymes are shown in Tables IIB V. Those in the art will recognize that while such examples are designed to one plant's 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 sequencesptcitt 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 bozymes and methods for their use in the inhibition of starch granule bound AD oD or their use n the ::::inhibition of starch graul bund ADP (UDP)-glucoe: a- 1 4 -D-glucan 4 -a-glucosyl transferase granule bound 'starch synthase (GBSS) activity i plants. Thiss accomplished through the inhibition of genetic expression, with ribozymes which results inthe 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 constftutive promoterl a tissue-specific promoter or an inducible promoter pro m ote r Modification of corn 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 corn has the waxy phenotype, but because of its reccssive nature the traditional waxy varieties are very difficult for the grower o h;in(dlc Ribozymes targeted to cleave the GBSS mRNA and thus reduce (C;SS activty i plnt1s, 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 fatty acid saturation profile in plants: 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: I) 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 stearoyl-ACP desaturase. This is followed by Simmediate 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 corn 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 transferred 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 11 and .12.

The three predominant fatty acids in corn seed oil are linoleic acid (18:2, oleic acid (18:1, and palmitic acid (16:0, These are average values and may be somewhat different depending on the genotype; however, composite samples of US Corn 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, 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 principlcs and modern practices (ed. Erickson, Am. Oil Chcmists Soc. Champaign, This predominance of C8 chain lengths may reflect the abundance and activity of several key enzymes, such as the fatty acid synthase responsible for production of C18 carbon chains, the stearoyl-ACP desaturase (A-9 desaturase) for production of 18:1 and a microsomal A-12 desaturase for conversion of 18:1 to 18:2.

A-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, 1982 J. Biol.

Chem. 257: 12141-12147). This contrasts to the mammalian, lower eukaryotic and cyanobacterial A-9 desaturases. Rat and yeast A-9 desaturases are membrane bound microsomal enzymes using acyl-CoA chains as substrates, whereas cyanobacterial A-9 desaturase uses acyl chains on diacyl glycerol as substrate. To date several A-9 desaturase cDNA clones from dicotelydenous plants have been isolated and characterized (Shanklin and Somerville, 1991 Proc. Nal. Acad Sci. USA 88: 2510-2514; Knutzon et al., 20 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 A-9 desaturase sequences suggests that this is a highly conserved enzyme, with high levels of identity both at the amino acid level and at the nucleotide level However, as might be expected from its very different physical and enzymologica properties, no sequence similarity exists between plant and other A-9 desaturases (Shanklin and Somerville, supra).

SPurification and characterization of the castor bean desaturase (and others) indicates that the A-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 diferric clusters, which in the presence of dithioni are reduced to the diferrous state. In the presence of stearoyl-CoA and 02 the clusters return to the diferric state. This suggests that the desaturase belongs to a group of 2 activating proteins containing diiron-oxo clusters. Other members of this group are ribonucleot reductase and methane monooxygenase hydroxylase. Comparison of the prcdictel primary structure for these catalytically diverse proteins shows that all contaice a conserved pair of amino acid sequences (Asp/Glu)-Glu-Xaa-Arg.-lis separated by 100 amino acids.

Traditional plant breeding programs have shown that increased stearate levels can be achieved without deleterious consequences to the plant. In safflower (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 A-9 desaturase activity have been achieved by the transformation of ~tobacco with the A-9 desaturase genes from yeast (Polashock et al., 1992 Plant Physiol.

100, 894) or rat (Grayburn 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.

Corn (maize) has been used minimally for the production of margarine products Sbecause 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, corn 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- 25 regulation of A-9 desaturase activity will make corn a viable alternative to soybean an 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 derived 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. A-9 desaturase introduces the first double bond into 18 car fkey step effecting the extent of desaturation of fatty acids. ac ds and s t e Thus, in a preferred embodiment, the invention concerns copositions (and methods for their use) for the modification of fatty acid c c ompolants. Thi o n s san accomplished through the inhibition of genetic expression with ribozyincs, p antisc is nucleic acid, cosuppression or triplex DNA, which results I the riducti, (o CIo y m liIaii()an of certain enzyme activities in plants, such as A-9 desaturase. Such activity is rcdiucd in monocotyledon plants, such as maize, wheat, rice, palm, coconut and others. A-9 desaturase activity may also be reduced in dicotyledon plants such as sunflower, safflower, cotton, peanut, -olive, sesame, cuphea, flax, ojoba, grape and othcrs.

Thus, in one aspect, the invention features ribozymes that inhibit enzymes involved in fatty acid unsaturation, by reducing Az-9 desaturase acivity. 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 th hammerhead or hairpin 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 increasedand unsaturated fatty acid production is reduced orinhibited Specific examples are provided below in the Tables listed directly below.

20 In preferred embodiments, the ribozymes have binding arms which ar complementary to the sequences in the Tables VI and VIII. Those in the art will recognize that while such examples are designed to one plant's corn) 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 25 RNA in a sequence-specific manner and enable the ribozyme to cause cleavage of a plant mRNA target. Examples of such ribo ri ozyme t o caus e cleavage o f a l a nt 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.

24 Those in the art will recognize that ribozyme sequences listed in th 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, sten-loo 11 sequence of hammerhead ribozymes listed in Table IV (S'-GGCGAAAGCC 3 can be altered (substitution, deletion, and/or insertion) to contain any scqucIc G A A A C C PcC b provided that a minimum of a two base-paired stei structure can form. Similarly, ste loop IV sequence of hairpin ribozymes listed in Tables V and VIII can be altered (substitution, deletion, and/or insertion) to ontain any sequence, preferably provided that a minimum of a two base-paired stem structure an form. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

In another aspect of the invention, ribozymes 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 mRNAs 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 20 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 A-9 desaturase in maize.

In preferred embodiments, hairpin and hammerhead ribozymes that cleave A-9 desaturase mRNA are also described. Those of ordinary skill in the ar will understand S. from the examples described below that other ribozymes that cleave target mRNAs required for A-9 desaturase activity may now be readily designed and are within the scope S of the invention.

While specific examples to corn RNA are provided, those in the art will recognize that the teachings are not limited to corn. 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 and teachings herein are meant to be non-limiting, and those skilled in te art will Srecognize that similar embodiments can be readily generated in a variety plants to modulate expression of a variety of different genes, using the teachings herein and are within the scope of the inventions.

e t Standard molecular biology techniques were followed in thie cxamplc,, hcrin.

Additional information may be found in Sambrook, Fritsch, l. ad Mali tis, T.

(1989), Molecular Cloning a Laboratory Manual, second edition, Cold Spring ilarbor: Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

Examples Example 1: Isolation of 9 desarase cDNA from Zea ma Degenerate PCR primers were designed and synthesized to two conserved peptides involved in diiron-oxo group binding of plant A-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 A-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 DNA; 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 A-9 desaturase gene for the predicted mature protein. The complete sequence is presented in Figure Example 2: Identification of Potential Ribome Cleava Site for 9 desaturase Approximately two hundred and fifty HH ribozyme sites and approximately forty three HP sites were identified in the corn A-9 desaturase mRNA. A HH site consists of a uridine and any nucleotide except guanosine Tables VI and VIII have a list of HH and HP ribozyme cleavage sites. The numbering system starts with I at the 5' end of a A- 9 desaturase cDNA clone having the sequence shown in Fig. 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 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 A9 desaturase The secondary structure of A-9 desarurase mRNA was assessed by computer analysis using algorithms, 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 nuclcotides and con c ain i l pdr y Po(tinl hammerhead ribozyme cleavage sites were identified.

These sites were assessed for oligonucleotide accessibility by RNase H assays (see Example 4 infra).ase assa see Example 4: RNaseH Assays for 9 desaturase Forty nine DNA oligonucleotides, each twenty one nucleotides long were used in 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 A-9 desaturase cDNA. RNA was screened for accessible cleavage sites by the method described generally in Draper et al., supra. Briefly, DNA oligonucleotides rprsenting ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to .generate a substrate for T7 RNA polymerase transcription from corn cDNA clones.

S. 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 37C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percetae of the substrate cleaved was determined by autoradiographic quantitation using a Molecular Dynamics phosphor imaging system (Figs. 13 and 14).osphor Example 5: Hammerhead and H nRibo es for 9 desaturase Hammerhead (HH) and hairpin (HP) ribozymes were designed to the sites covered 25 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 ribozymes were chemically synthesized. The general procedures for RNA synthesis have been described 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 Applied Biosystems, Inc. synthesizer using a modified 5 ol scale protocol ith a m in coupling step for alkylsilyl protected nucleotides and 2.5 min coupling step for methylated nucleotides. Table II outlines the amounts, and te contat ties, of the reagents used in the synthesis cycle. A 6.5-fold excess (163 .L of 0.1 M 163 imol) of phosphoramidite and a 24-fold excess of S-ethyl tetrazole (238 L of 0.25 M 595 o mol) relative to polymer-bound 5'-hydroxyl was used in eachi couling cyc. Avcr; gc coupling yields on the 394, determined by colorimetric quantitation ofthe trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride (ABI); capping was performed with 16% N- Methyl imidazole in THF (ABI) and 10% acetic anhydride/0% 2 6 -lutidine in THF (ABI); oxidation solution was 16.9 mM 12, 49 mM pyridinc, 9% water in TIF: (Millipore). B J Synthesis Grade acetonitrile was used directl from the reagent bottle.

S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from American International Chemical, Inc.

Deprotection of the RNA was performed as follows. The polymer-bound oligoribonucleotide, trityl-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 0 C for 10 min.

After cooling to -20 0 C, the supematant was removed from the polymer support. The support was washed three times with 1.0 mL of EtOH:MeCN:H20/3:I:I 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 L of a solution of 1.5 mL N-ethylpyrrolidin ne, 750L u TEA and 1.0 mL TEA*3HF to provide a 1.4 M HF concentration) and heated to 65 0 C for 25 1.5 h. The resulting, fully deprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.

SFor anion exchange desalting of the deprotected oligomer, the TEAB solution was loaded onto a Qiagen 5000 anion exchange cartridge (Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB mL), the RNA 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 4 (numbering from (Hertel, K. et al., 1992, Nucleic Acids Res., 20, 3252).

The hairpin ribozymes were synthesized as described above for the ammerhead RNAs. e lerhead Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, mlethodvs sEnzvno/. I 80, 51).

Ribozymes were purified by gel electrophoresis using general mcthlods or were purificc by high pressure liquid chromatography (IPLC; See Wincott t 1996, supra, the totality of.which is hereby incorporated herein by reference) and were resuspended in water. The sequences of the chemically synthesized ribozymes used in this study are shown below in Tables VII and VIII. tu c Example 6: Long substrate tests for A9 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 A-9 desaturase RNA. A template containing T7 RNA polymerase promoter upstream of A-9 desaturase target sequence, was PCR amplified from a cDNA clone. Target RNA was transcribed from thi s s amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [a- 3 2 p] CTP as one of the four ribonucleotide triphosphates The transcription mixture was treated with DNase-I, following transcription at 37C for 2 hours, to digest away the DNA template used in the transcription. The transcription S mixture was resolved on a denaturing polyacrylamide gel. Bands corresponding to full- 20 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/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.HC1, pH 7.5 and 10 mM MgC12. The RNAs were renatured by cooling to the reaction temperature (37 0 C, 26"C or 20C) for 10-20 min. Cleavage reaction was initiated by mixing the ribozyme and target f. .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 0 C, are summarized in Table IX and Figures 15 and 16. Of the ribozymes tested, seven hammerheads and two hairpins showed significant cleavage of A-9 desaturase RNA (Figures 15 and 16) Ribozymes to other sites showed varied levels of activity.

Example 7: Cleavage of the target RNA usin multiple ribo e combinations for A9 desaturase Several of the above ribozymes were incorporated into a Ilultimcr ribozymc 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 I intron (Dennis et al., 1984 Nucl. Acids Res. 12, 3983) and the Nos polyadenylation signal (DePicker et al, 1982 J. Molec. App. 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, an be readily generated using techniques known in the art and are within the scope of this "invention.

e @o o Ple 8: Construction ofRibo Example 8: Construction of Riboz xpressing transcription units for A9 desaturase 2Ribozymes targeted to cleave A-9 desaturase mRNA are endogenously expressed in plants, either from genes inserted into the plant genome (stable transformation) or from Sepisomal transcription units (transient expression) which are part of plasmid vectors or viral sequences. These ribozymes can be expressed via RNA polymerase 1, II, or III plant or plant virus promoters (such as CaMV). Promoters can be either constitutive, tissue specific, or developmentally expressed.

A9 259 Monomer Ribozyme Constructs (RPA 114 115) These are the A-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 115.

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 gel-purified and ligated into the pDAB 367 vector. Te constructs are hecked by Sdigestion with Sgf /Hind III and ba Sst I and confirmed by sequencing hecked b A9 453 Multimer Ribozvme Constructs (RpA 18. 1 19) These are the A-9 desaturase 453 Multimer ha rhad Figure 17). The ribozymes were designed with 3 bp long ste Ii rcgions zy Totr leg s (s the substrate binding arms of the multimer construct s bp. The aciv n vrsion is RPA 118, the inactive is 119. The conttnsruct w as 4 2 b p Te ac tive version is RPA 118 me. The muie 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 A-9 desaturase

RNA.

9 252 Multimer Ribozme ConsA 85. 113 These are the A-9 desaturase 252 multimer ribozye clones placed at the 3'end of bar (phosphoinothricin cetyl transferase ozym e cl on es p l a c e d a t t h e 3 'end of bar (phosphoinothrin acetyl transferase; Thompson et al., 1987 EMBO J 6: 2519-2523) open reading frame. The multimer contructs 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 constructd as follows: The parent plasmid pDAB 367 was partially digested with Bgl I and the single cut plasmid was gel-purified. This was recut with Eco RI and again gel-purified to isolate the correct Bgl/Eco RI cut fragment. The Bam H/ Eco RI inserts from the ribozme constructs were gel-isolated (this contains the ribozyme and the NOS po A region) and ligated into the 367 vector. The identitiy of positive pasmids wee co ed performing a Nco I Ss I digest and sequencing. con f by SUseful transgenic plants can be identified by standard assays. The transgenic plants can be evaluated for reduction in A-9 desaturase expression and A-9 desaturase activity as discussed in the examples infra.

Examle 9: Identification ofPotential Ribozvme Cleava e Sites in GBSS

RNA

Two hundred and forty one hammer-head ribozyme sites were identified in the corn GBSS mRNA polypeptide coding region (see table IIA). A hammer- iead site consists of a uridine and any nucleotide except guanine Following i the sequence of GBS coding region for corn (SEQ. I.D. No. 25). The numberig system starts with at the end of a GBSS cDNA clone having the following sequence to 3')

I

GAGACATGCATAGAACACCGCCGAGGCGACGCGACAGCCGCCA

73 144 14521 TCATCTCGTCGACGACCAGTGGATTAATCGGC 216CC-ACAG''(C GCTCGTCGCAACGCGCG

GCCTAGAG.CCA

21728 CGCCGGCGTCCCGGACGCGTCCACGTTCCGC 288CGGCGG'C GAGGGGGGGCCGGACGG

CCGGCCCGCC

28936 CGTCGGCGGCGGACACGCTCAGCATTCGGACAC360GCGGCCGC CCAGCACCAGCAGCAGC

CGCCGGCAGT

361 432 ACAGACGTCGCTCG

CAGTCCTCGCTCGTCGTGTGCGCCAGCGCCG

43350 TCGGCCAACTGGAG.CAAAC 504TGCGCTCT :20 CGGCCTGCCGCCGGCCA

CAGCTG

*::505 576 TGGCCGCGAATGGGCACCGTGTCATGGTCGCTCCCAGCATCA

GGACGCCTGGGACACCA

0.0. 57764 2'GCGTCGTGTCCGAGATCAAGATGGGAGACAGCAAGTAGTTCA a::.CTGCTACAAGCGCGGAG 649 720 TGGACCGCGTGTTCGTGACCACCCACTGTTCGflGTTGGAAA

CGAGGAGAAGATCTACG

a. 30 721 792 GGCCTGACc3CTGGAACGGACTACAGGGACAA

ACTGCGGTTCAGCCTGCTATG

CCAGGCAGCACTTGAAG

oa CCCAAGGATCCTGAGCCTCAACAACAACCCATCT864ACAACGG GGACGTCGTGTTCGTCT

AATCCGACTCGG

865 GCACGGGCACAGCTTCTCTACCTCAAGAGCAACTACCAGTcC O937

ACGCAAAGACCGCTTTCTGCATCCACAACATCTCCACAGGCGTCCCT

TCCGACTACCCGGAGC

1009 1 8 TGAACCTCCCGGAGAGATTCAAGCrc., l*ICAI' AClO(I*

C;()A

GCCCGTGGAAGGCCGGA

1081 1152 1 0 A G A T A A C T G T A G C G G A C T G G C G C A G G C T A C T A CCCCTACTACGCCGAGG

CTA

1153 1 2 AGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTG1224CTGGCTA CGGCATCACCGGCATCG

GCAACTCCTA

1225 1296 GtAAGTCGACTGCTCG GTGAC

AGCAGGGACAAGTACATCGCCGT

12 97 1 6

GGGCCGTGGAGGCCAAGGCGCTGAACAAGGAGGCTAGGAGCGC

20 TCCCGGTGGACCGGAACA *1369 14 TCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAG 1440CCGAGTA

GGCGGCCGCCATCCCGC

1441 1 1

AGAAGTTCGAGCGCATGC

1513 1584

TCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTCCCGGTAGTA

CGCGGCGCTGGCGCACC

.30 1585 1656

ACATCATGGCCGGCGCCGACGTGCTCGCCGTCACCGGTCACCGGC

CTCATCCAGCTGCAGG

1657 1 2 GGATGCGATACGGAACGCCCTGCGCCTGCGCGTCCACGTG1728CGCA CATCATCGAAGGCAAGA

~ACGGATGCAA

1729 1800 CCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACGTG GAC

GGA

CGTCAAGAAGGTGGCC A CTGGAGccGG CG GA 1801 CCACCTTGCAGCGCGCCATCAAGGTGGT 1872A

GAGGAACTGCATGATCC

1873 19)44 AGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGG1944CG'FC;C-I.(;C:-I'CA;C'C~l

CGGGGTCGCCGGCGGCG

1945 2016

AGCCAGGGGTCGAAGGCGAGGAGATCGCGCCGCTCGCCAAGGAGAACGTGGCCC

CGCCCTGAAGAGTTCGGC

2017

CTGCAGGCCCCCTGATCTCGCGCGIGGTGCAAACTGTTGGGACATCTTCTTTATT

ATGCTGTTTCGTTTAT

2089 2160 GTGATATGGACAAGTATGTGTA GCTGCTTGCTGTGCTAGTGTAATA TAGTGTA

G

TGGTGGCCAGTGGCACA

2161 ACCTAATAAGCGCATGAA CTAATT CTCTTT TA ACATT2232 GCTCGGGAGTATACCGATCGGTA ATTTTATATTGCGAGTA 2233 AATAAAT CCTGTAG:GTGGAAAAAAAA (SEQ I.D. NO. There are approximately 53 potential hairpin ribozyme Sites in the GBSS mRNA.

The ribozyne and target sequences are listed in Table

V.

Ribozymes can be readily designed and Synthesized to such sites with between and 100 or more bases as substrate binding arms (see Figs. I 5) as described above.

Exampie 10: Selection of Ribozvme Cleava"eSitesforGBSS The secondary structure of GBSS mRNA was assessed by computer analysis using folding algorithms, such as the ones developed by M. Zuker Zuker, 1989 Science, 244, 4852. Regions of the mRNA that did not form secondary folding structures with RNA/RNA stems of over eight nucleotides and contained potential hammerhead ribozyre cleavage sites were identified.

These sites w hich w ere then assessed fo h nu j 0 c esi i 1 wt R a c H assays (see Fig. FiftY-eight DNA Ol -o l 1'u leotide e actesslty O ,ne t n ceRisc long were used In these asas hs l gnceatides, each tw si na i n a s sa s Thes olig o tides covered 85 ts. Th an 1 e39, a 0 59f these 636,i78,n25, 41, 11, 5 9wer 195, 205, 240, 307, 390, 424, 472, 481, S99, 25, 6367, 6782, 75, 104, 811,05, 891, 897, 912, 91R, 928, 951, 958, 969, 99c), 999, 106, 127, 1032, 1056, 1384, 110, 1 156, 1168, 1186, 1195, 1204, 121., 1222, 124, 1926, 124, a198 Se345,r 1351 12471, 1533, 1563, 1714, 1750, 1786, 1806, 181 1 21, 195 nd 978 Se ond~ ites w ere also covered and included 202, 394, 384, 385, 484, 624, 627, 628, 679, 862, 901, 9.30, 950, 952, 967, 990, 991, 1026, 1035, 1108, 1159, 1225,1273, 1534, 1564, 1558, and 1717.

Exa.~1 I1 .ae~ sasfrG

S

RNase H- assays (Fig. 7) were perfor-med using a full length transcript of the

GBSS

coding region, 3'noncoding region, and part of the 5' noncoding region. TeGS

N

wa s srene fo cesbece g ites by the method described generally in Draper et sPr hereby incorporated by reference herein. Brietly, DNA oligonucleotide representing hammerhead ribozy 1 me cleavage sites were synthesized. A PolYmerase chain reaction was used to generate a substrate for T7 RNA Polymerase transcriptionfrmcr cDNA clones. Labeled RNA transc ip eesnh~ z~ i ir from hset mpats The Oligonucleotds n helble rnsrps o plts andth laeld tancritswere annealed, RNAsCH was added and the mixtures were incubated for 10 minutes at 3 7 0 C. Reactions wer t pe and.N separated Onsequencing P~~cyai gels. The percentage of the substrate cleaved .was determined byatrdorahcq e a autrdop. antitati 0 n using a phosphor imaging system (-Fig.

Exam le 12: Hammeread Ribo es for GBSs 25 Hammjerhlead ribozymes with 10/10 Ueable to form 10) base pairs on each arm of 'eribozyme) nucleotide binding arms were designed to the sites covered by'teois whc claedbs in the RNase H assays. These ribozYmes were then subjected to aalysis bycomputer fligand the ribozymes that had sign Ifcant secondary structure 3 0 m s t er r e c t d n s a r s l o f t i s s c r e e n i n g P r o c e d u r e 2 3 r b o z y nm e s w r e i n d t h most ope reg ons in t e G S NR'A the sequences o h s i o y~ 5 ae s o n i Table WV.Ofteerbzmsaesoni The ribozymes were chemically synthesized. The method of synthesis used follows the Procedure for normal RNA synthesis as described above (and in Usman et a., supra. Scaringe et al., and Wincott et al., supra) and are icorporatd by r cc 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 stepwi coupling yields were Inactive ribozymes were ynthesied by ubstituting a U for G5 and a U for A 14 (numbering from (Hertel et al., supra). Hairpin ribo7ymcs wccr synthesized in two parts and annealed to reconstruct th activ ribozy me (Chowri a Burke, 1992, Nucleic Acids Res., 20, 2835 All ribozy s we cte modifi l d o cnhwr ancc stability by modification of five ribonucleotides at both the 5' and 3' ends with 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 dcscribed abovc and wcrc 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 transcrbed ftsuc w a s

P

C R a m p l i f i ed f ro 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 S[as32p] CT as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase l, following transcription at 37 0 C for 2 -hours, to digest away the DNA template used in the transcription. The transcription mixture was resolved on a denaturing polyacylamide gel. Bands corresponding to full-length RNA was solated from a gel slice and the RNA was precipitated with isopropanol and the pellet was stored at 4 0

C.

