WO1998032843A2

WO1998032843A2 - Compositions and method for modulation of alkaloid biosynthesis and flower formation in plants

Info

Publication number: WO1998032843A2
Application number: PCT/US1998/000738
Authority: WO
Inventors: Michael G. Zwick; James A. Mcswiggen
Original assignee: Ribozyme Pharmaceuticals, Inc.
Priority date: 1997-01-28
Filing date: 1998-01-14
Publication date: 1998-07-30
Also published as: WO1998032843A3; AU5918398A

Abstract

An enzymatic nucleic acid molecule with RNA cleaving activity, wherein the nucleic acid molecule modulates the expression of a gene involved in the biosynthesis of alkaloid compounds and flower formation in a plant. A transgenic plant comprising nucleic acids encoding for an enzymatic nucleic acid molecule with RNA cleaving activity, wherein the nucleic acid molecule modulates the expression of a gene involved in the biosynthesis of alkaloid compounds of flower formation in a plant. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein the nucleic acid molecule modulates the expression of solanidine UDP-glucose glucosyl-transferase gene or citrate synthase in plants.

Description

DESCRIPTION

Compositions And Method For Modulation Of Alkaloid Biosynthesis And Flower Formation 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 co-suppression (reviewed in Jorgensen, 1995 Science 268, 686-691) approaches have been used to modulate gene expression. Insertion utagenesis 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 antisense RNA was first discovered in bacteria over a decade ago (Simons and Kleckner, 1983 Cell 34, 683-691). It is thought to be one way in which bacteria can regulate their gene expression (Green et al . , 1986 Ann. Rev. Biochem. 55: 567-597; Simons 1988 Gene 72: 35-44). The first demonstration of antisense- ediated inhibition of gene expression was reported in mammalian cells (Izant and eintraub 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:

Shewmaker et al. , U.S. Patent Nos . 5,107,065 and 5, 453,566 disclose methods for regulating gene expression in plants using antisense RNA.

It has been shown that an antisense gene expressed in plants can act as a dominant suppressor gene. Transgenic potato plants have been produced which express RNA antisense to potato or cassava granule bound starch synthase (GBSS) . In both of these cases, transgenic plants have been constructed which have reduced or no GBSS activity or protein. These transgenic plants give rise to potatoes containing starch with dramatically reduced amylose levels (Visser et al . 1991, Mol . Gen. Genet. 225: 2889-296; Salehuzzaman et al. 1993, Plant Mol. Biol. 23: 947-962).

Kull et al., 1995, J. Genet. & Breed. 49, 69-76 reported inhibition of amylose biosynthesis in tubers from transgenic potato lines mediated by the expression of antisense sequences of the gene for granule-bound starch synthase (GBSS) . The authors, however, indicated a failure to see any in vivo activity of ribozymes targeted against the GBSS RNA. Antisense RNA constructs targeted against Δ-9 desaturase enzyme in canola have been shown to increase the level of stearic acid (C18:0) from 2% to 40% (Knutzon et. al., 1992 Proc. Natl. Acad. Sci. 89, 2624). There was no decrease in total oil content or germination efficiency in one of the high stearate lines. Several recent reviews are available which illustrate the utility of plants with modified oil composition (Ohlrogge, J. B. 1994 Plant Physiol. 104, 821; 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 the transgene were down-regulated (Napoli et al., 1990 Plant Cell 2: 279-289). There appears to be at least two mechanisms for inactivation of homologous genetic sequences. One appears to be 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 interest (Ronchi et al 1995 EMBO J. 14: 5318-5328) . The other mechanism of co-suppression is post-transcriptional, 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 J. 6: 861-877). The exact molecular basis for co-suppression is unknown.

Unfortunately, both antisense and co-suppression technologies are subject to problems in heritability of the desired trait (Finnegan and McElroy 1994 Bio/Technology 12: 883-888) . Currently, there is no easy way to specifically inactivate a gene of interest at the DNA level in plants (Pazkowski et al., 1988 EMBO J. 7: 4021-4026) . 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 Mol. Biol. 20: 715-731; Kull et al., 1995 J. Genet. & Breed. 49: 69-76) . Although there are reports in the literature of ribozyme activity in plants cells, almost all of them involve down regulation of exogenously introduced genes, such as reporter genes in transient assays (Steinecke et al., 1992 EMBO J. 11:1525- 1530; Perriman et al., 1993 Antisense Res. Dev. 3: 253- 263; Perriman et al., 1995, Proc. Natl. Acad. Sci. USA, 92, 6165) .

There are also several publications, [e.g., Lamb and Hay, 1990, J. Gen. Virol. 71, 2257-2264; Gerlach et al., International PCT Publication No. WO 91/13994; Xu et al . , 1992, Science in China (Ser. B) 35, 1434-1443; Edington and Nelson, 1992, in Gene Regulation: Biology of antisense RNA and DNA, eds . R. P. Erickson and J. G. Izant, pp 209-221, Raven Press, NY.; Atkins et al., International PCT Publication No. WO 94/00012; Lenee et al. , International PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins et al., 1995, J. Gen. Virol. 76, 1781- 1790; Gruber et al., 1994, J. Cell. Biochem. Suppl . 18A, 110 (Xl-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 of ribozymes to modulate the expression of plant genes . Mazzolini et al . , 1992, Plant. Mol. Bio. 20, 715- 731; Steinecke et al., 1992, EMBO. J. 11, 1525-1530; Perriman et al., 1995, Proc. Natl. Acad. Sci. USA., 92, 6175-6179; Wegener et al., 1994, Mol. Gen. Genet. 245, 465-470; and Steinecke et al., 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 demonstrate hammerhead ribozyme-mediated in vitro cleavage of glna, glnb, glng and glnd RNA, coding for gluta ine synthetase enzyme in Phaseolus vulgaris.

Certain plants contain undesirable alkaloid compounds which, when present in excess, are undesirable for human or animal consumption (Valkonen et al . 1996 Crit. Rev. Plant Sci. 15, 1-20) . Potatoes and other sola- naceous plants contain steroidal glycoalkaloids, whose level is regulated by genetic, developmental and environmental signals (Bergenstrahle et al. 1992 J. Plant Phys. 140, 269-275; Sinden, 1984 Am. Potato J. 61, 141- 156) . Potato tubers synthesize the alkaloids solanine and chaconine in response to wounding, temperature, light and sprouting. These glycoalkaloids are thought to be responsible for preventing insect predation and resistance to infection by pathogenic fungi (Valkonen et al. supra) . The enzyme solanidine UDP-glucose glucosyl- transferase is implicated as the enzyme primarily responsible for the biosynthesis of both these alkaloid compounds (Stapleton et al. 1992 Prot . Exp. Purif. 3, 85- 92, 6; Stapleton et al . 1991 J. Agri . Food Chem. 39, 1187-1193) .

The mitochondrial tricarboxylic acid (TCA) cycle enzyme citrate synthase is implicated in the formation of flower buds in plants (Landshutze et al . , 1995 EMBO J. 14, 660-666). Experiments with antisense constructs have shown that inhibition of the expression of the gene for this enzyme can delay or eliminate flower bud formation. There were no visible effects on plant growth or yield. The ovaries in the transgenic antisense plants disintegrated, indicating that citrate synthase and the TCA cycle are important in the transition from vegetative to generative phase of plant growth. Cytoplasmic male sterility (CMS) has been associated with mitochondrial gene expression, but typically affects the ability of the plant to produce viable pollen, not affecting female fertility (Levings et al., 1993 Plant Cell 5, 1285-1290; Chaudhury, 1993 Plant Cell 5, 1277-1283) . Inhibition of expression of the citrate synthase gene by ribozymes should result in the delay or elimination of flower formation in plants. This would be very useful in preventing flowering in plant species that are vegetatively propagated or where the primary consumable part of the plant is root, stem or leaf. The enzyme is mitochondrial, but is encoded by a nuclear gene (Landshutze et al.,1995 Planta 196, 756-764) . Chemical inhibition of mitochondrial respiration is harmful (Kromer et al.,1991 Plant. Phys . 95, 1270-1276), thus the ribozyme genetic approach is potentially advantageous over other methods. The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the use of ribozymes to down regulate genes involved in the plant alkaloid biosynthesis 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, invention features inhibiting the expression of genes involved in the biosynthesis of certain alkaloid compounds using enzymatic nucleic acid molecules. That is, the inhibition of the gene product

(e.g., RNA) results in a lowering of the production of alkaloid in the plant. Limiting the levels of certain alkaloid compounds in commercial cultivars, especially reductions in alkaloid content in the tuber by use of tissue-specific promoters is disclosed. The isolation of the gene encoding solanidine glucosyltransferase now allows evaluation of the phenotype that results from down-regulation of this gene (Moehs et al . , 1997 Plant J. 11, 100-110) . This application further deals with methods to produce cultivars such as, potato, tomato, pepper, eggplant, ditura, and others, with low levels of the toxic alkaloids.

In another aspect, the invention features inhibiting the expression of genes involved in flower formation using enzymatic nucleic acid molecules. That is, the gene product (e.g., RNA) is inhibited to prevent formation of a flower by the plant modulating the expression of citrate synthase in commercial cultivars by use of enzymatic nucleic acid is disclosed as one example. Inhibition of expression of the citrate synthase gene by ribozymes may result in the delay or elimination of flower formation in plants. This would be very useful in preventing flowering in plant species that are vegetatively propagated or where the primary consumable part of the plant is root, stem or leaf. This application further deals with methods to produce cultivars such as, lettuce, spinach, cabbage, brussel sprouts, arugula, kale, collards, chard, beet, turnip, potato, sweet potato and turfgrass, with delayed or elimination of flower formation. Any gene in the flower formation pathway that does not effect vegetative growth can be targeted in this manner.

The enzymatic nucleic acid molecule with RNA cleaving activity may be in the form of, but 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 molecule with RNA cleaving activity may be encoded as a monomer or a multimer, preferably a multimer. The nucleic acids encoding_for the enzymatic nucleic acid molecule with RNA cleaving activity may be operably linked to an open reading frame. Gene expression in any plant species may be modified by transformation of the plant with the nucleic acid encoding the enzymatic nucleic acid molecules with RNA cleaving activity. There are also numerous technologies for transforming a plant: such technologies include but are not limited to transformation with Agrobacterium, bombarding with DNA coated microprojectiles, whiskers, or electroporation. Any target gene may be modified with the nucleic acids encoding the enzymatic nucleic acid molecules with RNA cleaving activity.

Ribozymes can be used to modulate flower formation of a plant, for example, by modulating the activity of an enzyme involved in a biochemical pathway. It may be desirable, 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 techniques were developed herein to allow directed modulation of gene expression to generate plant cells, plant tissues or plants with altered flowering phenotype.

In a preferred embodiment the invention features Ribozymes that 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 decrease the level of expression of a particular gene, rather than shutting down expression completely: ribozymes can be used to achieve this. Enzymatic nucleic acid-based techniques were developed herein to allow directed modulation of gene expression to generate plant cells, plant tissues or plants with altered phenotype.

Ribozymes (i.e., enzymatic nucleic acids) are nucleic acid molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules 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 in vitro and in vivo (Zaug et al., 1986, Nature 324, 429; Kim et al., 1987, Proc. Natl. Acad. Sci. USA 84, 8788; Dreyfus, 1988, Einstein Quarterly J. Bio. Med. , 6, 92; Haseloff and Gerlach, 1988, Nature 334 585; Cech, 1988, JAMA 260, 3030; Murphy and Cech, 1989, Proc. Natl. Acad. 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 abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a particular phenotype and/or disease state can be selectively inhibited. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Brief Description of the Figures

Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be > 2 base-pairs long. Each 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 Gerlach (1988, Nature, 334, 585-591) into two portions; and Figure 2d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl. Acids. Res., 17, 1371-1371) into two portions.

Figure 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs (i.e. , n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3 - 20 bases, i.e., m is from 1 - 20 or more) . Helix 2 and helix 5 may be covalently linked by one or more bases (i.e., r is > 1 base) . Helix 1, 4 or 5 may also be extended by 2 or more base pairs (e.g. , 4 - 20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site. In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate. Complete base- pairing is not required in the helices, but is preferred. Helix 1 and 4 can be of any size (i.e. , o and p is each independently from 0 to any number, e.g., 20) as long as some base-pairing is maintained. Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Helix 4 can be formed from two separate molecules, i.e., without a connecting loop. The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate.

"q" is > 2 bases. The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C. Y refers to pyrimidine bases.

" " refers to a covalent bond.

Figure 4 is a representation of the general structure of the hepatitis Δ virus ribozyme domain known in the art.

Figure 5 is a representation of the general structure of the self-cleaving VS RNA ribozyme domain.

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 solanidine UDP-glucose glucosyl-transferase, potato citrate synthase, or level of mRNAs encoded by these genes is reduced below that observed in the absence of an enzymatic nucleic acid and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA. By "enzymatic nucleic acid molecule" it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave that target. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA (or DNA) and thereby inactivate a target RNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA to allow the cleavage to occur. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, RNAzyme, polyribozymes, molecular scissors, self-splicing RNA, self-cleaving RNA, cis-cleaving RNA, autolytic RNA, endoribonuclease, minizyme, leadzy e, oligozyme 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 ribonucleotides and may include a majority of other types of nucleotides or abasic moieties, as described below.

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. By "gene" is meant a nucleic acid that encodes an RNA.

By "plant gene" is meant a gene encoded by a plant.

By "endogenous" gene is meant a gene normally found in a plant cell in its natural location in the genome.

By "foreign" or "heterologous" gene is meant a gene not normally found in the host plant cell, but that is introduced by standard gene transfer techniques.

By "nucleic acid" is meant a molecule which can be single-stranded or double-stranded, composed of nucleotides containing a sugar, a phosphate and either a purine or pyrimidine base which may be same or different, and may be modified or unmodified.

By "genome" is meant genetic material contained in each cell of an organism and/or a virus.

By "mRNA" is meant RNA that can be translated into protein by a cell.

By "cDNA" is meant 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 complementarity may exist with any part of the target RNA, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. Antisense RNA is normally a mirror image of the sense RNA.

By "expression", as used herein, is meant the transcription and stable accumulation of the enzymatic nucleic acid molecules, mRNA and/or the antisense RNA inside a plant cell. Expression of genes involves transcription of the gene and translation of the mRNA into precursor or mature proteins. By "cosuppression" is meant the expression of a foreign gene, which has substantial homology to an gene, and in a plant cell causes the reduction in activity ÷ft 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 promoters, such as the ribulose-1, 6- biphosphate (RUBP) carboxylase small subunit (ssu) , the beta-conglycinin promoter, the phaseolin promoter, the ADH promoter, heat-shock promoters, and tissue specific promoters. Promoter may also contain certain enhancer sequence elements that may improve the transcription efficiency.

By "enhancer" is meant nucleotide sequence element which can stimulate promoter activity (Adh) .

By "constitutive promoter" is meant promoter element that directs continuous gene expression in all cells types and at all times (actin, ubiquitin, CaMV 35S) . By "tissue-specific" promoter is meant promoter element responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (zein, oleosin, napin, ACP) . By "development-specific" promoter is meant promoter element responsible for gene expression at specific plant developmental stage, such as in early or late embryogenesis .

By "inducible promoter" is meant promoter element which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes) ; light (RUBP carboxylase) ; hormone (Em) ; metabolites; and stress.

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

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

By "gymnosperm" is meant a plant having its seed exposed and not enclosed in an ovary (e.g., pine, spruce) .

By "monocotyledon" is meant a plant characterized by the presence of only one seed leaf (primary leaf of the embryo). For example, maize, wheat, rice and others.

By "dicotyledon" is meant a plant producing seeds with two cotyledons (primary leaf of the embryo) . For example, coffee, canola, peas and others.

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 initiation and termination region.

The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule may be targeted to a highly specific sequence region of a target such that specific gene inhibition can be achieved. Alternatively, enzymatic nucleic acid can be targeted to a highly conserved region of a gene family to inhibit gene expression of a family of related enzymes. The ribozymes can be expressed in plants that have been transformed with vectors which express the nucleic acid of the present invention.

The enzymatic nature of a ribozyme is advantageous over other technologies, since the concentration of ribozyme necessary to affect a therapeutic treatment is lower. This advantage reflects the ability of the ribozyme to act enzymatically . Thus, a single ribozyme molecule is able to cleave many molecules of target RNA.

In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme.

Seven basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets . In one of the preferred embodiments of the inventions herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis Δ virus, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA.

Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al . , 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al. , EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al . , 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al . , 1990 Nucleic Acids Res. 18, 299; of the hepatitis Δ virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 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 Cell 61, 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. J. 14, 363); Group II introns are described by Griffin et al . , 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; and of the Group I intron by Cech et al . , U.S. Patent 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

The enzymatic nucleic acid molecules of the instant invention will be expressed within cells from eukaryotic promoters [e.g. , Gerlach et al. , International PCT Publication No. WO 91/13994; Edington and Nelson, 1992, in Gene Regulation: Biology of Antisense RNA and DNA, eds . R. P. Erickson and J. G. Izant, pp 209-221, Raven Press, NY.; Atkins et al . , International PCT Publication No. WO 94/00012; Lenee et al., International PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins et al., 1995, J. Gen. Virol. 76, 1781-1790; McElroy and Brettell, 1994, TIBTECH 12, 62; Gruber et al., 1994, J^ Cell. Biochem. Suppl . 18A, 110 (Xl-406)and Feyter et al., 1996, Mol. 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 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 ribozyme RNA is introduced into the plant. There are also numerous ways to transform plants; plants can be transformed using the gene gun (US Patents 4,945,050 to Cornell and 5,141,131 to DowElanco) ; plants may be transformed using Agrobacterium technology, see US Patent 5,177,010 to University of Toledo, 5,104,310 to Texas A&M, European Patent Application 0131624B1, 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, European Patent Applications 604662 and 627752 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and US Patent 5,231,019 all to Ciba Geigy, US Patents 5,463,174 and 4,762,785 both to Calgene, and US Patents 5,004,863 and 5,159,135 both to Agracetus; whiskers technology, see US Patents 5,302,523 and 5,464,765 both to Zeneca; electroporation technology, see WO 87/06614 to Boyce Thompson Institute, 5,472,869 and 5,384,253 both to Dekalb, WO9209696 and W09321335 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 include but would not be limited to embryogenic tissue, callus tissue type I and II, and any tissue which is receptive to transformation and subsequent regeneration into a transgenic plant. Another variable is 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 markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to chlorosulfuron, hygromyacin, PAT and/or bar, bromoxynil, kanamycin and the like. The bar gene may be isolated from Strptomuces, particularly from the hygroscopicus or viridochromogenes species. The bar gene codes for phosphinothricin acetyl transferase (PAT) that inactivates the active ingradient in the herbicide bialaphos phosphinothricin (PPT) . Thus, numerous combinations of technologies may be used in employing ribozyme mediated modulation.

The ribozymes may be expressed individually as monomers, i.e., one ribozyme targeted against one site is expressed per transcript. Alternatively, two or more ribozymes targeted against more than one target site are expressed as part of a single RNA transcript. A single RNA transcript comprising more than one ribozyme targeted against more than one cleavage site are readily generated to achieve efficient modulation of gene expression. Ribozymes within these multimer constructs are the same or different. For example, the multimer construct may comprise a plurality of hammerhead ribozymes or hairpin ribozymes or other ribozyme motifs. Alternatively, the multimer construct may be designed to include a plurality of different ribozyme motifs, such as hammerhead and hairpin ribozymes. More specifically, multimer ribozyme constructs are designed, wherein a series of ribozyme motifs are linked together in tandem in a single RNA transcript. The ribozymes are linked to each other by nucleotide linker sequence, wherein the linker sequence may or may not be complementary to the target RNA. Multimer ribozyme constructs (polyribozymes) are likely to improve the effectiveness of ribozyme-mediated modulation of gene expression.

The activity of ribozymes can also be augmented by their release from the primary transcript by a second ribozyme (Draper et al . , PCT WO 93/23569, and Sullivan et al. , PCT WO 94/02595, both hereby incorporated in their totality by reference herein; Ohkawa, J. , et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira, K. , et al . , 1991, Nucleic Acids Res., 19, 5125-30; Ventura, M. , et al. , 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al. , 1994 J. Biol. Chem. 269, 25856).

Ribozyme-mediated modulation of gene expression can be practiced in a wide variety of plants including but not limited to potato, lettuce spinach, cabbage, brussel sprouts, arugula, kale, collards, chard, beet, turnip, sweet potato and turfgrass. Following are a few non- limiting examples that describe the general utility of ribozymes in modulation of gene expression.

Thus, in one instance, the invention concerns compositions (and methods for their use) for the modulation of genes involved in the biosynthesis of undesirable alkaloid compounds in plants. This is accomplished through the inhibition of genetic expression, with ribozymes, which results in the reduction or elimination of certain gene activities in plants, such as solanidine UDP-glucose glucosyl-transferase. Such activity is reduced in plants, such as potato and other solanaceous plants. These endogenously expressed ribozyme molecules contain substrate binding domains that bind to accessible regions of the target RNA. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the 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, and/or if the level of expression of the target gene is significantly reduced, levels of undesirable alkaloids is reduced or inhibited. Specific examples are provided below in the Tables III and IV.

In one aspect, the ribozymes have binding arms which are complementary to the substrate sequences in Tables III and IV. Those in the art will recognize that while such examples are designed to one gene RNA (solanidine UDP-glucose glucosyl-transferase) of one plant (e.g. , potato) , similar ribozymes can be made complementary to other genes in other plant's RNA. By complementary is thus meant that the binding arms of the ribozymes are able to interact with the target RNA in a sequence- specific manner and enable the ribozyme to cause cleavage of a plant mRNA target. Examples of such ribozymes are typically sequences defined in Tables III and IV. 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.

In another instance, the invention features compositions (and methods for their use) for the modulation of genes involved in the flower formation in plants. This is accomplished through the inhibition of genetic expression, with ribozymes, which results in the reduction or elimination of certain gene activities in plants, such as citrate synthase. Such activity can be reduced in plants, such as lettuce, spinach, cabbage, brussel sprouts, arugula, kale, collards, chard, beet, turnip, potato, sweet potato and turfgrass. These endogenously expressed ribozyme molecules contain substrate binding domains that bind to accessible regions of the target RNA. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the 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, and/or if the level of expression of the target gene is significantly reduced, levels of undesirable alkaloids is reduced or inhibited. Specific examples are provided below in the Tables V and VI. In a non-limiting example, ribozymes have binding arms which are complementary to the substrate sequences shown in Tables V and VI are disclosed. Those in the art will recognize that while such examples are designed to one gene RNA (citrate synthase) of one plant (e.g. , potato) , similar ribozymes can be made complementary to other genes in other plant's RNA. By complementary is thus meant that the binding arms of the ribozymes are able to interact with the target RNA in a sequence-specific manner and enable the ribozyme to cause cleavage of a plant mRNA target. Examples of such ribozymes are typically sequences defined in Tables V and VI. 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 III-VI.

Those in the art will recognize that ribozyme sequences listed in the Tables are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Table III and V (5'- GGCGAAAGCC-3 ' ) can be altered (substitution, deletion, and/or insertion) to contain any sequences, preferably provided that a minimum of a two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Table IV and VI (5¹- CACGUUGUG-3' ) can be altered (substitution, deletion, and/or insertion) to contain any sequence, preferably provided that a minimum of a two base-paired stem structure can form. Such ribozymes are equivalent to the ribozymes described specifically in the Tables. 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 alkaloid production in 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 undesirable alkaloid profile is an important application of nucleic acid-based technologies which are capable of reducing specific gene expression. A high level of undesirable alkaloid compounds is undesirable in plants that produce products of commercial importance. In preferred embodiments, hairpin and hammerhead ribozymes that cleave solanidine UDP-glucose glucosyl- transferase RNA are described. Those of ordinary skill in the art will understand from the examples described below that other ribozymes that cleave target RNAs required for solanidine UDP-glucose glucosyl-transferase activity may now be readily designed and are within the scope of the invention.

Modification of flower formation is an important application of nucleic acid-based technologies which are capable of reducing specific gene expression. In preferred embodiments, hairpin and hammerhead ribozymes that cleave potato citrate synthase RNA are described. Those of ordinary skill in the art will understand from the examples described below that other ribozymes that cleave target RNAs required for potato citrate synthase activity may now be readily designed and are within the scope of the invention

While specific examples to potato RNA are provided, those in the art will recognize that the teachings are not limited to potato. Furthermore, the same or equivalent 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 the art will recognize that similar embodiments can be readily generated in a variety of different plants to modulate expression of a variety of different genes, using the teachings herein, and are within the scope of the inventions.

Standard molecular biology techniques were followed in the examples herein. Additional information may be found in Sambrook, J., Fritsch, E. F. , and Maniatis, T. (1989), Molecular Cloning a Laboratory Manual, second edition, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

Examples

Example 1: Identification of Potential Ribozyme Cleavage Sites for solanidine UDP-glucose glucosyl-transferase

Approximately 353 HH ribozyme cleavage sites and approximately 20 HP sites were identified in the potato solanidine UDP-glucose glucosyl-transferase RNA. A HH site consists of a uridine and any nucleotide except guanosine (UH) . Tables III and IV have a list of HH and HP ribozyme cleavage sites. The numbering system starts with 1 at the 5 ' end of a solanidine UDP-glucose glucosyl-transferase RNA having the sequence shown in Moehs et al. , supra.

Ribozymes, such as those listed in Tables III and IV, can be readily designed and synthesized to such cleavage sites with between 5 and 100 or more bases as substrate binding arms (see Figs. 1 - 5). These substrate binding arms within a ribozyme allow the ribozyme to interact with their target in a sequence- specific manner.

Example 2: Selection of Ribozyme Cleavage Sites for solanidine UDP-glucose glucosyl-transferase

The secondary structure of solanidine UDP-glucose glucosyl-transferase RNA 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 nucleotides and contained potential hammerhead ribozyme cleavage sites were identified.

Example 3 : Hammerhead and Hairpin Ribozymes for solanidine UDP-glucose glucosyl-transferase

Hammerhead (HH) and hairpin (HP) ribozymes are subjected to analysis by computer folding and the ribozymes that had significant secondary structure are rejected.

The ribozymes are 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 are conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 μmol scale protocol with a 5 in coupling step for alkylsilyl protected nucleotides and 2.5 min coupling step for 2' -O-methylated nucleotides. Table II outlines the amounts, and the contact times, of the reagents used in the synthesis cycle. A 6.5-fold excess (163 μL of 0.1 M = 16.3 μmol) of phosphoramidite and a 24-fold excess of S-ethyl tetrazole (238 μL of 0.25 M = 59.5 μmol) relative to polymer-bound 5 ' -hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by colorimetric quantitation of the 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/10% 2,6- lutidine in THF (ABI); oxidation solution is 16.9 mM I2,

49 mM pyridine, 9% water in THF (Millipore) . B & J Synthesis Grade acetonitrile is used directly 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 is performed as follows. The polymer-bound oligoribonucleotide, trityl-off, is transferred from the synthesis column to a 4 mL glass screw top vial and suspended in a solution of methylamine (MA) at 65°C for 10 min. After cooling to -20°C, the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H2θ/3: 1: 1, vortexed and the supernatant is then added to the first supernatant. The combined super- natants, containing the oligoribonucleotide, are dried to a white powder.

The base-deprotected oligoribonucleotide is resuspended in anhydrous TEAΗF/NMP solution (250 μL of a solution of 1.5 L N-methylpyrrolidinone, 750 μL TEA and 1.0 mL TEA»3HF to provide a 1.4 M HF concentration) and heated to 65°C for 1.5 h. The resulting, fully deprotected, oligomer is quenched with 50 mM TEAB (9 mL) prior to anion exchange desalting.

For anion exchange desalting of the deprotected

® oligomer, the TEAB solution is loaded onto a Qiagen 500 anion exchange cartridge (Qiagen Inc.) that is prewashed with 50 mM TEAB (10 mL) . After washing the loaded cartridge with 50 mM TEAB (10 mL) , the RNA is eluted with 2 M TEAB (10 mL) and dried down to a white powder.

Inactive hammerhead ribozymes are synthesized by substituting a U for G5 and a U for A 4 (numbering from

(Hertel, K. J. , et al. , 1992, Nucleic Acids Res., 20, 3252) .

The hairpin ribozymes are synthesized as described above for the hammerhead RNAs .

Ribozymes can also synthesized be from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51) .

Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography (HPLC; See Wincott et al. , 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 III and IV.

Example 4 : Construction of Ribozyme expressing transcription units for solanidine UDP-glucose glucosyl- transferase

Ribozymes targeted to cleave solanidine UDP-glucose glucosyl-transferase RNA can be endogenously expressed in plants, either from genes inserted into the plant genome (stable transformation) or from episomal transcription units (transient expression) which are part of plasmid vectors or viral sequences. These ribozymes can be expressed via RNA polymerase I, II, or III plant or plant virus promoters (such as CaMV) . Promoters can be either constitutive, tissue specific, or developmentally expressed.

Example 5: Identification of Potential Ribozyme Cleavage Sites for potato citrate synthase

Approximately 398 HH ribozyme cleavage sites and approximately 25 HP sites were identified in the potato citrate synthase RNA. A HH site consists of a uridine and any nucleotide except guanosine (UH) . Tables V and VI have a list of HH and HP ribozyme cleavage sites.

Ribozymes, such as those listed in Tables III and IV, can be readily designed and synthesized to such cleavage sites with between 5 and 100 or more bases as substrate binding arms (see Figs. 1 - 5) . These substrate binding arms within a ribozyme allow the ribozyme to interact with their target in a sequence- specific manner.

Example 6: Selection of Ribozyme Cleavage Sites for potato citrate synthase

The secondary structure of potato citrate synthase RNA 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 nucleotides and contained potential hammerhead ribozyme cleavage sites were identified. Example 7 : Hammerhead and Hairpin Ribozymes for potato citrate synthase

The ribozymes are synthesized as described above. The sequences of the chemically synthesized ribozymes used in this study are shown below in Tables V and VI.

Example 8 : Construction of Ribozyme expressing transcription units for potato citrate synthase

Ribozymes targeted to cleave potato citrate synthase RNA can be endogenously expressed in plants, either from genes inserted into the plant genome (stable transformation) or from episomal transcription units (transient expression) which are part of plasmid vectors or viral sequences. These ribozymes can be expressed via RNA polymerase I, II, or III plant or plant virus promoters (such as CaMV) . Promoters can be either constitutive, tissue specific, or developmentally expressed.

Example 9: Plant Transformation and Construction

There are several methods to genetically engineer plants (for a review see Gasser et al., 1989 Science 244, 1293-1299; Potrykus, 1991 Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 205-225; Gasser and Fraley, 1992 Scientific American June 1992 pp 62-69) . These methods can be used to introduce the above ribozymes directly or via exression vectors. These methods include the following: Helium blasting involves accelerating suspended DNA- coated gold particles towards and into prepared tissue targets. The device used was an earlier prototype to the one described in a DowElanco U.S. Patent (#5,141,131) which is incorporated herein by reference, although both function in a similar manner. The device consists of a high pressure helium source, a syringe containing the DNA/gold suspension, and a pneumatically-operated multipurpose valve which provides controlled linkage between the helium source and a loop of pre-loaded DNA/gold suspension. Prior to blasting, tissue targets are covered with a sterile 104 micron stainless steel screen, which holds the tissue in place during impact. Next, targets are placed under vacuum in the main chamber of the device. The DNA-coated gold particles are accelerated at the target 4 times using a helium pressure of 1500 psi. Each blast delivered 20 μl of DNA/gold suspension. Immediately post-blasting, the targets are placed back on maintenance medium plus osmoticum for a 16 to 24 hour recovery period.

Particle Bombardment-mediated transformation

(Gordon-Kam et al., 1990 The Plant Cell 2, 603-618; Potrykus, 1991 Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 205-225; Gasser and Fraley, 1992 Scientific American June 1992 pp 62-69; Vain et al., 1993 Plant Cell Rep. 12, 84-88; Weymann et al., 1993 In Vitro Cell. Dev. Biol. 29P, 33-37) : This strategy involves bombardment of plant cells with minute (1-2 microns in diameter) metal particles (for example tungsten or gold particles) using a "gene" gun (also referred to as "Biolistics" or "particle" gun) . The metal particles, coated with genetic material (ribozyme or ribozyme encoding plasmids) , can penetrate the cell wall, without causing any irreversible damage to the cell, and deliver the genetic material to the cytoplasm.

Electroporation-mediated transformation (Fromm et al., 1986 Nature 319, 791-793; Rhodes et al., 1988 Science 240, 204-207; Potrykus, 1991 Annu. Rev. Plant

Physiol. Plant Mol. Biol. 42, 205-225; Gasser and Fraley,

1992 Scientific American June 1992 pp 62-69; D'Halluin et al., 1992 The Plant Cell 4, 1495-1505; Sukhapinda et al . ,

1993 Plant Cell Rep. 13, 63-68; Laursen et al., 1994 Plant Mol. Biol. 24, 51-61) : This technique involves permeabilizing the target cell membrane by using short high voltage electric pulses. Nucleic acids (ribozyme encoding plasmids) can pass through a permeabilized cell membrane and potentially integrate into the host genome resulting in a transformed phenotype. Electroporation can be carried out on (a) plant protoplasts, plant cells lacking a cell wall, (Fromm et al., 1986 Nature 319, 791- 793; Rhodes et al . , 1988 Science 240, 204-207; Sukhapinda et al., 1993 Plant Cell Rep. 13, 63-68); (b) cultured cells ( Laursen et al., 1994 Plant Mol. Biol. 24, 51-61); (c) Plant tissue (D'Halluin et al., 1992 The Plant Cell 4, 1495-1505).

