Nucleic acid encoding delta-9 desaturase

Abstract

The present invention relates to nucleic acid molecules encoding delta 9 desaturase gene, and expression vectors, plant cells, and transgenic plants expressing delta 9 desaturase nucleic acid. The nucleic acid molecules of the present invention can be used, for example, to decrease delta 9 desaturase activity in plant cells, resulting in decreased unsaturated fatty acid production.

Description

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 mutagenesis of genes have also been used to silence gene expression. This approach, however, cannot be designed to specifically inactivate the gene of interest. Applicant believes that ribozyme technology offers an attractive new means to alter gene expression in plants.

Naturally occurring antisense RNA was first discovered in bacteria over a decade ago (Simons and Kleckner, 1983 Cell 34, 683-691). It is thought to be one way in which bacteria can regulate their gene expression (Green et al., 1986 Ann. Rev. Biochem . 55: 567-597; Simons 1988 Gene 72: 35-44). The first demonstration of antisense-mediated inhibition of gene expression was reported in mammalian cells (Izant and Weintraub 1984 Cell 36: 1007-1015). There are many examples in the literature for the use of antisense RNA to modulate gene expression in plants. Following are a few examples:

Shewmaker et al., U.S. Pat. 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 (X1-406) and Feyter et al., 1996 , Mol. Gen. Genet . 250, 329-338], that propose using hammerhead ribozymes to modulate: virus replication, expression of viral genes and/or reporter genes. None of these publications report the use 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 glutamine synthetase enzyme in Phaseolus vulgaris.

Hitz et al., (WO 91/18985) describe a method for using the soybean Δ-9 desaturasc enzyme to modify plant oil composition. The application describes the use of soybean Δ-9 desaturase sequence to isolate Δ-9 desaturase genes from other species.

The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the use of ribozymes in maize. Furthermore, Applicant believes that the references do not disclose and/or enable the use of ribozymes to down regulate genes in plant cells, let alone plants.

SUMMARY OF THE INVENTION

The invention features modulation of gene expression in plants specifically using enzymatic nucleic acid molecules. Preferably, the gene is an endogenous gene. The enzymatic nucleic acid molecule with RNA cleaving activity may be in the form of, but 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. Two targets which are exemplified herein are delta 9 desaturase and granule bound starch synthase (GBSS).

Until the discovery of the inventions herein, nucleic acid-based reagents, such as enzymatic nucleic acids (ribozymes), had yet to be demonstrated to modulate and/or inhibit gene expression in plants such as monocot plants (e.g., corn). Ribozymes can be used to modulate a specific trait of a plant cell, for example, by modulating the activity of an enzyme involved in a biochemical pathway. It may be desirable, in some instances, to 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

FIG. 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.

FIG. 2 a is a diagrammatic representation of the hammerhead ribozyme domain known in the art; FIG. 2 b is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987 , Nature , 327, 596-600) into a substrate and enzyme portion; FIG. 2 c is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988 , Nature , 334, 585-591) into two portions; and FIG. 2 d is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989 , Nucl. Acids. Res ., 17, 1371-1371) into two portions.

FIG. 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs (i.e., n is 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.

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

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

FIG. 6 is a schematic representation of an RNaseH accessibility assay. Specifically, the left side of FIG. 6 is a diagram of complementary DNA oligonucleotides bound to accessible sites on the target RNA. Complementary DNA oligonucleotides are represented by broad lines labeled A, B, and C. Target RNA is represented by the thin, twisted line. The right side of FIG. 6 is a schematic of a gel separation of uncut target RNA from a cleaved target RNA. Detection of target RNA is by autoradiography of body-labeled, T7 transcript. The bands common to each lane represent uncleaved target RNA; the bands unique to each lane represent the cleaved products.

FIG. 7 is a graphical representation of RNaseH accessibility of GBSS RNA.

FIG. 8 is a graphical representation of GBSS RNA cleavage by ribozymes at different temperatures.

FIG. 9 is a graphical representation of GBSS RNA cleavage by multiple ribozymes.

FIGS. 10A-C list the nucleotide sequence of Δ-9 desaturase cDNA isolated from Zea mays.

FIGS. 11 and 12 are diagrammatic representations of fatty acid biosynthesis in plants. FIG. 11 has been adapted from Gibson et al., 1994 , Plant Cell Envir . 17, 627.

FIGS. 13 and 14 are graphical representations of RNaseH accessibility of Δ-9 desaturase RNA.

FIG. 15 shows cleavage of Δ-9 desaturase RNA by ribozymes in vitro. 10/10 represents the length of the binding arms of a hammerhead (HH) ribozyme. 10/10 means helix 1 and helix 3 each form 10 base-pairs with the target RNA (FIG. 1 ). 4/6 and 6/6, represent helix2/helix1 interaction between a hairpin ribozyme and its target. 4/6 means the hairpin (HP) ribozyme forms four base-paired helix 2 and a six base-paired helix 1 complex with the target (see FIG. 3 ). 6/6 means, the hairpin ribozyme forms a 6 base-paired helix 2 and a six base-paired helix 1 complex with the target. The cleavage reactions were carried out for 120 min at 26° C.

FIG. 16 shows the effect of arm-length variation on the activity of HH and HP ribozymes in vitro. 7/7, 10/10 and 12/12 are essentially as described above for the HH ribozyme. 6/6, 6/8, 6/12 represents varying helix 1 length and a constant (6 bp) helix 2 for a hairpin ribozyme. The cleavage reactions were carried out essentially as described above.

FIGS. 17 , 18 , 19 and 23 are diagrammatic representations of non-limiting strategies to construct a transcript comprising multiple ribozyme motifs that are the same or different, targeting various sites within Δ-9 desaturase RNA.

FIGS. 20 and 21 show in vitro cleavage of Δ-9 desaturase RNA by ribozymes that are transcribed from DNA templates using bacteriophage T7 RNA polymerase enzyme.

FIG. 22 diagrammatic representation of a non-limiting strategy to construct a transcript comprising multiple ribozyme motifs that arc the same or different targeting various sites within GBSS RNA.

FIG. 24 shows cleavage of Δ-9 desaturase RNA by ribozymes. 453 Multimer, represents a multimer ribozyme construct targeted against hammerhead ribozyme sites 453, 464, 475 and 484. 252 Multimer, represents a multimer ribozyme construct targeted against hammerhead ribozyme sites 252, 271, 313 and 326. 238 Multimer, represents a multimer ribozyme construct targeted against three hammerhead ribozyme sites 252, 259 and 271 and one hairpin ribozyme site 238 (HP). 259 Multimer, represents a multimer ribozyme construct targeted against two hammerhead ribozyme sites 271 and 313 and one hairpin ribozyme site 259 (HP).

FIG. 25 illustrates GBSS mRNA levels in Ribozyme minus Controls (C, F, I, J, N, P, Q) and Active Ribozyme RPA63 Transformants (AA, DD, EE, FF, GG, HH, JJ, KK).

FIG. 26 illustrates Δ9 desaturase mRNA levels in Non-transformed plants (NT), 85-06 High Stearate Plants (1, 3, 5, 8, 12, 14), and Transformed (irrelevant ribozyme)

FIG. 27 illustrates Δ9 desaturase mRNA levels in Non-transformed plants (NTO), 85-15 High Stearate Plants (01, 06, 07, 10, 11, 12), and 85-15 Normal Stearate Plants (02, 05, 09, 14).

FIG. 28 illustrates Δ9 desaturase mRNA levels in Non-transformed plants (NTY), 113-06 Inactive Ribozyme Plants (02, 04, 07, 10, 11).

FIGS. 29 a and 29 b illustrate Δ9 desaturase protein levels in maize leaves (R0). (a) Line HiII, plants a-e nontransformed and ribozyme inactive line RPA113-17, plants 1-6. (b) Ribozyme active line RPA85-5, plants 1-15.

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

FIG. 31 illustrates stearic acid in leaves of RPA85-15 plants, results of three assays.

FIG. 32 illustrates stearic acid in leaves of RPA113-06 plants.

FIG. 33 illustrates stearic acid in leaves of RPA113-17 plants.

FIG. 34 illustrates stearic acid in leaves of control plants.

FIG. 35 illustrates leaf stearate in R1 plants from a high stearate plant cross (RPA85-15.07 self).

FIG. 36 illustrates Δ9 desaturase levels in next generation maize leaves (R1). * indicates those plants that showed a high stearate content.

FIG. 37 illustrates stearic acid in individual somatic embryos from a culture (308/430-012) transformed with antisense Δ9 desaturase.

FIG. 38 illustrates stearic acid in individual somatic embryos from a culture (308/430-015) transformed with antisense Δ9 desaturase.

FIG. 39 illustrates stearic acid in individual leaves from plants regenerated from a culture (308/430-012) transformed with antisense Δ9 desaturase.

FIG. 40 illustrates amylose content in a single kernel of untransformed control line (Q806 and antisense line 308/425-12.2.1.

FIG. 41 illustrates GBSS activity in single kernels of a southern negative line (RPA63-0306) and Southern positive line RPA63-0218.

FIG. 42 illustrates a transformation vector that can be used to express the enzymatic nucleic acid of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns compositions and methods for the modulation of gene expression in plants specifically using enzymatic nucleic acid molecules.

The Following Phrases and Terms are Defined Below

By “inhibit” or “modulate” is meant that the activity of enzymes such as GBSS and Δ-9 desaturase or level of mRNAs encoded by these genes is reduced below that observed in the absence of an enzymatic nucleic acid and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.

By “enzymatic nucleic acid molecule” it is meant a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave that target. That is, the enzymatic nucleic acid molecule is able to internolecularly cleave RNA (or DNA) acid thereby inactivate a target RNA molecule. This complementarty functions to allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA to allow the cleavage to occur. One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, RNAzyme, polyribozymes, molecular scissors, self-splicing RNA, self-cleaving RNA, cis-cleaving RNA, autolytic RNA, endoribonuclease, minizyme, leadzyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The term encompasses enzymatic RNA molecule which include one or more 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 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.

Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its 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. Pat. No. 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 (X1-406)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. Although examples are provided below for the construction of the plasmids used in the transformation experiments illustrated herein, it is well within the skill of an artisan to design numerous different types of plasmids which can be used in the transformation of plants, see Bevan, 1984 , Nucl. Acids Res . 12, 8711-8721, which is incorporated by reference. There are also numerous ways to transform plants. In the examples below embryogenic maize cultures were helium blasted. In addition to using the gene gun (U.S. Pat. No. 4,945,050 to Cornell and U.S. Pat. No. 5,141,131 to DowElanco), plants may be transformed using Agrobacterium technology, sec U.S. Pat. No. 5,177,010 to University of Toledo, U.S. Pat. No. 5,104,310 to Texas A&M, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 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 U.S. Pat. No. 5,231,019 all to Ciba Geigy, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135 both to Agracetus; whiskers technology, see U.S. Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca; electroporation technology, see WO 87/06614 to Boyce Thompson Institute, U.S. Pat. No. 5,472,869 and U.S. Pat. No. 5,384,253 both to Dekalb, WO9209696 and WO9321335 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 ingredient 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 arc 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 angiosperms, gymnosperms, monocotyledons, and dicotyledons. Plants of interest include but are not limited to: cereals, such as rice, wheat, barley, maize; oil-producing crops, such as soybean, canola, sunflower, cotton, maize, cocoa, safflower, oil palm, coconut palm, flax, castor, peanut; plantation crops, such as coffee and tea; fruits, such as pineapple, papaya, mango, banana, grapes, oranges, apples; vegetables, such as cauliflower, cabbage, melon, green pepper, tomatoes, carrots, lettuce, celery, potatoes, broccoli; legumes, such as soybean, beans, peas; flowers, such as carnations, chrysanthemum, daisy, tulip, gypsophila, alstromeria, marigold, petunia, rose; trees such as olive, cork, poplar, pine; nuts, such as walnut, pistachio, and others. Following are a few non-limiting examples that describe the general utility of ribozymes in modulation of gene expression.

Ribozyme-mediated down regulation of the expression of genes involved in caffeine synthesis can be used to significantly change caffeine concentration in coffee beans. Expression of genes, such as 7-methylxanthosine and/or 3-methyl transferase in coffee plants can be readily modulated using ribozymes to decrease caffeine synthesis (Adams and Zarowitz, U.S. Pat. No. 5,334,529; incorporated by reference herein).

Transgenic tobacco plants expressing ribozymes targeted against genes involved in nicotine production, such as N-methylputrescine oxidase or putrescine N-methyl transferase (Shewmaker et al., supra), would produce leaves with altered nicotine concentration.

Transgenic plants expressing ribozymes targeted against genes involved in ripening of fruits, such as ethylene-forming enzyme, pectin methyltransferase, pectin esterase, polygalacturonase, 1-amininocyclopropane carboxylic acid (ACC) synthase, ACC oxidase genes (Smith et al., 1988 , Nature , 334, 724; Gray et al., 1992 , Pl. Mol. Biol ., 19, 69; Tieman et al., 1992 , Plant Cell , 4, 667; Picton et al., 1993 , The Plant J . 3, 469; Shewmaker et al., supra; James et al., 1996 , Bio/Technology , 14, 56), would delay the ripening of fruits, such as tomato and apple.

Transgenic plants expressing ribozymes targeted against genes involved in flower pigmentation, such as chalcone synthase (CHS), chalcone flavanone isomerase (CHI), phenylalanine ammonia lyase, or dehydroflavonol (DF) hydroxylases, DF reductase (Krol van der, et al., 1988 , Nature , 333, 866; Krol van der, et al., 1990 , Pl. Mol. Biol ., 14, 457; Shewmaker et al., supra; Jorgensen, 1996 , Science , 268, 686), would produce flowers, such as roses, petunia, with altered colors.

Lignins are organic compounds essential for maintaining mechanical strength of cell walls in plants. Although essential, lignins have some disadvantages. They cause indigestibility of sillage crops and are undesirable to paper production from wood pulp and others. Transgenic plants expressing ribozymes targeted against genes involved in lignin production such as, O-methyltransferase, cinnamoyl-CoA:NADPH reductase or cinnamoyl alcohol dehydrogenase (Doorsselaere et al., 1995 , The Plant J . 8, 855; Atanassova et al., 1995 , The Plant J . 8, 465; Shewmaker et al., supra; Dwivedi et al., 1994 , Pl. Mol. Biol ., 26, 61), would have altered levels of lignin.

Other useful targets for useful ribozymes are disclosed in Draper et al., International PCT Publication No. WO 93/23569, Sullivan et al., International PCT Publication No. WO 94/02595, as well as by Stinchcomb et al., International PCT Publication No. WO 95/31541, and hereby incorporated by reference herein in totality.

Modulation of Granule Bound Starch Synthase Gene Expression in Plants

In plants, starch biosynthesis occurs in both chloroplasts (short term starch storage) and in the amyloplast (long term starch storage). Starch granules normally consist of a linear chain of α(1-4)-linked α-D-glucose units (amylose) and a branched form of amylose cross-linked by α(1-6) bonds (amylopectin). An enzyme involved in the synthesis of starch in plants is starch synthase which produces linear chains of α(1-4)-glucose using ADP-glucose. Two main forms of starch synthase are found in plants: granule bound starch synthase (GBSS) and a soluble form located in the stroma of chloroplasts and in amyloplasts (soluble starch synthase). Both forms of this enzyme utilize ADP-D-glucose while the granular bound form also utilizes UDP-D-glucose, with a preference for the former. The GBSS, known as waxy protein, has a molecular mass of between 55 to about 70 kDa in a variety of plants in which it has been characterized. Mutations affecting the GBSS gene in several plant species has resulted in the loss of amylose, while the total amount of starch has remained relatively unchanged. In addition to a loss of GBSS activity, these mutants also contain altered, reduced levels, or no GBSS protein (Mac Donald and Preiss, Plant Physiol. 78: 849-852 (1985), Sano, Theor. Appl. Genet. 68: 467-473 (1984), Hovenkamp-Hermelink et al. Theor. Appl. Genet. 75: 217-221 91987), Shure et al. Cell 35, 225-233 (1983), Echt and Schwartz Genetics 99: 275-284 (1981)). The presence of a branching enzyme as well as a soluble ADP-glucose starch glycosyl transferase in both GBSS mutants and wild type plants indicates the existence of independent pathways for the formation of the branched chain polymer amylopectin and the straight chain polymer amylose.

The Wx (waxy) locus encodes a granule bound glucosyl transferase involved in starch biosynthesis. Expression of this enzyme is limited to endosperm, pollen and the embryo sac in maize. Mutations in this locus have been termed waxy due to the appearance of mutant kernels, which is the phenotype resulting from an reduction in amylose composition in the kernels. In maize, this enzyme is transported into the amyloplast of the developing endosperm where it catalyses production of amylose. Corn kernels are about 70% starch, of which 27% is linear amylose and 73% is amylopectin. Waxy is a recessive mutation in the gene encoding granule bound starch synthase (GBSS). Plants homozygous for this recessive mutation produce kernels that contain 100% of their starch in the form of amylopectin.

Ribozymes, with their catalytic activity and increased site specificity (as described below), represent more potent and perhaps more specific inhibitory molecules than antisense oligonucleotides. Moreover, these ribozymes are able to inhibit GBSS activity and the catalytic activity of the ribozymes is required for their inhibitory effect. For those of ordinary skill in the art, it is clear from the examples that other ribozymes may be designed that cleave target mRNAs required for GBSS activity in plant species other than maize.

Thus, in a preferred embodiment, the invention features ribozymes that inhibit enzymes involved in amylose production, e.g., by reducing GBSS activity. These endogenously expressed RNA molecules contain substrate binding domains that bind to accessible regions of the target mRNA. The 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, amylose production is reduced or inhibited. Specific examples arc provided below infra.

Preferred embodiments include the ribozymes having binding arms which are complementary to the binding sequences in Tables IIIA, VA and VB. Examples of such ribozymes are shown in Tables IIIB-V. Those in the art will recognize that while such examples are designed to one plant's (e.g., maize) mRNA, similar ribozymes can be made complementary to other plant species' mRNA. By complementary is thus meant that the binding arms enable ribozymes to interact with the target RNA in a sequence-specific manner to cause cleavage of a plant mRNA target. Examples of such ribozymes consist essentially of sequences shown in Tables IIIB-V.

Preferred embodiments are the ribozymes and methods for their use in the inhibition of starch granule bound ADP (UDP)-glucose: α-1,4-D-glucan 4-α-glucosyl transferase i.e., granule bound starch synthase (GBSS) activity in plants. This is accomplished through the inhibition of genetic expression, with ribozymes, which results in the reduction or elimination of GBSS activity in plants.

In another aspect of the invention, ribozymes that cleave target molecules and inhibit amylose production are expressed from transcription units inserted into the plant genome. Preferably, the recombinant vectors capable of stable integration into the plant genome and selection of transformed plant lines expressing the ribozymes are expressed either by constitutive or inducible promoters in the plant cells. Once expressed, the ribozymes cleave their target mRNAs and reduce amylose production of their host cells. The ribozymes expressed in plant cells are under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter.

Modification of corn starch is an important application of ribozyme technology which is capable of reducing specific gene expression. A high level of amylopectin is desirable for the wet milling process of corn and there is also some evidence that high amylopectin corn leads to increased digestibility and therefore energy availability in feed. Nearly 10% of wet-milled corn has the waxy phenotype, but because of its recessive nature the traditional waxy varieties are very difficult for the grower to handle Ribozymes targeted to cleave the GBSS mRNA and thus reduce GBSS activity in plants and in particular, corn endosperm will act as a dominant trait and produce corn plants with the waxy phenotype that will be easier for the grower to handle.

Modification of Fatty Acid Saturation Profile in Plants

Fatty acid biosynthesis in plant tissues is initiated in the chloroplast. Fatty acids are synthesized as thioesters of acyl carrier protein (ACP) by the fatty acid synthase complex (FAS). Fatty acids with chain lengths of 16 carbons follow one of three paths: they are released, immediately after synthesis, and transferred to glycerol 3-phosphate (G3P) by a chloroplast acyl transferase for further modification within the chloroplast; 2) they are released and transferred to Co-enzyme A (CoA) upon export from the plastid by thioesterases; or 3) they are further elongated to C18 chain lengths. The C18 chains are rapidly desaturated at the C9 position by stearoyl-ACP desaturase. This is followed by immediate transfer of the oleic acid (18:1) group to G3P within the chloroplast, or by export from the chloroplast and conversion to oleoyl-CoA by thioesterases (Somerville and Browse, 1991 Science 252: 80-87). The majority of C16 fatty acids follow the third pathway.

In corn seed oil the predominant triglycerides are produced in the endoplasmic reticulum. Most oleic acids (18:1) and some palmitic acids (16:0) are transferred to G3P from phosphatidic acids, which are then converted to diacyl glycerides and phosphatidyl choline. Further desaturation of the acyl chains on phosphatidyl choline by membrane bound desaturases takes place in the endoplasmic reticulum. Di- and tri-unsaturated chains are then released into the acyl-CoA pool and transferred to the C3 position of the glycerol backbone in diacyl glycerol in the production of triglycerides (Frentzen, 1993 in Lipid Metabolism in Plants ., p.195-230, (ed. Moore, T. S.) CRC Press, Boca Raton, Fla.). A schematic of the plant fatty acid biosynthesis pathway is shown in FIGS. 11 and 12 . The three predominant fatty acids in corn seed oil are linoleic acid (18:2, ˜59%), oleic acid (18:1, ˜26%), and palmitic acid (16:0, ˜11%). These are average values and may be somewhat different depending on the genotype; however, composite samples of US Corn Belt produced oil analyzed over the past ten years have consistently had this composition (Glover and Mertz, 1987 in: Nutritional Quality of Cereal Grains: genetic and agronomic improvement., p.183-336, (eds. Olson, R. A. and Frey, K. J.) Am. Soc. Agronomy. Inc. Madison, Wis.; Fitch-Haumann, 1985 J. Am. Oil Chem. Soc . 62: 1524-1531; Strecker et al., 1990 in Edible fats and oils processing: basic principles and modern practices (ed. Erickson, D. R.) Am. Oil Chemists Soc. Champaign, Ill.). This predominance of C18 chain lengths may reflect the abundance and activity of several key enzymes, such as the fatty acid synthase responsible for production of C18 carbon chains, the stearoyl-ACP desaturase (Δ-9 desaturase) for production of 18:1 and a microsomal Δ-12 desaturase for conversion of 18:1 to 18:2.

Δ-9 desaturase (also called stearoyl-ACP desaturase) of plants is a soluble chloroplast enzyme which uses C18 and occasionally C16-acyl chains linked to acyl carrier protein (ACP) as a substrate (McKeon, T. A. and Stumpf, P. K., 1982 J. Biol. Chem . 257: 12141-12147). This contrasts to the mammalian, lower eukaryotic and cyanobacterial Δ-9 desaturases. Rat and yeast Δ-9 desaturases are membrane bound microsomal enzymes using acyl-CoA chains as substrates, whereas cyanobacterial Δ-9 desaturase uses acyl chains on diacyl glycerol as substrate. To date several Δ-9 desaturase cDNA clones from dicotelydenous plants have been isolated and characterized (Shanklin and Somerville, 1991 Proc. Natl. Acad. Sci. USA 88: 2510-2514; Knutzon et al., 1991 Plant Physiol . 96: 344-345; Sato et al., 1992 Plant Physiol . 99: 362-363; Shanklin et al., 1991 Plant Physiol . 97: 467-468; Slocombe et al., 1992 Plant. Mol. Biol . 20: 151-155; Tayloret al., 1992 Plant Physiol . 100: 533-534; Thompson et al., 1991 Proc. Natl. Acad. Sci. USA 88: 2578-2582). Comparison of the different plant Δ-9 desaturase sequences suggests that this is a highly conserved enzyme, with high levels of identity both at the amino acid level (˜90%) and at the nucleotide level (˜80%). However, as might be expected from its very different physical and enzymological properties, no sequence similarity exists between plant and other Δ-9 desaturases (Shanklin and Somerville, supra).

Purification and characterization of the castor bean desaturase (and others) indicates that the Δ-9 desaturase is active as a homodimer; the subunit molecular weight is ˜41 kDa. The enzyme requires molecular oxygen, NADPH, NADPH ferredoxin oxidoreductase and ferredoxin for activity in vitro. Fox et al. , 1993 ( Proc. Natl. Acad. Sci. USA 90: 2486-2490) showed that upon expression in E. coli the castor bean enzyme contains four catalytically active ferrous atoms per homodimer. The oxidized enzyme contains two identical diferric clusters, which in the presence of dithionite are reduced to the diferrous state. In the presence of stearoyl-CoA and O 2 the clusters return to the diferric state. This suggests that the desaturase belongs to a group of O 2 activating proteins containing diiron-oxo clusters. Other members of this group are ribonucleotide reductase and methane monooxygenase hydroxylase. Comparison of the predicted primary structure for these catalytically diverse proteins shows that all contain a conserved pair of amino acid sequences (Asp/Glu)-Glu-Xaa-Arg-His separated by ˜80-100 amino acids.

Traditional plant breeding programs have shown that increased stearate levels can be achieved without deleterious consequences to the plant. In safflower (Ladd and Knowles, 1970 Crop Sci . 10: 525-527) and in soybean (Hammond and Fehr, 1984 J. Amer. Oil Chem. Soc . 61: 1713-1716; Graef et al., 1985 Crop Sci . 25: 1076-1079) stearate levels have been increased significantly. This demonstrates the flexibility in fatty acid composition of seed oil.

Increases in Δ-9 desaturase activity have been achieved by the transformation of tobacco with the Δ-9 desaturase genes from yeast (Polashock et al., 1992 Plant Physiol . 100, 894) or rat (Grayburn et. al., 1992 BioTechnology 10, 675). Both sets of transgenic plants had significant changes in fatty acid composition, yet were phenotypically identical to control plants.

Corn (maize) has been used minimally for the production of margarine products because it has traditionally not been utilized as an oil crop and because of the relatively low seed oil content when compared with soybean and canola. However, corn oil has low levels of linolenic acid (18:3) and relatively high levels of palmitic (16:0) acid (desirable in margarine). Applicant believes that reduction in oleic and linoleic acid levels by down-regulation of Δ-9 desaturase activity will make corn a viable alternative to soybean and canola in the saturated oil market.

Margarine and confectionary fats are produced by chemical hydrogenation of oil from plants such as soybean. This process adds cost to the production of the margarine and also causes both cis and trans isomers of the fatty acids. Trans isomers are not naturally found in plant derived oils and have raised a concern for potential health risks. Applicant believes that one way to eliminate the need for chemical hydrogenation is to genetically engineer the plants so that desaturation enzymes are down-regulated. Δ-9 desaturase introduces the first double bond into 18 carbon fatty acids and is the key step effecting the extent of desaturation of fatty acids.

Thus, in a preferred embodiment, the invention concerns compositions (and methods for their use) for the modification of fatty acid composition in plants. This is accomplished through the inhibition of genetic expression, with ribozymes, antisense nucleic acid, cosuppression or triplex DNA, which results in the reduction or elimination of certain enzyme activities in plants, such as Δ-9 desaturase. Such activity is reduced in monocotyledon plants, such as maize, wheat, rice, palm, coconut and others. Δ-9 desaturase activity may also be reduced in dicotyledon plants such as sunflower, safflower, cotton, peanut, olive, sesame, cuphea, flax, jojoba, grape and others.

Thus, in one aspect, the invention features ribozymes that inhibit enzymes involved in fatty acid unsaturation, e.g., by reducing Δ-9 desaturase activity. These endogenously expressed RNA molecules contain substrate binding domains that bind to accessible regions of the target mRNA. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, stearate levels are increased and unsaturated fatty acid production is reduced or inhibited. Specific examples are provided below in the Tables listed directly below.

In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in the Tables VI and VIII. Those in the art will recognize that while such examples are designed to one plant's (e.g., corn) mRNA, similar ribozymes can be made complementary to other plant's mRNA. By complementary is thus meant that the binding arms of the ribozymes are able to interact with the target RNA in a sequence-specific manner and enable the ribozyme to cause cleavage of a plant mRNA target. Examples of such ribozymes are typically sequences defined in Tables VII and VIII. The active ribozyme typically contains an enzymatic center equivalent to those in the examples, and binding arms able to bind plant mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such binding and/or cleavage.

The sequences of the ribozymes that are particularly useful in this study, are shown in Tables VII and VIII.

Those in the art will recognize that ribozyme sequences listed in the Tables are representative only of many more such sequences where the enzymatic portion of the ribozyrne (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Table IV (5′-GGCGAAAGCC-3′)(SEQ ID NO. 1237) 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 Tables V and VIII (5′-CACGUUGUG-3′) (SEQ ID NO. 1238) 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.

In another aspect of the invention, ribozymes that cleave target molecules and reduce unsaturated fatty acid content in plants are expressed from transcription units inserted into the plant genome. Preferably, the recombinant vectors capable of stable integration into the plant genome and selection of transformed plant lines expressing the ribozymes are expressed either by constitutive or inducible promoters in the plant cells. Once expressed, the ribozymes cleave their target mRNAs and reduce unsaturated fatty acid production of their host cells. The ribozymes expressed in plant cells are under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter.

Modification of fatty acid profile is an important application of nucleic acid-based technologies which are capable of reducing specific gene expression. A high level of saturated fatty acid is desirable in plants that produce oils of commercial importance.

In a related aspect, this invention features the isolation of the cDNA sequence encoding Δ-9 desaturase in maize.

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

While specific examples to corn RNA are provided, those in the art will recognize that the teachings are not limited to corn. Furthermore, the same target may be used in other plant species. The complementary arms suitable for targeting the specific plant RNA sequences are utilized in the ribozyme targeted to that specific RNA. The examples and teachings herein are meant to be non-limiting, and those skilled in 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

Isolation of Δ9 Desaturase cDNA from Zea mays

Degenerate PCR primers were designed and synthesized to two conserved peptides involved in diiron-oxo group binding of plant Δ-9 desaturases. A 276 bp DNA fragment was PCR amplified from maize embryo cDNA and was cloned in to a vector. The predicted amino acid sequence of this fragment was similar to the sequence of the region separated by the two conserved peptides of dicot Δ-9 desaturase proteins. This was used to screen a maize embryo cDNA library. A total of 16 clones were isolated; further restriction mapping and hybridization identified one clone which was sequenced. Features of the cDNA insert are: a 1621 nt cDNA; 145 nt 5′ and 294 nt 3′ untranslated regions including a 18 nt poly A tail; a 394 amino acid open reading frame encoding a 44.7 kD polypeptide; and 85% amino acid identity with castor bean Δ-9 desaturase gene for the predicted mature protein. The complete sequence is presented in FIG. 10 .

Example 2

Identification of Potential Ribozyme Cleavage Sites for Δ9 Desaturase

Approximately two hundred and fifty HH ribozyme sites and approximately forty three HP sites were identified in the corn Δ-9 desaturase mRNA. A HH site consists of a uridine and any nucleotide except guanosine (UH). Tables VI and VIII have a list of HH and HP ribozyme cleavage sites. The numbering system starts with 1 at the 5′ end of a Δ-9 desaturase cDNA clone having the sequence shown in FIG. 10 .

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

Example 3

Selection of Ribozyme Cleavage Sites for Δ9 Desaturase

The secondary structure of Δ-9 desaturase mRNA was assessed by computer analysis using algorithms, such as those developed by M. Zuker (Zuker, M., 1989 Science , 244, 48-52). Regions of the mRNA that did not form secondary folding structures with RNA/RNA stems of over eight nucicotides and contained potential hammerhead ribozyme cleavage sites were identified.

These sites were assessed for oligonucleotide accessibility by RNase H assays (see Example 4 infra).

Example 4

RNaseH Assays for Δ9 Desaturase

Forty nine DNA oligonucleotides, each twenty one nucleotides long were used in RNase H assays. These oligonucleotides covered 108 sites within Δ-9 desaturase RNA. RNase H assays ( FIG. 6 ) were performed using a full length transcript of the Δ-9 desaturase cDNA. RNA was screened for accessible cleavage sites by the method described generally in Draper et al., supra. Briefly, DNA oligonucleotides representing ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to generate a substrate for T7 RNA polymerase transcription from corn cDNA clones. Labeled RNA transcripts were synthesized in vitro from these templates. The oligonucleotides and the labeled transcripts were annealed, RNAseH was added and the mixtures were incubated for 10 minutes at 37° C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a Molecular Dynamics phosphor imaging system (FIGS. 13 and 14 ).

Example 5

Hammerhead and Hairpin Ribozymes for Δ9 Desaturase

Hammerhead (HH) and hairpin (HP) ribozymes were designed to the sites covered by the oligos which cleaved best in the RNase H assays. These ribozymes were then subjected to analysis by computer folding and the ribozymes that had significant secondary structure were rejected.

The ribozymes were chemically synthesized. The general procedures for RNA synthesis have been described previously (Usman et al., 1987 , J. Am. Chem. Soc ., 109, 7845-7854 and in Scaringe et al., 1990 , Nucl. Acids Res ., 18, 5433-5341; Wincott et al., 1995 , Nucleic Acids Res . 23, 2677). Small scale syntheses were conducted on a 394 Applied Biosystems, Inc. synthesizer using a modified 2.5 μmol scale protocol with a 5 min 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 was 16.9 mM 12, 49 mM pyridine, 9% water in THF (Millipore). B & J Synthesis Grade acetonitrile was 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 was performed as follows. The polymer-bound oligoribonucleotide, trityl-off, was transferred from the synthesis column to a 4 mL glass screw top vial and suspended in a solution of methylamine (MA) at 65° C. for 10 min. After cooling to −20° C., the supernatant was removed from the polymer support. The support was washed three times with 1.0 mL of EtOH:MeCN:H 2 O/3:1:1, vortexed and the supernatant was then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder.

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

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

Inactive hammerhead ribozymes were synthesized by substituting a U for G 5 and a U for A 14 (numbering from (Hertel, K. J., et al., 1992 , Nucleic Acids Res ., 20, 3252).

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

Ribozymes were also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989 , Methods Enzymol . 180, 51). Ribozymes were purified by gel electrophoresis using general methods or were purified by high pressure liquid chromatography (HPLC; 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 VII and VIII.

Example 6

Long Substrate Tests for Δ9 Desaturase Ribozymes

Target RNA used in this study was 1621 nt long and contained cleavage sites for all the HH and HP ribozymes targeted against Δ-9 desaturase RNA. A template containing T7 RNA polymerase promoter upstream of Δ-9 desaturase target sequence, was PCR amplified from a cDNA clone. Target RNA was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [α- 32 P] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was treated with DNase-I, following transcription at 37° C. for 2 hours, to digest away the DNA template used in the transcription. The transcription mixture was resolved on a denaturing polyacrylamide gel. Bands corresponding to full-length RNA was isolated from a gel slice and the RNA was precipitated with isopropanol and the pellet was stored at 4° C.

Ribozyme cleavage reactions were carried out under ribozyme excess (k cat /K M ) conditions (Herschlag and Cech, 1990 , Biochemistry 29, 10159-10171). Briefly, 1 mM ribozyme and <10 nM internally labeled target RNA were denatured separately by heating to 65° C. for 2 min in the presence of 50 mM Tris.HCl, pH 7.5 and 10 mM MgCl 2 . The RNAs were renatured by cooling to the reaction temperature (37° C., 26° C. or 20° C.) for 10-20 min. Cleavage reaction was initiated by mixing the ribozyme and target RNA at appropriate reaction temperatures. Aliquots were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on 4% sequencing gel.

The results from ribozyme cleavage reactions, at 26° C. or 20° C., are summarized in Table IX and FIGS. 15 and 16 . Of the ribozymes tested, seven hammerheads and two hairpins showed significant cleavage of Δ-9 desaturase RNA (FIGS. 15 and 16 ). Ribozymes to other sites showed varied levels of activity.

Example 7

Cleavage of the Target RNA Using Multiple Ribozyme Combinations for Δ9 Desaturase

Several of the above ribozymes were incorporated into a multimer ribozyme construct which contains two or more ribozymes embedded in a contiguous stretch of complementary RNA sequence. Non-limiting examples of multimer ribozymes are shown in FIGS. 17 , 18 , 19 and 23 . The ribozymes were made by annealling complementary oligonucleotides and cloning into an expression vector containing the Cauliflower Mosaic Virus 35S enhanced promoter (Franck et al., 1985 Cell 21, 285), the maize Adh 1 intron (Dennis et al., 1984 Nucl. Acids Res . 12, 3983) and the Nos polyadenylation signal (DePicker et al., 1982 J. Molec. Appl. Genet . 1, 561). Cleavage assays with T7 transcripts made from these multimer-containing transcription units are shown in FIGS. 20 and 21 . These are non-limiting examples; those skilled in the art will recognize that similar embodiments, consisting of other ribozyme combinations, introns and promoter elements, can be readily generated using techniques known in the art and are within the scope of this invention.

Example 8

Construction of Ribozyme Expressing Transcription Units for Δ9 Desaturase

Ribozymes targeted to cleave Δ-9 desaturase mRNA are endogenously expressed in plants, either from genes inserted into the plant genome (stable transformation) or from episomal transcription units (transient expression) which are part of plasmid vectors or viral sequences. These ribozymes can be expressed via RNA polymerase I, II, or III plant or plant virus promoters (such as CaMV). Promoters can be either constitutive, tissue specific, or developmentally expressed.

Δ9 259 Monomer Ribozyme Constructs (RPA 114, 115)

These are the Δ-9 desaturase 259 monomer hammerhead ribozyme clones. The ribozymes were designed with 3 bp long stem II and 20 bp (total) long substrate binding arms targeted against site 259. The active version is RPA114, the inactive is RPA 115. The parent plasmid, pDAB367, was digested with Not I and filled in with Klenow to make a blunt acceptor site. The vector was then digested with Hind III restriction enzyme. The ribozyme containing plasmids were cut with Eco RI, filled-in with Klenow and recut with Hind III. The insert containing the entire ribozyme transcription unit was gel-purified and ligated into the pDAB 367 vector. The constructs are checked by digestion with SgfI/Hind III and Xba I/Sst I and confirmed by sequencing.

Δ9 453 Multimer Ribozyme Constructs (RPA 118, 119)

These are the Δ-9 desaturase 453 Multimer hammerhead ribozyme clones (see FIG. 17 ). The ribozymes were designed with 3 bp long stem II regions. Total length of the substrate binding anms of the multimer construct was 42 bp. The active version is RPA 118, the inactive is 119. The constructs were made as described above for the 259 monomer. The multimer construct was designed with four hammerhead ribozymes targeted against sites 453, 464, 475 and 484 within Δ-9 desaturase RNA.

Δ9 252 Multimer Ribozyme Constructs (RPA 85, 113)

These are the Δ-9 desaturase 252 multimer ribozyme clones placed at the 3′ end of bar (phosphoinothricin acetyl transferase; Thompson et al., 1987 EMBO J . 6: 2519-2523) open reading frame. The multimer contructs were designed with 3 bp long stem II regions. Total length of the substrate binding arms of the multimer construct was 91 bp. RPA 85 is the active ribozyme, RPA 113 is the inactive. The vector was constructed as follows: The parent plasmid pDAB 367 was partially digested with Bgl II and the single cut plasmid was gel-purified. This was recut with Eco RI and again gel-purified to isolate the correct Bgl II/Eco RI cut fragment. The Bam HI/Eco RI inserts from the ribozyme constructs were gel-isolated (this contains the ribozyme and the NOS poly A region) and ligated into the 367 vector. The identitiy of positive plasmids were confinned by performing a Nco I/Sst I digest and sequencing.

Useful transgenic plants can be identified by standard assays. The transgenic plants can be evaluated for reduction in Δ-9 desaturase expression and Δ-9 desaturase activity as discussed in the examples infra.