Ribozyme cleavage reactions were carried out under ribozyme excess (kcat/KM) conditions (Herschlag and Cech, supra). Briefly, 1000 nM ribozyme and 10 nM internally labeled target RNA were denatured Separately by heating to 90 n C for 2 in. in the presence of 50 mM Tris.HCI, pH 7.5 and 10 mM MgC12. The RNAs were renatured by cooling to the reaction temperature (37 0 C 26C and 20C) for 1 leavage 30 reaction was initiated by mixing the ribozyme and target RNA at appropriate reacvag 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.

36 The results from ribozyme cleavage reactions, att three different temperatures are summarized in Figure 8. Seven lead ribozymes were chosen (425, 892, 919, 959, 968, p era 1241, and 1787). One of the active ribozymes (811) produced a strange pattern of cleavage products; as a result, it was not chosen as one of our lead ribozym es Example 1 CleavaLe of the GBSS RNA Usin

M

Four of the lead ribozymes (892, 919, 959, 1241) wcre incubated with ilntrnally 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 carried out at 20 0 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: Consrucon of Ribozvme Exressin Transcrition 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: CGC GGA TCC TGG TAG GAC TGA TGA GGC CGA AAG GCC GAA ATG TTG TGC TGA TGA GGC CGA AAG GCC GAA ATG CAG AAA GCG GTC TTT GCG TCC CTG TAG ATG CCG TGG C Oligo 2: CGC GAG CTC GGC CCT CTC TTT CGG CCT TTC GGC CTC ATC AGG TGC TAC CTC AAG AGC AAC TAC CAG TTT CGG CCT TTC GGC CTC ATC AGC CAC GGC ATC TAC AGG

G

3 Inactive versions of the above were made by substituting T for G5 and T for A14 within the catalytic core of each ribozyme motif These were annealed in I X Klenow Buffer (Gibco/BRL) at 90 0 C for 5 minutes and slow cooled to room temperature (22 0 NTPs were added to 0.2 mM and the oligos extended with Klenow enzyme at lunit/ul for one hour at 37 0 C. This was phenol/chloroform extracted and ethanol precipitated then resuspended in IX React 3 buffer (Gibco/BRL) and digested with Bam HI and Sst I for 1 hour at 37 0 C. The DNA was gel purified on a 2% agarose gel using the Qiagen gel extraction kit.

The DNA fragments were ligated into BamHlllSs I digested pDAB 353. Th ligo w transformed into competent DH5c F' bacteria (Gibco/13RL). Potcntial clons were screened by digestion with Bam HIEco RI. Clones were confirmed by sequencng. 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 10/0 arms as follows. Two DNA oligos were ordered: w as c n st ed 10 0 rm s Oligo 1: GAT CCG ATG CCG TGG CTG ATG AGG CCG AAA GGC CGA AAC TGG TAG TT 0Oligo 2: AAC TAC CAG TTT CGG CCT TTC GGC CTC ATC AGC CAC GGC ATC 20 G be. I 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 The vector was prepared by digestion of pDAB 353 with Sst I and 25 blunting the ends with T4 DNA polymerase. The vector was redigested with Bam HI and (Gibco/BRL) at 16*C overnight. Potential clones were digested with Bam HI/Eco RI and confmed by sequencing.

Example 16: Plant Transformation Plasmids 9DAB Ribozme Experiments and pDAB353 used in the GBSS Ribozvme E ments

C.

Part A pDAB367 Plasmid pDAB367 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. and reading on the strand contiguous to the LacZ gene coding strand, the linker sequence

CTGCAGGCCGGCC

T A T A A G C G G C C G C G T T T A A A G C G G A T A A G C C C

G

GATCGTTGCAATCTCATGGTG, ucleoti-des 7093 to 7344 Of CaMV

DNA

the linker sequence CATCGATG, nucleotides 7093 to 7439 Of CaMV, the linker sequence GGGGACTCTAGAGGATCCG flucleotides 167 to 186 of MSVI(3), flucleotides 188 to 277 Of MSV a C residue followed by nUClcotldcs 119 to 209 of maize Adh IS containing -parts of exon I and ifltron 1 1I1UCICOtjiC s 7 containing parts of Adh IS intron I and exon 2 the inkcr scquclc GAcs 5551to.672 and nucleotides 278 to 31 7 Of MSV. This Is followed by a modified BAR coding region from pIJ4 104 having the AGC serine codon in the second Position replaced by a GCC alanine codon, an .d nucleotide 546 of the coding region changed from G to A to elliminate a Bgl 1I site. Next, the linker sequence TGAGATCTIGAGCTCGA

TCC,

nucleotides 1298 to 1554 of Nos and a G residue followed by the rest of the pUC 19 sequence (including the Eco R! site).

Part B DDAB353 Plasmid pDAB353 has the following DNA structure: beginning with the base after the final C residue of the Sph I site of pUG 19 (base 441; Ref. and reading on the strand contiguous to the LacZ gene coding 'Strand, the linker sequence CTGCAATCTCATGGTGnucleotides 7093 to 7344 of CaMV DNA the linker sequence CATCGATGj nucleotides. 7093 to 7439 of CaMV, the linker scquencc GGGGACTCTAGAG, nucleotides 11 9 to 209 of maize Adh IS containing parts of exon I and intlon 1 nlucleotides 555 to 672 containing parts of Adh IS intl-on I and exon 2 (4,and the lneseuneGCGT GCACwhere GGATCC represents the recognition sequence for BamH- I restriction enzyme. This is followed by the betaglucuronias (GUS) gene from pRAJ275 cloned as an Nco 1/Sac I fragment, the linker sequence GAATTTCCCC, the poly A region in nucleotides 1298 to 1554 of Nos ada G residue followed by the rest of the pUGC 19 sequence.(including the Eco

RI

site).

00*030 The following are herein incorporated by reference: .14. Messing, J. (1983) in "Methods in Enzymologyi (Wu, R. et Eds) 101:20-78.

2. Franck, Guilley, G. Jonard, K. Richards, and L. Hirth (1980) Nucleotide .sequence of Cauliflower Mosaic Virus DNA. Cell 21:285-294.

3. Mullineaux, P. J. Donson, B. A. M. Morris-Krsinich. M. I. Boulton and J.

Davies (1984) The nucleotide sequence of Maize Streak Virus DNA. EMBO J. 3:3063i 3068.

4. Dennis, E. W. L. Gerlach, A. J. Pryor, J. L. Bennetzen. A. Inglis, D. Llewellyn,

M.

M. Sachs, R. J. Ferl, and W. J. Peacock (1984) Molecular analysis of tile lCOhol dehydrogenase (Adhl) gene of maize. Nucl. Acids Res. 12:3983-4000.

White, S-Y Chang, M. J. Bibb, and M. J. Bibb (1990) A sscttc COntaining the bar gene of Strepomyces hygroscopicus.. a selectable marker for plant transformation Nucl.

Acids. Res. 18:1062.

6. DePicker, S. Stachel, P. Dhaese, P. Zambryski, and 11.. M. Goodman (1982) Nopaline Synthase: Transcript mapping and DNA sequence J. Molec. Appl. Gcnet.

1:561-573. se e. p G 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 DAB359 Plant Transformation Plasmid which Contains the Gamma-Zein Promoter, the Antisense GBSS. and a the Nos Polvadenvlation Seuence Plasmid pDAB359 is a 6702 bp double-stranded, circular DNA that contains the 20 following sequence elements: nucleotides 1-404 from pUC18 which include lac operon sequence from base 238 to base 404 and ends with the HindI c site of te Ml3mp8 polylinker the Nos polyadenylation sequence frm n ids to 6 (3) ^.heeun from u nucleotides 412 6 6 8 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

I-

2255 of SEQ. I.D. No. 25. The GBSS sequence in plasmid pDAB359 was modified from the original cDNA by the additiono s c p "the original cDNA by the addition ofa 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 Ssequences. Bases 2971 to 4453 are 5' untranslated sequence of the maize 7 kD gama- 30 zein gene corresponding to nucleotides 1078 to 2565 of the published sequence The gamma-zein sequence was modified to contain a 5' Kpn I site and 3' BamH/Sall/Nco sites. Additional changes in the gamma-zein sequence relative to the published sequence include a T deletion at nucleotide 104, a TACA deleton 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 puc 8 bases 456 to 2686 including the Kpn I/EcoRI/Sac I sites of the M13/mpl8 polylinker from 4454 to 4471, a lac operon fragEment fromn 4471 to 4697. and the 1 -acatmase genle from 5642 to 6433 2).

The following are incorporated by reference herein: pUCI18- Norrander, Kempe, Messing, J. Gene (1983) 26: 101-106, POLWCIS, 1.11.

Enger-Valk, Brammar, W. J. Cloning Vectors, Elscvicr 1 985 Mnd ulcet NosA DePicker, Stachel, Dhaese, Zambryski, and Goodman,

H-.M.

(1982) Nopaline Synthase: Transcript Mapping and DNA Sequence J. Mole. AppI.

Genet. 1:561-573.

Maize 27kD gammazein Das, Poliak, Ward, Messing, J. Nucleic Acids Research 19, 3325 -3330 (1991).

Example 18: Consruction of Plasmid DAB430 containing Antisense A9 Desaturase Expressed by the Ubiuitin Promote/intron (Ubi

I)

Part A Construction of plasmid pDAB421 Plasinid pDAB42 1 contains a unique blunt-end Srfl cloning site flanked by the maize *..Ubiquitin pooe/nr and the nopaline synthase polyadenylation sequences. PDAB42 I was prepared as follows: digestion of pDAB3355.with restriction enzymes KpnI and BamHIl drops out the R coding region on a 2.2 kB fragment. Following gel pturfication, the vector was ligated to an adapter composed of two annealed oligonucleotides 0F235 and 0F236. OF235 has the Sequence 5' GAT CCG CCC

GGG

GCC CGG GCG GTA C -3 Yand 0F236 has the sequence 5' CGC CCG GGC

CCC

GGG CG Clones containing this adapter were identified by digestion and linearization of plasmid DNA with the enzymes Srfl and SinaI which cut in the adapter, but not elsewhere in the plasmid. One representative clone wssqecdt eiyta only one adapter was inserted into the plasmid. The resulting plasmid pDAB342 I was used in subsequent construction of the A9 desaturase antisense plasmid pDAB43Q.

*Part B Construction of plasmid pDAB430 (antisense A9 desaturase) The antisense A9 desaturase construct present in plasmid pDAB43O was produced by cloning of an amplification product in the blunt-end cloning site of plasmid pDAB42

I.

Two constructs were produced simultaneously from the same experiment. The first constrt contains the iA9 desaturase gene in the sense orilentation behind thle ubiut 1 Promoter, and the c-myc tag fused to the C-terminus of tile A9 desaturase opell I eadfrl frame for Imnlgcldetection Ofoverproduced proreineain costuc wimuneogr oft nI)trngncl in rr n o tgeu i l es. T h is contmt wa itened or esing of ribozymes in a sYstemn which did no ress maize ,9 dsatra~ TIs construc t was never used, but thc prinr usI to pify and construct the A9 desaturase antisense gene were the saluc. The 69 Mczti.~ INA sequence described herein was amplified with two primlers. The N-tcriial primler 0F279 has the sequence GTG CCC ACA ATO GCG CTC CCC TC AAC GAC 3I* The underlined bases correspond to nucleotides 146-166 of the cDNA sequence.

C-

terminal Primer 0F280 has the sequence 5' TCA TCA CAG GTC CTC CTC

GCT

GAT CAG CTT CTC CTC CAG TTG GAO CTG CCT ACC GTA 3' and is the reverse complement of the sequence 5' TAC GGT ACGG GAO GTC CAA CTO

GAG

GAG AAG CTGA4TCA4GC GAG GAG GAC CTG TGA TGA 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-mye epitope. The 1179 bp of amplification product was purified through a 1.0% agarose gel, and ligated into plasmid pDA 8421 which was linearized with the restriction enzyme Sr/I. Colony hybridization was used to select clones containing the PDAB342l plasrnid with teis~Teoinaino h insrtwa deerind b rsticion digestion of plasmnid DNA with diagnosti C enzymes KpJand Barniff A clone containing the A9 desaturase coding sequence in thle sense orintaionrelative to the Ubiquitin Promoter/intron was recovered and was nlamed *pDAB429. An additional clone containing the iA9 desaturase coding sequence in the ant e s ore t t o e ai et*h r m t r a a e D B 3 P a d p A 3 ans s orientio re latve toly i the irmt e was naem ed PD B 3O P am i P AB 3 was sube~t d t se uen e a aly is nd t w s d te m ed that the sequence contained 25 three PCR induced errors compared to the expected sequence. One error was found in the sequence corresponding to primer 0F280 and two nucleotide changes were .observed inenlto the coding sqec.Tseerrors weentcorrected, because anrisense dOwnregulatij 0 doe not require 100% sequence identity between the antisense transcript and the dOwnregulation target.

Exam le 19: Helium Blastin of Embrvo enic Maize Cultures and the Subse uent e eeneratin o f Trans enic Prog enyteSus ~n *Part A Establishment of embryogenic maize cultures. The tissue cultures employed in transformation experiments were initiated from immature zygotic embryos of the genotype "Hi-Il". Hi-Il is a hybrid made by intermating 2 R3 lines derived from a B73xA188 cross (Armstrong et al. 1990). When cultured, this 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 cmbryos rcsultig from controlled pollinations of greenhouse grown Hi-11 plants. The initiation r su dinLm 1 l w r s m a N6 (Chu, 1978) which contained 1.0mg/L 2,4-D, 25 niM L-prolinc, 100 mg/il cascin hydrolysate, 10 mg/L AgNO 3 2.5 g/L gelrite and 2% sucrose adjusted to p 5.8. For approximately 2-8 weeks, selection occurred for Type callus and against nonembryogenic and/or Type I callus. Once Type II callus was sclcted, it was transferrcd to a maintenance medium in which AgNO 3 was omitted and L-proline rcduced to 6mM.

After approximately 3 months of subculture in which the quantity and quality of embryogenic cultures was increased, the cultures were deemed acceptablc for us a in transformation experiments.

em ed acc s in 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 rangig from 1.5-3.0 20 microns (Aldrich Chemical Co., Milwaukee, WI) ransfomation 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. Adsorption was accomplished by adding 74 gl of 2.5 M calcium chloride and 30 p.1 of 0.1 M spermidine to 300 jl of plasmid DNA and sterile H20. The concentration of plasmid DNA was 140 dodpacsrconcentration of plasmid DNA was 140 25 jig. The DNA-coated gold particles were immediately vortexed and allowed to settle out of suspension. The resulting clear supenatent 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 ofpetri 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).

Helium blasting involved accelerating the spended D coated gold and into prepared tissue targets. The device used W a a ea le particles to e described in a DowElanco U.S. Patent (#5,14 131) which is incorporated herein by reference, although both functon ncorporated herei b ressurence, although both function in a similar manner. The device consisted of a high pressure helium source, a Syringe containng the DNA/gold suspension, and a pneumatically-operated multipurpose valve which provided controlled linkage bet ween the helium source and a loop ofpre-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 during impact. Next, targets wcre placed under vacuum in the main chamber of the device. The DNA-coated gold paricles were accelerated at the target 4 times using a helium pressure of 1500 psi. Each blast delivered ,1 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 Selction of transformed tissue and the regeneati of plants from transgenic cultures After 16 to 24 hours Post-blasting, the tissue was divided into small pices and transferred to selection medium (maintenance medium plus 30 mgL BastaTM) Every 4 weeks for 3 months, the tissue pieces were non-selectively tr30 m BastafM) Everred to fresh 4election medium. After 8 weeks and up to 24 weeks, any sectors found Proliferating against a background ofgrowth inhibited tissue were removed and isolated. Putatively transformed tssue was subcultured onto fresh selection medium Transgenic cultures were s sed after 1 to 3 additional subcultures.ere establis *2 5 O n c e B a s tae resis ta n t S 25 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 ight (25 ft-candles) for one week followed by one week in high light ge325 ft-candSkoog, The induction medium was composed of MS salts and 0 bvitamins (Murashige and SkoogL 2 2. g/L sucrose, 100 mg/L myo-inositol, 5 mg/L 6benzylaminopurine, 0.025 mg/L 2,4-D, 2.5 g/L gelrite adjusted to pH 5.7. Following the S k induc period te tgelri te adj u st e d to p H 5.7. Following the two week induction period, the tissue was non-selectively transferred to hormone-free Sregeneraton medium and kept in high light. The regeneration medium as composed of MS salts and vitamins, 30 g/L sucrose and 2.5 gL gelrite adjusted to pH 5.7. Both induction and rgeneration media contained 30 mg/L Bastal. 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 I S re a ndI vitamins (Schenk and Hildebrandt, 1972). 10 g/L sucrose, 100 mgL o-iostol, 5 L FeEDTA and either 7 g/L Agar or 2.5 g Gelrite adjusted to pH 5.8 Plantlets were transferred to 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 transfelrce to gallon pots containing approximately 4 kg Mctro-Mix, 3I60 ani gown r t Il sf l til These RO plants were self-pollinated and/or cross-poll )lcl with o -tra sg w nic ibr to obtain transgenic progeny. In the case of transgenic plants produced for the GBSS target, RI seed produced from RO pollinations was replanted The RI plants were grown to maturity and pollinated to produce R2 seed in the uantities needed for the analyses.

Examle 20 Production and Reenerao of 9 Transenic Material.

Part A Transformation and isolation of embryogenic callus. Six ribozyme constructs, described previously, targeted to A9 desaturase were transformed into regenerabl Type II callus cultures as described herein. e w e re tran sf orm ed i n to re g e n era b le

T

y II callus cultures as described herein. These 6 constructs consisted of 3 active/inactive pairs; namely, RPA85/RPA113, RPAI14/RpAIIS, and RPA 18/RpA119. 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 20 isolated from selection. Approximately 50% of these lines were analyzed via DNA PCR or GCFAME as a means of determining which es w ere analy ze d v ia D N A

PCR

or GC/FAME as a means of determining which ones to move forward to regeneration and which ones to discard. The remaining 50% e not analyzed either because they had become non-embryogenic or contaminated. w t becau e Part B Regeneration of A9 plants from transgenic callus. Following analyses of the transgenic callus, twelve lines were chosen per ribozyme construct for regeneration, with Ro plants to be produced per line. These lines generally consisted of 10 analysispositive lines plus 2 negative controls, however, due to the poor regenerability of some of the cultures, plants were produced from less than 12 lines for constructs RA 113 RPA 15, RPA118, and RPAI 19. An overall toal of 854 Ro plants were regenerated from 66 individual lines (see Table When the plants reached maturity, self or sibpollinations. were given the highest priority, however, when this was not possible, crosspollinations 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 being comprised of self- or sib-pollinations and the minority being comprised ofFI crosses. R seed was collected aproxately days post-pollination. aaely Examle 21: Production d Reeneration of Transenc Maize for the GBSS Pan A Transformation of embryogenic maize callus and th establishment of transgencc cultures. the shscq t scl cc "oll and establishment of transgenic cultures. RPA63 and RPA64, an active/inactive pair of ribozyme multimers targeted to GBSS, were inserted along with bar selection pasrid pDAB308 into Type II callus as described herein. A total of 115 BastaM-rcsistant 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 R2 generationd Plnt Southern "positive" transgenic cultures and grown to maturity in a greenhouse The Primary renerates were pollinated to produ maturity in a g re en h o u se T he 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 RI plants, representing 16 original lines, were grown to sexual maturity anted. A 20 pollinated. Approximately 19 to 22 days after pollination, ears were harvstd and kernels were randomly excised per ear and frozen for later analysesd

*I

o mes in Maize Black Mexican Sweet (BMS) Stably Transformed Callus aribozymes. Bt M S does not produce a GBSS mRA hih is hGBSS-targeted endogenously in maize. Therefore, a double transformation system was developed to produce transformants which expressed both target and riboy st e w a s e e" l o

M

e d t o S 30 suspensions (obtained from Jack Widholm, University ofIllinois, also see W. F. Sheridan, "Black Mexican Sweet Corn: 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 vitamnns (Chu t al., 1978), 20 g'L sucrose, 1.5 mg/L 2 ,4-dichlorophenoxyacetic acid 25 mM L-prol ne; p= 19 5.8 before autoclaving 20 minutes at 121oC] solidified with 2% TC agar (JR

H

sciences 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 (JST/ar) w c re used Targets were individually shot using DowElanco Helium Blasting Device I. With a vacuum pressure of 6 5 0 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 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, 00 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 n

C)

solidified with 0.8% TC agar containing 6 mg/L Basta. Isolates were maintained by subculture to fresh medium every two weeks.

w ere int d 20 Basta®-resistant isolates which expressed GBSS wee subjected to second transformation. As with BMS suspensions, targets of transgeic 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 tranformants on AMCF-ARM selection media. Each sample was covered with a sterile 104 gm mesh screen and blasting was done at 1500 psi. Target tissue was co-bombarded with pDAB 319 (35S-ALS; GUS) and RPA63 (active ribozyme multimer) or pDAB319 and RPA64 (inactive Sribozyme 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 30 agents, 6 mg/L Basta® and 2 .g/L chlorsulfuron (CSN). The level of CSN was increased to 4 ug/L after 2 weeks. Continued transfer of the filters and genertion 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 tg/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 analysis for detection of a functional target gene (GBSS) and to detennine 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 prcsc fo r ribozyme transcript. Of 19 isolates tested from ne viously transl ormcd p lin c 1 expressed the active ribozyme, RPA63, and all expressed GoSS GBSS was detected n 1 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 G eSS transcript in the presence or absence of active ribozyme. GBSS values were normalized to an intenal control (A9 desaturase); Northern blot data is shown in Figure(25). Northe 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 and were significantly different from vector controls and similar to a nontransformed control.

Example 23: Analysis of Plant and Callus Materials 20 Plant material co-transformed with the pDAB308 and one f the followin ribozyme containing vectors, pRPA63, pRPA64, pRPA85, pRPAI 3, pRPA114, pRPA115, pRPA 18 or pRPA119 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 incorporated by reference herein.

SProbes 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 LKB, Piscataway, NJ) with 50 microcures of a 3 2 p.dCTLife Arlington Heights, IL). Probes were hybridized to the genoic DNA on tie nylon membranes. The membranes were washed at 60 0 C in 0.25X SSC and 0.2% SDS for 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 rcstrictioi C/ynes HindIII 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 whn hybridized to the RPA63 or RPA64 materials. The 1.3 kb hybridization product should contain the enhanced promoter, the AdhI intron, the ribozyme coding region and the nopaine synthase poy A 3' end.

