Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
COMPOSITIONS AND METHOD FOR MODULATIO~I OF GEi\E
EXPRESSION ~N PLA~\'TS
This application is a continuation-in-part of: l) a Non-l'rovisioll.ll a~ lic.l~ioll by
5 Edington, entitled "Method for the production of tralls~cllic l11.lllts clcflciclll ilm ~ rcll
granule bound glucose starch ~lycosyl transfcMsc activi~y" filc(l 011 ~Scr~lclllbcr 2, 1~)')4 ~lS
U.S.S.N. 08/300,726; and 2) a Provisional application by Zwick et al., entitled
"Composition and method for modification of fatty acid saturation profile in plants" filed
on July 13, 1995, as U.S.S.N 60/001,135. Both of thesc ar~plicatiolls in thcir cntircty,
10 including drawings, are hereby incorporated by reference herein.
Back~round of the Invention
The present invention concerns compositions and methods for the modulation of
gene c;Ay~es~ion in plants, specific~lly using enzymatic nucleic acid molecules.
The following is a brief description of regulation of gene e,~yl~s~ion in plants. The
;u~j~ion is not meant to be complete and is provided only for understanding of the
invention that follows. This s~ y is not an aAmic~ion that any of the work described
below is prior art to the claimed invention.
There are a variety of strategies for mod~ ting gene e~yl~ssion in plants.
Tr~ on~lly~ ~nti~?n~e RNA (reviewed in Bourque, 1995 Plant Sci 105, 125-149) and co-
su~pression (reviewed in Jorgel1sen, 1995 Science 268, 686-691) approaches have been
used to modulate gene e~ylession. Insertion mutagenesis of genes have also been used to
silence gene e,~,e;.~ion. This approach, however, cannot be ~esign.-d to specifically-
inactivate the gene of interest. Applicant believes that ribozyme technology offers an
attractive new means to alter gene expression in plants.
Naturally occumng ~nticence 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 e,~,ession (Green et al., 1986 Ann. Rev. Biochem. 55: 567-
597; Simons 1988 Gene 72: 35-44). The first demonstration of antisense-nle~ ted
rl 30 inhibitionofgenee~y-~ ionwasreportedinm~mm~ ncells(IzantandWeintraub 1984
Cell 36: 1007-1015). There are many examples in the literature for the use of antisense
A to modulate gene e~y~ession in plants. Following are a few examples:
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Shewmaker et al., U.s. Patent Nos 5,107.065 and 5. ~53,566 disclose methods for
regulating gene e~p~ssion in plants using antisense RNA.
It has been shown that an antisense gene expressed in plants can act as a dominallt
suppressor gene. Transgenic potato plants have been produced which exprcss RNA
5 an~icen~e to potato or cassava granule bound starch synthasc (~SS). hl holh ol ~hcsc
cases, transgenic plants have becn constructcd whicll havc rccl(lccd or no (il~ C~iVily 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;
~s7lleh~ A~n~ll et al. 1993, PlantMol. Biol. 23: 947-962).
Kull et al., 1995, J. Genet. & Breed 49, 69-~6 reported inhibition of amylose
biosynthesis in tubers from transgenic potato lines m~ t~d by the expression of
;c~ e sequences of the gene for granule-bound starch synthase (GBSS). The authors,
however, inrlic~ttod a failure to see any in vivo activity of ribozymes targeted against the
GBSS RNA.
,~nticence RNA constructs targeted against A-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 de~ ase in total oil content or
ion 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 doc~mçnted in plants as an unexpected
result of inserting a transgene in the sense orientation and finding that both the gene and
the ~ g~-e were down-regul~te~l (Napoli et al., 1990 Plant Cell 2: 279-289). There
appears to be at least two "~ for inactivation of homologous genetic sequences.
One appears to be llal1sc,i~tional inactivation via methylation, where duplicated DNA
regions signal endogenous mech~ni~m~ for gene silencing. This approach of gene
modt~ on involves either the introduction of multiple copies of transgenes or
l~a~sru~ ation 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-
,ls.,.;~lional, where the combined levels of expression from both the gene and the
,ansgel-e is thought to produce high levels of transcript which triggers threshold-induced
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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/Tec*nolo~y
12: 883-888). Currently, there is no easy way to specifically inactivatc a L~cnc nf in~crcst
~, at the DNA level in plants (Pazkowski et al., 1988 kM~O~/. 7: 4021-4026). l rallsposoll
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 "~ ;cm of action, have advantages over competing technologies.
However, there have been difficulties in demonstrating the effectiveness of ribozymes in
mo~ fing gene eA~ res.,ion 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 IiL~lUI~ of ribozyme activity in plants cells, almost all of them involve down
re~l~tion of exogenously introduced genes, such as reporter genes in transient assays
(S~e;~r~L~ et al., 1992 EMBO J. 1 1: 1525-1530; P~ .lilllall et al., 1993 Antisense Res. Dev.
3: 253-263; P.,.l;l~ et al., 1995, Proc. Natl. Acad. Sci. USA, 92, 6165).
There are also several publications, te.g, Lamb and Hay, 1990, J. Gen. Virol. 71,
2257-2264; Gerlach et al., T..l. .-~I;onal PCT Publication No. WO 91/13994; Xu et aL,
1992, Science in China ~Ser. BJ 35, 1434-1443; Edington and Nelson, 1992, in Gene
Regulation: Biology of antisense RNA and DNA, eds. R. P. Erickson and J. G. Izant, p p
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], tnat l)ro~ose using h~.. ~.l.P~cl ribozymes to modulate: virus replication,
~A~.cssion of viral genes and/or reporter genes. None of these publications report the use
of ribozymes to modulate the e~ ,sion of plant genes.
Mazzolini et al., 1992, Plant. Mol. Bio. 20, 715-731; Steinecke et al., 1992, EMBO.
J. Il, 1525-1530; Pc~ dl- et al., 1995, Proc. Natl. Aead. Sci. USA., 92, 6175-6179;
Wegener et al., 1994, Mol. Gen. Genet. 245, 465-470; and Stein~clce et al., 1994, Gene,
149, 47-54, describe the use of h~rnmerhead ribozymes to inhibit expression of reporter
genes in plant cells.
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Bennett and Cullimore, 1992 Nucleic Acids Res. 20, 831-837 demonstrate
h~mmrrhe~d ribozyme-mediated in vitro cleavage of glna, glnb, gl~.~g and glnd RNA,
coding for glut~mine svnthetase enzyme in Phaseolus v~lgaris.
Hitz et al., (WO 91/18985) describe a method for usl~ng the soybcall a-9 dcsaturasc
5 enzyme to modify plant oil composition. The applicatiol1 clcscrihcs tl-c usc of soybcal1
A-9 desaturase sequence to isolate ~-9 desaturasc gcncs f'rol1l oll1cl sr)ccics.
The lefe,~.lces 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 cnablc thc usc Or riboizylllcs
10 to down regulate genes in plant cells, let alone plants.
Summary Of The Invention
The invention fea~ s 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 forrn of, but
15 not limited to, a ha~ -l-ead, hairpin, hepatitis delta virus, group I intron, group ~1
intron, RNaseP RNA, Neurospora VS RNA and the like. The enzymatic nucleic acid
molecllle with RNA cleaving activity may be encoded as a monomer or a multimcr,
preferably a mllltim~n The nucleic acids encoding for the enzymatic nucleic acid molec~,.le
with RNA cleaving activity may be operably linked to an open reading frame. Gene20 e~yl~ssion in any plant species may be modified by transformation of the plant with the
nucleic acid encoding the enzymatic nucleic acid molec-lles with RNA cleaving activity.
There are also numerous technologies for tl~nsrol'l,illg a plant: such technologies include
but are not limited to transf~.llllation with Agrobacterium, bonlba,di~g with DNA coated
microprojectiles, whiskers, or electroporation. Any target gene may be modified with the
25 nucleic acids Pnrodin~ 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
30 inhibit gene ~ c~.sion in plants such as monocot plants (e.g., cornJ. 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
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decrease the level of expression of a particular gene, rather thall 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 exr~rcssion to
generate plant cells, plant tissues or plants with altered phenotype.
Ribozymes (i.e., enzymatic nucleic acids) arc nuclcic acid n1olcculcs h.lvil1g aenzymatic activity which is able to rcpcatcdly clcavc oll1cl sc~ c I~N/~ ok:e~llcs ilml
nucleotide base sequence-specific manner. Such cnzyl11atic RN/~ InOICcLllC~i cal~ bc
targeted to virtually any RNA transcript, and efficient cleava~e has been achieved in vitro
and in vivo (Zaug et al., 1986, Nature 324, 429; Kim et al., 1987, Proc. Nu~l. Acad. Sci.
USA 84, 8788; Dreyfus, 1988, Einstein Quarterly J. Bio. Med., 6, 92; Hascloff and
Gerlach, 1988, Nature 334 585; Cech, 1988, JAM~ 260, 3030; Murphy and Cech, 1989,
Proc. Natl. Acad. Sci. USA., 86, 9218; Jefferies et al., 1989, Nucleic Acids Research 17,
1371).
ReC~ce of their sequence-specificity, trans-cleaving ribozymes may be used as
efficient tools to modulate gene e~u,e~sion in a variety of organisms including plants,
~nim~lc snd h.~ c (Bennett et al., supra; F~lineton et al., supra; Usman & McSwiggen,
1995 Ann. ~ep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem.
38, 2023-2037). Ribozymes can be deci~cl to cleave specific RNA targets within the
background of cellular RNA. Such a cleavage event renders the mRNA non-functional and
abrogates protein ~,Lpre~sion from that RNA. In this manner, synthesis of a protein
associated with a particular phenotype and/or disease state can be selectively inhibited.
Other fealul~s and advantages of the invention will be apparent from the following
deswit,Lion ofthe plefel,ed embod;..,~ ; thereof, and from the claims.
Brief Descl i~,Lion of the Fi~ures
Figure 1 is a ~ ic repreSçnt~tion of the hammerhead ribozyme domain
known in the art. Stem II can be 2 2 base-pairs long. Each N is any nucleotide and each -
represents a base pair.
Figure 2a is a dia~ ;c .~.res~,llL~tion of the ha-,--..e-l-ead ribozyme domain
known in the art; Figure 2b is a diagrammatic le~.ese~ tion of the hammerhead ribozyme
as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzymeportion; Figure 2c is a similar ~ ~m showing the h~mmerhead divided by Haseloff and
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Gerlach (1988, Nature, 334, 585-~91) into t~o portions; and Figure 2d is a similar
diagram showing the hammerhead divided by Jeffries and Symolls (1989, Nucl. Acids.
Res., 17, 1371-1371) into two portions.
Figure 3 is a dia~-d~ -atic representation of the gcllcral struct~lre of a llairpin
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 Icngth 2 or morc b~scs (~rclcrably 3 - 20 b~l~cs, i.~.,
m is from 1 - 20 or more). Helix 2 and helix 5 may be covalcntly linkcd by onc or morc
bases (i.e., r is ~ I 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 r~rotein
binding site. In each instance, each N and N' independcntly is any nomlal or modificd
base and each dash lel,lescl.ts 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 plefe,.cd. Helix 1 and 4 can be of any size (i.e. o and p is each
in.l~pçndPntly from 0 to any number, e.g., 20) as long as some base-pairing is maintained.
F.~slonti~l bases are shown as specific bases in the structure, but those in the art will
rCCO~li~c that one or more may be modified chemic~lly (abasic, base, sugar and/or
phosph~fr modifications) or replaced with another base without significant effect. Helix
4 can be formed from two separate molecules, i.e., without a connecting loop. Thc
c~-nec~ g Ioop when present may be a ribonucleotide with or without modifications to
its base, sugar or phosphate. "q" is 2 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 topyrimidine bases. " " refers to a covalent bond.
.
Figure 4 is a representation of the general structure of the hepatitis ~ virus
ribozyme domain known in the art.
Figure S is a represlont~tion of the general structure of the self-cleaving VS RNA
ribozyme domain.
Figure 6 is a schematic rcp.es~ lion of an RNaseH accessibility assay.
Specifically, the left side of Figure 6 is a diagram of complementary DNA
oligonucleotides bound to arcessible sites on the target RNA. Complementary DNA
oligonucleotides are represented by broad lines labeled A, B, and C. Target RNA is
.c~.ci,e..ted by the thin, twisted line. The right side of Fi~lre 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
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represent uncleaved target RNA; the bands unique tO eacll lalle represellt the clcaved
products.
Figure 7 is a graphical representation of RNaseH accessibility of GBSS RNA.
Figure 8 is a graphical representation of GBSS RNA Clc.lvagC ~y ri~)OZylllC~i al5 different temperatures.
Figure 9 is a graphical representation of GBSS RNA cleavage by multiple
ribozymes.
Figure 10 lists the nucleotide sequence of A-9 desaturasc cDN~ isolatcd from 7,ea
mays.
Figures 11 and 12 are dia~ Lic Ic~,resentatiolls of fatty acid biosynthesis in
plants. Figure I I has been adapted from Gibson et al., 1994, Plant Cell f~;nvir. 17, 627.
Figures 13 and 14 are graphical ~ cs~.~t~tions of RNaseH accessibility of A-9
desaturase RNA.
Figure 15 shows cleavage of A-9 desaturase RNA by ribozymes in vitro. 10/10
15 r~,~Jles~ thelengthofthebindingarmsofah~ I.ead(HH)ribozyme. 10/10means
hdix 1 and helix 3 each form 10 base-pairs with the target RNA (Fig. 1). 4/6 and 6/6,
eyles~;nL helix2~elixl interaction between a hairpin ribozyme and its target. 4/6 means
the hairpin (HP) ribozyme forrns four base-paired helix 2 and a six base-paired helix I
complex with the target (see Fig. 3). 6/6 means, the hairpin ribozyme forms a 6 base-
20 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.
Figure 16 shows the effect of arm-length variation on the activity of HH and HP
ribozymes in vitro. 7/7, 10/10 and 12/12 are e~senti~lly as described above for the HH
ribozyme. 6/6, 6/8, 6/12 lepl~s, .-t~ varying helix 1 length and a constant (6 bp) helix 2 for
25 a hairpin ribozyme. The cleavage reactions were carried out essentially as described
above.
Figures 17, 18, 19 and 23 are diagrammatic representations of non-limiting strategies
to construct a transcript comprising multiple ribozyme motifs that are the same or
different, targeting various sites within A-9 desaturase RNA.
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Figures 20 and 21 show in vitro cleavage of a-s desaturase RNA bv ribozymes thatare transcribed from DNA templates using bacteriophage T7 RNA polymerase enzyme.
Figure 22 dia~,d-,llllatic representation of a non-limiting strategy to construct a
transcript comprising multiple ribozyme motifs that arc the samc or diffcrcnt tar~ctillL~
various sites within GBSS RNA. L
Figure 24 shows cleavage of a-g desaturase RNA by rihozyl1lcs. 453 M~ cr,
lep,esel-ls a multimer ribozyme construct targeted against ~l~mm~-rhead ribozyme sites
453, 464, 475 and 484. 252 Multimer, represents a multimer ribozyme construct targeted
againsth~mmçrhead ribozyme sites 252, 271, 313 and 326. 23Pi M ultimcr,rcprc~cllts a
multimer ribozyme construct targeted against three hammerhead ribozyme sites 252, 2S9
and 271 and one hairpin ribozyme site 238 (HP). 259 Multimer, represents a multimer
ribozyme construct targeted against two h~mmerhead ribozyme sites 271 and 313 and one
hairpin ribozyme site 259 (HP).
Figuré 25 illustrates GBSS mRNA levels in Ribozyme minus Controls (C, F, I, J,
N, P, Q) and Active Ribozyme ~PA63 Transformants (AA, DD, EE, FF, GG, HH, JJ,
KK).
Figure 26 illustrates ag desaturase mRNA levels in Non-transformed plants (NT),
85-06 High Stearate Plants (1, 3, 5, 8, 12, 14), and Transformed (irrélevant ribozyme)
Control Plants (RPA63-33, RPA63-51, RPA63-65).
Figure 27 illustrates ag desaLul~se mRNA levels in Non-transformed plants
(NTO), 85- 15 High Stearate Plants (01, 06, 07, 10, 11, 12), and 85- 15 ~'or nal Stearate
Plants (02, 05, 09, 14).
Figure 28 illustrates /~9 desaturase mRNA levels in Non-transformed plants (NTY),
113-06 Inactive Ribozyme Plants (02, 04, 07, 10,11).
Figures 29a and 29b illustrate ag desaturase protein levels in maize leaves (R0). (a)
Line HiII, plants a-e nontransforrned and ribozyme inactive line RPA113-17, plants 1-6.
(b) Ribozyme active line RPA85- I S, plants I - 15.
Figure 30 illustrates stearic acid in leaves of RPA85-06 plants.
Figure 31 illustrates stearic acid in leaves of RPA85-15 plants, results of three
assays.
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Figure 32 illustrates stearic acid in leaves of RPA 113-06 plants.
Figure 33 illustrates stearic acid in leaves of RPA113-17 plants.
Figure 3~ illustrates stearic acid in leaves of control plants.
Figure 35 illustrates leaf stearate in Rl plants from a lligll stearate r~lant cross
(RPA85-15.07 self).
. Figure 36 illustrates ~9 desaturase levels in next generation maize leaves (Rl).
* indicates those plants that showed a high stearate content.
Figure 37 illustrates stearic acid in individual somatic embryos from a culture
(308/430-012) transformed with ~nti~ence ~9 desaturase.
Figure 38 illustrates stearic acid in individual somatic cmbryos from a culture
(308/430-015) transformed with anticen~e A9 desaturase.
Figure 39 i~ sLlàles stearic acid in individual leaves from plants ~ atcd from aculture (308/430-012) transformed with ~ e~ce A9 desaturase.
Figure 40 illu~tlates amylose content in a single kernel of untransrul,llcd control line
(Q806 and ~ntjsence line 308/425-12.2.1.
Figure 41 ill~ ates GBSS activity in single kernels of a southern negative line
(RPA63-0306) and Southern positive line RPA63-0218.
Figure 42 ill-lsl~alts a Lldllsfullildlion vector that can be used to express the
enzymatic nucleic acid of the present invention.
Detailed Descli~.tion Of The Invention
The present invention collc~,.lls compositions and methods for the modulation ofgene eA~ sjion in plants ~pe~ ifically using enzymatic nucleic acid molecules.
The followin~ phrases and terrns are defined below:
By "inhibit" or "modulate" is meant that the activity of enzymes such as GBSS and
25 ~-9 desaturase or level of mRNAs encoded by these genes is reduced below that observed
in the ~bs~n~ e of an enzymatic nucleic acid and preferably is below that level observed in
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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 bindin~ region to a specificd gCIlC targct, and also llas an
5 enzymatic activity which is active to specifically clcavc that targcl. Tllat is, ll~c
enzymatic nucleic acid molecule is ablc to intcrmolccul~rly clc~lv(: I~NA (or I~NA) all(l
thereby inactivate a target RNA molecllle This complcmcntarity functions to allow
sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA to allow
the cleavage to occur. One hundred percent complementarity is preferrcd, but
10 complementarity as low as 50-75% may also be useful in tllis inventioll. Thc nuclcic
acids may be modified at the base, sugar, and/or phosphate groups. The tenn 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
15 RNA, autolytic RNA, endoribonuclease, minizyme, leadzyme or DNA enzyme. All of
these terminologies describe nucleic acid moleculfs with enzymatic activity. Thc term
encomp~sses enzymatic RNA molecule which include one or more ribonucleotides andmay include a majority of other types of nucleotides or abasic moieties, as described
below.
By "compk ~~f ~~ iLy" 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
30 plant cell, but that is introduced by standard gene transfer techniques.
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By ''nucleic acid is meant a molec-lle whicll can bc single-strallcied or double-
stranded, composed of nucleotidcs col~ail~ g a s-lL~ar, a phos~ ate and either a purine or
pyrhllidine base wllicll may be sallle or different, and may be modified or unmodified.
By "genome ' is meant genetic material contailled in each cell of an organism and/or a
5 virus.
By "mRNA" is meant RNA that can be translated hlto protein by a cell.
By "cDNA is meant DN~ that is complementaly to and dcrived 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
e~ylession 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
15 the coding seq~lence. ~ntis~n~e RNA is normally a mirror image of the sense RNA.
By "expression", as used herein, is meant the transcription and stable accum~ tion
of the enymatic 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
s~kst~ntial homology to an gene, and in a plant cell causes the reduction in activity of the
foreign and/or the endogellous 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 omlon-trallsgellic organism.
By "promoter' is meallt nucleotide sequence element within a gene which controlsthe expression of that gene. r'romoter sequence provides the recognition for RNApolymerase 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 orighl, such as the octopine synthetase promoter, the
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nopaline synthase promoter, the manopine synthetase promoter; promoters of viralorigin, such as the cauliflower mosaic vims (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
5 promoters. Promoter may also contain certain enhancer sequence elemcnts tllat may
improve the transcription efficiency.
By "enhancer is meant nucleotide sequence elcmcnt whicll can stimulatc l~romolc
activity (Adh).
By "constitutive promoter is meant promoter element that directs continuous gene10 ~ e;,~ion in all cells types and at all times (actin, ubiquitin, CaMV 35S).
By "tissue-specific promoter is meant promoter element responsible for gene
e,~c~lcs:,ion 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
15 gene e~ ress~on at specific plant developmental stage, such as in early or late
embryogenesls.
By "inducible promoter" is meant promoter element which is responsiblc for
e ,~ cs~-on 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 ol~allisln, either eukaryotic and
prokaryotic.
By "angiosperm" is meant a plant having its seed enclosed in an ovary (e.g., coffee,
tob~cco, 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
Ieaf of the embryo). For example, coffee, canola, peas and others.
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By "transgenic plant" is meant a plant expressing a foreign gene.
By "open reading frame is meant a nucleotide sequence, without introns, encodingan amino acid sequence, with a defined translation initiation and termination region.
The invention provides a method for producing a class of cnzylnatic clcaving a~c5 which exhibit a high degree of specificity for thc RNA of a dcsilc(l ~arucL. l llc cllzylllalic
nuc!eic acid molecule may be targeted to a highly specific se~uence region of a targct SLICIl
that specific gene inhibition can be achieved. Alternatively, enzymatic nucleic acid can be
La~ ,ted to a highly conserved region of a gene family to inhibit gene expression of a
family of related enzymes. The ribozymes can be expresscd in plants that havc bccn
10 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
15 ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not
only on the base-pairing ~I~f''f~ lll of binding to the target RNA, but also on the
n;~... of target RNA cleavage. Single mi~m~tches, or base-substitutions, near the
slte of cleavage can completely eli...;.-~t~ catalytic activity of a ribozyme.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently.
20 Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can
cleave other RNA molecules) under physiological conditions. Table I ~ ;S some
of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first
binding to a target RNA. Such binding occurs through the target binding portion of an
enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the
25 molerllle 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 enzym~tic~lly 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 targct, it is rclcascd from
30 that RNA to search for another target and can repeatedlybind and cleave new targets.
In one of the p,~if~ d embodiments of the inventions herein, the enzymatic nucleic
acid molecule is formed in a ~ e.llead or hairpin motif, but may also be formed in the
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motif of a hepatitis ~ virus, group I intron, group 11 intron or RNaseP RNA (in
association with an RNA guide sequence) or Ne~'rospora VS RNA. Examples of such
hammerhead motifs are described by Dreyfus, supra, Rossi e~ al., l 992, AIDS Research
and Human Retroviruses 8, 183; of hairpin motifs by Hampel e~ al., EP0360257, Hampel
and Tritz, 1989 Biochemistrv 28, 4929, Feldstein e~ al., 19~9, ~ nc~ ~2, 53, 1~ iclofI.IIld
Gerlach, 1989, Gene, ~2, 43, and Hampcl el al., 199n ~(IC~ .s. IX, -)')(); of 1l"~
hepatitis ~ virus motif is described by Perrotta and Bccn, l~)92 l~ioc*emi.slry 31, i6; of
the RNaseP motif by Guerrier-Takada et al., 1983 Cell 3~, 849; Forster and Altman,
1990, Science 249, 783; Li and Altman, 1996, ~ucleic Acids Res. 24, ~35; Neuro.~Jru VS
Rl~A ribozyme motif is described by Collins (Saville and Collins, 1990 Ccll ~ X5-h9~;
Saville and Collins, 1991 Proc. Na~l. Acad. Sci. USA 88, 8826-8830; Collins and Olive,
1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, ~;MBO. J. 14, 363); Group ~I
introns are described by Griffin et al. , 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995,
Biochemis-ry 34, 2965; and of the Group I intron by Cech et al., U.S. Patent 4,987,071.
These specific motifs are not limiting in the invention and those skilled in the art will
recognize that all that is h,l~oll~nt 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 exple;,sedwithin 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
~NA and DNA, eds. R. P. ~l;ck~ol1 and J. G. Izant, pp 209-221, Raven Press, NY.;Atkins et al., Tntf-m~tional PCT Publication No. WO 94/00012; Lenee et al., Intern~tion~l
PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins et al., 1995, J. Gen. Yirol.
76, 1781-1790; McElroy and Brettell, 1994, TIBTECH 12, 62; Gruber et al., 1994, J. Cell.
Biochem. Suppl. 18A, 110 (X1406)and Feyter et al., 1996, Mol. Gen. Ger~et. 250, 329-
338; all of these are incorporated by reference herein]. Those skilled in the art will realize
from the te~rhin~C herein that any ribozyme can be eAI,iessed in eukaryotic plant cells
from an a~lol,-iate promoter. The ribozymes ~ ssion is under the control of a
consliluli~e promoter, a tissue-specific promoter or an inducible promoter.
To obtain the ribozyme me~i~ted moA~ tion, the ribozyme RNA is introduced into
the plant. Although examples are provided below for the construction of the plasmids
used in the Ll~ rc,llllation eA~e,ilnents illustrated herein, it is well within the skill of an
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artisan to desi~n numerous different types of plasmids which can be used in the
lldllarollllation of plants,_see Bevan, 1984, Nucl. Acids ~2es. 12, 8711-8721, which is
incorporated by reference. There are also numerous ways to transform plants. ln the
examples below embryogenic maize cultures were helium blasted. In addition to ~lsin6 tllc
gene gun (US Patents 4,945,0S0 to Cornell and 5,141,13 1 to Dowl~lallco), plallLS m.ly bc
transformed usingAgrohacterium technology, scc US r'atcllt 5,177,()10 lo UlliVclsily ol
Toledo, 5,104,310 to Texas A&M, European Patent Application 0131-S24~1, LLlror)can
Patent Applications 120516, 159418Bl and 176,1 12 to Schilperoot, US Patents
5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, Europcan
Patent Applications 11 671 8, 290799, 320500 all to MaxPlanck, Europcan l'atcnt
Applications 604662 and 627752 to Japan Tobacco, European Patent Applications
0267159, and 0292435 and US Patent 5,231,019 all to Ciba Geigy, US Patents 5,463,174
and 4,762,785 both to C~le~ne~ and US Patents 5,004,863 and 5,159,135 both to
Agracetus; whiskers technology, see US Patents 5,302,523 and 5,464,765 both to 7.onPc~
electroporation technology, see WO 87/06614 to Boyce Thompson Institute, 5,472,869
and 5,384,253 both to Dekalb, W09209696 and W09321335 both to PGS; all of which
are incQrporated by reference herein in totality. In addition to numerous technologies for
transforming plants, the type of tissue which is cont~rte~ with the foreign material
(typically plasmids c~ ;..;..g RNA or DNA) may vary as well. Such tissue would
20 include but would not be limited to embryogenic tissue, callus tissue type I and II, and
any tissue which is receptive to transÇo, ll,alion and subsequent ~ ~gc~ dlion into a
transgenic plant. Another variable is the choice of a select~ble marker. The y~;r~ nce for
a particular marker is at the discretion of the artisan, but any of the following select~ble
",~ c,s may be used along with any other gene not listed herein which could function as a
25 select~kle marker. Such selectable ll~ include but are not limited to chlorosulfuron,
hygromyacin, PAT and/or bar, bromoxynil, kanamycin and the like. The bar gene may be
isolated from Strptomuces, particularly from the hygroscopicus or viridochromogenes
species. The bar gene codes for phosphinothricin acetyl transferase (PAT) that
inactivates the active ingradient in the herbicide bialaphos phosphinothricin (PPT). Thus,
30 llullle~olls 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 ~yl essed per transcript. Alternatively, two or more
ribozymes targeted against more than one target site are expressed as part of a single RNA
35 transcript. A single RNA tlanscl;yt comprising more than one ribozyme targeted against
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~nore than one cleavage site are readily generated to achieve efficient modulation of gene
e~y~ession~ Ribozymes within these multimer constructs are the same or different. For
example, the mIIItimer construct may comprise a plurality of hammerhead ribozymes or
hairpin ribozymes or other ribozyme motifs. Alternatively, the multimer construct may
5 be designed to include a plurality of different ribozyme motifs, sucll as llamIllcrllca(l an~l
hairpin ribozymes. More specifically, multimer rihozyllle constr~lc~s ~rc dcsigl7ccl,
wherein a series of ribozyme motifs are linked togctl1cr in tal1dclll in a SillL!IC I~N/\
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. Multimcr
10 ribozyme constructs (polyribozymes) are likely to improvc the cffcctivcncss of
ribozyme-mediated modulation of gene ek,ulession.
The activity of ribozymes can also be augmented by their release from the primary
kans.,lilJt 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.,etal., 1992, Nucleic~cidsSymp.Ser., 27, 15-6;Taira,K., etal., 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-rnedi~ted modulation of gene e~l"es~ion can be practiced in a wide
variety of plants inr!~I-ling angiosperms, gymnosperrns, monocotyledons, and
20 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; fmits, such as pincap~le, papaya, mango, banana, grapes, oranges, apples;
vegetables, such as cauliflower, c~bb~ge, melon, green pepper, tornatoes, carrots, lettuce,
25 celery, potatoes, broccoli; le~ c, such as soybean, beans, peas; flowers, such as
c~m~tions, chrys~nth~m~Im, daisy, tulip, gypsophila, al~l~o~ ia, marigold, petunia, rose;
trees such as olive, cork, poplar, pine; nuts, such as walnut, pistachio, and others.
