CA2257972A1 - Plant proteins - Google Patents
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- CA2257972A1 CA2257972A1 CA002257972A CA2257972A CA2257972A1 CA 2257972 A1 CA2257972 A1 CA 2257972A1 CA 002257972 A CA002257972 A CA 002257972A CA 2257972 A CA2257972 A CA 2257972A CA 2257972 A1 CA2257972 A1 CA 2257972A1
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- 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
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- 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
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- 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
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- 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
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Abstract
The invention relates to the isolation and characterization of a DNA sequence of plant cells which codes for a retinoblastoma protein. This finding is based on the structural and functional properties of the retinoblastoma protein of plants as a possible regulator of the cell cycle, the cell growth and the cell differentiation in plants. For all this, what are claimed, inter allia, are the use of the retinoblastoma protein or the DNA sequence which codes it in the control of the growth of plant cells, plants and/or plant viruses, as well as the use of vectors, cells, plants, or animals or animal cell's mofidied through manipulation of the control route based on the retinoblastoma protein of plants.
Description
~ CA 022~7972 1998-12-11 I
F ~ L ~ r~ 7 ! r; ~ r ~ r l ~ r r r PLANT PROTEINS
DESCRIPTION
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.
Cell cycle progression is regulated by positive and negative effectors. Among the latter, the product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. In mammalian cells, Rb regulates G1/S transit by inhibiting the function of the E2F family of transcription factors, known to interact with sequences in the promoter region of genes required for cellular DNA replication (see eg Weinberg, R.A. Cell 81,323 (1995); Nevins, J.R. Science 258,424 (1992)). 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-Lopez, P. and Gutiérrez, C. EMBO J. 14, 4073 (1995). The presence of the LXCXE
motif in plant D-type cyclins has also been reported (Soni, R., Carmichael, J. P., Shah, Z. H. an~ Murray, J.
CA 022~7972 1998-12-11 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 ZmRbl. ZmRbl 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 ZmRbl or human pl30, a member of the human Rb family.
Significantly the inventors work suggests that plant and animal cells may share fundamentally similar strategies for growth control, and thus human as well as plant Rb protein such as ZmRbl will be expected to have utility in, inter alia, plant therapeutics, diagnostics, growth control or investigations and many such plant proteins will have similar utility in animals.
In a first aspect of the present invention there is provided the use of retinoblastoma protein in controlling the growth of plant cells and/or plant viruses.
Particularly, 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 CA 022~7972 1998-12-11 their expression into the plant cell which it is desired to treat. By over expressing the retinoblastoma protein, or expressing an Rb protein or peptide fragment thereof that interacts with the LXCXE motif of the virus but does not affect the normal functioning of the cell, it is possible to inhibit normal virus growth and thus also to produce infection spreading from that cell to its neighbours.
Alternatively, by means of introducing 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.
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 CA 022~7972 1998-12-11 produced in conditions of high stringency.
The expressions "conditions 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 pl30, more preferably from 50% to 64%
homology. Particularly the plant Rb protein so encoded has the C706 amino acid of human Rb conserved. Preferably 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. Most preferably the protein so encoded has 80% or more homology with that of SEQ N0 2 of the sequence listing attached hereto, still more preferably 90% or more and most preferably 95% or more. Particularly provided is recombinant DNA of SEQ ID
No 1 bases 31 to 2079, or the entire SEQ ID No 1, or corresponding RNAs, encoding for maize cDNA clone encoding ZmRbl of SQ ID No 2.
In 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.
CA 022~7972 1998-12-11 In a fourth aspect there are provided vectors, cells and plants and animals comprising the recombinant DNA or RNA of correct sense or anti-sense, of the invention.
In a particularly preferred use of the first aspect there is provided a method of controlling cell or viral growth comprising administering the DNA, RNA or protein of the second or third aspects to the cell. Such 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.
Alternatively, 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.
The present invention will now be illustrated further by reference to the following non-limiting Examples.
Further embodiments falling within the scope of the claims attached hereto will occur to those skilled in the light of these.
Figures.
Fig. 1. The sub-figure A shows the relative lengths of the present ZmRbl protein and the human retinoblastoma proteins. The sub-figure B shows the alignment of the CA 022~7972 1998-12-11 amino acids sequences of the Pocket A and Pocket B of the ZmRbl with that of the Xenopus, chicken, rat and three human protein (Rb, plO7 and pl30).
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.
EXAMPLE 1.
Isolation of DNA and protein expressing clones.
Total RNA was isolated from maize root and mature leaves by grinding the material previously frozen in liquid nitrogen essentially as described in Soni et al (1995). The major and minor p75ZmRbl mRNAs were identified by hybridization to a random-primed 32P-labelled PstI internal fragment (1.4 kb).
A portion of a maize cDNA library (106 pfu) in lZAPII
(Stratagene) was screened by subsequent hybridization to 5'-labelled 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).
After the second round of screening, 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 p75ZmRbl 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 CA 022~7972 1998-12-11 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 ZmRbl cDNA contains a single open reading frame capable of encoding a protein of 683 amino acids (predicted Mr 75247, p75ZmRbl) 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.
et al, Proc. Natl. Acad. Sci. USA 86, 6474 (1989)).
Northern analysis indicates that maize cells derived from both root meristems and mature leaves contain a major message, ~2.7+0.2 kb in length. In addition, a minor ~3.7+0.2 kb message also appears. Heterogeneous transcripts have been detected in other species (Destrée, O. H. J. et al, Dev. Biol. 153, 141 (1992)).
Plasmid pWori~ was constructed by deleting in pWori most of the sequences encoding WDV proteins (Sanz and Gutierrez, unpublished). Plasmid p35S.Rbl was constructed by inserting the CaMV 35S promoter (obtained from pWDV3:35SGUS) upstream of the ZmRbl cDNA in the pBS
vector. Plasmid p35S.130 was constructed by introducing the complete coding sequence of human pl30 instead of ZmRbl sequences into p35S.Rbl. Plasmid p35.A+B was constructed by substituting sequences encoding the WDV
RepA and RepB ORFs instead of ZmRbl in p35S.Rbl plasmid.
(See Soni, R. and Murray, J. A. H. Anal. Biochem. 218, 474-476 (1994)).
The sequence around the methionine codon at nucleotide CA 022~7972 1998-12-11 position 31 contains a consensus translation start (Kozak, M. J. Mol. Biol. 196, 947 (1987)). To determine whether the cDNA contained the full-length ZmRbl coding region, the 5'-end of the mRNAs was amplified by RACE-PCR
using an oligonucleotide derived from a region close to the putative initiator AUG, which would produce a fragment of -150 bp. The results are consistent with the ZmRbl cDNA clone containing the complete coding region.
The ZmRbl 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. ZmRbl also contains non-conserved N-terminal and C-terminal domains. Overall, ZmRbl 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). Ewen, M. E., Xing, Y. Lawrence, J. B. and Livingston, D. M. Cell 66, 1155 (l991))(Lee W. L. et al, Science 235, 1394 (1987);
Bernards et al, Proc. Natl. Acad. Sci. USA 86, 6974 (1989)), with the A and B subdomains exhibiting the highest homology (~50-64%). Interestingly, amino acid C706 in human Rb, critical for its function (Kaye, F. J., Kratzke R. A., Gerster, J. L. and Horowitz, J. M. Proc.
Natl. Acad. Sci. USA 87, 6922 (1990)), is also conserved in maize p75ZmRbl.
Note: The 561-577 amino acids encompass a proline-rich domain.
ZmRbl 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. A nucleic acid preferred group which encodes proteins in which one or more of these sites are CA 022~7972 1998-12-11 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.
In vivo activity.
Replication of wheat dwarf geminivirus (WDV) is dependent upon an intact LXCXE motif of the viral RepA
protein. This motif can mediate interaction with a member of the human Rb family, pl30, in yeasts. Therefore, the inventors investigated whether p75ZmRbl could complex with WDV RepA by using the yeast two-hybrid system (Fields, S. and Song, O. Nature 340, 245-246 (1989)).