T

h e DNA from the RPA85 and ng region a nd the n o p aline sy nt h a se poly A 3' end. The DNA from the RPA85 and RPA 113 was digested with the restriction enzymes HindIII 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 RPA122 probe consists of the RPA122 ribozyme multimer coding region and the nopaline synthase 3' end and should produce a single 2.1 kb hybridization product when hybridized to the RPA85 or RPA 13 materials. The 2.1 kb hybridization product should contain the enhanced 35S promoter, the Adh intron, the bar gene, the ribozyme coding region and the nopaline synthase poly A 3' end. The DNA from the 20 RPA114 and RPA 115 was digested with the restriction enzymes Hindl and Sa and C. the blots containing these samples were hybridized to the RPA 115 probe. Th RPA 115 probe consist of the RPAI 15 ribozyme coding region and should produce a single 12 kb hybridization product when hybridized to the RPA 114 or RPA 11 materials. The 1.2 kb hybridization product should contain the enhanced 35S promoter, the Adh 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 Hind and Sa and .the blots containing these samples were hybridized to the RPA 118 probe. The RPA 118 probe consist of the RPA1 18 ribozyme cod A 8 probe. TheRPAll8 probe consist of the RPAI 18 ribozyme coding region and should produce a single 1.3 kb Shybridization product when hybridized to the RPA18 or RPA119 materials. The 1.3 kb hybridization product should contain the enhanced 35S promoter, the AdhI intron, the ribozyme coding region and the nopaline synthase poly A 3' end.

eo Example 24: Exraction of Genomic DNA from Transni Callus Three hundred mg of actively growing callus were quick frozen on dry ice. t was ground to a fine powder with a chilled Bessman Tissue Pulverizer (Spectrum, Houston, TX) and extracted with 4 0 0git of 2x CTAB buffer IFlexadeclriiethylainuio Bromide, 100 mM Ti-is pH 8.0, 20 MM EDTA, 1.4 M NaCI, 1% poIyvinylpyrroldone).

The suspension was lysed at 65 0 C for 25 minutes. then extracted with an equal volui-ne of chloroformn:isoamyi alcohol. To the aqueous phase was added 0. 1 volumes of CTAB buffer (10% Hexadecyltrimethylammonium Bromnide, 0.7 MI NaCI). Following extraction with an equal volume of chloroform: isoainyl alcohol, 0.6 volunics o)I coldI isopropyl alcohol was added to the aqueous phase, and placcd at -20-C for 30 minnutes.

After a 5 minute centrifugation at 14,000 rpm, the resulting precipitant was dried for minutes under vacuum. It was resuspended in 200 Al TE (10mM Tris, In1MEDTA, pl-l 8.0) at 65*C for 20 minutes. 20% Chelex (Biorad, was added to the DNA to a final concentration of 5% and incubated at 56'C for 15-30 minutes to remove irnpuritics. Thc 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 Method used to detect ribozyme DNA The Polymerase Chain Reaction (PCR) was performed as described in the suppliers protocol using AmpliTaq DNA Polymerase too. ~(GeneAmp PCR kit Perkin E m r Cetus). Aiquots of 300 ngof genomic cal sD

A

I. :l o M downstream prmer LGC AAG ACC GGC AAC AGG ]ofa 99** upstream primer and 191L of Perfect Match (Stratagene, Ca) PCR enhancer were mixed a 0. to: with tecomponents othki.The PRreaction wspromdfor 40 cyceusnth following parameters; denaturation at 94*C for I minute, annealing at 55"C for 2 minutes, and extension at 72*C 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 desaturase riboege s 999 R.PA85IRPAI 113 251 multimer fused to BAR 3' ORF RPA I 14/RPAI 115 258 ribozyme monomer 99: R.PAlI 18/RPAI 19452 ribozyme multimer TGG Afl 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. pe Example 26: Preparation of Total RNA from Transeenic Maize Calli and Plant Part A Preparation of total RNA from transgenic non-regencrable and regclncrable 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-HCI pH 8.0, 4% para-amino salicylic acid, 1% Tri-iso-propyinapthalenesulfonic acid, 10 mM dithiothreitol, and 10 mM Sodium meta-bisulfite) by vigorous vortexing. The homogenate 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 chloroform:isoamyl alcohol followed by extraction with chloroforn:octanol 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 rpm, RNA was resuspended in sterile water, precipitated with 2.5 M NH40Ac and 2 volumes of 100% ethanol and incubated overite at The harvested RNA pellet was washed with 70% ethanol and dried under vacuum.

RNA

was resuspended in sterile H20 and stored at 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 manufactorers 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 prewarmed (50*C) sterile water (30 pl each) and stored at 80*C.

30 Example 27: Use of RT-PCR Analysis to Demonstrae sion 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 tl of a 15 uM downstream primer CGC AAG ACC GGC AAC AGG w me components of the kit. The reverse transcription reaction was performed in a 3 step ramp up with 5 minute incubations at 60°C, 65 0 C, and 70'C. For the PCR reaction, I of upstream primer specific for the ribozyme RNA being analyzed was added to the RT reaction with the PCR components. The PCR reaction was pcrformcd for 35 cycles using the following parameters; incubation at 96 0 C for I minute, denmtrr;tiion at fir seconds, annealing at 50C for 30 seconds, and extension at 72 0 C for 3 mins. An alquot of0.2x vol. of each RT-PCR reaction was electrophoresed on a 2% 3 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 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 arm.

GBSS 918 Intron Monomer: 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.

S GBSS ribozyme expression in transgenic callus and plants was confirmed by RT-PCR.

S

5 GAT GAG ATC CGG TGG CAT TG 3' This primer spans the junction of th B A R gene an d he R A 85 1 13 ribozyme.

RPAI 14/RPA115 259 ribozyme monomer 5' ATC CCC TTG GTG GAC TGA TG 3' 30 This primer covers the 10 base pair ribozyme arm and the first 6 bases ofhe ribozyme catalytic domain. ases of the ozy RPA118/RPA119 453 ribozyme 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 arm.

Expression of A9 desaturase ribozymes in transgenic plant lines 85-06, 113-06 and 85-15 were confirmed by RT-PCR.

Primers were prepared using standard oligo synthesis protocols on an Applied Biosystems Model 394 DNA/RNA synthesizer.

Example 28: Demonstration of Ribozvme Mediated Reduction in Ta in Transeenic Maize Callus and Plants Part A Northern analysis method which was used to dcmonstrated rcductions i target mRNA levels. Five g 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 65C. Electrophoresis was at 50 volts through I agarose gel in 20 mM phosphate buffer (pH 6.8) with buffas recirculation. BR.phosphate buffer (pH 6.8) wit h 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 20 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 S: (Amersham Corporation, Arlington Heights r m with alpha 32 P dCTP.

Autoradiography was at -70" C with inten.ifi with alpha 32p dCTP.

Autoradiography was at 700 C with intensifying screens (DuPont, Wilmington DE) for 25 one to three days. Autoradiogram ignals for each probe were measured after a 24 hour ccexposure by densitometer and a ratio of target/internal control mRNA levels was b'calculated.

SQRibonuclease protection assays were performed as follows: RNA was prepared using the 30 Qiagen RNeasy Plant Total RNA Kit from either BMS protoplasts or callus The probes were made using the Ambion Maxiscript kit and were typically 10 s cpm/ 0 microgram or higher. The probes were made the same day they were used. They were gel S 5 purified, resuspended in RNase-freel0mM Tris (pH 8) and kept on ice. Probes were diluted to 5xl0 5 cpm/ul immediately before use. 5 jg of RNA derived from callus or 20 e ig of RNA derived from protoplasts was incubated with 5 x 1 05 cpm of probe in 4M Guanidine Buffer. [4M Guanidine Buffer: 4M Guanidine Sodium Citrate (pH 7.4)J. 40 ul of PCR mincral oil was added to cach tube to prevent evaporation. The samples were heated to 950 for 3 minutes and placed Simmediately into a 450 water bath. Incubation continued overnight 600 usl of RNase Treatment Mix was added persan u o ve n 8 h 6 0 0 p l o f R N ase Treatment Mix was added per sample and incubated for 30 minutes at 37 0 C. (RNase Treatment Mix: 4 0 0 mM NaCI, 40 units/mn RNase A and TI). 2I.tI 1 20'% SIS were added per tube, immediately followed by addition of 12 ul (20 mg/il) IPr 2 ocmI K io each tube. The tubes were vortexed gently and incubated for 30 minutes at 37O. 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.

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 Xylene Cyanol The sample was heated to 950 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 ulti mer 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 S transformants and 8 active RPA63 lines. Probes for this analysi were a full length maize GBSS cDNA and a maize A9 cDNA fragment. To distinguish variation in GBSS mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions, the level ofGBSS mRNA was compared to the level of A9 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/ A9 ratio was observed between ribozyme transgenics The target mRNA is produced by a transgene and may be subject to more variation in expression then the endogenous A9 mRNA. Active lines (RPA 63) AA, EE, KK, and JJ were shown to reduce the level of GBSS/A9 most signifcantly, as much as 10 fold as 35 compared to ribozyme control transgenics this is graphed in Figur 25. Those active lines were shown to be expressing GBSS targeted ribozyme by RT-PCR as described herein.

s escribed Reductions in GBSS mRNA compared to A9 mRNA were also seen by RNAse protection assay.Ase Part C Demonstration of reductions in A9 desaturase lcvcls in transgnc lants expressing ribozymes targeted to A9 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 ribozym RNA. Using the protocol described in Example 27. RPA85 ribozymc cxprcssion was demonstrated in plants of the 85-06 and 85-15 lines which contained high stearic 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 A9 desaturase cDNA and a maize actin cDNA. To distinguish variation in A9 mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions, the level of A9 mRNA was compared to the level of actin mRNA within that sample. Using densitometer readings described above a ratio was calculated for each sample. A 9 /actin ratio values ranging from 0.55 to 0.88 were calculated for the 85-06 plants. The average A9/actin value for nontransformed controls was 2.7. There is an apparent 4 fold reduction in A9/actin ratios between 85-06 and NT leaves. Comparing A9/actin values between 85-06 high stearate and TC plants, on average a 3 fold reduction in A9/actin was observed for the 85-06 lants. This data is graphed in Figure 26. Ranges in A9/actin 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 A9/actin ratio for the NT plants was 1.7. Comparing the average A9/actin ratio between NT controls and 85-15 high stearate plants, a 3.9 fold reduction in 85-15 A9 mRNA was demonstrated. An apparent 3 fold reduction in A9 mRNA level was observed for RPA85-15 high stearate transgenics when A9/actin ratios 30 were compared between 85-15 high stearate and normal stearate (TC) plants. These data are graphed in Figure 27. These data indicate ribozyme-mediatedreductio n of t9desaturase mRNA in transgenic plants expressing RPA85 ribozyme, and producing increased levels ofstearic acid in the leaves.

35 Example 29: Evidence ofA9 Desaturase Down Reulation in Maize Leaves as a Result of Active Ribozyme Activity Plants were produced which were transformed with inactive versions of the A9 desaturase ribozyme genes. Data was presented demonstrating control lcvels of leaf stearate in the inactive A9 ribozyme transgenic lines RPAI 13-06 and 113-17. Ribozyme expression and northern analysis was performed for the RPA 113-06 line. 9 (lesturias protein levels were determined in plants of the RPA 113-17 line. Ri bozy 1 ex c s pre-ssi was measured as described herein. Plants 113-06-04, -07, and -10 expressed dectccble levels of RPA 113 inactive A9 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 A9 desaturase mRNA correlating with ribozyme expression or elevations in leaf stearate were found in the RPA 113-06 plants as compared to controls, graphed in Figure 28. Protein analysis did not indicate any reduction in A9 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 RPA113 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 ibo Mdiatd Rduction in Staro-A &9 Desaturase levels in Maize Leaves (RO) A9 Desaturase Levels in Maize Leaves

(RO)

Part A Partial purification of stearoyl-ACP

A

9 -desaturase from maize leaves. All procedures were performed at 4 0 C unless stated otherwise. Maize leaves (50 mg) were harvested and ground to a fine powder in liquid N2 witha mortar and pestle. Proteins 25 were extracted in one equal volume of Buffer A consisting of 25 mM sodium-phosphate pH 6.5, 1 mM ethylenediamineteaacetic acid, 2 mM dithiothreitol, 10 mM phenylmethylsulfonyl fluoride, 5 mM leupeptin, and 5 mM antipapin. The crude homogenate was centrifuged for 5 minutes at 10,00 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 l in Buffer A, added to 50 pl 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 A9 desaturase-sepharose material was centrifuged (10,000 x g) for 10 seconds, decanted, 35 washed three times with Buffer A (500 p1), and washed one time with 200 mM sodium chloride (500 p1). Proteins were eluted by boiling in 50 ml of Treatment buffer (125 mM n pl of Treatment buffer (125 mM Tris-CI pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, and 10% 2 -ercapoethano) for 5 mintues. Samples were centrifuged (10,000 x g) for 5 minutes. The supe:ratant was saved for Western anaylsis and the pellet consisting of sepharose beads was discarded.

Part B Western analysis method which was used to dcmonstrate rductions i tcaroyl ACP A9 desaturase. Partially purified proteins were separated on sodium ddecyl (SDS)-polyacrylamide gels (10% PAGE) as described by Lacmmli, U. K. (1970) Clevage of structural proteins during assembly of the head of phage T4, Nature 227, 660-685. To distinguish variation in 69 desaturase levels, included on each blot as a reference was purified and quantified overexpressed A9 desaturase from E. coli as described hereforth Proteins were electrophoretically transferred to ECLTM nitrocellulose membranes (Amersham Life Sciences, Arling.on Heights, Illinois) using a Pharmacia Semi-Dry Blotter (Pharmacia Biotech Inc., Piscataway, NJ), using Towbin buffer (Towbin et at 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 T M Western Blotting Detection Reagent (Amersham Life Sciences, Arlington Heights, Illinois) with rabbit antiserum raised against E. coli expressed maize A9 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 A9 desaturase. These experiments were duplicated and the mean reduction was recorded.

.Part C Demonstration of Reductions in A9 desaturase levels in RO maize leaves S 25 expressing ribozymes targeted to A9 desaturase mRNA. The high stearate transgenic line, RPA85-15, contains an intact copy of the fused multimer gene. A9 desaturase was partially purified from RO maize leaves, using the protocol described herein. Western analysis was performed on ribozyme active (RPA85-15) and ribozyme inactive (RPA113-17) plants and nontransformed (Hill) plants as described above in pan B. The 30 natural variation of A9 desaturase was determined for the nontransformed line (Hill) by Western analysis see Figure 29 A. No reduction in A9 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 A9 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 A9 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 113-17) (Figures 29 A and B).

Collectively, thege results suggest that the ribozyme activity in the six plants from line RPA85-15 is responsible for the reduced A9 desaturase.

Example 31: E. coli Expression and Purification of Maizc A-9 dcsaturasc cnzynt Part A The mature protein encoding portion of the maize A-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 cDNA) was dcsignated as the first residue. This is found within the sequence Ala.Val.Ala.Ser.MetThr Restriction endonuclease Nhe I site was engineered into the maize sequence by PCR, modifying GCCTCC to GCTAGC and a BamHI 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 the Nhe I and Barn HI sites. The recombinant plasmid is designated as pDAB428. The maize A-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 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 37C. The cells were harvestnd by centrifugation at 1000xg at 4"C for 10 minutes. The cells were lysed by freezing and thawing the cell pellet 2X, followed by the addition of I ml lysis buffer (10 mM Tris-HCI pH 8.0, 1 mM EDTA, 150 mM NaCI, 0.1 Triton X 100 ug/ml DNAse I, 100 *-ug/ml RNAse A, and I mg/ml lysozyme). The mixture was incubated for 15 minutes at 25 37 0 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 was chilled on ice for I hr. Afterwards, the 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 S Basic proteins bound to the column matrix were eluted using a 0-500 mM NaCI 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 S. 35 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 A 9. Spectrophotometric analysis of the diiron-oxo co' ponent associated with the expressed protein (Fox et al., 1993 Proc. Natl. cad. S. US. 2486-2490), as well as identification using a specific nonheme iron stain (Leong et al., 1992 Anal. Biochem. 207, 317-320) confirmed that the purificd protein was a Part B Production ofpolyclonal antiserum The E. coli produced A-9 protein, as determined by amino terminal sequencing, was gel purified via SDS-PAGE, excised, and sent in the gel matrix to Berkelcy Antibody Co., Richmond, CA, for production of polyclonal sera in rabbits. Titers of the antibodies against A-9 were performed via western analysis using the ECL Detection system (Amersham, Inc.) Part C Purification of A9 desaturase from corn kernels Protein Precipitation: A9 was purified from corn kernels following homogenization using a Warring blender in 25 mM sodium phosphate buffer (pHi 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 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 v/v. In all cases, the A9 protein precipitated at either 80% ammonium sulfate or polyethylene glycol. The resulting pellets were then dialyzed extensively in sodium phosphate buffer (pH 6.0).

Cation Exchange Chromotography: The solubilized pellet material dscribed above was clarified via centrifugation and applied to Mono S HRIO/10 column equilibrated mM sodium phosphate buffer (pH After extensive column washing, basic proteins bound to the column matrix were eluted using a 0-500 mM NaCI gradient over I hr (2 ml/min: 2 ml fractions). Typically, the A9 protein eluted between 2 60-and 350 mM NaCI., as determined by enzymatic and western analysis After dialysis, this material 30 was further fracionated by acyl carrier protein (ACP)- sepharose and phenyl superose chromatography.

Acyl Carrier Protein-Sepharose Chromatographv: ACP was purchased from Silna Chemical Company and purified via precipitation at pH 4.1 (Rock and Cronan. 1981 J.

Biol. Chem. 254, 7116-7122) 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 described by Pharmacia, Inc., in the package insert. Afte linkage and blocking of the remaining sites with glycinc, the ACP-scpharoe msc nmrial wA s ackcd in 1 t a HR 5/5 column (Pharmacia, Inc.) and equilibrated in 25 mM sodiumr phosphate buffer (pH The dialyzed fractions identified above were then loaded onto the column (McKeon and Stumpf, 1982 J. Biol. Chem. 257, 12141-12147; Thompson e t 1991 Proc. Natl. Acad. Sci. USA 88, 2578-2582). After extensive column washin, ACPbinding proteins were eluted using I M NaCI. Enzymatic and western analysis, fllow c d by amino terminal sequencing, indicated that the eluent contained A-9 protein. The A-9 protein purified from corn 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 A9 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 A9 protein typically eluted between 60- and 30 mM ammonium sulfate as determined by enzymatic and western analysis.

Example 32: Evidence for the Increase -in Stearic Acidin Leaves as a Result of Transformation of Plants with Desaturase Ribozvmes 25 Part A Method used to determine the stearic acid levels in plant tissues The procedur for extraction and esterification of fatty acids from plant tissue was modified from a described procedure (Browse et. 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 I ml of methanolic HCL (Supeico, Bellefonte, PA), the tubes were purged with nitrogen gas and 30 sealed. The tubes were heated at 80 0 C for I hour and allowed to cool. The heating in the Spresence of the methanolic HCL results in the extraction as well as the esterification of the fatty acids. The fatty acid methyl esters were removed from the reaction mixture b extraction with hexane. One ml of hexane and, 1 ml of 0.9% NaCI was added Sfollowed by vigorous shaking of the test tubes. After centrifugation of the tubes at 2000 S. 35 rpm for 5 minutes the top hexane layer was removed and used for fatty acid methyl ester analysis. Gas chromatograph analysis was performed by ction of I of the sample on a Hewlett Packard (Wilmington, DE) Series II model 5890 gas of the sample equipped with a flame ionization detector and a J&W Scientific (Folso matograph) DB column. The oven temperature was 150 0 C throughout the run and the flow of the carrier gas (helium) was 80 cm/sec. The run time was 20 minutes. The conditions f alowc for the separation of the 5 fatty acid methyl esters of interest: C 6:0, panlityl mthyl ester; C18:0, stearyl methyl ester; C18:1, oleoyl methyl ester; C18:2, linolcoyl mcthyl ester; and C18:3, linolenyl methyl ester. Data collection and analysis was performed with a Hewlett Packard Series II Model 3396 integrator and a PE Nelson (Pcrki 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. ov ry of known amount of Part B Demonstration of an increase in stearic acid in leaves due to introduction of A9 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 25 active A9 desaturase ribozymes (RPA85, RPA114, RPAI18) and 406 plants from 31 lines tranformed with A9 desaturase inactive ribozymes (RPA 13, RPA 115, RPA 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 and 2% had levels greater than Only 3% of the plants from the inactive lines had stearic acid 30 levels greater than Two percent of the control plants had leaves with stearate greater than The controls included 49 non-transformed plants and 73 plants transformed .with a gene not related to 9 desaturase. There were no plants from the inactive lines or controls that had leaf stearate greater than Two of the lines transformed with the active A9 desaturase ribozyme RPA85 produced many plants which exhibited increased 9: 35 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 controls (Figure 30) The average stearic acid content of the control plants (122 plants) was 1.69% The average stearic acid content of leaves fron line RPA85-0 6 was 2.86% Line RPA85-15 had 6 out of 15 plants assaed ith 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 RPA85-15 was 3.

o When the leaf analysis was repeated for RPA85-15 plants the stcaric acid lcvcl in lcavcs from plants previously shown to have normal stearic acid lcvcls rmaincd normal and leaves from plants with high stearic acid were again found to be high (Figure 31). The stearic acid levels in leaves of plants from two lines which were transformed with an inactive A9 desaturase ribozyme, RPA 113, is shown in Figures 32 and 33. RPA I13-06 had three plants with a stearic acid content of 3% or highcr. The avcragc stcaric acid content of leaves from line RPA113-06 was 2.26% RPA 113-17 had no plants with leafstearic acid content greater than The average stearc acid content of leaves from line RPA 13-17 was 1.76% The stearic acid content of leaves from 15 control plants is shown in Figure 34. The average stearic acid content for these control plants was 1.70% When compared to the control and inactive A9 desaturase ribozyme data, the results obtained for stearic acid conent in RPA8-06 and RPA85-15 demonstrate an increase in stearic acid content due to the introduction of the A9 desaturase ribozyme. ton of the Example 33: Inheritance of the High Stearic Acid Trait in Leaves Part A Results obtained with stearic acid levels in leaves from offspring of high stearic acid plants. Plants from line RPA85-15 were pollinated as described herein. Twenty 25 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, RPA85-5.07 selfed. Fifty percent of the plants had high leaf stearic acid and 50% had normal leafstearic acid. Table XII shows the results from 5 different crosses of RPA85-5 plants. The number of plants :::with high stearic acid ranged from 20 to Part B Results demonstrating reductions in A9 desaturase levels in next generation

(R

35 maize leaves expressing ribozymes targeted to A9 desaturase mRNA. In next generation maize plants that showed a high stearate content (see above Part 9 desaturase was partially purified from RI maize leaves, usin the protocol described here esern analysis was performed on several of the high stearate plants. In leaves of next generation plants, a 40-50% reduction of A9 desaturase was observed in those plants that had high stearate content (Figure 36). The reduction was comparable to R m plan ts th le had hi reduction was observed in either OQ414 plants crossed with RPA85-1 5 pllein or RPA85-15 plants crossed with self or siblings. Therefore, this s s tt en encoding the ribozyme is heritable. is uggsts tha the gcne Eample 34: Increase in Stearic Acid in Plant Tissues Using Antisense- A9 Desaturase 1 0 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 the callus was transferred every two weeks. The somatic embryos in embryogenic callus continued to proliferate but usually remaied 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 b a r o o t and a s hoot, a nd ceases development as an embryo. Somatic embryos were made to develop as seed embryos, 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% sucrose and no plant hormones The callus was grown on the MS medium with 6% sucrose for 7 days and then the 25 somatic embryos were individually transferred to MS medium with 6% sucrose and RM abscisic acid (ABA). The m cd i um w ith 6 sucrose and M abscis d (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 30 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 C: and C18:2 The percentage of lipid represented by the fresh weight was different for the embryogenic callus when compared to the somatic embryos; 0.4% versus The fatty acid 35 composition of the zygotic embryos and somatic embryos were very similar and their ercentage of lipid represented by the fresh weigh were nearly identical. It was concluded that the somatic embryo culture system described above would be an useful in vitro system for testing the effect of certain genes on lipid snthesis in developing embryos of maize. synthesis in developing Part B Increase in stearic acid in somatic cmbryos of aizc as r lt o tle i'lrOc of an antisense- A9 desaturase gene. Somatic embryos wcrc prodluccld using the method described herein from embryogenic callus transformed with pDAB308/pDAB43 The somatic embryos from 16 different lines were assayed for fatty acid coposition. Two lines, 308/430-12 and 308/430-15, were found to produce somatic embryos with high levels ofstearic 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, searic acid in somatic embryos ranged from I to 23% while the controls ranged from 0.5 to For line 308/430-15, stearic acid in somatic embryos ranged from 2 to 15% while the controls ranged from 0.5 to More than 50% of the somatic embryos had stearic acid levels which were above the range of the controls in both the transformed lines. The above results indicate that an antisense- A9 desaturase gene can be used to raise the steaic acid levels in somatic embryos of maize. e used to cid 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 308/430were used to regenerate plants. Leaves from these plants were analyzed for fatty acid composition using the method previously described. Only plants were obtained from 25 the 308/430-15 culture and the stearic acid level in the aves of these plants were normal, The stearic acid levels in leaves from plants of line 308/43012 are shown in Figure 39. The stearic acid levels in leaves ranged from I to 13% in plants from line 308/430-12.