Following are a few non-1imitin~ examples that describe the general utility of ribozymes
inIno~ tionofgenee~l"e~ion.
Ribozyme-m~ t~d down regulation of the ex~leç~ion of genes involved in caffeine
synthesis can be used to signific~ntly change caffeine concentration in coffee beans.
Expression of genes, such as 7-methyl~nthosine and/or 3-methyl transferase in coffee
plants can be readily modulated using ribozymes to decrease caffeine synthesis (Adams
and Zarowitz, US Patent No. 5,334,529; incorporated by reference herein).
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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), ~ould produce leaves with altered nicotine
concentration.
Transgenic plants expressing ribozymes targeted against L~enes involvccl h1 ripcning
of fruits, such as ethylene-forming enzyme, pectin mcthyltransfcrasc, pCC~ill cstcrasc,
polyg~l~ct~ronase, l-aminocyclopropane carboxylic acid (/~CC) syntl1asc, ~CC oxiclasc
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 ~. 3, 469; Shewmaker
et al., supra; James et al., 1996, Bio/Technology, 14, 56), would dclay thc ripcnil1~ Or
fruits, such as tomato and apple.
Transgenic plants eAIJ~c~sillg ribozymes targeted against genes involved in flower
pi~ ;on, such as chalcone synthase (CHS), chalcone flavanone isomerase (CHI),
phenylalanine ~mmclni~ Iyase, 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; Jolgc.~sell, 1996, Science, 268, 686), would produce flowers,
such as roses, petunia, with altered colors.
Lignins are organic compounds Fc~f ll;~l for l~ lg l,.cc~ strength of cell
walls in plants. Although e ss~ l lignins have some disadvantages. They cause
indigestibility of sillage crops and are undesirable to paper production from wood pulp
and others. T.al~sgc.lic plants eAI,lcssil,g ribozymes targeted against genes involved in
lignin production such as, O-methyltransferase, cinnamoyl-CoA:NADPH reductase orcil~lallloyl alcohol dehydrogenase (Doorssel~ere 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.,
~nt~ tional 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.
30 Modulation of ~ranule bound starch svnthase ~ene expression in plants:
In plants, starch biosynthesis occurs in both chloroplasts (short term starch
storage) and in the amyloplast (long tenn starch storage). Starch granules normally
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consist of a linear chain of a(l-4)-linked a-D-glucose UllitS (amylose) and a brallchcd
form of amylose cross-linked by a(1-6) bonds (amylopectin). An enzyme involved in
the synthesis of starch in plants is starch synthase which produces linear chains of a (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 locatcd in tllc slrol1la of
chloroplasts and in amyloplasts (solublc starch syntllasc). I~otll lorms ol' ll)is e~ yl~c
utilize ADP-D-glucose while the granular bound form also ~lliliizcs lJl~ lcosc, wilh
a ~rcif~,.cnce 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 becn charactcrizcd.
Mutations affecting the GBSS gene in several plant spccics has rcsultcd in tllc loss Or
amylose, while the total amount of starch has remained relatively unchan~cd. 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, 22~-233 (1983), Echt and Scllwa~ Genetics 99: 275-
284 (1981) ). The presence of a br~cl,i,lg enzyme as well as a soluble ADP-glucose
starch glycosyl transferase in both GBSS mlltAntc and wild type plants indicates the
e~ tonA~e of independent pathways for the fommation of the branched chain polymer
amylopectin and the straight chain polymer amylose.
The Wx (waxy) locus encodes a granule bound glucosyl t,dn~rti,ase 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 temmed waxy due to the
appeA~ re of mutant k~rn~lc, which is the phenotype res~lting from an reduction in
amylose composition in the kern~lC. In maize, this enzyme is transported into the
amyloplast of the developing endosperm where it catalyses production of amylose. Com
kemels are about 70% starch, of which 27% is linear amylose and 73% is amylopectin.
Waxy is a recessive m-ltAtion in the gene enco~ling granule bound starch synthase (GBSS).
Plants homozygous for this recessive mutation produce kemels 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
Anti~en~e 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
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be designed that cleave target mRNAs required for GBSS activity in plant species othcr
than maize.
Thus, in a prefel.ed embodiment, the invention features ribozvmes that inhibit
enzymes involved in amylose production, e.g., by rcd~lcil1L~ G~3SS activity. Tl1c~;c
5 endogenously expressed RNA molecules contain substratc bin(lillg (lOm ~ s l~ hin(i lo
accessible regions of the target mRNA. Thc RNA molcc-llc~ ~lso COlltaill (It)lll~lillS t~
catalyze the cleavage of RNA. The RNA molecules are prefcrably ribozymcs of thc
han~ .llead or hairpin motif. Upon binding, the ribozyllles cleave the target mRN~s,
preventing translation and protein accumulation. In the absence of the expression of thc
10 target gene, amylose production is rcduccd or inhibitcd. Spccillc cxam~lc~; ~rc l~rovidcd
below infra.
Plcre.led embodiments include the ribozymes having binding arrns which are
compl~ .y 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
15 e~al--ples are rlesignçd to one plant's (e.g., maize) mRNA, similar ribozymes can be made
complem~nt~ry to other plant species' mRNA. By complc...l - .t~ ~ 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. E~ les of such ribozymes consist
ly of sequences shown in Tables IIIB - V.
P,c~.lcd embo~;.. L.I~ are the ribozymes and methods for their use in the
inhibition of starch granule bound ADP (UDP)-glucose: a-1,4-D-glucan 4-a-glucosyl
l,alls~.dse i.e., granule bound starch synthase (GBSS) activity in plants. This is
accomplished through the inhibition of genetic ek~l-,s~ion, with ribozymes, which results
in the reduction or elimin~tion of GBSS activity in plants.
In another aspect of the invention, ribozymes that cleave target molecules and
inhibit arnylose production are expressed from transcription units hls~ cd into the plant
genl7m~ Preferably, the lecullll,hldllL vectors capable of stable integration into the plant
genome and selection of transforrned plant lines e~lessi,lg the ribozymes are e~lcssed
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 riboymes ex~,lessed in plant cells are under the control of a constitutive promoter, a
tissue-specific promoter or an inducible promoter.
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Modification of corn starch is an important application of ribozyme tecllnology
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 enerL~y availability in fccd.
5 Nearly 10% of wet-milled corn has the waxy phenotypc, but bccausc of its rcccssivc
nature the traditional waxy varieties are very diffic~llt for thc ~rowcr t~ h,~
Ribozymes targeted to cleave the GBSS mRNA and tl1~ls rcclLlcc Cil~SS aclivily il1 p l.ll~ls,
and in particular, corn cndosperm will act as a dominal1L trait and produce corn plants
with the waxy phenotype that will be easier for the grower to handle.
10 Modification of fattv acid saturation profilc in plallts
Fatty acid biosynthesis in plant tissues is initiated in the chloroplast. Fatty acids
are synthesized as thioesters of acyl carrier protein (ACP) by the fatty acid synthase
complex (FAS). Fatty acids with chain lengths of 16 carbons follow one of three paths:
1) they are released, immediately after synthesis, and transferred to glycerol 3-phosphate
15 (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
thioe;".,.ases; or3) they are further elongated to C18 chain lengths. The C18 chains are
rapidly desaturated at the C9 position by stearoyl-ACP desaturase. This is fotlowed by
;.. ~eJ; ~le transfer of the oleic acid (18:1) group to G3P within thc 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
reticulllrn Most oleic acids (18:1) and some palmitic acids (16:0) are transferred to G3P
from phoSph~ti~ic acids, which are then converted to diacyl glycerides and phosphatidyl
choline. Further desatu~alion of the acyl chains on phosphatidyl choline by ~ b~bound desaLu~ases takes place in the endoplasmic retic~ m 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.l95-230, (ed. Moore,T.S.) CRC Press, Boca Raton, FA.).
A sch~om~tic of the plant fatty acid biosynthesis pathway is shown in Figures l l and 12.
The three prer~omin~nt fatty acids in com seed oil are linoleic acid (18:2, ~59%), oleic acid
(18:1, ~26%), and palmitic acid (16:0, ~11%). These are avesge values and may besomewhat dir~l~nt depending on the genotype; however, composite samples of US Corn
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Belt produced oil analyzed over the past ten years have consistently had this
composition (Glover and Mertz, 1987 in: Nutritional Quality of Cereal Grains: genetic
and agronomic improvement., p 183-336, (eds. Olson, R.A. and Frey, K.J.) Am. Soc.
Agronomy. Inc. Madison, WI; Fitch-H~ n, 1985 J Am. Oil Chem. Soc. 62: 1524-
1531; Strecker et al., 1990 in Edible fats and oils processin~: basic llrinciplc~ all(l modcrl1
practices (ed. Erickson, D.R.) ~m. Oil Chcmi~t~ Snc. C'll~lltllraiL~ll, Il,). l'lli~i
predominance of C18 chain lenL~ths lnay rcflcct ~hc abuncLll1~:c ancl ~l~;LiviLy ol ~ v~r.ll kcy
enzymes, such as the fatty acid synthase responsible for production of C18 carbon
chains, the stearoyl-ACP desaturase (~-9 desaturase) for production of 18: 1 and a
microsomal ~-12 desaturase forconversion of 18:1 to 18:2.
A_9 desaturase (also called stearoyl-ACP desaturase) of plants is a soluble
chloroplast enzyme which uses C18 and occasionally C16-acyl chains linked to acyl
ca~ier protein (ACP) as a substrate (McKeon, T.A. and Stumpf, P.K., 1982 J. Biol.
Chem. 257: 12141-12147). This contrasts to the "-~ "~ r" lower eukaryotic and
15 cyannb~cterial ~-9 desalu-ases. Rat and yeast ~-9 desaturases are mell-~lane bound
..l;c-osolnal enzymes using acyl-CoA chains as subsLlal.,s, whereas cyanobacterial ~-9
dejaLulase uses acyl chains on diacyl glycerol as substrate. To date several ~-9desalu-~se cDNA clones from dicotelydenous plants have been isolated and chara.;le.i~.ed
(Sh~nklin 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; Sh~n~lin et
al., 1991 PlantPhysiol. 97: 467-468; Sloconll~e et al., 1992 Plant. Mol. Biol. 20: 151-155;
Tayloretal., 1992PlantPhysiol. 100: 533-534; Thompson et al., 1991 Proc. Natl. Acad.
Sci. USA 88: 2578-2582). Comparison of the dirr~ ,.,t plant a-s desaturase sequ~orces
suE~estc that this is a highly conserved enzyme, with high levels of identity both at the
2~ amino acid level (~90%) and at the nucleotide level (--80%). However, as might be
e,.~e~,t~,d from its very dir~.l ~1t physical and enzymological l)lo~,lLies, no s~.lu~ ce
similarity exists ~ w.,~,.l plant and other ~-9 desaturases (~h~nklin and Somerville,
supra).
Purification and characte.i~lion of the castor bean desaturase (and others) indicates
that the ~-9 de-sa~u,ase is active as a homodimer; the subunit molecular weight is ~ 41
kDa. The enzyme le~ui.~s molecular oxygen, NADPH, NADPH ferredoxin
oxidored~ t~ce and ferredoxin for activity in vitro. Fox et al., 1993 (Proc. Matl. Acad. Sci.
USA 90: 2486-2490) showed that upon ~A~ ssion in E. coli the castor bean enzyme
cont~inC four catalytically active ferrous atoms per homodimer. The oxidized enzyme
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contains two identical diferric clusters, which in the presence of dithionite are reduced to
the diferrous state. In the presence of stearoyl-CoA and ~2 the clusters return to the
diferric state. This suggests that the desaturase belongs to a group of ~2 activating
proteins cont~inin~ diiron-oxo clusters. Other members of this group are ribonucleotide
5 reductase and methane monooxygenase hydroxylase. Comparison of thc r~rcdictc(lprimary structure for these catalytically diverse r~rotcins Sl10W.S thal all COllt~till .1
conserved pair of amino acid sequences (Asp/Glu)-Glu-Xaa-Arg-~-lis scparatcd by ~~0-
100 amino acids.
Traditional plant breeding programs have shown that increased stearate levels can
10 be achieved without deleterious consequences to the plant. In saMowcr (Lacld and
Knowles, 1970 Crop Sci. 10: ~25-~27) 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 cGl,.posilion of seed oil.
Increases in A-9 desaturase activity have been achieved by the transformation oftobacco with the A-9 desaturase genes from yeast (Polashock et al., 1992 Plan~ Physiol.
100, 894) or rat (Grayburn et. al., 1992 BioTechnology 10, 67~). Both sets of l~alls~enic
plants had si~r~ ch~nges in fatty acid composition, yet were phenotypically
Id~ntic~l to control plants.
Corn (maize) has been used minim~lly for the production of 1~ g~illC products
~ecau~e it has tr~ition~lly not been utilized as arl oil crop and because of the relatively
low seed oil content when COIllpds ed with soybean snd canola. However, corn oil has low
levels of linolenic acid (18:3) and relatively high levels of palmitic (16:0) acid (desirable in
C). Applicant believes that reduction in oleic and linoleic acid levels by down-re~ tion of ~-9 desaturase activity will make corn a viable alte~n~tive to soybean and
canola in the ~ oil marlcet.
Margarine and confectionary fats are produced by rh~mic~l hydrogenation of oil
from plants such as soybean. This process adds cost to the production of the ",a~""e
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 elimin~t~ the need for cl~ ..ic~l hydrogenation is to
g~nf-tic~lly ~ ;,.P~l the plants so that desaturation enzymes are down-regulated. ~-9
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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 concellls compositions (and
methods for their use) for the modification of fatty acid colllposition in E~lants. Tllic is
5 accomplished through the inhibition of genetic expression, Witll riboi!ymCS, al7~i!;cllsc
nucleic acid, cosuppression or triplcx DN~, Which rcsults ill Ihc lC(I~lC~iOl) or ~ illnlioll
of certain enzyme activities in plants, such as ~-9 desaturasc. Such activity is rc~uccd in
monocotyledon plants, such as maize, wheat, rice, palm, coconut and others. ~-9
desanlrase activity may also be reduced in dicotyledon plants such as sunflowcr,10 safflower, cotton, peanut, olive, sesame, cuphea, flax, jojoba, grapc and othcrs.
Thus, in one aspect, the invention features ribozymes that inhibit enzymes
involved in fatty acid unsaturation, e.g, by reducing A-9 desaturase activity. These
endogenously e~l.fessed RNA molecules contain substrate binding domains that bind to
aGcçssible regions of the target mRNA. The RNA molecules also contain ~lnrn~inc that
15 catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the
l-~.. head or hairpin motif. Upon binding, the ribozymes cleave the target mRNAs,
preventing tr~nCl~tion and protein accumulation. In the ~bs~once of the e~c~"~ ision 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 plefell~;d embo(~ r~l~, the ribozymes have binding arrns which are
comple~n~nt~ry to the sequences in the Tables VI and VIII. Those in the art willrecoE~i7e that while such examples are designe~ to one plant's (e.g, corn) mRNA, similar
ribozymes can be made compl~ y 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 ~ ne~ 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.cont~inc 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 h,.~.r~.~ 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.
,~
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Those in the art will recognize that ribozyme sequences listed in the Tables arelc:pl~selltative only of many more such sequences where the enzymatic portion of the
ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop
II sequence of hammerhead ribozymes listed in Table lV (5'-GGCG~AAGCC-3') can bcaltered (substitution, deletion, and/or inscrtioll) to conLIill ally ~cq~ ecs, pl-c~ ly
provided that a minimum of a two base-paircd stcm stmct~lrc c.ln form .Silllil.lrly, ~ilclll-
loop IV sequence of hairpin ribozymes listed in Tables V and Vlll (S'-C~CGUUC'JU~i-3'
can be altered (substitution, deletion, andtor insertion) to contain any scquence,
preferably provided that a minimum of a two base-paired stem structure can foml. Such
10 ribozymes are equivalent to the ribozymes described spccif~cally in thc Tahlc~
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
hlL~ dLion into the plant genome and selection of transformed plant lines e~yles~ g the
15 ribozymes are expressed either by co~lsliLL~ e or inducible promoters in the plant cells.
Once expressed, the ribozymes cleave their target mRNAs and reduce unsat~lralcd 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 nuclcic acid-based
20 technologies which are capable of reducing specific gene expression. A high level of
s;~ dted fatty acid is desirable in plants that produce oils of commercial importance.
In a related aspect, this invention fealules the isolation of the cDNA sequ~nre
~nrQding A-9 desaturase in maize.
In plerc.lGd embo-3;.. ~-- ~L~, hairpin and h~.. "llead ribozymes that cleave ~-9
25 de~alulase mRNA are also described. Those of ordillaly skill in the art will understand
from the eY~mples described below that other ribozymes that cleave target mRNAs
required for A-9 desaturase activity may now be readily designed and are within the scope
of the invention.
While specific examples to corn RNA are provided, those in the art will recognize
30 that the te?~cllingc 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
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and teachin~ herein are meant tO be non-limiting, and tilose skilled in tlle art will
. recognize that slmilar 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 followcd in tllc cxamr)lc.~ crcin.
Additional hlfol---ation may be found in Sambrook, J., I:ritscl1, 1~ ., al1d M,ll~
(1989), Molecular Cloning a Laboratory Manual, second cdition, Cold Sl~rin~ llarbor:
Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.
Examples
Example 1: Isolation of ~ 9 desaturase cDNA from Zea mavs
Degenerate PCR primers were designe~ and synthesi7ed to two conserved peptides
involved in diiron-oxo group binding of plant /~-9 desaturases. A 276 bp DNA fragment
was PCR amplified from maize embryo cDNA and was cloned in to a vector. The
predicted amino acid sequence of this fragment was similar to the sequence of the region
scpd~ate~ by the two conserved peptides of dicot ~-9 desaturase proteins. This was used
to screen a maize embryo cDNA library. A total of 16 clones were isolated; further
restriction mapping and hybridization identified one clone which was sequenced.
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 ~-9 desaturase gene for
the predicted mature protein. The complete sequence is plesen~d in Figure 10.
ExamPle 2~ entifiç~tion of Potential Ribo_vme Cleava~e Sites for ç~9 desaturase
Approxim~t~ly two hundred and fifty HH ribo_yme sites and approximately forty
three HP sites were identified in the corn ~-9 desaturase mRNA. A HH site consists of a
2S uridine and any nucleotide except guanosine (UH). Tables VI and VIII have a list of HH
and HP ribo yme cleavage sites. The numbering system starts with I at the ~' end of a ~-
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
syr~theci7~d to such cleavage sites with between 5 and 100 or more bases as substrate
binding arrns (see Figs. 1 - 5). These substrate binding arms within a ribozyme allow the
ribozyme to interact with their target in a sequence-specific manner.
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Example 3: Selection of RibozYme Cleava~e Sites for ~9 desaturase
The secondary structure of ~-9 desaturase mRNA was assessed by computer
analysis using algorithms, such as those developed by M. Zuker ( Zuker, M., 1989Science, 244, 48-52). Regions of the mRNA that did not fonn sccondary folding
5 structures with RNA/RNA stems of over ei~ht nucIcotidcs and cont<~ c(l l)otcnli.
h~mmerhead ribozyme clcavage SitcS wcrc idcnLiI;c(l.
These sites were ~sessed for oligonucleotide accessibility by RNase H assays (see
Example 4 infra).
Example 4: RNaseH Assavs for ~9 desaturase
Forty nine DNA oligonucleotides, each twenty one nucleotides long were used in
RNase H assays. These oligonucleotides covered 108 sites within A-g desaturase RNA.
RNase H assays (Fig. 6) were performed using a full length transcript of the J~-9
des~LulasecDNA. RNA was screened for acce~ihle cleavage sites by the method
described generally in Draper et al., supra. Briefly, DNA oligonucleotides reprcsentirlg
15 ribozyme cleavage sites were syrlthesi7e~ A polymerase chain reaction was used to
~er,e.~le a substrate for T7 RNA polymerase hanscl.l,tion from corn cDNA clones.T ~heled RNA transcripts were synshçsi~e~l in vitro from these templates. The
oligonucleotides and the labeled transcripts were ~nn~le~l RNAseH was added and the
IlliA~ were ;~ b~ted for 10 I-lillut~_s at 37~C. Reactions were stopped and RNA
20 seL~alated on seqU~rrin~ polyacrylamide gels. The pe.~ ge of the substrate cleaved
was deterInined by autoradiographic quantitation using a Molecular Dynamics phosphor
im~inE~ system (Figs. 13 and 14).
Exam~le 5: ~i.. ~. l.ead and Hairpin Ribozymes for ~9 desaturase
Ha.~ .llead (HH) and hairpin (HP) ribozymes were ~le~ d to the sites covered
by the oligos which cleaved best in the RNase H assays. These ribozymes were then
subjected to analysis by conlpuLtl folding and the ribozymes that had significant
second~y 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 ,4cids Res. 23, 2677). Small scale syntheses were con~ cted on a 394
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Applied Biosystems, Inc. synthesizer using a modified 2.5 ~mol 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 ~IL of 0.1 M = 16.3 ~mol) of
phosphoramidite and a 24-fold excess of S-ethyl tetra7.01c (23~ ~L of 0.25 M = 59.5
mol) relative to polymer-bound 5'-hydroxyl was uscd in cach couplillL~ cyclc. Avcr.lgc
coupling yields on the 394, determined by colorimetric qualltitatio1l of thc trityl liaclions,
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 imid~7Ole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF
(ABI); oxidation solution was 16.9 mM I2, 49 mM pyridinc, 9n/o watcr in Tl l l:
(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 Am~,.ical. International Chemical, Inc.
D~ c,tcction of the RNA was pc.ru~ ed as follows. The polymer-bound
oligoribonucleotide, trityl-off, was transferred from the synthesis column to a 4 mL L~lass
screw top vial and suspended in a solution of methylamine (MA) at 65~C for 10 min.
After cooling to -20~C, the sUpe~t~nt was removed from the polymer support. The
support was washed three times with l.0 mL of EtOH:MeCN:H20/3:l:1, vortexed and
the supernatant was then added to the first supernatant. The combined supernatants,
ct nt~inin~ the oligoribonucleotide, were dried to a white powder.
The base-deprotected oligoribon-lcleotide was res--spend~d in anhydrous
TEA-HF/NMP solution (250 ~LL of a solution of 1.5 rnL IV-methylpyrrolidinone, 750 ~lL
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 reSlllting~ fully deprotected, oligom~r was quenched with 50 mM TEAB (9
mL) prior to anion ~-Yeh~nge des~ltine
For arlion e~ .ge des~lting of the deprotected oligomer, the TEAB solution was
loaded onto a Qiagen 500~) anion ~Yrh~n~ cartridge (Qiagen Inc.) that was prewashed
with 50 mM TEAB (10 mL). After washing the loaded cartridge with 50 mM TEAB (10
mL), the R~A was eluted with 2 M TEAB (10 mL) and dried down to a white powder.
Inactive h~mm-orhead ribozymes were synthesized by substituting a U for Gs and aU for A 14 (numbering from (Hertel, K. J., et al., 1992, Nucleic Acids Res., 20, 3252).
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The hairpin ribozymes were synthesized as described above for the hammerhead
~As.
Ribozymes were also synthesized from DNA templates using bacteriophaL~e T7
R~A polymerase (Milligan and Uhlenbeck, 19~9, ~v~ethods Enzyn~o~ n 51).
Ribozymes were purified by gel electrophoresis using gcncral mclhods or wcrc pllriilc(l
by high pressure liquid chromatography (IIPLC; Scc Wincolt e~ fJl., 1')()~), .sU/Jrcl, lllc
totality of which is hereby incorporated herein by reference) and were resuspended in
water. The sequences of the chemically synthesized ribozyllles uscd in this stlldy arc
shown below in Tables Vll and VIII.
ExamDle 6: Lon~ substrate tests for ~9 desaturase ribozvmes
Target RNA used in this study was 1621 nt long and con-~intod cleavage sites for all
the HH and HP ribozymes targeted against ~-9 desaturase RNA. A template containing
T7 RNA polymerase promoter IlpsLr~anl of ~-9 desaturase target sequencel was PCR~mrlifif~d from a cDNA clone. Target RNA was transcribed from this PCR amplifiedtemplate using T7 RNA polymerase. The llanscli~t was internally labeled during
l~allscli~,tion by inr,l~ in8 [o~ 32p] CTP as one of the four ribonucleotide triphosphates.
The ll~lls~ yliOnl~ LLul~ was treated with DNase-I, following kal,s~iylion at 37~C for 2
hours, to digest away the DNA template used in the transcription. The transcription
mixture was resolved on a d~ a~ g polyacrylamide gel. Bands corresponding tO full-
length RNA was isolated from a gel slice and the RNA was ~lGci~ al. d with isopropanol
and the pellet was stored at 4~C.
Ribozyme cleavage reactions were carried out under ribozyme excess (kCat/KM)
conditions (Herschlag and Cech, 1990, Biochemistry 29, 10159-10171). Briefly, I mM
ribozyme and c 10 nM intern~lly labeled target RNA were del~alul~d separately byheating to 65~C for 2 min in the ~l~sencc of 50 mM Tris.HCI, pH 7.5 and 10 mM
MgC12. The RNAs were renatured by cooling to the reaction t~ ,.dLul~ (37~C, 26~C or
20~C) for 10-20 min. Cleavage reaction was initi~ted by mixing the ribozyme and target
RNA at applo~-iate reaction tt~ e~alLIlcs. Aliquots were taken at regular intervals of
time and the reaction was quenrhecl 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 inTable IX and Figures 1~ and 16. Of the ribozymes tested, seven h~mmerheads and two
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hairpins showed significant cleavage of ~-9 desaturase RNA (Figures 15 and 16).
Ribozymes to other sites showed varied levels of activity.
Example 7: Cleava~e of the tar~et RNA usin~ multiple ribozvme combinations for ~9
desaturase
Several of the above ribozymes were incorporatcd illtO a multimcl ribo;zylllc
construct which contains two or more ribozymes embeddcd in a contiguous strctcll Or
compl~ y RNA sequence. Non-limiting examples of multimer ribozymes are shown
in Figures 17, 18, 19 and 23. The ribozymes were made by annealling complementary
oligonucleotides and cloning into an ~ .res~ion vector containing thc Cauliflowcr Mosaic
Virus 35S ~r h~n~ed promoter (Franck et al., 1985 Cell 21, 285), the maize Adh I intron
(Dennis et al., 1984 Nucl. ~cids Res. 12, 3983) and the Nos polyadenylation signal
(DePicker et al., 1982 J. Molec. ~ppl. Genet. 1, 561). Cleavage assays with T7 transcripts
made from these multimer-cont~ining transcription units are shown in Figures 20 and 21.
These are non-limiting examples; those skilled in the art will r~,co~ that similar
embodi~ll.,.ll~, co~ .g of other ribozyme combinations, introns and promoter elements,
can be readily gellc,~ted using techniques known in the art and are within the scope of this
nvention.
Exam~le 8: Construction of Ribozvme e,~ sin~ Lldn~c~ lion units for ~9 desaturase
Ribozymes targeted to cleave ~\-9 desaturase mRNA are endogenously expressed in
plants, either from genes inserted into the plant genome (stable transforrnation) or from
episomal transcription units (transient e~y~s~ion) which are part of plasmid vectors or
viral sequences. These ribozymes can be e~p,essed via RNA polymerase I, II, or III
plant or plant virus promoters (such as CaMV). Promoters can be either constitutive,
tissue specific, or developm~.tS~lly e~ ,ssed.
Q9 259 Monomer Ribozvme Constructs (RPA 114, 115)
These are the ~-9 desaturase 259 monnrn~r h~.. l~ead ribozyme clones. The
ribozymes were d~o~igned with 3 bp long stem II and 20 bp (total) long substrate binding
arms targeted against site 259. The active version is RPA 114, the inactive is RPA 115.
The parent plasmid, pDAB367, was ~i~ect~d with Not I and filled in with Klenow to
30 make a blunt acceptor site. The vector was then fli~çsted with Hind III restriction
enzyme. The ribozyme containing plasmids were cut with ~:co R~, filled-in with Klenow
and recut with Hind III. The insert cont~inin~ the entire ribozyme transcription unit was
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gel-purified and ligated into the pDAB 367 vector. The constructs are checked by. digestion with Sgf I/Hind III and Xba I/Sst I and confirmed by sequencing.
~9 4~3 Multimer Ribozvme Constructs (RPA 118. 119)
These are the ~-9 desaturase 453 Multimer hammcrllcad ribozymc CIOllCS (SCC
Figure 17). The ribozymes were designed with 3 bp long stc111 il rcgions. Total lc~
the substrate binding arms of the m~lltimer construct was 42 bp. The aclivc vcrsion is
RPA 118, the inactive is 119. The constructs were made as described above for the 259
onolllei. The multimer construct was ~esigned with four hammerhead ribozymes
ed against sites 453, 464, 475 and 484 within ~-9 desaturase RNA.
~9 252 Multimer Ribozvme Constructs (RPA 85. 113)
These are the ~-9 desaturase 252 mllltimer 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 m-lltimPr contructs were d~ci~d with 3 bp long stem II
regions. Total length of the ~ul~hdte binding arms of the mlllti~er 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 ~igected with Bgl 11 and the single
cut plasmid was gel-purified. This was recut with ~:co Rl and again gel-purified to isolate
the correct Bgl ITJ~:co RI cut fr~gm~Pnt The Bam HI/ Eco R~ inserts from the ribozyme
corlshu~ 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 conr.-l,.ed by
pr ~ a Nco I / Sst I digest and sequ~n~ing
Useful l~dnsg~,lic plants can be j~A~ntified by standard assays. The l~dnsg~,.lic
plants can be evaluated for reduction in A-9 desaturase e~ ion and A 9 desaturase
activity as Ai~c~l~sed. in the examples infra. .
Fy~mr-le9~ Pntific~tion of Potential RibozYme Cleava~e Sites in GBSS RNA
Two hundred and forty one hammer-head ribozyme sites were identified in the com
GBSS mRNA polypeptide coding region (see table IIIA). A h~mmer-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 I at the 5'
end of a GBSS cDNA clone having the following sequence (5 ' to 3 '):
72
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GAccGATcGATcGccAcAGccAAcAccAcccGccGAGGcGAcGcGAcAGccGccA
GGAGGAAGGAATAAACT
73 144
CACTGCCAGCCAGTGAAGGGGGAGAAGTGTACTGcTccGTccAcCAGTGCGCGCA
CCGCCCGGCAGGGCTGC
145 21(.