Example 9

Identification of Potential Ribozyme Cleavage Sites in GBSS RNA

Two hundred and forty one hammer-head ribozyme sites were identified in the corn GBSS mRNA polypeptide coding region (see table IIIA). A hammer-head site consists of a uridine and any nucleotide except guanine (UH). Following is the sequence of GBSS coding region for corn (SEQ. I.D. No. 25). The numbering system starts with 1 at the 5′ end of a GBSS cDNA clone having the following sequence (5′ to 3′):

1	72	(SEQ I.D. NO. 25).
GACCGATCGATCGCCACAGCCAACACCACCCGCCGAGGCGACGCGACAGCCGCCAGGAGGAAGGAATAAACT
73	144
CACTGCCAGCCAGTGAAGGGGGAGAAGTGTACTGCTCCGTCCACCAGTGCGCGCACCGCCCGGCAGGGCTGC
145	216
TCATCTCGTCGACGACCAGTGGATTAATCGGCATGGCGGCTCTAGCCACGTCGCAGCTCGTCGCAACGCGCG
217	288
CCGGCCTGGGCGTCCCGGACGCGTCCACGTTCCGCCGCGGCGCCGCGCAGGGCCTGAGGGGGGGCCGGACGG
289	360
CGTCGGCGGCGGACACGCTCAGCATTCGGACCAGCGCGCGCGCGGCGCCCAGGCTCCAGCACCAGCAGCAGC
361	432
AGCAGGCGCGCCGCGGGGCCAGGTTCCCGTCGCTCGTCGTGTGCGCCAGCGCCGGCATGAACGTCGTCTTCG
433	504
TCGGCGCCGAGATGGCGCCGTGGAGCAAGACCGGCGGCCTCGGCGACGTCCTCGGCGGCCTGCCGCCGGCCA
505	576
TGGCCGCGAATGGGCACCGTGTCATGGTCGTCTCTCCCCGCTACGACCAGTACAAGGACGCCTGGGACACCA
577	648
GCGTCGTGTCCGAGATCAAGATGGGAGACAGGTACGAGACGGTCAGGTTCTTCCACTGCTACAAGCGCGGAG
649	720
TGGACCGCGTGTTCGTTGACCACCCACTGTTCCTGGAGAGGGTTTGGGGAAAGACCGAGGAGAAGATCTACG
721	792
GGCCTGACGCTGGAACGGACTACAGGGACAACCAGCTGCGGTTCAGCCTGCTATGCCAGGCAGCACTTGAAG
793	864
CTCCAAGGATCCTGAGCCTCAACAACAACCCATACTTCTCCGGACCATACGGGGAGGACGTCGTGTTCGTCT
865	936
GCAACGACTGGCACACCGGCCCTCTCTCGTGCTACCTCAAGAGCAACTACCAGTCCCACGGCATCTACAGGG
937	1008
ACGCAAAGACCGCTTTCTGCATCCACAACATCTCCTACCAGGGCCGGTTCGCCTTCTCCGACTACCCGGAGC
1009	1080
TGAACCTCCCGGAGAGATTCAAGTCGTCCTTCGATTTCATCGACGGCTACGAGAAGCCCGTGGAAGGCCGGA
1081	1152
AGATCAACTGGATGAAGGCCGGGATCCTCGAGGCCGACAGGGTCCTCACCGTCAGCCCCTACTACGCCGAGG
1153	1224
AGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAACATCATGCGCCTCACCGGCATCACCGGCATCG
1225	1296
TCAACGGCATGGACGTCAGCGAGTGGGACCCCAGCAGGGACAAGTACATCGCCGTGAAGTACGACGTGTCGA
1297	1368
CGGCCGTGGAGGCCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACA
1369	1440
TCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGACCCGACGTCATGGCGGCCGCCATCCCGC
1441	1512
AGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAAGAAGAAGTTCGAGCGCATGC
1513	1584
TCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAAGTTCAACGCGGCGCTGGCGCACC
1585	1656
ACATCATGGCCGGCGCCGACGTGCTCGCCGTCACCAGCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGG
1657	1728
GGATGCGATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGACACCATCATCGAAGGCAAGA
1729	1800
CCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGAGCCGGCGGACGTCAAGAAGGTGGCCA
1801	1872
CCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGATGGTGAGGAACTGCATGATCC
1873	1944
AGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGGAGAACGTGCTGCTCAGCCTCGGGGTCGCCGGCGGCG
1945	2016
AGCCAGGGGTCGAAGGCGAGGAGATCGCGCCGCTCGCCAAGGAGAACGTGGCCGCGCCCTGAAGAGTTCGGC
2017	2088
CTGCAGGCCCCCTGATCTCGCGCGTGGTGCAAACATGTTGGGACATCTTCTTATATATGCTGTTTCGTTTAT
2089	2160
GTGATATGGACAAGTATGTGTAGCTGCTTGCTTGTGCTAGTGTAATATAGTGTAGTGGTGGCCAGTGGCACA
2161	2232
ACCTAATAAGCGCATGAACTAATTGCTTGCGTGTGTAGTTAAGTACCGATCGGTAATTTTATATTGCGAGTA
2233
AATAAATGGACCTGTAGTGGTGGAAAAAAAAAAAA

There are approximately 53 potential hairpin ribozyme sites in the GBSS mRNA. The ribozyme and target sequences are listed in Table V.

Ribozymes can be readily designed and synthesized to such sites with between 5 and 100 or more bases as substrate binding arms (see FIGS. 1-5 ) as described above.

Example 10

Selection of Ribozyme Cleavage Sites for GBSS

The secondary structure of GBSS mRNA was assessed by computer analysis using folding algorithms, such as the ones developed by M. Zuker (Zuker, M., 1989 Science , 244, 48-52. Regions of the mRNA that did not form secondary folding structures with RNA/RNA stems of over eight nucleotides and contained potential hammerhead ribozyme cleavage sites were identified.

These sites which were then assessed for oligonucleotide accessibility with RNase H assays (see FIG. 6 ). Fifty-eight DNA oligonucleotides, each twenty one nucleotides long were used in these assays. These oligonucleotides covered 85 sites. The position and designation of these oligonucleotides were 195, 205, 240, 307, 390, 424, 472, 481, 539, 592, 625, 636, 678, 725, 741, 811, 859, 891, 897, 912, 918, 928, 951, 958, 969, 993, 999, 1015, 1027, 1032, 1056, 1084, 1105, 1156, 1168, 1186, 1195, 1204, 1213, 1222, 1240, 1269, 1284, 1293, 1345, 1351, 1420, 1471, 1533, 1563, 1714, 1750, 1786, 1806, 1819, 1921, 1954, and 1978. Secondary sites were also covered and included 202, 394, 384, 385, 484, 624, 627, 628, 679, 862, 901, 930, 950, 952, 967, 990, 991, 1026, 1035, 1108, 1159, 1225, 1273, 1534, 1564, 1558, and 1717.

Example 11

RNaseH Assays for GBSS

RNase H assays ( FIG. 7 ) were performed using a full length transcript of the GBSS coding region, 3′ noncoding region, and part of the 5′ noncoding region. The GBSS RNA was screened for accessible cleavage sites by the method described generally in Draper et al., supra. hereby incorporated by reference herein. Briefly, DNA oligonucleotides representing hammerhead ribozyme cleavage sites were synthesized. A polymerase chain reaction was used to generate a substrate for T7 RNA polymerase transcription from corn cDNA clones. Labeled RNA transcripts were synthesized in vitro from these templates. The oligonucleotides and the labeled transcripts were annealed, RNAseH was added and the mixtures were incubated for 10 minutes at 37° C. Reactions were stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved was determined by autoradiographic quantitation using a phosphor imaging system (FIG. 7 ).

Example 12

Hammerhead Ribozymes for GBSS

Hammerhead ribozymes with 10/10 (i.e., able to form 10 base pairs on each arm of the ribozyme) nucleotide binding arms were designed to the sites covered by the oligos which cleaved best in the RNase H assays. These ribozymes were then subjected to analysis by computer folding and the ribozymes that had significant secondary structure were rejected. As a result of this screening procedure 23 ribozymes were designed to the most open regions in the GBSS mRNA, the sequences of these ribozymes are shown in Table IV.

The ribozymes were chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described above (and in Usman et al., supra, Scaringe et al., and Wincott et al., supra) and are incorporated by reference herein, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The average stepwise coupling yields were >98%. Inactive ribozymes were synthesized by substituting a U for G 5 and a U for A 14 (numbering from (Hertel et al., supra). Hairpin ribozymes were synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 , Nucleic Acids Res ., 20, 2835-). All ribozymes were modified to enhance stability by modification of five ribonucleotides at both the 5′ and 3′ ends with 2′-O-methyl groups. Ribozymes were purified by gel electrophoresis using general methods. (Ausubel et al., 1990 Current Protocols in Molecular Biology Wiley & Sons, NY) or were purified by high pressure liquid chromatography, as described above and were resuspended in water.

Example 13

Long Substrate Tests for GBSS

Target RNA used in this study was 900 nt long and contained cleavage sites for all the 23 HH ribozymes targeted against GBSS RNA. A template containing T7 RNA polymerase promoter upstream of GBSS target sequence, was PCR amplified from a cDNA clone. Target RNA was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [α- 32 P] CTP as one of the four ribonucleotide tripbospbates. The transcription mixture was treated with DNase-1, following transcription at 37° C. for 2 hours, to digest away the DNA template used in the transcription. The transcription mixture was resolved on a denaturing polyacrylamide gel. Bands corresponding to full-length RNA was isolated from a gel slice and the RNA was precipitated with isopropanol and the pellet was stored at 4° C.

Ribozyme cleavage reactions were carried out under ribozyme excess (k cat /K M ) conditions (Herschlag and Cech, supra). Briefly, 1000 nM ribozyme and <10 nM internally labeled target RNA were denatured separately by heating to 90° C. for 2 min. in the presence of 50 mM Tris.HCl, pH 7.5 and 10 mM MgCl 2 . The RNAs were renatured by cooling to the reaction temperature (37° C., 26° C. and 20° C.) for 10-20 min. Cleavage reaction was initiated by mixing the ribozyme and target RNA at appropriate reaction temperatures. Alquots were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on 4% sequencing gel.

The results from ribozyme cleavage reactions, at the three different temperatures, summarized in FIG. 8 . Seven lead ribozymes were chosen (425, 892, 919, 959, 968, 1241, and 1787). One of the active ribozymes (811) produced a strange pattern of cleavage products; as a result, it was not chosen as one of our lead ribozymes.

Example 14

Cleavage of the GBSS RNA Using Multiple Ribozyme Combinations

Four of the lead ribozymes (892, 919, 959, 1241) were incubated with internally labeled target RNA in the following combinations: 892 alone; 892+919; 892+919+959; 892+919+959+1241. The fraction of RNA cleavage increased in an additive manner with an increase in the number of ribozymes used in the cleavage reaction (FIG. 9 ). Ribozyme cleavage reactions were carried out at 20° C. as described above. These data suggest that multiple ribozymes targeted to different sites on the same mRNA will increase the reduction of target RNA in an additive manner.

Example 15

Construction of Ribozvme Expressing Transcription Units for GBSS

Cloning of GBSS Multimer Ribozymes RPA 63 (active) and RPA 64 (inactive) A multimer ribozyme was constructed which contained four hammerhead ribozymes targeting sites 892, 919, 959 and 968 of the GBSS mRNA. Two DNA oligonucleotides (Macromolecular Resourses, Fort Collins, Colo.) were ordered which overlap by 18 nucleotides. The sequences were as follows:

Oligo 1: CGC GGA TCC TGG TAG GAC TGA TGA GGC CGA AAG GCC GAA ATG TTG TGC TGA TGA GGC CGA AAG GCC GAA ATG CAG AAA GCG GTC TTT GCG TCC CTG TAG ATG CCG TGG C (SEQ ID NO. 1238)

Oligo 2: CGC GAG CTC GGC CCT CTC TTT CGG CCT TTC GGC CTC ATC AGG TGC TAC CTC AAG AGC AAC TAC CAG TTT CGG CCT TTC GGC CTC ATC AGC CAC GGC ATC TAC AGG G (SEQ ID NO. 1239)

Inactive versions of the above were made by substituting T for G5 and T for A14 within the catalytic core of each ribozyme motif.

These were annealed in 1 × Klenow Buffer (Gibco/BRL) at 90° C. for 5 minutes and slow cooled to room temperature (22° C.). NTPs were added to 0.2 mM and the oligos extended with Klenow enzyme at 1 unit/ul for one hour at 37° C. This was phenol/chloroform extracted and ethanol precipitated, then resuspended in 1×React 3 buffer (Gibco/BRL) and digested with Bam HI and Sst I for 1 hour at 37° C. The DNA was gel purified on a 2% agarose gel using the Qiagen gel extraction kit.

The DNA fragments were ligated into BamHI/Sst I digested pDAB 353. The ligation was transformed into competent DH5α F′ bacteria (Gibco/BRL). Potential clones were screened by digestion with Bam HI/Eco RI. Clones were confirmed by sequencing. The total length of homology with the target sequence is 96 nucleotides.

919 Monomer Ribozyme (RPA 66)

A single ribozyme to site 919 of the GBSS mRNA was constructed with 10/10 arms as follows. Two DNA oligos were ordered:

Oligo 1: GAT CCG ATG CCG TGG CTG ATG AGG CCG AAA GGC CGA AAC TGG TAG TT (SEQ ID NO. 1240)

Oligo 2: AAC TAC CAG TTT CGG CCT TTC GGC CTC ATC AGC CAC GGC ATC G (SEQ ID NO. 1241)

The oligos are phosphorylated individually in 1×kinase buffer (Gibco/BRL) and heat denatured and annealed by combining at 90° C. for 10 min, then slow cooled to room temperature (22° C.). The vector was prepared by digestion of pDAB 353 with Sst I and blunting the ends with T4 DNA polymerase. The vector was redigested with Bam HI and gel purified as above. The annealed oligos are ligated to the vector in 1×ligation buffer (Gibco/BRL) at 16° C. overnight. Potential clones were digested with Bam HI/Eco RI and confirmed by sequencing.

Example 16

Plant Transformation Plasmids pDAB 367, Used in the Δ9 Ribozyme Experiments, and pDAB353 Used in the GBSS Ribozyme Experiments

Part A pDAB367

Plasmid pDAB367 has the following DNA structure: beginning with the base after the final C residue of the Sph I site of pUC 19 (base 441; Ref. 1), and reading on the strand contiguous to the LacZ gene coding strand, the linker sequence CTGCAGGCCGGCC TTAATTAAGCGGCCGCGTTTAAACGCCCGGGCATTTAAATGGCGCGCCGCGA TCGCTTGCAGATCTGCATGGGTG (SEQ ID NO. 1242), nucleotides 7093 to 7344 of CaMV DNA (2), the linker sequence CATCGATG, nucleotides 7093 to 7439 of CaMV, the linker sequence GGGGACTCTAGAGGATCCAG (SEQ ID NO. 1243), nucleotides 167 to 186 of MSV (3), nucleotides 188 to 277 of MSV (3), a C residue followed by nucleotides 119 to 209 of maize Adh 1S containing parts of exon 1 and intron 1 (4), nucleotides 555 to 672 containing parts of Adh 1S intron 1 and exon 2 (4), the linker sequence GACGGATCTG (SEQ ID NO. 1244), and nucleotides 278 to 317 of MSV. This is followed by a modified BAR coding region from pIJ4104 (5) having the AGC serine codon in the second position replaced by a GCC alanine codon, and nucleotide 546 of the coding region changed from G to A to eliminate a Bgl II site. Next, the linker sequence TGAGATCTGAGCTCGAATTTCCCC (SEQ ID NO. 1245), nucleotides 1298 to 1554 of Nos (6), and a G residue followed by the rest of the pUC 19 sequence (including the Eco RI site).

PartB pDAB353

Plasmid pDAB353 has the following DNA structure: beginning with the base after the final C residue of the Sph I site of pUC 19 (base 441; Ref. 1), and reading on the strand contiguous to the LacZ gene coding strand, the linker sequence CTGCAGATCTGCATGGGTG (SEQ ID NO. 1246), nucleotides 7093 to 7344 of CaMV DNA (2), the linker sequence CATCGATG, nucleotides 7093 to 7439 of CaMV, the linker sequence GGGGACTCTAGAG (SEQ ID NO. 1247), nucleotides 119 to 209 of maize Adh 1S containing parts of exon 1 and intron 1 (4), nucleotides 555 to 672 containing parts of Adh 1S intron 1 and exon 2 (4), and the linker sequence GACGGATCCGTCGACC (SEQ ID NO. 1248), where GGATCC represents the recognition sequence for BamH I restriction enzyme. This is followed by the beta-glucuronidase (GUS) gene from pRAJ275 (7), cloned as an Nco I/Sac I fragment, the linker sequence GAATTTCCCC (SEQ ID NO. 1249), the poly A region in nucleotides 1298 to 1554 of Nos (6), and a G residue followed by the rest of the pUC 19 sequence (including the Eco RI site).

The following are herein incorporated by reference:

1. Messing, J. (1983) in “Methods in Enzymology” (Wu, R. et al., Eds) 101:20-78.

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

3. Mullineaux, P. M., J. Donson, B. A. M. Morris-Krsinich, M. I. Boulton, and J. W. Davies (1984) The nucleotide sequence of Maize Streak Virus DNA. EMBO J . 3:3063-3068.

4. Dennis, E. S., W. L. Gerlach, A. J. Pryor, J. L. Bennetzen, A. Inglis, D. Llewellyn, M. M. Sachs, R. J. Ferl, and W. J. Peacock (1984) Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize. Nucl. Acids Res. 12:3983-4000.

5. White, J., S-Y Chang, M. J. Bibb, and M. J. Bibb (1990) A cassette containing the bar gene of Streptomyces hygroscopicus : a selectable marker for plant transformation. Nucl. Acids. Res. 18:1062.

6. DePicker, A., S. Stachel, P. Dhaese, P. Zambryski, and H. M. Goodman (1982) Nopaline Synthase: Transcript mapping and DNA sequence. J. Molec. Appl. Genet. 1:561-573.

7. Jefferson, R. A. (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Molec. Biol. Reporter 5:387-405.

Example 17

Plasmid pDAB359 a Plant Transformation Plasmid which Contains the Gamma-Zein Promoter, the Antisense GBSS, and a the Nos Polyadenylation Sequence

Plasmid pDAB359 is a 6702 bp double-stranded, circular DNA that contains the following sequence elements: nucleotides 1-404 from pUC18 which include lac operon sequence from base 238 to base 404 and ends with the HindIII site of the M13mp18 polylinker (1,2); the Nos polyadenylation sequence from nucleotides 412 to 668 (3); a synthetic adapter sequence from nucleotides 679-690 which converts a Sac I site to an Xho I site by changing GAGCTC to GAGCTT and adding CTCGAG; maize granule bound starch synthase cDNA from bases 691 to 2953, corresponding to nucleotides 1-2255 of SEQ. I.D. No. 25. The GBSS sequence in plasmid pDAB359 was modified from the original cDNA by the addition of a 5′ Xho I and a 3′ Nco I site as well as the deletion of internal Nco I and Xho I sites using Klenow to fill in the enzyme recognition sequences. Bases 2971 to 4453 are 5′ untranslated sequence of the maize 27 kD gamma-zein gene corresponding to nucleotides 1078 to 2565 of the published sequence (4). The gamma-zein sequence was modified to contain a 5′ Kpn I site and 3′ BamH/SalI/Nco I sites. Additional changes in the gamma-zein sequence relative to the published sequence include a T deletion at nucleotide 104, a TACA deletion at nucleotide 613, a C to T conversion at nucleotide 812, an A deletion at nucleotide 1165 and an A insertion at nucleotide 1353. Finally, nucleotides 4454 to 6720 of pDAB359 are identical to puc18 bases 456 to 2686 including the Kpn I/EcoRI/Sac I sites of the M13/mp18 polylinker from 4454 to 4471, a lac operon fragment from 4471 to 4697, and the β-lacatmase gene from 5642 to 6433 (1, 2).

The following are incorporated by reference herein:

pUC18—Norrander, J., Kempe, T., Messing, J. Gene (1983) 26: 101-106; Pouwels, P. H., Enger-Valk, B. E., Brammar, W. J. Cloning Vectors, Elsevier 1985 and supplements

NosA—DePicker, A., Stachel, S., Dhaese, P., Zambryski, P., and Goodman, H. M. (1982) Nopaline Synthase: Transcript Mapping and DNA Sequence J. Molec. Appl. Genet. 1:561-573.

Maize 27 kD gamma-zein—Das, O. P., Poliak, E. L., Ward, K., Messing, J. Nucleic Acids Research 19, 3325-3330 (1991).

Example 18

Construction of Plasmid pDAB430, Containing Antisense Δ9 Desaturase, Expressed by the Ubiquitin Promoter/intron (Ubil)

Part A Construction of plasmid pDAB421

Plasmid pDAB421 contains a unique blunt-end SrfI cloning site flanked by the maize Ubiquitin promoter/intron and the nopaline synthase polyadenylation sequences. pDAB421 was prepared as follows: digestion of pDAB355 with restriction enzymes KpnI and BamHI drops out the R coding region on a 2.2 kB fragment. Following gel purification, the vector was ligated to an adapter composed of two annealed oligonucleotides OF235 and OF236. OF235 has the sequence 5′-GAT CCG CCC GGG GCC CGG GCG GTA C-3′ (SEQ ID NO. 1250) and OF236 has the sequence 5′-CGC CCG GGC CCC GGG CG-3′ (SEQ ID NO. 1251). Clones containing this adapter were identified by digestion and linearization of plasmid DNA with the enzymes SrfI and SmaI which cut in the adapter, but not elsewhere in the plasmid. One representative clone was sequenced to verify that only one adapter was inserted into the plasmid. The resulting plasmid pDAB421 was used in subsequent construction of the Δ9 desaturase antisense plasmid pDAB430.

Part B Construction of plasmid pDAB430 (antisense Δ9 desaturase)

The antisense Δ9 desaturase construct present in plasmid pDAB430 was produced by cloning of an amplification product in the blunt-end cloning site of plasmid pDAB421. Two constructs were produced simultaneously from the same experiment. The first construct contains the Δ9 desaturase gene in the sense orientation behind the ubiquitin promoter, and the c-myc tag fused to the C-terminus of the Δ9 desaturase open reading frame for immunological detection of overproduced protein in transgenic lines. This construct was intended for testing of ribozymes in a system which did not express maize Δ9 desaturase. This construct was never used, but the primers used to amplify and construct the Δ9 desaturase antisense gene were the same. The Δ9 desaturase cDNA sequence described herein was amplified with two primers. The N-terminal primer OF279 has the sequence 5′-GTG CCC ACA ATG GCG CTC CGC CTC AAC GAC-3′ (SEQ ID NO. 1252). The underlined bases correspond to nucleotides 146-166 of the cDNA sequence. C-terminal primer OF280 has the sequence 5′-TCA TCA CAG GTC CTC CTC GCT GAT CAG CTT CTC CTC CAG TTG GAC CTG CCT ACC GTA-3′ (SEQ ID NO. 1253) and is the reverse complement of the sequence 5′-TAC GGT AGG GAC GTC CAA CTG GAG GAG AAG CTG ATC AGC GAG GAG GAC CTG TGA TGA-3′ (SEQ ID NO. 1254). In this sequence the underlined bases correspond to nucleotides 1304-1324 of the cDNA sequence, the bases in italics correspond to the sequence of the c-myc epitope. The 1179 bp of amplification product was purified through a 1.0% agarose gel, and ligated into plasmid pDAB421 which was linearized with the restriction enzyme Srf I. Colony hybridization was used to select clones containing the pDAB421 plasmid with the insert. The orientation of the insert was determined by restriction digestion of plasmid DNA with diagnostic enzymes KpnI and BamHI. A clone containing the Δ9 desaturase coding sequence in the sense orientation relative to the Ubiquitin promoter/intron was recovered and was named pDAB429. An additional clone containing the Δ9 desaturase coding sequence in the anitsense orientation relative to the promoter was named pDAB430. Plasmid pDAB430 was subjected to sequence analysis and it was determined that the sequence contained three PCR induced errors compared to the expected sequence. One error was found in the sequence corresponding to primer OF280 and two nucleotide changes were observed internal to the coding sequence. These errors were not corrected, because antisense downregulation does not require 100% sequence identity between the antisense transcript and the downregulation target.

Example 19

Helium Blasting of Embryogenic Maize Cultures and the Subsequent Regeneration of Transgenic Progeny

Part A Establishment of embryogenic maize cultures. The tissue cultures employed in transformation experiments were initiated from immature zygotic embryos of the genotype “Hi-II”. Hi-II is a hybrid made by intermating 2 R 3 lines derived from a B73×A188 cross (Armstrong et al. 1990). When cultured, this genotype produces callus tissue known as Type II. Type II callus is friable, grows quickly, and exhibits the ability to maintain a high level of embryogenic activity over an extended time period.

Type II cultures were initiated from 1.5-3.0 mm immature embryos resulting from controlled pollinations of greenhouse grown Hi-II plants. The initiation medium used was N6 (Chu, 1978) which contained 1.0 mg/L 2,4-D, 25 mM L-proline, 100 mg/L casein hydrolysate, 10 mg/L AgNO 3 , 2.5 g/L gelrite and 2% sucrose adjusted to pH 5.8. For approximately 2-8 weeks, selection occurred for Type II callus and against nonembryogenic and/or Type I callus. Once Type II callus was selected, it was transferred to a maintenance medium in which AgNO 3 was omitted and L-proline reduced to 6 mM.

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

Part B Preparation of plasmid DNA. Plasmid DNA was adsorbed onto the surface of gold particles prior to use in transformation experiments. The experiments for the GBSS target used gold particles which were spherical with diameters ranging from 1.5-3.0 microns (Aldrich Chemical Co., Milwaukee, Wis.). Transformation experiments for the Δ9 desaturase target used 1.0 micron spherical gold particles (Bio-Rad, Hercules, Calif.). All gold particles were surface-sterilized with ethanol prior to use. Adsorption was accomplished by adding 74 μl of 2.5 M calcium chloride and 30 μl of 0.1 M spermidine to 300 μl of plasmid DNA and sterile H 2 O. The concentration of plasmid DNA was 140 μg. The DNA-coated gold particles were immediately vortexed and allowed to settle out of suspension. The resulting clear supernatent was removed and the particles were resuspended in 1 ml of 100% ethanol. The final dilution of the suspension ready for use in helium blasting was 7.5 mg DNA/gold per ml of ethanol.

Part C Preparation and helium blasting of tissue targets. Approximately 600 mg of embryogenic callus tissue per target was spread over the surface of petri plates containing Type II callus maintenance medium plus 0.2 M sorbitol and 0.2 M mannitol as an osmoticum. After an approximately 4 hour pretreatment, all tissue was transferred to petri plates containing 2% agar blasting medium (maintenance medium plus osmoticum plus 2% agar).

Helium blasting involved accelerating the suspended DNA-coated gold particles towards and into prepared tissue targets. The device used was an earlier prototype to the one described in a DowElanco U.S. Pat. No. 5,141,131) which is incorporated herein by reference, although both function in a similar manner. The device consisted of a high pressure helium source, a syringe containing the DNA/gold suspension, and a pneumatically-operated multipurpose valve which provided controlled linkage between the helium source and a loop of pre-loaded DNA/gold suspension.

Prior to blasting, tissue targets were covered with a sterile 104 micron stainless steel screen, which held the tissue in place during impact. Next, targets w ere placed under vacuum in the main chamber of the device. The DNA-coated gold particles were accelerated at the target 4 times using a helium pressure of 1500 psi. Each blast delivered 20 μl of DNA/gold suspension. Immediately post-blasting, the targets were placed back on maintenance medium plus osmoticum for a 16 to 24 hour recovery period.

Part D Selection of transformed tissue and the regeneration of plants from transgenic cultures. After 16 to 24 hours post-blasting, the tissue was divided into small pieces and transferred to selection medium (maintenance medium plus 30 mg/L Basta™). Every 4 weeks for 3 months, the tissue pieces were non-selectively transferred to fresh selection medium. After 8 weeks and up to 24 weeks, any sectors found proliferating against a background of growth inhibited tissue were removed and isolated. Putatively transformed tissue was subcultured onto fresh selection medium. Transgenic cultures were established after 1 to 3 additional subcultures.

Once Basta™ resistant callus was established as a line, plant regeneration was initiated by transferring callus tissue to petri plate containing cytokinin-based induction medium which were then placed in low light (125 ft-candles) for one week followed by one week in high light (325 ft-candles). The induction medium was composed of MS salts and vitamins (Murashige and Skoog, 1962), 3 0 g/L sucrose, 100 mg/L myo-inositol, 5 mg/L 6-benzylaminopurine, 0.025 mg/L 2,4-D, 2.5 g/L gelrite adjusted to pH 5.7. Following the two week induction period, the tissue was non-selectively transferred to hormone-free regeneration medium and kept in high light. The regeneration medium was composed of MS salts and vitamins, 30 g/L sucrose and 2.5 g/L gelrite adjusted to pH 5.7. Both induction and regeneration media contained 30 mg/L Basta™. Tissue began differentiating shoots and roots in 2-4 weeks. Small (1.5-3 cm) plantlets were removed and placed in tubes containing SH medium. SH medium is composed of SH salts and vitamins (Schenk and Hildebrandt, 1972), 10 g/L sucrose, 100 mg/L myo-inositol, 5 mL/L FeEDTA, and either 7 g/L Agar or 2.5 g/L Gelrite adjusted to pH 5.8. Plantlets were transferred to 10 cm pots containing approximately 0.1 kg of Metro-Mix® 360 (The Scotts Co., Marysville, Ohio) in the greenhouse as soon as they exhibited growth and developed a sufficient root system (1-2 weeks). At the 3-5 leaf stage, plants were trans ferred to 5 gallon pots containing approximately 4 kg Metro-Mix® 360 and grown to maturity. These R 0 plants were self-pollinated and/or cross-pollinated with non-transgenic inbreds to obtain transgenic progeny. In the case of transgenic plants produced for the GBSS target, R 1 seed produced from R 0 pollinations was replanted. The R 1 plants were grown to maturity and pollinated to produce R 2 seed in the quantities needed for the analyses.

Example 20

Production and Regeneration of Δ9 Transgenic Material

Part A Transformation and isolation of embryogenic callus. Six ribozyme constructs, described previously, targeted to Δ9 desaturase were transformed into regenerable Type II callus cultures as described herein. These 6 constructs consisted of 3 active/inactive pairs; namely, RPA85/RPA113, RPA114/RPA115, and RPA118/RPA119. A total of 1621 tissue targets were prepared, blasted, and placed into selection. From these blasting experiments 334 independent Basta®-resistant transformation events (“lines”) were isolated from selection. Approximately 50% of these lines were analyzed via DNA PCR or GC/FAME as a means of determining which ones to move forward to regeneration and which ones to discard. The remaining 50% were not analyzed either because they had become non-embryogenic or contaminated.

Part B Regeneration of Δ9 plants from transgenic callus. Following analyses of the transgenic callus, twelve lines were chosen per ribozyme construct for regeneration, with 15 R 0 plants to be produced per line. These lines generally consisted of 10 analysis-positive lines plus 2 negative controls, however, due to the poor regenerability of some of the cultures, plants were produced from less than 12 lines for constructs RPA113, RPA115, RPA118, and RPA119. An overall total of 854 R 0 plants were regenerated from 66 individual lines (see Table X). When the plants reached maturity, self- or sib-pollinations were given the highest priority, however, when this was not possible, cross-pollinations were made using the inbreds CQ806, CS716, OQ414, or HO 1 as pollen donors, and occasionally as pollen recipients. Over 715 controlled pollinations have been made, with the majority (55%) being comprised of self- or sib-pollinations and the minority (45%) being comprised of F1 crosses. R 1 seed was collected approximately 45 days post-pollination.

Example 21

Production and Regeneration of Transgenic Maize for the GBSS

Part A Transformation of embryogenic maize callus and the subsequent selection and establishment of transgenic cultures. RPA63 and RPA64, an active/inactive pair of ribozyme multimers targeted to GBSS, were inserted along with bar selection plasinid pDAB308 into Type II callus as described herein. A total of 115 Basta™-resistant independent transformation events were recovered from the selection of 590 blasted tissue targets. Southern analysis was performed on callus samples from established cultures of all events to determine the status of the gene of interest.

Part B Regeneration of plants from cultures transformed with ribozymes targeted to GBSS as well as the advancement to the R 2 generation. Plants were regenerated from Southern “positive” transgenic cultures and grown to maturity in a greenhouse. The primary regenerates were pollinated to produce R 1 seed. From 30 to 45 days after pollination, seed was harvested, dried to the correct moisture content, and replanted. A total of 752 R 1 plants, representing 16 original lines, were grown to sexual maturity and pollinated. Approximately 19 to 22 days after pollination, ears were harvested and 30 kernels were randomly excised per ear and frozen for later analyses.

Example 22

Testing of GBSS-Targeted Ribozymes in Maize Black Mexican Sweet (BMS) Stably Transformed Callus

Part A Production of BMS callus stably transformed with GBSS and GBSS-targeted ribozymes. BMS does not produce a GBSS mRNA which is homologous to that found endogenously in maize. Therefore, a double transformation system was developed to produce transformants which expressed both target and ribozymes. “ZM” BMS suspensions (obtained from Jack Widholm, University of Illinois, also see W. F. Sheridan, “Black Mexican Sweet Corn: Its Use for Tissue Cultures” in Maize for Biological Research , W. F. Sheridan, editor. University Press. University of North Dakokta, Grand Forks, N. Dak., 1982, pp. 385-388) were prepared for helium blasting four days after subculture by transfer to a 100×20 mm Petri plate (Fisher Scientific, Pittsburgh, Pa.) and partial removal of liquid medium, forming a thin paste of cells. Targets consisted of 100-125 mg fresh weight of cells on a ½″ antibiotic disc (Schleicher and Schuell, Keene, N.H.) placed on blasting medium, DN6 [N6 salts and vitamins (Chu et al., 1978), 20 g/L sucrose, 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 25 mM L-proline; pH=5.8 before autoclaving 20 minutes at 121° C.] solidified with 2% TC agar (JRH Biosciences, Lenexa, Kas.) in 60×20 mm plates. DNA was precipitated onto gold particles. For the first transformation, pDAB 426 (Ubi/GBSS) and pDAB 308 (35T/Bar) were used. Targets were individually shot using DowElanco Helium Blasting Device I. With a vacuum pressure of 650 mm Hg and at a distance of 15.5 cm from target to device nozzle, each sample was blasted once with DNA/gold mixture at 500 psi. Immediately after blasting, the antibiotic discs were transferred to DN6 medium made with 0.8% TC agar for one week of target tissue recovery. After recovery, each target was spread onto a 5.5 cm Whatman #4 filter placed on DN6 medium minus proline with 3 mg/L Basta® (Hoechst, Frankfort, Germany). Two weeks later, the filters were transferred to fresh selection medium with 6 mg/L Basta®. Subsequent transfers were done at two week intervals. Isolates were picked from the filters and placed on AMCF-ARM medium (N6 salts and vitamins, 20 g/L sucrose, 30 g/L mannitol, 100 mg/L acid casein hydrolysate, and 1 mg/L 2,4-D, 24 mM L-proline; pH=5.8 before autoclaving 20 minutes at 121° C.) solidified with 0.8% TC agar containing 6 mg/L Basta®. Isolates were maintained by subculture to fresh medium every two weeks.

Basta®-resistant isolates which expressed GBSS were subjected to a second transformation. As with BMS suspensions, targets of transgenic callus were prepared 4 days after subculture by spreading tissue onto ½″ filters. However, AMCF-ARM with 2% TC agar was used for blasting, due to maintenance of transformants on AMCF-ARM selection media. Each sample was covered with a sterile 104 μm mesh screen and blasting was done at 1500 psi. Target tissue was co-bombarded with pDAB 319 (35S-ALS; 35T-GUS) and RPA63 (active ribozyme multimer) or pDAB3 19 and RPA64 (inactive ribozyme multimer), or shot with pDAB 319 alone. Immediately after blasting, all targets were transferred to nonselective medium (AMCF-ARM) for one week of recovery. Subsequently, the targets were placed on AMCF-ARM medium containing two selection agents, 6 mg/L Basta® and 2 μg/L chlorsulfuron (CSN). The level of CSN was increased to 4 ug/L after 2 weeks. Continued transfer of the filters and generation of isolates was done as described in the first transformation, with isolates being maintained on AMCF-ARM medium containing 6 mg/L Basta and 4 μg/L CSN.

Part B Analysis of BMS stable transformants expressing GBSS and GBSS-targeted ribozymes. Isolates from the first transformation were evaluated by Northern blot analysis for detection of a functional target gene (GBSS) and to detennine relative levels of expression. In 12 of 25 isolates analyzed, GBSS transcript was detected. A range of expression was observed, indicating an independence of transfornation events. Isolates generated from the second transformation were evaluated by Northern blot analysis for detection of continued GBSS expression and by RT-PCR to screen for the presence of ribozyme transcript. Of 19 isolates tested from one previously transformed line, 18 expressed the active ribozyme, RPA63, and all expressed GBSS. GBSS was detected in each of 6 vector controls; ribozyme was not expressed in these samples. As described herein, RNase protection assay (RPA) and Northern blot analysis were performed on ribozyme-expressing and vector control tissues to compare levels of GBSS transcript in the presence or absence of active ribozyme. GBSS values were normalized to an internal control (Δ9 desaturase); Northern blot data is shown in FIG. ( 25 ). Northern blot results revealed a significantly lower level of GBSS message in the presence of ribozyme, as compared to vector controls. RPA data showed that some of the individual samples expressing active ribozyme (“L” and “O”) were significantly different from vector controls and similar to a nontransformed control.

Example 23

Analysis of Plant and Callus Materials

Plant material co-transformed with the pDAB308 and one of the following ribozyme containing vectors, pRPA63, pRPA64, pRPA85, pRPA113, pRPA114, pRPA115, pRPA118 or pRPA119 were analyzed at the callus level, Ro level and select lines analyzed at the F1 level. Leaf material was harvested when the plantlets reached the 6-8 leaf stage. DNA from the plant and callus material was prepared from lyophilized tissue as described by Saghai-Maroof et al.(supra). Eight micrograms of each DNA was digested with the restriction enzymes specific for each construct using conditions suggested by the manufacturer (Bethesda Research Laboratory, Gaithersburg, Md.) and separated by agarose gel electrophoresis. The DNA was blotted onto nylon membrane as described by Southern, E. 1975 “Detection of specific sequences among DNA fragments separated by gel electrophoresis,” J Mol. Biol. 98:503 and Southern, E. 1980 “Gel electrophoresis of restriction fragments” Methods Enzmol. 69:152, which are incorporated by reference herein.

Probes specific for the ribozyme coding region were hybridized to the membranes. Probe DNA was prepared by boiling 50 ng of probe DNA for 10 minutes then quick cooling on ice before being added to the Ready-To-Go DNA labeling beads (Pharmacia LKB, Piscataway, N.J.) with 50 microcuries of α 32 P-dCTP (Amersham Life Science, Arlington Heights, Ill.). Probes were hybridized to the genomic DNA on the nylon membranes. The membranes were washed at 60° C. in 0.25×SSC and 0.2% SDS for 45 minutes, blotted dry and exposed to XAR-5 film overnight with two intensifying screens.

The DNA from the RPA63 and RPA64 was digested with the restriction enzymes HindIII and EcoRI and the blots containing these samples were hybridized to the RPA63 probe. The RPA63 probe consists of the RPA63 ribozyme multimer coding region and should produce a single 1.3 kb hybridization product when hybridized to the RPA63 or RPA64 materials. The 1.3 kb hybridization product should contain the enhanced 35S promoter, the AdhI intron, the ribozyme coding region and the nopaline synthase poly A 3′ end. The DNA from the RPA85 and RPA113 was digested with the restriction enzymes HindIII and EcoRI and the blots containing these samples were hybridized to the RPA122 probe. RPA122 is the 252 multimer ribozyme in pDAB 353 replacing the GUS reporter. The RPA122 probe consists of the RPA122 ribozyme multimer coding region and the nopaline synthase 3′ end and should produce a single 2.1 kb hybridization product when hybridized to the RPA85 or RPA113 materials. The 2.1 kb hybridization product should contain the enhanced 35S promoter, the AdhI intron, the bar gene, the ribozyme coding region and the nopaline synthase poly A 3′ end. The DNA from the RPA114 and RPA115 was digested with the restriction enzymes HindIII and SmaI and the blots containing these samples were hybridized to the RPA115 probe. The RPA115 probe consist of the RPA115 ribozyme coding region and should produce a single 1.2 kb hybridization product when hybridized to the RPA114 or RPA115 materials. The 1.2 kb hybridization product should contain the enhanced 35S promoter, the AdhI intron, the ribozyme coding region and the nopaline synthase poly A 3′ end. The DNA from the RPA118 and RPA119 was digested with the restriction enzymes HindIII and SmaI and the blots containing these samples were hybridized to the RPA118 probe. The RPA118 probe consist of the RPA118 ribozyme coding region and should produce a single 1.3 kb hybridization product when hybridized to the RPA118 or RPA119 materials. The 1.3 kb hybridization product should contain the enhanced 35S promoter, the Adhl intron, the ribozyme coding region and the nopaline synthase poly A 3′ end.