About 30% of the plants fom line 308/430-12 had stearic acid levels above the range observed in the controls, These results indicate that the stearic acid levels can be raised in leaves of maize by introduction of an antisense. A9 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 35 RNA (for a review see Stein and Cheng, 1993 Science 261, 1004).

Examole 35: Amvlose Content Assay of Maize Pooled Starcample ad S e Kerne The amylose content was assayed by the method of Hovenkamp-Hennelink et al.

(Potato Research 31:241-246) with modifications. For pooled starch sample 10 g to 100 mg starch was dissolved in 5 ml 45% perchloric acid in plastic culture tue. The solution was mixed occasionally by vortexing After one hour, 1ml o" t e surch solution was diluted to 10 ml by H20. 0.4 ml of the diluted solution was then mixd with 0.5 ml diluted Lugol's solution (Sigma) in ml cuvet. Readings at 618 nm and 550 nm were immediately taken and the R ratio (618 nm550 nm) was calculated. Using standard equation P (percentage of amylose) 4 .5R-2.6)/(7.33R) gcncr a tcd from potato amylose and maize amylopectin (Sigma, St. Louis), amylose contcnt was determincd For frozen single kernel sample, same procedure as above was used except it was extracted in perchloric acid for 20 min instead for one hour.

Example 36: Starch Purification and Granular Bound Starch Svnthas GBSS) Assa The purification 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 of 50 mM Tris- HCI, pH 8.0, 10 mM EDTA and filtrated through 120 m nylon membrane. The material was then centrifuged at 5000 g for 2 min and the supernatant wa discarded. The pellet was washed three times by resuspending in water and removing supernatant by centrifugation. After washing, the starch was filtrated through 20 m nylon membrane and centrifuged. Pellet was then lyophilized and stored in 20 "C until used for activity assay.

SA standard GBSS reaction mixture contained 0.2 M Tricine, pH 8.5, 25 mM Glutathione, 5 mM EDTA, I mM 14 C ADPG (6 nci/.mol), and 10 mg starch in a total volume of 200 Al. Reactions were conducted at 37 *C for 5 min and terminated by adding 30 200 p1 of 70% ethanol in 0.1 M KCI. The material was centrifuged and unincorporated ADPG in the supernatant is removed. The pellet was hen washed four ~time with Iml water each in the same fashion. After washing, pellet was suspended in 500 p1 water, placed into scintillation vial, and the incorporated ADPGwas counted by a Beckman (Fullerton, CA) scintillation counter. Specific activity was given as pmoles of 35 ADPG incorporated into starch per min per mg starch.

Example 37: Analysis ofAntisense-GBSS Plants Because of the segregation of R2 seeds, single kernels should therefore be analyzed for amylose content to identify phenotype. Because of the larg aount of samples generated in this study, a two-step screening strategy was used. In the first step, kernels were taken randomly from the same ear, freeze-dried and homogcllizcd ilto 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 in the lines with reduced amylose content was further analyzed (25 to 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 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 25 frequencies since endospern 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 distribution 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 30 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 transgeni:parental. The distribution for 308/425-12.2.1.1 is distinctly trimodal. The central mode is approximately twice the d e i s a p p ro x i m te t w i c t Ssize ofeither other mode. The two distal modes are of approximately equal size.

35 Goodness of fit to a 1:2:1 ratio was tested and the fit was excellent.

Further evidence was available demonstrating that the ode it e hes O 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 ombined 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 employd. The original data w; r Is s ed i a r 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 tice the size of the lowest amylose content mode. This would be expected ift te central mode includes two genetic classes: individuals with I or 2 copies of the antisens constr a l t od 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 GBSSgene as functioning in 308/425- 12.2.1.1 demonstrates a dosage dependent reduction in amylose content of maize kernels.

Example 38: Analysis-ofRibozyme-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 II transformation events were examined in the pooled starch level. Among the control lines (both untransformed line and Southern negative line), the amylose content varied from 28% to 19%. No significant reduction was observed among all lines carrying ribozyme gene (Southern 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 35 samples, and Southern analysis results. Three southern negative lines: RPA63.0283, RPA63.0236, and RPA63.0219 were used as control.

67 The GBSS activities of control lines RPA63.028 3 RPA63.0236, and RPA630219 werearond 30 uits/mg starch. In lines RPA63.021 RPA63.02 18, 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) indcicatedl that 11c rcduiccd l RSs ;tclivily was caused by the expression of ribozymc gcne incorporated Into the maji-e 9Clinmc.

GBSS activities at the single kernel level of line RPA 63.02 1 (Southerni positivc and reduced GBSS activity in pooled starch) was further examincd. using RPA63-0306 (Southern negative and GBSS activity normal in pooled starch) as control. About kernels from each line were taken, and starch samples were purified fromr each kernel individually. Figure 41 clearly indicated reduced GI3SS activit'y inln P .021 8 compared to RPA63.0306.

Other embodiments are within the following claims.

00* Table I 68 O 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: attack by the 3'-OH ofguanosine to generate cleavage products with 3'-OH and 5'-guanosine.

Additional protein cofactors required in some cases to help folding and maintainance of the Over 300 known members of this class. Found as an intervening sequence in Tetrahvmena thermophila rRNA. fungal mitochondria. chloroplasts, phage T4. blue-green algae. and others.

Major structural features largely established through phylo-enetic comparisons. mutaenesis.

and biochemical studies Complete kinetic framework established for one ribozyme Studies ofribozyme folding and substrate docking underway Chemical modification investigation of important residues well established 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-galactosdase message by the ligation of new b-galactosidase sequences onto the defective message RNAse P RNA (Ml RNA) Size: -290 to 400 nucleotides.

RNA portion of a ubiquitous ribonucleoprotein enzyme.

Cleaves tRNA precursors to form mature tRNA Reaction mechanism: possible attack by M2--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 S.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 6 Important phosphate and 2' OH contacts recently identified Group II Introns Size: >1000 nucleotides.

Trans cleavage of target RNAs recently demonstrated [9, 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 and a branch point.

Only natural ribozyme with demonstrated participation in DNA cleavage in addition to RNA cleavage and ligation.

Major structural features largely established through phylogenetic comparisons Table I 69 Important 2' OH contacts beginning to be identified [2] Kinetic framework under development Neurospora VS RNA Size: -144 nucleotides.

Trans cleavage of hairpin target RNAs recently demonstrated [26].

Sequence requirements not fully determined.

S 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 pathogens (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 [27,2,29, 5 0 Ligation activity (in addition to cleavage activity) makes ribozvme amenable to engineering through in vitro selection 31 Complete kinetic framework established for one ribozvme S" Chemical modification investigation of important residues begun

U

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 Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate cleavage products with 2'.3 -cyclic 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 [3] 1. Mohr, Caprara. Guo, Lambowitz. A.M. Nature, 1i0. 147-150(1994).

2. Michel. Francois: Westhof. Eric. Slippery substrates. Nat. Struct. Biol. (1994), 5-7.

3. Lisacek, Frederique; Diaz. Yolande: Michel. Francois. Automatic identification of group I intron coresin genomic DNA sequences. J. Mol. Biol. (1994), 235(4). 1206-17.

4. Herschlag, Daniel; Cech. Thomas 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.

Herschlag, Daniel; Cech. Thomas 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 Herschlag. Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent pKa. Biochemistry (1996). 35(5), 1560-70.

7. Bevilacqua. Philip Sugimoto. Naoki: Turner. Douglas A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58.

8. Li, Yi; Bevilacqua, Philip Mathews, David; Turner. Douglas Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change. Biochemistry (1995), 34(44), 14394-9.

9. Banerjee, Aloke Raj; Turner. Douglas The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12.

Zarrinkar, Patrick Williamson. James The P9.1 -P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996). 24(5). 854-8.

I1. Strobel. Scott Cech. Thomas Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena nbozyme reaction site. Science (Washington. D. (1995). 267(5198), 675-9.

12. Strobel. Scott Cech, Thomas Exocyclic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11.

13. Sullenger, Bruce Cech, Thomas Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature (London) (1994). 371(6498), 619-22.

14. Robertson. Altman, Smith, J.D. J. Biol. Chem., 247, 5243-5251 (1972).

15. Forster, Anthony Altman. Sidney. External guide sequences for an RNA enzyme. Science S* (Washington, D. 1883-) (1990), 249(4970), 783-6.

16. Yuan, Hwang, E. Altman. S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad.

.Sci. USA (1992) 89, 8006-10.

S17. Harris, Michael Pace, Norman Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 210-18.

18. Pan, Tao: Loria, Andrew; Zhong. Kun. Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts 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 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: Perlman. Philip 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 Jr.; Qin, Zhifeng; Michels. Williams 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(11), 761-70.

Table I 71 S23. Michel. Francois: Ferat. Jean Luc. Structure and activities of group 11 introns. Annu. Rev. Biochem.

(1995). 64. 435-61.

24. Abramovitz. Dana Friedman. Richard Pyle. Anna Marie. Catalytic role of 2'-hydroxyl groups within a group II intron active site. Science (Washington, D. (1996). 271(5254). 1410-13.

Daniels. Danette Michels, William Jr.; Pyle. Anna Marie. 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. 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 Berzal-Herranz, Alfredo; Burke. John Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2.

29. Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira. Bharat Butcher. Samuel Burke, John M..

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J. (1993), 12(6), 2567-73.

Joseph, Simpson: Berzal-Herranz, Alfredo: Chowrira, Bharat Butcher. Samuel Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation. and analysis of mismatched substrates. Genes Dev. (1993), 130-8.

31. Berzal-Herranz. Alfredo; Joseph, Simpson; Burke, John In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes Dev. (1992), 129-34.

32. Hegg. Lisa Fedor, Martha Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28.

33. Grasby, Jane Mersmann, Karin; Singh, Mohinder; Gait, Michael Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA. Biochemistry (1995), 34(12), 4068-76.

34. Schmidt. Sabine: Beigelman, Leonid; Karpeisky, Alexander; Usman. Nassim; Sorensen, Ulrik Gait, Michael Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure. Nucleic Acids Res. (1996), 24(4), 573-81.

Perrotta, Anne Been, Michael Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis .delta. virus RNA sequence. Biochemistry (1992), 31(1), 16-21.

36. Perrotta, Anne Been, Michael A pseudoknot-like structure required for efficient self-cleavage of ****hepatitis delta virus RNA. Nature (London) (1991), 350(6317), 434-6.

37. Puttaraju. Perrotta. Anne Been, Michael A circular trans-acting hepatitis delta virus "ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

a *o* Table 11 72 Table 11: 2.5,u.mol RNA Synthesis Cycle Reagent Equivalents Amount Wait Time* Phosphoramidites 6.5 163 uL S-Ethyl Tetrazole 23.8 238,uL Acetic Anhydride 100 233,uL 5 sec N-Methyl Imidazole 186 233,uL 5 sec TCA 83.2 1.73 mL 21 sec Iodine 8.0 1. 18mL 45 sec Acetonitrile NA 6.67 mL

NA

Wait time does not include contact time during delivery.

of.* Table IIA 73 Table IIIA: GBSS Hammerhead Substrate Sequence -,1 nt.

Position 12 68 73 103 109 113 146 149 151 154 169 170 173 188 188 196 203 206 230 241 247 248 292 308 314 315 3" 385 388 391 395 398 425 428 430 431 434 473 482 485 527 533 538 898 902 913 919 929 931 951 952 953 Substrate CGAUCGAUC

GCCACAGC

GAAGGAAUA

AACUCACU

AAUAAACUC

ACLJGCCAG

AGAAGUGUA

CUGCUCCG

GUACUGCUC

CGUCCACC

UGCUCCGUC

CACCAGUG

GGGCUGCUC

AUCUCGUC

CUGCUCAUC

UCGUCGAC

GCUCAUCUC

GUCGACGA

CAUCUCGUC

GACGACCA

CAGUGGAUu

AAUCGGCA

AGUGGAUUA

AUCGGCAU

GGAUUAAUC

'GGCAUGGC

UGGCGGCUC

UAGCCACG

GCGGCUCuA

GCCACGUC

AGCCACGUC

GCAGCUCG

UCGCAGCUC

GUCGCAAC

CAGCUCGUC

GCMACGCG

CUGGGCGUC

CCGGACGC

GGACGCGUC

CACGUUCC

GUCCACGUU

CCGCCGCG

UCCACGUUC

CGCCGCGG

GACGGCGUC

GGCGGCGG

GACACGCUC

AGCAUUCG

CUCAGCAJJU

CGGACCAG

UCAGCAUUC

GGACCAGC

CCCAGGCUC

CAGCACCA

GGCCAGGuU

CCCGUCGC

GCCAGGUUC

CCGUCGCU

GUUCCCGLJC

GCUCGUCG

CCGUCGCUC

GUCQUGUG

UCGCUCGUC

GUGUGCGC

AUGMACGUC

GUCUUCGU

MACGUCGUC

UUCGUCGG

CGUCGUCUU

CGUCGGCG

GUCGUCUUC

GUCGGCGC

GUCUUCGUC

GGCGCCGA

GGCGGCCUC

GGCGACGU

GGCGACGUC

CUCGGCGG

GACGuccuc

GGCGGCCU

CACCGUGUC

AUGGUCGU

GUCAUGGUC

GUCUCUCC

AUGGUCGUC

UCUCCCCG

CUCGUGCUA

CCUCMAGA

UGCUACCUC

MAGAGCAA

GAGCAACUA

CCAGUCCC

CUACCAGUC

CCACGGCA

CACGGCAUC

UACAGGGA

CGGCAUCUA

CAGGGACG

AGACCGCUu IJCuGCAuc GACCGCUUU

CUGCAUCC

ACCGCUUUC

UGCAUCCA

Seq. ID No.

26 28 30 32 34 36 38 40 42 44 48 52 5-4 58 58 80 62 64 66 68 70 72 74 76 78 80 82 84 88 88 90 92 94 96 98 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 nt.

Position Substrate 538 540 547 556 581 586 593 610 620 625 626 828 629 637 661 662 665 679 680 692 693 716 718 742 763 784 773 788 795 803 812 826 829 830 832 841 854 859 860 863 888 890 892 1241 1270 1274 1285 1294 1346 1352 1370 1384

S

C..

*CC.

C

C. C C C C. GGUCGUCUC

UCCCCGCU

UCGUCUCUC

CCCGCUAC

UCCCCGCUA

CGACCAGU

CGACCAGUA

CMAGGACG

ACCAGCGUC

GUGUCCGA

CGUCGUGUC

CGAGAUCA

UCCGAGAUC

AAGAUGGG

AGACAGGUA

CGAGACGG

GAGACGGUC

AGGUUCUU

GGUCAGGUU

CUUCCACU

GUCAGGUUC

UUCCACUG

CAGGUUCUU

CCACUGCU

AGGUUCUIJC

CACUGCUA

CCACUGCUA

CMAGCGCG

CCGCGUGUU

CGUUGACC

CGCGUGUUC

GUUGACCA

GUGUUCGUU

GCCACCC

CCCACUGUU

CCUGGAGA

CCACUGUUC

CUGGAGAG

GAGAGGGUU

UGGGGAAA

AGAGGGUUU

GGGGAAAG

GAGMAGAUC

UACGGGCC

GMAGAUCUA

CGGGCCUG

MACGGACUA

CAGGGACA

GCUGCGGUU

CAGCCUGC

CUGCGGUUC

AGCCUGCU

AGCCUGCUA

UGCCAGGC

GCAGCACUU

GMAGCUCC

UUGMAGCUC

CMGGAUC

CCMAGGAUC

CUGAGCCU

CUGAGCCUC

MACAACMA

CAACCCAUA

CUUCUCCG

CCCAUACUU

CUCCGGAC

CCAUACUUC

UCCGGACC

AUACUUCUC

CGGACCAU

CGGACCAUA

CGGGGAGG

GAGGACGUC

GUGUUCGU

CGUCGUGUU

CGUCUGCA

GUCGUGUUC

GUCUGCAA

GUGUUCGUC

UGCAACGA

CCGGCCCLJC

UCUCGUGC

GGCCCUCUC

UCGUGCUA

CCCUCUCUC

GUGCUACC

AUGGACGUC

AGCGAGUG

GGACMAGUA

CAUCGCCG

MAGUACAUC

GCCGUGAA

CGUGMAGUA

CGACGUGU

CGACGUGUC

GACGGCCG

GCGGAGGUC

GGGCLJCCC

GLJCGGGCUC

CCGGUGGA

CGGMCAUC

CCGCUGGU

GGUGGCGUU

CAUCGGCA

Seq. ID No.

27 29 31 33 37 39 41 43 47 49 51 53 57 59 61 63 67 69 71 73 77 79 81 83 87 89 91 93 97 99 101 103 105 107 109 ill 113 115 117 119 121 123 125 127 129 9

C

CCCC

CC

C

CC. a CC S a

CC

Table IIIA nt.

Position 959 968 970 973 985 986 991 992 994 1000 1016 1027 1028 1033 1038 1039 1040 1044 1045 1046 1049 1057 1085 1106 1109 1124 1127 1133 1141 1144 1157 1180 1182 ,1169 1187 1198 1205 1214 1223 1226 Substrate UUCUGCAUC CACAACAU CACAACAUC UCCUACCA CAACAUCUC CUACCAGG CAUCUCCUA CCAGGGCC GGGCCGGUU CGCCLJUCU GGCCGGUUC GCCUUCUC GUUCGCCUU CUCCGACU UUCGCCUUC UCCGACUA CGCCUUCUC CGACuACC CLJCCGACUA CCCGGAGC CUGAACCUC CCGGAGAG GGAGAGAUU CAAGUCGU GAGAGAUUC AAGUCGUC AUUCAAGUC GUCCUUCG CMAGUCGUC CUUCGAUU GUCGUCCUU CGAUUUCA UCGUCCUUC GAUUUCAU CCUUCGAUU UCAUCGAC CUUCGAUUU CAUCGACG UUCGAUUUC AUCGACGG GAUUUCAUC GACGGCUA CGACGGCUA CGAGAAGC CGGAAGAUC AACUGGAU GCCGGGAUC CUCGAGGC GGGAUCCUC GAGGCCGA GACAGGGUC CUCACCGU AGGGUCCUC ACCGUCAG CUCACCGOC AGCCCCUA CAGCCCCUA CUACGCCG CCCCUACUA CGCCGAGG GAGGAGCUC AUCUCCGG GAGCUCAUC UCCGGCAU GCUCAUCUC CGGCAUCG UCCGGCAUC GCCAGGGG UGCGAGCUC GACAACAu GACAACAUC AUGCGCCU AUGCGCCUC ACCGGCAU ACCGGCAUC ACCGGCAU ACCGGCAUC GUCMACGG GGCAUCGUC AACGGCAU Seq. ID No.

130 132 134 136 138 140 142 144 146 148 150 152 154 156 158 160 162 164 168 168 170 172 174 176 178 180 182 184 186 188 190 192 194 196 198 200 202 20-4 206 208 nit.

Position 1385 1388 1421 1436 1445 1472 1475 1476 1501 1502 1514 1534 1535 1559 1564 1565 1589 1610 1816 1627 1628 1643 1648 1666 1690 1703 1706 1715 1718 1735 1738 1751 1757 1769 1787 1807 1820 1829 1843 1871 Substrate GUGGCGUUC

AUCGGCAG

GCGUUCAUC

GGCAGGCU

CCCGACGUC

AUGGCGGC

GCCGCCAUC CCGCAGCU CCGCAGCUC

AUGGAGAU

GUGCAGAUC GUUCUGCU CAGAUCGUU

CUGCUGGG

AGAUCGUUC

UGCUGGGC

GMAGAAGUU

CGAGCGCA

AAGMAGUUC GAGCGCAU CGCAUGCUC AUGAGCGC GGAGAAGUU

CCCAGGCA

GAGMAGUUC

CCAGGCAA

GCCGUGGUC MAGUUCAA GGUCMAGUU

CMACGCGG

GUCMAGUUC

MACGCGGC

CACCACAUC AUGGCCGG GACGUGCUC GCCGUCAC CUCGCCGUC

ACCAGCCG

CAGCCGCUU CGAGCCCU AGCCGCUUC

GAGCCCUG

UGCGGCCUC AUCCAGCU GGCCUCAUC CAGCUGCA GAUGCGALJA CGGAACGC CUGCGCGJC CACCGGUG GGUGGACUC GUCGACAC GGACUCGUC

GACACCAU

GACACCAUC AUCGMAGG ACCAUCAUC GAAGGCA GACCGGGUU CCACAUGG ACCGGGUUC CACAUGGG GGCCGCCUC AGCGUCGA CUCAGCGUC GACUGCAA UGCMACGUC GUGGAGCC

GCGGACGUCAMGMAGGU

CACCACCUU GCAGCGCG

CGCGCCAUCAMGGUGGU

MAGGUGGUC GGCACGCC GCCGGCGUA CGAGGAGA UGCAUGAUC CAGGAUCU Seq. ID No.

131 133 135 137 139 141 143 145 147 149 151 153 155 157 159 161 163 165 167 169 171 173 175 177 179 181 183 185 187 189 191 193 195 197 199 201 203 205 207 209 S S.

S S. S S.

S

S

S..

*55*

S

S S. S

S

S.

*S S

S

S.