TCATCTCGTCGACGACCAGTGGATTAATCGGcA I GGCGGCTCl /~GCCACGTCC;~/~
GCTCGTCGCAACGCGCG
217 2%~
10 CCGGCCTGGGCGTCCCGGACGcGTccAcGTTccGccGcGGCGCCGCGCAGGGCCT
GAGGGGGGGCCGGACGG
289 360
CGTCGGCGGCGGACACGCTCAGCATTCGGACCAGCGCGCGCGCGGCGCCCAGGCT
CCAGCACCAGCAGCAGC
361 432
AGcAGGcGcGccGcGGGGccAGGTTcccGTcGcTcGTcGTGTGcGccAGcGccGG
CATGAACGTCGTCTTCG
433 504
TCGGCGCCGAGATGGCGCCGTGGAGCAAGACCGGCGGCCTCGGCGACGTCCTCGG
CGGCCTGCCGCCGGCCA
505 576
TGGCCGCGAATGGGCACCGTGTCATGGTCGTCTCTCCCCGCTACGACCAGTACAA
GGACGCCTGGGACACCA
577 648
2S GcGTcGTGTccGAGATcAAGATGGGAGAcAGGTAcGAGAcGGTcAGGTTcTTccA
CTGCTACAAGCGCGGAG
649 720
TGGACCGCGTGTTCGTTGACCACCCACTGTTCCTGGAGAGGGTTTGGGGAAAGAC
CGAGGAGAAGATCTACG
721 792
GGCCTGACGCTGGAACGGACTACAGGGACAACCAGCTGCGGTTCAGCCTGCTATG
CCAGGCAGCACTTGAAG
793 864
CTCCAAGGATCCTGAGCCTCAACAACAACCCATACTTCTCCGGACCATACGGGGA
GGACGTCGTGTTCGTCT
865
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GcAAcGAcTGGcAcAccGGcccTcTcTcGTGcTAccTcAAGAGcAAcTAccAGTcc
CACGGCATCTACAGGG
937 100
AcGcAAAGAccGcTTTcTGcATccAcAAcATcTccTAccAGGGccGGTTcGccTTc
TCCGACTACCCGGAGC
1009 I t)xt)
TGAAccTcccGGAGAGATTC~AGTcGTccT rCci~ r I I ~AIC'(;A~(i(i(lAC(iA(i~A
GCCCGTGGAAGGCCGGA
1081 1152
10 AGATcAAcTGGATGAAGGccGGGATccTcGAGGccGAcAGGGTccTcAccGTcAG
CCCCTACTACGCCGAGG
1153 1224
AGcTcATcTccGGcATcGccAGGGGcTGcGAGcTcGAcAAcATcATGcGccTcAc
CGGCATCACCGGCATCG
1225 1296
TCAACGGCATGGACGTCAGCGAGTGGGACCCCAGCAGGGACAAGTACATCGCCGT
GAAGTACGACGTGTCGA
1297 1368
CGGCCGTGGAGGCCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGC
TCCCGGTGGACCGGAACA
1369 . 1~0
TCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGACCCGACGTCAT
GGCGGCCGCCATC~
1441 . 1512
AGcTcATGGAGATGGTGGAGGAcGTGcAGATcGTTcTGcTGGGcAcGGGcAAGA
AGAAGTTCGAGCGCATGC
1513 . 1584
TCATGAGCGCCGAGGAGAAGTTCCCAGGcAAGGTGcGcGccGTGGTcAAGTTCAA
CGCGGCGCTGGCGCACC
1585 1656
AcATcATGGccGGcGccGAcGTGcTcGccGTcAccAGccGcTTcGAGcccTGcGGc
CTCATCCAGCTGCAGG
1657 1728
GGATGCGATACGGAACGCCCTGCGCcTGcGcGTccAccGGTGGAcTcGTCGACAC
CATCATCGAAGGCAAGA
1729 1800
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CCGGGTTCCACATGGGCcGccTcAGcGTcGAcTGcAAcGTcGTGGAGCCGGCGGA
CGTCAAGAAGGTGGCCA
1801 1872
CCACCTTGCAGCGCGCCATcAAGGTGGTcGGcAcGccGGcGTACGAGGAGATGGT
GAGGAACTGCATGATCC
18?3 1')44
AGGATcTcTccTGGAAGGGcccTGccAAGAAcTGGG/~GAAcGTGc~ c-l'c~ cT
CGGGGTCGCCGGCGGCG
1945 2016
10 AGccAGGGGTcGAAGGcGAGGAGATcGcGccGcTcGccAAGGAGAAcGTGc'Jccc'
CGCCCTGAAGAGTTCGGC
2017 2088
CTGCAGGCCCCCTGATCTCGCGCGTGGTGCAAACATGTTGGGACATCTTCTTATAT
ATGCTGTTTCGTTTAT
2089 2160
GTGATATGGACAAGTATGTGTAGCTGCTTGcTTGTGcTAGTGTAATATAGTGTAG
TGGTGGCCAGTGGCACA
2161 2232
AccTAATAAGcGcATGAAcTAATTGcTTGcGTGTGTAGTTAAGTAccGATcGGTA
ATTTTATATTGCGAGTA
2233
AATAAATGGAccTGTAGTGGTGGAAAAAAAAAAAA(sEQI.D~No.2s).
Thcre are a~yloxil~ s3 potential hairpin ribozyme sites in the GBSS mRNA.
25 The ribozyme and target sequences are listed in Table V.
Ribozymes can be readily d~Ci~n~d and s~ Pd to such sites with between 5
and 100 or more bases as aubaLIate binding arms (see Figs. 1 - 5) as described above.
30 r~ 10: Sel~ -- of Ribozvrne Cleava~e Sites for GBSS
The seco~ structure of GBSS mRNA was ~ssPcced by computer analysis using
foldingalg~ l",ls, 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
RNAtRNA stems of over eight nucleotides and cont~ined potential 1-,.. . " . . l,~:ad
35 ribozyme cleavage sites were id~PntifiP~l
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These sites which were then assessed for oligonucleotide accessibility with RNasc
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 decign~tion of these oligonucleotides were 195, 205, 240, 307, 390, 424, 472, 4~
5 539, 592, 625, 636, 678, 725, 741, 811, 859, 891, 897, 912, 91 ~, 92~, 951, 95~, 9(~9, 993,
999, 1015, 1027, 1032, 1056, 1084, 1105, 1156, 116~ )5, 12()4, 121~, 1222,1240, 1269, 1284, 1293, 1345, 1351, 1420, 1471, 1533, ~563, 1714, 1750, 17~6, 1806,
1819, 1921, 1954, and 1978. Secondary sites were also covered and included 202, 394,
384, 38S, 484, 624, 627, 628, 679, 862, 901, 930, 950, 952, 967, 990, 991, 1026, 1035,
101108, 1159, 1225,1273, 1534, 1564, 1558,and 1717.
Example 11: RNaseH Assavs for GBSS
RNase H assays (Fig. 7) were pc.rul.ned using a full length transcript of the GBSS
coding region,3' noncoding region, and part of the 5' noncoding region. The GBSS RNA
was s.irccned for ncc~ scible cleavage sites by the method described generally in Draper et
15 al., supra. hereby incorporated by reference herein. Briefly, DNA oligonucleotides
l~,plt,s~ h~....... ~,l,ead ribozyme cleavage sites were synth~osi7~ A polymerase chain
reaction was used to generate a substrate for T7 RNA polymerase transcription from corn
cl~A~cTones. Labeled RNA trans~ were synthesi7et~ in vitro from these templates.The oligonucleotides and the labeled ll~nscli~ts were annealed, RNAseH was added and
20 the llli~Lu-~_S were incubated for 10 min~-tes at 37~C. Reactions were stopped and RNA
s~pal~l~d on sequencing polyacrylamide gels. The pe..,~ ge of the substrate cleaved
was d~h-l--i-lcd by autoradiographic ~luantil~lion using a phosphor ill.a~illg system (Fig.
7).
Exarnple 12: E~ .. ..I ~F Pd Ribozymes for GBSS
."".~ d ribozymes with 10/10 (i.e., able to form 10 base pairs on each arrn of
the riboyme) mlcleotide binding arms were dloci~ç(l to the sites covered by the oligos
which cleaved best in the RNase H assays. These ribozymes were then subjected toanalysis by cor..~ hl folding and the ribozymes that had significant secondary structure
were rejected. As a result of this sc,e.,~ lg procedure 23 ribozymes were d~sign~od to the
30 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.,
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supra. Scarin~e et al., and Wincott el al, supra) and are incorpolatcd bv rcfcrcllcc l1crcill,
and makes use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise
coupling yields were >98%. Inactive ribozymes were synthesized by substituting a U for
G5 and a U for A14 (numbering from (Hertel e~ al., supra). I-Tairpill ribo~ymcs wcrc
synthesized in two parts and annealed to reconstruct thc activc rih07~ylnc ~Chowril;l all(i
Burke, 1992, NucleicAcids Res., 20, 2835-). All ribozymcs wcrc modificd to cnl)allcc
stability by modification of five ribonucleotides at both tllc 5' and 3' cnds with 2'-C)-
methyl groups. Ribozymes were purified by gel electrophoresis using general methods.
10 (Ausubel et al., 1990 Current Protocols in Molecular Biology Wiley & Sons, NY) or were
purified by high pres~u~ liquid chromatography, as dcscribcd abovc and wcrc
resuspended in water.
Example 13: Lone Substrate Tests for GBSS
Target RNA used in this shldy was 900 nt long and contained cleavage sites for all
15 the 23 HH ribozymes lal~.,t.d 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 T7RNA polymerase. The L.dlls~ t was internally labeled during transcription by incln~ine
[a-32P] CTP as one of the four ribnn~cleotide triphosphates. The l~al~sc~il lion mixture
20 was treated with DNase-l, 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
df~ lfr polyacrylamide gel. Bands cG"~ ,ollding to full-length RNA was isolated
from a gel slice and the RNA was pl~ . iyilated witb isoprol,allol and the pellet was stored
at4~C
Ribozyme cleavage reactions were carried out under ribozyme excess (kCat/KM)
c~n-TitiQr~c (Herschl~g and Cech, supra). Briefly, 1000 nM ribozyme and < 10 nM
intern~lly labeled target RNA were denatured separately by heating to 90~C for 2 min. in
the p~es~,.cc of 50 mM Tris.HCI, pH 7.5 and 10 mM MgC12. The RNAs were r~.,atul~d
by cooling to the reac~ion tcmp.,lalu,e (37~C, 26~C and 20~C) for 10-20 min. Cleavage
reaction was initi~ted by mixing the ribozyme and target RNA at appropriate reaction
t.,,~,l.e.a~ es. Alquots were taken at regular intervals of time and the reaction was
q". ~.~hF~ by adding equal volume of stop buffer. The samples were resolved on 4%
SC~ gel.
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36
The results from ribozyme cleavage reactions, at the three different temperatures,
are s~ A~i~ed in Figure 8. Seven lead ribozymes were chosen (425, 892, 919, 959, 968,
1241, and 1787). One of the active ribozymes (811) produced a strange pattern ofcleavage products; as a result, it was not chosen as one of our lead ribozymcs.
5 Example 14: Cleava~e of the GBSS RNA Usin~ Multiplc Ribozvmc Combinaliolls
Four of the lead ribozymes (892, 919, 959, 1241) wcrc incubatcci witll inlcrllally
labeled target RNA in the following combinations: 892 alone; 892 + 919; 892 + 919 +
959; 892 + 919 + 959 + 1241. The fraction of RNA cleavage increascd in an additivc
manner with an increase in the number of ribozymes used in the cleavagc rcaction (~ig. 9).
10 Ribozyme cleavage reactions were carried out at 20~C as described above. These data
suggest that multiple ribozymes targeted to dirr~,.e.lt sites on the same mRNA will
increase the reduction of target RNA in an additive manner.
Example 15: Construction of Ribozvme E~rcssill~ Transcription Units for GBSS
Cloning of GBSS Multimer Ribozymes RPA 63 (active) and RPA64 (inactive)
A mllltim~r ribozyme was constructed which contained four ha.",.,~,.l.ead ribozymes
targeting sites 892, 919, 959 and 968 of the GBSS mRNA. Two DNA oligonucleotides(Macromolecular Resourses, Fort Collins, CO) were ordered which overlap by 18
nucleotides~ The sequences were as follows:
Oligo 1: CGC GGA TCC TGG TAG GAC TGA TGA GGC CGA AAG GCC GAA
ATG TTG TGC TGA TGA GGC CGA AAG GCC GAA ATG CAG AAA GCG GTC
1 T ~ GCG TCC CTG TAG ATG CCG TGG C
25 Oligo 2: CGC GAG CTC GGC CCT CTC '1 TT 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
Inactive versions of the above were made by substituting T for G5 and T for A 14 within
30 the catalytic core of each ribozyme motif.
These were annealed in I X Klenow Buffer (Gibco/BRL) at 90~C for 5 minutes and slow
cooled to room t~ c~dture (22~C). ~TPs were added to 0.2 mM and the oligos
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extended with Klenow enzyme at lunitlul for one hour at 37~C. Tilis was
phenol/chloroform extracted and ethanol precipitated, then resuspended in lX React 3
buffer (Gibco/BRL) and digested with Bam HI and Sst I for I 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/S.st I digcstcd r~r)AI~ ~53. 'l'llc li~.tlioll W;lS
transformed into competent DHSa F' bactcria (Gibco/13RL). l'otcnLial cloncs wcrcscreened by digestion with Bam HJ/~co RI. Clones were confirrned by sequencing. The
total length of homology with the target sequence is 96 nucleotides.
919 Monomer Ribozyme (RPA66)
Asingle ribozyme to site 919 of the GBSS mRNA was constructed with 10/10 anns asfollows. Two DNA oligos were ordered:
Oligo 1: GAT CCG ATG CCG TGG CTG ATG AGG CCG AAA GGC CGA AAC
TGG TAG TT
Oligo 2: AAC TAC CAG TTT CGG CCT TTC GGC CTC ATC /~GC Cl~C GGC ATC
20 G
The oligos are phosphorylated individually in lX kinase buffer (Gibco/BRL) and heat
den~Lul~,d and ~nnP~l~(l by colllb;~ lg at 90~C for 10 min, then slow cooled to room
L~ .,.aLule (22~C). The vector was ,~,le~aled by digestion of pDAB 353 with Sst I and
25 blunting the ends with T4 DNA polymerase. The vector was redigested with Bam HI and
gd purified as above. The ~nnç~ oligos are ligated to the vector in lX ligation buffer
(GibcotBRL) at 16~C ovemight. Potential clones were digested with Bam HI/Eco RI and
c~ by sequ~n~inE~
FY~mrle 16: Plant Tlall,Çollllation Plasmids pDAB 367, Used in the ~9 Ribozyme
E,~e.illl~, ,t~ and pDAB353 used in the GBSS Ribozvme E~Je~;llle~lL~
Part A pDAB367
Plasmid pDAB367 has the following DNA structure: be~innin~ with the base after the
final C residue ofthe Sph I site of pUC 19 (base 441; Ref. 1), and reading on the strand
conti~ous to the LacZ gene coding strand, the linker sequence CTGCAGGCCGGCC
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TTAATTAAGcGGccGcGTTTAAAcGcccGGGcATTTAAATGGcGcGccGc
GATCGCTTGCAGATCTGCATGGGTG. nucleotides 7093 to 7344 of CaMV DNA
(2), the linker sequence CATCGATG, nucleotides 7093 to 7439 of CaMV, the linker
sequence GGGGACTCTAGAGGATCCAG, nucleotides 167 to 186 of MSV (3),
nucleotides 188 to 277 of MSV (3), a C residue followed by nuclcotidcs 119 to 209 of
maize Adh IS containing parts of exon I and intron I (4), nLlclcoli(lc.~i 5~S~ lo fi72 r
corlt~ining parts of Adh lS intron I and exon 2 (4), the linkcr scqucllcc G~CGCi~'l'C'I'Ci,
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
10 alanine codon, and nucleotide 546 of the coding region changed from G to A to eliminate a
Bgl II site. Next, the linker sequence TGAGATcTGAGcTcG~/\TTTcccc~
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).
15 Part B pDAB353
Plasmid pDAB353 has the following DNA structure: beginning with the base after the
final C residue ofthe Sph I site of pUC 19 (base 441; Ref. 1), and reading on the strand
contiguous to the LacZ gene coding strand, the linker sequellce
CTGCAGATCTGCATGGGTG, nucleotides 7093 to 7344 of CaMV DNA (2), the
20 linker se~ .re CATCGATG, nucleotides 7093 to 7439 of CaMV, the linker scq~lencc
GGGGACTCTAGAG, nucleotides 119 to 209 of maize Adh I S cont~inin~r parts of exonI and intron I (4), nucleotides 555 to 672 cont~inin~ parts of Adh IS intron I and exon 2
(4~, and the linker sequ~nre GACGGATCCGTCGACC, where GGATCC let,lesGnts the
recognihon se~ c for BamH I restriction enzyme. This is followed by the beta-
25 glu~:ulunidase (GUS) gene from pRAJ275 (7), cloned as an Nco I/Sac I fr~grn~nt, the
linker sequence GAAITTCCCC, 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).
30 The following are herein incoll~ulahd 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
sequenre of Cauliflower Mosaic Virus DNA. Cell 21 :285-294.
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3. ~lllline~ c, P. M., J. Donson, B. A. M. Morris-Krsinich. M. I. Boulton, and J W.
Davies (1984) The nucleotide sequence of Maize Streak Virus DN~. EMBO J. 3:3063-3068.
4. Dennis, E. S., W. L. Gerlach, A. J. Pryor, J. L. Bennetzen, A. In~lis, D. Llewellyn, M.
M. Sachs, R. J. Ferl, and W. J. Peacock (1984) Molccular analysis of ~hc alcol-ol
dehydrogenase (Adhl) gene of maize. Nucl. Acids Res. 12:39X3-4()()().
5. White, J., S-Y Chang, M. J. Bibb, and M. J. Bibb (1990) ~ casscttc containill~ ll1c ~ur
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 11. M. Goodm~ll (1982)Nopaline Synthase: Transcript mapping and DNA seq~lencc. J. Molec. ~ppl. Gcnct:
1 :561-573.
7. Jt;rr.,.~on, R. A. (1987) Assaying chimeric genes in plants: The GUS gene fusion
system. Plant Molec. Biol. Reporter ~:387-405.
Example 17: Plasmid pDAB359 a Plant Tran~ro"llalion Plasmid which Contains the
Gamma-Zein Promoter. the ~nti~n~e GBSS. and a the Nos Polvadenvlation Sen~l~nre
Plasmid pDAB359 is a 6702 bp double-stranded, circular DNA that contains the
following seqle~lce clf ~ : nucleotides 1-404 from pUC18 which include lac operon
se.~ e from base 238 to base 404 and ends with the HindIII site of the M13mpl8
polylinker (1,2); the Nos polyadenylation se~ cc from nucleotides 412 to 668 (3); a
synthetic adapter se.~ e from nucleotides 679-690 which converts a Sac I site to an
Xho I site by ch~n~ng GAGCTC to GAGCTT and adding CTCGAG: maize ~ranule
bound starch synthase cDNA from bases 691 to 2953, collej,uonding to nucleotides 1-
2255 of SEQ. I.D. No. 25. The GBSS sequence in plasmid pDAB359 was modified fromthe original cDNA by the ~dclition of a 5' Xho I and a 3' Nco I site as well as the deletion
of intemal Nco I and Xho I sites using Klenow to fill in the enzyme recognition
se.lu~,nccs. Bases 2971 to 4453 are 5' untr~ncl~t~d sequence of the maize 27 kD gamma-
zein gene coll~,s~nding to nucleotides 1078 to 2565 of the published sequence (4). The
gamrna-zein sequence was modified to contain a 5' Kpn I site and 3' BarnH/SalltNco 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 pucl8
bases 456 to 2686 including the Kpn I/Eco~/Sac I sites of the M13/mpl8 polylinker
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from 4454 to 4471, a lac operon fra_ment from 4471 to 4697,andthel3-lacatlllase L~ene
from 5642 to 6433 (1, 2).
The following are incorporated by reference herein:
pUC18- Norrander, J., Kempe, T., Messing, J. Gene (1983) 2t~: 101-10~; Pnuwcls. 1'.11
Enger-Valk, B.E., Brammar, W. J. Cloning Vectors, Elscvicr 19~5 and supr)lclllc
NosA - DePicker, A., Stachel, S., Dhaese, P., Zambryski, P., and Goodman, H.M.
(1982) Nopaline Synthase: Transcript Mapping and DNA Sequence J. Molec. Appl.
Genet. 1:561-573.
Maize 27kD gamma-~ein - Das, O.P., Poliak, E.L., Ward, K., Messing, J. Nucleic Acids
Research 19, 3325 - 3330 (1991).
Example 18: Construction of Plasmid pDAB430. containin~ Antisense ~9 Desaturase,E~ressed bv the Ubiquitin Promoter/intron (Ubi 1)
Part A Construction of plasmid pDAB421
Plasmid pDAB421 cont~inc a unique blunt-end Srp cloning site flanlced by the maize
Ubiquitin promote./i~ un and the nopaline synthase polyadenylation sequences
pDAB421 was p~ arcd as follows~ ction of pDAB355 with restriction enzymes
KpnI and BamHI drops out the R coding region on a 2.2 kB fr~gm~nt Following gel
purification, the vector was ligated to an adapter composed of two annP~I~d
oligonucleotides OF235 and OF236. OF235 has the sequence 5' - GAT CCG CCC GGG
GCC CGG GCG GTA C - 3' and OF236 has the seqlI~nre 5' - CGC CCG GGC CCC
GGG CG - 3'. Clones co..I;.;..;..g this adapter were id~ntifiecl by digestion and
lînca.,zation of plasmid DNA with the enzymes Srp and SmaI which cut in the adapter,
but not elsewhere in the plasmid. One rt,~les~ntative clone was sequenced to verify that
30 only one adapter was inserted into the plasmid. The resulting plasmid pDAB421 was
used in subsequent construction of the /~9 desaturase antisense plasmid pDAB430.
Part B Construction of plasmid pDAB430 (~nticence ~9 desaturase)
The antic~nce ~9 desaturase construct present in plasmid pDAB430 was produced by35 cloning of an amplification product in the blunt-end cloning site of plasmid pDAB421.
Two constructs were produced simult~r,eQusly from the same experiment. The first
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construct contains the ~9 desaturase gene in the sense orientation behilld the ubiquitin
promoter, and the c-myc tag fused to the C-terminus of the ~9 desaturase open reading
frame for imm--nological detection of overproduced protein il1 transgenic lines This
construct was int.-n-le(l for testing of ribozymes in a systeln which did not express maize
5 ~9 desaturase. This construct was never used, but thc primcrs uscd to amplify an(~
construct the ~9 desaturase antisense gene were thc sal11c Thc ~9 (lcs.lltll,l~c cl)NA
sequence described herein was amplified with two primcrs. Tl1c N-tcrmin~l pril1lcr
OF279 has the sequence 5'- GTG CCC ACA ATG GCG CTC CGC CTC AAC GAC -
3'. The underlined bases correspond to nucleotides 146-166 of the cDN~ scqucncc. C-
10 terminal primer OF280 has the sequence 5' - TCA TCA CAG GTC CTC CTC GCT
GAT CAG CTT CTC CTC CAG TTG GAC CTG CCT ACC GTA - 3' and is the
reverse complement of the sequence 5' - TAC GGT AGG GAC GTC CAA CTG GAG
GAGAAG CTGATCAGC GAG GAG GAC CTG TGA TGA - 3'. In this sequence the
underlined bases correspond to nucleotides 1304-1324 of the cDNA sequence, the bases
15 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 lineari~d with the restriction enzyme Srf I. Colony hybridization was used to
select clones c~ g the pDAB421 plasmid with the insert. The orientation of the
insert was deterrnined by restriction digestion of plasmid DNA with ~ nos~ic enzymes
20 KpnI and BamHI. A clone co..~ .;..g the A9 desaturase coding sequence in thc scnse
orientation relative to the Ubiquitin promote./inllon was recovered and was named
pDAB429. An additional clone co~ g the ~9 desaturase coding sequence in the
ce orientation relative to the promoter was named pDAB430. Plasmid pDAB430
was subjected to sequerl~e analysis and it was d. te.uli,.ed that the sequence co,l~ cd
25 three PCR indllce~l errors compared to the expected sequence. One error was found in the
S~ u~llCC co"~sponding to primer OF280 and two nucleotide çh~ng,~c were observedinternal to the coding se~ cc. These errors were not corrected, because antisense
dow~ tion does not require 100% sequence identity between the ~ntic~n~e transcript
and the dowl~e~ tion target.
Example 19: Helium Blastin~ of Embryo~enic Maize Cultures and the Subsequent
Re~e~ aliOn of Trans~enic Pro~eny
Part A Establi~hment of embryogenic maize cultures. The tissue cultures employed in
35 l.all:,Ç~llllalion e~ e,lts were initiated from imm~tl~re zygotic embryos of the
genotype "Hi-II". Hi-II is a hybrid made by illtellllalillg 2 R3 lines derived from a
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B73xA188 cross (Arrnstrong 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 eYtended time period.
5 Type II cultures were initiated from 1.5-3.0 mm immaturc cmbryos rcsulti~ fromcontrolled pollinations of greenhouse grown Hi-lI plants. Thc initiatiOIl mcdiulll u.~iccl w,l~
N6 (Chu, 1978) which contained 1 0mg/L 2,4-D, 25 mM L-prolinc, 1()0 m~/L casCillhydrolysate, 10 mg/L AL~No3~ 2.5 ~/L gelrite and 2% sucrose adjusted to pH 5.8. For
approximately 2-8 weeks, selection occurred for Type I1 callus and against non-
10 embryogenic and/or Type I callus. Once Type II callus was sclccted, it was transrcrrcd toa ,,~ lce medi~lln in which AgNO3 was omitted and L-prolinc reduccd to ~mM.
After approximately 3 months of subculture in which the quantity and quality of
embryogenic cultures was increased, the cultures were deemed acceptablc for usc in
15 L~ar,s~ll..alion e~c~.c,.,..~
Part B Preparation of plasmid DNA. Plasmid DNA was adsorbed onto the surface of
gold particles prior to use in transformation c~ ncl~La. The experiments for the GE~SS
target used gold particles which were sphclical with ~ met~ors ranging from 1.5-3.0
20 ~lliClOIls (Aldrich Chemical Co., Milwaukee, WI). T.a.,~rolnation e~,."i",c"ts for the ~9
dcs&L-I-dse target used 1.0 micron spherical gold particles (Bio-Rad, Hercules, CA). All
gold particles were surface-sterilized with ethanol prior to use. Adsorption wasaccomplished by adding ?4 ~LI of 2.5 M calcium chloride and 30 ~1 of 0.1 M spermidine to
300 ~1 of plasmid DNA and sterile H20. The co~ e~ aLion of plasmid DNA was 140
2~ ~g. The DNA-coated gold particles were ;.~ ccl;~ ly vortexed and allowed to settle out
of sUspen~ior~ The res~ ng clear sup~rn~ter~t was removed and the particles were-
c..us~ended in 1 ml of 100% ethanol. The final dilution of the suspension ready for usein helium blasting was 7.5 mg DNA/gold per ml of ethanol.
30 Part C Ple~aLion 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 m~ e ~ cd;~ plus 0.2 M sorbitol and 0.2 M mannitol as an
osmoticum. After an approximately 4 hour pr~hcaLl.lent, all tissue was transferred to
petri plates co-~ g 2% agar blasting ",cdiu", (m~inten~rlce medium plus osmoticum
35 plus 2% agar).
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Helium blasting involved accelerating the suspended DNA-coated ~old particles towards
and into ple~ared tissue targets The device used was an earlier prototype to the one
described in a DowElanco U.S. Patent (#5,141,131) which is incorporated herein by
reference, although both function in a similar manner. The device consisted of a high
5 pressure helium source, a syringe containing the DN~/L~old susr)cnsio~l, and .pJlrllm~tically-operated multipurpose valve which providcd conlrt llcd lillkaL~c bC~WCCIl
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
10 screen, which held the tissue in place during impact. Next, tar~ets wcrc l~laccd ulldcr
vacuum in the main clla,.,be. of the device. The DNA-coated L~old particlcs wcreaccelerated at the target 4 times using a helium pressure of 1500 psi. Each blast delivered
20 ~1 of DNA/gold ~u~ sion T.~ qdi~tely post-blasting, the targets were placed back
on .,.~;..t~ nce medium plus osmoticum for a 16 to 24 hour recovery period.
Part D Selection of transformed tissue and the r~.,"c.alion of plants from ll~nsg~,nic
cultures. After 16 to 24 hours post-blastin~, the tissue was dividcd Into small picccs and
transferred to selection .,~e~;u~ e ~-re ~,PAi.l~,- plus 30 mg/L BastaTM). Every 4
weeks for 3 months, the tissue pieces were non-selectively transferred to fresh selection
20 ,,.f~ "" After 8 weeks and up to 24 weeks, any sectors found proliferating against a
background of growth inhibited tissue were removed and isolated. Putatively transfonned
tissue was subc~ ed onto fresh selection medium. Transgenic cultures were established
after I to 3 additional subcultures.
