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WO1997047745A1 - Proteines vegetales associees au retinoblastome - Google Patents

Proteines vegetales associees au retinoblastome Download PDF

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Publication number
WO1997047745A1
WO1997047745A1 PCT/EP1997/003070 EP9703070W WO9747745A1 WO 1997047745 A1 WO1997047745 A1 WO 1997047745A1 EP 9703070 W EP9703070 W EP 9703070W WO 9747745 A1 WO9747745 A1 WO 9747745A1
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WIPO (PCT)
Prior art keywords
protein
plant
nucleic acid
dna
cell
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Ceased
Application number
PCT/EP1997/003070
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English (en)
Inventor
Crisanto Gutierrez-Armenta
Qi Xie
Andrés PELAYO SANZ-BURGOS
Paula Suarez Lopez
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Consejo Superior de Investigaciones Cientificas CSIC
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Consejo Superior de Investigaciones Cientificas CSIC
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Priority to NZ333100A priority Critical patent/NZ333100A/xx
Priority to JP10501212A priority patent/JP2001502522A/ja
Priority to AU32579/97A priority patent/AU721332B2/en
Priority to BR9710848-0A priority patent/BR9710848A/pt
Priority to EP97928187A priority patent/EP0914436A1/fr
Publication of WO1997047745A1 publication Critical patent/WO1997047745A1/fr
Anticipated expiration legal-status Critical
Priority to US09/213,293 priority patent/US6384299B1/en
Ceased 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates the proteins having biological activity in plant and animal systems, to polynucleotides encoding for the expression of such proteins, to oligonucleotides for use in identifying and synthesizing these proteins and polynucleotides, to vectors and cells containing the polynucleotides in recombinant form and to plants and animals comprising these, and to the use of the proteins and polynucleotides and fragments thereof in the control of plant growth and plant vulnerability to viruses.
  • Rb retinoblastoma susceptibility gene
  • DNA tumor viruses that infect animal cells express oncoproteins that interact with the Rb protein via a LXCXE motif, disrupting Rb-E2F complexes and driving cells into S-phase (Weinberg ibid; Ludlow, J. W FASEB J. 7, 866 (1993); Moran, E. FASEB J. 7, 880 (1993); Vousden, K. FASEB J. 7, 872 (1993)).
  • the present inventors have shown that efficient replication of a plant geminivirus requires the integrity of an LXCXE amino acid motif in the viral RepA protein and that RepA can interact with members of the human Rb family in yeast (Xie, Q., Suarez-L ⁇ pez, P. and Gutierrez, C. EMBO J. 14, 4073 (1995).
  • the presence of the LXCXE motif ni plant D-type cyclins has also been reported (Soni, R., Carmichael, J. P., Shah, Z. H. and Murray, J. A. H. Plant Cell 7, 85-103 (1995)).
  • the present inventors have now identified characteristic sequences of plant Rb proteins and corresponding encoding polynucleotides for the first time, isolated such a protein and polynucleotide, and particularly have identified sequences that distinguish it from known animal Rb protein sequences.
  • the inventors have determined that a known DNA sequence from the maize encoding a vegetable Rb plant protein and is hereinafter called ZmRb1.
  • ZmRb1 has been demonstrated by the inventors to interact in yeasts with RepA, a plant geminivirus protein containing LXCXE motif essential for its function.
  • the inventors have further determined that geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding either ZmRb1 or human pl30, a member of the human Rb family.
  • plant and animal cells may share fundamentally similar strategies for growth control, and thus human as well as plant Rb protein such as ZmRb1 will be expected to have utility in, in ter alia , plant therapeutics, diagnostics, growth control or investigations and many such plant proteins will have similar utility in animals.
  • retinoblastoma protein in controlling the growth of plant cells and/or plant viruses.
  • the present invention provides control of viral infection and/or growth in plant cells wherein the virus requires the integrity of an LXCXE amino acid motif in one of its proteins, particularly, e. g., in the viral RepA protein, for normal reproduction.
  • Particular plant viruses so controlled are Geminiviruses.
  • a preferred method of control using such proteins involves applying these to the plant cell, either directly or by introduction of DNA or RNA encoding for their expression into the plant cell which it is desired to treat.
  • anti-sense DNA or RNA in plant cells in vectors form that contain the necessary promoters for the DNA or RNA transcription, it will be possible to exploit the well known anti-sense mechanism in order to inhibit the expression of the Rb protein, and thus the S-phase.
