AU706185B2 - Antiviral transgenic plants, vectors, cells and methods - Google Patents

Description

WO 95/22245 PCT/US95/02058 -1- ANTIVIRAL TRANSGENIC PLANTS. VECTORS.

CELLS AND METHODS Related Application..

This application for U.S. ent is a continuation-in-part of patent application, which was as iged Serial No. 08/028,086 and filed on &ch W, 19-3.

Field of the Invention The present invention relates to isolated RNases having the ability to bind and/or cleave single stranded RNA when bound to encoding sequences therefor, recombinant nucleotide molecules, recombinant vectors, recombinant cells, and antiviral transgenic plants which express, for example, antiviral animal amino acid sequences which have activity similar or identical to RNase, 2-5A synthetase and/or PKR.

Background Control of RNA degradation is a critical cell function, and gene expression is often regulated 7- 0

J

WO 95/22245 PCT/US95/02058 -2at the level of RNA stability. See, Shaw, G.

and Kamen, Cell, 46:659-667 (1986). Nevertheless, relatively little is known about the biochemical pathways that mediate RNA degradation in mammalian or plant systems. For instance, most if not all of the ribonucleases responsible for mRNA turnover in mammalian or plant cells remain unidentified. This was reviewed in Brawerman, G., Cell, 57:9-10 (1989).

Presently, the 2-5A system is believed to be the only well-characterized RNA degradation pathway from higher animals including man. See FIG.

1. See also, Kerr, I.M. and Brown, Prod.

Natl. Acad. Sci. 75:256-260 (1978) and Cayley, P.J. et al., Biophvs Res. Commun., 108:1243-1250 (1982); reviewed in Sen, G.C. and Lengyel, J. Biol. Chem., 267:5017-5020 (1992).

The activity of the 2-5A system is believed to be mediated by an endoribonuclease known as dependent RNase. See Clemens, M.J. and Williams, Cell, 13:565-572 (1978). RNase is a unique enzyme in that it requires unusual oligoadenylates with phosphodiester linkages, Pn(A 2 'p)nA, for ribonuclease activity. See Kerr, I.M. and Brown, Prod. Natl. Acad. Sci.

75:256-260 (1978). 2-5A is produced from ATP by a family of synthetases in reactions requiring WO 95/22245 PCT/US95/02058 -3double-stranded RNA (dsRNA). See FIG. 1. See also Hovanessian, A.G. et al., Nature, 268:537-539 (1977); Marie, I. and Hovanessian, J. Biol. Chem., 267:9933-9939 (1992). 2-5A is unstable in cells and in cell-free systems due to the combined action of and 5'-phosphatase. See Williams, B.R.G. et al.; Eur. J. Biochem., 92:455-562 (1978); and Johnson, M.I. and Hearl, J. Biol.

Chem., 262:8377-8382 (1987). The interaction of 2-SA-dependent RNase and 2-5A(Kd 4 X 10 11

M),

Silverman, R.H. et al., Biol. Chem., 263:7336-7341 (1988), is highly specific. See Knight, M. et al., Nature, 288:189-192 (1980). 2-5A-dependent RNase is believed to have no detectable RNase activity until .it- is converted to its active state by binding to See Silverman, Anal. Biochem., 144:450-460 (1985). Activated 2-5A-dependent RNase cleaves single-stranded regions of RNA 3' of UpNp, with preference for UU and UA sequences. See Wreschner, D.H. et al., Nature, 289:414-417 (1981a); and Floyd-Smith, G. et al., Science, 212:1020-1032 (1981). Analysis of inactive 2-5A-dependent RNase from mouse liver revealed it to be a single polypeptide of approximately 80 kDa. See Silverman, R.H. et al., Biol. Chem., 263:7336-7341 (1988).

Although the full scope and biological significance of the 2-5A system remains unknown, WO 95/22245 PCT/US95/02058 -4studies on the molecular mechanisms of interferon action have provided at least some of the functions.

Interferons a, 0 or Y are believed to induce the accumulation of both 2-5A-dependent RNase, Jacobsen, H. et al., VirologY, 125:496-501 (1983A) and Floyd-Smith, J. Cellular Biochem., 38:12-21 (1988), and 2-5A synthetases, Hovanessian, A.G. et al., Nature, 268:537-539 (1977), reviewed in Sen, G.C. and Lengyel, J. Biol. Chem., 267:5017-5020 (1992). Furthermore, several investigations have implicated the 2-5A system in the mechanism by which interferon inhibits the replication of picornaviruses. Indeed, 2-5A per se and highly specific 2-5A mediated rRNA cleavage products were .induced in interferon-treated, encephalomyocarditis virus (EMCV)-infected cells. See Williams, B.R.G., Nature, 282:582-586 (1979); Wreschner, D.H. et al., Nucleic Acids Res., 9:1571-1581 (1981b); and Silverman, R.H. et al., Eur. J. Biochem., 124:131-138 (1982a). In addition, expression of 2-5A synthetase cDNA inhibited the replication of picornaviruses, Chebath, Nature, 330:587-588 (1987) and Rysiecki, E.F. et al., J. Interferon Res., 9:649-657 (1989), and the introduction of a 2-5A analogue inhibitor of RNase into cells reduced the interferon-mediated inhibition of EMCV replication.

See Watling, D. et al., EMBO 4:431-436 (1985).

WO 95/22245 PCT/US95/02058 Further, 2-5A-dependent RNase levels were correlated with the anti-EMCV activity of interferon, Kumar, R.

et al., J. Virol., 62:3175-3181 (1988), and EMCV-derived dsRNA both bound to and activated synthetase in interferon-treated, infected cells.

See Gribaudo, G. et al., J. Virol., 65:1948-1757 (1991).

The 2-5A system, however, almost certainly provides functions beyond the antipicornavirus activity of interferons. For instance, introduction of 2-5A into cells, Hovanessian, A.G. and Wood, J.N., Virology, 101:81-90 (1980), or expression of synthetase cDNA, Rysiecki, G. et al., J. Interferon Res., 9:649-657 (1989), inhibits cell growth rates.

4orbover, 2-5A-dependent RNase levels are elevated in growth arrested cells, Jacobsen, H. et al., Proc.

Natl. Acad. Sci. 80:4954-4958 (1983b), and synthetase, Stark, G. et al., Nature, 278:471-473 (1979), and 2-5A-dependent RNase levels are induced during cell differentiation. See, e.g., Krause, D. et al., Eur. J. Biochem., 146:611-618 (1985). Therefore, interesting correlations exist between 2-5A-dependent RNase and the fundamental control of cell growth and differentiation suggesting that the 2-5A system may function in general RNA metabolism. The ubiquitous presence of the system in reptiles, avians and mammalians certainly WO 95/22245 PCT/US95/02058 -6supports a wider role for the pathway. See, for example, Cayley, P.J. et al., Biochem. Biophy. Res.

Commun., 108:1243-1250 (1982).

While it is presently believed that the system is the only well-characterized RNA degradation pathway from higher animals, the dsRNA-dependent protein kinase enzyme, known as PKR, is also thought to have antiviral effects in higher animals. Like the 2-5A synthetase enzyme, it is believed that PKR is stimulated by dsRNA. It is believed that activated PKR phosphorylates the alpha subunit of translation factor elF 2 known as eIF 2 -alpha, which indirectly inhibits protein synthesis initiation. It is believed that interferons a, 13, and y induce the accumulation of PKR. See Hoavanessian et al.: J. Interferon Res., 9:641-647 (1989).

Like the 2-5A system, the PKR system is also likely to provide functions beyond the antipicornavirus activity of interferons. See Meurs, E.F. et al.: J. Virology, 66:5805-5814 (1992). For example, expression of mutant forms of PKR in NIH 3T3 cells resulted in tumor formation when injected into nude mice. See Meurs, E.F. et al.: Proc. Natl.

Acad. Sci 90:232-236 (1993).

In short, the 2-5A system and the PKR system inhibit viral protein synthesis. This is WO 95/22245 PCT/US95/02058 -7believed to be accomplished by the 2-5A system by degrading mRNA and rRNA whereas the PKR system is believed to accomplish this by indirectly inhibiting protein synthesis initiation.

Viral plant diseases are pandemic and their severity varies from mild symptoms to plant death.

The majority of plant viruses are believed to have single stranded RNA genomes. Moreover, it is currently believed that plants are void of the three enzymes discussed above, PKR, 2-5A synthetase and 2-5A-dependent RNase. See Cayley, P.J. et al.: Biochem. Biophvs Res. Commun., 108:1243-1250 (1982) and Devash, Y. et al.: Biochemistry, 24:593-599 (1985); but see Crum, C. et al.: J. Biol. Chem., 263513440-13443 (1988); Hiddinga, H.J. et al.: Science, 241:451-453 (1988); Sela, TIBS, pp.

31-33 (Feb 1981); and Devash, Y. et al.: Science, 216:1415-1416.

Notwithstanding the importance of dependent RNase to the 2-5A system, RNase enzymes having ribonuclease function have not been isolated, purified or sequenced heretofore.

Consequently, there is a demand for isolated, active RNases and their complete amino acid sequences, as well as a demand for encoding sequences for active 2-5A-dependent RNases. There is also a demand for plants which are resistant to viruses such as the picornaviruses.

Summary of the Invention In brief, the present invention alleviates and overcomes certain of the abovementioned problems and shortcomings of the present state of the art through the discovery of novel, isolated 2-5A-dependent RNases and encoding sequences therefor.

A first aspect of the present invention provides a transgenic plant all of whose cells contain at least one nucleotide sequence introduced into said transgenic plant, or ancestor of said transgenic plant, said introduced nucleotide sequence encoding an amino acid sequence having activity of an enzyme selected from the 2-5A-dependent RNase(s).

In a preferred embodiment of the first aspect of the invention, the nucleotide sequence introduced into the plant includes one or more sequences selected from the group consisting of the nucleotide sequences containing the complete or partial coding sequences for 2-5A-dependent RNases, 2-5A-synthetase and PKR.

A second aspect of the present invention provides a transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A-dependent RNase.

A third aspect of the present invention provides a transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an S.amino acid sequence having activity similar or identical to A fourth aspect of the present invention provides a transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an 25 amino acid sequence having activity similar or identical to PKR.

A fifth aspect of the present invention provides a plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to 2-5A-dependent RNase.

A sixth aspect of the present invention provides a plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to PKR.

A seventh aspect of the present invention provides a plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to 2-5A synthetase.

An eighth aspect of the present invention provides a method for producing genetically transformed plants which are resistant or immune to infection by a virus, said [N:\Liba]00099:SD method comprises the steps of: a) inserting into the genome of a plant cell of a plant susceptible to a virus a construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2-5A-dependent RNase; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the construct.

A ninth aspect of the present invention provides a method for producing genetically transformed plants which are resistant or immune to infection by a virus, said method lo comprises the steps of: a) inserting into the genome of a plant cell of a plant susceptible to a virus a construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to PKR; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the construct.

A tenth aspect of the present invention provides a method for producing genetically transformed plants which are resistant or immune to infection by a virus, said method comprises the steps of: i 20 a) inserting into the genome of a plant cell of a plant susceptible to a virus a construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2-5A synthetase; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant 25 which expresses the amino acid sequence encoded by the construct.

An eleventh aspect of the present invention provides a method for producing genetically transformed plants, which are resistant or immune to more than one viral infection, said method comprises the steps of: inserting into the genome of a plant cell of a plant susceptible to a virus a nucleotide sequence which encodes for an amino acid sequence having activity of an enzyme selected from the 2-SA-dependent RNase(s); b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the nucleotide sequence.

A twelfth aspect of the present invention provides a transgenic plant all of whose cells contain a nucleotide sequence introduced into said transgenic plant, or an ancestor of said transgenic plant, said introduced nucleotide sequence encoding an antisense 2-SA-dependent RNase amino acid sequence.

A thirteenth aspect of the present invention provides an isolated nucleotide sequence T [N:\Liba]00099:SSD which is identified as SEQ ID NO:3.

A fourteenth aspect of the present invention provides an amino acid sequence which is identified as SEQ ID NO:4.

A fifteenth aspect of the present invention provides an isolated 2 RNase, or an active fragment or analog thereof, said 2-5A-dependent RNase having a binding domain and the ability to cleave single stranded RNA when said 2 -5A-dependent RNase is bound to A sixteenth aspect of the present invention provides an isolated 2 RNase, or an active fragment or analog thereof, said 2-5A-dependent RNase having a 2-5A binding domain.

A seventeenth aspect of the present invention provides an isolated nucleotide sequence encoding 2 -5A-dependent RNase, or an active fragment or analog thereof, the 2 -5A-dependent RNase having a 2-5A binding domain and the ability to cleave single stranded RNA when the 2 -5A-dependent RNase is bound to An eighteenth aspect of the present invention provides an isolated nucleotide sequence encoding 2-5A-dependent RNase, or an active fragment or analog thereof, the S 2-5A-dependent RNase having a 2-5A binding domain.

A nineteenth aspect of the present invention provides a clone having a nucleotide sequence capable of expressing a murine 2-5A-dependent RNase having a 2-5A binding 20 domain, said clone being selected from a group consisting of ZB1, ZB2, ZB3, ZB5, ZB9, ZB11 and ZB13.

Broadly speaking, the novel 2-5A dependent RNases of the instant invention are involved in the fundamental control of single stranded RNA decay in animal cells, such as mammals, and are also present in animal cells, such as avian and reptilian cells. More 25 particularly, the novel 2-5A dependent RNases of the present invention have the ability to degrade single stranded RNA, mainly 3' of UpUp or UpAp sequences, after they are activated by binding to 5'-phosphorylated,2',5'-linked oligoadenylates (hereinafter As a result, it is believed that the novel 2-5A dependent RNases are useful in connection with inhibition of cell growth rates, viral replication and in connection with interferon treatment of viral infection and cancer. As used herein, the term dependent RNase(s)" is used in a broad sense and is meant to include any amino acid sequence which includes a 2-5A binding domain and/or [N:\Liba]00099:SSC WO 95/22245 PCT/US95/02058 -9ribonuclease function when the 2-5A-dependent RNase is activated by The novel 2-5A dependent RNases of the present invention are protein enzymes having molecular weights on the order of between about 74 KDa (murine) and about 84 KDa (human), as determined by gel electrophoresis migration and/or prediction from their respective encoding nucleotide sequences.

For example, a human 2-5A-dependent RNase of the instant invention has a molecular weight of about 83,539 Da as determined from the amino acid sequence predicted from the encoding sequence therefor, whereas the murine 2-5A-dependent RNase has a molecular weight of about 74 KDa as determined by gel electrophoresis migration and from prediction of the amino acid sequence from the encoding sequence.

While an about 74 KDa molecular weight is reported herein for a murine 2-5A-dependent RNase, it should nevertheless be appreciated that the reported molecular weight is for an incomplete murine RNase. It is nevertheless believed that once completely sequenced, when an about 84 amino acid end region is identified, the molecular weight of a complete murine 2-5A-dependent RNase will be similar to that of human, about 84 KDa.

It should also be readily apparent to those versed in this art, however, that since gel electro- WO 95/22245 PCTIUS95/02058 phoresis migration has been employed to determine molecular weight of a murine 2-5A-dependent RNase, the 74 KDa molecular weight is only an estimate based upon relative migration.

The amino acid sequence for human RNase protein is depicted in FIG. 3 and Table 1. The encoding sequence for the human RNase protein is also set forth in Table 1. The mRNA for human 2-5A-dependent RNase is about 5.0 Kb in size. The virtually complete amino acid sequence for the murine 2-5A-dependent RNase protein and the encoding sequence therefore is depicted in Table 2. The mRNA for murine RNase is about 5.7 Kb in size.

Analysis of the amino acid sequences of the RNases of the present invention have revealed several characteristics unique to the RNases. For example, it has been discovered that the novel 2-5A dependent RNases of the instant invention include the following unique domains which span between the amino terminus and the carboxy terminus. For instance, it has been discovered that there are at least four and possibly as many as nine or more ankyrin repeats, of which three lie closest to the amino terminus. However, while four ankyrin repeats have been discovered, it is believed that there may be additional ankyrin

I

WO 95/22245 PCTUS95/02058 -11repeats that may total, for instance, about eight or more when the amino acid sequences of the RNases of the present invention are further analyzed. It is believed that these ankyrin repeats may possibly function in protein-protein interaction. Ankyrin repeat 1 generally lies between amino acids designated as 58-90 in Tables 1 and 2 Ankyrin repeat 2 generally lies between amino acids designated as 91-123 in Tables 1 and 2. Ankyrin repeat 3 generally lies between amino acids designated as 124-156 in Tables 1 and 2 Ankyrin repeat 4 generally lies between amino acids designated as 238 and 270 in Tables 1 and 2 See also FIGS. 10A and It has also been discovered that the novel dependent RNases include a cysteine rich region (which has homology to zinc fingers) that lies closer to the carboxy terminus than the amino terminus which may possibly function in RNA recognition or in formation of protein dimers. The cysteine rich region is believed to include about 5 or 6 cysteine residues which generally lie between amino acids designated as 395-444 in the human sequence as reported in Table 1 and FIG. 4, or between amino acids designated as 401-436 in the murine sequence as reported in Table 2 and FIG. 4.

WO 95/22245 PCT/US95/02058 -12- Still further, it has been discovered that the novel 2-5A dependent RNases include a duplicated phosphate binding (2 P-loops) motif which lies generally within the ankyrin repeat motifs. It is believed that the two P-loops are in the same orientation and constitute the binding domain necessary for binding 2-5A. It is further believed that each P-loop motif includes a lysine residue which is essential for maximum 2-5A binding activity. The lysine residues are designated as 240 and 274 in Tables 1 and 2.

It has been further discovered that the RNase proteins contain an amino acid region which follows the cysteine rich region that is believed to be homologous to protein kinases. Within this region, there is believed to be separate domains designated as domains VI and VII which generally lie between amino acid residues designated as 470-504 in Tables 1 and 2 More particularly, as to the human sequence of 2-5A-dependent RNase, domain VI generally lies between amino acid residues designated as 471-491 and domain VII generally lies between amino acid residures designated as 501-504, as reported in Table 1 and FIG. 4. As to the murine sequence of the RNase, domain VI generally lies between amino acids designated as 470-489 and domain WO 95/22245 PCT/US95/02058 -13- VII generally lies between amino acid residues designated as 499-502, as reported in Table 2 and FIG. 4.

It has also been discovered that there is limited homology between the amino acid sequences for the 2-5A-dependent RNases of the present invention and RNase E, encoded by the altered mRNA stability (ams)/rne gene of E. Coli. Uniquely, the limited homology is generally conserved between the murine and human amino acid sequences for 2-SA-dependent RNases and generally lies between a 200 amino acid region. More particularly, for the human sequence, the amino acid region spans amino acid residues designated as 160-349 in Table 1 and FIGS. 9A and 9B. With respect to the murine sequence, the amino acid region spans amino acid residues designated as 160-348 in Table 2 and FIGS. 9A and 9B.

It has been further discovered and is believed that almost the entire, if not complete, amino acid sequences of the novel RNase proteins of the instant invention are necessary for ribonuclease function. For example, it is believed that, when an about 84 amino acid region at the carboxy terminus is present in the human RNase, the human 2-5A-dependent RNase has ribonuclease function in the presence of In contrast, when the murine 2-5A-dependent RNase F_ WO 95/22245 PCT/US95/02058 -14lacks the about 84 amino acid region at the carboxy terminus, it lacks ribonuclease function.

With respect to the binding activity of a murine 2-5A-dependent RNase protein to 2-5A, it has been discovered that, when one P-loop is deleted from the repeated P-loop motif of a murine RNase protein, nearly all 2-5A binding activity is lost, and that when both P-loops are deleted, virtually complete activity is lost. However, it has been found that, even though the carboxy terminus portion of the amino acid sequence of a murine RNase protein following the repeated P-loop motif has been deleted, partial 2-5A binding activity is maintained.

It has been further discovered that when lysine residues 240 and 274 are replaced with asparagine residues in both P-loop motifs, significant 2-5A binding activity of a murine RNase protein is lost. It has been further discovered, however, that when either lysine residue 240 or 274 is replaced in either P-loop motif, only partial 2-5A binding activity is lost.

It is therefore believed that the presence of both P-loop motifs in the amino acid sequences for the dependent RNases of the present invention plays an important role in 2-5A binding activity. It is further believed that the presence of lysine residues WO 95/22245 PCT/US95/02058 240 and 274 in each P-loop motif plays an important role for enhanced 2-5A binding activity. It is also believed that the presence of virtually the entire amino acid sequence of the 2-5A-dependent RNases of the present invention provides for even further enhanced 2-5A binding activity, as well as provides for ribonuclease function.

In addition, the present invention relates to the cloning of murine and human RNases and novel murine and human clones.

Recombinant and naturally occurring forms of RNase displayed virtually identical binding properties and ribonuclease specificities.

The present invention further contemplates the use of the novel isolated, 2-5A-dependent RNases and encoding sequences therefor, as well as analogs and active fragments thereof, for use, for instance, in gene therapy for human and animal diseases including viral disease and cancer, as genetic markers for human disease due to perhaps cancer or viral infection, to develop plants and animals resistant to certain viruses, and as enzymes in connection with research and development, such as for studying the structure of RNA. In one manner to accomplish the above, and as contemplated by the present invention, the encoding sequences of the

I

WO 95/22245 PCT/US95/02058 -16instant invention may be utilized in ex vivo therapy, to develop recombinant cells using the encoding sequence of the present invention using techniques known to those versed in this art. In another manner which may be employed to accomplish the above, the encoding sequences of the present invention may be combined with an appropriate promoter to form a recombinant molecule and inserted into a suitable vector for introduction into an animal, plant, or other lower life forms also using techniques known to those skilled in this art. Of course, other suitable methods or means known to those versed in this art may be selected to accomplish the above-stated objectives or other objectives for which the novel RNases and encoding sequences of the present invention are suited.

The present invention also contemplates novel transgenic plants, as indicated above, which are resistant to viruses such as the picornaviruses.

Generally speaking, the transgenic plants of the present invention include any inserted nucleotide sequence encoding any type of antiviral amino acid sequence, including proteins. Preferably, the antiviral nucleotide sequences introduced into plants in accordance with the present invention are animal antiviral genes, such as those genes which are stimulated in response to interferon production WO 95/22245 PCTIUS95/02058 -17and/or treatment. These include, for example, those animal antiviral genes that encode RNase, and PKR. These interferon-regulated proteins, RNase and PKR (the dsRNA-dependent protein kinase) have recognized antiviral effects in higher animals and are believed to have antiviral effects in the transgenic plants of the present invention. PKR is stimulated by dsRNA to phosphorylate translation factor eIF2 which indirectly inhibits protein synthesis intiation. On the other hand, 2-5A synthetase is activated by dsRNA resulting in the production of PxA( 2 wherein X about 1 to about 3 and Y Z about 2, from .ATP. The 2-5A then activates an endoribonuclease entitled 2-5A dependent RNase (also known as RNase L or nuclease The activated ribonuclease degrades mRNA and rRNA thus inhibiting protein synthesis.

These above-described pathways are particularly effective at inhibiting viruses in animals with single stranded RNA genomes that replicate through dsRNA intermediates, such as the picornaviruses, and are believed to be effective at inhibiting similar types of viruses that infect plants. This belief is premised upon the understanding that most single stranded RNA plant viruses produce double stranded structures during WO 95/22245 PCT/US95/02058 -18replication by their viral replicases, see Dawson, W.O. et al.: Acad. Press, 38:307-342 (1990), and that plant viruses are similar to animal viruses in structure, composition and mechanism of replication in cells. In addition, even viral so-called single-stranded RNA may contain secondary structures which could activate PKR and 2-5A synthetase leading to widespread plant protection against plant viruses. It is believed that co-expression of RNase and 2-5A-synthetase, will lead to the destruction of viral mRNA and viral genomic RNA thereby protecting the transgenic plants of the present invention from viruses. Moreover, it is believed that expression of PKR by the transgenic plants of the present invention will inhibit viral protein synthesis leading to inhibition of virus replication and protection of the transgenic plants.

The present invention is therefore premised in part upon the belief that plant virus RNAs activate and PKR in the transgenic plants of the instant invention leading to immunity against virus infection. Furthermore, expression of synthetase alone or 2-5A-dependent RNase alone or PKR alone may protect plants against viruses, perhaps by binding to viral RNA, such as viral replicative intermediates thereby blocking viral replication.

Moreover, expression of only the dsRNA binding WO 95122245 PCT/US95/02058 -19domains of PKR and/or of 2-5A-synthetase may similarly protect the transgenic plants of the present invention against viral infection.

It should therefore be appreciated by those versed in this art that novel transgenic plants which are resistant to viral infection can now be produced in accordance with the present invention. It is believed that the effectiveness of the anti-viral protection can be enhanced or even maximized when at least the three-above animal antiviral genes are inserted into plants to form exemplary transgenic plants of the present invention, since the animal antiviral proteins encoded by these three animal antiviral genes interfere with different stages of .the viral life cycles. Moreover, these animal antiviral proteins or amino acid sequences are believed likely to be safe to give or introduce into animals, including humans, since these antiviral proteins or amino acid sequences are naturally occurring in humans as well as in other mammals, avians and reptiles.

While the present invention is described herein with reference to the particular sequences disclosed, it should nevertheless be understood by those skilled in this art that the present invention contemplates variations to the amino acid and/or nucleotide sequences which do not destroy WO 95/22245 PCT/US95/02058 synthetase activity, PKR activity and/or ribonuclease activity. Therefore, the present invention contemplates any analogs, parts or fragments of 2-5A-dependent RNase, 2-5A synthetase, and PKR which are active, such as any active part, and any encoding sequences therefor. In other words, the present invention includes, among other things, any amino acid sequence, any nucleotide sequence and any transgenic plant which have the ability to accomplish the objectives of the instant invention.

For example, the instant invention includes any amino acid sequence which has antiviral activity and any nucleotide sequence which encodes therefor and those transgenic plants that express such nucleotide sequences. More specifically, the present invention includes, for instance: any animal amino acid sequence which has the ability to inhibit or interfere with viral replication such as those amino acid sequences that have activity similar or identical to PKR activity, 2-5A synthetase activity and/or 2-5A ribonuclease activity, and any nucleotide sequence which encodes for an amino acid sequence having any such activity; and any transgenic plant having any animal antiviral nucleotide sequence which encodes any such amino acid sequence which has any such antiviral activity.

WO 95/22245 PCT/US95/02058 -21- The above features and advantages of the present invention will be better understood with reference to the accompanying FIGS., Detailed Description and Examples. It should also be understood that the particular methods, amino acid sequences, encoding sequences, constructs, vectors, recombinant cells, and antiviral transgenic plants illustrating the invention are exemplary only and not to be regarded as limitations of the invention.

Brief Description of the FIGS.

Reference is now made to the accompanying FIGS. in which is shown illustrative embodiments of the present invention from which its novel features and advantages will be apparent.

FIG. 1 is the 2-5A system: a ribonuclease pathway which is believed to function in the molecular mechanism of interferon action.

p'tase; 2'-PDE.

FIGS. 2A and 2B is a comparison of binding activity of recombinant and naturally occurring forms of murine 2-5A-dependent RNase.

FIG. 2A is a specific affinity of truncated murine 2-5A-dependent RNase for 2-5A. UV covalent crosslinking of the 32 P-2-5A probe (lanes 1-7) to protein is performed after translation reactions in wheat germ extract (5 pl) with murine WO 95/22245 PCT/US95/02058 -22- RNase mRNA (from clone ZB1) (lanes 1-3) or without added RNA (lane 4) or in extract of interferon treated mouse L cells (100 jig of protein) (lanes Reactions are without added competitor (lanes 1, 4, and 5) or in the presence of either trimer core. (A2'p) 2 A, (100 nM) (lanes 2 and 6) or trimer p 3 (A2'p) 2 A (100 nM) (lanes 3 and Lanes 8 and 9 are produced by incubating the wheat germ extract with 35 S-methionine in the absence or presence of 2-5A-dependent RNase mRNA, respectively.

FIG. 2B are identical chymotrypsin cleavage products and are obtained from recombinant and naturally occurring form of 2-5A-dependent RNase.

Partial chymotrypsin digests (arrows) are performed .orr- truncated 2-5A-dependent RNase (clone ZB1) produced in wheat germ extract ("Recombinant") and murine L cell 2-5A-dependent RNase ("Naturally Occurring") after crosslinking to the 2-5A probe and purification from gels.

FIGS. 3A and 3B are clonings of the complete coding sequence for human RNase.

FIG. 3A is the construction of a human RNase clone. The initial human RNase cDNA clone, HZB1, is isolated from an adult human kidney cDNA library in Xgtl0 using radiolabeled murine 2-5A-dependent RNase cDNA WO 95/22245 PCTIUS95/02058 -23- (clone ZB1) as probe. See Example. Radiolabeled HZB1 DNA is used to isolate a partially overlapping cDNA clone, HZB22, which is fused to HZB1 DNA at the NcoI site to form clone ZC1. The 5'-region of the coding sequence is obtained from a genomic SacI fragment isolated using a radiolabeled HZB22 DNA fragment as probe. Fusion of the genomic SACI fragment with ZC1 at the indicated SacI site produces clone ZC3. The coding sequence with some flanking sequences is then subcloned as a HindIII fragment into pBluescript (Stratagene) resulting in clone ZC5. The restriction map for the composite clone, ZC5, is shown. Clone HZB1 includes nucleotides designated as 658-2223 in Table I which enc6de for amino acids designated as 220-741 in Table I. Clone HZB22 includes a nucleotide sequence which encodes for amino acids designated as 62-397 in Table I. Clone ZC1 includes a nucleotide sequence which encodes for amino acids designated as 62-741 in Table I. Clones ZC3 and ZC5 both include nucleotide sequences which encode for amino acids designated as 1-741 in Table I.

FIG. 3B is a nucleotide sequence and predicted amino acid sequence of human RNase with flanking nucleotide sequences. The numbers to the right indicate the positions of nucleotides and amino acid residues.

WO 95/22245 PCTfUS9/02058 -24- FIG. 4 is alignment of the predicted amino acid sequences for murine and human forms of RNase. The positions of the repeated P-loop motifs, the cysteine (Cys)-rich regions with homology to zinc fingers, and the regions of homology to protein kinase domains VI and VII are indicated.