Agrobacterium-mediated transformation : This method uses a disarmed (disease causing genes are deleted) species of Agrobacterium tumefaciens or Agrobacterium rizogenes (Potrykus, 1991 Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 205-225; Gasser and Fraley, 1992 Scientific American June 1992 pp 62-69) . This organism transfers part of its DNA into plant cells (T-DNA) . Ribozyme genes can be cloned into T-DNA fragments and Agrobacterium containing the recombinant T-DNA can be generated. Agrobacterium will infect and release the recombinant T- DNA into maize cells. The integration of T-DNA into host DNA will result in a transformed phenotype.

Other Uses: Potential usefulness of sequence-specific enzymatic nucleic acid molecules of the instant invention might have many of the same applications for the study of RNA that DNA restriction endonucleases have for the study of DNA (Nathans, D. and Smith, H. 0., (1975) Ann. Rev. Biochem. 44:273). For example, the pattern of restriction fragments could be used to establish sequence relationships between two related plant RNAs, and large plant RNAs could be specifically cleaved to fragments of a size more useful for study. The ability to engineer sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown sequence.

Ribozymes of this invention may be used as tools to examine genetic drift and mutations within plant cells. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the synthesis of undesirable alkaloids in plants. In this manner, other genetic targets may be defined as important mediators of alkaloid production. These experiments will lead to better modifications of the alkaloid production by affording the possibility of combinational concepts (e.g., multiple ribozymes targeted to different genes intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules) . Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNA associated with undesirable alkaloid production condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology. Other embodiments are within the following claims.

Table I

Table I:

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' -Oil of guanosine to generate cleavage products with 3' -OH and 5'- guanosine .

• Additional protein cofactors required in some cases to help folding and maintainance of the active structure t¹].

• Over 300 known members of this class. Found as an intervening sequence in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue- green algae, and others. • Major structural features largely established through phylogenetic comparisons, mutagenesis, and biochemical studies [2f 3] _#

• Complete kinetic framework established for one ribozyme [4,5,6,7] • Studies of ribozyme folding and substrate docking underway [8,9,10]_

• Chemical modification investigation of important residues well established [H'12]_

• The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA cleavage, however, the Tetrahymena group I intron has been used to repair a "defective" β-galactosidase message by the ligation Table I

of new β-galactosidase sequences onto the defective message [13] .

RNAse P RNA (Ml RNA) • Size: -290 to 400 nucleotides.

• RNA portion of a ubiquitous ribonucleoprotein enzyme.

• Cleaves tRNA precursors to form mature tRNA [1 ] .

• Reaction mechanism: possible attack by M +-0H to generate cleavage products with 3 ' OH and 5 ' -phosphate . • RNAse P is found throughout the prqkaryotes and eukaryotes. The RNA subunit has been sequenced from bacteria, yeast, rodents, and primates.

• Recruitment of endogenous RNAse P for therapeutic applications is possible through hybridization of an External Guide Sequence (EGS) to the target RNA

• Important phosphate and 2' OH contacts recently identified [17,18]

Group II Introns • Size: >1000 nucleotides.

• Trans cleavage of target RNAs recently demonstrated [19,20].

• Sequence requirements not fully determined.

• Reaction mechanism: 2 ' -OH of an internal adenosine generates cleavage products with 3' -OH and a "lariat" RNA containing a 3' -5' and a 2' -5' branch point.

• Only natural ribozyme with demonstrated participation in DNA cleavage [21,22] j__n addition to RNA cleavage and ligation. • Major structural features largely established through phylogenetic comparisons [23] . Table I

• Important 2' OH contacts beginning to be identified _[24]

• Kinetic framework under development [ 5]

Neurospora VS RNA

• Size: ~144 nucleotides.

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

• Sequence requirements not fully determined. • Reaction mechanism: attack by 2 ' -OH 5' to the scissile bond to generate cleavage products with 2 ',3' -cyclic phosphate and 5 ' -OH ends.

• Binding sites and structural requirements not fully determined. • Only 1 known member of this class. Found in Neurospora VS RNA.

Hammerhead Ribozyme (see text for references) • Size: -13 to 40 nucleotides.

• Requires the target sequence UH immediately 5' of the cleavage site.

• Binds a variable number nucleotides on both sides of the cleavage site. • Reaction mechanism: attack by 2 ' -OH 5' to the scissile bond to generate cleavage products with 2 ',3 '-cyclic phosphate and 5 ' -OH ends .

• 14 known members of this class. Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent.

• Essential structural features largely defined, including 2 crystal structures [] Table I

• 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 [27,28,29,30]

• Ligation activity (in addition to cleavage activity) makes ribozyme amenable to engineering through in vitro selection [31]

• Complete kinetic framework established for one ribozyme [31] .

• Chemical modification investigation of important residues begun [33,34] Table I

Hepatitis Delta Virus (HDV) Ribozyme

• Size: -60 nucleotides.

• Trans cleavage of target RNAs demonstrated [31] .

• Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required. Folded ribozyme contains a pseudoknot structure [36] .

• 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 [37]

1. Mohr, G.; Caprara, M.G.; Guo, Q. ; Lambowitz, A.M. Nature, 370,147-150. (1994) .

2. Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol. (1994), 1(1), 5-7. 3_ Lisacek, Frederique; Diaz, Yolande; Michel,

Francois. Automatic identification of group I intron cores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4), 1206-17.

⁴. Herschlag, Daniel; Cech, Thomas RCatalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1.

Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry (1990), 29(44), 10159-71.

5. Herschlag, Daniel; Cech, Thomas RCatalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2.

Kinetic description of the reaction of an RNA substrate Table I

that forms a mismatch at the active site. Biochemistry (1990), 29(44), 10172-80.

6. Knitt, Deborah S.; 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 C; Sugimoto, Naoki; Turner, Douglas HA mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58.

8. Li, Yi; Bevilacqua, Philip C; Mathews, David; Turner, Douglas HThermodynamic 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 HThe time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12. 0. Zarrinkar, Patrick P.; Williamson, James R., The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8. 11. Strobel, Scott A.; Cech, Thomas RMinor groove recognition of the conserved G.mtdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington, D.C.) (1995), 267(5198), 675-9.

12. Strobel, Scott A.; Cech, Thomas RExocychc 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. Table I

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

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

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

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

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

17. Harris, Michael E.; Pace, Norman R. , Identification of phosphates involved in catalysis by the ribozyme RNase P RNA. RNA (1995), 1(2), 210-18. 18. Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA: 2 ' -hydroxylbase 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 B., Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry (1994), 33(9), 2716-25. 0. 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 S . ; Lambowitz, Alan M. , A group 11 intron RNA is a catalytic component of a DNA Table I

endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38.

22. Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J. , Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2 ' -hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70.

23. Michel, Francois; Ferat, jean Luc. Structure and activities of group II introns. Annu. Rev. Biochem. (1995), 64, 435-61.

24. Abramovitz, Dana L.; Friedman, Richard A.; Pyle, Anna Marie. Catalytic role of T-hydroxyl groups within a group II intron active site. Science (Washington, D.C.) (1996), 271(5254), 1410-13. 25. Daniels, Danette L.; Michels, William J. , Jr.; Pyle, Anna 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. _{Gu0 / Hans} c. _τ.; Collins, Richard A. , Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995) , 14(2), 368-76.

27. Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. 'Hairpin' catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids Res. (1990), 18(2), 299-304.

28. Chowrira, Bharat M. ; Berzal-Herranz, Alfredo; Burke, John MNovel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2. Table I

29. Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M. ; Butcher, Samuel E.; Burke, John M., Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J. (1993), 12(6), 2567-73.

30. Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M. ; Butcher, Samuel E., Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. (1993), 7(1), 130-8.

31. Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John Min vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes Dev. (1992), 6(1), 129-34. 32. Hegg, Lisa A.; Fedor, Martha J. , Kinetics and Thermodynamics of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28.

33. Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait, Michael J., 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 S.; Gait, Michael J. , 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.

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

36. Perrotta, Anne T . ; Been, Michael DA pseudoknot- like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature (London) (1991), 350(6317), 434-6. 37. Puttaraju, M. ; Perrotta, Anne T . ; Been, Michael DA circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

Table II: 2.5 μmol RNA Synthesis Cycle

Reagent Equivalents Amount Wait

Time* Phosphoramidites 6.5 163 μL 2.5

S-Ethyl Tetrazole 23.8 238 μL 2.5

Acetic Anhydride 100 233 μL 5 sec

N-Methyl Imidazole 186 233 μL 5 sec

TCA 83.2 1.73 mL 21 sec Iodine 8.0 1.18 mL 45 sec

Acetonitrile NA 6.67 mL NA

* Wait time does not include contact time during delivery.