  • Such plants will be of use, among other aspects to replicate DNA or RNA until high levels, e.g. in yeasts.
  • the methods to introduce anti -sense DNA in cells are very well known for those skilled in the art: see for example "Principles of gene manipulation - An introduction to Genetic Engineering (1994) R.W. Old & S.B. Primrose; Oxford-Blackwell Scientific Publications Fifth Edition p398.
  • nucleic acid in a second aspect of the present invention there is provided recombinant nucleic acid, particularly in the form of DNA or cRNA (mRNA) , encoding for expression of Rb protein that is characteristic of plants.
  • This nucleic acid is characterised by one or more characteristic regions that differ from known animal Rb protein nucleic acid and is exemplified herein by SEQ ID No 1, bases 31- 2079.
  • the DNA or RNA can have a sequence that contains the degenerated substitution in the nucleotides of the codons in SEQ ID No. 1, and in where the RNA the T is U.
  • the most preferred DNA or RNA are capable of hybridate with the polynucleotide of the SEQ ID No. 1 in conditions of low stringency, preferably being the hybridization produced in conditions of high stringency.
  • condition of low stringency and “conditions of high stringency” are understood by those skilled, but are conveniently exemplified in US 5202257, Col-9-Col 10. If some modifications were made to lead to the expression of a protein with different amino acids, preferably of the same kind of the corresponding amino acids to the SEQ ID No 1; that is, are conservative substitutions. Such substitutions are known by those skilled, for example, see US 5380712, and it is only contemplated when the protein has activity with retinoblastoma protein.
  • Preferred DNA or cRNA encodes for a plant Rb protein having A and B pocket sub-domains having between 30% and 75% homology with human Rb protein, particularly as compared with p130, more preferably from 50% to 64% homology.
  • the plant Rb protein so encoded has the C706 amino acid of human Rb conserved.
  • the spacer sequence between the A and B pockets is not conserved with respect to animal Rb proteins, preferably being less than 50% homologous to the same region as found in such animal proteins
  • the protein so encoded has 80% or more homology with that of SEQ NO 2 of the sequence listing attached hereto, still more preferably 90% or more and most preferably 95% or more.
  • a third aspect of the present invention there is provided the protein expressed by the recombinant DNA or RNA of the second aspect, novel proteins derived from such DNA or RNA, and protein derived from naturally occurring DNA or RNA by mutagenic means such as use of mutagenic PCR primers.
  • vectors, cells and plants and animals comprising the recombinant DNA or
  • RNA of correct sense or anti-sense, of the invention RNA of correct sense or anti-sense, of the invention
  • a method of controlling cell or viral growth comprising administering the DNA, RNA or protein of the second or third aspects to the cell
  • administration may be direct in the case of proteins or may involve indirect means, such as electroporation of plant seed cells with DNA or by transformation of cells with expression vectors capable of expressing or over expressing the proteins of the invention or fragments thereof that are capable of inhibiting cell or viral growth
  • the method uses an expression vector capable of producing anti-sense RNA of the cDNA of the invention
  • Another one of the specific characteristics of the plants protein and of the nucleic acids includes a N- terminal domain corresponding in sequence to the amino acids 1 to 90 of the SEQ ID No 2 and a nucleotides sequence corresponding to the basis 31 to 300 of the SEQ ID No 1 These sequences are characterized by possessing less than 150 and less than 450 units that the animal sequences which possess more than 300 amino acids and 900 pairs of more bases.
  • Fig 1 The sub-figure a shows the relative lengths of the present ZmRb1 protein and the human retinoblastoma proteins
  • the sub-figure B shows the alignment of the amino acids sequences of the Pocket A and Pocket B of the ZmRb1 with that of the Xenopus, chicken, rat and three human protein (Rb, p107 and p130).
  • Fig. 2 This figure is a map of the main characteristics of the WDV virus and the pWori vector derived from WDV and the positions of the deletions and mutations used in order to establish that the LXCXE motif is required for its replication in plants cells.
  • oligonucleotides designed to be complementary to a known EST sequence of homologue maize of pl30. These oligonucleotides were 5'-AATAGACACATCGATCAA/G (M.5m, nt positions 1411-1438) and 5'-GTAATGATACCAACATGG (M.3c, nt positions 1606-1590) (Isogen Biosciences).