Amino acids residues which are important components of the indicated domains are represented in bold type and are italicized. Identical amino acid residues in murine and human 2-5A-dependent RNase are indicated with colon symbols adjacent therebetween.

FIGS. 5A and 5B are 2-5A binding properties and ribonuclease activity of recombinant human dependent RNase produced in vitro.

FIG. 5A is specific affinity of recombinant human 2-5A-dependent RNase for 2-5A. Crosslinking of the 2-5A probe (lanes 1-7) to protein is performed after translation reactions in wheat germ extract il) with human 2-5A-dependent RNase mRNA (lanes 1-3) or without added RNA (lane 4) or in extract of human interferon a treated (1000 units per ml for 16 h) human HeLa cells (350 pg of protein) (lanes 5-7).

Reactions were without added competitor (lanes 1, 4, and 5) or in the presence of either trimer core, (A2'p) 2 A, (100 nM) (lanes 2 and 6) or trimer p 3 (A2'p) 2 A (100 nM) (lanes 3 and Incubations with 35 S-methionine are shown in lanes 8 to 12. Lane WO 95/22245 PCT/US95/02058 8 is with wheat germ extract and human RNase mRNA. Reticulocyte lysate preadsorbed to is incubated with human 2-SA-dependent RNase mRNA in the absence (lane 9) or presence (lane of cycloheximide, or in the absence of added mRNA (lane 11). Lane 12 shows human 2-5A-dependent RNase which is produced in the nonadsorbed, crude reticulocyte lysate. The positions and relative molecular masses (in kDa) of the marker proteins are indicated.

FIG. 5B is reticulocyte lysate pretreated to remove endogeous 2-5A-dependent RNase and is incubated in the absence of added mRNA in the presence of human 2-5A-dependent RNase mRNA without inhibitor (0 E or in the presence of both RNase mRNA and cycloheximide (50 pg per ml See Example I. Subsequently, the recombinaht 2-5A-dependent RNase (or controls) is adsorbed to 2-5A-cellulose and ribonuclease assays are performed after extensive washing of the matrix to reduce general nuclease activity. Radiolabeled substrate RNA was either poly(U) or poly(C) FIGS. 6A, 6B and 6C show levels of RNase mRNA which are induced by interferon treatment of murine L929 cells even in the presence of cycloheximide.

WO 95/22245 PCT/US95/02058 -26- FIG. 6A is a northern blot prepared with poly(A)+RNA (4 pg per lane) that is isolated from murine L929 cells treated with murine interferon (a 3) (1000 units per ml) and/or cycloheximide (50 pg per ml) for different durations (indicated) which is probed with radiolabeled murine 2-5A-dependent RNase cDNA. Interferon, IFN; cycloheximide, CHI.

FIG. 6B shows levels of RNase which are estimated from the autoradiogram shown in panel with a video camera and QuickCapture and Image computer programs.

FIG. 6C shows levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as determined in the same blot shown in panel FIGS. 7A and 7B are the truncated, recombinant murine 2-5A-dependent RNase, clone ZB1, and murine L cell 2-5A-dependent RNase having identical 2-5A binding activities localized to a repeated P-loop motif.

FIG. 7A shows incubations of truncated RNase, clone ZB1, ("Recombinant") which is produced in wheat germ extract (upper panel) or of murine L cell 2-5A-dependent RNase (labeled "Naturally Occurring," lower panel) with the 32 probe, (2.4 nM), are in the absence of presence of unlabeled 2',5'-phosphodiester linked oligonucleotides (as indicated) followed by uv covalent WO 95/22245 PCTIUS95/02058 -27crosslinking. Autoradiograms of the dried polyacrylamide gels are shown. Concentrations of the oligonucleotide competitors are indicated. I is inosine.

FIG. 7B shows a truncated series of murine RNase mutants (ZB1 to ZB15) which is produced in wheat germ extract which are assayed for binding activity by a filter binding method.

See Example and Knight et al. 1980). The positions of the P-loop motifs and the lengths of the translation products are indicated. Clone ZB1 encodes for amino acids designated as 1-656 in Table 2 except for the last 5 amino acid residues which are Lys, Pro, Leu, Ser, and Gly. Clone ZB2 encodes .for- amino acids designated as 1-619 in Table 2 Clone ZB3 encodes for amino acids designated as 1-515 in Table 2 Clone ZB5 encodes for amino acids designated as 1-474 in Table 2 Clone ZB9 encodes for amino acids designated as 1-403 in Table 2 Clone ZB10 encodes for amino acids designated as 1-365 in Table 2 Clone ZB13 encodes for amino acids designated as 1-294 in Table 2 Clone ZB14 encodes for amino acids designated as 1-265 in Table 2 Clone ZB15 encodes for amino acids designated as 1-218 in Table 2 WO 95/22245 PCT/US95/02058 -28- FIGS. 8A and 8B are substitution mutations of the lysine residues in the P-loop motifs of RNase.

FIG. 8A shows the truncated murine RNase, clone ZB1, and lysine to asparagine substitution mutants of clone ZB1, which are synthesized in wheat germ extract. In (A) unlabeled translation products are covalently crosslinked to the bromine-substituted, 32 P-labeled probe, Br-2-5A-[ 32 p]pCp. See Nolan-Sorden et al., 1990.

FIG. 8B shows the mRNA species which are translated in the presence of 35 -S-methionine in separate reactions. Autoradiograms of the dried, SDS7polyacrylamide gels are shown. The order and positions of the translation products (labelled "RNase") and the relative molecular masses (in kDa) of the protein markers are indicated.

FIGS. 9A and 9B are a comparison of the amino acid sequences of RNase E and RNase.

FIG. 9A shows identical and conservative matches which are shown between E. coli RNase E and the murine and human forms of 2DR.

FIG. 9B is a model for the structure and function of 2DR. Abbreviations: P-loop motifs, a repeated sequence with homology to P-loops; Cys x a WO 95/22245 PCT/US95/02058 -29cysteine-rich region with homology to certain zinc fingers; PK, homology to protein kinase domains VI and VII.

FIGS. 10A and 10B are a comparison of the amino acid sequences of the ankyrin repeats in the human and murine 2-5A-dependent RNase proteins.

FIG. 10A shows murine and human forms of RNases containing four ankyrin repeats. Homology between the ankyrin consensus sequence and the murine and human forms of RNase are indicated. 4, hydrophobic amino acids.

FIG. 10B is a model showing the relative positions of the four ankyrin repeats in dependent RNase in comparison to the position of the proposed 2-5A binding domain (the repeated P-loop motif); Cysx, the cysteine-rich region; PK, the protein kinase homology region, and the carboxy-terminal region required for RNase activity.

FIG. 11 shows the role of RNase in the anti-viral response of cells to interferon treatment. Interferon binds to specific cell surface receptors resulting in the generation of a signal which activates a set of genes in the cell nucleus. The genes for 2-5A synthetase are thus activated producing inactive, native synthetase. Interferon treatment of the cell also WO 95/22245 PCT/US95/02058 activates the 2-5A-dependent RNase gene (not shown in the FIGure). Subsequently, the interferon-treated cells is infected by a virus. The virus produces double stranded RNA (dsRNA) during its replicative cycle. The viral dsRNA then activates the synthetase resulting in the production of 2-5A. The then activates the 2-5A-dependent RNase to degrade the viral RNA thus destroying the virus itself.

FIG. 12 depicts a physical map of T: based binary vector pAM943 which is about 12 Kbp.

Abbreviations: BL, left border; BR, right border; Kanr, kanamycin resistance; AMT, promoter of adenyl methyl transferase gene from Chlorella virus; promoter for 35S RNA from Cauliflower mosaic virus; TER, RNA termination signal; Ovi V and Ori K origins of DNA replication.

FIG. 13 depicts physical maps of portions of certain recombinant plasmid constructs containing cDNAs encoding mammalian antiviral proteins and showing the important DNA elements in between right border and left border of T-DNAs that are transferred to plant genomes. FIG. 13A depicts a certain portion of plasmid pAM943:PK68; FIG. 13B depicts a certain portion of plasmid pAM943:muPK68; FIG. 13C depicts a certain portion of plasmid pAM943:Synthetase; FIG.

13D depicts a certain portion of plasmid WO 95/22245 PCT/US95/02058 -31pAM943:2-5A-dep. RNase (sense); FIG. 13D/a depicts a certain portion of plasmid pAM943:2-5A-dep. RNase and FIG. 13E depicts pAM822:2-5A dep. RNase (antisense).

Abbreviations: BL, left border; BR, right border; Kanr, kanamycin resistance; Hygror, hygromycin resistance; AMT, promoter of adenyl methyl transferase gene from Chlorella virus; 35S, promoter for 35S RNA from Cauliflower mosaic virus; PKR, cDNA to human PKR; muPKR, cDNA to a lysine (amino acid 296) to arginine mutant form of PKR; Synthetase, cDNA to a low molecular weight form of human 2-5Adep. RNase, cDNA to human RNase; TER, RNA termination signal.

FIG. 14 shows a physical map of Ti based binary vector pAM822 which is about 14.6 Kbp.

Abbreviations: BL, left border; BR, right border; Kanr, kanamycin resistance; Hygror, hygromycin resistance; Tetr, tetracycline resistance; AMT, promoter of adenyl methyl transferase gene from Chlorella virus; 35S, promoter for 35S RNA from Cauliflower mosaic virus; TER, RNA termination signal; Ovi V, origin of DNA replication.

FIG. 15 shows expression of human cDNA intransgenic tobacco plants as determined by measuring mRNA levels in a Northern blot. Construct C (pAM943:Synthetase) was introduced into the plants. Total RNA was prepared from the WO 95/22245 PCT/US95/02058 -32leaves of control (labeled and transgenic plants using RNASTAT-60 (Tel-Test Inc.). Thirty pg of RNA was treated with glyoxal and separated in a agarose gel. After electrophoresis RNA was transferred to Magnagraph (MSI) Nylon membrane and probed with human 2-5A-synthetase cDNA labeled with [a- 32 P]dCTP by random priming. Autoradiograms were made from the dried blots.

FIG. 16 shows expression of mutant and wild type forms of human PKR cDNA in transgenic tobacco plants as determined by measuring mRNA levels in a Northern blot. Constructs A (pAM943:PK68) and B (pAM943:muPK68) encoding wild type and mutant (lysine at position 296 to arginine) forms of PKR, .respectively, were introduced into the plants. Total RNA was prepared from the leaves of control (labeled and transgenic plants using RNASTAT-60 (Tel-Test Inc.). Thirty pg of RNA was treated with glyoxal and separated in a 1.5% agarose gel. After electrophoresis RNA was transferred to Magnagraph(MSI) Nylon membrane and probed with human PKR cDNA labeled with [a- 32 p]dCTP by random priming.

Autoradiograms were made from the dried blots.

FIG. 17 shows a presence of RNase cDNA in transgenic plants as determined on a Southern blot. Genomic DNA was isolated from leaves of transgenic plants containing construct D/a WO 95/22245 PCT/US95/02058 -33- (pAM943:2-5A-dep.RNase, antisense) using CTAB (cetyltrimethylammonium bromide) following the method of Rogers and Bendich (1988, Plant Molecular Biology Manual, A6, pp. 1-10, Kluwar Academic Pulbisher, Dordrecht). Ten ig of genomic DNA was digested with HindIII for 5 h at 37*C and fractionated in a 1% agarose gel followed by transfer to Magnagraph (nylon transfer membrane, Micron Separations, Inc.) using a capillary transfer method. The cDNA for RNase (from plasmid pZC5) was labeled by random priming with [a-32P]dCTP (3,000 Ci/mmole) using a Prime-a-gene kit from (Promega) according to the protocol supplied by the company. The labeled RNase cDNA (Specific activity of 1.0 X 109' c.p.m. per ig DNA) was washed and an autoradiogram was made from the dried membrane. The sizes (in kilobases) and the positions of the DNA markers are indicated. The band indicated as RNase cDNA" (see arrow) was absent in Southern blots of control plants (data not shown).

FIG. 18 depicts a coding sequence for human p68 kinase mRNA (PKR).

FIG. 19 depicts a translation product of the complete coding sequence for human p68 kinase mRNA (PKR) of FIG. 18.

FIG. 20 depicts a coding sequence for human synthetase cDNA.

WO 95/22245 PCTfS95/02058 -34- FIG. 21 depicts a translation product of the coding sequence for human 2-5A-synthetase of FIG.

Detailed Description By way of illustrating and providing a more complete appreciation of the present invention and many of the attendant advantages thereof, the following Detailed Description and Examples are given concerning the novel 2-5A-dependent RNases, encoding sequences therefor, recombinant nucleotide molecules, constructs, vectors, recombinant cells, antiviral transgenic plants and methods.

Because 2-5A-dependent RNase is very low in abundance (one five-hundred-thousandth of the total protein in mouse liver, Silverman, R.H. et al., J.

Biol. Chem., 263:7336-7341 (1988)), its cloning requires the development of a sensitive screening method. Murine L929 cells are selected as the source of mRNA due to high basal levels of RNase. A protocol to enhance 2-5A-dependent RNase mRNA levels is developed based on the observation that optimal induction of 2-5A-dependent RNase is obtained by treating cells with both interferon and cycloheximide, then with medium alone. See Example.

The cDNA library is screened by an adaptation of techniques developed for cloning DNA binding proteins, Singh, H. et al., Cell, 52:415-423 (1988); WO 95/22245 PCTIUS95/02058 Singh H. et al., BioTechnicues, 7:252-261 (1989), in which a bromine-substituted 32p-labeled 2-5A analogue probe"), Example and Nolan-Sorden, N.L. et al., Anal. Biochem., 184:298-304 (1990), replaced a radiolabeled oligodeoxyribonucleotide. A single clone (ZB1) is thus isolated from about three million plaques. The protein expressed from the ZB1 clone, transferred from plaques to filter-lifts, shows reactivity to both the 2-5A probe and to a highly purified polyclonal antibody directed against RNase.

To obtain recombinant protein for characterization, the cDNA is transcribed and translated in cell-free systems. See Example. binding activity is then determined by covalently crosslinking the 2-5A probe to the protein with uv light, for example, Nolan-Sorden, N.L. et al., Anal.

Biochem., 184:298-304 (1990). The recombinant 74 kDa protein produced in a wheat germ extract shows specific affinity for the 2-5A probe. See FIG. 2A, lanes 1 to 3. A core derivative of 2-5A lacking groups, (A2'p) 2 A, fails to interfere with binding of the protein to the 2-5A probe whereas trimer 205A, p 3 (A2'p) 2 A, completely prevents probe binding. See FIG. 2A, lanes 2 and 3, respectively.

There is no detectable 2-5A binding proteins in the wheat germ extract as shown in the incubation without WO 95/22245 PCTfUS95/02058 -36added RNA, FIG. 2A, lane 4. For comparison, a similar profile of 2-5A binding activity is obtained for the 80 kDa 2-SA-dependent RNase from murine L929 cells, incubated without added oligonucleotide or with (A2'p) 2 A or p 3 (A2'p) 2 A as competitors. See FIG.

2A, lanes 5 to 7. The 35 S-labeled translation product is shown in FIG. 2A, lane 9. In a further comparison, covalent linkage of the 2-5A probe to the about 74 kDa protein and to murine L929 cell RNase followed by partial digestion with chymotrypsin produces an identical pattern of six labeled peptides. See FIG. 2B. Similarly, partial digestion of the two labeled proteins with S.

aureus V8 protease also produces identical patterns of-labeled cleavage products. These results and the apparent molecular weight of about 74 kDa for the recombinant protein, as compared to about 80 kDa for RNase, see FIG. 2A, suggests that the about 74 kDa protein is a truncated, or partial clone for 2-5A-dependent RNase.

To obtain the entire coding sequence for human 2-5A-dependent RNase, a composite DNA containing genomic and cDNA is constructed. See FIG.

3A. The initial cDNA portion of the human RNase clone (HZB1) is obtained by screening a human kidney cDNA library with radiolabeled murine 2-5A-dependent RNase cDNA. See WO 95/22245 PCT/US95/02058 -37- Example. A genomic clone, containing the 5'-part of the coding sequence, is isolated with radiolabeled human 2-5A-dependent RNase cDNA. The nucleotide and predicted amino acid sequences of human RNase are determined, FIG. 3B, resulting an open reading frame encoding a protein of 83,539 Da.

A comparison is made between the predicted amino acid sequences of the human and murine forms of RNase in order to identify and evaluate the conserved regions of the proteins. See FIG. 4. The murine cDNA, clone ZB1, contains about 88% of the coding sequence for 2-5A-dependent RNase to which an additional twenty-eight 3'-codons are added from a murine genomic clone. Alignment of the murine and human forms of 2-5A-dependent RNase indicates about 65% identity between the overlapping regions. See FIG. 4. In addition, there is 73% identity between the corresponding nucleotide sequences for murine and human 2-5A-dependent RNase.

The apparent translation start codons for both the murine and human 2-5A-dependent RNases, are in an appropriate context for translational initiation, namely ACCATGG and GTCATGG, respectively. See FIG.

3B. See also, for example, Kozak, Cell, 44:283-292 (1986). In addition, both the human and murine 2-5A-dependent RNase sequences contain WO 95/22245 PCT/US95/02058 -38in-frame stop codons upstream of the translation start sites. See FIG. 3B.

The 2-5A binding properties of the recombinant and naturally occurring forms of human RNase are compared by uv covalent crosslinking to the 2-5A probe. The recombinant human 2-5A-dependent RNase produces in wheat germ extract shows specific affinity for 2-5A. See FIG.

lanes 1 to 3. Radiolabeling of the cloned human RNase with the 2-5A probe is not prevented by (A2'p) 2 A. See FIG. 5A, lanes 1 and 2.

In contrast, addition of trimer 2-5A, p 3 (A2'p) 2

A,

effectively competes with the 2-5A probe for binding to the recombinant 2-5A-dependent RNase. See lane The same pattern of 2-5A binding activity is obtained with 2-5A-dependent RNase in an extract of interferon-treated human HeLa cells. See FIG. lanes 5 to 7. The apparent molecular weights of HeLa cell 2-5A-dependent RNase and 35 S-labeled recombinant human 2-5A-dependent RNase produced in reticulocyte lysate are believed to be exactly the same (about kDa). See FIG. 5A, lanes 5 and 9. The recombinant human 2-5A-dependent RNase produced in wheat germ extract migrates slightly faster probably due to post-translational modifications. See FIG. 5A, lanes 1, 2 and 8.

WO 95/22245 PCT/US95/02058 -39- To demonstrate and characterize the ribonuclease activity of the cloned RNase, translation is performed in a reticulocyte lysate instead of a wheat germ extract due to the substantially greater efficiency of protein synthesis in the former system. See FIG. 5A, compare lanes 9 and 8. Prior to translation, endogenous reticulocyte RNase is removed by adsorbing the lysate to the affinity matrix, 2-5A-cellulose. See Example. See also, Silverman, Anal. Biochem., 144:450-460 (1985). The treatment with effectively removes all measurable endogenous 2-5A-dependent RNase activity from the lysate, as determined by 2-5A-dependent ribonuclease .assays, and FIG. 5B. In addition, the adsorptiondepletion protocol did not reduce translational efficiency. FIG. 5A, lanes 9 and 12 show the 35 S-translation products produced in the and untreated lysates, respectively.

Ribonuclease assays with recombinant RNase are performed after immobilizing and purifying the translation product on the activating affinity matrix, 2-5A-cellulose. It was previously shown that murine L cell RNase bound to 2-SA-cellulose, resulting in ribonuclease activity against poly(U) but not WO 95/22245 PCT/US95/02058 poly(C). See Silverman, Anal. Biochem., 144:450-460 (1985). Furthermore, by washing RNase:2-5A-cellulose prior to adding the substrate the level of general, RNase, is greatly reduced. See Silverman, Anal. Biochem., 144:450-460 (1985).

Incubations of lysate in the absence of added mRNA or in the presence of both human 2-5A-dependent RNase mRNA and cycloheximide resulted in only low levels of poly(U) breakdown. See FIG. 5B. In addition, it is shown that cycloheximide completely prevented RNase synthesis. See FIG. 5A, lane In contrast, translation of the human RNase mRNA, in the absence of inhibitor, results in substantial ribonuclease activity against poly(U) but not against poly(C).

See FIG. 5B. The poly(U) is degraded with a half-life of about 10 minutes whereas only 20% of the poly(C) is degraded after one hour of incubation.

Binding of recombinant 2-5A-dependent RNase to the affinity matrix was also shown by monitoring the presence of the 35 S-labeled translation product.

These results are believed to demonstrate that the recombinant human 2-5A-dependent RNase produced in vitro is a functional and potent ribonuclease.

Furthermore, both recombinant and naturally occurring forms of 2-5A-dependent RNase are capable of cleaving WO 95/22245 PCT/US95/02058 -41poly(U) but not poly(C). See FIG. 5B. See also Silverman, Anal. Biochem., 144:450-460 (1985) and Floyd-Smith, G. et al., Science, 212:1020-1032 (1981).

To determine if 2-5A-dependent RNase mRNA levels are regulated by interferon, a northern blot from murine L929 cells treated with interferon and cycloheximide is probed with the radiolabeled murine RNase cDNA. See FIG. 6.

RNase mRNA levels are enhanced three-fold by interferon (a 13) treatment even in the presence of cycloheximide. See FIGS. 6A and B, compare lanes 1 and Regulation of RNase mRNA levels by interferon as a function of time is--demonstrated (FIGS. 6A and B, lanes 3 to 6.

Maximum 2-5A-dependent RNase mRNA levels are observed after 14 hours. of interferon treatment. See FIGS. 6A and B, lane 6. A similar increase in levels of RNase per se is observed after interferon treatment of the cells. Relatively invariant levels of GAPDH mRNA indicates that equivalent levels of RNA are present in every lane of the blot. See FIG. 6C. These results are believed to show that the induction of 2-5A-dependent RNase expression is a primary response to interferon treatment. The murine and human 2-5A-dependent RNase mRNAs are determined from northern blots to be 5.7 kb WO 95/22245 PCTIUS95/02058 -42and 5.0 kb in length, respectively. See FIG. 6A.

The 2-5A-dependent RNase coding sequences, therefore, comprise only about 40% the nucleotide sequences contained in the mRNAs.

The 2-5A binding functions of the recombinant and naturally occurring forms of murine RNase are characterized by covalent crosslinking to the 2-5A probe in the presence of unlabeled 2-5A or 2-5A analogues as competitors. See FIG. 7A. Interestingly, although the about 74 kDa truncated 2-5A-dependent RNase is missing about 84 amino acids from its carboxy-terminus, see FIG. 4, it nonetheless possesses a 2-5A binding activity indistinguishable from that of naturally occurring RNase. See FIG. 7A. Trimer 3 (A2'p) 2 at about 20 nM effectively prevents the 2-5A probe from binding to either protein. See FIG. 7A, lane 8. In comparison, a 500-fold higher concentration of (A2'p) 2 A (10 pM) is required to prevent probe binding to both proteins. See lane 13. The dimer species, p 3 A2'pA, is unable to prevent the 2-5A probe from binding to the proteins even at a concentration of 10pM (lane 18). However, the inosine analogue, p 3 12'pA2'pA, Imai, J. et al., J.

Biol. Chem., 260:1390-1393 (1985), is able to prevent probe binding to both proteins but only when added at a concentration of about 1.0 pM (lane 22).

WO 95/22245 PCT/US95/02058 -43- To further define sequences involved in binding, nested 3'-deletions of the murine RNase cDNA, clone ZB1, are constructed, transcribed in vitro, and expressed in a wheat germ extract. See FIG. 7B. The different deletion clones produces comparable amounts of polypeptide as monitored by incorporation of 35 S-methionine. The levels of 2-5A binding activity are determined with the 2-5A probe in both a filter binding assay, Knight, M. et al., Nature, 288:189-192 (1980), and the uv crosslinking assay, Nolan-Sorden, N.L. et al., Anal. Biochem., 184:298-304 (1990), with similar results. See FIG. 7B. Expression of clone ZB11, encoding amino acid residues 1 to 342, results in- a loss of only about 26% of the 2-5A binding activity as compared to clone ZB1 (amino acids 1 to 656). See FIG. 7B. Clones intermediate in length between ZB1 and ZB11 all result in significant levels of 2-5A binding activity. In contrast, protein produced from ZB13 (amino acids 1 to 294) results in only about 38.3% of the 2-5A binding activity of clone ZB1, suggesting that a region important for the binding function is affected. Indeed, clone ZB14 produced a protein encoding amino acids 1 to 265 which is nearly inactive in the 2-5A binding assay (only 1.9% of th activity of clone ZB1).

Interestingly, the significant decrease in WO 95/22245 PCT/US95/02058 -44binding activity observed with ZB14 occurs with the deletion of one of two P-loop motifs; nucleotide binding domains in many proteins. See FIGS. 4 and 7B. See also Saraste, M. et al., TIBS, 14:430-434 (1990). Deletion of both P-loop motifs in clone results in protein (amino acids 1 to 218) which is completely lacking in 2-5A binding activity. See FIG. 7B.

To probe the involvement of the consensus lysine residues in the P-loop motifs in 2-5A binding activity, site-directed mutagenesis is performed on the truncated form of murine 2-5A-dependent RNase encoded by clone ZB1. Previously, it is reported that substitution mutations of the conserved lysine residues in P-loop motifs of eucaryotic initiation factor 4A and for Bacillus anthracis adenylyl cyclase results in a loss of ATP binding and catalytic activities, respectively. See Rozen et al., Mol.

Cell. Biol., 9:4061-4063 (1989) and Xia, Z. and Storm, J. Biol. Chem., 265:6517-6520 (1990).

In the former study the invariant lysine residue is mutated to asparagine. See Rozen et al., Mol. Cell.

Biol., 9:4061-4063 (1989). We substituted, individually and together, the consensus lysines with asparagines at positions 240 and 274 in the two P-loop motifs of 2-5A-dependent RNase. See FIG. 8 and the Example. Analysis of the effects of these WO 95/22245 PCT/US95/02058 mutations on 2-5A binding activity is determined by covalently crosslinking the 32 P-2-5A probe to the in vitro translation products under uv light. See FIG.

8A. See also Nolan-Sorden, N.L. et al., Anal.

Biochem., 184:298-304 (1990). Similar levels of proteins are synthesized from the different mRNA species as shown in separate reactions containing 35 S-methionine. See FIG. 8B. The three mutant forms of 2-5A-dependent RNase shows reduced binding to the probe. See FIG. 8A, lanes 2 to 4. Clone ZBl(Lys 240 FIG. 8A, lane 2, expresses a mutant RNase with a substantially reduced affinity for 2-5A; about 48.4% of the activity of clone ZB1 as determined by phosphorimager analysis (Molecular Dynamics) of the dried gel. A more modest reduction in 2-5A binding activity, to 79% of the control value, is obtained from clone ZBl(Lys 274 See FIG. 8A, lane 3. In contrast, binding activity from clone ZB1(Lys 240 274 FIG. 8A, lane 4, in which both conserved lysine residues are replaced with asparagine residues, is reduced to only 12.2% of the activity of clone ZB1 (averaged from three separate experiments). These results suggest that the lysine residues at positions 240 and 274 function within the context of a repeated P-loop motif in the binding of to 2-5A-dependent RNase.

WO 95/22245 PCT/US95/02058 -46- The molecular cloning and expression of RNase, the terminal factor in the system and a key enzyme in the molecular mechanisms of interferon action is described. See FIG. 1. The recombinant proteins produced in vitro are demonstrated to possess 2-5A binding properties identical to naturally occurring forms of murine and human 2-5A-dependent RNase. See FIGS. 2, 5A, and 7.

In addition, linkage of a 32 P-2-5A analogue to a truncated murine 2-5A-dependent RNase and to murine L cell 2-5A-dependent RNase followed by partial proteolysis reveals identical patterns of labeled peptides. See FIG. 2B. Furthermore, the full-length recombinant human 2-5A-dependent RNase isolated on the- activating, affinity matrix, shows potent ribonuclease activity towards poly(U) but none against poly(C). See FIG. 5B. Similarly, it is previously demonstrated that murine L cell RNase was activated by resulting in the cleavage of poly(U), but not of poly(C). See Silverman, Anal. Biochem., 144:450-460 (1985). The full-length human RNase, which is produced in reticulocyte lysate, had the same apparent molecular weight as did naturally occurring RNase. See FIG. 5A. However, the actual molecular mass of human 2-5A-dependent RNase is determined from WO 95/22245 PCT/US95/02058 -47the predicted amino acid sequence, FIG. 3B, to be about 83,539 Da.

Previously, it was reported that interferon enhances levels of 2-5A-dependent RNase by between two- to twenty-fold depending on the cell type. See Silverman, R.H. et al., Eur. J. Biochem., 126:333-341 (1982b) and Jacobsen, H. et al., virology, 125:496-501 (1983a). Results presented herein suggest that the gene for 2-5A-dependent RNase may be an interferon-stimulated gene. See FIG. 6. Levels of 2-5A-dependent RNase mRNA in murine L929 cells are elevated as a function of time of interferon (a 3) treatment by a factor of about three. Furthermore, the induction appeared to be a primary response to interferon treatment because it is observed in the presence of cycloheximide. Therefore, interferon is believed to regulate the 2-5A pathway by elevating levels of both 2-5A synthetases, Hovanessian, A.G. et al., Nature, 268:537-539 (1977), and RNase, Jacobsen, H. et al., Virology, 125:496-501 (1983a). See. FIGS. 1, 6 and 11.

The cloning of 2-5A-dependent RNase reveals several features of the protein. The 2-5A binding domain is of particular interest because it is the ability of 2-5A-dependent RNase to be activated by that sets it apart from other nucleases. By expressing nested 3'-deletions of murine WO 95/22245 PCT/US95/02058 -48- RNase, a region between amino acids residues 218 and 294 which is believed to be critical for 2-5A binding activity is identified. See FIG.

7B. Interestingly, the identified region contains a repeated P-loop motif, one from residues 229 to 241 and another from residues 253 to 275. See FIG. 4 and Table 2 When the latter P-loop motif (amino acids 253-275) is partially deleted, there is a precipitous decline in 2-5A binding activity. See clone ZB14 in FIG. 7B.