Table I I I

Table III: Solanidine glucosyltransferase Hammerhead

Ribozyme and Target Sequences

Nt. Substrate Ribozyme Position

13 UCUUGGGUA GUAAAAAU AϋUUUUAC CUGAUGA X GAA ACCCAAGA

16 UGGGUAGUA AAAAUGGU ACCAUUUU CUGAUGA X GAA ACUACCCA

25 AAAAUGGUA GCAACCUG CAGGUUGC CUGAUGA X GAA ACCAUUUU

49 GGCGAAAUC CUCCAUGU ACAUGGAG CUGAUGA X GAA AUUUCGCC

52 GAAAUCCUC CAUGUUCU AGAACAUG CUGAUGA X GAA AGGAUUUC

58 CUCCAUGUU CUUUUCCU AGGAAAAG CUGAUGA X GAA ACAUGGAG

59 UCCAUGUUC uuuuccuu AAGGAAAA CUGAUGA X GAA AACAUGGA

61 CAUGUUCUU uuccuucc GGAAGGAA CUGAUGA X GAA AGAACAUG

62 AUGUUCUUU uccuuccc GGGAAGGA CUGAUGA X GAA AAGAACAU

63 UGUUCUUUU ccuucccu AGGGAAGG CUGAUGA X GAA AAAGAACA

64 GUUCUUUUC cuucccuu AAGGGAAG CUGAUGA X GAA AAAAGAAC

67 cuuuuccuu cccuucuu AAGAAGGG CUGAUGA X GAA AGGAAAAG

68 uuuuccuuc CCUUCUUA UAAGAAGG CUGAUGA X GAA AAGGAAAA

72 ccuucccuu CUUAUCCG CGGAUAAG CUGAUGA X GAA AGGGAAGG

73 cuucccuuc UUAUCCGC GCGGAUAA CUGAUGA X GAA AAGGGAAG

75 ucccuucuu AUCCGCUG CAGCGGAU CUGAUGA X GAA AGAAGGGA

76 CCCUUCUUA UCCGCUGG CCAGCGGA CUGAUGA X GAA AAGAAGGG

78 CUUCUUAUC CGCUGGUC GACCAGCG CUGAUGA X GAA AUAAGAAG

86 CCGCUGGUC AUUUCAUC GAUGAAAU CUGAUGA X GAA ACCAGCGG

89 CUGGUCAUU UCAUCCCA UGGGAUGA CUGAUGA X GAA AUGACCAG

90 UGGUCAUUU CAUCCCAU AUGGGAUG CUGAUGA X GAA AAUGACCA

91 GGUCAUUUC AUCCCAUU AAUGGGAU CUGAUGA X GAA AAAUGACC

94 CAUUUCAUC CCAUUAGU ACUAAUGG CUGAUGA X GAA AUGAAAUG

99 CAUCCCAUU AGUUAACG CGUUAACU CUGAUGA X GAA AUGGGAUG

100 AUCCCAUUA GUUAACGC GCGUUAAC CUGAUGA X GAA AAUGGGAU

103 CCAUUAGUU AACGCCGC GCGGCGUU CUGAUGA X GAA ACUAAUGG

104 CAUUAGUUA ACGCCGCA UGCGGCGU CUGAUGA X GAA AACUAAUG

118 GCAAGGCUA UUCGCCUC GAGGCGAA CUGAUGA X GAA AGCCUUGC

120 AAGGCUAUU CGCCUCCC GGGAGGCG CUGAUGA X GAA AUAGCCUU

121 AGGCUAUUC GCCUCCCG CGGGAGGC CUGAUGA X GAA AAUAGCCU

126 AUUCGCCUC CCGGGUGU ACACCCGG CUGAUGA X GAA AGGCGAAU

135 CCGGGUGUU AAAGCCAC GUGGCUUU CUGAUGA X GAA ACACCCGG

136 CGGGUGUUA AAGCCACA UGUGGCUU CUGAUGA X GAA AACACCCG

147 GCCACAAUC CUCACUAC GUAGUGAG CUGAUGA X GAA AUUGUGGC

150 ACAAUCCUC ACUACCCC GGGGUAGU CUGAUGA X GAA AGGAUUGU

154 UCCUCACUA CCCCUCAU AUGAGGGG CUGAUGA X GAA AGUGAGGA

160 CUACCCCUC AUAAUGCC GGCAUUAU CUGAUGA X GAA AGGGGUAG

163 CCCCUCAUA AUGCCUUA UAAGGCAU CUGAUGA X GAA AUGAGGGG

170 UAAUGCCUU ACUUUUUA UAAAAAGU CUGAUGA X GAA AGGCAUUA

171 AAUGCCUUA CUUUUUAG CUAAAAAG CUGAUGA X GAA AAGGCAUU

174 GCCUUACUU UUUAGAUC GAUCUAAA CUGAUGA X GAA AGUAAGGC

175 CCUUACUUU UUAGAUCU AGAUCUAA CUGAUGA X GAA AAGUAAGG

176 CUUACUUUU UAGAUCUA UAGAUCUA CUGAUGA X GAA AAAGUAAG

177 UUACUUUUU AGAUCUAC GUAGAUCU CUGAUGA X GAA AAAAGUAA

178 UACUUUUUA GAUCUACU AGUAGAUC CUGAUGA X GAA AAAAAGUA

182 UUUUAGAUC UACUAUUG CAAUAGUA CUGAUGA X GAA AUCUAAAA

184 UUAGAUCUA CUAUUGAC GUCAAUAG CUGAUGA X GAA AGAUCUAA

187 GAUCUACUA UUGACGAU AUCGUCAA CUGAUGA X GAA KGUAGAUC

189 UCUACUAUU GACGAUGA UCAUCGUC CUGAUGA X GAA AUAGUAGA

201 GAUGAUGUU CGAAUUUC GAAAUUCG CUGAUGA X GAA ACAUCAUC Table I I I

Nt. Substrate Ribozyme Position

202 AUGAUGUUC GAAUUUCC GGAAAUUC CUGAUGA X GAA AACAUCAU

207 GUUCGAAUU UCCGGAUU AAUCCGGA CUGAUGA X GAA AUUCGAAC

208 UUCGAAUUU CCGGAUUU AAAUCCGG CUGAUGA X GAA AAUUCGAA

209 UCGAAUUUC CGGAUUUC GAAAUCCG CUGAUGA X GAA AAAUUCGA

215 UUCCGGAUU UCCCAUUU AAAUGGGA CUGAUGA X GAA AUCCGGAA

216 UCCGGAUUU CCCAUUUC GAAAUGGG CUGAUGA X GAA AAUCCGGA

217 CCGGAUUUC CCAUUUCU AGAAAUGG CUGAUGA X GAA AAAUCCGG

222 UUUCCCAUU UCUAUCGU ACGAUAGA CUGAUGA X GAA AUGGGAAA

223 UUCCCAUUU CUAUCGUA UACGAUAG CUGAUGA X GAA AAUGGGAA

224 UCCCAUUUC UAUCGUAA UUACGAUA CUGAUGA X GAA AAAUGGGA

226 CCAUUUCUA UCGUAACU AGUUACGA CUGAUGA X GAA AGAAAUGG

228 AUUUCUAUC GUAACUAU AUAGUUAC CUGAUGA X GAA AUAGAAAU

231 UCUAUCGUA ACUAUUAA UUAAUAGU CUGAUGA X GAA ACGAUAGA

235 UCGUAACUA UUAAAUUC GAAUUUAA CUGAUGA X GAA AGUUACGA

237 GUAACUAUU AAAUUCCC GGGAAUUU CUGAUGA X GAA AUAGUUAC

238 UAACUAUUA AAUUCCCC GGGGAAUU CUGAUGA X GAA AAUAGUUA

242 UAUUAAAUU CCCCUCUG CAGAGGGG CUGAUGA X GAA AUUUAAUA

243 AUUAAAUUC CCCUCUGC GCAGAGGG CUGAUGA X GAA AAUUUAAU

248 AUUCCCCUC UGCUGAAG CUUCAGCA CUGAUGA X GAA AGGGGAAU

258 GCUGAAGUU GGGUUGCC GGCAACCC CUGAUGA X GAA ACUUCAGC

263 AGUUGGGUU GCCUGAAG CUUCAGGC CUGAUGA X GAA ACCCAACU

276 GAAGGAAUU GAGAGCUU AAGCUCUC CUGAUGA X GAA AUUCCUUC

284 UGAGAGCUU UAACUCUG CAGAGUUA CUGAUGA X GAA AGCUCUCA

285 GAGAGCUUU AACUCUGC GCAGAGUU CUGAUGA X GAA AAGCUCUC

286 AGAGCUUUA ACUCUGCC GGCAGAGU CUGAUGA X GAA AAAGCUCU

290 CUUUAACUC UGCCACUU AAGUGGCA CUGAUGA X GAA AGUUAAAG

298 CUGCCACUU CACCUGAA UUCAGGUG CUGAUGA X GAA AGUGGCAG

299 UGCCACUUC ACCUGAAA UUUCAGGU CUGAUGA X GAA AAGUGGCA

313 AAAUGCCUC AUAAAAUU AAUUUUAU CUGAUGA X GAA AGGCAUUU

316 UGCCUCAUA AAAUUUUU AAAAAUUU CUGAUGA X GAA AUGAGGCA

321 CAUAAAAUU UUUUAUGC GCAUAAAA CUGAUGA X GAA AUUUUAUG

322 AUAAAAUUU UUUAUGCU AGCAUAAA CUGAUGA X GAA AAUUUUAU

323 UAAAAUUUU UUAUGCUC GAGCAUAA CUGAUGA X GAA AAAUUUUA

324 AAAAUUUUU UAUGCUCU AGAGCAUA CUGAUGA X GAA AAAAUUUU

325 AAAUUUUUU AUGCUCUU AAGAGCAU CUGAUGA X GAA AAAAAUUU

326 AAUUUUUUA UGCUCUUU AAAGAGCA CUGAUGA X GAA AAAAAAUU

331 UUUAUGCUC UUUCUCUU AAGAGAAA CUGAUGA X GAA AGCAUAAA

333 UAUGCUCUU UCUCUUCU AGAAGAGA CUGAUGA X GAA AGAGCAUA

334 AUGCUCUUU CUCUUCUA UAGAAGAG CUGAUGA X GAA AAGAGCAU

335 UGCUCUUUC UCUUCUAC GUAGAAGA CUGAUGA X GAA AAAGAGCA

337 CUCUUUCUC UUCUACAA UUGUAGAA CUGAUGA X GAA AGAAAGAG

339 cuuucucuu CUACAAAA UUUUGUAG CUGAUGA X GAA AGAGAAAG

340 uuucucuuc UACAAAAG CUUUUGUA CUGAUGA X GAA AAGAGAAA

342 UCUCUUCUA CAAAAGCC GGCUUUUG CUGAUGA X GAA AGAAGAGA

361 UGGAAGAUA AAAUUCGU ACGAAUUU CUGAUGA X GAA AUCUUCCA

366 GAUAAAAUU CGUGAACU AGUUCACG CUGAUGA X GAA AUUUUAUC

367 AUAAAAUUC GUGAACUC GAGUUCAC CUGAUGA X GAA AAUUUUAU

375 CGUGAACUC CGUCCUGA UCAGGACG CUGAUGA X GAA AGUUCACG

379 AACUCCGUC CUGAUUGC GCAAUCAG CUGAUGA X GAA ACGGAGUU

385 GUCCUGAUU GCAUUUUU AAAAAUGC CUGAUGA X GAA AUCAGGAC

390 GAUUGCAUU UUUUCUGA UCAGAAAA CUGAUGA X GAA AUGCAAUC

391 AUUGCAUUU UUUCUGAU AUCAGAAA CUGAUGA X GAA AAUGCAAU

392 UUGCAUUUU UUCUGAUA UAUCAGAA CUGAUGA X GAA AAAUGCAA

393 UGCAUUUUU UCUGAUAU AUAUCAGA CUGAUGA X GAA AAAAUGCA

394 GCAUUUUUU CUGAUAUG CAUAUCAG CUGAUGA X GAA AAAAAUGC

395 CAUUUUUUC UGAUAUGU ACAUAUCA CUGAUGA X GAA AAAAAAUG

400 UUUCUGAUA UGUACUUC GAAGUACA CUGAUGA X GAA AUCAGAAA Table I I I

Nt. Substrate Ribozyme Position

404 UGAUAUGUA CUUCCCUU AAGGGAAG CUGAUGA X GAA ACAUAUCA

407 UAUGUACUU CCCUUGGA UCCAAGGG CUGAUGA X GAA AGUACAUA

408 AUGUACUUC CCUUGGAC GUCCAAGG CUGAUGA X GAA AAGUACAU

412 ACUUCCCUU GGACAGUA UACUGUCC CUGAUGA X GAA AGGGAAGU

420 UGGACAGUA GAUAUUGC GCAAUAUC CUGAUGA X GAA ACUGUCCA

424 CAGUAGAUA UUGCUGAU AUCAGCAA CUGAUGA X GAA AUCUACUG

426 GUAGAUAUU GCUGAUGA UCAUCAGC CUGAUGA X GAA AUAUCUAC

438 GAUGAGCUU CACAUCCC GGGAUGUG CUGAUGA X GAA AGCUCAUC

439 AUGAGCUUC ACAUCCCU AGGGAUGU CUGAUGA X GAA AAGCUCAU

444 CUUCACAUC CCUCGUAU AUACGAGG CUGAUGA X GAA AUGUGAAG

448 ACAUCCCUC GUAUUUUG CAAAAUAC CUGAUGA X GAA AGGGAUGU

451 UCCCUCGUA UUUUGUAC GUACAAAA CUGAUGA X GAA ACGAGGGA

453 CCUCGUAUU UUGUACAA UUGUACAA CUGAUGA X GAA AUACGAGG

454 CUCGUAUUU UGUACAAU AUUGUACA CUGAUGA X GAA AAUACGAG

455 UCGUAUUUU GUACAAUU AAUUGUAC CUGAUGA X GAA AAAUACGA

458 UAUUUUGUA CAAUUUGU ACAAAUUG CUGAUGA X GAA ACAAAAUA

463 UGUACAAUU UGUCUGCU AGCAGACA CUGAUGA X GAA AUUGUACA

464 GUACAAUUU GUCUGCUU AAGCAGAC CUGAUGA X GAA AAUUGUAC

467 CAAUUUGUC UGCUUACA UGUAAGCA CUGAUGA X GAA ACAAAUUG

472 UGUCUGCUU ACAUGUGC GCACAUGU CUGAUGA X GAA AGCAGACA

473 GUCUGCUUA CAUGUGCU AGCACAUG CUGAUGA X GAA AAGCAGAC

482 CAUGUGCUA CAGCAUUA UAAUGCUG CUGAUGA X GAA AGCACAUG

489 UACAGCAUU AUGCACAA UUGUGCAU CUGAUGA X GAA AUGCUGUA

490 ACAGCAUUA UGCACAAC GUUGUGCA CUGAUGA X GAA AAUGCUGU

501 CACAACCUU AAGGUUUA UAAACCUU CUGAUGA X GAA AGGUUGUG

502 ACAACCUUA AGGUUUAC GUAAACCU CUGAUGA X GAA AAGGUUGU

507 CUUAAGGUU UACAGACC GGUCUGUA CUGAUGA X GAA ACCUUAAG

508 UUAAGGUUU ACAGACCU AGGUCUGU CUGAUGA X GAA AACCUUAA

509 UAAGGUUUA CAGACCUC GAGGUCUG CUGAUGA X GAA AAACCUUA

517 ACAGACCUC ACAAGCAG CUGCUUGU CUGAUGA X GAA AGGUCUGU

529 AGCAGCCUA AUCUAGAC GUCUAGAU CUGAUGA X GAA AGGCUGCU

532 AGCCUAAUC UAGACGAA UUCGUCUA CUGAUGA X GAA AUUAGGCU

534 CCUAAUCUA GACGAAUC GAUUCGUC CUGAUGA X GAA AGAUUAGG

542 AGACGAAUC UCAAAGUU AACUUUGA CUGAUGA X GAA AUUCGUCU

544 ACGAAUCUC AAAGUUUC GAAACUUU CUGAUGA X GAA AGAUUCGU

550 CUCAAAGUU UCGUGGUU AACCACGA CUGAUGA X GAA ACUUUGAG

551 UCAAAGUUU CGUGGUUC GAACCACG CUGAUGA X GAA AACUUUGA

552 CAAAGUUUC GUGGUUCC GGAACCAC CUGAUGA X GAA AAACUUUG

558 UUCGUGGUU CCUGGUUU AAACCAGG CUGAUGA X GAA ACCACGAA

559 UCGUGGUUC CUGGUUUA UAAACCAG CUGAUGA X GAA AACCACGA

565 UUCCUGGUU UACCUGAU AUCAGGUA CUGAUGA X GAA ACCAGGAA

566 UCCUGGUUU ACCUGAUG CAUCAGGU CUGAUGA X GAA AACCAGGA

567 CCUGGUUUA CCUGAUGA UCAUCAGG CUGAUGA X GAA AAACCAGG

579 GAUGAGAUA AAGUUCAA UUGAACUU CUGAUGA X GAA AUCUCAUC

584 GAUAAAGUU CAAGUUAU AUAACUUG CUGAUGA X GAA ACUUUAUC

585 AUAAAGUUC AAGUUAUC GAUAACUU CUGAUGA X GAA AACUUUAU

590 GUUCAAGUU AUCCCAAC GUUGGGAU CUGAUGA X GAA ACUUGAAC

591 UUCAAGUUA UCCCAACU AGUUGGGA CUGAUGA X GAA AACUUGAA

593 CAAGUUAUC CCAACUGA UCAGUUGG CUGAUGA X GAA AUAACUUG

610 CAGAUGAUC UGAGAAAG CUUUCUCA CUGAUGA X GAA AUCAUCUG

620 GAGAAAGUC GGAUGACC GGUCAUCC CUGAUGA X GAA ACUUUCUC

639 AAGACUGUU UUUGACGA UCGUCAAA CUGAUGA X GAA ACAGUCUU

640 AGACUGUUU UUGACGAA UUCGUCAA CUGAUGA X GAA AACAGUCU

641 GACUGUUUU UGACGAAU AUUCGUCA CUGAUGA X GAA AAACAGUC

642 ACUGUUUUU GACGAAUU AAUUCGUC CUGAUGA X GAA AAAACAGU

650 UGACGAAUU GCUCGAAC GUUCGAGC CUGAUGA X GAA AUUCGUCA

654 GAAUUGCUC GAACAAGU ACUUGUUC CUGAUGA X GAA AGCAAUUC Table I I I

Nt. Substrate Ribozyme Position

663 GAACAAGUU GAAGAUUC GAAUCUUC CUGAUGA X GAA ACUUGUUC

670 UUGAAGAUU CGGAGGAA UUCCUCCG CUGAUGA X GAA AUCUUCAA

671 UGAAGAUUC GGAGGAAC GUUCCUCC CUGAUGA X GAA AAUCUUCA

686 ACGAAGCUA UGGCAUUG CAAUGCCA CUGAUGA X GAA AGCUUCGU

693 UAUGGCAUU GUUCAUGA UCAUGAAC CUGAUGA X GAA AUGCCAUA

696 GGCAUUGUU CAUGAUAC GUAUCAUG CUGAUGA X GAA ACAAUGCC

697 GCAUUGUUC AUGAUACA UGUAUCAU CUGAUGA X GAA AACAAUGC

703 UUCAUGAUA CAUUUUAU AUAAAAUG CUGAUGA X GAA AUCAUGAA

707 UGAUACAUU UUAUGAGC GCUCAUAA CUGAUGA X GAA AUGUAUCA

708 GAUACAUUU UAUGAGCU AGCUCAUA CUGAUGA X GAA AAUGUAUC

709 AUACAUUUU AUGAGCUA UAGCUCAU CUGAUGA X GAA AAAUGUAU

710 UACAUUUUA UGAGCUAG CUAGCUCA CUGAUGA X GAA AAAAUGUA

717 UAUGAGCUA GAACCUGC GCAGGUUC CUGAUGA X GAA AGCUCAUA

728 ACCUGCAUA UGUUGACU AGUCAACA CUGAUGA X GAA AUGCAGGU

