AU763096B2 - Improved methods and materials for transformation - Google Patents

Description

IMPROVED METHODS AND MATERIALS FOR

TRANSFORMATION

Background of the Invention RNA viruses have been found to be valuable tools in the phenotypic and genotypic transformation of targeted cells and tissues. See, e. U. S. Patent No. 5,500,360, which teaches novel viral RNA expression vectors. It has been shown that the RNA of the genome of an RNA virus can be modified to include an exogenous RNA segment and that the modified RNA can be introduced into a host cell, replicated therein, and thereby express the exogenous RNA segment.

Current methods of inoculating a host cell with modified RNA viruses involve the in vitro transcription of a particular strand followed by the introduction of the resulting RNA transcripts into the host cell. One problem with the current inoculation method is that the RNA rapidly degrades which causes a low efficiency of infection. In addition, the preparation of the in vitro RNA transcripts Sis expensive and time consuming.

Further, with the advent of transformation and the genetic engineering of plants, much eos 15 concern has arisen concerning the potential hazard of the dispersal of dangerous traits into the environment. For example, genes increasing the stress tolerance and/or herbicide resistance of an S.:i agriculturally important crop could theoretically "leak" to surrounding less desirable and damaging plants, e. through pollen, mechanical or insect dispersal. This phenomenon could create a novel species of "super-weed" which could wreak havoc on the agricultural industry. Existing RNA virusbased vectors can spread to non-target plants by mechanical means and/or by insects. Such spread can be prevented by using vectors that can replicate and/or move only in target plants S.expressing the appropriate trans-acting factors. Accordingly, there remains a need for less expensive and more efficient methods of transformation of target cells and tissues. Moreover, "0.0 there is a need for a novel method of transformation which alleviates the potential dangers 25 associated with the unwanted spread of engineered traits into the environment.

Brief Summary of the Invention According to a first embodiment of the invention, there is provided a DNA-launching platform comprising: a) a polynucleotide molecule encoding a modified viral RNA molecule, wherein said polynucleotide molecule is transcribed thereby forming a replicatable RNA transcript not capable of self-replication but replicatable in the presence of a trans-acting viral replication factor; and b) a DNA dependent RNA polymerase promoter.

[I:\DayLib\LIBFF]83079spec.doc:gcc 2 According to a second embodiment of the invention, there is provided a method of genotypically or phenotypically modifying one or more plant cells, said plant cell or cells having been tendered transgenic by stably comprising heterologous DNA encoding a trans-acting viral replication factor, said method comprising the following steps: a) obtaining a DNA-launching platform comprising a polynucleotide molecule encoding a modified viral RNA; and b) transfecting said one or more plant cells with said DNA-launching platform, wherein said polynucleotide molecule is transcribed thereby forming a replicatable RNA transcript not capable of self-replication but replicatable in the presence of said trans-acting viral replication lo factor, wherein expression of said RNA transcript confers a genotype or phenotype modification in said one or more plant cells.

According to a third embodiment of the invention, there is provided a method of producing a plant or plant tissue comprising at least one genotypically or phenotypically modified plant cell, said cell having been rendered transgenic by stably comprising heterologous DNA encoding a transacting viral replication factor, said method comprising transfecting cells of said plant or plant tissue with a DNA-launching platform, wherein said DNA-launching platform comprises a polynucleotide encoding a modified RNA molecule, such that said polynucleotide is transcribed to form a replicatable RNA transcript not capable of self-replication but replicatable in the presence of said trans-acting viral replication factor, and wherein expression of said RNA transcript confers a genotypic or phenotypic modification in at least one of said transfected cells.

According to a fourth embodiment of the invention, there is provided a method of producing a genotypically or phenotypically modified plant comprising obtaining at least one modified cell produced by the method in accordance with the second embodiment of the present invention; and subjecting said modified cell to conditions whereby a plant is regenerated therefrom.

According to a fifth embodiment of the invention, there is provided a plant produced by the method in accordance with the fourth embodiment of the present invention.

According to a sixth embodiment of the invention, there is provided a plant descended from the plant in accordance with the fifth embodiment of the present invention.

The disclosure herein pertains to improved materials and methods for transforming host cells which involve transfecting said cells with a DNA-launching platform. The disclosure herein further pertains to a DNA-launching platform which encodes a modified viral RNA molecule downstream of DNA-dependent RNA polymerase (pol) promoter, whereby the DNA-launching platform is capable of being introduced into a host cell and effectively "launching" said modified viral RNA molecule into [I:\DayLib\LIBFF]83079spec.doc:gcc 2a the host cell such that it is replicated and expressed therein. The term "modified viral RNA molecule" as used herein refers to a viral RNA which has been changed from its natural state.

Examples of changes of viral RNA include, but are not limited to, removal of a part of viral RNA genome, insertion or substitution of an exogenous RNA, etc. The exogenous RNA segment can be located in a region of the viral RNA molecule such that it does not disrupt the RNA replication.

Techniques for such manipulations have been well known to those of ordinary skill in the art for many years. Preferably, the modified viral RNA molecule further comprises a ribozyme which is located in the proximity of the 3' end of the modified viral RNA molecule. The viral segment may have the ability to be replicated with or, alternatively, without the presence of trans-acting viral replicating elements.

The disclosure herein also pertains to a method of genotypically or phenotypically modifying a host cell, comprising introducing a DNA-launching platform which encodes a viral RNA molecule and an exogenous RNA segment in a location which does not disrupt the replication of said viral RNA segment or said exogenous RNA segment, whereby the exogenous RNA segment confers a I s detectable trait in the host cell. The subject invention applies to a wide array of plant cells.

The disclosure herein also pertains to cells in which the DNA-launching platform of the subject invention has been introduced.

Yet another aspect of the disclosure herein also pertains to a plant comprising cells transfected with the DNA-launching platform.

The novel methods and materials of the subject invention provide a greater inoculation efficiency of RNA viruses because use of DNA-launching platforms of the subject invention are more resistant to degradation than RNA inocula, and because each DNA platform produces multiple RNA transcripts over an extended period of time. As the DNA-launching platform provides a genetically stable in planta archive copy of a desired vector construct, the continuing transcription 25 of said DNA platform will repeatedly reinoculate the host cell with the desired construct. This 0000 serves to counteract genetic instability problems that have inhibited the expression of some genes from vectors based on plant and animal RNA viruses. Further, the [I:\DayLib\LIBFF]83079spec.doc:gcc WO 99/61597 PCTIUS99/11250 3 inoculation methods of the subject invention provide a much simpler means of producing inocula in bulk for large scale use, which is cheaper and more efficient than inoculating with in vitro RNA transcripts.

Brief Description of the Drawings Figure 1 represents the schematic for producing the la and 2a proteins in the host cell.

Figure 2 illustrates an example of an Agrobacterium transformation vector containing an expression cassette capable of expressing la and/or 2a BMV proteins.

Figure 3 illustrates several Agrobacterium vectors that were produced to transform host plant cells (black rectangles indicate T-DNA borders).

Figure 4 represents the general mechanism of BMV RNA3 launching, and replication.

Figure 5 depicts DNA-launching platforms which can be used in accord with the teachings contained herein. The BMV and CCMV designations denote cis-acting elements.

Figure 6 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.

Figure 7 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.

Figure 8 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.

Figure 9 depicts Agrobacterium vector for delivery of DNA-launching platforms to plant cells (open triangles represent T-DNA borders).

Figure 10 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.

Legend For Figures 5-10: CaMV35S promoter t termination/polyA sequences Rz ribozyme NOS NOS promoter OOA origin of assembly FG foreign gene Figure 11 shows that BMV replication factors support efficient RNA3 replication in protoplasts.

WO 99/61597 PCT/US99/11250 4 Figure 12 shows the efficient replication of launched BMV RNA3 in protoplasts.

Figure 13 shows transgenic expression of BMV la and 2a mRNAs in N. tabacum and N. benthamiana.

Figure 14 shows the efficient replication of launched BMV RNA3 in (la 2a)-transgenic plants.

Figure 15 shows the successful GUS expression from the launched BMV RNA3 in (la 2a)- transgenic plants.

Figure 16 shows the successful GUS expression from the launched BMV RNA3 in protoplasts.

Figure 17 shows the successful GFP expression from the launched BMV RNA3 in (la 2a) transgenic plants.

Figure 18 shows the successful GFP expression from the launched BMV RNA3 in protoplasts.

Figure 19 shows the efficient replication of the launched BMV RNA3 in (la 2a)transgenic N. benthamiana using Agrobacterium inoculation.

Figure 20 shows the successful GUS expression from the launched BMV RNA3 having the SHMV coat protein in (la 2a)-transgenic plants.

Figure 21 shows that launched BMV replicates, moves cell-to-cell, and spreads long distances in (la+2a)-transgenic plants.

Figure 22 shows transfection of progeny from (1 a+2a)-transgenic N. benthamiana with BMV RNA3 DNA-launching platform and localization of the launched RNA3 to the roots.

Brief Description of the Sequences SEQ ID NO. 1: pB1LR2 partial nucleotide sequence includes BMV la expression cassette.

SEQ ID NO. 2: pBILR3 partial nucleotide sequence includes BMV la expression cassette.

SEQ ID NO. 3: pB2LR4 partial nucleotide sequence includes BMV 2a expression cassette.

SEQ ID NO. 4: pB2LR5 partial nucleotide sequence includes BMV 2a expression cassette.

SEQ ID NO. 5: pB12LR6 partial nucleotide sequence includes BMV la and 2a expression cassettes.

WO 99/61597 PCT/US99/1l250 SEQ ID NO. 6: pB 12LR7 partial nucleotide sequence includes BMV l a and 2a expression cassettes.

SEQ ID NO. 7: pB12LR8 partial nucleotide sequence includes BMV la and 2a expression cassettes.

SEQ ID NO. 8: pB12LR9 partial nucleotide sequence includes BMV la and 2a expression cassettes.

Detailed Disclosure of the Invention To facilitate understanding of the invention, certain terms used throughout are herein defined. The term "RNA virus" as used herein means a virus whose genome is RNA in a double-stranded or single-stranded form, the single strand being a strand or strand.

The terms "transfection" or "transfected" as used herein means an introduction of a foreign DNA or RNA into a cell by mechanical inoculation, electroporation, agroinfection, particle bombardment, microinjection, or by other known methods.

The terms "transformation" or "transformed" as used herein means a stable incorporation of a foreign DNA or RNA into the cell which results in a permanent, heritable alteration in the cell. Accordingly, the skilled artisan would understand that transfection of a cell may result in the transformation of that cell.

The term "launched" as used herein refers to a polynucleotide that has been transcribed from a DNA-launching platform, as described herein and, preferably, replicated.

The term "cis-acting element" as used herein denotes that portion of the RNA genome of an RNA virus which must be present in cis, that is, present as a part of each viral strand as a necessary condition for replication of that strand. Virus replication may depend upon the existence of one or more trans (diffusible) elements which interact with the cis-acting element to carry out RNA replication. If trans-acting elements are necessary for replication, they need not be present or coded for on the modified viral RNA provided, but may be made available within the infected cell by some other means. For example, the trans-acting replication functions may be provided by other, unmodified or modified, components of the viral genome transfected into the cells simultaneously with the modified RNA. The same approach can be used for other trans-acting functions including movement protein, coat protein, and other functions. The target cell may also be premodified, for example, cells may have been previously transformed to provide constitutive expression of the trans-acting functions from a chromosome. The cis-acting element is composed of one or more segments of viral RNA which must be present on any RNA molecule that is to be replicated within a host cell by RNA replication. The segment will most WO 99/61597 PCT/US99/11250 6 likely be the 5' and 3' terminal portions of the viral RNA molecule, and may include other portions and/or virus open reading frames as well. The cis-acting element is accordingly defined in functional terms: any modification which destroys the ability of the RNA to replicate in a cell known to contain the requisite trans-acting elements, is deemed to be a modification in the cisacting element. Conversely, any modification, such as deletion or insertion in a sequence region which is able to tolerate such deletion or insertion without disrupting replication, is a modification outside the cis-acting element. As is demonstrated herein, using the example of BMV which is known and accepted by those skilled in the art to be a functional example from which substantial portions of an RNA virus molecule may be modified, by deletion, insertion, or by a combination of deletion and insertion, without disrupting replication.

"Exogenous RNA" is a term used to describe a segment or component of RNA to be inserted into the virus RNA to be modified, the source of the exogenous RNA segment being different from the RNA virus itself. The source may be another virus, an organism such as a plant, animal, bacteria, virus, or fungus. The exogenous RNA may be a chemically synthesized RNA, derived from a native RNA, or it may be a combination of the foregoing. The exogenous RNA may provide any function which is appropriate and known to be provided by an RNA segment. Such functions include, but are not limited to, a coding function in which the RNA acts as a messenger RNA encoding a sequence which, when translated by the host cell, results in synthesis ofa peptide or protein having useful or desired properties; the RNA segment may also be structural, as for example in ribosomal RNA; it may be regulatory, as for example with small nuclear RNAs or anti-sense RNA; or it may be catalytic. One skilled in the art will understand that the exogenous RNA may encode, for example, a protein which is a key enzyme in a biochemical pathway, which upon expression effects a desirable phenotypic characteristic, such as altering cell metabolism. Further, the exogenous RNA may encode a protein involved in transcriptional regulation, such as zinc finger, winged-helix, and leucine-zipper proteins. A particularly interesting function is provided by anti-sense RNA, sometimes termed strand RNA, which is in fact a sequence complementary to another RNA sequence present in the target cell which can, through complementary base pairing, bind to and inhibit the function of the RNA in the target cell.

The term "non-viral" is used herein in a special sense to include any RNA segment which is not normally contained within the virus whose modification is exploited for replication and expression, and is therefore used synonymously with "exogenous". Accordingly, a gene derived from a different virus species than that which is modified is included within the meaning of the terms "non-viral" and "exogenous" for the purposes of describing the invention. For WO 99/61597 PCT/US99/11250 7 example, a non-viral gene as the term is used herein could include a gene derived from a bacterial virus, an animal virus, or a plant virus of a type distinguishable from the virus modified to effect transformation. In addition, a non-viral gene may be a structural gene derived from any prokaryotic or eukaryotic organism.

In one embodiment, the subject invention concerns a novel method oftransfecting a host cell which uses a DNA-launching platform to introduce viral RNA into the cell. The subject invention is directed towards a method of transfection employing a DNA-launching platform which encodes a modified viral RNA molecule comprising an RNA viral component attached to an exogenous RNA component and a DNA-dependent RNA poi promoter. The DNAdependent RNA pol promoter is preferably but not necessarily fused within up to 10 nucleotides of the 5' transcriptional start site of the modified viral RNA molecule, and more preferably within up to 5 nucleotides of the 5' transcriptional start site. Expression of the DNA-launching platform produces transcripts of the modified viral RNA molecule that are then capable of RNA replication in the presence of replication factors, which can be present in the modified viral RNA and/or may be supplied in trans by other means including expression from chromosome or supplied on different launching plasmids. When the modified viral RNA is replicated, the exogenous RNA can be replicated as well. Further, the exogenous RNA can be expressed in the cell, thereby providing a predetermined phenotypic characteristic. In a preferred embodiment, the DNA launching platform further comprises a nucleotide sequence encoding a self-cleavable ribozyme situated proximate to the 3' end of said RNA molecule. As would be readily apparent to those skilled in the art, known ribozymes may be used in accordance with the subject invention. In a preferred embodiment, the ribozyme cleaves the modified RNA viral molecule at the 3' region. The 3' region can consist of up to 30 nucleotides upstream or downstream of the 3' end; and preferably consists of up to 10 nucleotides upstream or downstream of the 3' end.

In a more preferred embodiment, the ribozyme cleaves the modified RNA viral molecule precisely at the 3' end. Other known regulatory sequences, promoters and/or termination sequences, may also be substituted for and/or included on the DNA-launching platform. A suitable restriction site can be introduced proximate to the 3' end of the modified viral RNA molecule sequence and the DNA molecule can be cleaved by an appropriate restriction enzyme prior to transfection. The term "DNA-launching platform" as used herein is intended to mean a DNA molecule, circular or linear, which has a coding region comprising a segment encoding a modified viral RNA segment, and further, which is capable of being delivered into a cell and subsequently transcribed.

WO 99/61597 PCT/US99/11250 8 Possible regulatory sequences can include, but are not limited to, any promoter already shown to be constitutive for expression, such as those of viral origin (CaMV 19S and 35S) or so-called "housekeeping" genes (ubiquitin, actin, tubulin) with their corresponding termination/polyA sequences. Also, seed-and/or developmentally-specific promoters, such as those from plant fatty acid/lipid biosynthesis genes (ACPs, acyltransferases, desaturases, lipid transfer protein genes) or from storage protein genes (zein, napin, cruciferin, conglycinin, phaseolin, or lectin genes, for example), with their corresponding termination/polyA sequences can be used for targeted expression. In addition, the gene can be placed under the regulation of inducible promoters and their termination sequences so that gene expression is induced by light (rbcS-3A, cab-1), heat (hsp gene promoters) or wounding (mannopine, HGPGs). It is clear to one skilled in the art that a promoter may be used either in native or truncated form, and may be paired with its own or a heterologous termination/polyA sequence.

In a particularly preferred embodiment, the subject invention is directed toward a method of genotypically or phenotypically modifying a cell comprising the following steps: a) forming a cDNA molecule of a virus RNA, or of at least one RNA component if the RNA virus is multipartite, the viral RNA having been modified to contain a DNA segment encoding a nonviral RNA component situated in a region able to tolerate such insertion without disrupting replication of the RNA product encoded thereby; b) cloning modified cDNA into a DNAlaunching platform; and c) transfecting a suitable host cell with said DNA-launching platform.

In a most preferred embodiment, the method further comprises pretransforming a plant with trans-acting viral replication factors and/or other trans-acting factors. Such trans-acting factors may include viral movement proteins(s), coat protein(s), viral protease(s), and other structural and nonstructural genes. In addition to stable expression of trans-acting factors, trans-acting factors may be introduced on separate expression plasmids or may be expressed from RNA transcripts. In a preferred embodiment such trans-acting factors do not replicate. Suitable host cells may include protoplasts, cells in suspension, or cells in tissues or whole organisms.

In a specific embodiment intended as an example of the broader teachings herein, the RNA viral segment can be derived from brome mosaic virus (BMV), whereby the DNAlaunching platform comprises DNA encoding the RNA3 segment of the virus. Brome mosaic virus (BMV) is a member of the a virus-like super family of positive-strand RNA viruses of animals and plants, and has a genome divided among three RNAs. RNA and RNA2 encode the la and 2a proteins, respectively, which are necessary for a genomic RNA replication and subgenomic mRNA synthesis (see, U.S. Patent No. 5,500,360, which to the extent not inconsistent herewith, is incorporated herein by reference). These proteins contain three WO 99/61597 PCT/US99/11250 9 domains conserved in all other members of the a virus-like super family. la (109kDa) contains a c-proximal helicase-like domain and an n-proximal domain implicated in RNA capping, and 2a (94kDa) contains a central polymerase-like domain. See, French and Ahlquist, (1988).

la and 2a interact with each other and with cell factors to form a membrane bound viral RNA replication complex associated with the endoplasmic reticulums of infected cells. BMV RNA3, a 2.1-kb RNA, encodes the 3a protein (32kDa) and coat protein (20kDa), which are involved in the spread of BMV infection in its natural plant hosts but are dispensable for RNA replication.

See U.S. Patent No. 5,500,360. The 3a or coat protein gene of the RNA3 viral segment can be replaced with exogenous RNA, whereby it does not interfere with the replication element.

Further, the exogenous RNA segment can be inserted downstream of an additional subgenomic promoter. Still further, cells or tissues can be pretransformed to express la, 2a, 3a, and coat protein, or any combination thereof, wherein DNA-launching platforms containing a foreign gene(s) with the necessary cis-acting components is transfected, such that the foreign gene is replicated and/or expressed.

In one embodiment, the host cell is pretransformed with BMVl or BMV2 such that it is transgenically engineered to express la and 2a proteins. Preferably, the 5' and 3' ends of BMV1 and BMV2 are removed such that they are incapable of replication, but can express la and 2a to form a viral RNA replication complex associated with the endoplasmic reticulum of the host cell. Subsequent transfection of a DNA-launching platform comprising the RNA3 viral replication segment, as well as the exogenous RNA of interest, can produce the expression of said exogenous RNA while also preventing the undesired and dangerous spread of viral RNA spillage into the environment. That is, because a plant must have all 3 segments to form infectious BMV particle(s), problems associated with the environmentally hazardous escape of foreign genes through mechanical or insect dispersal of RNA virus vectors are avoided. One skilled in the art will readily appreciate that in the example of BMV that DNA-launching platforms could be also derived from either RNA1 or RNA2. For example, the sequence encoding the la protein could be replaced with an exogenous RNA; replication would require the expression of la separate expression plasmid). In a preferred embodiment, the DNAlaunching platform also comprises a ribozyme situated proximate to the 3' end of the modified RNA3, wherein said ribozyme cleaves the RNA3 at the 3' end. As would be readily apparent to the skilled artisan with the teachings contained herein, viral segments from other known viruses, and/or subviral agents, can be used to formulate DNA-launching platforms of the subject invention. One skilled in the art will appreciate that BMV is merely one representative example of the many viruses suitable for practicing the subject invention. It is widely accepted that WO 99/61597 PCT/US99/11250 principles on which the subject invention is based are broadly applicable to a myriad of viruses.

Examples of other such viruses include, but are not limited to, alfalfa mosaic virus (AMV), barley stripe mosaic virus, cowpea mosaic virus, cucumber mosaic virus, reoviruses, polio virus, sindbis virus, vesicular stomatitis virus, influenza virus, retroviruses, and cowpea chiorotic mottle virus (CCMV) and any other viruses that replicate through RNA intermediates and from which a cDNA copy can be obtained. Specifically, as the other viruses are further characterized, those of skill in the art will readily appreciate the applicability of the teachings herein to other suitable viruses as well.

The skilled artisan would easily appreciate that known methods of introducing foreign DNA into cells can be used in accordance with the teachings of the subject disclosure. Such methods include, but are not limited to, mechanical inoculation, particle bombardment, agroinfection, electroporation, and microinjection, as well as other known methods.

Various aspects of the invention can be modified as needed, depending upon specific characteristics of the virus selected as the transforming and transfecting agent and of the RNA segment to be inserted. For example, the inserted gene need not be a naturally occurring gene, but may be modified, a composite of more than one coding segment, or it may encode more than one protein. The RNA may also be modified by combining insertions and deletions in order to control the total length or other properties of the modified RNA molecule. The inserted nonviral gene may be either prokaryotic or eukaryotic in origin. The inserted gene may contain its own translation start signals, for example, a ribosomal binding site and start (AUG) codon, or it may be inserted in a manner which takes advantage of one or more of these components preexisting in the viral RNA to be modified. Certain structural constraints must be observed to preserve correct translation of the inserted sequence, according to principles well understood in the art. For example, if it is intended that the exogenous coding segment is to be combined with an endogenous coding segment, the coding sequence to be inserted must be inserted in reading frame phase therewith and in the same translational direction.

It will be understood by those ordinarily skilled in the art that there may exist certain genes whose transfer does not result in obvious phenotypic modification of the recipient cell.

Such may occur, for example, if the translation product of the non-viral gene is toxic to the host cell, is degraded or processed in a manner which renders it non-functional or possesses structural features which render it impossible for the host cell to translate in sufficient quantities to confer a detectable phenotype on the transformed cells. However, the invention does not depend upon any specific property of an RNA segment or gene being transferred. Therefore, the possible existence of RNA segments or genes which fail to confer a readily observable phenotypic trait WO 99/61597 PCT/US99/11250 11 on recipient cells or plants is irrelevant to the invention, and in any case will be readily recognizable by those of ordinary skill in the art without undue experimentation.

An exogenous RNA segment may be inserted at any convenient insertion site in any of the cDNA sequences corresponding to a viral RNA, or component RNA of a multipartite RNA virus, provided the insertion does not disrupt a sequence essential for replication of the RNA within the host cell. For example, for a virus whose coat protein is not essential for replication, an exogenous RNA segment may be inserted within or substituted for the region which normally codes for coat protein. As desired, regions which contribute to undesirable host cell responses may be deleted or inactivated, provided such changes do not adversely affect the ability of the RNA to be replicated in the host cell. For many single component and multipartite RNA viruses, a reduction in the rate of normal RNA replication is tolerable and will in some instances be preferred, since the amount of RNA produced in a normal infection is more than enough to saturate the ribosomes of the transformed cell.

Plant cells which are inoculated in culture will normally remain transfected as the cells grow and divide since the RNA components expressed from the DNA-launching platform are able to replicate and thus become distributed to descendant cells upon cell division. Plants regenerated from phenotypically modified cells, tissues, or protoplasts remain phenotypically modified. Similarly, plants transfected as seedlings remain transfected during growth. Optimal timing of application of the transfecting components will be governed by the result which is intended and by variations in susceptibility to the transfecting components during various stages of plant growth.

Many plant RNA viruses are seed transmitted from one generation to the next. This property can be exploited to effect genotypic transformation of a plant. That is to say, the modified RNA remains transmissible from one generation to the next, just as seed-borne virus infections are transmitted from one generation to the next.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 Construction of Agrobacterium Vectors Binary vectors for expressing the BMV I a and 2a proteins in plants were constructed.

Starting with the pB1101.2 construct (Clontech, Palo Alto, CA), the GUS gene was removed by first cutting the construct with EcoR and SnaBI. The overhanging restriction fragment ends WO 99/61597 PCT/US99/11250 12 were filled in by treatment with Klenow fragments and dNTPs. The restriction fragment ends were religated forming the pBl01.2LRI.

The 2a expression cassette was inserted into pBIl01.2 LR1. First the pBI101.2LRI was cut with Hind III and dephosphorylated. Next, pB2PA 17 (Dinant et al., 1993) was cut with Hind III and the 2a insert was purified using a low melting agarose gel. The restriction fragment ends were ligated forming the pB2LR4 and pB2LR5 (Figures 3c and 3d).

The 1 a expression cassette was inserted into pBI 101.2LR1 by first cutting pBI 101.2LRI with SnaBI and dephosphorylated. pBIPA17 (Dinant et al., 1993) was cut with PstI and the extra nucleotides were removed with T4 DNA polymerase. The la insert was purified using a low melting agarose gel. The restriction fragment ends were ligated forming the pB 1LR2 and pB1LR3 vectors (Figures 3a and 3b).

The la expression cassette was inserted into pB2LR4 and pB2LR5 by cutting pB2LR4 or pB2LR5 with SnaBI and dephosphorylated. PBIPA17 (Dinant et 1993) was cut with PstI, and the extra nucleotides were removed with T4 DNA polymerase. The la insert was purified using low melting agarose gel and ligated with the cut pB2LR4 or pB2LR5 vectors to form pB12LR6, pB12LR7, pB12LR8, and pB12LR9 vectors (Figures 3e-3h).

Example 2 Construction of DNA-launching Platform for wtRNA3 of BMV and for RNA Derivatives Containing Foreign Sequences Vector pRT101 (T8pfer et al., 1987) was cut with PpuMI and the restriction fragment ends were filled in with Klenow fragment and dNTPs, and cut with BamHI and dephosphorylated. Vector pB3RQ39 (Ishikawa et al., 1997) was cut with SnaBI and BamHI; the B3 fragment was isolated from a low melting agarose gel. This fragment was ligated to the cut pRT101 thereby forming pB3LRIO (Figure The pB3LR15 (Figure 4) that is a pB3LR10 derivative has the ClaI-KpnI fragment replaced with the corresponding fragment from pB3TP8 (Janda et al., 1987).

PCR was performed on pRTIO1 to amplify an EcoRV and EcoRI fragment. To create a StuI site instead of a PpuMI site, a one nucleotide deletion was performed during the PCR process. The resulting PCR product was cut with EcoRV and EcoRI and inserted into dephosphorylated pRTIO1 cutwith EcoRV and EcoRI to form pRTIO1LRI 1. ThepRTIO1LRI was cut with StuI and BamHI and dephosphorylated. PB3RQ39 was cut with SnaBI and BamHI and a B3 fragment was isolated using a low melting agarose gel. The fragment was then ligated to pRTIO1LRl 1 to form pB3LR12 (Figure 4).

WO 99/61597 PCT/US99/11250 13 Another DNA-launching platform was constructed with wtRNA3 of BMV having a partially doubled CaMV35S promoter; thereby forming pB3LRI4 and pB3LRI6 (Figure 4).

A DNA-launching platform wherein the BMV RNA3 coat protein was replaced with GUS was also constructed. The pB3MI22 (Ishikawa et al., 1997) was cut with Clal and Stul and a B3GUS insert was isolated. The pB3LR10 or pB3LR14 DNA-launching constructs were cut with Clal and StuI and dephosphorylated. The B3GUS fragment was then ligated to the cut pB3LRI 0 or pB3LR14 thereby forming the pB3GUSLRI7 and pB3GUSLRI 8 DNA-launching constructs (Figure A DNA-launching platform having a BMV RNA3 with a GUS gene insertion wherein the GUS is downstream of an additional BMV subgenomic promoter was constructed. The pB3LR1 5 construct was cut with Aval and the restriction fragment ends were filled in with Klenow fragment and dNTPs. Construct was then cut with Clal and dephosphorylated. The pB3MI22 was cut with Clal and StuI and a B3GUS fragment was isolated. The isolated B3GUS fragment was then ligated to the cut pB3LRI5 construct to form a new construct of pB3GUSCPLRI9 (Figure A BMV RNA3 based DNA-launching platform with a CP gene inserted downstream of an additional cowpea chlorotic mottle virus (CCMV) subgenomic promoter was constructed.

The pB3GUSLRI7 construct was cut with StuI and KpnI and dephosphorylated. The pBC3AJ14 (Pacha and Ahlquist, 1991) was cut with NdeI, the ends were blunted by known methods in the art, and then cut with KpnI. A coat protein fragment was then isolated. The coat protein fragment was then ligated to the cut pB3GUSLRI 7 to form a new construct of pB3GUSCPLR22 (Figure A DNA-launching platform was constructed having a subgenomic RNA4. The pB4MK2 Kroll, personal communications) was cut with SnaBI and BamHI and a RNA4 fragment was then isolated. The pRTIO1LR11 construct was cut with StuI and BamHI and dephosphorylated.

The fragment and the cut pRTIOILRII construct were then ligated forming pB4LR20 (Figure A DNA-launching platform wherein the BMV coat protein was replaced with GFP was constructed. pEGFP (Clontech, CA) was cut with NotI, filled in with Klenow fragment and dNTPs, cut with Sail, and GFP insert was isolated using low-melting agarose gel. The pB3LR15 was cut with Sall and Stul and dephosphorylated. The GFP fragment was then ligated to the cut pB3LR15 thereby forming the pB3GFPLR48 (Figure 6e).

A DNA-launching platform having a BMV RNA3 with a GFP gene insertion wherein the CP is downstream of an additional CCMV subgenomic promoter was constructed. The WO 99/61597 PCT/US99/11250 14 pBC3AJ14 (Pacha and Ahlquist, 1991) was cut with Ndel and EcoRI and the ends were blunted by known methods in the art. The coat protein fragment was then isolated and ligated into dephosphorylated and blunted pEGFP cut with NotI and StuI forming pEGFPCPLR49.

pEGFPCPLR49 was cut with KpnI and the EGFPCP fragment was isolated using low-melting agarose gel. PB3GFPLR48 was cut with KpnI and dephosphorylated. The EGFPCP fragment was then ligated to the cut pB3GFPLR48 thereby forming the pB3GFPCPLR50 (Figure 6a).