S

55 Table EllA nit.

Position 1878 1880 1882 1922 1928 1934 1955 1970 1979 2012 2013 2033 2035 2055 2063 2065 2066 208 2069 2071 2073 2060 2081 2082 2085 2086 2087 2094 2104 2110 2117 2121 2127 2132 2135 2137 2162 2168 2181 2184 2188 2197 2200 2201 2205 2211 2215 2218 Substrate UCCAGGAUC

UCUCCUGG

CAGGAUCUC

UCCUGGAA

GGAUCUCUC

CUGGAAGG

GUGCUGCUC

AGCCUCGG

CUCAGCCUC

GGGGUCGC

CUCGGGGUC

GCCGGCGG

CCAGGGGUC

GAAGGCGA

GAGGAGAUC

GCGCCGCU

GCGCCGCUC

GCCAAGGA

UGAAGAGUU

CGGCCUGC

GAAGAGUUC

GGCCUGCA

CCCCUGAUC

UCGCGCGU

CCUGAUCUC

GCGCGUGG

AAACALJGUU

GGGACAUC

UGGGACAuc

UUCUUAUA

GGACAUCUU

CUUAUAUA

GACAUCUUC

UUAUAUAU

CAUCUUCUU

AUAUAUGC

AUCUUCUUA

UAUAUGCU

CUUCUUAUA

UAUGCUGU

UCUUAUAUA

UGCUGUUU

UAUGCUGUU

UCGUUUAU

AUGCUGUUU

CGUUUAUG

UGCUGUUUC

GUUUAUGU

UGUUUCGUU

UAUGUGAU

GUUUCGUUU

AUGUGAUA

UUUCGUUUA

UGUGAUAU

UAUGUGAUA

UGGACMAG

GGACMAGUA

UGUGUAGC

GUAUGUGUA

GCUGCUUG

UAGCUGCUU

GCUUGUGC

UGCUUGCUU

GUGCUAGU

CUUGUGCUA

GUGUMAUA

GCUAGUGUA

AUAUAGUG

AGUGUMAUA

UAGUGUAG

UGUMAUAUA

GUGUAGUG

UAUAGUGUA

GUGGUGGC

CACAACCUA

AUMAGCGC

MACCUAAUA

AGCGCAUG

CAUGMACUA

AUCLJUG

GMACUMAUU GCUUGCGU UMAUUGCUU

GCGUGUGU

GCGUGUGUA

GUUMAGUA

UGUGUAGUUAMGUACCG

GUGUAGUUA

AGUACCGA

AGUUMAGUA

CCGAUCGG

GUACCGAUC

GGUMAUUU

CGAUCGGUA

AUUUUAUA

UCGGUAAUU

UUAUAUUG

Seq. ID No.

210 212 214 216 218 220 222 224 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 25.4 255 256 257 258 259 260 261 262 263 264 265 266 'it.

Position 2219 2220 2221 2223 2225 2232 2236 2248 Substrate CGGUAAUUU

UAUAUUGC

GGUAAUUUUAUAUUGCG

GUMAUUUuA

UAUUGCGA

AAUUUUAUA

UUGCGAGU

UUUUAUAUU

GCGAGUMA

UUGCGAGUA

AAUAAAUG

GAGUAAAUA

AAUGGACC

GGACCUGUA GUGGUGGA Seq. ID No.

211 213 215 217 219 221 223 225 Table 11113 76 Table III B: Hammierhead. Ribozynie Sequence Targeted Against GBSS niRNA nt. Position HH Ribozynie Sequence Seq. ID No.

12 UGGCUGUGGC CUGAUGA X GAA AUCGAUCGGU 267 68 GCAGUGAGUU CUGAUGA X GMA AUUCCUUCCU 268 73 GGCUGGCAGU CUGAUGA X GMA AGUUUAUUCC 269 103 GACGGAGCAG CUGAUGA X GMA ACACUUCUCC 270 109 CUGGUGGACG CUGAUGA X GMA AGCAGUACAC 271 113 CGCACUGGUG CUGAUGA X GMAACGGAGCAGU 272 146 UCGACGAGAU CUGAUGA X GMA AGCAG3CCCUG 273 149 UCGUCGACGA CUGAUGA X GMA AUGAGCAGCC 274 151 GGUCGUCGAC CUGAUGA X GMA AGAUGAGOAG 275 154 ACUGGUCGUC CUGAUGA X GMA ACGAGAUGAG 276 169 CAUGCCGAUU CUGAUGA X GAA AUCCACUGGU 277 170 CCAUGCCGAU CUGAUGA X GAA AUCCACUGG 278 173 CCGCCAUGCC CUGAUGA X GMA AUUMAUCCAC 279 186 GACGUGGCUA CUGAUGA X GAA AGCCGCCAUG 280 188 GCGACGUGGC CUGAUGA X GMA AGAGCCGCCA 281 196 GACGAGCUGC CUGAUGA X GMA ACGUGGCUAG 282 203 GCGUUGCGAC CUGAUGA X GMA AGCUGCGACG 283 206 CGCGCGUUGC CUGAUGA X GMA ACGAGCUGCG 284 230 ACGCGUCCGG CUGAUGA X GMA ACGCCCAGGC 285 241 GCGGAACGUG CUGAUGA X GMA ACGCGUCCGG 286 247 GCCGCGGCGG CUGAUGA X GMA ACGUGGACGC 287 248 CGCCGCGGCG CUGAUGA X GMA MCGUGGACG 288 292 GUCCGCCGCC CUGAUGA X GMA ACGCCGUCCG 289 308 UCCGMAUGCU CUGAUGA X GMA AGCGUGUCCG 290 :314 CGCUGGUCCG CUGAUGA X GMA AUGCUGAGCG 291 *315 GCGCUGGUCC CUGAUGA X GMA AUGCUGAGC 292 344 GCUGGUGCUG CUGAUGA X GMA AGCCUGGGCG 293 385 GAGCGACGGG CUGAUGA X GMA ACCUGGCCCC 294 386 CGAGCGACGG CUGAUGA X GMA AACCUGGCCC. 295 391 CACGACGAGC CUGAUGA X GMA ACGGGMACCU 296 395 CGCACACGAC CUGAUGA X GMA AGCGACGGGA 297 398 UGGCGCACAC CUGAUGA X GMA ACGAGCGACG 298 425 CGACGMAGAC CUGAUGA X GMA ACGUUCAUGC 299 428 CGCCGACGAA CUGAUGA X GMA ACGACGUUCA 300 430 GGCGCCGACG CUGAUGA X GMA AGACGACGUU 301 431 CGGCGCCGAC CUGAUGA X GMA MGACGACGU 302 434 UCUCGGCGCC CUGAUGA X GMA ACGMAGACGA 303 *473 GGACGUCGCC CUGAUGA X GMA AGGCCGCCGG 304 482 GGCCGCCGAG CUGAUGA X GMA ACGUCGCCGA 305 485 GCAGGCCGCC CUGAUGA X GMA AGGACGUCGC 306 :..527 AGACGACCAU CUGAUGA X GMA ACACGGUGCC 307 *.:533 GGGGAGAGAC CUGAUGA X GMA ACCAUGACAC 308 *536 AGCGGGGAGA CUGAUGA X GMA ACGACCAUGA 309 538 GUAGCGGGGA CUGAUGA X GMA AGACGACCAU 310 540 UCGUAGCGGG CUGAUGA X GMA AGAGACGACC 311 Table 11113 nt. Position HH Riborvme Sequence 547 556 581 586 593 610 620 625 626 628 629 637 661 662 665 679 680 692 693 716 718 742 763 764 773 788 795 803 812 826 829 830 832 841 854 859 860 863 888 890 892 898 902 913 919 929 931 951 952 953 959 968 GUACUGGUCG CUGAUGA X GMA

AGCGGGGAGA

GGCGUCCUUG CUGAUGA X GMA

ACUGGUCGUA

UCUCGGACAC CUGAUGA X GMA ACGCUGGUGU CUUGAUCUCG CUGAUGA X GMA

ACACGACGCU

CUCCCAUCUU CUGAUGA X GMA

AUCUCGGACA

GACCGUCUCG CUGAUGA X GMA

ACCUGUCUCC

GGMAGMCCU CUGAUGA X GMA

ACCGUCUCGU

GCAGUGGMAG CUGAUGA X GMA

ACCUGACCGU

AGCAGUGGMA CUGAUGA X GMA MCCUGACCG GUAGCAGUGG CUGAUGA X GMA

AGMACCUGAC

UGUAGCAGUG CUGAUGA X GMA

MAGMCCUGA

UCCGCGCUUG CUGAUGA X GMA AGCAGUGGMA GUGGUCMACG CUGAUGA X GMA

ACACGCGGUC

GGUGGUCMAC CUGAUGA X GMA

MCACGCGGU

GUGGGUGGUC CUGAUGA X GMA

ACGMACACGC

CCUCUCCAGG CUGAUGA X GMA

ACAGUGGGUG

CCCUCUCCAG CUGAUGA X GMA

MCAGUGGGU

UCUUUCCCCA CUGAUGA X GMA ACCCUCUCCA GUCUUUCCCC CUGAUGA X GMA MCCCUCUCC CAGGCCCGUA CUGAUGA x GMA AUCUUCtJCCU GUCAGGCCCG CUGAUGA X GMA

AGAUCUUCUC

GUUGUCCCUG CUGAUGA X GMA

AGUCCGUUCC

UAGCAGGCUG CUGAUGA X GMA

ACCGCAGCUG

AUAGCAGGCU CUGAUGA X GMA

MCCGCAGCU

CUGCCUGGCA CUGAUGA X GMA

AGCAGGCUGA

UUGGAGCUUC CUGAUGA X GMA

AGUGCUGCCU

AGGAUCCUUG CUGAUGA X GMA

AGCUUCMAGU

UGAGGCUCAG CUGAUGA X GMA

AUCCUUGGAG

GGUUGUUGUU CUGAUGA X GMA

AGGCUCAGGA

UCCGGAGAAG CUGAUGA X GMA

AUGGGUUGUU

UGGUCCGGAG CUGAUGA X GM

AGUAUGGGUU

AUGGUCCGGA CUGAUGA X GMA

MGUAUGGGU

GUAUGGUCCG CUGAUGA X GMA

AGMAGUAUGG

GUCCUCCCCG CUGAIJGA X GMA

AUGGUCCGGA

AGACGMACAC CUGAUGA X GMA ACGUCCUCCC GUUGCAGACG CUGAUGA X GMA

ACACGACGUC

CGUUGCAGAC CUGAUGA X GMA

MCACGACGU

AGUCGUUGCA CUGAUGA X GMA ACGMACACGA UAGCACGAGA CUGAUGA X GMA

AGGGCCGGUG

GGUAGCACGA CUGAUGA X GMA

AGAGGGCCGG

GAGGUAGCAC CUGAUGA X GMA

AGAGAGGGCC

GCUCUUGAGG CUGAUGA X GMA

AGCACGAGAG

AGUUGCUCUU CUGAUGA X GMA

AGGUAGCACG

GUGGGACUGG CUGAUGA X GMA

AGUUGCUCUU

GAUGCCGUGG CUGAUGA X GMA ACUGGUAGUU CGUCCCUGUA CUGAUGA X GMA

AUGCCGUGGG

UGCGUCCCUG CUGAUGA X GMA AGAUGCCGUG UGGAUGCAGA CUGAUGA X GMA

AGCGGUCUUU

GUGGAUGCAG CUGAUGA X GMA MGCGGUCUU UGUGGAUGCA CUGAUGA X GMA

AAAGCGGUCU

AGAUGUUGUG CUGAUGA X GMA

AUGCAGAA&AG

CCUGGUAGGA CUGAUGA X GMA AUGUUGUGGA Seq.

ID

No.

312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363

S

S S 5* 5 S.

S.

S. S.

S

S

*S S SSe.

S

*5S*

*S

S S

S

S.

SS

S.

Table 11113 78 n t. Position HH Riboz vme Sequence Seq. ID No.

970 GCCCUGGUAG CUGAUGA X GMA AGAUGUUGUG 364 973 CCGGCCCUGG CUGAUGA X GMA AGGAGAUGUU 365 985 GGAGAAGGCG CUGAUGA X GMA ACCGGCCCUG 366 986 CGGAGAAGGC CUGAUGA X GMA MCCGGCCCU 367 991 GUAGUCGGAG CUGAUGA X GMA AGGCGMACCG 368 992 GGUAGUCGGA CUGAUGA X GMA MGGCGMACC 369 994 CGGGUAGUCG CUGAUGA X GMA AGAAGGCGAA 370 1000 CAGCUCCGGG CUGAUGA X GMA AGUCGGAGAA 371 1016 AUCUCUCCGG CUGAUGA X GAA AGGUUCAGCU 372 1027 GGACGACUUG CUGAUGA X GMA AUC 'UCUCCGG 373 1028 AGGACGACUU CUGAUGA X GMA MUCUCUCCG 374 1033 AUCGAAGGAC CUGAUGA X GMA ACUUGMAUCU 375 1036 GMAAUCGMAG CUJGAUGA X GMA ACGACUUGAA 376 1039 GAUGAAAUCG CUGAUGA X GMA AGGACGACUU 377 1040 CGAUGAAAUC CUGAUGA X GMA MGGACGACU 378 1044 CCGUCGAUGA CUGAUGA X GMA AUCGMAGGAC 379 1045 GCCGUCGAUG CUGAUGA X GMM AUCGMAGGA 380 1046 AGCCGUCGAU CUGAUGA X GMA AMAUCGMAGG. 381 1049 CGUAGCCGUC CUGAUGA X GMA AUGAAAUCGA 382 1057 GGGCUUCUCG CUGAUGA X GMA AGCCGUCGAU 383 1085 UCAUCCAGUU CUGAUGA X GMA AUCUUCCGGC 384 1106 CGGCCUCGAG CUGAUGA X GMA AUCCCGGCCU 385 1109 UGUCGGCCUC CUGAUGA X GMA AGGAUCCCGG 386 1124 UGACGGUGAG CUGAUGA X GMA ACCCUGUCGG 387 1127 GGCUGACGGU CUGAUGA X GMA AGGACCCUGU 388 1133 AGUAGGGGCU CUGAUGA X GMA ACGGUGAGGA 389 1141 CUCGGCGUAG CUGAUGA X GMA AGGGGCUGAC 390 1144 CUCCUCGGCG CUGAUGA X GMA AGUAGGGGCU 391 1157 UGCCGGAGAU CUGAUGA X GMA AGCUCCUCGG 392 1160 CGAUGCCGGA CUGAUGA X GMA AUGAGCUCCU 393 *1162 GGCGAUGCCG CUGAUGA X GMAAGAUGAGCUC 394 1169 AGCCCCUGGC CUGAUGA X GMA AUGCCGGAGA 395 :1187 UGAUGUUGUC CUGAUGA X GMA AGCUCGCAGC 396 1196 UGAGGCGCAU CUGAUGA X GMA AUGUUGUCGA 397 1205 UGAUGCCGGU CUGAUGA X GMA AGGCGCAUGA 398 **1214 CGAUGCCGGU CUGAUGA X GMA AUGCCGGUGA 399 1223 UGCCGUUGAC CUGAUGA X GMA AUGCCGGUGA 400 1226 CCAUGCCGUU CUGAUGA X GMA ACGAUGCCGG 401 1241 CCCACUCGCU CUGAUGA X GMA ACGUCCAUGC 402 1270 CACGGCGAuG CUGAUGA X GMA ACUUGUCCCU 403 1274 ACUUCACGGC CUGAUGA X GMA AUGUACUUGU 404 1285 CGACACGUCG CUGAUGA X GMA ACUUCACGGC 405 1294 CACGGCCGuC CUGAUGA X GMA ACACGUCGUA 406 1346 CCGGGAGCCC CUGAUGA X GMA ACCUCCGCCU 407 1352 GGUCCACCGG CUGAUGA X GMAAGCCCGACCU 408 1370 CCACCAGCGG CUGAUGA X GMA AUGUUCCGGU 409 1384 CCUGCCGAUG CUGAUGA X GMACGCCACCAG 410 *1385 GCCUGCCGAU CUGAUGA X GMA MCGCCACCA 411 *1388 CCAGCCUGCC CUGAUGA X GMAAUGMACGCCA 412 :1421 CGGCCGCCAU CUGAUGA X GMA ACGUCGGGUC 413 1436 UGAGCUGCGG CUGAUGA X GMA AUGGCGGCCG 414 1445 CCAUCUCCAU CUGAUGA X GMA AGCUGCGGGA 415 Table 11113 79 nt. Position HH Ribozyme Sequence Seq. ID No.

1472 CCAGCAGAAC CUGAUGA X GMA AUCUGCACGU 416 1475 UGCCCAGCAG CUGAUGA X GMA ACGAUCUGCA 417 1476 GUGCCCAGCA CUGAUGA X GMA MCGAUCUGC 418 1501 CAUGCGCUCG CUGAUGA X GMA ACUUCUUCUU 419 1502 GCAUGCGCUC CUGAUGA X GMA AACuUCUUCU 420 1514, CGGCGCUCAU CUGAUGA X GMA AGCAUGCGCU 421 1534 CUUGCCUGGG CUGAUGA X GMA ACUUCUCCUC 422 1535 CCUUGCCUGG CUGAUGA X GMA MCUUCUCCU 423 1559 CGUUGMACUU CUGAUGA X GMA ACCACGGCGC 424 1564 CGCCGCGUUG CUGAUGA X GMA ACUUGACCAC 425 1565 GCGCC:GCGUU CUGAUGA X GMA MCUUGACCA 426 1589 CGCCGGCCAU CUGAUGA-X GMA AUGUGGUGOG 427 1610 UGGUGACGGC CUGAUGA X GMA AGCACGUCGG 428 1616 AGCGGCUGGU CUGAUGA X GMA ACGGCGAGCA 429 1627 GCAGGGCUCG CUGAUGA X GMA AGCGGCUGGU 430 1628 CGCAGGGCUC CUGAUGA X GMA MGCGGCUGG 431 1643 GCAGCUGGAU CUGAUGA X GMA AGGCCGCAGG 432 1846 CCUGCAGCUG CUGAUGA X GMA AUGAGGCCGC433 1666 GGGCGUUCCG CUGAUGA X GMA AUCGCAUCCC 434 1690 UCCACCGGUG CUGAUGA X GMAACGCGCAGGC 435 1703 UGGUGUCGAC CUGAUGA X GMA AGUCCACCGG 436 1706 UGAUGGUGUC CUGAUGA X GMA ACGAGUCCAC 437 1715 UGCCUUCGAU CUGAUGA X GMA AUGGUGUCGA 438 1718 UCUUGCCUUC CUGAUGA X GMAAUGAUGGUGU 439 1735 GCCCAUGUGG CUGAUGA X GMA ACCCGGUCUU 440 1736 GGCCCAUGUG CUGAUGA X GMA MCCCGGUCU 441 1751 AGUCGACGCU CUGAUGA X GMAAGGCGGCCA 442 1757 CGUUGCAGUC CUGAUGA X GMA ACGCUGAGGC 443 1769 CCGGCUCCAC CUGAUGA X GMA ACGUUGCAGU 444 1787 CCACCUUCUU CUGAUGA X GMA ACGUCCGCCG 445 1807 GGCGCGCUGC CUGAUGA X GMA AGGUGGUGGC 446 1820 CGACCACCUU CUGAUGA X GMA AUGGCGCGCU 447 1829 CCGGCGUGCC CUGAUGA X GMA AccAcCUUGA 448 :::1843 CAUCUCCUCG CUGAUGA X GMA ACGCCGGCGU 449 1871 AGAGAUCCUG CUGAUGA X GMA AUCAUGCAGU 450 1878 UUCCAGGAGA CUGAUGA X GMA AUCCUGGAUC 451 ~*1880 CCUUCCAGGA CUGAUGA X GMA AGAUCCUGGA 452 1882 GCCCUUCCAG CUGAUGA X GMAAGAGAUCCUG 453 1922 CCCCGAGGCU CUGAUGA X GAA AGCAGCACGU 454 1928 CGGCGACCCC CUGAUGA X GMA AGGCUGAGCA 455 1934 CGCCGCCGGC CUGAUGA X GMAACCCCGAGGC 456 **1955 CCUCGCCUUC CUGAUGA X GMA ACCCCUGGCU 457 1970 CGAGCGGCGC CUGAUGA X GMA AUCUCCUCGC 458 1979 UCUCCUUGGC CUGAUGA X GMA AGCGGCGCGA 459 2012 CUGCAGGCCG CUGAUGA X GMA ACUCUUCAGG46 2013 CCUGCAGGCC CUGAUGA X GMA MCUCUUCAG 461 2033 CCACGCGCGA CUGAUGA X GMA AUCAGGGGGC 462 2035 CACCACGCGC CUGAUGA X GMA AGAUCAGGGG 463 :::2055 MAGAUGUCCC CUGAUGA X GMA ACAUGUUUGC 464 *.:2063 UAUAUMAGM CUGAUGA X GMA AUGUCCCMAC 465 *2065 CAUAUAUMAG CUGAUGA X GMA AGAUGUCCCA 466 2066 GCAUAUAUAA CUGAUGA X GMA MGAUGUCCC 467 Table 111B nt. Position HH Ribozvme Sequence Seq. ID No. 2068 CAGCAUAUAU CUGAUGA X GMA AGAAGAUGUC 468 2069 ACAGCAIJAUA CUGAUGA X GMA MGAAGAUGU 469 2071 AAACAGCAUA CUGAUGA X GMA AUMAGMGAU 470 2073 CGAAACAGCA CUGAUGA X GMA AUAUMAGMG 471 2080 ACAUAAACGA CUGAUGA X GMA ACAGCAUAUA 472 2081 CACAUAAACG CUGAUGA X GMA MCAGCAUAU 473 2082 UCACAUAAAC CUGAUGA X GMA AAACAGCAUA 474 2085 AUAUCACAUA CUGAUGA X GMA ACGAAACAGC 475 2086 CAUAUCACAU CUGAUGA X GMA MCGAAACAG 476 2087 CCAUAUCACA CUGAUGA X GMA AAACGAApCA 477 2094 UACUUGUCCA CUGAUGA X GMA AUCACAUAAA 478 2104 CAGCUACACA CUGAUGA X GMA ACUUIGUCCAU 479 2110 AGCMAGCAGC CUGAUGA X GMA ACACAUACU J 480 2-117 UAGCACMAGC CUGAUGA X GMA AGCAGCUACA 481 2121 ACACUAGCAC CUGAUGA X GMA AGCMAGCAGC 482 2127 UAUAUUACAC CUGAUGA X GMA AGCACMAGCA 483 2132 UACACUAUAU CUGAUGA X GMA ACACUAGCAC 484 2135 CACUACACUA CUGAUGA X GMA AUUACACUAG 485 2137 ACCACUACAC CUGAUGA X GMA AUAUUACACU 486 2142 UGGCCACCAC CUGAUGA X GMA ACACUAUAUU 487 2165 AUGCGCUUAU CUGAUGA XGAA AGGUUGUGCC 488 2168 UUCAUGCGCU CUGAUGA X GMA AUUAGGUUGU 489 2181 CGCMAGCMAU CUGAUGA X GMAGUUCAUGCG 490 2184 ACACGCMAGC CUGAUGA X GMA AUUAGUUCAU 491 2188 CUACACACGC CUGAUGA X GMA AGCMAUUAGU 492 2197 GGUACUUMAC CUGAUGA X GMA ACACACGCMA 493 2200 AUCGGUACUU CUGAUGA X GM. ACUACACACG 494 2201 GAUCGGUACU CUGAUGA X GMA MCUACACAC 495 2205 UACCGAUCGG CUGAUGA X GMA ACUUMACUAC 496 2211 UAAAAUUACC CUGAUGA X GMA AUCGGUACUU 497 '00. 2215 MAUAUAAAAU CUGAUGA X GMA ACCGAUCGGU 498 2218 CGCMAUAUMA CUGAUGA X GMA AUUACCGAUC 499 :2219 UCGCMAUAUA CUGAUGA X GMA MUUACCGAU 500 *2220 CUCGCMUAU CUGAUGA X GMA AAAUUACCGA 501 2221 ACUCGCMAUA CUGAUGA X GMA AAAAUUACCG 502 223 UUACUCGCMA CUGAUGA X GMA AUAAMAUUAC 503 ***2225 AUUUACUCGC CUGAUGA X GMA AUAUAAAAUU 504 2232 UCCAUUUAUU CUGAUGA X GMA ACUCGCMAUA 505 2236 CAGGUCCAUU CUGAUGA X GMA AUUUACUCGC 506 2248 UUUCCACCAC CUGAUGA X GMA ACAGGUCCAU 507 Where represents stem 11 region of a HH ribozyme (Hertel et al., 1992 N~ucleic Acids Res. 20 3252). The length of stem 11 may be 2 base-pairs.