732 GCAUAUGUU GACUACUA UAGUAGUC CUGAUGA X GAA ACAUAUGC

737 UGUUGACUA CUACCAGA UCUGGUAG CUGAUGA X GAA AGUCAACA

740 UGACUACUA CCAGAAAU AUUUCUGG CUGAUGA X GAA AGUAGUCA

749 CCAGAAAUU AAAGAAAC GUUUCUUU CUGAUGA X GAA AUUUCUGG

750 CAGAAAUUA AAGAAACC GGUUUCUU CUGAUGA X GAA AAUUUCUG

766 CAAAAUGUU GGCAUUUU AAAAUGCC CUGAUGA X GAA ACAUUUUG

772 GUUGGCAUU UUGGUCCG CGGAO-AA CUGAUGA X GAA AUGCCAAC

773 UUGGCAUUU UGGUCCGC GCGGACCA CUGAUGA X GAA AAUGCCAA

774 UGGCAUUUU GGUCCGCU AGCGGACC CUGAUGA X GAA AAAUGCCA

778 AUUUUGGUC CGCUCUCU AGAGAGCG CUGAUGA A GAA ACCAAAAU

783 GGUCCGCUC UCUCAUUU AAAUGAGA CUGAUGA X GAA AGCGGACC

785 UCCGCUCUC UCAUUUUG CAAAAUGA CUGAUGA X GAA AGAGCGGA

787 CGCUCUCUC AUUUUGCA UGCAAAAU CUGAUGA X GAA AGAGAGCG

790 UCUCUCAUU UUGCAUCC GGAUGCAA CUGAUGA X GAA AUGAGAGA

791 CUCUCAUUU UGCAUCCA UGGAUGCA CUGAUGA X GAA AAUGAGAG

792 UCUCAUUUU GCAUCCAA UUGGAUGC CUGAUGA X GAA AAAUGAGA

797 UUUUGCAUC CAAAUCCG CGGAUUUG CUGAUGA X GAA AUGCAAAA

803 AUCCAAAUC CGUAGUAA UUACUACG CUGAUGA X GAA AUUUGGAU

807 AAAUCCGUA GUAAGGAA UUCCUUAC CUGAUGA X GAA ACGGAUUU

810 UCCGUAGUA AGGAACUA UAGUUCCU CUGAUGA X GAA ACUACGGA

818 AAGGAACUA AUUUCUGA UCAGAAAU CUGAUGA X GAA AGUUCCUU

821 GAACUAAUU UCUGAGCA UGCUCAGA CUGAUGA X GAA AUUAGUUC

822 AACUAAUUU CUGAGCAU AUGCUCAG CUGAUGA X GAA AAUUAGUU

823 ACUAAUUUC UGAGCAUA UAUGCUCA CUGAUGA X GAA AAAUUAGU

831 CUGAGCAUA ACAACAAU AUUGUUGU CUGAUGA X GAA AUGCUCAG

845 AAUGAGAUU GUUAUAGA UCUAUAAC CUGAUGA X GAA AUCUCAUU

848 GAGAUUGUU AUAGAUUG CAAUCUAU CUGAUGA X GAA ACAAUCUC

849 AGAUUGUUA UAGAUUGG CCAAUCUA CUGAUGA X GAA AACAAUCU

851 AUUGUUAUA GAUUGGUU AACCAAUC CUGAUGA X GAA AUAACAAU

855 UUAUAGAUU GGUUGAAU AUUCAACC CUGAUGA X GAA AUCUAUAA

859 AGAUUGGUU GAAUGCAC GUGCAUUC CUGAUGA X GAA ACCAAUCU

876 AGAAACCUA AAUCGGUU AACCGAUU CUGAUGA X GAA AGGUUUCU

880 ACCUAAAUC GGUUCUCU AGAGAACC CUGAUGA X GAA AUUUAGGU

884 AAAUCGGUU CUCUAUGU ACAUAGAG CUGAUGA X GAA ACCGAUUU

885 AAUCGGUUC UCUAUGUA UACAUAGA CUGAUGA X GAA AACCGAUU

887 UCGGUUCUC UAUGUAUC GAUACAUA CUGAUGA X GAA AGAACCGA

889 GGUUCUCUA UGUAUCUU AAGAUACA CUGAUGA X GAA AGAGAACC

893 CUCUAUGUA UCUUUCGG CCGAAAGA CUGAUGA X GAA ACAUAGAG

895 CUAUGUAUC UUUCGGAA UUCCGAAA CUGAUGA X GAA AUACAUAG

897 AUGUAUCUU UCGGAAGC GCUUCCGA CUGAUGA X GAA AGAUACAU

898 UGUAUCUUU CGGAAGCA UGCUUCCG CUGAUGA X GAA AAGAUACA

899 GUAUCUUUC GGAAGCAU AUGCUUCC CUGAUGA X GAA AAAGAUAC Table III

Nt. Substrate Ribozyme Position

912 GCAUGGCUA GAUUUCCU AGGAAAUC CUGAUGA X GAA AGCCAUGC

916 GGCUAGAUU UCCUGAGA UCUCAGGA CUGAUGA X GAA AUCUAGCC

917 GCUAGAUUU CCUGAGAG CUCUCAGG CUGAUGA X GAA AAUCUAGC

918 CUAGAUUUC CUGAGAGC GCUCUCAG CUGAUGA X GAA AAAUCUAG

941 AAUGAAAUA GCCCAAGC GCUUGGGC CUGAUGA X GAA AUUUCAUU

951 CCCAAGCUC UGGAUGCU AGCAUCCA CUGAUGA X GAA AGCUUGGG

960 UGGAUGCUU CAAAUGUU AACAUUUG CUGAUGA X GAA AGCAUCCA

961 GGAUGCUUC AAAUGUUC GAACAUUU CUGAUGA X GAA AAGCAUCC

968 UCAAAUGUU CCUUUCAU AUGAAAGG CUGAUGA X GAA ACAUUUGA

969 CAAAUGUUC CUUUCAUU AAUGAAAG CUGAUGA X GAA AACAUUUG

972 AUGUUCCUU UCAUUUUU AAAAAUGA CUGAUGA X GAA AGGAACAU

973 UGUUCCUUU CAUUUUUG CAAAAAUG CUGAUGA X GAA AAGGAACA

974 GUUCCUUUC AUUUUUGU ACAAAAAU CUGAUGA X GAA AAAGGAAC

977 CCUUUCAUU UUUGUAUU AAUACAAA CUGAUGA X GAA AUGAAAGG

978 CUUUCAUUU UUGUAUUG CAAUACAA CUGAUGA X GAA AAUGAAAG

979 UUUCAUUUU UGUAUUGA UCAAUACA CUGAUGA X GAA AAAUGAAA

980 UUCAUUUUU GUAUUGAG CUCAAUAC CUGAUGA X GAA AAAAUGAA

983 AUULJUUGUA UUGAGGCC GGCCUCAA CUGAUGA X GAA ACAAAAAU

985 UUUUGUAUU GAGGCCUA UAGGCCUC CUGAUGA X GAA AUACAAAA

993 UGAGGCCUA AUGAAGAA UUCUUCAU CUGAUGA X GAA AGGCCUCA

1009 AACGGCGUC GUGGUUGC GCAACCAC CUGAUGA X GAA ACGCCGUU

1015 GUCGUGGUU GCCAGUUG CAACUGGC CUGAUGA X GAA ACCACGAC

1022 UUGCCAGUU GGUAAUUU AAAUUACC CUGAUGA X GAA ACUGGCAA

1026 CAGUUGGUA AUUUAGAG CUCUAAAU CUGAUGA X GAA ACCAACUG

1029 UUGGUAAUU UAGAGGAC GUCCUCUA CUGAUGA X GAA AUUACCAA

1030 UGGUAAUUU AGAGGACA UGUCCUCU CUGAUGA X GAA AAUUACCA

1031 GGUAAUUUA GAGGACAA UUGUCCUC CUGAUGA X GAA AAAUUACC

1044 ACAAGACUA AAAAGGGU ACCCUUUU CUGAUGA X GAA AGUCUUGU

1053 AAAAGGGUU UGUACAUC GAUGUACA CUGAUGA X GAA ACCCUUUU

1054 AAAGGGUUU GUACAUCA UGAUGUAC CUGAUGA X GAA AACCCUUU

1057 GGGUUUGUA CAUCAAAG CUUUGAUG CUGAUGA X GAA ACAAACCC

1061 UUGUACAUC AAAGGGUG CACCCUUU CUGAUGA X GAA AUGUACAA

1073 GGGUGGGUC CCACAGCU AGCUGUGG CUGAUGA X GAA ACCCACCC

1082 CCACAGCUU ACGAUCAU AUGAUCGU CUGAUGA X GAA AGCUGUGG

1083 CACAGCUUA CGAUCAUG CAUGAUCG CUGAUGA X GAA AAGCUGUG

1088 CUUACGAUC AUGGAACA UGUUCCAU CUGAUGA X GAA AUCGUAAG

1098 UGGAACAUU CAGCAACA UGUUGCUG CUGAUGA X GAA AUGUUCCA

1099 GGAACAUUC AGCAACAG CUGUUGCU CUGAUGA X GAA AAUGUUCC

1114 AGGCGGGUU CAUGACUC GAGUCAUG CUGAUGA X GAA ACCCGCCU

1115 GGCGGGUUC AUGACUCA UGAGUCAU CUGAUGA X GAA AACCCGCC

1122 UCAUGACUC AUUGUGGU ACCACAAU CUGAUGA X GAA AGUCAUGA

1125 UGACUCAUU GUGGUACU AGUACCAC CUGAUGA X GAA AUGAGUCA

1131 AUUGUGGUA CUAAUUCG CGAAUUAG CUGAUGA X GAA ACCACAAU

1134 GUGGUACUA AUUCGGUU AACCGAAU CUGAUGA X GAA AGUACCAC

1137 GUACUAAUU CGGTJUCUG CAGAACCG CUGAUGA X GAA AUUAGUAC

1138 UACUAAUUC GGUUCUGG CCAGAACC CUGAUGA X GAA AAUUAGUA

1142 AAUUCGGUU CUGGAAGC GCUUCCAG CUGAUGA X GAA ACCGAAUU

1143 AUUCGGUUC UGGAAGCC GGCUUCCA CUGAUGA X GAA AACCGAAU

1154 GAAGCCAUC ACUUUUGG CCAAAAGU CUGAUGA X GAA AUGGCUUC

1158 CCAUCACUU UUGGCGUG CACGCCAA CUGAUGA X GAA AGUGAUGG

1159 CAUCACUUU UGGCGUGC GCACGCCA CUGAUGA X GAA AAGUGAUO

1160 AUCACUUUU GGCGUGCC GGCACGCC CUGAUGA X GAA AAAGUGAU

1175 CCAAUGAUA ACAUGGCC GGCCAUGU CUGAUGA X GAA AUCAUUGG

1187 UGGCCACUU UAUGCUGA UCAGCAUA CUGAUGA X GAA AGUGGCCA

1188 GGCCACUUU AUGCUGAU AUCAGCAU CUGAUGA X GAA AAGUGGCC

1189 GCCACUUUA UGCUGAUC GAUCAGCA CUGAUGA X GAA AAAGUGGC

1197 AUGCUGAUC AAUUCUAC GUAGAAUU CUGAUGA X GAA AUCAGCAU Table I I I

Nt. Substrate Ribozyme Position

1201 UGAUCAAUU CUACAACG CGUUGUAG CUGAUGA X GAA AUUGAUCA

1202 GAUCAAUUC UACAACGA UCGUUGUA CUGAUGA X GAA AAUUGAUC

1204 UCAAUUCUA CAACGAGA UCUCGUUG CUGAUGA X GAA AGAAUUGA

1217 GAGAAGGUA GUCGAGGU ACCUCGAC CUGAUGA X GAA ACCUUCUC

1220 AAGGUAGUC GAGGUUAG CUAACCUC CUGAUGA X GAA ACUACCUU

1226 GUCGAGGUU AGGGGAUU AAUCCCCU CUGAUGA X GAA ACCUCGAC

1227 UCGAGGUUA GGGGAUUG CAAUCCCC CUGAUGA X GAA AACCUCGA

1234 UAGGGGAUU GGGAAUCA UGAUUCCC CUGAUGA X GAA AUCCCCUA

1241 UUGGGAAUC AAAAUCGG CCGAUUUU CUGAUGA X GAA AUUCCCAA

1247 AUCAAAAUC GGGAUAGA UCUAUCCC CUGAUGA X GAA AUUUUGAU

1253 AUCGGGAUA GAUGUAUG CAUACAUC CUGAUGA X GAA AUCCCGAU

1259 AUAGAUGUA UGGAAUGA UCAUUCCA CUGAUGA X GAA ACAUCUAU

1274 GAAGGGAUU GAGAUCAC GUGAUCUC CUGAUGA X GAA AUCCCUUC

1280 AUUGAGAUC ACGGGCCC GGGCCCGU CUGAUGA X GAA AUCUCAAU

1292 GGCCCUGUA AUAGAAAG CUUUCUAU CUGAUGA X GAA ACAGGGCC

1295 CCUGUAAUA GAAAGCGC GCGCUUUC CUGAUGA X GAA AUUACAGG

1310 GCCAAGAUU AGAGAAGC GCUUCUCU CUGAUGA X GAA AUCUUGGC

1311 CCAAGAUUA GAGAAGCA UGCUUCUC CUGAUGA X GAA AAUCUUGG

1322 GAAGCAAUU GAGAGACU AGUCUCUC CUGAUGA X GAA AUUGCUUC

1331 GAGAGACUA AUGAUCAG CUGAUCAU CUGAUGA X GAA AGUCUCUC

1337 CUAAUGAUC AGUAAUGG CCAUUACU CUGAUGA X GAA AUCAUUAG

1341 UGAUCAGUA AUGGUUCU AGAACCAU CUGAUGA X GAA ACUGAUCA

1347 GUAAUGGUU CUGAGGAA UUCCUCAG CUGAUGA X GAA ACCAUUAC

1348 UAAUGGUUC UGAGGAAA UUUCCUCA CUGAUGA X GAA AACCAUUA

1358 GAGGAAAUU AUAAAUAU AUAUUUAU CUGAUGA X GAA AUUUCCUC

1359 AGGAAAUUA UAAAUAUU AAUAUUUA CUGAUGA X GAA AAUUUCCU

1361 GAAAUUAUA AAUAUUAG CUAAUAUU CUGAUGA X GAA AUAAUUUC

1365 UUAUAAAUA UUAGGGAU AUCCCUAA CUGAUGA X GAA AUUUAUAA

1367 AUAAAUAUU AGGGAUAG CUAUCCCU CUGAUGA X GAA AUAUUUAU

1368 UAAAUAUUA GGGAUAGA UCUAUCCC CUGAUGA X GAA AAUAUUUA

1374 UUAGGGAUA GAGUAAUG CAUUACUC CUGAUGA X GAA AUCCCUAA

1379 GAUAGAGUA AUGGCUAU AUAGCCAU CUGAUGA X GAA ACUCUAUC

1386 UAAUGGCUA UGAGCAAA UUUGCUCA CUGAUGA X GAA AGCCAUUA

1401 AAAUGGCUC AGAAUGCA UGCAUUCU CUGAUGA X GAA AGCCAUUU

1426 AGGUGGAUC UUCGUGGA UCCACGAA CUGAUGA X GAA AUCCACCU

1428 GUGGAUCUU CGUGGAAC GUUCCACG CUGAUGA X GAA AGAUCCAC

1429 UGGAUCUUC GUGGAACA UGUUCCAC CUGAUGA X GAA AAGAUCCA

1440 GGAACAAUC UCACUGCU AGCAGUGA CUGAUGA X GAA AUUGUUCC

1442 AACAAUCUC ACUGCUCU AGAGCAGU CUGAUGA X GAA AGAUUGUU

1449 UCACUGCUC UCAUUCAA UUGAAUGA CUGAUGA X GAA AGCAGUGA

1451 ACUGCUCUC AUUCAACA UGUUGAAU CUGAUGA X GAA AGAGCAGU

1454 GCUCUCAUU CAACAUAU AUAUGUUG CUGAUGA X GAA AUGAGAGC

1455 CUCUCAUUC AACAUAUC GAUAUGUU CUGAUGA X GAA AAUGAGAG

1461 UUCAACAUA UCAAGAAU AUUCUUGA CUGAUGA X GAA AUGUUGAA

1463 CAACAUAUC AAGAAUUA UAAUUCUU CUGAUGA X GAA AUAUGUUG

1470 UCAAGAAUU AUAAUCUU AAGAUUAU CUGAUGA X GAA AUUCUUGA

1471 CAAGAAUUA UAAUCUUA UAAGAUUA CUGAUGA X GAA AAUUCUUG

1473 AGAAUUAUA AUCUUAAU AUUAAGAU CUGAUGA X GAA AUAAUUCU

1476 AUUAUAAUC UUAAUUAG CUAAUUAA CUGAUGA X GAA AUUAUAAU

1478 UAUAAUCUU AAUUAGUU AACUAAUU CUGAUGA X GAA AGAUUAUA

1479 AUAAUCUUA AUUAGUUG CAACUAAU CUGAUGA X GAA AAGAUUAU

1482 AUCUUAAUU AGUUGAAG CUUCAACU CUGAUGA X GAA AUUAAGAU

1483 UCUUAAUUA GUUGAAGA UCUUCAAC CUGAUGA X GAA AAUUAAGA

1486 UAAUUAGUU GAAGACAG CUGUCUUC CUGAUGA X GAA ACUAAUUA

1499 ACAGAAAUA AGUCCUUG CAAGGACU CUGAUGA X GAA AUUUCUGU

1503 AAAUAAGUC CUUGCAUU AAUGCAAG CUGAUGA X GAA ACUUAUUU

1506 UAAGUCCUU GCAUUGUA UACAAUGC CUGAUGA X GAA AGGACUUA Table I I I

Nt . Substrate Ribo zyme Position

1511 CCUUGCAUU GUAACAUG CAUGUUAC CUGAUGA X GAA AUGCAAGG

1514 UGCAUUGUA ACAUGGUG CACCAUGU CUGAUGA X GAA ACAAUGCA

1534 GUGUGUGUU UUUUUUCC GGAAAAAA CUGAUGA X GAA ACACACAC

1535 UGUGUGUUU UUUUUCCA UGGAAAAA CUGAUGA X GAA AACACACA

1536 GUGUGUUUU UUUUCCAC GUGGAAAA CUGAUGA X GAA AAACACAC

1537 UGUGUUUUU UUUCCACU AGUGGAAA CUGAUGA X GAA AAAACACA

1538 GUGUUUUUU UUCCACUU AAGUGGAA CUGAUGA X GAA AAAAACAC

1539 UGUUUUUUU UCCACUUA UAAGUGGA CUGAUGA X GAA AAAAAACA

1540 GUUUUUUUU CCACUUAA UUAAGUGG CUGAUGA X GAA AAAAAAAC

154 1 UUUUUUUUC CACUUAAU AUUAAGUG CUGAUGA X GAA AAAAAAAA

1546 UUUCCACUU AAUAAAAU AUUUUAUU CUGAUGA X GAA AGUGGAAA

1547 UUCCACUUA AUAAAAUG CAUUUUAU CUGAUGA X GAA AAGUGGAA

1550 CACUUAAUA AAAUGAAG CUUCAUUU CUGAUGA X GAA AUUAAGUG

1579 GGAUGGAUC UUAACUUU AAAGUUAA CUGAUGA X GAA AUCCAUCC

1581 AUGGAUCUU AACUUUAA UUAAAGUU CUGAUGA X GAA AGAUCCAU

1582 UGGAUCUUA ACUUUAAA UUUAAAGU CUGAUGA X GAA AAGAUCCA

1586 UCUUAACUU UAAAAAAA UUUUUUUA CUGAUGA X GAA AGUUAAGA

1587 CUUAACUUU AAAAAAAA UUUUUUUU CUGAUGA X GAA AAGUUAAG

1588 UUAACUUUA AAAAAAAA uuuuuuuu CUGAUGA X GAA AAAGUUAA

Where "X" represents stem II region of a HH ribozyme (Hertel et al . , 1992 Nucleic Acids Res. 20 3252). The length of stem II may be > 2 base-pairs.