25 Once BastaTM resistant callus was established as a line, plant regeneration was initiated by
han~r~ callus tissue to petri plate cc,..~ cytokinin-based induction l..ed;,.."
which were then placed in low light (125 ft-candles) for one week followed by one week
in high light (325 ft-candles). The intlllrtion .,~e~ " was composed of MS salts and
vitamins (Ml~chi~e and Skoog, 1962), 30 glL sucrose, 100 mg/L myo-inositol, S mg/L 6-
benzylh~ yuline~ 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 llan~.lcd to hormone-free
~e~ ion mP~illm and kept in high light. The regeneration mP~lium was composed ofMS salts and vit~min~, 30 g/L sucrose and 2.5 g/L gelrite adjusted to pH 5.7. Both
in-lnrtion and leg~..elalion media cnnt~ined 30 m~/L BastaTM. Tissue began dirr~ iating
shoots and roots in 2-4 weeks. Small (1.5-3 cm) plantlets were removed and placed in
~ tubes co~ g SH medium. SH medium is composed of SH salts and vitamins (Schenk
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and Hildebrandt, 1972). 10 g/L sucrose, 100 mg/L myo~ ositol, S mL/L FeEDTA, andeither 7 g/L Agar or 2.5 ~/L Gelrite adjusted to pH 5.8. Plantlets were transferred to 10
cm pots cont~ining approximately 0.1 kg of Metro-Mix(~) 360 (The Scotts Co.,
Marysville, OH) in the greenhouse as soon as they exhibited growth and developed a
sufficient root system (1-2 weeks). At the 3-5 leaf staL~e, plants wcrc tran.sfclrc(l to 5
gallon pots containing approximately 4 kg Metro-Mix~o ~ n(l L~IOWIl to Ill.l~lllily.
These Ro plants were sclf-pollinatcd and/or cross-pollinal-:cl willl l)oll-~ransL~ellie inbrc(ls
to obtain transgenic progeny. In the case of transgenic plants produced for the GBSS
target, Rl seed produced from Ro pollinations was replanted. The Rl plants were grown
0 tO maturity and pollinated to produce R2 seed in the quantities needcd for thc analyscs.
Example 20: Production and Re~eneration of ~9 Trans~enic Material.
Part A Tlallsçollllalion and isolation of embryo~enic callus. Six ribozyme constructs,
de,,r ;hcd previously, Lal~L._d to /\9 desaturase were transformed into .~ge.le.dble Type
II callus cultures as described herein. These 6 constructs consisted of 3 active/inactive
pairs; namely, RPA85/RPA113, RPA114/RPA115, and RPA118/RPAIl9. A total of
1621 tissue targets were p~ a ed, blasted, and placed into selection. From these blasting
w~ye~ e~ 334 independent Basta(~-resistant transformation events ("lines") were
isolated from selectisn Apprsxim~tely 50% of these lines were analyzed via DNA PCR
or GC/FAME as a means of deL~ ing which ones to move forward to ~g~"lc.a~ion andwhich ones to discard. The r~ g 50% were not analyzed either because they had
become non-embryogenic or C~ A
Part B Reg~ ,-alioll of ~9 plants from LI~ CgF~;C callus. Following analyses of the
Llal~SgC.liC callus, twelve lines were chosen per ribozyme construct for rcg~ ,.d.Lion, with
15 Ro plants to be produced per line. These lines generally conci~ted of 10 analysis-
positive lines plus 2 negative controls, however, due to the poor lc~ ability 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 Ro plants were ~ e~e~al~d
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-
pollin~tion~ were made using the inbreds CQ806, CS716, OQ414, or HOI as pollen
donors, and occasionally as pollen recipients. Over 715 controlled pollinations have been
made, with the majority (55%) being comprised of self- or sib-pollinations and the
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minority (45%) being comprised of Fl crosses. Rl seed was collected approximately 45
days post-pollination.
Example 21: Production and Re~eneration of Trans~enic Maize for the GBSS
Part A Transformation of embryogenic maize callus and thc suhscqucltt SclCCtioll .llld
establishment of transgenic cultures. RPA63 and RPA64, an active/inactivc r~air of
ribozyme multimers l~lgeLed to GBSS, were inserted along with bar selection plasmid
pDAB308 into Type II callus as described herein. A total of 115 BastaTM-rcsistant
independent tlal~srullllation events were recovered from the selection of 590 blastcd
tissue targets. Southern analysis was performed on callus samples from established
cultures of a!l events to deterrnine the status of the gene of interest.
Part B Reg~n~lalion of plants from cultures transformed with ribozymes targeted to
GBSS as well as the ad~ t to the R2 gellc,dtion. Plants were i~ge~ ated from
Southern "positive" transgenic cultures and grown to maturity in a greenhouse. The
primary lege,.~ t~,;, were pollinated to produce Rl seed. From 30 to 45 days after
pollination, seed was harvested, dried to the correct moisture content, and replanted. A
total of 752 Rl plants, r~ 5~ .g 16 original lines, were grown to sexual maturity and
pollin~tlo~ Approximately 19 to 22 days after pollination, ears wcrc harvcstcd and 30
kemels were randomly excised per ear and frozen for later analyses.
F~ mT~le 22: Testin~ of GBSS-Tar~eted Riboz~,rmes in Maize Black Mexican Sweet
(BMS) Stably T~ r,l,l.ed Callus
Part A Production of BMS callus stably transformed with GBSS and GBSS-~ .ted
ribozyrnes. BMS does not produce a GBSS mRNA which is homologous to that found
endor,.,-~o,J~Iy in maize. Therefore, a double transrulnlalion system was developed to
produce hal~rol~lal~L~ which e~lessed both target and ribozymes. "ZM" BMS
~u~ (obtained from Jack Widholm, University of Illinois, also see W. F. Sheridan,
"Black MeAi~l Sweet Corn: Its Use for Tissue Cultures in Maize for Biological
Research, W. F. Sheridan, editor. University Press. University of North Dakokta, Grand
Forks, ND, 1982, pp. 385-388) were p~ al~d for helium blasting four days after
sl~bculhl~e by h~n~r.,l to a 100 x 2û mm Petri plate (Fisher Scientific, PiU~buly,h, PA) and
partial removal of liquid medium, forming a thin paste of cells. Targets consisted of 100-
125 mg fresh weight of cells on a 1/2" antibiotic disc (Schleicher and Srlmell, Keene, NH)
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placed on blastin~g medium, DN6 ~N6 salts and vitamins (Chu el al., 197g), )0 g!L
sucrose, 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). 25 mM L-proline; pH= 5.~3
before autoclaving 20 minutes at 1~1~C] solidified with 2% TC agar (JRH Bioscicnces,
T en~ox~ Kansas) in 60 x 20 mm plates. DNA was precipitated onto gold particles. For
the first transformation, pDAB 426 (Ubi1GBSS) and pDAB 30% (35T/r3ar) wcrc uscd.Targets were individually shot using DowElanco Helium Blasting Dcvicc ~. Will1 avacuum pressure of 650 mm Hg and at a dict~nce of 15.5 cm from tar~ct to dcvicc noz~lc,
each sample was blasted once with DNA/gold mixture at 500 psi. Tmmedi~tely afterblasting, the antibiotic discs were llallsf~ d to DN6 medium made with O.g% 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
se~ecti-n ~n~ ,... with 6 mg/L Basta~). Subsequent transfers were done at two week
intervals. Isolates were picked from the filters and placed on AMCF-ARM mcriin-n (N6
salts and vil~ s, 20 g/L sucrose, 30 g/L .~ ;lQI~ 100 mg/L acid casein hydrolysate, and
1 mg/L 2,4-D, 24 mM L-proline; pH= 5.8 before autoclaving 20 ~ t~s at 121~C)
solidified with 0.8% TC agar co~ 6 m~/L Basta(l~. Isolates were m~int~ined by
s-lhc~ -re to fresh medium evcry two weeks.
Basta~)-.es~ t isolates which e,~l.leisGd GBSS were subjected to a second
hal,~r~ nalion. As with BMS suspensions, targets of transgenic callus were p.e~.aIed 4
days after subculture by spreading tissue onto 1/2" filters. However, AMCF-ARM with
2% TC agar was used for blacting, due to n~ t~ e of transforrnants on AMCF-ARM
s~ ction media. Each sample was covered with a sterile 104 llm 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 mlJltimPr) or pDAB319 and RPA64 (inactive
ribozyme ml-ltimf~r), or shot with pDAB 319 alone. Tmr~leAi~t~ly after blasting, all targets
were ll~lsr~ d to nol-c~ re ..~Ji~ . (AMCF-ARM) for one week of recovery.
Subsequently, the targets were placed on AMCF-ARM medium corlt~ining two selection
30 agents, 6 mg/L Basta~ and 2 ~Ig/L clllors-llfil~on (CSN). The level of CSN was h.w~ as~d
to 4 ug/L after 2 weeks. ContinlleA transfer of the filters and generation of isolates was
done as described in the first tran~rolmalion, with isolates being maintained on AMCF-
ARM ~ J;.~.. cu..~ 6 mg/L Basta and 4 ~Lg/L CSN.
35 Part B Analysis of BMS stable transformants e~cpl, ~si,.g GBSS and GBSS-targeted
ribozymes. Isolates from the first transforrnation were evaluated by Northern blot
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analysis for detection of a functional target gene (GBSS) and to detennine relative levels
of eA~;ssion. In 12 of 2~ isolates analyzed, GBSS transcript was detected. A range of
e~prGs~ion was observed, in-lic~tin~ an independence of transformation events. Isolates
generated from the second transformation were evaluated by Northern blot analysis for
detection of continued GBSS exprcssion and by RT-J'CR to ~crccll rOr thc l~r~ c ol
ribozyme transcript. O~ 19 isolatcs tested from onc r~rcvio~lsly lrallslornlc(l lhlc, IX
expressed the active ribozyme, RPA63, and all exprcssed GBSS. G13SS was dctcclcd in
each of 6 vector controls; ribozyme was not ex~rcssed in these samples. As described
herein, RNase protection assay (RPA) and Northern blot analysis were pcrformcd on
10 ribozyme-e~ ssillg and vector control tissues to comparc Icvcls of G~SS transcril~t in
the presence or absen~e of active ribozyme. GBSS values were normalizcd to an intcrnal
control (A9 desaturase); Northern blot data is shown in Figure (25). Northern blot results
revealed a significantly lower level of GBSS message in the presence of ribozyme, as
co".~a,cd to vector controls. RPA data showed that some of the individual samples
15 ~,~p,.,..si"g active ribozyme ("L" and "O") were significantly different from vector
controls and similar to a nontransformed control.
Exam~le 23: Analysis of Plant and Callus Materials
Plant material co-L~al~sfo~-"cd with the pDAB308 and one of the following
ribozyrne co..~ g vectors, pRPA63, pRPA64, pRPA85, pRPAI 13, pRPAI 14,
pRPAl 15, pRPAl 18 or pRPAl 19 were analyzed at the callus level, Ro level and select
lincs analyzed at the Fl level. Leaf material was harvested when the plantlets reached the
6-8 leaf stage. DNA from the plant and callus material was prepared from Iyophilized
tissue as described by Saghai-Maroof et al.(supra). Eight micrograms of each DNA was
~lig~ste~ with the restriction enzymes specific for each construct using conditions
5~g~,G~d by the m~mlf~c1~lrer (Bethçs~1~ Research Laboratory, Gaith~ ,~bu, ~" MD) and
s~ ak~d by agarose gel electrophoresis. The DNA was blotted onto nylon l"~ ."b,anc as
des~;,il,ed by Southern, E. 1975 "Detection of specific sequences among DNA fr~gm~nts
sG~Jalàlcd by gel clc~.l uphoresis, J Mol. Biol. 98:503 and Southern, E. 1980 "Gel
cle~l,o~horesis of restriction fr~gm~nt~ Methods Enzmol. 69:152, which are
incc"~o~ahd by reference herein.
Probes specific for the ribozyme coding region were hybridized to the membranes.Probe DNA was ~ret)artd 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
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LKB, Piscataway, NJ) with 50 microcuries of o!32P-dCTP (Amersham Life Science,
Arlington Heights, IL) Probes were hybridized to the genomic DNA on tlle nylon
membranes. The membranes were washed at 60~C in 0.25X Ssc and 0.2% SDS for 45
nlinutec, blotted drv and exposed to XAR-5 film overnight with two intensifying screens.
The DNA from the RPA63 and RP~tS4 was digcstccl witl~ tllc rcslri(:lioll CllZyll1cs
HindIII and EcoRI and the blots cont~ ing these samples were hybridized to the RPA63
probe. The RPA63 probe consists of the RPA63 ribozyme multimer coding region andshould produce a single 1.3 kb hybridization product when hybridized to thc RPA63 or
RPA64 materials. The 1.3 kb hybridization product should contain the enhanccd 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 cont~inin~ these samples were hybridized to
the RPA122 probe. RPA 122 is the 252 multimtor ribozyme in pDAB 353 replacing the
GUS r~p~lL~_~. The RPA122 probe consists of the RPA122 ribozyme mllltim~r codingregion 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 hybridi7~tiotl
product should contain the ~nh~nred 35S promoter, the AdhI ir.;ron, ~he bar gene, the
ribozyme coding region and the nopaline synthase poly A 3' end. The DNA from theRPA114 and RPA115 was digested with the restriction enzymes HindTII and SmaI andthe blots co~t~inin~ these samples were hybridized to the RPA115 probe. The RPA115
probe consist of the RPA 115 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 enh~n~e~l 35S promoter, the AdhI intron, the
ribozyrne 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 andthe blots Co~ g these samples were hybridized to the RPA 118 probe. The RPA 118
probe consist of the RPA 118 ribozyme coding region and should produce a single 1.3 kb
hybridization product when hybridized to the RPA 118 or RPAl l 9 materials. The I 3 kb
hybridization product should contain the tonh~nced 35S promoter, the AdhI intron, the
ribozyme coding region and the nopaline synthase poly A 3' end.
Exam~le 24: Extraction of Genomic DNA from Transeenic Callus
Three hundred mg of actively growing callus were quick frozen on dry ice. It wasground to a fine powder with a chilled Bessman Tissue Pulverizer (Spectrum, Houston,
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TX) and extracted with 4oolll of 2x CTAB buffer (2% ~lexadecyltrimet}lylamlllollium
Bromide, 100 mM Tris pH 8.0, 20 mM EDTA, 1.4 M NaCI, 1% polyvinylpyrrolidone).
The suspension was Iysed at 65~C for 25 minutes, then extracted witl1 an equal ~olume of
chloroform:isoamyl alcohol. To the aqueous phase was added 0.1 volumes of 10%
5 CTAB buffer (10% Hexadecyltrimethylammonium Bromidc, 0.7 M NaCI) ~ oliowin
extraction with an equal volume of chloroform:isoamyl alcohol, ().fi volumc~ of col(l
isopropyl alcohol was added to the aqueous phasc, and placcd at -20~C for 3() IllillUlCS.
After a 5 minute centrifugation at 14,000 rpm, the resulting precipitant was dried for 10
s undervacuum. It was resuspended in 200 ~11 TE (lOmM Tris, ImMEDTA, p1-1
8.0) at 65~C for 20 minutes. 20% Chelex (Biorad, ) was added to the DN~ to a final
co~ .l. ation of 5% and incuhat~1 at 56~C for 15-30 minutes to rcmovc impuritics. Thc
DNA co,~rc ~L~alion was measured on a Hoefer Fluorimeter (Hoefer, San Francisco).
Example 25: PCR Analvsis of Genomic Callus DNA
Use of Polymerase Chain Reaction (PCR) to demonstrate the stable insertion of
ribozyme genes into the clllul~oso..~c of l~ p.- ~ic maize calli.
Part A Method used to detect ribozvme DNA The Polymerase Chain Reaction (PCR)
was ~c.rolll,ed as described in the suppliers protocol using AmpliTaq DNA Polymerase
(GeneAmp PCR kit, Perkin Elmer, Cetus). Aliquots of 300 ng of g~ nic callus DNA,1 ~1 of a 50 ~lM do~ Ll~alll primer (5' CGC AAG ACC GGC AAC AGG 3' ), 1~1 of an
u~sl~ealll primer and 1~LI of Perfect Match (Str~t~nç, Ca) PCR t .h~r~ were mixed
with the co,l~o~ents of the kit. The PCR reaction was pc.r~,.llled for 40 cycles using the
following IJal~ dcllalulation at 94~C for 1 minute, ~nn~linE~ at 55~C for 2
...;...~lf s and eYt~ncion at 72~C for 3 mins. An aliquot of 0.2x vol. of each PCR reaction
was cle~,L u~uho,e~ised on a 2% 3:1 Agarose (FMC) gel using standard TAE agarose gel
contlition~
30 Part B U~slr~aln primer used for detection of A9 desaturase ribozyme ~enes
RPA85/RPA 113 251 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'
35 This primer is used to amplify across the Eco RV site in the 35S promoter.
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Primers were prepared using standard oligo synthesis protocols on an Applied
Biosystems Model 394 DNA/RNA synthesizer.
Example 26: Ple,,al~tion of Total RNA from Trans~enic Maize Calli and Plant
Part A Preparation of total RNA from transgenic non-regcncrablc alld rcgcl1crahlc callus
tissue. Three hundred milligrams of actively growin~ callus was quick frozcl1 Oll ~Iry icc.
The tissue was ground to a fine powder with a chilled Bessman Tissue Pulverizer
(~pccl-ull~, Houston, TX) and extracted with RNA Extraction Buffer (50 mM Tris-HCI
10 pH 8.0, 4% para-amino salicylic acid, 1% Tri-iso-propylnapthalenesulfonic acid, lO mM
dithiothreitol, and 10 mM Sodium meta-bisulfite) by vigorous vortcxin~. Thc homogcnatc
was then extracted with sn equal volume of phenol co~ 0.1% 8-hydroxyquinoline.
A*er centrifilgPtion~ the aqueous layer was exL~o~chd with an equal volume of phenol
co..l~;..;..g chlG,ofol~ isoamyl alcohol (24:1), followed by extraction with
chloroform:octanol (24:1). Subsequently, 7.5 M Ammonium acetate was added to a final
co.~ Lion of 2.S M, the RNA was precipitated for I to 3 hours at 4~C. Following
4~C centrifugation at 14,000 rpm, RNA was resuspended in sterile water, precipitated
with 2.5 M NH40Ac and 2 volumes of 100% ethanol and incubated overnite at -20~C.The harvested RNA pellet was washed with 70% ethanol and dried under vacuum. RNA20 was r~u~,e"ded in sterile H20 and stored at -80~C.
.
Part B Pr~aldLion 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 In~ lf~ctorers
25 instructions, total RNA was purified from the powder using a Qaigen RNeasy Plant Total
RNA kit (Qiagen Inc., ChaL~wollh, CA). Total RNA was released from the RNeasy
columns by two sequential elution spins of p~ led (50~C) sterile water (30 ~LI each)
and stored at - 80~C.
30 Example 27: Use of RT-PCR AnalYsis to Demonstrate Expression of Ribozvme RNA in
Trans~enic Maize Calli and Plants
Part A Method used to detect ribozyme RNA. The Reverse Transcription-Polymerase
Chain Reaction (RT-PCR) was performed as described in the suppliers protocol using a
35 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 ~1 of a 15 ~LM
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downstream primer (5' CGC AAG ACC GGC AAC=AGG 3' ) were mixed witll thc RT
components of the kit. The reverse transcription reactioll was perfonned in a 3 step
ramp up with 5 minute incubations at 60~C, 65~C, and 70~C. For the PCR reaction, ~
of upstream primer specific for the ribozyme RNA beillg analyzcd was addcd to tllc RT
reaction with the PCR components The PCR rcaction was r)cl formcd for 3~ cyclcs usillL
the following parameters; incubation at 96~C for I mill~ltc, clcn.~l~lr.~ti(ln .~t ~4'( lol 3()
seconds, ~nne~ling at 50~C for 30 seconds, and extension at 72~C for 3 mills. /~n aliquol
of O.2x vol. of each RT-PCR reaction was electrophoresed on a 2% 3:1 A~arose (FMC)
gel using standard TAE agarose gel conditions.
Part B Specific u~all~alll primers used for detection of GBSS ribozymes.
GBSS Active and Inactive Multimer
5' CAG ATC AAG TGC AAA GCT GCG GAC GGA TCT G 3'
This primer covers the Adh I intron footprint upsll~;a,~ of the first ribozyme ann.
GBSS 918 Intron (-) Monomer:
5' ATC CGA TGC CGT GGC TGA TG 3'
This primer covers the lO base pair ribozyme arm and the first 6 bases of the ribozyme
catalytic dom~in
GBSS ribozyme e~rc~,~ion in transgenic callus and plants was confirrned by RT-PCR.
GBSS mllltimçr ribozyme ~A~esjion in stably transforrned callus was also deterrnined by
Ribonuclease Protection Assay.
Part C Specific u~ ea~ lilllE~ used for detection of ~9 desaturase ribozymes.
RPA85/RPAl 13 252 multimer fused to BAR 3' ORF
5' GAT GAG ATC CGG TGG CAT TG 3'
This primer spans the junction of the BAR gene and the RPA85/l l3 ribozyme.
RPAl 14/RPAl 15 259 ribozyme monomer
5' ATC CCC TTG GTG GAC TGA TG 3'
This primer covers the lO base pair ribozyme arm and the first 6 bases of the ribozyme
catalytic ~om~in
RPA l l 8/RPA l l 9 453 ribozyme multimer
5' CAG ATC AAG TGC AAA GCT GCG GAC GGA TCT G 3'
This primer covers the Adh I intron footprint u,~.~k~alll of the first ribozyme arm.
Expression of ~9 desaturase ribozymes in transgenic plant lines 85-06, 1 l 3-06 and 85- l 5
were confirmed by RT-PCR.
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Primers were prepared using standard oligo synthesis protocols on an Applied
Biosystems Model 394 D~A/RNA synthesizer.
Example 28: Demonstration of Ribozvme Mediated Reduction in TarL~et mRNA Levels
5 in Trans~enic Maize Callus and Plants
Part A Northern analysis method which was uscd to dcmollstralccl rcclue~ ; in largel
mRNA levels. Five ~lg of total RNA was dried under vacuuln, resuspended in loading
buffer (20mM phosphate buffer pH 6.8, 5mM EDTA; 50% fonnamide: 16%
forrn~ yde: 10% glycerol) and denatured for 10 minutes at 65~C. Electrophoresis was
at 50 volts through I % agarose gel in 20 mM phosphate buffer (pll 6.8) with buffcr
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 ~ ...h".n~
filter ( DuPont NE?~, Boston MA) by capillary transfer with sterile water.
Hybridization was ~,,rulllled 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 fr~gm~nts from the target gene and an internal RNA control gene.
The internal RNA standard was utilized to distinguich variation in target mRNA levels
due to loading or h~n~l1ine errors from true ribozyme me~ tecl RNA reductions. For each
20 sample the level of target mRNA was con".a,ed to the level of control mRNA within that
sample. Fr~gm~nt~ were purified by Qiaex resin (Qaigen Inc. Chatsworth, CA) from lx
TAE agarose gels. They were nick-trancl~ted using an Amersham Nick Translation Kit
(Amersham Co~olation7 Arlington Heights, Ill.) with alpha 32p dCTP.
Autoradiography was at -70~ C with i-~Le~.sirying screens (DuPont, Wi1mingtnn DE) for
25 one to three days. Autoradiogram signals for each probe were IlleasulGd after a 24 hour
e~o~ul~ by d~ oll-eL~l and a ratio of l~lget/in~ al control mRNA levels was
c~lrlll~t~"l
Ribonuclease protection assays were pc.Çul...ed as follows: RNA was prcpar~d 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~ cpm/microgram or higher. The probes were made the same day they were used. They were gel
purified, resuspended in RNase-freelOmM Tris (pH 8) and kept on ice. Probes werediluted to Sx 1 05cpm/ul immerli~tely before use. 5 ~g of RNA derived from callus or 20 ~lg
of RNA derived from protoplasts was inc~bat~cl with 5 x 105 cpm of probe in 4M
Guanidine Buffer. ~4M Guanidine Buffer: 4M Guanidine Thiocyanate/0.5%
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Sarcosyl/25mM Sodium Citrate (pH 7.4)] 40 ul of PCR millcral oil ~tcls adclcd to cacl7
tube to prevent evaporation. The samples were heated tO 95~ for 3 minutes and placed
immedi~tely into a 45~ water bath. Incubation continued ovemight. 600 ,ul of RNase
Treatment Mix was added per sample and incubated for 30 minutes a~ 37~C (RNase
Treatment Mix: 400 mM NaCl, 40 units/m! RNa~c A .In(l 11). 12 ~1 ol 2()'~ i w~:r~
added per tube, immediately followed by addition of 12 Ul (2() Ill~/nll) l'rolcil~ K lo
each tube. The tubes were vortexed gently and incubatcd for 3() minulcs at 37~C. 7S0 ul
of room te,lll)c~ature RNase-free isopropanol was added to each tube, and mixed by
inverting repeatedly to get the SDS into solution. The samples were then microfu~cd at
top speed at room tc~ eldture for 20 minuteS The pellets were air dried for 45 minutes.
15 ul of RNA Running Buffer was added to each tube, and vortcxed hard for 30 seconds.
(RNA Running Buffer: 95% Form~nid~/20mM EDTA/0.1% Bromophenol Blue/0.1%
Xylene Cyanol ). The sample was heated to 95~ C for 3 minutes, and loaded onto an 8%
d~l~aLulillg acrylamide gel. The gel was vacuum dried and exposed to a phosphorimager
screens for 4 to l 2 hours.
Part B Results ~mcnctrating reductions in GBSS mRNA ievels in nonglonerable cal1us
CAplc~ g both a GBSS and GBSS targeted ribozyme RNA. Tbe productior, cf
nol~legell~,.dble callus e~le,,sillg RNAs for the GBSS target gene and an active multimer
ribozyme L~ GICd to GBSS mRNA was ptL~Ill-ed. Also produced were transgenics
e~ e;~ai~lg GBSS and a ribozyme (-) control RNA. Total RNA was ple~)ar-,d from the
Ll,.,-~gG,.;c lines. Northern analysis was pc.ru,llled on 7 ribozyme (-) controltlan:~rulll~anL~ and 8 active RPA63 lines. Probes for this analysis were a full length maize
GBSS cDNA and a maize ~9 cDNA fr~gm~-nt To distinguish variation in GBSS mRNA
levels due to loading or h~ntlling errors from ttue ribozyme me~i~ted RNA reductions, the
level of GBSS mRNA was cu.~lp~d to the level of ~9 mRNA within that sample. The
level of full length GBSS transcript was colll~alet between ribozyme ~ ,s~ g andribozyme minus calli to identify lines with ribozyme me~ teci target RNA reductions.
Blot to blot variation was controlled by performing duplicate analyses.
A range in GBSS/ ~9 ratio was observed b.,L~c_.1 ribozyme (-) transgenics. The
target mRNA is produced by a tr~nC~ne and may be subject to more variation in
e.~ .ssion then the endogenous ~9 mRNA. Active lines (RPA 63) AA, EE, KK, and JJwere shown to reduce the level of GBSS/~9 most significantly, as much as 10 fold as
compared to ribozyme (-) control transgenics this is graphed in Figure 25. Those active
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lines were shown to be ~,~pl~s~ g GBSS targeted ribozyme by RT-PCR as described
herein.
Reductions in GBSS mRNA compared to ~9 mRNA were also seen by RNAse
protection assay. v
Part C Demonstration of reductions in ~9 desaturasc lcvcls in transgcnic l~lantse,.~.e;,sil.g ribozymes targeted to t~9 desaturase mRNA. The high stearate tr~n~gerlics,
RPA85-06 and RPA85- 15, each contained an intact copy of the fused ribozyme multhner
gene. Within each line, plants were screened by RT-PCR for the presence of ribozymc
RNA. Using the protocol described in Example 27. RP~5 ribozymc cxprcssion was
tl~mo~cl~aLcd in plants of the 85-06 and 85-15 lines which cont~ined high stearic acid in
their leaves. Northern analysis was p~.lrc".led 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 fr~ nt~ from a maize ~9 desaturase cDNA and a maize
actin cDNA. To distinguish variation in A9 mRNA levels due to loading or h~n~ g
errors from true ribozYme ~-.eAiAlf~ RNA redllctionc, the level of a9 mRNA was
c~ alcid to the level of actin mRNA within that sample. Using densitometer ~~,adi-.~,~
described above a ratio was c~lc~ t~d for each sample. ~9/actin ratio values ranging from
0.55 to 0.88 were c~le~ te~l for the 85-06 plants. The average ~9/actin value for non-
n~fol-lled controls was 2.7. There is an appal~,A~t 4 fold reduction in ~9/actin ratios
~c~een 85-06 and NT leaves. Conlpalil.g ~9/actin values between 85-06 high stearate
and TC plants, on average a 3 fold reduct*on in /~9/actin was observed for the 85-06
plants. This data is gldi)hed in Figure 26. Ranges in ~9/actin ratios from 0.35 to 0.53,
with an average of 0.43 were c~ ted for the RPA85-15 high stearate transgenics. In
this ~ ,,hl~ the average /~9/actin ratio for the NT plants was 1.7. COlllpalillg the
average ~9/actin ratio between NT controls and 85-15 high stearate plants, a 3.9 fold
redl~c*on in 85-15 ~9 mRNA was ~emo~ .dted. An apparent 3 fold reduction in /~9
mRNA level was observed for RPA85-15 high stearate lldlls~,..ics when ~9/actin ratios
30 were co-llparcd between 85-15 high stearate and normal stearate (TC) plants. These data
are graphed in Figure 27. These data indicate ribozyme-medi~ted reduction of ag-desaturase mRNA in transgenic plants e~les~il-g RPA85 ribozyme, and producing
increased levels of stearic acid in the leaves.
35 Example 29: Evidence of ~9 Desaturase Down Re~ulation in Maize Leaves as a Result of
Active Ribozyme Activity
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Plants were produced which were transformed with inactive ~ersions of the ~9
desaturase ribozyme genes. Data ~as presented demo1lstratillg control Icvcls of Icaf
stearate in the inactive ~9 ribozylne transgenic lines RPA! 13-06 and 1 13-17. Ribozyme
5 e~pl~ssion and northern analysis was perfiorllled for thc RPA I 13-()~ linc. ~9 dc.c,llurasc
protein levels were detennined in r)lants of ~llC RPAI 1:~-17 lille. I~ ozylllc c.~ lc!iciol1
was rneasured as describcd herein. r'lants 113-06-04, -()7, alld -1() cxprcssc~ CLCC~ IC
levels of RPAl 13 inactive A9 ribozyme. Northern analysis was perfonned on 5 plants of
the 113-06 line with leaf stearate ranging from 1.8 - 3.9 %, all of which fall within the
10 range of controls. No reduction in A9 desaturase mRNA corrclating with ribozylIlc
exy~ssion or elevations in leaf stearate were found in the RPA I 13-06 plants as compared
to controls~ hcd in Figure 28. Protein analysis did not indicate any reduction in A9
desaturase protein levels correlating with elevated leaf stearate in the RPAl 13-17 plants.