An RNA transcription vector wherein the GFP gene is expressed as a translational fusion with BMV 3a was constructed. The pB3TP10 (Pacha and Ahlquist, 1991) was cut with BamHI and dephosphorylated. The GFP fragment was amplified from pEGFP (Clontech, CA) using PCR and the following primers: 5'GCAGTCGACGGTACCGCGGGCC3' and 5'CGCGGCCGCGGATCCTGTACAGCTCG3'.

The amplified product was cut with BamHI and purified using low-melting agarose gel. The GFP fragment was ligated to the cut pB3TP10 forming pB3GFPLR47 (Figure 6d). The pB3GFPLR47 was cut with EcoRI and transcribed using T7 RNA polymerase.

An Agrobacterium vector containing BMV RNA3 DNA-launching platform was constructed. The pBI101.2LR1 was cut with Smal and dephosphorylated. The pB3LR15 was cut with PvuII and the B3 fragment was purified using a low-melting agarose gel. The B3 fragment was then ligated to the cut pBI 101.2LR1 thereby forming pB3LR42 (Figure 9).

A DNA-launching platform wherein the BMV RNA3 coat protein was replaced with the SHMV (Sunn hemp mosaic virus) coat protein and the GUS gene was inserted downstream of an additional BMV subgenomic promoter was constructed. The pB3RS4 (Sacher et al., 1988) was cut with Aval, blunted with Klenow fragment and dNTPs, and cut with KpnI. The SHMV coat protein fragment was isolated using a low-melting agarose gel. The pB3GUSLRI7 was cut with StuI and Kpnl and dephosphorylated. The SHMV coat protein fragment was ligated to the cut pB3GUSLRI7 thereby forming pB3GUSCPLR24 (Figure 7).

Other permutations of DNA-launching platforms containing one or more foreign genes and the necessary cis-acting replication signals will be readily appreciated in view of the teachings herein. For examples, see Figures 5-10.

WO 99/61597 PCT[US99/11250 Example 3 Transfection ofN. tabacum Protoplasts with DNA-launching Platform Media: NT1 Medium (1 liter) was made with Gibco-BRL (MS salt, catalog #11118-031), 3ml of 6% KH2PO4, and 0.2 Mg/ml 2,4D (final concentration). The pH was adjusted to 5.5-5.7 using KOH, and the resulting mixture was autoclaved.

NTI Plating Medium (1 liter) was made with NTI medium and 72.86 g mannitol, the pH was adjusted to 5.5-5.7, and the resulting mixture was autoclaved.

Wash Solution (1 liter) was made with 72.86 g mannitol, the pH was adjusted to 5.5, and the resulting mixture was autoclaved.

Electroporation Buffer was made with 0.8% NaCI, 0.02% KCI, 0.02% KH2PO4, 0.11% Na2HPO4, and 0.4M mannitol. The pH was adjusted to 6.5, and the resulting mixture was autoclaved.

Enzyme Solution was made with 0.4M mannitol, and 20mM MES. The pH was adjusted to 5.5, and the resulting mixture was autoclaved.

Growth conditions: Cells (Nicotiana tabacum) were grown at room temperature in NTI media with constant shaking (about 200 rpm).

Preparation ofcultures for digestion: About 2-3 ml of one-week old suspension culture was subcultured into 50 ml of fresh NTI media 3 days before the enzyme digestion. The culture was maintained at 28 0 C under constant shaking.

Enzyme digestion: The enzyme digestion solution was prepared containing the following: 1% cellulysin (Calbiochem) and 0.3% macerase (Calbiochem) in the enzyme solution. The pH was adjusted to 5.5 and filter sterilized.

The cells were centrifuged at 800 rpm for 5 min. The supernatant was discarded. About ml of wash solution was added, cells were resuspended and were centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were then resuspended in three volumes of enzyme digestion solution, and incubated for 60 min. at room temperature.

Washing: The cells were transferred into 50 ml plastic tube and centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were resuspended in 40 ml of wash solution and centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were resuspended in 40ml of electroporation buffer and centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were resuspended in four volumes of electroporation buffer.

Electroporation: One ml of cells containing the RNA or DNA inocula was transferred into electroporation cuvettes and placed on ice for 10 min. The cells were then mixed and WO 99/61597 PCTIUS99/11250 16 electroporated at 500 microF, 250V. The cuvettes were placed on ice for 10 min. The cells were transferred into 10 ml ofNTI plating media.

Incubation and collection ofsamples: The cells were incubated at room temperature in dark. Samples were collected 24-48 hrs post inoculation.

RNA Analysis: RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization were performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 106 cpm/ml of radioactive probe in hybridization buffer was used per hybridization experiment. Replication of RNA3 was confirmed by detection of sgRNA4, thus showing that BMV RNA replication factors la and 2a expressed from expression plasmid(s) support efficient replication of RNA3 supplied as in vitro transcript (Figure 11) as well as launched from DNA-launching platform (Figure 12).

Example 4 Production of Transgenic N. tabacum Plants Once a desired molecule was constructed in E. coli, the molecule was transferred into Agrobacterium tumefaciens by the freeze-thaw method. Vectors pB 1LR2, pB2LR4, pB 12LR6, and pB12LR7 were all individually used. An Agrobacterium strain LBA 4404 containing an appropriate helper Ti plasmid was grown in 5 ml of YEP medium overnight at 28°C. Two ml of the overnight culture were added to 50 ml YEP medium in a 250-ml flask and shaken vigorously (250 rpm) at 28 0 C until the culture grew to an OD 5 oo of 0.5 to 1.0. The culture was chilled on ice. The cell suspension was centrifuged at 3000 g for 5 min. at 4°C. The supernatant solution was discarded. The cells were resuspended in 1 ml of ice-cold 20 mM CaCI, solution.

0.1-ml aliquots were dispensed into prechilled eppendorf tubes. About 1 sg of plasmid DNA was added to the cells. The cells were frozen in liquid nitrogen. The cells were thawed by incubating the test tube in a 37 C water bath for 5 min. 1 ml of YEP medium was added to the tube and incubated at 28 0 C for 2-4 h with gentle shaking to allow the bacteria to express the antibiotic resistance genes. The tubes were centrifuged for 30 s and the supernatant solution was discarded. The cells were resuspended in 0.1 ml YEP medium, plated on a YEP agar plate containing selection antibiotic(s), and incubated at 28 C. Transformed colonies appeared in 2-3 days.

In vitro clonal copies of approximately three week old Nicotina tabacum, Wisconsin No.

38, were used as the source of explants. Leaf explants were prepared from the second and third fully expanded leaves of in vitro cultures. The leaf pieces were cut into 1 cm x 1 cm squares and WO 99/61597 PCT/US99/11250 17 placed upon TBI (plus 2.0 mg/l 6-benzyl-aminopurine, and 0.1 mg/I -naphthalene acetic acid) media for 24 hours at 25 °C with a 16 hour photo period.

Agrobacterium tumefaciens strain LBA 4404 containing the preselected binary vector was used for plant transformation. Explants were placed in -10 ml of overnight grown Agrobacterium culture for 30 min. Leaf explants were then blotted on filter paper and placed on TB2 (plus 1.0 mg/I 6-benzyl-aminopurine and 0.1 mg/l -naphthalene acetic acid) media for 4 days, abaxial side down. Explants are then rinsed three times in sterile water, blotted on filter paper, and placed on TB2 media for regeneration with 100 mg/1 kanamycin and 400 mg/I carbenicillin at 25 C, 16 hour photo period, abaxial side down. Explants were transferred to fresh TB2 media with 100 mg/I kanamycin and 400 mg/I carbenicillin every 10 to 14 days until plantlets developed. Plantlets typically developed at 10-14 days. Plantlets were cut from the callus and placed on MST media containing 100 mg/1 kanamycin and 400 mg/l carbenicillin to induce rooting. Rooted plants were transferred to soil.

TBI (1 liter) included 4.30 g MS salts, 100 mg myo-inositol, 1.0 ml Nitsch and Nitsch vitamins, 30 g sucrose, 2 mg BAP, 0.10 mg of NAA, and 8g Noble agar. The media was adjusted to a pH 5.7 and autoclaved.

TB2 (1 liter) included 4.30 g MS salts, 100 mg myo-inositol, 1.0 ml Nitsch and Nitsch vitamins, 30 g sucrose, 1.0 mg BAP, 0.10 mg NAA, and 8 g Noble agar. The media was adjusted to pH 5.7 and autoclaved.

MST (1 liter) included 4.30 g MS salts, 1.0 ml Nitsch and Nitsch vitamins, 30 g sucrose, 100 mg myo-inositol, and 8.5 g Difco agar. The media was adjusted to pH 5.7 and autoclaved.

YEP (100 ml) included 1.0g Bacto-peptone, 1.0 g Bacto-yeast extract, and 0.5 g NaCI.

The media was autoclaved.

RNA Analysis: Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization was performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 106 cpm/ml of radioactive probe in hybridization buffer was used per hybridization experiment. Figure 13a shows the successful expression of BMV la and 2a mRNA in transgenic N. tabacum.

Example 5 Transfection of Transgenic N. tabacum Plants with DNA-launching Platform Precipitation ofDNA onto Microcarriers for Particle Bombardment: (Kikkert, 1993).

WO 99/61597 PCT/US99/11250 18 Sterilization ofMicrocarriers: 80 mg of gold microcarriers were resuspended in 1 ml of 70% ethanol, soaked for 15 min., and centrifuged at 13,000 x g for 5 min. The supernatant was carefully removed and discarded. Particles were resuspended in 1 ml of sterile distilled, deionized water and centrifuged at 13,000 x g for 5 min. The supernatant was carefully removed and discarded. Water washing of particles was repeated 2 more times. After final rinse, particles were resuspended in 1 ml of sterile 50% glycerol.

Coating Microcarriers with DNA: The following was sequentially and quickly added: DNA (lyg/pl), 50; 1 l of 2.5M CaCI 2 and 20,1 of 0.1M Spermidine.

The mixture was incubated for 10 min. on a vortex shaker at room temperature.

Particles were pelleted by centrifugation at 13,000 x g for 5 sec. Supernatant was carefully removed and discarded. Particles were resuspended in 140 pl of 70% ethanol and centrifuged at 13,000 x g for 5 sec. Supernatant was removed and discarded. Particles were resuspended in 140 gl of 100% ethanol and centrifuged at 13,000 x g for 5 sec. Supernatant was removed and discard. Particles were resuspended in 50pl of 100% ethanol.

Young leaves from tobacco plants grown in vitro on agar-solidified MS medium containing 30g/liter sucrose, were bombarded with 5-pl aliquots of resuspended DNA-coated particles using a PDS1000He biolistic gun (DuPont) and 1100 psi rupture disks (Bio-Rad).

RNA Analysis: Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization was performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 pzg) of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 106 cpm/ml of radioactive probe in hybridization buffer was used per hybridization experiment. Figure 14a shows that the launched BMV RNA3 replicates efficiently in transgenic plants expressing BMV replication factors la and 2a and that the launched RNA3 is unable to replicate in the absence of BMV la and/or 2a.

Example 6 Production of Transgenic N. benthamiana Plants Once a desired molecule was constructed in E. coli, the molecule was transferred into Agrobacterium tumefaciens. Vectors pB1LR2, pB2LR4, pB12LR6, and pB12LR7 were all individually used. An Agrobacterium strain LBA 4404 containing an appropriate helper Ti plasmid was grown in 5 ml of YEP medium overnight at 28 0 C. Two ml of the overnight culture were added to 50 ml YEP medium in a 250-ml flask and shaken vigorously (250 rpm) at 28 0

C

until the culture grew to an OD 5 oo of 0.5 to 1.0. The culture was chilled on ice. The cell suspension was centrifuged at 3000 g for 5 min. at 4 0 C. The supernatant solution was discarded.

WO 99/61597 PCT/US99/11250 19 The cells were resuspended in 1 ml of ice-cold 20 mM CaCI 2 solution. 0.1-ml aliquots were dispensed into prechilled eppendorf tubes. About 1 gg ofplasmid DNA was added to the cells.

The cells were frozen in liquid nitrogen. The cells were thawed by incubating the test tube in a 37 0 C water bath for 5 min. 1 ml of YEP medium was added to the tube and incubated at 28 0

C

for 2-4 h with gentle shaking to allow the bacteria to express the antibiotic resistance genes. The tubes were centrifuged for 30 s and the supernatant solution was discarded. The cells were resuspended in 0.1 mi YEP medium. The cells were plated on a YEP agar plate containing selection antibiotic(s) and incubated at 28 0 C. Transformed colonies appeared in 2-3 days.

In vitro clonal copies of approximately five-seven weeks old N. benthamiana were used as the source ofexplants. Leafexplants were prepared from the second and third fully expanded leaves of in vitro cultures. The leaf pieces were cut into 1cm x 1cm squares and placed upon MS104 media in 100 x 15 mm plates for 24 hours at 23°C with a 16 hour photo period.

Agrobacterium tumefaciens strain LBA 4404 containing the preselected binary vector was used. Explants were placed in 10ml of overnight grown Agrobacterium culture for 30 min.

Leaf explants were then blotted on filter paper and placed abaxial side down on MS 104 media for 4 days. Explants were then rinsed three times in sterile water, blotted on filter paper, and placed on MS104 media for regeneration with 300 mg/L kanamycin and 400 mg/L carbenicillin.

Explants were transferred to fresh MS104 media with 300 mg/L kanamycin and 400 mg/L carbenicillin every 10-14 days until plantlets developed. Plantlets typically developed at 31-50 days. Plantlets were cut from the callus and placed on MST media plus 300 mg/L kanamycin and 400 mg/L carbenicillin to induce rooting. Rooted plants were transferred to soil.

One liter of MS 104 included 4.3 g MS salt mixture, 1.0 ml B5 vitamin solution, 30 g sucrose, 1.0 mg BA, 0.1 mg NAA, and 8.0 g Phytagar. The media was adjusted to pH 5.8 and autoclaved.

100 ml of YEP included 1.0 g Bacto-peptone, 1.0 g Bacto-yeast extract, 0.5 g NaCI. The media was autoclaved.

One liter of MST included 4.3 g MS salt mixture, 1.0 ml Nitsch Nitsch vitamins, g sucrose, 100 mg myo-inositol, and 8.5 g Phytagar. The media was adjusted to pH 5.7 and autoclaved.

RNA Analysis: Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization was performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 106 cpm/ml of radioactive probe in WO 99/61597 PCTIUS99/11250 hybridization buffer was used per hybridization experiment. Figure 13b shows the successful expression of BMV la and 2a mRNA in transgenic N. benthamiana.

Example 7 Transfection of Transgenic N. benthamiana Plants Precipitation of DNA onto Microcarriers for Particle Bombardment: (From Kikkert (1993) "The biolistic PDS 1000/He device", Plant Cell Tiss. And Org. Cult. 33:221-226) Sterilization of Microcarriers: 80 mg of gold microcarriers were resuspended in 1 ml of 70% ethanol, soaked for 15 min., and centrifuged at 13,000 x g for 5 min. The supernatant was carefully removed and discarded. Particles were resuspended in 1 ml of sterile distilled, deionized water and centrifuged at 13,000 x g for 5 min. The supernatant was carefully removed and discarded. Water washing of particles was repeated 2 more times. After final rinse, particles were resuspended in 1 ml of sterile 50% glycerol.

Coating Microcarriers with DNA: To the 50 /l of particles the following was sequentially and quickly added: 5gl DNA (lbg/4l), 50g1 of 2.5M CaCI 2 and 20l1 of 0.1M Spermidine.

The mixture was incubated for 10 min. on a vortex shaker at room temperature.

Particles were pelleted by centrifugation at 13,000 x g for 5 sec. Supernatant was carefully removed and discarded. Particles were resuspended in 140 pl of 70% ethanol and centrifuged at 13,000 x g for 5 sec. Supernatant was removed and discarded. Particles were resuspended in 140 Al of 100% ethanol and centrifuged at 13,000 x g for 5 sec. Supernatant was removed and discarded. Particles were resuspended in 5 0ul of 100% ethanol.

Young leaves from N. benthamiana plants grown in vitro on agar-solidified MS medium containing 30g/liter sucrose, were bombarded with 5-k1 aliquots of resuspended DNA-coated particles using a PDSOOO1He biolistic gun (DuPont) and 1100 psi rupture disks (Bio-Rad).

RNA Analysis: Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization was performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 106 cpm/ml of radioactive probe in hybridization buffer was used per hybridization experiment. The launched BMV and RNA 3 showed efficient replication (Figure 14b) in transgenic N. benthamiana plants expressing BMV replication factors la and 2a and was unable to replicate in the absence of BMV la and/or 2a.

WO 99/61597 PCTIUS99/11250 21 Example 8 Transfection of Transgenic Plants with GUS Containing DNA-launching Platform Transgenic N. tabacum and N. benthaniana plants were produced according to the procedures discussed above. The plants were transfected with a DNA-launching platform containing a GUS gene (Figure 5a) by particle bombardment as described in Examples 5 and 7.

The plants were incubated for 3-5 days and then assayed for P-glucuronidase (GUS) activity using 1 mg/ml X-Gluc (5-bromo-4-chloro-3-indolyl glucucuronide) as substrate in 0.1M potassium phosphate buffer, pH 7.0, 50 /M potassium ferrocyanide, and 2% Triton® X-100.

Following an overnight incubation at 37 0 C, cells replicating launched RNA3 derivatives and expressing the GUS reporter gene from a subgenomic RNA4 gave rise to blue spots (Figure The launched RNA3 derivative did not replicate and express GUS reporter gene in the absence ofBMV RNA replication factors la and 2a in wt N. benthamiana and in wt N. tabacum).

Example 9 Transfection of Transgenic Plants Expressing BMV la. 2a. 3a. and CP A plant is transformed with BMV la, 2a, 3a, and CP genes whereby those genes are stably expressed in said plant. This can be done with the procedures outlined above. Any modifications that would be needed would be readily apparent to those skilled in the art in light of the teachings contained herein. A DNA-launching platform encoding an RNA replicon which contains a foreign gene and necessary BMV or CCMV cis-acting replication signals to replicate said replicon is constructed (Figure 10b). Foreign genes to be included in said replicon could include, for example, a Bacillus thuringiensis polynucleotide that codes for a B.t. protein. Other sequences would include, sequences that encode herbicide resistance, or any other known sequence that encodes peptides or proteins having desired qualities in plants.

Alternatively, plants can be transformed to express BMV la, 2a, 3a, and a TMV coat protein in place of the BMV coat protein. A DNA-launching platform is then made containing one or more foreign genes and the necessary cis-acting replication signals, either BMV or CCMV, and a TMV origin of assembly (Figures 8a, 8b, and 10a). This launching platform provides a distinct advantage as TMV is a rod-shaped virus which has no strict limit on the size of RNA that can be encapsidated. Alternatively, TMV movement protein can be used in place of BMV3a (Figure 7c). Hybrids between tobamo and bromoviruses were shown to be viable (Sacher et al., 1988; De Jong and Ahlquist, 1992).

Other permutations and combinations of genes pretransformed and those included in the DNA-launching platform will readily be appreciated by the skilled artisan in light of the teachings herein. (See, Figures 8c, 10b, and WO 99/61597 PCTIUS99/11250 22 As indicated above, CCMV subgenomic promoter can be substituted for BMV sequences in a desired DNA-launching platform. Because the sequence of CCMV subgenomic promoter differs from the sequence of BMV subgenomic promoter, the probability of recombination that would result in loss of a foreign gene is much lower in a construct having a combination of these two different promoters.

In the above examples, trans-acting components may include, but are not limited to, replication factors, components responsible for cell to cell movement, or components such as the coat protein which may be required for long distance spread, viral proteases responsible for post translational processing, or other known trans-acting functions.

Example 10 Transfection of N. tabacum Protoplasts with GUS Containing DNA-Launching Platforms N. tabacum protoplasts isolated using the above described methods were inoculated by electroporation with DNA-launching platforms for BMV RNA3 derivatives in the presence or absence of la and 2a expression plasmids. BMV RNA3 derivatives contained the GUS gene in place of the coat protein ORF (Figure 5a) (these were inoculated with or without coat protein expression plasmid, Figure 5b), or had the BMVCP gene translated from an additional subgenomic RNA driven from BMV or CCMV subgenomic promoter (Figures 5c and 5d), or had the SHMV coat protein translated from an additional BMV subgenomic RNA (Figure 7b).

Protoplasts were collected by centrifugation (800 rpm, 5 min.) 24 hours post inoculation. The chemiluminescent GUS assay was performed using GUS-Light T M (Tropix, MA) according to manufacturer's instructions. Protein concentrations were determined using the Bio-Rad protein kit (Bio-Rad Laboratories, Hercules, CA). The GUS values, determined by luminometer, were adjusted to the same total protein concentration. Figures 16a and 16b show successful GUS expression in protoplasts in the presence of trans-acting BMV replication factors la and 2a.

Example 11 Transfection ofN. tabacum Protoplasts with GFP Containing DNA-Launching Platform N. tabacum protoplasts isolated by using the above described methods were transfected by electroporation with expression plasmids for trans-acting BMV replication factors 1 a and 2a and with DNA-launching platforms for RNA3 derivatives having the GFP gene in place of BMV coat protein ORF (Figure 6e), the CP gene translated from an additional subgenomic RNA (Figure 6a) or with an RNA transcript having the GFP expressed as a fusion protein with BMV 3a ORF (Figure 6d). Protoplasts were incubated for 24 hrs and examined for GFP expression WO 99/61597 PCT/US99/11250 23 using a fluorescent microscope. Figure 18 shows the successful expression of GFP in protoplasts.

Example 12 Transfection of (la 2a)-Transgenic Plants with BMV RNA3-Based DNA- Launching Platform Containing GFP N. benthamiana plants were transfected using a particle bombardment as described above with a DNA-launching platform for BMV RNA3 having the GFP gene in place of BMV coat protein (Figure 6e). The GFP expression was determined 24 hrs post inoculation using a fluorescent microscope. Figure 17 shows the successful expression of GFP in (la 2a)transgenic N. benthamiana.

Example 13 Transfection of (Ia 2a)-Transgenic N. benthamiana with BMV RNA3 DNA- Launching Platform Using Agrobacterium N. benthamiana plants were inoculated with BMV RNA3 DNA-launching platform using Agrobacterium tumefaciens. Once the desired construct (pB3LR42) was obtained in E.

coli it was transferred to A. tumefaciens strain LBA4404 using a thaw-freeze method as described above. The Agrobacterium was grown overnight in 28 C under constant shaking. A single lower leaf of N benthamiana were punctured with a needle multiple times and submerged in Agrobacterium culture. The plants were grown at 23 0 C with a 16 hr photoperiod. The inoculated leaves were harvested 14 days post-inoculation. The total RNA extraction and northern blot hybridization were performed as described above. Figure 19 shows replication of launched BMV RNA3 in inoculated (la 2a)-transgenic N. benthamiana.

Example 14 Transfection of (la 2a)-Transgenic Plants with BMV RNA3-Based DNA- Launching Platform Containing GUS and SHMV Coat Protein N. benthamiana plants were transfected using a particle bombardment as described above with a DNA-launching platform for BMV RNA3 wherein the BMV coat protein was replaced with the SHMV coat protein (Sunn-hemp mosaic virus) and the GUS gene was inserted downstream of an additional BMV subgenomic promoter (Figure 7b). The GUS expression was determined by histochemical GUS assay described above. Figure 20 shows the successful expression of GUS in (la 2a)-transgenic plants.

WO 99/61597 PCT/US99/11250 24 Example 15 Movement of Launched BMV RNA 3 Fl progeny plants from self-fertilized (1a+2a)-transgenic N. benthamiana BP14 were inoculated with BMV RNA3 DNA launching platform using Agrobacterium tumefaciens.

Seedlings were germinated on Smurfmedia containing Kanamycin. Plants were grown at 23 C with a 16 hr photoperiod. Once the desired construct (pB3LR42) was obtained in E. coli it was transferred to A. tumefaciens strain LBA4404 using a thaw-freeze method as described above.

The Agrobacterium was grown overnight at 28 0 C under constant shaking. A single lower leaf ofN. benthamiana was punctured with a needle multiple times and submerged in Agrobacterium culture. The inoculated, middle, and upper leaves were harvested 14 days post-inoculation.

Total RNA extraction and northern blot hybridization were performed as described above.

RNA3 replication was detected in all leaves tested (Fig. 21). It shows that BMV RNA3 is able to replicate, move cell-to-cell and spread long distance in (la+2a)-transgenic plants.

Example 16 Transfection of Progeny From (1 a+2a)-Transgenic N. benthamiana With BMV RNA3 DNA-Launching Platform Progeny plants from self-fertilized (1a+2a)-transgenic N. benthamiana (designated BP14) were inoculated with BMV RNA3 DNA-launching platform using Agrobacterium as described in Example 13. Control plants (non-transgenic N. benthamiana) were inoculated with the sap from BMV infected barley using inoculation buffer composed of 50mM NaPO 4 and 1% celite. Root samples were harvested 6 weeks post inoculation. RNA extraction and northern blot hybridization were performed as described above. Figure 22 shows that BMV RNA3 replicated to very high levels in roots. In some (la+2a)-transgenic plants (Figure 22, lanes 2, 5, 6, 7, 8, 10) replication of launched RNA3 dramatically exceeded replication of wildtype BMV in non-transgenic N. benthamiana plants (Figure 22, lane This shows that this system can be used for delivery of RNA, proteins, peptides or other compounds to roots and enables testing of such compounds for various activities, for example, activities directed against root parasites. For example, proteins with anti-nematode activities can be inserted into RNA3 DNA-launching platform using the above described strategies and expressed in roots upon RNA3 replication. Such proteins can be engineered to be expressed in the cytoplasm or alternatively secreted into the surrounding soil.

WO 99/61597 PCT/US99/11250 Example 17 Barley Stripe Mosaic Virus Barley stripe mosaic virus (BSMV) has a tripartite genome (RNA alpha, beta, and gamma). These genomic RNAs have an m7Gppp cap at the 5' end and a t-RNA like structure at the 3' end (Jackson and Hunter, 1989).

A DNA-launching plasmid for BSMV RNA alpha, RNA beta, and RNA gamma containing BSMV RNA cDNA is constructed by precisely fusing at its 5' end to a DNAdependent RNA polymerase promoter and to a self-cleaving ribozyme at its 3' end. A polyadenylation signal may be also included. Alternatively, a convenient restriction site may be engineered at the 3' end of viral cDNAs. Foreign genes or sequences may be expressed in several ways. For example, DNA-launching plasmids based on BSMV RNA beta may contain a foreign gene or sequence expressed in place of ORF beta a.

Transgenic plants having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator are obtained. Such trans-acting factors may include parts of the viral RNA replicase (ORFs alpha a and/or gamma a) or other trans-acting factors.

The trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used. Cis-acting sequences necessary for BSMV RNA replication are removed from transgenes. Alternatively, the full-length RNA alpha is expressed from the chromosome. Alternatively, ORF gamma a including the 5' untranslated region and ORF gamma b from a seed transmitted strain, such as ND18, are also expressed (Edwards, 1995).

A DNA-launching plasmid is constructed containing the DNA-dependent RNA polymerase promoter precisely fused to the 5' end of the BSMV RNA beta, cis-acting elements important for BSMV RNA beta life cycle, such as the 5' and 3' ends, the intercistronic region between the beta a and beta b ORFs (Zhou and Jackson, 1996) and a foreign gene or sequence in place of ORF beta a (coat protein) which is dispensable for BSMV replication and movement (Petty and Jackson, 1990). Such DNA-launching plasmids may lack the internal poly(A) region as this region is dispensable for replication and contain a ribozyme or a convenient restriction site at the 3' end of the modified viral RNA. Alternatively, a DNA-launching plasmid is constructed from RNA gamma in which ORFs gamma a and/or gamma b are replaced with foreign genes or sequences which may also include the triple gene block genes (ORFs beta b, beta c, and beta d) or a heterologous movement protein (TMV 30K, RCNMV Example 18 Tobacco Mosaic Virus Tobacco mosaic virus (TMV) has a single-stranded positive sense RNA genome. The end has an m7Gppp cap and the 3' end contains a t-RNA like structure.

WO 99/61597 PCT/US99/11250 26 A DNA-launching plasmid is constructed based on TMV RNA containing TMV cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and at its 3' end to a self-cleaving ribozyme. A polyadenylation signal may be also included. Alternatively, a convenient restriction site may be engineered at the 3' end. Foreign gene may be expressed from an additional subgenomic RNA by including an additional subgenomic RNA promoter on the strand.

Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator. Such factors may include the viral replicase (126K/183K), movement protein (30K), or coat protein (17.6K). At least one cisacting sequence necessary for TMV RNA replication is removed from transgenes. The transacting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.

A DNA-launching plasmid is constructed containing the DNA-dependent RNA polymerase promoter precisely fused to the 5' end of the TMV cDNA, cis-acting elements important for the TMV life cycle, such as the 5' and 3' ends, origin of assembly, etc., at least one foreign gene or sequence in place of the trans-acting factor that is expressed from the chromosome, and a ribozyme or a convenient restriction site at the 3' end. Alternatively, the foreign gene sequence can be expressed from an additional subgenomic RNA promoter and the sequence coding for the trans-acting factor that is expressed from the transgene can be deleted from the DNA-launching plasmid. Preferably, if the viral replicase proteins are expressed in transgenic plants, the DNA-launching plasmid will have a deletion of nucleotides 3420-4902, which appears to be a region that inhibits replication in trans. (Lewandowski et al., 1998).

Example 19 Potato Virus X Potato virus X (PVX) has a single-stranded positive sense RNA genome. The 5' end has an m7Gppp cap and the 3' end is polyadenylated. A full-length cDNA clone of PVX has been constructed and infectious RNA transcripts obtained (Hemenway et al., 1990).

A DNA-launching plasmid is constructed based on PVX RNA containing PVX cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and having a polyadenylation site at its 3' end. A convenient restriction site may also be included at the 3' end. A foreign gene may be expressed from an additional subgenomic RNA.

Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator. Such factors may include the viral RNA polymerase gene (ORF1-147K), coat protein (ORF5-21K), or triple gene block (ORF2- WO 99/61597 PCT/US99/11250 27 ORF3-12K, ORF4-8K). The triple gene block genes can be expressed individually.

Alternatively, they can be expressed as negative sense transcripts from which plus sense subgenomic RNA for ORFs 2, 3, and 4 can be transcribed by the viral replicase. Such transgene will have a DNA-dependent RNA polymerase promoter fused to sequence of ORFs 2, 3, and 4 in the minus sense orientation and the transcribed sequence will include a subgenomic RNA promoter. At least one cis-acting sequence necessary for PVX RNA replication is removed from transgenes. The trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.