The homology with P-loops is believed to be highly conserved between the human and murine forms of 2-5A-dependent RNase; thus underscoring the belief of the importance of this region for 2-5A binding activity. See FIG. 4. The similarity to P-loops consists of the tripeptides, glycine-lysinethreonine, preceded by glycine-rich sequences. In this regard, the unusual feature of RNase is that the P-loop motif is repeated and are in the same orientation. Adenylyl cyclase from Bacillus anthracis also contains a duplicated P-loop motif, however, the two sequences are in opposite orientation and are overlapping. See Xia, Z. and Storm, J. Biol. Chem., 265:6517-6520 (1990).

The relative importance of the conserved P-loop lysines (at positions 240 and 274) are evaluated by site-directed mutagenesis of the murine WO 95/22245 PCT/US95/02058 -49- RNase, clone ZB1. Although individual substitution mutations of the two lysines significantly reduced 2-5A binding activity, replacing both of the lysines with asparagine residues in the same mutant RNase severely represses binding. See FIG. 8. Perhaps the trimer requirement for activation of most forms of RNase could be explained if the first and third adenylyl residues of 2-5A interact with the separate P-loop sequences inducing conformational changes in 2-5A-dependent RNase. In this regard, dimer 2-5A neither binds 2-5A-dependent RNase efficiently nor does it activate RNase, FIG. 7A; Kerr, I.M. and Brown, Prod.

Natl. Acad. Sci. 75:265-260 (1978) and Knight, M. et al., Nature, 288:189-192 (1980), perhaps because it is too short to span the two P-loop motifs. Alternately, the residual binding activity observed in the point mutants, ZBl(Lys 240 -)Asn) and ZBl(Lys 274 and the very low affinity of the double mutant, ZB1(Lys 240 274 -)Asn) for 2-5A, could indicate that the two P-loop motifs are parts of separate binding domains.

Homology with protein kinase domains VI and VII is also identified in 2-5A-dependent RNase. See FIG. 4. See also Hanks, S.K. et al., Science, WO 95/22245 PCT/US95/02058 241:42-52 (1988). Although domain VI is believed to be involved in ATP binding, this region in RNase is believed not to be important for 2-5A binding because its deletion caused only a minimal reduction in affinity for 2-5A. See FIG.

7B. However, a modest (two-fold) stimulatory effect of ATP on 2-5A-dependent RNase activity has been reported. See Wreschner, D.H. et al., Eur. J.

Biochem., 124:261-268 (1982) and Krause, D. et al., J. Biol. Chem., 261:6836-6839 (1986). The latter report indicated that ATP was not required for RNase activity but may act to stabilize the enzyme. Therefore, the region of homology with protein kinases could perhaps bind ATP resulting in stimulation of ribonuclease activity through stabilization of the enzyme.

A consensus zinc finger domain, reviewed in Evans, R.M. and Hollenberg, Cell, 52:1-3 (1988), consisting of six cysteine residues with the structure CX 4

CX

3

CX

17

CX

3

CX

3 C (amino acid residues 401-436 in Table 2 is identified in the murine form of 2-5A-dependent RNase. See FIG. 4. The homologous region in the human form of 2-5A-depenent RNase is

CX

11

CX

25

CX

3

CX

6 C (amino acid numbers 395 to 444 in Table 1 Because zinc fingers are nucleic acid binding domains, the cysteine-rich region in RNase could be involved in binding to WO 95/22245 PCT/US95/02058 -51the RNA substrate. Alternatively, the cysteine-rich domain in 2-5A-dependent RNase could mediate formation of 2-5A-dependent RNase dimers. Analysis of crude preparations of 2-5A-dependent RNase suggest that 2-5A-dependent RNase may form dimers in concentrated but not in dilute extracts. See Slattery, E. et al., Proc. Natl. Acad. Sci. U.S.A., 76:4778-4782 (1979) and Wreschner, D.H. et al., Eur.

J. Biochem., 124:261-268 (1982).

Comparison between the amino acid sequences of other ribonucleases with 2-5A-dependent RNase identifies some limited homology with RNase E, an endoribonuclease from E. coli. See FIG. 9A. See also Apirion D. and Lassar, J. Biol. Chem., .253i1738-1742 (1978) and Claverie-Martin, F. et al., J. Biol. Chem. 266:2843-2851 (1991). The homology with RNase E is relatively conserved between the human and murine forms of 2-5A-dependent RNase and spans a region of about 200 amino acid residues.

Within these regions there are 24 and 32% identical plus conservative matches, with some gaps, between RNase E and the human and murine forms of RNase, respectively. See FIG. 9A.

The me gene which encodes RNase E and the altered mRNA stability (ams) gene, Ono, M. and Kumano, Mol. Biol., 129:343-357 (1979), map to the same genetic locus. See Mudd E.A. et al., iol.

WO 95/22245 PCT/US95/02058 -52- Microbiol., 4:2127-2135 (1990); Babitzke, P. and Kushner, Proc. Natl. Acad. Sci. 88:1-5 (1991) and Taraseviciene, L. et al., Mol. Microbiol., 5:851-855 (1991). RNase E is required for both efficient mRNA turnover and rRNA processing in E.

coli. See Mudd E.A. et al., Mol. Microbiol., 4:2127-2135 (1990) and Babitzke, P. and Kushner, Proc. Natl. Acad. Sci. 88:1-5 (1991).

The cleavage specificities of 2-5A-dependent RNase and RNase E are similar in that 2-5A-dependent RNase cleaves mainly after UU or UA, Wreschner, D.H. et al., Nature, 289:414-417 (1981a) and Floyd-Smith, G.

et al., Science, 212:1020-1032 (1981), and RNase E usually cleaves within the central AUU sequence of (G or- A)AUU(A or Ehretsmann, C.P. et al., Genes Development, 6:149-159 (1992). The location of the RNase E homology and other identified features in RNase are shown. See FIG. 9B. These findings raise the possibility that RNase E may be the ancestral precursor of 2-5A-dependent RNase. In this regard, there are indications of in E. coli. See Brown, R.E.

and Kerr, Process in Clinical and Biological Research, 202:3-10 (1985) and Trujillo, M.A. et al., Eur. J. Biochem., 169:167-173 (1987). However, the evolutionary distribution of a complete 2-5A system 2-5A synthetase and 2-5A-dependent RNase) is WO 95/22245 PCT/US95/02058 -53reported to begin only with reptiles or possibly amphibia. See Cayley, P.J. et al., Biochem. Biophvs.

Res. Commun., 108:1243-1250 (1982).

Endoribonucleases play a controlling role in RNA metabolism by catalyzing the rate-limiting steps in RNA decay. See Brawerman, Cell, 57:9-10 (1989). 2-5A-dependent RNase is a uniquely regulated endoribonuclease which mediates effects of interferon against picornaviruses. It functions by binding and subsequently degrades both viral and cellular RNA. See Wreschner, D.H. et al., Nucleic Acids Res., 9:1571-1581 (1981b). In addition, the 2-5A system may be involved in the antiproliferative effects of interferon and in the fundamental control of RNA stability. Cellular levels of 2-5A-dependent RNase and/or 2-5A-synthetase are regulated during interferon-treatment, Hovanessian, A.G. et al., Nature, 268:537-539 (1977) and Jacobsen, H. et al., Virology, 125:496-501 (1983a), cell growth arrest, Stark, G. et al., Nature, 278:471-473 (1979) and Jacobsen, H. et al., Proc. Natl. Acad. Sci. U.S.A., 80:4954-4958 (1983b), cell differentiation, Krause, D. et al., Eur. J. Biochem., 146:611-618 (1985), changing hormone status, Stark, G. et al., Nature, 278:471-473 (1979), and liver regeneration, Etienne-Smekens, M. et al., Proc. Natl. Acad. Sci.

80:4609-4613 (1983). However, basal levels WO 95/22245 PCT/US95/02058 -54of 2-5A-dependent RNase and 2-5A synthetase are present in most if not all mammalian cells. The existence of multiple forms of 2-5A synthetase with different intracellular locations, Hovanessian, A.G.

et al., EMBO 6:1273-1280 (1987), could indicate diverse functions for the 2-5A system. Similarly, the ubiquitous presence of the 2-5A system in higher animals suggests an important function for RNase, Cayley, P.J. et al., Biochem.

Biophys. Res. Commun., 108:1243-1250 (1982). For instance, 2-5A-dependent RNase cleaves rRNA at specific sites in intact ribosomes, Wreschner, D.H.

et al., Nucleic Acids Res., 9:1571-1581 (1981b) and Silverman, R.H. et al., J. Virol., 46:1051-1055 (1983), possibly affecting translation rates. The transient nature of 2-5A, Williams, B.R.G. et al., Eur. J. Biochem., 92:455-562 (1978), and its growth inhibitory effect after introduction into cells, Hovanessian, A.G. and Wood, Virology, 101:81-89 (1980), indicate that the 2-5A system is a tightly regulated pathway.

EXAMPLE I The source of mRNA for preparing the cDNA library is murine L929 cells grown in EMEM (Whittaker, Inc.) and supplemented with about 10% FBS (Gibco-BRL), and antibiotics. The cells are treated with about 50 pig per ml of cycloheximide and 1000 ,11 -l--llllllllllllllll.lllllllllLIIIIILIIIIUm^^ WO 95/22245 PCT/US95/02058 units per ml of murine interferon (a 0) (1.3 X 107 units per mg protein: Lee Biomolecular) for about hours to increase levels of 2-5A-dependent RNase mRNA. Total RNA was then isolated, e.g. Chomczynski, P. and Sacchi, Anal. Biochem., 162:156-159 (1987), from which poly(A) RNA is prepared by oligo(dT)-cellulose chromatography as described. See Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989). Synthesis of the first strand of cDNA is done by using reverse transcriptase as described (Superscript; BRL) except that 5-methyl-dCTP is substituted for dCTP and an XhoI-oligo-dT adapter-primer (Stratagene) is used. Synthesis of the second strand of cDNA and ligation of EcoRI linker was as described (Stratagene). The cDNA is digested with EcoRI and XhoI and unidirectionally cloned into predigested XZAPII vector (Stratagene).

The library is packaged by using Giagpack Gold extract and titered on PLK-F bacteria.

The cDNA library is screened directly without prior amplification at a density of about 25,000 phage per 150 mm plate. Phage are grown for hours at about 42 0 C until plaques are visible.

Nitrocellulose filters saturated in IPTG (10 mM) and then dried, are overlaid on the plates and growth was continued for an additional 4 to 6 hours at 37 0

C.

The filters are processed by a modification of the WO 95/22245 PCT/US95/02058 -56methods of Singh, H. et al., Cell, 52:415-423 (1988) and Singh, H. et al., BioTechniques, 7:252-261 (1989). Filters are washed in ice-cold binding buffer (about 20 mM Tris-HCl, about pH 7.5, about mM magnesium acetate, about 50 mM potassium chloride, about 1 mM EDTA, about 50 mM 0-mercaptoethanol, about 0.1 mM PMSF, about 5% glycerol) containing about 6 M guanidine-HCl for about 20 min. The solution containing the filters is then diluted two-fold with binding buffer and washing on ice is continued for about an additional 5 minutes; serial two-fold dilutions were continued until the guanidine concentration was about 187 mM. The filters are then washed twice with binding buffer, and incubated with binding buffer containing about 5% nonfat milk for one hour at about room temperature. The filters are then washed twice with binding buffer and incubated in binding buffer (supplemented with about 0.25% nonfat dry milk and about 0.02% sodium azide) containing p(A2'p) 2 (br 8 A2'p) 2 A3'-[32P]Cp (the probe"), Nolan-Sorden, N.L. et al., Anal. Biochem., 184:298-304 (1990), at about 2 X 105 counts per minute per ml (about 3,000 Ci per mmole) at about 4 0

C

with shaking for about 24 hours. The filters are washed twice with binding buffer and then twice with water before air drying and exposing to film.

WO 95/22245 PCT/US95/02058 -57- Murine L929 cells are treated with about 1000 units per ml interferon (a 3) with or without about 50 pg per ml of cycloheximide and the total RNA is then isolated as described. See Chomczynski,

P.

and Sacchi, Anal. Biochem., 162:156-159 (1987).

Poly(A) RNA is prepared by oligo(dT) -cellulose chromatography, as described in Sambrook, J. et al., Cold Sprin Harbor Laboratory Press (1989), and is separated on glyoxal agarose gels and transferred to Nytran membranes. RNA is immobilized on the membrane by uv crosslinking (Stratalinker, Stratagene). The murine 2-5A-dependent RNase cDNA is 32 P-labeled by random priming and then hybridized to the filter [about 50% formamide, about 10% dextran sulphate, Denhardt's solution about 1% SDS, 6X SSPE, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989), about 250 pg per ml salmon sperm DNA] at about 42 0

C.

The Human 2-5A-dependent RNase cDNA clone, HZB1, is isolated from an adult human kidney cDNA library in Xgtlo with radiolabeled (random primed) murine 2-5A-dependent RNase cDNA (clone ZB1) as probe, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989). Clone HBZ22 is isolated using radiolabeled HZB1 DNA as probe. The genomic human 2-5A-dependent RNase clone is isolated from a human placenta cosmid library in vector WO 95/22245 PCTIUS95/02058 -58- (Stratagene) with a radiolabeled fragment of HZB22 DNA as probe. The murine genomic RNase clone is isolated from a mouse 129SV genomic library in vector XFIXII (Stratagene) with a radiolabeled fragment of 2-5A-BP cDNA (clone ZB1) as probe. Subcloning of DNA is in Bluescript vectors (Stratagene).

Transcription of plasmids with phage RNA polymerases is in the presence of mGppppG as described (Promega) except that reaction mixtures are supplemented with 15% dimethyl sulfoxide and incubations are at about 37 0 C for about 90 minutes.

RNA is purified through Sephadex G50 spun-columns and ethanol precipitated prior to translation. Protein synthesis was performed, as described (Promega), at about 30 0 C for about one hour in micrococcal nuclease-pretreated rabbit reticulocyte lysate or in an extract of wheat germ at about room temperature for about one hour and then at about 40 0 C for about 12 hours. Translation reactions contain about 50 iM zinc sulfate. Endogenous 2-5A-dependent RNase in the reticulocyte lysated is removed by adsorption to about 30 PM of P 2 (A2'p) 3 A covalently attached to cellulose (2-5A-cellulose), prepared as described in Wells, J.A. et al., J. Biol. Chem., 259:1363-1370 (1984) and Silverman, R.H. and Krause, I.R.L.

Press. Oxford. England, pp. 149-193 (1987), for about WO 95/22245 PCT/US95/02058 -59one hour on ice as described. See Silverman, R.H., Anal. Biochem., 144:450-460 (1985). The RNase:2-5A-cellulose complex is removed by twice centrifuging at about 400 x g for about 5 minutes at about 2 0 C. The supernatant completely lacking in measurable levels of RNase. See FIG. The set of nested 3'-deletions of the truncated murine 2-5A-dependent RNase cDNA, ZB1, is generated with exonuclease III/S1 nuclease digestion followed by filling-in with Klenow DNA Polymerase using the "Erase-A-Base" system (Promega).

The synthesis of the 2-5A probe, p(A2'p) 2 (br 8 A2'p) 2 A[32P]Cp, and its crosslinking to RNase is performed exactly as described. See Nolan-Sorden, N.L. et al., Anal.

Biochem., 184:298-304 (1990). Briefly, the probe, about 0.7 to 2.5 nM at 3,0009 Ci/mmole, is incubated for about one hour on ice with cell extract prepared as described, Silverman, R.H. and Krause, I.R.L. Press. Oxford, England, pp. 149-193 (1987), in the absence or presence of unlabeled oligonucleotide competitors. Covalent crosslinking is done under a uv lamp (308 nm) for one hour on ice and the proteins are separated on polyacrylamide gels. Filter assays for 2-5A binding activity using the 2-5A probe for about one hour on WO 95/22245 PCT/US5/02058 ice, as described in Knight, M. et al., Nature, 288:189-192 (1980).

Protease digestions are performed on gel-purified proteins in a gel, as described by Cleveland, D.W. et al., J. Biol. Chem., 252:1102-1106 (1977).

The ribonuclease assay with is performed, as described by Silverman, Anal.

Biochem., 144:450-460 (1985). Briefly, lysates are adsorbed to about 30 pM of 2-5A-cellulose on ice for about two hours. The matrix is then washed three times by centrifuging and resuspending in buffer A.

See Silverman, Anal. Biochem., 144:450-460 (1985). The matrix is then incubated with poly(U)-[ 32 p]Cp or poly(C)-[ 32 p]Cp (both at about 16 pM in nucleotide equivalents) at about 30 0 C and the levels of acid-precipitable radioactive RNA are determined by filtration on glass-fiber filters.

The Sanger dideoxy sequencing method is used to determine the DNA sequences (Sequenase, United States Biomedical).

The lysines in the truncated murine RNase, clone ZB1, at positions 240 and 274 are mutated, individually and together, to asparagine residues. Mutants ZBl(Lys 274 -)Asn) and the double mutant, ZBl(Lys 240 274 are obtained with mutant oligonucleotides after subcloning ZB1 WO 95/22245 PCTIUS95/02058 -61cDNA into pALTER-1 as described (Promega). Mutant ZBl(Lys 240 -)Asn) is obtained after polymerase chain reaction amplification of a segment of ZB1 with an upstream primer containing a unique HincII site attached to the mutant sequence and a second primer downstream of a unique BglII site. The HincII- and BG1II-digested polymerase chain reaction product and similarly-digested clone ZB1 are then ligated. The specific mutations are: for codon 240, AAA-)AAC and for codon 274, AAG-)AAC. Mutants are confirmed by DNA sequencing.

EXAMPLE II Seeds of tobacco (Nicotiana tabacum cv.

Wisconsin) and Ti based binary vectors pAM943 and pAM822 were obtained from Dr. Amit Mitra, Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska. The Argobacterium tumefaciens LBA4404 and the E. coli strains K802 and MM294 were purchased from Clonetech, Palo Alto, California and Stragene, LaJolla, California. The plant tissue culture medium Murashige and Skoog's ready mix (MS media) was purchased from Sigma Chemical Company, St. Louis, Missouri. The human cDNAs for PKR, the lysine arginine mutant PKR, and 2-5A synthetase were obtained from Dr. B.R.G. Williams, Department of Cancer Biology, The Cleveland Clinic Foundation.

See, for example, Meurs, E. et al.: Cell, 62:379-390 WO 95/22245 PCT/US95/02058 -62- (1990); Chong, K.L. et al.: EMBO 11:1553-1562 (1992); Rysieki, G. et al.: J. Interferon Res., 9:649-657 (1989); Benech, P. et al.: EMBO J., 4:2249-2256 (1985); and Saunders, M.E. et al.: EMBO 4:1761-1768 (1985). The human cDNA for dependent RNase, as shown in FIG. 3A, was cloned in Dr. R.H. Silverman's laboratory in the Department of Cancer Biology and is the property of The Cleveland Clinic Foundation. See, Zhou, A. et al.: Cell, 72:753-765 (1993).

The expression vector pAM943 is used to obtain Argobacterium-mediated transfer of T DNA containing the cDNAs and kanamycin resistance marker gene. The physical map of the plasmid vector pAM943 shows its elements. See FIG. 12. The plasmid pAM943 contains a dual promoter consisting of the adenyl methyl transferase (AMT) gene promoter of Chlorella virus and the wild type 35S promoter of Cauliflower mosaic virus. The vector also contains the gene for kanamycin resistance to select the transformed plants. Initially, the cDNAs are subcloned in pAM943 and amplified in E. coli strains K802 or MM294 using tetracycline resistance as the selectable marker.

The Argobacterium cells are transformed with the recombinant pAM943 plasmids and selected by growth in medium containing about 5 ig/ml of tetracycline, WO 95/22245 PCT/US95/02058 -63about 10 pg/ml of kanamycin and about 25 pg/ml of streptomycin.

To subclone cDNAs for PKR (PK68), a lysine arginine mutant PKR (muPk68; the mutant PKR protein binds to dsRNA but has no kinase activity and will thus function as a control), and a low molecular weight form of 2-5A-synthetase (synthetase), the plasmids pKS(+)PKR, pKS(+)muPKR, and pKS(+)synthetase are digested first with XbaI and than with Clal restriction endonucleases, the cDNA fragments are purified from low melting point agarose gels and subcloned in sense orientation at XbaI and Clal sites of pAM943. See FIG. 13. The recombinant plasmids, construct A, pAM943:PK68, construct B, pAMg43:muPK68, and contruct C, pAM943:synthetase, which correspond to the constructs depicted in FIG.

13A-C, respectively, are used to transform Araobacterium tumefaciens LBA4404. The resultant bacteria, identified as AG68, AGmu68 and AGsyn, respectively, are used for tobacco leaf disc transformations. Production of the recombinant plasmids, construct A, pAM943:PK68, construct B, pAM943:muPK68, and construct C pAM943:synthetase, is described in greater detail hereinafter.

To subclone cDNA for 2-5A-dependent RNase, the plasmid pKS(+)2C5 DNA is digested with HindIII enzyme and subcloned in the HindIII site of pAM943 in WO 95/22245 PCT/US95/02058 -64both orientations, see FIG. 13, and the recombinant plasmids, construct D, pAM943:2-5A-dep. RNase sense and construct D/a, pAM943:2-5A-dep. RNase antisense, both of which correspond to constructs D and D/a, respectively, in FIG. 13D and D/a, are used to transform Argobacterium to obtain the bacteria called AG2DR sense and AG2DR antisense, respectively.

Production of the recombinant plasmids, i.e., construct D, pAM943:2-5A-dep. RNase sense, construct D/a, pAM943:2-5A-dep. RNase antisense, and construct E, pAM822:2-5A dep. RNase antisense, is also described in greater detail hereinafter.

The competent Argobacterium cells are prepared and transformation follows the method of, for-example, An, G. et al.: Plant Molecular Biology Manual, AD:1-19 (1988). The presence of recombinant plasmids in the transformed Argobacterium cells is confirmed by preparing plasmid DNA and by performing PCR using specific complementary oligonucleotides and by observing restriction enzyme digests.

The physical map of plasmid pAM822, one of the vectors used to deliver the reverse orientation cDNA for 2-5A dependent RNase into plant cells by electroporation, is also shown. See FIGS. 13E and 14. To subclone cDNA for 2-5A-dependent RNase into pAM822 the entire coding region of RNase was PCR amplified using two oligonucleotide WO 95/22245 PCT/US95/02058 primers containing BamHI restriction sites before ATG (start codon) and after TGA (stop codon). The product was digested with BamHI and subcloned at BglII site of pAM822 vector. The cDNA used for RNase is in plasmid pZC5 referenced in Zhou et al. Cell 72, 753-765 (1994), the human form of the cDNA. The sequence is also disclosed herein.

The plasmid pAM822 contains a second selectable marker gene, the hygromycin resistance gene, permitting the construction of plants containing both and 2-5A-dependent RNase cDNAs.

Insertion of pAM822:2-5Adep. RNase (Fig. 13E), containing 2-5A-dependent RNase cDNA, into kanamycin-resistant, transgenic tobacco leaf discs containing 2-5A-synthetase cDNA is thus performed.

Tobacco plants are grown aseptically in Murashige and. Skoog's medium, known as MS medium, containing about 3% sucrose (MSO medium) and about 0.8% agar in plastic boxes (Phytatray) at about 28'C under cycles consisting of about 16 hr of light and about 8 hr of dark in a growth chamber. Leaves bigger than about 2" long are cut into about 2 to 3 cm 2 pieces under the MSO medium and 6-8 leaf pieces are placed in a 6 cm Petri dish containing about 2 ml of MSO medium and holes are made in the leaf pieces with a sterile pointed forcep. Overnight cultures of AG68, AGmu68, AGSyn, AG2DR sense and AG2DR antisense WO 95/22245 PCT/US95/02058 -66are grown in LB (L broth) containing about 50 iM of acetosyringone and appropriate antibiotics at about 28*C in a waterbath. One hundred microliter of overnight culture is added to each of the Petri dishes containing leaf pieces. Incubation is at about 28*C under diffuse light in the growth chamber for about 2 days. Leaf pieces are washed extensively with MSO medium and transferred to solid agar for selection in shoot regeneration medium [MSO; about mg/l BAP (benzylaminopurine); about 200 pg/ml kanamycin; about 200 pg/ml carbenicillin; and about 100 pg/ml of cefotaxine], under diffuse light at about 28*C in the growth chamber. Within about 3 weeks, regeneration of plantlets is observed. When the- plantlets are about 2-3cm long they are transferred to root-inducing, hormone-free MSO solid agar medium containing about 200 pg/ml kanamycin and about 200 pg/ml carbenicillin. The transgenic plants expressing 2-5A synthetase are substantially transformed to introduce the cDNA for RNase (with pAM943:2-5Adep.RNase sense, construct D; FIG. 13D). Alternatively, the vector pAM822 (FIG.

14) containing the 2-5A-dependent RNase cDNA in sense orientation and the hygromycin resistance gene is used to transform 2-5A-synthetase containing plants.

This allows selection in hygromycin containing MSO media. Tissue culture and regeneration of plants are WO 95/22245 PCT/US95/02058 -67done as described above. Transgenic plants are grown to produce flowers and seeds to demonstrate the transfer of the antiviral genes or nucleotide sequences to subsequent generations. Although specific plasmid constructs are described herein, the present invention is intended to include any plant vector including those with inducible promoters.

Expression of PKR, mutant PKR, and 2-5A-dependent RNase in plants that are 4" to 5" tall are tested in protein extracts of leaves (supernatant of 10,000 x g centrifugation). Results of Northern and Southern blot assays and functional binding assays for RNase are reported in Tables I-V. See also FIG. 15 wherein expression of human synthetase cDNA in transgenic tobacco plants as determined by measuring the mRNA levels in a Northern blot is shown. FIG. 16, on the other hand, shows expression of mutant and wild type forms of human PKR cDNA in transgenic tobacco plants as determined by measuring mRNA levels in a Northern blot. FIG. 17 depicts presence of 2-5A-dependent RNase cDNA in transgenic tobacco plants as determined on a Southern blot.

WO 95/22245 PCTfUS95/02058 -68- TABLE I Transgenic Tobacco Plants Expressing Wild Type and Mutant Forms of Human PKR cDNA (plasmid pAM943:PK6B) FIG. 13A (plasmid pAM943:muPK68) FIG. 13B Transgenic: Plant: (clone Southern Blot: (presence of DNA) Northern Blot: (expression of mRNA) Mutant PKR: (plasmid pAM943 :PK68) FIG. 13A Wild Type

PKR:

(plasmid pAM943 :muPK68) FIG. 13B N. T.

N. T.

N. T.

N. T.

N.T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N.T

N. T.

N.T

N.T

N. T.

Not Tested WO 95/22245 PTU9/25 PCTIUS95/02058 -69- TABLE II Transgenic Tobacco Plants Expressing Human 2-5A-Synthetase cDNA (Plasmid pAM943:synthetase FIG. 13C) Plant: (clone#) Southern Blot: (presence of DNA) Northern Blot: (expression of miRNA)

N.T.

4 6 7 8 9 12 13 14 16 17 18 a.

b

C

d N. T.

N. T.

N. T.

N.T

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

Not Tested.

WO 95/22245 PCTIUS95/02058 TABLE III Transgenic Tobacco Plants Containing Sense or Antisense Orientation Human RNase cDNA (plasmid pAM943:2-5A-dep. RNase sense -FIG. 13D) (plasmid pAM943:2-5A-dep. PNase antisense -FIG..13D/a) Transgenic: Southern- Northern Plant: (presence (expression (clone #1 of DNA)- -of xnRNA) 2 Assay: (protein activity Antisense: Sense: N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N Not Tested.

WO 95/22245 PCT/US95/02058 -71- TABLE IV Transgenic Tobacco Plants Containing Both Human and Human 2-5A-Dependent RNase cDNA (plasmid pAM943:synthetase FIG. 13C) (plasmid pAM943:2-5A-dep. RNase sense FIG. l3D) Plant: Southern Blots: (clone (2-5A-Syn (2-5A-Dep.

DNA RNase DNA) Northern Blot: (2-5A Syn. mRA RNase mRNA 14/1 14/2 14/3 14/4 14/5 14/6 15/1 15/2 15/3 15/4 15/5 15/6 15/7 N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N. T.

N.T.

N. T.

N. T.

N. T.

K. T.

N. T.

N. T.

N. T.

N.T.

N. T.

N. T.

N. T.

N.T.

N. T.

N. T.

N. T.

N.T.

N. T.

N.T Not Tested.

WO 95/22245 PCT/US95/02058 -72- Assays of dsRNA-dependent autophosphorylation of PKR, 2-5A synthetase activated with dsRNA, and 2-5A-dependent RNase by UV-crosslinking to radioactive 2-5A, see Nolan-Sorden et al.: Analytical Biochemists, (184):298-304 (1990), may be performed on the leaf extracts. The levels of the proteins may also be determined by Western blot analysis using the antibodies against PKR, 2-5A-synthetase and RNase.

To demonstrate the expression of RNase in transgenic plants containing construct D, pAM943:2-5A-dep. RNase sense, as depicted in FIG. 13D, functional assays that measure binding of radiolabeled 2-5A analog to RNase are performed. See Tables III and V. Results show the presence of 2-5A-dependent RNase in transgenic plants Z1, Z2, Z3, Z5 and Z6. It is believed that the highest levels of human, recombinant 2-5A dependent RNase are in plant See Table V.

WO 95/22245 PCT/US95/02058 -73- TABLE V Functional Expression of 2-5A-Dependent RNase in Transgenic Tobacco Plants ad Determined by a 2-5A Binding Assay (plasmid pAM943:2-5A-dep. RNase sense FIG. 13D) Plant; 2-5A Binding Activitya: Z1 662 22 1,618 Z3 1,545 2,575 Z6 1,547 Z7 31 aTobacco plants contain construct D, pAM943:2-5Adep. RNase (sense). 2-5A binding assays are performed by the filter binding method of Knight, M. et al. Nature (288):189-192 (1980) with modifications. A 32 P-labeled and bromine substituted 2-5A analog, p(A2'p) 2 (br8A2'p) 2 A3,_32p]Cp, about 15,000 counts per min per assay, at about 3,000 Ci per mmole, Nolan-Sorden, et al. Anal. Biochem., (184):298-304 (1990), is incubated with plant extracts, containing about 100 micrograms of protein per assay, on ice for about 4 h. The reaction mixtures are then transferred to nitrocellulose filteres which are washed twice in distilled water and dried and the amount of probe bound to the 2-5A-dependent RNase on the filters is measured by scintillation counting, Silverman, R.H. and Krause, In, Clemens, Morris, and Gearing.