This data is graphed in Figure 29(a). Taken together, the data from the two RPAI 13
15 inactive llans8~ lic lines inrlicate ribozyme activity is responsible for the high ~llG~late
phenotype observed in the RPA85 lines. The RPA8~ ribozyme is the active version of
the RPAI 13 ribozyme.
Example 30: Demonstration of Ribozyme Mediated Reduction in Stearoyl-ACP ~9
20 Desaturase levels in Maize Leaves (RO) ~A9 Desaturase Levels in Maize Leaves (R0)
Part A Partial purification of stearoyl-ACP /~9-desaturase from maize leaves. All
procedures were p~,~ru~ll-ed at 4~C unless stated otherwise. Maize leaves (50 mg) were
harvested and ground to a fine powder in liquid N2 with a mortar and pestle. Proteins
25 were extracted in one equal volume of Buffer A conci~ting of 25 mM sodium-phosphate
pH 6.5, 1 mM ethyle~rJ;~ el ~ l~aacetic acid, 2 mM dithiothreitol, 10 mM
phenylmethylsulfonyl fluoride, 5 mM l~u~c"lh~, and 5 mM antipapin. The crude
homogenate was centrifuged for 5 min~1tes at 10,û00 x g. The supernatant was assayed
for total protein co..~ .ation by Bio-Rad protein assay kit (Bio-Rad Laboratonesj
30 Hercules, CA). One hundred micrograms of total protein was brought up to a final
volume of 500 ~1 in Buffer A, added to 50 111 of mixed SP-sepharose beads (Pharrnacia
Biotech Inc., Piscataway, NJ), and resuspended by ~..lL~illg briefly. Proteins were
allowed to bind to sepharose beads for 10 minutes while on ice. After binding, the A9
desaturase-sepharose material was centrifuged (10,000 x g) for 10 seconds, dec~nted,
washed three times with Buffer A (500 11l), and washed one time with 200 mM sodium
chloride (500 ~LI). Proteins were eluted by boiling in 50 ~LI of Treatment buffer ( 125 m M
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Tris-CI pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol)
for ~ min1~1es. Samples were centrifuged (10,000 x g) for 5 minutes. The supernatant was
saved for Western anaylsis and the pellet consisting of sepharose beads was discarded.
5 Part B Western analysis method which was used to dcmo11stratc rcduc~ in xlcaroyl-
ACP ~9 desaturase. Partially purified proteins were separatcd on sodi~llll do(lccyl s~llf.llc
(SDS)-polyacrylamide gels ( 10% PAGE) as dcscribcd by Lacn1ll~ U. K. ( 197()) C'lcavagc
of structural proteins during assembly of the head of phage T4, Nc~re Z27, 660-685. To
distinguish variation in ~9 desaturase levels, included on each blot as a reference was
10 purified and quantified ove,~ ssed ~9 desaturase from E. coli as described hereforth.
Proteins were electrophoretically transferred to ECL nitrocellulose mcmbranes
(~...- .il.~... Life Sciences, Arlington Heights, Illinois) using a Pharmacia Semi-Dry Blotter
(Pllal-l'acia Biotech Inc., Piscataway, NJ), using Towbin buffer (Towbin et al. 1979).
The nolls~,c~ c binding sites were blocked with 10% dry milk in phosphate buffer saline
15 for 1 h. Tmmllnoreactive polypeptides were detect~d using the ECLTM Western Blotting
Detection Reagent (Amersham Life Sciences, Arlington Heights, Illinois) with rabbit
antiserum raised against E. coli e~ esjed maize ~9 desaturase. The antibody was
produced according to standard protocols by Berkeley Antibody Co. The secondary
antibody was goat antirabbit serum conjugated to horseradish peroxidase (BioRad).
20 Autoradiograms were scanned with a densito,lleter and quantified bascd on thc rclativc
amount of purified E. coli ~9 desaturase. These ex,~el;",ents were duplicated and the
mean reduction was l~,cGlded.
Part C Demonstration of Reductions in ~9 desaturase levels in R0 maize leaves
25 exl"cs~ing ribozymes lalgeh,d to A9 desaturase mRNA. The high stearate hànsg~l,ic line,
RPA85-15, cQr~t~inC an intact copy of the fused mllltim-or gene. ~9 desaturase was
partially purified from R0 maize leaves, using the protocol described herein. Western
analysis was pc.~lll.cd on ribozyme active (RPA85-1~) and ribozyme inactive
(RPAl 13-17) plants and nol~LIa~ ~llllcd (HiII) plants as described above in part B. The
30 natural variation of ~9 d~,SaLu~aSe was determined for the nontransformed line (HiII) by
Western analysis see Figure 29 A. No reduction in A9 desaturase was observed with the
ribozyme inactive line RPAI 13-17, all of which fell within the ran~e as comparcd to thc
nont~a~ orrned line (HiII). An apparent 50% reduction of ~9 desaturase was observed
in six plants of line RPA85-15 (Figure 29 B) as compared with the controls. Concurrent
35 with this, these same six plants also had increased stearate and reduced ~9 desaturase
mRNA (As described in Examples 28 and 32) However, nine active ribozyme plants
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from line RPA85- 15 did not have anv significant reduction as compared with
nontransformed line (HiII) and inactive ribozyme line (RPA 113- 17) (Figures 29 A and B).
Collectively, these results suggest that the ribozyme activity in tlle six plallts from linc
RPA85- 15 is responsible for the reduced ~9 desaturase.
Example 31: E. coli Expression and Purification of Maizc a-9 dc.saturclsc Cll;'.ylllC
Part A The mature protein encoding portion of the maize ~-9 desaturase cDNA was
inserted into the bacterial T7 e~,rcssion vector pET9D (Novagen Inc., Madison, WI).
The mature protein t~ncotlin~ region was de~1uced from the maturc castor bcan
polypeptide sequence. The alanine at position 32 (nts Z39-241 of cl)NA) was dcsiL~Ilatc(t
as the first residue. This is found within the sequence Ala.Val.Ala.Ser.Met.Thr.Restriction enflon~cle~ce Nhe I site was ~nein,~red 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 design~te~
as pDAB428. The maize ~-9 desaturase protein e~ sed in bacteria has an additional
m-~thiorlin~ residue at the S' end. This pDAB428 plasmid was transformed into the
bacterial strain BL21 (Novagen, Inc., Madison, WI) and plated on LB/kanamycin plates
(25 mg/ml). Colonies were resuspended in 10 ml LB with kanamycin (25 mg/ml) and
IPTG (ImM) and were grown in a shaker for 3 hours at 37~C. The cells were harvestcd
by centrifugation at lOOOxg at 4~C for 10 minlltes. The cells were Iysed by freezing and
thawing the cell pellet 2X, followed by the addition of 1 ml Iysis buffer (10 mM Tris-HCI
pH 8.0, 1 mM EDTA, 150 mM NaCI, 0.1 % Triton X100, 100 ug/ml DNAse I, 100
ug/ml RNAse A, and 1 mg/ml Iysozyme). The mixture was in~Ub~t~l for 15 minlltes at
37~C and then centrifuged at 1000 Xg for 10 mimltes at 4~C. The supernatant is used as
the soluble protein fraction.
The supematant, adjusted to 25 mM sodium phosphate buffer (pH 6.0), was
chilled on ice for I hr. An~ ds, the resulting flocculant precipitant was removed by
centrif~ tion The ice incubation step was rtpeatcd twice more after which the solution
~ -rd clear. The clarified solution was loaded onto a Mono S HR 10/10 column
(Ph~ ) 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 NaCI gradient
over 1 hr (2 mVmin; 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-terrninal sequence analysis. Comparison of the
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protein's amino terminal sequence to that encoded by the cDNA clone revcalcd that tllc
protein was indeed ~ 9. 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 nonhellle iron stain (Lcong et al.,
1992 ~nal. Biochem. 207, 317-320) confirmed that the purificd protcin W,IS a-s.
Part B Production of polyclonal antiserum
The E. coli produced ~-9 protein, as determined by amino terminal sequcncing, was
gel purified via SDS-PAGE, excised, and sent in the gel matrix to Bcrkclcy ~ntibody Co.,
Ric~lmon~ CA, for production of polyclonal sera in rabbits. Titcrs of tllc alltibodics
against ~-9 were performed via western analysis using thc ECL Dctcction systcm
(,~m~r~h~m? Inc.)
Part C Purification of ~9 desaturase from corn kernels
Protein Precipitation: ~\9 was purified from corn kemels following homogenization using
a Warring blender in 25 mM sodium phosphate buffer (pH 7.0) co~ ;";"g 25 mM
sodium bisulfite and a 2.5% polyvinylpolypyrrolidone. The crude homogenate was
filtered through cheesecloth, centrifuged ( I 0,OOOxg) 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 ~9 protein prc~ ted at either 80% a~.. o.. il.. sulfate or
25% polyethylene glycol. The resulting pellets were then dialyzed extensively in 25mM
sodium phosphate buffer (pH 6.0).
Cation Exchange Chromotography: The solubilized pellet material described above was
25 clarified via centrifugation and applied to Mono S HRI0/I0 colulnn equilibrated in 25
mM sodium phosph~te buffer (pH 6.0). After extensive column washing, basic proteins
bound to the column matrix were eluted using a 0-500 mM NaCI gradient over 1 hr (2
ml/min: 2 ml fractions). Typically, the ~9 protein eluted between 260-and 350 mMNaCI., as determined by enzymatic and western analysis. After dialysis, this material
30 was further fracionated by acyl carrier protein (ACP)- sepharose and phenyl superose
chromatography.
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,4c~yl Carrier Protein-Sepharose C hro~7~atography: ACP was purchased from Si~naCh.omi~l 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,
essenti~lly as described by Pharmacia, Inc., in the packa~c inscr~ ftcr link,lgc an(l
blocking of the remaining sites with glycinc, thc ACr-scr)l1.lrosc n7.llcri~l1 was p.lckc(l h)lo
a HR 5/5 column (Pharrnacia, Inc.) and equilibratcd in 25 mM so(liulll pllospllalc buf-~cr
(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 el al. 19910 Proc. Natl. Acad. Sci. USA 88, 2578-2582). After extensive column washillg, I~CP-
binding proteins were eluted using I M NaCI. Enzymatic and westcrn analysis, followcd
by amino terminal sequencing, intlic~ted that the eluent contained ~-9 protein. The A-9
protein purified from corn was del~,l..illed to have a molecular size of approximately 38
kDa by SDS-PAGE analysis (Hames, 1981 in Gel Electrophoresis of Proteins: A
Practical Approach, eds Hames BD and Rickwood, D., IRL Press, Oxford).
Phenyl Sepharose Chromatography: The fractions containing A9 obtained from the ACP-
Sepharose column were adjusted to 0.4 M ammonium sulfate (25 mM sodium phosphate,
pH 7.0) and loaded onto a Pharmacia Phenyl Superose column (HR 10/10). Proteins were
eluted by running a gradient (0.4 - 0.0 M ~mmonium sulfate) at 2 ml/min for I hour. The
~9 protein typically eluted between 60- and 30 mM ammonium sulfate as detennined by
enzymatic and wt~te.ll analysis.
F.Y~m~ le 32: Evidence for the Increase in Stearic Acid in Leaves as a Result ofTransformation of Plants with ~9 Desaturase Ribozvmes
PartA Method used to d~L~ c the stearic acid levels in plant tissues. The procedure
for extraction and esterification of fatty acids from plant tissue was modified from a
desclil)ed 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 I ml of
mçth~nolic 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 meth~n~ lic 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 a~nd I ml of 0 9% (w/v) NaCI was added
followed by vigorous ~h~kin~ of the test tubes. After centrifugation of the tubes at Z000
rpm for 5 minutes the top hexane layer was removed and used for fatty acid methyl ester
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analysis. Gas chromatograph analysis was performed by injection of I ~1 of the sample
on a Hewlett Packard (Wilmin~ton, DE) Series ~I model 5890 gas chromatograph
equipped with a flame ionization detector and a J&W Scientific (Folsom, CA) DB-23
column. The oven t~ .,.at~re was 150~C throughout the run and the flow of thc carrier
gas (helium) was 80 cm/sec. The run time was 20 minutc~s. Thc conditions ~ owc(l rOr r
the separation of the 5 fatty acid methyl esters of intcrcst: C l 6:(), r)allllityl mc~llyl cstcr;
C18:0, stearyl methyl ester; C18:1, oleoyl mcthyl estcr; C1~:2, linolcoyl mctl1yl cstcr;
and C18:3, linolenyl methyl ester. Data collection and analysis was performcd with a
Hewlett Packard Series II Model 3396 integrator and a PE Nclson (Pcrkin Elmcr,
Norwalk, CT) data collection system. The percentage of each fatty acid in the sample
was taken directly from the readouts of the data collection systcm. Quantitativc ~mounts
of each fatty acid were calculated using the peak areas of a standard (Matreya, Pleasant
Gap, PA) which col..ci~led of a known amount of the five fatty acid methyl esters. The
amount calculated was used to estimate the percentage, of total fresh weight, represcnted
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 A9
desaturase ri'oozymes. Leaf tissue from individual plants was assayed for stearic acid as
described in Part A. A total of 428 plants were assayed from 35 lines transformed with
active ~9 desat.-lase ribozymes (RPA85, *PA114, RPA118) and 406 plants from 31
lines transformed with ~9 desaturase inactive ribozymes (RPA113, RPA115, RPAI I9). .
Table XI ~7~ Gs 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 transforrned
with a gene not related to Ag 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 ~9 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
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controls (Figure 30) The average stearic acid content of the control plants (122 plants)
was 1.69% (SD+/-0.49%). The average stearic acid content of leaves from line RPA85-06
was 2.86% (+/-0.57%). Line RPA85-15 had 6 out of 15 plants assayed with stearic acid
levels which were approximately 4-fold greater than the average of the controls (Figure
31). The average leaf stearic acid content of line RPA85-15 was 3.83n/. ( 1/-2.5:~n/,).
When the leaf analysis was repeated for RPA85- 15 plants, thc stcaric aci~l lcvcl in lcavcs
from plants previously shown to havc normal stcaric aci(l lcvcls rcmaillccl norl1lal an~
leaves from plants with high stearic acid were again found to be high (Figure 31). The
stearic acid levels in leaves of plants from two lines which were transformed with an
inactive ~9 desaturase ribozyme, RPAI 13, is shown in Figures 32 and 33. Rr'A I 13-0
had three plants with a stearic acid content of 3% or lli~hcr. Tllc avcra~c stcaric acid
content of leaves from line RPA113-06 was 2.26% (+/-0.65%). RPA 113-17 had no
plants with leaf stearic acid content greater than 3%. The average stearic acid content of
leaves from line RPAI 13-17 was 1.76% (+/-0.29%). The stearic acid content of leaves
from lS control plants is shown in Figure 34. The average stearic acid content for these
15 control plants was 1.70% (+/-0.6%). When compared to the control and inactive ~9
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
~9 desaL~l,dse ribozyme.
Example 33: In}.e,;Lal,ce of the HiPh 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 imm~ re kernels from these
RPA85-15 plants and placed in a tube on media as described herein for growth of
l~f.,~ 1 plantlets. After the plants were transferred to the greenhouse, fatty acid
analysis was y~lrulllled on the leaf tissue. Figure 3S shows the stearic acid levels of
leaves from I û ~ cr~llt plants for one of the crosses, RPA85- 15.07 selfed. Fifty percent
of the plants had high leaf stearic acid and 50% had norrnal leaf stearic acid. Table XII
shows the results from 5 dirr~l~nt crosses of RPA8S-15 plants. The number of plants
with high stearic acid ranged from 20 to S0%.
- Part B Results demonstrating reductions in ~9 desaturase levels in next generation (Rl)
35 maize leaves ~x~le~sillg ribozymes targeted to ~9 desaturase mRNA. In next generation
maize plants that showed a high stearate content (see above Part A), ~9 desaturase was
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partially purified from Rl maize leaves, usin_ the protocol described hereil1. Western
analysis was performed on several of the high stearate plants. ~n leaves of next generation
plants, a 40-50% reduction of ~9 desaturase ~vas observed in those plants tllat had high
stearate content (Figure 36). The reduction was comparable to R0 maize leaves. This
reduction was observed in either OQ414 plants crosscd with RrA85-15 pt)llcl1 or
RPA85-1~ plants crossed with self or siblin~s. Thercfore, this sugL!cSts tllat thc gcnc
encoding the ribozyme is heritablc.
Example 34: Increase in Stearic Acid in Plant Tissues Usin~ Antisense- ~9 Desaturase
Part A Method for culturing somatic embryos of maize. The production and
ltg~ elalion of maize embryogenic callus has been described herein. Somatic embryos
make up a large part of this ernbryogenic callus. The somatic embryos continued to form
in callus becdusc 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 m~ m The somatic embryos
,~eg~ t~d into plantlets be~,d.l~e the callus was subjected to a regenc~alion procedure
described herein. Duting 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
,.lcldLion, by a specific medium tre~tment This medium treatment involved transfer
of the embryogenic callus to a Murashige and Skoog ".~.1;u~., (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 meclillm with 6% sucrose for 7 davs and then the
somatic emblyos were individually transferred to MS medium with 6% sucrose and 10
~LM 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 col.lpa.ed to the fatty
acid co.nl o~iLion of embryogenic callus and maize zygotic embryos 12 days afterpollination (Table XIII). The fatty acid composition of the somatic embryos was
dirr~ t than that of the embryogenic callus. The embryogenic callus had a higherpe.c~.lLage of C16:0 and C18:3, and a lower percentage of C18:1 and C18:2. The
pc.~;~nlage of lipid ,el,lesel1ted by the fresh weight was different for the embryo~enic
callus when co-"pa-ed 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 ~vere nearly identical. It was
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concluded that the somatic embryo culture sys~em described abo~e would be an useful in
vitro system for testin~ the effect of certain genes on lipid svnthesis in developing
embryos of maize.
5 Part B Increase in stearic acid in somatic cmbryos of m~i7c a.s .l rcs~ of' Illc inllodLlclion
of an antisense- ~9 desarurase gene Somatic cmbryos wcrc ~ro(lucccl ~ iinL~ thc IllC~IlOd
described herein from embryogenic callus transformed witll r)D~B308/pDAB43(). Tl~c
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 Witll lli~ll
10 levels of stearic acid. The stearic acid content of somatic embryos from these two lines is
coln~)a,~d to the stearic acid content of somatic embryos from their control lincs in
Figures 37 and 38. The control lines were from the same culture that tlle transformed
lines came from except that they were not transforrned. For line 308/430-12, s~earic acid
in somatic embryos ranged from I to 23% while the controls ranged from 0.5 to 3%. For
line 308/430-15, stearic acid in somatic emblyos 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- ~\9 desaturase gene can be used to raise the stearic acid
levels in somatic embryos of maize.
Part C Demonstration of an increase in stearic acid in leaves due to introduction of an
ic~n~e- A9 desaL~lase gene. Embryogenic cultures from lines 308/430-12 and 308/430-
15 were used to rege.,l,~ate plants. Leaves from these plants were analyzed for fatty acid
composition using the method previously described. Only 4 plants were obtained from
the 308/430-15 culture and the stearic acid level in the leaves of these plants were normal,
1-2%. The stearic acid levels in leaves from plants of line 308/430-12 are shown in Figure
39. The stearic acid levels in leaves ranged from 1 to 13% in plants from line 308/430-12.
About 30% of the plants from line 308/430-12 had stearic acid levels above the range
observed in the controls, 1-2%. These results iTl(liC~te that the stearic acid levels can be
raised in leaves of maize by introduction of an ~nt ~çnce- ~\9 desaturase gene.
By "~ntis~on~e~ 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;
- Egholrn 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).
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Exam~le 35: Amvlose Content Assav of Maize Pooled Starch Sample and Sin~!le Kernel
The amylose content was assayed by the method of Hovenkamp-Hennelink et al.
(Potato Research 31:241-246) with modifications. For pooled starch sample, 10 Illg to
100 mg starch was dissolved in 5 ml 45% perchloric acid in plastic cLtlturc tubc. Tllc
solution was mixed occasionally by vortexinL~ Aftcr onc hnLIr, ().2 nll of tllc slarcll
solution was diluted to 10 ml by H20. 0.4 ml of the dilutcd solution was thcn IlliXCd
with 0.5 ml diluted Lugol's solution (Sigma) in I ml cuvet. Readings at 618 nm and 550
nm were immediately taken and the R ratio (618 nm/550 nm) was calculatcd. Usin~
standard equation P (percentage of amylose) = (4.5R-2.6)/(7.3-3R) gcncratc(l from l-otato
amylose and maize amylopectin tsigma~ St. Louis), amylose contcnt was dctcrmillcd. 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 S~nthase (GBSS) Assay
The purification of starch and following GBSS activity assay were modified from
the ~etho~c 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-HCI, pH 8.0, 10 mM EDTA and filtrated through 120 ,um 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 ~Im nylon membrane
and centrifuged. Pellet was then Iyophilized and stored in - 20 ~C until used for activity
assay.
A standard GBSS reaction mi,.Lurt contained 0.2 M Tricine, pH 8.5, 25 mM
Glutathione, 5 mM EDTA, 1 mM 14C ADPG (6 nci/,umol), and 10 mg starch in a totalvolume of 200 ,ul. Reactions were c~nflucted at 37 ~C for S min and termi-l~ted by adding
200 ,ul of 70% ethanol (vfv) in 0.1 M KCI. The material was centrifuged and
unincol~,o~aled ADPG in the supernatant is removed. The pellet was then washed four
time with lml water each in the same fashion. After washing, pellet was suspended in
500 111 water, placed into scintillation vial, and the incorporated ADPG was counted by a
Beckman (Fullerton, CA) scintillation counter. Specific activity was given as pmoles of
ADPG incorporated into starch per min per mg starch.
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Example 37: Analvsis 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 lar~e amount of sampleso generated in this study, a two-step screening stratc~y was LlSC(I, ~ hC flrS~ SlCp, 3()
kernels were taken randomly from the same ear, freezc-dricd and 11ol11oL~CIliXC~ O St.lrCIl
flour. Amylose assays Oll the starch flours wcrc carricd Olll. Lincs will~ rc(luccd amylosc
content were identified by statistical analysis. ln the second step, amylose content of the
single kernels in the lines with reduced amylose content was further analyzed (25 to 50
10 Icernels per ear). Two sets of controls were used in the screening, one of the scts wcrc
untransforrned 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 ~c;pr~_s~l.ting 16 transformation events were examined at the pooled starch
15 level. Among those lines, six with si~ific~nt reduction of amylose content by statistical
analysis were identified for further single kernel analysis. One line, 308/425-12.2.1,
showed significant reduction of amylose content (Figure 40).
Twenty five individual kernels of CQ806, a conventional maize inbred line, were
analyzed. The amylose content of CQ806 ranged from 24.4% to 32.2%, av~ .. ,g
29.1%. The single kernel distribution of amylose content is skewed slightly towards
lower amylose co~ ts 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
e~r~ec~d distribution would consist of 4 distinct genetic classes present in equal
25 frequ~n~ies since endosperrn is a triploid tissue. The 4 genetic classes consist of
individuals carrying 0, 1, 2, and 3 copies of the ~nti~ence construct. If there is a lat~e
dosage effect for the t~ then the distribution of amylose contents would betetramodal. One of the modes of the resulting distribution should be indi~tinguich~hle
from the non-tr~n~ nic parent. If there is no dosage effect for the transgene (individuals
30 carrying 1, 2 or 3 copies of the llallsg.,.le 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
for308/42~-12.2.1.1 isdistinctlytrimodal. The central mode is approximately twice the
size of either other mode. The two distal modes are of approximately equal size.35 Goodness of fit to a 1 :2: 1 ratio was tested and the fit was excellent.
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Further evidence was available demonstrating tl1at the mode ~ i~l1 the hi~hest
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 usin~ amylose
contents only. The estimates of variance from the individ~lal allalyscs wcrc usc(l ill all r
tests. No pooled estimate of variance was emr)loyc(l. Thc origill,ll d.lt.l w".~. tcslc(l ~Or
reclassification. Based on the discriminant analysis, thc cnlirc IllOdC ol- tllC 3()X/42S-
12.2.1.1 distribution with the highest amylose content would be more appropriately
cl~ssifi~cl as parental. This is strong confirrnation that this mode of the distribution is
parental. Of the remaining two modes, the central mode is approxil1latcly twicc tllc sizc
of the lowest amylose content mode. This would be cxpcctcd if tl1c ccntlal mo~lcin~ (k-s 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 anti~ence 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 ~ senting 11 transformation events
were ~ in the pooled starch level. Among the control lines (both untransformed
line and Southem negative line), the amylose content varied from 28% to 19%. No
si~ific~n~ reduction was observed arnong all lines carrying ribozyme gene (Southern
positive line). More than 20 selected lines were further analyzed in the single kernel level,
no signifir~nt amylose reduction as well as se~l~,galion pattern were found. It was
ap~a~nL that ribozyme did not cause any alternation in the phenotypic level.
Tl~sns~,-,-ed lines were further examined by their GBSS activity (as described in
Example 36). For each line, 30 kemels were taken from the frozen ear and starcll was
purified. Table XIV shows the results of 9 plants r~pleSel1ting one transformation event
ofthe GBSS activity in the pooled starch samples, amylose content in the pooled starch
samples, and Southem analysis results. Three southern negative lines: RPA63.0283,
RPA63.0236, and RPA63.0219 were used as control.
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The GBSS activities of control lines RPA63.0283,RPA63.0236,andRPA63.0219
were around 300 units/mg starch In lines RPA63.0211,RPA63.0218,RPA63.0209, and
RPA63.0210, a reduction of GBSS activity to more than 30% was observed. The
5 correlation of varied GBSS activity to the Southcm analysis in tlli.s L~rour) (rrom
RPA63.0314 to RPA63.0210 of Tablc XlV) inclicatcd tl-a~ c rc(lllccd ~ .S.S .I~:livily
was caused by the expression of ribozymc ~cnc incorr)oralcd inlo Illc maii~c gcllolllc.
GBSS activities at the single kernel level of line RPA ~3.021~ (Soutllcrl1 r)ositivc
and reduced GBSS activity in pooled starch) was further examincd. using RPA63.030f
(Southern negative and GBSS activity normal in poolcd stalcll)ascolllrol. About30
kemels from each line were taken, and starch samples were purified from each kernel
individually. Figure 41 clearly indicated reduced GBSS activity in line RPA63.0218
co~ a~ ~d to RPA63.0306.
Other embo~ .L~i are within the following claims.
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TABLE 1
Characteristics of naturally OC~ur. illg ribozvmes
Group I Introns
~ Size: ~150 to >1000 nucleotides.
~ Requires a U in the target Sc4ucllCc imme~ tPIy 5' of the cleavage site.
~ Binds 4-6 nucleotides at the 5'-side of the cleavage site.
~ Reaction mech~nicm: attack by the 3'-OH of guanosine to ge~ alc cleavage pl~nlUI,,I:~ with
3'-OH and 5 -.,u~nos;..e,
ition~l protein cofactors required in some cases to help folding and m~int~in~n~e ofthe
active sll u-,lu- c ['].
~ Over 300 known members of this class. Found as an intervening sequence in ~I~uh,,..._,.a
thermophila rRNA. fungal mitochondria. chloroplasts. pha e T4. blue-green algae. and others.
~ Major structural features largely established through phylogenetic comparisons. mut~geneSiC
and biochPnnic~l studies [2,3].
~ Complete kinetic framework established for one ribozyme [4,5,6,7].
~ Studies of ribozyme folding and :.ub:,llale docking underway [~,9,~0].
~ ChPmi~l modification invPstig~tion of ;Illp~ residues well established [",~'].~ The small (4-6 nt) binding site may make this ribozyme too non-specific for targeted RNA
cleavage. however, the Tetrahymena group I intron has been used to repair a "defective"
b-g~l~rtc-si~ce ...- c5~p~, by the ligation of new b-g~ tosi~i~ce seyu- -~ es onto the defective
mecc~e [13].
RNAse P RNA (M1 RNA)
~ Size: ~290 to 400 nn~ leoti(ies
~ RNA portion of a ubiquitous ribonucleQ~ Ic;.. enzyme.
~ Cleaves tRNA ylc~.ulaOI~ to fomm mature tRNA [14].
~ Reaction me~-h~nicm: possible attack by M2 -OH to generate cleavage products with 3'-OH and
S -pho5ph:~tP
~ RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit has been
s~ ed from bacteria~ yeast, rodents, and primates.
~ Rcc. uil~ ,uL of endogenous RNAse P for IL~,,alJ~ LiC applications is possible through
hybridization of an Extemal Guide .Seq~Pnre (EGS) to the target RNA [15,'6]
~ I..lpGI~alll phosph~tP and 2' OH contacts recently identified [~ ]
Group ll Introns
~ Size: >1000 nucleotides.
~ Trans cleavage of target RNAs recentlv demoll~llalcd [~9,'0].
~ Sequence n,~uilc.llents not fully deterrnined.
~ Reaction ~..ecl-~ni~ 2'-OH of an internal ~f~Prlocine generates cleavage products with 3'-OH
and a "lariat" RNA cont~ining a 3'-5 and a 2'-5' branch point.
~ Only natural ribozyme with demonstrated pa.lic;~,ation in DNA cleavage [1l,''] in addition to
RNA cleavage and ligation.
~ Major structural features largelv established through phvlo_enetic cou.pa.,sons [' ].
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~ Important ' OH contacts besinnins to be identified [24]
~ Kinetic framework under development [25]
Neurospora VS RNA
~ Size: -144 nucleotides.
~ Trans cleavage of hairpin target RNAs recently demonstrated [26]
~ SequPn~e ~c.luilements not full,v determined.
~ Reaction me~h~nicm attack by 2'-OH 5' to the scissile bond to generate cleavage products
with 2',3'-cyclic phosrh~tP and 5'-OH ends.
