EP0753992A1 - Antiviral transgenic plants, vectors, cells and methods - Google Patents

Antiviral transgenic plants, vectors, cells and methods

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Publication number
EP0753992A1
EP0753992A1 EP95911802A EP95911802A EP0753992A1 EP 0753992 A1 EP0753992 A1 EP 0753992A1 EP 95911802 A EP95911802 A EP 95911802A EP 95911802 A EP95911802 A EP 95911802A EP 0753992 A1 EP0753992 A1 EP 0753992A1
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EP
European Patent Office
Prior art keywords
plant
leu
lys
glu
transgenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP95911802A
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German (de)
French (fr)
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EP0753992A4 (en
Inventor
Robert H. Silverman
Dibyendu N. Sengupta
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Cleveland Clinic Foundation
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Cleveland Clinic Foundation
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Publication of EP0753992A1 publication Critical patent/EP0753992A1/en
Publication of EP0753992A4 publication Critical patent/EP0753992A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates to isolated 2-5A-dependent RNases having the ability to bind 2-5A and/or cleave single stranded RNA when bound to 2-5A, 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 2-5A-dependent RNase, 2-5A synthetase and/or PKR.
  • RNA degradation is a critical cell function, and gene expression is often regulated at the level of RNA stability. See, e.g., Shaw, G and Ka en, R., Cell. 46:659-667 (1986). Nonethe less, relatively little is known about the bio chemical pathways that mediate RNA degradation i mammalian or plant systems. For instance, most i not all of the ribonucleases responsible for mRN turnover in mammalian or plant cells remai unidentified. This was reviewed in Brawerman, G. Cell. 57:9-10 (1989).
  • the 2-5A system is believed t be the only well-characterized RNA degradatio pathway from higher animals including man. See FIG 1. See also, e.g., Kerr, I.M. and Brown, R.E., Prod Natl. Acad. Sci. U.S.A.. 75:256-260 (1978) an Cayley, P.J. et al., Biophvs Res. Commun. 108:1243-1250 (1982); reviewed in Sen, G.C. an Lengyel, P., J. Biol. Chem.. 267:5017-5020 (1992)
  • the activity of the 2-5A system is believed to b mediated by an endoribonuclease known as 2-5A dependent RNase.
  • 2-5A-dependen RNase is a unique enzyme in that it requires 2-5A unusual oligoadenylates with 2',5' phosphodieste linkages, p n (A2 p) n A, for ribonuclease activity.
  • 2-5A is produced from AT by a family of synthetases in reactions requirin double-stranded RNA (dsRNA) . See FIG. 1. See als Hovanessian, A.G. et al.. Nature. 268:537-539 (1977) Marie, I. and Hovanessian, A.G., J. Biol. Chem. 267:9933-9939 (1992). 2-5A is unstable in cells an in cell-free systems due to the combined action o 2',5 '-phosphodiesterase and 5'-phosphatase.
  • 2-5A-dependent RNase i believed to have no detectable RNase activity unti .it- " is converted to its active state by binding t 2-5A. See Silverman, R.H. , Anal. Biochem. 144:450-460 (1985). Activated 2-5A-dependent RNas cleaves single-stranded regions of RNA 3' of UpNp with preference for UU and UA sequences. Se Wreschner, D.H. et al.. Nature. 289:414-417 (1981a) and Floyd-Smith, G. et al.. Science. 212:1020-103 (1981) .
  • Interferons ⁇ , ⁇ 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, G. , 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, P., J. Biol. Chem..
  • the 2-5A system however, almost certainl provides functions beyond the antipicornaviru activity of interferons.
  • introductio of 2-5A into cells Hovanessian, A.G. and Wood, J.N. Virology. 101:81-90 (1980), or expression of 2-5 synthetase cDNA, Rysiecki, G. et al., J. Interfero Res.. 9:649-657 (1989)
  • 2-5A-dependent RNase levels are elevated i growth arrested cells, Jacobsen, H. et al., Proc Natl. Acad. Sci. U.S.A..
  • PKR dsRNA-dependent protein kinase enzyme
  • PKR dsRNA-dependent protein kinase enzyme
  • PKR is stimulated by dsRNA.
  • activated PKR phosphorylates the alpha subunit of translation factor eIF 2 , known as eIF 2 -alpha, which indirectly inhibits protein synthesis initiation.
  • interferons ⁇ , f and ⁇ induce the accumulation of PKR. See Hoavanessian et al.: J. Interferon Res.. 9:641-647 (1989).
  • the PKR system is also likely to provide functions beyond the antipicornavirus activity of interferons. See Meurs, E.F. et al.: J. Virolo ⁇ v. 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 U.S.A.. 90:232-236 (1993).
  • the 2-5A system and the PK system inhibit viral protein synthesis. This is believed to be accomplished by the 2-5A system b degrading mRNA and rRNA whereas the PKR system i believed to accomplish this by indirectly inhibitin protein synthesis initiation.
  • Viral plant diseases are pandemic and thei severity varies from mild symptoms to plant death. The majority of plant viruses are believed to hav single stranded RNA genomes. Moreover, it i currently believed that plants are void of the thre enzymes discussed above, i.e., PKR, 2-5A synthetas 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. 24:593-59 (1985); but see Crum, C. et al.: J. Biol. Chem..
  • the present invention alleviates and overcomes certain of the above-mentioned problems and shortcomings of the present state of the art through the discovery of novel, isolated 2-5A-dependent RNases and encoding sequences therefor.
  • 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 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 "2-5A") . 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.
  • 2-5A 5'-phosphorylated,2',5'-linked oligoadenylates
  • H 2-5A-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 ribonuclease function when the 2-5A-dependent RNas is activated by 2-5A.
  • the novel 2-5A dependent RNases of th present invention are protein enzymes havin molecular weights on the order of between about 7 KDa (murine) and about 84 KDa (human) , as determine by gel electrophoresis migration and/or predictio from their respective encoding nucleotide sequences
  • a human 2-5A-dependent RNase of th instant invention has a molecular weight of abou 83,539 Da as determined from the amino acid sequenc predicted from the encoding sequence therefor whereas the murine 2-5A-dependent RNase has molecular weight of about 74 KDa as determined by ge electrophoresis migration and from prediction of th amino acid sequence from the encoding sequence
  • an about 74 KDa molecular weight is reporte herein for a murine 2-5A-dependent RNase, it shoul nevertheless be appreciated that the reporte molecular weight is for an incomplete murin 2-5A-dependent RNase.
  • the amino acid sequence for human 2-5A-dependent RNase protein is depicted in FIG. 3 and Table 1.
  • the encoding sequence for the human 2-5A-dependent 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 mRNA for murine 2-5A-dependent RNase is about 5.7 Kb in size.
  • the novel 2-5A dependent RNases of the instant invention include the following uniqu domains which span between the amino terminus and the carboxy terminus. For instance, it has bee discovered that there are at least four and possibl as many as nine or more ankyrin repeats, of whic three lie closest to the amino terminus.
  • ankyrin repeats there may be additional ankyri repeats that may total, for instance, about eight o more when the amino acid sequences of th 2-5A-dependent RNases of the present invention ar further analyzed. It is believed that these ankyri repeats may possibly function in protein-protei interaction.
  • Ankyrin repeat 1 generally lies betwee amino acids designated as 58-90 in Tables 1 and 2
  • Ankyrin repeat 2 generally lies between amino acid designated as 91-123 in Tables l and 2 •
  • Ankyri repeat 3 generally lies between amino acid designated as 124-156 in Tables i and 2•
  • Ankyri repeat 4 generally lies between amino acid designated as 238 and 270 in Tables ⁇ and 2 • Se also FIGS. 10A and 10B.
  • the nove 2-5A dependent RNases include a cysteine rich regio (which has homology to zinc fingers) that lies close to the carboxy terminus than the amino terminus whic may possibly function in RNA recognition or i formation of protein dimers.
  • the cysteine ric region is believed to include about 5 or 6 cystein residues which generally lie between amino acid designated as 395-444 in the human sequence a reported in Table 1 and FIG. 4, or between amin acids designated as 401-436 in the murine sequence a reported in Table 2 and FIG. 4.
  • the novel 2-5A dependent RNases include a duplicated phosphate binding (2 P-loops) motif which lies generally within the ankyrin repeat motifs.
  • 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 and 2.
  • 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 2-5A-dependent RNase, domain VI generally lies between amino acids designated as 470-489 and domain VII generally lies between amino acid residues desig ⁇ nated as 499-502, as reported in Table 2 and FIG. 4.
  • the limited homology is generally conserved between the murine and human amino acid sequences for 2-5A-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 i 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.
  • the present invention relates to the cloning of murine and human 2-5A-dependent RNases and novel murine and human clones.
  • Recombinant and naturally occurring forms of 2-5A-dependent RNase displayed virtually identical 2-5A binding properties and ribonucleas specificities.
  • the present invention further contemplates the use of the novel isolated, 2-5A-dependent RNases and encoding sequences therefor, as well as analog and active fragments thereof, for use, for instance, 1.) in gene therapy for human and animal disease including viral disease and cancer, 2.) as geneti markers for human disease due to perhaps cancer o viral infection, 3.) to develop plants and animal resistant to certain viruses, and 4.) as enzymes i connection with research and development, such as fo studying the structure of RNA.
  • the encoding sequences of th instant invention may be utilized in ex vivo therapy, i.e., to develop recombinant cells using the encoding sequence of the present invention using techniques known to those versed in this art.
  • 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.
  • 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 2-5A-dependent 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.
  • the transgenic plants of the present invention include any inserted nucleotide sequence encoding any type of antiviral amino acid sequence, including proteins.
  • 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 productio and/or treatment. These include, for example, thos animal antiviral genes that encode 2-5A-synthetase 2-5A-dependent RNase, and PKR.
  • Thes interferon-regulated proteins, 2-5A-synthetase 2-5A-dependent RNase and PKR have recognized antiviral effects i higher animals and are believed to have antivira effects in the transgenic plants of the presen invention.
  • PKR is stimulated by dsRNA t phosphorylate translation factor eIF2 whic indirectly inhibits protein synthesis intiation.
  • the 2-5A then activates an endoribonucleas entitled 2-5A dependent RNase (also known as RNase or nuclease F) .
  • the activated ribonuclease degrade mRNA and rRNA thus inhibiting protein synthesis.
  • tha plant viruses are similar to animal viruses i structure, composition and mechanism of replicatio in cells.
  • viral so-calle single-stranded RNA may contain secondary structure which could activate PKR and 2-5A synthetase leadin to widespread plant protection against plan viruses. It is believed that co-expression o
  • RNA thereby protecting the transgenic plants of th present invention from viruses. Moreover, it i believed that expression of PKR by the transgeni plants of the present invention will inhibit vira protein synthesis leading to inhibition of viru replication and protection of the transgenic plants.
  • the present invention is therefore premised in par upon the belief that plant virus RNAs activat
  • 2-5A-synthetase and PKR in the transgenic plants o the instant invention leading to immunity agains virus infection. Furthermore, expression of 2-5 synthetase alone or 2-5A-dependent RNase alone or PK alone may protect plants against viruses, perhaps b binding to viral RNA, such as viral replicativ intermediates thereby blocking viral replication.
  • expression of only the dsRNA bindin domains of PKR and/or of 2-5A-synthetase ma similarly protect the transgenic plants of th present invention against viral infection.
  • the present invention includes, among other things any amino acid sequence, any nucleotide sequence an any transgenic plant which have the ability t accomplish the objectives of the instant invention
  • the instant invention includes any amin acid sequence which has antiviral activity and an nucleotide sequence which encodes therefor and thos transgenic plants that express such nucleotid sequences.
  • the present inventio includes, for instance: 1.) any animal amino aci sequence which has the ability to inhibit o interfere with viral replication such as those amin acid sequences that have activity similar o identical to PKR activity, 2-5A synthetase activit and/or 2-5A ribonuclease activity, and any nucleotid sequence which encodes for an amino acid sequenc having any such activity; and 2.) any transgeni plant having any animal antiviral nucleotide sequenc which encodes any such amino acid sequence which ha any such antiviral activity.
  • FIGS in which is shown illustrative embodiments the present invention from which its novel featur and advantages will be apparent.
  • FIG. 1 is the 2-5A system: a ribonuclea pathway which is believed to function in t molecular mechanism of interferon actio 5'-phosphatase, p'tase; 2 , -5 -phosphodiesteras 2'-PDE.
  • FIGS. 2A and 2B is a comparison of 2- binding activity of recombinant and natural occurring forms of murine 2-5A-dependent RNase.
  • FIG. 2A is a specific affinity of truncat murine 2-5A-dependent RNase for 2-5A.
  • UV covale crosslinking of the 32 P-2-5A probe (lanes 1-7) protein is performed after translation reactions wheat germ extract (5 ⁇ l) with murine 2-5A-depende RNase mRNA (from clone ZBl) (lanes 1-3) or withou added RNA (lane 4) or in extract of interfero treated mouse L cells (100 ⁇ g of protein) (lane 5-7) . Reactions are without added competitor (lane 1, 4, and 5) or in the presence of either trime core.
  • Lanes and 9 are produced by incubating the wheat ger extract with 35 S-methionine in the absence o presence of 2-5A-dependent RNase mRNA, respectively.
  • FIG. 2B are identical chymotrypsin cleavag products and are obtained from recombinant an naturally occurring form of 2-5A-dependent RNase Partial chymotrypsin digests (arrows) are performe on- " truncated 2-5A-dependent RNase (clone ZBl produced in wheat germ extract (“Recombinant”) an murine L cell 2-5A-dependent RNase ("Naturall Occurring”) after crosslinking to the 2-5A probe an purification from gels.
  • FIGS. 3A and 3B are clonings of th complete coding sequence for human 2-5A-dependen RNase.
  • FIG. 3A is the construction of a huma 2-5A-dependent RNase clone.
  • the initial huma 2-5A-dependent RNase cDNA clone, HZB1 is isolate from an adult human kidney cDNA library in ⁇ gtl using radiolabeled murine 2-5A-dependent RNase cDN (clone ZBl) as probe. See Example.
  • Radiolabel HZBl DNA is used to isolate a partially overlappi cDNA clone, HZB22, which is fused to HZBl DNA at t Ncol site to form clone ZC1.
  • the 5'-region of t coding sequence is obtained from a genomic Sa fragment isolated using a radiolabeled HZB22 D fragment as probe.
  • FIG. 3B is a nucleotide sequence a predicted amino acid sequence of human 2-5A-depende RNase with flanking nucleotide sequences. T numbers to the right indicate the positions nucleotides and amino acid residues.
  • FIG. 4 is alignment of the predicted amin acid sequences for murine and human forms o 2-5A-dependent RNase. The positions of the repeate P-loop motifs, the cysteine (Cys)-rich regions wit homology to zinc fingers, and the regions of homolog to protein kinase domains VI and VII are indicated Amino acids residues which are important component of the indicated domains are represented in bold typ and are italicized. Identical amino acid residues i murine and human 2-5A-dependent RNase are indicate with colon (:) symbols adjacent therebetween.
  • FIGS. 5A and 5B are 2-5A binding propertie and ribonuclease activity of recombinant human 2-5A dependent RNase produced in vitro.
  • FIG. 5A is specific affinity of recombinan human 2-5A-dependent RNase for 2-5A.
  • Crosslinking o the 2-5A probe (lanes 1-7) to protein is performe after translation reactions in wheat germ extract ( ⁇ l) with human 2-5A-dependent RNase mRNA (lanes 1-3 or without added RNA (lane 4) or in extract of huma interferon ⁇ treated (1000 units per ml for 16 h human HeLa cells (350 ⁇ g 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 2-5A p 3 (A2'p) 2 A (100 nM) (lanes 3 and 7).
  • Lane 8 Incubation with 35 S-methionine are shown in lanes 8 to 12.
  • Lan 8 is with wheat germ extract and human 2-5A-dependent RNase mRNA.
  • Reticulocyte lysate preadsorbed to 2-5A-cellulose is incubated with human 2-5A-dependent RNase mRNA in the absence (lane 9) or presence (lane 10) 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 ( o , ⁇ ) or in the presence of both 2-5A-dependent RNase mRNA and cycloheximide (50 ⁇ g per ml (•) .
  • mRNA ⁇
  • human 2-5A-dependent RNase mRNA without inhibitor o , ⁇
  • both 2-5A-dependent RNase mRNA and cycloheximide 50 ⁇ g per ml (•) .
  • the recombinant 2-5A-dependent RNase or controls
  • Radiolabeled substrate RNA was either poly(U) (O, •,B) °r poly(C) (D).
  • FIGS. 6A, 6B and 6C show levels of 2-5A-dependent RNase mRNA which are induced by interferon treatment of murine L929 cells even in the presence of cycloheximide.
  • FIG. 6A is a northern blot prepared wit poly(A) + RNA (4 ⁇ g per lane) that is isolated fro murine L929 cells treated with murine interferon ( ⁇ ⁇ ) (1000 units per ml) and/or cycloheximide (50 ⁇ per ml) for different durations (indicated) which i probed with radiolabeled murine 2-5A-dependent RNas cDNA. Interferon, IFN; cycloheximide, CHI.
  • FIG. 6B shows levels of 2-5A-dependen RNase which are estimated from the autoradiogra shown in panel (a) with a video camera an QuickCapture and Image computer programs.
  • FIG. 6C shows levels o glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRN as determined in the same blot shown in panel (A) .
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • FIGS. 7A and 7B are the truncated recombinant murine 2-5A-dependent RNase, clone ZBl and murine L cell 2-5A-dependent RNase havin identical 2-5A binding activities localized to repeated P-loop motif.
  • FIG. 7A shows incubations of truncate 2-5A-dependent RNase, clone ZBl, ("Recombinant” which is produced in wheat germ extract (upper panel or of murine L cell 2-5A-dependent RNase (labele “Naturally Occurring,” lower panel) with the 3 P-2-5 probe, (2.4 nM) , are in the absence of presence o unlabeled 2',5 '-phosphodiester linked oligonucleo tides (as indicated) followed by uv covalen crosslinking. Autoradiograms of the dried SDS/1 polyacrylamide gels are shown. Concentrations of t oligonucleotide competitors are indicated. I inosine.
  • FIG. 7B shows a truncated series of muri 2-5A-dependent RNase mutants (ZBl to ZB15) which produced in wheat germ extract which are assayed f 2-5A binding activity by a filter binding metho See Example and Knight et al. 1980) .
  • the positio of the P-loop motifs and the lengths of t translation products are indicated.
  • Clone Z encodes for amino acids designated as 1-656 in Tab 2 , except for the last 5 amino acid residues whi are Lys, Pro, Leu, Ser, and Gly.
  • Clone ZB2 encod for " amino acids designated as 1-619 in Table 2
  • Clone ZB3 encodes for amino acids designated as 1-5 in Table 2 .
  • Clone ZB5 encodes for amino aci designated as 1-474 in Table 2.
  • Clone ZB9 encod for amino acids designated as 1-403 in Table 2
  • Clone ZB10 encodes for amino acids designated 1-365 in Table 2
  • Clone ZB13 encodes for ami acids designated as 1-294 in Table 2
  • Clone ZB encodes for amino acids designated as 1-265 in Tab 2
  • Clone ZB15 encodes for amino acids designated 1-218 in Table 2.
  • FIGS. 8A and 8B are substitution mutation of the lysine residues in the P-loop motifs o 2-5A-dependent RNase.
  • FIG. 8A shows the truncated murin 2-5A-dependent RNase, clone ZBl, and lysine t asparagine substitution mutants of clone ZBl, whic are synthesized in wheat germ extract.
  • a unlabeled translation products are covalentl crosslinked to the bromine-substituted, 32 P-labele 2-5A probe, Br-2-5A-[ 32 P]pCp. See Nolan-Sorden e al., 1990.
  • FIG. 8B shows the mRNA species which ar translated in the presence of 35 -S-methionine i separate reactions. Autoradiograms of the dried SDS/polyacrylamide gels are shown. The order an positions of the translation products (labelle "RNase") and the relative molecular masses (in kDa of the protein markers are indicated.
  • RNase Ribonucleic acid
  • FIGS. 9A and 9B are a comparison of th amino acid sequences of RNase E and 2-5A-dependen RNase.
  • FIG. 9A shows identical and conservativ matches which are shown between E. coli RNase E an the murine and human forms of 2DR.
  • FIG. 9B is a model for the structure an function of 2DR.
  • P-loop motifs repeated sequence with homology to P-loops
  • Cys ⁇ cysteine-rich region with homology to certain zi fingers
  • PK homology to protein kinase domains and VII.
  • FIGS. 10A and 10B are a comparison of t amino acid sequences of the ankyrin repeats in t human and murine 2-5A-dependent RNase proteins.
  • FIG. 10A shows murine and human forms 2-5A-dependent RNases containing four ankyr repeats. Homology between the ankyrin consens sequence and the murine and human forms 2-5A-dependent RNase are indicated. ⁇ , hydrophob amino acids.
  • FIG. 10B is a model showing the relati positions of the four ankyrin repeats in 2-5 .dependent RNase in comparison to the position of t proposed 2-5A binding domain (t) (the repeated P-lo motif) ; Cys ⁇ , the cysteine-rich region; PK, t protein kinase homology region, and t carboxy-terminal region required for RNase activity.
  • FIG. 11 shows the role of 2-5A-depende RNase in the anti-viral response of cells interferon treatment.
  • Interferon binds to specif cell surface receptors resulting in the generation a signal which activates a set of genes in the ce nucleus.
  • the genes for 2-5A synthetase are th activated producing inactive, native 2- synthetase.
  • Interferon treatment of the cell al activates the 2-5A-dependent RNase gene (not shown in the FIGure) .
  • 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 2-5A synthetase resulting in the production of 2-5A.
  • the 2-5A then activates the 2-5A-dependent RNase to degrade the viral RNA thus destroying the virus itself.
  • dsRNA double stranded RNA
  • FIG. 12 depicts a physical map of T: based binary vector p.AM943 which is about 12 Kbp.
  • B ⁇ left border
  • B j *> right border
  • Kan r kanamycin resistance
  • .AMT promoter of adenyl methyl transferase gene from Chlorella virus
  • 35S promoter for 35S RNA from Cauliflower mosaic virus
  • TER RNA termination signal
  • 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 pAM943:2-5A-dep.
  • RNase sense
  • FIG. 13D/a depicts certain portion of plasmid pAM943:2-5A-dep.
  • RNase an FIG. 13E depicts pAM822:2-5A dep.
  • RNase antisense
  • B L left border
  • Hygro r hygromyci resistance
  • AMT promoter of adenyl methy transferase gene from Chlorella virus
  • 35S promote for 35S RNA from Cauliflower mosaic virus
  • PKR cDN to human PKR
  • uPKR cDNA to a lysine (amino acid 296) to arginine mutant form of PKR
  • Synthetase cDN to a low molecular weight form of huma 2-5A-synthetase
  • 2-5Adep RNase, cDNA to huma 2-5A-dependent RNase
  • TER RNA termination signal.
  • FIG. 14 shows a physical map of Ti base binary vector pAM822 which is about 14.6 Kbp.
  • B L left border
  • Hygro r hygromyci resistance
  • Tet r tetracycline resistance
  • 35S promoter for 35S RNA fro Cauliflower mosaic virus
  • TER RNA terminatio signal
  • Ovi V origin of DNA replication.
  • FIG. 15 shows expression of huma 2-5A-synthetase cDNA intransgenic tobacco plants a determined by measuring mRNA levels in a Norther blot.
  • Construct C pAM943:Synthetase
  • Total RNA was prepared from th leaves of control (labeled "C") and transgenic plant using RNASTAT-60 (Tel-Test B. , Inc.). Thirty ⁇ g o RNA was treated with glyoxal and separated in a 1.5 agarose gel.
  • FIG. 16 shows expression of mutant and wil type forms of human PKR cDNA in transgenic tobacc plants as determined by measuring mRNA levels in Northern blot.
  • Constructs A pAM943:PK68
  • pAM943: uPK68 encoding wild type and mutant (lysin at position 296 to arginine) forms of PKR respectively, were introduced into the plants.
  • Tota RNA was prepared from the leaves of control (labele "C") and transgenic plants using RNASTAT-60 (Tel-Tes B. , Inc.). Thirty ⁇ g of RNA was treated with glyoxa and separated in a 1.5% agarose gel.
  • FIG. 17 shows a presence of 2-5A-dependen RNase cDNA in transgenic plants as determined on Southern blot.
  • Genomic DNA was isolated from leave of transgenic plants containing construct D/ (pAM943:2-5A-dep. Nase, antisense) using CTA (cetyltrimethylammonium bromide) following the metho of Rogers and Bendich (1988, Plant Molecular Biolog Manual, A6, pp. 1-10, Kluwar Academic Pulbisher Dordrecht) .
  • CTA cetyltrimethylammonium bromide
  • Ten ⁇ g of genomic DNA was digested wit Hindlll for 5 h at 37'C and fractionated in a 1 agarose gel followed by transfer to Magnagraph (nylo transfer membrane. Micron Separations, Inc.) using capillary transfer method.
  • the cDNA fo 2-5A-dependent RNase (from plasmid pZC5) was labele by random priming with [ ⁇ - 32 P]dCTP (3,000 Ci/mmole using a Prime-a-gene kit from (Promega) according t the protocol supplied by the company.
  • the labele 2-5A-dependent RNase cDNA (Specific activity of 1.0 10 9" c.p.m. per ⁇ g DNA) was washed and a autoradiogram was made from the dried membrane. Th sizes (in kilobases) and the positions of the DN markers are indicated. The band indicated a "2-5A-dep. RNase cDNA" (see arrow) was absent i Southern blots of control plants (data not shown) .
  • FIG. 18 depicts a coding sequence for hum p68 kinase mRNA (PKR) .
  • FIG. 19 depicts a translation product the complete coding sequence for human p68 kina mRNA (PKR) of FIG. 18.
  • FIG. 20 depicts a coding sequence for hum 2-5A synthetase cDNA.
  • FIG. 21 depicts a translation product the coding sequence for human 2-5A-synthetase of FI 20. Detailed Description
  • 2-5A-dependent RNase is very low abundance (one five-hundred-thousandth of the tot •protein in mouse liver, Silverman, R.H. et al., Biol. Chem.. 263:7336-7341 (1988)), its cloni requires the development of a sensitive screeni method.
  • Murine L929 cells are selected as the sour of mRNA due to high basal levels of 2-5A-depende RNase.
  • a protocol to enhance 2-5A-dependent RNa mRNA levels is developed based on the observati that optimal induction of 2-5A-dependent RNase obtained by treating cells with both interferon a cycloheximide, then with medium alone.
  • the cDNA is transcribed a translated in cell-free systems. See Example.
  • 2- ⁇ binding activity is then determined by covalent crosslinking the 2-5A probe to the protein with light, for example, Nolan-Sorden, N.L. et al., Ana Biochem.. 184:298-304 (1990).
  • the recombinant 74 k protein produced in a wheat germ extract sho specific affinity for the 2-5A probe. See FIG. 2 lanes 1 to 3.
  • a core derivative of 2-5A lacki 5'-phosphoryl groups, (A2 p) 2 A, fails to interfe with binding of the protein to the 2-5A probe where trimer 205A, p3(A2 , p) 2 , completely prevents pro binding. See FIG. 2A, lanes 2 and 3, respectivel There is no detectable 2-5A binding proteins in t wheat germ extract as shown in the incubation witho added RNA, FIG. 2A, lane 4.
  • a composite DN containing genomic and cDNA is constructed. See FIG 3A.
  • the initial cDNA portion of the huma 2-5A-dependent RNase clone (HZBl) is obtained b screening a human kidney cDNA library wit radiolabeled murine 2-5A-dependent RNase cDNA.
  • Se Example. A genomic clone, containing the 5'-part o the coding sequence, is isolated with radiolabele human 2-5A-dependent RNase cDNA.
  • the nucleotide an predicted amino acid sequences of huma 2-5A-dependent RNase are determined, FIG. 3B resulting an open reading frame encoding a protein o 83,539 Da.
  • FIG. 4 A comparison is made between the predicte amino acid sequences of the human and murine forms o 2-5A-dependent RNase in order to identify an evaluate the conserved regions of the proteins. Se FIG. 4.
  • the murine cDNA, clone ZBl contains abou 88% of the coding sequence for 2-5A-dependent RNas to which an additional twenty-eight 3'-codons ar added from a murine genomic clone. Alignment of th murine and human forms of 2-5A-dependent RNas indicates about 65% identity between the overlappin regions. See FIG. 4.
  • the 2-5A binding properties of th recombinant and naturally occurring forms of huma 2-5A-dependent RNase are compared by uv covalen crosslinking to the 2-5A probe.
  • the recombinan human 2-5A-dependent RNase produces in wheat ger extract shows specific affinity for 2-5A. See FIG 5A, lanes 1 to 3. Radiolabeling of the cloned huma 2-5A-dependent RNase with the 2-5A probe is no prevented by (A2 , p) 2 A. See FIG. 5A, lanes 1 and 2 In contrast, addition of trimer 2-5A, p 3 (A2 , p) A effectively competes with the 2-5A probe for bindin to the recombinant 2-5A-dependent RNase.
  • FIG. 5A lanes 9 and 12 show t 35 S-translation products produced in t 2-5A-cellulose-pretreated and untreated lysate respectively.
  • Ribonuclease assays with recombina 2-5A-dependent RNase are performed after immobilizi and purifying the translation product on t activating affinity matrix, 2-5A-cellulose. It w previously shown that murine L cell 2-5A-depende RNase bound to 2-5A-cellulose, resulting ribonuclease activity against poly(U) but n poly(C) . See Silverman, R.H., .Anal. Biochem. 144:450-460 (1985). Furthermore, by washin 2-5A-dependent RNase:2-5A-cellulose prior to addin the substrate the level of general non-2-5A-dependent RNase, is greatly reduced. Se Silverman, R.H. , .Anal. Biochem..