Lvymhokines and Interferons A Practical Approach, I.R.L. Press, Oxford, pp. 149-193 (1987). Data is presented as counts per min of labeled 2-5A bound to RNase expressed in the transgenic plants.

Background radioactivity from extracts of control plants, 705 counts per min, consisting of nonspecific binding of is subtracted from these data.

WO 95/22245 PCTIUS95/02058 -74- To further confirm that the transgenic plants containing 2-5A-dependent RNase cDNA express functional 2-5A-dependent RNase protein or an amino acid sequence, an affinity labeling method is performed (data not shown). In this method, activity is determined on a Western blot with a bromine-substituted, 32 P-labeled 2-5A analog (the "probe"), as described in Nolan-Sorden, N.L. et al.: Anal. Biochem., 184:298-304 (1990). More particularly, leaves are collected from transgenic plants containing 2-5A-dependent RNase cDNA and they are homogenized in NP40 lysis buffer, see Silverman, R.H. and Krause, D. (1987) In, Clemens, Morris, and Gearing, Lvmphokines and Interferons A Practical Approach, Press, Oxford, pp. 149-193, supplemented with about ascorbic acid, about 1 mM cysteine, about 2 pg per ml leupeptin, about 100 p per ml phenylmethyleulfonyl fluoride, and about 2 pg per ml pepstatin. Extracts are clarified by centrifugation at about 10,000 x g for about 10 min. Supernatants of the extracts, about 100 pg of protein per assay, are separated by polyacrylamide gel electrophoresis, followed by transfer of the proteins to Immobilon-P membrane filters (Millipore Corp., Bedford, MA). The filter is then incubated with about 4 X 105 c.p.m. per ml of 32 P-labeled 2-5A probe for about 24 h at about 4*C, WO 95/22245 PCT/US95/02058 according to Zhou, A. et al.: Cell 73:753-765 (1993). The autoradiograms of the washed and dried filters show the presence of functional human RNase visible to about 80 kDa bands, in plants Z3, Z5, and Z6 (data not shown).

Antiviral activity of the plants are determined by rubbing celite powder coated with Tobacco mosaic virus (ATCC) and Tobacco Etch virus (from Dr. Amit Mitra, Nebraska). The plants are monitored for symptoms of viral infection on leaves from control and transgenic plants and are documented in photographs.

The plasmids described and the transformed Argobacterium strains can be used to transform any other plants into virus-resistant plants. Exemplary of plants that may be transformed in accordance with the present invention include vegetable plants like corn, potato, carrot, lettuce, cabbage, broccoli, cauliflower, bean, squash, pumpkin, pepper, onion, tomato, pea, beet, celery, cucumber, turnip and radish plants, fruit plants like banana, apple, pear, plum apricot, peach, nectarine, cherry, key lime, orange, lemon, lime, grapefruit, grape, berry, and melon plants, grain plants like wheat, barley, rice, oat and rye plants, grass, flowers, trees, shrubs and weeds such as laboratory weeds like Arabidopsis. It should therefore be understood that the present WO 95/22245 PCT/US95/02058 -76invention includes any plant into which any nucleotide sequence encoding an amino acid having antiviral activity has been introduced to form transgenic plants having immunity or resistance against viral infection.

Construction of pAM943:PKR (Construct A) and pAM943;MuPKR (construct B) The plasmids pKS(+)PKR and pKS(+)muPKR, encoding wild type PKR and a lysine to arginine at codon 296 mutant form of PKR, respectively, present in E. coli cells (obtained from Dr. B.R.G. Williams, Cleveland Clinic, Cleveland, Ohio) are prepared by standard methods. See, for example, Katze, M.G. et al.: Mol. Cell Biol., 11:5497-5505 (1991) for generation of muPKR, lysine 296 arginine mutant (K296R), by site specific mutagenesis as described.

The PKR nucleotide sequence utilized to construct plasmids pKS(+)PKR and pKS(+)muPKR is depicted in FIG. 18. To determine the ability of a plant translation apparatus to synthesize PKR protein, capped PKR mRNA is produced from linearized pKS(+)PKR by in vitro transcription. The RNA is then translated in wheat germ extract (obtained from Promega Corp., Madison, in the presence of 35 S-methionine. Synthesis of the 35 S-labeled PKR is detected in an autoradiogram of the dried, SDS/polyacrylamide gel.

WO 95/22245 PCT/US95/02058 -77- The cDNAs encoding PKR and muPKR are excised from plasmids pKS(+)PKR and pKS(+)muPKR by digesting with KpnI and XbaI. The resulting DNA fragments containing the entire coding sequences for PKR and muPKR are purified from a low melting point agarose gel. To generate cDNAs containing at the end XbaI and at the 3' end Clal sites, the PKR cDNA and muPKR cDNA are then digested with Clal and purified. The resulting digested PKR cDNA and muPKR cDNA are then force cloned into XbaI and Clal digested pAM943 by DNA ligation. The resulting plasmids, FIG. 13, constructs A and B, are used to transform Araobacterium tumefaciens strain LBA4404 (Clonetech, Plao Alto, CA). Recombinant plasmids are prepared from transformed Argobacterium tumefaciens bacteria by standard methods and the presence of PKR and muPKR cDNA is confirmed by PCR analysis and restriction enzyme digests of the isolated plasmids.

Construction of pAM943:Synthetase (construct C) The plasmid ptac-15 containing the human cDNA illustrated in FIG. 20 for a small form of (producing a 1.8 kb mRNA) (obtained from Dr. B.R.G. Williams, Cleveland Clinic, Cleveland, Ohio) is prepared by standard methods and is digested with BamHI and EcoRI. The synthetase cDNA is purified from a low melting point agarose gel by standard methods and is then subcloned into WO 95/22245 PCT/US95/02058 -78plasmid pKS(+) (Strategene, La Jolla, CA) in BamHI and EcoRI sites. The resulting recombinant plasmid DNA (pKS(+)synthetase) is digested with XbaI and Clal and the 2-5A synthetase cDNA is purified from a low melting point agarose gel and is then subcloned into Xbal and Clal digested pAM943 to produce construct C (FIG. 13). Recombinant plasmids are prepared from transformed Araobacterium tumefaciens bacteria by standard methods and the presence of cDNA is confirmed by PCR analysis and by restriction enzyme digests of the isolated plasmids.

Construction of pAM943:2-5Adep.RNase sense (construct D) and pAM943:2-5Adep.RNase antisense (construct D/a) The plasmid pKS(+)ZC5 encoding a complete coding sequence for human 2-5A-dependent RNase is digested with HindIII. The 2.5kbp cDNA for RNase is purified in a low melting point agarose gel and is then subcloned in HindIII digested pAM943 in both sense (forward) and antisense (reverse) orientations to produce pAM943:2-5Adep.RNase sense (construct D) and pAM943:2-5Adep.RNase antisense (construct as depicted in FIG. 13D and D/a, respectively.

Transformed Araobacterium are determined to contain the 2-5A-dependent RNase cDNA by restriction enzyme digests and by PCR analysis.

WO 95/22245 WO 9522245PCT/US95/02058 -79- Construction of pA(B22 :2-SAdep Nase antisense (construct zi Polymerase chain reactions (PCR) are performed on plasmid pKS(+)ZC5 encoding human RNase to generate HindIII arnd BamHI sites on the two ends of the cDNA and to reduce and 3' untranslated sequences. The PCR primers used are: ID SEQ NO:7: 2 DR-5 5' -TCATGCTCGAGAAGCTTGGATCCACCATGGAGAGCAGGGATand ID SEQ NO:8: H2DR-4 5' -GATACTCGAGAAGCTTGCATCCTCATCAGCACCCAGGGCTGG The- PCR product (about 2.25 )cbp) is purified on a low melting point agarose gel and is then digested with HindIIl and is then subcloned into HindlIl digested plasmid pKS The resulting plasmid, pKS:pZC5 is digested with BamHI and the 2-5A-dependent Rliase cDNA fragment is purified and cloned into BglII digested pAM822. Recombinants isolated in the reverse (antisense) orientation give pAM822 antisense (construct See FIG. 13E.

WO 95/22245 PCT/US95/02058 As to the nucleotide sequences disclosed herein, A means adenine; C means cytosine; G means guanine; T means thymine; and U means uracil. With respect to the disclosed amino acid sequences, A means ala or alanine; R means arg or arginine; N means asn or asparagine; D means asp or aspartic acid; C means cys or cysteine; E means glu or glutamic acid; Q means gin or glutamine; G means gly or glycine; H means his or histidine; I means ile or isoleucine; L means leu or leucine; K means lys or Lysine; M means met or methionine; F means phe or phenylalanine; P means pro or proline; S means ser or serine; T means thr or threonine; W means trp or tryptophan; Y means tyr or tyrosine; and V means val or valine.

The following listed materials are on deposit under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, USA, and have been assigned the following Accession Numbers.

Plasmid DNA ATCC No. Deposit Date Viability Date pAM943:PK68 (Plasmid pA) 75996 21 Dec. 1994 13 Jan. 1995 pAM943:muPK68 (Plasmid pB) 75997 21 Dec. 1994 13 Jan. 1995 pAM943:Synthetase (Plasmid pC) 75998 21 Dec. 1994 13 Jan. 1995 pAM943:2-5Adep.RNase (Plasmid pD) 75999 21 Dec. 1994 13 Jan. 1995 Z9*, expressing, human 97047 01 Feb. 1995 07 Feb. 1995 RNase cDNA 15/2" expressing human 97041 01 Feb. 1995 07 Feb. 1995 cDNA *this seed contains construct D, shown in Fig. 13, which is pAM943:2-5Adep.RNase this seed contains construct C, shown in Fig. 13, which is pAM943:Synthetase WO 95/22245 WO 9522245PCTIUS95/02058 -81- Human 2-5A-devedent RNase SEQ ID NO:1-, SEQ ID NO:2:, SEQ ID NO:3: -103 aatcccaacttacactcaaagct tctttgattaagtgctaggagataaatttgcattttctca aggaaaaggctaaaagtggtagcaggtggcatttaccgtc and SEQ ID NO:4: ATG GAG AGC Met Giu Ser GAG GGA CCC Giu Gly Pro GCT GCA GTG Ala Ala Val AAA GCT GTT Lys Ala Val GTC CAG CAA Val Gin Gin GTT AAT TTC Val Asn Phe ACA-CCT CTG Thir Pro Leu AGG GAG GAC Arg Glu Asp CAT GGT GCT His Gly Ala AAT GGG GCC Asn Gly Ala ATT GCG GGG Ile Ala Gly TTC CTT TCT Phe Leu Ser TGT GAT TTT Cys Asp Plie AGG GAT CAT AAC AAC CCC CAG Arg Asp His Asn Asn Pro Gin ACG TCC TCC AGC GGT AGA AGG Thr Ser Ser Ser Gly Arg Arg GAA GAC AAT CAC TTG CTG ATT Giu Asp Asn His Leu Leu Ile CAA AAC GAA GAT GTT GAC CTG Gin Asn Glu Asp Val Asp Leu TTG CTG GAA GGT GGA GCC AAT Leu Leu Glu Gly Gly Ala Asn CAG GAA GAG GAA GGG GGC TGG Gin Giu Giu Giu Gly Gly Trp CAT AAC GCA GTA CAA ATG AGC His Asn Ala Val Gin Met Ser ATT GTG GAA CTT CTG CTT CGT Ile Val Giu Leu Leu Leu Arg GAC CCT GTT CTG AGG AAG AAG Asp Pro Val Leu Arg Lys Lys (CCT) ACG CTT TTT ATC CTC GCA GCG Thr Leu Phe Ile Leu Ala Ala (Pro) AGC GTG AAG CTG CTG AAA CTT Ser Val Lys Leu Leu Lys Leu AAA GGA GCA GAT GTC AAT GAG Lys Gly Ala Asp Val Asn Glu TAT GGC TTC ACA GCC TTC ATG Tyr Gly Phe Thr Ala Phe Met 120 150 180 210 240 270 300 100 330 110 360 120 390 130 420 140 GAA GCC GCT'GTG TAT GGT AAG GTC AAA GCC Glu Ala Ala Val Tyr Gly Lys Val Lys Ala WO 95/22245 PCTIUS95/02058 -82-

CTA

Leu

GTG

Val

CAA

Gin AAA TTC CTI! TAI Lys Phe Lou Tyx AAT TTG AGG CGA Asn Leu Arg Arg GAG CGG CTG AGG Glu Arg Leu Arg GCT CTC ATG, GAC Ala Leu Met Asp GTA GAG GTC Val Giu Val ATG GGG GCA Met Gly Ala ATG GGC AGA Met Gly Arg CTG AGC TCT Leu Ser Ser ATT ACG CAT Ile Thr His GAT GTC AAT Asp--Val Asn ACT CCC CTG Thr Pro Leu CAC TTG GGT His Leu Gly CAA GAG CAC Gin Giu His AGT GAT GGC Ser Asp Gly GTT GAA CTC Val Giu Leu TTG CTG TGC Leu Leu Cys TGT GGG GAT Cys Gly Asp AAT TAT GAC Asn Tyr Asp

TTG

Lou

GAT

Asp

AAT

Asn

GAC

Asp

CTG

Leu

GTG

Vai

ATC

Ile

TTG

Leu

ATA

Ile

AAA

Lys

AAA

Lys

AAA

Lys

CTT

Loeu

CAT

His

GCT

Ala

AAG

Lys

GTA

Val

GCC

Ala

GAT

Asp

CTG

Lou

AGG

Arg

CTG

Leu

GTG

Val

GAG

Giu

ACA

Thr

CTG

Leu

CGT

Arg

GTT

Val

TCC

Ser AAG AGA GGA Lys Arg Gly AAG ACA AAG Lys Thr Lys AAA GGA GGG Lys Gly Giy GCT GAA AAA Ala Glu Lys ATT CTC CTT Ile Lou Leu AAC GCC TGT Asn Ala Cys TTG ATC CAT Leu Ile His AGT GAT GTG Ser Asp Val CTG GAC CAT Lou Asp His GGA GAA AGA Gly Giu Arg GCA GTG GAG Ala Val Giu CAG AGG CTT Gin Arg Leu ATT AAT GAC Ile Asn Asp GCA CTG CTG Ala Leu Lou AAG, AAA ATC Lys Lys Ile GGA GCC AGT Gly Ala Ser ATG ACA GCG Met Thr Ala CTT GTG AAG Lou Val Lys GCA AAT Ala Asn GAG GAT Giu Asp GCC ACA Ala Thr GGA CAC Gly His GAT GAG Asp Giu GAC AAT Asp Asn GCT CTC Ala Lou GAG GCT Glu Ala GGG GCT Gly Ala GGG AAG Gly Lys AAG AAG Lys Lys CTG GAG Lou Glu ACA GAC Thr Asp CTT GCT Leu Ala GCC GAG Ala Giu ACA GAT Thr Asp AGG CGG Arg Arg GTT CTT Val Lou 450 150 480 160 510 170 540 180 570 190 600 200 630 210 660 220 690 230 720 240 750 250 780 260 810 270 840 280 870 290 900 300 930 310 960 320 WO 95/22245 PCTfUS9S,'02058 -83- CTC TCT CAT GGA GCC loeu Ser His Gly Ale CCT CCT GCT GAA GAC Pro Pro Ala Glu As; TCA CAC TGG Ser His Trp CAC AGA ATA His Arg Ile CTC AAG, TTC Leu Lys Phe ATT GCT GAT Ile Ala Asp CTG GGG TTC Leu Gly Phe GTG AAG ACG Vai Lys Thr GCA CAG CGG Ala Gin Arg AGC CGA GAG Ser -Arg Giu TAT GGG AGT Tyr Gly Ser TTT GTG TGT Phe Val Cys CTG GAA GCG Leu Giu Ala GAA GAT GTG Glu Asp Val GCC CGA AAT Ala Arg Asn GCT GTT CAA Ala Val Gin TAC ACC CAC4 Tyr Thr His GGG GCA Gly Aia TAC CGC Tyr Arg TTT ATT Phe le ACT TCA Thr Ser TAT GAG Tyr Giu TTC TGT Phe Cys GAA GTC Giu Val AAC AGT Asn Ser GAG AGC Glu Ser GTC ACC Val Thr TGT TTG Cys tLeu GAA AAT Glu Asn GTC CTG Val Leu GAA CTA Glu Leu CAG GAT Gln Asp AAA GAA GAT Lys Giu Asp TGG AAG CCT Trp Lys Pro GCC CTG AAG Ala Leu Lys CCT ATG ATT *Pro Met Ile *GAT GAA AAA Asp Giu Lys GAA GGA GGC Glu Giy Gly AAG CAA GAA Lys Gin Glu GAG GGC AGC Glu Gly Ser TCT TGT CTG Ser Cys Leu CAC TTG GTG His Leu Val CAC AGG GGC His Arg Gly CTC TGT GAG Leu Cys Giu GAT GTG CAC Asp Val His GAG GAA GAT Giu Giu Asp TCA TCT ATA Ser Ser Ile CAC TTG TCC His Leu Ser CTG CAA CCA Leu Gin Pro C Ph

CA(

Gir

GAIJ

AsF

GGC

Gly

TAC

Tyr

ATC

le

GTA

Val

CCA

Pro

CAA

Gin

ACA

Thr

CAC

H~is

CAG

G1n

ALGA

Akrg

GA.A

L'TT

?he

V'GT

ys

:AA

1n

[CAC

SHis

AGC

1Ser

CTC

Leu

:AAA

~Lys

:AAA

Lys

TAC

Tyr

GCT

Ala

CGT

Arg

AGC

Ser

TTC

Phe

TTG

Leu

ACT

Thr

GGG

Gly

TTT

Phe

AAG

Lys

GGA

Gly

AAC

Asn 990 330 1020 340 1050 350 1080 360 1110 370 1140 380 1170 390 1200 400 1230 410 1260 420 1290 430 1320 440 1350 450 1380 460 1410 470 1440 480 1470 490 1500 500 ATC TTA ATA GAT TCT Ile Leu Ile Asp Ser AAG AAA GCT GCT CAC Lys Lys Ala Ala His WO 95/22245 PCTIUS95/02058 -84- CTG GCA GAT TTT GAT AAG AGC Leu Ala Asp Phe Asp Lys Ser GCT GGA GAT CCA CAG GAA GTC Ala Gly Asp Pro Gin Giu Val

CTA

Leu

GTG

Val

GAT

Asp

GTT

Val

GAC

Asp

GAA

Glu

CTG

Leu

AGC

Ser-

AAT

Asn

GAA

Glu

GGG

Gly

AAG

Lys

ATG

Met

AGA

Arg

GAT

Asp

GAA

Glu

GAG

Glu

GTA

Val

CTG

Leu

CAA

Gin

CTC

Leu

CAT

His

GGT

Gly

CGC

-Arg

GAA

Glu

AGT

Ser

CCT

Pro

TGG

Trp

AAA

Lys

GGC

Gly

CTG

Leu

CAC

His GAC CTT Asp Leu AAG AAG Lys Lys AAA GCT Lys Ala CTT TCT Leu Ser ATT CAT Ile His GTG AGG Val Arg CAT CCC His Pro TAT AGG Tyr Arg TCC GAC Ser Asp GAG ATC Glu Ile TCT GAA Ser Glu ACG ACT Thr Thr AAA ATG Lys Met AAT TTC Asn Phe CTA AAG Leu Lys ATT GAT ile Asp GGA CGG Gly Arg GGA AGC Gly Ser CAA AGT Gin Ser CCA GAT Pro Asp CGT CTC Arg Leu GAC TGT Asp Cys TTC TTT Phe Phe ACG CTT Thr Loeu ATC AAA Ile Lys CTC AGA Leu Arg CAT TCC His Ser AAG ATT Lys Ile AAT AAG Asn Lys TAC CAG Tyr Gin TTC ATC Phe Ile GAA GAA Glu Glu

CTG

Leu

ATC

Ile

AAT

Asn

GAG

Glu

TTC

Phe

CTG

Leu

TGG

Trp

CGG

Arg

ACA

Thr

CTA

Leu

AAA

Lys

AAT

Asn

TTT

Phe

AAC

Asn

CGG

Arg

AAG

Lys ATC AAG TGG lie Lys Trp AAG AGA GAT Lys Arg Asp GTC CTC TAT Val Lou Tyr TCA TTT GAG Ser Phe Glu GAA GAG GTG Glu Giu Val GAA ACT AAG Glu Thr Lys CAT CCT GGG His Pro Gly AGT GAC CTG Ser Asp Leu ACT TGG GAG Thr Trp Glu AAT GTG GGA Asn Val Gly CGA AAA TCT Arg Lys Ser CTG CAA CCT Leu Gin Pro AGT TTT GAC Ser Phe Asp GAA TGT GTT Glu Cys Vai TAT GAA AAA Tyr Giu Lys ACT GTG GGT Thr Vai Gly AAT TTG GGA Asn Lou Gly CAT AAA AAG His Lys Lys 1530 510 1560 520 1590 530 1620 540 1650 550 1680 560 1710 570 1740 580 1770 590 1800 600 1830 610 1860 620 1890 630 1920 640 1950 650 1980 660 1210 670 2040 680 WO 95/22245 PCT/US95/02058 ATG AAA TTA AAA ATT GGA GAC CCT TCC CTG 2070 Met Lys Leu Lys le Gly Asp Pro Ser Leu 690 TAT TTT CAG AAG ACA TTT CCA GAT CTG GTG 2100 Tyr Phe Gin Lys Thr Phe Pro Asp Leu Val 700 ATC TAT GTC TAC ACA AAA CTA CAG AAC ACA 2130 Ile Tyr Val Tyr Thr Lys Leu Gin Asn Thr 710 GAA TAT AGA AAG CAT TTC CCC CAA ACC CAC 2160 Giu Tyr Arg Lys His Phe Pro Gin Thr His 720 AGT CCA AAC AAA CCT CAG TGT GAT GGA GCT 2190 Ser Pro Asn Lys Pro Gin Cys Asp Giy Ala 730 GGT GGG GCC AGT GGG TTG GCC AGC CCT GGG 2220 Gly Gly Ala Ser Gly Leu Ala Ser Pro Gly 740 TGC 2223 tgtgcgttcgatcggatc 2258 Cys 741 tatgttggcttgaaccaa 2292 tctgggccttttaactcaccaggttgcttgtgagggat 2330 gagttgcatagctgatatgtcagtccctggcatcgtg 2367 tattccatatgtctataacaaaagcaatatatacccag 2405 actacactagtccataagctttacccactaactggga 2442 ggctcgtaatcttgcatcca 248B0 aagaatgagtgccttgacccctaatgctgcatatgtt 2517 acaattctctcacttaattttcccaatgatcttgcaaa 2555 acagggattatcatccccatttaagaactgggaacc 2592 tggccggggggcatgcagta 2630 tcaatttatacctagcactttataaatttatgtggtg 2667 tttgtcttatggcctaacta 2705 tactcggttcaaggccaaa 2742 atataggggttccaggaatctcattcattcattcagta 2780 tttattgagcatctagtataagtctgggcactggatg 2817 catgaatt 2825 *It is believed that the original codon number i.e. CTT encoding the amino acid number 95, i.e.

leucine, is correct, however the alternative codon in parenthesis shown above codon number 95, i.e. CCT encoding the alternative amino acid in parenthesis shown below amino acid number 95, i.e. praline may also exist at this position (see page 81).

SEQ ID NO:1: represents the DNA encoding sequence for the human 2-SA-dependent RNase protein. SEQ ID NO:2: represents the amino acid sequence encoded by the DNA sequence designated SEQ ID NO:1:.

SEQ ID NO:3: represents the DNA sequence, represented by SEQ ID NO:1:, having the alternative codon number 95, CCT. SEQ ID NO: represents the 'amino acid sequence encoded by SEQ ID NO:3:, having the alternative amino acid number 95, praline.

I

WO 95/22245 PTU9/25 PCTfUS95/02058 -86- Murine 2-5A-debendent RNase (partial) SEQ ID NO:5: and SEQ ID NO:6: -163 attcggcacgaggaaggtgccaattactagctcccttctttattcgtgta ctgatgagatgtcagaagacagaacataatcagcccaacccctactccaa gactctcattgtgtcccaaagaaacacacgtgjtgcatttcccaaggaaaa ggcattgaggacc ATG GAG ACC CCG GAT TAT Met Giu Thr Pro Asp Tyr AAC ACA CCT CAG GGT GGA ACC CCA TCA GCG Asn Thr Pro Gin Giy Gly Thr Pro Ser Ala GGA AGT CAG AGG ACC GTT GTC GAA GAT GAT Giy Ser Gin Arg Thr Val Val Giu Asp Asp TCT TCG TTG ATC AAA GCT GTT CAG AAG GGA Ser Ser Leu le Lys Ala Val Gin Lys Gly GAT GTT GTC AGG GTC CAG CAA TTG TTA GAA Asp Val Val Arg Val Gin Gin Leu Leu Gi~u AIA-GGG GCT GAT GCC AAT GCC TGT GAA GAC Lys Gly Ala Asp Ala Asn Ala Cys Glu Asp ACC TGG GGC TGG ACA CCT TTG, CAC AAC GCA Thr Trp Gly.Trp Thr Pro Leu His Asn Ala GTG CAA GCT GGC AGG GTA GAC ATT GTG AAC Val Gin Ala Gly Arg Val Asp Ile Val Asn CTC CTG CTT AGT CAT GGT GCT GAC CCT CAT Leu Leu Leu Ser His Giy Ala Asp Pro His CGG AGG AAG AALG AAT GGG GCC ACC CCC TTC Arg Arg Lys Lys Asri Gly Ala Thr Pro Phe ATC ATT GCT GGG ATC CAG GGA GAT GTG AAA Ile Ile Ala Gly Ile Gin Gly Asp Val Lys CTG CTC GAG ATT CTC CTC TCT TGT GGT GCA Leu Leu Giu Ile Leu Leu Ser Cys Giy Ala GAC GTC AAT GAG TGT GAC GAG AAC GGA TTC Asp Val Asn Glu Cys Asp Giu Asn Gly Phe 18 6 48 16 78 26 108 36 138 46 168 56 198 66 228 76 258 86 288 96 318 106 348 116 378 126 WO 95/22245 PCT/US95/02058 -87- ACG GCT Thr Ala AAC GCT Asn Ala AAG GGA Lys Gly ACA ACG Thr Thr GGA GGC Gly Gly GAG AAG Giu Lys CTC CTC tLeu Leu

TTC

Phe

GAA

Glu

GCC

Ala

AAG

Lys

GCC

Ala

GGC

Gly

AAT

Asn GCT CGG GAC Ala Arg Asp ATC CGT ACT Ile Arg Thr AAT GTG GAG Asn-Val Glu CAG CAC GGG Gin His Gly GAA AGA GGG Glu Arg Gly GTG GAG AGG Val Giu Arg ATG CTC CTG Met Leu Leu GAT GCC AGG Asp Ala Arg CTG CTA ATT Leu Leu Ile GAA ATT GTC Giu Ile Val GCT GAT AAG Ala Asp Lys ATG GAA GCT GCT GAG CGT GGT Met Giu Ala Ala Giu Arg Gly GCC ?TA AGA TTC CTT TTT GCT Ala Leu Arg Phe Leu Phe Ala AAT GTG AAT TTG CGA CGA CAG Asn Val Asn Leu Arg Arg Gin GAC AAA AGG CGA TTG AAG CAA Asp Lys Arg Arg Leu Lys Gin ACA GCT CTC ATG AGC GCT GCT Thr Ala Leu Met Ser Ala Ala CAC CTG GAA GTC CTG AGA ATT His Leu Giu Val Leu Arg Ile GAC ATG AAG GCA GAA GTC GAT Asp Met Lys Ala Glu Val Asp AAC ATG GGC AGA AAT GCC CTG, Asn Met Gly Arg Asn Ala Leu CTG CTG AAC TGG GAT TGT GAA Leu Leu Asn Trp Asp Cys Glu GAG ATT ACT TCA ATC CTG ATT Glu Ile Thr Ser Ile Leu Ile GCT GAT GTT AAC GTG AGA GGA Ala Asp Val Asn Val Arg Gly AAA ACA CCC CTC ATC GCA GCA Lys Thr Pro Leu Ile Ala Ala AAG CAC ACA GGC TTG GTG CAG Lys His Thr Gly Leu Val Gin AGT CGG GAA GGC ATA AAC ATA Ser Arg Glu Gly Ile Asn Ile GAT AAC GAG GGC AAG ACA GCT Asp Asn Glu Gly Lys Thr Ala GCT GTT GAT AAA CAA CTG AAG Ala Val Asp Lys Gin Leu Lys CAG TTG CTT CTT GAA AAG GGA Gin Leu Leu Leu Glu Lys Gly 408 136 438 146 468 156 498 166 528 176 558 186 588 196 618 206 648 216 678 226 708 236 738 246 768 256 798 266 828 276 858 286 888 296 TGT GAC GAT CTT GTT Cys Asp Asp Leu Val TGG ATA Trp Ile 918 306 WO 95/22245 PCTIUS9/02058 -88- GCC AGG AGG Ala Arg Arg AAG CTT CTC Lys Leu Leu GAC ACC GAC Asp Thr Asp CCT CAC AGT Pro His Ser AMA AGC CTC Lys Ser Leu ATT GGC AAA Ile Gly Lys GAC TAT AMA Asp Tyr Lys GCT GTC TAC Ala Val Tyr GMA GTG GCT Giu Val Ala AGC CCA CGT Ser-Pro Arg CTG CGG GAC Leu Arg Asp GTG GCT TTC Val Ala Phe GGC TGT TTA Gly Cys Leu GAG TGG ACA Glu Trp Thr CCC AGA GAG Pro Arg Glu GAT MAG TTT Asp Lys Phe I ATA TTT GAG C Ile Phe Giu C CAT GGA TAT I1 His Gly Tyr S AA1 Asr