~ Binding sites and structural requh.~ not fully determined.
~ Only 1 known member of this class. Found in Neulu~Jola VS RNA.
I l~.. erl.~cl R;bG~ e
(see text for .ef."~,.ccs)
~ Size: ~13 to 40 nucleotides.
~ Requires the target sequence UH imm~di~t~Ply 5' of the cleavage site.
~ Binds a variable number nucleotides on both sides of the cleavage site.
~ Reaction ...e l.~ attack by 2'-OH 5' to the scissile bond to generate cleavage products
with 2',3'-cyclic phocl.l~ and 5'-OH ends.
~ 14 known members of this class. Found in a number of plant p~thngPnc (virusoids) that use
RNA as the infectious agent.
Fcc~nfi~l slru~,lul~l features largely defined, inrl~rling 2 crystal allu~;lulci~ []
~ Minimal ligation activity ~~emoricl ~ ah~d (for e,.gi..e~" ing through in vitro selection) []
~ Complete kinetic framework established for two or more ribozymes [].
~ . Ch~mi~ l modircdlion investig~tion of hllpollallL residues well established 1].
Ilai.~in Rit G~...e
~ Size: ~50 nucleQti~l~os
~ Requires the target seq~nee GUC immP~ t~ly 3' of the cleavage site.
~ Binds 4-6 n~rl~otirlPs at the 5'-side of the cleavage site and a variable number to the 3'-side of
the cleavage site.
~ Reaction meH.~ attack by 2'-OH 5' to the scissile bond to generate cleavage products
with 2',3'-cyclic phosrh~te and 5'-OH ends.
~ 3 kno vn members of this class. Found in three plant p~thngen (satellite RNAs of the tobacco
~;~7lJot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the
infectious agent.
O F.ccf~rlti~ llu~lulal features largely defined [27,28,29,30]
~ T .ig~tion activity (in a-l~iiti- n to cleavage activity) makes ribozvme amenable to en~,hle~"ing
through in vilro selection [31]
~ Complete kinetic framework established for one ribozyme [32]
~ Ch~mi~l modification investigation of hll~,ul~lll residues begun [33,34].
He~dlili:> Dettd Virus (HDV) Ribozyme
~ Size: ~60 nUcl~oti~pc
~ Trans cleavage of target RNAs demc,..sL~ al~d [35].
~ Binding sites and ~llu~;lulàl requh~nl.,.ll~ not fullv determined. ~Ithou~sh no se~ u~c 5~ of
cleavage site are required. Folded ribozvme contains a rceud~ n-~t ~llu~Lul~ t3~']
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~ Reaction me~~h~nicm: attack b~ 2'-OH 5' to the scissile bond to Penerate cleava~e products
with 3'-cyclic phosphate and o'-OH ends.
~ Only 2 known members of this class. Found in human HDV.
~ Circular forrn of HDV is active and shows i-l~,- . asc-d nuclease stability [37]
1. Mohr, G.: Caprara. M.G.: Guo, Q.; LalllbowiL~ A.M. Nature, ~Q. 147-150 (1994).
2. Michel. Francois: Westhof. Eric. Slippery aul,ahai.a. Nat. Struct. Biol. (1994), 1(1), 5-7.
3. Lisacek, Fn,d.,. iyu.,. Di~ Yolande: Michel. Francois. ~ntom~tir i~ -, ;on of group I intron
coresin genomic DNA se~ Pc J. Mol. Biol. (1994), 235(4). 1206-17.
4 ~Pr~crhla~ Daniel; Cech. Thomas R.. Catalysis of RNA cleavage by the Tetrahymena ~ ,hil~
ribozyme. I . Kinetic dea~ ion of the reaction of an RNA substrate cu...pl....~..~ y to the active site.
R~ l.y (1990), 29(44), 10159-71.
~lPtC~ hl~ Daniel; Cech. Thomas R.. Catalysis of RNA cleavage bv the Tetrahymena ~l-P ~..ot~
ribozyme. '7. Kinetic dea~ l iyliu-- of the reaction of an RNA substrate that forms a micm~mh at the active site.
Bio~ y (1990). 29(44), 10172-80.
6. Knitt, Deborah S.: Herschlag. Daniel. pH D~ of the Tetrahvmena Ribozyme Reveal an
U...,on~_..Liullal Origin of an Apparent pKa. BiorhPmictry ( 1996). 35(5). 1560-70.
7. Bevilacqua, Philip C.: Sugimotc~ Naoki: Turner. Douglas H.. A - ' - La l,_.. ulk for the second
s~ep of splicing catalyzed by the Tetrahvmena ribozyme. BiochPmictrv ( 1996), 35(2), 648-58.
8. Li, Yi; Bevilacqua, Philip C.: Mathews. David; Turner. Douglas H.. Thermodynamic and a~li./d~i
- for binding of a ~ ,.-c-labeled substrate by the Tetrahymena ribozyme: docking is not
diffusion-controlled and is driven by a L~,~,.able entropy change. Bio~h .~ . y (1995), 34(44), 14394-9.
9. Banerjee, Aloke Raj; Turner. Douglas H.. The time ~ ~, .,d~ ~e of chemical ~..n.l;r;. _l;.~., reveals slow
steps in the folding of a group I ribozyme. Bin- ~ -y (1995), 34(19), 6504-12.10. Zarrinkar, Patrick P.. Willi ~mcnn James R.. The P9. 1 -P9.2 ~.,. i,Jh~.al ~ . l~ -,, . helps guide folding of
the Tct~ahy..._..a ~ il,o ~..le. Nucleic Acids Res. ( 1996). 24(5). 854-8.
I I. Strobel. Scott A.; Cech. Thomas R.. Minor groove ~~cc~ ;GII of the con~ _d G.cntdot.U pair at the
Tetrahymena .il,u~...c reaction site. Science (WZ_I ;~ , 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 ofthe TLt al.~..._..a Ribo_yme Cu.n-iL to 5'-Splice Site Selectinn and Transition State
S~ ili7~inn E~;~l.. ~..;~-.y (1996), 35(4), 12ûl-11.
13. S~llPnpP~, Bruce A.: Cech, Thomas R.. Ribo_vme-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., _47. 5243-5251 (1972).
15. Forster, Anthony C.; Altman. Sidney. Extcrnal guide 5~ s for an RNA en_yme. Science
(W~ . 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.. kl~ -- of ~I-o~ . involved in catalysis by the
ribo_yme RNase P RNA. RNA (1995), 1(2). 210-18.
18. Pan, Tao: Loria. Andrew; Zhong. Kun. Probing of tertiary illt~a~,Liu~l~ in RNA: 2'-hydroxyl-base
contactcbetween she RNace P RNA and pre-tRNA Proc. Natl= Acad= Sci U. S. A (1995), 92(26). !25!0-!4.
19. Pyle, Anna Marie; Green. Justin B.. Building a ICinetic Fra...~ o-k for Group II Intron Ribozyme
Activity: Q ~~ --;ul, of Illte.-lulllaill Binding and Reaction Rate. Bio- hPmictTy (1994). 33(9). 2716-25.
20. Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group 11 Intron into a New
Multiple-Turnover Ribozyme that Selectively Cleaves OliPnn~cleoti~ Flllr~ n of Reaction Merh~nicm
and Structure/Function p~pl~ nchipc BiorhPmictTy (1995). 34(9). 2965-77.
21. Zimmerly, Steven; Guo. Huatao: Eskes. Robert; Yan~. Jian: Perlman. Philip S.: Lambowitz Alan M..
A group 11 intron RNA is a catalytic c ~ l of a DNA L lull~l~ Ir~c~= involved in intron mobility. Cell
(Cal..b.;dge. Mass.) (1995), 83(4). 5:29-38.
22. Griffin. Edmund A.. Jr.; Qin. Zhifeng; Michels. Williams J.. Jr.: Pyle. Anna Marie. Group 11 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.
SU~, 111 ~JTE SHEET (RULE 26)
CA 02226728 1998-01-13
WO 97/10328 PCTrUS96/l1689
TablCI
71
23. Michel. Francois: Ferat. Jean Luc. Structure and activities of _roup 11 introns. Annu. Rev. Biochem.
(1995). 6~. 435-61.
24. Abramovitz. Dana L.: Friedman. Richard A.; Pyle. Anna Marie. Catalvtic role of 2'-hvdroxyl groups
within a group 11 intron active site. Science (W~chin_ton D. C.) ( 1996). 271 (5254). 1410- 13.
25. Daniels. Danette L.: Michels, William J.. ~r.; Pyle. Anna Marie. Two cr....r~l;..g pathwavs for
self-splicing by group 11 introns: a ~ /e 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
ril~u~ c derived from NcL..va,uula VS RNA. EMBO J. (1995). 14(2). 368-76.
27. Hampel. Arnold; Tritz. Richard; Hicks. Ma.~a..~. Cruz. Phillip. 'Hairpin' catalytic RNA model:
evidence for helixes and sequence ~.~ui,~...c..~ for substrate RNA. Nucleic Acids Res. (1990). 18(2), 299-304.
28. Chowrira, Bharat M.: Berzal Ill..la-~ Alfredo; Burke. ~ohn M.. Novel gn~nnc~ cyuh~ for
câtalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351),320-2.
29. Berzal Ilc.la l~ Alfredo; Joseph, Simrson Chowrira. Bharat M.: Butcher. Samuel E.: Burke, John M..
Essential ~ ul ;~F 5~ 1 ~'f 5 and secu--.lcu y structure elements of the hairpin ribozyme. EMBO J. (1993),
12(6), 2567-73.
30. Joseph, Simrsnn Berzal Ilc~a~ Alfredo: Chowrira, Bharat M.: Butcher. Samuel E.. Substrate
selection rules for the hairpin ribozyme determined by in vitro selectinn mllt~tion and analysis of m;~ t~ I-rd
~uL.LIat~,. Genes Dev. (1993), 7(1), 130-8.
31. Berzal Ilc..anz. Alfredo; Joseph, Simpson, Burke. ~ohn M.. In vitro selection of active hairpin
libU~ s by 5~ RNA-catalyzed cleavage and ligation reactions. Genes Dev. (1992), 6(1). 129-34.
32. Hegg. Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of I~IL~ Oh l~r Catalysis by Hairpin
Ribozymes. Pjo. I.. ;~l.y (1995), 34(48), 15813-28.
33. Grasby, Jane A.; M~ ---- Karin; Singh, MoLiu.-l~.., Gait, Michael J.. Purine Fu-~ I Groups in
Essential Residues of *e Hairpin Ribozyme Required for Catalvtic C!eavage of RNA Bi~ (1995)
34(12), 4068-76.
34. Schmidt. Sabine; Re :~ f 1~ Leonid; Karpeisky, Alexander; Usman. Nassim; Sorensen, Ulrik S.; Gait,
Michael J.. Base and sugar r.,.lui~.l,e.~la for RNA cleavage of essential I~ 'IFO' '1~ residues in intemal loop B of
the hairpin ribozyme: implir~tir nc for seconda.y structure. Nucleic Acids Res. (1996), 24(4), 573-81.
35. Perrotta. Anne T.: Been, Michael D.. Cleavage of oli~c..;l,~,.,~.ck~,(id. ~ by a riboyrne derived from the
hepatitis .delta. virus RNA s~q~l~nre R;ocl~ y (1992), 31(1), 16-21.
36. Perrotta. Anne T.; Been, Michael D.. A 1~ -ot-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.
SUBSTITUTE SHEET ~RULE 26)
CA 02226728 1998-01-13
W O 97/10328 PCTrUS96/11689
Tablell
Table 11: 2.5 ,L~mol RNA Synthesis Cycle
Reagent Equivalents Amount Wait
Time~
Pllospl)u,d,, ' - 6.5 163 ~L 2.5
S-EthylTetrazole 23.8 238 ~L 2.5
Acetic Anhydride 100 233 ~L 5 sec
N-Methyl Irll ' '-. 186 233 ~lL 5 sec
TCA 83.2 1.73 mL 21 sec
lodine 8.0 1.18 mL 45 sec
Acelul ;'~ NA 6.67 mL NA
Wait time does not include contact time during delivery.
SUBSTITUTE SHEET (RULE 26)
CA 02226728 l998-0l-l3
WO 97/103Z8 PCT/US96111689
TablclllA
. 73
Table IIIA: GBSS H~ erhead Substrate SeclL~..ce
nt. Substrate Seq. ID nt. Substrate Seq.
Position No. Position No
12CGAUCGAUC GCCACAGC 26 538GGUCGUCUC UCCCCGCU 27
68GAAGGAAUAAACUCACU 28 540UCGUCUCUC CCCGCUAC 29
73AAUAAACUC ACUGCCAG 30 547UCCCCGCIJA CGACCAGU 31
103AGAAGUGUA CUGCUCCG 32 556CGACCAGUA CAAGGACG 33
109GUACUGCUC CGUCCACC 34 581ACCAGCGUC GUGUCCGA 35
113UGCUCCGUC CACCAGUG 36 586CGUCGUGUC CGAGAUCA 37
146Gr,GCUGCUC AUCUCGUC 38 593UCCGAGAUC AAGAUGGG 39
149CUGCUCAUC UCGUCGAC 40 610AGACAGGUA CGAGACGG 41
151GCUCAUCUC GUCGACGA 42 620GAGACGGUC AGGUUCUU 43
154CAUCUCGUC GACGACCA 44 625GGUCAGGUU CUUCCACU 45
169CAGUGGAUU AAUCGGCA 46 626GUCAGGUUC UUCCACUG 47
170AGUGGAUUA AUCGGCAU 48 628CAGGUUCUU CCACUGCU 49
173GGAUUAAUC GGCAUGGC 50 629AGGUUCUUC CACUGCUA 51
186UBGCGGCUC UAGCCACG 52 637CCACUGCUA CAAGCGCG 53
188r,cGr;cuCUA GCCACGUC 54 661cçGcGur;uu CGUUGACC 55
196AGCCACGUC GCAGCUCG 56 662CGCr,ur,uuc GUUGACCA 57
203UCGCAGCUC GUCGCAAC 58 665GUGUUCGUU GACCACCC 59
206CAGCUÇGUC GCAACGCG 60 679CCCACUGUU CCUGGAGA 61
230CUGGGCGUC CCGGACGC 62 680CCACUGUUC CUGGAGAG 63
241GGACGCGUC CACGUUCC 64 692GAGAGGGUU UGGGGAAA 65
247GUCCACGUU CCGCCGCr~ 66 693AGAGr;GUUU GGGGAAAG 67
248UCCACGUUC CGCCGCGI; 68 716GAGAAGAUC UACGGGCC 69
292GA~t;r~Cr;UC GGCC3GCGG 70 718GAAGAUCUA C~:3GGCCUG 71
308GACACGCUC AGCAUUCG 72 742AACGGACUA CAGGGACA 73
314CUCAGCAUU CGGACCAG 74 763GCUr,cl:;Guu CAr CCUGC 75
315UCAGCAUUC GGACCAGC 76 764cuGcr~Gl~JucAGccuGcu 77
344CCCAGGCUC CAGCACCA 78 773AGCCUGCIJAUGCCAGGC 79
385GGCCAGGUU CCCGUCGC 80 788GCAGCACUU GAAGCUCC 81
386GCCAGGUUC CCGUCGCU 82 795UUGAAGCUC CAAGGAUC 83
391GUUCCCGUC GCUCGUCG 84 803CCAAGGAUC CUGAGCCU 85
395CCGUCGCUC GUCGUGUG 86 812CUGAGCCUC AACAACAA 87
398UCGCUCGUC GUGUr;CGC 88 826CAACCCAUA CUUCUCCG 89
425AUGAACGUC GUCUUCGU 90 829CCCAUACUU CUCCGGAC 91
428A~.CGuçr,uc uucGucr~r~ 92 830CCAUACUUC UCCGGACC 93
430CGUCGUCUU CGUCGGCG 94 832AUACWCUC CGGACCAU 95
431GUCGUCUUC r UCGr~Gr~C 96 841CGGACCAUA CGGGGAGG 97
434GUCUUCr,UC G~CGCCr;~ 98 854GAGGACGUC GUGUUCGU 99
473GGGGGCCUC GGCGACGU 100 859CGUCGUGUU CGUCUGCA 101
482GGCGACGUC CUCG(3CGG 102 860GUCGUr;UUC GUCUGCM 103
485GACr;UCCUC GGCGGCCU 104 863GUGUUCr;UC UGCMCGA 105
527C:ACCI;UGUC AUGGUCGU 106 888CCG~,CCCLJC UCUCGUGC 107
533GUCAUGGUC GUCUCUCC 108 890GGCCCUCUC UCGUGCUA 109
536AUGGUCGUC UCUCCCCG 110 892CCCUCUCUC GUGCUACC 111
898CUCC;UrCU~CCUCAAGA 112 1241AUGGACGUCAGCGAGUG 113
~ 902UGCUACCUCAAGAGCAA 114 1270GGACAAGUACAUCGCCG 115
913GAGCAACUA CCAGUCCC 116 1274AAGUACAUC GCCGUGM 117
919CUACCAGUCCCACGGCA 118 1285CGUGAAGUACGACGUGU 119
929CACGGCAUC UACAGGGA 120 1294CGACGUGUC GACGGCCG 121
931CGGCAUCUA CAGGGACG 122 1346GCGGAGGUC r;GGcuccc 123
951AGACCGCUU UCUGCAUC 124 1352Guçr~r~Gcuc CCGGUGGA 125
952GACCGCUUU CUGCAUCC 126 1370CGGAACAUC CCGCUGGU 127
953ACCGCUUUC UGCAUCCA 128 1384GGUGGCGUU CAUCGGCA 129
SUBSTITUTE SHEET (RULE 26)
CA 02226728 l998-0l-l3
WO 97/l03Z8 PCI'/US96/11689
TablelllA
7~
nt. Substrate Seq. ID nt. Substrate Se4. ID
Position No. Position No.
959UUCUGCAUC CACAACAU 130 1385GUGGCGUUC AUCGGCAG 131
968CACAACAUC UCCUACCA 132 1388GCGUUCAUC GGCAGGCU 133
970CAACAUCUC CUACCAGG 134 1421CCCGACGUC AUGGCGGC 135
973CAUCUCCUA CCAGGGCC 136 1436GCCGCCAUC CCGCAGCU 137
985GGGCCGGUU CGCCUUCU 138 1445CCGCAGCUC AUGGAGAU 139
986GGCCr~GUUC GCCUUCUC 140 1472GUGCAGAUC GUUCUGCU 141
991GUUCGCCUU CUCCGACU 142 1475CAGAUCGUU CUGCUGGG 143
992UUCGCCUUC UCCGACUA 144 1476AGAUCGUUC UGCUGGGC 145
994CGCCUUCUC CGACUACC 146 1501GAAGAAGUU CGAGCGCA 147
1000CUCCGACUA CCCGGAGC 148 1502AAGAAGUUC GAGCGCAU 149
1016CUGAACCUC CCGGAGAG 150 1514CGCAUGCUC AUGAGCGC 151
1027GGAGAGAUU CAAGUCGU 152 1534GGAGAAGUU CCCAGGCA 153
1028GAGAGAUUC AAGUCGUC 154 1535GAGAAGUUC CCAGGCAA 155
1033AUUCAAGUC GUCCUUCG 156 1559GCCGUGGUC AAGUUCAA 157
1036CAAGUCGUC CUUCGAUU 158 1564GGUCAAGUU CAACGCGG 159
1039GUCGUCCUU CGAUUUCA 160 1565GUCAAGUUC A~CGCGGC 161
1040UCGUCCUUC GAUUUCAU 162 1589CACCACAUC AUGGCCGG 163
1044CCUUCGAUU UCAUCGAC 164 1610GACGUGCUC GCCGUCAC 165
1045CUUCGAUUU CAUCGACG 166 1616CUCGCCGUC ACCAGCCG 167
1046UUCGAUUUC AUCGACGG 168 1627CAGCCt;CUU CGAGCCCU 169
1049GAUUUCAUC GACGGCIJA 170 1628AGC::r~CVUC GAGCCCUC; 171
1057CGACGGCUA CGAGAAGC 172 1643UGCGGCCUC AUCCAGCU 173
1085CGGAAGAUC AACUGGAU 174 1646GGCCUrAUC CAGCUGr-~ 175
1106GCCGG~UC CUCGAGGC 176 1666GAUGCGAUA CGGAACGC 177
1109GGGAUCCUC GAGGCCGA 178 1690cur,cr,cr,uc CACCGGUG 179
1124GACAGGGUC CUCACCGU 180 1703GGUGGACUC GUCGACAC 181
1127AGr'GUCCUC ACCGUCAG 182 1706GGACUCGUC GACACCAU 183
1133CUCACCGUCAGCCCCIl~ 184 1715GACACCAUCAUCGAAGG 185
1141CAGCCCCUA CUACGCCG 186 1718ACCAUCAUC GAAGGCAA 187
1144CCCCUACUA CGCCGAGG 188 1735GArcGGr~uu CCACAUGG 189
1157GAGGAGCUC AUCUCCGG 190 1736~CCGr~r~UUC CACAUGGG 191
1160GAGCUCAUC UCCGGr~U 192 1751Gr'CCGCCUC AGcr-ucr~ 193
1162GCUCAUCUC CGGCAUCG 194 1757CUCAGCGUC GACUGCAA 195
1169UCCGGCAUC GCCAGGGG 196 1769UGCAACGUC GUGGAGCC 197
1187UGCGAGCUC GACAACAU 198 1787GCGGACGUC AAGAAGGU 199
1196GACAACAUC AUGCGCCU 200 1807CACCACCUU GCAGCGCG 201
1205AUGCGCCUC ACCGC'r,AU 202 1820cr~cr,cr.~uc AAGGUGGU 203
1214Ar-cGGrAuc ACCGGr~U 204 1829AAGGUGGUC GGCACGCC 205
1223ACCGr~r~UC GUCAACGG 206 1843GCCGGCGUA CGAGGAGA 207
1226GGCAUCGUC AACGGCAU 208 1871UGCAUGAUC CAGGAUCU 209
SUBSTITUTE SHEET (RULE 26)
CA 02226728 1998-01-13
WO 97/10328 PCTAUS96/11689
TablclllA
7~ -
nt. Substrate Seq. ID nt. Substrate Seq. ID
Position No. Position No.
1878UCCAGGAUC UCUCCUGG 210 2219CGGUAAUUU UAUAUUGC 211
1880CAGGAUCUC UCCUGGAA 212 2220GGUAAUUUU AUAUUGCG 213
1882GGAUCUCUC CUGGAAGG 214 2221GUAAUUUUA UAUUGCGA 215
19~GUGCUGCUC AGCCUCGG 216 2223AAUUUUAUA UUGCGAGU 217
1928CUCAGCCUC GGGGUCGC 218 2225UUUUAUAUU GCGAGUAA 219
1934CUCGGGGUC GCCGGCGG ~o 2232UUGCGAGUAAAUAAAUG ~1
1955CCAGGGGUC GAAGGCGA 222 2236GAGUAAAUA AAUGGACC 223
1970GAGGAGAUC rCt;CCGCU 224 2248GGACCUGUA GUGGUGGA 225
1979GCGCCI;CUC GCCAAGGA 226
2012UGAAGAGUU CC;GCCUGG 227
2013GAAGAGUUC GGCCUGCA 228
2033CCCCUGAUC UCGCGCC;U 229
2035CCUGAUCUC GCGCGUGG 230
2055AAACAUGUU GGGACAUC 231
2063UGGGACAUC UUCUUAUA 232
2065GGACAUCUU CUUAUAUA 233
2066GACAUCUUC UUAUAUAU 234
2068CAUCUUCUU AUAUAUGC 235
2069AUCUUCUUA UAUAUGCU 236
2071CUUCUUAUA UAUGCUGU 237
2073UCUUAUAUA UGCUGUUU 238
2080UAUGCUGUU UCr~UU!JAU 239
2081AUGCUGUUU CGUUUAUG 240
2082UGCUGUUUC GUUUAUGU 241
2085UGUUUCGUU UAUGUGAU 242
2086GUUUCGUUU AUGUGAUA 243
2087UUUCGUUUA UGUGAUAU 244
2094UAUGUGAUA UGGACAAG 245
2104GGACAAGUA UGUGUAGC 246
2110GUAUGUGUA GCUGCUUG 247
2117UAGCUGCUU GCUUGUGC 248
2121UGCUUGCUU GUGCUAGU 249
2127CUUGUGCUA GUGUAAUA 250
2132GCUAGUGUA AUAUAGUG 251
2135AGUGUAAUA UAGUGUAG 252
2137UGUAAUAUA GUGUAGUG 253
2142UAUAGUGUA GUGGUGGC 254
2165CACAACCUA AUAAGCGC 255
2168AACCUAAUA AGCGCAUG 256
2181CAUGAACUA AUUGCUUG 257
2184GAACUAAUU GCUUGCGU 258
2188UAAUUGCUU GCGUGUGU 259
2197GCC;U1:3UGUA GUUAAGUA 260
2200UGUGUAGUU AAGUACCG 261
2201GUGUAGUUA AGUACCGA 262
2205AGUUAAGUA CCGAUCGG 263
2211GUACCGAUC GGUAAUUU 264
2215CGAIlCGGUA AUUUUAUA 265
2218UCGGUAAUU UUAUAUUG 266
SUBSTITUTE SHEET (RULE 26)
CA 02226728 1998-01-13
WO97/10328 PCTrUS96/11689
TablelllB
76
Table III B: Hammerhead Ribozvme Sequence Targeted Against GBSS mRNA
nt. Position HH Ribo~c Sc.~._c ce Seq. ID
No.
12 UGGCUGUGGC CUGAUGA X GAA AUCGAUCGGU 267
68 GCAGUGAGUU CUGAUGA X GAA AUUCCUUCCU 268
73 GGCUGGCAGU CUGAUGA X GAA AGUUUAUUCC 269
103 GACGGAGCAG CUGAUGA X GAA ACACUUCUCC 270
109 CUGGUGGACG CUGAUGA X GAA AGCAGUACAC 271
113 CGCACUGGUG CUGAUGAXGAAACGGAGCAGU272
146 UCGACGAGAU CUGAUGA X GAA AGCAGCCCUG 273
149 UCGUCGACGA CUGAUGA X GAA AUGAGCAGCC 274
151 GGUCGUCGAC CUGAUGA X GAA AGAUGAGCAG 275
154 ACUGGUCGUC CUGAUGA X GAA ACGAGAUGAG 276
169 CAUGCCGAUU CUGAUGA X GAA AUCCACUGGU 277
170 CCAUGCCGAU CUGAUGA X GAA AAUCCACUGG 278
173 CCGCCAUGCC CUGAUGA X GAA AUUAAUCCAC 279
186 GACGUGGCUA CUGAUGA X GAA AGCCGCCAUG 280
188 GCGACGUGGC CUGAUGA X GAA AGAGCCGCCA 281
196 GACGAGCUGC CUGAUGA X GAA ACGUGGCUAG 282
203 GCGUUGCGAC CUGAUGA X GAA AGCUGCGACG 283
206 CGCGCGUUGC CUGAUGA X GAA ACGAGCUGCG 284
230 ACGCGUCCGG CUGAUGAXGAAACGCCCAGGC285
241 GCGGAACGUG CUGAUGA X GAA ACGCGUCCGG 286
247 GCCGCGGCGG CUGAUGA X GAA ACGUGGACGC 287
248 CGCCGCGGCG CUGAUGA X GAA AACGUGGACG 288
292 GUCCGCCGCC CUGAUGA X GAA ACGCCGUCCG 289
308 UCCGAAUGCU CUGAUGAX GAAAGCGUGUCCG 290
314 CGCUGGUCCG CUGAUGAX GAAAUGCUGAGCG 291
315 GCGCUGGUCC CUGAUGA X GAA AAUGCUGAGC 292
344 GCUGGUGCUG CUGAUGA X GAA AGCCUGGGCG 293
385 GAGCGACGGG CUGAUGA X GAA ACCUGGCCCC 294
386 CGAGCGACGG CUGAUGAX GAAAACCUGGCCC 295
391 CACGACGAGC CUGAUGAX GAAACGGGAACCU 296
395 CGCACACGAC CUGAUGA X GAA AGCGACGGGA 297
398 UGGCGCACAC CUGAUGAX GAAACGAGCGACG 298
425 CGACGAAGAC CUGAUGA X GAA ACGUUCAUGC 299
428 CGCCGACGAA CUGAUGA X GAA ACGACGUUCA 300
430 GGCGCCGACG CUGAUGA X GAA AGACGACGUU 301
431 CGGCGCCGAC CUGAUGA X GAA AAGACGACGU 302
434 UCUCGGCGCC CUGAUGA X GAA ACGAAGACGA 303
473 GGACGUCGCC CUGAUGA X GAA AGGCCGCCGG 304
482 GGCCGCCGAG CUGAUGA X GAA ACGUCGCCGA 305
485 GCAGGCCGCC CUGAUGA X GAA AGGACGUCGC 306
527 AGACGACCAU CUGAUGAX GAA ACACGGUGCC 307
533 GGGGAGAGAC CUGAUGA X GAA ACCAUGACAC 308
536 AGCGGGGAGA CUGAUGA X GAA ACGACCAUGA 309
538 GUAGCGGGGA CUGAUGA X GAA AGACGACCAU 310
540 UCGUAGCGGG CUGAUGA X GAA AGAGACGACC 311
SUBSTITUTESHEET(RULE26~
CA 02226728 l99X-01-13
WO 97/10328 PCI'/US96/11689
Tabie IIIB
nt.Position HH Ribo~ meSequence Seq.ID
No.