  • the murine and human 2-5A-dependent RNas mRNAs are determined from northern blots to be 5.7 k and 5.0 kb in length, respectively. See FIG. 6A.
  • the 2-5A-dependent RNase coding sequences therefore, comprise only about 40% the nucleotide sequence contained in the mRNAs.
  • the 2-5A binding functions of th recombinant and naturally occurring forms of murin 2-5A-dependent RNase are characterized by covalen crosslinking to the 2-5A probe in the presence o unlabeled 2-5A or 2-5A analogues as competitors.
  • Se FIG. 7A Interestingly, although the about 74 kD truncated 2-5A-dependent RNase is missing about 84 amino acids from its carboxy-terminus, see FIG. 4, i nonetheless possesses a 2-5A binding activit indistinguishable from that of naturally occurrin 2-5A-dependent RNase. See FIG. 7A. Trime 2-5A[p 3 (A2'p) 2 A] , at about 20 nM effectively prevent the 2-5A probe from binding to either protein.
  • FIG. 7B Expression of clon ZBll, encoding amino acid residues 1 to 342, result in- a loss of only about 26% of the 2-5A bindin activity as compared to clone ZBl (amino acids 1 t 656). See FIG. 7B. Clones intermediate in lengt between ZBl and ZBll all result in significant level of 2-5A binding activity. In contrast, protei produced from ZB13 (amino acids 1 to 294) results i only about 38.3% of the 2-5A binding activity o clone ZBl, suggesting that a region important for th 2-5A binding function is affected.
  • clon ZB14 produced a protein encoding amino acids 1 to 26 which is nearly inactive in the 2-5A binding assa (only 1.9% of th activity of clone ZBl)
  • the significant decrease in 2-5 binding activity observed with ZB14 occurs with t deletion of one of two P-loop motifs; nucleoti binding domains in many proteins.
  • FIGS. 4 a 7B See also Saraste, M. et al., TIBS. 14:430-4 (1990) .
  • Deletion of both P-loop motifs in clone ZB results in protein (amino acids 1 to 218) which completely lacking in 2-5A binding activity.
  • S FIG. 7B Deletion of both P-loop motifs in clone ZB results in protein (amino acids 1 to 218) which completely lacking in 2-5A binding activity.
  • FIG. 8B 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 2-5A probe. See FIG. 8A, lanes 2 to 4.
  • Clone ZBl(Lys 240 -)Asn) FIG. 8A, lane 2, expresses a mutant 2-5A-dependent RNase with a substantially reduced affinity for 2-5A; about 48.4% of the activity of clone ZBl as determined by phosphorimager analysis .(Molecular Dynamics) of the dried gel.
  • the full-length huma 2-5A-dependent RNase which is produced i reticulocyte lysate, had the same apparent molecula weight as did naturally occurring 2-5A-dependen RNase. See FIG. 5A. However, the actual molecula mass of human 2-5A-dependent RNase is determined fro the predicted amino acid sequence, FIG. 3B, to be about 83,539 Da.
  • interferon i believed to regulate the 2-5A pathway by elevatin levels of both 2-5A synthetases, Hovanessian, A.G. e al.. Nature, 268:537-539 (1977), and 2-5A-dependen RNase, Jacobsen, H. et al.. Virology. 125:496-50 (1983a). See. FIGS. 1, 6 and 11.
  • the cloning of 2-5A-dependent RNase reveal several features of the protein.
  • the 2-5A bindin domain is of particular interest because it is th ability of 2-5A-dependent RNase to be activated b 2-5A that sets it apart from other nucleases.
  • the identified region contains repeated P-loop motif, one from residues 229 to 24 and another from residues 253 to 275. See FIG. 4 an Table 2.
  • P-loop motif amino acid 253-275
  • the homology with P-loops is believed to b highly conserved between the human and murine form of 2-5A-dependent RNase; thus underscoring the belie of the importance of this region for 2-5A bindin activity. See FIG. 4.
  • the similarity to P-loop consists of the tripeptides, glycine-lysine threonine, preceded by glycine-rich sequences.
  • the unusual feature of 2-5A-dependen RNase is that the P-loop motif is repeated and are i the same orientation.
  • Adenylyl cyclase from Bacillu anthracis also contains a duplicated P-loop motif, however, the two sequences are in opposit orientation and are overlapping. See Xia, Z. an Storm, D.R. , J. Biol. Chem.. 265:6517-6520 (1990).
  • dimer 2-5A neither binds 2-5A-dependent RNas efficiently nor does it activate 2-5A-dependen RNase, FIG. 7A; Kerr, I.M. and Brown, R.E. , Prod Natl. Acad. Sci. U.S.A.. 75:265-260 (1978) an Knight, M. et al., Nature, 288:189-192 (1980) perhaps because it is too short to span the tw P-loop motifs.
  • the residual 2-5 binding activity observed in the point mutants ZBl(Lys 240 ->Asn) and ZBl(Lys 274 -)Asn) , and the ver low affinity of the double mutant ZBl(Lys 240 ' 274 ->Asn) for 2-5A, could indicate tha the two P-loop motifs are parts of separate 2-5 binding domains.
  • a consensus zinc finger domain reviewed i Evans, R.M. and Hollenberg, S.M. , Cell. 52:1- (1988) , consisting of six cysteine residues with th structure CX 4 CX 3 CX ⁇ 7 CX 3 CX 3 C (amino acid residue 401-436 in Table 2 ) is identified in the murine for of 2-5A-dependent RNase. See FIG. 4. The homologou region in the human form of 2-5A-depenent RNase i CX 11 CX 25 CX 3 CX ⁇ C (amino acid numbers 395 to 444 i Table 1 ) .
  • the cysteine-rich region i 2-5A-dependent RNase could be involved in binding t the RNA substrate.
  • the cysteine-ri domain in 2-5A-dependent RNase could media formation of 2-5A-dependent RNase dimers.
  • the homol with RNase E is relatively conserved between human and murine forms of 2-5A-dependent RNase spans a region of about 200 amino acid residu Within these regions there are 24 and 32% identi plus conservative matches, with some gaps, betw RNase E and the human and murine forms 2-5A-dependent RNase, respectively.
  • FIG. The me gene which encodes RNase E and the alte mRNA stability (ams) gene, Ono, M. and Kumano, M., Mol. Biol.. 129:343-357 (1979), map to the s genetic locus. See Mudd E.A. et al., M Microbiol.. 4:2127-2135 (1990); Babitzke, P. and Kushner, S.R. , Proc. Natl. Acad. Sci. U.S.A.. 88:1-5
  • 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, S.R. , Proc. Natl. Acad. Sci. U.S.A.. 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.
  • Endoribonucleases play a controlling role in RNA metabolism by catalyzing the rate-limiting steps in RNA decay. See Brawerman, G. , 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 2-5A and subsequently degrades both viral and cellular RNA. See Wreschner, D.H. et al., Nucleic Acids Res.. 9:1571-1581 (1981b).
  • the 2-5A system may be involved in the antiproliferative effects of interferon and in the fundamental control of RNA stability.
  • the source of mRNA for preparing the cDNA library is murine L929 cells grown in EMEM
  • 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 Xhol-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 Xhol and unidirectionally cloned into predigested ⁇ ZAPII 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 3.5 hours at about 42°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°C. The filters are processed by a modification of the ethods 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 20 mM magnesium acetate, about 50 mM potassium chloride, about 1 mM EDTA, about 50 mM ⁇ -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 "2-5A probe"), Nolan-Sorden, N.L. et al.. Anal. Biochem.. 184:298-304 (1990), at about 2 X 10 5 counts per minute per ml (about 3,000 Ci per mmole) at about 4°C with shaking for about 24 hours.
  • Murine L929 cells are treated with about 1000 units per ml interferon ( ⁇ + ⁇ ) with or without about 50 ⁇ g per ml of cycloheximide and the total RNA is then isolated as described. See Chomczynski, P. and Sacchi, N., Anal. Biochem.. 162:156-159 (1987).
  • Poly(A) + RNA is prepared by oligo(dT)-cellulose chromatography, as described in Sambrook, J. et al.. Cold Spring 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 ⁇ g per ml salmon sperm DNA] at about 42°C.
  • the Human 2-5A-dependent RNase cDNA clone, HZBl is isolated from an adult human kidney cDNA library in ⁇ gtlO with radiolabeled (random primed) murine 2-5A-dependent RNase cDNA (clone ZBl) as probe, Sambrook, J. et al.. Cold Spring Harbor Laboratory Press (1989) .
  • Clone HBZ22 is isolated using radiolabeled HZBl DNA as probe.
  • the genomic human 2-5A-dependent RNase clone is isolated from a human placenta cosmid library in vector pVE15 (Stratagene) with a radiolabeled fragment of HZB22 DNA as probe.
  • the murine genomic 2-5A-dependent RNase clone is isolated from a mouse 129SV genomic library in vector ⁇ FIXII (Stratagene) with a radiolabeled fragment of 2-5A-BP cDNA (clone ZBl) as probe. Subcloning of DNA is in Bluescript vectors (Stratagene) .
  • RNA polymerases 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°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°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°C for about 12 hours.
  • Translation reactions contain about 50 ⁇ M zinc sulfate.
  • Endogenous 2-5A-dependent RNase in the reticulocyte lysated is removed by adsorption to about 30 ⁇ M 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, D. , I.R.L. Press. Oxford. England, pp. 149-193 (1987) , for about one hour on ice as described. See Silverman, R.H. , .Anal. Biochem.. 144:450-460 (1985).
  • the 2-5A-dependent RNase:2-5A-cellulose complex is removed by twice centrifuging at about 400 x g for about 5 minutes at about 2°C. The supernatant completely lacking in measurable levels of 2-5A-dependent RNase. See FIG. 5.
  • the set of nested 3'-deletions of the truncated murine 2-5A-dependent RNase cDNA, ZBl is generated with exonuclease III/Sl 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) A[32P]Cp, and its crosslinking to 2-!-5A-dependent RNase is performed exactly as described. See Nolan-Sorden, N.L. et al., Anal. Biochem.. 184:298-304 (1990). Briefly, the 2-5A 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, D. , I.R.L. Press. Oxford. England, pp.
  • Covalent crosslinking is done under a uv lamp (308 nm) for one hour on ice and the proteins are separated on SDS/10% polyacrylamide gels. Filter assays for 2-5A binding activity using the 2-5A probe for about one hour on ice, as described in Knight, M. et al.. Nature, 288:189-192 (1980).
  • Protease digestions are performed o gel-purified proteins in a gel, as described b Cleveland, D.W. et al., J. Biol. Chem.. 252:1102-110 (1977).
  • the ribonuclease assay with 2-5A-cellulos is performed, as described by Silverman, R.H. , Anal. Biochem.. 144:450-460 (1985). Briefly, lysates ar adsorbed to about 30 ⁇ M of 2-5A-cellulose on ice fo about two hours. The matrix is then washed thre times by centrifuging and resuspending in buffer A. See Silverman, R.H. , Anal. Biochem.. 144:450-46 (1985).
  • the matrix is then incubated wit poly(U)-[ 32 P]Cp or poly(C)-[ 32 P]Cp (both at about 1 ⁇ M in nucleotide equivalents) at about 30°C and th levels of acid-precipitable radioactive RNA ar determined by filtration on glass-fiber filters.
  • the Sanger dideoxy sequencing method i used to determine the DNA sequences (Sequenase, United States Biomedical) .
  • the lysines in the truncated murin 2-5A-dependent RNase, clone ZBl, at positions 240 an 274 are mutated, individually and together, t asparagine residues. Mutants ZBl(Lys 274 ->Asn) an the double mutant, ZBl(Lys 240,274 -)Asn) , are obtaine with mutant oligonucleotides after subcloning ZB cDNA into pALTER-1 as described (Promega) .
  • Mutant ZBl(Lys 240 -)Asn) is obtained after polymerase chain reaction amplification of a segment of ZBl with an upstream primer containing a unique Hindi site attached to the mutant sequence and a second primer downstream of a unique Bglll site.
  • the Hindi- and BGlII-digested polymerase chain reaction product and similarly-digested clone ZBl 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 Kansas, Lincoln, Kansas.
  • 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 (1990); Chong, K.L. et al.: EMBO J.. 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.
  • 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.
  • 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 ⁇ g/ml of tetracycline. about 10 ⁇ g/ml of kanamycin and about 25 ⁇ g/ml of streptomycin.
  • PK68 a lysine •* arginine mutant PKR (muPk ⁇ ; the mutant PKR protein binds to dsRNA but has no kinase activity and will thus function as a control)
  • a low molecular weight form of 2-5A-synthetase (synthetase)
  • the plasmids pKS(+)PKR, pKS(+)muPKR, and pKS(+)synthetase are digested first with Xbal and than with Clal restriction endonucleases, the cDNA fragments are purified from low melting point agarose gels and subcloned in sense orientation at Xbal and Clal sites of pAM943.
  • the recombinant plasmids e.g., construct A, pAM943:PK68, construct B, pAM943:muPK68, and contruct C, pAM943:synthetase, which correspond to the constructs depicted in FIG. 13A-C, respectively, are used to transform Argobacterium tumefaciens LBA4404.
  • the resultant bacteria identified as AG68, AGmu68 and AGsyn, respectively, are used for tobacco leaf disc transformations.
  • the plasmid pKS(+)2C5 DNA is digested with Hindlll enzyme and subcloned in the Hindlll site of pAM943 in both orientations, see FIG. 13, and the recombinant plasmids, construct D, 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.
  • 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 cDNA used fo 2-5A-dependent RNase is in plasmid pZC5 referenced i Zhou et al. Cell 72, 753-765 (1994), the human for of the cDNA.
  • the sequence is also disclosed herein.
  • the plasmid pAM822 contains a second selectabl marker gene, the hygromycin resistance gene, permitting the construction of plants containing bot 2-5A-synthetase and 2-5A-dependent RNase cDNAs. Insertion of pAM822:2-5Adep.
  • RNase Fig. 13E
  • containing 2-5A-dependent RNase cDNA int kana ycin-resistant, transgenic tobacco leaf disc containing 2-5A-synthetase cDNA is thus performed.
  • Tobacco plants are grown aseptically i Murashige and. Skoog's medium, known as MS medium, containing about 3% sucrose (MSO medium) and abou 0.8% agar in plastic boxes (Phytatray) at about 28° under cycles consisting of about 16 hr of light an about 8 hr of dark in a growth chamber. Leave bigger than about 2" long are cut into about 2 to 3 cm 2 pieces under the MSO medium and 6-8 leaf piece are placed in a 6 cm Petri dish containing about 2 m of MSO medium and holes are made in the leaf piece with a sterile pointed forcep.
  • MS medium containing about 3% sucrose (MSO medium) and abou 0.8% agar in plastic boxes (Phytatray) at about 28° under cycles consisting of about 16 hr of light an about 8 hr of dark in a growth chamber.
  • Leave bigger than about 2" long are cut into about 2 to 3 cm 2 pieces under the MSO medium and 6-8 leaf piece are placed in a 6 cm
  • Leaf pieces are washed extensively with MSO medium and transferred to solid agar for selection in shoot regeneration medium [MSO; about 0.5 mg/1 BAP (benzylaminopurine) ; about 200 ⁇ g/ml kanamycin; about 200 ⁇ g/ml carbenicillin; and about 100 ⁇ g/ml of cefotaxine] , under diffuse light at about 28 ⁇ C in the growth chamber.
  • shoot regeneration medium [MSO; about 0.5 mg/1 BAP (benzylaminopurine) ; about 200 ⁇ g/ml kanamycin; about 200 ⁇ g/ml carbenicillin; and about 100 ⁇ g/ml of cefotaxine] , under diffuse light at about 28 ⁇ C in the growth chamber.
  • shoot regeneration medium [MSO; about 0.5 mg/1 BAP (benzylaminopurine) ; about 200 ⁇ g/ml kanamycin; about 200 ⁇ g/ml carbenicillin; and about 100 ⁇ g/ml of cefotaxine
  • the transgenic plants expressing 2-5A synthetase are substantially transformed to introduce the cDNA for 2-5A-dependent RNase (with pAM9 3:2-5Adep.RNase sense, construct D; FIG. 13D) .
  • the vector pAM822 FIG. 14
  • the vector pAM822 FIG. 14
  • the vector pAM822 FIG. 14
  • the vector pAM822 FIG. 14
  • Tissue culture and regeneration of plants are done 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.
  • specific plasmid constructs are described herein, the present invention is intended to include any plant vector including those with
  • 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.
  • FIG. 17 depicts presence of 2-5A-dependent RNase cDNA in transgenic tobacco plants as determined on a Southern blot.
  • FIG. 13A Plasmid pAM943:muPK68
  • Antisense 1 + N.T. N.T.
  • a Tobacco plants contain construct D, pAM943:2-5Adep. RNa (sense) .
  • 2-5A binding assays are performed by the filt binding method of Knight, M. et al. Nature (288) :189-1 (1980) with modifications.
  • a 32 P-labeled and bromi substituted 2-5A analog, p(A2'p) 2 (br 8 A2'p) 2 A3'- 32 p]C about 15,000 counts per min per assay, at about 3,000 per mmole, Nolan-Sorden, N.L. , et al. Anal. Biochem.
  • 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, M.J., Morris, A.G., and Gearing, A.J.H., (eds.), Lymphokines and Interferons - A Practical Approach. I.R.I. , Press, Oxford, pp.
  • Extracts are clarified by centrifugation at about 10,000 x g for about 10 min.
  • Supernatants of the extracts, about 100 ⁇ g of protein per assay, are separated by SDS/10% 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 10 5 c.p.m.
  • 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, Iowa) . The plants are monitored for symptoms of viral infection on leaves from control and transgenic plants and are documented in photographs.
  • ATCC Tobacco mosaic virus
  • Dr. Amit Mitra, Iowa Tobacco Etch virus
  • 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.
  • the present invention 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.
  • 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
  • the cDNAs encoding PKR and muPKR are excised from plasmids pKS(+)PKR and pKS(+)muPKR by digesting with Kpnl and Xbal. 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 5' end Xbal 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 Xbal and Clal digested pAM943 by DNA ligation.
  • the resulting plasmids FIG. 13, constructs A and B, are used to transform Argobacterium 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 Iconstruct C)
  • the plasmid ptac-15 containing the human cDNA illustrated in FIG. 20 for a small form of 2-5A-synthetase (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 plasmid pKS(+) (Strategene, La Jolla, CA) in BamHI and EcoRI sites.
  • Recombinant plasmids are prepared from transformed Argobacterium tumefaciens bacteria by standard methods and the presence of 2-5A-synthetase cDNA is confirmed by PCR analysis and by restriction enzyme digests of the isolated plasmids.
  • the plasmid pKS(+)ZC5 encoding a complete coding sequence for human 2-5A-dependent RNase is digested with Hindlll.
  • 2-5A-dependent RNase is purified in a low melting point agarose gel and is then subcloned in Hindlll digested pAM943 in both sense (forward) and antisense
  • Transformed Argobacterium are determined to contain the 2-5A-dependent RNase cDNA by restriction enzyme digests and by PCR analysis. Construction of pAM822:2-5Adep.RNase antisense fconstruct Ei
  • PCR Polymerase chain reactions
  • PCR primers 2-5A-dependent RNase to generate Hindlll and BamHI sites on the two ends of the cDNA and to reduce 5' and 3' untranslated sequences.
  • the PCR primers used are:
  • H2DR-4 5'-GATACTCGAGAAGCTTGCATCCTCATCAGCACCCAGGGCTGG -3'.
  • the PCR product (about 2.25 kbp) is purified on a low melting point agarose gel and is then digested with
  • 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 T.AB B 1 Hwna ⁇ -1 2-5A-depedent RNase
  • SEQ ID NO:l «, SEQ ID NO:2:, SEQ ID NO:3: and SEQ ID N0:
  • GCT CTC ATG GAC GCT GCT GAA AAA GGA CAC 540
  • Val Glu Val Leu Lys lie Leu Leu Asp Glu 190
  • ATT ACG CAT CTG CTG CTG GAC CAT GGG GCT 690 lie Thr His Leu Leu Leu Asp His Gly Ala 230
  • AAG AAA GCT GCT CAC 1500 lie Leu lie Asp Ser Lys Lys Ala Ala His 500 CTG GCA GAT TTT GAT AAG AGC ATC AAG TGG 1530
  • Lys Trp Thr Thr Lys lie Asn Glu Cys Val 640
  • Met Lys Leu Lys lie Gly Asp Pro Ser Leu 690
  • ATC TAT GTC TAC ACA AAA CTA CAG AAC ACA 2130 lie Tyr Val Tyr Thr Lys Leu Gin Asn Thr 710
  • SEQ ID NO:1 represents the DNA encoding sequence for the human 2-5A-dependent RNase protein.
  • SEQ ID NO:2 represents the amino acid sequence encoded by the DNA sequence designated SEQ ID N0:1:.
  • SEQ ID NO:3: represents the DNA sequence, represented by SEQ ID NO:l:, having the alternative codon number 95, CCT.
  • SEQ ID NO:4: represents the amino acid sequence encoded by SEQ ID NO:3: , having the alternative amino acid number 95, proline.
  • 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 N0:5:.
  • MOLECULE TYPE DNA (genomic)
  • GGG GCT GAT GTC AAT GTG AGG GGA GAA AGA GGG AAG ACT CCC CTG ATC 83 Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu lie 230 235 240
  • GCC AAA GAA GAT TTT CAC CCT CCT GCT GAA GAC TGG .AAG CCT CAG AGC 1123 Ala Lys Glu Asp Phe His Pro Pro Ala Glu Asp Trp Lys Pro Gin Ser 325 _ - 330 335 340
  • AAG CAT AAA AAG ATG AAA TTA AAA ATT GGA GAC CCT TCC CTG TAT TTT 2179 Lys His Lys Lys Met Lys Leu Lys He Gly Asp Pro Ser Leu Tyr Phe 680 685 690
  • MOLECULE TYPE DNA (genomic)
  • GGG GCT GAT GTC AAT GTG AGG GGA GAA AGA GGG AAG ACT CCC CTG ATC 83 Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu lie 230 235 240
  • GCC AAA GAA GAT TTT CAC CCT CCT GCT GAA GAC TGG AAG CCT CAG AGC 11 Ala Lys Glu Asp Phe His Pro Pro Ala Glu Asp Trp Lys Pro Gin Ser 325 330 335 340
  • AAG CAT AAA AAG ATG AAA TTA AAA ATT GGA GAC CCT TCC CTG TAT TTT 2179 Lys His Lys Lys Met Lys Leu Lys lie Gly .Asp Pro Ser Leu Tyr Phe 680 685 690
  • MOLECULE TYPE DNA (genomic)
  • ATC CAG GGA GAT GTG AAA CTG CTC GAG ATT CTC CTC TCT TGT GGT GCA 51 lie Gin Gly Asp Val Lys Leu Leu Glu lie Leu Leu Ser Cys Gly Ala 105 110 115
  • CAA CCA CAA AAC ATC TTA ATA GAT TCC AAG AAA GCT GTC CGG CTG GCA 166 Gln Pro Gin Asn lie Leu lie Asp Ser Lys Lys Ala Val Arg Leu Ala 485 490 495 500
  • MOLECULE TYPE protein
  • Glu Lys Gly His Leu-Glu Val Leu Arg lie Leu Leu Asn Asp Met Lys 180 185 190

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Abstract

Isolated 2-5A-dependent RNases, an interferon-induced enzyme which is activated by 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) and implicated in both the molecular mechanisms of interferon action and in the fundamental control of RNA stability in mammalian cells, and encoding sequences therefor are disclosed. The expression cloning and analysis of murine and human 2-5A-dependent RNases is also disclosed. In addition, recombinant nucleotide sequences, recombinant vectors, recombinant cells and antiviral plants which express, for example, 2-5A-dependent RNase, 2-5A synthetase and/or double-stranded RNA dependent protein kinase (PKR), or other amino acid sequences which have activity that interferes with or inhibits viral replication are disclosed.

Description

ANTIvTRAIi TRANSGENIC PLANTS. VECTORS. rvτ.τs. A D METHODS
Related Applications
This application for U.S. patent is a continuation-in-part of U.S. patent application, which was assigned Serial No. 08/028,086 and filed on March 8, 1993. Field of the Invention
The present invention relates to isolated 2-5A-dependent RNases having the ability to bind 2-5A and/or cleave single stranded RNA when bound to 2-5A, 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 2-5A-dependent RNase, 2-5A synthetase and/or PKR. Background
Control of RNA degradation is a critical cell function, and gene expression is often regulated at the level of RNA stability. See, e.g., Shaw, G and Ka en, R., Cell. 46:659-667 (1986). Neverthe less, relatively little is known about the bio chemical pathways that mediate RNA degradation i mammalian or plant systems. For instance, most i not all of the ribonucleases responsible for mRN turnover in mammalian or plant cells remai unidentified. This was reviewed in Brawerman, G. Cell. 57:9-10 (1989).
Presently, the 2-5A system is believed t be the only well-characterized RNA degradatio pathway from higher animals including man. See FIG 1. See also, e.g., Kerr, I.M. and Brown, R.E., Prod Natl. Acad. Sci. U.S.A.. 75:256-260 (1978) an Cayley, P.J. et al., Biophvs Res. Commun. 108:1243-1250 (1982); reviewed in Sen, G.C. an Lengyel, P., J. Biol. Chem.. 267:5017-5020 (1992) The activity of the 2-5A system is believed to b mediated by an endoribonuclease known as 2-5A dependent RNase. See Clemens, M.J. and Williams B.R.G., Cell. 13:565-572 (1978). 2-5A-dependen RNase is a unique enzyme in that it requires 2-5A unusual oligoadenylates with 2',5' phosphodieste linkages, pn(A2 p)nA, for ribonuclease activity. Se
Kerr, I.M. and Brown, R.E., Prod. Natl. Acad. Sci U.S.A.. 75:256-260 (1978). 2-5A is produced from AT by a family of synthetases in reactions requirin double-stranded RNA (dsRNA) . See FIG. 1. See als Hovanessian, A.G. et al.. Nature. 268:537-539 (1977) Marie, I. and Hovanessian, A.G., J. Biol. Chem. 267:9933-9939 (1992). 2-5A is unstable in cells an in cell-free systems due to the combined action o 2',5 '-phosphodiesterase and 5'-phosphatase. Se Williams, B.R.G. et al. ; Eur. J. Biochem.. 92:455-56 (1978); and Johnson, M.I. and Hearl, W.G., J. Biol Chem.. 262:8377-8382 (1987). The interaction o 2-5A-dependent RNase and 2-5A(Kd = 4 X 10""11 M) Silverman, R.H. et al., Biol. Chem. f 263:7336-734 (1988), is highly specific. See Knight, M. et al. Nature. 288:189-192 (1980). 2-5A-dependent RNase i believed to have no detectable RNase activity unti .it- "is converted to its active state by binding t 2-5A. See Silverman, R.H. , Anal. Biochem. 144:450-460 (1985). Activated 2-5A-dependent RNas cleaves single-stranded regions of RNA 3' of UpNp with preference for UU and UA sequences. Se Wreschner, D.H. et al.. Nature. 289:414-417 (1981a) and Floyd-Smith, G. et al.. Science. 212:1020-103 (1981) . Analysis of inactive 2-5A-dependent RNas from mouse liver revealed it to be a singl polypeptide of approximately 80 kDa. See Silverman R.H. et al., Biol. Chem.. 263:7336-7341 (1988).
Although the full scope and biologica significance of the 2-5A system remains unknown studies on the molecular mechanisms of interferon action have provided at least some of the functions. Interferons α, β 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, G. , 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, P., 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, J., 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 o 2-5A-dependent RNase into cells reduced th interferon-mediated inhibition of EMCV replication. See Watling, D. et al., EMBO J.. 4:431-436 (1985). Further, 2-5A-dependent RNase levels were correlate with the anti-EMCV activity of interferon, Kumar, R et al., J. Virol.. 62:3175-3181 (1988), an EMCV-derived dsRNA both bound to and activated 2-5 synthetase in interferon-treated, infected cells See Gribaudo, G. et al., J. Virol.. 65:1948-175 (1991) .
The 2-5A system, however, almost certainl provides functions beyond the antipicornaviru activity of interferons. For instance, introductio of 2-5A into cells, Hovanessian, A.G. and Wood, J.N. Virology. 101:81-90 (1980), or expression of 2-5 synthetase cDNA, Rysiecki, G. et al., J. Interfero Res.. 9:649-657 (1989), inhibits cell growth rates Moreover, 2-5A-dependent RNase levels are elevated i growth arrested cells, Jacobsen, H. et al., Proc Natl. Acad. Sci. U.S.A.. 80:4954-4958 (1983b), an 2-5A synthetase. Stark, G. et al.. Nature 278:471-473 (1979), and 2-5A-dependent RNase level are induced during cell differentiation. See, e.g. Krause, D. et al., Eur. J. Biochem.. 146:611-61 (1985) . Therefore, interesting correlations exis between 2-5A-dependent RNase and the fundamenta control of cell growth and differentiation suggestin that the 2-5A system may function in general R metabolism. The ubiquitous presence of the 2-5 system in reptiles, avians and mammalians certainl supports a wider role for the pathway. See, for example, Cayley, P.J. et al., Biochem. Bioohv. Res. Commun.. 108:1243-1250 (1982).
While it is presently believed that the 2-5A system is the only well-characterized RN 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 eIF2, known as eIF2-alpha, which indirectly inhibits protein synthesis initiation. It is believed that interferons α, f and γ 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. Viroloσv. 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 U.S.A.. 90:232-236 (1993).