CTC

Leu

CCI

Pro

TCA

Ser

CAC

His

CTC

Leu

ATT

Ile

CTA

Leu

GTG

Val

GGA

Gly rGC Cys

TAT

Iryr rAT ryr

:TG

Lieu flu ;cc lia

;GT

;ly 'cc ~er

I

CAT GAC His Asl CCT TAIJ Pro Tyz *CCT GCTI Pro Ala CGT TGG Arg Trp AGT ATG Ser Met MAG ATC Lys Ile GCT GOC Ala Gly GGG ATC Gly Ile MAG GTC Lys Val TGT MAG Cys Lys GGT GAC Gly Asp GGA AGA Gly Arg GTG TGT Val Cys GMA GAG Glu Glu CCT GTG Pro Val TAT CAC CTT GTA Tyr His Leu Val GTA GCT MAT CCT Val Ala Asn Pro GGA GAC TGG TCG Gly Asp Trp Ser GGG ACA GCC TTG, Gly Thr Ala Leu ACT CGA CCC ATG Thr Arg Pro Met TTC ATT CAT GAT Phe Ile His Asp ACT TCC GMA GGG Thr Ser Glu Gly TAT GAC MAT CGA Tyr Asp Asn Arg TTC CGT GAG MAT Phe Arg Glu Asn GMA GTC TCT TGT Giu Val Ser Cys CAC AGT MAC TTA His Ser Asn Leu GAG GAC GAT MAG Giu Asp Asp Lys GTG TCC CTG TGT Val Ser Leu Cys TTC CTG AGG TTG Pbhe Leu Arg Leu GAG MAC GGG GMA Glu Asn Gly Glu 948 316 978 326 1008 336 1038 346 1068 356 1098 366 1128 376 1158 386 1188 396 1218 406 1248 416 1278 426 1308 436 1338 446 1368 456 1398 466 1428 476 1458 486

CAC

His

GTT

Val

CAT

His AGC ATC CTA TTA TCT Ser le Leu Leu Ser CAA AAA CTA CAC TTG Gin Lys Leu His Leu CAG GAC CTG CMA CCA Gin Asp Leu Gin Pro WO 95/22245 PCTfUS95/02 0 5 8 -89- CAA AAC Gin Asn GTC CGG ATC TTA ATA lie Leu Ile CTG GCA GAT

GAT

Asp TCC AAG AAA

GCT

Ser Lys Lys Ala GAT CAG AGC ATC Asp Gin Ser Ile 1488 496 1518 506 Val Arg Leu Ala Asp Phe

CGA

Arg

AGA

TGG

Trp

GAC

ATG

Met

TTG

GGA

Gly

GAG

GA

Gi

GA

G

u

T

P

Arg Asp Leu Giu As CTC TAC Leu Tyr TTT GAG Phe Glu GTG CTG Val Leu ACT AAG Thr Lys CCT GGA Pro Gly GAC CTG Asp Leu TGG GAG Trp Glu CTG GGA Val Gly AAA TGTJ Lys Cys CAG CAT Gin His TTT GAC Phe Asp C AAT GTT ;Z Asn Val I GAA AAG A Glu Lys A

GTI

Va

AC)

Thl

CTI

Let

GAC

As;

GAA

Glu

CTT

Leu

AAC

Asn

AAT

Asn kAA Lys

:AG

Un

:AG

;in

LTG

let

LGA

rg GTA ATG i Val Met 9 CTA AAG Leu Lys ACA ATG Thr Met CTC ATT Leu Ile AAT GTC Asn Vai GGC CAT Gly His CGC TAT Arg Tyr GAA TCT Glu Ser AGT GAT 4 Ser Asp ACA CTT Thr Leu TGG ACA Trp Thr GAT GAA I Asp Giu 1! AAA AAA A Lys Lys A

TCA

Ser

CTT

Leu

AAA

Lys

ACT

Thr

TCT

Ser

CAT

His

AAG

Lys

CCT

Pro

AGA

Arg

GAC

Asp

CTT

Leu I ;AG C ;lu I CT A ;er i LTG A let A AC C sn P

CA

Gi

GG

Gi

GG

Gi'

CA

Gi,

CC)

Prc

TGC

Cyc

AAC

Asn

TTC

Phe kCA rhr kTC Ile

:TC

eu

CT

)ro

LAG

jys

AT

sn

CT

ro .G AT n Me

ACG

y Ar T GA y Gli 3 AA 1 Asi

SGA

Asj

CTC

SLet

TGC

Cys

TTI

Phe

CTC

Leu

AAA

Lys

AGA

Arg

CCC

Pro

ATC

Ile

CAT

His

TAT

Tyr G GTC t Val

GCTG

gLeu G ATC u Ile

PGAT

r Asp

['GAG

Glu

;TTT

iPhe, CTG 4 Leu

TGG

Trp CGG 2 Arg I GTA C Val I

CTAC

LeuL AGA A Arg S

GACA

AspL TTC T Phe? CAG G, Gin A!

AGG

Arg

GTT

Val

CCC

Pro

GAA

Glu

GAG

Glu

TCT

Ser

GTA

Val kCT Phr

AT

~sn

GG

rrg

TG

ieu

GC

er

AA

ys

AC

yr

AT

sp 1548 516 1578 526 1608 536 1638 546 1668 556 1698 566 1728 576 1758 586 1788 596 1818 606 1848 616 1878 626 1908 636 1938 646 1968 656 1998 666 ACT GTA GGT GAT CTG CTG AAG Thr Vai Gly Asp Leu Leu Lys TTT ATT CGG Phe Ile Arg WO 95/22245 PCT/US95/0205 8 AAT ATA GGC GAA CAC ATC AAT GAG GAA AAA 2028 Asn Ile Gly Glu His le Asn Giu Giu Lys 676 AAG CGG GGG 2037 Lys Arg Gly 679 SEQ ID NO:5: represents the DNA sequence encoding Murine 2 -5A-dependent RNase (partial). SEQ ID NO:6: represents the amino acid sequence encoded by SEQ ID NO:5:.

WO 95/22245 PCT/US95/02058 -91- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Silverman, Robert H.

SenGupta, Dibyendu N.

(ii) TITLE OF INVENTION: Antiviral Transgenic Plants, Vectors, Cells and Methods (iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Ruden, Barnett, McClosky, Smith, Schuster Russell STREET: 200 E. Broward Boulevard CITY: Fort Lauderdale STATE: Florida COUNTRY: USA ZIP: 33301 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US 08/198,973 FILING DATE: 18-FEB-1994 CLASSIFICATION: 1808 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Manso, Peter J.

REGISTRATION NUMBER: 32,264 REFERENCE/DOCKET NUMBER: CL11363-16 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 305/527/2498 TELEFAX: 305/764/4996 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2928 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 104..2326 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: WO 95/22245 PTU9/25 PCTIUS95/02058 -92- AATCCCAACT TACACTCAAA GCTTCTITMA TTAAGCTA GvGAGATAAAT TTGCAT=?C TCAAGGAAAA GGCTAAAADT GGTAGCAGGT GGCATTTACC G;TC ATO GAG AGC AGG Met Giu Ser Arg

I

TCC AGC Ser Ser

GAT

Asp

OCT

Ala

GAT

Asp

TTC

Phe

ATG

Met

CCT

Pro

ATT

Ile

GAT

Asp

OCT

Al a

GCA

Al a

AGO

Arg 165

GTA

Val

GCC

Ala

TCT

CAT AAC AAC His Asn Asn GCA GTG GAA Ala Val Qlu GTT GAC CTG Val Asp Leu CAG GAA GAG Gin Giu Glu AGC AGO GAG Ser Arg Glu GTT CTG AGO Val Leu Arg GCG GGG AGC Ala Gly Ser GTC AAT GAG Vai Asn Oiu 120 OTO TAT GOT Vai Tyr Gly 135 AAT GTO AAT Asn Val Asn 150 AAA OGA 000 Lys Gly Gly GAO OTC TTG Giu Val Leu

CCC

Pro

GAC

Asp

GTC

Val

GA.A

Glu

GAC

Asp

AAO

Lys

OTG

Val 105

TOT

Cys

AAG

Lys

TTG

Leu 0CC Ala

AAO

Lys 185

ATO

Met

GAT

CAG

Gin 10

AAT

An

CAG

Gin 000 Gly

AT?

Ile

AAG

Lys 90

AAG

Lys

.GAT

-Asp

GTC

Val

AGO

Arg

ACA

Thr 170

AT?

Ile

GGC

Oly

GTG

GAG

Giu

CAC

His

CAA

Gln

GOC

Gly

GTG

Val 75

AAT

Asn

CTG

Leu

TTT

Phe

AAA

Lys

CGA

Arg 155

OCT

Ala

CTC

Leu

AGA

Arg

GAG

GGA CCC ACG Gly Pro Thr TTG CTG AT? Leu Leu Ile 30 TG CTG GAA Leu Leu Giu 45 TGO ACA CCT Trp Thr Pro 60 G3AA CT? CTG Glu Leu Leu 000 Gly

CTG

Leu

TAT

Tyr

GCC

Ala 140

AAG

Lys

CTC

Leu

CT?

Leu

AAT

Asn

OCT

0CC Ala

AAA

Lys

GGC

Gly 125

CTA

Leu

ACA

Thr

ATG

Met

GAT

Asp

GCC

Ala 205

AT?

ACG

Thr

CT?

Leu 110

TC

Phe

AAA

Lys

AAO

Lys

GAC

Asp

GAG

Giu 190

TG

Leu

ACG

TCC

Ser 1s

AAA

Lys

GOT

Giy

CTG

Leu

CT?

Leu

CTT

Leu 95

TC

Phe

ACA

Thr

TC

Phe

GAG

Glu

OCT

Ala 175

ATO

Met

ATC

Ile

CAT

C

Ala

GGA

Gly

CAT

His

COT

Arg

TT

Phe Leu 0CC Ala arr Leu

GAT

Asp 160

OCT

Ala 000 Gly

CAT

His Cro

CT?

Val 0CC Ala

AAC

An

CAT

His

ATC

le

TCT

Ser

TC

Phe

TAT

Tyr 145

CAA

Gin

GAA

Oiu

GCA

Ala

OCT

Ala

CTG

GT

Giy

CAA

Gin

AAT

An

OCA

Ala

GOT

Giy

CTC

Leu

AAA

Lys

ATG

Met 130

AAO

Lys

GAG

Oiu

AAA

Lys

GA?

Asp

CTC

Leu 210 Cr0

AGA

Arg

AAC

An Val

O;TA

Val

GCT

Ala

OCA

Ala

OGA

Gly 115

GAA

Oiu

AGA

Arg

COO

Arg

GGA

Oly

GTA

Val 195 Cr0 Leu

GAC

AGO

Arg

OAA

Oiu

AAT

An

CAA

Gin

GAC

Asp

OCO

Ala 100

GCA

Ala 0CC Ala

GGA

Oly Cr0 Leu

CAC

His 180

AAC

An

AGC

Ser

CAT

115 163 211 259 307 355 403 451 499 547 595 643 691 739 787 Cys Asp GAC GAT WO 95122245 WO 9522245PCTfUS95/02058 -93- Ser Asp Asp Ser Asp Val Giu Ala Ile Th~r His Leu Leu Leu Asp His 215 220 225 GGG GCT GAT GTC AAT GTG AGG GGA GAA AGA GGG AAG ACT CCC CTG ATC 835 Gly Ala Asp Val Asn Val Ai-g Gly Glu Arg Gly Lys Thr Pro Leu Ile 230 235 240 CTG GCA GTG GAG AAG AAG CAC TTG GGT TTG GTG CAG AGG CTT CTG GAG B883 Leu Ala Val Giu Lys Lys His Leu Gly Leu Val Gin Arg Leu Leu Giu 245 250 255 260 CAA GAG CAC ATA GAG ATT AAT GAC ACA GAC AGT GAT 0CC AAA ACA GCA 931 Gin Giu His Ie Giu Ile Asn Asp Thr Asp Ser Asp Gly Lys Thr Ala 265 270 275 CTG CTG CTT GCT GTT GAA CTC AAA CTG AAG AAA ATC GCC GAG TTG CTG 979 Leu Leu Leu Ala Val Giu Leu Lys Leu Lys Lys Ile Ala Glu Leu Leu 280 285 290 TGC AAA COT GGA GCC AGT ACA GAT TGT GGG GAT CTT OTT ATG ACA GCG 1027 Cys Lys Arg Oly Ala Ser Thr Asp Cys Gly Asp Leu Val Met Thr Ala 295 300 305 AGG CGG AAT TAT GAC CAT TCC CTT GTG AAG OTT CTT CTC TCT CAT GGA 1075 Arg Arg Asn Tyr Asp His Ser Leu Val Lys Val Leu Leu Ser His Gly 310 315 320 GCC AAA GAA GAT TTT CAC CCT CCT GCT GAA GAC TGG AAG CCT CAG AGC 1123 Ala Lye Glu Asp Phe His Pro Pro Ala Giu Asp Trp Lys Pro Gin Ser 325 -330 335 340 TCA CAC TGG G GCA GCC CTG AAG GAT CTC CAC AGA ATA TAC CGC CCT 1171 Ser His Trp Gly Ala Ala Leu Lye Asp Leu His Arg Ile Tyr Arg Pro 345 350 355 ATG ATT GGC AAA CTC AAG TTC TTT ATT GAT GAA AAA TAC AAA ATT OCT 1219 Met Ile Gly Lye Leu-Lys Phe Phe Ile Asp Giu Lye Tyr Lye Ile Ala 360 365 370 GAT ACT TCA GAA GGA GGC ATC TAC CTG GOC TTC TAT GAG AAG CAA GAA 1267 Asp Thr Ser Glu Gly Gly Ile Tyr Leu Gly Phe Tyr Giu Lye Gin Giu 375 380 385 GTA GCT GTG AAG ACG TTC TOT GAG GGC AGC CCA COT GCA CAG CGG GAA 1315 Val Ala Val Lye Tkxr Phe Cys Giu Oly Ser Pro Arg Ala Gin Arg Giu 390 395 400 GTC TCT TOT CTG CAA AGC AGC CGA GAG AAC AGT CAC TTG GTG ACA TTC .1363 Val Ser Cys Leu Gin Ser Ser Arg Giu Asn Ser His Leu Val Thr Phe 405 410 415 420 TAT GGG AGT GAG AGC CAC AGG GGC CAC TTG TTT GTG TGT GTC ACC CTC 1411 Tyr Gly Ser Glu Ser His Arg Oly His Leu Phe Vai Cys Vai Thr Leu 425 430 435 TGT GAG CAG ACT CTG GAA GCG TGT TTG GAT GTG CAC AGA GOG GAA CAT 1459 Cys Glu Gin Thr Leu Giu Ala Cys Leu Asp Vai His Arg Gly Oiu Asp 440 445 450 GTG GAA AAT GAG GAA CAT GAA TTT GCC CGA AAT GTC CTG TCA TCT ATA 1507 WO 95122245 WO 9522245PCT/US95/02058 -94- Val Glu Asn Glu Glu Asp Qlu Phe Ala Arg Aen Vai Lou Ser Ser Ile 455 460 465 TTT AAG OCT OTT CAA GAA CTA CAC TrO TCC TOT GGA TAC ACC CAC CAG 1555 Phe Lys Ala Val Gin Giu Leu His Lou Ser Cys Gly Tyr Tbx His Gin 470 475 480 GAT CTG CAA CCA CAA AAC ATC TTA ATA GAT TCT AAG AAA GCT GCT CAC 1603 Asp Lou Gin Pro Gin Asn Ile Lou Ile Asp Ser Lys Lye Ala Ala His 485 490 495 500 CTG OCA OAT TTT GAT AAG AGC ATC AAG TOO OCT GGA GAT CCA CAG GAA 1651 Leu Ala Asp Phe Asp Lys Ser Ile Lys Trp Ala Gly Asp Pro Gin Giu 505 510 515 GTC AAG AGA GAT CTA GAG GAC CTT OGA CGG CTG GTC CTC TAT GTG GTA 1699 Val Lys Arg Asp Lou Giu Asp Lou Gly Arg Lou Val Leu Tyr Val Val 520 525 530 AAG AAG OGA AGC ATC TCA TTT GAG GAT CTG AAA OCT CAA AGT AAT GAA 1747 Lys Lys Gly Ser Ile Ser Phe Glu Asp Lou Lys Ala Gin Sor Asn Glu 535 540 545 GAG GTG GTT CAA CTI' TCT CCA OAT GAG GAA ACT AAG GAC CTC ATT CAT 1795 Giu Val Val Gin Leu 5cr Pro Asp Giu Glu Thr Lys Asp Lou Ile His 550 555 560 CGT CTC TTC CAT CCT 000 GAA CAT GTO AGO GAC TGT CTG AGT GAC CTG 1843 Arg Lou Phe His Pro Oly Giu His Val Arg Asp Cys Lou Ser Asp Lou 565 570 575 580 CTG GGT CAT CCC TTC TTT TOG ACT TGO GAG AOC COC TAT AGG, ACG CTT 1891 Leu Gly His Pro Pho Phe Trp Thr Trp Giu Ser Arg Tyr Arg Thr Leu 585 590 595 CGG AAT GTG GGA AAT OAA TCC GAC ATC AAA ACA CGA AAA TCT GAA ACT 1939 Arg Asn Val Gly Asn-Glu Ser Asp Ile Lye Thr Arg Lys Ser Giu 600 605 610 GAG ATC CTC AGA CTA CTG CAA CCT GGG CCT TCT GAA CAT TCC AAA AGT 1987 Glu Ile Lou Arg Leu Lou Gin Pro Gly Pro Ser Giu His 5cr Lys Ser 615 620 625 TTT GAC AAG TGG ACO ACT AAG ATT AAT GAA TGT OTT ATG AAA AAA ATG 2035 Phe Asp Lye Trp Thr Thr Lys Ile Aen Glu Cys Val Met Lys Lys Met 630 635 640 AAT AAG TT TAT GAA AAA AGA GGC AAT TTC TAC CAG AAC ACT GTO GOT 2083 Asn Lys Phe Tyr Oiu Lys Arg Oly Aen Phe Tyr Gin Asn Thr Val Oly 645 650 655 660 GAT CTG CTA AAG TTC ATC CGG AAT TTO GGA GAA CAC ATT OAT GAA GAA 2131 Asp Lou Leu Lys Phe Ile Arg Asn Leu Gly Oiu His Ile Asp Olu Olu 665 670 675 AAG CAT AAA AAG ATG AAA TTA AAA ATT GGA GAC CCT TCC CTG TAT TTT 2179 Lye His Lye Lye Met Lys Lou Lye Ile Gly Asp Pro 5cr Lou Tyr Phe 680 685 690 CAG AAG ACA TTT CCA OAT CTG GTG ATC TAT GTC TAC ACA AAA CTA CAG 22 2227 WO 95/22245 PCT/US95/02058 Gin Lys Thr Phe Pro Asp Lou Val le Tyr Val Tyr 695 700 AAC ACA GAA TAT AGA AAG CAT TTC CCC CAA ACC CAC Asn Thr Glu Tyr Arg Lys His Phe Pro Gin Thr His 710 715 720 CCT CAG TGT GAT OGA GCT GGT GGG GCC AGT COG TTG Pro Gin Cys Asp Gly Ala Gly Gly Ala Ser Gly Lou 725 730 735 Thr Lys Lou Gin 705 AGT CCA AAC AAA Ser Pro Asn Lys GCC AGC CCT Ala Ser Pro

TGC

Cys TGATGGACTG, ATTTGCTGGA GTTCAGGGAA CTACTTATTA GCTGTAGAGT C CTT GGCAAA TCACAACATT TTGCATAGCT GATATGTCAG ATATATACCC AGACTACACT TAAGATTCCT TTTGTCAATT TGTTACAATT CTCTCACTTA A TTT AAGAAC TGAGGAACCT ATTTATACCT AGCACTTTAT TTAAAACTTA ACTAT CTT CC CCAGGAATCT CATTCATTCA GATGCATGAA TT

CTGGGCCTTT

TCCC!TGGCAT

AGTCCATAAG

GCACCAAAAG

ATTTTCCCAA

GAGACTCAGA

AAATTTATGT

AGGGCTC

TTCAGTATTT

TAACTCACCA

CGTGTATTCC

CTTTACCCAC

AATG;AGTGCC

TGATCTTGCA

GAGTGTGAGC

GGTGTTATTG

CAGATGAGGC

ATTGAGCATC

GGTTGCTTGT

ATATGTCTAT

TAACTGGGAG

TTGACCCCTA

AAACAGGGAT

TACTGGCCCA

GTACCTCTCA

CCAAAACATA

TAGTATAAGT

GAGGGATGAG

AACAAAAGCA

GACATTCTGC

ATGCTGCATA

TATCATCCCC

AGATTATTCA

TTTGGGCACC

TATAGGGGTT

CTGGGCACTG,

2275 2323 2376 24.36 2496 2556 2616 2676 2736 2796 2856 2916 2928 INFORMATION FOR-SEQ ID NO:2: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 741 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Glu Ser Arg Asp His Asn Ann Pro Gin Glu Gly Pro Thr Ser Ser 1. 5 10 is Ser Gly Arg Arg Ala Ala Val Glu Asp Ann His Lou Lou 25 Val Gin Asn Glu Asp Val Asp Lou Val Gin Gin Lou Lou 40 Ala Asn Val Ann Phe Gin Giu Giu Giu Giy Gly Trp Thr 55 Ann Ala Val Gin Met Ser Arg Giu Asp Ile Val Giu Lou Ile Lys Ala Giu Gly Gly Pro Lou His Lou Lou Arg WO 95/22245 WO 9522245PCTfUS95/02058 -96- 70 75 s0 His Gly Ala Asp Pro Val Leu Arg Lys Lye Asn Gly Ala Thr Leu Phe 8590 Ile Leu Ala Ala Ile Ala Gly Ser Val Lys Leu Leu Lye Leu Phe Leu 100 105 110 Ser Lys Gly Ala Asp Val Aen Giu Cys Asp Phe Tyr Gly Phe Thr Ala 115 120 125 Phe Met Glu Ala Ala Val Tyr Gly Lye Val Lys Ala Leu Lys Phe Leu 130 135 140 Tyr Lys Arg Gly Ala Asn Val Asn Leu Arg Arg Lys Thr Lye Glu Asp 145 150 155 160 Gin Giu Arg Leu Arg Lys Gly Gly Ala Thr Ala Leu Met Asp Ala Ala 165 170 175 Giu Lye Gly His Val Glu Val Leu Lys Ile Leu Leu Asp Giu Met Gly 180 185 190 Ala Asp Val Asn Ala Cys Asp Asn Met Gly Arg Asn Ala Leu Ile His 195 200 205 Ala Leu Leu Ser Ser Asp Asp Ser Asp Val Giu Ala Ile Thr His Leu 210 215 220 Leu Leuf Asp His Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lye 225 230 235 240 Thr Pro Leu Ile Leu Ala Val Glu Lys Lye His Leu Giy Leu Val Gin 245 250 255 Arg Leu Leu Giu Gin Glu His Ile Glu Ile Asn Asp Thr Asp Ser Asp 260 -265 270 Gly Lys Thr Ala Leu Leu Leu Aia Val Giu Leu Lye Leu Lye Lye Ile 275 280 285 Ala Giu Leu Leu Cys Lye Arg Gly Al.a Ser Thr Asp Cys Gly Asp Leu 290 295 300 Val Met Thr Aia Arg Arg Ann Tyr Asp His Ser Leu Val Lye Val Leu 305 310 315 320 Leu Ser His Gly Ala Lye Glu Asp Phe His Pro Pro Ala Giu Asp Trp 325 330 335 Lys Pro Gin Ser Ser His Trp Gly Ala Ala Leu Lye Asp Leu His Arg 340 345 350 Ile Tyr Arg Pro Met Ile Gly Lye Leu Lye Phe Phe Ile Asp Giu Lye 355 360 365 Tyr Lye Ile Ala Asp Thr Ser Giu Gly Gly Ile Tyr Leu Gly Phe-Tyr 370 375 380 Glu Lys Gln Glu Val Ala Val Lys Thr Phe Cys Glu Gly Ser Pro Arg WO 95122245 WO 9522245PCTIUS95/02058 -97- 385 390 395 400 Ala Gin Arg Glu Val Ser Cys Leu Gin Ser Ser Arg Glu Asn Ser His 405 410 415 Leu Val Thr Phe Tyr Gly Ser Glu Ser His Arg Gly His Leu Phe Val 420 425 430 Cys Vai Thr Leu Cys Glu Gin Thr Leu Giu Ala Cys Leu Asp Val His 435 440 445 Arg Giy Giu Asp Val Giu Asn Giu Giu Asp Giu Phe Ala Arg Asn Val 450 455 460 Leu Ser Ser Ile Phe Lys Ala Val Gin Giu Leu His Leu Ser Cys Gly 465 470 475 480 Tyr Thr His Gin Asp Leu Gin Pro Gin Asn Ile Leu Ile Asp Ser Lys 485 490 495 Lys Ala Ala His Leu Ala Asp Phe Asp Lys Ser Ile Lys Trp Ala Gly 500 SO5 510 Asp Pro Gin Giu Val Lys Arg Asp Leu Giu Asp Leu Gly Arg Leu Val 515 520 525 Leu Tyr Vai Vai Lys Lys Gly Ser Ile Ser Phe Giu Asp Leu Lys Ala 530 -535 540 Gin Ser Asn Giu Giu Val Vai Gin Leu Scr Pro Asp Giu Giu Thr Lys 545 550 555 560 Asp Leu Ile His Arg Leu Phe His Pro Gly Giu His Vai Arg Asp Cys 565 -570 575 Leu Ser Asp Leu Leu Giy His Pro Phe Phe Trp Thr Trp Giu Ser Arg 580 585 590 Tyr Arg Thr Leu Arg Asn Vai Gly Asn Giu Ser Asp Ile Lys Thr Arg 595 600 605 Lys Ser Giu Ser Giu Ile Leu Arg Ley Leu Gin Pro Giy Pro Ser Giu 610 615 620 His Ser Lys Ser Phe Asp Lys Trp, Thr Thr Lys Ile Asn Giu Cys Val 625 630 635 640 Met Lys Lys Met Asn Lys Phe Tyr Giu Lys Arg Giy Asn Phe Tyr Gin 645 650 655 Asn Thr Val Giy Asp Leu Leu Lys Phe Ile Arg Asn Leu Gly Giu His 660 665 670 Ile Asp Giu Giu Lys His Lys Lys Met Lys Leu Lys Ile Giy Asp Pro 675 680 685 Ser Leu Tyr Phe Gin Lys Thr Phe Pro Asp Leu Vai Ile Tyr Val Tyr 690 695 700 Thr Lys Leu Gin Asn Thr Giu Tyr Arg Lys His Phe Pro Gin Thr His WO 95/22245 WO 9522245PCTJUS95/02058 -98- 720 705 Ser Pro Asn Ala Ser Pro Lys Pro Gin Cys Asp Gly Ala Gly Gly Ala Ser Gly Leu 725 730 .735 Gly Cys 740 INFORMATION FOR SEQ ID NO:3: Wi SEQUENCE CHARACTERISTICS: LENGTH: 2928 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE- NA1.E/KY: CDS LOCATION: 104. .2326 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AATCCCAACT TACACTCAAA GCTTCTZ'TGA TAAGTGCTA GGAGATAAAT TTGCATTTTC TCAAGGAAAA GGCTAAAAGT GGTAGCAGGT GGCATTTACC GTC ATG GAG AGC AGG Met Giu Ser Arg

I

115

GAT

Asp CAT AAC AAC CCC CAG GAG GGA CCC ACG His Asn Asn Pro Gin Giu Gly Pro Thr TCC AGC GGT AGA Ser Ser Gly Arg CCT GCA GTG GAA Ala Ala Val Glu GAC -AAT Asp Asn CAC TTG CTG His Leu Leu AAA GCT GTT CAA Lys Ala Val Gin AAC GAA Asn Glu CAT GTT GAC Asp Val Asp TTC CAG GAA Phe Gin Giu GTC CAG CAA TTG Val Gin Gin Leu GAA GGT GGA GCC Giu Gly Gly Ala AAT GTT AAT Asn Val Asn GCA GTA CAA Ala Val Gin GAG GAA GGG GGC Giu Giu Gly Gly ACA CCT CTG CAT Thr Pro Leu His ATG AGC Met Ser AGG GAG GAC ATT Arg Giu Asp Ile

GTG

Val GAA CTT CTG CTT Giu Leu Leu Leu CAT GGT GCT GAC His Gly Ala Asp 355 CCT GTT CTG AGG Pro Val Leu Arg ATT GCG GGG AGC Ile Ala Gly Ser AAG AAG AAT Lys Lys Asn 90 GOG GCC ACG Gly Ala Thr TTT ATC CTC GCA Phe Ile Leu Ala

GCG

Ala 100 GTG AAG CTG CTG AAA CTT TTC CTT TCT AAA GGA GCA 451 Val Lys Leu Leu Lys Lou Phe Lou Ser Lys Gly Ala 105 110 115 WO 95/22245 PCT/US95/02058 -99- GAT GTC AAT Asp Val Asn GCT GTG TAT Ala Val Tyr 135

GAG

Glu 120 TGT GAT TTT TAT Cys Asp Phe Tyr ,GOC TTC Gly Phe 125 ACA GCC TTC Thr Ala Phe ATG GAA GCC Met Giu Ala 130 AAG AGA GGA Lye Arg Gly GGT AAG GTC AAA Gly Lye Val Lye

GCC

Ala 140 CTA AAA TTC CTT Leu Lye Phe Leu

TAT

Tyr 145 GCA AAT Ala Asn 150 GTG AAT TTG AGO Val Asn leu Arg

CGA

Arg 155 AAG ACA AAO GAG Lye Thr Lys Glu

GAT

Asp 160 CAA GAG CGG CTG 595 Gin Giu Arg Leu AGG AAA OGA GGO CC ACA GCT CTC ATG GAC GCT GCT GAA AAA GGA CAC Arg Lys Gly Gly Ala Thr Ala Leu Met Asp Ala Ala Glu Lys Gly His 165 170 175 180 GTA GAG GTC TTG Val Glu Val leu