Example 24

Extraction of Genomic DNA from Transgenic Callus

Three hundred mg of actively growing callus were quick frozen on dry ice. It was ground to a fine powder with a chilled Bessman Tissue Pulverizer (Spectrum, Houston, Tex.) and extracted with 400 μl of 2×CTAB buffer (2% Hexadecyltrimethylammonium Bromide, 100 mM Tris pH 8.0, 20 mM EDTA, 1.4 M NaCl, 1% polyvinylpyrrolidone). The suspension was lysed at 65° C. for 25 minutes, then extracted with an equal volume of chloroform:isoamyl alcohol. To the aqueous phase was added 0.1 volumes of 10% CTAB buffer (10% Hexadecyltrimethylammonium Bromide, 0.7 M NaCl). Following extraction with an equal volume of chloroform:isoamyl alcohol, 0.6 volumes of cold isopropyl alcohol was added to the aqueous phase, and placed at −20° C. for 30 minutes. After a 5 minute centrifugation at 14,000 rpm, the resulting precipitant was dried for 10 minutes under vacuum. It was resuspended in 200 μl TE (10 mM Tris, 1 mM EDTA, pH 8.0) at 65° C. for 20 minutes. 20% Chelex (Biorad,) was added to the DNA to a final concentration of 5% and incubated at 56° C. for 15-30 minutes to remove impurities. The DNA concentration was measured on a Hoefer Fluorimeter (Hoefer, San Francisco).

Example 25

PCR Analysis of Genomic Callus DNA

Use of Polymerase Chain Reaction (PCR) to demonstrate the stable insertion of ribozyme genes into the chromosome of transgenic maize calli.

Part A Method used to detect ribozyme DNA

The Polymerase Chain Reaction (PCR) was performed as described in the suppliers protocol using AmpliTaq DNA Polymerase (GeneAmp PCR kit, Perkin Elmer, Cetus). Aliquots of 300 ng of genomic callus DNA, 1 μl of a 50 μM downstream primer (5′ CGC AAG ACC GGC AAC AGG 3′; SEQ ID NO. 1255), 1 μl of an upstream primer and 1 μl of Perfect Match (Stratagene, Calif.) PCR enhancer were mixed with the components of the kit. The PCR reaction was performed for 40 cycles using the following parameters; denaturation at 94° C. for 1 minute, annealing at 55° C. for 2 minutes, and extension at 72° C. for 3 mins. An aliquot of 0.2×vol. of each PCR reaction was electrophoresised on a 2% 3:1 Agarose (FMC) gel using standard TAE agarose gel conditions.

Part B Upstream primer used for detection of Δ9 desaturase ribozyme genes

RPA85/RPA113 251 multimer fused to BAR 3′ ORF

RPA114/RPA115 258 ribozyme monomer

RPA118/RPA119 452 ribozyme multimer 5′TGG ATT GAT GTG ATA TCT CCA C 3′ (SEQ ID NO. 1256) This primer is used to amplify across the Eco RV site in the 35S promoter. Primers were prepared using standard oligo synthesis protocols on an Applied Biosystems Model 394 DNA/RNA synthesizer.

Example 26

Preparation of Total RNA from Transgenic Maize Calli and Plant

Part A Preparation of total RNA from transgenic non-regenerable and regenerable callus tissue. Three hundred milligrams of actively growing callus was quick frozen on dry ice. The tissue was ground to a fine powder with a chilled Bessman Tissue Pulverizer (Spectrum, Houston, Tex.) and extracted with RNA Extraction Buffer (50 mM Tris-HCl pH 8.0, 4% para-amino salicylic acid, 1% Tri-iso-propylnapthalenesulfonic acid, 10 mM dithiothreitol, and 10 mM Sodium meta-bisulfite) by vigorous vortexing. The homogenate was then extracted with an equal volume of phenol containing 0.1% 8-hydroxyquinoline. After centrifugation, the aqueous layer was extracted with an equal volume of phenol containing chloroform:isoamyl alcohol (24:1), followed by extraction with chloroform:octanol (24:1). Subsequently, 7.5 M Ammonium acetate was added to a final concentration of 2.5 M, the RNA was precipitated for 1 to 3 hours at 4° C. Following 4° C. centrifugation at 14,000 rpm, RNA was resuspended in sterile water, precipitated with 2.5 M NH 4 OAc and 2 volumes of 100% ethanol and incubated ovemite at −20° C. The harvested RNA pellet was washed with 70% ethanol and dried under vacuum. RNA was resuspended in sterile H 2 O and stored at −80° C.

Part B Preparation of total RNA from transgenic maize plants. A five cm section (˜150 mg) of actively growing maize leaf tissue was excised and quick frozen in dry ice. The leaf was ground to a fine powder in a chilled mortar. Following manufactorers instructions, total RNA was purified from the powder using a Qaigen RNeasy Plant Total RNA kit (Qiagen Inc., Chatsworth, Calif.). Total RNA was released from the RNeasy columns by two sequential elution spins of prewarmed (50° C.) sterile water (30 μl each) and stored at −80° C.

Example 27

Use of RT-PCR Analysis to Demonstrate Expression of Ribozyme RNA in Transgenic Maize Calli and Plants

Part A Method used to detect ribozyme RNA. The Reverse Transcription-Polyinerase Chain Reaction (RT-PCR) was performed as described in the suppliers protocol using a thermostable rTth DNA Polymerase (rTth DNA Polymerase RNA PCR kit, Perkin Elmer Cetus). Aliquots of 300 ng of total RNA (leaf or callus) and 1 μl of a 15 μM downstream primer (5′ CGC AAG ACC GGC AAC AGG 3′; SEQ ID NO. 1257) were mixed with the RT components of the kit. The reverse transcription reaction was performed in a 3 step ramp up with 5 minute incubations at 60° C., 65° C., and 70° C. For the PCR reaction, 1 μl of upstream primer specific for the ribozyme RNA being analyzed was added to the RT reaction with the PCR components. The PCR reaction was performed for 35 cycles using the following parameters; incubation at 96° C. for 1 minute, denaturation at 94° C. for 30 seconds, annealing at 50° C. for 30 seconds, and extension at 72° C. for 3 mins. An aliquot of 0.2×vol. of each RT-PCR reaction was electrophoresed on a 2% 3:1 Agarose (FMC) gel using standard TAE agarose gel conditions.

Part B Specific upstream primers used for detection of GBSS ribozymes.

GBSS Active and Inactive Multimer

5′ CAG ATC AAG TGC AAA GCT GCG GAC GGA TCT G 3′ (SEQ ID NO. 1258). This primer covers the Adh I intron footprint upstream of the first ribozyme arm. GBSS 918 Intron (−) Monomer:

5′ ATC CGA TGC CGT GGC TGA TG 3′ (SEQ ID NO. 1259). This primer covers the 10 base pair ribozyme arm and the first 6 bases of the ribozyme catalytic domain. GBSS ribozyrne expression in transgenic callus and plants was confirmed by RT-PCR.

GBSS multimer ribozyme expression in stably transformed callus was also determined by Ribonuclease Protection Assay.

Part C Specific upstream primers used for detection of Δ9 desaturase ribozymes.

RPA85/RPA113 252 multimer fused to BAR 3′ ORF

5′ GAT GAG ATC CGG TGG CAT TG 3′ (SEQ ID NO. 1260)

This primer spans the junction of the BAR gene and the RPA85/113 ribozyme. RPA114/RPA115 259 ribozyme monomer

5′ ATC CCC TTG GTG GAC TGA TG 3′ (SEQ ID NO. 1261)

This primer covers the 10 base pair ribozyme arm and the first 6 bases of the ribozyme catalytic domain. RPA118/RPA119 453 ribozyme multimer

5′ CAG ATC AAG TGC AAA GCT GCG GAC GGA TCT G 3′ (SEQ ID NO. 1262)

This primer covers the Adh I intron footprint upstream of the first ribozyme arm. Expression of Δ9 desaturase ribozymes in transgenic plant lines 85-06, 113-06 and 85-15 were confirmed by RT-PCR.

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

Example 28

Demonstration of Ribozyme Mediated Reduction in Target mRNA Levels in Transgenic Maize Callus and Plants

Part A Northern analysis method which was used to demonstrated reductions in target mRNA levels. Five μg of total RNA was dried under vacuum, resuspended in loading buffer (20 mM phosphate buffer pH 6.8, 5 mM EDTA; 50% formamide: 16% formaldehyde: 10% glycerol) and denatured for 10 minutes at 65° C. Electrophoresis was at 50 volts through 1% agarose gel in 20 mM phosphate buffer (pH 6.8) with buffer recirculation. BRL 0.24-9.5 Kb RNA ladder (Gibco/BRL, Gaithersburg, Md.) were stained in gels with ethiduim bromide. RNA was transferred to GeneScreen membrane filter (DuPont NEN, Boston Mass.) by capillary transfer with sterile water. Hybridization was performed as described by DeLeon et al. (1983) at 42° C., the filters were washed at 55° C. to remove non-hybridized probe. The blot was probed sequentially with cDNA fragments from the target gene and an internal RNA control gene. The internal RNA standard was utilized to distinguish variation in target mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions. For each sample the level of target mRNA was compared to the level of control mRNA within that sample. Fragments were purified by Qiaex resin (Qaigen Inc. Chatsworth, Calif.) from 1× TAE agarose gels. They were nick-translated using an Amersham Nick Translation Kit (Amersham Corporation, Arlington Heights, Ill.) with alpha 32 P dCTP. Autoradiography was at −70° C. with intensifying screens (DuPont, Wilmington Del.) for one to three days. Autoradiogram signals for each probe were measured after a 24 hour exposure by densitometer and a ratio of target/internal control mRNA levels was calculated.

Ribonuclease protection assays were performed as follows: RNA was prepared using the Qiagen RNeasy Plant Total RNA Kit from either BMS protoplasts or callus material. The probes were made using the Ambion Maxiscript kit and were typically 10 8 cpm/microgram or higher. The probes were made the same day they were used. They were gel purified, resuspended in RNase-free 10 mM Tris (pH 8) and kept on ice. Probes were diluted to 5×10 5 cpm/ul immediately before use. 5 μg of RNA derived from callus or 20 μg of RNA derived from protoplasts was incubated with 5×10 5 cpm of probe in 4M Guanidine Buffer. [4M Guanidine Buffer: 4M Guanidine Thiocyanate/0.5% Sarcosyl/25 mM Sodium Citrate (pH 7.4)]. 40 ul of PCR mineral oil was added to each tube to prevent evaporation. The samples were heated to 95° for 3 minutes and placed immediately into a 45° water bath. Incubation continued overnight. 600 μl of RNase Treatment Mix was added per sample and incubated for 30 minutes at 37° C. (RNase Treatment Mix: 400 mM NaCl, 40 units/ml RNase A and T1). 12 μl of 20% SDS were added per tube, immediately followed by addition of 12 ul (20 mg/ml) Proteinase K to each tube. The tubes were vortexed gently and incubated for 30 minutes at 37° C. 750 ul of room temperature RNase-free isopropanol was added to each tube, and mixed by inverting repeatedly to get the SDS into solution. The samples were then microfuged at top speed at room temperature for 20 minutes. The pellets were air dried for 45 minutes. 15 ul of RNA Running Buffer was added to each tube, and vortexed hard for 30 seconds. (RNA Running Buffer: 95% Formamide/20 mM EDTA/0.1% Bromophenol Blue/0.1% Xylene Cyanol). The sample was heated to 95° C. for 3 minutes, and loaded onto an 8% denaturing acrylamide gel. The gel was vacuum dried and exposed to a phosphorimager screens for 4 to 12 hours.

Part B Results demonstrating reductions in GBSS mRNA levels in nongenerable callus expressing both a GBSS and GBSS targeted ribozyme RNA. The production of nonregenerable callus expressing RNAs for the GBSS target gene and an active multimer ribozyme targeted to GBSS mRNA was performed. Also produced were transgenics expressing GBSS and a ribozyme (−) control RNA. Total RNA was prepared from the transgenic lines. Northern analysis was performed on 7 ribozyme (−) control transformants and 8 active RPA63 lines. Probes for this analysis were a full length maize GBSS cDNA and a maize Δ9 cDNA fragment. To distinguish variation in GBSS mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions, the level of GBSS mRNA was compared to the level of Δ9 mRNA within that sample. The level of full length GBSS transcript was compared between ribozyme expressing and ribozyme minus calli to identify lines with ribozyme mediated target RNA reductions. Blot to blot variation was controlled by performing duplicate analyses.

A range in GBSS/Δ9 ratio was observed between ribozyme (−) transgenics. The target mRNA is produced by a transgene and may be subject to more variation in expression then the endogenous Δ9 mRNA. Active lines (RPA 63) AA, EE, KK, and JJ were shown to reduce the level of GBSS/Δ9 most significantly, as much as 10 fold as compared to ribozyme (−) control transgenics this is graphed in FIG. 25 . Those active lines were shown to be expressing GBSS targeted ribozyme by RT-PCR as described herein.

Reductions in GBSS mRNA compared to Δ9 mRNA were also seen by RNAse protection assay.

Part C Demonstration of reductions in Δ9 desaturasc levels in transgenic plants expressing ribozymes targeted to Δ9 desaturase mRNA. The high stearate transgenics, RPA85-06 and RPA85-15, each contained an intact copy of the fused ribozyme multimer gene. Within each line, plants were screened by RT-PCR for the presence of ribozyme RNA. Using the protocol described in Example 27. RPA85 ribozyme expression was demonstrated in plants of the 85-06 and 85-15 lines which contained high stearic acid in their leaves. Northern analysis was performed on the six high stearate plants from each line as well as non-transformed (NT) and transformed control (TC) plants. The probes for this analysis were cDNA fragments from a maize Δ9 desaturase cDNA and a maize actin cDNA. To distinguish variation in Δ9 mRNA levels due to loading or handling errors from true ribozyme mediated RNA reductions, the level of Δ9 mRNA was compared to the level of actin mRNA within that sample. Using densitometer readings described above a ratio was calculated for each sample. Δ9/actin ratio values ranging from 0.55 to 0.88 were calculated for the 85-06 plants. The average Δ9/actin value for non-transformed controls was 2.7. There is an apparent 4 fold reduction in Δ9/actin ratios between 85-06 and NT leaves. Comparing Δ9/actin values between 85-06 high stearate and TC plants, on average a 3 fold reduction in Δ9/actin was observed for the 85-06 plants. This data is graphed in FIG. 26 . Ranges in Δ9/actin ratios from 0.35 to 0.53, with an average of 0.43 were calculated for the RPA85-15 high stearate transgenics. In this experiment the average Δ9/actin ratio for the NT plants was 1.7. Comparing the average Δ9/actin ratio between NT controls and 85-15 high stearate plants, a 3.9 fold reduction in 85-15 Δ9 mRNA was demonstrated. An apparent 3 fold reduction in Δ9 mRNA level was observed for RPA85-15 high stearate transgenics when Δ9/actin ratios were compared between 85-15 high stearate and normal stearate (TC) plants. These data are graphed in FIG. 27 . These data indicate ribozyme-mediated reduction of Δ9-desaturase mRNA in transgenic plants expressing RPA85 ribozyme, and producing increased levels of stearic acid in the leaves.

Example 29

Evidence of Δ9 Desaturase Down Regulation in Maize Leaves as a Result of Active Ribozyme Activity

Plants were produced which were transformed with inactive versions of the Δ9 desaturase ribozyme genes. Data was presented demonstrating control levels of leaf stearate in the inactive Δ9 ribozyme transgenic lines RPA113-06 and 113-17. Ribozyme expression and northern analysis was performed for the RPA113-06 line. Δ9 desaturase protein levels were determined in plants of the RPA113-17 line. Ribozyme expression was measured as described herein. Plants 113-06-04, -07, and -10 expressed detectable levels of RPA 113 inactive Δ9 ribozyme. Northern analysis was performed on 5 plants of the 113-06 line with leaf stearate ranging from 1.8-3.9%, all of which fall within the range of controls. No reduction in Δ9 desaturase mRNA correlating with ribozyme expression or elevations in leaf stearate were found in the RPA113-06 plants as compared to controls, graphed in FIG. 28 . Protein analysis did not indicate any reduction in Δ9 desaturase protein levels correlating with elevated leaf stearate in the RPA113-17 plants. This data is graphed in FIG. 29 ( a ). Taken together, the data from the two RPA113 inactive transgenic lines indicate ribozyme activity is responsible for the high strearate phenotype observed in the RPA85 lines. The RPA85 ribozyme is the active version of the RPA113 ribozyme.

Example 30

Demonstration of Ribozyme Mediated Reduction in Stearoyl-ACP Δ9 Desaturase Levels in Maize Leaves (RO) Δ9 Desaturase Levels in Maize Leaves (R0)

Part A Partial purification of stearoyl-ACP Δ9-desaturase from maize leaves. All procedures were performed at 4° C. unless stated otherwise. Maize leaves (50 mg) were harvested and ground to a fine powder in liquid N 2 with a mortar and pestle. Proteins were extracted in one equal volume of Buffer A consisting of 25 mM sodium-phosphate pH 6.5, 1 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, 10 mM phenylmethylsulfonyl fluoride, 5 mM leupeptin, and 5 mM antipapin. The crude homogenate was centrifuged for 5 minutes at 10,000×g. The supernatant was assayed for total protein concentration by Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, Calif.). One hundred micrograms of total protein was brought up to a final volume of 500 μl in Buffer A, added to 50 μl of mixed SP-sepharose beads (Pharmacia Biotech Inc., Piscataway, N.J.), and resuspended by vortexing briefly. Proteins were allowed to bind to sepharose. beads for 10 minutes while on ice. After binding, the Δ9 desaturase-sepharose material was centrifuged (10,000×g) for 10 seconds, decanted, washed three times with Buffer A (500 μl), and washed one time with 200 mM sodium chloride (500 μl). Proteins were eluted by boiling in 50 μl of Treatment buffer (125 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol) for 5 mintues. Samples were centrifuged (10,000 ×g) for 5 minutes. The supernatant was saved for Western anaylsis and the pellet consisting of sepharose beads was discarded.

Part B Western analysis method which was used to demonstrate reductions in stearoyl-ACP Δ9 desaturase. Partially purified proteins were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels (10% PAGE) as described by Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of phage T4 , Nature 227, 660-685. To distinguish variation in Δ9 desaturase levels, included on each blot as a reference was purified and quantified overexpressed Δ9 desaturase from E. coli as described hereforth. Proteins were electrophoretically transferred to ECL™ nitrocellulose membranes (Amersham Life Sciences, Arlington Heights, Ill.) using a Phannacia Semi-Dry Blotter (Pharmacia Biotech Inc., Piscataway, N.J.), using Towbin buffer (Towbin et al. 1979). The nonspecific binding sites were blocked with 10% dry milk in phosphate buffer saline for 1 h. Immunoreactive polypeptides were detected using the ECL™ Western Blotting Detection Reagent (Amersham Life Sciences, Arlington Heights, Ill.) with rabbit antiserum raised against E. coli expressed maize Δ9 desaturase. The antibody was produced according to standard protocols by Berkeley Antibody Co. The secondary antibody was goat antirabbit serum conjugated to horseradish peroxidase (BioRad). Autoradiograms were scanned with a densitometer and quantified based on the relative amount of purified E. coli Δ9 desaturase. These experiments were duplicated and the mean reduction was recorded.

Part C Demonstration of Reductions in Δ9 desaturase levels in R0 maize leaves expressing ribozymes targeted to Δ9 desaturase mRNA. The high stearate transgenic line, RPA85-15, contains an intact copy of the fused multimer gene. Δ9 desaturase was partially purified from R0 maize leaves, using the protocol described herein. Western analysis was performed on ribozyme active (RPA85-15) and ribozyme inactive (RPA113-17) plants and nontransformed (HiII) plants as described above in part B. The natural variation of Δ9 desaturase was determined for the nontransformed line (HiII) by Western analysis see FIG. 29 A. No reduction in Δ9 desaturase was observed with the ribozyme inactive line RPA113-17, all of which fell within the range as compared to the nontransformed line (HiII). An apparent 50% reduction of Δ9 desaturase was observed in six plants of line RPA85-15 ( FIG. 29B ) as compared with the controls. Concurrent with this, these same six plants also had increased stearate and reduced Δ9 desaturase mRNA (As described in Examples 28 and 32). However, nine active ribozyme plants from line RPA85-15 did not have any significant reduction as compared with nontransformed line (HiII) and inactive ribozyme line (RPA113-17) (FIGS. 29 A and B). Collectively, these results suggest that the ribozyme activity in the six plants from line RPA85-15 is responsible for the reduced Δ9 desaturase.

Example 31

E. coli Expression and Purification of Maize Δ-9 Desaturase Enzyme

Part A The mature protein encoding portion of the maize Δ-9 desaturase cDNA was inserted into the bacterial T7 expression vector pET9D (Novagen Inc., Madison, Wis.). The mature protein encoding region was deduced from the mature castor bean polypeptide sequence. The alanine at position 32 (nts 239-241 of cDNA) was designated as the first residue. This is found within the sequence Ala.Val.Ala.Ser.Met.Thr. Restriction endonuclease Nhe I site was engineered into the maize sequence by PCR, modifying GCCTCC to GCTAGC and a BamHI site was added at the 3′ end. This does not change the amino acid sequence of the protein. The cDNA sequence was cloned into pET9d vector using the Nhe I and Bam HI sites. The recombinant plasmid is designated as pDAB428. The maize Δ-9 desaturase protein expressed in bacteria has an additional methionine residue at the 5′ end. This pDAB428 plasmid was transformed into the bacterial strain BL21 (Novagen, Inc., Madison, Wis.) and plated on LB/kanamycin plates (25 mg/ml). Colonies were resuspended in 10 ml LB with kanamycin (25 mg/ml) and IPTG (1 mM) and were grown in a shaker for 3 hours at 37° C. The cells were harvested by centrifugation at 1000×g at 4° C. for 10 minutes. The cells were lysed by freezing and thawing the cell pellet 2×, followed by the addition of 1 ml lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.1% Triton X100, 100 ug/mil DNAse I, 100 ug/mI RNAse A, and 1 mg/ml lysozyme). The mixture was incubated for 15 minutes at 37° C. and then centrifuged at 1000×g for 10 minutes at 4° C. The supernatant is used as the soluble protein fraction.

The supernatant, adjusted to 25 mM sodium phosphate buffer (pH 6.0), was chilled on ice for 1 hr. Afterwards, the resulting flocculant precipitant was removed by centrifugation. The ice incubation step was repeated twice more after which the solution remained clear. The clarified solution was loaded onto a Mono S HR 10/10 column (Pharmacia) that had been equilibrated in 25 mM sodium phosphate buffer (pH 6.0). Basic proteins bound to the column matrix were eluted using a 0-500 mM NaCl gradient over 1 hr (2 ml/min; 2 ml fractions). The putative protein of interest was subjected to SDS-PAGE, blotted onto PVDF membrane, visualized with coomassie blue, excised, and sent to Harvard Microchem for amino-terminal sequence analysis. Comparison of the protein's amino terminal sequence to that encoded by the cDNA clone revealed that the protein was indeed Δ9. Spectrophotometric analysis of the diiron-oxo component associated with the expressed protein (Fox et al., 1993 Proc. Natl. Acad. Sci. USA . 90, 2486-2490), as well as identification using a specific nonheme iron stain (Leong et al., 1992 Anal. Biochem . 207, 317-320) confirmed that the purified protein was Δ-9.

Part B Production of polyclonal antiserum

The E. coli produced Δ-9 protein, as determined by amino terminal sequencing, was gel purified via SDS-PAGE, excised, and sent in the gel matrix to Berkeley Antibody Co., Richmond, Calif., for production of polyclonal sera in rabbits. Titers of the antibodies against Δ-9 were performed via western analysis using the ECL Detection system (Amersham, Inc.)

Part C Purification of Δ9 desaturase from corn kernels

Protein Precipitation: Δ9 was purified from corn kernels following homogenization using a Warring blender in 25 mM sodium phosphate buffer (pH 7.0) containing 25 mM sodium bisulfite and a 2.5% polyvinylpolypyrrolidone. The crude homogenate was filtered through cheesecloth, centrifuged (10,000×g) for 0.25 h and the resulting supernatant was filtered once more through cheesecloth. In some cases, the supernatant was fractionated via saturated ammonium sulfate precipitation by precipitation at 20% v/v followed by 80% v/v. Extracts obtained from high oil germplasm were fractionated by adding a 50% polyethylene glycol solution (mw=8000) at final concentrations of 5- and 25% v/v. In all cases, the Δ9 protein precipitated at either 80% ammonium sulfate or 25% polyethylene glycol. The resulting pellets were then dialyzed extensively in 25 mM sodium phosphate buffer (pH 6.0).

Cation Exchange Chromotography: The solubilized pellet material described above was clarified via centrifugation and applied to Mono S HR10/10 column equilibrated in 25 mM sodium phosphate buffer (pH 6.0). After extensive column washing, basic proteins bound to the column matrix were eluted using a 0-500 mM NaCl gradient over 1 hr (2 ml/min: 2 ml fractions). Typically, the Δ9 protein eluted between 260- and 350 mM NaCl., as determined by enzymatic and western analysis. After dialysis, this material was further fracionated by acyl carrier protein (ACP)-sepharose and phenyl superose chromatography.

Acyl Carrier Protein - Sepharose Chromatography : ACP was purchased from Sigma Chemical Company and purified via precipitation at pH 4.1 (Rock and Cronan, 1981 J. Biol. Chem . 254, 7116-7122) before linkage to the beads. ACP-sepharose was prepared by covalently binding 100 mg of ACP to cyanogen bromide activated sepharose 4B beads, essentially as described by Pharmacia, Inc., in the package insert. After linkage and blocking of the remaining sites with glycine, the ACP-sepharose material was packed into a HR 5/5 column (Pharmacia, Inc.) and equilibrated in 25 mM sodium phosphate buffer (pH 7.0). The dialyzed fractions identified above were then loaded onto the column (McKeon and Stumpf, 1982 J. Biol. Chem . 257, 12141-12147; Thompson et al., 1991 , Proc. Natl. Acad. Sci. USA 88, 2578-2582). After extensive column washing, ACP-binding proteins were eluted using 1 M NaCl. Enzymatic and western analysis, followed by amino terminal sequencing, indicated that the eluent contained Δ-9 protein. The Δ-9 protein purified from corn was determined to have a molecular size of approximately 38 kDa by SDS-PAGE analysis (Hames, 1981 in Gel Electrophoresis of Proteins: A Practical Approach, eds Hames B D and Rickwood, D., IRL Press, Oxford).

Phenyl Sepharose Chromatography: The fractions containing Δ9 obtained from the ACP-Sepharose column were adjusted to 0.4 M ammonium sulfate (25 mM sodium phosphate, pH 7.0) and loaded onto a Pharmacia Phenyl Superose column (HR 10/10). Proteins were eluted by running a gradient (0.4-0.0 M ammonium sulfate) at 2 ml/min for 1 hour. The Δ9 protein typically eluted between 60- and 30 mM ammonium sulfate as determined by enzymatic and western analysis.

Example 32

Evidence for the Increase in Stearic Acid in Leaves as a Result of Transformation of Plants with Δ9 Desaturase Ribozymes

Part A Method used to determine the stearic acid levels in plant tissues. The procedure for extraction and esterification of fatty acids from plant tissue was modified from a described procedure (Browse et. al., 1986, Anal. Biochem. 152, 141-145). One to 20 mg of plant tissue was placed in Pyrex 13 mm screw top test tubes. After addition of 1 ml of methanolic HCL (Supelco, Bellefonte, Pa.), the tubes were purged with nitrogen gas and sealed. The tubes were heated at 80° C. for 1 hour and allowed to cool. The heating in the presence of the methanolic HCL results in the extraction as well as the esterification of the fatty acids. The fatty acid methyl esters were removed from the reaction mixture by extraction with hexane. One ml of hexane and 1 ml of 0.9% (w/v) NaCl was added followed by vigorous shaking of the test tubes. After centrifugation of the tubes at 2000 rpm for 5 minutes the top hexane layer was removed and used for fatty acid methyl ester analysis. Gas chromatograph analysis was performed by injection of 1 μl of the sample on a Hewlett Packard (Wilmington, Del.) Series II model 5890 gas chromatograph equipped with a flame ionization detector and a J&W Scientific (Folsom, Calif.) DB-23 column. The oven temperature was 150° C. throughout the run and the flow of the carrier gas (helium) was 80 cm/sec. The run time was 20 minutes. The conditions allowed for the separation of the 5 fatty acid methyl esters of interest: C16:0, palmityl methyl ester; C18:0, stearyl methyl ester; C18:1, oleoyl methyl ester; C18:2, linolcoyl methyl ester; and C18:3, linolenyl methyl ester. Data collection and analysis was performed with a Hewlett Packard Series II Model 3396 integrator and a PE Nelson (Perkin Elmer, Norwalk, Conn.) data collection system. The percentage of each fatty acid in the sample was taken directly from the readouts of the data collection system. Quantitative amounts of each fatty acid were calculated using the peak areas of a standard (Matreya, Pleasant Gap, Pa.) which consisted of a known amount of the five fatty acid methyl esters. The amount calculated was used to estimate the percentage, of total fresh weight, represented by the five fatty acids in the sample. An adjustment was not made for loss of fatty acids during the extraction and esterification procedure. Recovery of the standard sample, after subjecting it to the extraction and esterification procedure (with no tissue present), ranged from 90 to 100% depending on the original amount of the sample. The presence of plant tissue in the extraction mixture had no effect on the recovery of the known amount of standard.

Part B Demonstration of an increase in stearic acid in leaves due to introduction of Δ9 desaturase ribozymes. Leaf tissue from individual plants was assayed for stearic acid as described in Part A. A total of 428 plants were assayed from 35 lines transfonned with active Δ9 desaturase ribozymes (RPA85, RPA114, RPA118) and 406 plants from 31 lines transformed with Δ9 desaturase inactive ribozymes (RPA113, RPA115, RPA119). Table XI summarizes the results obtained for stearic acid levels in these plants. Seven percent of the plants from the active lines had stearic acid levels greater than 3%, and 2% had levels greater than 5%. Only 3% of the plants from the inactive lines had stearic acid levels greater than 3%. Two percent of the control plants had leaves with stearate greater than 3%. The controls included 49 non-transformed plants and 73 plants transformed with a gene not related to Δ9 desaturase. There were no plants from the inactive lines or controls that had leaf stearate greater than 4%. Two of the lines transformed with the active Δ9 desaturase ribozyme RPA85 produced many plants which exhibited increased stearate in their leaves. Line RPA85-06 had 6 out of the 15 plants assayed with stearic acid levels which were between 3 and 4%, about 2-fold greater than the average of the controls ( FIG. 30 ) The average stearic acid content of the control plants (122 plants) as 1.69% (SD+/−0.49%). The average stearic acid content of leaves from line RPA85-06 as 2.86% (+/−0.57%). Line RPA85-15 had 6 out of 15 plants assayed with stearic acid levels which were approximately 4-fold greater than the average of the controls (FIG. 31 ). The average leaf stearic acid content of line RPA85-15 was 3.83% (+/−2.53%). When the leaf analysis was repeated for RPA85-15 plants, the stearic acid level in leaves from plants previously shown to have normal stearic acid levels remained normal and leaves from plants with high stearic acid were again found to be high (FIG. 31 ). The stearic acid levels in leaves of plants from two lines which were transformed with an inactive Δ9 desaturase ribozyme, RPA113, is shown in FIGS. 32 and 33 . RPA113-06 had three plants with a stearic acid content of 3% or higher. The average stcaric acid content of leaves from line RPA113-06 was 2.26% (+/−0.65%). RPA113-17 had no plants with leaf stearic acid content greater than 3%. The average stearic acid content of leaves from line RPA113-17 was 1.76% (+/−0.29%). The stearic acid content of leaves from 15 control plants is shown in FIG. 34 . The average stearic acid content for these 15 control plants was 1.70% (+/−0.6%). When compared to the control and inactive Δ9 desaturase ribozyme data, the results obtained for stearic acid content in RPA85-06 and RPA85-15 demonstrate an increase in stearic acid content due to the introduction of the Δ9 desaturase ribozyme.

Example 33

Inheritance of the High Stearic Acid Trait in Leaves

Part A Results obtained with stearic acid levels in leaves from offspring of high stearic acid plants. Plants from line RPA85-15 were pollinated as described herein. Twenty days after pollination zygotic embryos were excised from immature kernels from these RPA85-15 plants and placed in a tube on media as described herein for growth of regenerated plantlets. After the plants were transferred to the greenhouse, fatty acid analysis was performed on the leaf tissue. FIG. 35 shows the stearic acid levels of leaves from 10 different plants for one of the crosses, RPA85-15.07 selfed. Fifty percent of the plants had high leaf stearic acid and 50% had normal leaf stearic acid. Table XII shows the results from 5 different crosses of RPA85-15 plants. The number of plants with high stearic acid ranged from 20 to 50%.

Part B Results demonstrating reductions in Δ9 desaturase levels in next generation (R1) maize leaves expressing ribozymes targeted to Δ9 desaturase mRNA. In next generation maize plants that showed a high stearate content (see above Part A), Δ9 desaturase was partially purified from R1 maize leaves, using the protocol described herein. Western analysis was performed on several of the high stearate plants. In leaves of next generation plants, a 40-50% reduction of Δ9 desaturase was observed in those plants that had high stearate content (FIG. 36 ). The reduction was comparable to R0 maize leaves. This reduction was observed in either OQ414 plants crossed with RPA85-15 pollcn or RPA85-15 plants crossed with self or siblings. Therefore, this suggests that the gene encoding the ribozyme is heritable.

Example 34

Increase in Stearic Acid in Plant Tissues Using Antisense-Δ9 Desaturase

Part A Method for culturing somatic embryos of maize. The production and regeneration of maize embryogenic callus has been described herein. Somatic embryos make up a large part of this embryogenic callus. The somatic embryos continued to form in callus because the callus was transferred every two weeks. The somatic embryos in embryogenic callus continued to proliferate but usually remained in an early stage of embryo development because of the 2,4-D in the culture medium. The somatic embryos regenerated into plantlets because the callus was subjected to a regeneration procedure described herein. During regeneration the somatic embryo formed a root and a shoot, and ceases development as an embryo. Somatic embryos were made to develop as seed embryos, i.e., beyond the early stage of development found in embryogenic callus and no regeneration, by a specific medium treatment. This medium treatment involved transfer of the embryogenic callus to a Murashige and Skoog medium (MS; described by Murashige and Skoog in 1962) which contains 6% (w/v) sucrose and no plant hormones. The callus was grown on the MS medium with 6% sucrose for 7 days and then the somatic embryos were individually transferred to MS medium with 6% sucrose and 10 μM abscisic acid (ABA). The somatic embryos were assayed for fatty acid composition as described herein after 3 to 7 days of growth on the ABA medium. The fatty acid composition of somatic embryos grown on the above media was compared to the fatty acid composition of embryogenic callus and maize zygotic embryos 12 days after pollination (Table XIII). The fatty acid composition of the somatic embryos was different than that of the embryogenic callus. The embryogenic callus had a higher percentage of C16:0 and C18:3, and a lower percentage of C18:1 and C18:2. The percentage of lipid represented by the fresh weight was different for the embryogenic callus when compared to the somatic embryos; 0.4% versus 4.0%. The fatty acid composition of the zygotic embryos and somatic embryos were very similar and their percentage of lipid represented by the fresh weight were nearly identical. It was concluded that the somatic embryo culture system described above would be an useful in vitro system for testing the effect of certain genes on lipid synthesis in developing embryos of maize.

Part B Increase in stearic acid in somatic embryos of maize as a result of the introduction of an antisense-Δ9 desaturase gene. Somatic embryos were produced using the method described herein from embryogenic callus transformed with pDAB308/pDAB430. The somatic embryos from 16 different lines were assayed for fatty acid composition. Two lines, 308/430-12 and 308/430-15, were found to produce somatic embryos with high levels of stearic acid. The stearic acid content of somatic embryos from these two lines is compared to the stearic acid content of somatic embryos from their control lines in FIGS. 37 and 38 . The control lines were from the same culture that the transformed lines came from except that they were not transformed. For line 308/430-12, stearic acid in somatic embryos ranged from 1 to 23% while the controls ranged from 0.5 to 3%. For line 308/430-15, stearic acid in somatic embryos ranged from 2 to 15% while the controls ranged from 0.5 to 3%. More than 50% of the somatic embryos had stearic acid levels which were above the range of the controls in both the transformed lines. The above results indicate that an antisense-Δ9 desaturase gene can be used to raise the stearic acid levels in somatic embryos of maize.

Part C Demonstration of an increase in stearic acid in leaves due to introduction of an antisense-Δ9 desaturase gene. Embryogenic cultures from lines 308/430-12 and 308/430-15 were used to regenerate plants. Leaves from these plants were analyzed for fatty acid composition using the method previously described. Only 4 plants were obtained from the 308/430-15 culture and the stearic acid level in the leaves of these plants were normal, 1-2%. The stearic acid levels in leaves from plants of line 308/430-12 are shown in FIG. 39 . The stearic acid levels in leaves ranged from 1 to 13% in plants from line 308/430-12. About 30% of the plants from line 308/430-12 had stearic acid levels above the range observed in the controls, 1-2%. These results indicate that the stearic acid levels can be raised in leaves of maize by introduction of an antisense-Δ9 desaturase gene.

By “antisense” is meant a non-enzymatic nucleic acid molecule that binds to a RNA (target RNA) by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004).

Example 35

Amylose Content Assay of Maize Pooled Starch Sample and Single Kernel

The amylose content was assayed by the method of Hovenkamp-Hermelink et al. (Potato Research 31:241-246) with modifications. For pooled starch sample, 10 mg to 100 mg starch was dissolved in 5 ml 45% perchloric acid in plastic culture tube. The solution was mixed occasionally by vortexing. After one hour, 0.2 ml of the starch solution was diluted to 10 ml by H 2 O. 0.4 ml of the diluted solution was then mixed with 0.5 ml diluted Lugol's solution (Sigma) in 1 ml cuvet. Readings at 618 nm and 550 nm were immediately taken and the R ratio (618 nm/550 nm) was calculated. Using standard equation P (percentage of amylose)=(4.5R-2.6)/(7.3-3R) generated from potato amylose and maize amylopectin (Sigma, St. Louis), ainylose content was determined. For frozen single kernel sample, same procedure as above was used except it was extracted in 45% perchloric acid for 20 min instead for one hour.

Example 36

Starch Purification and Granular Bound Starch Synthase (GBSS) Assay

The purification of starch and following GBSS activity assay were modified from the methods of Shure et al. (Cell, 35:225-233, 1983) and Nelson et al. (Plant Physiology, 62:383-386, 1978). Maize kernel was homogenized in 2 volume (v/w) of 50 mM Tris-HCl, pH 8.0, 10 mM EDTA and filtrated through 120 μm nylon membrane. The material was then centrifuged at 5000 g for 2 min and the supernatant was discarded. The pellet was washed three times by resuspending in water and removing supernatant by centrifugation. After washing, the starch was filtrated through 20 μm nylon membrane and centrifuged. Pellet was then lyophilized and stored in −20° C. until used for activity assay.

A standard GBSS reaction mixture contained 0.2 M Tricine, pH 8.5, 25 mM Glutathione, 5 mM EDTA, 1 mM 14 C ADPG (6 nci/μmol), and 10 mg starch in a total volume of 200 μl. Reactions were conducted at 37° C. for 5 min and terminated by adding 200 μl of 70% ethanol (v/v) in 0.1 M KCl. The material was centrifuged and unincorporated ADPG in the supernatant is removed. The pellet was then washed four time with 1 ml water each in the same fashion. After washing, pellet was suspended in 500 μl water, placed into scintillation vial, and the incorporated ADPG was counted by a Beckman (Fullerton, Calif.) scintillation counter. Specific activity was given as pmoles of ADPG incorporated into starch per min per mg starch.

Example 37

Analysis of Antisense-GBSS Plants

Because of the segregation of R2 seeds, single kernels should therefore be analyzed for amylose content to identify phenotype. Because of the large amount of samples generated in this study, a two-step screening strategy was used. In the first step, 30 kernels were taken randomly from the same ear, freeze-dried and homogenized into starch flour. Amylose assays on the starch flours were carried out. Lines with reduced amylose content were identified by statistical analysis. In the second step, amylose content of the single kernels in the lines with reduced amylose content was further analyzed (25 to 50 kernels per ear). Two sets of controls were used in the screening, one of the sets were untransformed lines with the same genetic background and the other were transformed lines which did not carry transgene due to segregation (Southern analysis negative line).

81 lines representing 16 transformation events were examined at the pooled starch level. Among those lines, six with significant reduction of amylose content by statistical analysis were identified for further single kernel analysis. One line, 308/425-12.2.1, showed significant reduction of amylose content (FIG. 40 ).