Yeast cells were co-transformed with a plasmid encoding the fusion GAL4BD-RepA protein and with plasmids encoding different GAL4AD fusion protein. The GAL4AD-p75ZmRbl fusion could also complex with GAL4BD-RepA to allow growth of the recipient yeast cells in the absence of histidine. This interaction was slightly stronger than that seen with the human pl30 protein. RepA could also bind to some extent to a N-terminally truncated form of p75ZmRbl. The role of the LXCXE motif in RepA-p75ZmRbl interaction was assessed using a point mutation in WDV
RepA (E198K) which we previously showed to destroy interaction with human pl30. Co-transformation of ZmRbl with a plasmid encoding the fusion GAL4BD-RepA(E198K) indicated that the interaction between RepA and p75ZmRbl occurred through the LXCXE motif.
In this respect, 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, .
CA 022~7972 1998-12-11 1091 (1987); DeCarpio, J. A. et al., ibid., p. 275 (1988)).
Specific interaction between maize p75ZmRbl and WDV
RepA in the yeast two-hybrid system (Fields et al) relied on the ability to reconstitute a functional GAL4 activity from two separated GAL4 fusion proteins containing the DNA binding domain (GAL4BD) and the activation domain (GAL4AD). Yeast HF7c cells were co-transformed with a plasmid expressing the GAL4BD-RepA or the GAL4BD-RepA(E198K) fusions and the plasmids expressing the GAL4AD alone (Vec) or fused to human pl30, maize p75 (p75ZmRbl) or a 69 amino acids N-terminal deletion of p75 (p75ZmRbl-DN). Cells were streaked on plates with or without histidine according to the distribution shown in the upper left corner. The ability to grow in the absence of histidine depends on the functional reconstitution of a GAL4 activity upon interaction of the fusion proteins, since this triggers expression of the HIS3 gene which is under the control of a GAL4 responsive element. The growth characteristics of these yeast co-transformants correlate with the levels of b-galactosidase activity.
Procedures for two-hybrid analysis are described in Xie et al (1995). The GAL4AD-ZmRbl fusions were construed in the pGAD424 vector.
In vivo activity.
Geminivirus DNA replication requires the cellular DNA
replication machinery as well as other S-phase specific factors (Davies, J. W. and Stanley, J. Trends Genet. 5, 77 (1989); Lazarowitz, S. Crit. Rev. Plant Sci. 11, 327 (1992)). Consistent with this requirement, geminivirus infection appears to drive non-proliferating cells into S-phase, as indicated by the accumulation of the proliferating cell nuclear antigen (PCNA), a protein which is not normally present in the nuclei of CA 022~7972 1998-12-11 differentiated cells (Nagar, S., Pedersen, T. J., Carrick, K. M., Hanley-Bowdoin, L. and Robertson, D.
Plant Cell 7, 705 (1995)). The inventors finding that efficient WDV DNA replication requires an intact LXCXE
motif in RepA coupled with the discovery of a plant homolog of Rb supports the model that, as in animal cells, sequestration of plant Rb by viral RepA protein promotes inappropriate entry of infected cells into S-phase. Therefore, one way to investigate the function of p75ZmRbl was to measure geminivirus DNA replication in cells transfected with a plasmid bearing the ZmRbl sequences under a promoter functional in plant cells, an approach analogous to that previously used in human cells (Uzvolgi, E. et al., Cell Growth Diff 2, 297 (1991)).
Accumulation of newly replicated viral plasmid DNA was impaired in wheat cells transfected with plasmids expressing p75ZmRbl or human pl30, when expression of WDV
replication protein(s) is directed wither by the WDV
promoter or by the CaMV 35S promoter.
Since WDV DNA replication requires an S-phase cellular environment, interference with viral DNA replication by p75ZmRbl and human pl30 strongly evidences a role for retinoblastoma protein in the control of the G1/S
transition in plants. The existence of a plant Rb homolog implies that despite their ancient divergence, plant and animal cells use, at least in part, similar regulatory proteins and pathways for cell cycle control.
Two lines of evidences reinforce this model. First, a gene encoding a protein that complements specifically the G1/S, but not the G2/M transition of the budding yeast cdc28 mutant has been identified in alfalfa cells (Hirt, H., Pay, A., Bogre, L., Meskiene, I. and Heberle-Bors, E.
Plant J. 4, 61 (1993)). Second, plant homologs of D-type cyclins have been isolated from Arabidopsis and these, like their mammalian relatives, contain LXCXE motifs. In CA 022~7972 1998-12-11 concert with plant versions of CDK4 and CDK6, plant D-type cyclins may regulate passage through G1 phase by controlling the phosphorylation state of Rb-like proteins.
SIn animal cells, the Rb family has been implicated in tumor suppression and in the control of differentiation and development. Thus, p75ZmRbl could also play key regulatory roles at other levels during the plant cell life. One key question that is raised by the existence of 10Rb homologs in plant cells in whether, as in animals disruption of the Rb pathway leads to a tumor-prone condition. In this regard, the inventors have noted that the VirB4 protein encoded by the Ti plasmids of both Agrobacterium tumefaciens and A. rhyzogenes contains an lSLXCXE motif. Although the VirB4 protein is required for tumor induction (Hooykas, P. J. J. and Beijersbergen, A.
G. M. Annu. Rev. Phytopathol. 32, lS7 (1994), the function of its LXCXE motif in this context remains to be examined. Geminivirus infection is not accompanied by 20tumor development in the infected plant, but in some cases an abnormal growth of enactions has been observed (G. Dafalla and B. Gronenborn, personal communication).
Inhibition of wheat dwarf geminivirus (WDV) DNA
replication by ZmRbl or human pl30 in cultured wheat 2Scells was carried out as follows. A. Wheat cells were transfected, as indicated, with pWori (Xie et al. l99S) alone (0.Sg), a replicating WDV-based plasmid which encodes WDV proteins required for viral DNA replication, and with control plasmid pBS (10 g) or p3SS.Rbl (10 g), 30which encodes ZmRbl sequences under the control of the CaMV 3SS promoter. Total DNA was purified one and two days after transfection, equal amounts fractionated in agarose gels and ethidium bromide staining and viral pWori DNA identified by Southern hybridization. Plasmid 35DNA represents exclusively newly-replicated plasmid DNA
CA 022~7972 1998-12-11 since it is fully resistant to DpnI digestion and sensitive to Mbol. Note that the MboI-digested samples were run for about half of the length than the undigested samples. B. To test the effect of human pl30 on WDV DNA
replication, wheat cells were co-transfected with pWori (0.5 g) and plasmids pBS (control), p35S.Rbl or p35S.130 (10 g in each case). Replication of the test plasmid (pWori) was analyzed two days after transfection and was detected as described in part A using ethidium bromide staining; and Southern hybridization. C. To test the effect of ZmRbl or human pl30 on WDV DNA replication when expression of viral proteins was directed by the CaMV 35S
promoter, the test plasmid pWoriAA (which does not encode functional WDV replication proteins but replicates when they are provided by a different plasmid, i. e. pWori) was used. Wheat cells were co-transfected, as indicated, with pWoriAA (0.25 g), pWori (0.25 g), p35S.A+B (6 g), p35S.Rbl (10 g) and/or p35S.130 (10 g). Replication of the test plasmid (pWoriAA) was analyzed 36 hours after transfection and was detected as described in part A
using ethidium bromide staining; Southern hybridization.
Plasmids pWori (M1) and pWoriAA (M2; Sanz and Gutiérrez, unpublished), 100 pg in each case, were used as markers.
Suspension cultures of wheat cells, transfection by particle bombardment and analysis of viral DNA
replication were carried out as described in (Xie et al.
1995), except that DNA extraction was modified as in (Soni and Murray. Arnal. Biochem. 218, 474-476 (1995).