A DNA-launching plasmid is constructed containing the DNA-dependent RNA polymerase promoter precisely fused to the 5' end of the PVX genome, cis-acting elements important for PVX life cycle, such as the 5' and 3' ends, origin of assembly, etc., at least one foreign gene or sequence in place of the trans-acting factor that is expressed from the chromosome and a polyadenylation signal. Alternatively, the foreign gene sequence can be expressed from an additional subgenomic RNA promoter and the sequence coding for the transacting factor that is expressed transgenically can be deleted from the DNA-launching plasmid.

Alternatively, a DNA-launching plasmid is constructed having a DNA-dependent RNA polymerase promoter, polyadenylation site, and the PVX cDNA sequence in which the ORF2 is replaced with a foreign gene or sequence. Alternatively, the ORF2 is deleted and the foreign gene is expressed from an additional subgenomic RNA promoter. Such a DNAlaunching plasmid is inoculated to transgenic plants expressing movement protein from heterologous virus, such as tobacco mosaic virus (TMV 30K), tomato mosaic virus (ToMV or red clover necrotic mosaic virus (RCNMV Example 20 Flock House Virus Flock house virus (FHV) has a genome consisting of two single stranded RNAs. RNAI encodes protein A, involved in RNA replication, and protein B that is translated from sg RNA3 and is dispensable for RNA replication. RNA2 encodes virion capsid precursor protein alpha.

FHV is infectious to insect, plant, mammalian, and yeast cells (Selling et al., 1990; Price et al., 1996).

A DNA-launching plasmid is constructed for FHV RNAl and RNA2 containing FHV RNA cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and at its 3' end to a self-cleaving ribozyme. A polyadenylation signal may be also included.

Alternatively, a convenient restriction site may be engineered at the 3' end. Foreign genes or sequences may be expressed in several ways. For example, DNA-launching plasmids based on WO 99/61597 PCT/US99/1 1250 28 FHV RNA1 may contain a foreign gene or sequence expressed from subgenomic RNA3 as ORF B replacement or as a translational fusion with ORF B. Alternatively, a foreign gene may be expressed from an additional sg RNA. DNA-launching plasmids based on FHV RNA2 may contain a foreign gene(s) or sequence(s) expressed as a part of polyprotein alpha. Foreign gene(s) in such construct may include sequences necessary for polyprotein clevage. DNAlaunching plasmids will preferably also express a movement protein of a heterologous plant virus, such as 30K of TMV or 35K of RCNMV. Alternatively, DNA-launching plasmids will be inoculated onto transgenic plants expressing such movement protein.

Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator. Such factors may include protein A or capsid protein precursor alpha, and preferably will also include a movement protein from a plant virus, such as 30K of TMV or 35K of RCNMV. Trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.

Transgenically expressed trans-acting factors preferably lack at least one cis-acting factor which is necessary for their replication, such as the 5' and/or 3' end.

A DNA-launching plasmid is constructed based on FHV RNAI or FHV RNA2 containing a DNA-dependent RNA polymerase promoter precisely fused to the 5' end of RNAI (or RNA2), cis-acting elements important for FHV RNAI (or RNA2) replication, such as the and 3' ends, at least one foreign gene or sequence and a self-cleaving ribozyme at the 3' end.

Polyadenylation signal may also be included. Alternatively, a convenient restriction site may be engineered at the 3' end of the modified viral RNA sequence of the DNA-launching plasmid.

DNA-launching plasmids based on FHV RNAI may contain a foreign gene or sequence in place of ORF A. Alternatively, the ORF A may be deleted and the foreign gene may be expressed from subgenomic RNA3, for example as an ORF B replacement or as a translational fusion with ORF B. Alternatively, DNA-launching plasmid may contain two exogenous RNA sequences, one in the place of ORF A and the other expressed from the subgenomic RNA3. DNAlaunching plasmids based on FHV RNA2 may contain a foreign gene(s) or sequence(s) in place of ORF alpha or expressed as a part of polyprotein alpha. Foreign gene(s) in such a construct may include sequences necessary for polyprotein clevage.

Example 21 Tomato Spotted Wild Virus Tomato spotted wild virus (TSWV) is a tripartite (RNA L, M, negative sense and ambisense, single stranded RNA virus.

WO 99/61597 PCT/US99/11250 29 Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator. Such factors include the putative TSWV polymerase gene (ORF ORF N, and possibly other trans-acting factors (NSm or NSs).

At least one cis-acting sequence, such as 5' and/or 3' ends, which are necessary for TSWV RNA replication are removed from the transgene. Trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.

A DNA-launching plasmid is constructed based on TSWV RNA M in which the GI and G2 coding sequences are replaced with at least one foreign gene or sequence. Such DNAlaunching plasmid contains a DNA-dependent RNA polymerase promoter and TSWV RNA M cDNA fused to the self-cleaving ribozymes at the 5' and 3' ends. Alternatively, a DNAlaunching plasmid is constructed based on TSWV RNA S in which the N coding region is replaced with a foreign gene or sequence.

Example 22 Barley Mild Mosaic Virus Genome of barley mild mosaic virus (BaMMV) consists of two positive sense, singlestranded, 3'-polyadenylated RNAs. The RNA1 encodes proteins related to the potyviral P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and capsid protein (Kashiwazaki et al., 1990). The RNA2 encodes P1 and P2 protein (Kashiwazaki et al., 1991). The P1 protein is related to the potyviral HC-Pro and the P2 protein is important for fungal transmission. An isolate was obtained containing a deletion in the P2 protein (Timpe and Kuhne, 1995) thus indicating that P2 is dispensable for viral RNA replication.

A DNA-launching plasmid is constructed for BaMMV RNA1 and RNA2 containing BaMMV RNA cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and a polyadenylation site at its 3' end. Foreign genes or sequences may be expressed in several ways. For example, DNA-launching plasmids based on BaMMV RNA2 may contain a foreign gene or sequence expressed as a part of polyprotein which can be cleaved and a foreign protein can be released.

Transgenic plants are obtained having the BaMMV RNAI cDNA lacking the 5' and 3' ends fused to the DNA-dependent RNA polymerase promoter and terminator.

A DNA-launching plasmid is constructed based on BaMMV (isolate M) RNA2. Such plasmid contains a DNA-dependent RNA polymerase promoter precisely fused to the 5' end of RNA2, RNA2 cis-acting replication signals located in the 5' and 3' ends, P1 ORF and a foreign gene in place of P2 ORF or expressed as a part of P1/P2 polyprotein which can be cleaved and a foreign protein can be released.

WO 99/61597 PCT/US99/11250 The contents of all references cited throughout are incorporated herein by this reference to the extent they are not inconsistent with the disclosure, teachings, and principles of the subject invention.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

WO 99/61597 PCT/US99/11250 31 References De Jong and Ahlquist (1992) "A hybrid plant RNA virus made by transferring the noncapsid movement protein from a rod-shaped to an icosahedral virus is competent for systemic infection," PNAS 89:6808-6812.

Dinant, Janda, Kroner, Ahlquist, P. (1993) "Bromovirus RNA replication and transcription requires compatibility between the polymerase- and helicase-like viral RNA synthesis proteins," J. Virol. 67:7181-7189.

Edwards, M.C. (1995) "Mapping of the seed transmission determinants of barley stripe mosaic virus," MPM 8:906-915.

French, R. and Ahlquist, P. (1988) "Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus RNA3," J Virol. 62(7):2411-2421.

Hemenway, Weiss, O'Connell, and Turer, N.E. (1990) "Characterization of infectious transcripts from a potato virus X cDNA clone," Virology 175:365-371.

Ishikawa, Diez, Restrepo-Hartwig, Ahlquist, P. (1997) "Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of viral polymerase-like gene," PNAS94:13810-13815.

Jackson, A.O. and Hunter, B.G. (1989) "Hordeivirus relationships and genome organization," Annu. Rev. Phytopathol. 27:95-121.

Janda, French, Ahlquist, P. (1987) "High efficiency T7 polymerase synthesis of infectious RNA from cloned brome mosaic virus cDNA and effect of 5' extensions on transcript infectivity," Virology 158:259-262.

Kashiwazaki, S. Minobe, Omura, Hibino, H. (1990) "Nucleotide sequence of barley yellow mosaic virus RNA1: a close evolutionary relationship with potyviruses," Journal of General Virology 71:2781-2790.

Kashiwazaki, Minobe, Hibino, H. (1991) "Nucleotide sequence of barley yellow mosaic virus RNA2," Journal of General Virology 72:995-999.

Kikkert (1993) "The biolistic PDS 1000/He device," Plant Cell Tiss. and Org. Cult. 33:221-226.

Lewandowski, Dennis Dawson, William 0. (1998) "Deletion of internal sequences results in tobacco mosaic virus defective RNAs that accumulate to high levels without interfering with replication of the helper virus," Virology 251(2):427-437.

Pacha, R.F. and Ahlquist, P. (1991) "Use of Bromovirus RNA3 hybrids to study template specificity in viral RNA amplification," Journal of Virology 65:3693-3703.

Petty, I.T.D. and Jackson, A.O. (1990) "Mutational analysis of barley stripe mosaic virus RNA beta," Virology 179:712-718.

WO 99/61597 PCT/US99/11250 32 Price, Rueckert, Ahlquist, P. (1996) "Complete replication of an animal virus and maintenance of expression vectors derived from it in Saccharomyces cerevisiae" PNAS 93:9465-9470.

Rasochova, L. and Miller, W.A. (1996) "Satellite RNA of barley yellow dwarf-RPV virus reduces accumulation of RPV helper virus RNA and attenuates RPV symptoms on oats," Molecular Plant-Microbe Interact 9:646-650.

Sacher, French. Ahlquist, P. (1988) "Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo," Virology 167:15-24.

Selling, Allison, Kaesberg, P. (1990) "Genomic RNA of an insect virus directs synthesis of infectious virions in plants," PNAS 87:434-438.

Timpe, U. and Kuhne, T. (1995) "In vitro transcript of a full-length cDNA of a naturally deleted RNA2 of barley mild mosaic virus (BaMMV) replicate in BaMMV-infected plants," Journal of General Virology 76:2619-2623.

T6pfer, Matzeit, Gronenborn, Schell, Steinbiss, H.H. (1987) "A set of plant expression vectors for transcriptional and translational fusions," Nucleic Acids Res.

15:5890.

U.S. Patent No. 5,500,360.

Zhou, H. and Jackson, A.O. (1996) "Analysis of cis-acting elements for replication of barley stripe mosaic virus RNA," Virology 219:150-160.

EDITORIAL NOTE APPLICATION NUMBER 43101/99 The following Sequence Listing pages 1 to 44 are part of the description. The claims pages follow on pages "33" to WO 99/61597 WO 9961597PCTIUS99/1 1250 SEQUENCE LISTING <110> WISCONSIN ALUMNI RESEARCH FOUNDATION Street Address: 614 Walnut Street City: Madison State: Wisconsin Country: us ZIP: 53705 Phone number: (608) 265-2135 Fax: (608) 263-1064 <120> Improved Methods and Materials for Transformation <130> WARF-lOOXC1 <140> <141> <150> <151> <160> <170> <210> <211> <212> <213> 60/086, 526 1998-05-22 8 Patentln Ver. 1 7074

DNA

Brome mosaic virus <400> 1

AAACACTGAT

AGGGAGTCAC

GACAGAACCG

AAGCACATAC

TAGCAAATAT

AATTAGNNNN

TGCACGCAGG

AGACAATCGG

TTTTTGTCAA

TATCGTGGCT

CGGGAAGGGA

AGTTTAAACT

GTTATGACCC

CAACGATTGA

GTCAGAAACC

TTCTTGTCAA.

NNI'NNNNNNN

TTCTCCGGCC

CTGCTCTGAT

GACCGACCTG

GGCCACGACG

CTGGCTGCTA.

GAAGGCGGGA

CCGCCGATGA

AGGAGCCACT

ATTATTGCGC

AAATGCTCCA

NN~NNNNNNN

GCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

AACGACAATC

CGCGGGACAA

CAGCCGCGGG

GTTCAAAAGT

CTGACGTTCC

GATCGTTTCG

AGAGGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

TGATCATGAG

GCCGTTTTAC

TTTCTGGAGT

CGCCTAAGGT

ATAAATTCCC

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

CGGAGAATTA

GTTTGGAACT

TTAATGAGCT

CACTATCAGC

CTCGGTATCC

CAAGATGGAT

TGGGCACAAC

CGCCCGGTTC

GCAGCGCGGC

GTCACTGAAG

TCATCTCACC

120 180 240 300 360 420 480 540 600 660 TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG CATACGCTTG 720 WO 99/61597 WO 9961597PCTIUS99/1 1250 ATCCGGCTAC CTGCCCATTC GGATGGAAGC CGGTCTTGTC CAGCCGAACT GTTCGCCAGG CCCATGGCGA TGCCTGCTTG TCGACTGTGG CCGGCTGGGT ATATTGCTGA AGAGCTTGGC CCGCTCCCGA TTCGCAGCGC I'NNNNNNINmN NNNNNNNNNN ATCCTGTTGC CGGTCTTGCG TAATAATTAA CATGTAATGC CGCAATTATA CATTTAATAC TATCGCGCGC GGTGTCATCT TCTGGTGGTG GTTCTGGTGG GGTGGCGGCT CTGAGGGAGG GAAAAGATGG CAAACGCTAA CAGTCTGACG CTAAAGGCAA GGTTTCATTG GTGACGTTTC GGCTCTAATT CCCAAATGGC TTCCGTCAAT ATTTACCTTC CCAATACGCA AACCGCCTCT 100 AGGTTTCCCG ACTGGAAAGC CATTAGOCAC CCCAGGCTTT 105 AGCGGATAAC AATTTCACAC CTGCAGGTCG ACTCTAGAGG TTATTGATAG AAGTATTTTA 110 ACATGAGCGA AACCCTATAA TCTGGAGAAA ATAGAGAGAG

GACCACCAAG

GATCAGGATG

CTCAAGGCGC

CCGAATATCA

CGAAACATCG

ATCTGGACGA

GCATGCCCGA

TGGTGGAAAA

CATCGAGCGA GCACGTACTC AGAGCATCAG GGGCTCGCGC CGGCGATGAT CTCGTCGTGA TGGCCGCTTT TCTGGATTCA 3TGGCGGACC GCTATCAGGA CATAGCGTTG

"GCGAATGGG

%TCGCCTTCT

GATCGTTCAA

ATGATTATCA

ATGACGTTAT

GCGATAGAAA

ATGTTACTAG

CGGCTCTGAG

CGGTTCCGGT

TAAGGGGGCT

ACTTGATTCT

CGGCCTTGCT

TCAAGTCGGT

CCTCCCTCAA

CCCCGCGCGT

GGGCAGTGAG

ACACTTTATG

AGGAAACAGC

ATCCCCGGTC

CAAATACAAA

GAACCCTAA'I

ATAGATTTG I

CTGACCGCTT

ATCGCCTTCT

ACATTTGGCA

TATAATTTCT

TTATGAGATG

ACAAAATATA

ATCGGGCCTC

GGTGGTGGCT

GGTGGCTCTG

ATGACCGAAA.

GTCGCTACTG

AATGGTAATG

GACGGTGATA

TCGGTTGAAT

TGGCCGATTC

CGCAACGCAA

CTTCCGGCTC

TATGACCATG

ACTGGATTTT

TACATACTAA

TCCCTTATCT

AGAGAGAGAC

'CTCGTGCTT

L'GACGAGTTC

),TAAAGTTTC

"TTGAATTAC

GGTTTTTATG

GCGCGCAAAC

CTGTCAATGC

CTGAGGGTGG

GTTCCGGTGA

ATGCCGATGA

ATTACGGTGC

GTGCTACTGG

ATTCACCTTT

GTCGCCCTTT

ATTAATGCAG

TTAATGTGAG

GTATGTTGTG

ATTACGCCAA

GGTTTTAGGA

GGGTTTCTTA

GGGAACTACT

TGGTGATTTG

GCTACCCGTG 1 rACGGTATCG1

TTCTGANNNNI

rTAAGATTGA

GTTAAGCATG

ATTAGAGTCC

TAGGATAAAT

TGGCGGCGGC

CGGTTCTGAG

TTTTGATTAT

AAACGCGCTA

TGCTATCGAT

TGATTTTGCT

AATGAATAAT

TGTCTTTGGC

CTGGCACGAC

TTAGCTCACT

TGGAATTGTG

GCTTGCATGC

ATTAGAAATT

TATGCTCAAC

CACACATTAT

CGGACTCTAG

780 840 900 960 .020 .080 L200 1260 1320 1380 1.440 1.500 1.560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 WO 99/61597 WO 9961597PCTIUS99/1 1250 115 AGGATCCCCG GGTACCGAGC TCACTTAACA CAATTAAAGA GGACTTCTTG TGTCGTGTTA 120 TTTGGTCGAC TTAAGCCGAA TACTGTTTTT ATGTGTCCAT 125 TTGAAGGGCC GCTTTATCAG GACCTGCAAA CCAGAAGTAA TGTCCAGGAT TGATACTTCG 130 AGCGATAACA TCTTGCGGAC CAGATTACCG TGGAGCAATT 135 GTCCCCAAAG GCAAGAACTT ATAATGTAGT AAACCAGCCT GATCGCAGAA TCCGCGGTGC 140 GAATAAAGCC ATCCTGACGT TCCCATACGG AATGCATCTT 145 CATGGAAATA TCGCATGTAG GTTAGAGCAT GCACATGTTT ACCATGCTCA TTATATCCAA 150 GGCCAAATGG ACATTCACCA TGCGATCTCA CTTCCGCGAC 155 GTTGTTATGT AATCTCTTAC GCCACGAGGG TCCGTTGGTA CGGTTTAACG TCATCACTGA 160 CTCCTGAGGT ATCTCAGCAG GGCTGTCTGC ACTTCATGCT 165 CAAACGTGAT ATCGGTACAG- AAACTTAAAA CTGTCCGCAI TCGAATTCTC C TCAAATCACC

AGGCAACCAA

CCAAAGTGAC

CAATCCAGTC

CTTGAGTCAT

TACGACGCTT

TGTCCCTATT

ACCGGTAAGT

TAAA~ACCCGC4

GTGAACATTT

CATCAACAAG

GCACAACGTC

CCTCGGCCGA

TTATGGCAGT

GGACAGAAAC

TATCAACAAT

CAGTGTAATC

TATCGTCTAT

CGCCGGAAAT

AATAACGCAC

CCTCAACAGA

CGGACTCAGG

CTGGCGGGAC

CCAGCCGGTC

AATCTTGCTC

AAGAATCCAG

;AGCAGAGGT C ~GCGAGCTCG C CAGTACTCC 'T TTGTCAACA C TTGCTCACG C GTAAGATAC C

CGTGAGACTT

A.CCGCATTTA

CTTGTGAACA

GTCACGAGAC

AGACAGAGCA

CAGCCTATGA

CAAAGCTACC

TTTACGATTC

GGTTTTACCG

TTTGGCGCTA

GTACGTTTCA

GTATTTTTTA

GCGATGGTAG

GTCCCATAGG

AAATTCCTTC

CACCTCGGCA

GCTCGAACTC

TTTCGCCTTC

GAATAAGTCC

AAATTCTTCA

ACGTAGTAGG

TCACACAGA

:CGTTAAAGC

'CATGTTTAA

;AGATCCCTT

;GAAAATCCT

~TTCTGTTCG

TAGAAACTT

'GCTTCAGCA

5,CGTCACGGC rTGAACGAAA

GCCACCACCA

CAGGACGGTA

TTGGAATTAT

GCCGTCACAA

CATCCCGCAA

GCTTCTGCAA

TCTGCGTGCT

GGATACAACC

GTCTCAAAGA

TGACGAAGAT

ATGGCTCCGT

TCCGGGACCA

TCAGGGGCAT

TTCTTCGAGC

TCTTCAGTCC

ACGTTTGAGz

GGAAATCTGC

GACAAGCGCA 2400 AATACTCAAA 2460 ACAAATCACA 2520 GCGCTTCGTG 2580 TAGCCCTCGT 2640 GATCAATAGT 2700 TGGACTCAGA 2760 GATTAACAGC 2820 GATCATATTG 2880 TCTGCTCTGT 2940 GGAGTTGACC 3000 CACCGTGCAT 3060 AAGTGTCAGG 3120 TTAGGTCCTC 3180 CTCCATCAAC 3240 TGTCCCTCAA 3300 TCGGACCTAA 3360 AGTTACCGTT 3420 TGCTCTTATT 3480 TAGACTCGGA 3540 GTCTAGATAT 3600 CATCAGTCAC 3660 CATGAAACTC 3720 GCTTGGTCTT 3780 *AAAACGTTCT 3840 GACGAGTCAG 3900 TAGCCAATGT 3960 WO 99/61597 WO 9961597PCT/US99/1 1250 TCTCAGCCAT CCTACTTTCG CCCTGGATGA ATCTCCACCC CACCAAAACC TAGTTTTGAA 4020 GTGATGGCAC CAACCTTTCC TTGATACAGA TTCAAAGTCA 175 CTCACCAGCC ATGATAGCCT AGATGCGACA GCTI-rCATGT TATCTCCTCT ACCTCTCTCA 180 ACTCCAGTCT TCAGGTATTG GTGACGTAGT GTCTCCCGGG 185 GTTACATTTC AGCATITCGC CTCGGTGAAA AATGAGCCCA TTCAAAATCG AATTTGATCA 190 AAGCAAGGGA AGAAAACCCT ACGTAGTACG CGTACTCCAT 195 ATAACCGCCG TGGATACAGA AAGACAAAAG TTCGGGACTT GCACATCCTC TCCTCATGTC 200 CACCCTTTTA TCCCTTCTTG GTCTTCAGGG GGAAAACTGT 205 CAGGCTATGG GGCGCATGAT GTCACGGAAG GCGTCAGCCT CCTTTTCGCG TATTCAATCT 210 TTGGGCACTC TGGCTGTCAG TGACATTTTG TTGGTGAAAA 215 AAATGAAATG AAC'FTCCTTA TCATCCCTTA CGTCAGTGGA ACGTCTTCTT TTTCCACGAT 220

ATTCCATCCC

AAGCAAAGGC

GACCGTTAAT

TCTCAGTCCA

CTGTGGTTTT

AGACCCCTAC

GGCAGCGTAA

GCTCCAACAG

AATCTTGCCA

CCTCATCCGC

CGCGGTCAAA

GCGAATGCAT

TAGCCCAATC

CATCGAAATC

GGGCAGCGTC

AAAAGTGATG

CGAGACAGTC

ACTGCTGAGT

CCTCAATAGA

GCGCAGATAA

CACCCTTCTC

ACAAAGAACA

TATAGAGGAA

GATATCACAT

GTTCCTCGTG

ATCGCGGAGG

CACTAGATGA

AATAACAGTC

TTCTTTACTT

GGCGACGCGC

GTACTTAGAT

ATTTGTAGCG

ATAGGTGGTT

TCCGTGGATG

GCCTGACCCG

CAACATGGCG

GGCGTCACAC

AGCTTGGACA

ATCGCTrTCT

TCTAACACCC

CCACCAAGAC

ATAATGCTCC

CAAATTTAAG

GAGCTTATTG

CTGTTGCGCA

AGCA7ATCAAC

AGTAGCAGAA

GGGTCTTGCG

CAATCCACTT

GGTGGGGGTC

GCCGTAAGCT

TAATCTTCAA

GACGACTTGG

TCCTTGAAAC

ACACATTTCC

ATGTCTTCAA

ATGATCTTAT

CCATCGATGC

TAAGATAATG

TCACGTTGCC

CCGTCGAACA

AGACCTTGGA

TCACAATCTT

TGCAAAATTT

AACACAGGAC

CCTCCGAAAT

GCTACACGCA

TCAAAGGCAC

CGAACGTTGA

ACCTGATTGT

TTCAGCAAAT

CCGTGGTCGA

AAGGATAGTG

GCTTTGAAGA

CATCTTTGGG

TTTCGTACTT 4080 TGTCTAAGCG 4140 CGGATAAGAT 4200 ATCTGAAAGC 4260 AGCGATTGAG 4320 ACCATACACA 4380 AGGTCATGAT 4440 AATGCACCGA 4500 TGCTTTCATT 4560 AGTGACATTT 4620 TAACGGTACC 4680 AGCCCATATC 4740 GAGCTCGGTT 4800 TTCGCATGCG 4860 AACAACTGTG 4920 CTATAACGGG 4980 GAGCACCAGC 5040 CACCATAACG 5100 TTTTCTTAGA 5160 CTACGATGTC 5220 CGATAGAACT 5280 GGTCCTCTCC 5340 GGATTGTGCG 5400 CGTGGTTGGA 5460 ACCACTGTCG 5520 GTAGAGGCAT TCTTGAACGA TAGCCTTTCC TTTATCGCAA TGATGGCATT TGTAGAAGCC 5580 WO 99/61597 WO 9961597PCTIUS99/1 1250 ATCTTCCTT TCTACTGTCC 225 GAGGTTTCCC GATATTACCC TGTATCT FIG ATATTCTTGG GTCAGTCCCT TATGTTACGT 230 GCCTGTGGGC ATTCAGTCTG GCGCGTTACA AGAAAGCCGG 235 ATGCAGATAT TCGTAATTAT AAGGTTGGGC AGGCCAGCGT TGTGGGTCAA TAATCAGGAA 240 ATGTCACGCC GTATGTTATT CGTCGTGACT GGGAA.AACCC 245 TTCGCCAGCT GGCGTAATAG AGCCTGAATG GCGAATGNNN AAGTTGTCTA AGCGTCAATT 250 GCTCCCCGAC CGGCAGCTCG NNNNNNNNN4IN NNNNN1NNNN 255 NNNN~ uiNNurNNNim NNNNNJ'NNNM NNNNNINN NNNI*W NNWNNNN 260 NNNNNINNN NI'JDNNNNNMD I'NNI'JI'NNN'J INNINNNNMN 265 NNNNNNNNmNN rNN ri NN'NN~NNMN NNNNiNNNNNN

TTTCGATGAA

TTTGTTGAAA

AGTAGACGAG

CCTGTAGAAA

GATCGCGAAA

GCAATTGCTG

GCGGGCAACG

ATCGTGCTGC

GTGATGGAGC

GCCGGGAAAA

TGGCGTTACC

CGAAGAGGCC

NNNNAATTCA

TGTTTACACC

GCACAAAATC

NNDJL'NNNNNN

NNNNNNNNN

NNNNNNINN

NN4INNNNNNN'

LJNNNNNNNNN

NNNNN'NNtN

NNNNNI~NNNN

GTGACAGATA

AGTCTCAATA

AGTGTCGTGC

CCCCAACCCG

ACTGTGGAAT

TGCCAGGCAG

TCTGGTATCA

GTTTCGATGC

ATCAGGGCGG

GTGTACAATT

CAACTTA1ATC

CGCACCGATC

GTACA ITAAA

ACAATATATC

ACCACTCGAT

NNNNNNNNI'J

NJINNDINNNN

L'JNNNINNNNN

rINNNNNN'NNN NNNNN1NL*1

JNNNNNNNNNN

NNNNNNN

NNNNNNI~NN

'CTGGGCAAT

3CCCTCTGGT

TCCACCATGT

TGAAATCAAA

rGATCAGCGT rTTTAACGAT

GCGCGAAGTC

GGTCACTCAT

CTATACGCCA

CACTGGCCGT

GCCTTGCAGC

GCCCTTCCCA

AACGTCCGCA

CTGCCACCAG

ACAGGCAGCC

NNN'NNNNNNN

NNNN'NNNNI'J

NNL'NNNNNNN

i'JNNN4INNNN D'1L'NNLNNNN

NN'NNNNNNNN

NNNNNN~NNNN

NL'NiNNNNNN~ GGAATCCGAG 5640 CTTCTGAGAC 5700 TGACCGGGTG 5760 AAACTCGACG 5820 TGGTGGGAAA 5880 CAGTTCGCCG 5940 TTTATACCGA 6000 TACGGCAAAG 6060 TTTGAAGCCG 6120 CGTTTTACAA 6180 ACATCCCCCT 6240 ACAGTTGCGC 6300 ATGTGTTATT 6360 CCAGCCAACA 6420 CATCAGNNNN 6480 NNNNNrH\NNN 6540 NNDNNNNNN 6600 NNNN~NNNNNN 6660 NNNNNNNNNN 6720 NNNN'NNNINJN 6780 NN NNNNNNNN 6840 NI'41\NNNNNNN 6900 NN'NNNNNNN 6960 270 NINNNUNNNM NNNNN'NNNNN NNNN NNNNNN NNNNN NNNNNNNNNN NINNJ'JNNNNM 7 02 0 NNNN.NNNNNN NNN~NNNNNNN NNN'NNDNNND NNx'NNNN NNNNNNNNN N1NNN 7074 <210> 2 275 <211> 6750 <212> DNA <213> Brome mosaic virus WO 99/61597 WO 9961597PCTIUS99/1 1250 <400> 2 AAACACTGAT AGTTTAAACT 280 AGGGAGTCAC GTTATGACCC GACAGAACCG CAACGATTGA 285 AAGCACATAC GTCAGAAACC TAGCAAATAT TTCTTGTCAA AATTAGNNNN NNNI'NNNNNJNI 290 TGCACGCAGG TTCTCCGGCC AGACAATCGG CTGCTCTGAT 295 TTTTTGTCAA GACCGACCTG TATCGTGGCT GGCCACGACG CGGGAAGGGA CTGGCTGCTA 300 TTGCTCCTGC CGAGAAAGTA ATCCGGCTAC CTGCCCATTC 305 GGATGGAAGC CGGTCTTGTC CAGCCGAACT GTTCGCCAGG CCCATGGCGA TGCCTGCTTG 310 TCGACTGTGG CCGGCTGGGT ATATTGCTGA AGAGCTTGGC 315 CCGCTCCCGA TTCGCAGCGC NNNNNNNNNN NUNflUNflNhNu ATCCTGTTGC CGGTCTTGCG 320 TAATAATTAA CATGTAATGC CGCAAFTATA CATTTAATAC 325 TATCGCGCGC GGTGTCATCT TCTGGTGGTG GTTCTGGTGG GGTGGCGGCT CTGAGGGAGG 330

GAAGGCGGGA

CCGCCGATGA

AGGAGCCACT

ATTATTGCGC

AAATGCTCCA

NNNNNNNI'NNN

GCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

TCCATCATGG

GACCACCAAG

GATCAGGATG

CTCAAGGCGC

AACGACAATC

CGCGGGACAA

CAGCCGCGGG

GTTCAAAAGT

CTGACGTTCC

GATCGTTTCG

AGAGGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

CTGATGCAAT

CGAAACATCG

ATCTGGACGA

GCATGCCCGA

TGATCATGAG

GCCGTTTTAC

TTTCTGGAGT

CGCCTAAGGT

ATAAATTCCC

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

GCGGCGGCTG

CATCGAGCGA

AGAGCATCAG

CGGCGATGAT

CGGAGAATTA GTTTGGAACT 120 TTAATGAGCT 180 CACTATCAGC 240 CTCGGTATCC 300 CAAGATGGAT 360 TGGGCACAAC 420 CGCCCGGTTC 480 GCAGCGCGGC 540 GTCACTGAAG 600 TCATCTCACC 660 CATACGCTTG 720 GCACGTACTC 780 GGGCTCGCGC 840 CTCGTCGTGA 900 CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA 960 GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG 1020