AAG

Lys 185 ATT CTC CTT GAT Ile leu leu Asp ATG GGG GCA GAT Met Gly Ala Asp GTA AAC 691 Val Asn 195 GCC TOT GAC A Ala Cys Asp A TCT GAC GAT I Ser Asp Asp S 215 hAT sn !00 ATG GGC AGA AAT Met Gly Arg Asn

GCC

Ala 205 TTG ATC CAT GCT Leu Ile His Ala CTC CTG AGC Leu leu Ser 210 CTG GAC CAT Leu Asp His 739 hGT GAT GTG GAG jer Asp Val Glu

GCT

Ala 220 ATT ACG CAT CTG Ile Thr His Leu

CTG

Leu 225 GGG GCT Gly Ala 230 GAT GTC AAT GTG Asp Val Asn Val

AGO

Arg 235 GGA GAA AGA GGG Gly Giu Arg Gly

AAG

Lys 240 ACT CCC CTG ATC Thr Pro Leu Ile

CTG

Zeu 245 GCA GTG GAG AAG Ala Val Giu Lys

AAG

Lys 250 CAC TTG GOT TTG His leu Gly Leu

GTG

Val 255 CAG AGO CTT CTG Gin Arg leu Leu CAA GAG CAC ATA Gln Giu His Ile GAO -ATT Glu Ile 265 AAT GAC ACA Asn Asp Thr AGT GAT GOC AAA Ser Asp Gly Lye ACA GCA Thr Ala 275 CTG CTG CTT Leu Leu Leu TGC AAA CGT Cys Lys Arg 295

GCT

Ala 280 GTT GAA CTC AAA Val Glu Leu Lye AAG AAA ATC GCC Lye Lye Ile Ala GAG TTG CTG Glu leu Leu 290 ATG ACA GCG Met Thr Ala GGA 0CC ACT ACA Oly Ala Ser Thr TGT GGG GAT CTT Cys Gly Asp leu

GTT

Val 305 AGG CGG Arg Arg 310 AAT TAT GAC CAT Asn Tyr Asp His CTT GTG AAG GTT Leu Vai Lye Val

CTT

Leu 320 CTC TCT CAT GGA leu Ser His Gly 1027 1075 1123 1171

GCC

Ala 325 AAA GAA GAT TTT Lys Olu Asp Phe CCT CC? GCT GAA Pro Pro Ala Giu

GAC

Asp 335 TGG AAG CCT CAG Trp Lys Pro Gin

AGC

Ser 340 TCA CAC TGG GG Ser His Trp Gly GCC CTG AAG CAT Ala Leu Lye Asp

CTC

Leu 350 CAC AGA ATA TAC His Arg Ile Tyr CGC CCT Arg Pro 355 WO 95/22245 WO 9522245PCTIUS95/02058 -100- ATG ATT GGC Met Ile Gly GAT ACT TCA Asp Thr Ser 375 Lys 360 CTC AAG TTC TTT Leu Lye Phe Phe ATT O3AT GAA AAA TAC )AAA ATT GCT Ile Asp Giu Lys Tyr Lye Ile Ala 365 370 GAA OGA GG;C ATC Giu Gly Gly Ile

TAC

Tyr 380 CTG GGG TTC TAT Leu Giy Phe Tyr

GAG

Giu 385 AAG CAA GAA Lys Gin Giu GTA GCT Val Ala 390 GTG AAG ACG TTC Val Lys Thr Phe

TGT

Cys 395 GAG GGC AGC CCA Giu Gly Ser Pro CGT GCA CAG CG Arg Ala Gin Arg 400 CAC TTG GTG ACA His Leu Vai Thr

GAA

Giu

TTC

Phe 420

GTC

Vai 405 TCT TOT Cro CAA Ser Cys Leu Gin

AOC

Ser 410 AGC CGA GAG AAC Scr Arg Giu Asn

AGT

Ser 41S TAT 000 AGT GAG AOC CAC AGO GGC CAC TTG TlT GTO TGT GTC ACC CTC Tyr Gly Ser Oiu Ser His Arg Giy His Leu Phe Vai Cys Vai Thr Leu 425 430 435 TGT GAG CAG Cys Giu Gin GTG GAA AAT Val Giu Asn 455 TTT AAG.GeT Phe Lys Ala 470

ACT

Thr 440 CTG OAA GCG TOT Leu Giu Ala Cys

TTG

Leu 445 OAT GTG CAC AGA Asp Vai His Arg 000 GAA GAT Gly Giu Asp 450 TCA TCT ATA Ser Ser Ile -'1219 1267 1315 1363 1411 1459 1507 1555 1603 1651 1699 1747 GAG GAA OAT GAA Giu Oiu Asp Giu

TTT

Phe 460 0CC CGA AAT GTC Ala Arg Asn Val

CTG

Leu 465 OTT CAA GAA Val Gin Giu

CTA

Leu 475 CAC TTO TCC TGT His Leu Ser Cys

OGA

Gly 480 TAC ACC CAC CAG Tyr Thr His Gin CTG CAA CCA CAA Leu Gin Pro Gin ATC TTA ATA GAT Ile Leu Ile Asp AAG AAA OCT GCT Lys Lys Ala Ala

CAC

His 500 CTG GCA GAT TTT Leu Ala Asp Phe GAT -AAG Asp Lys 505 AGC ATC AAG Ser Ile Lys

TG

Trp 510 OCT GGA GAT CCA Ala Gly Asp Pro CAG OAA Gin Giu 515 GTC AAG AGA Val Lys Arg AAG AAG OGA Lys Lys Giy 535 CTA GAG GAC CTT Leu Giu Asp Leu

GGA

Gly 525 CGG CTG GTC CTC Arg Leu Vai Leu TAT GTG OTA Tyr Val Vai 530 AGT ANT OAA Ser Asn Glu AGC ATC TCA TTT Ser Ile Ser Phe

GAG

Giu 540 GAT CTG AAA GCT Asp Leu Lys Ala

CAA

Gin 545 GAG OTG Giu Val 550 OTT CAA CTT TCT Val Gin Leu Ser

CCA

Pro 555 OAT GAG OAA ACT Asp Giu Giu Thr GAC CTC ATT CAT Asp Leu Ile His

CGT

Arg 565 CTC TTC CAT CCT Leu Phe His Pro 000 Gly 570 GAA CAT GTG AGO Giu His Vai Arg TOT CTG AGT GAC Cys Leu Ser Asp

CTG

Leu 580 1795 1843 1891 CTG GOT CAT CCC Leu Gly His Pro

TTC

Phe 585 TTT TOG ACT TG Phe Trp Thr Trp

GAG

Giu 590 AGC COC TAT AGO Scr Arg Tyr Arg ACO CTT Thr Leu 595 WO 95/22245 WO 9522245PCTIUS95/02058 -101- CGG AAT GTG GGA AAT GAA TCC GAC ATC Arg Asn Val Gly Asn Glu Ser Asp Ile 600 605 AAA ACA CGA AAA Lys Thr Arg Lys TCT GAA ACT Ser Giu Ser 610 TCC AAA ACT Ser Lys Ser GAG ATC CTC Glu Ile Leu 615 TTT GAC AAC Phe Asp Lys AGA CTA C"1G CAA Arg Leu Leu Gin

CCT

Pro 620 GGG CCT TCT GAA Gly Pro Ser Giu

CAT

His 625 MGG ACG ACT AAG A= AAT GAA =C =T ATC AAA AAA ATO Trp Tbr Thr Lys Ile Asn Giu Cys Val Met Lys Lys Met 635 640 630 AAT AAG Asn Lys TI'T TAT GAA Phe Tyr Clu AAA AGA Lys Arg 650 GCC AAT TTC Cly Asn Phe

TAC

Tyr 655 CAG AAC ACT GTC Gin Ann Thr Val

GGT

Cly 660 645

GAT

Asp CTG CTA AAG Leu Leu Lys ATC CCC AAT TTG Ile Arg Ann Leu

GGA

Gly 670 GAA CAC ATT GAT Glu His Ile Asp GAA GAA Ciu Glu 675 AAG CAT AAA Lys His Lys CAG AAG ACA Gin Lys Thr 695

AAC

Lys 680 ATO AAA TTA AAA Met Lys Leu Lys

ATT

Ile 685 GGA GAC CCT TCC Cly Asp Pro Ser CTG TAT TTT Leu Tyr Phe 690 AAA CTA CAG Lys Leu Gin CCA CAT CTG Phe Pro Asp Leu

GTG

Val 700 ATC TAT CTC TAC Ile Tyr Val Tyr

ACA

Thr 705 AAC ACA Asn Thr 710 CCT CAG Pro Gin 725 -GA TAT AGA Glu Tyr Arg TGT GAT GGA Cys Asp Gly AAG CAT Lys His 715 OCT COT Ala Cly 730 TTC CCC CAA ACC Phe Pro Gin Thr CAC ACT CCA AAC His Ser Pro Asn 720 TTC CCC ACC CCT Leu Ala Ser Pro

AAA

Lys

GGG

Gly 740 1939 1987 2035 2083 2131 2179 2227 2275 2323 2376 2436 2496 2556 2616 2676 2736 2796 2856 2916 2928 CCC CCC ACT Gly Ala Ser

TGC

Cys TGATGGACTG ATTTGCTGGA CTTCAGGGAA CTACTTATTA CCTCTAGACT

CTTGGCAAA

TTGCATAGCT

ATATATACCC

TAAGATTCCT

TGTTACAATT

ATTTAAGAAC

ATTTATACCT

TTAAAACTA

CCAGGAATCT

GATGCATGAA

TCACAACATT

GATATGTCAG

AGACTACACT

TTTGTCAATT

CTCTCACTA

TGAGGAACCT

AGCACTTTAT

ACTATCTTCC

CATTCATTCA

TT

CTGGGCCTT

TCCCTGGCAT

AGTCCATAAG

GCACCAAAAG

ATTTTCCCAA

GAGACTCAGA

AAATTTATGT

ACGGCTCTTC

TTCACTATT'

TAACTCACCA

dGTCTATTCC

CTTTACCCAC

AATGAGTGCC

TGATCTTGCA

GACTGTGAGC

GGTGTTATTG

CAGATGAGGC

ATTGAGCATC

GGTTGCTTGT

ATATGTCTAT

TAACTGGGAG

TTGACCCCTA

AAACACGGAT

TACTGGCCCA

CTACCTCTCA

CCAAAACATA

TACTATAAGT

GAGGGATGAG

AACAAAAGCA

GACATTCTC

ATGCTGCATA

TATCATCCCC

AGATTATTCA

TTTGGGCACC

TATACGCTT

CTGCACTG

WO 95/22245 WO 9522245PCTIUS95/02058 -102- INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 741. amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met Ser Val Ala Asn His Ile Ser Phe Tyr 145 Gin Glu Al a Al a Leu 225 Thr (xi) SEQUENCE Giu Ser Arg Asp Gly Arg Arg Ala Gin Asn Glu Asp Asn Val Asn Phe Ala Val Gin Met Gly-Aia Asp Pro Lieu Ala Ala Ile 100 Lys Gly Ala Asp 115 Met Giu Ala Ala- 130 Lys Arg Gly Ala Giu Arg Leu Arg 165 Lys Gly His Val 180 Asp Val Asn Ala 195 *Leu Leu Ser Ser 210 Leu Asp His Gly *Pro Leu Ile Leu 245 )ESCRIPTION: SEQ ID NO:4: liB Asn Asn Pro Gin Glu Gly Pro Thr Ser Ser kla Vl Ser 70 Val Ala Val Val Asn 150 Lys Giu Cys Asp Ala 230C Ala Val Asp Glu Arg Leu Gly Asn Tyr 135 Val Gly Val Asp Asp 215 Asp Val 1lu Lieu 40 Glu Arg Ser Glu 120 Gly Asn Gly Leu Asn 200 Ser Val Glu Asp 25 Val Giu Asp Lys Val 105 Cys Lys Leu Ala Lys 185 Met Asp Asn Lys Asn Gln Gly Ile Lys 90 Lys Asp Val Arg Thr 170 Ile Gly Val Val Lys 250 His Gin Gly Val 75 Asn Leu Phe Lys Arg 155 Ala Leu Arg *Glu *Arg 235 His Lieu Lieu rrp Giu Gly Lieu Tyr Ala 140 Lys Leu Leu Asn Ala 220 Gly Leu Leu Ile Lieu Giu Thr Pro Lieu Leu Ala Thr Lys Lieu 110 Giy Phe 125 Leu Lys Thr Lys Met Asp Asp Glu 190 Ala Leu 205 Ile Thr Giu Arg Gly Leu Lys Ala G1y Gly Lieu His Leu Arg Pro Phe Phe Lieu Thr Ala Phe Leu Glu Asp 160 Ala Ala 175 Met Gly Ile His His Lieu Gly Lys 240 Val- Gin 255 WO 95/22245 WO 9522245PCTIUS95/02058 -103- Arg Leu Leu Giu Gin Giu His Ile Giu Ile Asn Asp Thr Asp Ser Asp 260 265 270 Gly Lys Tbx Ala Leu Leu Lau Ala Val Giu Leu Lys Leu Lys Lys Ile 275 280 295 Ala Giu Leu Leu Cys Lys Arg Gly Ala Ser Thr Asp Cys Gly Asp Leu 290 295 300 Val Met Thr Ala Arg Arg Asn Tyr Asp His Ser Leu Vai Lys Vai Lcu 305 310 315 320 Leu Ser His Gly Ala Lys Giu Asp Phe His Pro Pro Ala Giu Asp Trp 325 330 .335 Lys Pro Gin 5cr Ser His Trp Gly Ala Ala Lcu Lys Asp Leu His Arg 340 345 350 Ile Tyr Arg Pro Met Ile Gly Lys Lcu Lys Phe Phe Ile Asp Giu Lys 355 360 365 Tyr Lys Ile Ala Asp Thr Ser Giu Gly Gly Ile Tyr Leu Gly Phe Tyr 370 375 380 Giu Lys Gin Giu Vai Ala Val Lys Thr Phe Cys Giu Gly Ser Pro Arg 385 390 395 400 Ala Gin Arg Giu Val 5cr Cys Leu Gin Ser 5cr Arg Giu Asn Ser His 405 410 415 Leu Val Thr Phe Tyr Gly Ser Giu Ser His Arg Gly His Leu Phe Val 420 425 430 Cys Val Thr Leu Cys Giu Gin Thr Lcu Giu Ala Cys Leu Asp Val His 435 440 445 Arg Gly Giu Asp Vai-Glu Asn Giu Giu Asp Giu Phe Ala Arg Asn Val 450 455 460 Leu Ser Ser Ile Phe Lys Ala Val Gin Giu Leu His Leu 5cr Cys Gly 465 470 475 480 Tyr Thr His Gin Asp Leu Gin Pro Gfn Asn Ile Leu Ile Asp 5cr Lys 485 490 495 Lys Ala Ala His Leu Ala Asp Phe Asp Lys 5cr Ile Lys Trp Ala Gly 500 505 510 Asp Pro Gin Giu Val Lys'Arg Asp Leu Giu Asp Leu Gly Arg Leu Val 515 520 525 Lcu Tyr Val Val Lys Lye Gly Ser Ile 5cr Phe Giu Asp Leu Lys Ala 530 535 540 Gin Ser Asn Giu Giu Vai Val Gin Lcu Ser Pro Asp Giu Giu Thr Lys 545 550 555 560 Asp Leu Ile His Arg Leu Phe His Pro Gly Giu His Vai Arg Asp Cys 565 570 575 WO 95/22245 WO 9522245PCT/US95/02058 -104- Leu Ser Asp Leu Leu Giy His Pro Phe Phe Trp Tbr Trp Giu Ser Arg 580 585 590 Tyr Arg Thr Leu Axg Asn Val Giy Ann Giu Ser Asp Ile Lys Thr Arg 595 600 605 Lys 3cr Giu Ser Giu Ile Leu Axg ILeu Leu Gin Pro Gly Pro Ser Giu 610 615 .620 His Ser Lys Ser Phe Asp Lys Tip, Thr Thr Lys Ile Ann Giu Cys Val 625 630 635 640 Met Lys Lys Met Ann Lys Ph. Tyr Giu Lye Arg Gly Ann Phe Tyr Gin 645 650 655 Asn Thr Val Gly Asp Leu Leu Lys Phe Ile Arg Ann Leu Gly Giu His 660 665 670 Ile Asp Giu Giu Lye His Lys Lys Met Lys Leu Lye Ile Gly Amp Pro 675 680 685 Ser Leu Tyr Phe Gin Lye Thr Phe Pro Amp Leu Val Ile Tyr Val Tyr 690 695 700 Thr Lys Leu Gin Ann Thr Giu Tyr Arg Lys His Phe Pro Gin Thr His 705 710 715 720 Ser Pro Asn Lye Pro Gin Cys Asp Gly Ala Gly Gly Ala Ser Gly Leu 725 730 735 Aia Ser Pro Gly Cys 740 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH-. 2200 base pairs TYPE: nucleic acid STRANflEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genornic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 164. .2200 (xi) SEQUENCE DESCRIPTION: SEQ ID ATTCGGCACG AGGAAGGTGC CAATTACTAG CTCCCTTCTT TATrCGTGTA CTGATGAGAT GTCAGAAGAC AGAACATAAT CAGCCCAATC CCTACTCCAA GACTCTCATr GTGTCCCAAA 120 GAAACACACG TGTGCATTTC CCAAGGAAAA GGCAT1'GAGG ACC ATG GAG ACC CCG 175 Met Glu Thr Pro 1 GAT TAT AAC ACA CCT CAG GG;T GGA ACC CCA TC& GCG GGA AGT CAG AGG 223 WO 95/22245 WO 9522245PCT/US95/02058 -105- Asp

ACC

Thr

GAT

Asp

GCC

Ala

GCT

Ala

CCT

Pro

ATC

Ile

GAC

Asp

GO-T

Al a

GC

Al a

AAG

Lys 165

CTG

Leu

GCT

Al a

TGG

Trp

GG

Gly Tyr

GTT

Val

GTT

Val

TOT

Cys

GGC

Gly

CAT

His

CAG

Gin

GTC

Vai

GAG

Giu

AAT

Asn

CAA

Gin

GAA

Glu

CG

Arg

GAT

Asp

OCT

Ala 230 Asn

GTC

Val

GTC

Val

GAA

Oiu 55

AG

Arg

CG

Arg

OGA

Gly

.A~T

Asn

COT

Arg 135

GTG

Val

GGA

Giy

OTC

Val

GAC

Asp

TOT

Cys 215

OAT

Asp Thr

GAA

Oiu

AGO

Arg 40

GAC

Asp

OTA

Val

AGG

Arg

OAT

Asp

GAG

Glu 120

GT

Gly

AAT

Asn 000 Oly

CTO

Leu

AAC

Asn 200

OAA

Oiu OTr Val Pro Gin Gly Gly Tbr Pro Ser Ala 10 i5 GAT GAT TCT TCG TTG ATC AAA GCT Asp Asp Ser Ser Leu Ile Lys Ala 25 30 OTC CAG CAA lTrG TTA OAA AAA 000 Val Gin Gin Leu Leu Giu Lys Gly 45 ACC TOG 000 TOO ACA CCT TrO CAC Thr Trp Oly Trp Thr Pro Leu His 60 GAC ATT OTG AAC CTC CTO CT!' AOT Asp Ile Val Asn Leu Leu Leu Ser 75 s0 AAO AAO AAT 000 0CC ACC CCC TTC Lys Lys Asn Gly Ala Thr Pro Phe 90 95 OTG AAA CTG CTC GAG ATT CTC CTC Val Lys Leu Leu Oiu Ile Leu Leu 105 110 TOT GAC GAG AAC GGA TTC ACO OCT Cys Asp Giu Asn Gly Phe Thr Ala 125 AAC OCT GAA GCC TTA AGA TTCCT Asn Ala Oiu Ala Leu Arg Phe Leu 140 TTG COA CGA CAG ACA ACG AAO GAC Leu-Arg Arg Oln Thr Thr Lys Asp 155 160 0CC ACA OCT CTC ATG AGC OCT OCT Ala Thr Ala Leu Met Ser Ala Ala 170 175 AGA ATT CTC CTC AAT GAC ATO AAG Arg Ile Leu Leu Asn Asp Met Lys 185 190 ATG OOC AGA AAT 0CC CTO ATC COT Met Oly Arg Asn Ala Leu Ile Arg 205 AAT OTO GAG GAG ATT ACT TCA ATC Asn Val Oiu Olu Ile Tlu- Ser Ile 220 AAC OTG AGA GGA GAA AGA 000 AAA Asn Val Arg Gly G1u Arg Giy Lys 235 240 Giy Val

OCT

Ala

AAC

CAT

His

ATC

Ile

TCT

Ser

TTC

Phe

TTT

Phe 145

AAA

Lys

GAO

Giu

GCA

Ala

ACT

Thr

CTG

Leu 225

ACA

Thr Ser

CAG

Gin

GAT

Asp

OCA

Ala

GGT

Gly

AT'

Ile

TOT

Cys

ATO

Met 130

OCT

Ala

AGO

Arg

AAO

Lys

GAA

Olu

CTO

Leu 210

ATT

Ile Gin Arg AAG GGA Lys Gly 0CC AAT Ala Asn OTO CAA Val Oin OCT GAC Ala Asp OCT 000 Ala Oly 100 00T GCA Gly Ala 115 GAA GCT Oiu Ala AAG GGA Lys Gly COA TTG Arg Leu 000 CAC Giy His 180 OTC OAT Val Asp 195 CTO AAC Leu Asn CAG CAC Gin His 271 319 367 415 463 511 559 607 655 703 '751 799 847 895 CCC CTC ATC Pro Leu Ile GCA GCA GTG GAO AGO AAG CAC ACA 00C TTG GTO CAG ATO CTC CTG AGT WO 95/22245 PCT/US95102058 -106- Ala 245 Ala Val Glu Arg Lye 250 His Thr Gly Leu Val Gin 255 Met Leu Leu Ser 260 CGG GAA GGC ATA Arg Glu Gly Ile

AAC

Asn 265 ATA GAT GCC AGG Ile Asp Ala Arg

GAT

Asp 270 MNC GAG GGC AAG Msn Giu Gly Lys ACA GCT Thr Ala 275 CTC CTA ATT Leu Ieu Ile CTT GAA AAG Leu Glu Lys 295 GTT GAT AAA CAA Val Asp Lys Gin CTG AAG GAA ATT GTC CAS TTG CTT Leu Lys Giu Ile Val Gin Leu Leu 285 290 GGA GCT GAT AAG Gly Ala Asp Lys

TGT

Cys 300 GAC GAT CTr GT Asp Asp Leu Val

TGG

Trp 305 ATA GCC AGG Ile Ala Arg AGG AAT Arg Asn 310 CAT GAC TAT CAC His Asp Tyr His GTA AAG CTT CTC Vai Lys Leu Leu

CTC

Leu 320 CCT TAT GTA GCT Pro Tyr Val Ala

AAT

Asn 325 CCT CAC ACC GAC Pro Asp Thr Asp

CCT

Pro 330 CCT GCT GGA GAC Pro Ala Gly Asp

TGG

Trp 335 TCG CCT CAC AGT Ser Pro His Ser

TCA

Ser 340 CGT TGG GGG ACA Arg Trp Gly Thr

GCC

Ala 345 TTG AAA AGC CTC Leu Lye Ser Leu

CAC

His 350 AGT ATC ACT CGA Ser Met Thr Arg CCC ATC Pro Met 355 ATT GGC AAA Ile Cly L y s- ACT TCC GAA Thr Ser Glu 375

CTC

Leu 360 AAG ATC TTC ATT Lys Ile Phe Ile

CAT

His 365 GAT GAC TAT AAA Asp Asp Tyr Lys ATT GCT GGC Ile Ala Gly 370 CGA GAA GTG Arg Clu Val GGG GCT GTC TAC Cly Ala Val Tyr

CTA

Leu 380 GGG ATC TAT GAC Cly Ile Tyr Asp

AAT

Asn 385 991 1039 1087 1135 1183 1231 1279 1327 1375 1423 1471 1519 1567 1615 1663 GCT GTG AAG GTC TTC CCT CAG AAT ACC CCA CCT GGA TCT AAG GAA GTC Ala Val Lys Val Phe-Arg Glu Asn Ser Pro Arg Cly Cys Lys Glu Val 390 395 400

TCT

Ser 405

GGA

Gly TGT CTG CGG CAC Cys Leu Arg Asp AGA GAG CAC CAT Arg Glu Asp Asp 425

TGC

Cys 410 GGT GAC CAC AGT Gly Asp His Ser

AAC

Asn 415 TTA GTG GCT TTC Leu Val Ala Phe

TAT

Tyr 420 AAG GGC TCT TTA Lye Cly Cys Leu

TAT

Tyr 430 GTG TCT GTC TCC Val Cys Val Ser CTG TGT Leu Cys 435 GAG TGC ACA CTG GAA GAG TTC CTG Glu Trp Thr Leu Glu Glu Phe Leu TTG CCC AGA GAG Leu Pro Arg Glu GAA CCT GTG Clu Pro Val 450 TCT ATA TTT Ser Ile Phe GAG AAC GGG Glu Asn Cly 455 GAA GAT AAG TTT CCC CAC ACC ATC Glu Asp Lye Phe Ala His Ser Ile CTA TTA Leu Leu 465 GAG GGT Clu Gly 470 CTT CAA AAA CTA Val Gin Lys Leu TTC CAT CGA TAT Leu His Cly Tyr

TCC

Ser 480 CAT CAC GAC CTC His Gin Asp Leu CAA CCA CAA AAC ATC TTA ATA CAT TCC AAG AAA OCT OTC CGG CTG GCA WO 95/22245 WO 9522245PCTIUS95/02058 -107- Gjln 485 Pro Gin Ann Ile Leu 490 le Asp Ser Lys Lys 495 Ala Val Arg Leu GAT TI'T GAT GAG Asp Phe Asp Gin

AGC

Ser.