Table IV: Solanidine glucosyltransferase Hairpin

Ribozyme and Target Sequences

79 AUGACC AGAA GAUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UAUCC GCU GGUCAU

211 UGGGAA AGAA GGAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUCCG GAU UUCCCA

249 AACUUC AGAA GAGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCUCϋ GCU GAAGUU

376 AAUCAG AGAA GAGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACUCC GUC CUGAUU

381 AAUGCA AGAA GGAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUCCU GAU UGCAUU

429 AAGCUC AGAA GCAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUGCO GAU GAGCUU

468 CAUGUA AGAA GACA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGUCU GCU UACAUG

511 UGUGAG AGAA GUAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUACA GAC CUCACA

524 AGAUUA AGAA GCUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AAGCA GCC UAAUCU

570 UAUCUC AGAA GGUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UACCU GAU GAGAUA

603 CAGAUC AGAA GUCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA OGACA GAU GAUCUG

621 UUGGUC AGAA GACU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGUCG GAU GACCAA

636 GUCAAA AGAA GUCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGACU GUU UUUGAC

779 UGAGAG AGAA GACC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGUCC GCU CUCUCA

881 AUAGAG AGAA GAUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AAUCG GUU CUCUAU

1019 AUUACC AGAA GGCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGCCA GUU GGUAAU

1078 AUCGUA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACA GCU UACGAU

1139 UUCCAG AGAA GAAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUUCG GUU CUGGAA

1193 GAAUUG AGAA GCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUGCO GAU CAAUUC

1445 AAUGAG AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCACU GCU CUCAUU Table V

Table V: Potato Citrate Synthase Hammerhead Ribozyme and Target Sequences

Nt. Substrate Ribozyme Position

9 UUUUUCGUU CCAUCAGC GCUGAUGG CUGAUGA X GAA ACGAAAAA

10 UUUUCGUUC CAUCAGCC GGCUGAUG CUGAUGA X GAA AACGAAAA

14 CGUUCCAUC AGCCUACU AGUAGGCU CUGAUGA X GAA AUGGAACG

20 AUCAGCCUA CUUGAGAU AUCUCAAG CUGAUGA X GAA AGGCUGAU

23 AGCCUACUU GAGAUGUA UACAUCUC CUGAUGA X GAA AGUAGGCU

31 UGAGAUGUA UUCCCACU AGUGGGAA CUGAUGA X GAA ACAUCUCA

33 AGAUGUAUU CCCACUGG CCAGUGGG CUGAUGA X GAA AUACAUCU

34 GAUGUAUUC CCACUGGU ACCAGUGG CUGAUGA X GAA AAUACAUC

43 CCACUGGUA AAAGUUAA UUAACUUU CUGAUGA X GAA ACCAGUGG

49 GUAAAAGUU AAUUUUUU AAAAAAUU CUGAUGA X GAA ACUUUUAC

50 UAAAAGUUA AUUUUUUU AAAAAAAU CUGAUGA X GAA AACUUUUA

53 AAGUUAAUU UUUUUGAU AUCAAAAA CUGAUGA X GAA AUUAACUU

54 AGUUAAUUU UUUUGAUU AAUCAAAA CUGAUGA X GAA AAUUAACU

55 GUUAAUUUU UUUGAUUU AAAUCAAA CUGAUGA X GAA AAAUUAAC

56 UUAAUUUUU UUGAUUUU AAAAUCAA CUGAUGA X GAA AAAAUUAA

57 UAAUUUUUU UGAUUUUC GAAAAUCA CUGAUGA X GAA AAAAAUUA

58 AAUUUUUUU GAUUUUCG CGAAAAUC CUGAUGA X GAA AAAAAAUU

62 UUUUUGAUU UUCGCGAG CUCGCGAA CUGAUGA X GAA AUCAAAAA

63 UUUUGAUUU UCGCGAGC GCUCGCGA CUGAUGA X GAA AAUCAAAA

64 UUUGAUUUU CGCGAGCA UGCUCGCG CUGAUGA X GAA AAAUCAAA

65 UUGAUUUUC GCGAGCAA UUGCUCGC CUGAUGA X GAA AAAAUCAA

80 AAUGGUGUU CUACCGUA UACGGUAG CUGAUGA X GAA ACACCAUU

81 AUGGUGUUC UACCGUAG CUACGGUA CUGAUGA X GAA AACACCAU

83 GGUGUUCUA CCGUAGCG CGCUACGG CUGAUGA X GAA AGAACACC

88 UCUACCGUA GCGUUUCG CGAAACGC CUGAUGA X GAA ACGGUAGA

93 CGUAGCGUU UCGUUGCU AGCAACGA CUGAUGA X GAA ACGCUACG

94 GUAGCGUUU CGUUGCUG CAGCAACG CUGAUGA X GAA AACGCUAC

95 UAGCGUUUC GUUGCUGU ACAGCAAC CUGAUGA X GAA AAACGCUA

98 CGUUUCGUU GCUGUCAA UUGACAGC CUGAUGA X GAA ACGAAACG

104 GUUGCUGUC AAAGCUCC GGAGCUUU CUGAUGA X GAA ACAGCAAC

111 UCAAAGCUC CGCUCUCG CGAGAGCG CUGAUGA X GAA AGCUUUGA

116 GCUCCGCUC UCGAGCGG CCGCUCGA CUGAUGA X GAA AGCGGAGC

118 UCCGCUCUC GAGCGGUC GACCGCUC CUGAUGA X GAA AGAGCGGA

126 CGAGCGGUC CAACAGUC GACUGUUG CUGAUGA X GAA ACCGCUCG

134 CCAACAGUC AAAUGUUA UAACAUUU CUGAUGA X GAA ACUGUUGG

141 UCAAAUGUU AGCAAUUC GAAUUGCU CUGAUGA X GAA ACAUUUGA

142 CAAAUGUUA GCAAUUCU AGAAUUGC CUGAUGA X GAA AACAUUUG

148 UUAGCAAUU CUGUGCGC GCGCACAG CUGAUGA X GAA AUUGCUAA

149 UAGCAAUUC UGUGCGCU AGCGCACA CUGAUGA X GAA AAUUGCUA

162 CGCUGGCUU CAAGUCCA UGGACUUG CUGAUGA X GAA AGCCAGCG

163 GCUGGCUUC AAGUCCAA UUGGACUU CUGAUGA X GAA AAGCCAGC

168 CUUCAAGUC CAAACCUC GAGGUUUG CUGAUGA X GAA ACUUGAAG

176 CCAAACCUC UUCCGGUC GACCGGAA CUGAUGA X GAA AGGUUUGG

178 AAACCUCUU CCGGUCUU AAGACCGG CUGAUGA X GAA AGAGGUUU

179 AACCUCUUC CGGUCUUG CAAGACCG CUGAUGA X GAA AAGAGGUU

184 CUUCCGGUC UUGAUCUG CAGAUCAA CUGAUGA X GAA ACCGGAAG

186 UCCGGUCUU GAUCUGCG CGCAGAUC CUGAUGA X GAA AGACCGGA

190 GUCUUGAUC UGCGUUCU AGAACGCA CUGAUGA X GAA AUCAAGAC

196 AUCUGCGUU CUGAGCUG CAGCUCAG CUGAUGA X GAA ACGCAGAU

197 UCUGCGUUC UGAGCUGG CCAGCUCA CUGAUGA X GAA AACGCAGA

207 GAGCUGGUA CAAGAAUU AAUUCUUG CUGAUGA X GAA ACCAGCUC Table V

Nt. Substrate Ribo∑ y e Position

215 ACAAGAAUU GAUUCCUG CAGGAAUC CUGAUGA X GAA AUUCUUGU

219 GAAUUGAUU CCUGAACA UGUUCAGG CUGAUGA X GAA AUCAAUUC

220 AAUUGAUUC CUGAACAA UUGUUCAG CUGAUGA X GAA AAUCAAUU

235 AACAGGAUC GCCUGAAA UUUCAGGC CUGAUGA X GAA AUCCUGUU

249 AAAAAGAUC AAGUCAGA UCUGACUU CUGAUGA X GAA AUCUUUUU

254 GAUCAAGUC AGAUAUGA UCAUAUCU CUGAUGA X GAA ACUUGAUC

259 AGUCAGAUA UGAAAGGU ACCUUUCA CUGAUGA X GAA AUCUGACU

268 UGAAAGGUU CAAUUGGG CCCAAUUG CUGAUGA X GAA ACCUUUCA

269 GAAAGGUUC AAUUGGGA UCCCAAUU CUGAUGA X GAA AACCUUUC

273 GGUUCAAUU GGGAACAU AUGUUCCC CUGAUGA X GAA AUUGAACC

282 GGGAACAUC ACAGUUGA UCAACUGU CUGAUGA X GAA AUGUUCCC

288 AUCACAGUU GAUAUGGU ACCAUAUC CUGAUGA X GAA ACUGUGAU

292 CAGUUGAUA UGGUUCUU AAGAACCA CUGAUGA X GAA AUCAACUG

297 GAUAUGGUU CUUGGUGG CCACCAAG CUGAUGA X GAA ACCAUAUC

298 AUAUGGUUC UUGGUGGA UCCACCAA CUGAUGA X GAA AACCAUAU

300 AUGGUUCUU GGUGGAAU AUUCCACC CUGAUGA X GAA AGAACCAU

326 GACAGGAUU ACUGUGGA UCCACAGU CUGAUGA X GAA AUCCUGUC

327 ACAGGAUUA CUGUGGAA UUCCACAG CUGAUGA X GAA AAUCCUGU

340 GGAAACCUC AUUACCUU AAGGUAAU CUGAUGA X GAA AGGUUUCC

343 AACCUCAUU ACCUUGAC GUCAAGGU CUGAUGA X GAA AUGAGGUU

344 ACCUCAUUA CCUUGACC GGUCAAGG CUGAUGA X GAA AAUGAGGU

348 CAUUACCUU GACCCUGA UCAGGGUC CUGAUGA X GAA AGGUAAUG

366 GAGGGAAUU CGCUUCCG CGGAAGCG CUGAUGA X GAA AUUCCCUC

367 AGGGAAUUC GCUUCCGG CCGGAAGC CUGAUGA X GAA AAUUCCCU

371 AAUUCGCUU CCGGGGGU ACCCCCGG CUGAUGA X GAA AGCGAAUU

372 AUUCGCUUC CGGGGGUU AACCCCCG CUGAUGA X GAA AAGCGAAU

380 CCGGGGGUU GUCUAUAC GUAUAGAC CUGAUGA X GAA ACCCCCGG

383 GGGGUUGUC UAUACCUG CAGGUAUA CUGAUGA X GAA ACAACCCC

385 GGUUGUCUA UACCUGAA UUCAGGUA CUGAUGA X GAA AGACAACC

387 UUGUCUAUA CCUGAAUG CAUUCAGG CUGAUGA X GAA AUAGACAA

405 CAAAAGGUA UUACCUGC GCAGGUAA CUGAUGA X GAA ACCUUUUG

407 AAAGGUAUU ACCUGCAG CUGCAGGU CUGAUGA X GAA AUACCUUU

408 AAGGUAUUA CCUGCAGC GCUGCAGG CUGAUGA X GAA AAUACCUU

437 UGAGCCCUU GCCUGAAG CUUCAGGC CUGAUGA X GAA AGGGCUCA

448 CUGAAGGUC UUCUCUGG CCAGAGAA CUGAUGA X GAA ACCUUCAG

450 GAAGGUCUU CUCUGGCU AGCCAGAG CUGAUGA X GAA AGACCUUC

451 AAGGUCUUC UCUGGCUU AAGCCAGA CUGAUGA X GAA AAGACCUU

453 GGUCUUCUC UGGCUUCU AGAAGCCA CUGAUGA X GAA AGAAGACC

459 CUCUGGCUU CUUUUAAC GUUAAAAG CUGAUGA X GAA AGCCAGAG

460 UCUGGCUUC UUUUAACA UGUUAAAA CUGAUGA X GAA AAGCCAGA

462 UGGCUUCUU UUAACAGG CCUGUUAA CUGAUGA X GAA AGAAGCCA

463 GGCUUCUUU UAACAGGA UCCUGUUA CUGAUGA X GAA AAGAAGCC

464 GCUUCUUUU AACAGGAA UUCCUGUU CUGAUGA X GAA AAAGAAGC

465 CUUCUUUUA ACAGGAAA UUUCCUGU CUGAUGA X GAA AAAAGAAG

482 GGUGCCAUC AAAAGAGC GCUCUUUU CUGAUGA X GAA AUGGCACC

499 AAGUGAAUU CAAUUGUC GACAAUUG CUGAUGA X GAA AUUCACUU

500 AGUGAAUUC AAUUGUCU AGACAAUU CUGAUGA X GAA AAUUCACU

504 AAUUCAAUU GUCUCAGG CCUGAGAC CUGAUGA X GAA AUUGAAUU

507 UCAAUUGUC UCAGGAAU AUUCCUGA CUGAUGA X GAA ACAAUUGA

509 AAUUGUCUC AGGAAUUG CAAUUCCU CUGAUGA X GAA AGACAAUU

516 UCAGGAAUU GCAGAGUC GACUCUGC CUGAUGA X GAA AUUCCUGA

524 UGCAGAGUC GGGCAUCA UGAUGCCC CUGAUGA X GAA ACUCUGCA

531 UCGGGCAUC AUAUCCCU AGGGAUAU CUGAUGA X GAA AUGCCCGA

534 GGCAUCAUA UCCCUGAU AUCAGGGA CUGAUGA X GAA AUGAUGCC

536 CAUCAUAUC CCUGAUCA UGAUCAGG CUGAUGA X GAA AUAUGAUG

543 UCCCUGAUC AUCAUGUA UACAUGAU CUGAUGA X GAA AUCAGGGA

546 CUGAUCAUC AUGUAUAC GUAUACAU CUGAUGA X GAA AUGAUCAG Table V

Table V

Nt. Substrate Ribozyme Position

831 CUUGGUUUC AGUAGCUC GAGCUACU CUGAUGA X GAA AAACCAAG

835 GUUUCAGUA GCUCUGAA UUCAGAGC CUGAUGA X GAA ACUGAAAC

839 CAGUAGCUC UGAAAUGC GCAUUUCA CUGAUGA X GAA AGCUACUG

855 CAUGAACUU CUUAUGAG CUCAUAAG CUGAUGA X GAA AGUUCAUG

856 AUGAACUUC UUAUGAGG CCUCAUAA CUGAUGA X GAA AAGUUCAU

858 GAACUUCUU AUGAGGCU AGCCUCAU CUGAUGA X GAA AGAAGUUC

859 AACUUCUUA UGAGGCUC GAGCCUCA CUGAUGA X GAA AAGAAGUU

867 AUGAGGCUC UAUGUAAC GUUACAUA CUGAUGA X GAA AGCCUCAU

869 GAGGCUCUA UGUAACAA UUGUUACA CUGAUGA X GAA AGAGCCUC

873 CUCUAUGUA ACAAUACA UGUAUUGU CUGAUGA X GAA ACAUAGAG

879 GUAACAAUA CACAGUGA UCACUGUG CUGAUGA X GAA AUUGUUAC

889 ACAGUGAUC AUGAAGGU ACCUUCAU CUGAUGA X GAA AUCACUGU

901 AAGGUGGUA AUGUCAGU ACUGACAU CUGAUGA X GAA ACCACCUU

906 GGUAAUGUC AGUGCUCA UGAGCACU CUGAUGA X GAA ACAUUACC

913 UCAGUGCUC ACACCGGU ACCGGUGU CUGAUGA X GAA AGCACUGA

922 ACACCGGUC ACUUGGUU AACCAAGU CUGAUGA X GAA ACCGGUGU

926 CGGUCACUU GGUUGCUA UAGCAACC CUGAUGA X GAA AGUGACCG

930 CACUUGGUU GCUAGUGC GCACUAGC CUGAUGA X GAA ACCAAGUG

934 UGGUUGCUA GUGCUUUG CAAAGCAC CUGAUGA X GAA AGCAACCA

940 CUAGUGCUU UGUCUGAU AUCAGACA CUGAUGA X GAA AGCACUAG

941 UAGUGCUUU GUCUGAUC GAUCAGAC CUGAUGA X GAA AAGCACUA

944 UGCUUUGUC UGAUCCUU AAGGAUCA CUGAUGA X GAA ACAAAGCA

949 UGUCUGAUC CUUACCUC GAGGUAAG CUGAUGA X GAA AUCAGACA

952 CUGAUCCUU ACCUCUCC GGAGAGGU CUGAUGA X GAA AGGAUCAG

953 UGAUCCUUA CCUCUCCU AGGAGAGG CUGAUGA X GAA AAGGAUCA

957 CCUUACCUC UCCUUUGC GCAAAGGA CUGAUGA X GAA AGGUAAGG

959 UUACCUCUC CUUUGCUG CAGCAAAG CUGAUGA X GAA AGAGGUAA

962 CCUCUCCUU UGCUGCUG CAGCAGCA CUGAUGA X GAA AGGAGAGG

963 CUCUCCUUU GCUGCUGC GCAGCAGC CUGAUGA X GAA AAGGAGAG

973 CUGCUGCUU UGAAUGGU ACCAUUCA CUGAUGA X GAA AGCAGCAG

974 UGCUGCUUU GAAUGGUU AACCAUUC CUGAUGA X GAA AAGCAGCA

982 UGAAUGGUU UAGCCGGA UCCGGCUA CUGAUGA X GAA ACCAUUCA

983 GAAUGGUUU AGCCGGAC GUCCGGCU CUGAUGA X GAA AACCAUUC

984 AAUGGUUUA GCCGGACC GGUCCGGC CUGAUGA X GAA AAACCAUU

996 GGACCACUU CAUGGUUU AAACCAUG CUGAUGA X GAA AGUGGUCC

997 GACCACUUC AUGGUUUA UAAACCAU CUGAUGA X GAA AAGUGGUC

600 UUCAUGGUU UAGCCAAU AUUGGCUA CUGAUGA X GAA ACCAUGAA

1004 UCAUGGUUU AGCCAAUC GAUUGGCU CUGAUGA X GAA AACCAUGA

1005 CAUGGUUUA GCCAAUCA UGAUUGGC CUGAUGA X GAA AAACCAUG

1012 UAGCCAAUC AGGAAGUU AACUUCCU CUGAUGA X GAA AUUGGCUA

1020 CAGGAAGUU UUGCUAUG CAUAGCAA CUGAUGA X GAA ACUUCCUG

1021 AGGAAGUUU UGCUAUGG CCAUAGCA CUGAUGA X GAA AACUUCCU

1022 GGAAGUUUU GCUAUGGA UCCAUAGC CUGAUGA X GAA AAACUUCC

1026 GUUUUGCUA UGGAUAAA UUUAUCCA CUGAUGA X GAA AGCAAAAC

1032 CUAUGGAUA AAAUCUGU ACAGAUUU CUGAUGA X GAA AUCCAUAG

1037 GAUAAAAUC UGUUGUAG CUACAACA CUGAUGA X GAA AUUUUAUC

1041 AAAUCUGUU GUAGAAGA UCUUCUAC CUGAUGA X GAA ACAGAUUU

1044 UCUGUUGUA GAAGAAUG CAUUCUUC CUGAUGA X GAA ACAACAGA

1065 GAGAACAUU UCCAAAGA UCUUUGGA CUGAUGA X GAA AUGUUCUC

1066 AGAACAUUU CCAAAGAG CUCUUUGG CUGAUGA X GAA AAUGUUCU

1067 GAACAUUUC CAAAGAGC GCUCUUUG CUGAUGA X GAA AAAUGUUC

1079 AGAGCAGUU GAAAGACU AGUCUUUC CUGAUGA X GAA ACUGCUCU

1088 GAAAGACUA UGUUUGGA UCCAAACA CUGAUGA X GAA AGUCUUUC

1092 GACUAUGUU UGGAAAAC GUUUUCCA CUGAUGA X GAA ACAUAGUC