GGCGAATGGG

ATCGCCTTCT

GATCGTTCAA

ATGATTATCA

ATGACGTTAT

GCGATAGAAA

ATGTTACTAG

CGGCTCTGAG

CGGTTCCGGT

CTGACCGCTT

ATCGCCTTCT

ACATTTGGCA

TATAATTTCT

TTATGAGATG

ACAAAATATA

ATCGGGCCTC

GGTGGTGGCT

GGTGGCTCTG

CCTCGTGCTT

TGACGAGTTC

ATAAAGTTTC

GTTGAATTAC

GGTTTTTATG

GCGCGCAAAC

CTGTCAATGC

CTGAGGGTGG

TACGGTATCG

TTCTGANNNN

TTAAGATTGA

GTTAAGCATG

ATTAGAGTCC

TAGGATAAAT

TGGCGGCGGC

CGGTTCTGAG

1080 1140 1200 1260 1320 1380 1440 1500 GTTCCGGTGA TTTTGATTAT 1560 GAAAAGATGG CAAACGCTAA TAAGGGGGCT ATGACCGAAA ATGCCGATGA AAACGCGCTA 1620 WO 99/61597 WO 9961597PCTIUS99/1 1250 7 CAGTCTGACG CTAAAGGCAA ACTTGATTCT GTCGCTACTG ATTACGGTGC TGCTATCGAT 1680 335 GGTTTCATTG GTGACGTTTC CGGCCTTGCT GGCTCTAATT CCCAAATGGC TCAAGTCGGT C TTCCGTCAAT ATTTACCTTC CCTCCCTCAA I] 340 CCAATACGCA AACCGCCTCT CCCCGCGCGT 9J AGGTTTCCCG ACTGGAAAGC GGGCAGTGAGC 345 CATTAGGCAC CCCAGGCTTT ACACTTTATG AGCGGATAAC AATTTCACAC AGGAAACAGC CTGCAGGTCG ACTCTAGAGG ATCCCCGGTC 350 TCCAAGAATA TCAAAGATAC AGTCTCAGAA AGGGTAATAT CGGGAAACCT CCTCGGATTC 355 AGGACAGTAG AAAAGGAAGA TGGCTTCTAC ATCGTTCAAG AATGCCTCTA CCGACAGTGG CATCGTGGAA AAAGAAGACG TTCCAACCAC 360 CTCCACTGAC GTAAGGGATG ACGCACAATC ATAAGGAAGT TCA'ITTCATT TGGAGAGGAC 365 TTTTTCACCA ACAAAATGTC AAGTTCTATC GCTGACAGCC AGAGTGCCCA AGACATCGTA CAGATTGAAT ACGCGAAAAG GTCTAAGAAA 370 GAGGCTGACG CCTTCCGTGA CCGTTATGGT TATCATGCGC CCCATAGCCT GGCTGGTGCT 375 GACAGITTTC CCCCTGAAGA CCCCGTTATA TCAAGAAGGG ATAAAAGGGT GCACAGTTGT CGACATGAGG AGAGGATGTG CCGCATGCGA 380 GAAGTCCCGA ACTTTTGTCT TAACCGAGCT ATCTGTATCC ACGGCGGTTA TGATATGGGC 385 CATGGAGTAC GCGTACTACG TGGTACCGTT ~ATGGTAATG G

;ACGGTGATA

'CGGTTGAAT G 'GGCCGATTC T 'GCAACGCAA I TTCCGGCTC C rATGACCATG V CATGGTGG

GACCAGAGGG

CATTGCCCAG

A4AATGCCATC

TCCCAAAGAT

GTCTTCAAAG

CCACTATCCT

CTCGACCACG

GATTTGCTGA

GACAATCAGG

ATCAACGTTC

GGTGCCTTTG

CTGCGTGTAG

GATTTCGGAG

TGTCCTGTGT

AAAATTTTGC

CAAGATTGTG

TTCCAAGGTC

ATGTTCGACG

ITGCTACTGG TI LTTCACCTTT ;TCGCCCTTT I LTTAATGCAG C 'TAATGTGAG 9]

;TATGTTGTG

kTTACGCCAAC

!LGCACGACAC

:TATTGAGAC

CTATCTGTCA

rTTGCGATAA

GGACCCCCAC

CAAGTGGATT

TCGCAAGACC

GTTCTGCTAC

AGTTGATTGC

TTGCGCAACA

GCAATAAGCT

ACTTAAATTT

CGGAGCATTA

GGTCTTGGTG

TGGGTGTTAG

AAGAAAGCGA

ATGTCCAAGC

TGTGTGACGC

GCGCCATGTT

GATTTTGCT 1740 LATGAATAAT 1800 'GTCTTTGGC 1860 ~TGGCACGAC 1920 ETAGCTCACT 1980 [GGAATTGTG 2040 3CTTGCATGC 2100 rCTCGTCTAC 2160 TTTTCAACAA 2220 CTTCATCGAA 2280 PGGAAAGGCT 2340 CCACGAGGAA 2400 GATGTGATAT 2460 CTTCCTCTAT 2520 TTGTTCTTTG 2580 TGAGAAGGGT 2640 GTTATCTGCG 2700 CTCTATTGAG 2760 GACTCAGCAG 2820 TGACTGTCTC 2880 GCATCACTTT 2940 AGACGCTGCC 3000 TGATTTCGAT 3060 TGATTGGGCT 3120 CATGCATTCG 3180 GTTTGACCGC 3240 WO 99/61597 WO 9961597PCTfUS99/1 1250 GAGGGTTTTC TTCCCTTGCT GTGATCAAAT TCGATTTTGA 390 TTGGGCTCAT TTTTCACCGA C CGCGAAATGC TGAAATGTAAC 395 CCCCGGGAGA CACTACGTCA TCAATACCTG AAGACTGGAG GTGAGAGAGG TAGAGGAGAT 400 AACATGAAAG CTGTCGCATC CAGGCTATCA TGGCTGGTGA 405 TTGACTTTGA ATCTGTATCA TGGAAAGGTT GGTGCCATCA GCGAAAGTAG GATGGCTGAG 410 TATGCGGACA GTT'ITAAGTT TCTGTACCGA TATCACGTTT 415 GAGCATGAAG TGCAGACAGC GCTGCTGAGA TACCTCAGGA GTCAGTGATG ACGITAAACC 420 GTACCAACGG ACCCrCGTGG TGTAAGAGAT TACATAACAA 425 GGTCGCGGAA GTGAGATCGC ATGGTGAATG TCCATTTGGC GTTGGATATA ATGAGCATGG 430 AAAACATGTG CATGCTCTAA CCTACATGCG ATA~rTCCAT 435 AAAGATGCAT TCCGTATGGG GACGTCAGGA TGGC=TATT CGCACCGCGG ATTCTGCGAT .AAATGTCAC TI

AATGAAAGC

TCGGTGCAT I 'ATCATGACC 'I :TGTGTATGG I] ECTCAATCGC I1 kGCTTTCAGA rATCTTATC

GCGCTTAGAC

AAAGTACGAA

CTTCAAAACT

AACATTGGCT

TCTGACTCGT

GAGAACGTTT

CAAGACCAAG

GGAGTTTCAT

GGTGACTGAT

CATATCTAGA

CTCCGAGTCT

AAATAAGAGC

CAACGGTAAC

TTTAGGTCCG

CTTGAGGGAC

GGTTGATGGA

AGAGGACCTA

CCCTGACACT

CATGCACGGT

'GGCAACGTG1 LCATTATCTT2

GCATCGATG

'ATAAGATCA

~TTGAAGACA

'GGAAATGTG

CGTTTCAAGG

3CCAAGTCGT

%.TTGAAGATT

V GCTTACGG

%GGTTTTGGT

PGCAGATTTC

CTCTCAAACG

TGGACTGAAG

CGCTCGAAGA

GATGCCCCTG

GTGGTCCCGG

CACGGAGCCA

AATCTTCGTC

ATCTTTGAGA

TGGTTGTATC

AAGCACGCAG

ATTGCAGAAG

GTTGCGGGAT

ATTGTGACGG

TATAATTCCA

GTACCGTCCT

kCGGGTCAGG

%CATCCACGG

3AACCACCTA

TCGCTACAAA

TATCTAAGTA

TGCGCGTCGC

AAAGTAAAGA

CGACTGTTAT

ATCATCTAGT

CCCTCCGCGA

GGGGTGGAGA

CCCTACTACG

TTGAAGAATT

AGGACTTATT

AGAAGGCGAA

AGAGTTCGAG

ATGCCGAGGT

TGAAGGAATT

ACCTATGGGA

CCTACCATCG

CTAAAAAATA

ATGAAACGTA

CTAGCGCCAP.

GCGGTAAAAC

CGAATCGTA;

AGGTAGCTT]

GTCATAGGC9 CGCGGATGAG 3300 ATGGCAAGAT 3360 TCTGTTGGAG 3420 TTTACGCTGC 3480 CGTAGGGGTC 3540 CAAAACCACA 3600 ATGGACTGAG 3660 TATTAACGGT 3720 GGCCTTTGCT 3780 TGGGATGGAA 3840 TTCATCCAGG 3900 TCTGGATTCT 3960 TGAGCAAGAT 4020 CGACCGGCTG 4080 AGTCCCGCCA 4140 CCCTGAGTCC 4200 GTCTGTTGAG 4260 TGTGCGTTAT 4320 CATTTCCGGC 4380 CATAGACGAT 4440 CGATTACACT 4500 CATTGTTGAT 4560 AGTTTCTGTC 4620 *CACTGCCATA 4680 ATCGGCCGAG 4740 *GGACGTTGTG 4800 SGCTTGTTGAT 4860 WO 99/61597 WO 9961597PCTIUS99/1 1250 GAGGCTGGTT TACTACATTA CAAGTTCTTG CCTTTGGGGA 445 AAATTGCTCC ACGGTAATCT TGTCCGCAAG ATGTTATCGC ACGAAGTATC AATCCTGGAC 450 ATTACTTCTG GTTTGCAGGT GCTGATAAAG CGGCCCTTCA 455 GATGGACACA TAAAAACAGT GTTCGGCTTA AGTCGACCAA TTAACACGAC ACAAGAAGTC 460 ATCTTTAATT GTGFTAAGTG AGCTCGGTAC CCGGGGATCC 465 TCTCTCTCTA TTTTCTCCAG CTTATAGGGT TTCGCTCATG GTAAAATACT TCTATCAATA 470 GTCAGTCCCT TATGTTACGT GCCTGTGGGC ATTCAGTCTG 475 GCGCGTTACA AGAAAGCCGG ATGCAGATAT TCGTAATTAI AAGGTTGGGC AGGCCAGCGI 480 TGTGGGTCAA TAATCAGGAP ATGTCACGCC GTATGTTATI 485 CGTCGTGACT GGGAAAACCC TTCGCCAGCT GGCGTAATAC AGCCTGAATG GCGAATGNNI 490 AAGTTGTCTA AGCGTCAAT' GCTCCCCGAC CGGCAGCTC(

TGGTCAACTC

CACAGAGCAG

GCAATATGAT

TGCTGTTAAT

ATCTGAGTCC

CACTATTGAT

AACGAGGGCT

ACACGAAGCG

ATGTGATTTG

CTTTGAGTAT

ATGCGCTTGT

TCTAGAGTCC

AATAATGTGT

TGTTGAGCAT

AAATTTCTAA

CCTGTAGAAA

GATCGCGAAA

GCAATTGCTG

GCGGGCAACG

ATCGTGCTGC

GTGATGGAGC

GCCGGGAAA-P

TGGCGTTACC

CGAAGAGGCC

~I NNNNAATTC] P TGTTTACAC( 3 GCACAAAAT( CTGGTGGTGG C ATTTCGTTCA CGCCGTGACG I] CTGCTGAAGC C AAAGTTTCTA C

CCGAACAGAA

AAGGATTTTC(

CAAGGGATCT

TTTAAACATG

TGCTTTAACG

CTCTGTGTGA

GCAAATCACC

GAGTAGTTCC

ATAAGAAACC

TTCCTAAAAC

CCCCAACCCG

ACTGTGGAAT

TGCCAGGCAG

TCTGGTATCA

GTTTCGATGC

ATCAGGGCGG

LGTGTACAATT

CAACTTAATC

CGCACCGATC

kGTACATTAAA

'ACAATATATC

:ACCACTCGAT

TGCTCTGTC ~GTCTCGTGA C ~TGTTCACAA C

;TAAATGCGG

AAGTCTCAC

'GTATCTTAC

CGTGAGCAA

CTGTTGACAA

%GGAGTACTG

GCGAGCTCGC

GACCTCTGCT

PGTCTCTCTC

CAGATAAGGG

CTTAGTATGT

CAAAATCCAG

TGAAATCAAA

TGATCAGCGT

TTTTAACGAT

GCGCGAAGTC

GGTCACTCAT

CTATACGCCA

CACTGGCCGT

GCCTTGCAGC

GCCCTTCCCA

AACGTCCGCA

CTGCCACCAG

ACAGGCAGCC

.AAATGTTCA 4920 GCGGGTTTT 4980 ACTTACCGG 5040 LAATAGGGAC 5100 NAAGCGTCGT 5160 GATGACTCAA 5220 GGACTGGATT 5280 CGTCACTTTG 5340 TTTGGTTGCC 5400 TGGTGATTTG 5460 CGAGAATTCG 5520 TACAAATCTA 5580 AATTAGGGTT 5640 ATTTGTATTT 5700 TGACCGGGTG 5760 AAACTCGACG 5820 TGGTGGGAAA 5880 CAGTTCGCCG 5940 TTTATACCGA 6000 TACGGCAAAG 6060 TTTGAAGCCG 6120 CGTTTTACAA 6180 ACATCCCCCT 6240 ACAGTTGCGC 6300 ATGTGTTATT 6360 CCAGCCAACA 6420 CATCAGNNNN 6480 WO 99/61597 PCT/US99/1 1250 495 1NNNNDN'~NNNNN

NNNDNNDNNNN

NNNNNNNI'NNN

500

NNNNNNNNN

NNNNNJNN

NNNNNNNNNN NNI'NNNNNNM' N.NDJ~NLNNNNN NNNN~NNNNNN NINNNNNNNN NNNNNNNNNN NN~NNNNNNNN NNNNr'NNLNNM DNNNNrNINNM NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNrINI'rl~ rlNNNNDNNNM NNNJNN'NNM NNNNNNNNNN NNNNNNNNN NNINrNNNNNNN NND4NNNNNNN NNNNNI'NNNN NNNNNNNlNN NI'JNNNNNNN 6540 6600 6660 6720 6750 <210> 3 <211> 6426 <212> DNA <213> Brome mosaic virus <400> 3

AAACACTGAT

AGGGAGTCAC

515

GACAGAACCG

AAGCACATAC

520 TAG CAAATAT

AATTAGNNNN

TGCACGCAGG

525

AGACAATCGG

TTTTTGTCAA

530 TATCGTGGCT

CGGGAAGGGA

TTGCTCCTGC

535 AT CCGGCTAC

GGATGGAAGC

540 CAGCCGAACT

CCCATGGCGA

TCGACTGTGG

545

ATATTGCTGA

CCGCTCCCGA

AGTTTAAACT

GTTATGACCC

CAACGATTGA

GTCAGAAACC

TTCTTGTCAA

NNNI'NNNNNNN

TTCTCCGGCC

CTGCTCTGAT

GACCGACCTG

GGCCACGACG

CTGGCTGCTA

CGAGAAAGTA

CTGCCCATTC

CGGTCTTGTC

GTTCGCCAGG

TGCCTGCTTG

CCGGCTGGGT

AGAGCTTGGC

TTCGCAGCGC

GAAGGCGGGA

CCGCCGATGA

AGGAGCCACT

ATTATTGCGC

AAATGCTCCA

NNNI'I'NNNNN

GCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

TCCATCATGG

GACCACCAAG

GATCAGGATG

CTCAAGGCGC

CCGAATATCA

GTGGCGGACC

GGCGAATGGG

ATCGCCTTCT

AACGACAATC

CGCGGGACAA

CAGCCGCGGG

GTTCAAAAGT

CTGACGTTCC

GATCGTTTCG

AGAGGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

CTGATGCAAT

CGAAACATCG

ATCTGGACGA

GCATGCCCGA

TGATCATGAG

GCCGTTTTAC

TTTCTGGAGT

CGCCTAAGGT

ATAAATTCCC

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

GCGGCGGCTG

CATCGAGCGA

AGAGCATCAG

CGGCGATGAT

CGGAGAATTA

GTTTGGAACT

TTAATGAGCT

CACTATCAGC

CTCGGTATCC

CAAGATGGAT

TGGGCACAAC

CGCCCGGTTC

GCAGCGCGGC

GTCACTGAAG

TCATCTCACC

CATACGCTTG

GCACGTACTC

GGGCTCGCGC

CTCGTCGTGA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 TGGTGGAAAA TGGCCGCTTT TCTGGATTCA 960 GCTATCAGGA CATAGCGTTG GCTACCCGTG CTGACCGCTT CCTCGTGCTT TACGGTATCG ATCGCCTTCT TGACGAGTTC TTCTGANNNN 1020 1080 1140 WO 99/61597 WO 9961597PCT/US99/1 1250 550 NNNNNIN DINNNNN'Nr4N ATCCTGTTGC CGGTCT1'GCG TAATAATTAA CATGTAATGC 555 CGCAATTATA CATTTAATAC TATCGCGCGC GGTGTCATCT 560 TCTGGTGGTG GTTCTGGTGG GGTGGCGGCT CTGAGGGAGG GAAAAGATGG CAAACGCTAA 565 CAGTCTGACG CTAAAGGCAA GGTTTCATTG GTGACGTTTC 570 GGCTCTAATT CCCAAATGGC TTCCGTCAAT ATTTACCTTC CCAATACGCA AACCGCCTCT 575 AGGTTTCCCG ACTGGAAAGC CATTAGGCAC CCCAGGCTTT 580 AGCGGATAAC AATTTCACAC CTGCAGGTCA CTGGATTTTG AAATACAAAT ACATACTAAG 585 AACCCTAATT CCCTTATCTG TAGATTTGTA GAGAGAGACT 590 TAGCCAAAGT GGTCTGCCTG TTAGGTTCAT ATATCATAGA GGGACTTCAC GAGCAAAGCA 595 CGACCTTCTT TTTCAAGCGT GCTCAAGGGC CTCATGTAAA 600 CCGAGGAATA AATTCCAATG TCTTCCAGTT GTGAA.ACCAG

GATCGTTCAA

ATGATTATCA

ATGACGTTAT

GCGATAGAAA

ATGTTACTAG

CGGCTCTGAG

CGGTTCCGGT

TA7AGGGGGCT

ACTTGATTCT

CGGCCTTGCT

TCAAGTCGGT

CCTCCCTCAA

CCCCGCGCGT

GGGCAGTGAG

ACACTTTATG

AGGAAACAGC

GTTTTAGGAA

GGTTTCTTAT

GGAACTACTC

GGTGATTTGC

ACCAGGAGTT

ACCGATCATC

TCAACTGACG

TGTGTGGTCC

CGTCCGATCC

GTTCTGTAGA

TCACCATCAG

ACATTTGGCA

TATAATTTCT

TTATGAGATG

ACAAAATATA

ATCGGGCCTC

GGTGGTGGCT

GGTGGCTCTG

ATGACCGAAA

GTCGCTACTG

AATGGTAATG

GACGGTGATA

TCGGTTGAAT

TGGCCGATTC

CGCAACGCAA

CTTCCGGCTC

TATGACCATG

TTAGAAATTT

ATGCTCAACA

ACACATTIATT

GGACTCTAGA

TTTAACCTTA

TCAGATCAGA

TTAGGCCTCC

TACGATCATT

GTTTGACAGG

CTTTGTCTAA

TTTTACGCTC

ATAAAGTTTC

GTTGAATTAC

GGTTTTTATG

GCGCGCAAAC

CTGTCAATGC

CTGAGGGTGG

GTTCCGGTGA

ATGCCGATGA

ATTACGGTGC

GTGCTACTGG

ATTCACCTTT

GTCGCCCTTT

ATTAATGCAG

TTAATGTGAG

GTATGTTGTG

ATTACGCCAA

TATTGATAGA

CATGAGCGAA

CTGGAGAAAA

GGATCCCCAG

ACCAAAGGGC

GGGCTTAAAA

TCTACCGGTA

AGCTAATTTG

GAGCTCCTTA

CACACCAGGA

TTCCGTGGTG

TTAAGATTGA 1200 GTTAAGCATG 1260 ATTAGAGTCC 1320 TAGGATAAAT 1380 TGGCGGCGGC 1440 CGGTTCTGAG 1500 TTTTGATTAT 1560 AAACGCGCTA 1620 TGCTATCGAT 1680 TGATTTTGCT 1740 AATGAATAAT 1800 TGTCTTTGGC 1860 CTGGCACGAC 1920 TTAGCTCACT 1980 TGGAATTGTG 2040 GCTTGCATGC 2100 AGTATTTTAC 2160 ACCCTATAAG 2220 TAGAGAGAGA 2280 CTTTTAAACT 2340 TGTTCACAGC 2400 GTCTCACAAT 2460 GCGTAATCGT 2520 AGTGACTCAC 2580 GTACTACAGT 2640 AACTTTGGAT 2700 CGTTTGAACT 2760 WO 99/61597 WO 9961597PCTIUJS99/1 1250 TACATACAGG ATCGCTCATC 605 ACCTCAGGAA ATTCTCGGAG AAATCCAAGG TTTITCTTTC 610 TCTTCTCATC AAGIWGATTA TGAGCATCTG TTCATCZACGC GATCTGGTAC AGACACCAAA 615 AGGGCACACT AGGGTCCATG TATCCAGGAC TGGCTTAACT 620 AGTCACAGTC ACTTAGATCA CGAAATATGT AAACGCGTCA CATGAGGGTC TGATAAATAA 625 CTGGAAAGCC CAGCGCAAGG ATTTATCAAA TTTGITrAGG 630 TAAGCTCCAG AGAGGATATC ATAACTTCTC GAAACAAGCA GAAATGTTAT AGTAGCTGCT 635 TTACGTCAGA CTTCAGCATA CACCTTGCAA GTCCATGTGC 640 CATCCATAGA AGCTCTCAGA GCTTTGCTAT AGCTTTCGCA CCGCGTTTCG CTT'I=GAGT 645 CACTCCCGAT ATTCTTTT CACTTTGCTT AAGTCTGATC 650 CTTGATGGTA CGAATCATCG CTGTTCTAGC AAGGCTGACT CTGGATGGCA GTGTGTATCA 655

TGATAAACTC

TATAAAGAAA

CCATATTTCA

ATAAACTTCA

AGAATCTTTC

TTGCCCATTT

ACTTTTATCT

TTAGAGATGA

GAGGCATATG

CCAGTTCTGC

GAATCGCGAT

AGTATCTCTC

TCCGCTTCAA

TTTCCGATAG

GTGAAAAAGG

ACTGCTCGTT

TGCTGGTACC

TTACCCCACT

CAGTCTTCAC

GTGTCCTTCA

GCGGTTAAGA

GGTTGAAAAT

CTATCAAAGT

AAATAAGCAT

CTCTCCATAT

ATAACACGCG

TGATGCCTTC

AAGCCGCAAG

GATAAACAAA

TTCGATCACA

GCTTAGCTAA

CAGTTTCGAC

CCATATTGAA

TTAAAGAATC

CAATCATAGC

GTTGGAAGGA

GAAAATCAGA

TCTGAAACTC

GAAAGTATCT

GCACAATGAA

GTGAP.AAATT

CCAAGTGAAG

GACATAAATC

TCTTATGGTA

CGTCAACATT

TGTTAATCGC

CTTCTTTCTG

ATTTATCGGG

CCATGGAATA

GGGTTGGCAG

AAATTGGCCC

AAACCCTATG

GGTACAGTAG

AGCAGCTCTA

ATGACAGAGC

GAAGGAAACG

GCGCTGGATC

GAGAAACTTA

GAGAGACGTA

ATCTCCTGAA

CATAGTGACA

AACGGACATT

CCACCAATTC

TAGGTGCAGC

GTTATTCAAG

CCTGGATTTC

ACTAGTCACA

GGTGTCAGTT

AGTCTCTGCT

CTCAAGACAT

AAGGAATGTG

GTCTCCCATT

AGTACCAACT

GTCCCTATAC

ATCACCATTT

TATGGAATGA

AGTAGAGATG

TTCAATAGGG

CAATCAGAGA 2820 1CCTCCTCGA 2880 GTCGTAATCA 2940 AAATGTGCTC 3000 TCTCTCAGAG 3060 CTACAAACGT 3120 AACATATCGG 3180 AATATTGCAC 3240 AGAGTATTAC 3300 CCCACCTTGG 3360 GTCAGCGGCG 3420 TCACCCTGAG 3480 CGGACATTCT 3540 AGGGCCAGTG 3600 CCTTTACTAT 3660 ACAACAGGTT 3720 GCCACATTCA 3780 TTAGTCATGA 3840 CTACGAAAGC 3900 TCTGGAACGT 3960 CTTCGCTGAG 4020 CAGTCTACAT 4080 TCAACAAGGG 4140 CTGGTCGCTT 4200 TCAGGGTTAT 4260 TTCATGATTT 4320 GAAGAGTGAT GTCGTAATCA GTATTAGTAG TCTGAAACTC TTCATCAATG CCCATGTACC 4380 WO 99/61597 WO 9961597PCTIUS99/1 1250

TATCTCCAAG

660 AGTTCGAGGA

ATACACCGTA

CGAAAGAGCC

665

GTCTATCGGT

CTCGGTCAAA

670 AACTCGCGAG

CAGCCTCCAC

GAACGAAATC

GGTCAGCTCC

ATCACTGGCG

ACCATCACTA

CGGAATGTCA

GAACTGTTGA

AGGGGGATCG

AGATCCGTCA

CTCGTCTTCT

ATCATCCCAG

CACAACAACA ACAAGGTCAG

CTCCAAATGA

680 TGCGTCATCC

TGGAACGTCT

GTCGGTAGAG

685

AGCCATCITC

CGAGGAGGTT

690 AGACTGTATC

GCAGGCAGCA

CTTATGTTAC

695

GCATTCAGTC

CAAGAAAGCC

700 ATTCGTAATT

GCAGGCCAGC

AATAATCAGG

705

CCGTATGT'A

CTGGGAAAAC

710 CTGGCGTAAT

AATGAACTTC

CTTACGTCAG

TCTTTTTCCA

GCA ITCTTGA C~T'rCTACT

TCCCGATATT

TTTGATATTC

AGC 1TGCATG

GTCCTGTAGA

TGGATCGCGA

GGGCAATTGC

ATGCGGGCAA

GTATCGTGCT

AAGTGATGGA

TTGCCGGGAA

CCTGGCGTTA

AGCGAAGAGG

TTGGGGGTAT

AGTTTTAGAT

GTATCCTCGG

AGATATAACA

ACCATTTGTT

AATACCCCTC

ATGGCAACTC

AAGGATTGAT

GTTTTCGAAG

ATGTGTGTTG

CTTATATAGA

TGGAGATATC

CGATGTTCCT

ACGATAGCCT

GTCCTTTCGA

ACCCTTTGTT

TTGGAGTAGA

CCTGCAGGTC

AACCCCAACC

AAACTGTGGA

TGTGCCAGGC

CGTCTGGTAT

GCGTTTCGAT

GCATCAGGGC

AAGTGTACAA

CCCAACTTAA

CCCGCACCGA

CTCCAGTAAC

CGCTCGCATG

GATACCAGTC

TCCGTGCCAT

GGACGGTGTC

CTATCTCCAA

CGTCTGCCGG

CTATGATCCA

ACATCTTGGT

CGGGTACCGA

GGAAGGGTCT

ACATCAATCC

CGTGGGTGGG

TTCCTTTATC

TGAAGTGACA

GAAAAGTCTC

CGAGAGTGTC

GACTCTAGAG

CGTGAAATCA

ATTGATCAGC

AGTTTTAACG

CAGCGCGAAG

GCGGTCACTC

GGCTATACGC

TTCACTGGCC

TCGCCTTGCA

TCGCCCTTCC

ACGAACTTCC

ATCTTCATCG

ATCAATTTCA

TTCAGCTTGA

GCAAATAGAG

GGGCGCTATA

CTCCTGCACC

TTGGAAAGAC

GATAGTAGAA

GCTCGAATTC

TGCGAAGGAT

ACTTGCTTTG

GGTCCATCTT

GCAATGATGG

GATAGCTGGG

AATAGCCCTC

GTGCTCCACC

GATCCCCGGG

AAAAACTCGA

GTTGGTGGGA

ATCAGTTCGC

TCTTTATACC

ATTACGGCAA

CATTTGAAGC

GTCGTTTTAC

GCACATCCCC

CAACAGTTGC

TCAATTTCAC 4440 GCGGCAAACG 4500 TCTTCGAGCA 4560 GGAATCAGCG 4620 CCCCAGCGCA 4680 GCTAATTTAA 4740 TGAAGGCTAG 4800 GGGACCTGGC 4860 AGAACAAGCA 4920 TCGAGGTCCT 4980 AGTGGGATTG 5040 AAGACGTGGT 5100 TGGGACCACT 5160 CATTTGTAGA 5220 CAATGGAATC 5280 TGGTCTTCTG 5340 ATGTTGACCT 5400 TGGTCAGTCC 5460 CGGCCTGTGG 5520 AAGCGCGTTA 5580 CGATGCAGAT 5640 GAAAGGTTGG 5700 AGTGTGGGTC 5760 CGATGTCACG 5820 AACGTCGTGA 5880 CTTTCGCCAG 5940 GCAGCCTGAA 6000 WO 99/61597 WO 9961597PCTIUS99/1 1250

TGGCGAATGN

TAAGCGTCAA

715

ACCGGCAGCT

LNNI'lNINNNN

NNNNNNNNNN

725

NNNM

NNNNNNAATT

TTTGTTTACA

CGGCACAAA

NNNNNNNNNN

NNNI'NNNNJI J

NNDNNNNNNN

DJDUNNNNNN'N

CAGTACATTA AAAACGTCCG CCACAATATA TCCTGCCACC TCACCACTCG ATACAGGCAG NNNNNNN NNNNN NNNNNNNNr.N NNNNNNNNNN NNNNNNN NNNNNN NNNNNN NNNNNNN

CAATGTGTTA

AGCCAGCCAA

CCCATCAGNN

1NINNINN'rl~N

NNDNNN!NNDN

NN\NNNN'NNNN

NNNN'L\NNN

TTAAGTTGTC

CAGCTCCCCG

NN'NNI'JINNN

NNNNNlNNNNN'

NNNNLNNNNN

NNDNNNNNNNN

6060 6120 6180 6240 6300 6360 6420 6426 <210> 4 730 <211> 6500 <212> DNA <213> Brome mosaic virus <400> 4 735 AAACACTGAT