CA 022~7972 1998-12-11 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: CRISANTO GUTIERREZ ARMENTA
(A) NAME: QI XIE
(A) NAME: ANDRES PELAYO SANZ-BURGOS
(A) NAME: PAULA SUAREZ-LOPEZ
(B) STREET: CSIC-UAM, UNIVERSIDAD AUTONOMA, CANTOBLANCO
(C) CITY: MADRID
(E) COUNTRY: SPAIN
(F) POSTAL CODE (ZIP): 28049 (ii) TITLE OF THE INVENTION: PLANT PROTEINS
(iii) NUMBER OF SEQUENCES: 2 (iv) 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 #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3747 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea mays (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 31..2079 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Glu Cys Phe Gln Ser Asn Leu Glu Lys Met Glu Lys Leu Cys Afin Ser Asn Ser Cys Lys Gly Glu Leu Asp Phe Lys Ser Ile Leu Ile Asn Asn Asp Tyr Ile Pro Tyr Asp Glu Asn Ser Thr Gly Al3p Ser Thr Asn Leu Gly His Ser Lys Cys Ala Phe Glu Thr Leu Ala Ser Pro Thr Lys Thr Ile Lys Asn Met Leu Thr Val Pro Ser Ser Pro L~u Ser Pro Ala Thr Gly Gly Ser Val Lys Ile Val Gln Met Thr Pro Val Thr Ser Ala Met Thr Thr Ala Lys Trp ~eu Arg Glu Val Ile Ser Ser Leu Pro Asp Lys Pro Ser Ser Lys Leu Gln Gln Phe Leu Ser Ser Cys Asp Arg Asp Leu Thr Asn Ala Val Thr Glu Arg 1~5 130 135 Val Ser Ile Val ~u Glu Ala Ile Phe Pro Thr Lys Ser Ser Ala Asn Arg Gly Val Ser L~u Gly Leu Asn Cys Ala Asn Ala Phe Asp Ile Pro Trp Ala Glu Ala Arg Lys Val Glu Ala Ser Lys Leu Tyr Tyr Arg Val CA 022~7972 l998-l2-ll Leu Glu Ala Ile Cy6 Arg Ala Glu Leu Gln Aen Ser A6n Val A6n Asn Leu Thr Pro Leu Leu Ser A6n Glu Arg Phe Hi6 Arg Cy6 Leu Ile Ala 2l~5 210 215 Cys Ser Ala Asp Leu Val Leu Ala Thr Hie Lye Thr Val Ile Met Met Phe Pro Ala Val Leu Glu Ser Thr Gly Leu Thr Ala Phe A6p Leu Ser A~A ATA ATT GAG AAC TTT GTG AGA CAT GAA GAG ACC CTC CCA AGA GAA 822 Ly6 Ile Ile Glu A6n Phe Val Arg Hie Glu Glu Thr Leu Pro Arg Glu Leu Lys Arg Hi6 Leu A6n Ser Leu Glu Glu Gln Leu Leu Glu Ser Met Ala Trp Glu Lys Gly Ser Ser Leu Tyr Aen Ser Leu Ile Val Ala Arg Pro Ser Val Ala Ser Glu Ile A6n Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser Leu Al;p A6p Leu Val Ser Arg Gln Asn Val Arg Ile Glu Gly Leu Pro Ala Thr Pro Ser Lye Lye Arg Ala Ala Gly Pro Asp Asp A6n Ala Asp Pro Arg Ser Pro Ly6 Arg Ser Cy6 A6n Glu Ser Arg A6n Thr Val Val Glu Arg Aen Leu Gln Thr Pro Pro Pro Ly6 Gln Ser Hi6 3l;5 370 375 Met Val Ser Thr Ser Leu Lys Ala Lys Cys His Pro Leu Gln Ser Thr .
CA 022~7972 1998-12-11 Phe Ala Ser Pro Thr Val Cys Asn Pro Val Gly Gly Asn Glu Lys Cys Ala Asp Val Thr Ile His Ile Phe Phe Ser Lys Ile Leu Lys Leu Ala Ala Ile Arg Ile Arg Asn Leu Cys Glu Arg Val Gln Cy6 Val Glu Gln Thr Glu Arg Val Tyr Asn Val Phe Ly6 Gln I le Leu Glu Gln Gln Thr Thr Leu Phe Phe Asn Arg His Ile Asp Gln Leu Ile Leu Cy6 Cys Leu TAT GGT GTT GCA Ai~G GTT TGT CAA TTA GAA CTC ACA TTC AGG GAG ATA 1494 Tyr Gly Val Ala Lys Val Cys Gln Leu Glu Leu Thr Phe Arg Glu Ile Leu Asn Asn Tyr Lys Arg Glu Ala Gln Cys Lys Pro Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr Asn Arg Asn Gly Val Leu Val Ser Arg His Val Gly Ile Ile Thr Phe Tyr Asn Glu val Phe Val Pro Ala Ala Lys Pro Phe Leu V.~l Ser Leu Ile Ser Ser Gly Thr His Pro Glu Asp Lys Lys Asn Ala Ser Gly Gln Ile Pro Gly Ser Pro Lys Pro Ser Pro Phe Pro Asn Leu Pro Asp Met Ser Pro Lys Lys Val Ser Ala Ser His Asn Val Tyr Val Ser Pro Leu Arg Gln Thr Lys Leu Asp Leu Leu Leu CA 022~7972 l998-l2-ll Ser Pro Ser Ser Arg Ser Phe Tyr Ala Cys Ile Gly Glu Gly Thr His 6~5 610 615 Ala Tyr Gln Ser Pro Ser Lys Asp Leu Ala Ala Ile Asn Ser Arg Leu Asn Tyr Asn Gly Arg Lys Val Asn Ser Arg Leu Asn Phe Asp Met Val Ser Asp Ser Val Val Ala Gly Ser Leu Gly Gln Ile Asn Gly Gly Ser Thr Ser Asp Pro Ala Ala Ala Phe Ser Pro Leu Ser Lys Lys Arg Glu 665 670 ~75 680 ACA GAT ACT TGATCAATTA TAAATGGTGG C~l~l~l~l ATATAGCTCA 2119 Thr Asp Thr TGAATCTTTA GTTTTCATTG GGCTGACATA ACAAATCTTT ATCCTAGTTG G~l W'l"l'~'l-l' 2359 AGTACTTCTC r~l~ GCT ACTTTTGTAC TGTATATTTC CAGCTTCTCC ATCAGACTGA 2599 TTCCCAAATT GCAATTAATC CAGAAGTCTA C--ll~ll-ll A TTCTATTAGT TCTCAGCAAC 2899 GACATCGCAT CT-lTll'TGC AAGTGAGATG AAGAAAACCT GAAATGCTAT CACCATTTAA 3019 CA 022~7972 1998-12-11 T~lC~ll~AC ATGTCAACAG ATTAGTGTTG GGTTGCAGTC ATGTGGTGTT TAAGTCTTGG 3439 TCTATATACA AGCAGCGGAT lll~lllAGA GTTAGCTTTT GAGATGCATC A11-1~11-1~A 3619 CATCTGATTC I~ L ~ 1 ~ 11 ~ 1 AACTCGGAGT CGCGTAGAAG TTAGAATGCT AACTGACCTT 3679 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 683 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Glu Cys Phe Gln Ser Asn Leu Glu Lys Met Glu Lys Leu Cy6 Asn Ser Asn Ser Cys Lys Gly Glu Leu Asp Phe Lys Ser Ile Leu Ile Asn Asn Asp Tyr Ile Pro Tyr Asp Glu Asn Ser Thr Gly Asp Ser Thr Asn Leu Gly Hls Ser Lys Cys Ala Phe Glu Thr Leu Ala Ser Pro Thr Lys CA 022~7972 1998-12-11 Thr Ile Lys Asn Met Leu Thr Val Pro Ser Ser Pro Leu Ser Pro Ala ~hr Gly Gly Ser Val Lys Ile Val Gln Met Thr Pro Val Thr Ser Ala ~et Thr Thr Ala Lys Trp Leu Arg Glu Val Ile Ser Ser Leu Pro Asp Lys Pro Ser Ser Lys Leu Gln Gln Phe Leu Ser Ser Cys Asp Arg Asp Leu Thr Asn Ala Val Thr Glu Arg Val Ser Ile Val Leu Glu Ala Ile Phe Pro Thr Lys Ser Ser Ala Asn Arg Gly Val Ser Leu Gly Leu Asn ~ys Ala Asn Ala Phe A8p Ile Pro Trp Ala Glu Ala Arg Lys Val Glu ~la Ser Lys Leu Tyr Tyr Arg Val Leu Glu Ala Ile Cys Arg Ala Glu Leu Gln Asn Ser Asn Val Asn Asn Leu Thr Pro Leu Leu Ser Asn Glu Arg Phe His Arg Cys Leu Ile Ala Cys Ser Ala Asp Leu Val Leu Ala Thr His Lys Thr Val Ile Met Met Phe Pro Ala Val Leu Glu Ser Thr ~ly Leu Thr Ala Phe Asp Leu Ser Lys Ile Ile Glu Asn Phe Val Arg ~is Glu Glu Thr Leu Pro Arg Glu Leu Lys Arg His Leu Asn Ser Leu Glu Glu Gln Leu Leu Glu Ser Met Ala Trp Glu Lys Gly Ser Ser Leu Tyr Asn Ser Leu Ile Val Ala Arg Pro Ser Val Ala Ser Glu Ile Asn Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser Leu Asp Asp Leu Val ~er Arg Gln Asn Val Arg Ile Glu Gly Leu Pro Ala Thr Pro Ser Lys ~ys Arg Ala Ala Gly Pro Asp Asp Asn Ala A6p Pro Arg Ser Pro Lys CA 022~7972 1998-12-11 Arg Ser Cy6 A6n Glu Ser Arg A6n Thr Val Val Glu Arg A6n Leu Gln Thr Pro Pro Pro Ly6 Gln Ser Hie Met Val Ser Thr Ser Leu Lys Ala Lys Cys His Pro Leu Gln Ser Thr Phe Ala Ser Pro Thr Val Cys Asn ~ro Val Gly Gly Asn Glu Lys Cys Ala Asp Val Thr Ile His Ile Phe ~he Ser Lys Ile Leu Lyg Leu Ala Ala Ile Arg Ile Arg A6n Leu Cys Glu Arg Val Gln Cy6 Val Glu Gln Thr Glu Arg Val Tyr A6n Val Phe Lys Gln Ile Leu Glu Gln Gln Thr Thr Leu Phe Phe Asn Arg Hls Ile Asp Gln Leu Ile Leu Cys Cys Leu Tyr Gly Val Ala Ly6 Val Cys Gln ~eu Glu Leu Thr Phe Arg Glu Ile Leu Asn Asn Tyr Lys Arg Glu Ala ~ln Cys Lys Pro Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr Asn Arg Asn Gly Val Leu Val Ser Arg His Val Gly Ile Ile Thr Phe Tyr Asn Glu Val Phe Val Pro Ala Ala Lys Pro Phe I,eu Val Ser Leu Ile Ser Ser Gly Thr Hls Pro Glu Asp Lys Ly6 A6n Ala Ser Gly Gln Ile ~ro Gly Ser Pro Lys Pro Ser Pro Phe Pro Asn Leu Pro A6p Met Ser ~ro Lys Lys Val Ser Ala Ser His Asn Val Tyr Val Ser Pro Leu Arg Gln Thr Lys Leu Asp Leu Leu Leu Ser Pro Ser Ser Arg Ser Phe Tyr Ala Cys Ile Gly Glu Gly Thr His Ala Tyr Gln Ser Pro Ser Lys Asp Leu Ala Ala Ile A~n Ser Arg Leu A6n Tyr A6n Gly Arg Lys Val A6n CA 022~7972 1998-12-11 Ser Arg Leu A~n Phe Asp Met Val Ser A6p Ser Val Val Ala Gly Ser ~eu Gly Gln Ile Asn Gly Gly Ser Thr Ser A6p Pro Ala Ala Ala Phe Ser Pro ~eu Ser ~ys ~y8 Arg Glu Thr A~p Thr INDICATION REGARDING THE DEPOSIT OF A MICRO-ORGANISM