In short, the 2-5A system and the PK system inhibit viral protein synthesis. This is believed to be accomplished by the 2-5A system b degrading mRNA and rRNA whereas the PKR system i believed to accomplish this by indirectly inhibitin protein synthesis initiation.
Viral plant diseases are pandemic and thei severity varies from mild symptoms to plant death. The majority of plant viruses are believed to hav single stranded RNA genomes. Moreover, it i currently believed that plants are void of the thre enzymes discussed above, i.e., PKR, 2-5A synthetas 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. 24:593-59 (1985); but see Crum, C. et al.: J. Biol. Chem.. .263:13440-13443 (1988); Hiddinga, H.J. et al.: Science. 241:451-453 (1988); Sela, I.: TIBS. pp 31-33 (Feb 1981); and Devash, Y. et al.: Science 216:1415-1416.
Notwithstanding the importance of 2-5A dependent RNase to the 2-5A system, 2-5A-dependen RNase enzymes having ribonuclease function have no been isolated, purified or sequenced heretofore Consequently, there is a demand for isolated, activ 2-5A-dependent RNases and their complete amino aci sequences, as well as a demand for encoding sequence for active 2-5A-dependent RNases. There is also demand for plants which are resistant to viruses such as the picornaviruses. fiιτnmι*τγ of the Invention
In brief, the present invention alleviates and overcomes certain of the above-mentioned problems and shortcomings of the present state of the art through the discovery of novel, isolated 2-5A-dependent RNases and encoding sequences therefor.
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 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 "2-5A") . 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 H2-5A-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 ribonuclease function when the 2-5A-dependent RNas is activated by 2-5A.
The novel 2-5A dependent RNases of th present invention are protein enzymes havin molecular weights on the order of between about 7 KDa (murine) and about 84 KDa (human) , as determine by gel electrophoresis migration and/or predictio from their respective encoding nucleotide sequences For example, a human 2-5A-dependent RNase of th instant invention has a molecular weight of abou 83,539 Da as determined from the amino acid sequenc predicted from the encoding sequence therefor whereas the murine 2-5A-dependent RNase has molecular weight of about 74 KDa as determined by ge electrophoresis migration and from prediction of th amino acid sequence from the encoding sequence While an about 74 KDa molecular weight is reporte herein for a murine 2-5A-dependent RNase, it shoul nevertheless be appreciated that the reporte molecular weight is for an incomplete murin 2-5A-dependent RNase. It is nevertheless believe that once completely sequenced, i.e., when an abou 84 amino acid end region is identified, the molecula weight of a complete murine 2-5A-dependent RNase wil be similar to that of human, i.e. , about 84 KDa.
It should also be readily apparent to thos versed in this art, however, that since gel electro 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 2-5A-dependent RNase protein is depicted in FIG. 3 and Table 1. The encoding sequence for the human 2-5A-dependent 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 2-5A-dependent RNase is about 5.7 Kb in size.
Analysis of the amino acid sequences of the 2-5A-dependent RNases of the present invention have revealed several characteristics unique to the 2-5A-dependent RNases. For example, it has been discovered that the novel 2-5A dependent RNases of the instant invention include the following uniqu domains which span between the amino terminus and the carboxy terminus. For instance, it has bee discovered that there are at least four and possibl as many as nine or more ankyrin repeats, of whic three lie closest to the amino terminus. However, while four ankyrin repeats have been discovered, i is believed that there may be additional ankyri repeats that may total, for instance, about eight o more when the amino acid sequences of th 2-5A-dependent RNases of the present invention ar further analyzed. It is believed that these ankyri repeats may possibly function in protein-protei interaction. Ankyrin repeat 1 generally lies betwee amino acids designated as 58-90 in Tables 1 and 2 Ankyrin repeat 2 generally lies between amino acid designated as 91-123 in Tables l and 2 • Ankyri repeat 3 generally lies between amino acid designated as 124-156 in Tables i and 2• Ankyri repeat 4 generally lies between amino acid designated as 238 and 270 in Tables ± and 2 • Se also FIGS. 10A and 10B.
It has also been discovered that the nove 2-5A dependent RNases include a cysteine rich regio (which has homology to zinc fingers) that lies close to the carboxy terminus than the amino terminus whic may possibly function in RNA recognition or i formation of protein dimers. The cysteine ric region is believed to include about 5 or 6 cystein residues which generally lie between amino acid designated as 395-444 in the human sequence a reported in Table 1 and FIG. 4, or between amin acids designated as 401-436 in the murine sequence a reported in Table 2 and FIG. 4. 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 and 2.
It has been further discovered that the 2-5A-dependent 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 2-5A-dependent RNase, domain VI generally lies between amino acids designated as 470-489 and domain VII generally lies between amino acid residues desig¬ nated 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 2-5A-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 i 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 2-5A-dependent 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 2-5A-dependent RNase, the human 2-5A-dependent RNase has ribonuclease function in the presence of 2-5A. In contrast, when the murine 2-5A-dependent RNase lacks the about 84 amino acid region at the carbox terminus, it lacks ribonuclease function.
With respect to the binding activity of murine 2-5A-dependent RNase protein to 2-5A, it ha been discovered that, when one P-loop is deleted fro the repeated P-loop motif of a murine 2-5A-dependen RNase protein, nearly all 2-5A binding activity i lost, and that when both P-loops are deleted virtually complete activity is lost. However, it ha been found that, even though the carboxy terminu portion of the amino acid sequence of a murin 2-5A-dependent RNase protein following the repeate P-loop motif has been deleted, partial 2-5A bindin activity is maintained.
. ~ ~ It has been further discovered that whe lysine residues 240 and 274 are replaced wit asparagine residues in both P-loop motifs significant 2-5A binding activity of a murin 2-5A-dependent RNase protein is lost. It has bee further discovered, however, that when either lysin residue 240 or 274 is replaced in either P-loo motif, only partial 2-5A binding activity is lost It is therefore believed that the presence of bot P-loop motifs in the amino acid sequences for th 2-5A dependent RNases of the present invention play an important role in 2-5A binding activity. It i further believed that the presence of lysine residue 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 2-5A-dependent RNases and novel murine and human clones. Recombinant and naturally occurring forms of 2-5A-dependent RNase displayed virtually identical 2-5A binding properties and ribonucleas specificities.
. -" The present invention further contemplates the use of the novel isolated, 2-5A-dependent RNases and encoding sequences therefor, as well as analog and active fragments thereof, for use, for instance, 1.) in gene therapy for human and animal disease including viral disease and cancer, 2.) as geneti markers for human disease due to perhaps cancer o viral infection, 3.) to develop plants and animal resistant to certain viruses, and 4.) as enzymes i connection with research and development, such as fo studying the structure of RNA. In one manner t accomplish the above, and as contemplated by th present invention, the encoding sequences of th instant invention may be utilized in ex vivo therapy, i.e., to develop recombinant cells using the encoding sequence of the present invention using techniques known to those versed in this art. In another manne 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 2-5A-dependent 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 productio and/or treatment. These include, for example, thos animal antiviral genes that encode 2-5A-synthetase 2-5A-dependent RNase, and PKR. Thes interferon-regulated proteins, 2-5A-synthetase 2-5A-dependent RNase and PKR (the dsRNA-dependen protein kinase) have recognized antiviral effects i higher animals and are believed to have antivira effects in the transgenic plants of the presen invention. PKR is stimulated by dsRNA t phosphorylate translation factor eIF2 whic indirectly inhibits protein synthesis intiation. O the other hand, 2-5A synthetase is activated by dsRN resulting in the production of n2-5A," pχA(2'p5'A) wherein X = about 1 to about 3 and Y ≥ about 2, fro AT?. The 2-5A then activates an endoribonucleas entitled 2-5A dependent RNase (also known as RNase or nuclease F) . The activated ribonuclease degrade mRNA and rRNA thus inhibiting protein synthesis.
These above-described pathways ar particularly effective at inhibiting viruses i animals with single stranded RNA genomes tha replicate through dsRNA intermediates, such as th picornaviruses, and are believed to be effective a inhibiting similar types of viruses that infec plants. This belief is premised upon th understanding that most single stranded RNA plan viruses produce double stranded structures durin replication by their viral replicases, see Dawson,
W.O. et al.: Acad. Press. 38:307-342 (1990), and tha plant viruses are similar to animal viruses i structure, composition and mechanism of replicatio in cells. In addition, even viral so-calle single-stranded RNA may contain secondary structure which could activate PKR and 2-5A synthetase leadin to widespread plant protection against plan viruses. It is believed that co-expression o
2-5A-dependent RNase and 2-5A-synthetase, will lea to the destruction of viral mRNA and viral genomi
RNA thereby protecting the transgenic plants of th present invention from viruses. Moreover, it i believed that expression of PKR by the transgeni plants of the present invention will inhibit vira protein synthesis leading to inhibition of viru replication and protection of the transgenic plants.
The present invention is therefore premised in par upon the belief that plant virus RNAs activat
2-5A-synthetase and PKR in the transgenic plants o the instant invention leading to immunity agains virus infection. Furthermore, expression of 2-5 synthetase alone or 2-5A-dependent RNase alone or PK alone may protect plants against viruses, perhaps b binding to viral RNA, such as viral replicativ intermediates thereby blocking viral replication.
Moreover, expression of only the dsRNA bindin domains of PKR and/or of 2-5A-synthetase ma similarly protect the transgenic plants of th present invention against viral infection.
It should therefore be appreciated by thos versed in this art that novel transgenic plants whic are resistant to viral infection can now be produce in accordance with the present invention. It i believed that the effectiveness of the anti-vira protection can be enhanced or even maximized when a least the three-above animal antiviral genes ar inserted into plants to form exemplary transgeni plants of the present invention, since the anima antiviral proteins encoded by these three anima antiviral genes interfere with different stages o the" viral life cycles. Moreover, these anima antiviral proteins or amino acid sequences ar believed likely to be safe to give or introduce int animals, including humans, since these antivira proteins or amino acid sequences are naturall occurring in humans as well as in other mammals avians and reptiles.
While the present invention is describe herein with reference to the particular sequence disclosed, it should nevertheless be understood those skilled in this art that the present inventi contemplates variations to the amino acid and/ nucleotide sequences which do not destroy 2- synthetase activity, PKR activity and/o 2-5A-dependent ribonuclease activity. Therefore, th present invention contemplates any analogs, parts o 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 an any transgenic plant which have the ability t accomplish the objectives of the instant invention For example, the instant invention includes any amin acid sequence which has antiviral activity and an nucleotide sequence which encodes therefor and thos transgenic plants that express such nucleotid sequences. More specifically, the present inventio includes, for instance: 1.) any animal amino aci sequence which has the ability to inhibit o interfere with viral replication such as those amin acid sequences that have activity similar o identical to PKR activity, 2-5A synthetase activit and/or 2-5A ribonuclease activity, and any nucleotid sequence which encodes for an amino acid sequenc having any such activity; and 2.) any transgeni plant having any animal antiviral nucleotide sequenc which encodes any such amino acid sequence which ha any such antiviral activity. The above features and advantages of t present invention will be better understood wi reference to the accompanying FIGS. , Detail Description and Examples. It should also understood that the particular methods, amino ac sequences, encoding sequences, constructs, vector recombinant cells, and antiviral transgenic plan illustrating the invention are exemplary only and n to be regarded as limitations of the invention. Brief Description of the FIGS.
Reference is now made to the accompanyi FIGS, in which is shown illustrative embodiments the present invention from which its novel featur and advantages will be apparent.
FIG. 1 is the 2-5A system: a ribonuclea pathway which is believed to function in t molecular mechanism of interferon actio 5'-phosphatase, p'tase; 2,-5 -phosphodiesteras 2'-PDE.
FIGS. 2A and 2B is a comparison of 2- binding activity of recombinant and natural occurring forms of murine 2-5A-dependent RNase.
FIG. 2A is a specific affinity of truncat murine 2-5A-dependent RNase for 2-5A. UV covale crosslinking of the 32P-2-5A probe (lanes 1-7) protein is performed after translation reactions wheat germ extract (5 μl) with murine 2-5A-depende RNase mRNA (from clone ZBl) (lanes 1-3) or withou added RNA (lane 4) or in extract of interfero treated mouse L cells (100 μg of protein) (lane 5-7) . Reactions are without added competitor (lane 1, 4, and 5) or in the presence of either trime core. (A2'p)2A, (100 nM) (lanes 2 and 6) or trime 2-5A, p3(A2'p)2A (100 nM) (lanes 3 and 7). Lanes and 9 are produced by incubating the wheat ger extract with 35S-methionine in the absence o presence of 2-5A-dependent RNase mRNA, respectively.
FIG. 2B are identical chymotrypsin cleavag products and are obtained from recombinant an naturally occurring form of 2-5A-dependent RNase Partial chymotrypsin digests (arrows) are performe on- " truncated 2-5A-dependent RNase (clone ZBl produced in wheat germ extract ("Recombinant") an murine L cell 2-5A-dependent RNase ("Naturall Occurring") after crosslinking to the 2-5A probe an purification from gels.
FIGS. 3A and 3B are clonings of th complete coding sequence for human 2-5A-dependen RNase.
FIG. 3A is the construction of a huma 2-5A-dependent RNase clone. The initial huma 2-5A-dependent RNase cDNA clone, HZB1, is isolate from an adult human kidney cDNA library in λgtl using radiolabeled murine 2-5A-dependent RNase cDN (clone ZBl) as probe. See Example. Radiolabel HZBl DNA is used to isolate a partially overlappi cDNA clone, HZB22, which is fused to HZBl DNA at t Ncol site to form clone ZC1. The 5'-region of t coding sequence is obtained from a genomic Sa fragment isolated using a radiolabeled HZB22 D fragment as probe. Fusion of the genomic SA fragment with ZC1 at the indicated Sad site produc clone ZC3. The coding sequence with some flanki sequences is then subcloned as a Hindlll fragme into pBluescript KS(+) (Stratagene) resulting clone ZC5. The restriction map for the composi clone, ZC5, is shown. Clone HZBl includ nucleotides designated as 658-2223 in Table I whi encode for amino acids designated as 220-741 in Tab I. Clone HZB22 includes a nucleotide sequence whi encodes for amino acids designated as 62-397 in Tab I. Clone ZC1 includes a nucleotide sequence whi encodes for amino acids designated as 62-741 in Tab I. Clones ZC3 and ZC5 both include nucleoti sequences which encode for amino acids designated 1-741 in Table I.
FIG. 3B is a nucleotide sequence a predicted amino acid sequence of human 2-5A-depende RNase with flanking nucleotide sequences. T numbers to the right indicate the positions nucleotides and amino acid residues. FIG. 4 is alignment of the predicted amin acid sequences for murine and human forms o 2-5A-dependent RNase. The positions of the repeate P-loop motifs, the cysteine (Cys)-rich regions wit homology to zinc fingers, and the regions of homolog to protein kinase domains VI and VII are indicated Amino acids residues which are important component of the indicated domains are represented in bold typ and are italicized. Identical amino acid residues i murine and human 2-5A-dependent RNase are indicate with colon (:) symbols adjacent therebetween.
FIGS. 5A and 5B are 2-5A binding propertie and ribonuclease activity of recombinant human 2-5A dependent RNase produced in vitro.
. - ~ FIG. 5A is specific affinity of recombinan human 2-5A-dependent RNase for 2-5A. Crosslinking o the 2-5A probe (lanes 1-7) to protein is performe after translation reactions in wheat germ extract ( μl) with human 2-5A-dependent RNase mRNA (lanes 1-3 or without added RNA (lane 4) or in extract of huma interferon α treated (1000 units per ml for 16 h human HeLa cells (350 μg 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)2A, (100 nM) (lanes 2 and 6) or trimer 2-5A p3(A2'p)2A (100 nM) (lanes 3 and 7). Incubation with 35S-methionine are shown in lanes 8 to 12. Lan 8 is with wheat germ extract and human 2-5A-dependent RNase mRNA. Reticulocyte lysate preadsorbed to 2-5A-cellulose is incubated with human 2-5A-dependent RNase mRNA in the absence (lane 9) or presence (lane 10) 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 ( o , □ ) or in the presence of both 2-5A-dependent RNase mRNA and cycloheximide (50 μg per ml (•) . See Example I. Subsequently, the recombinant 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) (O, •,B) °r poly(C) (D).
FIGS. 6A, 6B and 6C show levels of 2-5A-dependent RNase mRNA which are induced by interferon treatment of murine L929 cells even in the presence of cycloheximide. FIG. 6A is a northern blot prepared wit poly(A)+RNA (4 μg per lane) that is isolated fro murine L929 cells treated with murine interferon (α β) (1000 units per ml) and/or cycloheximide (50 μ per ml) for different durations (indicated) which i probed with radiolabeled murine 2-5A-dependent RNas cDNA. Interferon, IFN; cycloheximide, CHI.
FIG. 6B shows levels of 2-5A-dependen RNase which are estimated from the autoradiogra shown in panel (a) with a video camera an QuickCapture and Image computer programs.
FIG. 6C shows levels o glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRN as determined in the same blot shown in panel (A) .
FIGS. 7A and 7B are the truncated recombinant murine 2-5A-dependent RNase, clone ZBl and murine L cell 2-5A-dependent RNase havin identical 2-5A binding activities localized to repeated P-loop motif.
FIG. 7A shows incubations of truncate 2-5A-dependent RNase, clone ZBl, ("Recombinant" which is produced in wheat germ extract (upper panel or of murine L cell 2-5A-dependent RNase (labele "Naturally Occurring," lower panel) with the 3 P-2-5 probe, (2.4 nM) , are in the absence of presence o unlabeled 2',5 '-phosphodiester linked oligonucleo tides (as indicated) followed by uv covalen crosslinking. Autoradiograms of the dried SDS/1 polyacrylamide gels are shown. Concentrations of t oligonucleotide competitors are indicated. I inosine.
FIG. 7B shows a truncated series of muri 2-5A-dependent RNase mutants (ZBl to ZB15) which produced in wheat germ extract which are assayed f 2-5A binding activity by a filter binding metho See Example and Knight et al. 1980) . The positio of the P-loop motifs and the lengths of t translation products are indicated. Clone Z encodes for amino acids designated as 1-656 in Tab 2 , except for the last 5 amino acid residues whi are Lys, Pro, Leu, Ser, and Gly. Clone ZB2 encod for" amino acids designated as 1-619 in Table 2 Clone ZB3 encodes for amino acids designated as 1-5 in Table 2 . Clone ZB5 encodes for amino aci designated as 1-474 in Table 2. Clone ZB9 encod for amino acids designated as 1-403 in Table 2 Clone ZB10 encodes for amino acids designated 1-365 in Table 2 . Clone ZB13 encodes for ami acids designated as 1-294 in Table 2 . Clone ZB encodes for amino acids designated as 1-265 in Tab 2 . Clone ZB15 encodes for amino acids designated 1-218 in Table 2. FIGS. 8A and 8B are substitution mutation of the lysine residues in the P-loop motifs o 2-5A-dependent RNase.
FIG. 8A shows the truncated murin 2-5A-dependent RNase, clone ZBl, and lysine t asparagine substitution mutants of clone ZBl, whic are synthesized in wheat germ extract. In (A unlabeled translation products are covalentl crosslinked to the bromine-substituted, 32P-labele 2-5A probe, Br-2-5A-[32P]pCp. See Nolan-Sorden e al., 1990.
FIG. 8B shows the mRNA species which ar translated in the presence of 35-S-methionine i separate reactions. Autoradiograms of the dried SDS/polyacrylamide gels are shown. The order an positions of the translation products (labelle "RNase") and the relative molecular masses (in kDa of the protein markers are indicated.
FIGS. 9A and 9B are a comparison of th amino acid sequences of RNase E and 2-5A-dependen RNase.
FIG. 9A shows identical and conservativ matches which are shown between E. coli RNase E an the murine and human forms of 2DR.
FIG. 9B is a model for the structure an function of 2DR. Abbreviations: P-loop motifs, repeated sequence with homology to P-loops; Cysχ, cysteine-rich region with homology to certain zi fingers; PK, homology to protein kinase domains and VII.
FIGS. 10A and 10B are a comparison of t amino acid sequences of the ankyrin repeats in t human and murine 2-5A-dependent RNase proteins.
FIG. 10A shows murine and human forms 2-5A-dependent RNases containing four ankyr repeats. Homology between the ankyrin consens sequence and the murine and human forms 2-5A-dependent RNase are indicated. ψ, hydrophob amino acids.
FIG. 10B is a model showing the relati positions of the four ankyrin repeats in 2-5 .dependent RNase in comparison to the position of t proposed 2-5A binding domain (t) (the repeated P-lo motif) ; Cysχ, the cysteine-rich region; PK, t protein kinase homology region, and t carboxy-terminal region required for RNase activity.
FIG. 11 shows the role of 2-5A-depende RNase in the anti-viral response of cells interferon treatment. Interferon binds to specif cell surface receptors resulting in the generation a signal which activates a set of genes in the ce nucleus. The genes for 2-5A synthetase are th activated producing inactive, native 2- synthetase. Interferon treatment of the cell al 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 2-5A synthetase resulting in the production of 2-5A. The 2-5A 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 p.AM943 which is about 12 Kbp. Abbreviations: B^, left border; Bj*>, right border; Kanr, kanamycin 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 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 pAM943:2-5A-dep. RNase (sense); FIG. 13D/a depicts certain portion of plasmid pAM943:2-5A-dep. RNase an FIG. 13E depicts pAM822:2-5A dep. RNase (antisense). Abbreviations: BL, left border; B-R, right border Kanr, kanamycin resistance; Hygror, hygromyci resistance; AMT, promoter of adenyl methy transferase gene from Chlorella virus; 35S, promote for 35S RNA from Cauliflower mosaic virus; PKR, cDN to human PKR; uPKR, cDNA to a lysine (amino acid 296) to arginine mutant form of PKR; Synthetase, cDN to a low molecular weight form of huma 2-5A-synthetase; 2-5Adep. RNase, cDNA to huma 2-5A-dependent RNase; TER, RNA termination signal.
FIG. 14 shows a physical map of Ti base binary vector pAM822 which is about 14.6 Kbp. Abbreviations: BL, left border; BR, right border Kanr, kanamycin resistance; Hygror, hygromyci resistance; Tetr, tetracycline resistance; AMT promoter of adenyl methyl transferase gene fro Chlorella virus; 35S, promoter for 35S RNA fro Cauliflower mosaic virus; TER, RNA terminatio signal; Ovi V, origin of DNA replication.
FIG. 15 shows expression of huma 2-5A-synthetase cDNA intransgenic tobacco plants a determined by measuring mRNA levels in a Norther blot. Construct C (pAM943:Synthetase) was introduce into the plants. Total RNA was prepared from th leaves of control (labeled "C") and transgenic plant using RNASTAT-60 (Tel-Test B. , Inc.). Thirty μg o RNA was treated with glyoxal and separated in a 1.5 agarose gel. After electrophoresis RNA wa transferred to Magnagraph (MSI) Nylon membrane an probed with human 2-5A-synthetase cDNA labeled wit [α-32P]dCTP by random priming. Autoradiograms wer made from the dried blots.
FIG. 16 shows expression of mutant and wil type forms of human PKR cDNA in transgenic tobacc plants as determined by measuring mRNA levels in Northern blot. Constructs A (pAM943:PK68) and (pAM943: uPK68) encoding wild type and mutant (lysin at position 296 to arginine) forms of PKR respectively, were introduced into the plants. Tota RNA was prepared from the leaves of control (labele "C") and transgenic plants using RNASTAT-60 (Tel-Tes B. , Inc.). Thirty μg of RNA was treated with glyoxa and separated in a 1.5% agarose gel. Afte electrophoresis RNA was transferred t Magnagraph(MSI) Nylon membrane and probed with huma PKR cDNA labeled with [α-32P]dCTP by random priming Autoradiograms were made from the dried blots.
FIG. 17 shows a presence of 2-5A-dependen RNase cDNA in transgenic plants as determined on Southern blot. Genomic DNA was isolated from leave of transgenic plants containing construct D/ (pAM943:2-5A-dep. Nase, antisense) using CTA (cetyltrimethylammonium bromide) following the metho of Rogers and Bendich (1988, Plant Molecular Biolog Manual, A6, pp. 1-10, Kluwar Academic Pulbisher Dordrecht) . Ten μg of genomic DNA was digested wit Hindlll for 5 h at 37'C and fractionated in a 1 agarose gel followed by transfer to Magnagraph (nylo transfer membrane. Micron Separations, Inc.) using capillary transfer method. The cDNA fo 2-5A-dependent RNase (from plasmid pZC5) was labele by random priming with [α-32P]dCTP (3,000 Ci/mmole using a Prime-a-gene kit from (Promega) according t the protocol supplied by the company. The labele 2-5A-dependent RNase cDNA (Specific activity of 1.0 109" c.p.m. per μg DNA) was washed and a autoradiogram was made from the dried membrane. Th sizes (in kilobases) and the positions of the DN markers are indicated. The band indicated a "2-5A-dep. RNase cDNA" (see arrow) was absent i Southern blots of control plants (data not shown) .
FIG. 18 depicts a coding sequence for hum p68 kinase mRNA (PKR) .
FIG. 19 depicts a translation product the complete coding sequence for human p68 kina mRNA (PKR) of FIG. 18.
FIG. 20 depicts a coding sequence for hum 2-5A synthetase cDNA. FIG. 21 depicts a translation product the coding sequence for human 2-5A-synthetase of FI 20. Detailed Description
By way of illustrating and providing a mo complete appreciation of the present invention a many of the attendant advantages thereof, t following Detailed Description and Examples are giv concerning the novel 2-5A-dependent RNases, encodi sequences therefor, recombinant nucleotide molecule constructs, vectors, recombinant cells, antivir transgenic plants and methods.
Because 2-5A-dependent RNase is very low abundance (one five-hundred-thousandth of the tot •protein in mouse liver, Silverman, R.H. et al., Biol. Chem.. 263:7336-7341 (1988)), its cloni requires the development of a sensitive screeni method. Murine L929 cells are selected as the sour of mRNA due to high basal levels of 2-5A-depende RNase. A protocol to enhance 2-5A-dependent RNa mRNA levels is developed based on the observati that optimal induction of 2-5A-dependent RNase obtained by treating cells with both interferon a cycloheximide, then with medium alone. See Exampl The cDNA library is screened by an adaptation techniques developed for cloning DNA bindi proteins, Singh, H. et al., Cell. 52:415-423 (1988) Singh H. et al., BioTechniques. 7:252-261 (1989), which a bromine-substituted 32P-labeled 2-5A analog ("2-5A probe"). Example and Nolan-Sorden, N.L. al.. Anal. Biochem.. 184:298-304 (1990), replaced radiolabeled oligodeoxyribonucleotide. A sing clone (ZBl) is thus isolated from about three milli plaques. The protein expressed from the ZBl clon transferred from plaques to filter-lifts, sho reactivity to both the 2-5A probe and to a high purified polyclonal antibody directed again 2-5A-dependent RNase.
To obtain recombinant protein f characterization, the cDNA is transcribed a translated in cell-free systems. See Example. 2- ■binding activity is then determined by covalent crosslinking the 2-5A probe to the protein with light, for example, Nolan-Sorden, N.L. et al., Ana Biochem.. 184:298-304 (1990). The recombinant 74 k protein produced in a wheat germ extract sho specific affinity for the 2-5A probe. See FIG. 2 lanes 1 to 3. A core derivative of 2-5A lacki 5'-phosphoryl groups, (A2 p)2A, fails to interfe with binding of the protein to the 2-5A probe where trimer 205A, p3(A2,p)2 , completely prevents pro binding. See FIG. 2A, lanes 2 and 3, respectivel There is no detectable 2-5A binding proteins in t wheat germ extract as shown in the incubation witho added RNA, FIG. 2A, lane 4. For comparison, similar profile of 2-5A binding activity is obtaine for the 80 kDa 2-5A-dependent RNase from murine L9 cells, incubated without added oligonucleotide o with (A2,p)2A or p3(A2,p)2A as competitors. See FIG 2A, lanes 5 to 7. The 35S-labeled translatio product is shown in FIG. 2A, lane 9. In a furthe comparison, covalent linkage of the 2-5A probe to th about 74 kDa protein and to murine L929 cel 2-5A-dependent RNase followed by partial digestio with chymotrypsin produces an identical pattern o six labeled peptides. See FIG. 2B. Similarly partial digestion of the two labeled proteins with S aureus V8 protease also produces identical pattern •of- labeled cleavage products. These results and th apparent molecular weight of about 74 kDa for th recombinant protein, as compared to about 80 kDa fo 2-5A-dependent RNase, see FIG. 2A, suggests that th about 74 kDa protein is a truncated, or partial clon for 2-5A-dependent RNase.
To obtain the entire coding sequence fo human 2-5A-dependent RNase, a composite DN containing genomic and cDNA is constructed. See FIG 3A. The initial cDNA portion of the huma 2-5A-dependent RNase clone (HZBl) is obtained b screening a human kidney cDNA library wit radiolabeled murine 2-5A-dependent RNase cDNA. Se Example. A genomic clone, containing the 5'-part o the coding sequence, is isolated with radiolabele human 2-5A-dependent RNase cDNA. The nucleotide an predicted amino acid sequences of huma 2-5A-dependent RNase are determined, FIG. 3B resulting an open reading frame encoding a protein o 83,539 Da.