AGGGAGTCAC

GACAGAACCG

740

AAGCACATAC

TAGCAAATAT

745 AATTAGNNNN

TGCACGCAGG

AGACAATCGG

750

TTTTTGTCAA

TATCGTGGCT

755 CGGGAAGGGA

TTGCTCCTGC

ATCCGGCTAC

760

GGATGGAAGC

CAGCCGAACT

765 CCCATGGCGA

AGTTTAAACT

GTTATGACCC

CAACGATTGA

GTCAGAAJACC

TTCTTGTCAA

NNINNNNNNNN~

TTCTCCGGCC

CTGCTCTGAT

GACCGACCTG

GGCCACGACG

CTGGCTGCTA

CGAGAAAGTA

CTGCCCATTC

CGGTCTTGTC

GTTCGCCAGG

TGCCTGCTTG

GAAGGCGGGA

CCGCCGATGA

AGGAGCCACT

ATTATTGCGC

AAATGCTCCA

NNNNNNNNNN

GCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

TCCATCATGG

GACCACCAAG

GATCAGGATG

CTCAAGGCGC

CCGAATATCA

AACGACAATC

CGCGGGACAA

CAGCCGCGGG

GTTCAAAAGT

CTGACGTTCC

GATCGTTTCG

AGAGGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

CTGATGCAAT

CGAAACATCG

ATCTGGACGA

GCATGCCCGA

TGGTGGAAAA

TGATCATGAG

GCCGTTTTAC

TTTCTGGAGT

CGCCTAAGGT

ATAAATTCCC

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

GCGGCGGCTG

CATCGAGCGA

AGAGCATCAG

CGGCGATGAT

TGGCCGCTTT

CGGAGAATTA GTTTGGAACT 120 TTAATGAGCT 180 CACTATCAGC 240 CTCGGTATCC 300 CAAGATGGAT 360 TGGGCACAAC 420 CGCCCGGTTC 480 GCAGCGCGGC 540 GTCACTGAAG 600 TCATCTCACC 660 CATACGCTTG 720 GCACGTACTC 780 GGGCTCGCGC 840 CTCGTCGTGA 900 TCTGGATTCA 960 WO 99/61597 WO 9961597PCT/US99/1 1250 TCGACTGTGG CCGGCTGGGT ATATTGCTGA AGAGCTTGGC 770 CCGCTCCCGA TTCGCAGCGC NNNNNNNNNN NNNirNxNu',u 775 ATCCTGTTGC CGGTCTTGCG TAATAATTAA CATGTAATGC CGCAATTATA CATTTAATAC 780 TATCGCGCGC GGTGTCATCT TCTGGTGGTG GTTCTGGTGG 785 GGTGGCGGCT CTGAGGGAGG GAAAAGATGG CAAACGCTAA CAGTCTGACG CTAAAGGCAA 790 GGTTTCATTG GTGACGTTTC GGCTCTAATT CCCAAATGGC 795 TTCCGTCAAT A'ITTACCTTC CCPJATACGCA AACCGCCTCT AGGTTTCCCG ACTGGAAAGC 800 CATTAGGCAC CCCAGGCTTT AGCGGATAAC AATTTCACAC 805 TGCAGGTCAA CATGGTGGAG TCTCAGAAGA CCAGAGGGCT TCGGATTCCA TTGCCCAGCT 810 GCTTCTACAA ATGCCATCAT GACAGTGGTC CCAAAGATGG 815 CCAACCACGT CTTCAAAGCA GCACAATCCC ACTATCCTTC

GTGGCGGACC

GGCGAATGGG

ATCGCCTTCT

GATCGTTCAA

ATGATTATCA

ATGACGTTAT

GCGATAGAAA

ATGTTACTAG

CGGCTCTGAG

CGGTTCCGGT

TAAGGGGGCT

ACTTGATTCT

CGGCCTTGCT

TCAAGTCGGT

CCTCCCTCAA

CCCCGCGCGT

GGGCAGTGAG

ACACTTTATG

AGGAAACAGC

CACGACACTC

ATTGAGACTT

ATCTGTCACT

TGCGATAAAG

ACCCCCACCC

AGTGGATTGA

GCAAGACCCT

GCTATCAGGA

CTGACCGCTT

PTCGCCTTCT

ACATTTGGCA

TATAATTTCT

TTATGAGATG

ACAAAATATA

ATCGGGCCTC

GGTGGTGGCT

GGTGGCTCTG

ATGACCGAAA

GTCGCTACTG

AATGGTAATG

GACGGTGATA

TCGGTTGAAT

TGGCCGATTC

CGCJ4ACGCAA

CTTCCGGCTC

TATGACCATG

TCGTCTACTC

TTCAACAAAG

TCATCGAAAG

GAAAGGCTAT

ACGAGGAACA

TGTGATATCT

TCCTCTATAT

CATAGCGTTG

CCTCGTGCTT

TGACGAGTTC

ATAAAGTTTC

GTTGAATTAC

GGTTTTTATG

GCGCGCAALAC

CTGTCAATGC

CTGAGGGTGG

GTTCCGGTGA

ATGCCGATGA

ATTACGGTGC

GTGCTACTGG

ATTCACCTTT

GTCGCCCTTT

ATTAATGCAG

TTAATGTGAG

GTATGTTGTG

ATTACGCCAA

CAAGAATATC

GGTAATATCG

GACAGTAGAA

CGTTCAAGAA

TCGTGGA.AA

CCACTGACGT

AAGGAAGTTC

GCTACCCGTG 1020 TACGGTATCG 1080 TTCTGANNNN 1140 TTAAGATTGA 1200 GTTAAGCATG 1260 ATTAGAGTCC 1320 TAGGATAAAT 1380 TGGCGGCGGC 1440 CGGTTCTGAG 1500 TTTTGATTAT 1560 AAACGCGCTA 1620 TGCTATCGAT 1680 TGATTTTGCT 1740 AATGAATAAT 1800 TGTCTTTGGC 1860 CTGGCACGAC 1920 TTAGCTCACT 1980 TGGAATTGTG 2040 GCTTGCTGCC 2100 AAAGATACAG 2160 GGAAACCTCC 2220 AAGGAAGATG 2280 TGCCTCTACC 2340 AGAAGACGTT 2400 AAGGGATGAC 2460 ATTTCATTTG 2520 GAGAGGACCT CGAGAATTCG AGCTCGGTAC CCGCAACACA CATCTGACCT TGTTGTTGTT 2580 WO 99/61597 WO 9961597PCT/US99/1 1250 GTGTGCTTGT TCTTTCTACT TTCGCCAGGT CCCGTCTTTC 825 CTGCTAGCCT TCAGGTGCAG GTTTTAAATT AGCTATAGCG GAGTGCGCTG GGGCTCTATT 830 GACCGCTGAT TCCTCAAGCT TCGTGCTCGA AGATGAAATT 835 TATCGTTTGC CGCCGATGAA ACTGTGAAAT TGAGGAAGTT ATAGOTACAT GGGCAITGAT 840 TTCAAATCAT GAACCCTATT CAGATAACCC TGACATCTCT 845 CAGAAGCGAC CAGTCATTCC AAGCCCTTGT TGAAAATGGT GTGATGTAGA CTGGTATAGG 850 GTGCTCAGCG AAGAGTTGGT CGGACGTTCC AGAAATGGGP 855 AGCGCTTTCG TAGCACATTC ATGTCATGAC TAAATGTCT I GTGTGAATGT GGCAGCAGAC 860 TAAAACCTGT TGTAACTGAC TTCATAGTAA AGGTGTGAC'.

865 TATCACTGGC CCTGAAATC( TTAAGAATGT CCGCTTGAA! AATCTCAGGG TGAGCTGCA 870 CAGCGCCGCT GACGAATTG ATGCCAAGGT GGGAATGTC

ATCACCAAGA

CAATGGATCA

GAGCCGGCAG

CCCTTGGAGA

TGCGACACCG

GAAATGGCAC

GATGACTGGT

GATCATGCGA

CGTGTTACTG

GAAGAGTTTC

GAACATAGGG

ACTGGGCCAA

ATACTGCCAA

GATTATTCCP

GACCCCGATP

ACTCAGAAAC

GACGCGATTI

CTTAATGTT(

GAGTACCATI

ACTGATTTK,

ACCCTTCAC'

r' AGTAATTTT

-AGGTTCATT

r AACAGATAC

TGTCTTCGAA

TAGATCAATC

ACGGAGTTGC

TAGGAGGGGT

TCCAACAAAT

GGATG'ITATA

ATCCCGAGGA

GCGATCTAAA

GAGATACCCC

AGACTACTAA

TTTCGCGTGT

TTTATATGGA

CCCATGCTTA

TGGACTTTGA

LAATATTTTCA

;AAGTCTTAAC

LACATGAAGGA

ACGGTGAAGP

k. AGAAGTGGGC r GTCGGTACCI r TGGAACGAG( r CACCCTTTT F G TGCCTATCG( AACCTGGGAT G CTTAGAAGAC C CATTGACGGA 'I ATTCGATCCC C GGTTCAACAG 9 TCTTGACATT TACTAGTGAT C ACTCGCCAGT CAAGGAGCTGj

TACTGATTAC

TATTGATACA

GAGAGTCAGC

TTTCGATGAT

TAGGATCAGA

ACCAAAAATG

CGCACTCAAA

CACTGCGAAA

LCTGTCTGAGA

TAAGCACATG

GCATATGCTG

AGTAGCAGCT

P CACTGCTTGT 3AAAGATATCC ATGATTTCG 2640 AGGTGGAGG 2700 CTCTCGCGA 2760 CTTTTGACC 2820 TCACCGATA 2880 CGGGCTCTT 2940 ;GTTACGGTG 3000 ;ATTCCTCGA 3060 LCCCTTGGAG 3120 ;ACATCACTC 3180 'ACTGCCATC 3240 .TTGCTAGAA 3300 rCGTACCATC 3360 ZTTAAGCAAA 3420 AATATCGGGA 3480 PAGCGAAACG 3540 GCTATAGCAA 3600 GCTTCTATGG 3660 GACTTGCAAG 3720 AAGTCTGACG 3780 ACTATAACAT 3840 TTCGAGAAGT 3900 TCTCTGGAGC 3960 AAATTTGATA 4020 CTGGGCTTTC 4080 TCAGACCCTC 4140 TTTACATATT 4200 TTCTTGAAGC GGACCTAAGC CTAGAGTTTC AGAGAGAGAT ACTCCTTGCG

TGGTCTGATT

GTTTCCTTCC

TTCATCGCGA

AACGCAGAAC

TTCTTATTTA

TGGTGACGCG

WO 99/61597 WO 9961597PCTIUS99/1 1250 17 875 TCGGTAATAC TCTTGTCACT ATGGCTATGA TTGCATATGC CTCTGATCTA AGTGACTGTG 4260 ACTGTGCAAT ATTTTCAGGA G ATACCGATAT GTTTACGTCT C 880 CCTACGTTTG TAGTAAGTTT C ATCCTCTGAG AGAGATCCAG C 885 TCAGAGCACA TTTCGTTTCC T AGATGATTAC GACGCTCTGT C TTTTCGAGGA GGTTAGAGCT G 890 GGTTCTCTGA TTGCTACTGT GTAAGTTCAA ACGCACCACGC 895 AGAATCCAAA GTTTCCTGGT CGGACTGTAG TACTAAGGAG AGCGTGAGTC ACTCAAATTA 900 TCGACGATTA CGCTACCGGT CCCATTGTGA GACTT'ITAAG 905 TAAGCTGTGA ACAGCCCTTT CTAAGTTTAA AAGCTGGGGA CTATCTCTCT CTATTTrCTC 910 GTTCTTATAG GGT'ITCGCTC TTTGTAAAAT ACTTCTATCA 915 CAGGCATGCA AGCT]TGCATG CTTATGTTAC GTCCTGTAGA GCATTCAGTC TGGATCGCGA 920 CAAGAAAGCC GGGCAATTGC ATTCGTAATT ATGCGGGCAA 925 GCAGGCCAGC GTATCGTGCT ATGATTCTT T TCTTCAATA T TCGTCGAAA C GCTTAGCTA J TCTGTGATC G ATTTTGTTT ;CTCTTGCGG C CCGAAGGCA I1

;AAGAGCGTA

3TGTTAGACA

CTCCCTGTCA

GCTAATGATC

AGAGGAGGCC

CCCTCTGATC

GGTTAAGGTT

TCCTCTAGAG

CAGAATAATG

ATGTGTTGAG

ATAAAATTTC

CCTGCAGGTC

AACCCCAACC

AAACTGTGGA

TGTGCCAGGC

AATCATCTC TAAAGTTAAG CCAGTCCTGG 4320 GGAGATAAA 'TGAAATGGG C ,GCGAAA.GAT 'I IAATGAAGTT TCTGAAATA I)

TTTTTCTTT

[CAGAGTTTA

!.AACTGATGG

),AGTCTACAG

V CGGATCGG

GTAGGACCAC

TAACGTCAGT

TGAGATGATC

AAAAACTCCT

TCCGCAAATC

TGTGAGTAGT

CATATAAGAA

TAATTCCTAA

GACTCTAGAG

CGTGAAATCA

ATTGATCAGC

GTCATGGAC C :AATTTGGTG TI

CTGCGTGATC

'ATTAATCAA C ~GGGAAA.GAA

%.TACTCCGAG

ECAGATGAGC

rGACTGGTTT

AACCATTGGA

ACGTTTACAT

ACAACGCTTG

TGATGCTTTG

GGTTCTATGA

GGTCAGGCAG

ACCAGTCTCT

TCCCAGATAA

ACCCTTAGTA

AACCAAAATC

GATCCCCGGG

AAAAACTCGA

GTTGGTGGGA

CTAGTGTGC 4380 'CTGTACCAG 4440 ,AACAGATGC 4500 TTGATGAGA 4560 LZLACCTTGGA 4620 ATTTCTGA 4680 3ATCCTGTAT 4740 CACAACTGGA 4800 %TTTATTCCT 4860 GAGGCCCTTG 4920 AAAAAGAAGG 4980 CTCGTGP-AGT 5040 TATATGAACC 5100 ACCACTTTGG 5160 CTCTACAAAT 5220 GGGAATTAGG 5280 TGTATTTGTA 5340 CAGTGACCTG 5400 TGGTCAGTCC 5460 CGGCCTGTGG 5520 AAGCGCGTTA 5580 CGATGCAGAT 5640 GAAAGGTTGG 5700 AGTTTTAACG ATCAGTTCGC CGTCTGGTAT CAGCGCGAAG TCTTTATACC GCGTTTCGAT GCGGTCACTC ATTACGGCAA AGTGTGGGTC 5760 AATAATCAGG AAGTGATGGA GCATCAGGGC GGCTATACGC CATTTGAAGC CGATGTCACG 5820 WO 99/61597 WO 9961597PCTIUS99/1 1250

CCGTATGITA

930

CTGGGAAAAC

CTGGCGTAAT

935 TGGCGAATGN

TAAGCGTCAA

ACCGGCAGCT

940 1'NNNNNNNNN NNNNNtNNM 945 N~NNNNNN1'NNN

N'NNNNNNNNN

TTGCCGGGAA

CCTGGCGTTA

AGCGAAGAGG

NNNNNNAATT

TTTGTTTACA

CGGCACAAAA

NNNNNN

NN'NNNNr.NNNN

NNNINNINNN

NDJNNNNNNNN

NNNNNNN

AAGTGTACAA

CCCAACTTAA

CCCGCACCGA

CAGTACATTA

CCACAATATA

TCACCACTCG

NNLJNNNNNNN

1'NINNNNNNI'J

NNNDNL'INNN

N'NNNNNNNNN

TTCACTGGCC

TCGCCTTGCA

TCGCCCTTCC

AAAACGTCCG

TCCTGCCACC

ATACAGGCAG

NNNL'NNNNNN

NNNNNI'NNNNND

NNNNN'I'NNNN

NNNNNNNNNN

GTCGTTTTAC

GCACATCCCC

CAACAGTTGC

CAATGTGTTA

AGCCAGCCAA

CCCATCAGN

NNN1\NrNNNNN

NNNNNNNN

NNNNNNNNDNN

NNLJNNNNNNN

AACGTCGTGA

CTTTCGCCAG

GCAGCCTGAA

TTAAGTTGTC

CAGCTCCCCG

DNNNNNNNNN

I'NNN1NN~NN

N'NNNNI'NNINN

NNNNNNNNNN

NNNuNNNNDNNN 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 NNk4DNNNNNL'J NNNNNNNNNN NNNI'NNNWNN NNNDNNNNNNN 6480 950 NL'NNNNNDNIN NNNNNNNNMJ <210> <212> DNA <213> Brome mosaic virus 6500 <400> 960 AAACACTGAT

AGGGAGTCAC

GACAGAACCG

965

AAGCACATAC

TAGCAAATAT

970 AATTAGNNNN

TGCACGCAGG

AGACAATCGG

975

TTTTTGTCAA

TATCGTGGCT

980 CGGGAAGGGA

TTGCTCCTGC

AGTTTAAACT

GTTATGACCC

CAACGATTGA

GTCAGAAACC

TTCTTGTCAA

TTCTCCGGCC

CTGCTCTGAT

GACCGACCTG

GGCCACGACG

CTGGCTGCTA

CGAGAAAGTA

GAAGGCGGGA AACGACAATC TGATCATGAG CGGAGAATTA

CCGCCGATGA

AGGAGCCACT

ATTATTGCGC

AAATGCTCCA

NN'NNNNNNNN'

GCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

TCCATCATGG

CGCGGGACAA

CAGCCGCGGG

GTTCAAAAGT

CTGACGTTCC

GATCGTTTCG

AGAGGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

CTGATGCAAT

GCCGTTTTAC

TTTCTGGAGT

CGCCTAAGGT

ATAAATTCCC

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

GCGGCGGCTG

GTTTGGAACT 120 TTAATGAGCT 180 CACTATCAGC 240 CTCGGTATCC 300 CAAGATGGAT 360 TGGGCACAAC 420 CGCCCGGTTC 480 GCAGCGCGGC 540 GTCACTGAAG 600 TCATCTCACC 660 CATACGCTTG 720 WO 99/61597 WO 9961597PCTIUS99/1 1250 ATCCGGCTAC CTGCCCATTC 985 GGATGGAAGC CGGTCTTGTC CAGCCGAACT GTTCGCCAGG 990 CCCATGGCGA TGCCTGCTTG TCGACTGTGG CCGGCTGGGT ATATTGCTGA AGAGCTTGGC 995 CCGCTCCCGA TTCGCAGCGC NN~NNNNNNNN NNNNNNNNNN 1000 ATCCTGTTGC CGGTCITGCG TAATAATTAA CATGTAATGC CGCAATTATA CATTTAATAC 1005 TATCGCGCGC GGTGTCATCT TCTGGTGGTG GTTCTGGTGG 1010 GGTGGCGGCT CTGAGGGAGG GAAAAGATGG CAAACGCTAA CAGTCTGACG CTAAAGGCAA 1015 GGTTTCATTG GTGACGTTTC GGCTCTAATT CCCAAATGGC 1020 TTCCGTCAAT ATTTACCTTC CCAATACGCA AACCGCCTCT AGGTTTCCCG ACTGGAAAGC 1025 CATTAGOCAC CCCAGGCTTT AGCGGATAAC AATTrCACAC 1030 CTGCAGGTCA CTGGATTTTG AAATACAAAT ACATACTAAG AACCCTAATT CCCTTATCTG 1035 TAGATTTGTA GAGAGAGACT GACCACCAAG CGAAACATCG CATCGAGCGA GCACGTACTC GATCAGGATG ATCTGGACGA AGAGCATCAG GGGCTCGCGC CTCAAGGCGC GCATGCCCGA CGGCGATGAT CTCGTCGTGA CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA 780 840 900 960 GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG 1020

GGCGAATGGG

ATCGCCTTCT

GATCGTTCAA

ATGATTATCA

ATGACGTTAT

GCGATAGAAA

ATGTTACTAG

CGGCTCTGAG

CGGTTCCGGT

TAAGGGGGCT

ACTTGATTCT

CGGCCTTGCT

TCAAGTCGGT

CCTCCCTCAA

CCCCGCGCGT

GGGCAGTGAG

ACACTTTATG

AGGAAACAGC

GTTTTAGGAA

GGTTTCTTAT

GGAACTACTC

GGTGATTTGC

CTGACCGCTT

ATCGCCTTCT

ACATTTGGCA

TATAATTTCT

TTATGAGATG

ACAAAATATA

ATCGGGCCTC

GGTGGTGGCT

GGTGGCTCTG

ATGACCGAAA

GTCGCTACTG

AATGGTAATG

GACGGTGATA

TCGGTTGAAT

TGGCCGATTC

CGCAACGCAA

CTTCCGGCTC

TATGACCATG

TTAGAAATTT

ATGCTCAACA

ACACATTATT

GGACTCTAGA

CCTCGTGCTT

TGACGAGTTC

ATAAAGTTTC

GTTGAATTAC

GGTTTTTATG

GCGCGCAAAC

CTGTCAATGC

CTGAGGGTGG

GTTCCGGTGA

ATGCCGATGA

ATTACGGTGC

GTGCTACTGG

ATTCACCTTT

GTCGCCCTTT

ATTAATGCAG

TTAATGTGAG

GTATGTTGTG

ATTACGCCAA

TATTGATAGA

CATGAGCGAA

CTGGAGAAAA

GGATCCCCAG

TACGGTATCG 1080 TTCTGANNNN 1140 TTAAGATTGA 1200 GTTAAGCATG 1260 ATTAGAGTCC 1320 TAGGATAAAT 1380 TGGCGGCGGC 1440 CGGTTCTGAG 1500 TTTTGATTAT 1560 AAACGCGCTA 1620 TGCTATCGAT 1680 TGATTTTGCT 1740 AATGAATAAT 1800 TGTCTTTGGC 1860 CTGGCACGAC 1920 TTAGCTCACT 1980 TGGAATTGTG 2040 GCTTGCATGC 2100 AGTATTTTAC 2160 ACCCTATAAG 2220 TAGAGAGAGA 2280 CTTTTAAACT 2340 WO 99/61597PC/S/125 PCTIUS99/11250 TAGCCAAAGT GGTCTGCCTG 1040 TTAGGTTCAT ATATCATAGA GGGACTTCAC GAGCAAAGCA CGACCTTCTT TTTCAAGCGT 1045 GCTCAAGGGC CTCATGTAAA CCGAGGAATA AATTCCAATG 1050 TCTTCCAGTT GTGAAACCAG TACATACAGG ATCGCTCATC ACCTCAGGAA ATTCTCGGAG 1055 AAATCCAAGG TTTTTCTTTC TCTTCTCATC AAGTTGATTA 1060 TGAGCATCTG TTCATCACGC GATCTGGTAC AGACACCAAA AGGGCACACT AGGGTCCATG 1065 TATCCAGGAC TGGCTTAACT AGTCACAGTC ACTTAGATCA 1070 CGAAATATGT AAACGCGTCA CATGAGGGTC TGATAAATAA CTGGAAAGCC CAGCGCAAGG 1075 ATTTATCAAA TTTGCTTAGG TAAGCTCCAG AGAGGATATC 1080 ATAACTTCTC GAAACAAGCA GAAATGTTAT AGTAGCTGCT TTACGTCAGA CTTCAGCATA 1085 CACCTTGCAA GTCCATGTGC CATCCATAGA AGCTCTCAGA

ACCAGGAGTT

ACCGATCATC

TCAACTGACG

TGTGTGGTCC

CGTCCGATCC

GTTCTGTAGA

TCACCATCAG

TGATAAACTC

TATAAAGAAA

CCATATTTCA

ATAAACTTCA

AGAATCTTTC

TTGCCCATTT

ACTTTTATCT

TTAGAGATGA

GAGGCATATG

CCAGTTCTGC

GAATCGCGAT

AGTATCTCTC

TCCGCTTCAA

TTTCCGATAG

GTGAAAAAGG

ACTGCTCGTT

TGCTGGTACC

TTACCCCACT

CAGTCTTCAC

TTTAACCTTA

TCAGATCAGA

TTAGGCCTCC

TACGATCATT

GTTTGACAGG

CTTTGTCTAA

TTTTACGCTC

TGATGCCTTC

AAGCCGCAAG

GATAAACAAA

TTCGATCACA

GCTTAGCTAA

CAGTTTCGAC

CCATATTGAA

TTAAAGAATC

CAATCATAGC

GTTGGAAGGA

GAAAATCAGA

TCTGAAACTC

GAAAGTATCT

GCACAATGAA

GTGAAAAATT

CCAAGTGAAG

GACATAAATC

TCTTATGGTA

CGTCAACATT

ACCAAAGGGC

GGGCTTAAAA

TCTACCGGTA

AGCTAATTTG

GAGCTCCTTA

CACACCAGGA

TTCCGTGGTG

GGTACAGTAG

AGCAGCTCTA

ATGACAGAGC

GAAGGAAACG

GCGCTGGATC

GAGAAACTTA

GAGAGACGTA

ATCTCCTGAA

CATAGTGACA

AACGGACATT

CCACCAATTC

TAGGTGCAGC

GTTATTCAAG

CCTGGATTTC

ACTAGTCACA

GGTGTCAGTT

AGTCTCTGCT

CTCAAGACAT

AAGGAATGTG

TGTTCACAGC

GTCTCACAAT

GCGTAATCGT

AGTGACTCAC

GTACTACAGT

AACTTTGGAT

CGTTTGAACT

CAATCAGAGA

ACCTCCTCGA

GTCGTAATCA

AAATGTGCTC

TCTCTCAGAG

CTACAAACGT

AACATATCGG

AATATTGCAC

AGAGTATTAC

CCCACCTTGG

GTCAGCGGCG

TCACCCTGAG

CGGACATTCT

AGGGCCAGTG

CCTTTACTAT

ACAACAGGTT

GCCACATTCA

TTAGTCATGA

CTACGAAAGC

2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 1090 GCTTTGCTAT AGCTTTCGCA GTGTCCTTCA TGTTAATCGC GTCTCCCATT TCTGGAACGT 3960 WO 99/61597 WO 9961597PCTIUS99/1 1250 CCGCGTTTCG CTTTTTGAGT CACTCCCGAT ATTCATTTTT 1095 CACTTTGCTT AAGTCTGATC CTTGATGGTA CGAATCATCG 1100 CTGTTCTAGC AAGGCTGACT CTGGATGGCA GTGTGTATCA GAAGAGTGAT GTCGTAATCA 1105 TATCTCCAAG GGTCAGCTCC AGTTCGAGGA ATCACTGGCG 1110 ATACACCGTA ACCATCACTA CGAAAGAGCC CGGAATGTCA GTCTATCGGT GAACTGTTGA 1115 CTCGGTCAAA AGGGGGATCG AACTCGCGAG AGATCCGTCA 1120 CAGCCTCCAC CTCGTCTTCT GAACGAAATC ATCATCCCAG CACAACAACA ACAAGGTCAG 1125 CTCCAAATGA AATGAACTTC TGCGTCATCC CTTACGTCAG 1130 TGGAACGTCT TCTTTCCA GTCGGTAGAG GCA FTC=TGA AGCCATCTTC CTTTTCTACT 1135 CGAGGAGGTT TCCCGATATT AGACTGTATC TTTGATATTC 1140 GCAGGCAGCA AGCFTGCATG GAGCACGACA CTCTCGTCTA GCTATTGAGA Crr'FrCAACA 1145

GCGGTTAAGA

GGTTGAAAAT2

CTATCAAAGT

AAATAAGCAT

CTCTCCATAT

ATAACACGCG

GTATT1AGTAG'

TTGGGGGTAT

AGTTTTAGAT

GTATCCTCGG

AGATATAACA

ACCATTTGTT

AATACCCCTC

ATGGCAACTC

AAGGATTGAT

GTTTTCGAAG

ATGTGTGTTG

CTTATATAGA

TGGAGATATC

CGATGTTCCT

ACGATAGCCT

GTCCTTTCGA

ACCC TTGTT

TTGGAGTAGA

CCTGCAGGTC

CTCCAAGAAT

.AAGGGTAATA

'TTCTTTCTG

kTTTATCGGGC =CAGGAATA2

'GGTTGGCAG

B.AATTGGCCC

k.AACCCTATG

TCTGAAACTC

CTCCAGTAAC

CGCTCGCATG

GATACCAGTC

TCCGTGCCAT

GGACGGTGTC

CTATCTCCAA

CGTCTGCCGG

CTATGATCCA

ACATCTTGGT

CGGGTACCGA

GGAAGGGTCT

ACATCAATC C

CGTGGGTGGG

TTCCTTTATC

TGAAGTGACA

GAAAAGTCTC

CGAGAGTGTC

GACTCTAGAG

ATCAAAGATA

TCGGGAAACC

k.GTACCAACT 7TCCCTATAC kTCACCATTT rATGGAATGA

%.GTAGAGATG

TTCAATAGGG

TTCATCAATG

A.CGAACTTCC

PTCTTCATCG

ATCAATTTCA

TTCAGCTTGA

GCAAATAGAG

GGGCGCTATA

CTCCTGCACC

TTGGAAAGAC

GATAGTAGAA

GCTCGAATTC

TGCGAAGGAT

ACTTGCTTTG

GGTCCATCTT

GCAATGATGG

GATAGCTGGG

AATAGCCCTC

GTGCTCCACC

GATCCCCGGT

CAGTCTCAGA

TCCTCGGATT

CTTCGCTGAG 4020 CAGTCTACAT 4080 TCAACAAGGG 4140 CTGGTCGCTT 4200 TCAGGGTTAT 4260 TTCATGATTT 4320 CCCATGTACC 4380 TCAATTTCAC 4440 GCGGCAAACG 4500 TCTTCGAGCA 4560 GGAATCAGCG 4620 CCCCAGCGCA 4680 GCTAATTTAA 4740 TGAAGGCTAG 4800 GGGACCTGGC 4860 AGAACAAGCA 4920 TCGAGGTCCT 4980 AGTGGGATTG 5040 AAGACGTGGT 5100 TGGGACCACT 5160 CATTTGTAGA 5220 CAATGGAATC 5280 TGGTCTTCTG 5340 ATGTTGACCT 5400 CAACATGGTG 5460 AGACCAGAGG 5520 CCATTGCCCA 5580 WO 99/61597 WO 9961597PCT/US99/1 1250 GCTATCTGTC ACTTCATCGA AAGGACAGTA CATTGCGATA AAGGAAAGGC TATCGTTCAA 1150 TGGACCCCCA CCCACGAGGA ACATCGTGGA GCAAGTGGAT TGATGTGATA TCTCCACTGA TTCGCAAGAC CCTTCCTCTA TATAAGGAAG 1155 GGTTCTGCTA CTTGTTCTTT GTTTTTCACC AAGTTGATTG CTGAGAAGGG TGCTGACAGC 1160 GTTGCGCAAC AGTTATCTGC GCAGA'ITGAA CGCAATAAGC TCTCTATTGA GGAGGCTGAC GACTTAAATT TGACTCAGCA GTATCATGCG 1165 GCGGAGCATT ATGACTGTCT CGACAGTTTT GGGTCTTGGT GGCATCACTT TTCAAGAAGG 1170 TTGGGTGTTA GAGACGCTGC CCGACATGAG CAAGAAAGCG ATGATTTCGA TGAAGTCCCG GATGTCCAAG CTGATTGGGC TATCTGTATC 1175 CTGTGTGACG CCATGCATTC GCATGGAGTA GGCGCCATGT TGTTTGACCG CGAGGGTTTT 1180 GACGGGTCAG GCGCGGATGA GGTGATCAAA TACATCCACG GATGGCAAGA TTTGGGCTCA GGAACCACCT ATCTGTTGGA GCGCGAAATG 1185 ATCGCTACAA ATTTACGCTG CCCCCGGGAG ATATCTAAGT ACGTAGGGGT CTCAATACCT 1190 GTGCGCGTCG CCAAAACCAC AGTGAGAGAG GAAAGTAAAG AATGGACTGA GAACATGAAA TCGACTGTTA TTATTAACGG TCAGGCTATC 1195 TATCATCTAG TGGCCTTTGC TTTGACTTTG GCCCTCCGCG ATGGGATGGA ATGGAAAGGT