Table IV 0 Table IV: HH Ribozymne Sequences Tested against GBSS mRNA nt. EM Ribozyme Sequence Sequece Position.

I.D.

425 CGACGAAGAC CUGAUGAGGCCGAAGGCCGAA ACGUC:AUG;C 2 593 CUCCCAUCUU CUGAUGAGGCCGACA AUCUCGGAA 3 742 GUUJGUCCCUG CUGAUGAGCCGAGC AGUCCGTJTCC 4 812 GGUUGUUGUU CUGAUGAGGCCGAWCA AGGCUCAGA 892 GAGGUJAGCAC CUG.AUGAGGCCGA .GGCCGAA AGAGAGGGCC 6 913 GUGGGACUG CtUGAGG JkCGCAA AGUUGCUCUU 7 919 GAUGCCGUGG CUGAUGAGG GGCCGAA ACUGGUAGUU 8 953 U'GuGGAuGCA ctJGAUAGGccC GAAAGcc. AAGGucT 9 959 AGAUGUUGUG CUGAUGAGGCCG flQGCA AUGCAGAAAG 968 CCU)GGUAGGA CUGAUGAGGCCCAG CCAA~ AUGUUGUGGA 11 1016 AUCUCUCCO CUGAUGAWAAGGCC CAi AGGUUCAGCU 12 1028 AGGACGAcuu cu~GGcGA~cA AIucucuccG, 13 1085 UCAUCCAGUU CUGUGAMGG C CG AAA CCQk AUCUUCCGGC 14

C

CCC.

CC..

CCC.

C

1187 UGaAUGUUGUC 1196 UGAGGCGCAU 1226 CCAUG3CCGUU 1241

CCCA.CUCGCU

1270 CACGGCGAUG 1352

GGUCCACCGG

1421 CGGCCGCCAU 1534 CUUJGCCUGGG 1715 UGCCUUCGAU CUG-AUGAG-GCCGAAAGGCCGAA

AGCUCG;CAGC

CUAt'G;CGAGCGC

AUGUUGUCCGA

CUGAUGAGGCCG1AGGCCJ-,A

ACGAUJGCCG

CUGAUGAlGCCGACCC-,

ACGUCCAUGC

CUGAUAGGCCGAGCCA ACUtJGUCCU CUGAUGAGCCAAGGCCGr-%

AG;CCCGACCU

CUGAU-AGGCCGAGCM.;:,

ACGUCGGGUC.

CUGAUGAGG AGCM-L ACUCCC CUGAUGAGGCCAGGCGry;

AUGGUGUCGA

1787 CCACCUUCEJ CUGAUGAGGCCGAAAGGCCGAA,

ACG;UCCGCC.

C.

C C CC. C CC C C

CC

9 9* 9 eq. 9.

9 *tu 9 .9 9 99 *9 9 s S. *9 9 9 99~..

S 9 eq I[Ale

VA

Table V A: GBSS Hairpin Ribozyme and Substrate Sequences fit.

l'osifijun 48 129 468 489 496 676 737 760 1298 1427 1601 1638 1746 1781 2077 liairpin Ribozynie Sequgence CUCCUGGC AGAA GUCG

ACCAGAGMAACACACGUUGUGGUACAUACGA

CCCUGCCG AGAA GUGC

ACCAGAGAAACACACGUUGUGGUAC

GUCGCCGA AGAA GCCG

ACCAGAGAAACACACGUUGIJGGUACAUUACGU

CGGCGGCA AGMA GCCG

ACCAGAGAAACACACGUUGUGGUACAUACGU

CCAUGGCC AGMA GCAG

ACCAGAGMACACACGUUGUGGUACAUACGA

UCUCCAGG AGAA GUGG

ACCAGAGAAACACACGUUGUGGUACAUUACGU

UCCCUGUA AGAA GUUC

ACCAGAGAAACACACGUUGUGGUACAUACGU

GCAGGCUG AGAA GCAG

ACCAGAGMAACACACGUUGUGGUACAUACGU

GCCUCCAC AGAA GUCG ACCAGAGAAACACACGUUGUGGUACAUAcGU GGGAUGGC AGMA GCCA

ACCAGAGAAACACACGUUGUGGUACAUACGU

GCGAGCAC AGAA GCGC

ACCAGAGAAACACACGIJUGUGGUACAUCGGA

CUGGAIJGA AGMA OCAG

ACCAGAGAAACACACGIJUGUGGIJACAUACGU

GACGCUGA AGAA GCCC

ACCAGAGAAACACACGUUGUGGUACAUACGU

UUCUUGAC AGAA GCCG

ACCAGAGAAACACACGUUGUGGUACAUACGA

AUAAACGA AGAA GCAU

ACCAGAGAAACACACGUUGUGGUACAUACGU

Seq. I D No.

508 510 512 514 516 518 520 522 524 526 528 530 532 534 536 C AASubstrae CG CAGC

GOCAGGAG

GCACC GCC

CGGCAGGG

CGGCG GCC UCGGCGAC CGGCG GCC

UGCCGCCG

CUGCC GCC GGCCAUGG CCACU GUU

CCUGGAGA

GAACG GAC

UACAGGGA

CUGCG GUU CAGCCUGC CGACG GCC

GUGGAGGO

UGGCG GCC GCCAUCCC GCGCC GAC

GUGCUCGC

CUGCG GCC

UCAUCCAG

GGGCC GCC

UCAGCGUC

CGGCG GAC GUCAAGAA AUGCU GUU

UCGUUUAU

Seq. 11) No.

509 511 513 515 517 519 521 523 525 527 529 531 533 535 537 C C Table VI Table VB: GBSS Hairpin Ribozyme and Substrate Sequences t. Position Ribozyme sequence 31 GUCGCCUC AGAA GGUGGU

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

48 CUCCUGGC AGAA GUCGCG ACCAGAGAAACACACGUUGUGGUACAUtJACCUGGIJ 105 GUGGACGG AGAA GUACAC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

110 CACUGGUG AGMA GAGCAG

ACCAGAGMACACACGUUGUGGUACAUUACCUGGUA

129 CCCUGCCG AGAA GUGCGC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

142 ACGAGAUG AGAA GCCCUG

ACCAGAGMACACACGUUGUGGUACAUUACCUGGUA

182 GUGGCUAc3 AGAA GCCAUG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

199 UUGCGACG AGAA GCGACG

ACCAGAGMAACACACGUUGUGGUACAUUACCUGGUA

219 GACGCCCA AGMA GGCGCG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

233 GUGGACGC AGAA GGGACG

ACCAGAGMAACACACGUUGUGGUACAUUACCUGGUA

249 GGCGCCGC AGMA GMACGU

ACCAGAGMACACACGUUGUGGUACAUUACCUGGUA

283 CCGACGCC AGAA GGCCCC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

316 GCGCGCUG AGMA GAAUGC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

388 CGACGAGC AGAA GGMACC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

468 GUCGCCGA AGAA GCCGGU

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

489 CGGCGGCA AGAA GCCGAG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

493 UGGCCGGC AGAA GGCCGC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

496 CCAUGGCC AGAA GCAGGC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

676 UCUCCAGG AGAA GUGGGU

ACCAGAGMACACACGUUGUGGUACAUUACCUGGUA

725 GUUCCAGC AGAA GGCCCG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

737 UCCCUGUA AGAA GUUCCA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

754 UGAACCGC AGAA GGUUGU

ACCAGAGMACACACGUUGUGGUACAUUACCUGGUA

760 GCAGGCUG AGAA GCAGCU

ACCAGAGAAACACACGUUGIJGGUACAUUACCUGGUA

765 GCAUAGCA AGAA GAACCG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

834 CCCGUAUG AGAA GGAGAA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

882 CGAGAGAG AGAA GGUGUG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

916 UGCCGUGG AGAA GGUAGU

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

947 AUGCAGAA AGAA GUCUUU

ACCAGAGWACACACGUUGUGGUACAUUACCUGGUA

982 AGAAGGCG AGAA GGCCCU

ACCAGAGMAACACACGUUGUGGUACAUUACCUGGUA

995 UCCGGGUA AGAA GAGAAG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

1134 GUAGUAGG AGAA GACGGU

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

1298 GCCUCCAC AGAA GUCGAC

ACCAGAGMAACACACGUUGUGGUACAUUACCUGGUA

Seq. *ID

NO.

538 540 542 544 546 548 550 552 554 556 558 560 562 564 566 568 570 572 574 576 578 580 582 584 586 588 590 592 594 596 598 600 Substrate ACCACCC GCC GAGGOGAC CGCGACA GCC GCCAGGAG GUGUACU GCU CCGUCCAC CUGCUCC GUC CACCAGUG GCGCACC GCC OGGCAGGG CAGGGCU GCU CAUCUCGU CAUGGCG GCU CUAGCCAC CIGUCGCA GCU CGUCGCMA CGCGCCG GCC UGGGCGUC CGUCCCG GAC GCGUCCAC ACGUUCC GCC GCGGCGCC GGGGCCG GAC GGCGUCGG GCAUUCG GAC CAGCGCGC GGUUCCC GUC GCUCGUCG ACCGGCG GCC UCGGCGAC CUCGGCG GOC UGCCGCCG GCGGCCU GCC GCCGGCCA GCCUGCC GCC GGCCAUGG ACCCACU GUU CCUGGAGA CGGGCCU GAC GCUGGAAC UGGMACG GAC UACAGGGA ACMACCA GCU GCGGUUCA AGCUGCG GUU CAGCCUGC CGGUUCA GCC UGCUAUGC UUCUCCG GAC CAUACGGG CACACCG GCC CUCUCUCG ACUACCA GUC CCACGGCA AAAGACC GCU UUCUGCAU AGGGCOG GUU CGCCUUCU CUUCUCC GAC UACCCGGA ACCGUCA 0CC CCUACUAC GUCGACG GCC GUGGAGGC Seq. ID No.

539 541 543 545 547 549 551 553 555 557 559 561 563 565 567 569 571 573 575 577 579 581 583 585 587 589 591 593 595 597 599 601 a.

a a a. a a a a a. a a Tablc VB .Position Ribozyme Sequence 1372 1415 1427 1441 1468 1477 1601 1620 1623 1638 1648 1746 1781 1918 1923 1975 2014 2029 2077 2113 2207 ACGCCACC AGAA GGAUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCAUGAC AGAA GGUCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGAUGGC AGMA GCCAUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCLICCAUG AGAA GCGGGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCAGMACG AGMA GCACGU ACCAGAGAMACACACGUUGUGGUACAUUACCUGGUA CCGUGCCC AGMA GMACGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGAGCAC AGMA GCGCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUCGAAGC AGAA GGUGAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCUCGA AGMA GCUGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGGAUGA AGMA GCAGGG ACCAGAGA.AACACACGUUGUGGUACAUUACCUGGUA UCCCCUGC AGMA GGAUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GACGCUGA AGAA GCCCALJ ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUCUUGAC AGAA GCCGGC

ACCAGAGMACACACGUUGUGGUACAUUACCUGGUA

CGAGGCUG AGAA GCACGU ACCAGAGAMACACACGUUGUGGUACAUUACCUGGUA GACCCCGA AGAA GAGCAG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CCUUGGCG AGMA GCGCGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGCCUGCA AGAA GAACUC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CGCGCGAG AGAA GGGGGC ACCAGAGWACACACGUUGUGGUACAUUACCUGGUA AUAAACGA AGMA GCAUAU ACCAGAGAAACACACtPUUGUGGUACAUUACCUGGUA CACAAGCA AGAA OCUACA ACCAGAGA.AACACACGUUGUGGUACAUUACCUGGUA AAUUACCG AGAA GUACUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA Seq. ID No.

602 604 606 608 610 612 614 816 618 620 622 624 626 628 630 632 634 636 638 640 642 Substrate ACAUCCC GCU GGUGGCGU GGGACCC GAC GUCAUGGC CAUGGCG GCC GCCAUCCC LJCCCGCA GCU CAUGGAGA ACGUGCA GAU CGUUCUGC UCGUUCU GCU GGGCACGG CGGCGCC GAC GUGCUCGC GUCACCA GCC GCUUCGAG ACCAGCC GCU UCGAGCCC CCCUGCG GCC UCAUCCAG UCAUCCA GCU GCAGGGGA AUGGGCC GCC UCAGCGUC GCCGGCG GAC GUCMAGAA ACGUGCU GCU CAGCCUCG CUGCUCA GCC UCGGGGUC UCGCGCC GCU CGCCAAGG GAGUUCG GCC UGCAGGCC GCCCCCU GAU CUCGCGCG A'JAUGCU GUU UCGUUL)AU UGUAGCU GCU UGCUUGUG MAGUACC GAU CGGUAAUU Seq. ID No.

603 605 607 609 611 613 615 617 619 621 623 625 627 629 631 633 635 637 639 641 643 Table VI Table VI: Delta-9 Desaturase HH Ribozyme Target Sequences nt.

Position Substrate Seq. ID nt.

No. Position Substrate S. S

S.

*SS S. *5 13 21 24 28 29 32 38 63 69 71 92 117 118 124 129 130 135 141 154 180 169 175 181 183 193 228 229 232 238 243 252 259 261 271 278 288 289 293 296 307 313 528 544 545 557 559 567 575 580 581 589 598 CGCGCCCUC

UGCCGCUU

CUGCCGCUU

GUUCGUUC

CCGCUUGUU

CGUUCCUC

CGCUUGUUC

GUUCCUCG

UUGUUCGUU

CCUCGCGC

UGUUCGUUC

CUCGCGCU

UCGUUCCUC

GCGCUCGC

CUCGCGCUC

GCCACCAG

ACACACAUC

CCAAUCUC

AUCCCMAUC

UCGCGAGG

CCCAAUCUC

GCGAGGGC

AGCAGGGUC

UGCGGCGG

GCCGCGCUU

CCGGCUCC

CCGCGCUUC

CGGCUCCC

UUCCGGCUC

CCCUUCCC

GCUCCCCUU

CCCAUUGG

CUCCCCUUC

CCAUUGGC

CUUCCCAUU

GGCCUCCA

AUUGGCCUC

CACGAUGG

AUGGCGCUC

CGCCUCAA

CUCCGCCUC

AACGACGU

MACGACGUC

GCGCUCUG

GUCGCGCUC

UGCCUCUC

CUCUGCCUC

UCCCCGCC

CUGCCUCUC

CCCGCCGC

CCGCCGCUC

GCCGCCCG

CGGCAGGLJU

CGUCGCCG

GGCAGIUC

GUCGCCGU

AGGUUCGUC

GCCGUCGC

GUCGCCGUC

GCCUCCAU

CGUCGCCUC

CAUGACGU

CAUGACGUC

CGCCGUCU

UCCGCCGUC

UCCACCAA

CGCCGUCUC

CACCAAGG

ACCAAGGUC

GAGAAUAA

UCGAGMAUA

AGAAGCCA

GMAGCCAUU

UGCUCCUC

MGCCAUUU

GCUCCUCC

CAUUUGCUC

CUCCMAGG

UUGCUCCUC

CAAGGGAG

AGGGAGGUA

CAUGUCCA

GUACAUGUC

CAGGUUAC

UGUUUGUUU

GGUGGGAG

GACAUGALUU

ACCGAGGA

ACAUGAUUA

CCGAGGAA

AGGMAGCUC

UACCMACA

GMAGCUCUA

CCAACAUA

ACCAACAUA

CCAGACUA

ACCAGACUA

UGCUUAAC

ACUAUGCUU

AACACCCU

CUAUGCUUA

ACACCCUC

AACACCCUC

GACGGUGU

GACGGUGUC

AGAGAUGA

644 646 648 650 652 854 656 658 660 662 664 666 668 670 672 674 676 678 680 682 884 688 688 690 692 694 698 698 700 702 704 706 708 710 712 714 716 718 720 722 724 726 728 730 732 734 736 738 740 742 744 746 748 319 320 326 327 338 346 352 353 354 355 360 384 371 377 383 388 388, 390 398 400 409 419 434 435 436 439 453 482 483 464 475 476 484 505 515 516 518 519 520 523 524 527 857 860 873 874 882 883 889 898 907 910 915 GUCCAGGUU

ACACAUUC

UCCAGGUUA

CACAUUCA

UUACACAUU

CMAUGCCA

UACACAUUC

MAUGCCAC

UGCCACCUC

ACAAGAUU

CACAAGAUU GAAAuuuu AUUGAAAUu

UUCAAGUC

UUGAAAUUU

UCMAGUCG

UGMAUUUU

CAAGUCGC

GAAAUUUUC

AAGUCGCU

UUUCMAGUC

GCUUGAUG

IAAGUCGCUU

GAUGAUUG

UUGAUGAUU

GGGCUAGA

AUUGGGCUA

GAGAUMAU

CUAGAGAUA

AUAUCUUG

GAGAUAAUA

UCUUGACG

GAUMAUAUC

UUGACGCA

UMAUAUCUU

GACGCAUC

UGACGCAUC

UCAAGCCA

ACGCAUCUC

AAGCCAGU

AAGCCAGUC

GAGMOGUG

AGMAGUGUU

GGCAGCCA

CACAGGAUU

UCCUCCCG

ACAGGAUUU

CCUCCCGG

CAGGAUUUC

CUCCCGGA

GAUUUCCUC

CCGGACCC

CCCAGCAUC

UGAAGGAU

UGAAGGAUU

UCAUGAUG

GAAGGAUUU

CAUGAUGA

AAGGAUUUC

AUGAUGMA

GAUGMAGUU

MAGGAGCU

AUGMAGUUA

AGGAGCUC

AAGGAGCUC

AGAGMACG

AAGGAAAUC

CCUGAUGA

CUGAUGAUU

AUUUUGUU

UGAuGAUuA

UUUUGUUU

AUGAUUAUU

UUGUUUGU

UGAUUAUUU

UGUUUGUU

GAUUAUUUU

GUUUGUUU

UAUUUUGUU

UGUUUGGU

AUUUUGUUU

GUUUGGUG

UUGUUUGUU

UGGUGGGA

ACACUGCUC

GUCACGCC

CUGCUCGUC

ACGCCAAG

CAAGGACUU

UGGCGACU

AAGGACUUU

GGCGACUU

UGGCGACUU

AAAGCUUG

GGCGACUUAAMGCUUGC

UUAAAGCUU

GCACAAAU

GCACAAAUC

UGCGGCAU

UGCGGCAUCAUCGCCUC

GGCAUCAUC

GCCUCAGA

CAUCGCCUC

AGAUGAGA

Seq. ID No.

645 647 649 651 653 655 657 659 661 663 665 667 669 671 673 675 677 679 681 683 685 687 689 691 693 695 697 699 701 703 705 707 709 711 713 715 717 719 721 723 725 727 729 731 733 735 737 739 741 743 745 747 749 Table VI nt Position Substrate Seq. ID nt.

No. Position Substrate 637 638 680 685 693 m9 699 703 719 730 742 743 747 749 751 752 754 759 770 773 785 788 791 792 794 796 800 801 802 805 807 813 816 817 834 835 838 840 1292 1293 1294 1303 1305 1310 1318 1331 1348 1353 1354 1372 1378 1379 1383 1387 1389 1395 1396 UGGGCUGUU

UGGACGAG

GGGCUGUUU

GGACGAGG

AUGGUGAUC

UGCUCAAC

GAUCUGCUC

AACAAGUA

CAACAAGUA

UAUGUACC

ACMAGUAUA

UGUACCUC

GUAUAUGUA

CCUCACUG

AUGUACCUC

ACUGGGAG

GGGUGGAUA

UGAGGCAG

AGGCAGAUU

GAGAAGAC

MAGACAAUU

CAGUAUCU

AGACAAUUC

AGUAUCUU

AAUUCAGUA

UCUUAUUG

UUCAGUAUC

UUAUUGGC

CAGUAUCUU

AUUGGCUC

AGUAUCUUA

UUGGCUCU

UAUCUUAUU

GGCUCUGG

UAUUGGCUC

UGGMAUGG

GAAUGGAUC

CUAGGACU

UGGAUCCUA

GGACUGAG

CUGAGAAUA

AUCCUUAU

AGMAUMUC

CUUAUCUU

AUMAUCCUU

AUCUUGGU

UMAUCCUUA

UCUUGGUU

AUCCUUAUC

UUGGUUUC

CCUUAUCUU GGUUUCAu AUCUUGGUU

UCAUCUAC

UCUUGGUUO

CAUCUACA

CUUGGUUUC

AUCUACAC

GGUUUCAUC

UACACCUC

UUUCAUCUA

CACCUCCU

CUACACCUC

CUUCCMAG

CACCUCCUU CCiAAGAGC ACCUCCUUC

CAAGAGCG

GGCGACCUU

CAUCUCAC

GCGACCUUC

AUCUCACA

ACCUUCAUC

UCACACGG

CUUCAUCUC

ACACGGGA

CGCUGCCUU

UCAGCUGG

GCUGCCUUU

CAGCUGGG

CUGCCUUUC

AGCUGGGU

AGCUGGGUA

UACGGUAG

CUGGGUAUA

CGGUAGGG

UAUACGGUA

GGGACGUC

AGGGACGUC

CAACUGUG

UGUGAGAUC

GGAMACCU

GCUGCGGUC

UGCUUAGA

GGUCUGCUU

AGACMAGA

GUCUGCUUA

GACAAGAC

UGCUGUGUC

UGCGUUAC

GUCUGCGUU

ACAUAGGU

UCUGCGUUA

CAUAGGUC

CGUUACAUA

GGUCUCCA

ACAUAGGUC

UCCAGGUU

AUAGGUCUC

CAGGUUUU

CUCCAGGUU

UUGAUCAA

UCCAGGuuu UGAUCAAA 750 752 754 756 758 760 762 764 766 768 770 772 774 776 778 780 782 784 786 788 790 792 794 796 798 800 802 804 808 808 810 812 814 816 818 820 822 824 826 828 830 832 834 836 838 840 842 844 846 848 850 852 854 856 858 860 862 942 AACUGCGUA

CACCAAGA

952 ACCAAGAUC

GUGGAGAA

966 GAAGCUGUU

UGAGAUCG

967 AAGCUGUUU

GAGAUCGA

973 UUUGAGALJC

GACCCUGA

986 CUGAUGGUA

CCGUGGUC

994 ACCGUGGUC

GCUCUGGC

998 UGGUCGCUC

UGGCUGAC

1024 AAGMAGAUC

UCAAUGCC

1026 GAAGAUCUC

MAUGCCUG

1047 CCUGAUGUU

UGACGGGC

1048 CUGAUGUUU

GACGGGCA

1071 CAAGCUGUU CGAGCACu 1072 AAGCUGUUC

GAGCACUU

1080 CGAGCACUU

CUCCAUGG

1081 GAGCACUUC

UCCAUGGU

1083 GCACUUCUC

CAUGGUCG

1090 UCCAUGGUC

GCGCAGAG

1102 CAGAGGCUU

GGCGUUUA

1108 CUUGGCGUU

UACACCGC

1109 UUGGCGUUU

ACACCGCC

1110 UGGCGUUUA

CACCGCCA

1125 CAGGGACUA

CGCCGACA

1135 GCCGACAUC CUCGAGuu 1138 GACAUCCUC

GAGUUCCU

1143 CCUCGAGUU

CCUCGUCG

1144 CUCGAGUUC

CUCGUCGA

1147 GAGUUCCUC

GUCGACAG

1150 UUCCUCGUC

GACAGGUG

1181 UGACUGGUC

UGUCGGGU

1185 UGGUCUGUC

GGGUGMAG

1212 GCAGGACUA

CCUUUGCA

1216 GACUACCUU

UGCACCCU

1217' ACUACCUUU

GCACCCUU

1225 UGCACCCUU

GCUUCMAG

1229 CCCUUGCUU

CMAGMUC

1230 CCUUGCUUC

MAGAAUCA

1237 UCMAGAAUC

AGGAGGCU

1494 UUUGAUGUA

CAACCUGU

1546 CAUGCCGUA'CUUUGUCU 1549 GCCGUACUU

UGUCUGUC

1550 CCGUACUUU

GUCUGUCG

1553 UACUUUGUC

UGUCGCUG

1557 UUGUCUGUC

GCUGGCGG

1571 CGGUGUGUU

UCGGUAUG

1572 GGUGUGUUU

CGGUAUGU

1573 GUGUGUUUC

(SGUAUGUU

1577 GUUUCGGUA

UGUUAUUU

1581 CGGUAUGUU

AUUUGAGU

1582 GGUAUGUUA

UUUGAGUU

1584 UAUGUUAUU

UGAGUUGC

1585 AUGUUAUUU GAGUUGCu 1590 AUUUGAGUU

GCUCAGAU

1594 GAGUUGCUC

AGAUCUGU

1599 GCUCAGAUC

UGUUAAAA

1603 AGAUCUGUU

AAAA

1604 GAUCUGUUA AAA.AAA Seq. ID No.

751 753 755 757 759 761 763 765 767 769 771 773 775 777 779 781 783 785 787 789 791 793 795 797 799 801 803 805 807 809 811 813 815 817 819 821 823 825 827 829 831 833 835 837 839 841 843 845 847 849 851 853 855 857 859 861 863 Table VI 87 nt. Substrate Seq. ID Position No.