SOS

ATC CGA TGG ATG Ile Arg Trp Met

GGA

Gly 510 GAG TCA CAG ATG Giu Ser Gin Met GTC AGG Val Arg 515 AGA GAC TTG Arg Asp Leu GGT GAG ATC Gly Giu Ile 535 GAG GAT CTT GGA CGG CTG GTT CTC TAC GT GCTA ATG AMA Giu. Asp Leu Gly Arg Leu Val Leu Tyr Val Val Met Lys 520 525 530 CCC TTT GAG ACA Pro Phe Giu Thr

CTA

Leu 540 AAG ACT CAG, AAT Lys Thr Gin An GAA GTG CTG Giu Val Leu CTT ACA Leu Thr 550 ATG TCT CCA CAT Met Ser Pro Asp

GAG

Giu 555 GAG ACT AAG GAC Giu Thr Lys Asp

CTC

Leu 560 ATT CAT TOC CTG Ile His Cys Leu TCT CCT CGA GAA Ser Pro Gly Giu GTC AAG AAC TGC Val Lys Ann Cys

CTG

Leu 575 GTA GAC CTG CTT Vai Asp Leu Leu

GCC

Cly 580 CAT CCT TTC TTT His Pro Phe Phe

TGG

Trp 585 ACT TGG GAG AAC Thr Trp Ciu An TAT AGA ACA CTC Tyr Arg Thr Leu CGG AAT Arg An 595 1711 1759 1807 1855 1903 1951 1999 2047 2095 2143 2191 2200 GTG GGA AAT Val Gly Asnr CTC AGA CTA Leu Arg Leu 615

GAA

Giu 600 TCT GAC ATC AAA Ser Asp Ile Lys CGG AAA TGT AAA Arg Lys Cys Lys ACT CAT CTT Ser Asp Leu 610 AGC 'I"IT GAC Ser Phe Asp CTG CAG CAT CAG Leu Gin His Gin CTT GAG CCT CCC Leu Giu Pro Pro

AGA

Arg 625 CAG TGG Gln Trp 630 ACA TCT AAG ATC Thr Ser Lys-Ilie GAC AAA AAT GTT ATG GAT CAA ATG AAT CAT Asp Lys Ann Vai Met Asp Giu Met Asn His 635 640 77TC TAC GAA AAG AGA AAA AAJ. AAC CCT TAT CAG GAT ACT GTA GGT Phe Tyr Glu Lys Arg Lys Lys Ann Pro Tyr Gin Asp Thr Val Gly 645 650 655

GAT

Asp 660 CTG CTG AAG TTT Leu Leu Lys Phe

ATT

Ile 665 CGG AAT ATA GGC Arg Ann Ile Gly

CAA

Giu 670 CAC ATC ANT GAG His Ile Ann Giu GAN AA Ciu LYS 675 AAG CGG GGG Lys Arg Giy INFORMATION FOR SEQ ID NO:6: Wi SEQUENCE CHARACTERISTICS: LEN GTH 679 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein WO 95/22245 WO 9522245PCTIUS95/02058 8- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Glu Thr Pro Asp 1 5 Gly Ser Gin Arg Thr Val Gin Lys Gly Asp Ala Asp Ala Asn Ala Asn Ala Val Gin Ala His Gly Ala Asp Pro Ile Ile Ala Gly Ile 100 Ser Cys Gly Ala Asp 115 Phe Met Giu Ala Ala 130 Phe Ala Lys Gly Ala 145 Lys Arg Arg Leu Lys 165 Glu Lys Gly His Leu 180 Ala Giu Val Asp Ala 195 Thr Leu Leu Asn Trp 210 Leu Ilie Gin His Gly 225 Thr Pro Leu Ile Ala 245 Met Leu Leu Ser Arg 260 Gly Lys Thr Ala Let~ 275 Tyr Asn Thr Pro Val Val Cys Gly 70 His Gin Val Giu Asn iso Gin -Giu Arg Asp Ala 230 Ala Giu Leu Val Val Giu 55 Arg Arg Gly Asn Arg 135 Val Gly Val Asp Cys 215 Asp Val Gly Ile Glu Arg' 40 Asp Val Arg Asp Giu 120 Gly Asn Gly Leu Asn 200 Glu Val Glu Ile Ala 280 Asp 25 V7al Thr Asp Lys Val 105 Cys Asn Leu Ala Arg 185 Met Asn Asn Arg Asn 265 Val Gin 10 Asp TrpI Ile Lys 90 Lys Asp Ala Arg Th? 170 Ile Gly Val Val Lys 250 Ile Asp 9cr Gly Val 75 Asn Leu Giu Giu Arg 155 Ala Leu Arg Giu Arg 235 His Asp

LYE

Ser Leu Trp Asn Gly Leu Asn Ala 140 Gin Leu Leu Asn Giu 220 Gly Thr Ala Gin Gly Gly Thr Pro Leu Leu Thr Leu Ala Giu Gly 125 Leu Thr Met Asn Ala 205 Ile Giu Gly Arg Leu 285 Ile Giu Pro Leu Thr Ile 110 Phe Arg Thr Ser Asp 190 Lieu Thr Arg Leu Asp 270 Lys Ser is Lays Lys Leu Leu Pro Leu Thr Phe Lys Ala 175 Met Ile Ser Gly Val 255 Asfl Glu Ala Ala Gly His Ser Phe Leu Al a Leu Asp 160 Ala Lys Arg Ile Lys 240 Gin Giu Ile Val Gin 290 Leu Leu Leu Giu Lys 295 Gly Ala Asp Lys Asp Asp Leu-Val

I

WO 95/22245 WO 9522245PCT/US95/02058 -109- *Trp Ile Ala 305 pro Tyr Val Pro His Ser Thr Arg Pro 355 Lys Ile Ala 370 Asn Arg Giu 385 Cys Lys Glu Val Ala Phe Val Ser Leu 435 Giu Giu-Pro 4 50 Leu Ser Ile 465 His Gin Asp Val Arg Leu Gin Met Vai 515 Val Val Met 530 Asp Giu Vai 545 Ile His Cys Asp Leu Leu Thr Leu Arg 595 Arg Ala Ser 340 Met Gly Val Val Tyr 420 Cys Val Phe Leu Al a 500 Arg Lys Leu Leu Gly 580 Asn Arg An 325 Arg Ile Thr Ala Ser 405 Gly Giu Giu Giu Gin 485 Asp Arg Gly Leu Phe 565 His Val Ann His 310 Pro Asp Trp Gly Gly Lys Ser Giu 375 Val Lys 390 Cys Leu Arg Glu Trp Thr Ann Gly 455 Gly Val 470 Pro Gin -Phe Asp Asp Leu Glu Ile 535 Thr Met 550 Ser Pro Pro Phe Gly An Asp Tbr Thr Lou 360 Gly Val Arg Asp Leu 440 Giu Gin An Gin Giu 520 Pro Ser Gly Phe Glu 600 Tyr His Lou 315 Asp Pro Pro 330 Ala Lou Lys 345 Lys Ile Phe Ala Val Tyr Phe Arg Giu 395 Asp Cys Gly 410 Asp Lys Gly 425 Giu Giu Phe Asp Lys Phe Lys Leu His 475 Ile Leu Ile 490 Scr Ile Arg 505 Asp Leu Giy Ph~e Giu Thr Pro Asp Giu 555 Giu Asn Val 570 Trp Thr Trp 585 Ser Asp Ile Val Ala Ser le Leu 380 An Asp Cys Lou Ala 460 Leu Asp Trp Arg Leu 540 Glu Lys Giu Lys Lys Gly Lou His 365 Gly Ser His Lou Arg 445 His His Ser Met Leu 525 Lys Thr Asn Asn Val 605 Leu Asp His 350 Asp Ile Pro Ser Tyr 430 Leu Ser Gly Lys Gly 510 Val Thr Lys Cys Arg 590 Arg Leu Trp 335 Ser Asp Tyr Arg Asn 41i5 Val Pro Ile Tyr Lys 495 Giu Leu Gin Asp Leu 575 Tyr Lys Lou 320 Ser Met Tyr Asp Gly 400 Leu Cys Arg Lou Ser 480 Ala Tyr Asn Lou 560 Val Arg Cl's Lys Ser .610 sSrAsp Lou Lou Arg Lou Lou Gin His Gin 610615 Thr Lou Giu Pro Pro 620 WO 95/22245 WO 9522245PCTIUS95/02058 Arg Ser Phe Asp Gin Trp Thr Ser Lys Ile Asp Lys Asn Vai Met Asp 625 630 635 640 Glu Met Ann His Phe Tyr Giu Lys Arg Lys Lys Ann Pro* Tyr Gin Asp 645 650 655 Thr Val Gly Asp Leu Leu Lys Ph Ile Arg. Ann Ile Gly Giu His Ile 660 665 670 Asn Giu Glu Lys Lys Arg Gly 675 INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE CHARACTERISTICS: LENGTH: 190 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Asp Arg Arg Lys Pro Arg Gin Ann An Arg Asp Asn Arg Giu Ser Glu Gin Lys Arg Ser Val Arg Arg Val Ala 130 Ala Giu 145 Thr Arg Arg Gin Gin Gin Lys Glu Pro Arg An Gin Ala Ala Giu 100 Gin Giu Ile Ser Arg Gin Pro Gin Thr Arg Ala Val Giu Arg Ala Arg 70 Gin Giu Gin Val Gin 150 Val Arg Gin Glu 55 Arg Glu Gin Leu Val 135 Glu Val Thr Ala 40 Val Giu Ala Giu An 120 Ala Ala Ala Giu 25 Gin Thr Arg Lys Glu 105 Gin Pro Pro Gin Arg.

10 Gly Gin Giu Ser Ala 90 Arg Lys Val Ala Thr Arg Asp Arg An Ser Gin Lys Arg 75 Leu Val Val Val Pro 155 Ala Asp Thr Al a Arg An Arg Arg Giu 140 Arg Pro An Ala Arg Arg Val Pro Tyr 125 Giu Thr Giu Arg Giu Thr An Giu Val 110 Giu Thr Glu Gin Giu Arg Giu Giu Thr Arg Ala Asp Asp Asp Giu Gin Gin Pro Gin Val Ala Leu Val 160 Gin Glu 175 Lys Val Pro Leu Pro 165 170 Giu Ann Ann Ala Asp Ann Arg Asp Ann Gly Gly Met Pro Ser WO 95/22245 WO 9522245PCT1US95/02058 -111- ISO 185 INFORMATION FOR SEQ ID NOE-S: SEQUENCE

CHARACTERISTICS:

LENGTH: 2562 base pairs TYPE: nucleic acid sTE.AZDED1NEss: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 190 CAGTTTCTGG AGCAAATTCA

GTTTGCCTTC

CTTTAGCAGT TCTTCCATCT

GACTCAGGTT

CCACACTTCC GTGATTATCT

GCGTGCATTT

GAAGAAATGG CTGGTGATCT

TTCAGCAGGT

CAGAAGCAGG GAGTAGTACT

TAAATATCAA

AGGAGGTTTA CATTTCAAGT

TATAATAGAT

TCAAAGAAGG AAGCAAAAAA

TGCCGCAGCC

AAGAAGGCAG TTAGTCCTTT

ATTATTGACA,

-GGAATTACA TAGGCCTTAT

CAATAGAATT

GAACAGTGTG CATCGGGGGT

GCATGGGCCA

CAGAAAGAAT ATAGTATTGG TACAGGTrC'I AAACTTG CAT ATCTTCAGAT

ATTATCAGAA

7TGGTTCTT TTGCTACTAC

GTGTGAGTCC

GCTTCTGAAT CATCATCTGA

AGGTGACTTC

AOTGACAGTT TAAACAGTTC

TTCGTTG=?

GCAAAAAGAT CTTTGGCACC

CAGATTTGA(

GTGGACAAGA. GGTTTGGCAT

GGATTTTAAJ

GGCCAAGTTT TCAAAGCAAA

ACACAGAAT

AAATATAATA ACGAGAAGGC

GGAGCGTGAX

AATATTGTTC ACTACAATGG

CTGTTGGGA

GATTCTCTTG AGAGCAGTGA

TTATGATCC

CTGGATrGT

TGCTTCTCTG

TGGACAAAGC

TTCTTCATGG

GAACTGCCTA

GGAAGAGAAT

AAATTAGCTG

ACAACGAATT

GCCCAGAAGA

GAAGGATTTC

ACTAAACAGG

GAAACCTCAG

CAAAGCAACT

TCAGCAGATA

ATGAATGGTC

:CTTCCTGA.CA

~GAAATAGAXI

rGACGGAAAG; PGTAAAAGCA1

TGGATTTGAT'

T GAGALCAGC]

AAATTGTAAT

GCGGTCTTCA

TTCCAACCAGC

AGGAACTTAA

ATTCAGGACC

I

TTCCAGAAGG I

TTGAGATACT'

CTTCAGAAGG

AAAGACTAAC

ATTATAAATG

AAGCAAAACA.

TGAA6ATCTGA

CTTTAGTGAC

CATCAGAGAT

TCAGAAATAA

TGAAAGAAAC

TAATTGGCTC

CTTACGTTAT

7TGGCAAAACT rATGATCCTGA k. AAAATAGTTC

~CCTCAAAA

MATCAACAT

;ATACGGGAA

'ACATACCGT

~CCACATGAT

EGAAGGTAGA

FAATAAGGAA

IL.TTATCCATG

rGTAAATTAT.

CAAAATGGGA

ATTGGCCGCT

CTACCTGTCC

CAGCACACTC

AAATTCTAAC

TCAAAGGAAG

AAAGTATACT

AGGTGGATTT

TAAACGTGTT

TGATCATGTA

GACCAGTGAT

AAGGTCAAAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 ACTAAGTGCC TTTCATCCA AATGGAATTC TGTGATAAAG GGACCTTGGA

ACAATGGATT

WO 95/22245 WO 9522245PCT/US95/02058 -112- GAAAAAAGAA GAGGCGAGAA

ACTAGACAAA

ACAAAAGGGG TGGATTATAT

ACATTCAAAA

AATATATTCT TAGTAGATAC

AAAACAAGTA

CTAAAAATG ATGGAAAGCG AACAAGGAGT CAGATTTCrT CGCAAGACTA TGGAAAGGAA GCTGAACTTC TTCATGTATG

TGACACTGCT

CGGGATGGCA TCATCTCAGA

TATATTTGAT

CTCTCAAAGA AACCTGAGGA

TCGACCTAAC

TGGAAGAAAA GCCCAGAGAA

AAATGAACGA

TCCTGCTTCT GATATGCAGT

TTTCCTTAAA

GATATTTACC TTTTATTTTA

ATGTTTCCTT

GcAGAAACAG AAAGGTrTTC TTCTTT TGC CTGGCTCATC TCTTTATTTT TTTTT 1rT 711 AGGCTGGAGT GCAATGACAC

AGTCTTGGCT

GATTCTCCTG CCTCAGCCTC

CTGAGTAGCT

AATTTTTGTG TTTTTAATAA

AGACAGGGTT

CCTGACCTCA AGTAATCCAC

CTGCCTCGGC

CCACCGCGCC CAGCCTCATC

TCTTTGTTCI

TTTTATACTA TTAATGAATC

AATCAXTTCA

GGCCAAAAAA ATGTAAGATC

GTTCTCTGCC

GTTTTGGCTT

AAATTAATTC

AAGATTGGAG

AGGGGPJACTT

GTGGACCTCT

TrTGAAACAT

AAAAAAGAAA

ACATCTGAAA

CACACATGTT

TTATCTAAAA

TAATTTTTTA

TTCAAAAACA

TTTAiAGAC

CACTGCAACT

GGATTACAGG

TcACCALTGTT

CTCCCAAAGT

AAAGATGGAA

TATCTATTTA

TCACATAGCI1

TGGAACTCTT

ATAGAGATCT

ACTTTGGACT

TGCGATACAT

ACGCTTGGG

CAAAGTTTTT

AAACTCTTCT

TACTAAGGAC

AGAGCCCTTC

TCTGCTAGGG

CTATTTTTAC

TrCTTACATT

AGAGTCTCGC

TCTGCCTCTT

CATGTGCCAC

GGCCAGGCTG

GCTGGGATTA

AAACCACCCC

TTAAATTTCI

'TACAAGCCAC

TGAACAAATA

TAAGCCAAGT

TGTAACATCT

GAGCCCAGAA

GCTAA.TTCTT

CACAGACCTA

ACAGAA.ATTA

CTrGACTGTG

TGAAAAAGTA

AATATCAATA

TAALTCTTTCT

TTACTTTTT C

TCTGTTGCCC

GGGTTCAAGT

CCACCCAACT

GTCTCAAACT

CAGGGATGAG

CAAATTTTCT

ACCGCTTTTA

CTGGAGAAAT

1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2562 ATGGTACTCA TTAAAAAAAA

AAAAAAAAAG

INFORMATION FOR SEQ ID NO:9: TGATGTACAA CC Ci) SEQUENCE CHARACTERISTICS: LENGTH: 551 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Ala Gly Asp Leu Ser Ala Gly Phe Phe Met Glu Glu Leu Asn Thr 1 5 10 Tyr Arg Gin Lys Gin Gly Val Val Leu Lys Tyr Gin Glu Leu Pro Asn WO 95/22245 WO 9522245PCTIUS95/02058 -113ser Gly Asn Ala Ser Arg Glu Gly 145 Al a Leu Leu Se r Ser 225 Arg Tyr Ile Asp Al a 305 Val Gly Pro Arg Giu Ala -Ala Val Ser Met Gly Leu Thr Gly Phe 130 Thr Gly Tyr Leu Ser Ser Val Thr 195 Ala Asp 210 Ser Leu Ser Leu Thr Val Gly Ser 275 Gly Lys 290 Glu Arg His Tyr Pro Phe Ala Pro Asn 100 Val His Ser Gin Gly 180 Ser Thr Leu Al a Asp 260 Gly Thr Glu Asn His Pro Lys Leu Tyr Asn Tyr Thr Ile 165 Ser Thr Ser Met Pro 245 Ly's Gly Tyr Val Gly 325 Asp Giu Leu 70 Leu Ile Tyr Lys Lys 150 Leu Phe Leu Giu Asn 230 Arg Arg Phe Val Lys 310 Cye Arg Gly Ala Leu Gly Glu Cys 135 Gin Ser Ala Ala Ile 215 Gly Phe Phe Giy Ile 295 Ala Trp Arg 40 Glu Val Thr Leu Gin 120 Lys Giu Giu Thr Ser 200 Asn Leu Asp Gly Gin 280 Lys Leu Asp 25 Phe Gly Giu Thr Ile 105 Cye Met Ala Giu Thr 185 Glu Ser Arg Leu Met 265 Val Arg Ala Gly Thr Arg Ile Thr 90 Asn Ala Gly Lys Thr 170 Cys 5cr Asn Asn Pro 250 Asp Phe Val Lys Phe 330 Phe Ser Leu 75 Asn Arg Ser Gin Gin 155 Ser Glu Ser Scr Asn 235 Asp Phe Lys Lys Leu 315 Asp Gin Vai Lys Lys Asn Lys Ser Ser Ile Ala Gly Val 125 Lys Giu 140 Leu Ala Val Lye Ser Gin Ser Giu 205 Asp Ser 220 Gin Arg Met Lys Lys Giu Ala Lye 285 Tyr Asn 300 Asp His Tyr Asp Ile Ile Giu Ala Giu Lye Giu Gly Gin Lye 110 His Gly Tyr Ser Ala Lye Ser Asp 175 Ser Aen 190 Gly Asp Leu Asn Lye Ala Giu Thr 255 Ile Glu 270 His Arg Aen Giu Val Aen Pro Giu 335 Asp Lye Lys Leu Lys Pro Ile Leu 160 Tyr Ser Phe Ser Lys 240 Lys Leu Ile Lye Ile 320 Thr Ser Asp Asp Ser Leu Giu Ser Ser Asp Tyr Asp Pro Giu Aen Ser Lye WO 95/22245 WO 9522245PCTIEJS95/02058 -114- Asn Ser Ser Arg Ser Lys Thr Cys Lys 385 Giy Pro Phe Lys Tyr 465 Leu Asp Thr Thr Lys 545 Gly Lys Tyr Ile 420 Val Leu Giu Vai Asp 500 Gin Ile Tbr Val Ile 405 Phe Thr Arg Val Cys 485 Gly Lys Leu Leu Giu 375 Leu Ala 390 His Ser Leu Vai Ser Leu Tyr Met 455 Asp Leu 470 Asp Thr Ile Ile Leu Leu Arg Thr 535 Thr Cys 550

LYS

360 Gin Leu Lys

ASP

Lys 440 Ser Ala Ser Ser 520 Leu Glu Phe 395 Ile Gin Gly Gin Giy 475 Thr Phe Pro Trp Lys 380 Giu His Val Lys Ile 460 Leu Ser Asp Glu Lys 540 365 Arg Gin Arg Lys Arg 445 Ser Ile Lys Lys Asp 525 Lys Cys Leu Phe Ile Gin Met Giu Phe Arg Ile Asp Ile 430 Thr Ser Leu Phe Lys 510 Arg Ser Giy Thr Leu 415 Giy Arg Gin Ala Phe 495 Giu Pro Pro Glu Lys 400 Lys Asp Ser Asp Giu 480 Thr Lys An Giu Asn Giu Arg His INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1650 base pairs TYPE: nucieic acid STRANDEDNESS: singie TOPOLOGY: iinear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:iO: AACTGAAACC AACAGCAGTC CAAGCTCAGT CAGCAGAAGA GATAAAAGCA AACAGGTCTG GGAGGCAGTT CTGTTGCCAC TCTCTCTCCT GTCAA TGATG GATCTCAGAA. ATACCCCAGC CAAATCTCTG GACAAGTTCA TTGAAGACTA TCTCTTGCCk QACACGTGTT TCCGCATGCA 120 180 WO 95/22245 PCTIUS95/02058 -115-

AATCGACCAT

CTCCTACCCT

CCTCAGAGGC

GGATCAGTTA

TCAAAGAGAG

CCGTGCGCTC

GCTGCCTGCC

CTATGTC&AG

CTTCACAGAA

CCGCCTAGTC

GTATGCCCTG,

CAACACAGCC

CATCTACTGG

GCAGCTCACG

TGGTGGAGAC

CC-CATGCTTT

CAACAGTACA

AACACATGAG

ACAGGCAGAA

GCTCCAGT

AGACAAAGCT

CTGGCCTTCT

CACCTATTCT

ATTCCAGCCT

ATAACAATAA

GCCATTGACA

KuTTG

CGA.TCTGACG

AATCGCCGGG

AGAGCACTT

AGCTTCGTAC

TTTG.ATGCCC

CTCATCGAGG

CTACAGAGAG

AAGCACTGGT

GAGCTCCTGA

CAAGGATTTC

ACAAALGTATT

AAACCCAGGC

~CCAAAGGGTT

AAGAATTGGG

GACGATGAGA

TACCCTCATT

GAGGACTGGA

GTTATCTGGA

CCTCAGTGAG

ATGCCCTCTA

CTGAAAATAT

TGACTTTCTT

AAATAAAGCA

TCALTCTGTGG

CCAAGGTGGT

CTGACCTGGT

GAGAGTTCAT

CCGTGAAAGTT

TGAGTTCGCT

TGGGTCAGTT

AGTGCACCGA

ACTTCCTGAA

ACCAAAATTG

CGGTCTATGC

GGACGGTCT T

ATGACTTTAA

CTGTGATCCT

GGAGGCAGCT

ATGGGTCCCC

CCGACGATCC

TCTCTCATAG

CCTGCACCAT

CCAGTTCCTT

CTGGTGTATA

TCCTATCATA

TCCCTGAGAG

CTGTGCACCT

AATACCAAAA

GTCCTGAAC

AAAGGGTGGC

TGTCTTCCTC

CCAGGAAATT

TGAGGTCCAG

CCAGCTCGGG

GACTGGCAGC

CCTGCAGAAA

GCAGCGCCCC

TAAGAAGAAG

TrGGGAGCGA GGAATrAGTC

AAACCCCATT

GGACCC!GGCG

GGCACA.AGAG

AGTGAGCTCC

CAGGACGTAT

ACCCAGCACG

CCTCTGAATG

CATTTTCAGG

ATCCAAGACA

GATAP.CATTC

AGAACAGAGA

GATGGGAGGG

GAAAGGTGCT

TCCTCAGGCA

AGTCCTCTCA

AGGAGACAGC

GCTCCACGCT

GAGGGGGTGG

TATAAACCTA

GAGGGCGAGT

ACCAAGCTCA

CTTGGGA.AGC

GGGAGCATGA

ATAAACTACC

ATTGAAAAGT

GACCCTACAG

GCTGAGGCCT

TGGATTCTGC

CAGAAATATG

CTCCAGGCAG

CCAGTGCATC

TGGGACTCTT

GAACCCAAGT

TCCACAGCCT

GATTTAGATA

TAATGTCTAA

TCCGAGGTAG

AGGGCACCAC

GCACTITTTCA

TGGAAGCCTG

GGGGCAACCC

AGTTCGATGT

ACCCCCAAAT

TCTCCACCTG

AGAGCCTCAT

TGCCACCTCA

AAACACATTT

AGCAACTCTG

ACCTGAGAAG

GAAACTTGGG

GGCTGAATTA

TGGCTGAAAG

GTTACATTGG

CATCCACCCC

TTGGGGGAAA

GATCCAGAGA

CTCCTGACTC

CACTTCATTC

AGAGAATGAA

TGTATTATCA

240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1650 INFORMATION FOR SEQ ID NO:11: Wi SEQUENCE CHARACTERISTICS: LENGTH: 400 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 95/22245 WO 9522245PCTIUS95/02058 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ii: Met

I

Met Asp Lieu Arg 5 Asn Thr Pro Ala Giu Asp Tyr Ala Ile Asp Ser Ser Tyr Gly Lys Gly Phe Leu Ser Giu Phe Thr Arg Ala Leu 115 Pro Arg Ala 130 Val Giu Phe 145 Gly Ser Tyr, Cys Thr Asp Leu Gin Arg 195 Ile Arg Leu 210 Lys Leu Pro 225 Giu Arg Gly Thr Val Leu Ile Lys Tyr 275 Leu Leu Ile Ile Pro Val Thx Thr Pro Leu Gin Giu 100 Ser Val Leu Ser Asp Val Lys Pro 165 Leu Gin 180 Asp Phe Val Lys Pro Gin Ser Met 245 Giu Leu 260 Tyr Asp Pro CyB Cys Leu 70 Thr Ile Lys Phe Leu 150 Asn Lys Leu His Tyr 230 Lys Val Phe Asp Gly Val Arg Th' Arg Phe Vai 135 Pro Pro Giu Lys Trp 215 Ala Thr Ile Lys Thr Phe 40 Ser Gly Phe Arg Giu 120 Leu Ala Gin Gly G ln 200 Thr Leu His Asn Asn Cys 25 Leu Lys Arg Gin Gin Val Ser Phe Ile Giu 185 Arg Gin Giu Phe Tyr 265 Pro

LYS

10 Ph.

Lys Vai Ser Asp 90 Leu Gin Ser Asp Tyr 170 Phe Pro Asn Leu Asn 250 Gin Ile Arg Giu Val Asp 75 Gin Giu Ala Leu Ala 155 Vai Scr Thr Cys Leu 235 Thr Gin Ile met Arg Lys Ala Leu Ala Pro Gin 140 Leu Lys Thr Lye Lye 220 Thr Ala Leu Giu Gin Cys Gly Asp Asn Cys Arg 125 Leu Gly Leu Cys Leu 205 Lys Val Gin Cys Lys 285 Ser Leu Asp Lye Ile Ph.

Gly Leu Arg Gin 110 Trp Gly Gin Ile Gly 190 Lys Lye Tyr Gly Ile 270 Tyr Phe Asp Arg Ser Val Arg Arg Gly Giu Leu Giu 175 Thr Ser Leu Ala Phe 255 Tyr Leu Ile His Gly Ser Val s0 Gly Giu Asn Gly Thr 160 Giu Glu Leu Gly Trp 240 Arg Trp Arg 280 Arg Gin Leu Thr Lys Pro Arg Pro Val Ile Leu Lye Pro Ala Asp Pro WO 95/22245 PCTIUS95/02058 -117- 290 295' 300 Thx Gly Ann Leu Gly*GlY Giy ASP Pro Lys Gly Trp Arg Gin Lau Ala 305 310 315 320 Gin Giu Ala Giu Ala Trp Leu Asn Tyr Pro Cys Phe Lys Ann Trp Asp 325 330 335 Gly Ser Pro Vai Ser Ser Trp Ile Leu Leu Ala Giu Ser Ann Ser Thr 340 345 350 Asp Asp Giu Thr Asp Asp Pro Arg Thr Tyr Gin Lys Tyr Gly Tyr Ile 355 360 36S Gly Thr His Glu Tyr Pro His Phe Ser His Arg Pro Ser Thr Leu Gin 370 375 380 Ala Ala Ser Thr Pro Gin Ala Giu Giu Asp Trp Thr Cys Thr Ile Leu 385 390 395 400 WO 95/22245 PCTIUS95/02058 -118- The present invention may, of course, be carried out in other specific ways than those herein set forth without departing from the spirit and essential characteristics of the invention. For example, the nucleotide sequences disclosed herein may be combined with other nucleotide sequences to generate heterologous nucleotide sequences for introduction into the genomes of plants to form transgenic plants. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced herein.

Having described our invention, we claim:

Claims (164)

1. A transgenic plant all of whose cells contain at least one nucleotide sequence introduced into said transgenic plant, or ancestor of said transgenic plant, said introduced nucleotide sequence encoding an amino acid sequence having activity of an enzyme selected from the 2-5A-dependent RNase(s).

2. A transgenic plant of claim 1, said nucleotide sequence includes the nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

3. A transgenic plant of claim 1, said nucleotide sequence being selected from a o1 group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

4. A transgenic plant of claim 1, said nucleotide sequence includes the nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR. S. S o o *S [N:\Liba]00099:SSD WO 95/22245 PCT/US95/02058 -120- A transgenic plant of claim 1, said nucleotide sequence includes the nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of

6. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to RNase.

7. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to synthetase.

8. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to PKR.

9. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to RNase, said nucleotide sequence further encoding a second amino acid sequence, said second amino acid sequence having activity similar or identical to 2-5A synthetase. WO 95/22245 PCT/US95/02058 -121- A transgenic plant of claim 9, said nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase and nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for or the double stranded RNA binding domain of

11. A transgenic plant of claim 9, said nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase and nucleotides selected from the group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

12. A transgenic plant of claim 9, said nucleotide sequence further encoding a third amino acid sequence, said third amino acid sequence having activity similar or identical to PKR. WO 95/22245 PCT/US95/02058 -122-

13. A transgenic tobacco plant of claim 12, said nucleotide sequence including nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase, nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for or the double stranded RNA binding domain of 2-5A-synthetase and nucleotides designated as 1-2562 in FIG. 18 or any part of said nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR. WO 95/22245 PCT/US95/02058 -123-

14. A transgenic plant of claim 11, said nucleotide sequence including nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR and nucleotides selected from the group of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028, and 1-884 in Table 2. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to synthetase, said nucleotide sequence further encoding a second amino acid sequence, said amino acid sequence having activity similar or identical to PKR. WO 95/22245 PCT/US95/02058 -124-

16. A transgenic plant of claim 15, said nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase and nucleotides designated as 1-2562 in FIG. 18 or any part of said nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

17.- A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to RNase, said nucleotide sequence further encoding a second amino acid sequence, said amino acid sequence having activity similar or identical to PKR. 125

18. A transgenic plant of claim 17, said nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase and nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

19. A transgenic plant of claim 17, said nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding 1 o domain of PKR and nucleotides selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2. A transgenic plant of claim 1 which is immune to or resistant against more than one viral infection, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. C CC C CC« IN:\Liba]00099:SSD WO 95/22245 PCT/US95/02058 -126-

21. A transgenic plant of claim 2, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

22. A transgenic plant of claim 3, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

23. A transgenic plant of claim 4, said transgenic plant being selected from the group of tranfsgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

24. A transgenic plant of claim 5, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -127- A transgenic plant of claim 6, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

26. A transgenic plant of claim 7, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

27. A transgenic plant of claim 8, said transgenic plant being selected from the group of tran-sgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

28. A transgenic plant of claim 9, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -128-

29. A transgenic plant of claim 12, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. A transgenic plant of claim 15, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

31. A transgenic plant of claim 17, said transgenic plant being selected from the group of trarsgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -129-

32. A transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A-dependent RNase.

33. A transgenic tobacco plant of claim 32, said nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

34. A transgenic tobacco plant of claim 32, said nucleotide sequence includes nucleotides selected from the group of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2. WO 95/22245 PCT/US95/02058 -130- A transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an amino acid sequence having activity similar or identical to

36. A transgenic tobacco plant of claim said nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of WO 95/22245 PCTIUS95/02058 -131-

37. A transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR.

38. A transgenic tobacco plant of claim 37, said nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR. WO 95/22245 PCT/US95/02058 -132-

39. A transgenic plant of claim 1, said transgenic plant all of whose cells contain at least three nucleotide sequences, each said nucleotide sequence being introduced into said transgenic plant, or an ancestor of said transgenic plant, said first introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to RNase, said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to synthetase, and said third introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR. A transgenic plant of claim 39, said transgenic plant being a transgenic tobacco plant.

41. A transgenic plant of claim 39, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -133-

42. A transgenic plant of claim 39, said first nucleotide sequence including nucleotides designated as 1-2223 in Table I or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

43. A transgenic plant of claim 42, said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase and said third nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nuoGeotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

44. A transgenic plant of claim 39, said first nucleotide sequence includes nucleotides selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2. WO 95/22245 PCT/US95/02058 -134- A transgenic plant of claim 44, said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase, and said third nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of said nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

46. A transgenic plant of claim 42, said transgenic plant being selected from the group of trarsgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

47. A transgenic plant of claim 43, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -135-

48. A transgenic plant of claim 44, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

49. A transgenic plant of claim 45, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCTUS95/02058 -136- A transgenic plant of claim 1, said transgenic plant all of whose cells contain at least two nucleotide sequences, each said nucleotide sequence being introduced into said transgenic plant, or an ancestor of said transgenic plant, said first introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to RNase, and said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to synthetase.