A comparison is made between the predicte amino acid sequences of the human and murine forms o 2-5A-dependent RNase in order to identify an evaluate the conserved regions of the proteins. Se FIG. 4. The murine cDNA, clone ZBl, contains abou 88% of the coding sequence for 2-5A-dependent RNas to which an additional twenty-eight 3'-codons ar added from a murine genomic clone. Alignment of th murine and human forms of 2-5A-dependent RNas indicates about 65% identity between the overlappin regions. See FIG. 4. In addition, there is 73 identity between the corresponding nucleotid sequences for murine and human 2-5A-dependent RNase The apparent translation start codons for both th murine and human 2-5A-dependent RNases, are in a appropriate context for translational initiation namely ACCATGG and GTCATGG. respectively. See FIG 3B. See also, for example, Kozak, M. , Cell 44:283-292 (1986). In addition, both the human an murine 2-5A-dependent RNase sequences contai in-frame stop codons upstream of the translatio start sites. See FIG. 3B.
The 2-5A binding properties of th recombinant and naturally occurring forms of huma 2-5A-dependent RNase are compared by uv covalen crosslinking to the 2-5A probe. The recombinan human 2-5A-dependent RNase produces in wheat ger extract shows specific affinity for 2-5A. See FIG 5A, lanes 1 to 3. Radiolabeling of the cloned huma 2-5A-dependent RNase with the 2-5A probe is no prevented by (A2,p)2A. See FIG. 5A, lanes 1 and 2 In contrast, addition of trimer 2-5A, p3(A2,p) A effectively competes with the 2-5A probe for bindin to the recombinant 2-5A-dependent RNase. See lan 3.-~ The same pattern of 2-5A binding activity i obtained with 2-5A-dependent RNase in an extract o interferon-treated human HeLa cells. See FIG. 5A lanes 5 to 7. The apparent molecular weights of HeL cell 2-5A-dependent RNase and 35S-labeled recombinan human 2-5A-dependent RNase produced in reticulocyt lysate are believed to be exactly the same (about 8 kDa). See FIG. 5A, lanes 5 and 9. The recombinan human 2-5A-dependent RNase produced in wheat ger extract migrates slightly faster probably due t post-translational modifications. See FIG. 5A, lane 1, 2 and 8. To demonstrate and characterize t ribonuclease activity of the cloned 2-5A-depende RNase, translation is performed in a reticulocy lysate instead of a wheat germ extract due to t substantially greater efficiency of protein synthes in the former system. See FIG. 5A, compare lanes and 8. Prior to translation, endogenous reticulocy 2-5A-dependent RNase is removed by adsorbing t lysate to the affinity matrix, 2-5A-cellulose. S Example. See also, Silverman, R.H. , Anal. Biochem 144:450-460 (1985). The treatment wi 2-5A-cellulose effectively removes all measurab endogenous 2-5A-dependent RNase activity from t lysate, as determined by 2-5A-dependent ribonuclea assays, and FIG. 5B. In addition, the adsorptio depletion protocol did not reduce translation efficiency. FIG. 5A, lanes 9 and 12 show t 35S-translation products produced in t 2-5A-cellulose-pretreated and untreated lysate respectively.
Ribonuclease assays with recombina 2-5A-dependent RNase are performed after immobilizi and purifying the translation product on t activating affinity matrix, 2-5A-cellulose. It w previously shown that murine L cell 2-5A-depende RNase bound to 2-5A-cellulose, resulting ribonuclease activity against poly(U) but n poly(C) . See Silverman, R.H., .Anal. Biochem. 144:450-460 (1985). Furthermore, by washin 2-5A-dependent RNase:2-5A-cellulose prior to addin the substrate the level of general non-2-5A-dependent RNase, is greatly reduced. Se Silverman, R.H. , .Anal. Biochem.. 144:450-460 (1985) Incubations of lysate in the absence of added mRNA o in the presence of both human 2-5A-dependent RNas mRNA and cycloheximide resulted in only low levels o poly(U) breakdown. See FIG. 5B. In addition, it i shown that cycloheximide completely prevente 2-5A-dependent RNase synthesis. See FIG. 5A, lan 10. In contrast, translation of the huma 2-5A-dependent RNase mRNA, in the absence o .inhibitor, results in substantial ribonucleas activity against poly(U) but not against poly(C) See FIG. 5B. The poly(U) is degraded with half-life of about 10 minutes whereas only 20% of th poly(C) is degraded after one hour of incubation Binding of recombinant 2-5A-dependent RNase to th affinity matrix was also shown by monitoring th presence of the 35S-labeled translation product These results are believed to demonstrate that th recombinant human 2-5A-dependent RNase produced i vitro is a functional and potent ribonuclease Furthermore, both recombinant and naturally occurrin forms of 2-5A-dependent RNase are capable of cleavin poly(U) but not poly(C) . See FIG. 5B. See als Silverman, R.H., .Anal. Biochem.. 144:450-460 (1985 and Floyd-Smith, G. et al.. Science. 212:1020-103 (1981).
To determine if 2-5A-dependent RNase mRN levels are regulated by interferon, a northern blo from murine L929 cells treated with interferon an cycloheximide is probed with the radiolabeled murin 2-5A-dependent RNase cDNA. See FIG. 6 2-5A-dependent RNase mRNA levels are enhance three-fold by interferon (α + β) treatment even i the presence of cycloheximide. See FIGS. 6A and B compare lanes 1 and 2) . Regulation of 2-5A-dependen RNase mRNA levels by interferon as a function of tim .is- "demonstrated (FIGS. 6A and B, lanes 3 to 6 Maximum 2-5A-dependent RNase mRNA levels are observe after 14 hours, of interferon treatment. See FIGS. 6 and B, lane 6. A similar increase in levels o 2-5A-dependent RNase per se is observed afte interferon treatment of the cells. Relativel invariant levels of GAPDH mRNA indicates tha equivalent levels of RNA are present in every lane o the blot. See FIG. 6C. These results are believe to show that the induction of 2-5A-dependent RNas expression is a primary response to interfero treatment. The murine and human 2-5A-dependent RNas mRNAs are determined from northern blots to be 5.7 k and 5.0 kb in length, respectively. See FIG. 6A. The 2-5A-dependent RNase coding sequences, therefore, comprise only about 40% the nucleotide sequence contained in the mRNAs.
The 2-5A binding functions of th recombinant and naturally occurring forms of murin 2-5A-dependent RNase are characterized by covalen crosslinking to the 2-5A probe in the presence o unlabeled 2-5A or 2-5A analogues as competitors. Se FIG. 7A. Interestingly, although the about 74 kD truncated 2-5A-dependent RNase is missing about 84 amino acids from its carboxy-terminus, see FIG. 4, i nonetheless possesses a 2-5A binding activit indistinguishable from that of naturally occurrin 2-5A-dependent RNase. See FIG. 7A. Trime 2-5A[p3(A2'p)2A] , at about 20 nM effectively prevent the 2-5A probe from binding to either protein. Se FIG. 7A, lane 8. In comparison, a 500-fold highe concentration of (A2'p)2A (10 μM) is required t prevent probe binding to both proteins. See lan 13. The dimer species, p3A2'pA, is unable to preven the 2-5A probe from binding to the proteins even at concentration of lOμM (lane 18) . However, th inosine analogue, p3I2 pA2,pA, Imai, J. et al., J. Biol. Chem.. 260:1390-1393 (1985), is able to preven probe binding to both proteins but only when added a a concentration of about 1.0 μM (lane 22). To further define sequences involved i 2-5A binding, nested 3'-deletions of the murin 2-5A-dependent RNase cDNA, clone ZBl, ar constructed, transcribed in vitro, and expressed in wheat germ extract. See FIG. 7B. The differen deletion clones produces comparable amounts o polypeptide as monitored by incorporation o 35S-methionine. The levels of 2-5A binding activit are determined with the 2-5A probe in both a filte binding assay. Knight, M. et al.. Nature, 288:189-19 (1980) , and the uv crosslinking assay, Nolan-Sorden N.L. et al.. Anal. Biochem.. 184:298-304 (1990), wit similar results. See FIG. 7B. Expression of clon ZBll, encoding amino acid residues 1 to 342, result in- a loss of only about 26% of the 2-5A bindin activity as compared to clone ZBl (amino acids 1 t 656). See FIG. 7B. Clones intermediate in lengt between ZBl and ZBll all result in significant level of 2-5A binding activity. In contrast, protei produced from ZB13 (amino acids 1 to 294) results i only about 38.3% of the 2-5A binding activity o clone ZBl, suggesting that a region important for th 2-5A binding function is affected. Indeed, clon ZB14 produced a protein encoding amino acids 1 to 26 which is nearly inactive in the 2-5A binding assa (only 1.9% of th activity of clone ZBl) Interestingly, the significant decrease in 2-5 binding activity observed with ZB14 occurs with t deletion of one of two P-loop motifs; nucleoti binding domains in many proteins. See FIGS. 4 a 7B. See also Saraste, M. et al., TIBS. 14:430-4 (1990) . Deletion of both P-loop motifs in clone ZB results in protein (amino acids 1 to 218) which completely lacking in 2-5A binding activity. S FIG. 7B.
To probe the involvement of the consens lysine residues in the P-loop motifs in 2-5A bindi activity, site-directed mutagenesis is performed the truncated form of murine 2-5A-dependent RNa encoded by clone ZBl. Previously, it is report that substitution mutations of the conserved lysi residues in P-loop motifs of eucaryotic initiati factor 4A and for Bacillus anthracis adenylyl cycla results in a loss of ATP binding and catalyt activities, respectively. See Rozen et al., Mo Cell. Biol.. 9:4061-4063 (1989) and Xia, Z. a Storm, D.R., J. Biol. Chem.. 265:6517-6520 (1990) In the former study the invariant lysine residue mutated to asparagine. See Rozen et al., Mol. Cel Biol.. 9:4061-4063 (1989). We substitute individually and together, the consensus lysines wi asparagines at positions 240 and 274 in the t P-loop motifs of 2-5A-dependent RNase. See FIG. and the Example. Analysis of the effects of the mutations on 2-5A binding activity is determined by covalently crosslinking the 32P-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 35S-methionine. See FIG. 8B. The three mutant forms of 2-5A-dependent RNase shows reduced binding to the 2-5A probe. See FIG. 8A, lanes 2 to 4. Clone ZBl(Lys240-)Asn) , FIG. 8A, lane 2, expresses a mutant 2-5A-dependent RNase with a substantially reduced affinity for 2-5A; about 48.4% of the activity of clone ZBl 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(Lys274->Asn) . See FIG. 8A, lane 3. In contrast, 2-5A binding activity from clone ZBl(Lys240'274->Asn) , 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 ZBl (averaged from three separate experiments) . These results suggest that the lysine residues at positions 240 and 274 function within th context of a repeated P-loop motif in the binding o 2-5A to 2-5A-dependent RNase. The molecular cloning and expression o 2-5A-dependent RNase, the terminal factor in the 2-5 system and a key enzyme in the molecular mechanism of interferon action is described. See FIG. 1. Th recombinant proteins produced in vitro ar demonstrated to possess 2-5A binding propertie identical to naturally occurring forms of murine an human 2-5A-dependent RNase. See FIGS. 2, 5A, and 7. In addition, linkage of a 32P-2-5A analogue to truncated murine 2-5A-dependent RNase and to murine cell 2-5A-dependent RNase followed by partia proteolysis reveals identical patterns of labele peptides. See FIG. 2B. Furthermore, the full-lengt recombinant human 2-5A-dependent RNase isolated o the" activating, affinity matrix, 2-5A-cellulose, shows potent ribonuclease activity towards poly(U) but none against poly(C). See FIG. 5B. Similarly, it is previously demonstrated that murine L cel 2-5A-dependent RNase was activated by 2-5A-cellulos resulting in the cleavage of poly(U) , but not o poly(C). See Silverman, R.H. , Anal. Biochem.. 144:450-460 (1985). The full-length huma 2-5A-dependent RNase, which is produced i reticulocyte lysate, had the same apparent molecula weight as did naturally occurring 2-5A-dependen RNase. See FIG. 5A. However, the actual molecula mass of human 2-5A-dependent RNase is determined fro the 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 (α + β) treatment by a factor of about three. Furthermore, the induction appeared to be a primary response t interferon treatment because it is observed in th presence of cycloheximide. Therefore, interferon i believed to regulate the 2-5A pathway by elevatin levels of both 2-5A synthetases, Hovanessian, A.G. e al.. Nature, 268:537-539 (1977), and 2-5A-dependen RNase, Jacobsen, H. et al.. Virology. 125:496-50 (1983a). See. FIGS. 1, 6 and 11.
The cloning of 2-5A-dependent RNase reveal several features of the protein. The 2-5A bindin domain is of particular interest because it is th ability of 2-5A-dependent RNase to be activated b 2-5A that sets it apart from other nucleases. B expressing nested 3'-deletions of murin 2-5A-dependent RNase, a region between amino acid residues 218 and 294 which is believed to be critica for 2-5A binding activity is identified. See FIG. 7B. Interestingly, the identified region contains repeated P-loop motif, one from residues 229 to 24 and another from residues 253 to 275. See FIG. 4 an Table 2. When the latter P-loop motif (amino acid 253-275) is partially deleted, there is a precipitou decline in 2-5A binding activity. See clone ZB14 i FIG. 7B.
The homology with P-loops is believed to b highly conserved between the human and murine form of 2-5A-dependent RNase; thus underscoring the belie of the importance of this region for 2-5A bindin activity. See FIG. 4. The similarity to P-loop consists of the tripeptides, glycine-lysine threonine, preceded by glycine-rich sequences. I this regard, the unusual feature of 2-5A-dependen RNase is that the P-loop motif is repeated and are i the same orientation. Adenylyl cyclase from Bacillu anthracis also contains a duplicated P-loop motif, however, the two sequences are in opposit orientation and are overlapping. See Xia, Z. an Storm, D.R. , J. Biol. Chem.. 265:6517-6520 (1990).
The relative importance of the conserve P-loop lysines (at positions 240 and 274) ar evaluated by site-directed mutagenesis of the murin 2-5A-dependent RNase, clone ZBl. Although individua substitution mutations of the two lysine significantly reduced 2-5A binding activity replacing both of the lysines with asparagin residues in the same mutant RNase severely represse 2-5A binding. See FIG. 8. Perhaps the trimer 2-5 requirement for activation of most forms o 2-5A-dependent RNase could be explained if the firs and third adenylyl residues of 2-5A interact with th separate P-loop sequences inducing conformationa changes in 2-5A-dependent RNase. In this regard dimer 2-5A neither binds 2-5A-dependent RNas efficiently nor does it activate 2-5A-dependen RNase, FIG. 7A; Kerr, I.M. and Brown, R.E. , Prod Natl. Acad. Sci. U.S.A.. 75:265-260 (1978) an Knight, M. et al., Nature, 288:189-192 (1980) perhaps because it is too short to span the tw P-loop motifs. Alternately, the residual 2-5 binding activity observed in the point mutants ZBl(Lys240->Asn) and ZBl(Lys274-)Asn) , and the ver low affinity of the double mutant ZBl(Lys240'274->Asn) for 2-5A, could indicate tha the two P-loop motifs are parts of separate 2-5 binding domains.
Homology with protein kinase domains VI an VII is also identified in 2-5A-dependent RNase. Se FIG. 4. See also Hanks, S.K. et al.. Science 241:42-52 (1988). Although domain VI is believed t be involved in ATP binding, this region i 2-5A-dependent RNase is believed not to be importan for 2-5A binding because its deletion caused only minimal reduction in affinity for 2-5A. See FIG 7B. However, a modest (two-fold) stimulatory effec of ATP on 2-5A-dependent RNase activity has bee 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 latte report indicated that ATP was not required fo 2-5A-dependent RNase activity but may act t stabilize the enzyme. Therefore, the region o homology with protein kinases could perhaps bind AT resulting in stimulation of ribonuclease activit through stabilization of the enzyme.
A consensus zinc finger domain, reviewed i Evans, R.M. and Hollenberg, S.M. , Cell. 52:1- (1988) , consisting of six cysteine residues with th structure CX4CX3CX^7CX3CX3C (amino acid residue 401-436 in Table 2 ) is identified in the murine for of 2-5A-dependent RNase. See FIG. 4. The homologou region in the human form of 2-5A-depenent RNase i CX11CX25CX3CXβC (amino acid numbers 395 to 444 i Table 1 ) . Because zinc fingers are nucleic aci binding domains, the cysteine-rich region i 2-5A-dependent RNase could be involved in binding t the RNA substrate. Alternatively, the cysteine-ri domain in 2-5A-dependent RNase could media formation of 2-5A-dependent RNase dimers. Analys of crude preparations of 2-5A-dependent RNase sugge that 2-5A-dependent RNase may form dimers concentrated but not in dilute extracts. S Slattery, E. et al., Proc. Natl. Acad. Sci. U.S.A 76:4778-4782 (1979) and Wreschner, D.H. et al., Eu J. Biochem.. 124:261-268 (1982).
Comparison between the amino acid sequenc of other ribonucleases with 2-5A-dependent RNa identifies some limited homology with RNase E, endoribonuclease from E. coli. See FIG. 9A. S also Apirion D. and Lassar, A.B., J. Biol. Che 253:1738-1742 (1978) and Claverie-Martin, F. et al J. Biol. Chem. 266:2843-2851 (1991). The homol with RNase E is relatively conserved between human and murine forms of 2-5A-dependent RNase spans a region of about 200 amino acid residu Within these regions there are 24 and 32% identi plus conservative matches, with some gaps, betw RNase E and the human and murine forms 2-5A-dependent RNase, respectively. See FIG. The me gene which encodes RNase E and the alte mRNA stability (ams) gene, Ono, M. and Kumano, M., Mol. Biol.. 129:343-357 (1979), map to the s genetic locus. See Mudd E.A. et al., M Microbiol.. 4:2127-2135 (1990); Babitzke, P. and Kushner, S.R. , Proc. Natl. Acad. Sci. U.S.A.. 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, S.R. , Proc. Natl. Acad. Sci. U.S.A.. 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 U) , Ehretsmann, C.P. et al.. Genes & Development. 6:149-159 (1992). The location of the RNase E homology and other identified features in 2-5A-dependent 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 2',5,-oligoadenylates in E. coli. See Brown, R.E. and Kerr, I.M., 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
(i.e. 2-5A synthetase and 2-5A-dependent RNase) is reported to begin only with reptiles or possibly amphibia. See Cayley, P.J. et al., Biochem. Biophys. 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, G. , 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 2-5A 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., Natur . 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, e.g.. Stark, G. et al.. Nature, 278:471-473 (1979), and liver regeneration, Etienne-S ekens, M. et al., Proc. Natl. Acad. Sci. U.S.A.. 80:4609-4613 (1983). However, basal levels of 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 J.. 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 2-5A-dependent 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, J.N. , 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 μg per ml of cycloheximide and 1000 units per ml of murine interferon (α + β) (1.3 X 107 units per mg protein: Lee Biomolecular) for about 2.5 hours to increase levels of 2-5A-dependent RNase mRNA. Total RNA was then isolated, e.g. Chomczynski, P. and Sacchi, N. , 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 Xhol-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 Xhol and unidirectionally cloned into predigested λZAPII 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 3.5 hours at about 42°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°C. The filters are processed by a modification of the ethods 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 20 mM magnesium acetate, about 50 mM potassium chloride, about 1 mM EDTA, about 50 mM β-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(br8A2'p)2A3'-[32P]Cp (the "2-5A 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°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. Murine L929 cells are treated with about 1000 units per ml interferon (α + β) with or without about 50 μg per ml of cycloheximide and the total RNA is then isolated as described. See Chomczynski, P. and Sacchi, N., Anal. Biochem.. 162:156-159 (1987). Poly(A)+ RNA is prepared by oligo(dT)-cellulose chromatography, as described in Sambrook, J. et al.. Cold Spring 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 32P-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 μg per ml salmon sperm DNA] at about 42°C.
The Human 2-5A-dependent RNase cDNA clone, HZBl, is isolated from an adult human kidney cDNA library in λgtlO with radiolabeled (random primed) murine 2-5A-dependent RNase cDNA (clone ZBl) as probe, Sambrook, J. et al.. Cold Spring Harbor Laboratory Press (1989) . Clone HBZ22 is isolated using radiolabeled HZBl DNA as probe. The genomic human 2-5A-dependent RNase clone is isolated from a human placenta cosmid library in vector pVE15 (Stratagene) with a radiolabeled fragment of HZB22 DNA as probe. The murine genomic 2-5A-dependent RNase clone is isolated from a mouse 129SV genomic library in vector λFIXII (Stratagene) with a radiolabeled fragment of 2-5A-BP cDNA (clone ZBl) 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°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°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°C for about 12 hours. Translation reactions contain about 50 μM zinc sulfate. Endogenous 2-5A-dependent RNase in the reticulocyte lysated is removed by adsorption to about 30 μM of p2(A2'p)3A 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, D. , I.R.L. Press. Oxford. England, pp. 149-193 (1987) , for about one hour on ice as described. See Silverman, R.H. , .Anal. Biochem.. 144:450-460 (1985). The 2-5A-dependent RNase:2-5A-cellulose complex is removed by twice centrifuging at about 400 x g for about 5 minutes at about 2°C. The supernatant completely lacking in measurable levels of 2-5A-dependent RNase. See FIG. 5.
The set of nested 3'-deletions of the truncated murine 2-5A-dependent RNase cDNA, ZBl, is generated with exonuclease III/Sl 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(br8A2'p) A[32P]Cp, and its crosslinking to 2-!-5A-dependent RNase is performed exactly as described. See Nolan-Sorden, N.L. et al., Anal. Biochem.. 184:298-304 (1990). Briefly, the 2-5A 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, D. , 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 SDS/10% polyacrylamide gels. Filter assays for 2-5A binding activity using the 2-5A probe for about one hour on ice, as described in Knight, M. et al.. Nature, 288:189-192 (1980).
Protease digestions are performed o gel-purified proteins in a gel, as described b Cleveland, D.W. et al., J. Biol. Chem.. 252:1102-110 (1977).
The ribonuclease assay with 2-5A-cellulos is performed, as described by Silverman, R.H. , Anal. Biochem.. 144:450-460 (1985). Briefly, lysates ar adsorbed to about 30 μM of 2-5A-cellulose on ice fo about two hours. The matrix is then washed thre times by centrifuging and resuspending in buffer A. See Silverman, R.H. , Anal. Biochem.. 144:450-46 (1985). The matrix is then incubated wit poly(U)-[32P]Cp or poly(C)-[32P]Cp (both at about 1 μM in nucleotide equivalents) at about 30°C and th levels of acid-precipitable radioactive RNA ar determined by filtration on glass-fiber filters.
The Sanger dideoxy sequencing method i used to determine the DNA sequences (Sequenase, United States Biomedical) .
The lysines in the truncated murin 2-5A-dependent RNase, clone ZBl, at positions 240 an 274 are mutated, individually and together, t asparagine residues. Mutants ZBl(Lys274->Asn) an the double mutant, ZBl(Lys240,274-)Asn) , are obtaine with mutant oligonucleotides after subcloning ZB cDNA into pALTER-1 as described (Promega) . Mutant ZBl(Lys240-)Asn) is obtained after polymerase chain reaction amplification of a segment of ZBl with an upstream primer containing a unique Hindi site attached to the mutant sequence and a second primer downstream of a unique Bglll site. The Hindi- and BGlII-digested polymerase chain reaction product and similarly-digested clone ZBl 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 (1990); Chong, K.L. et al.: EMBO J.. 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 J.. 4:1761-1768 (1985). The human cDNA for 2-5A 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 μg/ml of tetracycline. about 10 μg/ml of kanamycin and about 25 μg/ml of streptomycin.
To subclone cDNAs for PKR (PK68) , a lysine •* arginine mutant PKR (muPkβδ; 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 Xbal and than with Clal restriction endonucleases, the cDNA fragments are purified from low melting point agarose gels and subcloned in sense orientation at Xbal and Clal sites of pAM943. See FIG. 13. The recombinant plasmids, e.g., construct A, pAM943:PK68, construct B, pAM943:muPK68, and contruct C, pAM943:synthetase, which correspond to the constructs depicted in FIG. 13A-C, respectively, are used to transform Argobacterium tumefaciens LBA4404. The resultant bacteria, identified as AG68, AGmu68 and AGsyn, respectively, are used for tobacco leaf disc transformations. Production of the recombinant plasmids, i.e., 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 Hindlll enzyme and subcloned in the Hindlll site of pAM943 in both 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 2-5A-dependent RNase was PCR amplified using two oligonucleotide primers containing BamHI restriction sites before AT (start codon) and after TGA (stop codon) . Th product was digested with BamHI and subcloned a Bglll site of pAM822 vector. The cDNA used fo 2-5A-dependent RNase is in plasmid pZC5 referenced i Zhou et al. Cell 72, 753-765 (1994), the human for of the cDNA. The sequence is also disclosed herein. The plasmid pAM822 contains a second selectabl marker gene, the hygromycin resistance gene, permitting the construction of plants containing bot 2-5A-synthetase and 2-5A-dependent RNase cDNAs. Insertion of pAM822:2-5Adep. RNase (Fig. 13E) , containing 2-5A-dependent RNase cDNA, int kana ycin-resistant, transgenic tobacco leaf disc containing 2-5A-synthetase cDNA is thus performed.
Tobacco plants are grown aseptically i Murashige and. Skoog's medium, known as MS medium, containing about 3% sucrose (MSO medium) and abou 0.8% agar in plastic boxes (Phytatray) at about 28° under cycles consisting of about 16 hr of light an about 8 hr of dark in a growth chamber. Leave bigger than about 2" long are cut into about 2 to 3 cm2 pieces under the MSO medium and 6-8 leaf piece are placed in a 6 cm Petri dish containing about 2 m of MSO medium and holes are made in the leaf piece with a sterile pointed forcep. Overnight cultures o AG68, AGmu68, AGSyn, AG2DR sense and AG2DR antisens are grown in LB (L broth) containing about 50 μM 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 28βC 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 0.5 mg/1 BAP (benzylaminopurine) ; about 200 μg/ml kanamycin; about 200 μg/ml carbenicillin; and about 100 μg/ml of cefotaxine] , under diffuse light at about 28βC 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 μg/ml kanamycin and about 200 μg/ml carbenicillin. The transgenic plants expressing 2-5A synthetase are substantially transformed to introduce the cDNA for 2-5A-dependent RNase (with pAM9 3:2-5Adep.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 done 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, 2-5A-synthetase, and 2-5A-dependent RNase in plants that are 4" to 5M 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 2-5A-dependent RNase are reported in Tables I-V. See also FIG. 15 wherein expression of human 2-5A 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. TABLE I
Transgenic Tobacco Plants Expressing Wild Type and Mutant Forms of Human PKR cDNA
(plasmid pAM943:PK68) FIG. 13A (plasmid pAM943:muPK68) FIG. 13B
Transgenic: Plant: Southern Blot: Northern Blot:
(clone #) (presence of DNA) (expression of mRNA
Mutant PKR: 1 + N.T.
(plasmid 2 ++ + pAM943:PK68) 4 N.T N.T.
FIG. 13A 6 N.T +
7 N.T +
10 N.T +
11 N.T +
12 N.T +
17 N.T +
Wild Type 1 N.T +
PKR: 2 N.T N.T.
(plasmid 5 N.T + pAM943:muPK68) 6 N.T N.T.
FIG. 13B 7 N.T N.T.
-*_ 8 N.T +
10 N.T +
20 N.T N.T.
22 N.T N.T.
N.T. , Not Tested
T.ABLE II
Transgenic Tobacco Plants Expressing Human 2-5A-Synthetase cDNA
(Plasmid pAM943:synthetase - FIG. 13C)
Plant: Southern Blot: Northern Blot:
(clone#) (presence Of DNA) (expression of mRN
1 ++ +
3 ± N.T.
4 + ++
5 •* N.T.
6 •*; N.T.
7 •£ N.T.
8 +++ +
9 + N.T.
10 + +
12 + N.T.
13 + N.T.
14 ++ -
15 +
16 + ~
17 N.T. ++
18 N.T. ++ a". N.T. N.T. b N.T. N.T. c N.T. N.T. d N.T. N.T.
N.T. , Not Tested.
TABLE III
Transgenic Tobacco Plants Containing
Sense or Antisense Orientation Human
2-5A-Dependent RNase cDNA
(plasmid pAM943:2-5A-dep. RNase sense - FIG. 13D) (plasmid pAM943:2-5A-dep. RNase antisense - FIG. 13D/a)
Transgenic: Southern Northern 2-5A-Binding
Plant: (presence (expression Assay: (pro¬
(clone #) of DNA> of mRNAϊ tein activit
Antisense: 1 + N.T. N.T.
2 + N.T. N.T.
3 + N.T. N.T.
4 + N.T. N.T.
5 + N.T. N.T. a N.T. N.T. N.T. b N.T. N.T. N.T. c N.T. N.T. N.T.
Sense: Zl + — +
Z2 ++ - ++
Z3 ++ N.T. ++
Z4 + N.T. N.T.
Z5 N.T. N.T. +++
Z6 N.T. N.T. ++
Z7 N.T. N.T. +/-
N.T. , Not Tested.
TABLE IV
Transgenic Tobacco Plants Containing Both Human 2-5A-Synthetase and Human 2-5A-Dependent RNase cDNA
(plasmid pAM943:synthetase - FIG. 13C) (plasmid pAM943:2-5A-dep. RNase sense - FIG. 13D)
Plant: Southern Blots: Northern Blot:
(clone #) (2-5A-Syn (2-5A-Dep. (2- -5A Syn. (2-5A-de
DNAΪ RNase DNA) mRNA) RNase mR
14/1 N.T. - + -
14/2 N.T. — + —
14/3 N.T. N.T. N.T. N.T.
14/4 N.T. N.T. N.T. N.T.
14/5 N.T. N.T. N.T. N.T.
14/6 N.T. N.T. N.T. N.T.