The micro-organism referred to on page 7 of the description has been deposited in the following institution:
COLECCION ESPANOLA DE CULTIVOS TIPO (CECT) Departamento de Microbiologia Facultad de Ciencias Biologicas 46100 BURJASOT (Valencia) Spain Identification of the Micro-organism deposited: pBS.Rbl Date of Deposit: 12 June 1996 Order number: 4699 These indications are reflected on form PCE/RO/134, enclosed with the request.
F ~ L ~ r~ 7 ! r; ~ r ~ r l ~ r r r PLANT PROTEINS
DESCRIPTION
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.
Cell cycle progression is regulated by positive and negative effectors. Among the latter, the product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. In mammalian cells, Rb regulates G1/S transit by inhibiting the function of the E2F family of transcription factors, known to interact with sequences in the promoter region of genes required for cellular DNA replication (see eg Weinberg, R.A. Cell 81,323 (1995); Nevins, J.R. Science 258,424 (1992)). 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-Lopez, P. and Gutiérrez, C. EMBO J. 14, 4073 (1995). The presence of the LXCXE
motif in plant D-type cyclins has also been reported (Soni, R., Carmichael, J. P., Shah, Z. H. an~ Murray, J.
CA 022~7972 1998-12-11 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 ZmRbl. ZmRbl 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 ZmRbl or human pl30, a member of the human Rb family.
Significantly the inventors work suggests that plant and animal cells may share fundamentally similar strategies for growth control, and thus human as well as plant Rb protein such as ZmRbl will be expected to have utility in, inter alia, plant therapeutics, diagnostics, growth control or investigations and many such plant proteins will have similar utility in animals.
In a first aspect of the present invention there is provided the use of retinoblastoma protein in controlling the growth of plant cells and/or plant viruses.
Particularly, 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 CA 022~7972 1998-12-11 their expression into the plant cell which it is desired to treat. By over expressing the retinoblastoma protein, or expressing an Rb protein or peptide fragment thereof that interacts with the LXCXE motif of the virus but does not affect the normal functioning of the cell, it is possible to inhibit normal virus growth and thus also to produce infection spreading from that cell to its neighbours.
Alternatively, by means of introducing 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.
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 CA 022~7972 1998-12-11 produced in conditions of high stringency.
The expressions "conditions 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 pl30, more preferably from 50% to 64%
homology. Particularly the plant Rb protein so encoded has the C706 amino acid of human Rb conserved. Preferably 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. Most preferably the protein so encoded has 80% or more homology with that of SEQ N0 2 of the sequence listing attached hereto, still more preferably 90% or more and most preferably 95% or more. Particularly provided is recombinant DNA of SEQ ID
No 1 bases 31 to 2079, or the entire SEQ ID No 1, or corresponding RNAs, encoding for maize cDNA clone encoding ZmRbl of SQ ID No 2.
In 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.
CA 022~7972 1998-12-11 In a fourth aspect there are provided vectors, cells and plants and animals comprising the recombinant DNA or RNA of correct sense or anti-sense, of the invention.
In a particularly preferred use of the first aspect there is provided a method of controlling cell or viral growth comprising administering the DNA, RNA or protein of the second or third aspects to the cell. Such 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.
Alternatively, 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.
The present invention will now be illustrated further by reference to the following non-limiting Examples.
Further embodiments falling within the scope of the claims attached hereto will occur to those skilled in the light of these.
Figures.
Fig. 1. The sub-figure A shows the relative lengths of the present ZmRbl protein and the human retinoblastoma proteins. The sub-figure B shows the alignment of the CA 022~7972 1998-12-11 amino acids sequences of the Pocket A and Pocket B of the ZmRbl with that of the Xenopus, chicken, rat and three human protein (Rb, plO7 and pl30).
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.
EXAMPLE 1.
Isolation of DNA and protein expressing clones.
Total RNA was isolated from maize root and mature leaves by grinding the material previously frozen in liquid nitrogen essentially as described in Soni et al (1995). The major and minor p75ZmRbl mRNAs were identified by hybridization to a random-primed 32P-labelled PstI internal fragment (1.4 kb).
A portion of a maize cDNA library (106 pfu) in lZAPII
(Stratagene) was screened by subsequent hybridization to 5'-labelled 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).
After the second round of screening, 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 p75ZmRbl 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 CA 022~7972 1998-12-11 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 ZmRbl cDNA contains a single open reading frame capable of encoding a protein of 683 amino acids (predicted Mr 75247, p75ZmRbl) 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.
et al, Proc. Natl. Acad. Sci. USA 86, 6474 (1989)).
Northern analysis indicates that maize cells derived from both root meristems and mature leaves contain a major message, ~2.7+0.2 kb in length. In addition, a minor ~3.7+0.2 kb message also appears. Heterogeneous transcripts have been detected in other species (Destrée, O. H. J. et al, Dev. Biol. 153, 141 (1992)).