GAAAAGGAAG

GAATGCCTCT

AAAAGAAGAC

CGTAAGGGAT

TTCATTTCAT

AACAAAATGT

CAGAGTGCCC

TACGCGAAAA

GCCTTCCGTG

CCC CATAGC C

CCCCCTGAAG

GATAAAAGGG

GAGAGGATGT

AACTTTTGTC

CACGGCGGTT

CGCGTACTAC

CTTCCCTTGC

TTCGATTTTG

TTTTTCACCG

CTGAAATGTA

ACACTACGTC

GAAGACTGGA

GTAGAGGAGA

GCTGTCGCAT

ATGGCTGGTG

AATCTGTATC

TGGTGCCATC

ATGGCTTCTA

ACCGACAGTG

GTTCCAACCA

GACGCACAAT

TTGGAGAGGA

CAAGTTCTAT

AAGACATCGT

GGTCTAAGAA

ACCGTTATGG

TGGCTGGTGC

ACCCCGTTAT

TGCACAGTTG

GCCGCATGCG

TTAACCGAGC

ATGATATGGG

GTGGTACCGT

TTAAATGTCA

AAAATGAAAG

AGTCGGTGCA

ACATCATGAC

ACTGTGTATG

GTCTCAATCG

TAGCTTTCAG

CTATCTTATC

AGCGCTTAGA

AAAAGTACGA

ACTTCAAAAC

CAAATGCCAT 5640 GTCCCAAAGA 5700 CGTCTTCAAA 5760 CCCACTATCC 5820 CCTCGACCAC 5880 CGATTTGCTG 5940 AGACAATCAG 6000 AATCAACGTT 6060 TGGTGCCTTT 6120 TCTGCGTGTA 6180 AGATTTCGGA 6240 TTGTCCTGTG 6300 AAAAATTTTG 6360 TCAAGATTGT 6420 CTTCCAAGGT 6480 TATGTTCGAC 6540 CTGGCAACGT 6600 CACATTATCT 6660 TTGCATCGAT 6720 CTATAAGATC 6780 GTTTGAAGAC 6840 CTGGAAATGT 6900 ATGTTTCAAG 6960 CGCCAAGTCG 7020 CATTGAAGAT 7080 AAAGCTTACG 7140 TAGGTTTTGG 7200 WO 99/61597 WO 9961597PCT/US99/1 1250 1200 TGGGGTGGAG ATTCATCCAG CCCCTACTAC GTCTGGATTC GTTGAAGAAT TTGAGCAAGA 1205 GAGGACTTAT TCGACCGGCT AAGAAGGCGA AAGTCCCGCC 1210 GAGAGTTCGA GCCCTGAGTC GATGCCGAGG TGTCTGTTGA ATGAAGGAAT TTGTGCGTTA 1215 CACCTATGGG ACATTTCCGG ACCTACCATC GCATAGACGA 1220 CCTAAAAAAT ACGATTACAC GATGAAACGT ACATTGTTGA GCTAGCGCCA AAGTTTCTGT 1225 TGCGGTAAAA CCACTGCCAT GCGAATCGTA AATCGGCCGA 1230 AAGGTAGCTT TGGACGTTGT TGTCATAGGC TGCTTGTTGA GCTGCTCTGT CTAAATGTTC 1235 AAGTCTCGTG ACGCGGGTTT GTTGTTCACA AGACTTACCG 1240 CGTAAATGCG GTAATAGGGA AGAAGTCTCA CGAAGCGTCG ACGTATCTTA CGATGACTCA 1245 CCCGTGAGCA AGGACTGGAT TCTGTTGACA ACGTCACTTT 1250 GAGGAGTACT GTTTGGTTGC GGCGAGCTCG CTGGTGATTT

GGCGAAAGTA

TTATGCGGAC

TTCTGTACCG

GGAGCATGAA

AGCTGCTGAG

CGTCAGTGAT

GGTACCAACG

TTGTAAGAGA

CGGTCGCGGA

TATGGTGAAT

TGTTGGATAT

TAAAACATGT

CCCTACATGC

AAAAGATGCA

GGACGTCAGG

GCGCACCGCG

TGAGG~TGGT

ACAAGTTCTT

TAAATTGCTC

GTGTCCGCAA

CACGAAGTAT

TATTACTTCT

AGCTGATAAA

TGATGGACAC

GGTTCGGCTT

CTTAACACGA

GATCTTTAAT

GGATGGCTGA

AGTTTTAAGT

ATATCACGTT

GTGCAGACAG

PTACCTCAGG

GACGTTAAAC

GACCCTCGTG

TTACATAACA.

AGTGAGATCG

GTCCATTTGG

AATGAGCATG

GCATGCTCTA

GATAT TCCA

TTCCGTATGG

ATGGCTTTAT

GATTCTGCGA

TTACTACATT

GCCTTTGGGG

CACGGTAATC

GATGTTATCG

CAATCCTGGA

GGTTTGCAGG

GCGGCCCTTC

ATAAAAACAG

AAGTCGACCA

CACAAGAAGT

TGTGTTAAGT

GAACATTGGC

TCTGACTCG

TGAGAACGTT

CCAAGACCAA~

AGGAGTTTCA

CGGTGACTGA

GCATATCTAG

ACTCCGAGTC

CAAATAAGAG

CCAACGGTAA

GTTTAGGTCC

ACTTGAGGGA

TGGTTGATGG

GAGAGGACCT

TCCCTGACAC

TCATGCACGG

ATGGTCAACT

ACACAGAGCA

TGCAATATGA

CTGCTGTTAA

CATCTGAGTC

TCACTATTGA

AAACGAGGGC

TACACGAAGC

AATGTGATTT

CCTTTGAGTA

GATGCGCTTG

LAGCAGATTT 7260 rCTCTCAAAC 7320 rTGGACTGAA 7380 GCGCTCGAAG 7440 TGATGCCCCT 7500 TGTGGTCCCG 7560 AJCACGGAGCC 7620 TAATCTTCGT 7680 CATCTTTGAG 7740 CTGGTTGTAT 7800 GAAGCACGCA 7860 CATTGCAGAA 7920 AGTTGCGGGA 7980 AATTGTGACG 8040 TTATAATTCC 8100 TGTACCGTCC 8160 CCTGGTGGTG 8220 GATTTCGTTC 8280 TCGCCGTGAC 8340 TCTGCTGAAG 8400 CAAAGTTTCT 8460 TCCGAACAGA 8520 TAAGGATTTT 8580 GCAAGGGATC 8640 GTTTAAACAT 8700 TTGCTTTAAC 8760 TCTCTGTGTG 8820 WO 99/61597 WO 9961597PCT/US99/1 1250

AGACCTCTGC

1255

CAGTCTCTCT

CCAGATAAGG

1260 CCTTAGTATG

CCAAAATCCA

GTGAAATCAA

1265

TTGATCAGCG

GTTTTAACGA

1270 AGCGCGAAGT

CGGTCACTCA

GCTATACGCC

1275

TCACTGGCCG

CGCCTTGCAG

1280 CGCCCTTCCC

AAACGTCCGC

CCTGCCACCA

1285

TACAGGCAGC

NNNNDNNI'J1\I 1290 NNNDNN'DNNNNJ NNNN4NNNNNNJ

TCGAGAATTC

CTACAAATCT

GAATTAGGGT

TATTTGTATT

GTGACCGGGT

AAAACTCGAC

TTGGTGGGAA

TCAGTTCGCC

CTTTATACCG

TTACGGCAAA

ATTTGAAGCC

TCGTTTTACA

CACATCCCCC

AACAGITrGCG

AAJTGTGTTAT

GCCAGCCAAC

CCATCAGNNN

NNNNuuiNNNNN NNNFNNNiNINNN

NN~NNNN

GAGCTCGGTA

ATCTCTCTCT

TCTTATAGGG

TGTAAAATAC

GGTCAGTCCC

GGCCTGTGGG

AGCGCGTTAC

GATGCAGATA

AAAGGTTGGG

GTGTGGGTCA

GATGTCACGC

ACGTCGTGAC

TTI'CGCCAGC

CAGCCTGAAT

TAAGTTGTCT

AGCTCCCCGA

ufluNNNNluuu

NNNNNNNNNNI~

NNNNNNNN

N'NNNNNNNNN

CCCGGGGATC

ATTTTCTCCA

TTTCGCTCAT

TTCTATCAAT

TTATGTTACG

CATTCAGTCT

AAGAAAGCCG

TTCGTAATTA

CAGGCCAGCG

ATAATCAGGA

CGTATGTTAT

TGGGAAAACC

TGGCGTAATA

GGCGAATGNN

AAGCGTCAAT

CCGGCAGCTC

NNNNNmNNN

NNNNNNLNN

CTCTAGAGTC

GAATAATGTG

GTGTTGAGCA

AAAATTTCTA

TCCTGTAGAA

GGATCGCGAA

GGCAATTGCT

TGCGGGCAAC

TATCGTGCTG

AGTGATGGAG

TGCCGGGAAA

CTGGCGTTAC

GCGAAGAGGC

NNNNNAATTC

TTGTTTACAC

GGCACAAAAT

NNNDNN'Ni\ID1'

I'I'.NNNNDNNNN

CGCAAATCAC

TGAGTAGTTC

TATAAGAAAC

ATTCCTAAAA

ACCCCAACCC

AACTGTGGAA

GTGCCAGGCA

GTCTGGTATC

CGTTTCGATG

CATCAGGGCG

AGTGTACAAT

CCAACTTAAT

CCGCACCGAT

AGTACATTAA

CACAATATAT

CACCACTCGA

NNDNIINNNNN

NNNDJNNDNN

8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 NNNNJNNNNN NNNNNNNNN NNNDJNI'4fNN 9960 NNNNNNNNN NNN1'NNNNNNN NNNNNNNNN 10020 NNLJIJ1'NNNNN NNNNNN N DJNNN NLNNNNN NNNNNNN NNNNNNNN 10080 1295 NNNNNNNNNN I'NNNNNNNM 10100 <210> 6 <211> 10240 1300 <212> DNA <213> Brorne mosaic virus <400> 6 AAACACTGAT AGTTTAAACT GAAGGCGGGA AACGACAATC TGATCATGAG CGGAGAATTA 1305 AGGGAGTCAC GTTATGACCC CCGCCGATGA CGCGGGACAA GCCGTTTTAC GTTTGGAACT 120 WO 99/61597 WO 9961597PCTIUS99/1 1250 GACAGAACCG CAACGATTGA 1310 AAGCACATAC GTCAGAAACC TAGCAAATAT TTCTTGTCAA AATTAGNNNN NNNNNNNNNN 1315 TGCACGCAGG TTCTCCGGCC AGACAATCGG CTGCTCTGAT 1320 TTTTTGTCAA GACCGACCTG TATCGTGGCT GGCCACGACG CGGGAAGGGA CTGGCTGCTA 1325 TTGCTCCTGC CGAGAAAGTA ATCCGGCTAC CTGCCCATTC 1330 GGATGGAAGC CGGTCTTGTC CAGCCGAACT GTTCGCCAGG CCCATGGCGA TGCCTGCTTG 1335 TCGACTGTGG CCGGCTGGGT ATATTGCTGA AGAGCTTGGC 1340 CCGCTCCCGA TTCGCAGCGC N~L4DNN DNNN NNNNNNNNNN ATCCTGTTGC CGGTCTTGCG 1345 TAATAATTAA CATGTAATGC CGCAATTATA CATTTAATAC 1350 TATCGCGCGC GGTGTCATCT TCTGGTGGTG GTTCTGGTGG GGTGGCGGCT CTGAGGGAGG 1355 GAAAAGATGG CAAACGCTAA CAGTCTGACG CTAAAGGCAA 1360 GGTTTCATTG GTGACGTTTC

AGGAGCCACT

ATTATTGCGC

AAATGCTCCA

NNNNNNINNNN

GCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

TCCATCATGG

GACCACCAAG

GATCAGGATG

CTCAAGGCGC

CAGCCGCGGG TTTCTGGAGT TTAATGAGCT 180

GTTCAAAAGT

CTGACGTTCC

GATCGTTTCG

AGAGGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

CTGATGCAAT

CGAAACATCG

ATCTGGACGA

GCATGCCCGA

CGCCTAAGGT

ATAAATTCCC

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

GCGGCGGCTG

CATCGAGCGA

AGAGCATCAG

CGGCGATGAT

CACTATCAGC

CTCGGTATCC

CAAGATGGAT

TGGGCACAAC

CGCCCGGTTC

GCAGCGCGGC

GTCACTGAAG

TCATCTCACC

CATACGCTTG

GCACGTACTC

GGGCTCGCGC

CTCGTCGTGA

240 300 360 420 480 540 600 660 720 780 840 900 CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA 960 GTGGCGGACC GCTATCAGGA CATAGCGTTG

GGCGAATGGG

ATCGCCTTCT

GATCGTTCAA

ATGATTATCA

ATGACGTTAT

GCGATAGAAA

ATGTTACTAG

CGGCTCTGAG

CGGTTCCGGT

TAAGGGGGCT

ACTTGATTCT

CGGCCTTGCT

CTGACCGCTT

ATCGCCTTCT

ACATTTGGCA

TATAATTTCT

TTATGAGATG

ACAAAATATA

ATCGGGCCTC

GGTGGTGGCT

GGTGGCTCTG

ATGACCGAAA

GTCGCTACTG

AATGGTAATG

CCTCGTGCTT

TGACGAGTTC

ATAAAGTTTC

GTTGAATTAC

GGTTTTTATG

GCGCGCAAAC

CTGTCAATGC

CTGAGGGTGG

GTTCCGGTGA

ATGCCGATGA

ATTACGGTGC

GTGCTACTGG

GCTACCCGTG 1020 TACGGTATCG 1080 TTCTGANNNN 1140 TTAAGATTGA 1200 GTTAAGCATG 1260 ATTAGAGTCC 1320 TAGGATAAAT 1380 TGGCGGCGGC 1440 CGGTTCTGAG 1500 TTTTGATTAT 1560 AAACGCGCTA 1620 TGCTATCGAT 1680 TGATTTTGCT 1740 WO 99/61597 WO 9961597PCTIUS99/1 1250 GGCTCTAATT CCCAAATGGC TTCCGTCAAT ATTTACCTTC 1365 CCAATACGCA AACCGCCTCT AGG ITTCCCG ACTGGAAAGC 1370 CATTAGGCAC CCCAGGCTTT AGCGGATAAC AATTTCACAC CTGCAGGTCA CTGGATTTTG 1375 AAATACAAAT ACATACTAAG AACCCTAATT CCCTTATCTG 1380 TAGATITGTA GAGAGAGACT TAGCCAAAGT GGTCTGCCTG TTAGGTTCAT ATATCATAGA 1385 GGGACTTCAC GAGCA~AAGCA CGACCTTCTT TTTCAAGCGT 1390 GCTCAAGGGC CTCATGTAAA CCGAGGAATA AATTCCAATG TCTTCCAGTT GTGAAACCAG 1395 TACATACAGG ATCGCTCATC ACCTCAGGAA XrCTCGGAG 1400 AAATCCAAGG TTTI'CT ITC TCTTCTCATC AAGTTGATTA TGAGCATCTG TTCATCACGC 1405 GATCTGGTAC AGACACCAAA AGGGCACACT AGGGTCCATG 1410 TATCCAGGAC TGGCTTAACT AGTCACAGTC ACTTAGATCA CGAAATATGT AAACGCGTCA 1415

TCAAGTCGGT

CCTCCCTCAA

CCCCGCGCGT

GGGCAGTGAG

ACACTTTATG

AGGAAACAGC

GTTTTAGGAA

GGTTTCTTAT

GGAACTACTC

GGTGATTTGC

ACCAGGAGTT

ACCGATCATC

TCAACTGACG

TGTGTGGTCC

CGTCCGATCC

GTTCTGTAGA

TCACCATCAG

TGATAAACTC

TATAAAGAAA

CCATATTTCA

ATAAACTTCA

AGAATCTTTC

TTGCCCATTT

ACTTTTATCT

TTAGAGATGA

GAGGCATATG

CCAGTTCTGC

GACGGTGATA

TCGGTTGAAT

TGGCCGATTC

CGCAACGCAA

CTTCCGGCTC

TATGACCATG

TTAGAAATTT

ATGCTCAACA

ACACATTATT

GGACTCTAGA

TTTAACCTTA

TCAGATCAGA

TTAGGCCTCC

TACGATCATT

GTTTGACAGG

CTTTGTCTAA

TTTTACGCTC

TGATGCCTTC

AAGCCGCAAG

GATAALACAAA

TTCGATCACA

GCTTAGCTAA

CAGTTTCGAC

CCATATTGAA

TTAAAGAATC

CAATCATAGC

GTTGGAAGGA

PITTCACCTTT

GTCGCCCTTT

ATTAA FGCAG

TTAATGTGAG

GTATGTTGTG

ATTACGCCAA

TATTGATAGA

CATGAGCGAA

CTGGAGAAAA

GGATCCCCAG

ACCAAAGGGC

GGGCTTAAAA

TCTACCGGTA

AGCTAATTTG

GAGCTCCTTA

CACACCAGGA

TTCCGTGGTG

GGTACAGTAG

AGCAGCTCTA

ATGACAGAGC

GAAGGAAACG

GCGCTGGATC

GAGAAACTTA

GAGAGACGTA

ATCTCCTGAA

CATAGTGACA

AACGGACATT

AATGAATAAT 1800 TGTCTTTGGC 1860 CTGGCACGAC 1920 TTAGCTCACT 1980 TGGAATTGTG 2040 GCTTGCATGC 2100 AGTATTTTAC 2160 ACCCTATAAG 2220 TAGAGAGAGA 2280 CTTTTAAACT 2340 TGTTCACAGC 2400 GTCTCACAAT 2460 GCGTAATCGT 2520 AGTGACTCAC 2580 GTACTACAGT 2640 AACTTTGGAT 2700 CGTTTGAACT 2760 CAATCAGAGA 2820 ACCTCCTCGA 2880 GTCGTAATCA 2940 AAATGTGCTC 3000 TCTCTCAGAG 3060 CTACAAACGT 3120 AACATATCGG 3180 AATATTGCAC 3240 AGAGTATTAC 3300 CCCACCTTGG 3360 WO 99/61597 WO 9961597PCTIUS99/1 1250 27 CATGAGGGTC TGATAAATAA GAATCGCGAT GAAAATCAGA CCACCAATTC GTCAGCGGCG 3420 CTGGAAAGCC CAGCGCAAGG 1420 ATTTATCAAA TTTGCTTAGG TAAGCTCCAG AGAGGATATC ATAACTTCTC GAAACAAGCA 1425 GAAATGTTAT AGTAGCTGCT TTACGTCAGA CTTCAGCATA 1430 CACCTTGCAA GTCCATGTGC CATCCATAGA AGCTCTCAGA GCTTTGCTAT AGCTTTCGCA 1435 CCGCGTTTCG CTTTTTGAGT CACTCCCGAT ATTCATTTTT 1440 CACTTTGCTT AAGTCTGATC CTTGATGGTA CGAATCATCG CTGTTCTAGC AAGGCTGACT 1445 CTGGATGGCA GTGTGTATCA GAAGAGTGAT GTCGTAATCA 1450 TATCTCCAAG GGTCAGCTCC AGTTCGAGGA ATCACTGGCG ATACACCGTA ACCATCACTA 1455 CGAAAGAGCC CGGAATGTCA GTCTATCGGT GAACTGTTGA 1460 CTCGGTCAAA AGGGGGATCG AACTCGCGAG AGATCCGTCA CAGCCTCCAC CTCGTCTTCT 1465 GAACGAAATC ATCATCCCAG CACAACAACA ACAAGGTCAG

AGTATCTCTC

TCCGCTTCAA

TTTCCGATAG

GTGAAAAAGG

ACTGCTCGTT

TGCTGGTACC

TTACCCCACT

CAGTCTTCAC

GTGTCCTTCA

GCGGTTAAGA

GGTTGAAAAT

CTATCAAAGT

AAATAAGCAT

CTCTCCATAT

ATAACACGCG

GTATTAGTAG

TTGGGGGTAT

AGTTTTAGAT

GTATCCTCGG

AGATATAACA

ACCATTTGTT

AATACCCCTC

ATGGCAACTC

AAGGATTGAT

GTTTTCGAAG

ATGTGTGTTG

rCTGAAACTC

'AAAGTATCT

GCACAATGAA

GTGAAAAATT

CCAAGTGAAG

GACATAAATC

ICTTATGGTA

CGTCAACATT

TGTTAATCGC

CTTCTTTCTG

ATTTATCGGG

CCATGGAATA

GGGTTGGCAG

AAATTGGCCC

AAACCCTATG

TCTGAAACTC

CTCCAGTAAC

CGCTCGCATG

GATACCAGTC

TCCGTGCCAT

GGACGGTGTC

CTATCTCCAA

CGTCTGCCGG

CTATGATCCA

ACATCTTGGT

CGGGTACCGA

rAGGTGCAGC

GTTATTCAAG

CCTGGATTTC

PCTAGTCACA

GGTGTCAGTT

AGTCTCTGCT

CTCAAGACAT

AAGGAATGTG

GTCTCCCATT

AGTACCAACT

GTCCCTATAC

ATCACCATTT

TATGGAATGA

AGTAGAGATG

TTCAATAGGG

TTCATCAATG

ACGAACTTCC

ATCTTCATCG

ATCAATTTCA

TTCAGCTTGA

GCAAATAGAG

GGGCGCTATA

CTCCTGCACC

TTGGAAAGAC

GATAGTAGAA

GCTCGAATTC

TCACCCTGAG 3480 CGGACATTCT 3540 AGGGCCAGTG 3600 CCTTTACTAT 3660 ACAACAGGTT 3720 GCCACATTCA 3780 TTAGTCATGA 3840 CTACGAAAGC 3900 TCTGGAACGT 3960 CTTCGCTGAG 4020 CAGTCTACAT 4080 TCAACAAGGG 4140 CTGGTCGCTT 4200 TCAGGGTTAT 4260 TTCATGATTT 4320 CCCATGTACC 4380 TCAATTTCAC 4440 GCGGCAAACG 4500 TCTTCGAGCA 4560 GGAATCAGCG 4620 CCCCAGCGCA 4680 GCTAATTTAA 4740 TGAAGGCTAG 4800 GGGACCTGGC 4860 AGAACAAGCA 4920 TCGAGGTCCT 4980 WO 99/61597 WO 9961597PCT[US99/1 1250 1470 CTCCAAATGA AATGAACTTC TGCGTCATCC CTTACGTCAG TGGAACGTCT TCTITTTCCA 1475 GTCGGTAGAG GCATTCTTGA AGCCATCTTC CTTTTCTACT 1480 CGAGGAGGTT TCCCGATATT AGACTGTATC TFTGATATTC GCAGGCAGCA AGCTTGCATG 1485 TGGTTTTAGG AATTAGAAAT AGGGTTTCTT ATATGCTCAA 1490 TGGGAACTAC TCACACATTA CTGGTGATTT GCGGACTCTA TCTCACACAG AGACAAGCGC 1495 GCCGTTAAAG CAATACTCAA CTCATGTTTA AACAAATCAC 1500 AGAGATCCCT TGCGCTTCGT GGGAAAATCC TTAGCCCTCG CGTTCTGTTC GGATCAATAG 1505 TCTAGAAACT TTGGACTCAG ACGCTTCAGC AGATTAACAG 1510 AACGTCACGG CGATCATATT CTTGAACGAA ATCTGCTCTG AGCCACCACC AGGAGTTGAC 1515 ACAGGACGGT ACACCGTGCA CTTGGAATTA TAAGTGTCAG 1520 CGCCGTCACA ATTAGGTCCT GCATCCCGCA ACTCCATCAA :TTATATAGA C EGGAGATATC I

GATGTTCCTC

2LCGATAGCCT

GTCCTTTCGA

kCCCTTTGTT

TTGGAGTAGA

CCTGCAGGTC

TTTATTGATA

CACATGAGCG

TTCTGGAGAA

GAGGATCCCC

ATCACTTAAC

AGGACTTCTT

ATTTGGTCGA

GTACTGTTTT

TTTGAAGGGC

TGACCTGCAA

ATGTCCAGGA

CAGCGATAAC

GCAGATTACC

TGTCCCCAAA

CATAATGTAG

TGATCGCAGA

GGAATAAAGC

CTCCCATACG

CCATGGAAAT

;GAAGGGTCT I ~CATCAATCC I

GTGGGTGGGC

E'TCCTTTATC

[GAAGTGACA

'AAAAGTCTC

CGAGAGTGTC

GACTCTAGAG

GAAGTATTTT

1A'AACCCTATA

P.ATAGAGAGA

GGGTACCGAG

ACAATTAAAG

GTGTCGTGTT

CTTAAGCCGA

TATGTGTCCA

CGCTTTATCA

ACCAGAAGTA

TTGATACTTC

ATCTTGCGGA

GTGGAGCAAT

GGCAAGAACT

TAAACCAGCC

ATCCGCGGTG

CATCCTGACG

GAATGCATCT

ATCGCATGTA

rGCGAAGGAT

~CTTGCTTTG

3GTCCATCTT I

'CAATGATGG

3ATAGCTGGG

ATAGCCCTC

3TGCTCCACC

GATCCCCGGT

PCAAATACAA

AGAACCCTAA

GATAGATTTG

CTCGAATTCT

ATCAAATCAC

AAGGCAACCA

ACCAAAGTGA

TCAATCCAGT

GCTTGAGTCA

ATACGACGCT

GTGTCCCTAT

CACCGGTAAG

TTAAAACCCG

TGTGAACATT

TCATCAACAA

CGCACAACGT

TCCTCGGCCG

TTTATGGCAG

GGGACAGAAA

GTGGGATTG 5040 AGACGTGGT 5100 rGGGACCACT 5160 :ATTTGTAGA 5220 7AATGGAATC 5280 LGGTCTTCTG 5340 kTGTTGACCT 5400 C.ACTGGATTT 5460 ATACATACTA 5520 TTCCCTTATC 5580 TAGAGAGAGA 5640 CGAGCAGAGG 5700 CAGCGAGCTC 5760 AACAGTACTC 5820 CGTTGTCAAC 5880 CCTTGCTCAC 5940 TCGTAAGATA 6000 TCGTGAGACT 6060 TACCGCATTT 6120 TCTTGTGAAC 6180 CGTCACGAGA 6240 TAGACAGAGC 6300 GCAGCCTATG 6360 CCAAAGCTAC 6420 ATTTACGATT 6480 TGGTTTTACC 6540 CTTTGGCGCT 6600 WO 99/61597 WO 9961597PCTIUS99/1 1250 AGCTTCTGCA ATGTCCCTCA 1525 ATCTGCGTGC TTCGGACCTA AGGATACAAC CAGTTACCGT 1530 GGTCTCAAAG ATGCTCTTAT GTGACGAAGA TTAGACTCGG CATGGCTCCG TGTCI'AGATA 1535 ATCCGGGACC ACATCAGTCA CTCAGGGGCA TCATGAAACT 1540 CTTCTTCGAG CGcrGGTCT CTCTTCAGTC CAAAACGTTC AACGTTTGAG AGACGAGTCA 1545 GGGAAATCTG CTAGCCAATG CCACCAAAAC CTAGTTTTGA 1550 GGCCGTAAGC TTTTCGTACT ATAATCTTCA ATGTCTAAGC CGACGAC~rG GCGGATAAGA 1555 TTCCTTGAAA CATCTGAALAG CACACATITC CAGCGATTGA 1560 TATGTCTTCA AACCATACAC GATGATCTTA TAGGTCATGA TCCATCGATG CAATGCACCG 1565 GTAAGATAAT GTGCTTTCAT GTCACGTTGC CAGTGACATT 1570 GCCGTCGAAC ATAACGGTAC CAGACCTTGG AAGCCCATAT ATCACAATCT TGAGCTCGGT 1575

AGTTAGAGCA

AACCATGCTC

TGGCCAAATG

TTGCGATCTC

AGTTGTTATG

TGCCACGAGG

CCGGTTTIAAC

CCTCCTGAGG

TGGCTGTCTG

TCAAACGTGA

GAAACTTAAA

TTCTCAGCCA

AGTGATGGCA

TTTGATACAG

GCTCACCAGC

TAGATGCGAC

CTATCTCCTC

GACTCCAGTC

AGTGACGTAG

TGTTACATTT

ACTCGGTGAA

TTTCAAAATC

TAAGCAAGGG

CACGTAGTAC

CATAACCGCC

TGCACATGTT

ATTATATCCA

GACATTCACC

ACTTCCGCGA

TAATCTCTTA

GTCCGTTGGT

GTCATCACTG

TATCTCAGCA

CACTTCATGC

TATCGGTACA

ACTGTCCGCA

TCCTACTTTC

CCAACCTTTC

ATTCAAAGTC

CATGATAGCC

AGCTTTCATG

TACCTCTCTC

TTCAGGTATT

TGTCTCCCGG

CAGCATTCG

AAATGAGCCC

GAATTTGATC

AAGAAAACCC

GCGTACTCCA

GTGGATACAG

TTATCAACAA I

FCAGTGTAATC

PTATCGTCTAJ

CCGCCGGAAA

CAATAACGCA

ACCTCAACAG

ACGGACTCAG

GCTGGCGGGA

TCCAGCCGGT

GAATCTTGCT

TAAGAATCCA

GCCCTGGATG

CATTCCATCC

AAAGCAAAGG

TGACCGTTAA

TTCTCAGTCC

ACTGTGGTTT

GAGACCCCTA

GGGCAGCGTA

CGCTCCAACA

AAATCTTGCC

ACCTCATCCG

TCGCGGTCAA

TGCGAATGCA

ATAGCCCAAT

TCATCGAAAT

[GTACGTTTC 6660 .GTATTTTTT 6720 LGCGATGGTA 6780 PGTCCCATAG 6840 AAATTCCTT 6900 kCACCTCGGC 6960 GGCTCGAACT 7020 CTTTCGCCTT 7080 CGAATAAGTC 7140 CAAATTCTTC 7200 GACGTAGTAG 7260 AATCTCCACC 7320 CATCGCGGAG 7380 CCACTAGATG 7440 TAATAACAGT 7500 ATTCTTTACT 7560 TGGCGACGCG 7620 CGTACTTAGA 7680 AATTTGTAGC 7740 GATAGGTGGT 7800 ATCCGTGGAT 7860 CGCCTGACCC 7920 ACAACATGGC 7980 TGGCGTCACA 8040 CAGCTTGGAC 8100 CATCGCTTTC 8160 TAAGACAAAA GTTCGGGACT TTGCAAAATT TTTCGCATGC GGCACATCCT CTCCTCATGT CGGGCAGCGT CTCTAACACC 8220 WO 99/61597 WO 9961597PCT/UJS99/1 1250

CAACACAGGA

1580 CCCTCCGAAA

CGCTACACGC

GTCAALAGGCA

1585

GCGAACGTTG

AACCTGATTG

1590 CTTCAGCAAA

ACCGTGGTCG

GAAGGATAGT

1595

TGCTTTGAAG

CCATCTTTGG

1600 ATGATGGCAT

AGCTGGGCAA

AGCCCTCTGG

1605

CTCCACCATG

GTGAAATCAA

1610 TTGATCAGCG

GTTTTAACGA

AGCGCGAAGT

1615

CGGTCACTCA

GCTATACGCC

1620 TCACTGGCCG

CGCCTTGCAG

CGCCCTTCCC

1625

AAACGTCCGC

CCTGCCACCA

1630 TACAGGCAGC

CAACAACTGT

TCTATAACGG

AGAGCACCAG

CCACCATAAC

ATT'ITCTTAG

TCTACGATGT

TCGATAGAAC

AGGTCCTCTC

GGGATTGTGC

ACGTGGTTGG

GACCACTGTC

TTGTAGAAGC

TGGAATCCGA

TCTTCTGAGA

TTGACCGGGT

AAAACTCGAC

TTGGTGGGAA

TCAGTTCGCC

CTTTATACCG

TTACGGCAAA

ATTTGAAGCC

TCGT=~ACA

CACATCCCCC

AACAGTTGCG

AATGTGTTAT

GCCAGCCAAC

CCATCAGNNN

GCACCCTTTT

GGTCTTCAGG

CCAGGCTATG

GGTCACGGAA

ACCTTTTCGC

CTTGGGCACT

TTGACATTTT

CAAATGAAAT

GTCATCCCTT

AACGTCTTCT

GGTAGAGGCA

CATCTTCCTT

GGAGGTTTCC

CTGTATCTTT

GGTCAGTCCC

GGCCTGTGGG

AGCGCGTTAC

GATGCAGATA

AAAGGTTGGG

GTGTGGGTCA

GATGTCACGC

ACGTCGTGAC

TTTCGCCAGC

CAGCCTGAAT

TAAGTTGTCT

AGCTCCCCGA

NNNNNN

ATCCCTTCTT

GGGAAAACTG

GGGCGCATGA

GGCGTCAGCC

GTATTCAATC

CTGGCTGTCA

G ITGGTGAAA

GAACTTCCTT

ACGTCAGTGG

TTTTCCACGA

TTCTTGAACG

TTCTACTGTC

CGATATTACC

GATATTCTTG

TTATGTTACG

CATTCAGTCT

AAGAAAGCCG

TTCGTAATTA

CAGGCCAGCG

ATAATCAGGA

CGTATGTTAT

TGGGAAAACC

TGGCGTAATA

GGCGAATGNN

AAGCGTCAAT

CCGGCAGCTC

NNNL'NNNNt'NNN

GAAAAGTGAT

TCGAGACAGT

TACTGCTGAG

TCCTCAATAG

TGCGCAGATA

GCACCCTTCT

AACALAAGAAC

ATATAGAGGA

AGATATCACA

TGTTCCTCGT

ATAGCCTTTC

CTTTCGATGA

CTTTGTTGAP.