547 GUACUGGUCGCUGAUGAXGAAAGCGGGGAGA 312
556 GGCGUCCUUGCUGAUGAXGAAACUGGUCGUA 313
581 UCUCGGACACCUGAUGAXGAAACGCUGGUGU 314
586 CUUGAUCUCGCUGAUGAXGAAACACGACGCU 315
593 CUCCCAUCUUCUGAUGAXGAAAUCUCGGACA 316
610 GACCGUCUCGCUGAUGAXGAAACCUGUCUCC 317
620 GGAAGAACCUCUGAUGAXGAAACCGUCUCGU 318
625 GCAGUGGAAGCUGAUGAXGAAACCUGACCGU 319
626 AGCAGUGGAACUGAUGAXGAAAACCUGACCG 320
628 GUAGCAGUGGCUGAUGAXGAAAGAACCUGAC 321
629 UGUAGCAGUGCUGAUGAXGAAAAGAACCUGA 322
637 UCCGCGCUUGCUGAUGAXGAAAGCAGUGGAA 323
661 GUGGUCAACGCUGAUGAXGAAACACGCGGUC 324
662 GGUGGUCAACCUGAUGAXGAAAACACGCGGU 325
665 GUGGGUGGUCCUGAUGAXGAAACGAACACGC 326
679 CCUCUCCAGGCUGAUGAXGAAACAGUGGGUG 327
680 CCCUCUCCAGCUGAUGAXGAAAACAGUGGGU 328
692 UCUUUCCCCACUGAUGAXGAAACCCUCUCCA 329
693 GUCUUUCCCCCUGAUGAXGAAAACCCUCUCC 330
716 CAGGCCCGUACUGAUGAXGAAAUCUUCUCCU 331
718 GUCAGGCCCGCUGAUGAXGAAAGAUCUUCUC 332
742 GUUGUCCCUGCUGAUGAXGAAAGUCCGUUCC 333
763 UAGCAGGCUGCUGAUGAXGAAACCGCAGCUG 334
764 AUAGCAGGCUCUGAUGAXGAAAACCGCAGCU 335
773 CUGCCUGGCACUGAUGAXGAAAGCAGGCUGA 336
788 UUGGAGCUUCCUGAUGAXGAAAGUGCUGCCU 337
795 AGGAUCCUUGCUGAUGAXGAAAGCUUCAAGU 338
803 UGAGGCUCAGCUGAUGAXGAAAUCCUUGGAG 339
812 GGUUGUUGUUCUGAUGAXGAAAGGCUCAGGA 340
826 UCCGGAGAAGCUGAUGAXGAAAUGGGUUGUU 341
829 UGGUCCGGAGCUGAUGAXGAAAGUAUGGGUU 342
830 AUGGUCCGGACUGAUGAXGAAAAGUAUGGGU 343
832 GUAUGGUCCGCUGAUGAXGAAAGAAGUAUGG 344
841 GUCCUCCCCGCUGAUGAXGAAAUGGUCCGGA 345
854 AGACGAACACCUGAUGAXGAAACGUCCUCCC 346
859 GUUGCAGACGCUGAUGAXGAAACACGACGUC 347
860 CGUUGCAGACCUGAUGAXGAAAACACGACGU 348
863 AGUCGUUGCACUGAUGAXGAAACGAACACGA 349
888 UAGCACGAGACUGAUGAXGAAAGGGCCGGUG 350
890 GGUAGCACGACUGAUGAXGAAAGAGGGCCGG 351
892 GAGGUAGCACCUGAUGAXGAAAGAGAGGGCC 352
898 GCUCUUGAGGCUGAUGAXGAAAGCACGAGAG 353
902 AGUUGCUCUUCUGAUGAXGAAAGGUAGCACG 354
913 GUGGGACUGGCUGAUGAXGAAAGUUGCUCUU 355
919 GAUGCCGUGGCUGAUGAXGAAACUGGUAGUU 356
929 CGUCCCUGUACUGAUGAXGAAAUGCCGUGGG 357
931 UGCGUCCCUGCUGAUGAXGAAAGAUGCCGUG 358
951 UGGAUGCAGACUGAUGAXGAAAGCGGUCUUU 359
952 GUGGAUGCAGCUGAUGAXGAAAAGCGGUCUU 360
953 UGUGGAUGCACUGAUGAXGAAAAAGCGGUCU 361
959 AGAUGUUGUGCUGAUGAXGAAAUGCAGAAAG 362
968 CCUGGUAGGACUGAUGAXGAAAUGUUGUGGA 363
SUBSTITUTESHEET(RULE26)
CA 02226728 1998-01-13
WO 97/10328 PCTAUS96/11689
TablelllB
78
nt.Position HH Ribo~meSequence Seq.ID
No.
970 GCCCUGGUAGCUGAUGAXGAAAGAUGUUGUG 364
973 CCGGCCCUGGCUGAUGAXGAAAGGAGAUGUU 365
985 GGAGAAGGCGCUGAUGAXGAAACCGGCCCUG 366
986 CGGAGAAGGCCUGAUGAXGAAAACCGGCCCU 367
991 GUAGUCGGAGCUGAUGAXGAAAGGCGAACCG 368
992 GGUAGUCGGACUGAUGAXGAAAAGGCGAACC 369
994 CGGGUAGUCGCUGAUGAXGAAAGAAGGCGAA 370
1000 CAGCUCCGGGCUGAUGAXGAAAGUCGGAGAA 371
1016 AUCUCUCCGGCUGAUGAXGAAAGGUUCAGCU 372
1027 GGACGACUUGCUGAUGAXGAAAUCUCUCCGG 373
1028 AGGACGACUUCUGAUGAXGAA MUCUCUCCG 374
1033 AUCGAAGGACCUGAUGAXGAAACUUGAAUCU 375
1036 GAAAUCGAAGCUGAUGAXGAAACGACUUGAA 376
1039 GAUGAAAUCGCUGAUGAXGAAAGGACGACUU 377
1040 CGAUGAAAUCCUGAUGAXGAAAAGGACGACU 378
10~ CCGUCGAUGACUGAUGAXGAAAUCGAAGGAC 379
1045 GCCGUCGAUGCUGAUGAXGAAAAUCGAAGGA 380
1046 AGCCGUCGAUCUGAUGAXGAAAAAUCGAAGG 381
1049 CGUAGCCGUCCUGAUGAXGAAAUGAAAUCGA 382
1057 GGGCUUCUCGCUGAUGAXGAAAGCCGUCGAU 383
1085 UCAUCCAGUUCUGAUGAXGAAAUCUUCCGGC 384
1106 CGGCCUCGAGCUGAUGAXGAAAUCCCr~GCCU 385
1109 UGUCGGCC~'~ ~J~AU&A X ~AA ~GGAUeCCGG 886
1124 UGACGGUGAGCUGAUGAXGAAACCCUGUCGG 387
1127 GGCUGACGGUCUGAUGAXGAAAGGACCCUGU 388
1133 AGUAGGGGCUCUGAUGAXGAAACGGUGAGGA 389
1141 CUCGGCGUAGCUGAUGAXGAAAGGGGCUGAC 390
1144 CUCCUCGGCGCUGAUGAXGAAAGUAGGGGCU 391
1157 UGCCGGAGAUCUGAUGAXGAAAGCUCCUCGG 392
1160 CGAUGCCGGACUGAUGAXGAAAUGAGCUCCU 393
1162 GGCGAUGCCGCUGAUGAXGAAAGAUGAGCUC 394
1169 AGCCCCUGGCCUGAUGAXGAAAUGCCGGAGA 395
1187 UGAUGUUGUCCUGAUGAXGAAAGCUCGCAGC 396
1196 UGAGGCGCAUCUGAUGAXGAAAUGUUGUCGA 397
1205 UGAUGCCGGUCUGAUGAXGAAAGGCGCAUGA 398
1214 CGAUGCCGGUCUGAUGAXGAAAUGCCGGUGA 399
1223 UGCCGUUGACCUGAUGAXGAAAUGCCGGUGA 400
1226 CCAUGCCGUUCUGAUGAXGAAACGAUGCCGG 401
1241 CCCACUCGCUCUGAUGAXGAAACGUCCAUGC 402
1270 CACGGCGAUGCUGAUGAXGAAACUUGUCCCU 403
1274 ACUUCACGGCCUGAUGAXGAAAUGUACUUGU 404
1285 CGACACGUCGCUGAUGAXGAAACUUCACGGC 405
1294 CACGGCCGUCCUGAUGAXGAAACACGUCGUA 406
1346 CCGGGAGCCCCUGAUGAXGAAACCUCCGCCU 407
1352 GGUCCACCGGCUGAUGAXGAAAGCCCGACCU 408
1370 CCACCAGCGGCUGAUGAXGAAAUGUUCCGGU 409 r
1384 CCUGCCGAUGCUGAUGAXGAAACGCCACCAG 410
1385 GCCUGCCGAUCUGAUGAXGAAAACGCCACCA 411
1388 CCAGCCUGCCCUGAUGAXGAAAUGAACGCCA 412
1421 CGGCCGCCAUCUGAUGAXGAAACGUCGGGUC 413
1436 UGAGCUGCGGCUGAUGAXGAAAUGGCGGCCG 414
1445 CCAUCUCCAUCUGAUGAXGAAAGCUGCGGGA 415
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TablelllB
79
nt.Position HH Ribo~meSe~ cc Seq.ID
No.
1472 CCAGCAGAACCUGAUGAXGAAAUCUGCACGU 416
1475 UGCCCAGCAGCUGAUGAXGAAACGAUCUGCA 417
1476 GUGCCCAGCACUGAUGAXGAAAACGAUCUGC 418
1501 CAUGCGCUCGCUGAUGAXGAAACUUCUUCUU 419
1502 GCAUGCGCUCCUGAUGAXGAAAACUUCUUCU 420
1514 CGGCGCUCAUCUGAUGAXGAAAGCAUGCGCU 421
1534 CUUGCCUGGGCUGAUGAXGM ACUUCUCCUC 422
1535 CCUUGCCUGGCUGAUGAXGAAAACUUCUCCU 423
1559 CGUUGAACUUCUGAUGAXGAAACCACGGCGC 424
1564 CGCCGCGUUGCUGAUGAXGAAACUUGACCAC 425
1565 GCGCCGCGUUCUGAUGAXGAAAACUUGACCA 426
1589 CGCCGGCCAUCUGAUGA.XGAAAUGUGGUGCG 427
1610 UGGUGACGGCCUGAUGAXGAAAGCACGUCGG 428
1616 AGCGGCUGGUCUGAUGAXGAAACGGCGAGCA 429
1627 GCAGGGCUCGCUGAUGAXGAAAGCGGCUGGU 430
1628 CGCAGGGCUCCUGAUGAXGAAAAGCGGCUGG 431
1643 GCAGCUGGAUCUGAUGAXGAAAGGCCGCAGG 432
1646 CCUGCAGCUGCUGAUGAXGAAAUGAGGCCGC433
1666 GGGCGUUCCGCUGAUGAXGAAAUCGCAUCCC 434
1690 UCCACCGGUGCUGAUGAXGAAACGCGCAGGC 435
1703 UGGUGUCGACCUGAUGAXGAAAGUCCACCGG 436
1706 UGAUGGUGUCCUGAUGAXGAAACGAGUCCAC 437
1715 UGCCUUCGAUCUGAUGAXGAAAUGGUGUCGA 438
1718 UCUUGCCUUCCUGAUGAXGAAAUGAUGGUGU 439
1735 GCCCAUGUGGCUGAUGAXGAAACCCGGUCUU 440
1736 GGCCCAUGUGCUGAUGAXGAAAACCCGGUCU 441
1751 AGUCGACGCUCUGAUGAXGAAAGGCGG~CCA 442
1757 CGUUGCAGUCCUGAUGAXGAAACGCUGAGGC 443
1769 CCG~CUCCACCUGAUGAXGAAACGUUGCAGU 444
1787 CCACCUUCUUCUGAUGAXGAAACGUCCGCCG 445
1807 GGCGCGCUGCCUGAUGAXGAAAGGUGGUGGC 446
1820 CGACCACCUUCUGAUGAXGAAAUGGCGCGCU 447
1829 CCGGCGUGCCCUGAUGAXGAAACCACCUUGA 448
1843 CAUCUCCUCGCUGAUGAXGAAACGCCGGCGU 449
1871 AGAGAUCCUGCUGAUGAXGAAAUCAUGCAGU 450
1878 UUCCAGGAGACUGAUGAXGAAAUCCUGGAUC 451
1880 CCUUCCAGGACUGAUGAXGAAAGAUCCUGGA 452
1882 GCCCUUCCAGCUGAUGAXGAAAGAGAUCCUG 453
1922 CCCCGAGGCUCUGAUGAXGAAAGCAGCACGU 454
1928 CGGCGACCCCCUGAUGAXGAAAGGCUGAGCA 455
1934 CGCCGCCGGCCUGAUGAXGAAACCCCGAGGC 456
1955 CCUCGCCUUCCUGAUGAXGAAACCCCUGGCU 457
1970 CGAGCGGCGCCUGAUGAXGAAAUCUCCUCGC 458
1979 UCUCCUUGGCCUGAUGAXGAAAGCGGCGCGA 459
2012 CUGCAGGCCGCUGAUGAXGAAACUCUUCAGG 460
2013 CCUGCAGGCCCUGAUGAXGAAAACUCUUCAG 461
2033 CCACGCGCGACUGAUGAXGAAAUCAGGGGGC 462
2035 CACCACGCGCCUGAUGAXGAAAGAUCAGGGG 463
2055 AAGAUGUCCCCUGAUGAXGAAACAUGUUUGC 464
2063 UAUAUAAGAACUGAUGAXGAAAUGUCCCAAC 465
2065 CAUAUAUAAGCUGAUGAXGAAAGAUGUCCCA 466
2066 GCAUAUAUAACUGAUGAXGAAAAGAUGUCCC 467
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Table IIIB
nt. Position HH Ribozvme Sequence Seq ID
2068 CAGCAUAUAU CUGAUGA X GAAAGAAGAUGUC 468
2069 ACAGCAUAUA CUGAUGA X GAA AAGAAGAUGU 469
2071 AAACAGCAUA CUGAUGA X GAA AUAAGAAGAU 470
2073 CGAAACAGCA CUGAUGA X GAA AUAUAAGAAG 471
2080 ACAUAAACGA CUGAUGA X GAA ACAGCAUAUA 472
2081 CACAUAAACG CUGAUGA X GAA AACAGCAUAU 473
2082 UCACAUAAAC CUGAUGA X GAA AAACAGCAUA 474
2085 AUAUCACAUA CUGAUGA X GAA ACGAAACAGC 475
2086 CAUAUCACAU CUGAUGA X GAA AACGAAACAG 476
2087 CCAUAUCACA CUGAUGA X GAA AAACGAAACA 477
2094 UACUUGUCCA CUGAUGA X GAA AUCACAUAAA 478
2104 CAGCUACACA CUGAUGA X GAA ACUUGUCCAU 479
2110 AGCAAGCAGC CUGAUGAXGAAACACAUACUU 480
2117 UAGCACAAGC CUGAUGAXGAAAGCAGCUACA 481
2121 ACACUAGCAC CUGAUGA X GAA AGCAAGCAGC 482
2127 UAUAUUACAC CUGAUGA X GAA AGCACAAGCA 483
2132 UACACUAUAU CUGAUGA X GAA ACACUAGCAC 484
2135 CACUACACUA CUGAUGA X GAA AUUACACUAG 485
2137 ACCACUACAC CUGAUGA X GAA AUAUUACACU 486
2142 UGGCCACCAC CUGAUGA X GAA ACACUAUAUU 487
2165 AUGCGCUUAU CUGAUGA X GAA AGGUUGUGCC 488
2168 UUCAUGCGCU CUGAUGA X GAA AUUAGGUUGU 489
2181 CGCAAGCAAU CUGAUGA X GAA AGUUCAUGCG 490
2184 ACACGCAAGC CUGAUGA X GAA AUUAGUUCAU 491
2188 CUACACACGC CUGAUGA X GAA AGCAAUUAGU 492
2197 GGUACUUAAC CUGAUGA X GAA ACACACGCAA 493
2200 AUCGGUACUU CUGAUGA X GAA ACUACACACG 494
2201 GAUCGGUACU CUGAUGAX GAAAACUACACAC 495
2205 UACCGAUCGG CUGAUGA X GAA ACUUAACUAC 496
2211 UAAAAUUACC CUGAUGA X GAA AUCGGUACUU 497
2215 AAUAUAAAAU CUGAUGA X GAA ACCGAUCGGU 498
2218 CGCAAUAUAA CUGAUGA X GAA AUUACCGAUC 499
2219 UCGCAAUAUA CUGAUGA X GAA AAUUACCGAU 500
2~o CUCGCAAUAU CUGAUGA X GAA AAAUUACCGA 501
2221 ACUCGCAAUA CUGAUGA X GAA AMAUUACCG 502
~2~3 UUACUCGCAA CUGAUGA X GAA AUAAAAUUAC 503
2~5 AUUUACUCGC CUGAUGA X GAA AUAUAAAAUU 504
2232 UCCAUUUAUU CUGAUGA X GAA ACUCGCAAUA 505
2236 CAGGUCCAUU CUGAUGA X GAA AUUUACUCGC 506
2248 UUUCCACCAC CUGAUGA X GAA ACAGGUCCAU 507
Where "X" ,c~l~;sclll~ stem II region of a HH riboyme (Hertel et al., 199~ I~ ucleic
Acids Res. 20 3252). The length of stem II may be 2 2 base-pairs.
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Table IV
Table IV: HH Ribozyme Sequences Tested ~in.ct GBSS rnRNA
nt. HH I~ e Sequence ~}1 ~
Posif;ion I.D.
425 CGACGAAGAC CUGAUGAGGCCGAAAGGCCGAA ACGUUCAUGC 2
593 CUCCCAUCUU CUGAUG~c-.GCCc-.~AAGGCCGAA ArJcucc-~rArA 3
742 Cwuciucc~uci Cur~ArTr~AGGccGAAAGGccGAA AC;U~_CCjUUC C 4
812 ~uuciuucjuu cur-2~rr~rGccr~AAGGccGAA ArGcur~r~-A 5
892 GAr~jrTAC-r~r CU~AUG~G&CCGAAAGGCCGAA AGAGAGGGCC 6
913 GUGGGACUGG cu~ rr~ r~Gccr~AAAr~iccr~A Acjuucjc-uc uu 7
919 c.AIlcjccciu~j CUGAUGAc-,GCCc-.A~AGGCCGAA ACUGGUAGUU 8
953 uwr,r.~TTGrP, ~ ~CGAA A~jC~iu~-U 9
959 AGAu~uu~iu~j r-ur~TTr~rrccrz~AAGGccGAA AUGCAGAAAG 10
968 C~uw~GGA cuGAlTr~rGccrAAAGGccGAA A~KjUU~ '3 11
1016 Al ILuuuCC~G cuGAuG~rGcrr-~AAGGccG~A AGGWCAGCU 12
1028 ~r~ rr~ArqTu cur~rJrz~rrccr-~r-r~ccr~A AAU~ lCC~ 13
1085 UCAUCCAGW cur~rTr~r~iccrA~AGGccG~ Iuuu~l'GGC 14
1187 U~ ~uwu~ CUG~Ur-Ar~iCrr~AAr~GCCG~A ~ -uCG~ 15
1196 ur.~r~Gr~rT cuGATTr-~rGcr-r~AAAGGccG~ Au~juu~i~J~:G~. 16
1226 cr~rl(j~C-~uu cur~rTr~rricrrA~AcGccG~A ACGMTGCCGG 17
1241 CCr2~rUCGCU CUG~TTr-~r~iCrr-~AAGGCCGaA ACGUCCAUGC 18
1270 r~rr,Grr.ArTG cur-AT7r-Ar~cr~AAAr~GccG2~A A~:UU(jUCC--u 19
1352 r~;UcrP~rrc~i c~ur2~TTr2~rGccr~Ac~Gccr~A Ar~cccrArcu20
1421 ~ C-I~ArT cur~ Tr~r~Gccr~Ap~r~r~ccc~ C(jU~W~ 21
1534 ~ uu~u~i CUr-Z~lTr~ArGcc'r~A~Ar~ic~~ A~'UU~.'UL~'~' 22
1715 u~:~:uuu-GAU CUr-2~JrArGCC'r-~AAGGCCC~ A~Jwu~ju~-~' 23
1787 rr~ ~uu~uu cur~TTrz~rr~cr~AAGGccG~ ACGuu-CGCC~ 24
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83
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SUBSTITUTE SHEET (RULE 26)
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W O 97tlO328 84 PCT~US96/~1689
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TableVI
8~ ~,
Table Vl: Delta-9 Desdlu~dse HH RiLG~"...e Target Sequences
nt. S~bal~ Seq. ID nt. Substrate Seq. ID
P~siLion No.Posilion No.
13 CGCGCCCUC UGCCGCUU 644 319 GUCCAGGUU ACACAUUC 645
21 CUGCCGCUU GUUCGUUC 646 320 UCCAGGUUACACAUUCA 647
24 CCGCUUGUU CGUUCCUC 648 326 UUACACAUU CAAUGCCA 649
CGCUUGUUC GUUCCUCG 650 327 UACACAUUC AAUGCCAC 651
28 UUGUUCGUU CCUCGCGC 652 338 UGCCACCUCACAAGAUU 653
29 UGUUCGUUC CUCGCGCU 654 346 CACAAGAUU GAAAUUUU 655
32 UCGUUCCUC GCGCUCGC 656 352 AUUGAAAUU UUCAAGUC 657
38 CUCGCGCUC GCCACCAG 658 353 UUGAAAUUU UCAAGUCG 659
63 ACACACAUC CCAAUCUC 660 354 UGAAAUUUU CAAGUCGC 661
69 AUCCCAAUC UCGCGAGG 662 355 GAAAUUUUCAAGUCGCU 663
71 CCCAAUCUC GCGAGGGC 664 360 UUUCAAGUC GCUUGAUG 665
92 AGCAGGGUC UGCGGCGG 666 364 AAGUCGCUU GAUGAUUG 667
117 GCCGCGCUU CCGGCUCC 668 371 UUGAUGAUU GGGCUAGA 669
118 CCGCGCUUC CGGCUCCC 670 377 AUUGGGCUA GAGAUAAU 671
124 UUCCGGCUC CCCUUCCC 672 383 CUAGAGAUA AUAUCUUG 673
129 GCUCCCCUU CCCAUUGG 674 386 GAGAUAAUA UCUUGACG 675
130 CUCCCCUUC CCAUUGGC 676 388 GAUAAUAUC UUGACGCA 677
135 CUUCCCAUU GGCCUCCA 678 390 UAAUAUCUU GACGCAUC 679
141 AUUGGCCUC CACGAUGG 680 398 UGACGCAUC UCAAGCCA 681
154 AUGGCGCUC CGCCUCAA 682 400 ACGCAUCUC AAGCCAGU 683
160 CUCCGCCUC AACGACGU 684 409 AAGCCAGUC GAGAAGUG 685
169 AACGACGUCC;C5CUCU5 686 419 AGAAGUGUU GGCAGCCA 687
175 GUCGCt--CUC UGCCUCUC 688 434 CACAGGAUU UCCUCCCG 689
181 CUCUGCCUC UCCCCGCC 690 435 ACAGGAUUU CCUCCCGG 691
183 CUGCCUCUC CCCGCCGC 692 436 CAGGAUUUC CUCCCGGA 693
193 CCGCCGCUC GCCGCCCG 694 439 GAUUUCCUC CCGGACCC 695
228 CGGCAGGUU CGUCGCCG 696 453 CCCAGCAUC UGAAGGAU 697
229 GGCAGGUUC GUCGCCI-~U 698 462 UGAAGGAUU UCAUGAUG 699
232 AGGUUCGUC GCCI;UCl;C 700 463 GAAGGAUUU CAUGAUGA 701
238 GUCGCCGUC GCCUCCAU 702 464 AAGGAUUUC AUGAUGAA 703
243 CGUCGCCUC CAUGACGU 704 475 GAUGAAGUU AAGGAGCU 705
252 CAUGACGUC CGCCGUCU 706 476 AUGAAGUUAAGGAGCUC 707
259 UCCGCCGUC UCCACCAA 708 484 AAGGAGCUC AGAGAACG 709
261 CGCCGUCUC CACCAAGG 710 505 AAGGAAAUC CCUGAUGA 711
271 ACCAAGGUC GAGAAUAA 712 515 CUGAUGAUU AUUUUGUU 713
278 UCGAGAAUA AGAAGCCA 714 516 UGAUGAUUA UUUUGUUU 715
288 GAAGCCAUU UGCUCCUC 716 518 AUGAUUAUU UUGUUUGU 717
289 AAGCCAUUU GCUCCUCC 718 519 UGAUUAUUU UGUUUGUU 719
293 CAUUUGCUC CUCCAAGG 720 520 GAUUAUUUU GUUUGUUU 721
296 UUGCUCCUC CAAGGGAG 722 523 UAUUUUGUU UGUUUGGU 723
307 AGGGAGGUACAUGUCCA 724 524 AUUUUGUUU GUUUGGUG 725
313 GUACAUGUC CAGGUUAC 726 527 UUGUUUGUU UGGUGGGA 727
528 UGUUUGUUU GGUGGGAG 728 857 ACACUGCUC GUCACGCC 729
544 GACAUGAUU ACCGAGGA 730 860 CUGCUCGUC ACGCCAAG 731
545 ACAUGAUUA CCGAGGAA 732 873 CAAGGACUU UGGCGACU 733
557 AGGAAGCUC UACCAACA 734 874 AAGGACUUU GGCGACUU 735
559 GMGCUCUACCAACAUA736 882UGGCGACUU AAAGCUUG 737
567 ACCAACAUA CCAGACUA 738 883 GGCGACUUA AAGCUUGC 739
575 ACCAGACUA UGCUUAAC 740 889 UUAAAGCUU GCACAAAU 741
580 ACUAUGCUU AACACCCU 742 898 GCACAAAUC UGCGGCAU 743
581 CUAUGCUUAACACCCUC 744 907 UGCGGCAUC AUCGCCUC 745
589 AACACCCUC GACGGUGU 746 910 GGCAUCAUC GCCUCAGA 747
598 GACGGUGUC AGAGAUGA 748 915 CAUCGCCUC AGAUGAGA 749
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TableVI
. 86
nt. Substrate Seq. ID nt. Substrate Seq. ID
Posilion No.Posilion No.
637UGGGCUGUU UGGACGAG750 942AACUGCGUA CACCAAGA 751
638GGGCUGUUU GGACGAGG752 952ACCAAGAUC GUGGAGAA 753
680AUGGUGAUC UGCUCAAC754 966GAAGCUGUU UGAGAUCG 755 "
685GAUCUGCUC AACAAGUA756 967AAGCUGUUU GAGAUCGA 757
693CAACAAGUA UAUGUACC758 973UUUGAGAUC GACCCUGA 759
695ACAAGUAUA UGUACCUC760 986CUGAUGGUA CCGUGGUC 761
699GUAUAUGUACCUCACUG 762 994ACCGUGGUC GCUCUGGC 763
703AUGUACCUCACUGGGAG 764 998UGGUCGCUC UGGCUGAC 765
719GGGUGGAUA UGAGGCAG766 1024AAGAAGAUC UCAAUGCC 767
730AGGCAGAUU GAGAAGAC768 1026GAAGAUCUC AAUGCCUG 769
742AAGACAAUU CAGUAUCU770 1047CCUGAUGUU UGACGGGC 771
743AGACAAUUC AGUAUCUU772 1048CUGAUGUUU GACGGGCA 773
747AAUUCAGUA UCUUAUUG774 1071CAAGCUGUU CGAGCACU 775
749UUCAGUAUC UUAUUGGC776 1072AAGCUGUUC GAGCACUU 777
751CAGUAUCUU AUUGGCUC778 1080CGAGCACUU CUCCAUGG 779
752AGUAUCUUA UUGGCUCU780 1081GAGCACUUC UCCAUGGU 781
754UAUCUUAUU GGCUCUGG782 1083GCACUUCUC CAUGGUCG 783
759UAUUGGCUC UGGAAUGG784 1090UCCAUGGUC GCGCAGAG 785
770GAAUGGAUC CUAGGACU786 1102CAGAGGCUU GGCGUUUA 787
773UGGAUCCUA GGACUGAG788 1108CUUGGCGUU UACACCGC 789
785CUGAGAAUA AUCCUUAU790 1109UUGGCGUUU ACACCGCC 791
788AGAAUAAUC CUUAUCUU792 1110Ut:GCGUUIJ~ CACCGCCA 793
791AUAAUCCUU AUCUUGGU794 1125CAGGGACUA CGCCGACA 795
792UAAUCCUUA UCUUGGUU796 1135GCCGACAUC CUCGAGUU 797
794AUCCUUAUC UUGGUUUC798 1138GACAUCCUC GAGUUCCU 799
796CCUUAUCUU GGUUUCAU800 1143CCUCGAGUU CCUCGUCG 801
800AUCUUGGUU UCAUCUAC802 1144CUCGAGUUC CUCGUCGA 803
801UCUUGGUUU CAUCUACA804 1147GAGUUCCUC GUCGACAG 805
802CUUGGUUUC AUCUACAC806 1150UUCCUCGUC GACAGGUG 807
805GGUUUCAUC UACACCUC808 1181UGACUGGUC UGUCGGGU 809
807UUUCAUCUA CACCUCCU810 1185UGGUCUGUC GGGUGAAG 811
813CUACACCUC CUUCCAAG812 1212GCAGGACUA CCUUUGCA 813
816C~CCUCCUU CCAAGAGC814 1216GACUACCUU UGCACCCU 815
817ACCUCCUUC CAAGAGCG816 1217ACUACCUUU GCACCCUU 817
834GGCGACCUU CAUCUCAC818 1225UGCACCCUU GCUUCAAG 819
835GCGACCUUCAUCUCACA 820 1~9CCCUUGCUU CAAGAAUC 821
838ACCUUCAUC UCACACGG822 1230CCUUGCUUC AAGAAUCA 823
840CUUCAUCUC ACACGGGA824 1237UCAAGAAUC AGGAGGCU 825
1292CGCU~CCUU UCAGCUGG 826 1494 UUUGAUGUACAACCUGU 827
1293GCUGCCUUU CAGCUGGG 828 1546 CAUGCCGUA CUUUGUCU 829
1294CUGCCUUUC AGCUGGGU 830 1549 GCCGUACUU UGUCUGUC 831
1303AGCUGGGUA UACGGUAG 832 1550 CCGUACUUU GUCUGUCG 833
1305CUGGGUAUA CGGUAGGG 834 1553 UACUUUGUC UGUCGCUG 835
1310UAUACGGUA GGGACGUC 836 1557 UUGUCUGUC GCUGGCGG 837
1318AGGGACGUC CAACUGUG 838 1571 CGGUGUGUU UCGGUAUG 839
1331UGUGAGAUC GGAAACCU 840 1572 GGUGUGUUU CGGUAUGU 841
1348GCUGCGGUC UGCUUAGA 842 1573 GUGUGUUUC GGUAUGUU 843
1353GGUCUGCUU AGACAAGA 844 1577 GUUUCGGUA UGUUAUUU 845
1354GUCUGCUUA GACAAGAC 846 1581 CGGUAUGUU AUUUGAGU 847
1372UGCUGUGUC UGCGUUAC 848 1582 GGUAUGUUA UUUGAGUU 849 J
1378GUCUGCGUU ACAUAGGU 850 1584 UAUGUUAUU UGAGUUGC 851
1379UCUGCGUUA CAUAGGUC 852 1585 AUGUUAUUU GAGUUGCU 853
1383CGUUACAUA GGUCUCCA 854 1590 AUUUGAGUU GCUCAGAU 855
1387ACAUAGGUC UCCAGGUU 856 1594 GAGUUGCUC AGAUCUGU 857
1389AUAGGUCUC CAGGUUUU 858 1599 GCUCAGAUC UGUUAAAA 859
1395CUCCAGGUU UUGAUCAA 860 1603 AGAUCUGUU AAAAAA~ 861
1396UCCAGGUUU UGAUCAAA 862 1604 GAUCUGUUA AAA~AAA 863
SUBSTITUTE SHEET (RULE 26)
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WO 97/10328 PCTrUS96/11689
TableVI 87
nt. Substrate Seq. ID
Fosition No.