  • pBluescript SK- (pBS) phagemids from positive clones were isolated by in vivo excision with ExAssist helper phage (Stratagene) according to protocols recommended by the manufacturer. DNA sequencing was carried out using a SequenaseTM Kit (USB).
  • the 5'-end of the mRNAs encoding p75ZmRb1 was determined by RACE-PCR.
  • Poly-A+mRNA was purified by chromatography on oligo-dT-cellulose (Amersham).
  • the first strand was synthesized using oligonucleotide DraI35 (5'-GATTTAAAATCAAGCTCC, nt positions 113-96). After denaturation at 90°C for 3 min, RNA was eliminated by RNase treatment, the cDNA recovered and 5'-tailed with terminal transferase and dATP. Then a PCR fragment was amplified using primer DraI35 and the linker-primer (50 bp) of the Stratagene cDNA synthesis kit.
  • One of the positive clones so produced contained a -4 kb insert that, according to restriction analysis, extended both 5' and 3' of the region contained in the Expressed Sequence Tag used.
  • the nucleotide sequence corresponding to the longest cDNA insert (3747 bp) is shown in SEQ ID No. 1.
  • This ZmRb1 cDNA contains a single open reading frame capable of encoding a protein of 683 amino acids (predicted Mr 75247, p75ZmRb1) followed by a 1646 bp 3'-untranslated region. Untranslated regions of similar length have been also found in mammalian Rb cDNAs (Lee, W.-L. et al, Science 235, 1394 (1987); Bernards, R.
  • Plasmid pWori ⁇ was constructed by deleting in pWori most of the sequences encoding WDV proteins (Sanz and Gutierrez, unpublished).
  • Plasmid p35S . Rb1 was constructed by inserting the CaMV 35S promoter (obtained from pWDV3 :35SGUS) upstream of the ZmRb1 cDNA in the pBS vector.
  • Plasmid p35S.130 was constructed by introducing the complete coding sequence of human pl30 instead of ZmRb1 sequences into p35S.Rb1.
  • Plasmid p35.A+B was constructed by substituting sequences encoding the WDV RepA and RepB ORFs instead of ZmRb1 in p35S.Rb1 plasmid. (See Soni, R. and Murray, J. A. H. Anal. Biochem. 218, 474-476 (1994)).
  • sequence around the methionine codon at nucleotide position 31 contains a consensus translation start
  • the ZmRb1 protein contains segments homologous to the A and B subdomains of the "pocket" that is present in all members of the Rb family. These subdomains are separated by a non-conserved spacer. ZmRb1 also contains non- conserved N-terminal and C- terminal domains. Overall, ZmRb1 shares ⁇ 28-30% amino acid identity (-50% similarity) with the Rb family members (Hannon, G. J., Demetrick, D. & Beach, D. Genes Dev. 7, 2378 (1993); Cobrinik, D., Whyte, P., Peeper, D.S., Jacks, T. & Weinberg, R. A. ibid., p. 2392 (1993).
  • the 561-577 amino acids encompass a proline-rich domain.
  • ZmRb1 contains 16 consensus sites, SP or TP for phosphorilation by cyclins dependant kinases (CDKs) with one of the 5'-tail of the sub-domain A and several in the C-terminal area which are potential sites of phosphorilation.
  • CDKs cyclins dependant kinases
  • a nucleic acid preferred group which encodes proteins in which one or more of these sites are changed or deleted, making the protein more resistant to the phosphorilation and thus, to its functionality, for example linking to E2F or similar This can be easily carried out by means of mutagenesis conducted by means of PCR.
  • the E198K mutant of WDV RepA behaves similarly to analogous point mutants of animal virus oncoproteins (Moran, E , Zerler, B , Harrison, T M and Mathews, M B Mol Cell Biol. 6, 3470 (1986), Cherington, V et al ibid , p 1380 (1988), Lillie, J W , Lowenstein, P M , Green, M R and Green, M Cell 50, 1091 (1987); DeCarpio, J. A. et al., ibid., p. 275 (1988)).
  • PCNA proliferating cell nuclear antigen
  • the Rb family has been implicated in tumor suppression and in the control of differentiation and development.
  • p75ZmRb1 could also play key regulatory roles at other levels during the plant cell life.
  • the inventors have noted that the V ⁇ rB4 protein encoded by the Ti plasmids of both Agrobacteri uw tumefaciens and A . rhyzogenes contains an LXCXE motif. Although the V ⁇ rB4 protein is required for tumor induction (Hooykas, P. J. J and Beijersbergen, A G. M.
  • WDV wheat dwarf geminivirus
  • Plasmid DNA represents exclusively newly-replicated plasmid DNA since it is fully resistant to Dpnl digestion and sensitive to Mbol. Note that the Mbol-digested samples were run for about half of the length than the undigested samples.
  • test plasmid pWori ⁇ (which does not encode functional WDV replication proteins but replicates when they are provided by a different plasmid, i. e.
  • pWori pWori ⁇
  • pWori ⁇ pWori ⁇
  • p35S.A+B 6 g
  • p35S.Rb1 10 g
  • p35S.130 10 g
  • Replication of the test plasmid (pWori ⁇ ) was analyzed 36 hours after transfection and was detected as described in part A using ethidium bromide staining; Southern hybridization.