51. A transgenic plant of claim 50, said transgenic plant being a transgenic tobacco plant.

52. A transgenic plant of claim 50, said first nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase. I WO 95/22245 PCT/US9502058 -137-

53. A transgenic plant of claim 52, said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of

54. A transgenic plant of claim 50, said first nucleotide sequence includes nucleotides selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2. A transgenic plant of claim 54, said second nuoaeotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of

56. A transgenic plant of claim 50, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -138-

57. A transgenic plant of claim 52, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

58. A transgenic plant of claim 53, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

59. A transgenic plant of claim 54, said transgenic plant being selected from the group of trarsgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. A transgenic plant of claim 55, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -139-

61. A transgenic plant of claim 1, said transgenic plant all of whose cells contain at least two nucleotide sequences, each said nucleotide sequence being introduced into said transgenic plant, or an ancestor of said transgenic plant, said first introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR, and said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A synthetase.

62. A transgenic plant of claim 61, said transgenic plant being a transgenic tobacco plant. 63,- A transgenic plant of claim 61, said first nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of said nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR and said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for or the double stranded RNA binding domain of WO 95/22245 PCT/US95/02058 -140-

64. A transgenic plant of claim 61, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. A transgenic plant of claim 63, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -141-

66. A transgenic plant of claim 1, said transgenic plant all of whose cells contain at least two nucleotide sequences, each said nucleotide sequence being introduced into said transgenic plant, or an ancestor of said transgenic plant, said first introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to RNase and said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR.

67. A transgenic plant of claim 66, said transgenic plant being a transgenic tobacco plant.

68.- A transgenic plant of claim 66, said first nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

69. A transgenic plant of claim 68, said second nucleotide sequence including nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR. WO 95/22245 PCT/US95/02058 -142- A transgenic plant of claim 66, said first nucleotide sequence includes nucleotides selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

71. A transgenic plant of claim 70, said second nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

72. A transgenic plant of claim 66, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

73. A transgenic plant of claim 68, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. WO 95/22245 PCT/US95/02058 -143-

74. A transgenic plant of claim 69, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. A transgenic plant of claim 70, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

76. A transgenic plant of claim 71, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants, WO 95/22245 PCT/US95/02058 -144-

77. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to 2-5A-dependent RNase.

78. A plant transformation vector of claim 77, said nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

79. A plant transformation vector of claim 77, said nucleotide sequence includes nucleotides selected from the group consisting of nucleotides des-ignated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, q-1028 and 1-884 in Table 2. A plant transformation vector of claim 77, said vector being plasmid pAM943:2-5A-dep. RNA sense.

81. A plant cell containing said plant transformation vector of claim 77.

82. A plant cell of claim 81, said plant transformation vector being plasmid pAM943:2-5A-dep. RNase sense. WO 95/22245 PCT/US95/02058 -145-

83. A plant cell of claim 81, said plant cell being a tobacco plant cell.

84. A differentiated tobacco plant comprising said tobacco plant cell of claim 83. A differentiated tobacco plant of claim 84, said plant transformation vector being plasmid pAM943:2-5A-dep. RNase sense.

86. A plant cell of claim 81, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells.

87. A bacterial cell containing said plant transformation vector of claim 77.

88. A bacterial cell of claim 87, said bacterial cell being an Argobacterium tumefaciens bacterial cell. WO 95/22245 PCTIUS95/02058 -146-

89. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to PKR. A plant transformation vector of claim 89, said nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

91. A plant transformation vector of claim 89, said vector being plasmid pAM943:PK68.

92. A plant cell containing said plant transformation vector of claim 89.

93. A plant cell of claim 92, said plant cell being a tobacco plant cell.

94. A tobacco plant comprising said tobacco plant cell of claim 93. A tobacco plant of claim 94, said plant transformation vector being plasmid pAM943:PK68. WO 95/22245 PCT/US95/02058 -147-

96. A plant cell of claim 92, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells. WO 95/22245 PCT/US95/02058 -148-

97. A comprises a amino acid identical to plant transformation vector which nucleotide sequence which encodes an sequence having activity similar or 2-5A synthetase.

98. A plant transformation vector of claim 97, said nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of

99. A plant transformation vector of claim 97, said vector being plasmid pAM943:2-5A synthetase.

100. A plant cell containing transformation vector of claim 97. said plant

101. A plant cell of claim 100, said plant cell being a tobacco plant cell.

102. A plant cell of claim 100, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells. WO 95/22245 PCT/US95/02058 -149-

103. A tobacco plant comprising said tobacco plant cell of claim 101.

104. A tobacco plant of claim 94, said plant transformation vector being plasmid pAM943:synthetase.

105. A bacterial cell containing said plant transformation vector of claim 97.

106. A bacterial cell of claim 105, said bacterial cell being an Argobacterium tumefaciens bacterial cell. WO 95/22245 PCT/US95/02058 -150-

107. A plant cell of claim 81, said plant cell containing a second plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to 2-5A synthetase.

108. A plant cell of claim 107, said plant cell containing a third plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to PKR.

109. A plant cell of claim 107, said plant cell being a tobacco plant cell.

110. A plant cell of claim 108, said plant cell being a tobacco plant cell.

111. A plant cell of claim 107, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells. -151- S 112. A plant cell of claim 108, said plant cell being selected from the group consisting of vegetable, fruit, grain tree, flower, grass, weed and shrub plant cells.

113. A bacterial cell containing said plant transformation vector and said second plant transformation vector of claim 107.

114. A bacterial cell of claim 113, said bacterial cell being an Argobacterium tumefaciens bacterial cell.

115. A bacterial cell containing said plant transformation vector, said second plant transformation vector and said third plant transformation .vector of claim 108. .t A bacterial cell of claim 115, said bacterial cell being an Argobacterium tumefaciens bacterial cell.

117. A transgenic plant comprising said tobacco plant cell of claim 109. -0T O -152-

118. A transgenic plant comprising said tobacco plant cell of claim Iho.

119. A transgenic plant comprising said plant cell of claim 81.

120. cell of

121. Cell of A transgenic plant comprising said plant claim 109. A transgenic plant comprising said plant claim 110. 9 *ee .4 .9 9 4 49 4 9. 9499 0* .9 9. .099. 9 9 9 9t *9*S 9 00 9 .4 4. 99 0 9 90 .9 9 94 004009 4

122. A transgenic plant comprising said plant cell of claim 111.

123. A transgenic plant comprising said plant cell of claim 112. 153

124. A method for producing genetically transformed plants which are resistant or immune to infection by a virus, said method comprises the steps of: a) inserting into the genome of a plant cell of a plant susceptible to a virus a constructhaving a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2 -5A-dependent RNase; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the construct. e a. a a [N:\Liba]00099:SSC 154

125. A method of claim 124, said method including the further step of inserting into said genome of said plant cell a second construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to synthetase.

126. A method of claim 124, said method including the further step of inserting into said genome of said plant cell a second construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to PKR. S. o o a a a [N:\Liba]00099:SSC

127. A method for producing genetically transformed plants which are resistant or immune to infection by a virus, said method comprises the steps of: a) inserting into the genome of a plant cell of a plant susceptible to a virus a constructhaving a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to PKR; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the construct.

128. A method of claim 127, said method including the further step of inserting 0o into said genome of said plant cell a second construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to synthetase. 0 S* ID [N:\Liba]00099:SSC [N:\Liba]00099:5SC 156

129. A method for producing genetically transformed plants which are resistant or immune to infection by a virus, said method comprises the steps of: a) inserting into the genome of a plant cell of a plant susceptible to a virus a construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2-5A synthetase; b) obtaining a transformed plant cell; and c) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the construct.

130. A method of claim 124 in which the plant is a tobacco plant.

131. A method of claim 125 in which the plant is a tobacco plant.

132. A method of claim 126 in which the plant is a tobacco plant. o* V V* V o [N:\Liba]00099:SSC [N:\Liba]00099:55C 157

133. A method of claim 127 in which the plant is a tobacco plant.

134. A method of claim 128 in which the plant is a tobacco plant.

135. A method of claim 129 in which the plant is a tobacco plant. 136, A method of claim 124 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

137. A method of claim 125 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants. C CC C C *9 [N:\Liba]00099:SSC 158

138. A method of claim 126 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

139. A method of claim 127 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

140. A method of claim 128 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

141. A method of claim 129 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants. [N:\Liba]00099:SSC 159

142. A method for producing genetically transformed plants, which are resistant or immune to more than one viral infection, said method comprises the steps of: a) inserting into the genome of a plant cell of a plant susceptible to a virus a nucleotide sequence which encodes for an amino acid sequence having activity of an enzyme selected from the 2-5A-dependent RNase(s); b) obtaining a transformed plant cell; and c) regenerating from the transformed plant which expresses the amino acid sequence encoded by

143. A method of claim 142, the amino acid 1lo identical to 2-5A-dependent RNase.

144. A method of claim 142, the amino acid identical to

145. A method of claim 142, the amino acid identical to PKR. cell a genetically transformed plant the nucleotide sequence. sequence 'having activity similar or sequence having activity similar or sequence having activity similar or ~[N:\Liba]00099:SSD 160

146. A transgenic plant all of whose cells contain a nucleotide sequence introduced into said transgenic plant, or an ancestor of said transgenic plant, said introduced nucleotide sequence encoding an antisense 2 -5A-dependent RNase amino acid sequence. 147, A plant transformation vector which comprises said nucleotide sequence of claim 146.

148. A plant transformation vector of claim 147, said plant transformation vector being plasmid pAM943:2-5A-dep. RNase antisense.

149. A plant transformation vector of claim 147, said plant transformation vector being plasmid pAM822:2-5A-dep. RNase antisense.

150. A construct which comprises said nucleotide sequence of claim 146, said construct being the construct as described in FIG. 13 D/a.

151. A construct which comprises said nucleotide sequence of claim 146, said construct being the construct as described in FIG. 13E. S S. Sr S S., S S [N:\Liba]00099:SSC 161

152. A plant cell containing said plant transformation vector of claim 147.

153. A plant cell of claim 152, said plant cell being a tobacco plant cell.

154. A plant cell of claim 152, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells. S *r S S. S S S S. 4iA% -sc 0 0' [N:\Liba]00099:SSC 162

155. A bacterial cell containing said plant transformation vector of claim 147.

156. A bacterial cell of claim 155, said bacterial cell being an Argobacterium tumefaciens bacterial cell.

157. A transgenic plant of claim 146, said transgenic plant being a tobacco plant.

158. A transgenic plant of claim 146, said transgenic plant being selected from a group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants. a a. [N:\Liba]00099:SSC -163-

159. An isolated nucleotide sequence encoding an amino acid sequence having human 2 -5A-dependent RNAse activity, or an active fragment or analog thereof, said nucleotide sequence being identified as SEQ ID NO:3: and comprising: ATG GAG AGC AGO GAT CAT AAC AAC CCc C S S S .*r a. S a S TCC AAA GAA ACA OAA AAT AAG TGT GGT GTG AAAi GTA 4 AAC CTG I CTG ACT C CAG I AGT C AAG A TGT G CTT G CCT C GCC C' CTC A. GAAG4 GTG Al TCT T( TAT G( CTC TC GAA Gz TCA TC TAC AC AAG AA OCT 00 CGG CT Al G( CC C1 GG CT GA AA AA LOC A ;T TG C(C LGC LT AA ;GG TG CT TG A 0G TT TT TT CC G 3C GGT CT OTT ;T GA :T CTG 'T CTG GO 0CC '0 CTG T TTT O OTC T TTG I A GGG GTC 'I CTT C TCT C CAT C CTG A CTT C GGC A ATC 0 OAT C' AAG G OCT 0; AAG 01 TTC T1 GGC Ai ACO TI CTO CA AGT GA GAG CA OTG GA ATA TT CAC CA OCT GC OAT CC GTC CTi A' 0 C1 CI AC AA rA kG ;CI LTi ;A IT LT( 'C LA A T rT CC CC T T GA AGO NA AAC C AAT iT AAC 'T CGT G CCT A CTT *T GGC A 0CC G CGA C ACA G AAG I C AAT 1 C GAT I G OCT C CTG G GAO C ACA G GAG T OTT A CTT C' GAC Ti CTC C ATT OJ TAC C TGT G2 AGC AC AGC CA ACT CT AAT GA AAG GC OAT CT CAC CT CAG GA 0G G; Cj TI TI TT A C Tc LT C A2E C2 T TC GG G 0 GG T G G CT 0G AAOQ TA :A G' ~T O( 'T Al 'C CI 'C AC A AA O AC T CT T CT G GO! r GA' C OT( G OT( ~GA( CTC CTG SACA TCT AAG AGA GAA GGG GGC CGA AGO GAA GAA OTT CAA GCA GTC CA 0 NT 0 T T T Gi 'C C: 'T T( 'A GC A TI A AP C AT C CT C AG T GT C AA' ;GAI CAI CT TG( GC( 'CAI CCI ATA AAA TTC AGC GAG GGC GCG OAT CAA CCA OAT AAG TG GAA TT GAC TC CAG AA ATG CT GAC rC OCA :T AAA C TTC 'C CTT LO GAO 4 G GAC 'T OAT A AAT O GAG C T OTO I C AAG A C ATA 0 CTT 0 AAA C AGO C OGA G 'CAG Ai TAC Ci TAC Al TAT G2 CCA CC AAC AC CAC TT TGT TT GAA TT GAA CT CAA AA TTT GA AGA GA' C C G A C G( G( LA C G( ;C C rA GI G( Ct T AA :AG GAG 'AC AAT 'TG GTC AA GAO GC AGO CT GTT CO ATT 3A OCA CG GAA T AAG LT CAA T OCT 4 L ATO 'C TTG 2 T ATT I O OGA C O CAC 'I C ATT A T GTT 0 r OGA G AAT T AAA G TCA C CCT A' ATT AAG Cl OCA C1 CAC TI TTT 01 OAT GT GCC CC CAC TT ATC TT AAG AG CTA GA 0 C C 0; C' 3G 01 OC A A7 cc Al AC CT A AA C GG GG GG GG C GA C AC AG C PLA NG G. rG Ac 'G 04 ~T G C GC A C G0 CC A AA G G C CA G CA A AG GO' r GAI CT AG1 GAC G A' TGC SATT GAT GAA CGG GTG TGT CAC AAT TCC ATA ATC GAC :CC ACG ?G CTO AA TTO GG GOC AC ATT CO AAO GO AGO IC AAT 'T GTG A CA ;o CTO JGGA A OAT C *T OCT C T CTG C A GGG A T TTG G C ACA 0 C AAA C V ACA 0 CAT T TTT C. GGG G GGC A ACT T OTA GC OAA GI1 ACA TT GTC AC AGA GO GTC CT TGT GG OAT TC AAG TG( CTT M~ T A C' Ti G' Al 01 GI TI A N CA T. T TT LA( A( T( Al CC AC NA :A TC 'C C 0 GG A T cc TT TG 135 GG 180 TO 225 G. 270 GO 315 LO 360 T 405 LT 450 G 495 C 540 A 585 C 630 G 675 C720 765 810 855 900 945 990 1035 1080 1125 1170 1215 1260 1305 1350 1395 1440 1485 1530 1575 1620 a A CAG G OTC AAGAGA GA T CTA GA G AAG TG GAC C CTT 00;A OTA AAG AAG OGA AGO ATC TCA TTT GAG -164- GAT CTG AAA GCT CAA AGT AAT GAA GAG GTG GTT CAA CTT TCT CCA 1665 GAT GAG GMA ACT AAG GAC CTC ATT CAT CGT CTC TTC CAT CCT GGG 1710 GMA CAT GTG AGG GAC TGT CTG AGT GAC CTG CTG GOT CAT CCC TTC 1755 TTT TOG ACT TOG GAG AGC CGC! TAT AGO ACG CTT COG AAT GTG OGA .1800 MAT GMA TCC GAC ATC MAA ACA COA AMA TCT GMA AGT GAG ATC CTC 1845 AGA CTA CTG CMA CCT 000 CCT TCT GMA CAT TCC AMA AGT TTT GAC 1890 MAG TOG ACG ACT MAG ATT MAT GMA TGT GTT ATG AMA AMA ATG AAT 1935 MAG TTT TAT GMA AMA AGA GGC MAT TTC TAC CAG MAC ACT GTG GGT 1980 OAT CTG CTA MAG TTC ATC COG MAT TTG GGA GMA CAC ATT OAT GMA 2025 GMA MO CAT AMA MG ATG AMA TTA MAA ATT GGA GAC CCT TCC CTG 2070 TAT TTT CAG MAG ACA TTT CCA OAT CTO OTG ATC TAT GTC TAC ACA 2115 AMA CTA CAG MAC ACA GMA TAT AGA MAG CAT TTC CCC CMA ACC CAC 2160 AOT CCA MAC AMA CCT CAG TOT OAT GGA OCT GOT 000 0CC AGT 000 2205 TTO GCC AOC CCT 000 TOC 2223

160. -165- An amino acid sequence having human 2-SA- depende-tt RNAse activity, or an active fragment or analog thereof, said amino acid sequence being identified as SEQ ID NO:4: and comprising: *r V V V V *r V V V. Met Sex Lys Glu Thr Glu Asn Lys Cys Gly Val Lys Val Asn Leu Leu Thr Gin Ser 2 Lys I Cys C Leu Pro I Ala I Leu L Glu G Val L Ser C Tyr G Leu C Glu A Ser S Tyr T Lys L Ala G Arg L Asp L Se Al Gi Pri Le Gil Let As! LyE Asr G15 Glu Ala Ser Asp Pro krg ksp .ys ;ly Tal 'ro aeu 'ys ily 'yB ly ys sp er hr ye ly eu eu r Gi' a Va y G1' Let 1 Let r Al i Let Phc Val 1 Leu Gly Val I Cys Ser His Leu Leu Gly lie Asp Lys Ala Lys Phe Gly Thr Leu Ser Glu Val Ile His Ala Asp Val Lys Y Arg Arg 1 Gin Asn F Ala Asn I His Asn I Leu Arg x Thr Pro Lys Leu Tyr Gly Lys Ala i Arg Arg Ala Thr Leu Lys Asp Asn Asp Asp Gly Ala. lie Leu Lett Glu Lys Thr Ala Glu Leu Val I Val Leu i Glu Asp Asp Leu I Phe lie I Ile Tyr I Phe Cys C Gin Ser S Glu Ser E Gin Thr L Glu Asn G Phe Lye A Gin Asp L Ala His L Pro Gin G Leu Tyr V Ala Gin S Al. Gl Va Al. Hi Ph Ph( Phc Let Lys Ala Ile Met Ser Asp Ala Gin kla Leu iet Leu Vrp lis sp .eu ;lu er [is leu Ilu ,la leu ;lu al a Ala Asp 1 Asn Val Gly Ile 3 Leu Thr I Lye Thr Leu Leu Gly Asp Val Val 4 Glu Leui Leti Thr 2 Ser I Lye I Arg I Glu I Gly I Gly S Arg G Arg G Glu A Glu A Val G Gin p Ala A Val L Val L~ Va Va Ph G1 Al Le Se: Al Ph Ly Met Let Ar Val Asn Glu His Leu :ys kla lis 'ro le ys ,he ;er ;lu ily .1a .sp in ro sp ys ys 1 Gi lAs e G1 n Me' a As] u Al r Lyu a Phe Let Git Asn I Asn Glu Val Lys Ile Leu Lys Arg Gly Gin Tyr Tyr Tyr Pro Asn His Cys Glu Glu Gin Phe Arg Lys u Asp p Leu n Glu t Ser p Pro a Ala s Gly a Met 1 Tyr i Asp Glu Ala Ala Arg Lys Glu Ala I Arg 4 Arg 2 Ala I Ser I Arg I Lys I Glu I Arg I Ser F Leu P Leu A Phe A Leu H Asn I Asp L Asp L Gly SI Asn Val Glu Arg Val Ile Ala Glu Lys Gin Ala Met Leu Ile Gly 4 His lie 2 Val ;iy .1 ksn I ys( jer F ?ro2 le A 4's G lia G [is L 'he V *sp V la A is L le L ys S eu G er I: Hi GI Gl G1 Le Al As Al Ar Gli Gli G1 Ilt Th Let ksr ;la Ua 'yr ;lu [is let la ;In ;In eu 'al al rg eu eu er lu le Giu Sex Arg Asp His Asn Asn Pro Gin Glu Gl -y Pro .S Leu .n Gin u Gly u Asp u Arg a Gly p Val a Ala g Gly u Arg Lys F Ala 3 His c His 1 Arg I Gly Asp Leu Ser Asp I Asp I Trp C Ile C Asp Glu Arg G Val T Cys V His A Asn V Ser C lie A Ile L Asp L Ser P1 Th Le Le GI 11 Ii Ly Se As; Va Al Lel Gl As! Alz Let Glj Leu rhr LYs 'hr ie ?he ly ily 'hr ral ;lu hr 'al .g 'al ys ep Ye eu *x Ser iu lie U Leu y Trp e Val Lys r Val Glu 1 Tyr Asn Arg His Val t Leu 1 Leu Lye Val Asp Leu Asp Sex 3 His 3 Ala 3 Lye 3 Ser 3 Ala 3 Val 4 Phe 4 Thr 4 Gly 4 Leu 4 Gly 4' Ser 4! Trp 5; Gly 5: Glu 51 105 120 135 150 165 180 195 210 225 240 255 270 285 100 115 to S V S r P h e G U 5 I er Asn Giu Giti Val Val Gin Leu Sex Pro 555 -166- ASP Giu Giu Thr Lye Asp Leu Ile His Arg Leu Phe His Pro Gly 570 Giu His i A 'Phe Asn Arg Lys Lys Asp Glu Tyr Lys Ser Leu Trp Giu Leu Trp Phe Leu Lys Phe Leu Pro Ala Thr Ser Leu Thr Tyr Leu His Gin Gin Asn Ser Trp Asp Gin Thr Giu Lys Lys Lys Asn Lye Pro Giu Ile Pro Lye Lye Phe Lye Thr Thr Pro Gly Ser Lye Giy Ile Arg Ile Met Phe Giu Gin Cys jjeu Arg Thr Pro Asn Gly Arg Lye Pro Tyr Cys Ser Tyr Arg Ser Giu Asn Asn Leu Asp Arg Asp Asp Arg Lys Giu Cys Phe Leu Lye Leu Lye Gly Leu Thr Ser His Val Tyr Gly Ile Val His Ala Leu Leu Giu Ser Met Gin Giu Gly Ile Phe Giy Arg Ser Lye Lye As n His Asp Tyr His Aen Giu Ser Lye Thr Ile Pro Vai Pro Vai Ile Phe Met Val Asp Ser Tyr Phe Giy Leu Asp As n Gly Giu Leu Thr 585 600 615 630 645 660 675 690 705 720 735 741 Pro Gin Thr His Gly Giy Aia Ser Giy 9*9* 9 9 9 9* 0 9. 9* 9 9 9 9 9 9. 9 9* 9 9 9 9 99 9 9* 9. a 9**999 9 167 9* S C S

161. A transgenic plant all of whose cells contain at least one nucleotide sequence introduced into said transgenic plant, or ancestor of said transgenic plant, said introduced nucleotide sequence encoding an amino acid sequence having antiviral activity, substantially as hereinbefore described with reference to any one of the Examples.

162. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to dependent RNase.

163. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to PKR.

164. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to synthetase.

165. A method for producing genetically transformed plants which are resistant or immune to infection by a virus, substantially as hereinbefore described with reference to any one of the Examples.

166. An isolated nucleotide sequence encoding an amino acid sequence having human 2 -5A-dependent RNAse activity, or an active fragment or analog thereof, substantially as hereinbefore described with reference to any one of the Examples.

167. An amino acid sequence having human 2 -5A-dependent RNAse activity, or an active fragment or analog thereof, substantially as hereinbefore described with reference to any one of the Examples.

168. An isolated 2 -5A-dependent RNase, or an active fragment or analog thereof, said 2 -5A-dependent RNase having a 2-5A binding domain and the ability to cleave single stranded RNA when said 2 -5A-dependent RNase is bound to

169. An isolated 2-5A dependent RNase of claim 168, said 2-5A binding domain includes amino acids designated as 229-275 in Table I or Table II.

170. An isolated 2 -5A-dependent RNase of claim 168, said 2-5A binding domain comprises a duplicated phosphate binding P-loop motif.

171. An isolated 2 -5A-dependent RNase of claim 170, said first P-loop motif having an amino acid sequence designated as amino acids 229-241 in Table I and said second P-loop motif having an amino acid sequence designated as amino acids 253-275 in Table I or Table II.

172. An isolated 2 -5A-dependent RNase of claim 168, said dependent RNase being a human 2 -5A-dependent RNase.

173. An isolated 2 -5A-dependent RNase of claim 168, said dependent RNase being a murine 2 -5A-dependent RNase.

174. An isolated 2 -5A-dependent RNase of claim 168, said dependent RNase further including four ankyrin repeats. IN:\LIBUU101092:SSC r RA TF 168

175. An isolated 2 -5A-dependent RNase of claim 168, said 2 -5A-dependent RNase further including a cysteine-rich region, said cysteine-rich region containing between about 5 and about 6 cysteine amino acid residues.

176. An isolated 2 -5A-dependent RNase of claim 175, said cysteine-rich region includes amino acids designated as 395-444 in Table I.

177. An isolated 2 -5A-dependent RNase of claim 175, said cysteine region includes amino acids designated as amino acids 401-436 in Table II.

178. An isolated 2 -5A-dependent RNase of claim 168, said 2 -5A-dependent RNase having an amino acid sequence which includes amino acid residues designated as 1-741 in Table I.

179. An isolated 2 -5A-dependent RNase of claim 178, wherein either or both lysine amino acid residues designated as amino acid residues 240 and 274 in Table I are substituted with an asparagine amino acid residue.

180. An isolated 2 -5A-dependent RNase, or an active fragment or analog thereof, said 2 -5A-dependent RNase having a 2-5A binding domain.

181. An isolated 2 -5A-dependent RNase of claim 180, said 2 -5A-dependent RNase having an amino acid sequence which includes amino acids S designated as 1-679 in Table II. S

182. An isolated 2 -5A-dependent RNase of claim 181, wherein either or 20 both lysine amino acid residues designated as amino acid residues 240 and 274 in Table II are substituted with an asparagine amino acid residue.

183. An isolated 2 -5A-dependent RNase of claim 180, said 2-5A-dependent RNase having an amino acid sequence which includes amino acid residues selected from the group consisting of amino acid members designated as o 25 1-619, 1-515, 1-474, 1-403, 1-365, 1-342, and 1-294 in Table II.

184. An isolated nucleotide sequence encoding 2 -5A-dependent RNase, or S an active fragment or analog thereof, the 2 -5A-dependent RNase having a 2-5A binding domain and the ability to cleave single stranded RNA when the S 2 -5A-dependent RNase is bound to

185. An isolated nucleotide sequence of claim 184, said sequence includes the nucleotides designated as 1-2223 in Table I.

186. An isolated nucleotide sequence of claim 184, said sequence includes nucleotides which encodes for the 2-5A binding domain which are designated as

685-825 in Table I. 187. An isolated nucleotide sequence of claim 186, said nucleotide sequence encoding for the 2-5A binding domain includes two separate nucleotide sequences which encode for a duplicated phosphate binding P-loop motif, said two separate nucleotide sequences include nucleotides designated as 685-723 and

757-825, respectively, in Table I or Table II. IN:\LIBUU01092:SSC [N:\LjBUuJOlog2.ssc 169 188. An isolated nucleotide sequence of claim 184, said sequence includes a translational initiation sequence designated as nucleotides -3 to 4 in Table I. 189. An isolated nucleotide sequence of claim 184, said sequence includes nucleotides designated as nucleotides -103 to 2825 in Table I. 1 0. An isolated nucleotide sequence of claim 184, said nucleotide sequence encoding human 2 -5A-dependent RNase. 191. A recombinant DNA molecule containing the nucleotide sequence of claim 184. 192. A recombinant DNA molecule containing the nucleotide sequence of claim 185. 193. A recombinant vector comprising a vector and the nucleotide sequence claim 184. 194. A recombinant vector comprising a vector and the nucleotide sequence of claim 185. 195. A recombinant cell capable of expressing a 2 -5A-dependent RNase, or an active fragment or analog thereof, which has a 2-5A binding domain and the S ability to cleave single stranded RNA when the 2-5A dependent RNase is bound to 2-5A, said recombinant host cell comprising a host cell, a promoter and said nucleotide sequence of claim 184 which is subject to control of said promoter. 20 196. A recombinant cell of claim 195, said nucleotide sequence including I nucleotides designated as 1-2223 in Table I. 197. An isolated nucleotide sequence encoding 2 -5A-dependent RNase, or an active fragment or analog thereof, the 2 -5A-dependent RNase having a binding domain. 198. An isolated nucleotide sequence of claim 197, said nucleotide S sequence being selected from a group consisting of nucleotides designated as S 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 11. cm 199. A recombinant DNA molecule containing said nucleotide sequence of claim 197. 200. A recombinant DNA molecule containing said nucleotide sequence of claim 198. 201. A recombinant vector comprising a vector and the nucleotide sequence of claim 197. 202. A recombinant vector comprising a vector and the nucleotide sequence of claim 198. 203. A recombinant cell capable of expressing a 2 -5A-dependent RNase, or an active fragment or analog thereof, which has a 2-5A binding domain, said recombinant host cell comprising a cell, a promoter and said nucleotide sequence of claim 197. [N:\LIBUU101092:SSC [N:\LIBUUIO1 092:SSC 204. A recombinant cell capable of expressing a 2 -5A-dependent RNase, or an active fragment or analog thereof, which has a 2-5A binding domain, said recombinant cell comprising a host cell, a promoter and said nucleotide sequence of claim 198. 6 205. A clone having a nucleotide sequence capable of expressing a murine 2 -5A-dependent RNase having a 2-5A binding domain, said clone being selected from a group consisting of ZB1, ZB2, ZB3, ZB5, ZB9, ZB10, ZB11 and ZB13. 206. A clone having a nucleotide sequence capable of expressing a human 2 -5A-dependent RNase having a 2-5A binding domain and ribonuclease activity, said clone being ZC5. 207. An isolated 2 -5A-dependent RNase, or an active fragment or analog thereof, said RNase substantially as hereinbefore described with reference to any one of the Examples. Dated 11 August, 1998 15 Cleveland Clinic Foundation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 9 9 9 9 [N:\LIBUU01092:SSC fN~~LiBUUJo1O92:6sc