Twenty five individual kernels of CQ806, a conventional maize inbred line, were analyzed. The amylose content of CQ806 ranged from 24.4% to 32.2%, averaging 29.1%. The single kernel distribution of amylose content is skewed slightly towards lower amylose contents. Forty nine single kernels of 308/425-12.2.1.1 were analyzed. Given that 308/425-12.2.1.1 resulted from self pollination of a hemizygous individual, the expected distribution would consist of 4 distinct genetic classes present in equal frequencies since endosperm is a triploid tissue. The 4 genetic classes consist of individuals carrying 0, 1, 2, and 3 copies of the antisense construct. If there is a large dosage effect for the transgene, then the distribution of amylose contents would be tetramodal. One of the modes of the resulting distribution should be indistinguishable from the non-transgenic parent. If there is no dosage effect for the transgene (individuals carrying 1, 2 or 3 copies of the transgene are phenotypically equivalent), then the distribution should be bimodal with one of the modes identical to the parent. The number of individuals included in the modes should be 3:1 of transgenic:parental. The distribution for 308/425-12.2.1.1 is distinctly trimodal. The central mode is approximately twice the size of either other mode. The two distal modes are of approximately equal size. Goodness of fit to a 1:2:1 ratio was tested and the fit was excellent.

Further evidence was available demonstrating that the mode with the highest amylose content was identical to the non-transgenic parent. This was done using discriminant analysis. The CQ806 and 308/425-12.2.1.1 data sets were combined for this analysis. The distance metrics used in the analysis were calculated using amylose contents only. The estimates of variance from the individual analyses were used in all tests. No pooled estimate of variance was employed. The original data was tested for reclassification. Based on the discriminant analysis, the entire mode of the 308/425-12.2.1.1 distribution with the highest amylose content would be more appropriately classified as parental. This is strong confirmation that this mode of thc distribution is parental. Of the remaining two modes, the central mode is approximately twice the size of the lowest amylose content mode. This would be expected if the central mode includes two genetic classes: individuals with 1 or 2 copies of the antisense construct. The mode with the lowest amylose content thus represents those individuals which are fully homozygous (3 copies) for the antisense construct. The 2:1 ratio was tested and could not be rejected on the basis of the data.

This analysis indicates that the antisense GBSS gene as functioning in 308/425-12.2.1.1 demonstrates a dosage dependent reduction in amylose content of maize kernels.

Example 38

Analysis of Ribozyme-GBSS Plants

The same two-step screening strategy as in the antisense study (Example 37) was used to analyze ribozyme-GBSS plants. 160 lines representing 11 transformation events were examined in the pooled starch level. Among the control lines (both untransformed line and Southern negative line), the amylose content varied from 28% to 19%. No significant reduction was observed among all lines carrying ribozyme gene (Southern positive line). More than 20 selected lines were further analyzed in the single kernel level, no significant amylose reduction as well as segregation pattern were found. It was apparent that ribozyme did not cause any alternation in the phenotypic level.

Transformed lines were further examined by their GBSS activity (as described in Example 36). For each line, 30 kernels were taken from the frozen ear and starch was purified. Table XIV shows the results of 9 plants representing one transformation event of the GBSS activity in the pooled starch samples, amylose content in the pooled starch samples, and Southern analysis results. Three southern negative lines: RPA63.0283, RPA63.0236, and RPA63.0219 were used as control.

The GBSS activities of control lines RPA63.0283, RPA63.0236, and RPA63.0219 were around 300 units/mg starch, In lines RPA63.021 1, RPA63.021 8, RPA63.0209, and RPA63.0210, a reduction of GBSS activity to more than 30% was observed. The correlation of varied GBSS activity to the Southern analysis in this group (from RPA63.0314 to RPA63.0210 of Table XIV) indicated that the reduced GBSS activity was caused by the expression of ribozyme gene incorporated into the maize genome.

GBSS activities at the single kernel level of line RPA63.0218 (Southern positive and reduced GBSS activity in pooled starch) was further examined, using RPA63.0306 (Southern negative and GBSS activity normal in pooled starch) as control. About 30 kernels from each line were taken, and starch samples were purified from each kernel individually. FIG. 41 clearly indicated reduced GBSS activity in line RPA63.0218 compared to RPA63.0306.

Other embodiments are within the following claims.

Characteristics of naturally occurring ribozymes
Group I Introns
Size: ˜150 to >1000 nucleotides.
Requires a U in the target sequence immediately 5′ of the cleavage site.
Binds 4-6 nucleotides at the 5′-side of the cleavage site.
Reaction mechanism: attack by the 3′-OH 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 [1].
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 [2, 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 [11, 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 of new β-galactosidase sequences
onto the defective message [13].
RNAse P RNA (M1 RNA)
Size: ˜290 to 400 nucleotides.
RNA portion of a ubiquitous ribonucleoprotein enzyme.
Cleaves tRNA precursors to form mature tRNA [14].
Reaction mechanism: possible attack by M                                                                        2+                                                                    -OH to generate cleavage
products with 3′-OH and 5′-phosphate.
RNAse P is found throughout the prokaryotes and eukaryotes. The RNA
subunit has been sequenced from bacteria, yeast, rodents, and primates.
Recruitment of endogenous RNAse P for therapeutic applications is
possible through hybridization of an External Guide Sequence (EGS) to
the target RNA [15, 16]
Important phosphate and 2′ OH contacts recently identified [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] in addition to RNA cleavage and ligation.
Major structural features largely established through phylogenetic
comparisons [23].
Important 2′ OH contacts beginning to be identified [24]
Kinetic framework under development [25]
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 [ ]
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 [32].
Chemical modification investigation of important residues begun [33, 34].
Hepatitis Delta Virus (HDV) Ribozyme
Size: ˜60 nucleotides.
Trans cleavage of target RNAs demonstrated [35].
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 R.. Catalysis of RNA cleavage by the                                                                                     Tetrahymena thermophila                                                                                 ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry (1990), 29(44), 10159-71.
5. Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the                                                                                     Tetrahymena thermophila                                                                                 ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site. Biochemistry (1990), 29(44), 10172-80.
6. Knitt, Deborah S.; 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 H.. A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58.
8. Li, Yi; Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H.. Thermodynamic and activation parameters for binding of a pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not diffusion-controlled and is driven by a favorable entropy change. Biochemistry (1995), 34(44), 14394-9.
9. Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19), 6504-12.
10. 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 R.. Minor groove recognition of the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction site. Science (Washington, D.C.) (1995), 267(5198), 675-9.
12. Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5′-Splice Site Selection and Transition State Stabilization. Biochemistry (1996), 35(4), 1201-11.
13. Sullenger, Bruce 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′-hydroxyl-base contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U.S.A. (1995), 92(26), 12510-14.
19. Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for Group II Intron Ribozyme Activity: Quantitation of Interdomain Binding and Reaction Rate. Biochemistry (1994), 33(9), 2716-25.
20. Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of Reaction Mechanism and Structure/Function Relationships. Biochemistry (1995), 34(9), 2965-77.
21. Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian; Perlman, Philip S.; Lambowitz, Alan M.. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
22. Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2′-hydroxyl groups. Chem. Biol. (1995), 2(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 2′-hydroxyl groups within a group II intron active site. Science (Washington, D.C.) (1996), 271(5254), 1410-13.
25. Daniels, Danette L.; Michels, William J., Jr.; Pyle, Anna Marie. Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J. Mol. Biol. (1996), 256(1), 31-49.
26. Guo, Hans C. T.; Collins, Richard A.. Efficient trans-cleavage of a stem-loop RNA substrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368-76.
27. Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. ‘Hairpin’ catalytic RNA model: evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids Res. (1990), 18(2), 299-304.
28. Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M.. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2.
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 M.. In 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.
36. Perrotta, Anne T.; Been, Michael D.. A 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 D.. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

TABLE II
2.5 μmol RNA Synthesis Cycle
				Wait
	Reagent	Equivalents	Amount	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 IIIA
GBSS Hammerhead Substrate Sequence
nt.
Position Substrate
12       CGAUCGAUC GCCACAGC
68       GAAGGAAUA AACUCACU
73       AAUAAACUC ACUGCCAG
103      AGAAGUGUA CUGCUCCG
109      GUACUGCUC CGUCCACC
113      UGCUCCGUC CACCAGUG
146      GGGCUGCUC AUCUCGUC
149      CUGCUCAUC UCGUCGAC
151      GCUCAUCUC GUCGACGA
154      CAUCUCGUC GACGACCA
169      CAGUGGAUU AAUCGGCA
170      AGUGGAUUA AUCGGCAU
173      GGAUUAAUC GGCAUGGC
186      UGGCGGCUC UAGCCACG
188      GCGGCUCUA GCCACGUC
196      AGCCACGUC GCAGCUCG
203      UCGCAGCUC GUCGCAAC
206      CAGCUCGUC GCAACGCG
230      CUGGGCGUC CCGGACGC
241      GGACGCGUC CACGUUCC
247      GUCCACGUU CCGCCGCG
248      UCCACGUUC CGCCGCGG
292      GACGGCGUC GGCGGCGG
308      GACACGCUC AGCAUUCG
314      CUCAGCAUU CGGACCAG
315      UCAGCAUUC GGACCAGC
344      CCCAGGCUC CAGCACCA
385      GGCCAGGUU CCCGUCGC
386      GCCAGGUUC CCGUCGCU
391      GUUCCCGUC GCUCGUCG
395      CCGUCGCUC GUCGUGUG
398      UCGCUCGUC GUGUGCGC
425      AUGAACGUC GUCUUCGU
428      AACGUCGUC UUCGUCGG
430      CGUCGUCUU CGUCGGCG
431      GUCGUCUUC GUCGGCGC
434      GUCUUCGUC GGCGCCGA
473      GGCGGCCUC GGCGACGU
482      GGCGACGUC CUCGGCGG
485      GACGUCCUC GGCGGCCU
527      CACCGUGUC AUGGUCGU
533      GUCAUGGUC GUCUCUCC
536      AUGGUCGUC UCUCCCCG
538      GGUCGUCUC UCCCCGCU
540      UCGUCUCUC CCCGCUAC
547      UCCCCGCUA CGACCAGU
556      CGACCAGUA CAAGGACG
581      ACCAGCGUC GUGUCCGA
586      CGUCGUGUC CGAGAUCA
593      UCCGAGAUC AAGAUGGG
610      AGACAGGUA CGAGACGG
620      GAGACGGUC AGGUUCUU
625      GGUCAGGUU CUUCCACU
626      GUCAGGUUC UUCCACUG
628      CAGGUUCUU CCACUGCU
629      AGGUUCUUC CACUGCUA
637      CCACUGCUA CAAGCGCG
661      CCGCGUGUU CGUUGACC
662      CGCGUGUUC GUUGACCA
665      GUGUUCGUU GACCACCC
679      CCCACUGUU CCUGGAGA
680      CCACUGUUC CUGGAGAG
692      GAGAGGGUU UGGGGAAA
693      AGAGGGUUU GGGGAAAG
716      GAGAAGAUC UACGGGCC
718      GAAGAUCUA CGGGCCUG
742      AACGGACUA CAGGGACA
763      GCUGCGGUU CAGCCUGC
764      CUGCGGUUC AGCCUGCU
773      AGCCUGCUA UGCCAGGC
788      GCAGCACUU GAAGCUCC
795      UUGAAGCUC CAAGGAUC
803      CCAAGGAUC CUGAGCCU
812      CUGAGCCUC AACAACAA
826      CAACCCAUA CUUCUCCG
829      CCCAUACUU CUCCGGAC
830      CCAUACUUC UCCGGACC
832      AUACUUCUC CGGACCAU
841      CGGACCAUA CGGGGAGG
854      GAGGACGUC GUGUUCGU
859      CGUCGUGUU CGUCUGCA
860      GUCGUGUUC GUCUGCAA
863      GUGUUCGUC UGCAACGA
888      CCGGCCCUC UCUCGUGC
890      GGCCCUCUC UCGUGCUA
892      CCCUCUCUC GUGCUACC
898      CUCGUGCUA CCUCAAGA
902      UGCUACCUC AAGAGCAA
913      GAGCAACUA CCAGUCCC
919      CUACCAGUC CCACGGCA
929      CACGGCAUC UACAGGGA
931      CGGCAUCUA CAGGGACG
951      AGACCGCUU UCUGCAUC
952      GACCGCUUU CUGCAUCC
953      ACCGCUUUC UGCAUCCA
959      UUCUGCAUC CACAACAU
968      CACAACAUC UCCUACCA
970      CAACAUCUC CUACCAGG
973      CAUCUCCUA CCAGGGCC
985      GGGCCGGUU CGCCUUCU
986      GGCCGGUUC GCCUUCUC
991      GUUCGCCUU CUCCGACU
992      UUCGCCUUC UCCGACUA
994      CGCCUUCUC CGACUACC
1000     CUCCGACUA CCCGGAGC
1016     CUGAACCUC CCGGAGAG
1027     GGAGAGAUU CAAGUCGU
1028     GAGAGAUUC AAGUCGUC
1033     AUUCAAGUC GUCCUUCG
1036     CAAGUCGUC CUUCGAUU
1039     GUCGUCCUU CGAUUUCA
1040     UCGUCCUUC GAUUUCAU
1044     CCUUCGAUU UCAUCGAC
1045     CUUCGAUUU CAUCGACG
1046     UUCGAUUUC AUCGACGG
1049     GAUUUCAUC GACGGCUA
1057     CGACGGCUA CGAGAAGC
1085     CGGAAGAUC AACUGGAU
1106     GCCGGGAUC CUCGAGGC
1109     GGGAUCCUC GAGGCCGA
1124     GACAGGGUC CUCACCGU
1127     AGGGUCCUC ACCGUCAG
1133     CUCACCGUC AGCCCCUA
1141     CAGCCCCUA CUACGCCG
1144     CCCCUACUA CGCCGAGG
1157     GAGGAGCUC AUCUCCGG
1160     GAGCUCAUC UCCGGCAU
1162     GCUCAUCUC CGGCAUCG
1169     UCCGGCAUC GCCAGGGG
1187     UGCGAGCUC GACAACAU
1196     GACAACAUC AUGCGCCU
1205     AUGCGCCUC ACCGGCAU
1214     ACCGGCAUC ACCGGCAU
1223     ACCGGCAUC GUCAACGG
1226     GGCAUCGUC AACGGCAU
1241     AUGGACGUC AGCGAGUG
1270     GGACAAGUA CAUCGCCG
1274     AAGUACAUC GCCGUGAA
1285     CGUGAAGUA CGACGUGU
1294     CGACGUGUC GACGGCCG
1346     GCGGAGGUC GGGCUCCC
1352     GUCGGGCUC CCGGUGGA
1370     CGGAACAUC CCGCUGGU
1384     GGUGGCGUU CAUCGGCA
1385     GUGGCGUUC AUCGGCAG
1388     GCGUUCAUC GGCAGGCU
1421     CCCGACGUC AUGGCGGC
1436     GCCGCCAUC CCGCAGCU
1445     CCGCAGCUC AUGGAGAU
1472     GUGCAGAUC GUUCUGCU
1475     CAGAUCGUU CUGCUGGG
1476     AGAUCGUUC UGCUGGGC
1501     GAAGAAGUU CGAGCGCA
1502     AAGAAGUUC GAGCGCAU
1514     CGCAUGCUC AUGAGCGC
1534     GGAGAAGUU CCCAGGCA
1535     GAGAAGUUC CCAGGCAA
1559     GCCGUGGUC AAGUUCAA
1564     GGUCAAGUU CAACGCGG
1565     GUCAAGUUC AACGCGGC
1589     CACCACAUC AUGGCCGG
1610     GACGUGCUC GCCGUCAC
1616     CUCGCCGUC ACCAGCCG
1627     CAGCCGCUU CGAGCCCU
1628     AGCCGCUUC GAGCCCUG
1643     UGCGGCCUC AUCCAGCU
1646     GGCCUCAUC CAGCUGCA
1666     GAUGCGAUA CGGAACGC
1690     CUGCGCGUC CACCGGUG
1703     GGUGGACUC GUCGACAC
1706     GGACUCGUC GACACCAU
1715     GACACCAUC AYCGAAGG
1718     ACCAYCAYC GAAGGCAA
1735     GACCGGGUU CCACAUGG
1736     ACCGGGUUC CACAUGGG
1751     GGCCGCCUC AGCGUCGA
1757     CUCAGCGUC GACUGCAA
1769     UGCAACGUC GUGGAGCC
1787     GCGGACGUC AAGAAGGU
1807     CACCACCUU GCAGCGCG
1820     CGCGCCAUC AAGGUGGU
1829     AAGGUGGUC GGCACGCC
1843     GCCGGCGUA CGAGGAGA
1871     UGCAUGAUC CAGGAUCU
1878     UCCAGGAUC UCUCCUGG
1880     CAGGAUCUC UCCUGGAA
1882     GGAUCUCUC CUGGAAGG
1922     GUGCUGCUC AGCCUCGG
1928     CUCAGCCUC GGGGUCGC
1934     CUCGGGGUC GCCGGCGG
1955     CCAGGGGUC GAAGGCGA
1970     GAGGAGAUC GCGCCGCU
1979     GCGCCGCUC GCCAAGGA
2012     UGAAGAGUU CGGCCUGC
2013     GAAGAGUUC GGCCUGCA
2033     CCCCUGAUC UCGCGCGU
2035     CCUGAUCUC GCGCGUGG
2055     AAACAUGUU GGGACAUC
2063     UGGGACAUC UUCUUAUA
2065     GGACAUCUU CUUAUAUA
2066     GACAUCUUC UUAUAUAU
2068     CAUCUUCUU AUAUAUGC
2069     AUCUUCUUA UAUAUGCU
2071     CUUCUUAUA UAUGCUGU
2073     UCUUAUAUA UGCUGUUU
2080     UAUGCUGUU UCGUUUAU
2081     AUGCUGUUU CGUUUAUG
2082     UGCUGUUUC GUUUAUGU
2085     UGUUUCGUU UAUGUGAU
2086     GUUUCGUUU AUGUGAUA
2087     UUUCGUUUA UGUGAUAU
2094     UAUGUGAUA UGGAVAAG
2104     GGACAAGUA UGUGUAGC
2110     GAUAGUGUA GCUGCUUG
2117     UAGCUGCUU GCUUGUGC
2121     UGCUUGCUU GUGCUAGU
2127     CUUGUGCUA GUGUAAUA
2132     GCUAGUGUA AUAUAGUG
2135     AGUGUAAUA UAGUGUAG
2137     UGUAAUAUA GUGUAGUG
2142     UAUAGUGUA GUGGUGGC
2165     CACAACCUA AUAAGCGC
2168     AACCUAAUA AGCGCAUG
2181     CAUGAACUA AUUGCUUG
2184     GAACUAAUU GCUUGCGU
2188     UAAUUGCUU GCGUGUGU
2197     GCGUGUGUS GUUAAGUA
2200     UGUGUAGUU AAGUACCG
2201     GUGUAGUUA AGUACCGA
2205     AGUUAAGUA CCGAUCGG
2211     GUACCGAUC GGUAAUUU
2215     CGAUCGGUA AUUUUAUA
2218     UCGGUAAUU UUAUAUUG
2219     CGGUAAUUU UAUAUUGC
2220     GGUAAUUUU AUAUUGCG
2221     GUAAUUUUA UAUUGCGA
2223     AAUUUUAUA UUGCGAGU
2225     UUUUAUAUU GCGAGUAA
2232     UUGCGAGUA AAUAAAUG
2236     GAGUAAAUA AAUGGACC
2248     GGACCUGUA GUGGUGGA

TABLE III B
Hammerhead Robozyme Sequence Targeted
Against GBSS mRNA
nt.
Position HH Ribozyme Sequence
12       UGGCUGUGGC CUGAUGA X GAA AUCGAUCGGU
68       GCAGUGAGUU CUGAUGA X GAA AUUCCUUCCU
73       GGCUGGCAGU CUGAUGA X GAA AGUUUAUUCC
103      GACGGAGCAG CUGAUGA X GAA ACACUUCUCC
109      CUGGUGGACG CUGAUGA X GAA AGCAGUACAC
113      CGCACUGGUG CUGAUGA X GAA ACGGAGCAGU
146      UCGACGAGAU CUGAUGA X GAA AGCAGCCCUG
149      UCGUCGACGA CUGAUGA X GAA AUGAGCAGCC
151      GGUCGUCGAC CUGAUGA X GAA AUGAGCAGCC
154      ACUGGUCGUC CUGAUGA X GAA ACGAGAUGAG
169      CAUGCCGAUU CUGAUGA X GAA AUCCACUGGU
170      CCAUGCCGAU CUGAUGA X GAA AUCCACUGGU
173      CCGCCAUGCC CUGAUGA X GAA AUUAAUCCAC
186      GACGUGGCUA CUGAUGA X GAA AGCCGCCAUG
188      GCGACGUGGC CUGAUGA X GAA AGAGCCGCCA
196      GACGAGCUGC CUGAUGA X GAA ACGUGGCUAG
203      GCGUUGCGAC CUGAUGA X GAA AGCUGCGACG
206      CGCGCGUUGC CUGAUGA X GAA ACGAGCUGCG
230      ACGCGUCCGG CUGAUGA X GAA ACGCCCAGGC
241      GCGGAACGUG CUGAUGA X GAA ACGCGUCCGG
247      GCCGCGGCGG CUGAUGA X GAA ACGUGGACGC
248      CGCCGCGGCG CUGAUGA X GAA AACGUGGACG
292      GUCCGCCGCC CUGAUGA X GAA ACGCCGUCCG
308      UCCGAAUGCU CUGAUGA X GAA AGCGUGUCCG
314      CGCUGGUCCG CUGAUGA X GAA AUGCUGAGCG
315      GCGCUGGUCC CUGCUGA X GAA AAUGCUGAGC
344      GCUGGUGCUG CUGAUGA X GAA AGCCUGGGCG
385      GAGCGACGGG CUGAUGA X GAA ACCUGGCCCC
386      CGAGCGACGG CUGAUGA X GAA AACCUGGCCC
391      CACGACGAGC CUGAUGA X GAA ACGGGAACCU
395      CGCACACGAC CUGAUGA X GAA AGCGACGGGA
398      UGGCGCACAC CUGAUGA X GAA ACGAGCGACG
425      CGACGAAGAC CUGAUGA X GAA ACGUUCAUGC
428      CGCCGACGAA CUGAUGA X GAA AGACGACGUU
430      GGCGCCGACG CUGAUGA X GAA AGACGACGUU
431      CGGCGCCGAC CUGAUGA X GAA AAGACGACGU
434      UCUCGGCGCC CUGAUGA X GAA ACGAAGACGA
473      GGACGUCGCC CUGAUGA X GAA AGGCCGCCGG
482      GGCCGCCGAG CUGAUGA X GAA ACGUCGCCGA
485      GCAGGCCGCC CUGAUGA X GAA AGGACGUCGC
527      AGACGACCAU CUGAUGA X GAA ACACGGUGCC
533      GGGGAGAGAC CUGAUGA X GAA ACCAUGACAC
536      AGCGGGGAGA CUGAUGA X GAA ACGACCAUGA
538      GUAGCGGGGA CUGAUGA X GAA AGACGACCAU
540      UCGUAGCGGG CUGAUGA X GAA AGAGACGACC
547      GUACUGGUCG CUGAUGA X GAA AGCGGGGAGA
556      GGCGUCCUUG CUGCUGA X GAA ACUGGUCGUA
581      UCUCGGACAC CUGAUGA X GAA ACGCUGGUGU
586      CUUGAUCUCG CUGAUGA X GAA ACACGACGCU
593      CUCCCAUCUU CUGAUGA X GAA AUCUCGGACA
610      GACCGUCUCG CUGAUGA X GAA ACCUGUCUCC
620      GGAAGAACCU CUGAUGA X GAA ACCGUCUCGU
625      GCAGUGGAAG CUGAUGA X GAA ACCUGACCGU
626      AGCAGUGGAA CUGAUGA X GAA AACCUGACCG
628      GUAGCACUGG CUGAUGA X GAA AGAACCUGAC
629      UGUAGCAGUG CUGAUGA X GAA AAGAACCUGA
637      UCCGCGCUUG CUGAUGA X GAA AGCAGUGGAA
661      GUGGUCAACG CUGAUGA X GAA ACACGCGGUC
662      GGUGGUCAAC CUGAUGA X GAA AACACGCGGU
665      GUGGGUGGUC CUGAUGA X GAA ACGAACACGC
679      CCUCUCCAGG CUGAUGA X GAA ACAGUGGGUG
680      CCCUCUCCAH CUGAUGA X GAA AACAGUGGGU
692      UCUUUCCCCA CUGAUGA X GAA ACCCUCUCCA
693      GUCUUUCCCC CUGAUGA X GAA AACCCUCUCC
716      CAGGCCCGUA CUGAUGA X GAA AUCUUCUCCU
718      GUCAGGCCCG CUGAUGA X GAA AGAUCUUCUC
742      GUUGUCCCUG CUGAUGA X GAA AGUCCGUUCC
763      UAGCAGGCUG CUGAUGA X GAA ACCGCAGCUG
764      AUAGCAGGCU CUGAUGA X GAA AACCGCAGCU
773      CUGCCUGGCA CUGAUGA X GAA AGCAGGCUGA
788      UUGGAGCUUC CUGAUGA X GAA AGUGCUGCCU
795      AGGAUCCUUG CUGAUGA X GAA AGCUUCAAGU
803      UGAGGCUCAG CUGAUGA X GAA AUCCUUGGAG
812      GGUUGUUGUU CUGAUGA X GAA AGGCUCAGGA
826      UCCGGAGAAG VUGAUGA X GAA AUGGGUUGUU
829      UGGUCCGGAG CUGAUGA X GAA AGUAUGGGUU
830      AUGGUCCGGA CUGAUGA X GAA AAGUAUGGGC
832      GUAUGGUCCG CUGAUGA X GAA AGAACUAUGG
841      GUCCUCCCCG CUGAUGA X GAA AUGGUCCGGA
854      AGACGAACAC CUGAUGA X GAA ACGUCCUCCC
859      GUUGCAGACG CUGAUGA X GAA ACACGACGUC
860      CGUUGCAGAC CUGAUGA X GAA AACACGACGU
863      AGUCGUUGCA CUGAUGA X GAA ACGAACACGA
888      UAGCACGAGA CUGAUGA X GAA AGGGCCGGUG
890      GGUAGCACGA CUGAUGA X GAA AGAGGGCCGG
892      GAGGUAGCAC CUGUAGA x GAA AGAGAGGGCC
898      GCUCUUGAGG CUGAUGA X GAA AGCACGAGAG
902      AGUUGCUCUU CUGAUGA X GAA AGGUAGCACG
913      GUGGGACUGG CUGAUGA X GAA AGUUGCUCUU
919      GAUGCCGUGG CUGAUGA X GAA ACUGGUAGUU
929      CGUCCCUGUA CUGAUGA X GAA AUGCCGUGGG
931      UGCGUCCCUG CUGAUGA X GAA AGAUGCCGUG
951      UGGAUGCAGA CUGAUGA X GAA AGCGGUCUUU
952      GUGGAUGCAG CUGAUGA X GAA AAGCGGUCUU
953      UGUGGAUGCA CUGAUGA X GAA AAAGCGGUCU
959      AGAUGUUGUG CUGAUGA X GAA AUGCAGAAAG
968      CCUGGUAGGA CUGAUGA X GAA AUGUUGUGGA
970      GCCCUGGUAG CUGAUGA X GAA AGAUGUUGUG
973      CCGGCCCUGG CUGAUGA X GAA AGGAGAUGUU
985      GGAGAAGGCG CUGAUGA X GAA ACCGGCCCUG
986      CGGAGAAGGC CUGAUGA X GAA AACCGGCCCU
991      GUAGUCGGAG CUGAUGA X GAA AGGCGAACCG
992      GGUAGUCGGA CUGAUGA X GAA AAGGCGAACC
994      CGGGAUGUCG CUGAUGA X GAA AGAAGGCGAA
1000     CAGCUCCGGG CUGAUGA X GAA AGUCGGAGAA
1016     AUCUCUCCGG CUGAUGA X GAA AGGUUCAGCU
1027     GGACGACUUG CUGAUGA X GAA AUCUCUCCGG
1028     AGGACGACUU CUGAUGA X GAA AAUCUCUCCG
1033     AUCGAAGGAC CUGAUGA X GAA ACUUGAAUCU
1036     GAAAUCGAAG CUGAUGA X GAA ACGACUUGAA
1039     GAUGAAAUCG CUGAUGA X GAA AGGACGACUU
1040     CGAUGAAAUC CUGAUGA X GAA AAGGACGACU
1044     CCGUCGAUGA CUGAUGA X GAA AUCGAAGGAC
1045     GCCGUCGAUG CUGAUGA X GAA AAUCGAAGGA
1046     AGCCGUCGAU CUGAUGA X GAA AAAUCGAAGG
1049     CGUAGCCGUC CUGAUGA X GAA AUGAAAUCGA
1057     GGGCUUCUCG CUGAUGA X GAA AGCCGUCGAU
1085     UCAUCCAGUU CUGAUGA X GAA AUCUUCCGGC
1106     CGGCCUCGAG CUGAUGA X GAA AUCCCGGCCU
1109     UGUCGGCCUC CUGAUGA X GAA AGGAUCCCGG
1124     UGACGGUGAG CUGAUGA X GAA ACCCUGUCGG
1127     GGCUGACGGU CUGAUGA X GAA AGGACCCUGU
1133     AGUAGGGGCU CUGAUGA X GAA ACGGUGAGGA
1141     CUCGGCGUAG CUGAUGA X GAA AGGGGCUGAC
1144     CUCCUCGGCG CUGAUGA X GAA AGUAGGGGCU
1157     UGCCGGAGAU CUGAUGA X GAA AGCUCCUCGG
1160     CGAUGCCGGA CUGAUGA X GAA AUGAGCUCCU
1162     GGCGAUGCCG CUGAUGA X GAA AGAUGAGCUC
1169     AGCCCCUGGC CUGAUGA X GAA AUGCCGGAGA
1187     UGAUGUUCUG CUGAUGA X GAA AGCUCGCAGC
1196     UGAGGCGCAU CUGAUGA X GAA AUGUUGUCGA
1205     UGAUGCCGGU CUGAUGA X GAA AGGCGCAUGA
1214     CGAUGCCGGU CUGAUGA X GAA AUGCCGGUGA
1223     UGCCGUUGAC CUGAUGA X GAA AUGCCGGUGA
1226     CCAUGCCGUU CUGAUGA X GAA ACGAUGCCGG
1241     CCCACUCGCU CUGAUGA X GAA ACGUCCAUGC
1270     CACGGCGAUG CUGAUGA X GAA ACUUCUCCCU
1274     ACUUCACGGC CUGAUGA X GAA AUGUACUUGU
1285     CGACACGUCG CUGAUGA X GAA ACUUCACGGC
1294     CACGGCCGUC CUGAUGA X GAA ACACGUCGUA
1346     CCGGGAGCCC CUGAUGA X GAA ACCUCCGCCU
1352     GGUCCACCGG CUGAUGA X GAA AGCCCGACCU
1370     CCACCAGCGG CUGAUGA X GAA AUGUUCCGGU
1384     CCUGCCGAUG CUGAUGA X GAA ACGCCACCAG
1385     GCCUGCCGAU CUGAUGA X GAA AACGCCACCA
1388     CCAGCCUGCC CUGAUGA X GAA AUGAACGCCA
1421     CGGCCGCCAU CUGAUGA X GAA ACGUCGGGUC
1436     UGAGCUGCGG CUGAUGA X GAA AUGGCGGCCG
1445     CCAUCUCCAU CUGAUGA X GAA AGCUGCGGGA
1472     CCAGCAGAAC CUGAUGA X GAA AUCUGCACGU
1475     UGCCCAGCAG CUGAUGA X GAA ACGAUCUGCA
1476     GUGCCCAGCA CUGAUGA X GAA AACGAUCUGC
1501     CAUGCGCUCG CUGAUGA X GAA ACUUCUUCUU
1502     GCAUGCGCUC CUGAUGA X GAA AACUUCUUCU
1514     CGGCGCUCAU CUGAUGA X GAA ACUUCUCCUC
1534     CUUGCCUGGG CUGAUGA X GAA ACUUCUCCUC
1535     CCUUGCCUGG CUGAUGA X GAA AACUUCUCCU
1559     CGUUGAACUU CUGAUGA X GAA ACCACGGCGC
1564     CGCCGCGUUG CUGAUGA C GAA ACUUGACCAC
1565     GCGCCGCGUU CUGAUGA X GAA AACUUGACCA
1589     CGCCGGCCAU CUGAUGA X GAA AUGUGGUGCG
1610     UGGUGACGGC CUGAUGA C GAA AGCACGUCGG
1616     AGCGGCUGGU CUGAUGA X GAA ACGGCGAGCA
1627     GCAGGGCUCG CUGAUGA X GAA AGCGGCUGGU
1628     CGCAGGGCUC CUGAUGA X GAA AAGCGGCUGG
1643     GCAGCUGGAU CUGAUGA X GAA AGGCCGCAGG
1646     CCUGCAGCUG CUGAUGA X GAA AUGAGGCCGC
1666     GGGCGUUCCG CUGAUGA X GAA AUCGCAUCCC
1690     UCCACCGGUG CUGAUGA X GAA ACGCGCAGGC
1703     UGGUGUCGAC CUGAUGA X GAA AGUCCACCGG
1706     UGAUGGUGUC CUGAUGA X GAA ACGAGUCCAC
1715     UGCCUUCGAU CUGAUGA X GAA AUGGUGUCGA
1718     UCUUGCCUUC CUGAUGA X GAA AUGAUGGUGU
1735     GCCCAUGUGG CUGAUGA X GAA ACCCGGUCUU
1736     GGCCCAUGUG CUGAUGA X GAA AACCCGGUCU
1751     AGUCGACGCU CUGAUGA X GAA AGGCGGCCCA
1757     CGUUGCAGUC CUGAUGA X GAA ACGCUGAGGC
1769     CCGGCUCCAC CUGAUGA X GAA ACGUUGCAGU
1787     CCACCUUCUU CUGAUGA X GAA ACGUCCGCCG
1807     GGCGCGCUGC CUGAUGA X GAA AGGUGGUGGC
1820     CGACCACCUU CUGAUGA X GAA AUGGCGCGCU
1829     CCGGCGUGCC CUGAUGA X GAA ACCACCUUGA
1843     CAUCUCCUCG CUGAUGA X GAA ACGCCGGCGU
1871     AGAGAUCCUG CUGAUGA X GAA AUCAUGCAGU
1878     UUCCAGGAGA CUGAUGA X GAA AUCCUGGAUC
1880     CCUUCCAGGA CUGAUGA X GAA AGAUCCUGGA
1882     GCCCUUCCAG CUGAUGA X GAA AGAGAUCCUG
1922     CCCCGAGGCU CUGAUGA X GAA AGCAGCACGU
1928     CGGCGACCCC CUGAUGA X GAA AGGCUGAGCA
1934     CGCCGCCGGC CUGAUGA X GAA ACCCCGAGGC
1955     CCUCGCCUUC CUGAUGA X GAA ACCCCUGGCU
1970     CGAGCGGCGC CUGAUGA X GAA AUCUCCUCGC
1979     UCUCCUUGGC CUGAUGA X GAA AGCGGCGCGA
2012     CUGCAGGCCG CUGAUGA X GAA ACUCUUCAGG
2013     CCUGCAGGCC CUGAUGA X GAA AACUCUUCAG
2033     CCACGCGCGA CUGAUGA X GAA AUCAGGGGGC
2035     CACCACGCGC CUGAUGA X GAA AGAUCAGGGG
2055     AAGAUGUCCC CUGAUGA X GAA ACAUGUUUGC
2063     UAUAUAAGAA CUGAUGA X GAA AUGUCCCAAC
2065     CAUAUAUAAG CUGAUGA X GAA AGAUGUCCCA
2066     GCAUAUAUAA CUGAUGA X GAA AAGAUGUCCC
2068     CAGCAUAUAU CUGAUGA X GAA AGAAGAUGUC
2069     ACAGCAYAYA CYGAYGA X GAA AAGAAGAUGU
2071     AAACAGCAUA CUGAUGA X GAA AUAAGAAGAU
2073     CGAAACAGCA CUGAUGA X GAA AUAUAAGAAG
2080     ACAUAAACGA CUGAUGA X GAA ACAGCAUAUA
2081     CACAUAAACG CUGAUGA X GAA AACAGCAUAU
2082     UCACAUAAAC CUGAUGA X GAA AAACAGCAUA
2085     AUAUCACAUA CUGAUGA X GAA ACGAAACAGC
2086     CAUAUCACAU CUGAUGA X GAA AACGAAACAG
2087     CCAUAUCACA CUGAUGA X GAA AAACGAAACA
2094     UACUUGUCCA CUGAUGA X GAA AUCACAUAAA
2104     CAGCUACACA CUGAUGA X GAA ACUUGUCCAU
2110     AGCAAGCAGC CUGAUGA X GAA ACACAUACUU
2117     UAGCACAAGC CUGAUGA X GAA AGCAGCUACA
2121     ACACUAGCAC CUGAUGA X GAA AGCAAGCAGC
2127     UAUAUUACAC CUGAUGA X GAA AGCACAAGCA
2132     UACACUAUAU CUGAUGA X GAA ACACUAGCAC
2135     CACUACUCUA CUGAUGA X GAA AUUACACUAG
2137     ACCACUACAC CUGAUGA X GAA AUAUUACACU
2142     UGGCCACCAC CUGAUGA X GAA ACACUAUAUU
2165     AUGCGCUUAU CUGAUGA X GAA AGGUUGUGCC
2168     UUCAUGCGCU CUGAUGA X GAA AUUAGGUUGU
2181     CGCAAGCAAU CUGAUGA X GAA AGUUCAUGCG
2184     ACACGCAAGC CUGAUGA X GAA AUUAGUUCAU
2188     CUACACACGC CUGAUGA X GAA AGCAAUUAGU
2197     GGUACUUAAC CUGAUGA X GAA ACACACGCAA
2200     AUCGGUACUU CUGAUGA X GAA ACUACACACG
2201     GAUCGGUACU CUGAUGA X GAA AACUACACAC
2205     UACCGAUCGG CUGAUGA X GAA ACUUAACUAC
2211     UAAAAUUACC CUGAUGA X GAA AUCGGUACUU
2215     AAUAUAAAAU CUGAUGA X GAA ACCGAUCGGU
2218     CGCAAUAUAA CUGAUGA X GAA AUUACCGAUC
2219     UCGCAAUAUA CUGAUGA X GAA AAUUACCGAU
2220     CUCGCAAUAU CUGAUGA X GAA AAAUUACCGA
2221     ACUCGCAAUA CUGAUGA X GAA AAAAUUACCG
2223     UUACUCGCAA CUGAUGA X GAA AUAAAAUUAC
2225     AUUUACUCGC CUGAUGA X GAA AUAUAAAAUU
2232     UCCAUUUAUU CUGAUGA X GAA ACUCGCAAUA
2236     CAGGUCCAUU CUGAUGA X GAA AUUUACUCGC
2248     UUUCCACCAC CUGAUGA X GAA ACAGGUCCAU
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
HH Ribozyme Sequences Tested against GBSS mRNA
nt.		Sequence
Position	HH Ribozyme Sequence	I.D.
425	CGACGAAGAC CUGAUGAGGCCGAAAGGCCGAA ACGUUCAUGC	2
593	CUCCCAUCUU CUGAUGAGGCCGAAAGGCCGAA AUCUCGGACA	3
742	GUUGUCCCUG CUGAUGAGGCCGAAAGGCCGAA AGUCCGUUCC	4
812	GGUUGUUGUU CUGAUGAGGCCGAAAGGCCGAA AGGCUCAGAA	5
892	GAGGUAGCAC CUGAUGAGGCCGAAAGGCCGAA AGAGAGGGCC	6
913	GUGGGACUGG CUGAUGAGGCCGAAAGGCCGAA AGUUGCUCUU	7
919	GAUGCCGUGG CUGAUGAGGCCGAAAGGCCGAA ACUGGUAGUU	8
953	UGUGGAUGCA CUGAUGAGGCCGAAAGGCCGAA AAAGCGGUCU	9
959	AGAUGUUGUG CUGAUGAGGCCGAAAGGCCGAA AUGCAGAAAG	10
968	CCUGGUAGGA CUGAUGAGGCCGAAAGGCCGAA AUGUUGUGGA	11
1016	AUCUCUCCGG CUGAUGAGGCCGAAAGGCCGAA AGGUUCAGCU	12
1028	AGGACGACUU CUGAUGAGGCCGAAAGGCCGAA AAUCUCUCCG	13
1085	UCAUCCAGUU CUGAUGAGGCCGAAAGGCCGAA AUCUUCCGGC	14
1187	UGAUGUUGUC CUGAUGAGGCCGAAAGGCCGAA AGCUCGCAGC	15
1196	UGAGGCGCAU CUGAUGAGGCCGAAAGGCCGAA AUGUUGUCGA	16
1226	CCAUGCCGUU CUGAUGAGGCCGAAAGGCCGAA ACGAUGCCGG	17
1241	CCCACUCGCU CUGAUGAGGCCGAAAGGCCGAA ACGUCCAUGC	18
1270	CACGGCGAUG CUGAUGAGGCCGAAAGGCCGAA ACUUGUCCCU	19
1352	GGUCCACCGG CUGAUGAGGCCGAAAGGCCGAA AGCCCGACCU	20
1421	CGGCCGCCAU CUGAUGAGGCCGAAAGGCCGAA ACGUCGGGUC	21
1534	CUUGCCUGGG CUGAUGAGGCCGAAAGGCCGAA ACUUCUCCUC	22
1715	UGCCUUCGAU CUGAUGAGGCCGAAAGGCCGAA AUGGUGUCGA	23
1787	CCACCUUCUU CUGAUGAGGCCGAAAGGCCGAA ACGUCCGCCG	24