1397 CCAGGUUUU GAUCAAAU 864 1401 GUUUUGAUC AAAUGGUC 865 1409 CMAUGGUC CCGUGUCG 866 1416 UCCCGUGUC GUCUUAUA 867 1419 CGUGUCGUC UUAUAGAG 868 1421 UGUCGUCUU AUAGAGCG 869 1422 GUCGUCUUA UAGAGCGA 870 1424 CGUCUUAUA GAGCGAUA 871 1432 AGAGCGAUA GGAGAACG 872 1444 GAACGUGUU GGUCUGUG 873 1448 GUGUUGGUCUGUGGUGU 874 1457 UGUGGUGUA GCUUUGUU 875 1461 GUGUAGCUU UGUUUUUA 876 1462 UGUAGCUUU GUUUUUAU 877 1465 AGCUUUGUU UUUAUUUU 878 1466 GCUUUGUUU UUAUUUUG 879 1467 CUUUGUUUU UAUUUUGU 880 1468 UUUGUUUUu AUUiJUGUA 881 1469 UUGUUUUUA UUUUGUAU 882 1471 GUUUUUAUU UUGUAUUU 883 1472 UUUUUAUUU UGUAUUUU 884 1473 UUUUAUUUU GUAUUUUU 885 1476 UAUUUUGUA UUUUUCUG 886 1478 UUUUGUAUU UUUCUGCU 887 1479 UUUGUAUUU UUCUGCUU 888 *1480 UUGUAUUUU UCUGCUUU 889 **1481 UGUAUUUUU CUGCUUUG 890 1482 GUAUUUUUC UGCUUUGA 891 ***:1487 UUUCUGCUU UGAUGUAC 892 1488 UUCUGCUUU GAUGUACA 893 Table VII 88 Table VII: Delta-9 Desaturase HH Ribozyme Sequences n.Ribozyme sequence Seq. ID No.

Position 13 AAGCGGCA CUGAUGA XGAA AGGGCGCG 894 21 GMACGMAC CUGAUGA X GAA AGCGGCAG 895 24 GAGGAACG CUGAUGA X GAA ACAAGCGG 896 CGAGGMAC CUGAUGA X GAA AACMAGCG 897 28 GCGCGAGG CUGAUGA X GAA ACGMACM 898 29 AGCGCGAG CUGAUGA X GAA MACGAACA 899 32 GCGAGCGC CUGAUGA X GAA AGGMACGA 900 38 CUGGUGGC CUGAUGA X GMAGCGCGAG 901 63 GAGAUUGG CUGAUGA X GMA AUGUGUGU 902 69 CCUCGCGA CUGAUGA X GM4 71 GCCCUCGC CUGAUGA X GMA 92 CCGCCGCA CUGAUGA X GMA ACCCUGCU 905 117 GGAGCCGG CUGAUGA X GMA AGCGCGGC 906 118 GGGAGCCG CUGAUGA X GMM GCGCGG 907 124 GGGMAGGG CUGAUGA X GMA AGCCGGMA 908 129 CCMAUGGG CUGAUGA X GMA AGGGGAGC 909 130 GCCMAUGG CUGAUGA X GMA MGGGGAG 910 135 UGGAGGCC CUGAUGA X GMA AUGGGAAG 911 141 CCAUCGUG CUGAUGA X GMA AGGCCAAU 912 154 UUGAGGCG CUGAUGA X GAAGCGCCAU 913 160 ACGUCGUU CUGAUGA X GMA AGGCGGAG 914 169 CAGAGCGC CUGAUGA X. GMA ACGUCGUU 915 175 GAGAGGCA CUGAUGA X GMA AGCGCGAC 916 181 GGCGGGGA CUGAUGA X GMA AGGCAGAG 917 183 GCGGCGGG CUGAUGA X GMA AGAGGCAG 918 193 CGGGCGGC CUGAUGA X GMA AGCGGCGG 919 *228 CGGCGACG CUGAUGA X GMA ACCUGCCG 920 229 ACGGCGAC CUGAUGA X GMA MCCUGCC 921 :232 GCGACGGC CUGAUGA X GMA ACGMACCU 922 238 AUGGAGGC CUGAUGA X GMA ACGGCGAC 923 243 ACGUCAUG CUGAUGA X GMA AGGCGACG 924 *252 AGACGGCG CUGAUGA X GMA ACGUCAUG 925 259 UUGGUGGA CUGAUGA X GMA ACGGCGGA 926 261 CCUUGGUG CUGAUGA X GMA AGACGGCG 927 *271 UUAUUCUC CUGAUGA X GMA ACCUUGGU 928 278 UGGCUUCU CUGAUGA X GMA AUUCUCGA 929 288 GAGGAGCA CUGAUGA X GMA AUGGCUUC 930 289 GGAGGAGC CUGAUGA X GMA MUGGCUU 931 293 CCUUGGAG CUGAUGA X GMA AGCAAAUG 932 *296 CUCCCUUG CUGAUGA X GMA AGGAGCAA 933 307 UGGACAUG CUGAUGA X GMA ACCUCCCU 934 313 GUMACCUG CUGAUGA X GMA ACAUGUAC 935 319 GMAUGUGU CUGAUGA X GMA ACCUGGAC 936 320 UGMAUGUG CUGAUGA X GMA MCCUGGA 937 32 U G C GA G A A G G A 3 :::326 GUGGCAUU CUGAUGA X GMA AUGUGUA 938 338 MUCUUGU CUGAUGA X GMA AGGUGGCA 940 346 AAAAUUUC CUGAUGA X GMA AUCUUGUG 941 352 GACUUGMA CUGAUGA X GMA AUUUCMAU 942 353 CGACUUGA CUGAUGA X GMA MUUUCMA 943 354 GCGACUUG CUGAUGA X GAAUUUCA 944 355 AGCGACUU CUGAUGA X GA AUUUC 945 360 CAUCMAGC CUGAUGA X GMA ACUUGAAA 946 364 CAAUCAUC CUGAUGA X GAAGCGACUU 947 Table VI I 89 nt. Ribozvme sequenceSe.INo PositionSe.INo 371 UCUAGCCC CUGAUGA X GMAUCAUCAA 948 377 AUUAUCUC CUGAUGA X GAAGCCCMAU 949 383 CAAGAUAU CUGAUGA X GMA AUCUCUAG 950 386 CGUCAAGA CUGAUGA X GMA AUUAuCUC 951 388 UGCGUCAA CUGAUGA X GMA AUAUUAUC 952 390 GAUGCGUC CUGAUGA X GMA AGAUAUUA 953 398 UGGCUUGA CUGAUGA X GMA AUGCGUCA 954 4003 ACUGGCUU CUGAUGA X GMAGAUGCGU 955 409 CACUUCUC CUGAUGA X GMA ACUGGCUU 956 419 UGGCUGCC CUGAUGA X GMA ACACUUCU 957 434 CGjGGAGGA CUGAUGA X GMA AUCCUGUG 958 4315 CCGGGAGG CUGAUGA X GMA MUCCUGU 959 436 UCCGGGAG CUGAUGA X GMWAUCCUG 960 439 GGGUCCGG CUGAUGA X GMA AGGMAAUC 961 453 AUCCUUCA CUGAUGA X GMA AUGCUGGG 962 462 CAUCAUGA CUGAUGA X GMA AUCCUUCA 963 463 UCAUCAUG CUGAUGA X GMA MUCCUUC 964 464 UUCAUCAU CUGAUGA X GMA AAAUCCUU 965 475 AGCUCCUU CUGAUGA X GMA ACUUCAUC 966 476 GAGCUCCU CUGAUGA X GMA MCUUCAU 967 484 CGUUCUCU CUGAUGA X GMA AGCUCCUU 968 505 UCAUCAGG CUGAUGA x GMA AUUUCCUU 969 515 AACAAAAU CUGAUGA X GMA AUCAUCAG 970 516 AAACAAAA CUGAUGA X GMA MUCAUCA 971 518 ACAAACAA CUGAUGA X GMA AUMAUCAU 972 519 AACAAACA CUGAUGA X GMA MUAAUCA 973 520 AMCAAAC CUGAUGA X GMA AAAuAAuc 974 523 ACCAAACA CUGAUGA X GMA AcAAAAUA 975 524 CACCAAAC CUGAUGA X GMA MACMAAU 976 *527 UCCCACCA CUGAUGA X GMA AcAAACMA 977 528 CUCCCACC CUGAUGA X GMA MCAAACA 978* 544 UCCUCGGU CUGAUGA X GMA AUCAUGUC 979 545 UUCCUCGG CUGAUGA X GMA AAUCAUGU 980 557 UGUUGGUA CUGAUGA X GMA AGCUUCCu 981 :559 UAUGUUGG CUGAUGA X GMA AGAGCUUC 982 567 UAGUfCUGG CUGAUGA X GMA AUGUUGGU 983 575 GUUAAGCA CUGAUGA X GMA AGUCUGGU 984 580 AGGGUGUU CUGAUGA X GMA AGCAUAGU 985 581 GAGGGUGU CUGAUGA X GMA MGCAUAG 986 *589 ACACCGUC CUGAUGA X GMA AGGGUGUU 987 598 UCAUCUCU CUGAUGA X GMA ACACCGUC 988 637 CUCGUCCA CUGAUGA X GMA ACAGCCCA 989 638 CCUCGUCC CUGAUGA X GMA MCAGCCC 990 680 GUUGAGCA CUGAUGA X GMA AUCACCAU 991 685 UACUUGUU CUGAUGA X GMA AGCAGAUC 992 693 GGUACAUA CUGAUGA X GMA ACUUGUUG 993 695 GAGGUACA CUGAUGA X GMA AUACUUGU 994 :::699 CAGUGAGG CUGAUGA X GMA ACAUAUAC 995 **:703 CCCG UA GM A AGGUACAU 998 730 GUCUUCUC CUGAUGA X GMA AUCUGCCU 998 742 AGAUACUG CUGAUGA X GMA AUUGUCUu 999 743 MAGAUACU CUGAUGA X GMA MUUGUCU 1000 747 CMAUMGA CUGAUGA X GMA ACUGAAUU 1001 749 GCCMAUM CUGAUGA X GMA AUACUGMA 1002 751 GAGCCMAU CUGAUGA X GMA AGAUACUG 1003 752 AGAGCCAA CUGAUGA X GMA MGAUACU 1004 Table VII nt Rioym eqec Position Rbzm eeneSeq. ID No.

754 CCAGAGCC CUGAUGA X GMA AUAAGAUA 759 CCAUUCCA CUGAUGA X GMA AGCCAAUA 1006 770AGUCCUAG CUGAUGA X GMA AUCCAUUC 1007' 773 CUCAGUCC CUGAUGA X GMA AGGAUCCA 1008 785 AUSAAGGAU CUGAUGA X GMA AUUCUCAG 1009 788 MAGAUMAG CUGAUGA X GMA AUUAUUCU 1010 791 ACCAAGAU CUGAUGA X GMA AGGAUUAU 1011 792 AACCAAGA CUGAUGA X GMA AAGGAUUA 1012 794 GAAACCAA CUGAUGA X GMA AUMAGGAU 1013 796 AUGAAACC CUGAUGA X GMA AGAUMAGG 1014 800 GUAGAUGA CUGAUGA X GMA ACCAAGAU 1015 801 UGUAGAUG CUGAUGA X GMA MCCAAGA 1016 802 GUGUAGAU CUGAUGA X GMA AMACCMG 1017 805 GAGGUGUA CUGAUGA X GMA AUGAAACC 11 807 AGGAGGUG CUGAUGA X GMA AGAL'GAAA 1018 813 CUUGGAAG CUGAUGA X GMA AGGUGUAG 1020 816 GCUCUUGG CUGAUGA X GMA AGGAGGUG 1021 817 CGCUCUUG CUGAUGA X GMA MGGAGGU 1022 834 GUGAGAUG CUGAUGA X GMA AGGUCGCC 1023 835 UGUGAGAU CUGAUGA X GMA MGGUCGC 1024 838 CCGUGUGA CUGAUGA X GMA AUGMAGGU 1025 840 UCCCGUGU CUGAUGA x GMA AGAUGMAG 1026 857 GGCGUGAC CUGAUGA X GMA AGCAGUGU 1027 860 CUUGGCGU CUGAUGA X GMA ACGAGCAG 1028 873 AGUCGCCA CUGAUGA X GMA AGUCCUUG 1029 874 AAGUCGCC CUGAUGA x GMA MGUCCUU 1030 882 CAAGCUUU CUGAUGA X GMA AGUCGCCA 1031 883 GCAAGCUU CUGAUGA X GMA MGUCGCC 1032 889 AUUUGUGC CUGAUGA X GMA AGCUUUMA 1033 *898 AUGCCGCA CUGAUGA x GMA AUUUGUGC 1034 *907 GAGGCGAU CUGAUGA X GMA AUGCCGCA 1035 :910 UCUGAGGC CUGAUGA X GMA AUGAUGCC 13 915 UCUCAUCU CUGAUGA X GMA AGGCGAUG 1037 942 UCUUGGUG CUGAUGA x GMA ACGCAGUU 1038 952 UUCUCCAC CUGAUGA X GMA AUCUUGGU 1039 966 CGAUCUCA CUGAUGA X GMA ACAGCUUC 10-40 967 UCGAUCUC CtJGAUGA X GMA MCAGCUU 1041 973 UCAGGGUC CUGAUGA X GMA AUCUCAAA 1042 988 GACCACGG CUGAUGA x GMA ACCAUCAG 1043 *994 GCCAGAGC CUGAUGA X GMA ACCACGGU 1044 998 GUCAGCCA CUGAUGA X GMA AGCGACCA 1045 1024 GGCAUUGA CUGAUGA X GMA AUCUUCUU 1046 1026 CAGGCAUU CUGAUGA X GMA AGAUCUUC 1047 1047 GCCCGUCA CUGAUGA x GMA ACAUCAGG 1048 1048 UGCCCGUC CUGAUGA X GMA MCAUCAG 1049 1071 AGUGCUCG CUGAUGA X GMAACAGCUUG 1050 1072 MAGUGCUC CUGAUGA X GMA MCAGCUU 1051 108 *.UGGCGUAX A 1080 CCAUGGA CUGAUGA X GMA AAGUGCUC 1052 *.:1083 CGACCAUG CUGAUGA X GMA AGMAGUGC 1054 *1090 CUCUGCGC CUGAUGA X GMA ACCAUGGA 1055 1102 UAAACGCC CUGAUGA X GMA AGCCUCUG 1056 1108 GCGGUGUA CUGAUGA X GMA ACGCCMAG 1057 1109 GGCGGUGU CUGAUGA X GMA ACGCCAA 1058 1110 UGGCGGUG CUGAUGA X GMM AACGCCA 1059 1125 UGUCGGCG CUGAUGA X GMA AGUCCCUG 1060 1135 AACUCGAG CUGAUGA X GMA AUGUCGGC 1061 Table VII 91 n t. Ribozvme sequence Seq. ID No.

Position 1138 AGGMACUC CUGAUGA X GAA AGGAUGUC 16 1143 CGACGAGG CUGAUGA X GAA ACUCGAGG 1063 1144 UCGACGAG CUGAUGA X GAA CUCGAG 1064 1147 CUGUCGAC CUGAUGA X GAA AGGAACUC 1065 1150 CACCUGUC CUGAUGA X GAA ACGAGGMA 1066 1181 ACCCGACA CUGAUGA X GAA ACCAGUCA 1067 1185 CUUCACCC CUGAUGA X GAA ACAGACCA 1068 1212 UGCAAAGG CUGAUGA X GAA AGUCCUGC 1069 1216 AGGGUGCA CUGAUGA X GAA AGGUAGLIC 1070 1217 GGGUGC CUGAUGA X GAA AAGGUAGU 1071 1225 CUUGAAGC CUGAUGA X GAA AGGGUGCA 17 1229 GAUUCUUG CUGAUGA X GAA AGCMAGGG 1073 1230 UGAUUCUU CUGAUGA X GMA AGCAAGG 1074 1237 AGCCUCCU CUGAUGA X GMA AUUCUUGA 1075 1292 CCAGCUGA CUGAUGA X GMA AGGCAGCG 1076 1293 CCCAGCUG CUGAUGA X GMA AAGGCAGC 1077 124ACCCAGCU CUGAUGA X GMA AAAGGCAG 1078 1303 CUACCGUA CUGAUGA X GMA ACCCAGCU 17 1305 CCCUACCG CUGAUGA X GMA AUACCCAG 1080 1310 GACGUCCC CUGAUGA X GMA ACCGUAUA 1081 1318 CACAGUUG CUGAUGA x GAA ACGUCCCU 1082 1331 AGGUUUCC CUGAUGA X GMA AUCUCACA 1083 1348 UCUMAGCA CUGAUGA X GMA ACCGCAGC 1084 1353 UCUUGUCU CUGAUGA X GMA AGCAGACC 1085 1354 GUCUUGUC CUGAUGA X GMA AGCAGAC 1088 132GUMACGCA CUGAUGA x GMA ACACAGCA 1087 1378 ACCUAUGU CUGAUGA X GMA ACGCAGAC 18 179GACCUAUG CUGAUGA X GMA ACGCAGA 1089 11383 UGGAGACC CUGAUGA X GMA AUGUMACG 1090 *1387 MCCUGGA CUGAUGA X GMA ACCUAUGU 1091 138 AAAACCG UGUA GAAGCCA UU1389 A CUGAUGA X GMA ACCUAG 1092 1395 UUUGAUCM CUGAUGA X GMA ACCUGGA 1093 *1397 AUUUGAUC CUGAUGA X GMA AACCUGG 1095 1401 GACCAUUU CUGAUGA X GMA AUCAAAAC 10196 1409 CGACACGG CUGAUGA X GMA ACCAUUUG 1097 1416 UAUMAGAC CUGAUGA X GMA ACACGGGA 1098 *1419 CUCUAUMA CUGAUGA X GMA ACGACACG 1099 1421 CGCUCUAU CUGAUGA X GMA AGACGACA 1100 *1422 UCGCUCUA CUGAUGA X GMA MGACGAC 1101 1424 UAUCGCUC CUGAUGA X GMA AUMAGACG 1102 1432 CGUUCUCC CUGAUGA X GMAAUCGCUCU 1103 *1444 CACAGACC CUGAUGA X GMA ACACGUUC 1104 1448 ACACCACA CUGAUGA X GMA ACCMACAC 1105 1457 MACAAAGC CUGAUGA X GMA ACACCACA 1106 1461 UMAAAACA CUGAUGA X GAAGCUACAC 1107 1462 AUMAAAAC CUGAUGA X GMA AGCUACA 1108 1465 AAAAUAAA CUGAUGA X GMA ACMAAGCU 1109 46CAAAAUMA CUGAUGA X GMA MCAMAGC 11110 *1467 ACWAAUA CUGAUGA X GMA AAACMAAG 1111 1468 UACAAAAU CUGAUGA X GMA MAACAAA 1112 1469 AUACMAAA CUGAUGA X GMA AAAACMA 1113 1471 AAAUACMA CUGAUGA X GMA AUAAAAAC 1114 1472 AAAAUACA CUGAUGA X GMA AUAAAAA 1115 1473 AAAAAUAC CUGAUGA X GMA AAAUMAAA 1116 1476 CAGMAAAA CUGAUGA X GMA ACAAAAUA 1117 1478 AGCAGAMA CUGAUGA X GMA AUACAAAA 1118 Table VII 92 nt. ioy esqec Position ibvmseeneSeq. ID No.

1479 AAGCAGAA CUGAUGA X GAA AAUACAAA 1119 1480 AAAGCAGA CUGAUGA X GAA MAUACAA 1120 1481 CAAAGCAG CUGAUGA X GAA AAAAUACA 1121 1482 UCAAAGCA CUGAUGA X GMA AAAAAUAC 1122 1487 GUACAUCA CUGAUGA X GMA AGCAGWA 1123 1488 UGUACAUC CUGAUGA X GMA MGCAGMA 1124 1494 ACAGGUUG CUGAUGA X GMA ACAUCAAA 12 156AGACAAAG CUGAUGA X GMA ACGGCAUG 1125 1549 GACAGACA CUGAUGA X GMA AGUACGGC 1127 1550 CGACAGAC CUGAUGA X GMA MGUACGG 1128 1553 CAGCGACA CUGAUGA X GMA ACAAAGUA 1129 -1557 CCGCCAGC CUGAUGA X GMA ACAGACAA 1130 1571 CAUACCGA CUGAUGA X GMA ACACACCG 1131 1572 ACAUACCG CUGAUGA X GMA MCACACC 1132 1573 MACAUACC CUGAUGA X GMA AAACACAC 1133 1577 AAAUMACA CUGAUGA X GMA ACCGAAAC 1134 1581 ACUCAAAU CUGAUGA X GMA ACAUACCG 1135 1582 MACUCAAA CUGAUGA X GMA MCAUACC 1136 1584 GCMACUCA CUGAUGA X GMA AUMACAUA 1137 1585 AGCAACUC CUGAUGA X GMA MAUMCAU 1138 1590 AUCUGAGC CUGAUGA X GMA ACUCAAAU 1139 1594 ACAGAUCU CUGAUGA X GMA AGCMACUC 1140 1599 UUUUMACA CUGAUGA X GMA AUCUGAGC 1141 1603 UUUUUUUU CUGAUGA X GMA ACAGAUCU 14 1604 UUUUUUUU CUGAUGA X GMA MCAGAUC 1142 Where represents stem I I region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 3252). The length of stem II may be 2t 2 base-pairs.

e Ilible Vill Table VIII: Det. 9 Desatuirase H~airpin, Rlbozyme and Substrate Sequences ft.