15/1 N.T. — + -
15/2 N.T. — + —
15/3 N.T. — + -
15/4 N.T. N.T. + —
15/5 N.T. N.T. N.T. N.T.
15/6 N.T. - + -
15/7 N.T. — N.T. N.T.
N.T7-, Not Tested.
Assays of dsRNA-dependent autophosphoryl- ation 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 2-5A-dependent RNase.
To demonstrate the expression of 2-5A-dependent 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 2-5A-dependent RNase are performed. See Tables III and V. Results show the presence of 2-5A-dependent RNase in transgenic plants Zl, Z2, Z3, Z5 and Z6. It is believed that the highest levels of human, recombinant 2-5A dependent RNase are in plant Z5. See Table V.
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:
Zl 662
Z2 1,618
Z3 1,545
Z5 2,575
Z6 1,547
Z7 31
aTobacco plants contain construct D, pAM943:2-5Adep. RNa (sense) . 2-5A binding assays are performed by the filt binding method of Knight, M. et al. Nature (288) :189-1 (1980) with modifications. A 32P-labeled and bromi substituted 2-5A analog, p(A2'p)2(br8A2'p)2A3'-32p]C about 15,000 counts per min per assay, at about 3,000 per mmole, Nolan-Sorden, N.L. , et al. Anal. Biochem. (184) :298-304 (1990), is incubated with plant extract containing about 100 micrograms of protein per assay, ice for about 4 h. The reaction mixtures are th transferred to nitrocellulose filteres which are wash twice in distilled water and dried and the amount of 2- probe bound to the 2-5A-dependent RNase on the filters measured by scintillation counting, Silverman, R.H. a Krause, D. , In, Clemens, M.J., Morris, A.G., and Gearin A.J.H., (eds.), Lvmohokines and Interferons - A Practic Approach. I.R.L. Press, Oxford, pp. 149-193 (1987) . Da is presented as counts per min of labeled 2-5A bound 2-5A-dependent RNase expressed in the transgenic plant Background radioactivity from extracts of control plant 705 counts per min, consisting of nonspecific binding 2-5A, is subtracted from these data. 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, 2-5A-binding activity is determined on a Western blot with a bromine-substituted, 3 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, M.J., Morris, A.G., and Gearing, A.J.H., (eds.), Lymphokines and Interferons - A Practical Approach. I.R.I. , Press, Oxford, pp. 149-193, supplemented with about 5mM ascorbic acid, about 1 mM cysteine, about 2 μg per ml leupeptin, about 100 μ per ml phenylmethyleulfonyl fluoride, and about 2 μg per ml pepstatin. Extracts are clarified by centrifugation at about 10,000 x g for about 10 min. Supernatants of the extracts, about 100 μg of protein per assay, are separated by SDS/10% 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 32P-labeled 2-5A probe for about 24 h at about 4βC, 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 2-5A-dependent 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 invention 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, W.I.) in the presence of
35S-methionine. Synthesis of the 35S-labeled PKR is detected in an autoradiogram of the dried,
SDS/polyacrylamide gel. The cDNAs encoding PKR and muPKR are excised from plasmids pKS(+)PKR and pKS(+)muPKR by digesting with Kpnl and Xbal. 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 5' end Xbal 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 Xbal and Clal digested pAM943 by DNA ligation. The resulting plasmids, FIG. 13, constructs A and B, are used to transform Argobacterium 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 Iconstruct C)
The plasmid ptac-15 containing the human cDNA illustrated in FIG. 20 for a small form of 2-5A-synthetase (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 plasmid pKS(+) (Strategene, La Jolla, CA) in BamHI and EcoRI sites. The resulting recombinant plasmid
DNA (pKS(+)synthetase) is digested with Xbal 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 Argobacterium tumefaciens bacteria by standard methods and the presence of 2-5A-synthetase 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. Nase antisense (construct D/a)
The plasmid pKS(+)ZC5 encoding a complete coding sequence for human 2-5A-dependent RNase is digested with Hindlll. The 2.5kbρ cDNA for
2-5A-dependent RNase is purified in a low melting point agarose gel and is then subcloned in Hindlll digested pAM943 in both sense (forward) and antisense
(reverse) orientations to produce pAM9 3:2-5Adep.RNase sense (construct D) and pAM943:2-5Adep.RNase antisense (construct D/a), as depicted in FIG. 13D and D/a, respectively.
Transformed Argobacterium are determined to contain the 2-5A-dependent RNase cDNA by restriction enzyme digests and by PCR analysis. Construction of pAM822:2-5Adep.RNase antisense fconstruct Ei
Polymerase chain reactions (PCR) are performed on plasmid pKS(+)ZC5 encoding human
2-5A-dependent RNase to generate Hindlll and BamHI sites on the two ends of the cDNA and to reduce 5' and 3' untranslated sequences. The PCR primers used are:
ID SEQ NO:7:
2DR-5 5'-TCATGCTCGAGAAGCTTGGATCCACCATGGAGAGCAGGGAT- 3' ; and
ID SEQ NO:8:
H2DR-4 5'-GATACTCGAGAAGCTTGCATCCTCATCAGCACCCAGGGCTGG -3'.
The PCR product (about 2.25 kbp) is purified on a low melting point agarose gel and is then digested with
Hindlll and is then subcloned into Hindlll digested plasmid pKS(+). The resulting plasmid, pKS:pZC5 is digested with BamHI and the 2-5A-dependent RNase cDNA fragment is purified and cloned into Bglll digested pAM822. Recombinants isolated in the reverse
(antisense) orientation give pAM822:2-5Adep.RNase antisense (construct E) . See FIG. 13E. 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 2-5A-dependent RNase cDNA
15/2* * expressing human 97041 01 Feb. 1995 07 Feb. 1995 2-5A-synthetase 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 T.AB B 1 Hwnaτ-1 2-5A-depedent RNase
SEQ ID NO:l«, SEQ ID NO:2:, SEQ ID NO:3: and SEQ ID N0:
-103 aatcccaacttacactcaaagct tctttgattaagtgctaggagataaatttgcattttctca aggaaaaggctaaaagtggtagcaggtggcatttaccgtc
ATG GAG AGC AGG GAT CAT AAC AAC CCC CAG 30
Met Glu Ser Arg Asp His Asn Asn Pro Gin 10
GAG GGA CCC ACG TCC TCC AGC GGT AGA AGG 60
Glu Gly Pro Thr Ser Ser Ser Gly Arg Arg 20
GCT GCA GTG GAA GAC AAT CAC TTG CTG ATT 90
Ala Ala Val Glu Asp Asn His Leu Leu lie 30
AAA GCT GTT CAA AAC GAA GAT GTT GAC CTG 120
Lys Ala Val Gin Asn Glu Asp Val Asp Leu 40
GTC CAG CAA TTG CTG GAA GGT GGA GCC AAT 150
Val Gin Gin Leu Leu Glu Gly Gly Ala Asn 50
GTT AAT TTC CAG GAA GAG GAA GGG GGC TGG 180
Val Asn Phe Gin Glu Glu Glu Gly Gly Trp 60
ACA-"CCT CTG CAT AAC GCA GTA CAA ATG AGC 210
Ttir Pro Leu His Asn Ala Val Gin Met Ser 70
AGG GAG GAC ATT GTG GAA CTT CTG CTT CGT 240
Arg Glu Asp lie Val Glu Leu Leu Leu Arg 80
CAT GGT GCT GAC CCT GTT CTG AGG AAG AAG 270
His Gly Ala Asp Pro Val Leu Arg Lys Lys 90
(CCT)*
AAT GGG GCC ACG CTT TTT ATC CTC GCA GCG 300
Asn Gly Ala Thr Leu Phe lie Leu Ala Ala 100
(Pro)*
ATT GCG GGG AGC GTG AAG CTG CTG AAA CTT 330 lie Ala Gly Ser Val Lys Leu Leu Lys Leu 110
TTC CTT TCT .AAA GGA GCA GAT GTC AAT GAG 360
Phe Leu Ser Lys Gly Ala Asp Val Asn Glu 120
TGT GAT TTT TAT GGC TTC ACA GCC TTC ATG 390
Cys Asp Phe Tyr Gly Phe Thr Ala Phe Met 130
GAA GCC GCT GTG TAT GGT AAG GTC AAA GCC 420
Glu Ala Ala Val Tyr Gly Lys Val Lys Ala 140 CTA .AAA TTC CTT TAT AAG AGA GGA GCA AAT 450
Leu Lys Phe Leu Tyr Lys Arg Gly Ala Asn 150
GTG AAT TTG AGG CGA AAG ACA AAG GAG GAT 480
Val Asn Leu Arg Arg Lys Thr Lys Glu Asp 160
CAA GAG CGG CTG AGG AAA GGA GGG GCC ACA 510
Gin Glu Arg Leu .Arg Lys Gly Gly Ala Thr 170
GCT CTC ATG GAC GCT GCT GAA AAA GGA CAC 540
Ala Leu Met Asp Ala Ala Glu Lys Gly His 180
GTA GAG GTC TTG AAG ATT CTC CTT GAT GAG 570
Val Glu Val Leu Lys lie Leu Leu Asp Glu 190
ATG GGG GCA GAT GTA AAC GCC TGT GAC AAT 600
Met Gly Ala Asp Val Asn Ala Cys Asp Asn 200
ATG GGC AGA AAT GCC TTG ATC CAT GCT CTC 630
Met Gly Arg Asn Ala Leu lie His Ala Leu 210
CTG AGC TCT GAC GAT AGT GAT GTG GAG GCT 660
Leu Ser Ser Asp Asp Ser Asp Val Glu Ala 220
ATT ACG CAT CTG CTG CTG GAC CAT GGG GCT 690 lie Thr His Leu Leu Leu Asp His Gly Ala 230
GAT GTC AAT GTG AGG GGA GAA AGA GGG AAG 720
As Val Asn Val Arg Gly Glu Arg Gly Lys 240
ACT CCC CTG ATC CTG GCA GTG GAG AAG AAG 750
Thr Pro Leu lie Leu Ala Val Glu Lys Lys 250
CAC TTG GGT TTG GTG CAG AGG CTT CTG GAG 780
His Leu Gly Leu Val Gin Arg Leu Leu Glu 260
CAA GAG CAC ATA GAG ATT AAT GAC ACA GAC 810
Gin Glu His lie Glu lie Asn Asp Thr Asp 270
AGT GAT GGC AAA ACA GCA CTG CTG CTT GCT 840
Ser Asp Gly Lys Thr Ala Leu Leu Leu Ala 280
GTT GAA CTC AAA CTG AAG AAA ATC GCC GAG 870
Val Glu Leu Lys Leu Lys Lys lie Ala Glu 290
TTG CTG TGC AAA CGT GGA GCC AGT ACA GAT 900
Leu Leu Cys Lys Arg Gly Ala Ser Thr Asp 300
TGT GGG GAT CTT GTT ATG ACA GCG AGG CGG 930
Cys Gly Asp Leu Val Met Thr Ala Arg Arg 310
AAT TAT GAC CAT TCC CTT GTG AAG GTT CTT 960
Asn Tyr Asp His Ser Leu Val Lys Val Leu 320 CTC TCT CAT GGA GCC AAA GAA GAT TTT CAC 990
Leu Ser His Gly Ala Lys Glu Asp Phe His 330
CCT CCT GCT GAA GAC TGG AAG CCT CAG AGC 1020
Pro Pro Ala Glu Asp Trp Lys Pro Gin Ser 340
TCA CAC TGG GGG GCA GCC CTG AAG GAT CTC 1050
Ser His Trp Gly Ala Ala Leu Lys Asp Leu 350
CAC AGA ATA TAC CGC CCT ATG ATT GGC AAA 1080
His Arg lie Tyr .Arg Pro Met lie Gly Lys 360
CTC AAG TTC TTT ATT GAT GAA AAA TAC AAA 1110
Leu Lys Phe Phe lie Asp Glu Lys Tyr Lys 370
ATT GCT GAT ACT TCA GAA GGA GGC ATC TAC 1140 lie Ala Asp Thr Ser Glu Gly Gly lie Tyr 380
CTG GGG TTC TAT GAG AAG CAA GAA GTA GCT 1170
Leu Gly Phe Tyr Glu Lys Gin Glu Val Ala 390
GTG AAG ACG TTC TGT GAG GGC AGC CCA CGT 1200
Val Lys Thr Phe Cys Glu Gly Ser Pro Arg 400
GCA CAG CGG GAA GTC TCT TGT CTG CAA AGC 1230
Ala Gin Arg Glu Val Ser Cys Leu Gin Ser 410
AGC CGA GAG AAC AGT CAC TTG GTG ACA TTC 1260
Ser-"Arg Glu Asn Ser His Leu Val Thr Phe 420
TAT GGG AGT GAG AGC CAC AGG GGC CAC TTG 1290
Tyr Gly Ser Glu Ser His Arg Gly His Leu 430
TTT GTG TGT GTC ACC CTC TGT GAG CAG ACT 1320
Phe Val Cys Val Thr Leu Cys Glu Gin Thr 440
CTG GAA GCG TGT TTG GAT GTG CAC AGA GGG 1350
Leu Glu Ala Cys Leu Asp Val His Arg Gly 450
GAA GAT GTG GAA AAT GAG GAA GAT GAA TTT 1380
Glu Asp Val Glu Asn Glu Glu Asp Glu Phe 460
GCC CGA AAT GTC CTG TCA TCT ATA TTT AAG 1410
Ala Arg Asn Val Leu Ser Ser lie Phe Lys 470
GCT GTT CAA GAA CTA CAC TTG TCC TGT GGA 1440
Ala Val Gin Glu Leu His Leu Ser Cys Gly 480
TAC ACC CAC CAG GAT CTG CAA CCA CAA AAC 1470
Tyr Thr His Gin Asp Leu Gin Pro Gin Asn 490
ATC TTA ATA GAT TCT .AAG AAA GCT GCT CAC 1500 lie Leu lie Asp Ser Lys Lys Ala Ala His 500 CTG GCA GAT TTT GAT AAG AGC ATC AAG TGG 1530
Leu Ala Asp Phe Asp Lys Ser lie Lys Trp 510
GCT GGA GAT CCA CAG GAA GTC AAG AGA GAT 1560
Ala Gly Asp Pro Gin Glu Val Lys Arg Asp 520
CTA GAG GAC CTT GGA CGG CTG GTC CTC TAT 1590
Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr 530
GTG GTA AAG AAG GGA AGC ATC TCA TTT GAG 1620
Val Val Lys Lys Gly Ser lie Ser Phe Glu 540
GAT CTG AAA GCT CAA AGT AAT GAA GAG GTG 1650
Asp Leu Lys Ala Gin Ser Asn Glu Glu Val 550
GTT CAA CTT TCT CCA GAT GAG GAA ACT AAG 1680
Val Gin Leu Ser Pro Asp Glu Glu Thr Lys 560
GAC CTC ATT CAT CGT CTC TTC CAT CCT GGG 1710
Asp Leu lie His Arg Leu Phe His Pro Gly 570
GAA CAT GTG AGG GAC TGT CTG AGT GAC CTG 1740
Glu His Val Arg Asp Cys Leu Ser Asp Leu 580
CTG GGT CAT CCC TTC TTT TGG ACT TGG GAG 1770
Leu Gly His Pro Phe Phe Trp Thr Trp Glu 590
AGC CGC TAT AGG ACG CTT CGG AAT GTG GGA 1800
Ser-"Arg Tyr Arg Thr Leu Arg Asn Val Gly 600
AAT GAA TCC GAC ATC AAA ACA CGA AAA TCT 1830
Asn Glu Ser Asp lie Lys Thr Arg Lys Ser 610
GAA AGT GAG ATC CTC AGA CTA CTG CAA CCT 1860
Glu Ser Glu lie Leu Arg Leu Leu Gin Pro 620
GGG CCT TCT GAA CAT TCC AAA AGT TTT GAC 1890
Gly Pro Ser Glu His Ser Lys Ser Phe Asp 630
AAG TGG ACG ACT AAG ATT AAT GAA TGT GTT 1920
Lys Trp Thr Thr Lys lie Asn Glu Cys Val 640
ATG AAA AAA ATG AAT AAG TTT TAT GAA AAA 1950
Met Lys Lys Met Asn Lys Phe Tyr Glu Lys 650
AGA GGC AAT TTC TAC CAG AAC ACT GTG GGT 1980
Arg Gly Asn Phe Tyr Gin Asn Thr Val Gly 660
GAT CTG CTA AAG TTC ATC CGG AAT TTG GGA 1210
Asp Leu Leu Lys Phe lie Arg Asn Leu Gly 670
GAA CAC ATT GAT GAA GAA AAG CAT AAA AAG 2040
Glu His lie Asp Glu Glu Lys His Lys Lys 680 ATG AAA TTA AAA ATT GGA GAC CCT TCC CTG 2070
Met Lys Leu Lys lie 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 lie Tyr Val Tyr Thr Lys Leu Gin Asn Thr 710
GAA TAT AGA AAG CAT TTC CCC CAA ACC CAC 2160
Glu 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 Gly 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 tgatggactgatttgctggagttcagggaactact 2258 Cys 741 tattagctgtagagtccttggcaaatcacaacat 2292 tctgggccttttaactcaccaggttgcttgtgagggat 2330 gagttgcatagctgatatgtcagtccctggcatcgtg 2367 tattccatatgtctataacaaaagcaatatatacccag 2405 actacactagtccataagctttacccactaactggga 2442 ggacattctgctaagattccttttgtcaattgcaccaa 2480 aagaatgagtgccttgacccctaatgctgcatatgtt 2517 aca-attctctcacttaattttcccaatgatcttgcaaa 2555 acagggattatcatccccatttaagaactgaggaacc 2592 tgagactcagagagtgtgagctactggcccaagattat 2630 tcaatttatacctagcactttataaatttatgtggtg 2667 ttattggtacctctcatttgggcaccttaaaacttaac 2705 tatcttccagggctcttccagatgaggcccaaaacat 2742 atataggggttccaggaatctcattcattcattcagta 2780 tttattgagcatctagtataagtctgggcactggatg 2817 catgaatt 2825
*It is believed that the original codon number 95, 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. proline may also exist at this position (see page 81) .
SEQ ID NO:1: represents the DNA encoding sequence for the human 2-5A-dependent RNase protein. SEQ ID NO:2: represents the amino acid sequence encoded by the DNA sequence designated SEQ ID N0:1:. SEQ ID NO:3: represents the DNA sequence, represented by SEQ ID NO:l:, having the alternative codon number 95, CCT. SEQ ID NO:4: represents the amino acid sequence encoded by SEQ ID NO:3: , having the alternative amino acid number 95, proline. TABLE 2 f-Tgyine 2-5A-dependent RNase (partial)
SEQ ID NO:5: and SEQ ID NO:6:
-163 attcggcacgaggaaggtgccaattactagctcccttctttattcgtgta ctgatgagatgtcagaagacagaacataatcagcccaatccctactccaa gactctcattgtgtcccaaagaaacacacgtgtgcatttcccaaggaaaa ggcattgaggacc ATG GAG ACC CCG GAT TAT 18
Met Glu Thr Pro Asp Tyr 6
AAC ACA CCT CAG GGT GGA ACC CCA TCA GCG 48
Asn Thr Pro Gin Gly Gly Thr Pro Ser Ala 16
GGA AGT CAG AGG ACC GTT GTC GAA GAT GAT 78
Gly Ser Gin Arg Thr Val Val Glu Asp Asp 26
TCT TCG TTG ATC AAA GCT GTT CAG AAG GGA 108
Ser Ser Leu lie Lys Ala Val Gin Lys Gly 36
GAT GTT GTC AGG GTC CAG CAA TTG TTA GAA 138
Asp Val Val Arg Val Gin Gin Leu Leu Glu 46
AAA-"GGG GCT GAT GCC AAT GCC TGT GAA GAC 168
Lys Gly Ala Asp Ala Asn Ala Cys Glu Asp 56
ACC TGG GGC TGG ACA CCT TTG CAC AAC GCA 198
Thr Trp Gly Trp Thr Pro Leu His Asn Ala 66
GTG CAA GCT GGC AGG GTA GAC ATT GTG AAC 228
Val Gin Ala Gly Arg Val Asp He Val Asn 76
CTC CTG CTT AGT CAT GGT GCT GAC CCT CAT 258
Leu Leu Leu Ser His Gly Ala Asp Pro His 86
CGG AGG AAG AAG AAT GGG GCC ACC CCC TTC 288
Arg Arg Lys Lys Asn Gly Ala Thr Pro Phe 96
ATC ATT GCT GGG ATC CAG GGA GAT GTG AAA 318
He He Ala Gly He Gin Gly Asp Val Lys 106
CTG CTC GAG ATT CTC CTC TCT TGT GGT GCA 348
Leu Leu Glu He Leu Leu Ser Cys Gly Ala 116
GAC GTC AAT GAG TGT GAC GAG AAC GGA TTC 378
Asp Val Asn Glu Cys Asp Glu Asn Gly Phe 126 ACG GCT TTC ATG GAA GCT GCT GAG CGT GGT 408
Thr Ala Phe Met Glu Ala Ala Glu Arg Gly 136
AAC GCT GAA GCC TTA AGA TTC CTT TTT GCT 438
Asn Ala Glu Ala Leu Arg Phe Leu Phe Ala 146
AAG GGA GCC AAT GTG AAT TTG CGA CGA CAG 468
Lys Gly Ala Asn Val Asn Leu Arg Arg Gin 156
ACA ACG AAG GAC AAA AGG CGA TTG AAG CAA 498
Thr Thr Lys Asp Lys Arg Arg Leu Lys Gin 166
GGA GGC GCC ACA GCT CTC ATG AGC GCT GCT 528
Gly Gly Ala Thr Ala Leu Met Ser Ala Ala 176
GAG AAG GGC CAC CTG GAA GTC CTG AGA ATT 558
Glu Lys Gly His Leu Glu Val Leu Arg He 186
CTC CTC AAT GAC ATG AAG GCA GAA GTC GAT 588
Leu Leu Asn Asp Met Lys Ala Glu Val Asp 196
GCT CGG GAC AAC ATG GGC AGA AAT GCC CTG 618
Ala Arg Asp Asn Met Gly Arg Asn Ala Leu 206
ATC CGT ACT CTG CTG AAC TGG GAT TGT GAA 648
He Arg Thr Leu Leu Asn Trp Asp Cys Glu 216
AAT GTG GAG GAG ATT ACT TCA ATC CTG ATT 678
Asn"Val Glu Glu He Thr Ser He Leu He 226
CAG CAC GGG GCT GAT GTT .AAC GTG AGA GGA 708
Gin His Gly Ala Asp Val Asn Val Arg Gly 236
GAA AGA GGG AAA ACA CCC CTC ATC GCA GCA 738
Glu Arg Gly Lys Thr Pro Leu He Ala Ala 246
GTG GAG AGG AAG CAC ACA GGC TTG GTG CAG 768
Val Glu Arg Lys His Thr Gly Leu Val Gin 256
ATG CTC CTG AGT CGG GAA GGC ATA AAC ATA 798
Met Leu Leu Ser Arg Glu Gly He Asn He 266
GAT GCC AGG GAT AAC GAG GGC AAG ACA GCT 828
Asp Ala Arg Asp Asn Glu Gly Lys Thr Ala 276
CTG CTA ATT GCT GTT GAT AAA CAA CTG AAG 858
Leu Leu He Ala Val Asp Lys Gin Leu Lys 286
GAA ATT GTC CAG TTG CTT CTT G.AA .AAG GGA 888
Glu He Val Gin Leu Leu Leu Glu Lys Gly 296
GCT GAT AAG TGT GAC GAT CTT GTT TGG ATA 918
Ala Asp Lys Cys Asp Asp Leu Val Trp He 306 GCC AGG AGG AAT CAT GAC TAT CAC CTT GTA 948
Ala Arg Arg Asn His Asp Tyr His Leu Val 316
AAG CTT CTC CTC CCT TAT GTA GCT AAT CCT 978
Lys Leu Leu Leu Pro Tyr Val Ala Asn Pro 326
GAC ACC GAC CCT CCT GCT GGA GAC TGG TCG 1008
Asp Thr Asp Pro Pro Ala Gly Asp Trp Ser 336
CCT CAC AGT TCA CGT TGG GGG ACA GCC TTG 1038
Pro His Ser Ser Arg Trp Gly Thr Ala Leu 346
AAA AGC CTC CAC AGT ATG ACT CGA CCC ATG 1068
Lys Ser Leu His Ser Met Thr Arg Pro Met 356
ATT GGC AAA CTC AAG ATC TTC ATT CAT GAT 1098
He Gly Lys Leu Lys He Phe He His Asp 366
GAC TAT AAA ATT GCT GGC ACT TCC GAA GGG 1128
Asp Tyr Lys He Ala Gly Thr Ser Glu Gly 376
GCT GTC TAC CTA GGG ATC TAT GAC AAT CGA 1158
Ala Val Tyr Leu Gly He Tyr Asp Asn Arg 386
GAA GTG GCT GTG AAG GTC TTC CGT GAG AAT 1188
Glu Val Ala Val Lys Val Phe Arg Glu Asn 396
AGC CCA CGT GGA TGT AAG GAA GTC TCT TGT 1218
Ser"Pro Arg Gly Cys Lys Glu Val Ser Cys 406
CTG CGG GAC TGC GGT GAC CAC AGT AAC TTA 1248
Leu Arg Asp Cys Gly Asp His Ser Asn Leu 416
GTG GCT TTC TAT GGA AGA GAG GAC GAT AAG 1278
Val Ala Phe Tyr Gly Arg Glu Asp Asp Lys 426
GGC TGT TTA TAT GTG TGT GTG TCC CTG TGT 1308
Gly Cys Leu Tyr Val Cys Val Ser Leu Cys 436
GAG TGG ACA CTG GAA GAG TTC CTG AGG TTG 1338
Glu Trp Thr Leu Glu Glu Phe Leu Arg Leu 446
CCC AGA GAG GAA CCT GTG GAG AAC GGG GAA 1368
Pro Arg Glu Glu Pro Val Glu Asn Gly Glu 456
GAT AAG TTT GCC CAC AGC ATC CTA TTA TCT 1398
Asp Lys Phe Ala His Ser He Leu Leu Ser 466
ATA TTT GAG GGT GTT CAA AAA CTA CAC TTG 1428
He Phe Glu Gly Val Gin Lys Leu His Leu 476
CAT GGA TAT TCC CAT CAG GAC CTG CAA CCA 1458
His Gly Tyr Ser His Gin Asp Leu Gin Pro 486 CAA AAC ATC TTA ATA GAT TCC AAG AAA GCT 1488
Gin Asn He Leu He Asp Ser Lys Lys Ala 496
GTC CGG CTG GCA GAT TTT GAT CAG AGC ATC 1518
Val Arg Leu Ala Asp Phe Asp Gin Ser He 506
CGA TGG ATG GGA GAG TCA CAG ATG GTC AGG 1548
Arg Trp Met Gly Glu Ser Gin Met Val Arg 516
AGA GAC TTG GAG GAT CTT GGA CGG CTG GTT 1578
Arg Asp Leu Glu Asp Leu Gly Arg Leu Val 526
CTC TAC GTG GTA ATG AAA GGT GAG ATC CCC 1608
Leu Tyr Val Val Met Lys Gly Glu He Pro 536
TTT GAG ACA CTA .AAG ACT CAG AAT GAT GAA 1638
Phe Glu Thr Leu Lys Thr Gin Asn Asp Glu 546
GTG CTG CTT ACA ATG TCT CCA GAT GAG GAG 1668
Val Leu Leu Thr Met Ser Pro Asp Glu Glu 556
ACT AAG GAC CTC ATT CAT TGC CTG TTT TCT 1698
Thr Lys Asp Leu He His Cyc Leu Phe Ser 566
CCT GGA GAA AAT GTC AAG AAC TGC CTG GTA 1728
Pro Gly Glu Asn Val Lys Asn Cys Leu Val 576
GAC CTG CTT GGC CAT CCT TTC TTT TGG ACT 1758
Asp"Leu Leu Gly His Pro Phe Phe Trp Thr 586
TGG GAG AAC CGC TAT AGA ACA CTC CGG AAT 1788
Trp Glu Asn Arg Tyr Arg Thr Leu Arg Asn 596
GTG GGA AAT GAA TCT GAC ATC AAA GTA CGG 1818
Val Gly Asn Glu Ser Asp He Lys Val Arg 606
AAA TGT AAA AGT GAT CTT CTC AGA CTA CTG 1848
Lys Cys Lys Ser Asp Leu Leu Arg Leu Leu 616
CAG CAT CAG ACA CTT GAG CCT CCC AGA AGC 1878
Gin His Gin Thr Leu Glu Pro Pro Arg Ser 626
TTT GAC CAG TGG ACA TCT AAG ATC GAC AAA 1908
Phe Asp Gin Trp Thr Ser Lys He Asp Lys 636
AAT GTT ATG GAT GAA ATG AAT CAT TTC TAC 1938
Asn Val Met Asp Glu Met Asn His Phe Tyr 646
GAA AAG AGA AAA AAA AAC CCT TAT CAG GAT 1968
Glu Lys Arg Lys Lys Asn Pro Tyr Gin Asp 656
ACT GTA GGT GAT CTG CTG AAG TTT ATT CGG 1998
Thr Val Gly Asp Leu Leu Lys Phe He Arg 666 AAT ATA GGC GAA CAC ATC AAT GAG GAA AAA 2028
Asn He Gly Glu His He Asn Glu Glu 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 N0:5:.