TABLE V A
GBSS Hairpin Ribozyme and Substrate Sequences
nt.
Position	Hairpin Ribozyme Sequence	Substrate
48	CUCCUGGC AGAA GUCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGACA GCC GCCAGGAG
129	CCCUGCCG AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GCACC GCC CGGCAGGG
468	GUCGCCGA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGGCG GCC UCGGCGAC
489	CGGCGGCA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGGCG GCC UGCCGCCG
496	CCAUGGCC AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CUGCC GCC GGCCAUGG
676	UCUCCAGG AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CCACU GUU CCUGGAGA
737	UCCCUGUA AGAA GUUC ACCAGAGAAACACACGUUCUGGUACAUUACCUGGUA	GAACG GAC AUCAGGGA
760	GCAGGCUG AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CUGCG GUU CAGCCUGC
1298	GCCUCCAC AGAA GUCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGACG GCC GUGGAGGC
1427	GGGAUGGC AGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	UGGCG GCC GCCAUCCC
1601	GCGAGCAC AGAA GCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GCGCC GAC GUGCUCGC
1638	CUGGAUGA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CUGCG GCC UCAUCCAG
1746	GACGCUGA AGAA GCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GGGCC GCC UCAGCGUC
1781	UUCUUGAC AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGGCG GAC GUCAAGAA
2077	AUAAACGA AGAA GCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	AUGCU GUU UCGUUUAU

TABLE VB
GBSS Hairpin Ribozyme and Substrate Sequences
nt.
Position	Ribozyme Sequence	Substrate
31	GUCGCCUC AGAA GGUGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACCACCC GCC GAGGCGAC
48	CUCCUGGC AGAA GUCGCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGCGACA GCC GCCAGGAG
105	GUGGACGG AGAA GUACAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GUGUACU GCU CCGUCCAC
110	CACUGGUG AGAA GAGCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CUGCUCC GUC CACCAGUG
129	CCCUGCCG AGAA GUGCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GCGCACC GCC CGGCAGGG
142	ACGAGAUG AGAA GCCCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CAGGGCU GCU CAUCUCGU
182	GUGGCUAG AGAA GCCUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CAUGGCG GCU CUAGCCAC
199	UUGCGACG AGAA GCGACG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGUCGCA GCU CGUCGCAA
219	GACGCCCA AGAA GGCGCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGCGCCG GCC UGGGCGUC
233	GUGGACGC AGAA GGGACG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGUCCCG GAC GCGUCCAC
249	GGCGCCGC AGAA GAACGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACGUUCC GCC GCGGCGCC
283	CCGACGCC AGAA GGCCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GGGGCCG GAC GGCGUCGG
316	GCGCGCUG AGAA GAAUGC ACGAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GCAUUCG GAC CAGCGCGC
388	CGACGAGC AGAA GGAACC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GGUUCCC GUC GCUCGUCG
468	GUCGCCGA AGAA GCCGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACCGGCG GCC ACGGCGAC
489	CGGCGGCA AGAA GCCGAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CUCGGCG GCC UGCCGCCG
493	UGGCCGGC AGAA GGCCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GCGGCCU GCC GCCGGCCA
496	CCAUGGCC AGAA GCAGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GCCUGCC GCC GGCCAUGG
676	UCUCCAGG AGAA GUGGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACCCACU GUU CCUGGAGA
725	GUUCCAGC AGAA GGCCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGGGCCU GAC GCUGGAAC
737	UCCCUGUA AGAA GUUCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	UGGAACG GAC AUCAGGGA
754	UGAACCGC AGAA GGUUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACAACCA GCU GCGGUUCA
760	GCAGGCUG AGAA GCAGCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	AGCUGCG GUU CAGCCUGC
765	GCAUAGCA AGAA GAACCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGGUUCA GCC UGCUAUGC
834	CCCGUAUG AGAA GGAGAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	UUCUCCG GAC CAUACGGG
882	CGAGAGAG AGAA GGUGUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CACACCG GCC CUCUCUCG
916	UGCCGUGG AGAA GGUAGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACUACCA GUC CCACGGCA
947	AUGCAGAA AGAA GUCUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	AAAGACC GCU UUCUGCAU
982	AGAAGGCG AGAA GGCCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	AGGGCCG GUU CGCCUUCU
995	UCCGGGUA AGAA GAGAAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CUUCUCC GAC UACCCGGA
1134	GUAGUAGG AGAA GACGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACCGUCA GCC CCUACUAC
1298	GCCUCCAC AGAA GUCGAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GUCGACG GCC GUGGAGGC
1372	ACGCCACC AGAA GGAUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACAUCCC GCU GGUGGCGU
1415	GCCAUGAC AGAA GGUCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GGGACCC GAC GUCAUGGC
1427	GGGAUGGC AGAA GCCAUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CAUGGCG GCC GCCAUCCC
1441	UCUCCAUG AGAA GCGGGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	UCCCGCA GCU CAUGGAGA
1468	GCAGAACG AGAA GCACGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACGUGCA GAU CGUUCUGC
1477	CCGUGCCC AGAA GAACGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	UCGUUCU GCU GGGCACGG
1601	GCGAGCAC AGAA GCGCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CGGCGCC GAC GUGCUCGC
1620	CUCGAAGC AGAA GGUGAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GUCACCA GCC GCUUCGAG
1623	GGGCACGA AGAA GCUGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACCAGCC GCU UCGAGCCC
1638	CUGGAUGA AGAA GCAGGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CCCUGCG GCC UCAUCCAG
1648	UCCCCUGC AGAA GGAUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	UCAUCCA GCU GCAGGGGA
1746	GACGCUGA AGAA GCCCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	AUGGGCC GCC UCAGCGUC
1781	UUCUUGAC AGAA GCCGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GCCGGCG GAC GUCAAGAA
1918	CGAGGCUG AGAA GCACGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	ACGUGCU GCU CAGCCUCG
1923	GACCCCGA AGAA GAGCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	CUGCUCA GCC UCGGGGUC
1975	CCUUGGCG AGAA GCGCGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	UCGCGCC GCU CGCCAAGG
2014	GGCCUGCA AGAA GAACUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GAGUUCG GCC UGCAGGCC
2029	CGCGCGAG AGAA GGGGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	GCCCCCU GAU CUCGCGCG
2077	AUAAACGA AGAA GCAUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	AUAUGCU GUU UVHUUUAU
2113	CACAAGCA AGAA GCUACA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	UGUAGCU GCU UGCUUGUG
2207	AAUUACCG AGAA GUACUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA	AAGUACC GAU CGGUAAUU

TABLE VI
Delta-9 Desaturase HH Ribozyme Target Sequences
nt.
Position Substrate
13       CGCGCCCUC UGCCGCUU
21       CUGCCGCUU GUUCGUUC
24       CCGCUUGUU CGUUCCUC
25       CGCUUGUUC GUUCCUCG
28       UUGUUCGUU CCUCGCGC
29       UGUUCGUUC CUCGCGCU
32       UCGUUCCUC GCGCUCGC
38       CUCGCGCUC GCCACCAG
63       ACACACAUC CCAAUCUC
69       AUCCCAAUC UCGCGAGG
71       CCCAAUCUC GCGAGGGC
92       AGCAGGGUC UGCGGCGG
117      GCCGCGCUU CCGGCUCC
118      CCGCGCUUC CGGCUCCC
124      UUCCGGCUC CCCUUCCC
129      GCUCCCCUU CCCAUUGG
130      CUCCCCUUC CCAUUGGC
135      CUUCCCAUU GGCCUCCA
141      AUUGGCCUC CACGAUGG
154      AUGGCGCUC CGCCUCAA
160      CUCCGCCUC AACGACGU
169      AACGACGUC GCGCUCUG
175      GUCGCGCUC UGCCUCUC
181      CUCUGCCUC UCCCCGCC
183      CUGCCUCUC CCCGCCGC
193      CCGCCGCUC GCCGCCCG
228      CGGCAGGUU CGUCGCCG
229      GGCAGGUUC GUCGCCGU
232      AGGUUCGUC GCCGUCGC
238      GUCGCCGUC GCCUCCAU
243      CGUCGCCUC CAUGACGU
252      CAUGACGUC CGCCGUCU
259      UCCGCCGUC UCCACCAA
261      CGCCGUCUC CACCAAGG
271      ACCAAGGUC GAGAAUAA
278      UCGAGAAUA AGAAGCCA
288      GAAGCCAUU UGCUCCUC
289      AAGCCAUUU GCUCCUCC
293      CAUUUGCUC CUCCAAGG
296      UUGCUCCUC CAAGGGAG
307      AGGGAGGUA CAUGUCCA
313      GUACAUGUC CAGGUUAC
319      GUCCAGGUU ACACAUUC
320      UCCAGGUUA CACAUUCA
326      UUACACAUU CAAUGCCA
327      UACACAUUC AAUGCCAC
338      UGCCACCUC ACAAGAUU
346      CACAAGAUU GAAAUUUU
352      AUUGAAAUU UUCAAGUC
353      UUGAAAUUU UCAAGUCG
354      UGAAAUUUU CAAGUCGC
355      GAAAUUUUC AAGUCGCU
360      UUUCAAGUC GCUUGAUG
364      AAGUCGCUU GAUGAUUG
371      UUGAUGAUU GGGCAUGA
377      AUUGGGCUA GAGAUAAU
383      CUAGAGAUA AUAUCUUG
386      GAGAUAAUA UCUUGACG
388      GAUAAUAUC UUGACGCA
390      UAAUAUCUU GACGCAUC
398      UGACGCAUC UCAAGCCA
400      ACGCAUCUC AAGCCAGU
409      AAGCCAGUC GAGAAGUG
419      AGAAGUGUU GGCAGCCA
434      CACAGGAUU UCCUCCCG
435      ACAGGAUUU CCUCCCGG
436      CAGGAUUUC CUCCCGGA
439      GAUUUCCUC CCGGACCC
453      CCCAGCAUC UGAAGGAU
462      UGAAGGAUU UCAUGAUG
463      GAAGGAUUU CAUGAUGA
464      AAGGAUUUC AUGAUGAA
475      GAUGAAGUU AAGGAGCU
476      AUGAAGUUA AGGAGCUC
484      AAGGAGCUC AGAGAACG
505      AAGGAAAUC CCUGAUGA
515      CUGAUGAUU AUUUUGUU
516      UGAUGAUUA UUUUGUUU
518      AUGAUUAUU UUGUUUGU
519      UGAUUAUUU UGUUUGUU
520      GAUUAUUUU GUUUGUUU
523      UAUUUUGUU UGUUUGGU
524      AUUUUGUUU GUUUGGUG
527      UUGUUUGUU UGGUGGGA
528      UGUUUGUUU GGUGGGAG
544      GACAUGAUU ACCGAGGA
545      ACAUGAUUA CCGAGGAA
557      AGGAAGCUC UACCAACA
559      GAAGCUCUA CCAACAUA
567      ACCAACAUA CCAGACUA
575      ACCAGACUA UGCUUAAC
580      ACUAUGCUU AACACCCU
581      CUAUGCUUA ACACCCUC
589      AACACCCUC GACGGUGU
598      GACGGUGUC AGAGAUGA
637      UGGGCUGUU UGGACGAG
638      GGGCUGUUU GGACGAGG
680      AUGGUGAUC UGCUCAAC
685      CAACAAGUA UAUGUACC
693      CAACAAGUA UAUGUACC
695      ACAAGUAUA UGUACCUC
699      GUAUAUGUA CCUCACUG
703      AUGUACCUC ACUGGGAG
719      GGGUGGAUA UGAGGCAG
730      AGGCAGAUU GAGAAGAC
742      AAGACAAUU CAGUAUCU
743      AGACAAUUC AGUAUCUU
747      AAUUCAGUA UCUUAUUG
749      UUCAGUAUC UUAUUGGC
751      CAGUAUCUU AUUGGCUC
752      AGUAUCUUA UUGGCUCU
754      UAUCUUAUU GGCUCUGG
759      UAUUGGCUC UGGAAUGG
770      GAAUGGAUC CUAGGACU
773      UGGAUCCUA GGACUGAG
785      CUGAGAAUA AUCCUUAU
788      AGAAUAAUC CUUAUCUU
791      AUAAUCCUU AUCUUGGU
792      UAAUCCUUA UCUUGGUU
794      AUCCUUAUC UUGGUUUC
796      CCUUAUCUU GGUUUCAU
800      AUCUUGGUU UCAUCUAC
801      UCUUGGUUU CAUCUACA
802      CUUGGUUUC AUCUACAC
805      GGUUUCAUC UACACCUC
807      UUUCAUCUA CACCUCCU
813      CUACACCUC CUUCCAAG
816      CACCUCCUU CCAAGAGC
817      ACCUCCUUC CAAGAGCG
834      GGCGACCUU CAUCUCAC
835      GCGACCUUC AUCUCACA
838      ACCUUCAUC UCACACGG
840      CUUCAUCUC ACACGGGA
857      ACACUGCUC GUCACGCC
860      CUGCUCGUC ACGCCAAG
873      CAAGGACUU UGGCGACU
874      AAGGACUUU GGCGACUU
882      UGGCGACUU AAAGCUUG
883      GGCGACUUA AAGCUUGC
889      UUAAAGCUU GCACAAAU
898      GCACAAAUC UGCGGCAU
907      UGCGGCAUC AUCGCCUC
910      GGCAUCAUC GCCACAGA
915      CAUCGCCUC AGAUGAGA
942      AACUGCGUA CACCAAGA
952      ACCAAGAUC GUGGAGAA
966      GAAGCUGUU UGAGAUCG
967      AAGCUGUUU GAGAUCGA
973      UUUGAGAUC GACCCUGA
986      CUGAUGGUA CCGUGGUC
994      ACCGUGGUC GCUCUGGC
998      UGGUCGCUC UGGCUGAC
1024     AAGAAGAUC UCAAUGCC
1026     GAAGAUCUC AAUGCCUG
1047     CCUGAUGUU UGACGGGC
1048     CUGAUGUUU GACGGGCA
1071     CAAGCUGUU CGAGCACU
1072     AAGCUGUUC GAGCACUU
1080     CGAGCACUU CUCCAUGG
1081     GAGCACUUC UCCAUGGU
1083     GCACUUCUC CAUGGUCG
1090     UCCAUGGUC GCGCAGAG
1102     CAGAGGCUU GGCGUUUA
1108     CUUGGCGUU UACACCGC
1109     UUGGCGUUU ACACCGCC
1110     UGGCGUUUA CACCGCCA
1125     CAGGGACUA CGCCGACA
1135     GCCGACAUC CUCGAGUU
1138     GACAUCCUC GAGUUCCU
1143     CCUCGAGUU CCUCGUCG
1144     CUCGAGUUC CUCGUCGA
1147     GAGUUCCUC GUCGACAG
1150     UUCCUCGUC GACAGGUG
1181     UGACUGGUC UGUCGGGU
1185     UGGUCUGUC GGGUGAAG
1212     GCAGGACUA CCUUUGCA
1216     GACUACCUU UGCACCCU
1217     ACUACCUUU GCACCCUU
1225     UGCACCCUU GCUUCAAG
1229     CCCUUGCUU CAAGAAUC
1230     CCUUGCUUC AAGAAUCA
1237     UCAAGAAUC AGGAGGCU
1292     CGCUGCCUU UCAGCUGG
1293     GCUGCCUUU CAGCUGGG
1294     CUGCCUUUC AGCUGGGU
1303     AGCUGGGUA UACGGUAG
1305     CUGGGUAUA CGGUAGGG
1310     UAUACGGUA GGGACGUC
1318     AGGGACGUC CAACUGUG
1331     UGUGAGAUC GGAAACCU
1348     GCUGCGGUC UGCUUAGA
1353     GGUCUGCUU AGACAAGA
1354     GUCUGCUUA GACAAGAC
1372     UGCUGUGUC UGCGUUAC
1378     GUCUGCGUU ACAUAGGU
1379     UCUGCGUUA CAUAGGUC
1383     CGUUACAUA GGUCUCCA
1387     ACAUAGGUC UCCAGGUU
1389     AUAGGUCUC CAGGUUUU
1395     CUCCAGGUU UUGAUCAA
1396     UCCAGGUUU UGAUCAAA
1397     CCAGGUUUU GAUCAAAU
1401     GUUUUGAUC AAAUGGUC
1409     CAAAUGGUC CCGUGUCG
1416     UCCCGUGUC GUCUUAUA
1419     CGUGUCGUC UUAUAGAG
1421     UGUCGUCUU AUAGAGCG
1422     GUCGUCUUA UAGAGCGA
1424     CGUCUUAUA GAGCGAUA
1432     AGAGCGAUA GGAGAACG
1444     GAACGUGUU GGUCUGUG
1448     GUGUUGGUC UGUGGUGU
1457     UGUGGUGUA GCUUUGUU
1461     GUGUAGCUU UGUUUUUA
1462     UGUAGCUUU GUUUUUAU
1465     AGCUUUGUU UUUAUUUU
1466     GCUUUGUUU UUAUUUUG
1467     CUUUGUUUU UAUUUUGU
1468     UUUGUUUUU AUUUUGUA
1469     UUGUUUUUA UUUUGUAU
1471     GUUUUUAUU UUGUAUUU
1472     UUUUUAUUU UGUAUUUU
1473     UUUUAUUUU GUAUUUUU
1476     UAUUUUGUA UUUUUCUG
1478     UUUUGUAUU UUUGUGCU
1479     UUUGUAUUU UUCUGCUU
1480     UUGUAUUUU UVUGCUUU
1481     UGUAUUUUU CUGCUUUG
1482     GUAUUUUUC UGCUUUGA
1487     UUUCUGCUU UGAUGUAC
1488     UUCUGCUUU GAUGAUCA
1494     UUUGAUGUA CAACCUGU
1546     CAUGCCGUA CUUUGUCU
1549     GCCGUACUU UGUCUGUC
1550     CCGUACUUU GUCUGUCG
1553     UACUUUGUC UGUCGCUG
1557     UUGUCUGUC GCUGGCGG
1571     CGGUGUGUU UCGGUAUG
1572     GGUGUGUUU CGGUAUGU
1573     GUGUGUUUC GGUAUGUU
1577     GUUUCGGUA UGUUAUUU
1581     CGGUAUGUU AUUUGAGU
1582     GGUAUGUUA UUUGAGUU
1584     UAUGUUAUU UGAGUUGC
1585     AUGUUAUUU GAGUUGCU
1590     AUUUGAGUU GCUCAGAU
1594     GAGUUGCUC AGAUCUGU
1599     GCUCAGAUC UGUUAAAA
1603     AGAUCUGUU AAAAAAAA
1604     GAUCUGUUA AAAAAAAA

TABLE VII
Delta-9 Desaturase HH Ribozyme Sequences
nt.
Position Ribozyme sequence
13       AAGCGGCA CUGAUGA X GAA AGGGCGCG
21       GAACGAAC CUGAUGA X GAA AGCGGCAG
24       GAGGAACG CUGAUGA X GAA ACAAGCGG
25       CGAGGAAC CUGAUGA X GAA AACAAGCG
28       GCGCGAGG CUGAUGA X GAA ACGAACAA
29       AGCGCGAG CUGAUGA X GAA AACGAACA
32       GCGAGCGC CUGAUGA X GAA AGGAACGA
38       CUGGUGGC CUGAUGA X GAA AGCGCGAG
63       GAGAUUGG CUGAUGA X GAA AUGUGUGU
69       CCUCGCGA CUGAUGA X GAA AUUGGGAU
71       GCCCUCGC CUGAUGA X GAA AGAUUGGG
92       CCGCCGCA CUGAUGA X GAA ACCCUGCU
117      GGAGCCGG CUGAUGA X GAA AGCGCGGC
118      GGGAGCCG CUGAUGA X GAA AAGCGCGG
124      GGGAAGGG CUGAUGA X GAA AGCCGGAA
129      CCAAUGGG CUGAUGA X GAA AGGGGAGC
130      GCCAAUGG CUGAUGA X GAA AAGGGGAG
135      UGGAGGCC CUGAUGA X GAA AUGGGAAG
141      CCAUCGUG CUGAUGA X GAA AGGCCAAU
154      UUGAGGCG CUGAUGA X GAA AGCGCCAU
160      ACGUCGUU CUGAUGA X GAA AGGCGGAG
169      CAGAGCGC CUGAUGA X GAA ACGUCGUU
175      GAGAGGCA CUGAUGA X GAA AGCGCGAC
181      GGCGGGGA CUGAUGA X GAA AGGCAGAG
183      GCGGCGGG CUGAUGA X GAA AGAGGCAG
193      CGGGCGGC CUGAUGA X GAA AGCGGCGG
228      CGGCGACG CUGAUGA X GAA ACCUGCCG
229      ACGGCGAC CUGAUGA X GAA AACCUGCC
232      GCGACGGC CUGAUGA X GAA ACGAACCU
238      AUGGAGGC CUGAUGA X GAA ACGGCGAC
243      ACGUCAUG CUGAUGA X GAA AGGCGACG
252      AGACGGCG CUGAUGA X GAA ACGUCAUG
259      UUGGUGGA CUGAUGA X GAA ACGGCGGA
261      CCUUGGUG CUGAUGA X GAA AGACGGCG
271      UUAUUCUC CUGAUGA X GAA ACCUUGGU
278      UGGCUUCU CUGAUGA X GAA AUUCUCGA
288      GAGGAGCA CUGAUGA X GAA AUGGCUUC
289      GGAGGAGC CUGAUGA X GAA AAUGGCUU
293      CCUUGGAG CUGAUGA X GAA AGCAAAUG
296      CUCCCUUG CUGAUGA X GAA AGGAGCAA
307      UGGACAUG CUGAUGA X GAA ACCUCCCU
313      GUAACCUG CUGAUGA X GAA ACAUGUAC
319      GAAUGUGU CUGAUGA X GAA ACCUGGAC
320      UGAAUGUG CUGAUGA X GAA AACCUGGA
326      UGGCAUUG CUGAUGA X GAA AUGUGUAA
327      GUGGCAUU CUGAUGA X GAA AAUGUGUA
338      AAUCUUGU CUGAUGA X GAA AGGUGGCA
346      AAAAUUUC CUGAUGA X GAA AUCUUGUG
352      GACUUGAA CUGAUGA X GAA AUUUCAAU
353      CGACUUGA CUGAUGA X GAA AAUUUCAA
354      GCGACUUG CUGAUGA X GAA AAAUUUCA
355      AGCGACUU CUGAUGA X GAA AAAAUUUC
360      CAUCAAGC CUGAUGA X GAA ACUUGAAA
364      CAAUCAUC CUGAUGA X GAA AGCGACUU
371      UCUAGCCC CUGAUGA X GAA AUCAUCAA
377      AUUAUCUC CUGAUGA X GAA AGCCCAAU
383      CAAGAUAU CUGAUGA X GAA AUCUCUAG
386      CGUCAAGA CUGAUGA X GAA AUUAUCUC
388      UGCGUCAA CUGAUGA X GAA AUAUUAUC
390      GAUGCGUC CUGAUGA X GAA AGAUAUUA
398      UGGCUUGA CUGAUGA X GAA AUGCGUCA
400      ACUGGCUU CUGAUGA X GAA AGAUGCGU
409      CACUUCUC CUGAUGA X GAA ACUGGCUU
419      UGGCUGCC CUGAUGA X GAA ACACUUCU
434      CGGGAGGA CUGAUGA X GAA AUCCUGUG
435      CCGCGAGG CUGAUGA X GAA AAUCCUGU
436      UCCGGGAG CUGAUGA X GAA AAAUCCUG
439      GGGUCCGG CUGAUGA X GAA AGGAAAUC
453      AUCCUUCA CUGAUGA X GAA AUGCUGGG
462      CAUCAUGA CUGAUGA X GAA AUCCUUCA
463      UCAUCAUG CUGAUGA X GAA AAUCCUUC
464      UUCAUCAU CUGAUGA X GAA AAAUCCUU
475      AGCUCCUU CUGAUGA X GAA ACUUCAUC
476      GAGCUCCU CUGAUGA X GAA AACUUCAU
484      CGUUCUCU CUGAUGA X GAA AGCUCCUU
505      UCAUCAGG CUGAUGA X GAA AUUUCCUU
515      AACAAAAU CUGAUGA X GAA AUCAUCAG
516      AAACAAAA CUGAUGA X GAA AAUCAUCA
518      ACAAACAA CUGAUGA X GAA AUAAUCAU
519      AACAAACA CUGAUGA X GAA AAUAAUCA
520      AAACAAAC CUGAUGA X GAA AAAUAAUC
523      ACCAAACA CUGAUGA X GAA ACAAAAUA
524      CACCAAAC CUGAUGA X GAA AACAAAAU
527      UCCCACCA CUGAUGA X GAA ACAAACAA
528      CUCCCACC CUGAUGA X GAA AACAAACA
544      UCCUCGGU CUGAUGA X GAA AUCAUGUC
545      UUCCUCGG CUGAUGA X GAA AAUCAUGU
557      UGUUGGUA CUGAUGA X GAA AGCUUCCU
559      UAUCUUGG CUGAUGA X GAA AGAGCUUC
567      UAGUCUGG CUGAUGA X GAA AUGUUGGU
575      GUUAAGCA CUGAUGA X GAA AGUCUGGU
580      AGGGUGUU CUGAUGA X GAA AGCAUAGU
581      GAGGGUGU CUGAUGA X GAA AAGCAUAG
589      ACACCGUC CUGAUGA X GAA AGGGUGUU
598      UCAUCUCU CUGAUGA X GAA ACACCGUC
637      CUCGUCCA CUGAUGA X GAA ACAGCCCA
638      CCUCGUCC CUGAUGA X GAA AACAGCCC
680      GUUGAGCA CUGAUGA X GAA AUCACCAU
685      UAGUUGUU CUGAUGA X GAA AGCAGAUC
693      GGUACAUA CUGAUGA X GAA ACUUGUUG
695      GAGGUACA CUGAUGA X GAA AUACUUGU
699      CAGUGAGG CUGAUGA X GAA ACAUAUAC
703      CUCCCAGU CUGAUGA X GAA AGGUACAU
719      CUGCCUCA CUGAUGA X GAA AUCCACCC
730      GUCUUCUC CUGAUGA X GAA AUCUGCCU
742      AGAUACUG CUGAUGA X GAA AUUGUCUU
743      AAGAUACU CUGAUGA X GAA AAUUGUCU
747      CAAUAAGA CUGAUGA X GAA ACUGAAUU
749      GCCAAUAA CUGAUGA X GAA AUACUGAA
751      GAGCCAAU CUGAUGA X GAA AGAUACUG
752      AGAGCCAA CUGAUGA X GAA AAGAUACU
754      CCAGAGCC CUGAUGA x GAA AUAAGAUA
759      CCAUUCCA CUGAUGA X GAA AGCCAAUA
770      AGUCCUAG CUGAUCA X GAA AUCCAUUC
773      CUCAGUCC CUGAUGA X GAA AGGAUCCA
785      AUAAGGAU CUGAUGA X GAA AUUCUCAG
788      AAGAUAAG CUGAUGA X GAA AUUAUUCU
791      ACCAAGAU CUGAUGA X GAA AGGAUUAU
792      AACCAAGA CUGAUGA X GAA AAGGAUUA
794      GAAACCAA CUGAUGA X GAA AUAAGGAU
796      AUGAAACC CUGAUGA X GAA AGAUAAGG
800      GUAGAUGA CUGAUGA X GAA ACCAAGAU
801      UGUAGAUG CUGAUGA X GAA AACCAAGA
802      GUGUAGAU CUGAUGA X GAA AAAGCAAG
805      GAGGUGUA CUGAUGA X GAA AUGAAACC
807      AGGAGGUG CUGAUGA X GAA AGAUGAAA
813      CUUGGAAG CUGAUGA X GAA AGGUGUAG
816      GCUCUUGG CUGAUGA X GAA AGGAGGUG
817      CGCUCUUG CUGAUGA X GAA AAGGAGGU
834      GUGAGAUG CUGAUGA X GAA AGGUCGCC
835      UGUGAGAU CUGAUGA X GAA AAGGUCGC
838      CCGUGUGA CUGAUGA X GAA AUGAAGGU
840      UCCCGUGU CUGAUGA X GAA AGAUGAAG
857      GGCGUGAC CUGAUGA X GAA AGCAGUGU
860      CUUGGCGU CUGAUGA X GAA ACGAGCAG
873      AGUCGCCA CUGAUGA X GAA AGUGCUUG
874      AAGUCGCC CUGAUGA X GAA AAGUCCUU
882      CAAGCUUU CUGAUGA X GAA AGUCGCCA
883      GCAAGCUU CUGAUGA X GAA AAGUCGCC
889      AUUUGUGC CUGAUGA X GAA AGCUUUAA
898      AUGCCGCA CUGAUGA X GAA AUUUGUGC
907      GAGGCGAU CUGAUGA X GAA AUGCCGCA
910      UCUGAGGC CUGAUGA X GAA AUGAUGCC
915      UCUCAUCU CUGAUGA X GAA AGGCGAUG
942      UCUUGGUG CUGAUGA X GAA ACGCAGUU
952      UUCUCCAC CUGAUGA X GAA AUCUUGGU
966      CGAUCUCA CUGAUGA X GAA ACAGCUUC
967      UCGAUCUC CUGAUGA X GAA AACAGCUU
973      UCAGGGUC CUGAUGA X GAA AUCUCAAA
986      GACCACGG CUGAUGA X GAA ACCAUCAG
994      GCCAGAGC CUGAUGA X GAA ACCACGGU
998      GUCAGCCA CUGAUGA X GAA AGCGACCA
1024     GGCAUUGA CUGAUGA X GAA AUCUUCUU
1026     CAGGCAUU CUGAUGA X GAA AGAUCUUC
1047     GCCCGUCA CUGAUGA X GAA ACAUCAGG
1048     UGCCCGUC CUGAUGA X GAA AACAUCAG
1071     AGUGCUCG CUGAUGA X GAA ACAGCUUG
1072     AAGUGCUC CUGAUGA X GAA AACAGCUU
1080     CCAUGGAG CUGAUGA X GAA AGUGCUCG
1081     ACCAUGGA CUGAUGA X GAA AAGUGCUC
1083     CGACCAUG CUGAUGA X GAA AGAAGUGC
1090     CUCUGCGC CUGAUGA X GAA ACCAUGGA
1102     UAAACGCC CUGAUGA X GAA AGCCUCUG
1108     GCGGUGUA CUGAUGA X GAA ACGCCAAG
1109     GGCGGUGU CUGAUGA X GAA AACGCCAA
1110     UGGCGGUG CUGAUGA X GAA AAACGCCA
1125     UGUCGGCG CUGAUGA X GAA AGUCCCUG
1135     AACUCGAG CUGAUGA X GAA AUGUCGGC
1138     AGGAACUC CUGAUGA X GAA AGGAUGUC
1143     CGACGAGG CUGAUGA X GAA ACUCCAGG
1144     UCGACGAG CUGAUGA X GAA AACUCGAG
1147     CUGUCGAC CUGAUGA X CAA AGGAACUC
1150     CACCUGUC CUGAUGA X GAA ACGAGGAA
1181     ACCCGACA CUGAUGA X GAA ACCAGUCA
1185     CUUCACCC CUGAUGA X GAA ACAGACCA
1212     UGCAAAGG CUGAUGA X GAA AGUCCUGC
1216     AGGGUGCA CUGAUGA X GAA AGGUAGUC
1217     AAGGGUGC CUGAUGA X GAA AAGGUAGU
1225     CUUGAAGC CUGAUGA X GAA AGGGUGCA
1229     GAUUCUUG CUGAUGA X GAA AGCAAGGG
1230     UGAUUCUU CUGAUGA X GAA AAGCAAGG
1237     AGCCUCCU CUGAUGA X GAA AUUCUUGA
1292     CCAGCUGA CUGAUGA X GAA AGGCAGCG
1293     CCCAGCUG CUGAUGA X GAA AAGGCAGC
1294     ACCCAGCU CUGAUGA X GAA AAAGGCAG
1303     CUACCGUA CUGAUGA X GAA ACCCAGCU
1305     CCCUACCG CUGAUGA X GAA AUACCCAG
1310     GACGUCCC CUGAUGA X GAA ACCGUAUA
1318     CACAGUUG CUGAUGA X GAA ACGUCCCU
1331     AGGUUUCC CUGAUGA X GAA AUCUCACA
1348     UCUAAGCA CUGAUGA X GAA ACCGCAGC
1353     UCUUGUCU CUGAUGA X GAA AGCAGACC
1354     GUCUUGUC CUGAUGA X GAA AAGCAGAC
1372     GUAACGCA CUGAUGA X GAA ACACAGCA
1378     ACCUAUGU CUGAUGA X GAA ACGCAGAC
1379     GACCUAUG CUGAUGA x GAA AACGCAGA
1383     UGGAGACC CUGAUGA X GAA AUGUAACG
1387     AACCUGGA CUGAUGA X GAA ACCUAUGU
1389     AAAACCUG CUGAUGA X GAA AGACCUAU
1395     UUGAUCAA CUGAUGA X GAA ACCUGGAG
1396     UUUGAUCA CUGAUGA X GAA AACCUGGA
1397     AUUUGAUC CUGAUGA X GAA AAACCUGG
1401     GACCAUUU CUGAUGA X GAA AUCAAAAC
1409     CGACACGG CUGAUGA X GAA ACCAUUUG
1416     UAUAAGAC CUGAUGA X GAA ACACGGGA
1419     CUCUAUAA CUGAUGA x GAA ACGACACG
1421     CGCUCUAU CUGAUGA X GAA AGACGACA
1422     UCGCUCUA CUGAUGA X GAA AAGACGAC
1424     UAUCGCUC CUGAUGA x GAA AUAAGACG
1432     CGUUCUCC CUGAUGA X GAA AUCGCUCU
1444     CACAGACC CUGAUGA X GAA ACACGUUC
1448     ACACCACA CUGAUGA X GAA ACCAACAC
1457     AACAAAGC CUGAUGA X GAA ACACCACA
1461     UAAAAACA CUGAUGA X GAA AGCUACAC
1462     AUAAAAAC CUGAUGA X GAA AAGCUACA
1465     AAAAUAAA CUGAUGA X GAA ACAAAGCU
1466     CAAAAUAA CUGAUGA X GAA AACAAAGC
1467     ACAAAAUA CUGAUGA X GAA AAACAAAG
1468     UACAAAAU CUGAUGA X GAA AAAACAAA
1469     AUACAAAA CUGAUGA X GAA AAAAACAA
1471     AAAUACAA CUGAUGA X GAA AUAAAAAC
1472     AAAAUACA CUGAUGA X GAA AAUAAAAA
1473     AAAAAUAC CUGAUGA X GAA AAAUAAAA
1476     CAGAAAAA CUGAUGA X GAA ACAAAAUA
1478     AGCAGAAA CUGAUGA X GAA AUACAAAA
1479     AAGCAGAA CUGAUGA X GAA AAUACAAA
1480     AAAGCAGA CUGAUGA X GAA AAAUACAA
1481     CAAAGCAG CUGAUGA X GAA AAAAUACA
1482     UCAAAGCA CUGAUGA X GAA AAAAAUAC
1487     GUACAUCA CUGAUGA X GAA AGCAGAAA
1488     UGUACAUC CUGAUGA X GAA AAGCAGAA
1494     ACAGGUUG CUGAUGA X GAA ACAUCAAA
1546     AGACAAAG CUGAUGA X GAA ACGGCAUG
1549     GACAGACA CUGAUGA X GAA AGUACGGC
1550     CGACAGAC CUGAUGA X GAA AAGUACGG
1553     CAGCGACA CUGAUGA X GAA ACAAAGUA
1557     CCGCCAGC CUGAUGA X GAA ACAGAGAA
1571     CAUACCGA CUGAUGA X GAA ACACACCG
1572     ACAUACCG CUGAUGA X GAA AACACACC
1573     AACAUACC CUGAUGA X GAA AAACACAC
1577     AAAUAACA CUGAUGA X GAA ACCGAAAC
1581     ACUCAAAU CUGAUGA X GAA ACAUACCG
1582     AACUCAAA CUGAUGA X GAA AACAUACC
1584     GCAACUCA CUGAUGA X GAA AUAACAUA
1585     AGCAACUC CUGAUGA X GAA AAUAACAU
1590     AUCUGAGC CUGAUGA X GAA ACUCAAAU
1594     ACAGAUCU CUGAUGA X GAA AGCAACUC
1599     UUUUAACA CUGAUGA X GAA AUCUGAGC
1603     UUUUUUUU CUGAUGA X GAA ACAGAUCU
1604     UUUUUUUU CUGAUGA X GAA AACAGAUC
Where “X”  represents stem II region of a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252). The length of stem II may be ≧2 base-pairs.