1093 ACUAUGUUU GGAAAACA UGUUUUCC CUGAUGA X GAA AACAUAGU

1103 GAAAACAUU GAACAGUG CACUGUUC CUGAUGA X GAA AUGUUUUC

1119 GGCAAGGUU GUCCCUGG CCAGGGAC CUGAUGA X GAA ACCUUGCC Table V

Nt. Substrate Ribo∑ .yme Position

1122 AAGGUUGUC CCUGGUUU AAACCAGG CUGAUGA X GAA ACAACCUU

1129 UCCCUGGUU UUGGACAU AUGUCCAA CUGAUGA X GAA ACCAGGGA

1130 CCCUGGUUU UGGACAUG CAUGUCCA CUGAUGA X GAA AACCAGGG

1131 CCUGGUUUU GGACAUGG CCAUGUCC CUGAUGA X GAA AAACCAGG

1143 CAUGGAGUU CUGCGAAA UUUCGCAG CUGAUGA X GAA ACUCCAUG

1144 AUGGAGUUC UGCGAAAG CUUUCGCA CUGAUGA X GAA AACUCCAU

1158 AAGACUGUA CCAAGAUA UAUCUUGG CUGAUGA X GAA ACAGUCUU

1166 ACCAAGAUA UACAUGCC GGCAUGUA CUGAUGA X GAA AUCUUGGU

1168 CAAGAUAUA CAUGCCAG CUGGCAUG CUGAUGA X GAA AUAUCUUG

1184 GAGAGAGUU CGCUAUGA UCAUAGOG CUGAUGA X GAA ACUCUCUC

1185 AGAGAGUUC GCUAUGAA UUCAUAGC CUGAUGA X GAA AACUCUCU

1189 AGUUCGCUA UGAAGCAU AUGCUUCA CUGAUGA X GAA AGCGAACU

1198 UGAAGCAUU UGCCUGAA UUCAGGCA CUGAUGA X GAA AUGCUUCA

1199 GAAGCAUUU GCCUGAAG CUUCAGGC CUGAUGA X GAA AAUGCUUC

1210 CUGAAGAUC CACUGUUU AAACAGUG CUGAUGA X GAA AUCUUCAG

1217 UCCACUGUU UCAACUGG CCAGUUGA CUGAUGA X GAA ACAGUGGA

1218 CCACUGUUU CAACUGGU ACCAGUUG CUGAUGA X GAA AACAGUGG

1219 CACUGUUUC AACUGGUU AACCAGUU CUGAUGA X GAA AAACAGUG

1227 CAACUGGUU UCAAAACU AGUUUUGA CUGAUGA X GAA ACCAGUUG

1228 AACUGGUUU CAAAACUC GAGUUUUG CUGAUGA X GAA AACCAGUU

1229 ACUGGUUUC AAAACUCU AGAGUUUU CUGAUGA X GAA AAACCAGU

1236 UCAAAACUC UACGAAGU ACUUCGUA CUGAUGA X GAA AGUUUUGA

1238 AAAACUCUA CGAAGUUU AAACUUCG CUGAUGA X GAA AGAGUUUU

1245 UACGAAGUU UUCCUCCU AGGAGGAA CUGAUGA X GAA ACUUCGUA

1246 ACGAAGUUU UCCUCCUG CAGGAGGA CUGAUGA X GAA AACUUCGU

1247 CGAAGUUUU CCUCCUGU ACAGGAGG CUGAUGA X GAA AAACUUCG

1248 GAAGUUUUC CUCCUGUU AACAGGAG CUGAUGA X GAA AAAACUUC

1251 GUUUUCCUC CUGUUCUU AAGAACAG CUGAUGA X GAA AGGAAAAC

1256 CCUCCUGUU CUUACAGA UCUGUAAG CUGAUGA X GAA ACAGGAGG

1257 CUCCUGUUC UUACAGAA UUCUGUAA CUGAUGA X GAA AACAGGAG

1259 CCUGUUCUU ACAGAACU AGUUCUGU CUGAUGA X GAA AGAACAGG

1260 CUGUUCUUA CAGAACUU AAGUUCUG CUGAUGA X GAA AAGAACAG

1268 ACAGAACUU GGCAAAGU ACUUUGCC CUGAUGA X GAA AGUUCUGU

1277 GGCAAAGUU AAAACCUU AAGGUUUU CUGAUGA X GAA ACUUUGCC

1278 GCAAAGUUA AAACCUUG CAAGGUUU CUGAUGA X GAA AACUUUGC

1285 UAAAACCUU GGCCAAAU AUUUGGCC CUGAUGA X GAA AGGUUUUA

1296 CCAAAUGUU GAUGCCCA UGGGCAUC CUGAUGA X GAA ACAUUUGG

1316 UGGUGUGUU GUUGAACU AGUUCAAC CUGAUGA X GAA ACACACCA

1319 UGUGUUGUU GAACUAUU AAUAGUUC CUGAUGA X GAA ACAACACA

1325 GUUGAACUA UUAUGGUU AACCAUAA CUGAUGA X GAA AGUUCAAC

1327 UGAACUAUU AUGGUUUA UAAACCAU CUGAUGA X GAA AUAGUUCA

1328 GAACUAUUA UGGUUUAA UUAAACCA CUGAUGA X GAA AAUAGUUC

1333 AUUAUGGUU UAACUGAA UUCAGUUA CUGAUGA X GAA ACCAUAAU

1334 UUAUGGUUU AACUGAAG CUUCAGUU CUGAUGA X GAA AACCAUAA

1335 UAUGGUUUA ACUGAAGC GCUUCAGU CUGAUGA X GAA AAACCAUA

1349 AGCAAGAUA UUAUACGG CCGUAUAA CUGAUGA X GAA AUCUUGCU

1351 CAAGAUAUU AUACGGUC GACCGUAU CUGAUGA X GAA AUAUCUUG

1352 AAGAUAUUA UACGGUCC GGACCGUA CUGAUGA X GAA AAUAUCUU

1354 GAUAUUAUA CGGUCCUC GAGGACCG CUGAUGA X GAA AUAAUAUC

1359 UAUACGGUC CUCUUUGG CCAAAGAG CUGAUGA X GAA ACCGUAUA

1362 ACGGUCCUC UUUGGCGU ACGCCAAA CUGAUGA X GAA AGGACCGU

1364 GGUCCUCUU UGGCGUAU AUACGCCA CUGAUGA X GAA AGAGGACC

1365 GUCCUCUUU GGCGUAUC GAUACGCC CUGAUGA X GAA AAGAGGAC

1371 UUUGGCGUA UCAAGAGC GCUCUUGA CUGAUGA X GAA ACGCCAAA

1373 UGGCGUAUC AAGAGCUC GAGCUCUU CUGAUGA X GAA AUACGCCA

1381 CAAGAGCUC UUGGCAUU AAUGCCAA CUGAUGA X GAA AGCUCJUG

1383 AGAGCUCUU GGCAUUUG CAAAUGCC CUGAUGA X GAA AGAGCUCU Table V

Nt. Substrate Ribo∑ .yme Position

1389 CUUGGCAUU UGCUCUCA UGAGAGCA CUGAUGA X GAA AUGCCAAG

1390 UUGGCAUUU GCUCUCAG CUGAGAGC CUGAUGA X GAA AAUGCCAA

1394 CAUUUGCUC UCAGCUAA UUAGCUGA CUGAUGA X GAA AGCAAAUG

1396 UUUGCUCUC AGCUAAUU AAUUAGCU CUGAUGA X GAA AGAGCAAA

1401 UCUCAGCUA AUUUGGGA UCCCAAAU CUGAUGA X GAA AGCUGAGA

1404 CAGCUAAUU UGGGACCG CGGUCCCA CUGAUGA X GAA AUUAGCUG

1405 AGCUAAUUU GGGACCGA UCGGUCCC CUGAUGA X GAA AAUUAGCU

1417 ACCGAGCUC UUGGAUUG CAAUCCAA CUGAUGA X GAA AGCUCGGU

1419 CGAGCUCUU GGAUUGCC GGCAAUCC CUGAUGA X GAA AGAGCUCG

1424 UCUUGGAUU GCCGCUAG CUAGCGGC CUGAUGA X GAA AUCCAAGA

1431 UUGCCGCUA GAGAGGCC GGCCUCUC CUGAUGA X GAA AGCGGCAA

1449 AAGAGUGUC ACAAUGGA UCCAUUGU CUGAUGA X GAA ACACUCUU

1464 GAGUGGCUU GAGAACCA UGGUUCUC CUGAUGA X GAA AGCCACUC

1491 GCAUGAAUU GUUUGAAA UUUCAAAC CUGAUGA X GAA AUUCAUGC

1494 UGAAUUGUU UGAAAUCU AGAUUUCA CUGAUGA X GAA ACAAUUCA

1495 GAAUUGUUU GAAAUCUC GAGAUUUC CUGAUGA X GAA AACAAUUC

1501 UUUGAAAUC UCGCGAGC GCUCGCGA CUGAUGA X GAA AUUUCAAA

1503 UGAAAUCUC GCGAGCAU AUGCUCGC CUGAUGA X GAA AGAUUUCA

1512 GCGAGCAUA AAACACAA UUGUGUUU CUGAUGA X GAA AUGCUCGC

1524 CACAAUGUA UAAUCUCU AGAGAUUA CUGAUGA X GAA ACAUUGUG

1526 CAAUGUAUA AUCUCUAU AUAGAGAU CUGAUGA X GAA AUACAUUG

1529 UGUAUAAUC UCUAUGAA UUCAUAGA CUGAUGA X GAA AUUAUACA

1531 UAUAAUCUC UAUGAAUA UAUUCAUA CUGAUGA X GAA AGAUUAUA

1533 UAAUCUCUA UGAAUAAU AUUAUUCA CUGAUGA X GAA AGAGAUUA

1539 CUAUGAAUA AUUGCUUG CAAGCAAU CUGAUGA X GAA AUUCAUAG

1542 UGAAUAAUU GCUUGACA UGUCAAGC CUGAUGA X GAA AUUAUUCA

1546 UAAUUGCUU GACAAAGC GCUUUGUC CUGAUGA X GAA AGCAAUUA

1558 AAAGCACUC CUUUCUUG CAAGAAAG CUGAUGA X GAA AGUGCUUU

1561 GCACUCCUU UCUUGGGG CCCCAAGA CUGAUGA X GAA AGGAGUGC

1562 CACUCCUUU CUUGGGGG CCCCCAAG CUGAUGA X GAA AAGGAGUG

1563 ACUCCUUUC UUGGGGGA UCCCCCAA CUGAUGA X GAA AAAGGAGU

1565 UCCUUUCUU GGGGGACA UGUCCCCC CUGAUGA X GAA AGAAAGGA

1578 GACAAGAUA GGUCGGCC GGCCGACC CUGAUGA X GAA AUCUUGUC

1582 AGAUAGGUC GGCCCUUC GAAGGGCC CUGAUGA X GAA ACCUAUCU

1589 UCGGCCCUU CAAUGGGU ACCCAUUG CUGAUGA X GAA AGGGCCGA

1590 CGGCCCUUC AAUGGGUU AACCCAUU CUGAUGA X GAA AAGGGCCG

1598 CAAUGGGUU AACGAACU AGUUCGUU CUGAUGA X GAA ACCCAUUG

1599 AAUGGGUUA ACGAACUU AAGUUCGU CUGAUGA X GAA AACCCAUU

1607 AACGAACUU CAGUUCAA UUGAACUG CUGAUGA X GAA AGUUCGUU

1608 ACGAACUUC AGUUCAAA UUUGAACU CUGAUGA X GAA AAGUUCGU

1612 ACUUCAGUU CAAACUUC GAAGUUUG CUGAUGA X GAA ACUGAAGU

1613 CUUCAGUUC AAACUUCA UGAAGUUU CUGAUGA X GAA AACUGAAG

1619 UUCAAACUU CACUGAAU AUUCAGUG CUGAUGA X GAA AGUUUGAA

1620 UCAAACUUC ACUGAAUU AAUUCAGU CUGAUGA X GAA AAGUUUGA

1628 CACUGAAUU UGUGUGAA UUCACACA CUGAUGA X GAA AUUCAGUG

1629 ACUGAAUUU GUGUGAAU AUUCACAC CUGAUGA X GAA AAUUCAGU

1638 GUGUGAAUU GUAUGGUU AACCAUAC CUGAUGA X GAA AUUCACAC

1641 UGAAUUGUA UGGUUUCU AGAAACCA CUGAUGA X GAA ACAAUUCA

1646 UGUAUGGUU UCUCGAGA UCUCGAGA CUGAUGA X GAA ACCAUACA

1647 GUAUGGUUU CUCGAGAC GUCUCGAG CUGAUGA X GAA AACCAUAC

1648 UAUGGUUUC UCGAGACU AGUCUCGA CUGAUGA X GAA AAACCAUA

1650 UGGUUUCUC GAGACUUG CAAGUCUC CUGAUGA X GAA AGAAACCA

1657 UCGAGACUU GUCCUGAA UUCAGGAC CUGAUGA X GAA AGUCUCGA

1660 AGACUUGUC CUGAAUUU AAAUUCAG CUGAUGA X GAA ACAAGUCU

1667 UCCUGAAUU UUGAACUU AAGUUCAA CUGAUGA X GAA AUUCAGGA

1668 CCUGAAUUU UGAACUUA UAAGUUCA CUGAUGA X GAA AAUUCAGG

1669 CUGAAUUUU GAACUUAG CUAAGUUC CUGAUGA X GAA AAAUUCAG Table V

Nt. Substrate Ribo∑ .yme Position

1675 UUUGAACUU AGUCUAGU ACUAGACU CUGAUGA X GAA AGUUCAAA

1676 UUGAACUUA GUCUAGUG CACUAGAC CUGAUGA X GAA AAGUUCAA

1679 AACUUAGUC UAGUGGAU AUCCACUA CUGAUGA X GAA ACUAAGUU

1681 CUUAGUCUA GUGGAUUC GAAUCCAC CUGAUGA X GAA AGACUAAG

1668 UAGUGGAUU CAUUUUUC GAAAAAUG CUGAUGA X GAA AUCCACUA

1689 AGUGGAUUC AUUUUUCU AGAAAAAU CUGAUGA X GAA AAUCCACU

1692 GGAUUCAUU UUUCUUCA UGAAGAAA CUGAUGA X GAA AUGAAUCC

1693 GAUUCAUUU UUCUUCAU AUGAAGAA CUGAUGA X GAA AAUGAAUC

1694 AUUCAUUUU UCUUCAUU AAUGAAGA CUGAUGA X GAA AAAUGAAU

1695 UUCAUUUUU CUUCAUUC GAAUGAAG CUGAUGA X GAA AAAAUGAA

1696 UCAUUUUUC UUCAUUCC GGAAUGAA CUGAUGA X GAA AAAAAUGA

1698 AUUUUUCUU CAUUCCGA UCGGAAUG CUGAUGA X GAA AGAAAAAU

1699 uuuuucuuc AUUCCGAA UUCGGAAU CUGAUGA X GAA AAGAAAAA

1702 UUCUUCAUU CCGAAUUC GAAUUCGG CUGAUGA X GAA AUGAAGAA

1703 UCUUCAUUC CGAAUUCC GGAAUUCG CUGAUGA X GAA AAUGAAGA

1709 UUCCGAAUU CCUCACAC GUGUGAGG CUGAUGA X GAA AUUCGGAA

1710 UCCGAAUUC CUCACACG CGUGUGAG CUGAUGA X GAA AAUUCGGA

1713 GAAUUCCUC ACACGCUG CAGCGUGU CUGAUGA X GAA AGGAAUUC

1724 ACGCUGAUC CAGCAUGU ACAUGCUG CUGAUGA X GAA AUCAGCGU

1733 CAGCAUGUA AAAAUUAA UUAAUUUU CUGAUGA X GAA ACAUGCUG

1739 GUAAAAAUU AAUAGGUC GACCUAUU CUGAUGA X GAA AUUUUUAC

1740 UAAAAAUUA AUAGGUCA UGACCUAU CUGAUGA X GAA AAUUUUUA

1743 AAAUUAAUA GGUCAAUG CAUUGACC CUGAUGA X GAA AUUAAUUU

1747 UAAUAGGUC AAUGCUAU AUAGCAUU CUGAUGA X GAA ACCUAUUA

1754 UCAAUGCUA UUAAUCGC GCGAUUAA CUGAUGA X GAA AGCAUUGA

1756 AAUGCUAUU AAUCGCGU ACGCGAUU CUGAUGA X GAA AUAGCAUU

1757 AUGCUAUUA AUCGCGUU AACGCGAU CUGAUGA X GAA AAUAGCAU

1760 CUAUUAAUC GCGUUCUU AAGAACGC CUGAUGA X GAA AUUAAUAG

1765 AAUCGCGUU CUUGGUUG CAACCAAG CUGAUGA X GAA ACGCGAUU

1766 AUCGCGUUC UUGGUUGC GCAACCAA CUGAUGA X GAA AACGCGAU

1768 CGCGUUCUU GGUUGCCA UGGCAACC CUGAUGA X GAA AGAACGCG

1772 UUCUUGGUU GCCAUUAG CUAAUGGC CUGAUGA X GAA ACCAAGAA

1778 GUUGCCAUU AGACUUGU ACAAGUCU CUGAUGA X GAA AUGGCAAC

1779 UUGCCAUUA GACUUGUG CACAAGUC CUGAUGA X GAA AAUGGCAA

1784 AUUAGACUU GUGAAUGA UCAUUCAC CUGAUGA X GAA AGUCUAAU

1795 GAAUGACUU CCUUUGCU AGCAAAGG CUGAUGA X GAA AGUCAUUC

1796 AAUGACUUC CUUUGCUG CAGCAAAG CUGAUGA X GAA AAGUCAUU

1799 GACUUCCUU UGCUGGAA UUCCAGCA CUGAUGA X GAA AGGAAGUC

1800 ACUUCCUUU GCUGGAAA UUUCCAGC CUGAUGA X GAA AAGGAAGU

1811 UGGAAAGUU AGUAAUCG CGAUUACU CUGAUGA X GAA ACUUUCCA

1812 GGAAAGUUA GUAAUCGG CCGAUUAC CUGAUGA X GAA AACUUUCC

1815 AAGUUAGUA AUCGGCUG CAGCCGAU CUGAUGA X GAA ACUAACUU

1818 UUAGUAAUC GGCUGAUU AAUCAGCC CUGAUGA X GAA AUUACUAA

1826 CGGCUGAUU CACGCAAU AUUGCGUG CUGAUGA X GAA AUCAGCCG

1827 GGCUGAUUC ACGCAAUA UAUUGCGU CUGAUGA X GAA AAUCAGCC

1835 CACGCAAUA AACUGCAA UUGCAGUU CUGAUGA X GAA AUUGCGUG

1845 ACUGCAAUU GUGUAGUU AACUAC C CUGAUGA X GAA AUUGCAGU

1850 AAUUGUGUA GUUUCUUA UAAGAAAC CUGAUGA X GAA ACACAAUU

1853 UGUGUAGUU UCUUAAAU AUUUAAGA CUGAUGA X GAA ACUACACA

1854 GUGUAGUUU CUUAAAUU AAUUUAAG CUGAUGA X GAA AACUACAC

1855 UGUAGUUUC UUAAAUUU AAAUUUAA CUGAUGA X GAA AAACUACA

1857 UAGUUUCUU AAAUUUGC GCAAAUUU CUGAUGA X GAA AGAAACUA

1858 AGUUUCUUA AAUUUGCU AGCAAAUU CUGAUGA X GAA AAGAAACU

1862 UCUUAAAUU UGCUAAUU AAUUAGCA CUGAUGA X GAA AUUUAAGA

1863 CUUAAAUUU GCUAAUUC GAAUUAGC CUGAUGA X GAA AAUUUAAG

1867 AAUUUGCUA AUUCUUAU AUAAGAAU CUGAUGA X GAA AGCAAAUU

1870 UUGCUAAUU CUUAUUUG CAAAUAAG CUGAUGA X GAA AUUAGCAA Table V

Where "X" represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252). The length of stem II may be > 2 base-pairs.

Table VI: Potato Citrate Synthase Hairpin Ribozyme and Target Sequences

Claims

1. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a plant gene involved in the biosynthesis of alkaloid compounds.

2. The enzymatic nucleic acid molecule of claim

1, wherein said plant is a solanaceous plant.

3. The enzymatic nucleic acid molecule of claim

2, wherein said plant is selected from a group consisting of potato, tomato, pepper, eggplant and ditura.

4. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid is in a hammerhead configuration .

5. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid is in a hairpin configuration.

6. The enzymatic nucleic acid molecule of claim 1, wherein said nucleic acid is in a hepatitis δ virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration.

7. The enzymatic nucleic acid of claim 1, wherein said nucleic acid comprises between 12 and 100 bases complementary to RNA of said gene.