Plasmid pWori~ was constructed by deleting in pWori most of the sequences encoding WDV proteins (Sanz and Gutierrez, unpublished). Plasmid p35S.Rbl was constructed by inserting the CaMV 35S promoter (obtained from pWDV3:35SGUS) upstream of the ZmRbl cDNA in the pBS
vector. Plasmid p35S.130 was constructed by introducing the complete coding sequence of human pl30 instead of ZmRbl sequences into p35S.Rbl. Plasmid p35.A+B was constructed by substituting sequences encoding the WDV
RepA and RepB ORFs instead of ZmRbl in p35S.Rbl plasmid.
(See Soni, R. and Murray, J. A. H. Anal. Biochem. 218, 474-476 (1994)).
The sequence around the methionine codon at nucleotide CA 022~7972 1998-12-11 position 31 contains a consensus translation start (Kozak, M. J. Mol. Biol. 196, 947 (1987)). To determine whether the cDNA contained the full-length ZmRbl coding region, the 5'-end of the mRNAs was amplified by RACE-PCR
using an oligonucleotide derived from a region close to the putative initiator AUG, which would produce a fragment of -150 bp. The results are consistent with the ZmRbl cDNA clone containing the complete coding region.
The ZmRbl 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. ZmRbl also contains non-conserved N-terminal and C-terminal domains. Overall, ZmRbl 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). Ewen, M. E., Xing, Y. Lawrence, J. B. and Livingston, D. M. Cell 66, 1155 (l991))(Lee W. L. et al, Science 235, 1394 (1987);
Bernards et al, Proc. Natl. Acad. Sci. USA 86, 6974 (1989)), with the A and B subdomains exhibiting the highest homology (~50-64%). Interestingly, amino acid C706 in human Rb, critical for its function (Kaye, F. J., Kratzke R. A., Gerster, J. L. and Horowitz, J. M. Proc.
Natl. Acad. Sci. USA 87, 6922 (1990)), is also conserved in maize p75ZmRbl.
Note: The 561-577 amino acids encompass a proline-rich domain.
ZmRbl 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. A nucleic acid preferred group which encodes proteins in which one or more of these sites are CA 022~7972 1998-12-11 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.
In vivo activity.
Replication of wheat dwarf geminivirus (WDV) is dependent upon an intact LXCXE motif of the viral RepA
protein. This motif can mediate interaction with a member of the human Rb family, pl30, in yeasts. Therefore, the inventors investigated whether p75ZmRbl could complex with WDV RepA by using the yeast two-hybrid system (Fields, S. and Song, O. Nature 340, 245-246 (1989)).
Yeast cells were co-transformed with a plasmid encoding the fusion GAL4BD-RepA protein and with plasmids encoding different GAL4AD fusion protein. The GAL4AD-p75ZmRbl fusion could also complex with GAL4BD-RepA to allow growth of the recipient yeast cells in the absence of histidine. This interaction was slightly stronger than that seen with the human pl30 protein. RepA could also bind to some extent to a N-terminally truncated form of p75ZmRbl. The role of the LXCXE motif in RepA-p75ZmRbl interaction was assessed using a point mutation in WDV
RepA (E198K) which we previously showed to destroy interaction with human pl30. Co-transformation of ZmRbl with a plasmid encoding the fusion GAL4BD-RepA(E198K) indicated that the interaction between RepA and p75ZmRbl occurred through the LXCXE motif.
In this respect, 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, .
CA 022~7972 1998-12-11 1091 (1987); DeCarpio, J. A. et al., ibid., p. 275 (1988)).
Specific interaction between maize p75ZmRbl and WDV
RepA in the yeast two-hybrid system (Fields et al) relied on the ability to reconstitute a functional GAL4 activity from two separated GAL4 fusion proteins containing the DNA binding domain (GAL4BD) and the activation domain (GAL4AD). Yeast HF7c cells were co-transformed with a plasmid expressing the GAL4BD-RepA or the GAL4BD-RepA(E198K) fusions and the plasmids expressing the GAL4AD alone (Vec) or fused to human pl30, maize p75 (p75ZmRbl) or a 69 amino acids N-terminal deletion of p75 (p75ZmRbl-DN). Cells were streaked on plates with or without histidine according to the distribution shown in the upper left corner. The ability to grow in the absence of histidine depends on the functional reconstitution of a GAL4 activity upon interaction of the fusion proteins, since this triggers expression of the HIS3 gene which is under the control of a GAL4 responsive element. The growth characteristics of these yeast co-transformants correlate with the levels of b-galactosidase activity.
Procedures for two-hybrid analysis are described in Xie et al (1995). The GAL4AD-ZmRbl fusions were construed in the pGAD424 vector.
In vivo activity.
Geminivirus DNA replication requires the cellular DNA
replication machinery as well as other S-phase specific factors (Davies, J. W. and Stanley, J. Trends Genet. 5, 77 (1989); Lazarowitz, S. Crit. Rev. Plant Sci. 11, 327 (1992)). Consistent with this requirement, geminivirus infection appears to drive non-proliferating cells into S-phase, as indicated by the accumulation of the proliferating cell nuclear antigen (PCNA), a protein which is not normally present in the nuclei of CA 022~7972 1998-12-11 differentiated cells (Nagar, S., Pedersen, T. J., Carrick, K. M., Hanley-Bowdoin, L. and Robertson, D.
Plant Cell 7, 705 (1995)). The inventors finding that efficient WDV DNA replication requires an intact LXCXE
motif in RepA coupled with the discovery of a plant homolog of Rb supports the model that, as in animal cells, sequestration of plant Rb by viral RepA protein promotes inappropriate entry of infected cells into S-phase. Therefore, one way to investigate the function of p75ZmRbl was to measure geminivirus DNA replication in cells transfected with a plasmid bearing the ZmRbl sequences under a promoter functional in plant cells, an approach analogous to that previously used in human cells (Uzvolgi, E. et al., Cell Growth Diff 2, 297 (1991)).
Accumulation of newly replicated viral plasmid DNA was impaired in wheat cells transfected with plasmids expressing p75ZmRbl or human pl30, when expression of WDV
replication protein(s) is directed wither by the WDV
promoter or by the CaMV 35S promoter.
Since WDV DNA replication requires an S-phase cellular environment, interference with viral DNA replication by p75ZmRbl and human pl30 strongly evidences a role for retinoblastoma protein in the control of the G1/S
transition in plants. The existence of a plant Rb homolog implies that despite their ancient divergence, plant and animal cells use, at least in part, similar regulatory proteins and pathways for cell cycle control.
Two lines of evidences reinforce this model. First, a gene encoding a protein that complements specifically the G1/S, but not the G2/M transition of the budding yeast cdc28 mutant has been identified in alfalfa cells (Hirt, H., Pay, A., Bogre, L., Meskiene, I. and Heberle-Bors, E.
Plant J. 4, 61 (1993)). Second, plant homologs of D-type cyclins have been isolated from Arabidopsis and these, like their mammalian relatives, contain LXCXE motifs. In CA 022~7972 1998-12-11 concert with plant versions of CDK4 and CDK6, plant D-type cyclins may regulate passage through G1 phase by controlling the phosphorylation state of Rb-like proteins.
SIn animal cells, the Rb family has been implicated in tumor suppression and in the control of differentiation and development. Thus, p75ZmRbl could also play key regulatory roles at other levels during the plant cell life. One key question that is raised by the existence of 10Rb homologs in plant cells in whether, as in animals disruption of the Rb pathway leads to a tumor-prone condition. In this regard, the inventors have noted that the VirB4 protein encoded by the Ti plasmids of both Agrobacterium tumefaciens and A. rhyzogenes contains an lSLXCXE motif. Although the VirB4 protein is required for tumor induction (Hooykas, P. J. J. and Beijersbergen, A.
G. M. Annu. Rev. Phytopathol. 32, lS7 (1994), the function of its LXCXE motif in this context remains to be examined. Geminivirus infection is not accompanied by 20tumor development in the infected plant, but in some cases an abnormal growth of enactions has been observed (G. Dafalla and B. Gronenborn, personal communication).