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Abstract

La présente invention est fondée sur l'isolation et la caractérisation d'une séquence d'ADN de cellules végétales codant une protéine de rétinoblastome. Une telle découverte se base sur les propriétés structurelles et fonctionnelles de la protéine de rétinoblastome végétale comme un régulateur possible du cycle cellulaire, de la croissance cellulaire et de la différenciation cellulaire de la plante. Pour cette raison, on revendique, en autres, l'utilisation de la protéine de rétinoblastome, ou de la séquence d'ADN qui la code, dans la régulation de la croissance de cellules végétales, de plantes et/ou de virus végétaux, ainsi que l'utilisation de vecteurs, cellules, plantes ou animaux, ou cellules animales, présentant une modification obtenue par la manipulation de la voie de régulation basée sur la protéine de rétinoblastome végétale.
PCT/EP1997/003070 1996-06-13 1997-06-12 Proteines vegetales associees au retinoblastome Ceased WO1997047745A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
NZ333100A NZ333100A (en) 1996-06-13 1997-06-12 Plant retinoblastoma-associated proteins and their use in modulating the cell cycle of a plant
JP10501212A JP2001502522A (ja) 1996-06-13 1997-06-12 網膜芽細胞腫関連植物蛋白
AU32579/97A AU721332B2 (en) 1996-06-13 1997-06-12 Plant retinoblastoma-associated proteins
BR9710848-0A BR9710848A (pt) 1996-06-13 1997-06-12 Proteìnas de retinoblastoma associado vegetal.
EP97928187A EP0914436A1 (fr) 1996-06-13 1997-06-12 Proteines vegetales associees au retinoblastome
US09/213,293 US6384299B1 (en) 1996-06-13 1998-12-14 Plant retinoblastoma-associated gene

Applications Claiming Priority (3)

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PCT/ES1996/000130 WO1997047647A1 (fr) 1996-06-13 1996-06-13 Proteines vegetales
CA002257972A CA2257972A1 (fr) 1996-06-13 1996-06-13 Proteines vegetales
ESPCT/ES96/00130 1996-06-13

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US09/213,293 Continuation US6384299B1 (en) 1996-06-13 1998-12-14 Plant retinoblastoma-associated gene

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WO1997047745A1 true WO1997047745A1 (fr) 1997-12-18

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PCT/EP1997/003070 Ceased WO1997047745A1 (fr) 1996-06-13 1997-06-12 Proteines vegetales associees au retinoblastome

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EP (1) EP0914436A1 (fr)
CN (1) CN1227605A (fr)
AU (1) AU721332B2 (fr)
CA (2) CA2257972A1 (fr)
WO (2) WO1997047647A1 (fr)
ZA (1) ZA975202B (fr)

Cited By (14)