TABLE VIII
Delta-9 Desaturase Hairpin Ribozyme and Substrate Sequences
nt. Position	Ribozyme	Substrate
14	GAACAAGC AGAA GAGGGC	GCCCUCU GCC GCUUGUUC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
17	AACGAACA AGAA GCAGAG	CUCUGCC GCU UGUUCGUU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
108	GGAAGCGC AGAA GCCGCC	GGCGGCG GCC GCGCUUCC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
120	GGAAGGGG AGAA GGAAGC	GCUUCCG GCU CCCCUUCC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
155	GUCGUUGA AGAA GAGCGC	GCGCUCC GCC UCAACGAC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
176	CGGGGAGA AGAA GAGCGC	GCGCUCU GCC UCUCCCCG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
186	CGGCGAGC AGAA GGGAGA	UCUCCCC GCC GCUCGCCG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
189	GGGCGGCG AGAA GCGGGG	CCCCGCC GCU CGCCGCCC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
196	CGGCGGCG AGAA GCGAGC	GCUCGCC GCC CGCCGCCG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
200	GCGGCGGC AGAA GGCGGC	GCCGCCC GCC GCCGCCGC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGCUA
203	GCGGCGGC AGAA GCGGGC	GCCCGCC GCC GCCGCCGC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
206	GCUGCGGC AGAA GCGGCG	CGCCGCC GCC GCCGCAGC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
209	GCUGCUGC AGAA GCGGCG	CGCCGCC GCC GCAGCAGC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
235	AUGGAGGC AGAA GCGACG	CGUCGCC GUC GCCUCCAU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
253	GUGGAGAC AGAA GACGUC	GACGUCC GCC GUCUCCAC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
256	UUGGUGGA AGAA GCGGAC	GUCCGCC GUC UCCACCAA
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
406	CACUUCUC AGAA GGCUUG	CAAGCCA GUC GAGAAGUG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
442	GAUGCUGG AGAA GGGAGG	CCUCCCG GAC CCAGCAUC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGGA
508	AAAUAAUC AGAA GGGAUU	AAUCCCU GAU GAUUAUUU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUCGUA
570	UAAGCAUA AGAA GGUAUG	CAUACCA GAC UAUGCUUA
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
625	ACAGCCCA AGAA GUGGGG	CCCCACU GCC UGGGCUGU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
634	CUCGUCCA AGAA GCCCAG	CUGGGCU GUU UGGACGAG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
655	UUCUCCUC AGAA GUCCAU	AUGGACU GCU GAGGAGAA
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
681	ACUUGUUG AGAA GAUCAC	GUGAUCU GCU CAACAAGU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
726	UCUUCUCA AGAA GCCUCA	UGAGGCA GAU UGAGAAGA
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
853	GCGUGACG AGAA GUGUUC	GAACACU GCU CGUCACGC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
916	CGCUUCUC AGAA GAGGCG	CGCCUCA GAU GAGAAGCG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
963	CGAUCUCA AGAA GCUUCU	AGAAGCU GUU UGAGAUCG
	ACCAGAGAAACACACCUUGUCGUACAUUACCUGGUA
979	ACGGUACC AGAA GGGUCG	CGACCCU GAU GGUACCGU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1033	AUCAGGUG AGAA GGCAUU	AAUGCCU GCC CACCUGAU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1041	CGUCAAAC AGAA GGUGGG	CCCACCU GAU GUUUGACG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1068	AGUGCUCG AGAA GCUUGU	ACAAGCU GUU CGAGCACU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1173	ACAGACCA AGAA GGCUCG	CGAGCCU GAC UGGUCUGU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1182	CUUCACCC AGAA GACCAG	CUGGUCU GUC GGGUGAAG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1287	AGCUGAAA AGAA GCGUGC	GCACGCU GCC UUUCAGCU
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1295	GUAUACCC AGAA GAAAGG	CCUUUCA GCU GGGUAUAC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1339	CAGACCGC AGAA GGUUUC	GAAACCU GCU GCGGUCUG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1345	UCUAAGCA AGAA GCAGCA	UGCUGCG GUC UGCUUAGA
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1349	CUUGUCUA AGAA GACCGC	GCGGUCU GCU UAGACAAG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1364	GCAGACAC AGAA GGUCUU	AAGACCU GCU GUGUCUGC
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1483	UACAUCAA AGAA GAAAAA	UUUUUCU GCU UUGAUGUA
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1554	CCGCCAGC AGAA GACAAA	UUUGUCU GUC GCUGGCGG
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA
1595	UUUAACAG AGAA GAGCAA	UUGCUCA GAU CUGUUAAA
	ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

TABLE IX
Cleavage of Delta-9 Desaturase RNA by HH Ribozymes
	Percent Cleaved
	20° C.		26° C.
nt. Position	10 min	120 min	10 min	120 min
183	6.3	7.0	10.45	11.8
252	25.2	51.2	33.1	52.9
259	20.3	41.3	24.8	44.0
271	17.2	52.4	21.5	56.3
278	9.9	25.7	13.3	33.6
307	10.3	24.2	9.2	32.4
313	16.9	43.0	23.8	53.4
320	10.6	23.6	15.0	31.3
326	5.7	14.6	8.0	17.1
338	10.0	17.5	10.4	12.9
353	10.2	11.3	10.7	14.7
390	8.6	8.9	7.8	9.8
419	6.3	10.1	5.8	10.9
453	7.3	29.0	8.0	33.8
484	7.8	28.9	6.9	29.2
545	4.8	8.5	3.6	8.9
773	4.5	11.5	4.4	8.9
1024	11.9	17.1	13.3	23.8
1026	11.6	12.6	13.1	17.2
1237	23.1	32.4	13.8	28.6

TABLE X
Construct	Targets	Isolates	Greenhouse	Plants
Number	Blasted	Recovered	Lines	Produced
RPA85	231	70	13	161
RPA113	292	82	9	116
RPA114	244	35	12	152
RPA115	285	42	11	165
RPA118	268	38	10	125
RPA119	301	67	11	135
Totals	1621	334	66	854

TABLE XI
Stearic acid levels in leaves from plants transformed with active
and inactive ribozymes compared to control leaves.
Stearic Acid in Leaves Transformed with Active and Inactive
Ribozymes
(Percentage of total plants with certain levels of leaf stearic acid)
		Ribozyme
	Ribozyme Actives	Inactives
	(428 plants	(406 plants	Controls
Stearic Acid	from 35 lines)	from 31 lines)	(122 plants)
>3%	7%	3%	2%
>5%	2%	0	0
>10%	0	0	0

TABLE XII
Inheritance of the high stearic acid trait in leaves from crosses
of high stearic acid plants.
Inheritance of high stearate in leaves.
	R1 Plants	R1 Plants	% of Plants
	with Normal	with High	with High
Cross	Leaf Stearate	Leaf Stearate	Stearate
RPA85-15.06 × RPA85-15.12	6	3	33%
RPA85-15.07 self	5	5	50%
RPA85-15.10 self	8	2	20%
OQ414 × RPA85-15.06	5	3	38%
OQ414 × RPA85-15.11	6	4	40%

TABLE XIII
Comparison of fatty acid composition of embryogenic callus,
somatic embryos and zygotic embryos.
Tissue and/						% Lipid
or Media	Fatty Acid Composition	of Fresh
Treatment	C16:0	C18:0	C18:1	C18:2	C18:3	Weight
embryogenic	19.4	1.1	6.2	55.7	8.8	0.4
callus	+/−	+/−	+/−	+/−	+/−	+/−
	0.9	0.1	2.0	3.1	2.0	0.1
somatic	12.6	1.6	18.2	60.7	1.9	4.0
embryo grown	+/−	+/−	+/−	+/−	+/−	+/−
on MS + 6%	0.7	0.8	4.9	5.1	0.3	1.1
sucrose +
10 mM ABA
zygotic embryo	14.5	1.1	18.5	60.2	1.4	3.9
12 days after	+/−	+/−	+/−	+/−	+/−	+/−
pollination	0.4	0.1	1.0	1.5	0.2	0.6

TABLE XIV
GBSS activity, amylose content, and Southern analysis results of
selected Ribozyme Lines
	GBSS activity	Amylose Content
Line	(Units/mg starch)	(%)	Southern
RPA63.0283	321.5 ± 31.2	23.3 ± 0.5	−
RPA63.0236	314.6 ± 9.2	27.4 ± 0.3	−
RPA63.0219	299.8 ± 10.4	21.5 ± 0.3	−
RPA63.0314	440.4 ± 17.1	19.1 ± 0.8	−
RPA63.0316	346.5 ± 8.5	17.9 ± 0.5	−
RPA63.0311	301.5 ± 17.4	19.5 ± 0.4	−
RPA63.0309	264.7 ± 19	21.7 ± 0.1	+
RPA63.0218	190.8 ± 7.8	21.0 ± 0.3	+
RPA63.0209	203 ± 2.4	22.6 ± 0.6	+
RPA63.0306	368.2 ± 7.5	19.0 ± 0.4	−
RPA63.0210	195.1 ± 7	22.1 ± 0.2	+