GAGTAGACGA

TCCTGTAGAA

GGATCGCGAA

GGCAATTGCT

TGCGGGCAAC

TATCGTGCTG

AGTGATGGAG

TGCCGGGAAA

CTGGCGTTAC

GCGAAGAGGC

NNNNNAATTC

TTGTTTACAC

GGCACAAAAT

GCCACCAAGA 8280 CATAATGCTC 8340 TCAAATTTAA 8400 AGAGCTTATT 8460 ACTGTTGCGC 8520 CAGCAATCAA 8580 AAGTAGCAGA 8640 AGGGTCTTGC 8700 TCAATCCACT 8760 GGGTGGGGGT 8820 CTTTATCGCA 8880 AGTGACAGAT 8940 AAGTCTCAAT 9000 GAGTGTCGTG 9060 ACCCCAACCC 9120 AACTGTGGAA 9180 GTGCCAGGCA 9240 GTCTGGTATC 9300 CGTTTCGATG 9360 CATCAGGGCG 9420 AGTGTACAAT 9480 CCAACTTAAT 9540 CCGCACCGAT 9600 AGTACATTAA 9660 CACAATATAT 9720 CACCACTCGA 9780 ?NNNJL'iNJDNN 9840 WO 99/61597 PCTJUS99/1 1250 NLINI'NNN NI'NNNNDNi'4DJ NNNNI'NNNNI\J NNNNNNNNNN 1635 NNNNNNNL'NNN NNNNNNPNNNN INt.1NNNN1 I'JDNNNDINNNN 1640 NNNNN~1i~NNN NNUNI'NNN NNNNNDI4NM NNNNNNNNNN NNI'NNNN'NNM 1'JNNJsNNNI 1645 <210> 7 <211> 10272 <212> DNA <213> Brome mosaic vi 1650 <400> 7 NNNNNNNNNN' NNNNNNNNNN NNL'NNNNNNN NrNNNNrNNNNN 9900 NNI'NNNNNNJSI NNNNNNNNNN NNNNNNNNNN N1NNNNNNNNN 9960 NINNNNNNN'JD N'NNNNNJNNLJN NINNL'JNN NNNNNNNNNN 10020 NNNNNNNNNN NNNNNNNNNN NNI'NrNNNNN NNNNN'NNNN 10080 NNNNNNNNN NNNNNNNNN PNNNNNNNNN DJNNNNNNNNN 10140 NNNNNNNNNL'N NNNNNNNNNN NNNI'NNNNNNN NNNNNNNNNN 10200 NYNNNNNNNNN NNNNINNNNN 10240 rus GAAGGCGGGA AACGACAATC TGATCATGAG CGGAGAATTA AAACACTGAT

AGGGAGTCAC

1655

GACAGAACCG

AAGCACATAC

1660 TAGCAAATAT

AATTAGNNNN

TGCACGCAGG

1665

AGACAATCGG

TTTTTGTCAA

1670 TATCGTGGCT

CGGGAAGGGA

TTGCTCCTGC

1675

ATCCGGCTAC

GGATGGAAGC

1680 CAGCCGAACT

AGTTTAAACT

GTTATGACCC

CAACGATTGA

GTCAGAAACC

TTCTTGTCAA

NNNNNNN

TTCTCCGGCC

CTGCTCTGAT

GACCGACCTG

GGCCACGACG

CTGGCTGCTA

CGAGAAAGTA

CTGCCCATTC

CGGTCTTGTC

GTTCGCCAGG

CCGCCGATGA

AGGAGCCACT

ATTATTGCGC

AAATGCTCCA

NNNNNNNN'NN

GCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

TCCATCATGG

GACCACCAAG

GATCAGGATG

CTCAAGGCGC

CGCGGGACAA

CAGCCGCGGG

GTTCAAAAGT

CTGACGTTCC

GATCGTTTCG

AGAGGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

CTGATGCAAT

CGAAACATCG

ATCTGGACGA

GCATGCCCGA

GCCGTTTTAC

TTTCTGGAGT

CGCCTAAGGT

ATAAATTCCC

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

GCGGCGGCTG

CATCGAGCGA

AGAGCATCAG

CGGCGATGAT

GTTTGGAACT 120 TTAATGAGCT 180 CACTATCAGC 240 CTCGGTATCC 300 CAAGATGGAT 360 TGGGCACAAC 420 CGCCCGGTTC 480 GCAGCGCGGC 540 GTCACTGAAG 600 TCATCTCACC 660 CATACGCTTG 720 GCACGTACTC 780 GGGCTCGCGC 840 CTCGTCGTGA 900 CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA 960 TCGACTGTGG CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG 1020 1685 WO 99/61597 WO 9961597PCTIUS99/1 1250 ATATTGCTGA AGAGCTTGGC CCGCTCCCGA TTCGCAGCGC 1690 NNNNNNNN NNNNNNNNNN ATCCTGTTGC CGGTCTTGCG TAATAATTAA CATGTAATGC 1695 CGCAATTATA CATTTAATAC TATCGCGCGC GGTGTCATCT 1700 TCTGGTGGTG GTTCTGGTGG GGTGGCGGCT CTGAGGGAGG GAAAAGATGG CAAACGCTAA 1705 CAGTCTGACG CTAAAGGCAA GGTTTCATTG GTGACGTTTC 1710 GGCTCTAATT CCCAAATGGC TTCCGTCAAT A ITTACCTTC CCAATACGCA AACCGCCTCT 1715 AGGTTTCCCG ACTGGAAAGC CATTAGGCAC CCCAGGCTTT 1720 AGcGGATAAc AATTTcAcAc TGCAGGTCAA CATGGTGGAG TCTCAGAAGA CCAGAGGGCT 1725 TCGGATTCCA TTGCCCAGCT GCTTCTACAA ATGCCATCAT 1730 GACAGTGGTC CCAAAGATGG CCAACCACGT CTTCAAAGCA GCACAATCCC ACTATCCT'rC 1735 GAGAGGACCT CGAGAATTCG GTGTGCTTGT TCTTTCTACT

GGCGAATGGG

ATCGCCTTCT

GATCGTTCAA

ATGATTATCA

ATGACGTTAT

GCGATAGAAA

ATGTTACTAG

CGGCTCTGAG

CGGTTCCGGT

TAAGGGGGCT

ACTTGATTCT

CGGCCTTGCT

TCAAGTCGGT

CCTCCCTCAA

CCCCGCGCGT

GGGCAGTGAG

ACACTTTATG

AGGAAACAGC

CACGACACTC

ATTGAGACTT

ATCTGTCACT

TGCGATAAAG

ACCCCCACCC

AGTGGATTGA

GCAAGACCCT

AGCTCGGTAC

ATCACCAAGA

CTGACCGCTT

ATCGCCTTCT

ACATTTGGCA

TATAATTTCT

TTATGAGATG

ACAAAATATA

ATCGGGCCTC

GGTGGTGGCT

GGTGGCTCTG

ATGACCGAAA

GTCGCTACTG

AATGGTAATG

GACGGTGATA

TCGGTTGAAT

TGGCCGATTC

CGCAACGCAA

CTTCCGGCTC

TATGACCATG

TCGTCTACTC

TTCAACAAAG

TCATCGAAAG

GAAAGGCTAT

ACGAGGAACA

TGTGATATCT

TCCTCTATAT

CCGCAACACA

TGTCTTCGAA

CCTCGTGCTT

TGACGAGTTC

1TAAAGTTTC

GTTGAATTAC

GGTTTTTATG

GCGCGCAAAC

CTGTCAATGC

CTGAGGGTGG

GTTCCGGTGA

ATGCCGATGA

ATTACGGTGC

GTGCTACTGG

ATTCACCTTT

GTCGCCCTTT

ATTAATGCAG

TTAATGTGAG

GTATGTTGTG

ATTACGCCAA

CAAGAATATC

GGTAATATCG

GACAGTAGAA

CGTTCAAGAA

TCGTGGAAAA

CCACTGACGT

AAGGAAGTTC

CATCTGACCT

AACCTGGGAT

TACGGTATCG 1080 TTCTGANNNN 1140 TTAAGATTGA 1200 GTTAAGCATG 1260 ATTAGAGTCC 1320 TAGGATAAAT 1380 TGGCGGCGGC 1440 CGGTTCTGAG 1500 TTTTGATTAT 1560 AAACGCGCTA 1620 TGCTATCGAT 1680 TGATTTTGCT 1740 AATGAATAAT 1800 TGTCTTTGGC 1860 CTGGCACGAC 1920 TTAGCTCACT 1980 TGGAATTGTG 2040 GCTTGCTGCC 2100 AAAGATACAG 2160 GGAAACCTCC 2220 AAGGAAGATG 2280 TGCCTCTACC 2340 AGAAGACGTT 2400 AAGGGATGAC 2460 ATTTCATTTG 2520 TGTTGTTGTT 2580 GATGATTTCG 2640 WO 99/61597 WO 9961597PCTIUS99/1 1250 1740 TTCGCCAGGT

CTGCTAGCCT

GTTTTAAATT

1745

GAGTGCGCTG

GACCGCTGAT

1750 TCGTGCTCGA

CCCGTCTTTC

TCAGGTGCAG

AGCTATAGCG

GGGCTCTATT

TCCTCAAGCT

AGATGAALATT

TATCGTTTGC CGCC2GATGAA ACTGTGAAAT TGAGGAAGTT 1755 ATAGGTACAT GGGCATTGAT TTCAAATCAT GAACCCTATT 1760 CAGATAACCC TGACATCTCT CAGAAGCGAC CAGTCATTCC AAGCCCTTGT TGAAAATGGT 1765 GTGATGTAGA CTGGTATAGG GTGCTCAGCG AAGAGTTGGT 1770 CGGACGTTCC AGAAATGGGA AGCGCTTTCG TAGCACATTC ATGTCATGAC TAAATGTCTT 1775 GTGTGAATGT GGCAGCAGAG TAAAACCTGT TGTAACTGAC 1780 TTCATAGTAA AGGTGTGACT TATCACTGGC CCTGAAATCC TTAAGAATGT CCGCTTGAAT 1785 AATCTCAGGG TGAGCTGCAC

CAATGGATCA

GAGCCGGCAG

CCCTTGGAGA

rGCGACACCG

GAAATGGCAC

GATGACTGGT

GATCATGCGA

CGTGTTACTG

GAAGAGTTTC

GAACATAGGG

ACTGGGCCAA

ATACTGCCAA

GATTATTCCA

GACCCCGATA

ACTCAGAAAG

GACGCGATTA

CTTAATGTTG

GAGTACCATA

ACTGATTTAT

ACCCTTCACT

AGTAATTTTT

AGGTTCATTG

AACAGATACT

CTAGAGTTTC

TGGTCTGAT'I

GTTTCCTTCC

TAGATCAATC

ACGGAGTTGC

TAGGAGGGGT

TCCAACAAAT

GGATGTTATA

ATCCCGAGGA

GCGATCTAAA

GAGATACCCC

AGACTACTAA

TTTCGCGTGT

TTTATATGGA

CCCATGCTTA

TGGAC ITTGA

AATATTTTCA

AAGTCTTAAC

ACATGAAGGA

ACGGTGAAGA

AGAAGTGGGG

GTCGGTACCA

TGGAACGAGC

CACCCTTT

TGCCTATCGG

TTCTTGAAGC

AGAGAGAGAT

TTCATCGCGA

AACGCAGAAC

CTTAGAAGAC C

CATTGACGGAJ

ATTCGATCCCC

GGTTCAACAG

TCTTGACATT

TACTAGTGAT

ACTCGCCAGT

CAAGGAGCTG

TACTGATTAC

TATTGATACA

GAGAGTCAGC

TTTCGATGAT

TAGGATCAGA

ACCAAAAATG

CGCACTCAAA

CACTGCGAAA

CTGTCTGAGA

TAAGCACATG

GCATATGCTG

AGTAGCAGCT

CACTGCTTGT

AAAGATATCC

GGACCTAAGC

ACTCCTTGCG

TTCTTATTTA

TGGTGACGCG

~AGGTGGAGG 2700 ECTCTCGCGA 2760 :CTTTTGACC 2820 rTCACCGATA 2880 CCGGGCTCTT 2940 3GTTACGGTG 3000 GATTCCTCGA 3060 ACCCTTGGAG 3120 GACATCACTC 3180 CACTGCCATC 3240 CTTGCTAGAA 3300 TCGTACCATC 3360 CTTAAGCAAA 3420 AATATCGGGA 3480 AAGCGAAACG 3540 GCTATAGCAA 3600 GCTTCTATGG 3660 GACTTGCAAG 3720 AAGTCTGACG 3780 ACTATAACAT 3840 TTCGAGAAGT 3900 TCTCTGGAGC 3960 AAATTTGATA 4020 CTGGGCTTTC 4080 TCAGACCCTC 4140 TTTACATATT 4200

CAGCGCCGCT

1790 ATGCCAAGGT

GACGAATTGG

GGGAATGTCC

TCGGTAATAC TCTTGTCACT ATGGCTATGA TTGC-ATATGC CTCTGATCTA AGTGACTGTG 4260 WO 99/61597 WO 9961597PCT[US99/1 1250 34 ACTGTGCAAT ATT ETCAGGA GATGATTCTT TAATCATCTC TAAAGTTAAG CCAGTCCTGG 4320 1795 ATACCGATAT GTrTACGTCT CCTACGTTTG TAGTAAGTTT 1800 ATCCTCTGAG AGAGATCCAG TCAGAGCACA TTTCGTrTCC AGATGATTAC GACGCTCTGT 1805 TTTTCGAGGA GGTTAGAGCT GGTTCTCTGA TTGCTACTGT 1810 GTAAGTTCAA ACGC-ACCACG AGAATCCAAA GTTTCCTGGT CGGACTGTAG TACTAAGGAG 1815 AGCGTGAGTC ACTCA7AATTA TCGACGATTA CGCTACCGGT 1820 CCCATTGTGA GACTTTI'AAG TAAGCTGTGA ACAGCCCTTT CTAAGTTTAA AAGCTGGGGA 1825 CTATCTCTCT CTATTTTCTC GTTCTTATAG GGT'ITCGCTC 1830 TTTGTAAAAT ACTTCTATCA CAGGCATGCA AGC FrGCATG- GAGCACGACA CTCTCGTCTP 1835 GCTATTGAGA CTTTTCAACz GCTATCTGTC ACTTCATCG; 1840 CATTGCGATA AAGGAAAGGC TGGACCCCCA CCCACGAGGI GCAAGTGGAT TGATGTGAT) 1845

CTCTTCAATA

CTCGTCGAAA

CGCTTAGCTA

TTCTGTGATC

CATTTTGTTT

GCTCTTGCGG

ACCGAAGGCA

GAAGAGCGTA

GTGTTAGACA

CTCCCTGTCA

GCTAATGATC

AGAGGAGGCC

CCCTCTGATC

GGTTAAGGTT

TCCTCTAGAG

CAGAATAATG-

ATGTGTTGAG-

ATAAAATTTC

CCTGCAGGTC

CTCCAAGAA]

AAGGGTAATI

SAAGGACAGTI

TATCGTTC.A)

k. ACATCGTGGj TGGAGATAAA I

CTGAAATGGG

AGCGAAAGAT

GAATGAAGTT

ATCTGAAATA

CTTTTTCTTT

TCAGAGTTTA

AAACTGATGG

AAGTCTACAG

AACGGATCGG

GTAGGACCAC

TAACGTCAGT

TGAGATGATC

AAAAACTCCT

TCCGCAAATC

1TGTGAGTAGT

CATATAAGAAI

TAATTCCTAA

GACTCTAGAG

ATCAAAGATA

~TCGGGAAACC

k. GAAAAGGAAG k~ GAATGCCTCT k. AAAAGAAGAC

~GTCATGGACC

:AATTTGGTG

LCTGCGTGATC

E'ATTAATCAA

TGGGAAAGAA

kTACTCCGAG

TCAGATGAGC

TGACTGGTTT

1AACCATTGGA

ACGTTTACAT

ACAACGCTTG

TGATGCTTTG

GGTTCTATGA

GGTCAGGCAG

ACCAGTCTCT

TCCCAGATAA

ACCCTTAGTA

AACCAAAATC

GATCCCCGGT

CAGTCTCAGA

TCCTCGGATT

ATGGCTTCTA

ACCGACAGTG

GTTCCAACCA

.CTAGTGTGC 4380 LCTGTACCAG 4440 3AACAGATGC 4500 7TTGATGAGA 4560 kAACCTTGGA 4620 kATTTCCTGA 4680 GATCCTGTAT 4740 CACAACTGGA 4800 ATTTATTCCT 4860 GAGGCCCTTG 4920 AAAAAGAAGG 4980 CTCGTGAAGT 5040 TATATGAACC 5100 ACCACTTTGG 5160 CTCTACAAAT 5220 GGGAATTAGG 5280 TGTATTTGTA 5340 CAGTGACCTG 5400 CAACATGGTG 5460 AGACCAGAGG 5520 CCATTGCCCA 5580 CAAATGCCAT 5640 GTCCCAAAGA 5700 CGTCTTCAAA 5760 CCCACTATCC 5820 TCTCCACTGA CGTAAGGGAT GACGCACAAT TTCGCAAGAC CCTTCCTCTA TATAAGGAA.G TTCATTTCAT TTGGAGAGGA CCTCGACCAC 5880 WO 99/61597 WO 9961597PCT/US99/1 1250 GGTTCTGCTA CTTGTTCTTT 1850 AAGTTGATTG CTGAGAAGGG GTTGCGCAAC AGTTATCTGC CGCAATAAGC TCTCTATTGA 1855 GACTTAAATT TGACTCAGCA GCGGAGCATT ATGACTGTCT 1860 GGGTCTTGGT GGCATCACTT TTGGGTGTTA GAGACGCTGC CAAGAAAGCG ATGATTTCGA 1865 GATGTCCAAG CTGATTGGGC CTGTGTGACG CCATGCATTC 1870 GGCGCCATGT TGTTTGACCG GACGGGTCAG GCGCGGATGA TACATCCACG GATGGCAAGA 1875 GGAACCACCT ATCTGTTGGA ATCGCTACAA ATTTACGCTG 1880 ATATCTAAGT ACGTAGGGGT GTGCGCGTCG CCAAAACCAC GAAAGTAAAG AATGGACTGA 1885 TCGACTGTTA TTATTAACGG TATCATCTAG TGGCCTTTGC 1890 GCCCTCCGCG ATGGGATGGA TGGGGTGGAG ATTCATCCAG CCCCTACTAC GTCTGGATTC 1895 GTTGAAGAAT TTGAGCAAGA GAGGACTTAT TCGACCGGC7 1900 AAGAAGGCGA AAGTCCCGCC GTTTTTCACC1

TGCTGACAGC

GCAGATTGAA

GGAGGCTGAC

GTATCATGCG

CGACAGTTTT

TTCAAGAAGG

CCGACATGAG

TGAAGTCCCG

TATCTGTATC

GCATGGAGTA

CGAGGGTTTT

GGTGATCAAA

TTTGGGCTCA

GCGCGAAATG

CCCCCGGGAG

CTCAATACCT

AGTGAGAGAG

GAACATGAAA

TCAGGCTATC

TTTGACTTTG

ATGGAAAGGT'

GGCGAAAGTA

TTATGCGGAC

TTCTGTACCG

GGAGCATGAA

AGCTGCTGAG

k.ACAAAATGT

:AGAGTGCCC

E'ACGCGAAAA

3CCTTCCGTG

CCCCATAGCC

CCCCCTGAAG

GATAAAAGGG

GAGAGGATGT

AACTTTTGTC

CACGGCGGTT

CGCGTACTAC

CTTCCCTTGC

TTCGATTTTG

TTTTTCACCG

CTGAAATGTA

ACACTACGTC

GAAGACTGGA

GTAGAGGAGA

GCTGTCGCAT

ATGGCTGGTG

AATCTGTATC

TGGTGCCATC

GGATGGCTGA

AGTTTTAAGT

ATATCACGTT

GTGCAGACAG

ATACCTCAGG

ZAAGTTCTAT

kAGACATCGT 3GTCTAAGAA kCCGTTATGG

TGGCTGGTGC

PCCCCGTTAT

TGCACAGTTG

GCCGCATGCG

TTAACCGAGC

ATGATATGGG

GTGGTACCGT

TTAAATGTCA

AAAATGAAAG

AGTCGGTGCA

ACATCATGAC

ACTGTGTATG

GTCTCAATCG

TAGCTTTCAG

CTATCTTATC

AGCGCTTAGA

AAAAGTACGA

ACTTCAAAAC

GAACATTGGC

TTCTGACTCG

TGAGAACGTT

CCAAGACCAA

AGGAGTTTCP

CGATTTGCTG 5940 AGACAATCAG 6000 AATCAACGTT 6060 TGGTGCCTTT 6120 TCTGCGTGTA 6180 AGATTTCGGA 6240 TTGTCCTGTG 6300 AAAAATTTTG 6360 TCAAGATTGT 6420 CTTCCAAGGT 6480 TATGTTCGAC 6540 CTGGCAACGT 6600 CACATTATCT 6660 TTGCATCGAT 6720 CTATAAGATC 6780 GTTTGAAGAC 6840 CTGGAAATGT 6900 ATGTTTCAAG 6960 CGCCAAGTCG 7020 CATTGAAGAT 7080 AAAGCTTACG 7140 TAGGTTTTGG 7200 TAGCAGATTT 7260 TCTCTCAAAC 7320 TTGGACTGAA 7380 GCGCTCGAAG 7440 ,TGATGCCCCT 7500 WO 99/61597 WO 9961597PCTIUS99/1 1250 GAGAGTTCGA GCCCTGAGTC GATGCCGAGG TGTCTGTTGA 1905 ATGAAGGAA.T TTGTGCGTTA CACCTATGGG ACATTTCCGG 1910 ACCTACCATC GCATAGACGA CCTAAAAAAT ACGATTACAC GATGAAACGT ACATTGTTGA 1915 GCTAGCGCCA AAGTTTCTGT TGCGGTAAAA CCACTGCCAT 1920 GCGAATCGTA AATCGGCCGA AAGGTAGCTT TGGACGTTGT TGTCATAGGC TGCTTGTTGA 1925 GCTGCTCTGT CTAAATGTTC AAGTCTCGTG ACGCGGGT'rT 1930 GTTGTTCACA AGACTTACCG CGTAAATGCG GTAATAGGGA AGAAGTCTCA CGAAGCGTCG 1935 ACGTATCTTA CGATGACTCA CCCGTGAGCA AGGACTGGAT 1940 TCTGTTGACA ACGTCACTTT GAGGAGTACT GTTTGGTTGC GGCGAGCTCG CTGGTGATTT 1945 AGACCTCTGC TCGAGAATTC CAGTCTCTCT CTACAAATCI 1950 CCAGATAAGG GAATTAGGGI CCTTAGTATG TATTTGTAT I

CGTCAGTGAT

GGTACCAACG

TTGTAAGAGA'

CGGTCGCGGA

TATGGTGAAT

TGTTGGATAT

TAAAACATGT

CCCTAC-ATGC

AAAAGATGCA

GGACGTCAGG

GCGCACCGCG

TGAGGCTGGT

ACAAGTTCTT

TAAATTGCTC

GTGTCCGCAA

CACGAAGTAT

TATTACTTCT

AGCTGATAAA

TGATGGACAC

GGTTCGGCTT

CTTAACACGA

GATCTTTAAT

GAGCTCGGTA

ATCTCTCTCT

TCTTATAGGG

TGTAAAATAC

GACGTTAAAC GACCCTCGTG C TTACATAACA

AGTGAGATCG

GTCCATTTGG

A.ATGAGCATG

GCATGCTCTA

GATATTTCCA

TTCCGTATGG

ATGGCTTTAT

GATTCTGCGA

TTACTACATT

GCCTTTGGGG

CACGGTAATC

GATGTTATCG

CAATCCTGGA

GGTTTGCAGG

GCGGCCCTTC

ATAAAAACAG

AAGTCGACCA

CACAAGAAGT

TGTGTTAAGT

CCCGGGGATC

ATTTTCTCCA

TTTCGCTCAT

TTCTATCAAT

~GGTGACTGA

CATATCTAG

kCTCCGAGTC

'AAATAAGAG

:CAACGGTAA

3TTTAGGTCC

OLCTTGAGGGA

TGGTTGATGG

GAGAGGACCT

rCCCTGACAC

ICATGCACGG

ATGGTCAACT

ACACAGAGCA

TGCAATATGA

CTGCTGTTAA

CATCTGAGTC

TCACTATTGA

AAACGAGGGC

TACACGAAGC

AATGTGATTT

CCTTTGAGTA

GATGCGCTTG

CTCTAGAGTC

GAATAATGTG

GTGTTGAGCA

AAAATTTCTA

PGTGGTCCCG 7560 kCACGGAGCC 7620 IAATCTTCGT 7680 CATCTTTGAG 7740 CTGGTTGTAT 7800 GAAGCACGCA 7860 CATTGCAGAA 7920 A~GTTGCGGGA 7980 AATTGTGACG 8040 TTATAATTCC 8100 TGTACCGTCC 8160 CCTGGTGGTG 8220 GATTTCGTTC 8280 TCGCCGTGAC 8340 TCTGCTGAAG 8400 CAAAGTTTCT 8460 TCCGAACAGA 8520 TAAGGATTTT 8580 GCAAGGGATC 8640 GTTTAAACAT 8700 TTGCTTTAAC 8760 TCTCTGTGTG 8820 CGCAAATCAC 8880 TGAGTAGTTC 8940 TATAAGAAAC 9000 ATTCCTAAAA 9060 CCAAA.ATCCA GTGACCGGGT GGTCAGTCCC TTATGTTACG TCCTGTAGAA ACCCCAACCC 9120 1955 WO 99/61597 WO 9961597PCTlUS99/1 1250

GTGAAATCAA

TTGATCAGCG

1960 GTTTTAACGA AGCGCG7AAGT

CGGTCACTCA

1965

GCTATACGCC

TCACTGGCCG

1970 CGCCTTGCAG

CGCCCTTCCC

AAACGTCCGC

1975

CCTGCCACCA

TACAGGCAGC

1980 NNNN um

NNNNNN

NNNNNN

1985

NNNNN~N

NNNNNNN

1990 NNNNNNNN

AAAACTCGAC

TTGGTGGGAA

TCAGTTCGCC

CTTTATACCG

TTACGGCAAA

ATTTGAAGCC

TCGTTTTACA

CACATCCCCC

AACAGTTGCG

AATGTGTTAT

GCCAGCCAAC

CCATCAGNNN

i'NNN'NNNNNNN NNI'NNNN~1JNN

GGCCTGTGGG

AGCGCGTTAC

GATGCAGATA

AAAGGTTGGG

GTGTGGGTCA

GATGTCACGC

ACGTCGTGAC

TTTCGCCAGC

CAGCCTGAAT

TAAGTTGTCT

AGCTCCCCGA

NNNNNNNNNI4

NNNDNNJNN

NNNNJ'rJNDJ

UNUUINNNNNNU

NNNNNNN

IsNI'NNNNNNN NNN4NNNi'I'