Inhibition of wheat dwarf geminivirus (WDV) DNA
replication by ZmRbl or human pl30 in cultured wheat 2Scells was carried out as follows. A. Wheat cells were transfected, as indicated, with pWori (Xie et al. l99S) alone (0.Sg), a replicating WDV-based plasmid which encodes WDV proteins required for viral DNA replication, and with control plasmid pBS (10 g) or p3SS.Rbl (10 g), 30which encodes ZmRbl sequences under the control of the CaMV 3SS promoter. Total DNA was purified one and two days after transfection, equal amounts fractionated in agarose gels and ethidium bromide staining and viral pWori DNA identified by Southern hybridization. Plasmid 35DNA represents exclusively newly-replicated plasmid DNA
CA 022~7972 1998-12-11 since it is fully resistant to DpnI digestion and sensitive to Mbol. Note that the MboI-digested samples were run for about half of the length than the undigested samples. B. To test the effect of human pl30 on WDV DNA
replication, wheat cells were co-transfected with pWori (0.5 g) and plasmids pBS (control), p35S.Rbl or p35S.130 (10 g in each case). Replication of the test plasmid (pWori) was analyzed two days after transfection and was detected as described in part A using ethidium bromide staining; and Southern hybridization. C. To test the effect of ZmRbl or human pl30 on WDV DNA replication when expression of viral proteins was directed by the CaMV 35S
promoter, the test plasmid pWoriAA (which does not encode functional WDV replication proteins but replicates when they are provided by a different plasmid, i. e. pWori) was used. Wheat cells were co-transfected, as indicated, with pWoriAA (0.25 g), pWori (0.25 g), p35S.A+B (6 g), p35S.Rbl (10 g) and/or p35S.130 (10 g). Replication of the test plasmid (pWoriAA) was analyzed 36 hours after transfection and was detected as described in part A
using ethidium bromide staining; Southern hybridization.
Plasmids pWori (M1) and pWoriAA (M2; Sanz and Gutiérrez, unpublished), 100 pg in each case, were used as markers.
Suspension cultures of wheat cells, transfection by particle bombardment and analysis of viral DNA
replication were carried out as described in (Xie et al.
1995), except that DNA extraction was modified as in (Soni and Murray. Arnal. Biochem. 218, 474-476 (1995).
CA 022~7972 1998-12-11 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: CRISANTO GUTIERREZ ARMENTA
(A) NAME: QI XIE
(A) NAME: ANDRES PELAYO SANZ-BURGOS
(A) NAME: PAULA SUAREZ-LOPEZ
(B) STREET: CSIC-UAM, UNIVERSIDAD AUTONOMA, CANTOBLANCO
(C) CITY: MADRID
(E) COUNTRY: SPAIN
(F) POSTAL CODE (ZIP): 28049 (ii) TITLE OF THE INVENTION: PLANT PROTEINS
(iii) NUMBER OF SEQUENCES: 2 (iv) 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 #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3747 base pairs (B) TYPE: nucleic acid .
(C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea mays (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 31..2079 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Glu Cys Phe Gln Ser Asn Leu Glu Lys Met Glu Lys Leu Cys Afin Ser Asn Ser Cys Lys Gly Glu Leu Asp Phe Lys Ser Ile Leu Ile Asn Asn Asp Tyr Ile Pro Tyr Asp Glu Asn Ser Thr Gly Al3p Ser Thr Asn Leu Gly His Ser Lys Cys Ala Phe Glu Thr Leu Ala Ser Pro Thr Lys Thr Ile Lys Asn Met Leu Thr Val Pro Ser Ser Pro L~u Ser Pro Ala Thr Gly Gly Ser Val Lys Ile Val Gln Met Thr Pro Val Thr Ser Ala Met Thr Thr Ala Lys Trp ~eu Arg Glu Val Ile Ser Ser Leu Pro Asp Lys Pro Ser Ser Lys Leu Gln Gln Phe Leu Ser Ser Cys Asp Arg Asp Leu Thr Asn Ala Val Thr Glu Arg 1~5 130 135 Val Ser Ile Val ~u Glu Ala Ile Phe Pro Thr Lys Ser Ser Ala Asn Arg Gly Val Ser L~u Gly Leu Asn Cys Ala Asn Ala Phe Asp Ile Pro Trp Ala Glu Ala Arg Lys Val Glu Ala Ser Lys Leu Tyr Tyr Arg Val CA 022~7972 l998-l2-ll Leu Glu Ala Ile Cy6 Arg Ala Glu Leu Gln Aen Ser A6n Val A6n Asn Leu Thr Pro Leu Leu Ser A6n Glu Arg Phe Hi6 Arg Cy6 Leu Ile Ala 2l~5 210 215 Cys Ser Ala Asp Leu Val Leu Ala Thr Hie Lye Thr Val Ile Met Met Phe Pro Ala Val Leu Glu Ser Thr Gly Leu Thr Ala Phe A6p Leu Ser A~A ATA ATT GAG AAC TTT GTG AGA CAT GAA GAG ACC CTC CCA AGA GAA 822 Ly6 Ile Ile Glu A6n Phe Val Arg Hie Glu Glu Thr Leu Pro Arg Glu Leu Lys Arg Hi6 Leu A6n Ser Leu Glu Glu Gln Leu Leu Glu Ser Met Ala Trp Glu Lys Gly Ser Ser Leu Tyr Aen Ser Leu Ile Val Ala Arg Pro Ser Val Ala Ser Glu Ile A6n Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser Leu Al;p A6p Leu Val Ser Arg Gln Asn Val Arg Ile Glu Gly Leu Pro Ala Thr Pro Ser Lye Lye Arg Ala Ala Gly Pro Asp Asp A6n Ala Asp Pro Arg Ser Pro Ly6 Arg Ser Cy6 A6n Glu Ser Arg A6n Thr Val Val Glu Arg Aen Leu Gln Thr Pro Pro Pro Ly6 Gln Ser Hi6 3l;5 370 375 Met Val Ser Thr Ser Leu Lys Ala Lys Cys His Pro Leu Gln Ser Thr .