1263 1621 base pairs nucleic acid single linear Coding Sequence 146...1324 1CGCACGCGCC CTCTGCCGCT TGTTCGTTCC TCGCGCTCGC CACCAGGCAC CACCACACAC 60ATCCCAATCT CGCGAGGGCA AGCAGCAGGG TCTGCGGCGG CGGCGGCGGC CGCGCTTCCG 120GCTCCCCTTC CCATTGGCCT CCACG ATG GCG CTC CGC CTC AAC GAC GTC GCG 172 Met Ala Leu Arg Leu Asn Asp Val Ala 1 5CTC TGC CTC TCC CCG CCG CTC GCC GCC CGC CGC CGC CGC CGC AGC AGC 220Leu Cys Leu Ser Pro Pro Leu Ala Ala Arg Arg Arg Arg Arg Ser Ser10 15 20 25GGC AGG TTC GTC GCC GTC GCC TCC ATG ACG TCC GCC GTC TCC ACC AAG 268Gly Arg Phe Val Ala Val Ala Ser Met Thr Ser Ala Val Ser Thr Lys 30 35 40GTC GAG AAT AAG AAG CCA TTT GCT CCT CCA AGG GAG GTA CAT GTC CAG 316Val Glu Asn Lys Lys Pro Phe Ala Pro Pro Arg Glu Val His Val Gln 45 50 55GTT ACA CAT TCA ATG CCA CCT CAC AAG ATT GAA ATT TTC AAG TCG CTT 364Val Thr His Ser Met Pro Pro His Lys Ile Glu Ile Phe Lys Ser Leu 60 65 70GAT GAT TGG GCT AGA GAT AAT ATC TTG ACG CAT CTC AAG CCA GTC GAG 412Asp Asp Trp Ala Arg Asp Asn Ile Leu Thr His Leu Lys Pro Val Glu 75 80 85AAG TGT TGG CAG CCA CAG GAT TTC CTC CCG GAC CCA GCA TCT GAA GGA 460Lys Cys Trp Gln Pro Gln Asp Phe Leu Pro Asp Pro Ala Ser Glu Gly90 95 100 105TTT CAT GAT GAA GTT AAG GAG CTC AGA GAA CGT GCC AAG GAA ATC CCT 508Phe His Asp Glu Val Lys Glu Leu Arg Glu Arg Ala Lys Glu Ile Pro 110 115 120GAT GAT TAT TTT GTT TGT TTG GTG GGA GAC ATG ATT ACC GAG GAA GCT 556Asp Asp Tyr Phe Val Cys Leu Val Gly Asp Met Ile Thr Glu Glu Ala 125 130 135CTA CCA ACA TAC CAG ACT ATG CTT AAC ACC CTC GAC GGT GTC AGA GAT 604Leu Pro Thr Tyr Gln Thr Met Leu Asn Thr Leu Asp Gly Val Arg Asp 140 145 150GAG ACA GGT GCA AGC CCC ACT GCC TGG GCT GTT TGG ACG AGG GCA TGG 652Glu Thr Gly Ala Ser Pro Thr Ala Trp Ala Val Trp Thr Arg Ala Trp 155 160 165ACT GCT GAG GAG AAC AGG CAT GGT GAT CTG CTC AAC AAG TAT ATG TAC 700Thr Ala Glu Glu Asn Arg His Gly Asp Leu Leu Asn Lys Tyr Met Tyr170 175 180 185CTC ACT GGG AGG GTG GAT ATG AGG CAG ATT GAG AAG ACA ATT CAG TAT 748Leu Thr Gly Arg Val Asp Met Arg Gln Ile Glu Lys Thr Ile Gln Tyr 190 195 200CTT ATT GGC TCT GGA ATG GAT CCT AGG ACT GAG AAT AAT CCT TAT CTT 796Leu Ile Gly Ser Gly Met Asp Pro Arg Thr Glu Asn Asn Pro Tyr Leu 205 210 215GGT TTC ATC TAC ACC TCC TTC CAA GAG CGG GCG ACC TTC ATC TCA CAC 844Gly Phe Ile Tyr Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile Ser His 220 225 230GGG AAC ACT GCT CGT CAC GCC AAG GAC TTT GGC GAC TTA AAG CTT GCA 892Gly Asn Thr Ala Arg His Ala Lys Asp Phe Gly Asp Leu Lys Leu Ala 235 240 245CAA ATC TGC GGC ATC ATC GCC TCA GAT GAG AAG CGA CAT GAA ACT GCG 940Gln Ile Cys Gly Ile Ile Ala Ser Asp Glu Lys Arg His Glu Thr Ala250 255 260 265TAC ACC AAG ATC GTG GAG AAG CTG TTT GAG ATC GAC CCT GAT GGT ACC 988Tyr Thr Lys Ile Val Glu Lys Leu Phe Glu Ile Asp Pro Asp Gly Thr 270 275 280GTG GTC GCT CTG GCT GAC ATG ATG AGG AAG AAG ATC TCA ATG CCT GCC 1036Val Val Ala Leu Ala Asp Met Met Arg Lys Lys Ile Ser Met Pro Ala 285 290 295CAC CTG ATG TTT GAC GGG CAG GAC GAC AAG CTG TTC GAG CAC TTC TCC 1084His Leu Met Phe Asp Gly Gln Asp Asp Lys Leu Phe Glu His Phe Ser 300 305 310ATG GTC GCG CAG AGG CTT GGC GTT TAC ACC GCC AGG GAC TAC GCC GAC 1132Met Val Ala Gln Arg Leu Gly Val Tyr Thr Ala Arg Asp Tyr Ala Asp 315 320 325ATC CTC GAG TTC CTC GTC GAC AGG TGG AAG GTG GCG AGC CTG ACT GGT 1180Ile Leu Glu Phe Leu Val Asp Arg Trp Lys Val Ala Ser Leu Thr Gly330 335 340 345CTG TCG GGT GAA GGG AAC AAG GCG CAG GAC TAC CTT TGC ACC CTT GCT 1228Leu Ser Gly Glu Gly Asn Lys Ala Gln Asp Tyr Leu Cys Thr Leu Ala 350 355 360TCA AGA ATC AGG AGG CTG GAG GAG AGG GCC CAG AGC AGA GCC AAG AAA 1276Ser Arg Ile Arg Arg Leu Glu Glu Arg Ala Gln Ser Arg Ala Lys Lys 365 370 375GCC GGC ACG CTG CCT TTC AGC TGG GTA TAC GGT AGG GAC GTC CAA CTG TG 1326Ala Gly Thr Leu Pro Phe Ser Trp Val Tyr Gly Arg Asp Val Gln Leu 380 385 390AGATCGGAAA CCTGCTGCGG TCTGCTTAGA CAAGACCTGC TGTGTCTGCG TTACATAGGT 1386CTCCAGGTTT TGATCAAATG GTCCCGTGTC GTCTTATAGA GCGATAGGAG AACGTGTTGG 1446TCTGTGGTGT AGCTTTGTTT TTATTTTGTA TTTTTCTGCT TTGATGTACA ACCTGTGGCC 1506GCATGAACTG GGGCGTGGAG ATGGGAGCGA CCATGCCGTA CTTTGTCTGT CGCTGGCGGT 1566GTGTTTCGGT ATGTTATTTG AGTTGCTCAG ATCTGTTAAA AAAAAAAAAA AAAAA 1621 42 base pairs nucleic acid single linear 2CGACGAAGAC CUGAUGAGGC CGAAAGGCCG AAACGUUCAU GC 42 42 base pairs nucleic acid single linear 3CUCCCAUCUU CUGAUGAGGC CGAAAGGCCG AAAUCUCGGA CA 42 42 base pairs nucleic acid single linear 4GUUGUCCCUG CUGAUGAGGC CGAAAGGCCG AAAGUCCGUU CC 42 42 base pairs nucleic acid single linear 5GGUUGUUGUU CUGAUGAGGC CGAAAGGCCG AAAGGCUCAG GA 42 42 base pairs nucleic acid single linear 6GAGGUAGCAC CUGAUGAGGC CGAAAGGCCG AAAGAGAGGG CC 42 42 base pairs nucleic acid single linear 7GUGGGACUGG CUGAUGAGGC CGAAAGGCCG AAAGUUGCUC UU 42 42 base pairs nucleic acid single linear 8GAUGCCGUGG CUGAUGAGGC CGAAAGGCCG AAACUGGUAG UU 42 42 base pairs nucleic acid single linear 9UGUGGAUGCA CUGAUGAGGC CGAAAGGCCG AAAAAGCGGU CU 42 42 base pairs nucleic acid single linear 10AGAUGUUGUG CUGAUGAGGC CGAAAGGCCG AAAUGCAGAA AG 42 42 base pairs nucleic acid single linear 11CCUGGUAGGA CUGAUGAGGC CGAAAGGCCG AAAUGUUGUG GA 42 42 base pairs nucleic acid single linear 12AUCUCUCCGG CUGAUGAGGC CGAAAGGCCG AAAGGUUCAG CU 42 42 base pairs nucleic acid single linear 13AGGACGACUU CUGAUGAGGC CGAAAGGCCG AAAAUCUCUC CG 42 42 base pairs nucleic acid single linear 14UCAUCCAGUU CUGAUGAGGC CGAAAGGCCG AAAUCUUCCG GC 42 42 base pairs nucleic acid single linear 15UGAUGUUGUC CUGAUGAGGC CGAAAGGCCG AAAGCUCGCA GC 42 42 base pairs nucleic acid single linear 16UGAGGCGCAU CUGAUGAGGC CGAAAGGCCG AAAUGUUGUC GA 42 42 base pairs nucleic acid single linear 17CCAUGCCGUU CUGAUGAGGC CGAAAGGCCG AAACGAUGCC GG 42 42 base pairs nucleic acid single linear 18CCCACUCGCU CUGAUGAGGC CGAAAGGCCG AAACGUCCAU GC 42 42 base pairs nucleic acid single linear 19CACGGCGAUG CUGAUGAGGC CGAAAGGCCG AAACUUGUCC CU 42 42 base pairs nucleic acid single linear 20GGUCCACCGG CUGAUGAGGC CGAAAGGCCG AAAGCCCGAC CU 42 42 base pairs nucleic acid single linear 21CGGCCGCCAU CUGAUGAGGC CGAAAGGCCG AAACGUCGGG UC 42 42 base pairs nucleic acid single linear 22CUUGCCUGGG CUGAUGAGGC CGAAAGGCCG AAACUUCUCC UC 42 42 base pairs nucleic acid single linear 23UGCCUUCGAU CUGAUGAGGC CGAAAGGCCG AAAUGGUGUC GA 42 42 base pairs nucleic acid single linear 24CCACCUUCUU CUGAUGAGGC CGAAAGGCCG AAACGUCCGC CG 42 2267 base pairs nucleic acid single linear 25GACCGATCGA TCGCCACAGC CAACACCACC CGCCGAGGCG ACGCGACAGC CGCCAGGAGG 60AAGGAATAAA CTCACTGCCA GCCAGTGAAG GGGGAGAAGT GTACTGCTCC GTCCACCAGT 120GCGCGCACCG CCCGGCAGGG CTGCTCATCT CGTCGACGAC CAGTGGATTA ATCGGCATGG 180CGGCTCTAGC CACGTCGCAG CTCGTCGCAA CGCGCGCCGG CCTGGGCGTC CCGGACGCGT 240CCACGTTCCG CCGCGGCGCC GCGCAGGGCC TGAGGGGGGG CCGGACGGCG TCGGCGGCGG 300ACACGCTCAG CATTCGGACC AGCGCGCGCG CGGCGCCCAG GCTCCAGCAC CAGCAGCAGC 360AGCAGGCGCG CCGCGGGGCC AGGTTCCCGT CGCTCGTCGT GTGCGCCAGC GCCGGCATGA 420ACGTCGTCTT CGTCGGCGCC GAGATGGCGC CGTGGAGCAA GACCGGCGGC CTCGGCGACG 480TCCTCGGCGG CCTGCCGCCG GCCATGGCCG CGAATGGGCA CCGTGTCATG GTCGTCTCTC 540CCCGCTACGA CCAGTACAAG GACGCCTGGG ACACCAGCGT CGTGTCCGAG ATCAAGATGG 600GAGACAGGTA CGAGACGGTC AGGTTCTTCC ACTGCTACAA GCGCGGAGTG GACCGCGTGT 660TCGTTGACCA CCCACTGTTC CTGGAGAGGG TTTGGGGAAA GACCGAGGAG AAGATCTACG 720GGCCTGACGC TGGAACGGAC TACAGGGACA ACCAGCTGCG GTTCAGCCTG CTATGCCAGG 780CAGCACTTGA AGCTCCAAGG ATCCTGAGCC TCAACAACAA CCCATACTTC TCCGGACCAT 840ACGGGGAGGA CGTCGTGTTC GTCTGCAACG ACTGGCACAC CGGCCCTCTC TCGTGCTACC 900TCAAGAGCAA CTACCAGTCC CACGGCATCT ACAGGGACGC AAAGACCGCT TTCTGCATCC 960ACAACATCTC CTACCAGGGC CGGTTCGCCT TCTCCGACTA CCCGGAGCTG AACCTCCCGG 1020AGAGATTCAA GTCGTCCTTC GATTTCATCG ACGGCTACGA GAAGCCCGTG GAAGGCCGGA 1080AGATCAACTG GATGAAGGCC GGGATCCTCG AGGCCGACAG GGTCCTCACC GTCAGCCCCT 1140ACTACGCCGA GGAGCTCATC TCCGGCATCG CCAGGGGCTG CGAGCTCGAC AACATCATGC 1200GCCTCACCGG CATCACCGGC ATCGTCAACG GCATGGACGT CAGCGAGTGG GACCCCAGCA 1260GGGACAAGTA CATCGCCGTG AAGTACGACG TGTCGACGGC CGTGGAGGCC AAGGCGCTGA 1320ACAAGGAGGC GCTGCAGGCG GAGGTCGGGC TCCCGGTGGA CCGGAACATC CCGCTGGTGG 1380CGTTCATCGG CAGGCTGGAA GAGCAGAAGG GACCCGACGT CATGGCGGCC GCCATCCCGC 1440AGCTCATGGA GATGGTGGAG GACGTGCAGA TCGTTCTGCT GGGCACGGGC AAGAAGAAGT 1500TCGAGCGCAT GCTCATGAGC GCCGAGGAGA AGTTCCCAGG CAAGGTGCGC GCCGTGGTCA 1560AGTTCAACGC GGCGCTGGCG CACCACATCA TGGCCGGCGC CGACGTGCTC GCCGTCACCA 1620GCCGCTTCGA GCCCTGCGGC CTCATCCAGC TGCAGGGGAT GCGATACGGA ACGCCCTGCG 1680CCTGCGCGTC CACCGGTGGA CTCGTCGACA CCATCATCGA AGGCAAGACC GGGTTCCACA 1740TGGGCCGCCT CAGCGTCGAC TGCAACGTCG TGGAGCCGGC GGACGTCAAG AAGGTGGCCA 1800CCACCTTGCA GCGCGCCATC AAGGTGGTCG GCACGCCGGC GTACGAGGAG ATGGTGAGGA 1860ACTGCATGAT CCAGGATCTC TCCTGGAAGG GCCCTGCCAA GAACTGGGAG AACGTGCTGC 1920TCAGCCTCGG GGTCGCCGGC GGCGAGCCAG GGGTCGAAGG CGAGGAGATC GCGCCGCTCG 1980CCAAGGAGAA CGTGGCCGCG CCCTGAAGAG TTCGGCCTGC AGGCCCCCTG ATCTCGCGCG 2040TGGTGCAAAC ATGTTGGGAC ATCTTCTTAT ATATGCTGTT TCGTTTATGT GATATGGACA 2100AGTATGTGTA GCTGCTTGCT TGTGCTAGTG TAATATAGTG TAGTGGTGGC CAGTGGCACA 2160ACCTAATAAG CGCATGAACT AATTGCTTGC GTGTGTAGTT AAGTACCGAT CGGTAATTTT 2220ATATTGCGAG TAAATAAATG GACCTGTAGT GGTGGAAAAA AAAAAAA 2267 17 base pairs nucleic acid single linear 26CGAUCGAUCG CCACAGC 17 17 base pairs nucleic acid single linear 27GGUCGUCUCU CCCCGCU 17 17 base pairs nucleic acid single linear 28GAAGGAAUAA ACUCACU 17 17 base pairs nucleic acid single linear 29UCGUCUCUCC CCGCUAC 17 17 base pairs nucleic acid single linear 30AAUAAACUCA CUGCCAG 17 17 base pairs nucleic acid single linear 31UCCCCGCUAC GACCAGU 17 17 base pairs nucleic acid single linear 32AGAAGUGUAC UGCUCCG 17 17 base pairs nucleic acid single linear 33CGACCAGUAC AAGGACG 17 17 base pairs nucleic acid single linear 34GUACUGCUCC GUCCACC 17 17 base pairs nucleic acid single linear 35ACCAGCGUCG UGUCCGA 17 17 base pairs nucleic acid single linear 36UGCUCCGUCC ACCAGUG 17 17 base pairs nucleic acid single linear 37CGUCGUGUCC GAGAUCA 17 17 base pairs nucleic acid single linear 38GGGCUGCUCA UCUCGUC 17 17 base pairs nucleic acid single linear 39UCCGAGAUCA AGAUGGG 17 17 base pairs nucleic acid single linear 40CUGCUCAUCU CGUCGAC 17 17 base pairs nucleic acid single linear 41AGACAGGUAC GAGACGG 17 17 base pairs nucleic acid single linear 42GCUCAUCUCG UCGACGA 17 17 base pairs nucleic acid single linear 43GAGACGGUCA GGUUCUU 17 17 base pairs nucleic acid single linear 44CAUCUCGUCG ACGACCA 17 17 base pairs nucleic acid single linear 45GGUCAGGUUC UUCCACU 17 17 base pairs nucleic acid single linear 46CAGUGGAUUA AUCGGCA 17 17 base pairs nucleic acid single linear 47GUCAGGUUCU UCCACUG 17 17 base pairs nucleic acid single linear 48AGUGGAUUAA UCGGCAU 17 17 base pairs nucleic acid single linear 49CAGGUUCUUC CACUGCU 17 17 base pairs nucleic acid single linear 50GGAUUAAUCG GCAUGGC 17 17 base pairs nucleic acid single linear 51AGGUUCUUCC ACUGCUA 17 17 base pairs nucleic acid single linear 52UGGCGGCUCU AGCCACG 17 17 base pairs nucleic acid single linear 53CCACUGCUAC AAGCGCG 17 17 base pairs nucleic acid single linear 54GCGGCUCUAG CCACGUC 17 17 base pairs nucleic acid single linear 55CCGCGUGUUC GUUGACC 17 17 base pairs nucleic acid single linear 56AGCCACGUCG CAGCUCG 17 17 base pairs nucleic acid single linear 57CGCGUGUUCG UUGACCA 17 17 base pairs nucleic acid single linear 58UCGCAGCUCG UCGCAAC 17 17 base pairs nucleic acid single linear 59GUGUUCGUUG ACCACCC 17 17 base pairs nucleic acid single linear 60CAGCUCGUCG CAACGCG 17 17 base pairs nucleic acid single linear 61CCCACUGUUC CUGGAGA 17 17 base pairs nucleic acid single linear 62CUGGGCGUCC CGGACGC 17 17 base pairs nucleic acid single linear 63CCACUGUUCC UGGAGAG 17 17 base pairs nucleic acid single linear 64GGACGCGUCC ACGUUCC 17 17 base pairs nucleic acid single linear 65GAGAGGGUUU GGGGAAA 17 17 base pairs nucleic acid single linear 66GUCCACGUUC CGCCGCG 17 17 base pairs nucleic acid single linear 67AGAGGGUUUG GGGAAAG 17 17 base pairs nucleic acid single linear 68UCCACGUUCC GCCGCGG 17 17 base pairs nucleic acid single linear 69GAGAAGAUCU ACGGGCC 17 17 base pairs nucleic acid single linear 70GACGGCGUCG GCGGCGG 17 17 base pairs nucleic acid single linear 71GAAGAUCUAC GGGCCUG 17 17 base pairs nucleic acid single linear 72GACACGCUCA GCAUUCG 17 17 base pairs nucleic acid single linear 73AACGGACUAC AGGGACA 17 17 base pairs nucleic acid single linear 74CUCAGCAUUC GGACCAG 17 17 base pairs nucleic acid single linear 75GCUGCGGUUC AGCCUGC 17 17 base pairs nucleic acid single linear 76UCAGCAUUCG GACCAGC 17 17 base pairs nucleic acid single linear 77CUGCGGUUCA GCCUGCU 17 17 base pairs nucleic acid single linear 78CCCAGGCUCC AGCACCA 17 17 base pairs nucleic acid single linear 79AGCCUGCUAU GCCAGGC 17 17 base pairs nucleic acid single linear 80GGCCAGGUUC CCGUCGC 17 17 base pairs nucleic acid single linear 81GCAGCACUUG AAGCUCC 17 17 base pairs nucleic acid single linear 82GCCAGGUUCC CGUCGCU 17 17 base pairs nucleic acid single linear 83UUGAAGCUCC AAGGAUC 17 17 base pairs nucleic acid single linear 84GUUCCCGUCG CUCGUCG 17 17 base pairs nucleic acid single linear 85CCAAGGAUCC UGAGCCU 17 17 base pairs nucleic acid single linear 86CCGUCGCUCG UCGUGUG 17 17 base pairs nucleic acid single linear 87CUGAGCCUCA ACAACAA 17 17 base pairs nucleic acid single linear 88UCGCUCGUCG UGUGCGC 17 17 base pairs nucleic acid single linear 89CAACCCAUAC UUCUCCG 17 17 base pairs nucleic acid single linear 90AUGAACGUCG UCUUCGU 17 17 base pairs nucleic acid single linear 91CCCAUACUUC UCCGGAC 17 17 base pairs nucleic acid single linear 92AACGUCGUCU UCGUCGG 17 17 base pairs nucleic acid single linear 93CCAUACUUCU CCGGACC 17 17 base pairs nucleic acid single linear 94CGUCGUCUUC GUCGGCG 17 17 base pairs nucleic acid single linear 95AUACUUCUCC GGACCAU 17 17 base pairs nucleic acid single linear 96GUCGUCUUCG UCGGCGC 17 17 base pairs nucleic acid single linear 97CGGACCAUAC GGGGAGG 17 17 base pairs nucleic acid single linear 98GUCUUCGUCG GCGCCGA 17 17 base pairs nucleic acid single linear 99GAGGACGUCG UGUUCGU 17 17 base pairs nucleic acid single linear 100GGCGGCCUCG GCGACGU 17 17 base pairs nucleic acid single linear 101CGUCGUGUUC GUCUGCA 17 17 base pairs nucleic acid single linear 102GGCGACGUCC UCGGCGG 17 17 base pairs nucleic acid single linear 103GUCGUGUUCG UCUGCAA 17 17 base pairs nucleic acid single linear 104GACGUCCUCG GCGGCCU 17 17 base pairs nucleic acid single linear 105GUGUUCGUCU GCAACGA 17 17 base pairs nucleic acid single linear 106CACCGUGUCA UGGUCGU 17 17 base pairs nucleic acid single linear 107CCGGCCCUCU CUCGUGC 17 17 base pairs nucleic acid single linear 108GUCAUGGUCG UCUCUCC 17 17 base pairs nucleic acid single linear 109GGCCCUCUCU CGUGCUA 17 17 base pairs nucleic acid single linear 110AUGGUCGUCU CUCCCCG 17 17 base pairs nucleic acid single linear 111CCCUCUCUCG UGCUACC 17 17 base pairs nucleic acid single linear 112CUCGUGCUAC CUCAAGA 17 17 base pairs nucleic acid single linear 113AUGGACGUCA GCGAGUG 17 17 base pairs nucleic acid single linear 114UGCUACCUCA AGAGCAA 17 17 base pairs nucleic acid single linear 115GGACAAGUAC AUCGCCG 17 17 base pairs nucleic acid single linear 116GAGCAACUAC CAGUCCC 17 17 base pairs nucleic acid single linear 117AAGUACAUCG CCGUGAA 17 17 base pairs nucleic acid single linear 118CUACCAGUCC CACGGCA 17 17 base pairs nucleic acid single linear 119CGUGAAGUAC GACGUGU 17 17 base pairs nucleic acid single linear 120CACGGCAUCU ACAGGGA 17 17 base pairs nucleic acid single linear 121CGACGUGUCG ACGGCCG 17 17 base pairs nucleic acid single linear 122CGGCAUCUAC AGGGACG 17 17 base pairs nucleic acid single linear 123GCGGAGGUCG GGCUCCC 17 17 base pairs nucleic acid single linear 124AGACCGCUUU CUGCAUC 17 17 base pairs nucleic acid single linear 125GUCGGGCUCC CGGUGGA 17 17 base pairs nucleic acid single linear 126GACCGCUUUC UGCAUCC 17 17 base pairs nucleic acid single linear 127CGGAACAUCC CGCUGGU 17 17 base pairs nucleic acid single linear 128ACCGCUUUCU GCAUCCA 17 17 base pairs nucleic acid single linear 129GGUGGCGUUC AUCGGCA 17 17 base pairs nucleic acid single linear 130UUCUGCAUCC ACAACAU 17 17 base pairs nucleic acid single linear 131GUGGCGUUCA UCGGCAG 17 17 base pairs nucleic acid single linear 132CACAACAUCU CCUACCA 17 17 base pairs nucleic acid single linear 133GCGUUCAUCG GCAGGCU 17 17 base pairs nucleic acid single linear 134CAACAUCUCC UACCAGG 17 17 base pairs nucleic acid single linear 135CCCGACGUCA UGGCGGC 17 17 base pairs nucleic acid single linear 136CAUCUCCUAC CAGGGCC 17 17 base pairs nucleic acid single linear 137GCCGCCAUCC CGCAGCU 17 17 base pairs nucleic acid single linear 138GGGCCGGUUC GCCUUCU 17 17 base pairs nucleic acid single linear 139CCGCAGCUCA UGGAGAU 17 17 base pairs nucleic acid single linear 140GGCCGGUUCG CCUUCUC 17 17 base pairs nucleic acid single linear 141GUGCAGAUCG UUCUGCU 17 17 base pairs nucleic acid single linear 142GUUCGCCUUC UCCGACU 17 17 base pairs nucleic acid single linear 143CAGAUCGUUC UGCUGGG 17 17 base pairs nucleic acid single linear 144UUCGCCUUCU CCGACUA 17 17 base pairs nucleic acid single linear 145AGAUCGUUCU GCUGGGC 17 17 base pairs nucleic acid single linear 146CGCCUUCUCC GACUACC 17 17 base pairs nucleic acid single linear 147GAAGAAGUUC GAGCGCA 17 17 base pairs nucleic acid single linear 148CUCCGACUAC CCGGAGC 17 17 base pairs nucleic acid single linear 149AAGAAGUUCG AGCGCAU 17 17 base pairs nucleic acid single linear 150CUGAACCUCC CGGAGAG 17 17 base pairs nucleic acid single linear 151CGCAUGCUCA UGAGCGC 17 17 base pairs nucleic acid single linear 152GGAGAGAUUC AAGUCGU 17 17 base pairs nucleic acid single linear 153GGAGAAGUUC CCAGGCA 17 17 base pairs nucleic acid single linear 154GAGAGAUUCA AGUCGUC 17 17 base pairs nucleic acid single linear 155GAGAAGUUCC CAGGCAA 17 17 base pairs nucleic acid single linear 156AUUCAAGUCG UCCUUCG 17 17 base pairs nucleic acid single linear 157GCCGUGGUCA AGUUCAA 17 17 base pairs nucleic acid single linear 158CAAGUCGUCC UUCGAUU 17 17 base pairs nucleic acid single linear 159GGUCAAGUUC AACGCGG 17 17 base pairs nucleic acid single linear 160GUCGUCCUUC GAUUUCA 17 17 base pairs nucleic acid single linear 161GUCAAGUUCA ACGCGGC 17 17 base pairs nucleic acid single linear 162UCGUCCUUCG AUUUCAU 17 17 base pairs nucleic acid single linear 163CACCACAUCA UGGCCGG 17 17 base pairs nucleic acid single linear 164CCUUCGAUUU CAUCGAC 17 17 base pairs nucleic acid single linear 165GACGUGCUCG CCGUCAC 17 17 base pairs nucleic acid single linear 166CUUCGAUUUC AUCGACG 17 17 base pairs nucleic acid single linear 167CUCGCCGUCA CCAGCCG 17 17 base pairs nucleic acid single linear 168UUCGAUUUCA UCGACGG 17 17 base pairs nucleic acid single linear 169CAGCCGCUUC GAGCCCU 17 17 base pairs nucleic acid single linear 170GAUUUCAUCG ACGGCUA 17 17 base pairs nucleic acid single linear 171AGCCGCUUCG AGCCCUG 17 17 base pairs nucleic acid single linear 172CGACGGCUAC GAGAAGC 17 17 base pairs nucleic acid single linear 173UGCGGCCUCA UCCAGCU 17 17 base pairs nucleic acid single linear 174CGGAAGAUCA ACUGGAU 17 17 base pairs nucleic acid single linear 175GGCCUCAUCC AGCUGCA 17 17 base pairs nucleic acid single linear 176GCCGGGAUCC UCGAGGC 17 17 base pairs nucleic acid single linear 177GAUGCGAUAC GGAACGC 17 17 base pairs nucleic acid single linear 178GGGAUCCUCG AGGCCGA 17 17 base pairs nucleic acid single linear 179CUGCGCGUCC ACCGGUG 17 17 base pairs nucleic acid single linear 180GACAGGGUCC UCACCGU 17 17 base pairs nucleic acid single linear 181GGUGGACUCG UCGACAC 17 17 base pairs nucleic acid single linear 182AGGGUCCUCA CCGUCAG 17 17 base pairs nucleic acid single linear 183GGACUCGUCG ACACCAU 17 17 base pairs nucleic acid single linear 184CUCACCGUCA GCCCCUA 17 17 base pairs nucleic acid single linear 185GACACCAUCA UCGAAGG 17 17 base pairs nucleic acid single linear 186CAGCCCCUAC UACGCCG 17 17 base pairs nucleic acid single linear 187ACCAUCAUCG AAGGCAA 17 17 base pairs nucleic acid single linear 188CCCCUACUAC GCCGAGG 17 17 base pairs nucleic acid single linear 189GACCGGGUUC CACAUGG 17 17 base pairs nucleic acid single linear 190GAGGAGCUCA UCUCCGG 17 17 base pairs nucleic acid single linear 191ACCGGGUUCC ACAUGGG 17 17 base pairs nucleic acid single linear 192GAGCUCAUCU CCGGCAU 17 17 base pairs nucleic acid single linear 193GGCCGCCUCA GCGUCGA 17 17 base pairs nucleic acid single linear 194GCUCAUCUCC GGCAUCG 17 17 base pairs nucleic acid single linear 195CUCAGCGUCG ACUGCAA 17 17 base pairs nucleic acid single linear 196UCCGGCAUCG CCAGGGG 17 17 base pairs nucleic acid single linear 197UGCAACGUCG UGGAGCC 17 17 base pairs nucleic acid single linear 198UGCGAGCUCG ACAACAU 17 17 base pairs nucleic acid single linear 199GCGGACGUCA AGAAGGU 17 17 base pairs nucleic acid single linear 200GACAACAUCA UGCGCCU 17 17 base pairs nucleic acid single linear 201CACCACCUUG CAGCGCG 17 17 base pairs nucleic acid single linear 202AUGCGCCUCA CCGGCAU 17 17 base pairs nucleic acid single linear 203CGCGCCAUCA AGGUGGU 17 17 base pairs nucleic acid single linear 204ACCGGCAUCA CCGGCAU 17 17 base pairs nucleic acid single linear 205AAGGUGGUCG GCACGCC 17 17 base pairs nucleic acid single linear 206ACCGGCAUCG UCAACGG 17 17 base pairs nucleic acid single linear 207GCCGGCGUAC GAGGAGA 17 17 base pairs nucleic acid single linear 208GGCAUCGUCA ACGGCAU 17 17 base pairs nucleic acid single linear 209UGCAUGAUCC AGGAUCU 17 17 base pairs nucleic acid single linear 210UCCAGGAUCU CUCCUGG 17 17 base pairs nucleic acid single linear 211CGGUAAUUUU AUAUUGC 17 17 base pairs nucleic acid single linear 212CAGGAUCUCU CCUGGAA 17 17 base pairs nucleic acid single linear 213GGUAAUUUUA UAUUGCG 17 17 base pairs nucleic acid single linear 214GGAUCUCUCC UGGAAGG 17 17 base pairs nucleic acid single linear 215GUAAUUUUAU AUUGCGA 17 17 base pairs nucleic acid single linear 216GUGCUGCUCA GCCUCGG 17 17 base pairs nucleic acid single linear 217AAUUUUAUAU UGCGAGU 17 17 base pairs nucleic acid single linear 218CUCAGCCUCG GGGUCGC 17 17 base pairs nucleic acid single linear 219UUUUAUAUUG CGAGUAA 17 17 base pairs nucleic acid single linear 220CUCGGGGUCG CCGGCGG 17 17 base pairs nucleic acid single linear 221UUGCGAGUAA AUAAAUG 17 17 base pairs nucleic acid single linear 222CCAGGGGUCG AAGGCGA 17 17 base pairs nucleic acid single linear 223GAGUAAAUAA AUGGACC 17 17 base pairs nucleic acid single linear 224GAGGAGAUCG CGCCGCU 17 17 base pairs nucleic acid single linear 225GGACCUGUAG UGGUGGA 17 17 base pairs nucleic acid single linear 226GCGCCGCUCG CCAAGGA 17 17 base pairs nucleic acid single linear 227UGAAGAGUUC GGCCUGC 17 17 base pairs nucleic acid single linear 228GAAGAGUUCG GCCUGCA 17 17 base pairs nucleic acid single linear 229CCCCUGAUCU CGCGCGU 17 17 base pairs nucleic acid single linear 230CCUGAUCUCG CGCGUGG 17 17 base pairs nucleic acid single linear 231AAACAUGUUG GGACAUC 17 17 base pairs nucleic acid single linear 232UGGGACAUCU UCUUAUA 17 17 base pairs nucleic acid single linear 233GGACAUCUUC UUAUAUA 17 17 base pairs nucleic acid single linear 234GACAUCUUCU UAUAUAU 17 17 base pairs nucleic acid single linear 235CAUCUUCUUA UAUAUGC 17 17 base pairs nucleic acid single linear 236AUCUUCUUAU AUAUGCU 17 17 base pairs nucleic acid single linear 237CUUCUUAUAU AUGCUGU 17 17 base pairs nucleic acid single linear 238UCUUAUAUAU GCUGUUU 17 17 base pairs nucleic acid single linear 239UAUGCUGUUU CGUUUAU 17 17 base pairs nucleic acid single linear 240AUGCUGUUUC GUUUAUG 17 17 base pairs nucleic acid single linear 241UGCUGUUUCG UUUAUGU 17 17 base pairs nucleic acid single linear 242UGUUUCGUUU AUGUGAU 17 17 base pairs nucleic acid single linear 243GUUUCGUUUA UGUGAUA 17 17 base pairs nucleic acid single linear 244UUUCGUUUAU GUGAUAU 17 17 base pairs nucleic acid single linear 245UAUGUGAUAU GGACAAG 17 17 base pairs nucleic acid single linear 246GGACAAGUAU GUGUAGC 17 17 base pairs nucleic acid single linear 247GUAUGUGUAG CUGCUUG 17 17 base pairs nucleic acid single linear 248UAGCUGCUUG CUUGUGC 17 17 base pairs nucleic acid single linear 249UGCUUGCUUG UGCUAGU 17 17 base pairs nucleic acid single linear 250CUUGUGCUAG UGUAAUA 17 17 base pairs nucleic acid single linear 251GCUAGUGUAA UAUAGUG 17 17 base pairs nucleic acid single linear 252AGUGUAAUAU AGUGUAG 17 17 base pairs nucleic acid single linear 253UGUAAUAUAG UGUAGUG 17 17 base pairs nucleic acid single linear 254UAUAGUGUAG UGGUGGC 17 17 base pairs nucleic acid single linear 255CACAACCUAA UAAGCGC 17 17 base pairs nucleic acid single linear 256AACCUAAUAA GCGCAUG 17 17 base pairs nucleic acid single linear 257CAUGAACUAA UUGCUUG 17 17 base pairs nucleic acid single linear 258GAACUAAUUG CUUGCGU 17 17 base pairs nucleic acid single linear 259UAAUUGCUUG CGUGUGU 17 17 base pairs nucleic acid single linear 260GCGUGUGUAG UUAAGUA 17 17 base pairs nucleic acid single linear 261UGUGUAGUUA AGUACCG 17 17 base pairs nucleic acid single linear 262GUGUAGUUAA GUACCGA 17 17 base pairs nucleic acid single linear 263AGUUAAGUAC CGAUCGG 17 17 base pairs nucleic acid single linear 264GUACCGAUCG GUAAUUU 17 17 base pairs nucleic acid single linear 265CGAUCGGUAA UUUUAUA 17 17 base pairs nucleic acid single linear 266UCGGUAAUUU UAUAUUG 17 31 base pairs nucleic acid single linear The letter “N” stands for any base. 267UGGCUGUGGC CUGAUGANGA AAUCGAUCGG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 268GCAGUGAGUU CUGAUGANGA AAUUCCUUCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 269GGCUGGCAGU CUGAUGANGA AAGUUUAUUC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 270GACGGAGCAG CUGAUGANGA AACACUUCUC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 271CUGGUGGACG CUGAUGANGA AAGCAGUACA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 272CGCACUGGUG CUGAUGANGA AACGGAGCAG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 273UCGACGAGAU CUGAUGANGA AAGCAGCCCU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 274UCGUCGACGA CUGAUGANGA AAUGAGCAGC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 275GGUCGUCGAC CUGAUGANGA AAGAUGAGCA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 276ACUGGUCGUC CUGAUGANGA AACGAGAUGA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 277CAUGCCGAUU CUGAUGANGA AAUCCACUGG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 278CCAUGCCGAU CUGAUGANGA AAAUCCACUG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 279CCGCCAUGCC CUGAUGANGA AAUUAAUCCA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 280GACGUGGCUA CUGAUGANGA AAGCCGCCAU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 281GCGACGUGGC CUGAUGANGA AAGAGCCGCC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 282GACGAGCUGC CUGAUGANGA AACGUGGCUA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 283GCGUUGCGAC CUGAUGANGA AAGCUGCGAC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 284CGCGCGUUGC CUGAUGANGA AACGAGCUGC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 285ACGCGUCCGG CUGAUGANGA AACGCCCAGG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 286GCGGAACGUG CUGAUGANGA AACGCGUCCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 287GCCGCGGCGG CUGAUGANGA AACGUGGACG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 288CGCCGCGGCG CUGAUGANGA AAACGUGGAC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 289GUCCGCCGCC CUGAUGANGA AACGCCGUCC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 290UCCGAAUGCU CUGAUGANGA AAGCGUGUCC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 291CGCUGGUCCG CUGAUGANGA AAUGCUGAGC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 292GCGCUGGUCC CUGAUGANGA AAAUGCUGAG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 293GCUGGUGCUG CUGAUGANGA AAGCCUGGGC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 294GAGCGACGGG CUGAUGANGA AACCUGGCCC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 295CGAGCGACGG CUGAUGANGA AAACCUGGCC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 296CACGACGAGC CUGAUGANGA AACGGGAACC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 297CGCACACGAC CUGAUGANGA AAGCGACGGG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 298UGGCGCACAC CUGAUGANGA AACGAGCGAC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 299CGACGAAGAC CUGAUGANGA AACGUUCAUG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 300CGCCGACGAA CUGAUGANGA AACGACGUUC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 301GGCGCCGACG CUGAUGANGA AAGACGACGU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 302CGGCGCCGAC CUGAUGANGA AAAGACGACG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 303UCUCGGCGCC CUGAUGANGA AACGAAGACG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 304GGACGUCGCC CUGAUGANGA AAGGCCGCCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 305GGCCGCCGAG CUGAUGANGA AACGUCGCCG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 306GCAGGCCGCC CUGAUGANGA AAGGACGUCG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 307AGACGACCAU CUGAUGANGA AACACGGUGC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 308GGGGAGAGAC CUGAUGANGA AACCAUGACA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 309AGCGGGGAGA CUGAUGANGA AACGACCAUG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 310GUAGCGGGGA CUGAUGANGA AAGACGACCA U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 311UCGUAGCGGG CUGAUGANGA AAGAGACGAC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 312GUACUGGUCG CUGAUGANGA AAGCGGGGAG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 313GGCGUCCUUG CUGAUGANGA AACUGGUCGU A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 314UCUCGGACAC CUGAUGANGA AACGCUGGUG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 315CUUGAUCUCG CUGAUGANGA AACACGACGC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 316CUCCCAUCUU CUGAUGANGA AAUCUCGGAC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 317GACCGUCUCG CUGAUGANGA AACCUGUCUC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 318GGAAGAACCU CUGAUGANGA AACCGUCUCG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 319GCAGUGGAAG CUGAUGANGA AACCUGACCG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 320AGCAGUGGAA CUGAUGANGA AAACCUGACC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 321GUAGCAGUGG CUGAUGANGA AAGAACCUGA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 322UGUAGCAGUG CUGAUGANGA AAAGAACCUG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 323UCCGCGCUUG CUGAUGANGA AAGCAGUGGA A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 324GUGGUCAACG CUGAUGANGA AACACGCGGU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 325GGUGGUCAAC CUGAUGANGA AAACACGCGG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 326GUGGGUGGUC CUGAUGANGA AACGAACACG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 327CCUCUCCAGG CUGAUGANGA AACAGUGGGU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 328CCCUCUCCAG CUGAUGANGA AAACAGUGGG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 329UCUUUCCCCA CUGAUGANGA AACCCUCUCC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 330GUCUUUCCCC CUGAUGANGA AAACCCUCUC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 331CAGGCCCGUA CUGAUGANGA AAUCUUCUCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 332GUCAGGCCCG CUGAUGANGA AAGAUCUUCU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 333GUUGUCCCUG CUGAUGANGA AAGUCCGUUC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 334UAGCAGGCUG CUGAUGANGA AACCGCAGCU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 335AUAGCAGGCU CUGAUGANGA AAACCGCAGC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 336CUGCCUGGCA CUGAUGANGA AAGCAGGCUG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 337UUGGAGCUUC CUGAUGANGA AAGUGCUGCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 338AGGAUCCUUG CUGAUGANGA AAGCUUCAAG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 339UGAGGCUCAG CUGAUGANGA AAUCCUUGGA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 340GGUUGUUGUU CUGAUGANGA AAGGCUCAGG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 341UCCGGAGAAG CUGAUGANGA AAUGGGUUGU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 342UGGUCCGGAG CUGAUGANGA AAGUAUGGGU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 343AUGGUCCGGA CUGAUGANGA AAAGUAUGGG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 344GUAUGGUCCG CUGAUGANGA AAGAAGUAUG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 345GUCCUCCCCG CUGAUGANGA AAUGGUCCGG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 346AGACGAACAC CUGAUGANGA AACGUCCUCC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 347GUUGCAGACG CUGAUGANGA AACACGACGU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 348CGUUGCAGAC CUGAUGANGA AAACACGACG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 349AGUCGUUGCA CUGAUGANGA AACGAACACG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 350UAGCACGAGA CUGAUGANGA AAGGGCCGGU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 351GGUAGCACGA CUGAUGANGA AAGAGGGCCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 352GAGGUAGCAC CUGAUGANGA AAGAGAGGGC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 353GCUCUUGAGG CUGAUGANGA AAGCACGAGA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 354AGUUGCUCUU CUGAUGANGA AAGGUAGCAC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 355GUGGGACUGG CUGAUGANGA AAGUUGCUCU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 356GAUGCCGUGG CUGAUGANGA AACUGGUAGU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 357CGUCCCUGUA CUGAUGANGA AAUGCCGUGG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 358UGCGUCCCUG CUGAUGANGA AAGAUGCCGU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 359UGGAUGCAGA CUGAUGANGA AAGCGGUCUU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 360GUGGAUGCAG CUGAUGANGA AAAGCGGUCU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 361UGUGGAUGCA CUGAUGANGA AAAAGCGGUC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 362AGAUGUUGUG CUGAUGANGA AAUGCAGAAA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 363CCUGGUAGGA CUGAUGANGA AAUGUUGUGG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 364GCCCUGGUAG CUGAUGANGA AAGAUGUUGU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 365CCGGCCCUGG CUGAUGANGA AAGGAGAUGU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 366GGAGAAGGCG CUGAUGANGA AACCGGCCCU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 367CGGAGAAGGC CUGAUGANGA AAACCGGCCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 368GUAGUCGGAG CUGAUGANGA AAGGCGAACC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 369GGUAGUCGGA CUGAUGANGA AAAGGCGAAC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 370CGGGUAGUCG CUGAUGANGA AAGAAGGCGA A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 371CAGCUCCGGG CUGAUGANGA AAGUCGGAGA A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 372AUCUCUCCGG CUGAUGANGA AAGGUUCAGC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 373GGACGACUUG CUGAUGANGA AAUCUCUCCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 374AGGACGACUU CUGAUGANGA AAAUCUCUCC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 375AUCGAAGGAC CUGAUGANGA AACUUGAAUC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 376GAAAUCGAAG CUGAUGANGA AACGACUUGA A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 377GAUGAAAUCG CUGAUGANGA AAGGACGACU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 378CGAUGAAAUC CUGAUGANGA AAAGGACGAC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 379CCGUCGAUGA CUGAUGANGA AAUCGAAGGA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 380GCCGUCGAUG CUGAUGANGA AAAUCGAAGG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 381AGCCGUCGAU CUGAUGANGA AAAAUCGAAG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 382CGUAGCCGUC CUGAUGANGA AAUGAAAUCG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 383GGGCUUCUCG CUGAUGANGA AAGCCGUCGA U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 384UCAUCCAGUU CUGAUGANGA AAUCUUCCGG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 385CGGCCUCGAG CUGAUGANGA AAUCCCGGCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 386UGUCGGCCUC CUGAUGANGA AAGGAUCCCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 387UGACGGUGAG CUGAUGANGA AACCCUGUCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 388GGCUGACGGU CUGAUGANGA AAGGACCCUG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 389AGUAGGGGCU CUGAUGANGA AACGGUGAGG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 390CUCGGCGUAG CUGAUGANGA AAGGGGCUGA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 391CUCCUCGGCG CUGAUGANGA AAGUAGGGGC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 392UGCCGGAGAU CUGAUGANGA AAGCUCCUCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 393CGAUGCCGGA CUGAUGANGA AAUGAGCUCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 394GGCGAUGCCG CUGAUGANGA AAGAUGAGCU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 395AGCCCCUGGC CUGAUGANGA AAUGCCGGAG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 396UGAUGUUGUC CUGAUGANGA AAGCUCGCAG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 397UGAGGCGCAU CUGAUGANGA AAUGUUGUCG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 398UGAUGCCGGU CUGAUGANGA AAGGCGCAUG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 399CGAUGCCGGU CUGAUGANGA AAUGCCGGUG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 400UGCCGUUGAC CUGAUGANGA AAUGCCGGUG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 401CCAUGCCGUU CUGAUGANGA AACGAUGCCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 402CCCACUCGCU CUGAUGANGA AACGUCCAUG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 403CACGGCGAUG CUGAUGANGA AACUUGUCCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 404ACUUCACGGC CUGAUGANGA AAUGUACUUG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 405CGACACGUCG CUGAUGANGA AACUUCACGG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 406CACGGCCGUC CUGAUGANGA AACACGUCGU A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 407CCGGGAGCCC CUGAUGANGA AACCUCCGCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 408GGUCCACCGG CUGAUGANGA AAGCCCGACC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 409CCACCAGCGG CUGAUGANGA AAUGUUCCGG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 410CCUGCCGAUG CUGAUGANGA AACGCCACCA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 411GCCUGCCGAU CUGAUGANGA AAACGCCACC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 412CCAGCCUGCC CUGAUGANGA AAUGAACGCC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 413CGGCCGCCAU CUGAUGANGA AACGUCGGGU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 414UGAGCUGCGG CUGAUGANGA AAUGGCGGCC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 415CCAUCUCCAU CUGAUGANGA AAGCUGCGGG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 416CCAGCAGAAC CUGAUGANGA AAUCUGCACG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 417UGCCCAGCAG CUGAUGANGA AACGAUCUGC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 418GUGCCCAGCA CUGAUGANGA AAACGAUCUG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 419CAUGCGCUCG CUGAUGANGA AACUUCUUCU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 420GCAUGCGCUC CUGAUGANGA AAACUUCUUC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 421CGGCGCUCAU CUGAUGANGA AAGCAUGCGC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 422CUUGCCUGGG CUGAUGANGA AACUUCUCCU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 423CCUUGCCUGG CUGAUGANGA AAACUUCUCC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 424CGUUGAACUU CUGAUGANGA AACCACGGCG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 425CGCCGCGUUG CUGAUGANGA AACUUGACCA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 426GCGCCGCGUU CUGAUGANGA AAACUUGACC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 427CGCCGGCCAU CUGAUGANGA AAUGUGGUGC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 428UGGUGACGGC CUGAUGANGA AAGCACGUCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 429AGCGGCUGGU CUGAUGANGA AACGGCGAGC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 430GCAGGGCUCG CUGAUGANGA AAGCGGCUGG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 431CGCAGGGCUC CUGAUGANGA AAAGCGGCUG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 432GCAGCUGGAU CUGAUGANGA AAGGCCGCAG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 433CCUGCAGCUG CUGAUGANGA AAUGAGGCCG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 434GGGCGUUCCG CUGAUGANGA AAUCGCAUCC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 435UCCACCGGUG CUGAUGANGA AACGCGCAGG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 436UGGUGUCGAC CUGAUGANGA AAGUCCACCG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 437UGAUGGUGUC CUGAUGANGA AACGAGUCCA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 438UGCCUUCGAU CUGAUGANGA AAUGGUGUCG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 439UCUUGCCUUC CUGAUGANGA AAUGAUGGUG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 440GCCCAUGUGG CUGAUGANGA AACCCGGUCU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 441GGCCCAUGUG CUGAUGANGA AAACCCGGUC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 442AGUCGACGCU CUGAUGANGA AAGGCGGCCC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 443CGUUGCAGUC CUGAUGANGA AACGCUGAGG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 444CCGGCUCCAC CUGAUGANGA AACGUUGCAG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 445CCACCUUCUU CUGAUGANGA AACGUCCGCC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 446GGCGCGCUGC CUGAUGANGA AAGGUGGUGG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 447CGACCACCUU CUGAUGANGA AAUGGCGCGC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 448CCGGCGUGCC CUGAUGANGA AACCACCUUG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 449CAUCUCCUCG CUGAUGANGA AACGCCGGCG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 450AGAGAUCCUG CUGAUGANGA AAUCAUGCAG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 451UUCCAGGAGA CUGAUGANGA AAUCCUGGAU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 452CCUUCCAGGA CUGAUGANGA AAGAUCCUGG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 453GCCCUUCCAG CUGAUGANGA AAGAGAUCCU G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 454CCCCGAGGCU CUGAUGANGA AAGCAGCACG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 455CGGCGACCCC CUGAUGANGA AAGGCUGAGC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 456CGCCGCCGGC CUGAUGANGA AACCCCGAGG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 457CCUCGCCUUC CUGAUGANGA AACCCCUGGC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 458CGAGCGGCGC CUGAUGANGA AAUCUCCUCG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 459UCUCCUUGGC CUGAUGANGA AAGCGGCGCG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 460CUGCAGGCCG CUGAUGANGA AACUCUUCAG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 461CCUGCAGGCC CUGAUGANGA AAACUCUUCA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 462CCACGCGCGA CUGAUGANGA AAUCAGGGGG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 463CACCACGCGC CUGAUGANGA AAGAUCAGGG G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 464AAGAUGUCCC CUGAUGANGA AACAUGUUUG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 465UAUAUAAGAA CUGAUGANGA AAUGUCCCAA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 466CAUAUAUAAG CUGAUGANGA AAGAUGUCCC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 467GCAUAUAUAA CUGAUGANGA AAAGAUGUCC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 468CAGCAUAUAU CUGAUGANGA AAGAAGAUGU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 469ACAGCAUAUA CUGAUGANGA AAAGAAGAUG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 470AAACAGCAUA CUGAUGANGA AAUAAGAAGA U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 471CGAAACAGCA CUGAUGANGA AAUAUAAGAA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 472ACAUAAACGA CUGAUGANGA AACAGCAUAU A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 473CACAUAAACG CUGAUGANGA AAACAGCAUA U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 474UCACAUAAAC CUGAUGANGA AAAACAGCAU A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 475AUAUCACAUA CUGAUGANGA AACGAAACAG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 476CAUAUCACAU CUGAUGANGA AAACGAAACA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 477CCAUAUCACA CUGAUGANGA AAAACGAAAC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 478UACUUGUCCA CUGAUGANGA AAUCACAUAA A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 479CAGCUACACA CUGAUGANGA AACUUGUCCA U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 480AGCAAGCAGC CUGAUGANGA AACACAUACU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 481UAGCACAAGC CUGAUGANGA AAGCAGCUAC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 482ACACUAGCAC CUGAUGANGA AAGCAAGCAG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 483UAUAUUACAC CUGAUGANGA AAGCACAAGC A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 484UACACUAUAU CUGAUGANGA AACACUAGCA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 485CACUACACUA CUGAUGANGA AAUUACACUA G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 486ACCACUACAC CUGAUGANGA AAUAUUACAC U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 487UGGCCACCAC CUGAUGANGA AACACUAUAU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 488AUGCGCUUAU CUGAUGANGA AAGGUUGUGC C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 489UUCAUGCGCU CUGAUGANGA AAUUAGGUUG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 490CGCAAGCAAU CUGAUGANGA AAGUUCAUGC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 491ACACGCAAGC CUGAUGANGA AAUUAGUUCA U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 492CUACACACGC CUGAUGANGA AAGCAAUUAG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 493GGUACUUAAC CUGAUGANGA AACACACGCA A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 494AUCGGUACUU CUGAUGANGA AACUACACAC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 495GAUCGGUACU CUGAUGANGA AAACUACACA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 496UACCGAUCGG CUGAUGANGA AACUUAACUA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 497UAAAAUUACC CUGAUGANGA AAUCGGUACU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 498AAUAUAAAAU CUGAUGANGA AACCGAUCGG U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 499CGCAAUAUAA CUGAUGANGA AAUUACCGAU C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 500UCGCAAUAUA CUGAUGANGA AAAUUACCGA U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 501CUCGCAAUAU CUGAUGANGA AAAAUUACCG A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 502ACUCGCAAUA CUGAUGANGA AAAAAUUACC G 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 503UUACUCGCAA CUGAUGANGA AAUAAAAUUA C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 504AUUUACUCGC CUGAUGANGA AAUAUAAAAU U 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 505UCCAUUUAUU CUGAUGANGA AACUCGCAAU A 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 506CAGGUCCAUU CUGAUGANGA AAUUUACUCG C 31 31 base pairs nucleic acid single linear The letter “N” stands for any base. 507UUUCCACCAC CUGAUGANGA AACAGGUCCA U 31 52 base pairs nucleic acid single linear 508CUCCUGGCAG AAGUCGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 509CGACAGCCGC CAGGAG 16 52 base pairs nucleic acid single linear 510CCCUGCCGAG AAGUGCACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 511GCACCGCCCG GCAGGG 16 52 base pairs nucleic acid single linear 512GUCGCCGAAG AAGCCGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 513CGGCGGCCUC GGCGAC 16 52 base pairs nucleic acid single linear 514CGGCGGCAAG AAGCCGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 515CGGCGGCCUG CCGCCG 16 52 base pairs nucleic acid single linear 516CCAUGGCCAG AAGCAGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 517CUGCCGCCGG CCAUGG 16 52 base pairs nucleic acid single linear 518UCUCCAGGAG AAGUGGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 519CCACUGUUCC UGGAGA 16 52 base pairs nucleic acid single linear 520UCCCUGUAAG AAGUUCACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 521GAACGGACUA CAGGGA 16 52 base pairs nucleic acid single linear 522GCAGGCUGAG AAGCAGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 523CUGCGGUUCA GCCUGC 16 52 base pairs nucleic acid single linear 524GCCUCCACAG AAGUCGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 525CGACGGCCGU GGAGGC 16 52 base pairs nucleic acid single linear 526GGGAUGGCAG AAGCCAACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 527UGGCGGCCGC CAUCCC 16 52 base pairs nucleic acid single linear 528GCGAGCACAG AAGCGCACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 529GCGCCGACGU GCUCGC 16 52 base pairs nucleic acid single linear 530CUGGAUGAAG AAGCAGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 531CUGCGGCCUC AUCCAG 16 52 base pairs nucleic acid single linear 532GACGCUGAAG AAGCCCACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 533GGGCCGCCUC AGCGUC 16 52 base pairs nucleic acid single linear 534UUCUUGACAG AAGCCGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 535CGGCGGACGU CAAGAA 16 52 base pairs nucleic acid single linear 536AUAAACGAAG AAGCAUACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 537AUGCUGUUUC GUUUAU 16 54 base pairs nucleic acid single linear 538GUCGCCUCAG AAGGUGGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 539ACCACCCGCC GAGGCGAC 18 54 base pairs nucleic acid single linear 540CUCCUGGCAG AAGUCGCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 541CGCGACAGCC GCCAGGAG 18 54 base pairs nucleic acid single linear 542GUGGACGGAG AAGUACACAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 543GUGUACUGCU CCGUCCAC 18 54 base pairs nucleic acid single linear 544CACUGGUGAG AAGAGCAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 545CUGCUCCGUC CACCAGUG 18 54 base pairs nucleic acid single linear 546CCCUGCCGAG AAGUGCGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 547GCGCACCGCC CGGCAGGG 18 54 base pairs nucleic acid single linear 548ACGAGAUGAG AAGCCCUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 549CAGGGCUGCU CAUCUCGU 18 54 base pairs nucleic acid single linear 550GUGGCUAGAG AAGCCAUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 551CAUGGCGGCU CUAGCCAC 18 54 base pairs nucleic acid single linear 552UUGCGACGAG AAGCGACGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 553CGUCGCAGCU CGUCGCAA 18 54 base pairs nucleic acid single linear 554GACGCCCAAG AAGGCGCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 555CGCGCCGGCC UGGGCGUC 18 54 base pairs nucleic acid single linear 556GUGGACGCAG AAGGGACGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 557CGUCCCGGAC GCGUCCAC 18 54 base pairs nucleic acid single linear 558GGCGCCGCAG AAGAACGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 559ACGUUCCGCC GCGGCGCC 18 54 base pairs nucleic acid single linear 560CCGACGCCAG AAGGCCCCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 561GGGGCCGGAC GGCGUCGG 18 54 base pairs nucleic acid single linear 562GCGCGCUGAG AAGAAUGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 563GCAUUCGGAC CAGCGCGC 18 54 base pairs nucleic acid single linear 564CGACGAGCAG AAGGAACCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 565GGUUCCCGUC GCUCGUCG 18 54 base pairs nucleic acid single linear 566GUCGCCGAAG AAGCCGGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 567ACCGGCGGCC UCGGCGAC 18 54 base pairs nucleic acid single linear 568CGGCGGCAAG AAGCCGAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 569CUCGGCGGCC UGCCGCCG 18 54 base pairs nucleic acid single linear 570UGGCCGGCAG AAGGCCGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 571GCGGCCUGCC GCCGGCCA 18 54 base pairs nucleic acid single linear 572CCAUGGCCAG AAGCAGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 573GCCUGCCGCC GGCCAUGG 18 54 base pairs nucleic acid single linear 574UCUCCAGGAG AAGUGGGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 575ACCCACUGUU CCUGGAGA 18 54 base pairs nucleic acid single linear 576GUUCCAGCAG AAGGCCCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 577CGGGCCUGAC GCUGGAAC 18 54 base pairs nucleic acid single linear 578UCCCUGUAAG AAGUUCCAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 579UGGAACGGAC UACAGGGA 18 54 base pairs nucleic acid single linear 580UGAACCGCAG AAGGUUGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 581ACAACCAGCU GCGGUUCA 18 54 base pairs nucleic acid single linear 582GCAGGCUGAG AAGCAGCUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 583AGCUGCGGUU CAGCCUGC 18 54 base pairs nucleic acid single linear 584GCAUAGCAAG AAGAACCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 585CGGUUCAGCC UGCUAUGC 18 54 base pairs nucleic acid single linear 586CCCGUAUGAG AAGGAGAAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 587UUCUCCGGAC CAUACGGG 18 54 base pairs nucleic acid single linear 588CGAGAGAGAG AAGGUGUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 589CACACCGGCC CUCUCUCG 18 54 base pairs nucleic acid single linear 590UGCCGUGGAG AAGGUAGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 591ACUACCAGUC CCACGGCA 18 54 base pairs nucleic acid single linear 592AUGCAGAAAG AAGUCUUUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 593AAAGACCGCU UUCUGCAU 18 54 base pairs nucleic acid single linear 594AGAAGGCGAG AAGGCCCUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 595AGGGCCGGUU CGCCUUCU 18 54 base pairs nucleic acid single linear 596UCCGGGUAAG AAGAGAAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 597CUUCUCCGAC UACCCGGA 18 54 base pairs nucleic acid single linear 598GUAGUAGGAG AAGACGGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 599ACCGUCAGCC CCUACUAC 18 54 base pairs nucleic acid single linear 600GCCUCCACAG AAGUCGACAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 601GUCGACGGCC GUGGAGGC 18 54 base pairs nucleic acid single linear 602ACGCCACCAG AAGGAUGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 603ACAUCCCGCU GGUGGCGU 18 54 base pairs nucleic acid single linear 604GCCAUGACAG AAGGUCCCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 605GGGACCCGAC GUCAUGGC 18 54 base pairs nucleic acid single linear 606GGGAUGGCAG AAGCCAUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 607CAUGGCGGCC GCCAUCCC 18 54 base pairs nucleic acid single linear 608UCUCCAUGAG AAGCGGGAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 609UCCCGCAGCU CAUGGAGA 18 54 base pairs nucleic acid single linear 610GCAGAACGAG AAGCACGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 611ACGUGCAGAU CGUUCUGC 18 54 base pairs nucleic acid single linear 612CCGUGCCCAG AAGAACGAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 613UCGUUCUGCU GGGCACGG 18 54 base pairs nucleic acid single linear 614GCGAGCACAG AAGCGCCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 615CGGCGCCGAC GUGCUCGC 18 54 base pairs nucleic acid single linear 616CUCGAAGCAG AAGGUGACAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 617GUCACCAGCC GCUUCGAG 18 54 base pairs nucleic acid single linear 618GGGCUCGAAG AAGCUGGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 619ACCAGCCGCU UCGAGCCC 18 54 base pairs nucleic acid single linear 620CUGGAUGAAG AAGCAGGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 621CCCUGCGGCC UCAUCCAG 18 54 base pairs nucleic acid single linear 622UCCCCUGCAG AAGGAUGAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 623UCAUCCAGCU GCAGGGGA 18 54 base pairs nucleic acid single linear 624GACGCUGAAG AAGCCCAUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 625AUGGGCCGCC UCAGCGUC 18 54 base pairs nucleic acid single linear 626UUCUUGACAG AAGCCGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 627GCCGGCGGAC GUCAAGAA 18 54 base pairs nucleic acid single linear 628CGAGGCUGAG AAGCACGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 629ACGUGCUGCU CAGCCUCG 18 54 base pairs nucleic acid single linear 630GACCCCGAAG AAGAGCAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 631CUGCUCAGCC UCGGGGUC 18 54 base pairs nucleic acid single linear 632CCUUGGCGAG AAGCGCGAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 633UCGCGCCGCU CGCCAAGG 18 54 base pairs nucleic acid single linear 634GGCCUGCAAG AAGAACUCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 635GAGUUCGGCC UGCAGGCC 18 54 base pairs nucleic acid single linear 636CGCGCGAGAG AAGGGGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 637GCCCCCUGAU CUCGCGCG 18 54 base pairs nucleic acid single linear 638AUAAACGAAG AAGCAUAUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 639AUAUGCUGUU UCGUUUAU 18 54 base pairs nucleic acid single linear 640CACAAGCAAG AAGCUACAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 641UGUAGCUGCU UGCUUGUG 18 54 base pairs nucleic acid single linear 642AAUUACCGAG AAGUACUUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 643AAGUACCGAU CGGUAAUU 18 17 base pairs nucleic acid single linear 644CGCGCCCUCU GCCGCUU 17 17 base pairs nucleic acid single linear 645GUCCAGGUUA CACAUUC 17 17 base pairs nucleic acid single linear 646CUGCCGCUUG UUCGUUC 17 17 base pairs nucleic acid single linear 647UCCAGGUUAC ACAUUCA 17 17 base pairs nucleic acid single linear 648CCGCUUGUUC GUUCCUC 17 17 base pairs nucleic acid single linear 649UUACACAUUC AAUGCCA 17 17 base pairs nucleic acid single linear 650CGCUUGUUCG UUCCUCG 17 17 base pairs nucleic acid single linear 651UACACAUUCA AUGCCAC 17 17 base pairs nucleic acid single linear 652UUGUUCGUUC CUCGCGC 17 17 base pairs nucleic acid single linear 653UGCCACCUCA CAAGAUU 17 17 base pairs nucleic acid single linear 654UGUUCGUUCC UCGCGCU 17 17 base pairs nucleic acid single linear 655CACAAGAUUG AAAUUUU 17 17 base pairs nucleic acid single linear 656UCGUUCCUCG CGCUCGC 17 17 base pairs nucleic acid single linear 657AUUGAAAUUU UCAAGUC 17 17 base pairs nucleic acid single linear 658CUCGCGCUCG CCACCAG 17 17 base pairs nucleic acid single linear 659UUGAAAUUUU CAAGUCG 17 17 base pairs nucleic acid single linear 660ACACACAUCC CAAUCUC 17 17 base pairs nucleic acid single linear 661UGAAAUUUUC AAGUCGC 17 17 base pairs nucleic acid single linear 662AUCCCAAUCU CGCGAGG 17 17 base pairs nucleic acid single linear 663GAAAUUUUCA AGUCGCU 17 17 base pairs nucleic acid single linear 664CCCAAUCUCG CGAGGGC 17 17 base pairs nucleic acid single linear 665UUUCAAGUCG CUUGAUG 17 17 base pairs nucleic acid single linear 666AGCAGGGUCU GCGGCGG 17 17 base pairs nucleic acid single linear 667AAGUCGCUUG AUGAUUG 17 17 base pairs nucleic acid single linear 668GCCGCGCUUC CGGCUCC 17 17 base pairs nucleic acid single linear 669UUGAUGAUUG GGCUAGA 17 17 base pairs nucleic acid single linear 670CCGCGCUUCC GGCUCCC 17 17 base pairs nucleic acid single linear 671AUUGGGCUAG AGAUAAU 17 17 base pairs nucleic acid single linear 672UUCCGGCUCC CCUUCCC 17 17 base pairs nucleic acid single linear 673CUAGAGAUAA UAUCUUG 17 17 base pairs nucleic acid single linear 674GCUCCCCUUC CCAUUGG 17 17 base pairs nucleic acid single linear 675GAGAUAAUAU CUUGACG 17 17 base pairs nucleic acid single linear 676CUCCCCUUCC CAUUGGC 17 17 base pairs nucleic acid single linear 677GAUAAUAUCU UGACGCA 17 17 base pairs nucleic acid single linear 678CUUCCCAUUG GCCUCCA 17 17 base pairs nucleic acid single linear 679UAAUAUCUUG ACGCAUC 17 17 base pairs nucleic acid single linear 680AUUGGCCUCC ACGAUGG 17 17 base pairs nucleic acid single linear 681UGACGCAUCU CAAGCCA 17 17 base pairs nucleic acid single linear 682AUGGCGCUCC GCCUCAA 17 17 base pairs nucleic acid single linear 683ACGCAUCUCA AGCCAGU 17 17 base pairs nucleic acid single linear 684CUCCGCCUCA ACGACGU 17 17 base pairs nucleic acid single linear 685AAGCCAGUCG AGAAGUG 17 17 base pairs nucleic acid single linear 686AACGACGUCG CGCUCUG 17 17 base pairs nucleic acid single linear 687AGAAGUGUUG GCAGCCA 17 17 base pairs nucleic acid single linear 688GUCGCGCUCU GCCUCUC 17 17 base pairs nucleic acid single linear 689CACAGGAUUU CCUCCCG 17 17 base pairs nucleic acid single linear 690CUCUGCCUCU CCCCGCC 17 17 base pairs nucleic acid single linear 691ACAGGAUUUC CUCCCGG 17 17 base pairs nucleic acid single linear 692CUGCCUCUCC CCGCCGC 17 17 base pairs nucleic acid single linear 693CAGGAUUUCC UCCCGGA 17 17 base pairs nucleic acid single linear 694CCGCCGCUCG CCGCCCG 17 17 base pairs nucleic acid single linear 695GAUUUCCUCC CGGACCC 17 17 base pairs nucleic acid single linear 696CGGCAGGUUC GUCGCCG 17 17 base pairs nucleic acid single linear 697CCCAGCAUCU GAAGGAU 17 17 base pairs nucleic acid single linear 698GGCAGGUUCG UCGCCGU 17 17 base pairs nucleic acid single linear 699UGAAGGAUUU CAUGAUG 17 17 base pairs nucleic acid single linear 700AGGUUCGUCG CCGUCGC 17 17 base pairs nucleic acid single linear 701GAAGGAUUUC AUGAUGA 17 17 base pairs nucleic acid single linear 702GUCGCCGUCG CCUCCAU 17 17 base pairs nucleic acid single linear 703AAGGAUUUCA UGAUGAA 17 17 base pairs nucleic acid single linear 704CGUCGCCUCC AUGACGU 17 17 base pairs nucleic acid single linear 705GAUGAAGUUA AGGAGCU 17 17 base pairs nucleic acid single linear 706CAUGACGUCC GCCGUCU 17 17 base pairs nucleic acid single linear 707AUGAAGUUAA GGAGCUC 17 17 base pairs nucleic acid single linear 708UCCGCCGUCU CCACCAA 17 17 base pairs nucleic acid single linear 709AAGGAGCUCA GAGAACG 17 17 base pairs nucleic acid single linear 710CGCCGUCUCC ACCAAGG 17 17 base pairs nucleic acid single linear 711AAGGAAAUCC CUGAUGA 17 17 base pairs nucleic acid single linear 712ACCAAGGUCG AGAAUAA 17 17 base pairs nucleic acid single linear 713CUGAUGAUUA UUUUGUU 17 17 base pairs nucleic acid single linear 714UCGAGAAUAA GAAGCCA 17 17 base pairs nucleic acid single linear 715UGAUGAUUAU UUUGUUU 17 17 base pairs nucleic acid single linear 716GAAGCCAUUU GCUCCUC 17 17 base pairs nucleic acid single linear 717AUGAUUAUUU UGUUUGU 17 17 base pairs nucleic acid single linear 718AAGCCAUUUG CUCCUCC 17 17 base pairs nucleic acid single linear 719UGAUUAUUUU GUUUGUU 17 17 base pairs nucleic acid single linear 720CAUUUGCUCC UCCAAGG 17 17 base pairs nucleic acid single linear 721GAUUAUUUUG UUUGUUU 17 17 base pairs nucleic acid single linear 722UUGCUCCUCC AAGGGAG 17 17 base pairs nucleic acid single linear 723UAUUUUGUUU GUUUGGU 17 17 base pairs nucleic acid single linear 724AGGGAGGUAC AUGUCCA 17 17 base pairs nucleic acid single linear 725AUUUUGUUUG UUUGGUG 17 17 base pairs nucleic acid single linear 726GUACAUGUCC AGGUUAC 17 17 base pairs nucleic acid single linear 727UUGUUUGUUU GGUGGGA 17 17 base pairs nucleic acid single linear 728UGUUUGUUUG GUGGGAG 17 17 base pairs nucleic acid single linear 729ACACUGCUCG UCACGCC 17 17 base pairs nucleic acid single linear 730GACAUGAUUA CCGAGGA 17 17 base pairs nucleic acid single linear 731CUGCUCGUCA CGCCAAG 17 17 base pairs nucleic acid single linear 732ACAUGAUUAC CGAGGAA 17 17 base pairs nucleic acid single linear 733CAAGGACUUU GGCGACU 17 17 base pairs nucleic acid single linear 734AGGAAGCUCU ACCAACA 17 17 base pairs nucleic acid single linear 735AAGGACUUUG GCGACUU 17 17 base pairs nucleic acid single linear 736GAAGCUCUAC CAACAUA 17 17 base pairs nucleic acid single linear 737UGGCGACUUA AAGCUUG 17 17 base pairs nucleic acid single linear 738ACCAACAUAC CAGACUA 17 17 base pairs nucleic acid single linear 739GGCGACUUAA AGCUUGC 17 17 base pairs nucleic acid single linear 740ACCAGACUAU GCUUAAC 17 17 base pairs nucleic acid single linear 741UUAAAGCUUG CACAAAU 17 17 base pairs nucleic acid single linear 742ACUAUGCUUA ACACCCU 17 17 base pairs nucleic acid single linear 743GCACAAAUCU GCGGCAU 17 17 base pairs nucleic acid single linear 744CUAUGCUUAA CACCCUC 17 17 base pairs nucleic acid single linear 745UGCGGCAUCA UCGCCUC 17 17 base pairs nucleic acid single linear 746AACACCCUCG ACGGUGU 17 17 base pairs nucleic acid single linear 747GGCAUCAUCG CCUCAGA 17 17 base pairs nucleic acid single linear 748GACGGUGUCA GAGAUGA 17 17 base pairs nucleic acid single linear 749CAUCGCCUCA GAUGAGA 17 17 base pairs nucleic acid single linear 750UGGGCUGUUU GGACGAG 17 17 base pairs nucleic acid single linear 751AACUGCGUAC ACCAAGA 17 17 base pairs nucleic acid single linear 752GGGCUGUUUG GACGAGG 17 17 base pairs nucleic acid single linear 753ACCAAGAUCG UGGAGAA 17 17 base pairs nucleic acid single linear 754AUGGUGAUCU GCUCAAC 17 17 base pairs nucleic acid single linear 755GAAGCUGUUU GAGAUCG 17 17 base pairs nucleic acid single linear 756GAUCUGCUCA ACAAGUA 17 17 base pairs nucleic acid single linear 757AAGCUGUUUG AGAUCGA 17 17 base pairs nucleic acid single linear 758CAACAAGUAU AUGUACC 17 17 base pairs nucleic acid single linear 759UUUGAGAUCG ACCCUGA 17 17 base pairs nucleic acid single linear 760ACAAGUAUAU GUACCUC 17 17 base pairs nucleic acid single linear 761CUGAUGGUAC CGUGGUC 17 17 base pairs nucleic acid single linear 762GUAUAUGUAC CUCACUG 17 17 base pairs nucleic acid single linear 763ACCGUGGUCG CUCUGGC 17 17 base pairs nucleic acid single linear 764AUGUACCUCA CUGGGAG 17 17 base pairs nucleic acid single linear 765UGGUCGCUCU GGCUGAC 17 17 base pairs nucleic acid single linear 766GGGUGGAUAU GAGGCAG 17 17 base pairs nucleic acid single linear 767AAGAAGAUCU CAAUGCC 17 17 base pairs nucleic acid single linear 768AGGCAGAUUG AGAAGAC 17 17 base pairs nucleic acid single linear 769GAAGAUCUCA AUGCCUG 17 17 base pairs nucleic acid single linear 770AAGACAAUUC AGUAUCU 17 17 base pairs nucleic acid single linear 771CCUGAUGUUU GACGGGC 17 17 base pairs nucleic acid single linear 772AGACAAUUCA GUAUCUU 17 17 base pairs nucleic acid single linear 773CUGAUGUUUG ACGGGCA 17 17 base pairs nucleic acid single linear 774AAUUCAGUAU CUUAUUG 17 17 base pairs nucleic acid single linear 775CAAGCUGUUC GAGCACU 17 17 base pairs nucleic acid single linear 776UUCAGUAUCU UAUUGGC 17 17 base pairs nucleic acid single linear 777AAGCUGUUCG AGCACUU 17 17 base pairs nucleic acid single linear 778CAGUAUCUUA UUGGCUC 17 17 base pairs nucleic acid single linear 779CGAGCACUUC UCCAUGG 17 17 base pairs nucleic acid single linear 780AGUAUCUUAU UGGCUCU 17 17 base pairs nucleic acid single linear 781GAGCACUUCU CCAUGGU 17 17 base pairs nucleic acid single linear 782UAUCUUAUUG GCUCUGG 17 17 base pairs nucleic acid single linear 783GCACUUCUCC AUGGUCG 17 17 base pairs nucleic acid single linear 784UAUUGGCUCU GGAAUGG 17 17 base pairs nucleic acid single linear 785UCCAUGGUCG CGCAGAG 17 17 base pairs nucleic acid single linear 786GAAUGGAUCC UAGGACU 17 17 base pairs nucleic acid single linear 787CAGAGGCUUG GCGUUUA 17 17 base pairs nucleic acid single linear 788UGGAUCCUAG GACUGAG 17 17 base pairs nucleic acid single linear 789CUUGGCGUUU ACACCGC 17 17 base pairs nucleic acid single linear 790CUGAGAAUAA UCCUUAU 17 17 base pairs nucleic acid single linear 791UUGGCGUUUA CACCGCC 17 17 base pairs nucleic acid single linear 792AGAAUAAUCC UUAUCUU 17 17 base pairs nucleic acid single linear 793UGGCGUUUAC ACCGCCA 17 17 base pairs nucleic acid single linear 794AUAAUCCUUA UCUUGGU 17 17 base pairs nucleic acid single linear 795CAGGGACUAC GCCGACA 17 17 base pairs nucleic acid single linear 796UAAUCCUUAU CUUGGUU 17 17 base pairs nucleic acid single linear 797GCCGACAUCC UCGAGUU 17 17 base pairs nucleic acid single linear 798AUCCUUAUCU UGGUUUC 17 17 base pairs nucleic acid single linear 799GACAUCCUCG AGUUCCU 17 17 base pairs nucleic acid single linear 800CCUUAUCUUG GUUUCAU 17 17 base pairs nucleic acid single linear 801CCUCGAGUUC CUCGUCG 17 17 base pairs nucleic acid single linear 802AUCUUGGUUU CAUCUAC 17 17 base pairs nucleic acid single linear 803CUCGAGUUCC UCGUCGA 17 17 base pairs nucleic acid single linear 804UCUUGGUUUC AUCUACA 17 17 base pairs nucleic acid single linear 805GAGUUCCUCG UCGACAG 17 17 base pairs nucleic acid single linear 806CUUGGUUUCA UCUACAC 17 17 base pairs nucleic acid single linear 807UUCCUCGUCG ACAGGUG 17 17 base pairs nucleic acid single linear 808GGUUUCAUCU ACACCUC 17 17 base pairs nucleic acid single linear 809UGACUGGUCU GUCGGGU 17 17 base pairs nucleic acid single linear 810UUUCAUCUAC ACCUCCU 17 17 base pairs nucleic acid single linear 811UGGUCUGUCG GGUGAAG 17 17 base pairs nucleic acid single linear 812CUACACCUCC UUCCAAG 17 17 base pairs nucleic acid single linear 813GCAGGACUAC CUUUGCA 17 17 base pairs nucleic acid single linear 814CACCUCCUUC CAAGAGC 17 17 base pairs nucleic acid single linear 815GACUACCUUU GCACCCU 17 17 base pairs nucleic acid single linear 816ACCUCCUUCC AAGAGCG 17 17 base pairs nucleic acid single linear 817ACUACCUUUG CACCCUU 17 17 base pairs nucleic acid single linear 818GGCGACCUUC AUCUCAC 17 17 base pairs nucleic acid single linear 819UGCACCCUUG CUUCAAG 17 17 base pairs nucleic acid single linear 820GCGACCUUCA UCUCACA 17 17 base pairs nucleic acid single linear 821CCCUUGCUUC AAGAAUC 17 17 base pairs nucleic acid single linear 822ACCUUCAUCU CACACGG 17 17 base pairs nucleic acid single linear 823CCUUGCUUCA AGAAUCA 17 17 base pairs nucleic acid single linear 824CUUCAUCUCA CACGGGA 17 17 base pairs nucleic acid single linear 825UCAAGAAUCA GGAGGCU 17 17 base pairs nucleic acid single linear 826CGCUGCCUUU CAGCUGG 17 17 base pairs nucleic acid single linear 827UUUGAUGUAC AACCUGU 17 17 base pairs nucleic acid single linear 828GCUGCCUUUC AGCUGGG 17 17 base pairs nucleic acid single linear 829CAUGCCGUAC UUUGUCU 17 17 base pairs nucleic acid single linear 830CUGCCUUUCA GCUGGGU 17 17 base pairs nucleic acid single linear 831GCCGUACUUU GUCUGUC 17 17 base pairs nucleic acid single linear 832AGCUGGGUAU ACGGUAG 17 17 base pairs nucleic acid single linear 833CCGUACUUUG UCUGUCG 17 17 base pairs nucleic acid single linear 834CUGGGUAUAC GGUAGGG 17 17 base pairs nucleic acid single linear 835UACUUUGUCU GUCGCUG 17 17 base pairs nucleic acid single linear 836UAUACGGUAG GGACGUC 17 17 base pairs nucleic acid single linear 837UUGUCUGUCG CUGGCGG 17 17 base pairs nucleic acid single linear 838AGGGACGUCC AACUGUG 17 17 base pairs nucleic acid single linear 839CGGUGUGUUU CGGUAUG 17 17 base pairs nucleic acid single linear 840UGUGAGAUCG GAAACCU 17 17 base pairs nucleic acid single linear 841GGUGUGUUUC GGUAUGU 17 17 base pairs nucleic acid single linear 842GCUGCGGUCU GCUUAGA 17 17 base pairs nucleic acid single linear 843GUGUGUUUCG GUAUGUU 17 17 base pairs nucleic acid single linear 844GGUCUGCUUA GACAAGA 17 17 base pairs nucleic acid single linear 845GUUUCGGUAU GUUAUUU 17 17 base pairs nucleic acid single linear 846GUCUGCUUAG ACAAGAC 17 17 base pairs nucleic acid single linear 847CGGUAUGUUA UUUGAGU 17 17 base pairs nucleic acid single linear 848UGCUGUGUCU GCGUUAC 17 17 base pairs nucleic acid single linear 849GGUAUGUUAU UUGAGUU 17 17 base pairs nucleic acid single linear 850GUCUGCGUUA CAUAGGU 17 17 base pairs nucleic acid single linear 851UAUGUUAUUU GAGUUGC 17 17 base pairs nucleic acid single linear 852UCUGCGUUAC AUAGGUC 17 17 base pairs nucleic acid single linear 853AUGUUAUUUG AGUUGCU 17 17 base pairs nucleic acid single linear 854CGUUACAUAG GUCUCCA 17 17 base pairs nucleic acid single linear 855AUUUGAGUUG CUCAGAU 17 17 base pairs nucleic acid single linear 856ACAUAGGUCU CCAGGUU 17 17 base pairs nucleic acid single linear 857GAGUUGCUCA GAUCUGU 17 17 base pairs nucleic acid single linear 858AUAGGUCUCC AGGUUUU 17 17 base pairs nucleic acid single linear 859GCUCAGAUCU GUUAAAA 17 17 base pairs nucleic acid single linear 860CUCCAGGUUU UGAUCAA 17 17 base pairs nucleic acid single linear 861AGAUCUGUUA AAAAAAA 17 17 base pairs nucleic acid single linear 862UCCAGGUUUU GAUCAAA 17 17 base pairs nucleic acid single linear 863GAUCUGUUAA AAAAAAA 17 17 base pairs nucleic acid single linear 864CCAGGUUUUG AUCAAAU 17 17 base pairs nucleic acid single linear 865GUUUUGAUCA AAUGGUC 17 17 base pairs nucleic acid single linear 866CAAAUGGUCC CGUGUCG 17 17 base pairs nucleic acid single linear 867UCCCGUGUCG UCUUAUA 17 17 base pairs nucleic acid single linear 868CGUGUCGUCU UAUAGAG 17 17 base pairs nucleic acid single linear 869UGUCGUCUUA UAGAGCG 17 17 base pairs nucleic acid single linear 870GUCGUCUUAU AGAGCGA 17 17 base pairs nucleic acid single linear 871CGUCUUAUAG AGCGAUA 17 17 base pairs nucleic acid single linear 872AGAGCGAUAG GAGAACG 17 17 base pairs nucleic acid single linear 873GAACGUGUUG GUCUGUG 17 17 base pairs nucleic acid single linear 874GUGUUGGUCU GUGGUGU 17 17 base pairs nucleic acid single linear 875UGUGGUGUAG CUUUGUU 17 17 base pairs nucleic acid single linear 876GUGUAGCUUU GUUUUUA 17 17 base pairs nucleic acid single linear 877UGUAGCUUUG UUUUUAU 17 17 base pairs nucleic acid single linear 878AGCUUUGUUU UUAUUUU 17 17 base pairs nucleic acid single linear 879GCUUUGUUUU UAUUUUG 17 17 base pairs nucleic acid single linear 880CUUUGUUUUU AUUUUGU 17 17 base pairs nucleic acid single linear 881UUUGUUUUUA UUUUGUA 17 17 base pairs nucleic acid single linear 882UUGUUUUUAU UUUGUAU 17 17 base pairs nucleic acid single linear 883GUUUUUAUUU UGUAUUU 17 17 base pairs nucleic acid single linear 884UUUUUAUUUU GUAUUUU 17 17 base pairs nucleic acid single linear 885UUUUAUUUUG UAUUUUU 17 17 base pairs nucleic acid single linear 886UAUUUUGUAU UUUUCUG 17 17 base pairs nucleic acid single linear 887UUUUGUAUUU UUCUGCU 17 17 base pairs nucleic acid single linear 888UUUGUAUUUU UCUGCUU 17 17 base pairs nucleic acid single linear 889UUGUAUUUUU CUGCUUU 17 17 base pairs nucleic acid single linear 890UGUAUUUUUC UGCUUUG 17 17 base pairs nucleic acid single linear 891GUAUUUUUCU GCUUUGA 17 17 base pairs nucleic acid single linear 892UUUCUGCUUU GAUGUAC 17 17 base pairs nucleic acid single linear 893UUCUGCUUUG AUGUACA 17 27 base pairs nucleic acid single linear The letter “N” stands for any base. 894AAGCGGCACU GAUGANGAAA GGGCGCG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 895GAACGAACCU GAUGANGAAA GCGGCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 896GAGGAACGCU GAUGANGAAA CAAGCGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 897CGAGGAACCU GAUGANGAAA ACAAGCG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 898GCGCGAGGCU GAUGANGAAA CGAACAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 899AGCGCGAGCU GAUGANGAAA ACGAACA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 900GCGAGCGCCU GAUGANGAAA GGAACGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 901CUGGUGGCCU GAUGANGAAA GCGCGAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 902GAGAUUGGCU GAUGANGAAA UGUGUGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 903CCUCGCGACU GAUGANGAAA UUGGGAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 904GCCCUCGCCU GAUGANGAAA GAUUGGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 905CCGCCGCACU GAUGANGAAA CCCUGCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 906GGAGCCGGCU GAUGANGAAA GCGCGGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 907GGGAGCCGCU GAUGANGAAA AGCGCGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 908GGGAAGGGCU GAUGANGAAA GCCGGAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 909CCAAUGGGCU GAUGANGAAA GGGGAGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 910GCCAAUGGCU GAUGANGAAA AGGGGAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 911UGGAGGCCCU GAUGANGAAA UGGGAAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 912CCAUCGUGCU GAUGANGAAA GGCCAAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 913UUGAGGCGCU GAUGANGAAA GCGCCAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 914ACGUCGUUCU GAUGANGAAA GGCGGAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 915CAGAGCGCCU GAUGANGAAA CGUCGUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 916GAGAGGCACU GAUGANGAAA GCGCGAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 917GGCGGGGACU GAUGANGAAA GGCAGAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 918GCGGCGGGCU GAUGANGAAA GAGGCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 919CGGGCGGCCU GAUGANGAAA GCGGCGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 920CGGCGACGCU GAUGANGAAA CCUGCCG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 921ACGGCGACCU GAUGANGAAA ACCUGCC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 922GCGACGGCCU GAUGANGAAA CGAACCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 923AUGGAGGCCU GAUGANGAAA CGGCGAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 924ACGUCAUGCU GAUGANGAAA GGCGACG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 925AGACGGCGCU GAUGANGAAA CGUCAUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 926UUGGUGGACU GAUGANGAAA CGGCGGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 927CCUUGGUGCU GAUGANGAAA GACGGCG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 928UUAUUCUCCU GAUGANGAAA CCUUGGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 929UGGCUUCUCU GAUGANGAAA UUCUCGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 930GAGGAGCACU GAUGANGAAA UGGCUUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 931GGAGGAGCCU GAUGANGAAA AUGGCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 932CCUUGGAGCU GAUGANGAAA GCAAAUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 933CUCCCUUGCU GAUGANGAAA GGAGCAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 934UGGACAUGCU GAUGANGAAA CCUCCCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 935GUAACCUGCU GAUGANGAAA CAUGUAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 936GAAUGUGUCU GAUGANGAAA CCUGGAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 937UGAAUGUGCU GAUGANGAAA ACCUGGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 938UGGCAUUGCU GAUGANGAAA UGUGUAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 939GUGGCAUUCU GAUGANGAAA AUGUGUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 940AAUCUUGUCU GAUGANGAAA GGUGGCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 941AAAAUUUCCU GAUGANGAAA UCUUGUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 942GACUUGAACU GAUGANGAAA UUUCAAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 943CGACUUGACU GAUGANGAAA AUUUCAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 944GCGACUUGCU GAUGANGAAA AAUUUCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 945AGCGACUUCU GAUGANGAAA AAAUUUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 946CAUCAAGCCU GAUGANGAAA CUUGAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 947CAAUCAUCCU GAUGANGAAA GCGACUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 948UCUAGCCCCU GAUGANGAAA UCAUCAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 949AUUAUCUCCU GAUGANGAAA GCCCAAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 950CAAGAUAUCU GAUGANGAAA UCUCUAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 951CGUCAAGACU GAUGANGAAA UUAUCUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 952UGCGUCAACU GAUGANGAAA UAUUAUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 953GAUGCGUCCU GAUGANGAAA GAUAUUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 954UGGCUUGACU GAUGANGAAA UGCGUCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 955ACUGGCUUCU GAUGANGAAA GAUGCGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 956CACUUCUCCU GAUGANGAAA CUGGCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 957UGGCUGCCCU GAUGANGAAA CACUUCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 958CGGGAGGACU GAUGANGAAA UCCUGUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 959CCGGGAGGCU GAUGANGAAA AUCCUGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 960UCCGGGAGCU GAUGANGAAA AAUCCUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 961GGGUCCGGCU GAUGANGAAA GGAAAUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 962AUCCUUCACU GAUGANGAAA UGCUGGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 963CAUCAUGACU GAUGANGAAA UCCUUCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 964UCAUCAUGCU GAUGANGAAA AUCCUUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 965UUCAUCAUCU GAUGANGAAA AAUCCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 966AGCUCCUUCU GAUGANGAAA CUUCAUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 967GAGCUCCUCU GAUGANGAAA ACUUCAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 968CGUUCUCUCU GAUGANGAAA GCUCCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 969UCAUCAGGCU GAUGANGAAA UUUCCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 970AACAAAAUCU GAUGANGAAA UCAUCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 971AAACAAAACU GAUGANGAAA AUCAUCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 972ACAAACAACU GAUGANGAAA UAAUCAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 973AACAAACACU GAUGANGAAA AUAAUCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 974AAACAAACCU GAUGANGAAA AAUAAUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 975ACCAAACACU GAUGANGAAA CAAAAUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 976CACCAAACCU GAUGANGAAA ACAAAAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 977UCCCACCACU GAUGANGAAA CAAACAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 978CUCCCACCCU GAUGANGAAA ACAAACA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 979UCCUCGGUCU GAUGANGAAA UCAUGUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 980UUCCUCGGCU GAUGANGAAA AUCAUGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 981UGUUGGUACU GAUGANGAAA GCUUCCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 982UAUGUUGGCU GAUGANGAAA GAGCUUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 983UAGUCUGGCU GAUGANGAAA UGUUGGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 984GUUAAGCACU GAUGANGAAA GUCUGGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 985AGGGUGUUCU GAUGANGAAA GCAUAGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 986GAGGGUGUCU GAUGANGAAA AGCAUAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 987ACACCGUCCU GAUGANGAAA GGGUGUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 988UCAUCUCUCU GAUGANGAAA CACCGUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 989CUCGUCCACU GAUGANGAAA CAGCCCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 990CCUCGUCCCU GAUGANGAAA ACAGCCC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 991GUUGAGCACU GAUGANGAAA UCACCAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 992UACUUGUUCU GAUGANGAAA GCAGAUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 993GGUACAUACU GAUGANGAAA CUUGUUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 994GAGGUACACU GAUGANGAAA UACUUGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 995CAGUGAGGCU GAUGANGAAA CAUAUAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 996CUCCCAGUCU GAUGANGAAA GGUACAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 997CUGCCUCACU GAUGANGAAA UCCACCC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 998GUCUUCUCCU GAUGANGAAA UCUGCCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 999AGAUACUGCU GAUGANGAAA UUGUCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1000AAGAUACUCU GAUGANGAAA AUUGUCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1001CAAUAAGACU GAUGANGAAA CUGAAUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1002GCCAAUAACU GAUGANGAAA UACUGAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1003GAGCCAAUCU GAUGANGAAA GAUACUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1004AGAGCCAACU GAUGANGAAA AGAUACU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1005CCAGAGCCCU GAUGANGAAA UAAGAUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1006CCAUUCCACU GAUGANGAAA GCCAAUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1007AGUCCUAGCU GAUGANGAAA UCCAUUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1008CUCAGUCCCU GAUGANGAAA GGAUCCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1009AUAAGGAUCU GAUGANGAAA UUCUCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1010AAGAUAAGCU GAUGANGAAA UUAUUCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1011ACCAAGAUCU GAUGANGAAA GGAUUAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1012AACCAAGACU GAUGANGAAA AGGAUUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1013GAAACCAACU GAUGANGAAA UAAGGAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1014AUGAAACCCU GAUGANGAAA GAUAAGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1015GUAGAUGACU GAUGANGAAA CCAAGAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1016UGUAGAUGCU GAUGANGAAA ACCAAGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1017GUGUAGAUCU GAUGANGAAA AACCAAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1018GAGGUGUACU GAUGANGAAA UGAAACC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1019AGGAGGUGCU GAUGANGAAA GAUGAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1020CUUGGAAGCU GAUGANGAAA GGUGUAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1021GCUCUUGGCU GAUGANGAAA GGAGGUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1022CGCUCUUGCU GAUGANGAAA AGGAGGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1023GUGAGAUGCU GAUGANGAAA GGUCGCC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1024UGUGAGAUCU GAUGANGAAA AGGUCGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1025CCGUGUGACU GAUGANGAAA UGAAGGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1026UCCCGUGUCU GAUGANGAAA GAUGAAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1027GGCGUGACCU GAUGANGAAA GCAGUGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1028CUUGGCGUCU GAUGANGAAA CGAGCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1029AGUCGCCACU GAUGANGAAA GUCCUUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1030AAGUCGCCCU GAUGANGAAA AGUCCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1031CAAGCUUUCU GAUGANGAAA GUCGCCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1032GCAAGCUUCU GAUGANGAAA AGUCGCC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1033AUUUGUGCCU GAUGANGAAA GCUUUAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1034AUGCCGCACU GAUGANGAAA UUUGUGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1035GAGGCGAUCU GAUGANGAAA UGCCGCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1036UCUGAGGCCU GAUGANGAAA UGAUGCC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1037UCUCAUCUCU GAUGANGAAA GGCGAUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1038UCUUGGUGCU GAUGANGAAA CGCAGUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1039UUCUCCACCU GAUGANGAAA UCUUGGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1040CGAUCUCACU GAUGANGAAA CAGCUUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1041UCGAUCUCCU GAUGANGAAA ACAGCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1042UCAGGGUCCU GAUGANGAAA UCUCAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1043GACCACGGCU GAUGANGAAA CCAUCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1044GCCAGAGCCU GAUGANGAAA CCACGGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1045GUCAGCCACU GAUGANGAAA GCGACCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1046GGCAUUGACU GAUGANGAAA UCUUCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1047CAGGCAUUCU GAUGANGAAA GAUCUUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1048GCCCGUCACU GAUGANGAAA CAUCAGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1049UGCCCGUCCU GAUGANGAAA ACAUCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1050AGUGCUCGCU GAUGANGAAA CAGCUUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1051AAGUGCUCCU GAUGANGAAA ACAGCUU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1052CCAUGGAGCU GAUGANGAAA GUGCUCG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1053ACCAUGGACU GAUGANGAAA AGUGCUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1054CGACCAUGCU GAUGANGAAA GAAGUGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1055CUCUGCGCCU GAUGANGAAA CCAUGGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1056UAAACGCCCU GAUGANGAAA GCCUCUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1057GCGGUGUACU GAUGANGAAA CGCCAAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1058GGCGGUGUCU GAUGANGAAA ACGCCAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1059UGGCGGUGCU GAUGANGAAA AACGCCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1060UGUCGGCGCU GAUGANGAAA GUCCCUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1061AACUCGAGCU GAUGANGAAA UGUCGGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1062AGGAACUCCU GAUGANGAAA GGAUGUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1063CGACGAGGCU GAUGANGAAA CUCGAGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1064UCGACGAGCU GAUGANGAAA ACUCGAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1065CUGUCGACCU GAUGANGAAA GGAACUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1066CACCUGUCCU GAUGANGAAA CGAGGAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1067ACCCGACACU GAUGANGAAA CCAGUCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1068CUUCACCCCU GAUGANGAAA CAGACCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1069UGCAAAGGCU GAUGANGAAA GUCCUGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1070AGGGUGCACU GAUGANGAAA GGUAGUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1071AAGGGUGCCU GAUGANGAAA AGGUAGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1072CUUGAAGCCU GAUGANGAAA GGGUGCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1073GAUUCUUGCU GAUGANGAAA GCAAGGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1074UGAUUCUUCU GAUGANGAAA AGCAAGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1075AGCCUCCUCU GAUGANGAAA UUCUUGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1076CCAGCUGACU GAUGANGAAA GGCAGCG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1077CCCAGCUGCU GAUGANGAAA AGGCAGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1078ACCCAGCUCU GAUGANGAAA AAGGCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1079CUACCGUACU GAUGANGAAA CCCAGCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1080CCCUACCGCU GAUGANGAAA UACCCAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1081GACGUCCCCU GAUGANGAAA CCGUAUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1082CACAGUUGCU GAUGANGAAA CGUCCCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1083AGGUUUCCCU GAUGANGAAA UCUCACA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1084UCUAAGCACU GAUGANGAAA CCGCAGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1085UCUUGUCUCU GAUGANGAAA GCAGACC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1086GUCUUGUCCU GAUGANGAAA AGCAGAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1087GUAACGCACU GAUGANGAAA CACAGCA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1088ACCUAUGUCU GAUGANGAAA CGCAGAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1089GACCUAUGCU GAUGANGAAA ACGCAGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1090UGGAGACCCU GAUGANGAAA UGUAACG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1091AACCUGGACU GAUGANGAAA CCUAUGU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1092AAAACCUGCU GAUGANGAAA GACCUAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1093UUGAUCAACU GAUGANGAAA CCUGGAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1094UUUGAUCACU GAUGANGAAA ACCUGGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1095AUUUGAUCCU GAUGANGAAA AACCUGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1096GACCAUUUCU GAUGANGAAA UCAAAAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1097CGACACGGCU GAUGANGAAA CCAUUUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1098UAUAAGACCU GAUGANGAAA CACGGGA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1099CUCUAUAACU GAUGANGAAA CGACACG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1100CGCUCUAUCU GAUGANGAAA GACGACA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1101UCGCUCUACU GAUGANGAAA AGACGAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1102UAUCGCUCCU GAUGANGAAA UAAGACG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1103CGUUCUCCCU GAUGANGAAA UCGCUCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1104CACAGACCCU GAUGANGAAA CACGUUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1105ACACCACACU GAUGANGAAA CCAACAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1106AACAAAGCCU GAUGANGAAA CACCACA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1107UAAAAACACU GAUGANGAAA GCUACAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1108AUAAAAACCU GAUGANGAAA AGCUACA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1109AAAAUAAACU GAUGANGAAA CAAAGCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1110CAAAAUAACU GAUGANGAAA ACAAAGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1111ACAAAAUACU GAUGANGAAA AACAAAG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1112UACAAAAUCU GAUGANGAAA AAACAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1113AUACAAAACU GAUGANGAAA AAAACAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1114AAAUACAACU GAUGANGAAA UAAAAAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1115AAAAUACACU GAUGANGAAA AUAAAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1116AAAAAUACCU GAUGANGAAA AAUAAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1117CAGAAAAACU GAUGANGAAA CAAAAUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1118AGCAGAAACU GAUGANGAAA UACAAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1119AAGCAGAACU GAUGANGAAA AUACAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1120AAAGCAGACU GAUGANGAAA AAUACAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1121CAAAGCAGCU GAUGANGAAA AAAUACA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1122UCAAAGCACU GAUGANGAAA AAAAUAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1123GUACAUCACU GAUGANGAAA GCAGAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1124UGUACAUCCU GAUGANGAAA AGCAGAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1125ACAGGUUGCU GAUGANGAAA CAUCAAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1126AGACAAAGCU GAUGANGAAA CGGCAUG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1127GACAGACACU GAUGANGAAA GUACGGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1128CGACAGACCU GAUGANGAAA AGUACGG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1129CAGCGACACU GAUGANGAAA CAAAGUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1130CCGCCAGCCU GAUGANGAAA CAGACAA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1131CAUACCGACU GAUGANGAAA CACACCG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1132ACAUACCGCU GAUGANGAAA ACACACC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1133AACAUACCCU GAUGANGAAA AACACAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1134AAAUAACACU GAUGANGAAA CCGAAAC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1135ACUCAAAUCU GAUGANGAAA CAUACCG 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1136AACUCAAACU GAUGANGAAA ACAUACC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1137GCAACUCACU GAUGANGAAA UAACAUA 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1138AGCAACUCCU GAUGANGAAA AUAACAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1139AUCUGAGCCU GAUGANGAAA CUCAAAU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1140ACAGAUCUCU GAUGANGAAA GCAACUC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1141UUUUAACACU GAUGANGAAA UCUGAGC 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1142UUUUUUUUCU GAUGANGAAA CAGAUCU 27 27 base pairs nucleic acid single linear The letter “N” stands for any base. 1143UUUUUUUUCU GAUGANGAAA ACAGAUC 27 54 base pairs nucleic acid single linear 1144GAACAAGCAG AAGAGGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1145GCCCUCUGCC GCUUGUUC 18 54 base pairs nucleic acid single linear 1146AACGAACAAG AAGCAGAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1147CUCUGCCGCU UGUUCGUU 18 54 base pairs nucleic acid single linear 1148GGAAGCGCAG AAGCCGCCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1149GGCGGCGGCC GCGCUUCC 18 54 base pairs nucleic acid single linear 1150GGAAGGGGAG AAGGAAGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1151GCUUCCGGCU CCCCUUCC 18 54 base pairs nucleic acid single linear 1152GUCGUUGAAG AAGAGCGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1153GCGCUCCGCC UCAACGAC 18 54 base pairs nucleic acid single linear 1154CGGGGAGAAG AAGAGCGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1155GCGCUCUGCC UCUCCCCG 18 54 base pairs nucleic acid single linear 1156CGGCGAGCAG AAGGGAGAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1157UCUCCCCGCC GCUCGCCG 18 54 base pairs nucleic acid single linear 1158GGGCGGCGAG AAGCGGGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1159CCCCGCCGCU CGCCGCCC 18 54 base pairs nucleic acid single linear 1160CGGCGGCGAG AAGCGAGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1161GCUCGCCGCC CGCCGCCG 18 54 base pairs nucleic acid single linear 1162GCGGCGGCAG AAGGCGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1163GCCGCCCGCC GCCGCCGC 18 54 base pairs nucleic acid single linear 1164GCGGCGGCAG AAGCGGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1165GCCCGCCGCC GCCGCCGC 18 54 base pairs nucleic acid single linear 1166GCUGCGGCAG AAGCGGCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1167CGCCGCCGCC GCCGCAGC 18 54 base pairs nucleic acid single linear 1168GCUGCUGCAG AAGCGGCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1169CGCCGCCGCC GCAGCAGC 18 54 base pairs nucleic acid single linear 1170AUGGAGGCAG AAGCGACGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1171CGUCGCCGUC GCCUCCAU 18 54 base pairs nucleic acid single linear 1172GUGGAGACAG AAGACGUCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1173GACGUCCGCC GUCUCCAC 18 54 base pairs nucleic acid single linear 1174UUGGUGGAAG AAGCGGACAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1175GUCCGCCGUC UCCACCAA 18 54 base pairs nucleic acid single linear 1176CACUUCUCAG AAGGCUUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1177CAAGCCAGUC GAGAAGUG 18 54 base pairs nucleic acid single linear 1178GAUGCUGGAG AAGGGAGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1179CCUCCCGGAC CCAGCAUC 18 54 base pairs nucleic acid single linear 1180AAAUAAUCAG AAGGGAUUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1181AAUCCCUGAU GAUUAUUU 18 54 base pairs nucleic acid single linear 1182UAAGCAUAAG AAGGUAUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1183CAUACCAGAC UAUGCUUA 18 54 base pairs nucleic acid single linear 1184ACAGCCCAAG AAGUGGGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1185CCCCACUGCC UGGGCUGU 18 54 base pairs nucleic acid single linear 1186CUCGUCCAAG AAGCCCAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1187CUGGGCUGUU UGGACGAG 18 54 base pairs nucleic acid single linear 1188UUCUCCUCAG AAGUCCAUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1189AUGGACUGCU GAGGAGAA 18 54 base pairs nucleic acid single linear 1190ACUUGUUGAG AAGAUCACAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1191GUGAUCUGCU CAACAAGU 18 54 base pairs nucleic acid single linear 1192UCUUCUCAAG AAGCCUCAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1193UGAGGCAGAU UGAGAAGA 18 54 base pairs nucleic acid single linear 1194GCGUGACGAG AAGUGUUCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1195GAACACUGCU CGUCACGC 18 54 base pairs nucleic acid single linear 1196CGCUUCUCAG AAGAGGCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1197CGCCUCAGAU GAGAAGCG 18 54 base pairs nucleic acid single linear 1198CGAUCUCAAG AAGCUUCUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1199AGAAGCUGUU UGAGAUCG 18 54 base pairs nucleic acid single linear 1200ACGGUACCAG AAGGGUCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1201CGACCCUGAU GGUACCGU 18 54 base pairs nucleic acid single linear 1202AUCAGGUGAG AAGGCAUUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1203AAUGCCUGCC CACCUGAU 18 54 base pairs nucleic acid single linear 1204CGUCAAACAG AAGGUGGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1205CCCACCUGAU GUUUGACG 18 54 base pairs nucleic acid single linear 1206AGUGCUCGAG AAGCUUGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1207ACAAGCUGUU CGAGCACU 18 54 base pairs nucleic acid single linear 1208ACAGACCAAG AAGGCUCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1209CGAGCCUGAC UGGUCUGU 18 54 base pairs nucleic acid single linear 1210CUUCACCCAG AAGACCAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1211CUGGUCUGUC GGGUGAAG 18 54 base pairs nucleic acid single linear 1212AGCUGAAAAG AAGCGUGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1213GCACGCUGCC UUUCAGCU 18 54 base pairs nucleic acid single linear 1214GUAUACCCAG AAGAAAGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1215CCUUUCAGCU GGGUAUAC 18 54 base pairs nucleic acid single linear 1216CAGACCGCAG AAGGUUUCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1217GAAACCUGCU GCGGUCUG 18 54 base pairs nucleic acid single linear 1218UCUAAGCAAG AAGCAGCAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1219UGCUGCGGUC UGCUUAGA 18 54 base pairs nucleic acid single linear 1220CUUGUCUAAG AAGACCGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1221GCGGUCUGCU UAGACAAG 18 54 base pairs nucleic acid single linear 1222GCAGACACAG AAGGUCUUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1223AAGACCUGCU GUGUCUGC 18 54 base pairs nucleic acid single linear 1224UACAUCAAAG AAGAAAAAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1225UUUUUCUGCU UUGAUGUA 18 54 base pairs nucleic acid single linear 1226CCGCCAGCAG AAGACAAAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1227UUUGUCUGUC GCUGGCGG 18 54 base pairs nucleic acid single linear 1228UUUAACAGAG AAGAGCAAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1229UUGCUCAGAU CUGUUAAA 18 11 base pairs nucleic acid single linear The letter “N” stands for any base. The letter “H” stands for A, U or C. 1230NNNNUHNNNN N 11 28 base pairs nucleic acid single linear The letter “N” stands for any base. 1231NNNNNCUGAN GAGNNNNNNC GAAANNNN 28 15 base pairs nucleic acid single linear The letter “N” stands for any base. The leter “Y” stands for U or C. The letter “H” stands for A, U or C. 1232NNNNNNNYNG HYNNN 15 47 base pairs nucleic acid single linear The letter “N” stands for any base. 1233NNNNGAAGNN NNNNNNNNNA AAHANNNNNN NACAUUACNN NNNNNNN 47 49 base pairs nucleic acid single linear The letter “N” stands for any base. 1234CUCCACCUCC UCGCGGUNNN NNNNGGGCUA CUUCGGUAGG CUAAGGGAG 49 176 base pairs nucleic acid single linear 1235GGGAAAGCUU GCGAAGGGCG UCGUCGCCCC GAGCGGUAGU AAGCAGGGAA CUCACCUCCA 60AUUUCAGUAC UGAAAUUGUC GUAGCAGUUG ACUACUGUUA UGUGAUUGGU AGAGGCUAAG 120UGACGGUAUU GGCGUAAGUC AGUAUUGCAG CACAGCACAA GCCCGCUUGC GAGAAU 176 91 base pairs nucleic acid double linear 1236AAGCTTGCAT GCCTGCAGGC CGGCCTTAAT TAAGCGGCCG CGTTTAAACG CCCGGGCATT 60TTCGAACGTA CGGACGTCCG GCCGGAATTA ATTCGCCGGC GCAAATTTGC GGGCCCGTAATAAATGGCGC GCCGCGATCG CTTGCAGATC T 91ATTTACCGCG CGGCGCTAGC GAACGTCTAG A 10 base pairs nucleic acid single linear 1237GGCGAAAGCC 10 109 base pairs nucleic acid single linear 1238CGCGGATCCT GGTAGGACTG ATGAGGCCGA AAGGCCGAAA TGTTGTGCTG ATGAGGCCGA 60AAGGCCGAAA TGCAGAAAGC GGTCTTTGCG TCCCTGTAGA TGCCGTGGC 109 106 base pairs nucleic acid single linear 1239CGCGAGCTCG GCCCTCTCTT TCGGCCTTTC GGCCTCATCA GGTGCTACCT CAAGAGCAAC 60TACCAGTTTC GGCCTTTCGG CCTCATCAGC CACGGCATCT ACAGGG 106 47 base pairs nucleic acid single linear 1240GATCCGATGC CGTGGCTGAT GAGGCCGAAA GGCCGAAACT GGTAGTT 47 43 base pairs nucleic acid single linear 1241AACTACCAGT TTCGGCCTTT CGGCCTCATC AGCCACGGCA TCG 43 88 base pairs nucleic acid single linear 1242CTGCAGGCCG GCCTTAATTA AGCGGCCGCG TTTAAACGCC CGGGCATTTA AATGGCGCGC 60CGCGATCGCT TGCAGATCTG CATGGGTG 88 20 base pairs nucleic acid single linear 1243GGGGACTCTA GAGGATCCAG 20 10 base pairs nucleic acid single linear 1244GACGGATCTG 10 24 base pairs nucleic acid single linear 1245TGAGATCTGA GCTCGAATTT CCCC 24 19 base pairs nucleic acid single linear 1246CTGCAGATCT GCATGGGTG 19 13 base pairs nucleic acid single linear 1247GGGGACTCTA GAG 13 16 base pairs nucleic acid single linear 1248GACGGATCCG TCGACC 16 10 base pairs nucleic acid single linear 1249GAATTTCCCC 10 25 base pairs nucleic acid single linear 1250GATCCGCCCG GGGCCCGGGC GGTAC 25 17 base pairs nucleic acid single linear 1251CGCCCGGGCC CCGGGCG 17 30 base pairs nucleic acid single linear 1252GTGCCCACAA TGGCGCTCCG CCTCAACGAC 30 57 base pairs nucleic acid single linear 1253TCATCACAGG TCCTCCTCGC TGATCAGCTT CTCCTCCAGT TGGACCTGCC TACCGTA 57 57 base pairs nucleic acid single linear 1254TACGGTAGGG ACGTCCAACT GGAGGAGAAG CTGATCAGCG AGGAGGACCT GTGATGA 57 18 base pairs nucleic acid single linear 1255CGCAAGACCG GCAACAGG 18 22 base pairs nucleic acid single linear 1256TGGATTGATG TGATATCTCC AC 22 18 base pairs nucleic acid single linear 1257CGCAAGACCG GCAACAGG 18 31 base pairs nucleic acid single linear 1258CAGATCAAGT GCAAAGCTGC GGACGGATCT G 31 20 base pairs nucleic acid single linear 1259ATCCGATGCC GTGGCTGATG 20 20 base pairs nucleic acid single linear 1260GATGAGATCC GGTGGCATTG 20 20 base pairs nucleic acid single linear 1261ATCCCCTTGG TGGACTGATG 20 31 base pairs nucleic acid single linear 1262CAGATCAAGT GCAAAGCTGC GGACGGATCT G 31 6 amino acids amino acid single linear peptide 1263Ala Val Ala Ser Met Thr 1 5