SEQDENCE LISTING
(1) GENERAL INFORMATION:
(i) .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:
(A) ADDRESSEE: Ruden, Barnett, McClosky, Smith, Schuster &
Russell
(B) STREET: 200 E. Bro ard Boulevard
(C) CITY: Fort Lauderdale
(D) STATE: Florida
(E) COUNTRY: USA
(F) ZIP: 33301
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
<vi)_ CURRENT APPLICATION DATA:
• " (A) APPLICATION NUMBER: US 08/198,973
(B) FILING DATE: 18-FEB-1994
(C) CLASSIFICATION: 1808
(viii) ATTORNE /AGENT INFORMATION:
(A) NAME: Manso, Peter J.
(B) REGISTRATION NUMBER: 32,264
(C) REFERENCE/DOCKET NUMBER: CL11363-16
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 305/527/2498
(B) TELEFAX: 305/764/4996
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2928 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 104..2326
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AATCCCAACT TACACTCAAA GCTTCTTTGA TTAAGTGCTA GGAGATAAAT TTGCATTTTC 60
TCAAGGAAAA GGCTAAAAGT GGTAGCAGGT GGCATTTACC GTC ATG GAG AGC AGG 115
Met Glu Ser Arg
1
GAT CAT AAC AAC CCC CAG GAG GGA CCC ACG TCC TCC AGC GGT AGA AGG 163 Asp His Asn Asn Pro Gin Glu Gly Pro Thr Ser Ser Ser Gly Arg Arg 5 10 15 20
GCT GCA GTG GAA GAC AAT CAC TTG CTG ATT AAA GCT GTT CAA AAC GAA 211 Ala Ala Val Glu Asp Asn His Leu Leu lie Lys Ala Val Gin Asn Glu 25 30 35
GAT GTT GAC CTG GTC CAG CAA TTG CTG GAA GGT GGA GCC AAT GTT AAT 259 Asp Val Asp Leu Val Gin Gin Leu Leu Glu Gly Gly Ala Asn Val Asn 40 45 50
TTC CAG GAA GAG GAA GGG GGC TGG ACA CCT CTG CAT AAC GCA GTA CAA 307 Phe Gin Glu Glu Glu Gly Gly Trp Thr Pro Leu His .Asn Ala Val Gin 55 60 65
ATG AGC AGG GAG GAC ATT GTG GAA CTT CTG CTT CGT CAT GGT GCT GAC 355 Met Ser Arg Glu Asp He Val Glu Leu Leu Leu Arg His Gly Ala Asp 70 75 80
CCT GTT CTG AGG AAG AAG AAT GGG GCC ACG CTT TTT ATC CTC GCA GCG 403 Pro Val Leu Arg Lys Lys Asn Gly Ala Thr Leu Phe He Leu Ala Ala 85 _ _ 90 95 100
ATT GCG GGG AGC GTG AAG CTG CTG AAA CTT TTC CTT TCT AAA GGA GCA 451 He Ala Gly Ser Val Lys Leu Leu Lys Leu Phe Leu Ser Lys Gly Ala 105 110 115
GAT GTC AAT GAG TGT GAT TTT TAT GGC TTC ACA GCC TTC ATG GAA GCC 499 Asp Val Asn Glu Cys -ftsp Phe Tyr Gly Phe Thr Ala Phe Met Glu Ala 120 125 130
GCT GTG TAT GGT AAG GTC AAA GCC CTA AAA TTC CTT TAT AAG AGA GGA 547 Ala Val Tyr Gly Lys Val Lys Ala Leu Lys Phe Leu Tyr Lys Arg Gly 135 140 . 145
GCA AAT GTG AAT TTG AGG CGA AAG ACA AAG GAG GAT CAA GAG CGG CTG 595 Ala Asn Val Asn Leu Arg Arg Lys Thr Lys Glu Asp Gin Glu Arg Leu 150 155 160
AGG AAA GGA GGG GCC ACA GCT CTC ATG GAC GCT GCT GAA AAA GGA CAC 643 Arg Lys Gly Gly Ala Thr Ala Leu Met Asp Ala Ala Glu Lys Gly His 165 170 175 180
GTA GAG GTC TTG AAG ATT CTC CTT GAT GAG ATG GGG GCA GAT GTA AAC 691 Val Glu Val Leu Lys He Leu Leu Asp Glu Met Gly Ala Asp Val Asn 185 190 195
GCC TGT GAC AAT ATG GGC AGA AAT GCC TTG ATC CAT GCT CTC CTG AGC 739 Ala Cys Asp Asn Met Gly Arg Asn Ala Leu He His Ala Leu Leu Ser 200 205 210
TCT GAC GAT AGT GAT GTG GAG GCT ATT ACG CAT CTG CTG CTG GAC CAT 787 Ser Asp Asp Ser Asp Val Glu Ala lie Thr 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 83 Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu lie 230 235 240
CTG GCA GTG GAG AAG AAG CAC TTG GGT TTG GTG CAG AGG CTT CTG GAG 883 Leu Ala Val Glu Lys Lye His Leu Gly Leu Val Gin Arg Leu Leu Glu 245 250 255 260
CAA GAG CAC ATA GAG ATT AAT GAC ACA GAC AGT GAT GGC AAA ACA GCA 931 Gin Glu His lie Glu He .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 Glu Leu Lys Leu Lys Lys He Ala Glu Leu Leu 280 285 290
TGC AAA CGT GGA GCC AGT ACA GAT TGT GGG GAT CTT GTT ATG ACA GCG 1027 Cys Lys Arg Gly 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 GTT 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 Lys Glu Asp Phe His Pro Pro Ala Glu Asp Trp Lys Pro Gin Ser 325 _ - 330 335 340
TCA CAC TGG GGG GCA GCC CTG -AAG GAT CTC CAC AGA ATA TAC CGC CCT 1171 Ser His Trp Gly Ala Ala Leu Lys Asp Leu His Arg He Tyr Arg Pro 345 350 355
ATG ATT GGC AAA CTC AAG TTC TTT ATT GAT GAA AAA TAC AAA ATT GCT 1219 Met He Gly Lys Leu-Lys Phe Phe He Asp Glu Lys Tyr Lys He Ala 360 365 370
GAT ACT TCA GAA GGA GGC ATC TAC CTG GGG TTC TAT GAG AAG CAA GAA 1267 Asp Thr Ser Glu Gly Gly He Tyr Leu Gly Phe Tyr Glu Lys Gin Glu 375 380 . 385
GTA GCT GTG AAG ACG TTC TGT GAG GGC AGC CCA CGT GCA CAG CGG GAA 1315 Val Ala Val Lys Thr Phe Cys Glu Gly Ser Pro Arg Ala Gin Arg Glu 390 395 400
GTC TCT TGT CTG CAA AGC AGC CGA GAG AAC AGT CAC TTG GTG ACA TTC 1363 Val Ser Cys Leu Gin Ser Ser Arg Glu 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 141 Tyr Gly Ser Glu Ser His Arg Gly His Leu Phe Val Cys Val Thr Leu 425 430 435
TGT GAG CAG ACT CTG GAA GCG TGT TTG GAT GTG CAC AGA GGG GAA GAT 145 Cys Glu Gin Thr Leu Glu Ala Cys Leu Asp Val His Arg Gly Glu Asp 440 445 450
GTG GAA AAT GAG GAA GAT GAA TTT GCC CGA AAT GTC CTG TCA TCT ATA 150 Val Glu Asn Glu Glu Asp Glu Phe Ala Arg Asn Val Leu Ser Ser lie 455 460 465
TTT AAG GCT GTT CAA GAA CTA CAC TTG TCC TGT GGA TAC ACC CAC CAG 1555 Phe Lys Ala Val Gin Glu Leu His Leu Ser Cys Gly Tyr Thr His Gin 470 475 480
GAT CTG CAA CCA CAA AAC ATC TTA ATA GAT TCT AAG AAA GCT GCT CAC 1603 Asp Leu Gin Pro Gin Asn He Leu He Asp Ser Lys Lys Ala Ala His 485 490 495 500
CTG GCA GAT TTT GAT AAG AGC ATC AAG TGG GCT GGA GAT CCA CAG GAA 1651 Leu Ala Asp Phe Asp Lys Ser He Lys Trp Ala Gly Asp Pro Gin Glu 505 510 515
GTC AAG AGA GAT CTA GAG GAC CTT GGA CGG CTG GTC CTC TAT GTG GTA 1699 Val Lys Arg Asp Leu Glu Asp Leu Gly .Arg Leu Val Leu Tyr Val Val 520 525 530
AAG AAG GGA AGC ATC TCA TTT GAG GAT CTG AAA GCT CAA AGT AAT GAA 1747 Lys Lys Gly Ser He Ser Phe Glu Asp Leu Lys Ala Gin Ser Asn Glu 535 540 545
GAG GTG GTT CAA CTT TCT CCA GAT GAG GAA ACT AAG GAC CTC ATT CAT 1795 Glu Val Val Gin Leu Ser Pro Asp Glu Glu Thr Lys Asp Leu He His 550 555 560
CGT CTC TTC CAT CCT GGG GAA CAT GTG AGG GAC TGT CTG AGT GAC CTG 1843 Arg Leu Phe His Pro Gly Glu His Val Arg Asp Cys Leu Ser Asp Leu 565 570 575 580 •
CTG GGT CAT CCC TTC TTT TGG ACT TGG GAG AGC CGC TAT AGG ACG CTT 1891 Leu Gly His Pro Phe Phe Trp Thr Trp Glu Ser Arg Tyr Arg Thr Leu 585 590 595
CGG AAT GTG GGA AAT GAA TCC GAC ATC AAA ACA CGA AAA TCT GAA AGT 1939 Arg Asn Val Gly Asn-Glu Ser Asp He Lys Thr Arg Lys Ser Glu Ser 600 605 610
GAG ATC CTC AGA CTA CTG CAA CCT GGG CCT TCT GAA CAT TCC AAA AGT 1987 Glu He Leu Arg Leu Leu Gin Pro Gly Pro Ser Glu His Ser Lys Ser 615 620 625
TTT GAC AAG TGG ACG ACT AAG ATT AAT GAA TGT GTT ATG AAA AAA ATG 2035 Phe Asp Lys Trp Thr Thr Lys He Asn Glu Cys Val Met Lys Lys Met 630 635 640
AAT AAG TTT TAT GAA AAA AGA GGC AAT TTC TAC CAG AAC ACT GTG GGT 2083 Asn Lys Phe Tyr Glu Lys Arg Gly Asn Phe Tyr Gin Asn Thr Val Gly 645 650 655 660
GAT CTG CTA AAG TTC ATC CGG AAT TTG GGA GAA CAC ATT GAT GAA GAA 2131 Asp Leu Leu Lys Phe He Arg Asn Leu Gly Glu His He Asp Glu Glu 665 670 675
AAG CAT AAA AAG ATG AAA TTA AAA ATT GGA GAC CCT TCC CTG TAT TTT 2179 Lys His Lys Lys Met Lys Leu Lys He Gly Asp Pro Ser Leu Tyr Phe 680 685 690
CAG AAG ACA TTT CCA GAT CTG GTG ATC TAT GTC TAC ACA AAA CTA CAG 2227 Gln Lys Thr Phe Pro .Asp Leu Val He Tyr Val Tyr Thr Lys Leu Gin 695 700 705
AAC ACA GAA TAT AGA AAG CAT TTC CCC CAA ACC CAC AGT CCA AAC AAA 227 Asn Thr Glu Tyr Arg Lys His Phe Pro Gin Thr His Ser Pro Asn Lys 710 715 720
CCT CAG TGT GAT GGA GCT GGT GGG GCC AGT GGG TTG GCC AGC CCT GGG 2323 Pro Gin Cys Asp Gly Ala Gly Gly Ala Ser Gly Leu .Ala Ser Pro Gly 725 730 735 740
TGC TGATGGACTG ATTTGCTGGA GTTCAGGGAA CTACTTATTA GCTGTAGAGT 2376
Cys
CCTTGGCAAA TCACAACATT CTGGGCCTTT TAACTCACCA GGTTGCTTGT GAGGGATGAG 2436
TTGCATAGCT GATATGTCAG TCCCTGGCAT CGTGTATTCC ATATGTCTAT AACAAAAGCA 2496
ATATATACCC AGACTACACT AGTCCATAAG CTTTACCCAC TAACTGGGAG GACATTCTGC 2556
TAAGATTCCT TTTGTCAATT GCACCAAAAG AATGAGTGCC TTGACCCCTA ATGCTGCATA 2616
TGTTACAATT CTCTCACTTA ATTTTCCCAA TGATCTTGCA AAACAGGGAT TATCATCCCC 2676
ATTTAAGAAC TGAGGAACCT GAGACTCAGA GAGTGTGAGC TACTGGCCCA AGATTATTCA 2736
ATTTATACCT AGCACTTTAT AAATTTATGT GGTGTTATTG GTACCTCTCA TTTGGGCACC 2796
TTAAAACTTA ACTATCTTCC AGGGCTCTTC CAGATGAGGC CCAAAACATA TATAGGGGTT 2856
CCAGGAATCT CATTCATTCA TTCAGTATTT ATTGAGCATC TAGTATAAGT CTGGGCACTG 2916
GATGCATGAA TT 2928
(2) INFORMATION FOR'SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 741 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear _
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Ser Arg Asp His Asn Asn Pro Gin Glu Gly Pro Thr Ser Ser 1 5 10 15
Ser Gly Arg Arg Ala Ala Val Glu Asp Asn His Leu Leu He Lys Ala 20 25 30
Val Gin Asn Glu Asp Val Asp Leu Val Gin Gin Leu Leu Glu Gly Gly 35 40 45
Ala Asn Val Asn Phe Gin Glu Glu Glu Gly Gly Trp Thr Pro Leu His 50 55 60
Asn Ala Val Gin Met Ser Arg Glu Asp He Val Glu Leu Leu Leu Arg 65 70 75 80
His Gly Ala Asp Pro Val Leu Arg Lys Lys Asn Gly Ala Thr Leu Phe 85 90 95
He Leu Ala Ala He Ala Gly Ser Val Lys Leu Leu Lys Leu Phe Leu 100 105 110
Ser Lys Gly Ala Asp Val Asn Glu Cys Asp Phe Tyr Gly Phe Thr Ala 115 120 125
Phe Met Glu Ala Ala Val Tyr Gly Lys Val Lys Ala Leu Lys Phe Leu 130 135 140
Tyr Lys Arg Gly Ala Asn Val Asn Leu Arg Arg Lys Thr Lys Glu Asp 145 150 155 160
Gin Glu Arg Leu Arg Lys Gly Gly Ala Thr .Ala Leu Met Asp Ala Ala 165 170 175
Glu Lys Gly His Val Glu Val Leu Lys He Leu Leu Asp Glu Met Gly 180 185 190
Ala Asp Val Asn Ala Cys Asp Asn Met Gly Arg Asn Ala Leu He His 195 200 205
Ala Leu Leu Ser Ser Asp Asp Ser Asp Val Glu Ala He Thr His Leu 210 215 220
Leu Leu"Asp His Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys 225 230 235 240
Thr Pro Leu He Leu Ala Val Glu Lys Lys His Leu Gly Leu Val Gin 245 250 255
Arg Leu Leu Glu Gin Glu His He Glu He Asn Asp Thr Asp Ser Asp 260 - 265 270
Gly Lys Thr Ala Leu Leu Leu Ala Val Glu Leu Lys Leu Lys Lys He 275 280 285
Ala Glu 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 Val Lys Val Leu 305 310 315 320
Leu Ser His Gly Ala Lys Glu Asp Phe His Pro Pro Ala Glu Asp Trp 325 330 335
Lys Pro Gin Ser Ser His Trp Gly Ala Ala Leu Lys Asp Leu His Arg 340 345 350
He Tyr Arg Pro Met He Gly Lys Leu Lys Phe Phe He Asp Glu Lys 355 360 365
Tyr Lys He Ala Asp Thr Ser Glu Gly Gly He Tyr Leu Gly Phe Tyr 370 375 380
Glu Lys Gin Glu Val Ala Val Lys Thr Phe Cys Glu Gly Ser Pro Arg 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 Val Thr Leu Cys Glu Gin Thr Leu Glu Ala Cys Leu Asp Val His 435 440 445
Arg Gly Glu Asp Val Glu Asn Glu Glu Asp Glu Phe Ala Arg Asn Val 450 455 460
Leu Ser Ser He Phe Lys Ala Val Gin Glu Leu His Leu Ser Cys Gly 465 470 475 480
Tyr Thr His Gin Asp Leu Gin Pro Gin Asn He Leu He Asp Ser Lys 485 490 495
Lys Ala Ala His Leu Ala Asp Phe Asp Lys Ser He Lys Trp Ala Gly 500 505 510
Asp Pro Gin Glu Val Lys Arg Asp Leu Glu Asp Leu Gly Arg Leu Val 515 520 525
Leu Tyr Val Val Lys Lys Gly Ser He Ser Phe Glu Asp Leu Lys Ala 530 _ 535 540
Gin Ser Asn Glu Glu Val Val Gin Leu Ser Pro Asp Glu Glu Thr Lys 545 550 555 560
Asp Leu He His Arg Leu Phe His Pro Gly Glu His Val Arg Asp Cys 565 570 575
Leu Ser Asp Leu .Leu Gly His Pro Phe Phe Trp Thr Trp Glu Ser Arg 580 " 585 590
Tyr Arg Thr Leu Arg Asn Val Gly Asn Glu Ser Asp He Lys Thr Arg 595 600 605
Lys Ser Glu Ser Glu He Leu Arg Leu Leu Gin Pro Gly Pro Ser Glu 610 615 620
His Ser Lys Ser Phe Asp Lys Trp Thr Thr Lys He Asn Glu Cys Val 625 630 635 640
Met Lys Lys Met Asn Lys Phe Tyr Glu Lys Arg Gly Asn Phe Tyr Gin 645 650 655
Asn Thr Val Gly Asp Leu Leu Lys Phe He Arg Asn Leu Gly Glu His 660 665 670
He Asp Glu Glu Lys His Lys Lys Met Lys Leu Lys He Gly Asp Pro 675 680 685
Ser Leu Tyr Phe Gin Lys Thr Phe Pro Asp Leu Val He Tyr Val Tyr 690 695 700
Thr Lys Leu Gin Asn Thr Glu Tyr Arg Lys His Phe Pro Gin Thr His 705 710 715 720
Ser Pro Asn Lys Pro Gin Cys Asp Gly Ala Gly Gly Ala Ser Gly Leu 725 730 735
Ala Ser Pro Gly Cys 740
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2928 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 104..2326
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AATCCCAACT TACACTCAAA GCTTCTTTGA TTAAGTGCTA GGAGATAAAT TTGCATTTTC
TCAAGGAAAA GGCTAAAAGT GGTAGCAGGT GGCATTTACC GTC ATG GAG AGC AGG 1
Met Glu Ser Arg
1
GAT CAT AAC AAC CCC CAG GAG GGA CCC ACG TCC TCC AGC GGT AGA AGG 1 Asp His Asn Asn Pro Gin Glu Gly Pro Thr Ser Ser Ser Gly Arg Arg 5 10 15 20
GCT GCA GTG GAA GAC'AAT CAC TTG CTG ATT AAA GCT GTT CAA AAC GAA 2 Ala Ala Val Glu Asp Asn His Leu Leu lie Lys Ala Val Gin Asn Glu 25 30 35
GAT GTT GAC CTG GTC CAG CAA TTG CTG GAA GGT GGA GCC AAT GTT AAT 2 Asp Val Asp Leu Val Gin Gin Leu Leu Glu Gly Gly Ala Asn Val Asn 40 45 50
TTC CAG GAA GAG GAA GGG GGC TGG ACA CCT CTG CAT AAC GCA GTA CAA 3 Phe Gin Glu Glu Glu Gly Gly Trp Thr Pro Leu His Asn Ala Val Gin 55 60 65
ATG AGC AGG GAG GAC ATT GTG GAA CTT CTG CTT CGT CAT GGT GCT GAC 3 Met Ser Arg Glu Asp lie Val Glu Leu Leu Leu Arg His Gly Ala Asp 70 75 80
CCT GTT CTG AGG AAG AAG AAT GGG GCC ACG CCT TTT ATC CTC GCA GCG Pro Val Leu Arg Lys Lys Asn Gly Ala Thr Pro Phe lie Leu Ala Ala 85 90 95 100
ATT GCG GGG AGC GTG AAG CTG CTG AAA CTT TTC CTT TCT AAA GGA GCA 4 lie Ala Gly Ser Val Lys Leu Leu Lys Leu Phe Leu Ser Lys Gly Ala 105 110 115 -99-
GAT GTC AAT GAG TGT GAT TTT TAT GGC TTC ACA GCC TTC ATG GAA GCC 4 Asp Val Asn Glu Cys Asp Phe Tyr Gly Phe Thr Ala Phe Met Glu Ala 120 125 130
GCT GTG TAT GGT AAG GTC AAA GCC CTA AAA TTC CTT TAT AAG AGA GGA 5 Ala Val Tyr Gly Lys Val Lys Ala Leu Lys Phe Leu Tyr Lys Arg Gly 135 140 145
GCA AAT GTG AAT TTG AGG CGA AAG ACA AAG GAG GAT CAA GAG CGG CTG 5 Ala Asn Val Asn Leu Arg Arg Lys Thr Lys Glu Asp Gin Glu Arg Leu 150 155 160
AGG AAA GGA GGG GCC ACA GCT CTC ATG GAC GCT GCT GAA AAA GGA CAC 64 Arg Lys Gly Gly Ala Thr Ala Leu Met Asp Ala Ala Glu Lys Gly His 165 170 175 180
GTA GAG GTC TTG AAG ATT CTC CTT GAT GAG ATG GGG GCA GAT GTA AAC 6 Val Glu Val Leu Lys lie Leu Leu Asp Glu Met Gly Ala Asp Val Asn 185 190 195
GCC TGT GAC AAT ATG GGC AGA AAT GCC TTG ATC CAT GCT CTC CTG AGC 73 Ala Cys Asp Asn Met Gly Arg Asn Ala Leu lie His Ala Leu Leu Ser 200 205 210
TCT GAC GAT AGT GAT GTG GAG GCT ATT ACG CAT CTG CTG CTG GAC CAT 78 Ser Asp Asp Ser Asp Val Glu Ala lie Thr 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 83 Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu lie 230 235 240
CTG GCA GTG GAG AAG AAG CAC TTG GGT TTG GTG CAG AGG CTT CTG GAG 8 Leu Ala Val Glu Lys Lys His Leu Gly Leu Val Gin Arg Leu Leu Glu 245 250 255 260
CAA GAG CAC ATA GAG "ATT AAT GAC ACA GAC AGT GAT GGC AAA ACA GCA 93 Gin Glu His He Glu He 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 9 Leu Leu Leu Ala Val Glu Leu Lys Leu Lys Lys He Ala Glu Leu Leu 280 285 290
TGC AAA CGT GGA GCC AGT ACA GAT TGT GGG GAT CTT GTT ATG ACA GCG 10 Cys Lys Arg Gly 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 GTT CTT CTC TCT CAT GGA 10 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 11 Ala Lys Glu Asp Phe His Pro Pro Ala Glu Asp Trp Lys Pro Gin Ser 325 330 335 340
TCA CAC TGG GGG GCA GCC CTG AAG GAT CTC CAC AGA ATA TAC CGC CCT 11 Ser His Trp Gly .Ala Ala Leu Lys Asp Leu His Arg He Tyr Arg Pro 345 350 355 ATG ATT GGC AAA CTC AAG TTC TTT ATT GAT GAA AAA TAC AAA ATT GCT '12 Met lie Gly Lys Leu Lys Phe Phe lie Asp Glu Lys Tyr Lys lie Ala 360 365 370
GAT ACT TCA GAA GGA GGC ATC TAC CTG GGG TTC TAT GAG AAG CAA GAA 12 Asp Thr Ser Glu Gly Gly lie Tyr Leu Gly Phe Tyr Glu Lys Gin Glu 375 380 385
GTA GCT GTG .AAG ACG TTC TGT GAG GGC AGC CCA CGT GCA CAG CGG GAA 13 Val Ala Val Lys Thr Phe Cys Glu Gly Ser Pro Arg Ala Gin Arg Glu 390 395 400
GTC TCT TGT CTG CAA AGC AGC CGA GAG AAC AGT CAC TTG GTG ACA TTC. 13 Val Ser Cys Leu Gin Ser Ser Arg Glu 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 14 Tyr Gly Ser Glu Ser His Arg Gly His Leu Phe Val Cys Val Thr Leu 425 430 435
TGT GAG CAG ACT CTG GAA GCG TGT TTG GAT GTG CAC AGA GGG GAA GAT 145 Cys Glu Gin Thr Leu Glu Ma Cys Leu Asp Val His Arg Gly Glu Asp 440 445 450
GTG GAA AAT GAG GAA GAT GAA TTT GCC CGA AAT GTC CTG TCA TCT ATA 150 Val Glu Asn Glu Glu Asp Glu Phe Ala Arg Asn Val Leu Ser Ser lie 455 460 465
TTT AAG. GCT GTT CAA GAA CTA CAC TTG TCC TGT GGA TAC ACC CAC CAG 155 Phe Lys Ala Val Gin Glu Leu His Leu Ser Cys Gly Tyr Thr His Gin 470 475 480
GAT CTG CAA CCA CAA AAC ATC TTA ATA GAT TCT -AAG AAA GCT GCT CAC 160 Asp Leu Gin Pro Gin Asn lie Leu lie Asp Ser Lys Lys Ala Ala His 485 490 495 500
CTG GCA GAT TTT GAT-AAG AGC ATC AAG TGG GCT GGA GAT CCA CAG GAA 165 Leu Ala Asp Phe Asp Lys Ser lie Lys Trp Ala Gly Asp Pro Gin Glu 505 510 515
GTC AAG AGA GAT CTA GAG GAC CTT GGA CGG CTG GTC CTC TAT GTG GTA 169 Val Lys Arg Asp Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr Val Val 520 525 530
AAG AAG GGA AGC ATC TCA TTT GAG GAT CTG AAA GCT CAA AGT AAT GAA 174 Lys Lys Gly Ser lie Ser Phe Glu Asp Leu Lys Ala Gin Ser Asn Glu 535 540 545
GAG GTG GTT CAA CTT TCT CCA GAT GAG GAA ACT AAG GAC CTC ATT CAT 179 Glu Val Val Gin Leu Ser Pro Asp Glu Glu Thr Lys Asp Leu lie His 550 555 560
CGT CTC TTC CAT CCT GGG GAA CAT GTG AGG GAC TGT CTG AGT GAC CTG 184 Arg Leu Phe His Pro Gly Glu His Val Arg Asp Cys Leu Ser Asp Leu 565 570 575 580
CTG GGT CAT CCC TTC TTT TGG ACT TGG GAG AGC CGC TAT AGG ACG CTT 189 Leu Gly His Pro Phe Phe Trp Thr Trp Glu Ser Arg Tyr Arg Thr Leu 585 590 595 CGG AAT GTG GGA AAT GAA TCC GAC ATC .AAA ACA CGA AAA TCT GAA AGT 1939 Arg Asn Val Gly Asn Glu Ser Asp lie Lys Thr Arg Lys Ser Glu Ser 600 605 610
GAG ATC CTC AGA CTA CTG CAA CCT GGG CCT TCT GAA CAT TCC AAA AGT 1987 Glu lie Leu Arg Leu Leu Gin Pro Gly Pro Ser Glu His Ser Lys Ser 615 620 625
TTT GAC AAG TGG ACG ACT AAG ATT AAT GAA TGT GTT ATG AAA AAA ATG 2035 Phe Asp Lys Trp Thr Thr Lys lie Asn Glu Cys Val Met Lys Lys Met 630 635 640
AAT AAG TTT TAT GAA AAA AGA GGC AAT TTC TAC CAG AAC ACT GTG GGT 2083 Asn Lys Phe Tyr Glu Lys Arg Gly Asn Phe Tyr Gin Asn Thr Val Gly 645 650 655 660
GAT CTG CTA AAG TTC ATC CGG AAT TTG GGA GAA CAC ATT GAT GAA GAA 2131 Asp Leu Leu Lys Phe lie Arg Asn Leu Gly Glu His lie Asp Glu Glu 665 670 675
AAG CAT AAA AAG ATG AAA TTA AAA ATT GGA GAC CCT TCC CTG TAT TTT 2179 Lys His Lys Lys Met Lys Leu Lys lie Gly .Asp Pro Ser Leu Tyr Phe 680 685 690
CAG AAG ACA TTT CCA GAT CTG GTG ATC TAT GTC TAC ACA AAA CTA CAG 2227 Gin Lys Thr Phe Pro Asp Leu Val lie Tyr Val Tyr Thr Lys Leu Gin 695 700 705
AAC ACA-GAA'TAT AGA AAG CAT TTC CCC CAA ACC CAC AGT CCA AAC AAA 2275 Asn Thr Glu Tyr Arg Lys His Phe Pro Gin Thr His Ser Pro Asn Lys 710 715 720
CCT CAG TGT GAT GGA GCT GGT GGG GCC AGT GGG TTG GCC AGC CCT GGG 2323 Pro Gin Cys Asp Gly Ala Gly Gly Ala Ser Gly Leu Ala Ser Pro Gly 725 730 735 740
TGC TGATGGACTG ATTTGCTGGA GTTCAGGGAA CTACTTATTA GCTGTAGAGT 2376
Cys
CCTTGGCAAA TCACAACATT CTGGGCCTTT TAACTCACCA GGTTGCTTGT GAGGGATGAG 2436
TTGCATAGCT GATATGTCAG TCCCTGGCAT CGTGTATTCC ATATGTCTAT AACAAAAGCA 2496
ATATATACCC AGACTACACT AGTCCATAAG CTTTACCCAC TAACTGGGAG GACATTCTGC 2556
TAAGATTCCT TTTGTCAATT GCACCAAAAG AATGAGTGCC TTGACCCCTA ATGCTGCATA 2616
TGTTACAATT CTCTCACTTA ATTTCCCAA TGATCTTGCA AAACAGGGAT TATCATCCCC 2676
ATTTAAGAAC TGAGGAACCT GAGACTCAGA GAGTGTGAGC TACTGGCCCA AGATTATTCA 2736
ATTTATACCT AGCACTTTAT AAATTTATGT GGTGTTATTG GTACCTCTCA TTTGGGCACC 2796
TTAAAACTTA ACTATCTTCC AGGGCTCTTC CAGATGAGGC CCAAAACATA TATAGGGGTT 2856
CCAGGAATCT CATTCATTCA TTCAGTATTT ATTGAGCATC TAGTATAAGT CTGGGCACTG 2916
GATGCATGAA TT 2928 (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 741 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Glu Ser Arg Asp His Asn Asn Pro Gin Glu Gly Pro Thr Ser Ser 1 5 10 15
Ser Gly Arg Arg Ala Ala Val Glu Asp Asn His Leu Leu He Lys Ala 20 25 30
Val Gin Asn Glu Asp Val Asp Leu Val Gin Gin Leu Leu Glu Gly Gly 35 40 45
Ala Asn Val Asn Phe Gin Glu Glu Glu Gly Gly Trp Thr Pro Leu His 50 55 60
Asn Ala Val Gin Met Ser Arg Glu Asp He Val Glu Leu Leu Leu Arg 65 70 75 80
His Gly-Ala Asp Pro Val Leu Arg Lys Lys Asn Gly Ala Thr Pro Phe 85 90 95
He Leu Ala Ala He Ala Gly Ser Val Lys Leu Leu Lys Leu Phe Leu 100 105 110
Ser Lys Gly Ala Asp Val Asn Glu Cys Asp Phe Tyr Gly Phe Thr Ala 115 120 125
Phe Met Glu Ala Ala"Val Tyr Gly Lys Val Lys Ala Leu Lys Phe Leu 130 135 140
Tyr Lys Arg Gly Ala Asn Val Asn Leu Arg Arg Lys Thr Lys Glu Asp 145 150 155 160
Gin Glu Arg Leu Arg Lys Gly Gly Ala Thr Ala Leu Met Asp Ala Ala 165 170 175
Glu Lys Gly His Val Glu Val Leu Lys He Leu Leu Asp Glu Met Gly 180 185 190
Ala Asp Val Asn Ala Cys Asp Asn Met Gly Arg Asn Ala Leu He His 195 200 205
Ala Leu Leu Ser Ser Asp Asp Ser Asp Val Glu Ala He Thr His Leu 210 215 220
Leu Leu Asp His Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys 225 230 235 240
Thr Pro Leu He Leu Ala Val Glu Lys Lys His Leu Gly Leu Val' Gin 245 250 255 Arg Leu Leu Glu Gin Glu His He Glu He .Asn Asp Thr .Asp Ser Asp 260 265 270
Gly Lys Thr a Leu Leu Leu Ala Val Glu Leu Lys Leu Lys Lys He 275 280 285