CA 022~7972 1998-12-11 Phe Ala Ser Pro Thr Val Cys Asn Pro Val Gly Gly Asn Glu Lys Cys Ala Asp Val Thr Ile His Ile Phe Phe Ser Lys Ile Leu Lys Leu Ala Ala Ile Arg Ile Arg Asn Leu Cys Glu Arg Val Gln Cy6 Val Glu Gln Thr Glu Arg Val Tyr Asn Val Phe Ly6 Gln I le Leu Glu Gln Gln Thr Thr Leu Phe Phe Asn Arg His Ile Asp Gln Leu Ile Leu Cy6 Cys Leu TAT GGT GTT GCA Ai~G GTT TGT CAA TTA GAA CTC ACA TTC AGG GAG ATA 1494 Tyr Gly Val Ala Lys Val Cys Gln Leu Glu Leu Thr Phe Arg Glu Ile Leu Asn Asn Tyr Lys Arg Glu Ala Gln Cys Lys Pro Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr Asn Arg Asn Gly Val Leu Val Ser Arg His Val Gly Ile Ile Thr Phe Tyr Asn Glu val Phe Val Pro Ala Ala Lys Pro Phe Leu V.~l Ser Leu Ile Ser Ser Gly Thr His Pro Glu Asp Lys Lys Asn Ala Ser Gly Gln Ile Pro Gly Ser Pro Lys Pro Ser Pro Phe Pro Asn Leu Pro Asp Met Ser Pro Lys Lys Val Ser Ala Ser His Asn Val Tyr Val Ser Pro Leu Arg Gln Thr Lys Leu Asp Leu Leu Leu CA 022~7972 l998-l2-ll Ser Pro Ser Ser Arg Ser Phe Tyr Ala Cys Ile Gly Glu Gly Thr His 6~5 610 615 Ala Tyr Gln Ser Pro Ser Lys Asp Leu Ala Ala Ile Asn Ser Arg Leu Asn Tyr Asn Gly Arg Lys Val Asn Ser Arg Leu Asn Phe Asp Met Val Ser Asp Ser Val Val Ala Gly Ser Leu Gly Gln Ile Asn Gly Gly Ser Thr Ser Asp Pro Ala Ala Ala Phe Ser Pro Leu Ser Lys Lys Arg Glu 665 670 ~75 680 ACA GAT ACT TGATCAATTA TAAATGGTGG C~l~l~l~l ATATAGCTCA 2119 Thr Asp Thr TGAATCTTTA GTTTTCATTG GGCTGACATA ACAAATCTTT ATCCTAGTTG G~l W'l"l'~'l-l' 2359 AGTACTTCTC r~l~ GCT ACTTTTGTAC TGTATATTTC CAGCTTCTCC ATCAGACTGA 2599 TTCCCAAATT GCAATTAATC CAGAAGTCTA C--ll~ll-ll A TTCTATTAGT TCTCAGCAAC 2899 GACATCGCAT CT-lTll'TGC AAGTGAGATG AAGAAAACCT GAAATGCTAT CACCATTTAA 3019 CA 022~7972 1998-12-11 T~lC~ll~AC ATGTCAACAG ATTAGTGTTG GGTTGCAGTC ATGTGGTGTT TAAGTCTTGG 3439 TCTATATACA AGCAGCGGAT lll~lllAGA GTTAGCTTTT GAGATGCATC A11-1~11-1~A 3619 CATCTGATTC I~ L ~ 1 ~ 11 ~ 1 AACTCGGAGT CGCGTAGAAG TTAGAATGCT AACTGACCTT 3679 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 683 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Glu Cys Phe Gln Ser Asn Leu Glu Lys Met Glu Lys Leu Cy6 Asn Ser Asn Ser Cys Lys Gly Glu Leu Asp Phe Lys Ser Ile Leu Ile Asn Asn Asp Tyr Ile Pro Tyr Asp Glu Asn Ser Thr Gly Asp Ser Thr Asn Leu Gly Hls Ser Lys Cys Ala Phe Glu Thr Leu Ala Ser Pro Thr Lys CA 022~7972 1998-12-11 Thr Ile Lys Asn Met Leu Thr Val Pro Ser Ser Pro Leu Ser Pro Ala ~hr Gly Gly Ser Val Lys Ile Val Gln Met Thr Pro Val Thr Ser Ala ~et Thr Thr Ala Lys Trp Leu Arg Glu Val Ile Ser Ser Leu Pro Asp Lys Pro Ser Ser Lys Leu Gln Gln Phe Leu Ser Ser Cys Asp Arg Asp Leu Thr Asn Ala Val Thr Glu Arg Val Ser Ile Val Leu Glu Ala Ile Phe Pro Thr Lys Ser Ser Ala Asn Arg Gly Val Ser Leu Gly Leu Asn ~ys Ala Asn Ala Phe A8p Ile Pro Trp Ala Glu Ala Arg Lys Val Glu ~la Ser Lys Leu Tyr Tyr Arg Val Leu Glu Ala Ile Cys Arg Ala Glu Leu Gln Asn Ser Asn Val Asn Asn Leu Thr Pro Leu Leu Ser Asn Glu Arg Phe His Arg Cys Leu Ile Ala Cys Ser Ala Asp Leu Val Leu Ala Thr His Lys Thr Val Ile Met Met Phe Pro Ala Val Leu Glu Ser Thr ~ly Leu Thr Ala Phe Asp Leu Ser Lys Ile Ile Glu Asn Phe Val Arg ~is Glu Glu Thr Leu Pro Arg Glu Leu Lys Arg His Leu Asn Ser Leu Glu Glu Gln Leu Leu Glu Ser Met Ala Trp Glu Lys Gly Ser Ser Leu Tyr Asn Ser Leu Ile Val Ala Arg Pro Ser Val Ala Ser Glu Ile Asn Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser Leu Asp Asp Leu Val ~er Arg Gln Asn Val Arg Ile Glu Gly Leu Pro Ala Thr Pro Ser Lys ~ys Arg Ala Ala Gly Pro Asp Asp Asn Ala A6p Pro Arg Ser Pro Lys CA 022~7972 1998-12-11 Arg Ser Cy6 A6n Glu Ser Arg A6n Thr Val Val Glu Arg A6n Leu Gln Thr Pro Pro Pro Ly6 Gln Ser Hie Met Val Ser Thr Ser Leu Lys Ala Lys Cys His Pro Leu Gln Ser Thr Phe Ala Ser Pro Thr Val Cys Asn ~ro Val Gly Gly Asn Glu Lys Cys Ala Asp Val Thr Ile His Ile Phe ~he Ser Lys Ile Leu Lyg Leu Ala Ala Ile Arg Ile Arg A6n Leu Cys Glu Arg Val Gln Cy6 Val Glu Gln Thr Glu Arg Val Tyr A6n Val Phe Lys Gln Ile Leu Glu Gln Gln Thr Thr Leu Phe Phe Asn Arg Hls Ile Asp Gln Leu Ile Leu Cys Cys Leu Tyr Gly Val Ala Ly6 Val Cys Gln ~eu Glu Leu Thr Phe Arg Glu Ile Leu Asn Asn Tyr Lys Arg Glu Ala ~ln Cys Lys Pro Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr Asn Arg Asn Gly Val Leu Val Ser Arg His Val Gly Ile Ile Thr Phe Tyr Asn Glu Val Phe Val Pro Ala Ala Lys Pro Phe I,eu Val Ser Leu Ile Ser Ser Gly Thr Hls Pro Glu Asp Lys Ly6 A6n Ala Ser Gly Gln Ile ~ro Gly Ser Pro Lys Pro Ser Pro Phe Pro Asn Leu Pro A6p Met Ser ~ro Lys Lys Val Ser Ala Ser His Asn Val Tyr Val Ser Pro Leu Arg Gln Thr Lys Leu Asp Leu Leu Leu Ser Pro Ser Ser Arg Ser Phe Tyr Ala Cys Ile Gly Glu Gly Thr His Ala Tyr Gln Ser Pro Ser Lys Asp Leu Ala Ala Ile A~n Ser Arg Leu A6n Tyr A6n Gly Arg Lys Val A6n CA 022~7972 1998-12-11 Ser Arg Leu A~n Phe Asp Met Val Ser A6p Ser Val Val Ala Gly Ser ~eu Gly Gln Ile Asn Gly Gly Ser Thr Ser A6p Pro Ala Ala Ala Phe Ser Pro ~eu Ser ~ys ~y8 Arg Glu Thr A~p Thr INDICATION REGARDING THE DEPOSIT OF A MICRO-ORGANISM
The micro-organism referred to on page 7 of the description has been deposited in the following institution:
COLECCION ESPANOLA DE CULTIVOS TIPO (CECT) Departamento de Microbiologia Facultad de Ciencias Biologicas 46100 BURJASOT (Valencia) Spain Identification of the Micro-organism deposited: pBS.Rbl Date of Deposit: 12 June 1996 Order number: 4699 These indications are reflected on form PCE/RO/134, enclosed with the request.
Claims (21)
1. A method of controlling the growth of a plant cell or a plant virus within that cell comprising increasing or decreasing the level and/or activity or retinoblastoma protein in that plant cell by incorporation therein of a recombinant nucleic acid.
2. A method as claimed in claim 1 characterised in that the nucleic acid is such as to increase or inhibit expression of a retinoblastoma protein in the cell.
3. A method as claimed in claim 1 characterised in that the nucleic acid is such as to express a retinoblastoma protein or peptide fragment of a retinoblastoma protein that interacts with viral LXCXE motif without affecting the normal functioning of the cell.
4. A method as claimed in claim 3 characterised in that the retinoblastoma protein has been rendered resistant to phosphorylation by cyclin dependent kinases by change or deletion of one or more consensus SP or TP sites found in the SEQ ID No. 2.
5. A method as claimed in claim 2 characterised in that the DNA or RNA is antisense to retinoblastoma protein encoding DNA or RNA and inhibits retinoblastoma protein expression.
6. A method of transforming a plant cell such that it is directed into the S phase of the cell cycle comprising introducing a nucleic acid encoding antisense RNA to a plant retinoblastoma protein.
7. Recombinant nucleic acid encoding for expression of a retinoblastoma protein characterised in that the retinoblastoma protein has an amino acid sequence having 80% or more homology with that of SEQ No. 2 of the sequence listing attached hereto.
8. Recombinant nucleic acid as claimed n claim 7 characterised in that it comprises SEQ ID no. 1, bases 31-207, sequences only having degenerate substitutions thereof or sequences capable of hybridizing with a polynucleotide of SEQ ID No. 1 under conditions of high stringency.