8. The enzymatic nucleic acid of claim 1, wherein said nucleic acid comprises between 14 and 24 bases complementary to RNA of said gene.

9. The enzymatic nucleic acid of claim 4, wherein said hammerhead comprises a stem II region of length greater than on equal to two base-pairs.

10. The enzymatic nucleic acid of claim 5, wherein said hairpin comprises a stem II region of length between three and seven base-pairs.

11. The enzymatic nucleic acid of claim 5, wherein said hairpin comprises a stem IV region of length greater than or equal to two base-pairs.

12. The enzymatic nucleic acid of claim 1, wherein said gene is solanidine UDP-glucose glucosyl-transferase.

13. The enzymatic nucleic acid molecule of claim 12, wherein said nucleic acid specifically cleaves any of sequences shown in Table III, wherein said nucleic acid is in a hammerhead configuration.

14. The enzymatic nucleic acid molecule of claim 12, wherein said nucleic acid specifically cleaves any of sequences shown in Table IV, wherein said nucleic acid is in a hairpin configuration.

15. The enzymatic nucleic acid molecule of any of claims 13 or 14, consisting essentially of one or more sequences selected from the group shown in Tables III and IV.

16. A plant cell comprising the enzymatic nucleic acid molecule of claim 1.

17. A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid molecule of claim 1.

18. An expression vector comprising nucleic acid encoding the enzymatic nucleic acid molecule of claim 1, in a manner which allows expression and/or delivery of that enzymatic nucleic acid molecule within a plant cell.

19. An expression vector comprising nucleic acid encoding a plurality of enzymatic nucleic acid molecules of claim 1, in a manner which allows expression and/or delivery of said enzymatic nucleic acid molecules within a plant cell.

20. A plant cell comprising the expression vector of claim 18.

21. A plant cell comprising the expression vector of claim 19.

22. A transgenic plant and the progeny thereof, comprising the expression vector of claim 18.

23. A transgenic plant and the progeny thereof, comprising the expression vector of claim 19.

24. A method for modulating expression of an gene in a plant by administering to said plant the enzymatic nucleic acid molecule of claim 1.

25. The method of claim 24, wherein said plant is a potato plant.

26. The method of claim 24, wherein said gene is solanidine UDP-glucose glucosyl-transferase.

27. The expression vector of claim 18, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) a gene encoding at least one said enzymatic nucleic acid molecule; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.

28. The expression vector of claim 18, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said enzymatic nucleic acid molecule, wherein said gene is operably linked to the 3 '-end of said open reading frame; and wherein said gene is operably linked to said initiation region, 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.

29. The expression vector of claim 18, 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 .

30. The expression vector of claim 18, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said enzymatic nucleic acid molecule, wherein said gene is operably linked to the 3 ' -end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.

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

32. The transgenic plant of Claim 31, wherein said gene is solanidine UDP-glucose glucosyl-transferase.

33. The transgenic plant of Claim 31, wherein the plant is transformed with Agrobacterium, bombarding with DNA coated microprojectiles, whiskers, or electro- poration.

34. The transgenic plant of Claim 33, wherein said bombarding with DNA coated microprojectiles is done with the gene gun.

35. The transgenic plant of Claim 31, wherein said plant contains a selectable marker selected from the group consisting of chlorosulfuron, hygromycin, bar gene, bromoxynil, and kanamycin and the like.

36. The transgenic plant of Claim 31, wherein said nucleic acid is operably linked to a promoter selected from the group consisting of octopine synthetase, the nopaline synthase, the manopine synthetase, cauliflower mosaic virus (35S) ; ribulose-1, 6-biphosphate (RUBP) carboxylase small subunit (ssu) , the beta-conglycinin, the phaseolin promoter, napin, gamma zein, globulin, the ADH promoter, heat-shock, actin, and ubiquitin.

37. The transgenic plant of Claim 31, said enzymatic nucleic acid molecule is in a hammerhead, hairpin, hepatitis Δ virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration

38. The transgenic plant of Claim 31, wherein said enzymatic nucleic acid with RNA cleaving activity encoded as a monomer.

39. The transgenic plant of Claim 31, wherein said enzymatic nucleic acid with RNA cleaving activity encoded as a multimer.

40. The transgenic plant of Claim 31, wherein the nucleic acids encoding for said enzymatic nucleic acid molecule with RNA cleaving activity is operably linked to the 3' end of an open reading frame.

41. An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a plant gene involved in the flower formation.

42. The enzymatic nucleic acid molecule of claim 41, wherein said plant is a potato plant.

43. The enzymatic nucleic acid molecule of claim 41, wherein said plant is selected from a group consisting of Lettuce, spinach, cabbage, brussel sprouts, arugula, kale, collards, chard, beet, turnip, sweet potato and turfgrass.

44. The enzymatic nucleic acid molecule of claim 41, wherein said nucleic acid is in a hammerhead configuration.

45. The enzymatic nucleic acid molecule of claim 41, wherein said nucleic acid is in a hairpin configuration.

46. The enzymatic nucleic acid molecule of claim 41, wherein said nucleic acid is in a hepatitis δ virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration.

47. The enzymatic nucleic acid of claim 41, wherein said nucleic acid comprises between 12 and 100 bases complementary to RNA of said gene.

48. The enzymatic nucleic acid of claim 41, wherein said nucleic acid comprises between 14 and 24 bases complementary to RNA of said gene.

49. The enzymatic nucleic acid of claim 44, wherein said hammerhead comprises a stem II region of length greater than on equal to two base-pairs.

50. The enzymatic nucleic acid of claim 45, wherein said hairpin comprises a stem II region of length between three and seven base-pairs.

51. The enzymatic nucleic acid of claim 45, wherein said hairpin comprises a stem IV region of length greater than or equal to two base-pairs.

52. The enzymatic nucleic acid of claim 41, wherein said gene is citrate synthase.

53. The enzymatic nucleic acid molecule of claim 52, wherein said nucleic acid specifically cleaves any of sequences shown in Table V, wherein said nucleic acid is in a hammerhead configuration.

54. The enzymatic nucleic acid molecule of claim 52, wherein said nucleic acid specifically cleaves any of sequences shown in Table VI, wherein said nucleic acid is in a hairpin configuration.

55. The enzymatic nucleic acid molecule of any of claims 53 or 54, consisting essentially of one or more sequences selected from the group shown in Tables V and VI.

56. A plant cell comprising the enzymatic nucleic acid molecule of claim 41.

57. A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid molecule of claim

41.

58. An expression vector comprising nucleic acid encoding the enzymatic nucleic acid molecule of claim 41, in a manner which allows expression and/or delivery of that enzymatic nucleic acid molecule within a plant cell.

59. An expression vector comprising nucleic acid encoding a plurality of enzymatic nucleic acid molecules of claim 41, in a manner which allows expression and/or delivery of said enzymatic nucleic acid molecules within a plant cell.

60. A plant cell comprising the expression vector of claim 58.

61. A plant cell comprising the expression vector of claim 59.

62. A transgenic plant and the progeny thereof, comprising the expression vector of claim 58.

63. A transgenic plant and the progeny thereof, comprising the expression vector of claim 59.

64. A method for modulating expression of an gene in a plant by administering to said plant the enzymatic nucleic acid molecule of claim 41.

65. The method of claim 64, wherein said plant is a potato plant.

66. The method of claim 64, wherein said gene is citrate synthase.

67. The expression vector of claim 58, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) a gene encoding at least one said enzymatic nucleic acid molecule; and wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.

68. The expression vector of claim 58, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; d) a gene encoding at least one said enzymatic nucleic acid molecule, wherein said gene is operably linked to the 3 ' -end of said open reading frame; and wherein said gene is operably linked to said initiation region, 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.

69. The expression vector of claim 58, 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.

70. The expression vector of claim 58, wherein said vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding at least one said enzymatic nucleic acid molecule, wherein said gene is operably linked to the 3 ' -end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.

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

72. The transgenic plant of Claim 71, wherein said gene is citrate synthase.

73. The transgenic plant of Claim 71, wherein the plant is transformed with Agrobacterium, bombarding with DNA coated microprojectiles, whiskers, or electro- poration.

74. The transgenic plant of Claim 73, wherein said bombarding with DNA coated microprojectiles is done with the gene gun.

75. The transgenic plant of Claim 71, wherein said plant contains a selectable marker selected from the group consisting of chlorosulfuron, hygromycin, bar gene, bromoxynil, and kanamycin and the like.

76. The transgenic plant of Claim 71, wherein said nucleic acid is operably linked to a promoter selected from the group consisting of octopine synthetase, the nopaline synthase, the manopine synthetase, cauliflower mosaic virus (35S) ; ribulose-1, 6-biphosphate (RUBP) carboxylase small subunit (ssu) , the beta-conglycinin, the phaseolin promoter, napin, gamma zein, globulin, the ADH promoter, heat-shock, actin, and ubiquitin.

77. The transgenic plant of Claim 71, said enzymatic nucleic acid molecule is in a hammerhead, hairpin, hepatitis δ virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration

78. The transgenic plant of Claim 71, wherein said enzymatic nucleic acid with RNA cleaving activity encoded as a monomer.

79. The transgenic plant of Claim 71, wherein said enzymatic nucleic acid with RNA cleaving activity encoded as a multimer.

80. The transgenic plant of Claim 71, wherein the nucleic acids encoding for said enzymatic nucleic acid molecule with RNA cleaving activity is operably linked to the 3' end of an open reading frame.