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Publication number Priority date Publication date Assignee Title
WO1998042851A1 (fr) * 1997-03-26 1998-10-01 Cambridge University Technical Services Ltd. Plantes presentant une croissance modifiee
WO1998056811A3 (fr) * 1997-06-12 1999-03-04 Consejo Superior Investigacion Proteines vegetales grab
WO1999013083A3 (fr) * 1997-09-05 1999-05-06 Cropdesign Nv Methode et dispositif de modulation de proteines de cycle cellulaire vegetal et leur utilisation dans la regulation de la croissance de cellules vegetales
WO1999058681A3 (fr) * 1998-05-08 1999-12-29 Consejo Superior Investigacion Cellules de plantes transgeniques exprimant un peptide e2f vegetal recombinant
WO1999066055A3 (fr) * 1998-06-15 2000-03-02 Cropdesign Nv Sequences de regulation inductibles par des pathogenes de vegetaux, liees de maniere operationnelle a des genes du cycle cellulaire, et utilisation desdites sequences
WO1999053075A3 (fr) * 1998-04-09 2000-03-23 Du Pont Proteines de regulation du cycle cellulaire cdc-16, dp-1, dp-2 et e2f tirees de plantes
WO2000053784A1 (fr) * 1999-03-12 2000-09-14 Pioneer Hi-Bred International, Inc. Methodes d'utilisation d'une replicase virale
WO2000050614A3 (fr) * 1999-02-25 2000-12-14 Pioneer Hi Bred Int Procedes d'utilisation de polynucleotides et de polypeptides de replicase virale
US6696560B1 (en) 1999-03-19 2004-02-24 The United States Of America As Represented By The United States Department Of Energy Retinoblastoma-like RRB gene of arabidopsis thaliana
WO2004016775A3 (fr) * 2002-08-14 2004-05-06 Cropdesign Nv Vegetaux a croissance modifiee et leur methode d'elaboration
US7888460B2 (en) 1999-10-01 2011-02-15 Isis Innovation Limited Diagnostic and therapeutic epitope, and transgenic plant
US9017690B2 (en) 2004-04-28 2015-04-28 Btg International Limited Epitopes related to coeliac disease
US10053497B2 (en) 2002-06-05 2018-08-21 Oxford University Innovation Limited Therapeutic epitopes and uses thereof
US10105437B2 (en) 2004-04-28 2018-10-23 Btg International Limited Epitopes related to coeliac disease

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
US20030167505A1 (en) * 2001-03-16 2003-09-04 Dilkes Brian R. Cell cycle nucleic acids, polypeptides and uses thereof
CN112119163A (zh) * 2018-02-16 2020-12-22 首尔大学校产学协力团 产量提高的转基因植物

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WO1992005272A1 (fr) * 1990-09-17 1992-04-02 The Regents Of The University Of California Procede et composition permettant de maitriser la proliferation cellulaire
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WO1999053069A3 (fr) * 1998-04-09 2000-03-23 Du Pont Proteines de regulation du cycle cellulaire
WO1999058681A3 (fr) * 1998-05-08 1999-12-29 Consejo Superior Investigacion Cellules de plantes transgeniques exprimant un peptide e2f vegetal recombinant
WO1999066055A3 (fr) * 1998-06-15 2000-03-02 Cropdesign Nv Sequences de regulation inductibles par des pathogenes de vegetaux, liees de maniere operationnelle a des genes du cycle cellulaire, et utilisation desdites sequences
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WO2000050614A3 (fr) * 1999-02-25 2000-12-14 Pioneer Hi Bred Int Procedes d'utilisation de polynucleotides et de polypeptides de replicase virale
US7309813B2 (en) 1999-02-25 2007-12-18 Pioneer Hi-Bred International, Inc. Methods of using viral replicase polynucleotides and polypeptides
WO2000053784A1 (fr) * 1999-03-12 2000-09-14 Pioneer Hi-Bred International, Inc. Methodes d'utilisation d'une replicase virale
US6770800B2 (en) 1999-03-12 2004-08-03 Pioneer Hi-Bred International, Inc. Methods of using viral replicase polynucleotides and polypeptides
US6696560B1 (en) 1999-03-19 2004-02-24 The United States Of America As Represented By The United States Department Of Energy Retinoblastoma-like RRB gene of arabidopsis thaliana
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AU721332B2 (en) 2000-06-29

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