Ala Glu 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 Val Lys Val Leu 305 310 315 320
Leu Ser His Gly .Ala Lys Glu Asp Phe His Pro Pro Ala Glu Asp Trp 325 330 335
Lys Pro Gin Ser Ser His Trp Gly Ala Ala Leu Lys Asp Leu His Arg 340 345 350
He Tyr Arg Pro Met He Gly Lys Leu Lys Phe Phe He Asp Glu Lys 355 360 365
Tyr Lys He Ala Asp Thr Ser Glu Gly Gly He Tyr Leu Gly Phe Tyr 370 375 380
Glu Lys Gin Glu Val .Ala Val Lys Thr Phe Cys Glu Gly Ser Pro Arg 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 Val Thr Leu Cys Glu Gin Thr Leu Glu .Ala Cys Leu Asp Val His 435 440 445
Arg Gly Glu Asp Val"Glu Asn Glu Glu Asp Glu Phe Ala Arg Asn Val 450 455 460
Leu Ser Ser He Phe Lys Ala Val Gin Glu Leu His Leu Ser Cys Gly 465 470 475 480
Tyr Thr His Gin Asp Leu Gin Pro Gin Asn He Leu He Asp Ser Lys 485 490 495
Lys Ala Ala His Leu Ala Asp Phe Asp Lys Ser He Lys Trp Ala Gly 500 505 510
Asp Pro Gin Glu Val Lys Arg Asp Leu Glu Asp Leu Gly Arg Leu Val 515 520 525
Leu Tyr Val Val Lys Lys Gly Ser He Ser Phe Glu Asp Leu Lys Ala 530 535 540
Gin Ser Asn Glu Glu Val Val Gin Leu Ser Pro .Asp Glu Glu Thr Lys 545 550 555 560
Asp Leu He His Arg Leu Phe His Pro Gly Glu His Val Arg Asp Cys 565 570 575 Leu Ser Asp Leu Leu Gly His Pro Phe Phe Trp Thr Trp Glu Ser Arg 580 585 590
Tyr Arg Thr Leu .Arg Asn Val Gly Asn Glu Ser Asp lie Lys Thr Arg 595 600 605
Lys Ser Glu Ser Glu lie Leu Arg Leu Leu Gin Pro Gly Pro Ser Glu 610 615 620
His Ser Lys Ser Phe .Asp Lys Trp Thr Thr Lys lie Asn Glu Cys Val 625 630 635 640
Met Lys Lys Met Asn Lys Phe Tyr Glu Lys Arg Gly Asn Phe Tyr Gin 645 650 655
Asn Thr Val Gly Asp Leu Leu Lys Phe lie Arg Asn Leu Gly Glu His 660 665 670 lie Asp Glu Glu Lys His Lys Lys Met Lys Leu Lys lie Gly Asp Pro 675 680 685
Ser Leu Tyr Phe Gin Lys Thr Phe Pro Asp Leu Val lie Tyr Val Tyr 690 695 700
Thr Lys Leu Gin Asn Thr Glu Tyr .Arg Lys His Phe Pro Gin Thr His 705 710 715 720
Ser Pro Asn Lys Pro Gin Cys Asp Gly Ala Gly Gly Ala Ser Gly Leu 725 730 735
Ala Ser Pro Gly Cys 740
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH": 2200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 164..2200
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATTCGGCACG AGGAAGGTGC CAATTACTAG CTCCCTTCTT TATTCGTGTA CTGATGAGAT 60
GTCAGAAGAC AGAACATAAT CAGCCCAATC CCTACTCCAA GACTCTCATT GTGTCCCAAA 120
GAAACACACG TGTGCATTTC CCAAGGAAAA GGCATTGAGG ACC ATG GAG ACC CCG 175
Met Glu Thr Pro
1
GAT TAT AAC ACA CCT CAG GGT GGA ACC CCA TCA GCG GGA AGT CAG AGG 223 Asp Tyr -Asn Thr Pro Gin Gly Gly Thr Pro Ser Ala Gly Ser Gin Arg 5 10 15 20
ACC GTT GTC GAA GAT GAT TCT TCG TTG ATC AAA GCT GTT CAG AAG GGA 27 Thr Val Val Glu Asp Asp Ser Ser Leu lie Lys Ala Val Gin Lys Gly 25 30 35
GAT GTT GTC AGG GTC CAG CAA TTG TTA GAA AAA GGG GCT GAT GCC AAT 31 Asp Val Val Arg Val Gin Gin Leu Leu Glu Lys Gly a .Asp Ala Asn 40 45 50
GCC TGT GAA GAC ACC TGG GGC TGG ACA CCT TTG CAC AAC GCA GTG CAA 36 Ala Cys Glu Asp Thr Trp Gly Trp Thr Pro Leu His Asn Ala Val Gin 55 60 65
GCT GGC AGG GTA GAC ATT GTG AAC CTC CTG CTT AGT CAT GGT GCT GAC 41 Ala Gly Arg Val Asp lie Val Asn Leu Leu Leu Ser His Gly Ala Asp 70 75 80
CCT CAT CGG AGG AAG AAG AAT GGG GCC ACC CCC TTC ATC ATT GCT GGG 46 Pro His Arg Arg Lys Lys Asn Gly .Ala Thr Pro Phe lie lie Ala Gly 85 90 95 100
ATC CAG GGA GAT GTG AAA CTG CTC GAG ATT CTC CTC TCT TGT GGT GCA 51 lie Gin Gly Asp Val Lys Leu Leu Glu lie Leu Leu Ser Cys Gly Ala 105 110 115
GAC GTC-ART GAG TGT GAC GAG AAC GGA TTC ACG GCT TTC ATG GAA GCT 55 Asp Val Asn Glu Cys Asp Glu Asn Gly Phe Thr Ala Phe Met Glu Ala 120 125 130
GCT GAG CGT GGT AAC GCT GAA GCC TTA AGA TTC CTT TTT GCT AAG GGA 60 Ala Glu Arg Gly Asn Ala Glu Ala Leu Arg Phe Leu Phe Ala Lys Gly 135 140 145
GCC AAT GTG AAT TTG CGA CGA CAG ACA ACG AAG GAC AAA AGG CGA TTG 65 Ala Asn Val Asn Leu *Arg Arg Gin Thr Thr Lys Asp Lys Arg Arg Leu 150 155 160
AAG CAA GGA GGC GCC ACA GCT CTC ATG AGC GCT GCT GAG AAG GGC CAC 70 Lys Gin Gly Gly Ala Thr Ala Leu Met Ser Ala Ala Glu Lys Gly His 165 170 . 175 180
CTG GAA GTC CTG AGA ATT CTC CTC AAT GAC ATG AAG GCA GAA GTC GAT 75 Leu Glu Val Leu Arg lie Leu Leu Asn Asp Met Lys Ala Glu Val Asp 185 190 195
GCT CGG GAC AAC ATG GGC AGA AAT GCC CTG ATC CGT ACT CTG CTG AAC 79 Ala Arg Asp Asn Met Gly Arg Asn Ala Leu lie Arg Thr Leu Leu Asn 200 205 210
TGG GAT TGT GAA AAT GTG GAG GAG ATT ACT TCA ATC CTG ATT CAG CAC 84 Trp Asp Cys Glu Asn Val Glu Glu lie Thr Ser lie Leu lie Gin His 215 220 225
GGG GCT GAT GTT AAC GTG AGA GGA GAA AGA GGG AAA ACA CCC CTC ATC 89 Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu lie 230 235 240
GCA GCA GTG GAG AGG AAG CAC ACA GGC TTG GTG CAG ATG CTC CTG AGT 94 Ala Ala Val Glu Arg Lys His Thr Gly Leu Val Gin Met Leu Leu Ser 245 250 255 260
CGG GAA GGC ATA AAC ATA GAT GCC AGG GAT AAC GAG GGC AAG ACA GCT 99 Arg Glu Gly lie Asn lie Asp Ala Arg Asp Asn Glu Gly Lys Thr Ala 265 270 275
CTG CTA ATT GCT GTT GAT AAA CAA CTG AAG GAA ATT GTC CAG TTG CTT 103 Leu Leu lie Ala Val Asp Lys Gin Leu Lys Glu lie Val Gin Leu Leu 280 285 290
CTT GAA AAG GGA GCT GAT AAG TGT GAC GAT CTT GTT TGG ATA GCC AGG 108 Leu Glu Lys Gly Ala Asp Lys Cys Asp Asp Leu Val Trp lie Ala Arg 295 300 305
AGG AAT CAT GAC TAT CAC CTT GTA AAG CTT CTC CTC CCT TAT GTA GCT 113 Arg Asn His Asp Tyr His Leu Val Lys Leu Leu Leu Pro Tyr Val Ala 310 315 320
AAT CCT GAC ACC GAC CCT CCT GCT GGA GAC TGG TCG CCT CAC AGT TCA 118 Asn Pro Asp Thr Asp Pro Pro Ala Gly Asp Trp Ser Pro His Ser Ser 325 330 335 340
CGT TGG GGG ACA GCC TTG AAA AGC CTC CAC AGT ATG ACT CGA CCC ATG 123 Arg Trp Gly Thr Ala Leu Lys Ser Leu His Ser Met Thr Arg Pro Met 345 350 355
ATT GGC AAA CTC AAG ATC TTC ATT CAT GAT GAC TAT AAA ATT GCT GGC 127 lie Gly Lys. Leu Lys lie Phe lie His .Asp Asp Tyr Lys lie Ala Gly 360 365 370
ACT TCC GAA GGG GCT GTC TAC CTA GGG ATC TAT GAC AAT CGA GAA GTG 132 Thr Ser Glu Gly Ala Val Tyr Leu Gly lie Tyr Asp Asn Arg Glu Val 375 380 385
GCT GTG AAG GTC TTC CGT GAG AAT AGC CCA CGT GGA TGT AAG GAA GTC 137 Ala Val Lys Val Phe -Arg Glu Asn Ser Pro Arg Gly Cys Lys Glu Val 390 395 400
TCT TGT CTG CGG GAC TGC GGT GAC CAC AGT AAC TTA GTG GCT TTC TAT 142 Ser Cys Leu Arg Asp Cys Gly Asp His Ser Asn Leu Val Ala Phe Tyr 405 410 415 420
GGA AGA GAG GAC GAT AAG GGC TGT TTA TAT GTG TGT GTG TCC CTG TGT 147 Gly Arg Glu Asp Asp Lys Gly Cys Leu Tyr Val Cys Val Ser Leu Cys 425 430 435
GAG TGG ACA CTG GAA GAG TTC CTG AGG TTG CCC AGA GAG GAA CCT GTG 151 Glu Trp Thr Leu Glu Glu Phe Leu Arg Leu Pro Arg Glu Glu Pro Val 440 445 450
GAG AAC GGG GAA GAT AAG TTT GCC CAC AGC ATC CTA TTA TCT ATA TTT 156 Glu Asn Gly Glu Asp Lys Phe Ala His Ser lie Leu Leu Ser He Phe 455 460 465
GAG GGT GTT CAA AAA CTA CAC TTG CAT GGA TAT TCC CAT CAG GAC CTG 161 Glu Gly Val Gin Lys Leu His Leu His Gly Tyr Ser His Gin Asp Leu 470 475 480
CAA CCA CAA AAC ATC TTA ATA GAT TCC AAG AAA GCT GTC CGG CTG GCA 166 Gln Pro Gin Asn lie Leu lie Asp Ser Lys Lys Ala Val Arg Leu Ala 485 490 495 500
GAT TTT GAT CAG AGC ATC CGA TGG ATG GGA GAG TCA CAG ATG GTC AGG 171 Asp Phe Asp Gin Ser* lie Arg Trp Met Gly Glu Ser Gin Met Val Arg 505 510 515
AGA GAC TTG GAG GAT CTT GGA CGG CTG GTT CTC TAC GTG GTA ATG AAA 175 Arg Asp Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr Val Val Met Lys 520 525 530
GGT GAG ATC CCC TTT GAG ACA CTA AAG ACT CAG AAT GAT GAA GTG CTG 180 Gly Glu lie Pro Phe Glu Thr Leu Lys Thr Gin .Asn Asp Glu Val Leu 535 540 545
CTT ACA ATG TCT CCA GAT GAG GAG ACT AAG GAC CTC ATT CAT TGC CTG 185 Leu Thr Met Ser Pro Asp Glu Glu Thr Lys Asp Leu lie His Cys Leu 550 555 560
TTT TCT CCT GGA GAA AAT GTC AAG AAC TGC CTG GTA GAC CTG CTT GGC 190 Phe Ser Pro Gly Glu Asn Val Lys Asn Cys Leu Val Asp Leu Leu Gly 565 570 575 580
CAT CCT TTC TTT TGG ACT TGG GAG AAC CGC TAT AGA ACA CTC CGG AAT 195 His Pro Phe Phe Trp Thr Trp Glu .Asn Arg Tyr Arg Thr Leu Arg Asn 585 590 595
GTG GGA AAT GAA TCT GAC ATC AAA GTA CGG .AAA TGT AAA AGT GAT CTT 199 Val Gly Asn Glu Ser Asp lie Lys Val Arg Lys Cys Lys Ser Asp Leu 600 605 610
CTC AGA CTA CTG CAG CAT CAG ACA CTT GAG CCT CCC AGA AGC TTT GAC 204 Leu Arg Leu Leu Gin His Gin Thr Leu Glu Pro Pro Arg Ser Phe Asp 615 620 625
CAG TGG ACA TCT AAG ATC GAC AAA AAT GTT ATG GAT GAA ATG AAT CAT 209 Gin Trp Thr Ser Lys -lie Asp Lys Asn Val Met Asp Glu Met Asn His 630 635 640
TTC TAC GAA AAG AGA AAA AAA AAC CCT TAT CAG GAT ACT GTA GGT GAT 214 Phe Tyr Glu Lys Arg Lys Lys Asn Pro Tyr Gin Asp Thr Val Gly Asp 645 650 655 660
CTG CTG AAG TTT ATT CGG AAT ATA GGC GAA CAC ATC AAT GAG GAA AAA 219 Leu Leu Lys Phe lie Arg Asn lie Gly Glu His lie Asn Glu Glu Lys 665 670 675
AAG CGG GGG 220
Lys Arg Gly
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 679 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Glu Thr Pro Asp Tyr .Asn Thr Pro Gin Gly Gly Thr Pro Ser Ala 1 5 10 15
Gly Ser Gin Arg Thr Val Val Glu Asp Asp' Ser Ser Leu lie Lys Ala 20 25 30
Val Gin Lys Gly Asp Val Val Arg Val Gin Gin Leu Leu Glu Lys Gly 35 40 45
Ala Asp Ala Asn Ala Cys Glu Asp Thr Trp Gly Trp Thr Pro Leu His 50 55 60
Asn Ala Val Gin Ala Gly Arg Val Asp lie Val Asn Leu Leu Leu Ser 65 70 75 80
His Gly Ala Asp Pro His Arg Arg Lys Lys Asn Gly Ala Thr Pro Phe 85 90 95 lie lie Ala Gly lie Gin Gly Asp Val Lys Leu Leu Glu lie Leu Leu 100 105 110
Ser Cys Gly Ala Asp Val Asn Glu Cys Asp Glu Asn Gly Phe Thr Ala 115 120 125
Phe Met Glu Ala Ala Glu Arg Gly Asn Ala Glu Ala Leu Arg Phe Leu 130 135 140
Phe Ala Lys Gly Ala Asn Val Asn Leu Arg Arg Gin Thr Thr Lys Asp 145 150 155 160
Lys Arg Arg Leu Lys Gin Gly Gly Ala Thr Ala Leu Met Ser Ala Ala 165 170 175
Glu Lys Gly His Leu-Glu Val Leu Arg lie Leu Leu Asn Asp Met Lys 180 185 190
Ala Glu Val Asp Ala Arg Asp Asn Met Gly Arg Asn Ala Leu lie Arg 195 200 205
Thr Leu Leu Asn Trp Asp Cys Glu Asn Val Glu Glu lie Thr Ser lie 210 215 220
Leu lie Gin His Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys 225 230 235 240
Thr Pro Leu lie Ala Ala Val Glu Arg Lys His Thr Gly Leu Val Gin 245 250 255
Met Leu Leu Ser Arg Glu Gly lie Asn lie Asp Ala Arg Asp Asn Glu 260 265 270
Gly Lys Thr Ala Leu Leu lie Ala Val Asp Lys Gin Leu Lys Glu lie 275 280 285
Val Gin Leu Leu Leu Glu Lys Gly Ala Asp Lys Cys Asp Asp Leu'Val 290 295 300 Trp He Ala Arg Arg Asn His Asp Tyr His Leu Val Lys Leu Leu Leu 305 310 315 320
Pro Tyr Val Ala Asn Pro Asp Thr Asp Pro Pro Ala Gly Asp Trp Ser 325 330 335
Pro His Ser Ser Arg Trp Gly Thr Ala Leu Lys Ser Leu His Ser Met 340 345 350
Thr Arg Pro Met He Gly Lys Leu Lys He Phe He His Asp Asp Tyr 355 360 365
Lys He Ala Gly Thr Ser Glu Gly Ala Val Tyr Leu Gly He Tyr Asp 370 375 380
Asn Arg Glu Val Ala Val Lys Val Phe Arg Glu Asn Ser Pro Arg Gly 385 390 395 400
Cys Lys Glu Val Ser Cys Leu Arg Asp Cys Gly Asp His Ser Asn Leu 405 410 415
Val Ala Phe Tyr Gly Arg Glu Asp Asp Lys Gly Cys Leu Tyr Val Cys 420 425 430
Val Ser Leu Cys Glu Trp Thr Leu Glu Glu Phe Leu Arg Leu Pro Arg 435 440 445
Glu Glu. ro Val Glu Asn Gly Glu Asp Lys Phe Ala His Ser He Leu 450 455 460
Leu Ser He Phe Glu Gly Val Gin Lys Leu His Leu His Gly Tyr Ser 465 470 475 480
His Gin Asp Leu Gin Pro Gin Asn He Leu He Asp Ser Lys Lys Ala 485 490 495
Val Arg Leu Ala Asp "Phe Asp Gin Ser He Arg Trp Met Gly Glu Ser 500 505 510
Gin Met Val Arg Arg Asp Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr 515 520 525
Val Val Met Lys Gly Glu He Pro Phe Glu Thr Leu Lys Thr Gin Asn 530 535 540
Asp Glu Val Leu Leu Thr Met Ser Pro Asp Glu Glu Thr Lys Asp Leu 545 550 555 560
He His Cys Leu Phe Ser Pro Gly Glu Asn Val Lys Asn Cys Leu Val 565 570 575
Asp Leu Leu Gly His Pro Phe Phe Trp Thr Trp Glu Asn Arg Tyr Arg 580 585 590
Thr Leu Arg Asn Val Gly Asn Glu Ser Asp He Lys Val Arg Lys Cys 595 600 605
Lys Ser Asp Leu Leu Arg Leu Leu Gin His Gin Thr Leu Glu Pro Pro • 610 615 620 Arg Ser Phe Asp Gin Trp Thr Ser Lys He Asp Lys Asn Val Met Asp 625 630 635 640
Glu Met Asn His Phe Tyr Glu Lys Arg Lys Lys Asn Pro Tyr Gin Asp 645 650 655
Thr Val Gly Asp Leu Leu Lye Phe He Arg Asn He Gly Glu His He 660 665 670
Asn Glu Glu Lys Lys Arg Gly 675
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 190 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Asp Arg Arg Lys Pro Arg Gin Asn Asn Arg Arg Asp Arg Asn Glu Arg 1 - - 5 10 15
Arg Asp Thr Arg Ser Glu Arg Thr Glu Gly Ser Asp Asn Arg Glu Glu 20 25 30
Asn Arg Arg Asn Arg Arg Gin Ala Gin Gin Gin Thr Ala Glu Thr Arg 35 40 45
Glu Ser Arg Gin Gin Ala Glu Val Thr Glu Lys Ala Arg Thr Ala Asp 50 - 55 60
Glu Gin Gin Ala Pro Arg Arg Glu Arg Ser Arg Arg Arg Asn Asp Asp 65 70 75 80
Lys Arg Gin Ala Gin Gin Glu Ala Lys Ala Leu Asn Val Glu Glu Gin 85 90 95
Ser Val Gin Glu Thr Glu Gin Glu Glu Arg Val Arg Pro Val Gin Pro 100 105 110
Arg Arg Lys Gin Arg Gin Leu Asn Gin Lys Val Arg Tyr Glu Gin Ser 115 120 125
Val Ala Glu Glu Ala Val Val Ala Pro Val Val Glu Glu Thr Val Ala 130 135 140
Ala Glu Pro He Val Gin Glu Ala Pro Ala Pro Arg Thr Glu Leu Val 145 150 155 160
Lys Val Pro Leu Pro Val Val Ala Gin Thr Ala Pro Glu Gin Gin Glu 165 170 175
Glu Asn Asn Ala Asp Asn Arg Asp Asn Gly Gly Met Pro Ser 180 185 190
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2562 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CAGTTTCTGG AGCAAATTCA GTTTGCCTTC CTGGATTTGT AAATTGTAAT GACCTCAAAA 6
CTTTAGCAGT TCTTCCATCT GACTCAGGTT TGCTTCTCTG GCGGTCTTCA GAATCAACAT 12
CCACACTTCC GTGATTATCT GCGTGCATTT TGGACAAAGC TTCCAACCAG GATACGGGAA 18
GAAGAAATGG CTGGTGATCT TTCAGCAGGT TTCTTCATGG AGGAACTTAA TACATACCGT 24
CAGAAGCAGG GAGTAGTACT TAAATATCAA GAACTGCCTA ATTCAGGACC TCCACATGAT 30
AGGAGGTTTA CATTTCAAGT TATAATAGAT GGAAGAGAAT TTCCAGAAGG TGAAGGTAGA' 36
TCAAAGAAGG AAGCAAAAAA TGCCGCAGCC AAATTAGCTG TTGAGATACT TAATAAGGAA 42
AAGAAGGCAG TTAGTCCTTT ATTATTGACA ACAACGAATT CTTCAGAAGG ATTATCCATG 48
GGGAATTACA TAGGCCTTAT CAATAGAATT GCCCAGAAGA AAAGACTAAC TGTAAATTAT . 54
GAACAGTGTG CATCGGGGGT GCATGGGCCA GAAGGATTTC ATTATAAATG CAAAATGGGA 60
CAGAAAGAAT ATAGTATTGG TACAGGTTCT ACTAAACAGG AAGCAAAACA ATTGGCCGCT 66
AAACTTGCAT ATCTTCAGAT ATTATCAGAA GAAACCTCAG TGAAATCTGA CTACCTGTCC 72
TCTGGTTCTT TTGCTACTAC GTGTGAGTCC CAAAGCAACT CTTTAGTGAC CAGCACACTC 78
GCTTCTGAAT CATCATCTGA AGGTGACTTC TCAGCAGATA CATCAGAGAT AAATTCTAAC 84
AGTGACAGTT TAAACAGTTC TTCGTTGCTT ATGAATGGTC TCAGAAATAA TCAAAGGAAG 90
GCAAAAAGAT CTTTGGCACC CAGATTTGAC CTTCCTGACA TGAAAGAAAC AAAGTATACT 96
GTGGACAAGA GGTTTGGCAT GGATTTTAAA GAAATAGAAT TAATTGGCTC AGGTGGATTT 102
GGCCAAGTTT TCAAAGCAAA ACACAGAATT GACGGAAAGA CTTACGTTAT TAAACGTGTT 108
AAATATAATA ACGAGAAGGC GGAGCGTGAA GTAAAAGCAT TGGCAAAACT TGATCATGTA 114
AATATTGTTC ACTACAATGG CTGTTGGGAT GGATTTGATT ATGATCCTGA GACCAGTGAT 120
GATTCTCTTG AGAGCAGTGA TTATGATCCT GAGAACAGCA AAAATAGTTC AAGGTCAAAG 126
ACTAAGTGCC TTTTCATCCA AATGGAATTC TGTGATAAAG GGACCTTGGA ACAATGGATT 132 GAAAAAAGAA GAGGCGAGAA ACTAGACAAA GTTTTGGCTT TGGAACTCTT TGAACAAATA 1380
ACAAAAGGGG TGGATTATAT ACATTCAAAA AAATTAATTC ATAGAGATCT TAAGCCAAGT 1440
AATATATTCT TAGTAGATAC AAAACAAGTA AAGATTGGAG ACTTTGGACT TGTAACATCT 1500
CTGAAAAATG ATGGAAAGCG AACAAGGAGT AGGGGAACTT TGCGATACAT GAGCCCAGAA 1560
CAGATTTCTT CGCAAGACTA TGGAAAGGAA GTGGACCTCT ACGCTTTGGG GCTAATTCTT 1620
GCTGAACTTC TTCATGTATG TGACACTGCT TTTGAAACAT CAAAGTTTTT CACAGACCTA 1680
CGGGATGGCA TCATCTCAGA TATATTTGAT AAAAAAGAAA AAACTCTTCT ACAGAAATTA 1740
CTCTCAAAGA AACCTGAGGA TCGACCTAAC ACATCTGAAA TACTAAGGAC CTTGACTGTG 1800
TGGAAGAAAA GCCCAGAGAA AAATGAACGA CACACATGTT AGAGCCCTTC TGAAAAAGTA 1860
TCCTGCTTCT GATATGCAGT TTTCCTTAAA TTATCTAAAA TCTGCTAGGG .AATATCAATA 1920
GATATTTACC TTTTATTTTA ATGTTTCCTT TAATTTTTTA CTATTTTTAC TAATCTTTCT 1980
GCAGAAACAG AAAGGTTTTC TTCTTTTTGC TTCAAAAACA TTCTTACATT TTACTTTTTC 2040
CTGGCTCATC TCTTTATTTT TTTTTTTTTT TTTT.AAAGAC AGAGTCTCGC TCTGTTGCCC 2100
AGGCTGGAGT GCAATGACAC AGTCTTGGCT CACTGCAACT TCTGCCTCTT GGGTTCAAGT 2160
GATTCTCCTG"CCTCAGCCTC CTGAGTAGCT GGATTACAGG CATGTGCCAC CCACCCAACT 2220
AATTTTTGTG TTTTTAATAA AGACAGGGTT TCACCATGTT GGCCAGGCTG GTCTCAAACT 2280
CCTGACCTCA AGTAATCCAC CTGCCTCGGC CTCCCAAAGT GCTGGGATTA CAGGGATGAG 2340
CCACCGCGCC CAGCCTCATC TCTTTGTTCT AAAGATGGAA AAACCACCCC CAAATTTTCT 2400
TTTTATACTA TTAATGAATC AATCAATTCA TATCTATTTA TTAAATTTCT ACCGCTTTTA 2460
GGCCAAAAAA ATGTAAGATC GTTCTCTGCC TCACATAGCT TACAAGCCAG CTGGAGAAAT 2520
ATGGTACTCA TTAAAAAAAA AAAAAAAAAG TGATGTACAA CC 2562 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 551 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) 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 15
Tyr Arg Gin Lys Gin Gly Val Val Leu Lys Tyr Gin Glu Leu Pro Asn 20 25 30
Ser Gly Pro Pro His .Asp Arg Arg Phe Thr Phe Gin Val He He Asp 35 40 45
Gly Arg Glu Phe Pro Glu Gly Glu Gly Arg Ser Lys Lys Glu Ala Lys 50 55 60
Asn Ala Ala Ala Lys Leu Ala Val Glu He Leu Asn Lys Glu Lys Lys 65 70 75 80
Ala Val Ser Pro Leu Leu Leu Thr Thr Thr Asn Ser Ser Glu Gly Leu 85 90 95
Ser Met Gly Asn Tyr He Gly Leu He Asn Arg He Ala Gin Lys Lys 100 105 110
Arg Leu Thr Val Asn Tyr Glu Gin Cys Ala Ser Gly Val His Gly Pro 115 120 125
Glu Gly Phe His Tyr Lys Cys Lys Met Gly Gin Lys Glu Tyr Ser He 130 135 140
Gly Thr Gly Ser Thr Lys Gin Glu Ala Lys Gin Leu .Ala Ala Lys Leu 145 150 155 160
Ala Tyr Leu Gin He Leu Ser Glu Glu Thr Ser Val Lys Ser Asp Tyr 165 170 175
Leu Ser Ser Gly Ser Phe Ala Thr Thr Cys Glu Ser Gin Ser Asn Ser 180 185 190
Leu Val Thr Ser Thr Leu Ala Ser Glu Ser Ser Ser Glu Gly Asp Phe 195 200 205
Ser Ala Asp Thr Ser Glu He Asn Ser Asn Ser Asp Ser Leu Asn Ser 210 - 215 220
Ser Ser Leu Leu Met Asn Gly Leu Arg Asn Asn Gin Arg Lys Ala Lys 225 230 235 240
Arg Ser Leu Ala Pro Arg Phe Asp Leu Pro Asp Met Lys Glu Thr Lys 245 250 255
Tyr Thr Val Asp Lys Arg Phe Gly Met Asp Phe Lys Glu He Glu Leu 260 265 270
He Gly Ser Gly Gly Phe Gly Gin Val Phe Lys Ala Lys His Arg He 275 280 285
Asp Gly Lys Thr Tyr Val He Lys Arg Val Lys Tyr Asn Asn Glu Lys 290 295 300
Ala Glu Arg Glu Val Lys Ala Leu Ala Lys Leu Asp His Val Asn He 305 310 315 320
Val His Tyr Asn Gly Cys Trp Asp Gly Phe Asp Tyr Asp Pro Glu Thr 325 330 335
Ser Asp Asp Ser Leu Glu Ser Ser Asp Tyr Asp Pro Glu Asn Ser Lys 340 345 350
Asn Ser Ser Arg Ser Lys Thr Lys Cys Leu Phe He Gin Met Glu Phe 355 360 365
Cys Asp Lys Gly Thr Leu Glu Gin Trp He Glu Lys Arg Arg Gly Glu 370 375 380
Lys Leu Asp Lys Val Leu Ala Leu Glu Leu Phe Glu Gin He Thr Lys 385 390 395 400
Gly Val Asp Tyr He His Ser Lys Lys Leu He His Arg Asp Leu Lys 405 410 415
Pro Ser Asn He Phe Leu Val Asp Thr Lys Gin Val Lys He Gly Asp 420 425 430
Phe Gly Leu Val Thr Ser Leu Lys .Asn Asp Gly Lys Arg Thr Arg Ser 435 440 445
Lys Gly Thr Leu Arg Tyr Met Ser Pro Glu Gin He Ser Ser Gin Asp 450 455 460
Tyr Gly Lys Glu Val Asp Leu Tyr Ala Leu Gly Leu He Leu Ala Glu 465 470 475 480
Leu Leu His Val Cys Asp Thr Ala Phe Glu Thr Ser Lys Phe Phe Thr • " 485 490 495
Asp Leu Arg Asp Gly He He Ser Asp He Phe Asp Lys Lys Glu Lys 500 505 510
Thr Leu Leu Gin Lys Leu Leu Ser Lys Lys Pro Glu Asp Arg Pro Asn 515 520 525
Thr Ser Glu He Leu Arg Thr Leu Thr Val Trp Lys Lys Ser Pro Glu 530 - 535 540
Lys Asn Glu Arg His Thr Cys 545 550
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1650 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
AACTGAAACC AACAGCAGTC CAAGCTCAGT CAGCAGAAGA GATAAAAGCA AACAGGTCTG 60
GGAGGCAGTT CTGTTGCCAC TCTCTCTCCT GTCAATGATG GATCTCAGAA ATACCCCAGC 120
CAAATCTCTG GACAAGTTCA TTGAAGACTA TCTCTTGCCA GACACGTGTT TCCGCATGCA 180 AATCGACCAT GCCATTGACA TCATCTGTGG GTTCCTGAAG GAAAGGTGCT TCCGAGGTAG 240
CTCCTACCCT GTGTGTGTGT CCAAGGTGGT AAAGGGTGGC TCCTCAGGCA AGGGCACCAC 300
CCTCAGAGGC CGATCTGACG CTGACCTGGT TGTCTTCCTC AGTCCTCTCA GCACTTTTCA 360
GGATCAGTTA .AATCGCCGGG GAGAGTTCAT CCAGGAAATT AGGAGACAGC TGGAAGCCTG 420
TCAAAGAGAG AGAGCACTTT CCGTGAAGTT TGAGGTCCAG GCTCCACGCT GGGGCAACCC 480
CCGTGCGCTC .AGCTTCGTAC TGAGTTCGCT CCAGCTCGGG GAGGGGGTGG AGTTCGATGT 540
GCTGCCTGCC TTTGATGCCC TGGGTCAGTT GACTGGCAGC TATAAACCTA ACCCCCAAAT 600
CTATGTCAAG CTCATCGAGG AGTGCACCGA CCTGCAGAAA GAGGGCGAGT TCTCCACCTG 660
CTTCACAGAA CTACAGAGAG ACTTCCTGAA GCAGCGCCCC ACCAAGCTCA AGAGCCTCAT 720
CCGCCTAGTC AAGCACTGGT ACCAAAATTG TAAGAAGAAG CTTGGGAAGC TGCCACCTCA 780
GTATGCCCTG GAGCTCCTGA CGGTCTATGC TTGGGAGCGA GGGAGCATGA AAACACATTT 840
CAACACAGCC CAAGGATTTC GGACGGTCTT GGAATTAGTC ATAAACTACC AGCAACTCTG 900
CATCTACTGG ACAAAGTATT ATGACTTTAA AAACCCCATT ATTGAAAAGT ACCTGAGAAG 960
GCAGCTCACG AAACCCAGGC CTGTGATCCT GGACCCGGCG GACCCTACAG GAAACTTGGG 1020
TGGTGGAGAC "CCAAAGGGTT GGAGGCAGCT GGCACAAGAG GCTGAGGCCT GGCTGAATTA 1080
CCCATGCTTT AAGAATTGGG ATGGGTCCCC AGTGAGCTCC TGGATTCTGC TGGCTGAAAG 1140
C.AACAGTACA GACGATGAGA CCGACGATCC CAGGACGTAT CAGAAATATG GTTACATTGG 1200
AACACATGAG TACCCTCATT TCTCTCATAG ACCCAGCACG CTCCAGGCAG CATCCACCCC 1260
ACAGGCAGAA GAGGACTGGA CCTGCACCAT CCTCTGAATG CCAGTGCATC TTGGGGGAAA 1320
GGGCTCCAGT GTTATCTGGA CCAGTTCCTT CATTTTCAGG TGGGACTCTT GATCCAGAGA 1380
AGACAAAGCT CCTCAGTGAG CTGGTGTATA ATCCAAGACA GAACCCAAGT CTCCTGACTC 1440