1397CCAGGUUUU GAUCAAAU864
1401GUUUUGAUC AAAUGGUC865
1409CAAAUGGUC CCGUGUCG866
1416UCCCGUGUC GUCUUAUA867
1419CGUGUCGUC UUAUAGAG868
1421UGUCGUCUU AUAGAGCG869
1422GUCGUCUUA UAGAGCGA870
1424CGUCUUAUA GAGCGAUA871
1432AGAGCGAUA GGAGAACG872
1444GAACGUGUU GGUCUGUG873
1448GUGUUGGUC UGUGGUGU874
1457UGUGGUGUA GCUUUGUU875
1461GUGUAGCUU UGUUUUUA876
1462UGUAGCUUU GUUUUUAU877
1465AGCUUUGUU UUUAUUUU878
1466GCUUUGUUU UUAUUUUG879
1467CUUUGUUUU UAUUUUGU880
1468UUUGUUUUU AUUUUGUA881
1469UUGUUUUUA UUUUGUAU882
1471GUUUUUAUU UUGUAUUU883
1472UUUUUAUUU UGUAUUUU884
1473UUUUAUUUU GUAUUUUU885
1476UAUUUUGUA UUUUUCUG886
1478UUUUGUAUU UUUCUGCU887
1479UUUGUAUUU UUCUGCUU888
1480UUGUAUUUU UCUGCUUU889
1481UGUAUUUUU CUGCUUUG890
1482GUAUUUUUC UGCUUUGA891
1487UUUCUGCUU UGAUGUAC892
1488UUCUGCUUU GAUGUACA893
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Tablc Vll
88
Table Vll: Delta-9 Dtsalu...se HH Ribozvme Sc.~. ~r--
nt. Ribozvmese~ Se~. ID No.
Position
13 AAGCGGCA CUGAUGA X GAA AGGGCGCG 894
21 GAACGAAC CUGAUGAX GAA AGCGGCAG 895
24 GAGGAACG CUGAUGA X GAA ACAAGCGG 896
CGAGGAAC CUGAUGA X GAA AACAAGCG 897
28 GCGCGAGG CUGAUGA X GAAACGAACAA 898
29 AGCGCGAG CUGAUGA X GAA AACGAACA 899
32 GCGAGCGC CUGAUGA X GAA AGGAACGA 900
38 CUGGUGGC CUGAUGA X GAA AGCGCGAG 901
63 GAGAUUGG CUGAUGAX GAAAUGUGUGU 902
69 CCUCGCGA CUGAUGA X GAA AUUGGGAU 903
71 GCCCUCGC CUGAUGAX GAAAGAUUGGG 904
92 CCC;CCGCA CUGAUGA X GAA ACCCUGCU 905
117 GGAGCCGG CUGAUGA X GAA AGCGCGGC 906
118 GGGAGCCG CUGAUGA X GAA AAGCGCGG 907
124 GGGAAGGG CUGAUGA X GAA AGCCGGAA 908
129 CCAAUGGG CUGAUGA X GAA AGGGGAGC 909
130 GCCAAUGG CUGAUGA X GAA AAGGGGAG 910
135 UGGAGGCCCUGAUGAXGAAAUGGGAAG911
141 CCAUCGUG CUGAUGA X GAA AGGCCAAU 912
154 UUGAGGCG CUGAUGA X GAA AGCGCCAU 913
160 AÇS3U5GUU GUS~ ;A~rJAAA!:3~Cr-r-4-- ;.14
169 CAGAGCGC CUGAUGA X GAA ACGUCGUU 915
175 GAGAGGCA CUGAUGA X GAA AGCGCGAC 916
181 GGCGGGI:A CUGAUGA X GAA AGGCAGAG 917
183 GC~;GCGC;G CUGAUGA X GAA AGAGGCAG 918
193 CGGGCGGC CUGAUGA X GAA AGCGGCGG 919
~8 CGGCGACG CUGAUGA X GAA ACCUGCCG 920
~g ACGGCGAC CUGAUGA X GAA AACCUGCC 921
232 GCGACGGC CUGAUGA X GAA ACGAACCU 922
238 AUGGAGGC CUGAUGA X GAA ACGGCGAC 923
243 ACGUCAUG CUGAUGA X GAA AGGCGACG 924
252 AGACGGCG CUGAUGA X GAA ACGUCAUG 925
259 UUGGUGGA CUGAUGA X GAA ACGGCGGA 926
261 CCUUGGUG CUGAUGA X GAA AGACGGCG 927
271 UUAUUCUC CUGAUGA X GAA ACCUUGGU 928
278 UGGCUUCU CUGAUGAX GAAAUUCUCGA 929
288 GAGGAGCA CUGAUGA X GAA AUGGCUUC 930
289 GGAGGAGC CUGAUGA X GAA AAUGGCUU 931
293 CCUUGGAG CUGAUGAX GAA AGCAAAUG 932
296 CUCCCUUG CUGAUGA X GAA AGGAGCAA 933
307 UGGACAUG CUGAUGAX GAAACCUCCCU 934
313 GUAACCUG CUGAUGA X GAA ACAUGUAC 935
319 GAAUGUGU CUGAUGA X GAA ACCUGGAC 936
320 UGAAUGUG CUGAUGAX GAAAACCUGGA 937
326 UGGCAUUG CUGAUGA X GAA AUGUGUAA 938
327 GUGGCAUU CUGAUGA X GAA AAUGUGUA 939
338 AAUCUUGU CUGAUGAX GAAAGGUGGCA 940
346 AAAAUUUC CUGAUGA X GAA AUCUUGUG 941
352 GACUUGAA CUGAUGA X GAA AUUUCAAU 942
353 CGACUUGA CUGAUGAX GAA AAUUUCAA 943
354 GCGACUUG CUGAUGA X GAA AAAUUUCA 944
355 AGCGACUU CUGAUGA X GAA AAAAUUUC 945
360 CAUCAAGC CUGAUGA X GAA ACUUGAAA 946
364 CAAUCAUC CUGAUGA X GAAAGCGACUU 947
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CA 02226728 1998-01-13
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TableVII
89
nt. Ribozyme s~l. ~ e Seq. ID No.
Position
371 UCUAGCCC CUGAUGAX GAAAUCAUCAA 948
377 AUUAUCUC CUGAUGA X GAA AGCCCAAU 949
383 CAAGAUAU CUGAUGA X GAAAUCUCUAG 950
386 CGUCAAGA CUGAUGA X GAA AUUAUCUC 951
388 UGCGUCAA CUGAUGA X GAA AUAUUAUC 952
390 GAUGCGUC CUGAUGA X GAA AGAUAUUA 953
398 UGGCUUGA CUGAUGA X GAA AUGCGUCA 954
400 ACUGGCUU CUGAUGA X GAA AGAUGCGU 955
409 CACUUCUC CUGAUGA X GAA ACUGGCUU 956
419 UGGCUGCC CUGAUGA X GAA ACACUUCU 957
434 CGGGAGGA CUGAUGA X GAA AUCCUGUG 958
435 CCGGGAGG CUGAUGA X GAA AAUCCUGU 959
436 UCCGGGAG CUGAUGAX GAAAAAUCCUG 960
439 GGGUCCGG CUGAUGA X GAA AGGAAAUC 961
453 AUCCUUCA CUGAUGA X GAA AUGCUGGG 962
462 CAUCAUGA CUGAUGA X GAA AUCCUUCA 963
463 UCAUCAUG CUGAUGA X GAA AAUCCUUC 964
464 UUCAUCAU CUGAUGA X GAA AAAUCCUU 965
475 AGCUCCUU CUGAUGA X GAA ACUUCAUC 966
476 GAGCUCCU CUGAUGA X GAA AACUUCAU 967
484 CGUUCUCU CUGAUGA X GAA AGCUCCUU 968
505 UCAUCAGG CUGAUGA X GAA AUUUCCUU 969
515 AACAAAAU CUGAUGA X GAA AUCAUCAG 970
516 AAACAAAA CUGAUGA X GAA AAUCAUCA 971
518 ACAAACAA CUGAUGA X GAA AUAAUCAU 972
519 AACAAACA CUGAUGA X GAA AAUAAUCA 973
520 AAACAAAC CUGAUGA X GAA AAAUAAUC 974
523 ACCAAACA CUGAUGA X GAA ACAAAAUA 975
524 CACCAAAC CUGAUGA X GAA AACAAAAU 976
527 UCCCACCA CUGAUGAX GAA ACAAACAA 977
528 CUCCCACC CUGAUGAX GAAAACAAACA 978
544 UCCUCGGU CUGAUGAX GAAAUCAUGUC 979
545 UUCCUCGG CUGAUGA X GAA AAUCAUGU 980
557 UGUUGGUA CUGAUGA X GAA AGCUUCCU 981
559 UAUGUUGG CUGAUGA X GAA AGAGCUUC 982
567 UAGUCUGG CUGAUGA X GAA AUGUUGGU 983
575 GUUAAGCA CUGAUGA X GAA AGUCUGGU 984
580 AGGGUGUU CUGAUGA X GAA AGCAUAGU 985
581 GAGGGUGU CUGAUGA X GAA AAGCAUAG 986
589 ACACCGUC CUGAUGA X GAA AGGGUGUU 987
598 UCAUCUCU CUGAUGA X GAA ACACCGUC 988
637 CUCGUCCA CUGAUGA X GAA ACAGCCCA 989
638 CCUCGUCC CUGAUGA X GAA AACAGCCC 990
680 GUUGAGCA CUGAUGA X GAA AUCACCAU 991
685 UACUUGUU CUGAUGA X GAA AGCAGAUC 992
693 GGUACAUA CUGAUGA X GAA ACUUGUUG 993
695 GAGGUACA CUGAUGA X GAA AUACUUGU 994
699 CAGUGAGG CUGAUGA X GAA ACAUAUAC 995
703 CUCCCAGU CUGAUGA X GAA AGGUACAU 996
719 CUGCCUCA CUGAUGA X GAA AUCCACCC 997
730 GUCUUCUC CUGAUGA X GAA AUCUGCCU 998
742 AGAUACUG CUGAUGA X GAA AUUGUCUU 999
743 AAGAUACU CUGAUGA X GAA AAUUGUCU 1000
747 CAAUAAGA CUGAUGA X GAA ACUGAAUU 1001
749 GCCAAUAA CUGAUGA X GAA AUACUGAA 1002
751 GAGCCAAU CUGAUGA X GAA AGAUACUG 1003
752 AGAGCCAA CUGAUGA X GAA AAGAUACU 1004
Sll~S 111 I.ITE SHEET (RULE 26)
CA 02226728 1998-01-13
W O97/10328 PCTrUS96/11689
TableVII
nt. Ribo~ meseq-:r-e Seq.lD No.
Position
754CCAGAGCC CUGAUGA X GAA AUAAGAUA1005
759CCAUUCCA CUGAUGA X GAA AGCCAAUA1006
770AGUCCUAG CUGAUGA X GAA AUCCAUUC1007
773CUCAGUCC CUGAUGA X GAA AGGAUCCA1008
785AUAAGGAU CUGAUGA X GAA AUUCUCAG1009
788AAGAUAAG CUGAUGA X GAA AUUAUUCU1010
791ACCAAGAU CUGAUGAX GAAAGGAUUAU 1011
792AACCAAGA CUGAUGA X GAA AAGGAUUA101Z
794GAAACCAA CUGAUGA X GAA AUMGGAU 1013
796AUGAAACC CUGAUGA X GAA AGAUAAGG1014
800GUAGAUGA CUGAUGA X GAA ACCAAGAU1015
801UGUAGAUG CUGAUGA X GAA AACCAAGA1016
802GUGUAGAU CUGAUGA X GAA AAACCAAG1017
805GAGGUGUA CUGAUGA X GAA AUGAAACC1018
807AGGAGGUG CUGAUGA X GAA AGAUGAAA1019
813CUUGGAAG CUGAUGA X GAA AGGUGUAG1020
816GCUCUUGG CUGAUGAX GAAAGGAGGUG 1021
817CGCUCUUG CUGAUGA X GAA AAGGAGGU 10~
834GUGAGAUG CUGAUGA X GAA AGGUCGCC1023
83~iUGUGAGAU CUGAUGA X GAA AAGGUCGC1024
838CCGUGUGA CUGAUGA X GAA AUGAAGGU1025
840UCCCGUGU CUGAUGA X GAA AGAUGAAG1026
857GGCGUGAC CUGAUGA X GAA AGCAGUGU1027
860CUUGGCGU CUGAUGA X GAA ACGAGCAG1028
873AGUCGCCA CUGAUGA X GAA AGUCCUUG1029
874AAGUCGCC CUGAUGA X GAA AAGUCCUU1030
882CAAGCUUU CUGAUGA X GAA AGUCGCCA1031
883GCAAGCUU CUGAUGA X GAA MGUCGCC 1032
889AUUUGUGC CUGAUGA X GAA AGCUUUAA1033
898AUGCCGCA CUGAUGA X GAA AUUUGUGC1034
907CA~GC~'AU CUGAUGA X GAA AUGCCGCA1035
910UCUGAGGC CUGAUGA X GAA AUGAUGCC1036
915UCUCAUCU CUGAUGA X GAA AGGCGAUG1037
942UCUUGGUG CUGAUGA X GAA ACGCAGUU1038
952UUCUCCAC CUGAUGA X GAA AUCUUGGU1039
966CGAUCUCA CUGAUGA X GAA ACAGCUUC1040
967UCGAUCUC CUGAUGA X GAA AACAGCUU1041
973UCAGGGUC CUGAUGA X GAA AUCUCAAA1042
986GACCACGG CUGAUGA X GAA ACCAUCAG1043
994GCCAGAGC CUGAUGA X GAA ACCACGGU1044
998GUCAGCCA CUGAUGA X GAA AGCGACCA1045
1024GGCAUUGA CUGAUGA X GAA AUCUUCUU1046
1026CAGGCAUU CUGAUGA X GAA AGAUCUUC1047
1047GCCCGUCA CUGAUGA X GAA ACAUCAGG1048
1048UGCCCGUC CUGAUGA X GAA AACAUCAG1049
1071AGUGCUCG CUGAUGAX GAAACAGCUUG 1050
1072AAGUGCUC CUGAUGA X GAA AACAGCUU1051
1080CCAUGGAG CUGAUGA X GAA AGUGCUCG1052
1081ACCAUGGA CUGAUGA X GAA AAGUGCUC1053
1083CGACCAUG CUGAUGA X GAA AGAAGUGC1054 "
1090CUCUGCGC CUGAUGA X GAA ACCAUGGA1055
1102UAAACGCCCUGAUGAXGAAAGCCUCUG 1056
1108GCGGUGUACUGAUGAXGAAACGCCAAG 1057
1109GGCGGUGU CUGAUGA X GAA AACGCCAA1058
1110UGGCGGUG CUGAUGAX GAAAAACGCCA 1059
1125UGUCGGCG CUGAUGAX GAAAGUCCCUG 1060
1135AACUCGAG CUGAUGAX GAAAUGUCGGC 1061
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TableVII
91
nt. Ribozvmeseqn~ ~e Seq.ID No.
Position
1138 AGGAACUCCUGAUGAXGAAAGGAUGUC 1062
1143CGACGAGG CUGAUGA X GAA ACUCGAGG 1063
1144UCGACGAG CUGAUGA X GAA AACUCGAG 1064
1147CUGUCGAC CUGAUGA X GAA AGGAACUC 1065
1150CACCUGUC CUGAUGA X GAA ACGAGGAA 1066
1181ACCCGACA CUGAUGA X GAA ACCAGUCA 1067
1185 CUUCACCCCUGAUGAXGAAACAGACCA 1068
1212UGCAAAGG CUGAUGA X GAA AGUCCUGC 1069
Z16AGGGUGCA CUGAUGA X GAA AGGUAGUC 1070
1217AAGGGUGC CUGAUGA X GAA AAGGUAGU 1071
1225CUUGAAGC CUGAUGA X GAA AGGGUGCA 1072
1229GAUUCUUG CUGAUGA X GAA AGCAAGGG 1073
1230UGAUUCUU CUGAUGA X GAA AAGCAAGG 1074
1237AGCCUCCU CUGAUGA X GAA AUUCUUGA 1075
1292CCAGCUGA CUGAUGA X GAA AGGCAGCG 1076
1293CCCAGCUG CUGAUGA X GAA AAGGCAGC 1077
1294ACCCAGCU CUGAUGA X GAA AAAGGCAG 1078
1303CUACCGUA CUGAUGA X GAA ACCCAGCU 1079
1305CCCUACCG CUGAUGA X GAA AUACCCAG 1080
1310GACGUCCC CUGAUGA X GAA ACCGUAUA 1081
1318CACAGUUG CUGAUGA X GAA ACGUCCCU 1082
1331AGGUUUCC CUGAUGA X GAA AUCUCACA 1083
1348UCUAAGCA CUGAUGA X GAA ACCGCAGC 1084
1353UCUUGUCU CUGAUGA X GAA AGCAGACC 1085
1354GUCUUGUC CUGAUGA X GAA AAGCAGAC 1086
1372GUAACGCA CUGAUGA X GAA ACACAGCA 1087
1378ACCUAUGU CUGAUGA X GAA ACGCAGAC 1088
1379GACCUAUG CUGAUGA X GAA AACGCAGA 1089
1383UGGAGACC CUGAUGA X GAA AUGUAACG 1090
1387AACCUG~A CUGAUGA X GAA ACCUAUGU 1091
1389AAAACCUG CUGAUGA X GAA AGACCUAU 1092
1395UUGAUCAA CUGAUGA X GAA ACCUGGAG 1093
1396UUUGAUCA CUGAUGA X GAA AACCUGGA 1094
1397AUUUGAUC CUGAUGA X GAA AAACCUGG 1095
1401GACCAUUU CUGAUGA X GAA AUCAAAAC 1096
1409CGACACGG CUGAUGA X GAA ACCAUUUG 1097
1416UAUAAGAC CUGAUGA X GAA ACACGGGA 1098
1419CUCUAUAA CUGAUGA X GAA ACGACACG 1099
1421CGCUCUAU CUGAUGA X GAA AGACGACA 1100
1422UCGCUCIJACUGAUGAXGAAAAGACGAC 1101
1424 UAUCGCUCCUGAUGAXGAAAUAAGACG 1102
1432 CGUUCUCCCUGAUGAXGAAAUCGCUCU 1103
1444CACAGACC CUGAUGA X GAA ACACGUUC 1104
1448 ACACCACACUGAUGAXGAAACCAACAC 1105
1457AACAAAGC CUGAUGAX GAAACACCACA 1106
1461UAAAAACA CUGAUGA X GAA AGCUACAC 1107
1462AUAAAAAC CUGAUGAX GAAAAGCUACA 1108
1465 AAAAUAAACUGAUGAXGAAACAAAGCU 1109
1466CAAAAUAA CUGAUGA X GAA AACAAAGC 1110
1467ACAAAAUA CUGAUGA X GAA AAACAAAG 1111
1468 UACAAAAUCUGAUGAXGAAAAAACAAA 1112
1469AUACAAAA CUGAUGA X GAA AAAAACAA 1113
1471AAAUACAA CUGAUGA X GAA AUAAAAAC 1114
1472AAAAUACA CUGAUGA X GAA AAUAAAAA 1115
1473AAAAAUAC CUGAUGA X GAA AAAUAAAA 1116
1476CAGAAAAA CUGAUGA X GAA ACAAAAUA 1117
1478AGCAGAAA CUGAUGA X GAA AUACAAAA 1118
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TableVII
9,
nt. Ribozvmeseqa- -e Seq.ID~io.
Position
1479 AAGCAGAA CUGAUGA X GAA AAUACAAA 1119
1480 AAAGCAGA CUGAUGA X GAA AAAUACAA 1120
1481 CAAAGCAG CUGAUGA X GAA AAAAUACA 1121
1482 UCAAAGCA CUGAUGA X GAA AAAAAUAC 1122
1487 GUACAUCA CUGAUGA X GAA AGCAGAAA 1123
1488 UGUACAUC CUGAUGA X GAA AAGCAGAA 1124
1494 ACAGGUUG CUGAUGA X GAA ACAUCAAA 1125
1546 AGACAAAG CUGAUGA X GAA ACGGCAUG 1126
1549 GACAGACA CUGAUGA X GAA AGUACGGC 1127
1550 CGACAGAC CUGAUGA X GAA AAGUACGG 1128
1553 CAGCGACA CUGAUGA X GAA ACAAAGUA 1129
1557 CCGCCAGC CUGAUGA X GAA ACAGACAA 1130
1571 CAUACCGA CUGAUGA X GAA ACACACCG 1131
1572 ACAUACCG CUGAUGA X GAA AACACACC 1132
1573 AACAUACC CUGAUGA X GAA AAACACAC 1133
1577 AAAUAACA CUGAUGA X GAA ACCGAAAC 1134
1581 ACUCAAAU CUGAUGA X GAA ACAUACCG 1135
1582 AACUCAAACUGAUGAXGAAAACAUACC 1136
1584 GCAACUCA CUGAUGA X GAA AUAACAUA 1137
1585 AGCAACUC CUGAUGA X GAA AAUAACAU 1138
1590 AUCUGAGCCUGAUGAXGAAACUCAAAU 1139
1594 ACAGAUCUCUGAUGAXGAAAGCAACUC 1140
1599 UUUUAACA CUGAUGA X GAA AUCUGAGC 1141
1603 UUUUUUUU CUGAUGA X GAA ACAGAUCU 1142
1604 UUUUUUUU CUGAUGA X GAA AACAGAUC 1143
~ere''X''~ c.l~stemllregionofaHHribv~...c(Herteletal., 1992 Nucleic Acids Res. 20
3252). The length of stem 11 may be 2 2 base-pairs.
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-- ---- = = = = = = = = = = = = = =
C~ ~ C C~ V
~ ~ C C, ~ 3 'C ~ ~" v ~ ~
c~c~ C~ '3 U 3 ~ ~ ~ ~ ~ ~ ~ '~ ~ ~ ~ V
~ ~ ~ ~ ~ ~ ~ ~ 3 V ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~3
3 ~ c V r~ V~ ~ S 8 3 ~ ~ cj 3 3 ~c
~ ~ ~ ~ 3 ~ ~ 3 ~ C ~ ~3'3 ~ ~ ~ ~ ~ ~ C
2 tJ ~ 3 '~ ~ 8
'S CC> 0 0 N ~t CD 0 0 ~1 ~r CD 0 0 r.~ D 0 0 C~l ~ CD 0 0 C.~l ~ tD
o ~ er ~ 1~ 1~ 1' 0 0 0 0 0 rJ~ rJ~ rJ~ a~
.
~ ~ ~ t t v ~ v t t t o ~ ~ tv ~ t v v ~ o t
~ ~- V ~ ~ t~ ~ y t~ V, ~ t~ t,~ O y ~ V ~0 ~ ~ ~
o ~ ~ ~ O o o V ~ v ~O 3 c~ ~ ~ u ~ ~ u
r ) ~S C) r~ ~S C ~ r~ er ~ r) ~ ~ ~S C~ C~ S C~
r~ ~ O ~ ~ ~ ~ C~ ~ ~r5 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ _ ~ ~
c~ ~ ~c~ ~ ~ ~ ~ rJ ~ ~ ~ ~ ~rJ ~ ~ ~ rJ
~ ~ r~ o ~ r~ c~ c~ ) ~ C ~ ~ ~) ~ r~ J C)
r~ ~ ~ ~ ~ ~ r~ ~ ~ ~ ~ ~5 ~ O~
8 8 ~ 8 ~ ' ' 3 ~
o
c --~r 1~ o r~~~J ~ tD D a0) CD oO o ~D o ~ 0 a~ ~ o ~~~ ~n ~ 0 _ rD C~ ~D
l N N C~l N C~l N ~ D CD CD ~D N tr~ --
SUBSTITUTE SHEET (RULE 26)
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WO 97/10328 9~ PCTAUS96/11689
o D o o o o o ~ tJ~ C~) ~ ~ tD
~ Z -- ~ ~1 ~J ~ ~ ~ t~ N ~ ~ 'J t~
C~ --------------_ _ _
~S y ~ ~ 5 3 ~ 3
3 ~ ~ 3 ~) 3 ~ ~:
3 3 ~ . 3 ~ ~ g ~ ?
~ J _
~ ~ ~ '~ 3 ~,
o cn 8 o o oD ~o O N ~r D ~ O N ~ tD ~D
~ Z -- N N N N N N N N N N N N N N N
~ ~ ~ ~ ~ ~ Q ~ ~ ~ ,
iy c~ ~ c~ o- o o y o'
~OOoO~O~OO~
~tJ ~ - ~ X
3 8 3 ~ 8 ~
~)
~ C~ C~ C~ C~ ~ C~ ~ C~ 3 ~ C~
c~c~c~c~c~c~c~c~c~c~c~c~c~c~
C~ ~ 5 Cl ~ ~ ~ C
C~) ~ C~C C~ ~ V
-
~a ,~ ~ t.~ t.~ -- ~ ~ N r-- U~ tD 1~ tD ~ ~ et U~
o D 5~ ~ ~ ~ -- -- C~l t.~ C~ t~ ~ C~ ~ ~ It~
SUBSTITUTESHEET(RULE26)
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TableLK
Table IX: Cleavage of D elta-9 Desaturase RNA by HH ~ibozymes
Percent Cleaved
20~C 26~C
nt. P~* -~ 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
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96
TABLE X:
ConstruaTargets Blasted Isolates RecoveredGreo~~o -se Lines Plants ~r. :' ~c~
Number
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
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Table XI Stearic acid levels in leaves from plants transformed with active and inactive
ribo~vmes compared to control leaves.
-
Stearic Acid in Leaves Transformed with Active and Inactive Ribozvmes
(Pcrc~..lage of total plants with certain levels of leaf stearic acid)
Stearic AcidRibozyme Actives RibozymeControls
Inactives
(428 plants (406 plants(122 plants)
from 35 lines) from 31 lines)
>3% 7% 3% 2%
> 5% 2% 0 0
10% 0 0 0
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98
Table XII Inheritance of the high stearic acid trait in leaves from crosses of high stearic
acid plants.
Inheritance of high stearate in leaves.
CrossRl Plants with RI Plantswith % of Plants with
Normal High High Stearate
Leaf StearateLeaf Stearate
RPA85-15.06 x 6 3 33%
RPA85-15.12
RPA85-15.07 self 5 5 50%
RPA85-15.10 self 8 2 20%
OQ414 x RPA85-15.06 5 3 38%
OQ414 x RPA85-15.11 6 4 40%
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W O 97/10328 PCTAUS96/11689
99
Table XIII Comparison of fatty acid composition of embryogenic callus, somatic
embrvos and zygotic embrvos.
Tissue and/or Media Fatty Acid Composition % Lipid
Treatment of Fresh
C16:0 C18:0 C18:1 C18:2C18:3Weight
embryogenic callus 19.4 1.1 6.2 55.7 8.8 0.4
+/ +/ +/ +/_
0.9 0.1 2.0 3.1 2.0 0.1
somatic embryo grown12.6 1.6 18.2 60.7 1.9 4.0
on
MS + 6~ sucrose + 10 +/- +/- +/- +/- +/- +/-
mM ABA 0.7 0.8 4.9 5.1 0.3 1.1
zygotic embryo 14.5 1.1 18.5 60.2 1.4 3.9
12 days a~ter +/- +/- +/- +/- +/- +/_
pollination 0.4 0.1 1.0 1.5 0.2 0.6
SUBSTITUTE SHEET (RULE 26)
CA 02226728 1998-01-13
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100
Table XIV: GBSS activity, amylose content, and Southern analysis results of selcLIed Ribozyme Lim
LineGBSS activity AmyloseContent Southern
(Units/mg starch) (~/0)
RPA63.0283321.5 _ 31.2 23.3 + 0.5
RPA63.0236314.6 _ 9.2 27.4 + 0.3
RPA63.0219299.8 _ 10.4 21.5 + 0.3
RPA63.0314440.4 _ 17.1 19.1 + 0.8
RPA63.0316346.5 _ 8.5 17.9 +0.5
RPA63.0311301.5 _ 17.4 19.5 + 0.4
RPA63.0309264.7 _ 19 21.7 _ 0.1 +
RPA63.0218190.8 _ 7.8 21.0 _ 0.3 +
RPA63.0209203 _ 2.4 22.6 + 0.6 +
RPA63.0306368.2 _ 7.5 19.0 + 0.4
RPA63.0210195.1 _ 7 22.1 + 0.2 +
SUBSTITUTE SHEET (RULE 26)