Claims

1. An isolated nucleic acid fragment comprising SEQ ID NO. 1.

2. A maize plant transformed with a construct comprising in the 5′ to 3′ direction of Transcription:a promoter functional in said plant; a double strand DNA (dsDNA) comprising SEQ ID NO. 1, wherein the transcript strand of said dsDNA is complementary to RNA endogenous to said plant; and a termination region functional in said plant.

3. A transgenic plant that is a progeny of the maize plant of claim 2.

4. An expression vector comprising a nucleic acid sequence encoding at least one nucleic acid of claim 1, in a manner which allows expression of said nucleic acid.

5. A plant cell comprising the expression vector of claim 4.

6. A maize plant transformed with the expression vector of claim 4.

7. A transgenic plant that is a progeny of the maize plant of claim 6.

8. The transgenic plant of claim 2, 3, 6, or 7, wherein the plant is transformed by Agrobacterium, electroporation, whiskers, or by bombardment with DNA coated microprojectiles.

9. The transgenic plant of claim 8, wherein said bombardment with DNA coated microprojectiles is done with a gene gun.

10. The transgenic plant of claim 2, 3, 6, or 7, wherein the plant contains a selectable marker comprising the bar gene or a gene encoding resistance to a selection agent selected from the group consisting of chlorosulfuron, hygromycin, bromoxynil, and kanamycin.

11. The transgenic plant of claim 2 or 3, wherein the double strand DNA is operably linked to a cauliflower mosaic virus (35S) promoter or a promoter from a gene encoding a protein selected from the group consisting of octopine synthetase, nopaline synthase, mannopine synthetase, ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin, phaseolin, napin, gamma zein, globulin, ADH, heat shock protein, actin, and ubiquitin.