Position 14 17 108 120 155 176 186 189 196 200 203 206 209 235 253 256 406 442 508 570 625 634 655 661 726 853 916 Ribozyme GAACAAGC AGAA GAGGGC

ACCAGAGAAALCACACGUUGUGGUACAUUACCUGGUA

MCGAACA AGAA GCAGAG

ACCAGAGWACACACGUUGUGUACAUUACCUGGUA

GGAAGCGC AGMA GCCGCC

ACCAGAGMAACACACGUUGUGGUACAUUACCUGGUA

GGAAGGGG AGAA GGMAGC

ACCAGAGMAACACACGUUGUGGUACAUUACCUGGUA

GUCGUUGA AGMA GAGCGC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CGGGGAGA AGAA GAGCGC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CGGCGAGC AGAA GGGAGA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

GGGCGGCG AGMA GCGGGG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CGGCGGCG AGAA GCGAGC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

GCGGCGGC AGMA GGCGGC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

GCGGCGGC AGMA GCGGGC

ACCAGAGWACACACGUUGUGGUACAUUACCUGGUA

GCUGCGGC AGMA GCGGCG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

GCUGCUGC AGMA GCGGCG

ACCAGAGAAACACACGUUGUGGUACAIJUACCUGGUA

AUGGAGGC AGMA GCGACG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

GUGGAGAC AGMA GACGUC

ACCAGAGAAACACACGUUGUGGUACAUI.ACCUGGUA

UUGGUGGA AGAA GCGGAC

ACCAGAGMAACACACGUUGUGGUACAUUACCUGGUA

CACULJCUC AGAA GGCUUG

ACCAGAGMAACACACGUUGUGGUACAUUACCUGGIJA

GAUGCUGG AGAA GGGAGG ACCAGAGAAACACACGUjUGUGGUACAUUACCUGGUA AAAUAAUC AGAA GGGAUU

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

UMAGCAUA AGAA GGUAUG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACAGOCCA AGMA GUGGGG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

CUCGUCCA AGAA GCCCAG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGU

UUCUCCUC AGMA GUCCAU

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACUUGUUG AGMA GAUCAC

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

UCUUCLICA AGAA GCCUCA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGU

GCGUGACG AGAA GUGUUC

ACCAGAGWACACACGUUGUGGUACAUUACCUGGU

CGCUUCUC AGMA GAGGCG

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

Seq. ID No.

1144 1146 1148 1150 1152 1154 1156 1158 1160 1162 1164 1166 1168 1170 1172 1174 1176 1178 1180 1182 1184 1186 118 1190 1192 1194 1196 Substrate GCCCUCU GCC GCUUGUUC CUCUGCC GCU UGUUCGUU GGCGGCG GOC GCGCUUCC GCUUCCG GCU CCCCUUCC GCGCUCC GCC UCAACGAC GCGCUCU GCC UCUCCCCG UCUCCCC GCC GCUCGCCG CCCCGCC GCU CGCCGCC GCUCGCC GCC CGCCGCCG GCCGCCC GCC GCCGCCGC GCCCGCC GCC GCCGCCGC CGCCGCC GCC GCCGCAGC CGCCGCC GCC GCAGCAGC CGUCGCC GUC GCCUCCAU GACGUCC GCC GUCUCCAC GUCCGCC GUC UCCACCAA CAAGCCA GUC GAGAAGUG CCUCCCG GAC CCAGCAUC MUCCCU GAU GAUUAUUU CAUACCA GAC UAUGCUUA CCCCACU GCC UGGGCUGU CUGGGCU GUU UGGACGAG AUGGACU GCU GAGGAGMA GUGAUCU GCU CAACAAGU UGAGGCA GAU UGAGAAGA GAACACU GCU CGUCACGC CGCCUCA GAU GAGMAGCG Seq. 11) No.

1145 1147 1149 1151 1153 1155 1157 1159 1161 1163 1165 1167 1169 1171 1173 1175 1177 1179 1181 1183 1185 1187 1189 1191 1193 1195 1197

S..

5 0*

S

St

S

S S S S to S9 S 5 *5 S S@ S S S S. S S S S S 5555 S S 555 5*S *S S 0 I able VIII nt.

Position Ribozyme Seq. ID No.

Substrate Seq. I D NoU.

963 979 1033 1041 1068 1173 1182 1287 1295 1339 1345 1349 1364 1483 1554 1595 CGAUCUCA AGMA GCUUCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGGUACC AGAA GGGUCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUCAGGUG AGMA GGCAUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGUCAAAC AGAA GGUGGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGUGCUCG AGMA GCUUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACAGACCA AGAA GGCUCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUUCACCC AGAA GACCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGCUGAAA AGAA GCGUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUAUACCC AGMA GAAAGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGACCGC AGAA GGUUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCUAAGCA AGAA GCAGCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUUGUCUA AGAA GACCOC ACCAGAGAMACACACGUUGUGGUACAUUACCUGGUA GCAGACAC AGMA GGUCUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UACAUCMA AGAA GAAAAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCGCCAGC AGMA GACMAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUUMACAG AGAA GAGCMA ACCAGAGAAACACACGULJGUGGUACAUUACCUGGUA 1198 1200 1202 1204 1206 1208 1210 1212 1214 1216 1218 1220 1222 1224 1226 1228 AGMAGCU GUU UGAGAUCG CGACCCU GAU GGUACCGU AAUGCCU GCC CACCUGAU CCCACCU GAU GUUUGACG ACMAGCU GUU CGAGCACU CGAGCCU GAC UGGUCUGU CUGGUCU GUC GGGUGAAG GCACGCU GCC UUUCAGCU CCUUUCA GCU GGGUAUAC GAAACCU GCU GCGGUCUG UGCUGCG GUC UGCUUAGA GCGGUCU GCU UAGACAAG AAGACCU GCU GUGUCUGC UUUUUCU GCU UUGAUGUA UUUGUCU GUC GCUGGCGG UUGCUCA GAU CUGUUAAA 1199 1201 1203 1205 1207 1209 1211 1213 1215 1217 1219 1221 1223 1225 1227 1229 Table IX Table MX Cleavage of D elta-9 Desaturase RNA by HH Ribozynies Percent Cleaved 0

C

0#*S 0* 6e a if if.

if if if...

nt Position 183 252 259 271 278 307 313 320 326 338 353 390 419 453 484 545 773 1024 1026 1237 10min 6.3 25.2 20.3 17.2 9.9 10.3 16.9 10.6 5.7 10.0 10.2, 8.6 6.3 7.3 7.8 4.8 4.5 11.9 11.6 23.1 120 m 7.0 51.2 41.3 52.4 25.7 24.2 43.0 23.6 14.6 17.5 11.3 8.9 10.1 29.0 28.9 8.5 11.5 17.1 12 .6 32.4 10 min 10.45 33.1 24.8 21.5 13.3 9.2 23.8 15.0 8.0 10.4 10.7 7.8 5.8 8.0 6.9 3.6 4.4 13.3 13.1 13.8 26 0

C

120 m 11.8 52.9 44.0 56.3 33.6 32.4 53.4 31.3 17.1 12.9 14.7 9.8 10.9 33.8 29.2 8.9 8.9 23.8 17.2 28.6 if.

if S a. 4if ifO TABLE X: Table XI Stearic acid levels in leaves from plants transformed with active and inactive ribozYmes compared to control leaves.

Stearic Acid in Leaves Transformed with Active and Inactive Ribozvnies (Percentage of total plants with certain levels of leaf stearic acid)' Stearic Acid Ribozyme Actives Ribozynie Controls Inactives (428 plants (406 plants (122 plants) from 35 lines3) frunw 31 lines) 3% 7% 3% 2% 5% 2% .00 0 0 q 0

S.

a a a a.

0 Table XII Inheritance of the high steanic acid trait in leaves from crosses of high stearic acid plants.

Cross Inheritance of high stearate in leaves.

RI Plants with RI Plants with of Plants with I Nnrrnal I I..L- ILeaf Stearate Leaf Stearate, 3 RPA85-1 5.06 x IRPA85-1 5.12 IRPA85-1 5.07 self RPA85-15. 10 self 0Q414 x RPA85-1 5.06 0Q414 x RPA85-15.i1 T

I-

5 5 8 2 5 3 6 1 4 M1gt Stearate 33% 38% 99 Table XIII Comparison of fatty acid composition of embryogenic callus, somatic embryos and zygotic embryos.

Tissue and/or Media Fatty Acid Composition Lipid Treatment o rs C18:0 C18:1 C18:2 C18:3 Weight enryogenic callus 19.4 1.1 6.2 55.7 8.8 0.4 0.9 0.1 2.0 3.1 2.0 0.1 somatic embryo grown 12.6 1.6 18.2 60.7 1.9 4 .0 on MS 6*1 sucrose 10 +1 +1 4/ mM ABA 07.. 4.9 5.1 0.3 1 zygotic embryo 14.5 1.1 18.5 60.2 14 3.9 12 days after4/ 4/ 1 4pollination 0.4 0.1 1.0 1.5 0.2 0.6 Table XIV: GBSS activity, ainylose content, and Southern analysis results of selected Ribozyme Line RPA63 .0283 RPA63 .0236 RPA63 .0219 RPA63 .0314 RPA63 .0316 RPA63 .0311 RPA63 .0309 RPA63 .0218 RPA63 .0209 RPA63 .0306 RPA63 .0210 GBSS activity (Units/mg starch) 321.5 31.2 314.6 9,2 299.8 10.4 440.4 +17.1 346. 5 8 8.5 301.5 +17..4 264.7 19 190. 8 8 203 +2.4 368.2 7.5 195.1 7 Amylose Content 23.3 27.4 0.3 21.5 0.3 19.1 0.8 17.9 19.5 0.4 21.7 0.1 21.0 0.3 22.6 +I 0.6 19.0 0.4 22.1 0.2 Southern a.

Claims (89)

1. An enzymatic nucacid moleculeic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression ofa plant g w ne.

2. The enzymatic nucleic acid molecule of claim I, whercin ad pl;n I a monocotyledon. The enzymatic nucleic acid molecule of claim 1, wherin said plant is a dicotyledon.

4. The enzymatic nucleic acid molecule of claim wherein said plant is a gymnosperm.

5. The enzymatic nucleic acid molecule of claim wherein said plant is an angiosperm.

6. The enzymatic nucleic acid molecule of claim I, wherein said nucleic acid is in a hammerhead configuration.

7. The enzymatic nucleic acid molecule of claim I, wherein said nucleic acid is in a hairpin configuration.

8. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid is in a hepatitis A 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 20 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.

11. The enzymatic nucleic acid of claim 6, wherein said hammerhead comprises a stem II region of length greater than or equal to two base-pairs. 12 The enzymatic nucleic acid of claim 7, wherein said hairpin comprises a stem II region of length between three and seven base-pairs. a S.

13. The enzymatic nucleic acid of claim 7. wherein said hairpin comprises a stei 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 dicotylcdon plnt is selected from a group consisting of canola, sunflower, safflower, 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. e s iv d f

17. The enzymatic nucleic acid of claim 16, wherein said gene is A-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, safflower, soybean and rice.

19. The enzymatic nucleic acid of claim 1, wherein said gene is involved in starch biosynthesis in said plant. The enzymatic nucleic acid of claim 19, wherein said gene is granule bound starch -synthase. ene s granul d starch

21. The enzymatic nucleic acid of any of claims 19 or 20, wherein said plant is Sselected from a group consisting of maize, potato, wheat, and cassava. 20

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. S:. 2

24. The enzymatic nucleic acid of any of claims 22 or 23 wherin said plan is a coffee plant.

25. The enzymatic nucleic acid of claim 1, wherein said gene is involved in nicotine production in said plant. 103

26. The enzymatic nucleic acid of claim 25, wherein said gne s elected fro a group consisting of N-methylputrescine oxidase and pu sel ec ted fro ethyl transferase. n e ethyl

27. The enzymatic nucleic acid of any of claims 25 or 26, whrc said lant s tobacco plant. 2 w h rc n i s

28. The enzymatic nucleic acid of claim 1, wherein said gene is in ripening process in said plant. nv ol v e d n fruit

29. The enzymatic nucleic acid of claim 28, wherein said gene is selectcd from a group consisting of ethylene-forming enzyme, pectin ncthyltransferasc, pectin esterase, polygalacturonase, l-aminocyclopropane carboxylic acid (ACC) synthase, and ACC oxidase. 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, phenylalanine ammonia lyase, dehydroflavonol hydroxylases, and dehydroflavonol reductase. 20

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 marigold.

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, cinnamoyl-CoA:NADPH 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. 104

37. A nucleic acid fragment comprising a cDNA sequeize desaturase, wherein said sequence is represented by t nce co D. No. 1.

38. The enzymatic nucleic acid molecule of claim 17, wherein said nucleic acid specifically cleaves any of sequences defined in Table V, 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 hairpin configuration. The enzymatic nucleic acid molecule of any of claims 38 or 39, consistng 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 liIA, 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 hairpin configuration.

43. The enzymatic nucleic acid molecule of any of claims 41 or 42, consisting Sessentially of one or more sequences selected from the group son in s 20 IIIB, IV, VA and VB. r oup show n i Tables

44. The enzymatic nucleic acid molecule of claim 41, consisting essentially of sequences defined as any of SEQ. I.D. NOS. 2-24. 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 orany of claims 144.

46. A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid molecule of any of claims 1-8, 1-17, 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, 105 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 -17, 19-20, 22-23, 25-26, 28-29, 31-32, 34-35, 37-39, 41-42 or 44, in a manner which allows cxprcssion and/or delivery of said enzymatic nucleic acid molccules within a plant cell.

49. A plant cell comprising the expression vector of claim 47. A plant cell comprising the expression vector of claim 48.

51. A transgenic plant and the progeny thereof, comprising 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 comprising the enzymatic nucleic acid of any of claims 16 or 17.

54. The plant cell of claim 53, wherein 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, comprising 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 comprising the enzymatic nucleic acid of any of claims 19 or

60. The plant cell of claim 59, wherein said cell is a maize cell. S

61. A transgenic plant and the progeny thereof, comprising the enzymatic nucleic 25 acid of any of claims 19 or .o S. S. 106

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 of any of claims -8.

64. The method of claim 63, wherein said plant is a nmonocnt plant. 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 A-9 desaturase.

69. The method of claim 68, wherein said plant is a maize plant. 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 0 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; 107 c) an open reading frame; S 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 rgion, said opn rcadin frame and said termination region, in a manncr which allows Cxprcssi n and/or delivery of said enzymatic molecule within said plant cell. d/ 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 tosaid 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. y o f sa i S 15

76. The expression vector of claim 47, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; Sd) an-open reading frame; 20 e) a gene encoding at least one said e) a gene encodg at least onesaid 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. 108

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 acid 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 15 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, 20 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), 25 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, hairpin, hepatitis A virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration 109

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 multimcr.

89. The transgenic plant of Claim 78, wherein the nucleic acids encoding lor said enzymatic nucleic acid molecule with RNA cleavin activity is oprably linked to the 3' end of an open reading frame. is ope ly l 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; c 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 whereisaid gene is a i, wherein said gene is an 94 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. 110

96. An enzymatic nucleic acid molecule with RNA cleaving activity, substantially as hereinbefore described with reference to any one of the examples.

97. A nucleic acid fragment comprising a cDNA sequence coding for maize A-9 desaturase, substantially as hereinbefore described with reference to any one of the examples.

98. A method for modulating expression of a gene in a plant, substantially as hereinbefore described with reference to any one of the examples.

99. A transgenic plant comprising nucleic acids encoding for an enzymatic nucleic acid molecule with RNA cleaving activity, substantially as hereinbefore described io with reference to any one of the examples.

100. A transgenic maize plant, substantially as hereinbefore described with reference to any one of the examples. o* IR \I.IRZ1529)3 doc ecf 111 The claims defining the invention are as follows: 1. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a plant gene selected from the group consisting of granule bound starch synthase, delta-9 desaturase, 7-methylguanosine, 3-methyl transferase, N- methylputrescine oxidase, putrescine N-methyl transferase, ethylene-forming enzyme, pectin methyltransferase, pectin esterase, polygalacturonase, 1-aminocyclopropane carboxylic acid (ACC) synthase. ACC oxidase, chalcone synthase, chalcone flavanone isomerase, phenylalanine ammonia lyase, dehydroflavonol hydroxylases, dehydroflavonol reductase, O-methyltransferase, cinnamoyl-CoA:NADPH reductase, and cinnamoyl alcohol dehydrogenase. 2. The enzymatic nucleic acid molecule of claim 1, wherein said plant is a monocotyledon. 3. The enzymatic nucleic acid molecule of claim 1, wherein said plant is a dicotyledon. 4. The enzymatic nucleic acid molecule of claim 1, wherein said plant is a gymnosperm. 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 hairpin configuration. 8. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid is in a m, hepatitis A virus, group 1 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. 11. The enzymatic nucleic acid of claim 6, wherein said hammerhead comprises a stem II o.. region of length greater than or equal to two base-pairs. 12 The enzymatic nucleic acid of claim 7, wherein said hairpin comprises a stem II region of length between three and seven base-pairs. 112 13. The enzymatic nucleic acid of claim 7. wherein said hairpin prises a stem IV region of length greater than or equal to two base-pairs. 14. The enzymatic nucleic acid of claim 2 wherein said m cotyledon plant is selected from a group consisting of maize, rice, wheat, antid harley. 15. The enzymatic nucleic acid of claim 3, wherein said dicotyledon plant is selected from a group consisting ofcanola, sunflower, safflower, soybean, cotton, peanut olive, sesame, cuphea, flax, jojoba, and grape. 16. The enzymatic nucleic acid of claim wherei said gene is nvolved in fatty acid biosynthesis in said plant. 17. The enzymatic nucleic acid of claim 16, herein said gene is desaturase 18. The enzymatic nucleic acid of any of claims 16 or 17, whercin said plant is selected from a group consisting of maize, canola, flax, sunflower, cotton, peanuts, safflower, soybean and rice. 19. The enzymatic nucleic acid of claim wherein said gene is involved in starch 15 biosynthesis in said plant. said is in l v d srch .i 20. The enzymatic nucleic acid of claim 19, wherein said geneis granule bound starch -synthase. s granule bound starch 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 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. S24. The enzymatic nucleic acid of any of claims 22 or 23, wherein said plant is a coffee plant. The enzymatic nucleic acid of claim 1, wherein said gene is involved in nicotine production in said plant. 26. The enzymatic nucleic acid of claim 25, wherein said gene is selected from a Sgroup consisting of N-methylputrescine dase pr e et transferase. y 27. The enzymatic nucleic acid of any of claims 25 or 26, wr said la tobacco plant. is a 28. The enzymatic nucleic acid of claim I, wherein said gene is involved in fruit ripening process in said plant. ene s nv ol ved 29. The enzymatic nucleic acid of claim 28, wherein said gene is sctd from a group consisting of ethylene-forming enzyme, pectin metihy trans frasc pectin esterase, polygalacturonase I-aminocyclopropane carboxylic acid (ACC) synthase, and ACC oxidase. 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. 15 pigmentation in said plant. sa d is 32. The enzymatic nucleic acid of claim 31, wherein said gene is selected from a group consisting of chalcone synthase, chalcone flavanone isomerase, phenylalanine ammonia lyase, dehydroflavonol hydroxylases, and dehydroflavonol reductase. 20 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 marigold. 34. The enzymatic nucleic acid of claim wherein said gene is involved in lignin production in said plant. The enzymatic nucleic acid of claim 34, wherein said gene is selected from a group consisting of O-methyltransferase, cinnamoyl-CoA:NADPH 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. 114 37. A nucleic acid fragment comprising a cDNA sequence coding for maize Sdesaturase, wherein said sequence is represented by tle or maize No 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 hairpin configuration. 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 VII. show i Tables 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. 15 42. The enzymatic nucleic acid molecule of claim 20, whrein said nucleic acid specifically cleaves any of sequences defined in Tables VA and VB, wherein said .nucleic acid is in a hairpin 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. e group shown in Tables 44. The enzymatic nucleic acid molecule of claim 41, consisting essentially of sequences defined as any ofSEQ. 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 or44. 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- 37-39, 41-42 or44. 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, 115 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, 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 said enzymatic nucleic acid molecules within a plant cell. 49. A plant cell comprising the expression vector of claim 47. A plant cell comprising the expression vector of claim 48. 51. A transgenic plant and the progeny thereof, comprising 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 comprising the enzymatic nucleic acid of any of claims 16 or 17. 54. The plant cell of claim 53, wherein said cell is a maize cell. 15 55. The plant cell of claim 53, wherein said cell is a canola cell. 56. A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid of any of claims 16 or 17. *o*o 57. The transgenic plant and the progeny thereof of claim 56, wherein said plant is a maize plant. 20 58. The transgenic plant and the progeny thereof of claim 56, wherein said plant is a canola plant 59. A plant cell comprising the enzymatic nucleic acid of any of claims 19 or The plant cell of claim 59, wherein said cell is a maize cell. 61. A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid of any of claims 19 or S S C. 116 62. The transgenic plant and progeny thereof of claim 61, wherein said plant is maize plant. 63. A method for modulating expression of an gene in a plant by administering to said plant the enzymatic nucleic acid molecule of'any of claims 1-8. 64. The method of claim 63, wherein said plant is a imlnocol plant. 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 A-9 desaturase. 69. The method of claim 68, wherein said plant is a maize plant. 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 0 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; 117 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 rgion, said on radi frame and said termination region, in a manner which allows cxpression and/or delivery of said enzymatic molecule within said plant cell. 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. S 15 76. The expression vector of claim 47, wherein said vector comprises: a) a transcription initiation region; o 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 intronr 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. 118 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, whcrcin said nucleic acid m1olecule 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 15 electroporation. 83. The transgenic plant of Claim 82, wherein said bombarding with DNA coated Smicroprojectiles is done with the gene gun. 84. The transgenic plant of any of Claims 78 or 82, wherein said plant contains a selectable marker seleted 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-l, 6-biphosphate (RUBP) carboxylase small subunit (ssu), 25 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, hairpin, hepatitis A virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration 119 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 nuclcic acids encoding lor said enzymatic nucleic acid molecule with RNA cleaving activity is operably linked to the 3' end of an open reading frame. 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. 96. An enzymatic nucleic acid molecule with RNA cleaving activity, substantially 9 as hereinbefore described with reference to any one of the examples. 97. A nucleic acid fragment comprising a cDNA sequence coding for maize A-9 desaturase, substantially as hereinbefore described with reference to any one of the s examples. 98. A method for modulating expression of a gene in a plant, substantially as hereinbefore described with reference to any one of the examples. 99. A transgenic plant comprising nucleic acids encoding for an enzymatic nucleic acid molecule with RNA cleaving activity, substantially as hereinbefore described with reference to any one of the examples. 100. A transgenic maize plant, substantially as hereinbefore described with reference to any one of the examples. DATED this twenty-fifth Day of September, 2000 Ribozyme Pharmaceuticals, Incorporated iDow AgroSciences LLC Patent Attorneys for the Applicants/Nominated Persons SPRUSON FERGUSON 0 *000 SOO .6 0 [R \IJBZ15293.docmef