9. Recombinant nucleic acid as claimed in claim 7 or 8 characterised in that it encodes for a retinoblastoma protein conservatively substituted with respect to SEQ ID
No. 2.
No. 2.
10. Recombinant nucleic acid characterised in that it comprises antisense DNA or RNA to a plant retinoblastoma protein.
11. Recombinant nucleic acid as claimed in claim 10 characterised in that it comprises antisense DNA or RNA
to a plant retinoblastoma protein comprising SEQ ID No.
2 or a sequence having at least 80% homology thereto.
to a plant retinoblastoma protein comprising SEQ ID No.
2 or a sequence having at least 80% homology thereto.
12. Recombinant nucleic acid as claimed in claim 10 or 11 characterised in that it comprises antisense DNA or RNA to that of SEQ ID No. 1 or a sequence having at least 80% homology thereto.
13. Recombinant nucleic acid characterised in that it encodes for a retinoblastoma protein or a peptide fragment of a retinoblastoma protein that interacts with viral LXCXE motif without affecting the normal functioning of a plant cell.
14. Recombinant nucleic acid as claimed in claim 13 characterised in that it encodes for a plant retinoblastoma protein in which one or more consensus SP
or TP sites found in the SEQ ID No. 2 have been changed or deleted.
or TP sites found in the SEQ ID No. 2 have been changed or deleted.
15. A protein produced by the expression of a recombinant DNA or RNA as claimed in any one of claims 7 to 9, 13 and 14.
16. A protein as claimed in claim 15 characterised in that one or more consensus ST or TP sites found in the SEQ ID No. 2 have been changed or deleted.
17. A recombinant vector characterised in that it comprises a recombinant nucleic acid as claimed in any one of claims 7 to 9, 13 and 14.
18. A plant cell characterised in that it comprises a recombinant nucleic acid encoding for expression of a retinoblastoma protein.
19. A plant cell as claimed in claim 18 characterised in that it comprises a recombinant nucleic acid as claimed in any one of claims 7 to 9, 13 and 14.
20. A plant cell as claimed in claim 18 or 19 characterised in that it expresses a retinoblastoma protein from said nucleic acid.
21. A transgenic plant characterised in that it comprises a cell as claimed in any one of claims 18 to 20.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/ES1996/000130 WO1997047647A1 (en) | 1996-06-13 | 1996-06-13 | Plant proteins |
| CA002257972A CA2257972A1 (en) | 1996-06-13 | 1996-06-13 | Plant proteins |
| EP97928187A EP0914436A1 (en) | 1996-06-13 | 1997-06-12 | Plant retinoblastoma-associated proteins |
| ZA975202A ZA975202B (en) | 1996-06-13 | 1997-06-12 | Plants proteins |
| CN97197141A CN1227605A (en) | 1996-06-13 | 1997-06-12 | Plant retinoblastoma-associated proteins |
| PCT/EP1997/003070 WO1997047745A1 (en) | 1996-06-13 | 1997-06-12 | Plant retinoblastoma-associated proteins |
| AU32579/97A AU721332B2 (en) | 1996-06-13 | 1997-06-12 | Plant retinoblastoma-associated proteins |
| CA002257828A CA2257828A1 (en) | 1996-06-13 | 1997-06-12 | Plant retinoblastoma-associated proteins |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/ES1996/000130 WO1997047647A1 (en) | 1996-06-13 | 1996-06-13 | Plant proteins |
| CA002257972A CA2257972A1 (en) | 1996-06-13 | 1996-06-13 | Plant proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2257972A1 true CA2257972A1 (en) | 1997-12-18 |
Family
ID=25680704
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002257972A Abandoned CA2257972A1 (en) | 1996-06-13 | 1996-06-13 | Plant proteins |
| CA002257828A Abandoned CA2257828A1 (en) | 1996-06-13 | 1997-06-12 | Plant retinoblastoma-associated proteins |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002257828A Abandoned CA2257828A1 (en) | 1996-06-13 | 1997-06-12 | Plant retinoblastoma-associated proteins |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0914436A1 (en) |
| CN (1) | CN1227605A (en) |
| AU (1) | AU721332B2 (en) |
| CA (2) | CA2257972A1 (en) |
| WO (2) | WO1997047647A1 (en) |
| ZA (1) | ZA975202B (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2282715A1 (en) * | 1997-03-26 | 1998-10-01 | Cambridge University Technical Services Ltd. | Plants with modified growth |
| ES2132025B1 (en) * | 1997-06-12 | 2000-12-01 | Consejo Superior Investigacion | URAG PROTEINS OF PLANTS. |
| CA2301830A1 (en) * | 1997-09-05 | 1999-03-18 | Cropdesign N.V. | Method and means for modulating plant cell cycle proteins and their use in controlling plant cell growth |
| AU3478599A (en) * | 1998-04-09 | 1999-11-01 | E.I. Du Pont De Nemours And Company | Cell cycle regulatory proteins |
| CA2327546A1 (en) * | 1998-05-08 | 1999-11-18 | Consejo Superior De Investigaciones Cientificas | Transgenic plant cells expressing a recombinant plant e2f peptide |
| EP1088091A2 (en) * | 1998-06-15 | 2001-04-04 | CropDesign N.V. | Plant pathogen inducible control sequences operably linked to cell cycle genes and the uses thereof |
| US6284947B1 (en) * | 1999-02-25 | 2001-09-04 | Pioneer Hi-Bred International, Inc. | Methods of using viral replicase polynucleotides and polypeptides |
| 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 |
| GB9923306D0 (en) | 1999-10-01 | 1999-12-08 | Isis Innovation | Diagnostic and therapeutic epitope, and transgenic plant |
| US20030167505A1 (en) * | 2001-03-16 | 2003-09-04 | Dilkes Brian R. | Cell cycle nucleic acids, polypeptides and uses thereof |
| GB0212885D0 (en) | 2002-06-05 | 2002-07-17 | Isis Innovation | Therapeutic epitopes and uses thereof |
| AU2003255457A1 (en) * | 2002-08-14 | 2004-03-03 | Cropdesign N.V. | Plants having modified growth and a method for making the same |
| CN1976715B (en) | 2004-04-28 | 2014-07-30 | 英国技术集团国际有限公司 | Epitopes related to coeliac disease |
| US10105437B2 (en) | 2004-04-28 | 2018-10-23 | Btg International Limited | Epitopes related to coeliac disease |
| WO2019159003A1 (en) * | 2018-02-16 | 2019-08-22 | Corvalan Claudia | Transgenic plants with increased yields |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06501160A (en) * | 1990-09-17 | 1994-02-10 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Methods and compositions for controlling cell proliferation |
| ZA946595B (en) * | 1993-09-03 | 1996-02-28 | Res Dev Foundation | Mutants of the retinoblastoma and P53 genes and uses thereof |
| ZA947065B (en) * | 1993-09-13 | 1995-05-03 | Univ California | Therapeutic use of the retinoblastoma susceptibility gene product |
-
1996
- 1996-06-13 CA CA002257972A patent/CA2257972A1/en not_active Abandoned
- 1996-06-13 WO PCT/ES1996/000130 patent/WO1997047647A1/en active Search and Examination
-
1997
- 1997-06-12 AU AU32579/97A patent/AU721332B2/en not_active Ceased
- 1997-06-12 CA CA002257828A patent/CA2257828A1/en not_active Abandoned
- 1997-06-12 EP EP97928187A patent/EP0914436A1/en not_active Withdrawn
- 1997-06-12 CN CN97197141A patent/CN1227605A/en active Pending
- 1997-06-12 WO PCT/EP1997/003070 patent/WO1997047745A1/en not_active Application Discontinuation
- 1997-06-12 ZA ZA975202A patent/ZA975202B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CN1227605A (en) | 1999-09-01 |
| AU721332B2 (en) | 2000-06-29 |
| CA2257828A1 (en) | 1997-12-18 |
| EP0914436A1 (en) | 1999-05-12 |
| WO1997047647A1 (en) | 1997-12-18 |
| AU3257997A (en) | 1998-01-07 |
| WO1997047745A1 (en) | 1997-12-18 |
| ZA975202B (en) | 1998-12-14 |
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