CATTCAGTCT

AAGAAAGCCG

TTCGTAATTA

CAGGCCAGCG

ATAATCAGGA

CGTATGTTAT

TGGGAAAACC

TGGCGTAATA

GGCGAATGNN

AAGCGTCAAT

CCGGCAGCTC

DNNtJNNNNDNN

N'NNNNNNNNN

NNNNNNN

NNNNNNNNNN

i~NNrNN1'NN

GGATCGCGAA

GGCAATTGCT

TGCGGGCAAC

TATCGTGCTG

AGTGATGGAG

TGCCGGGAAA

CTGGCGTTAC

GCGAAGAGGC

NNNNNAATTC

TTGTTTACAC

GGCACAAAAT

NND4NN'NNNI'J NNI'Ni'LNNN

AACTGTGGAA

GTGCCAGGCA

GTCTGGTATC

CGTTTCGATG

CATCAGGGCG

AGTGTACAAT

CCAACTTAAT

CCGCACCGAT

AGTACATTAA

CACAATATAT

CACCACTCGA

NDNNNNNNNN

L'N1'NIN4I'NNN 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 NNNNNNNNN NNL'NNNNNNN 9960 NNNNN.1'NNNN

NNLNDNNNN

INi]NNNrNNN

NDNNNNI'NI'JD

NNi'NNNNNNNN 1'NI'NNNNNNDJ 10020 10080 10140 10200 10260 10.272 IrNINhNNNNN NI'NNNNL'I'NNNNN NNNNNNNN NN 1995 <210> 8 <211> 10166 <212> DNA <213> Brome mosaic virus 2000 NNNNNMNN1I'NNNNI'JNNN NNNNL'NNNNM NNI'NN'NNNM <400> 8 AAACACTGAT AGTTTAAACT GAAGGCGGGA AACGACAATC TGATCATGAG CGGAGAATTA AGGGAGTCAC GTTATGACCC CCGCCGATGA CGCGGGACAA GCCGTTTTAC GTTTGGAACT 120 2005 GACAGAACCG CAACGATTGA AGGAGCCACT CAGCCGCGGG TTTCTGGAGT TTAATGAGCT 180 AAGCACATAC GTCAGAAACC ATTATTGCGC GTTCAAAAGT CGCCTAAGGT CACTATCAGC 240 WO 99/61597 WO 9961597PCT/US99/111250 38 2010 TAGCAAATAT TTCTTGTCAA AAATGCTCCA CTGACGTTCC ATAAATTCCC CTCGGTATCC 300

AATTAGNNNN

TGCACGCAGG

2015

AGACAATCGG

TTTTTGTCAA

L'NNLNNNNNNN'

TTCTCCGGCC

CTGCTCTGAT

GACCGACCTG

2020 TATCGTGGCT GGCCACGACG CGGGAAGGGA CTGGCTGCTA TTGCTCCTGC CGAGAAAGTA 2025 ATCCGGCTAC CTGCCCATTC GGATGGAAGC CGGTCTTGTC 2030 CAGCCGAACT GTTCGCCAGG CCCATGGCGA TGCCTGCTTG TCGACTGTGG CCGGCTGGGT 2035 ATATTGCTGA AGAGCTTGGC CCGCTCCCGA TTCGCAGCGC ATCCTGTTGC CGGTCTTGCG TAATAATTAA CATGTAATGC 2045 CGCAA'rrATA CAT'FrAATAC TATCGCGCGC GGTGTCATCT 2050 TCTGGTGGTG GTTCTGGTGG GGTGGCGGCT CTGAGGGAGG GAAAAGATGG CAAACGCTAA 2055 CAGTCTGACG CTAAAGGCAA GGTTTCATTG GTGACGTTTC NNNDJNN4NN

GCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

TCCATCATGG

GACCACCAAG

GATCAGGATG

CTCAAGGCGC

CCGAATATCA

GTGGCGGACC

GGCGAATGGG

ATCGCCTTCT

GATCGTTCAA

ATGATTATCA

ATGACGTTAT

GCGATAGAAA

ATGTTACTAG

CGGCTCTGAG

CGGTTCCGGT

TAAGGGGGCT

ACTTGATTCT

CGGCCTTGCT

GATCGTTTCG

AGAGGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

CTGATGCAAT

CGAAACATCG

ATCTGGACGA

GCATGCCCGA

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

GCGGCGGCTG

CATCGAGCGA

AGAGCATCAG

CGGCGATGAT

CAAGATGGAT 360 TGGGCACAAC 420 CGCCCGGTTC 480 GCAGCGCGGC 540 GTCACTGAAG 600 TCATCTCACC 660 CATACGCTTG 720 GCACGTACTC 780 GGGCTCGCGC 840 CTCGTCGTGA 900 TCTGGATTCA 960 TGGTGGAAAA TGGCCGCTTT GCTATCAGGA CATAGCGTTG GCTACCCGTG 1020

CTGACCGCTT

ATCGCCTTCT

ACATTTGGCA

TATAATTCT

TTATGAGATG

ACAAAATATA

ATCGGGCCTC

GGTGGTGGCT

GGTGGCTCTG

ATGACCGAAA

GTCGCTACTG

AATGGTAATG

GACGGTGATA

CCTCGTGCTT

TGACGAGTTC

ATAAAGTTTC

GTTGAATTAC

GGTTTTTATG

GCGCGCAAAC

CTGTCAATGC

CTGAGGGTGG

GTTCCGGTGA

ATGCCGATGA

ATTACGGTGC

GTGCTACTGG

ATTCACCTTT

TACGGTATCG 1080 TTCTGANNNN 1140 TTAAGATTGA 1200 GTTAAGCATG 1260 ATTAGAGTCC 1320 TAGGATAAAT 1380 TGGCGGCGGC 1440 CGGTTCTGAG 1500 TTTTGATTAT 1560 AAACGCGCTA 1620 TGCTATCGAT 1680 TGATTTTGCT 1740 AATGAATAAT 1800 2060 GGCTCTAATT CCCAAATGGC TCAAGTCGGT TTCCGTCAAT A'PrTACCTTC CCTCCCTCAA TCGGTTGAAT GTCGCCCTTT TGTCTTTGGC 1860 WO 99/61597 WO 9961597PCT/US99/1 1250 CCAATACGCA AACCGCCTCT 2065 AGGTTTCCCG ACTGGAAAGC CATTAGGCAC CCCAGGCTTT 2070 AGCGGATAAC AATTTCACAC TGCAGGTCAA CATGGTGGAG TCTCAGAAGA CCAGAGGGCT 2075 TCGGATTCCA TTGCCCAGCT GCTTCTACAA ATGCCATCAT 2080 GACAGTGGTC CCAAAGATGG CCAACCACGT CTTCAAAGCA GCACAATCCC ACTATCCTTC 2085 GAGAGGACCT CGAGAATTCG GTGTGCTTGT TCTITCTACT 2090 TTCGCCAGGT CCCGTCTTTC CTGCTAGCCT TCAGGTGCAG GTTTTAAATT AGCTATAGCG 2095 GAGTGCGCTG GGGCTCTATT GACCGCTGAT TCCTCAAGCT 2100 TCGTGCTCGA AGATGAAATT TATCGTTTGC CGCCGATGAA ACTGTGAAAT TGAGGAAGTT 2105 ATAGGTACAT GGGCATTGAT TTCAAATCAT GAACCCTATT 2110 CAGATAACCC TGACATCTCT CAGAAGCGAC CAGTCATTCC AAGCCCTTGT TGAAAATGGT 2115

CCCCGCGCGT

GGGCAGTGAG

ACACTTTATG

AGGAAACAGC

CACGACACTC

ATTGAGACTT

ATCTGTCACT

TGCGATAAAG

ACCCCCACCC

AGTGGATTGA

GCAAGACCCT

AGCTCGGTAC

ATCACCAAGA

CAATGGATCA

GAGCCGGCAG

CCCTTGGAGA

TGCGACACCG

GAAATGGCAC

GATGACTGGT

GATCATGCGA

CGTGTTACTG

GAAGAGTTTC

GAACATAGGG

ACTGGGCCAA

ATACTGCCAA

GATTATTCCA

TGGCCGATTC

CGCAACGCAA

CTTCCGGCTC

TATGACCATG

TCGTCTACTC

TTCAACAAAG

TCATCGAAAG

GAAAGGCTAT

ACGAGGAACA

TGTGATATCT

TCCTCTATAT

CCGCAACACA

TGTCTTCGAA

TAGATCAATC

ACGGAGTTGC

TAGGAGGGGT

TCCAACAAAT

GGATGTTATA

ATCCCGAGGA

GCGATCTAAA

GAGATACCCC

AGACTACTAA

TTTCGCGTGT

TTTATATGGA

*CCCATGCTTA

TGGACTTTGA

A~TTAATGCAG

TTAATGTGAG

GTATGTTGTG

ATTACGCCAA

CAAGAATATC

GGTAATATCG

GACAGTAGAA

CGTTCAAGAA

TCGTGGAAAA

CCACTGACGT

AAGGAAGTTC

CATCTGACCT

AACCTGGGAT

CTTAGAAGAC

CATTGACGGA

ATTCGATCCC

GGTTCAACAG

TCTTGACATT

TACTAGTGAT

ACTCGCCAGT

CAAGGAGCTG

TACTGATTAC

TATTGATACA

GAGAGTCAGC

TTTCGATGAT

TAGGATCAGA

CTGGCACGAC 1920 TTAGCTCACT 1980 TGGAATTGTG 2040 GCTTGCTGCC 2100 AAAGATACAG 2160 GGAAACCTCC 2220 AAGGAAGATG 2280 TGCCTCTACC 2340 AGAAGACGTT 2400 AAGGGATGAC 2460 ATTTCATTTG 2520 TGTTGTTGTT 2580 GATGATTTCG 2640 GAGGTGGAGG 2700 TCTCTCGCGA 2760 CCTTTTGACC 2820 TTCACCGATA 2880 CCGGGCTCTT 2940 GGTTACGGTG 3000 GATTCCTCGA 3060 ACCCTTGGAG 3120 GACATCACTC 3180 CACTGCCATC 3240 CTTGCTAGAA 3300 TCGTACCATC 3360 CTTAAGCAAA 3420 GTGATGTAGA CTGGTATAGG GACCCCGATA AATATTTTCA ACCAAAAATG AATATCGGGA 3480 WO 99/61597 WO 9961597PCTIUS99/1 1250 GTGCTCAGCG AAGAGTTGGT 2120 CGGACGTTCC AGAAATGGGA AGCGCTTTCG TAGCACATTC ATGTCATGAC TAAATGTCTT 2125 GTGTGAATGT GGCAGCAGAG TAAAACCTGT TGTAACTGAC 2130 TTCATAGTAA AGGTGTGACT TATCACTGGC CCTGAAATCC TTAAGAATGT CCGCTTGAAT 2135 AATCTCAGGG TGAGCTGCAC CAGCGCCGCT GACGAATTGG 2140 ATGCCAAGGT GGGAATGTCC TCGGTAATAC TCTTGTCACT ACTGTGCAAT ATT'rrCAGGA 2145 ATACCGATAT GTTTACGTCT CCTACGTTTG TAGTAAGTTT 2150 ATCCTCTGAG AGAGATCCAG TCAGAGCACA TTTCG'ITTCC AGATGATTAC GACGCTCTGT 2155 TTTTCGAGGA GGTTAGAGCT GGTTCTCTGA TTGCTACTGT 2160 GTAAGTTCAA ACGCACCACG AGAATCCAAA GTTTCCTGGT CGGACTGTAG TACTAAGGAG 2165 AGCGTGAGTC ACTCAAATTA TCGACGA'rrA CGCTACCGGT 2170 CCCATTGTGA GACTTTAAG ACTCAGAAAG AAGTCTTAAC GACGCGATTA ACATGAAGGA CTTAATGTTG ACGGTGAAGA GAGTACCATA AGAAGTGGGG ACTGATTTAT GTCGGTACCA

ACCCTTCACT

AGTAATTTTT

AGGTTCATTG

AACAGATACT

CTAGAGTTTC

TGGTCTGATT

GTTTCCTTCC

ATGGCTATGA

GATGATTCTT

CTCTTCAATA

CTCGTCGAAA

CGCTTAGCTA

TTCTGTGATC

CATTTTGTTT

GCTCTTGCGG

ACCGAAGGCA

GAAGAGCGTA

GTGTTAGACA

CTCCCTGTCA

GCTAATGATC

AGAGGAGGCC

CCCTCTGATC

TGGAACGAGC

CACCCTTTTT

TGCCTATCGG

TTCTTGAAGC

AGAGAGAGAT

TTCATCGCGA

AACGCAGAAC

TTGCATATGC

TAATCATCTC

TGGAGATAAA

CTGAAATGGG

AGCGAAAGAT

GAATGAAGTT

ATCTGAAATA

CTTTTTCTTT

TCAGAGTTTA

AAACTGATGG

AAGTCTACAG

AACGGATCGG

GTAGGACCAC

TAACGTCAGT

TGAGATGATC

CGCACTCAAA

CACTGCGAAA

CTGTCTGAGA

TAAGCACATG

GCATATGCTG

AGTAGCAGCT

CACTGCTTGT

AAAGATATCC

GGACCTAAGC

ACTCCTTGCG

TTCTTATTTA

TGGTGACGCG

CTCTGATCTA

TAAAGTTAAG

AGTCATGGAC

CAATTTGGTG

TCTGCGTGAT

TATTAATCAA

TGGGAAAGAA

ATACTCCGAG

TCAGATGAGC

TGACTGGTTT

AACCATTGGA

ACGTTTACAT

ACAACGCTTG

TGATGCTTTG

GGTTCTATGA

AAGCGAAACG

GCTATAGCAA

GCTTCTATGG

GACTTGCAAG

AAGTCTGACG

ACTATAACAT

TTCGAGAAGT

TCTCTGGAGC

AAATTTGATA

CTGGGCTTTC

TCAGACCCTC

TTTACATATT

AGTGACTGTG

CCAGTCCTGG

CCTAGTGTGC

TCTGTACCAG

GAACAGATGC

CTTGATGAGA

AAACCTTGGA

AATTTCCTGA

GATCCTGTAT

CACAACTGGA

ATTTATTCCT

GAGGCCCTTG

AAAAAGAAGG

CTCGTGAAGT

TATATGAACC

3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 WO 99/61597 WO 9961597PCTIUS99/1 1250 TAAGCTGTGA ACAGCCCTT CTAAGTTTAA AAGCTGGGGA 2175 CTATCTCTCT CTATTTTCTC GTTCTTATAG GGT ITCGCTC 2180 TTTGTAAAAT ACTTCTATCA CAGGCATGCA AGCTTGCATG TGGTTTTAGG AATTAGAAAT 2185 AGGGTTTCTT ATATGCTCAA TGGGAACTAC TCACACATTA 2190 CTGGTGA=T GCGGACTCTA TCTCACACAG AGACAAGCGC GCCGTTAAAG CAATACTCAA 2195 CTCATGTTTA AACAAATCAC AGAGATCCCT TGCGCTTCGT 2200 GGGAAAATCC TTAGCCCTCG CGTTCTG'rrC GGATCAATAG TCTAGAAACT TTGGACTCAG 2205 ACGCTTCAGC AGATTAACAG AACGTCACGG CGATCATATT 2210 CTTGAACGAA ATCTGCTCTG AGCCACCACC AGGAGTTGAC ACAGGACGGT ACACCGTGCA 2215 CTTGGAATTA TAAGTGTCAG CGCCGTCACA ATTAGGTCCT 2220 GCATCCCGCA ACTCCATCAA AGCTTCTGCA ATGTCCCTCA ATCTGCGTGC TTCGGACCTA 2225

GGTTAAGGTT

TCCTCTAGAG

CAGAATAATG

ATGTGTTGAG

ATAAAATTTC

CCTGCAGGTC

T ITATTGATA

CACATGAGCG

TTCTGGAGAA

GAGGATCCCC

ATCACTTAAC

AGGACTTCTT

ATTTGGTCGA

GTACTGTTTT

TTTGAAGGGC

TGACCTGCAA

ATGTCCAGGA

CAGCGATAAC

GCAGATTACC

TGTCCCCAAA

CATAATGTAG

TGATCGCAGA

GGAATAAAGC

CTCCCATACG

CCATGGAAAT

AGTTAGAGCA

AACCATGCTC

AAAAACTCCT GGTCAGGCAG ACCACTTTGG 5160

TCCGCAAATC

TGTGAGTAGT

CATATAAGAA.

TAATTCCTAA

GACTCTAGAG

GAAGTATTTT

AAACCCTATA

A.ATAGAGAGA

GGGTACCGAG

ACAATTAAAG

GTGTCGTGTT

CTTAAGCCGA

TATGTGTCCA

CGCTTTATCA

ACCAGAAGTA

TTGATACTTC

ATCTTGCGGA

GTGGAGCAAT

GGCAAGAACT

TAAACCAGCC

ATCCGCGGTG

CATCCTGACG

GAATGCATCT

ATCGCATGTA

TGCACATGTT

ATTATATCCA

ACCAGTCTCT

TCCCAGATAA

ACCCTTAGTA

AACCAAAATC

GATCCCCGGT

ACAAATACAA

AGAACCCTA1A

GATAGATTTG

CTCGAATTCT

ATCAAATCAC

AAGGCAACCA

ACCAAAGTGA

TCAATCCAGT

GCTTGAGTCA

ATACGACGCT

GTGTCCCTAT

CACCGGTAAG

TTAAAACCCG

TGTGAACATT

TCATCAACAA

CGCACAACGT

TCCTCGGCCG

TTTATGGCAG

GGGACAGAAA

TTATCAACAA

ACAGTGTAAT

CTCTACAAAT 5220 GGGAATTAGG 5280 rGTATTTGTA 5340 CAGTGACCTG 5400 CACTGGATTT 5460 ATACATACTA 5520 TTCCCTTATC 5580 TAGAGAGAGA 5640 CGAGCAGAGG 5700 CAGCGAGCTC 5760 AACAGTACTC 5820 CGTTGTCAAC 5880 CCTTGCTCAC 5940 TCGTAAGATA 6000 TCGTGAGACT 6060 TACCGCATTT 6120 TCTTGTGAAC 6180 CGTCACGAGA 6240 TAGACAGAGC 6300 GCAGCCTATG 6360 CCAAAGCTAC 6420 ATTTACGATT 6480 TGGTTTTACC 6540 CTTTGGCGCT 6600 TGTACGTTTC 6660 CGTATTTTTT 6720 WO 99/61597 WO 9961597PCTIUS99/1 1250 AGGATACAAC CAGTTACCGT GGTCTCAAAG ATGCTCTTAT 2230 GTGACGAAGA TTAGACTCGG CATGGCTCCG TGTCTAGATA ATCCGGGACC ACATCAGTCA 2235 CTCAGGGGCA TCATGAAACT CTTCTTCGAG CGC'ITGGTCT 2240 CTCTTCAGTC CAAAACGTTC AACGTTTGAG AGACGAGTCA GGGAAATCTG CTAGCCAATG 2245 CCACCAAAAC CTAGTTTTGA GGCCGTAAGC TTTTCGTACT 2250 ATAATCTTCA ATGTCTAAGC CGACGACTTG GCGGATAAGA TTCCTTGAAA CATCTGAAAG 2255 CACACATTTC CAGCGATTGA TATGTCTTCA AACCATACAC 2260 GATGATCTTA TAGGTCATGA TCCATCGATG CAATGCACCG GTAAGATAAT GTGCTTTCAT 2265 GTCACGTTGC CAGTGACATT GCCGTCGAAC ATAACGGTAC 2270 CAGACCTTGG AAGCCCATAT ATCACAATCT TGAGCTCGGT TTGCAAAATT TTTCGCATGC 2275 CAACACAGGA CAACAACTGT

['GGCCAAATGC

rTGCGATCTC2 k.GTTGTTATG rGCCACGAGG

CCGGTTTAAC

CCTCCTGAGG

rGGCTGTCTG

TCAAACGTGA

GAAACTTAAA

TTCTCAGCCA

AGTGATGGCA

TTTGATACAG

GCTCACCAGC

TAGATGCGAC

CTATCTCCTC

GACTCCAGTC

AGTGACGTAG

TGTTACATTT

ACTCGGTGAA

TTTCAAAATC

TAAGCAAGGG

CACGTAGTAC

CATAACCGCC

TAAGACAAAA

GGCACATCCT

GCACCCTTTT

ACATTCACC

kCTTCCGCGA

PAATCTCTTA

3TCCGTTGGT 3TCATCACTG

TATCTCAGCA

CACTTCATGC

TATCGGTACA

PCTGTCCGCA

TCCTACTTTC

CCAACCTTTC

ATTCAAAGTC

CATGATAGCC

AGCTTTCATG

TACCTCTCTC

TTCAGGTATT

TGTCTCCCGG

CAGCATTTCG

AAATGAGCCC

GAATTTGATC

AAGAAAACCC

GCGTACTCCA

GTGGATACAG

GTTCGGGACT

CTCCTCATGT

ATCCCTTCTT

kTATCGTCTA

"CGCCGGAAA

CAATAACGCA

%'CCTCAACAG

kCGGACTCAG

GCTGGCGGGA

TCCAGCCGGT

GAATCTTGCT

TAAGAATCCA

GCCCTGGATG

CATTCCATCC

AAAGCAAAGG

TGACCGTTAA

TTCTCAGTCC

ACTGTGGTTT

GAGACCCCTA

GGGCAGCGTA

CGCTCCAACA

AAATCTTGCC

ACCTCATCCG

TCGCGGTCAA

TGCGAATGCA

ATAGCCCAAT

TCATCGAAAT

CGGGCAGCGT

GAAAAGTGAT

TGCGATGGTA 6780 rGTCCCATAG 6840 CAAATTCCTT 6900 ACACCTCGGC 6960 GGCTCGAACT 7020 CTTTCGCCTT 7080 CGAATAAGTC 7140 CAAATTCTTC 7200 GACGTAGTAG 7260 AATCTCCACC 7320 CATCGCGGAG 7380 CCACTAGATG 7440 TAATAACAGT 7500 ATTCTTTACT 7560 TGGCGACGCG 7620 CGTACTTAGA 7680 AATTTGTAGC 7740 GATAGGTGGT 7800 ATCCGTGGAT 7860 CGCCTGACCC 7920 ACAACATGGC 7980 TGGCGTCACA 8040 CAGCTTGGAC 8100 CATCGCTTTC 8160 CTCTAACACC 8220 GCCACCAAGA 8280 CCCTCCGAAA TCTATAACGG GGTCTTCAGG GGGAAAACTG TCGAGACAGT CATAATGCTC 8340 WO 99/61597 WO 9961597PCTIUS99/1 1250 2280 CGCTACACGC AGAGCACCAG GTCAAAGGCA CCACCATAAC GCGAACGTTG ATTTTCTTAG 2285 AACCTGATTG TCTACGATGT CTTCAGCAAA TCGATAGAAC 2290 ACCGTGGTCG AGGTCCTCTC GAAGGATAGT GGGATTGTGC TGCTrTGAAG ACGTGGTTGG 2295 CCATCTTTGG GACCACTGTC ATGATGGCAT TTGTAGAAGC 2300 AGCTGGGCAA TGGAATCCGA AGCCCTCTGG TCTTCTGAGA CTCCACCATG TTGACCGGGT 2305 GTGAAATCAA AAAACTCGAC TTGATCAGCG TTGGTGGGAA 2310 GTTTTAACGA TCAGTTCGCC AGCGCGAAGT CTTTATACCG CGGTCACTCA TTACGGCAA 2315 GCTATACGCC ATTTGAAGCC TCACTGGCCG TCGTTACA 2320 CGCCTTGCAG CACATCCCCC CGCCCTTCCC AACAGTTGCG AAACGTCCGC AATGTGTTAT 2325 CCTGCCACCA GCCAGCCAAC TACAGGCAGC CCATCAGNNN 2330 NNNNm NNNNNNNNMNN iUhJUNNh1I

CCAGGCTATG

GGTCACGGAA

ACCTTTTCGC

CTTGGGCACT

TTGACATTTT

CAAATGAAAT

GTCATCCCTT

AACGTCTTCT

GGTAGAGGCA

CATCTTCCTT

GGAGGTTTCC

CTGTATCTTT

GGTCAGTCCC

GGCCTGTGGG

AGCGCGTTAC

GATGCAGATA

AAAGGTTGGG

GTGTGGGTCA

GATGTCACGC

ACGTCGTGAC

TTTCGCCAGC

CAGCCTGAAT

TAAGTTGTCT

AGCTCCCCGA

NNNNI4NNiNN

NNNNNI'DJNN

NNIINIUIUU

GGGCGCATGA

GGCGTCAGCC

GTATTCAATC

CTGGCTGTCA

GTTGGTGAAA

GAACTTCCTT

ACGTCAGTGG

TTTTCCACGA

TTCTTGAACG

TTCTACTGTC

CGATATTACC

GATATTCTTG

TTATGTTACG

CATTCAGTCT

AAGAAAGCCG

TTCGTAATTA

CAGGCCAGCG

ATAATCAGGA

CGTATGTTAT

TGGGAAAACC

TGGCGTAATA

GGCGAATGNN

AAGCGTCAAT

CCGGCAGCTC

£'NNI'JNNNNNN

i'NDJ1NNI'INN

TACTGCTGAG

TCCTCAATAG

TGCGCAGATA

GCACCCTTCT

AACAAAGAAC

ATATAGAGGA

AGATATCACA

TGTTCCTCGT

ATAGCCTTTC

CTTTCGATGA

CTTTGTTGAA

GAGTAGACGA

TCCTGTAGAA

GGATCGCGAA

GGCAATTGCT

TGCGGGCAAC

TATCGTGCTG

AGTGATGGAG

TGCCGGGAAA

CTGGCGTTAC

GCGAAGAGGC

NIINNNAATTC

TTGTTTACAC

GGCACAAAAT

NIJNNNNNN

NNNN.NNNNNN'

TCAAATTTAA 8400 PGAGCTTATT 8460 PCTGTTGCGC 8520 CAGCAATCAA 8580 AAGTAGCAGA 8640 AGGGTCTTGC 8700 TCAATCCACT 8760 GGGTGGGGGT 8820 CTTTATCGCA 8880 AGTGACAGAT 8940 AAGTCTCAAT 9000 GAGTGTCGTG 9060 ACCCCAACCC 9120 AACTGTGGAA 9180 GTGCCAGGCA 9240 GTCTGGTATC 9300 CGTTTCGATG 9360 CATCAGGGCG 9420 AGTGTACAAT 9480 CCAACTTAAT 9540 CCGCACCGAT 9600 AGTACATTAA 9660 CACAATATAT 9720 CACCACTCGA 9780 ?NI'JNNNNNNN' 9840 NNNN'J'JLiDNN 9900 NNNNNNNNN NNNNNNNJNN1' NNNNNNNNNN 9960 NNNNNNNNNN NNNNumiiNNNN UNNNNUNNN NIJNNNNNNNN NNNNNNNN NNNNNNNNN 10020 WO 99/61597 PCTIUS99/1 1250 44 2335 NNNNNNNNNN NNNNNNNNNN NN1 NNNNM NNNNNNNNNN NNNNNNNNN 10080 NNt'JNNNNJNN NNNNNNNNNN NNIJNNNNNNN NNNNNNNNNN NNNLNL'NNrJN NNNNNNNNN~ 10140 NL'NNNNNNNN NNNNNNNNNNl~hi NNNNNN 10166 2340

Claims (27)

1. A DNA-launching platform comprising: a) a polynucleotide molecule encoding a modified viral RNA molecule, wherein said polynucleotide molecule is transcribed thereby forming a replicatable RNA transcript not capable of self-replication but replicatable in the presence of a trans-acting viral replication factor; and b) a DNA dependent RNA polymerase promoter.

2. The DNA-launching platform of claim 1 further comprising a sequence encoding at least one cis-acting element.

3. The DNA-launching platform of claim 1 further comprising a ribozyme sequence.

4. The DNA-launching platform of claim 1 further comprising a termination sequence. The DNA-launching platform of claim 1 further comprising a restriction site.

6. The DNA-launching platform of claim 1 wherein said modified RNA molecule comprises an exogenous RNA segment. i 7. The DNA-launching platform of claim 1 wherein said DNA dependent RNA polymerase promoter is capable of functioning in a plant cell. S8. A method of genotypically or phenotypically modifying one or more plant cells, said plant cell or cells having been tendered transgenic by stably comprising heterologous DNA encoding a trans-acting viral replication factor, said method comprising the following steps: a) obtaining a DNA-launching platform comprising a polynucleotide molecule encoding a modified viral RNA; and b) transfecting said one or more plant cells with said DNA-launching platform, wherein said polynucleotide molecule is transcribed thereby forming a replicatable RNA transcript not capable of self-replication but replicatable in the presence of said trans-acting viral replication factor, wherein expression of said RNA transcript confers a genotype or phenotype modification in said one or more plant cells.

9. The method of claim 8 further comprising pre-transforming said cell with at least one polynucleotide molecule encoding at least one trans-acting factor. The method of claim 8 further comprising introducing a trans-acting factor.

11. The method of claim 10 wherein said introducing a trans-acting factor comprises co- transfection of an expression plasmid comprising a nucleotide sequence encoding said transacting factor.

12. The method of claim 10 wherein said introducing a trans-acting factor comprises co- transfection of an RNA transcript encoding said trans-acting factor. [I:\DayLib\LIBFF]83079spec.doc:gcc 34

13. The method of claim 10 wherein said trans-acting factor is stably expressed.

14. The method of claim 8 wherein said modified viral RNA comprises an exogenous RNA segment. The method of claim 8 wherein said DNA-launching platform comprises a ribozyme sequence.

16. The method of claim 8 wherein said DNA-launching platform comprises a promoter.

17. The method of claim 8 wherein said DNA-launching platform comprises a termination sequence.

18. The method of claim 8 wherein said DNA-launching platform comprises a restriction site.

19. The modified cell produced by the method of claim 8. A method of producing a plant or plant tissue comprising at least one genotypically or phenotypically modified plant cell, said cell having been rendered transgenic by stably comprising heterologous DNA encoding a trans-acting viral replication factor, said method comprising transfecting cells of said plant or plant tissue with a DNA-launching platform, wherein said DNA- launching platform comprises a polynucleotide encoding a modified RNA molecule, such that said polynucleotide is transcribed to form a replicatable RNA transcript not capable of self-replication but replicatable in the presence of said trans-acting viral replication factor, and wherein expression of said RNA transcript confers a genotypic or phenotypic modification in at least one of said transfected cells.

21. The method of claim 20 wherein said modified RNA molecule comprises an exogenous RNA segment.

22. The method of claim 20 wherein said DNA-launching platform comprises a ribozyme sequence. 25 23. The method of claim 20 wherein said DNA-launching platform comprises a promoter. S.l.

24. The method of claim 20 wherein said DNA-launching platform comprises a termination sequence. The method of claim 20 wherein said DNA-launching platform comprises a restriction site.

26. A method of producing a genotypically or phenotypically modified plant comprising obtaining at least one modified cell produced by the method of claim 8; and subjecting said modified cell to conditions whereby a plant is regenerated therefrom.

27. A plant produced by the method of claim 26.

28. A plant descended from the plant of claim 27. [1:\DayLib\LIBFF]83079spec.doc:gcc L 1

29. The method of claim 20, wherein said plant or plant tissue comprises one or more cells transformed with a polynucleotide molecule encoding at least one trans-acting factor, wherein said polynucleotide molecule is expressed. The method of claim 20, wherein said modified viral RNA molecule is capable of replication only in said one or more cells transformed with a polynucleotide molecule encoding at least one trans-acting factor.

31. A DNA-launching platform, substantially as hereinbefore described with reference to any one of the examples.

32. A method of genotypically or phenotypically modifying one or more plant cells, substantially as hereinbefore described with reference to any one of the examples.

33. A method of producing a plant or plant tissue comprising at least one genotypically or phenotypically modified plant cell, substantially as hereinbefore described with reference to any one of the examples.

34. A method of producing a genotypically or phenotypically modified plant comprising S* 15 obtaining at least one modified cell produced by the method of claim 32. A plant produced by the method of claim 34.

36. A plant descended from the plant of claim

37. Seed of the plant of claim 27, 28, 35 or 36. Dated 9 May, 2003 Wisconsin Alumni Research Foundation Patent Attorneys for the Applicant/Nominated Person S: SPRUSON FERGUSON [I:\DayLib\LIBFF]83079spec.doc:gcc