CTGGCCTTCT ATGCCCTCTA TCCTATCATA GATAACATTC TCCACAGCCT CACTTCATTC 1500
CACCTATTCT CTGAAAATAT TCCCTGAGAG AGAACAGAGA GATTTAGATA AGAGAATGAA 1560
ATTCCAGCCT TGACTTTCTT CTGTGCACCT GATGGGAGGG TAATGTCTAA TGTATTATCA 1620
ATAACAATAA AAATAAAGCA AATACCAAAA 1650 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 400 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Met Asp Leu Arg Asn Thr Pro Ala Lys Ser Leu Asp Lys Phe He 1 5 10 15
Glu Asp Tyr Leu Leu Pro Asp Thr Cys Phe Arg Met Gin He Asp His 20 25 30
Ala He Asp He He Cys Gly Phe Leu Lys Glu .Arg Cys Phe Arg Gly 35 40 45
Ser Ser Tyr Pro Val Cys Val Ser Lys Val Val Lys Gly Gly Ser Ser 50 55 60
Gly Lys Gly Thr Thr Leu Arg Gly Arg Ser Asp Ala Asp Leu Val Val 65 70 75 80
Phe Leu Ser Pro Leu Thr Thr Phe Gin Asp Gin lieu Asn Arg Arg Gly 85 90 95
Glu Phe Thr Gin Glu He Arg Arg Gin Leu Glu .Ala Cys Gin .Arg Glu 100 105 110
Arg Ala Leu Ser Val Lys Phe Glu Val Gin Ala Pro Arg Trp Gly Asn 115 120 125
Pro Arg Ala Leu Ser Phe Val Leu Ser Ser Leu Gin Leu Gly Glu Gly 130 135 140
Val Glu Phe Asp Val Leu Pro Ala Phe Asp Ala Leu Gly Gin Leu Thr 145 150 155 160
Gly Ser Tyr Lys Pro Asn Pro Gin He Tyr Val Lys Leu He Glu Glu -165 170 175
Cys Thr Asp Leu Gin Lys Glu Gly Glu Phe Ser Thr Cys Gly Thr Glu 180 185 190
Leu Gin Arg Asp Phe Leu Lys Gin Arg Pro Thr Lys Leu Lys Ser Leu 195 200 205
He Arg Leu Val Lys His Trp Thr Gin Asn Cys Lys Lys Lys Leu Gly 210 215 220
Lys Leu Pro Pro Gin Tyr Ala Leu Glu Leu Leu Thr Val Tyr Ala Trp 225 230 235 240
Glu Arg Gly Ser Met Lys Thr His Phe Asn Thr Ala Gin Gly Phe Arg 245 250 255
Thr Val Leu Glu Leu Val He Asn Tyr Gin Gin Leu Cys He Tyr Trp 260 265 270
He Lys Tyr Tyr Asp Phe Lys Asn Pro He He Glu Lys Tyr Leu Arg 275 280 285
Arg Gin Leu Thr Lys Pro Arg Pro Val He Leu Lys Pro Ala Asp Pro 290 295 300
Thr Gly Asn Leu Gly Gly Gly Asp Pro Lys Gly Trp Arg Gin Leu Ala 305 310 315 320
Gin Glu Ala Glu Ala Trp Leu .Asn Tyr Pro Cys Phe Lys Asn Trp Asp 325 * 330 335
Gly Ser Pro Val Ser Ser Trp He Leu Leu Glu Ser Asn Ser Thr 340 345 350
Asp Asp Glu Thr Asp Asp Pro Arg Thr Tyr Gin Lys Tyr Gly Tyr He 355 360 365
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 Glu Glu Asp Trp Thr Cys Thr He Leu 385 390 395 400
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

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 antiviral activity for conferring to the transgenic plant immunity or resistance against viral infection.
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 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.
5. 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 2-5A-synthetase.
6. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to 2-5A-dependent RNase.
7. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to 2-5A 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 2-5A-dependent 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.
10. 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 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.
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 an 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 i Table 2.
12. A transgenic plant of claim 9, sai nucleotide sequence further encoding a third amin acid sequence, said third amino acid sequence havin activity similar or identical to PKR.
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 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.
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 2-5A-synthetase, 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.
15.-" A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to 2-5A synthetase, said nucleotide sequence further encoding a second amino acid sequence, said amino acid sequence having activity similar or identical to PKR.
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 2-5A-dependent RNase, said nucleotide sequence further encoding a second amino acid sequence, said amino acid sequence having activity similar or identical to PKR.
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 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 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.
20. A transgenic plant of claim 1, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.
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 transgenic 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.
25. 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 plant .
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 transgenic 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.
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.
30. 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 transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.
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.
35. 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-synthetase.
36. A transgenic tobacco plant of claim 35, 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.
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.
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 2-5A-dependent RNase, said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A synthetase, and said third introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR.
40.-" 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.
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 nucleotide 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.
45. 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 transgenic 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.
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.
50. 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 2-5A-dependent RNase, and said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A 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.
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 2-5A-synthetase.
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.
55. A transgenic plant of claim 54, said second nucreotide 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.
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.
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 transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.
60. 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.
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.
63Ϊ " 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 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.
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.
65. 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.
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 2-5A-dependent 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 RN binding domain of PKR.
70. 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.
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.
75. 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.
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 designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, q-1028 and 1-884 in Table 2.
80. 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.
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.
85. 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.
89. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to PKR.
90. 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 p.AM943: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.
95. A tobacco plant of claim 94, said plant transformation vector being plasmid pAM943:PK68.
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.
97. A plant transformation vector whic comprises a nucleotide sequence which encodes a amino acid sequence having activity similar o identical to 2-5A synthetase.
98. A plant transformation vector of claim 97, said nucleotide sequence includes nucleotide designated as 1-1650 in FIG. 20 or any part of thi nucleotide sequence which contains the complete o partial coding sequence for 2-5A-synthetase or th double stranded RNA binding domain of 2-5A-synthetase.
99. A plant transformation vector of claim 97, said vector being plasmid pAM943:2-5A synthetase.
100. A plant cell containing said plan transformation vector of claim 97.
101. A plant cell of claim 100, said plant cel being a tobacco plant cell.
102. A plant cell of claim 100, said plant cel being selected from the group consisting o vegetable, fruit, grain, tree, flower, grass, wee and shrub plant cells.
103. A tobacco plant comprising said tobac plant cell of claim 101.
104. A tobacco plant of claim 94, said pla transformation vector being plasmid pAM943:synthetas
105. A bacterial cell containing said pla transformation vector of claim 97.
106. A bacterial cell of claim 105, sai bacterial cell being an Argobacterium tumefacie bacterial cell.
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.
112. A plant cell of claim 108, said plant cel being selected from the group consisting o vegetable, fruit, grain tree, flower, grass, weed an shrub plant cells.
113. A bacterial cell containing said plan transformation vector and said second plan transformation vector of claim 107.
114. A bacterial cell of claim 113, sai bacterial cell being an Argobacterium tumefacien bacterial cell.
115. A bacterial cell containing said plan transformation vector, said second plan transformation vector and said third plan transformation vector of claim 108.
116. A bacterial cell of claim 114, sai bacterial cell being an Argobacterium tumefacien bacterial cell.
117. A transgenic plant comprising said tobacc plant cell of claim 109.
118. A transgenic plant comprising said tobacco plant cell of claim 110.
119. A transgenic plant comprising said plant cell of claim 31.
120. A transgenic plant comprising said plant cell of claim 109.
121. A transgenic plant comprising said plant cell of claim 110.
122. A transgenic plant comprising said plant cell of claim 111.
123. A transgenic plant comprising said plant cell of claim 112.
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 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.
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 2-5A synthetase.
126. A method of claim 125, 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 2-5A synthetase.
127. 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.
128. 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 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.
129. A method of claim 128, 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 2-5A synthetase.
130. 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.
131. A method of claim 124 in which the plant is a tobacco plant.
132. A method of claim 125 in which the plant is a tobacco plant.
133. A method of claim 126 in which the plant is a tobacco plant.
134. A method of claim 127 in which the plant is a tobacco plant.
135. A method of claim 128 in which the plant is a tobacco plant.
136. A method of claim 129 in which the plant is a tobacco plant.
137. A method of claim 130 in which the plant is a tobacco plant.
138. 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.
1.39-." 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.
140. 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.
141. 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.
142. 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.
143. 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.
144. A method of claim 130 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.
145. 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 nucleotide sequence which encodes for an amino acid sequence having the ability to inhibit or interfere with viral replication; 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.
146. A method of .claim 145, the amino acid sequence having activity similar or identical to 2-5A-dependent RNase.
147. A method of claim 145, the amino acid sequence having activity similar or identical to 2-5A-synthetase.
148. A method of claim 145, the amino acid sequence having activity similar or identical to PKR.
149. 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.
150. A plant transformation vector which comprises said nucleotide sequence of claim 149.
151. A plant transformation vector of claim 150, said plant transformation vector being plasmid p.AM943:2-5A-dep. RNase antisense.
152. A plant transformation vector of claim 150, said" plant transformation vector being plasmid pAM822:2-5A-dep. RNase antisense.
153. A construct which comprises said nucleotide sequence of claim 149, said construct being the construct as described in FIG. 13 D/a.
154. A construct which comprises said nucleotide sequence of claim 149, said construct being the construct as described in FIG. 13E.
155. A plant cell containing said plant transformation vector of claim 150.
156. A plant cell of claim 155, said plant cell being a tobacco plant cell.
157. A plant cell of claim 155, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells.
158. A bacterial cell containing said plant transformation vector of claim 150.
159. A bacterial cell of claim 158, said bacterial cell being an Argobacterium tumefaciens bacterial cell.
160. A transgenic plant of claim 149, said transgenic plant being a tobacco plant.
161. A transgenic plant of claim 149, said transgenic plant being selected from a group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.
162. 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 AGG GAT CAT AAC AAC CCC CAG GAG GGA CCC ACG TCC 45 TCC AGC GGT AGA AGG GCT GCA GTG GAA GAC AAT CAC TTG CTG ATT 90 AAA GCT GTT CAA AAC GAA GAT GTT GAC CTG GTC CAG CAA TTG CTG 135 GAA GGT GGA GCC AAT GTT AAT TTC CAG GAA GAG GAA GGG GGC TGG 180 ACA CCT CTG CAT AAC GCA GTA CAA ATG AGC AGG GAG GAC ATT GTG 225 GAA CTT CTG CTT CGT CAT GGT GCT GAC CCT GTT CTG AGG AAG AAG 270 AAT GGG GCC ACG CCT TTT ATC CTC GCA GCG ATT GCG GGG AGC GTG 315 AAG CTG CTG AAA CTT TTC CTT TCT AAA GGA GCA GAT GTC AAT GAG 360 TGT GAT TTT TAT GGC TTC ACA GCC TTC ATG GAA GCC GCT GTG TAT 405 GGT AAG GTC AAA GCC CTA AAA TTC CTT TAT AAG AGA GGA GCA AAT 450 GTG AAT TTG AGG CGA AAG ACA AAG GAG GAT CAA GAG CGG CTG AGG 495 AAA GGA GGG GCC ACA GCT CTC ATG GAC GCT GCT GAA AAA GGA CAC 540 GTA GAG GTC TTG AAG ATT CTC CTT GAT GAG ATG GGG GCA GAT GTA 585 AAC GCC TGT GAC AAT ATG GGC AGA AAT GCC TTG ATC CAT GCT CTC 630 CTG AGC TCT GAC GAT AGT GAT GTG GAG GCT ATT ACG CAT CTG CTG 675 CTG -GAC CAT GGG GCT GAT GTC AAT GTG AGG GGA GAA AGA GGG AAG 720 ACT CCC CTG ATC CTG GCA GTG GAG AAG AAG CAC TTG GGT TTG GTG 765 CAG AGG CTT CTG GAG CAA GAG CAC ATA GAG ATT AAT GAC ACA GAC 810 AGT GAT GGC AAA ACA GCA CTG CTG CTT GCT GTT GAA CTC AAA CTG 855 AAG AAA ATC GCC GAG TTG CTG TGC AAA CGT GGA GCC AGT ACA GAT 900 TGT GGG GAT CTT GTT ATG ACA GCG AGG CGG AAT TAT GAC CAT TCC 945 CTT GTG AAG GTT CTT CTC TCT CAT GGA GCC AAA GAA GAT TTT CAC 990 CCT CCT GCT GAA GAC TGG AAG CCT CAG AGC TCA CAC TGG GGG GCA 1035 GCC CTG AAG GAT CTC CAC AGA ATA TAC CGC CCT ATG ATT GGC AAA 1080 CTC AAG TTC TTT ATT GAT GAA AAA TAC AAA ATT GCT GAT ACT TCA 1125 GAA GGA GGC ATC TAC CTG GGG TTC TAT GAG AAG CAA GAA GTA GCT 1170 GTG AAG ACG TTC TGT GAG GGC AGC CCA CGT GCA CAG CGG GAA GTC 1215 TCT TGT CTG CAA AGC AGC CGA GAG AAC AGT CAC TTG GTG ACA TTC 1260 TAT GGG AGT GAG AGC CAC AGG GGC CAC TTG TTT GTG TGT GTC ACC 1305 CTC TGT GAG CAG ACT CTG GAA GCG TGT TTG GAT GTG CAC AGA GGG 1350 GAA GAT GTG GAA AAT GAG GAA GAT GAA TTT GCC CGA AAT GTC CTG 1395 TCA TCT ATA TTT AAG GCT GTT CAA GAA CTA CAC TTG TCC TGT GGA 1440 TAC ACC CAC CAG GAT CTG CAA CCA CAA AAC ATC TTA ATA GAT TCT 1485 AAG AAA GCT GCT CAC CTG GCA GAT TTT GAT AAG AGC ATC AAG TGG 1530 GCT GGA GAT CCA CAG GAA GTC AAG AGA GAT CTA GAG GAC CTT GGA 1575 CGG CTG GTC CTC TAT GTG GTA AAG AAG GGA AGC ATC TCA TTT GAG 1620 GAT CTG AAA GCT CAA AGT AAT GAA GAG GTG GTT CAA CTT TCT CCA 1665 GAT GAG GAA ACT AAG GAC CTC ATT CAT CGT CTC TTC CAT CCT GGG 1710 GAA CAT GTG AGG GAC TGT CTG AGT GAC CTG CTG GGT CAT CCC TTC 1755 TTT TGG ACT TGG GAG AGC CGC TAT AGG ACG CTT CGG AAT GTG GGA 1800 AAT GAA TCC GAC ATC AAA ACA CGA AAA TCT GAA AGT GAG ATC CTC 1845 AGA CTA CTG CAA CCT GGG CCT TCT GAA CAT TCC AAA AGT TTT GAC 1890 AAG TGG ACG ACT AAG ATT AAT GAA TGT GTT ATG AAA AAA ATG AAT 1935 AAG TTT TAT GAA AAA AGA GGC AAT TTC TAC CAG AAC ACT GTG GGT 1980 GAT CTG CTA AAG TTC ATC CGG AAT TTG GGA GAA CAC AΪT GAT GAA 2025 GAA AAG CAT AAA AAG ATG AAA TTA AAA ATT GGA GAC CCT TCC CTG 2070 TAT TTT CAG .AAG ACA TTT CCA GAT CTG GTG ATC TAT GTC TAC ACA 2115 AAA CTA CAG AAC ACA GAA TAT AGA AAG CAT TTC CCC CAA ACC CAC 2160 AGT CCA AAC AAA CCT CAG TGT GAT GGA GCT GGT GGG GCC AGT GGG 2205 TTG GCC AGC CCT GGG TGC 2223
163. .An amino acid sequence having human 2-5A- dependent RNAse activity, or an active fragment or analog thereof, said amino acid sequence being identified as SEQ ID NO:4: and comprising:
Met Glu Ser Arg Asp His Asn Asn Pro Gin Glu Gly Pro Thr Ser 15 Ser Ser Gly Arg Arg Ala Ala Val Glu Asp Asn His Leu Leu He 30 Lys Ala Val Gin Asn Glu Asp Val Asp Leu Val Gin Gin Leu Leu 45 Glu Gly Gly Ala Asn Val Asn Phe Gin Glu Glu Glu Gly Gly Trp 60 Thr Pro Leu His Asn Ala Val Gin Met Ser Arg Glu Asp He Val 75 Glu Leu Leu Leu Arg His Gly Ala Asp Pro Val Leu Arg Lys Lys 90 Asn Gly Ala Thr Pro Phe He Leu Ala Ala He Ala Gly Ser Val 105 Lys Leu Leu Lys Leu Phe Leu Ser Lys Gly Ala Asp Val Asn Glu 120 Cys Asp Phe Tyr Gly Phe Thr Ala Phe Met Glu Ala Ala Val Tyr 135 Gly Lys Val Lys Ala Leu Lys Phe Leu Tyr Lys Arg Gly Ala Asn 150 Val Asn Leu Arg Arg Lys Thr Lys Glu Asp Gin Glu Arg Leu Arg 165 Lys Gly Gly Ala Thr Ala Leu Met Asp Ala Ala Glu Lys Gly His 180 Val Glu Val Leu Lys He Leu Leu Asp Glu Met Gly Ala Asp Val 195 Asn Ala Cys Asp Asn Met Gly Arg Asn Ala Leu He His Ala Leu 210 Leu Ser Ser Asp Asp Ser Asp Val Glu Ala He Thr His Leu Leu 225 Leu Asp His Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys 240 Thr Pro Leu He Leu Ala Val Glu Lys Lys His Leu Gly Leu Val 255 Gin Arg Leu Leu Glu Gin Glu His He Glu He Asn Asp Thr Asp 270 Ser Asp Gly Lys Thr Ala Leu Leu Leu Ala Val Glu Leu Lys Leu 285 Lys Lys He Ala Glu Leu Leu Cys Lys Arg Gly Ala Ser Thr Asp 300 Cys Gly Asp Leu Val Met Thr Ala Arg Arg Asn Tyr Asp His Ser 315 Leu Val Lys Val Leu Leu Ser His Gly Ala Lys Glu Asp Phe His 330 Pro Pro Ala Glu Asp Trp Lys Pro Gin Ser Ser His Trp Gly Ala 345 Ala Leu Lys Asp Leu His Arg He Tyr Arg Pro Met He Gly Lys 360 Leu Lys Phe Phe He Asp Glu Lys Tyr Lys He Ala Asp Thr Ser 375 Glu Gly Gly He Tyr Leu Gly Phe Tyr Glu Lys Gin Glu Val Ala 390 Val Lys Thr Phe Cys Glu Gly Ser Pro Arg Ala Gin Arg Glu Val 405 Ser Cys Leu Gin Ser Ser Arg Glu Asn Ser His Leu Val Thr Phe 420 Tyr Gly Ser Glu Ser His Arg Gly His Leu Phe Val Cys Val Thr 435 Leu Cys Glu Gin Thr Leu Glu Ala Cys Leu Asp Val His Arg Gly 450 Glu Asp Val Glu Asn Glu Glu Asp Glu Phe Ala Arg Asn Val Leu 465 Ser Ser He Phe Lys Ala Val Gin Glu Leu His Leu Ser Cys Gly 480 Tyr Thr His Gin Asp Leu Gin Pro Gin Asn He Leu He Asp Ser 495 Lys Lys Ala Ala His Leu Ala Asp Phe Asp Lys Ser He Lys Trp 510 Ala Gly Asp Pro Gin Glu Val Lys Arg Asp Leu Glu Asp Leu Gly 525 Arg Leu Val Leu Tyr Val Val Lys Lys Gly Ser He Ser Phe Glu 540 Asp Leu Lys Ala Gin Ser Asn Glu Glu Val Val Gin Leu Ser Pro 555 Asp Glu Glu Thr Lys Asp Leu He His Arg Leu Phe His Pro Gly 570 Glu His Val Arg Asp Cys Leu Ser Asp Leu Leu Gly His Pro Phe 585 Phe Trp Thr Trp Glu Ser Arg Tyr Arg Thr Leu Arg Asn Val Gly 600 Asn Glu Ser Asp He Lys Thr Arg Lys Ser Glu Ser Glu He Leu 615 Arg Leu Leu Gin Pro Gly Pro Ser Glu His Ser Lys Ser Phe Asp 630 Lys Trp Thr Thr Lys He Asn Glu Cys Val Met Lys Lys Met Asn 645 Lys Phe Tyr Glu Lys Arg Gly Asn Phe Tyr Gin Asn Thr Val Gly 660 Asp Leu Leu Lys Phe He Arg Asn Leu Gly Glu His He Asp Glu 675 Glu Lys His Lys Lys Met Lys Leu Lys He Gly Asp Pro Ser Leu 690 Tyr Phe Gin Lys Thr Phe Pro Asp Leu Val He Tyr Val Tyr Thr 705 Lys Leu Gin Asn Thr Glu Tyr Arg Lys His Phe Pro Gin Thr His 720 Ser Pro Asn Lys Pro Gin Cys Asp Gly Ala Gly Gly Ala Ser Gly 735 Leu Ala Ser Pro Gly Cys 741
EP95911802A 1994-02-18 1995-02-16 Antiviral transgenic plants, vectors, cells and methods Withdrawn EP0753992A4 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US19897394A 1994-02-18 1994-02-18
PCT/US1995/002058 WO1995022245A1 (en) 1994-02-18 1995-02-16 Antiviral transgenic plants, vectors, cells and methods
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UA95446C2 (en) 2005-05-04 2011-08-10 Іллюміджен Байосайєнсіз, Інк. Mutations in oas1 genes
CN114591978B (en) * 2021-09-30 2023-07-18 湖南大学 Application of OsFLR14 gene in improving weed resistance of rice

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