CA2257828A1 - Plant retinoblastoma-associated proteins - Google Patents

Abstract

The present invention is based on the isolation and characterization of a plant cell DNA sequence encoding for a retinoblastoma protein. Such finding is based on the structural and functional properties of the plant retinoblastoma protein as possible regulator of the cellular cycle, of the cellular growth and of the plant cellular differentiation. For this reason, among other aspects, it is claimed the use of retinoblastoma protein or the DNA sequence which encodes for it in the growing control of vegetable cells, plants and/or vegetable virus, as well as the use of vectors, cells, plants or animals, or animal cells modified through the manipulation of the control route based on plant retinoblastoma protein.

Description

CA 022~7828 1998-12-11 W 097/47745 PCT~EP97/03070 PLANT RETTNOBLASTOMA-ASSOC~ATED PROT~nNS
DESCRIPTION
The present invention relates the protelns having r biological activity in plant and animal systems, to polynucleotides encoding ~or the expression o~ such proteins, to oligonucleotides ~or use in identi~ying and synthesizing these proteins and polynucleotides, to vectors and cells containing the polynucleotides in recombinant ~orm and to plants and animals comprising these, and to the use of the proteins and polynucleotides and ~ragments thereo~ in the control o~ plant growth and plant vulnerability to viruses.
Cell cycle progression is regulated by positive and negative e~ectors. Among the latter, the product o~ the retinoblastoma susceptibility gene (Rb) controls the passage o~ mammalian cells through G1 phase. In mammalian cells, Rb regulates G1/S tra~sit by lnhibiting the ~unction o~ the E2F ~amily o~ transcription ~actors, ]cnown to interact with sequences in the promoter region o~ genes required ~or cellular DNA replication (see eg Weinberg, R.A. Cell 81,323 (1995); Nevins, J.R. Science 258,424 (1992)). DNA tumor viruses that in~ect animal cells express oncoproteins that interact with the Rb protein via a LXCXE moti~, disrupting Rb-E2F comple~es 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 e~icient replication o~ a plant geminivirus requires the integrity o~ an LXCXE amino acid moti~ in the viral RepA protein and that RepA can interact with members o~ the human Rb ~amily in yeast (Xie, Q., Suarez-Lopez, P. and Gutiérrez, C. EMBO J. 14, 4073 (1995). The presence o~ the LXCXE
moti~ in plant D-type cyclins has also been reported (Soni, R., Carmichael, J. P., Shah, Z. H. and Murray, J.

CONFIRMATION COPY

CA 022~7828 1998-12-11 A. H. Plant Cell 7, 85-103 tl995)).
The present inventors have now identified characteristic sequences of plant Rb proteins and corresponding encoding polynucleotides for the ~irst 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 veyetable 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 mo~i~ essential ~or 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 o~ the human Rb ~amily.
Significantly the inventors work suggests that plant and animal cells may share fl7n~m~ntally similar strategies ~or 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 ~n~m~l s .
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 o~
viral infection and/or growth in plant ceils 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 ~eminiviruses.
A preferred method of control using such proteins involves applying these to the plant cell, either directly or by introduction of DNA or RNA encodiny for CA 022~7828 1998-12-11 W O 97/47745 PCT~EP97/03070 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 ~ragment thereo~
that interacts with the LXCXE moti~ o~ the virus but does not a~ect the normal ~unctioning o~ the cell, it is possible to inhibit normal virus growth and thus also to produce in~ection spreading ~rom that cell to its neighbours.
Alternatively, by means o~ introducing anti-sense DNA
or RNA in plant cells in vectors ~orm that contain the necessary promoters ~or the DNA or RNA transcription, it will be possible to exploit the well known anti-sense mechanism in order to inhibit the expression o~ the Rb protein, and thus the S-phase. Such plants will be o~
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 ~or those skilled in the art: see ~or example "Principles o~ gene manipulation - An introduction to Genetic Engineering (1994) R.W. Old & S.B. Primrose; Ox~ord-Blackwell Scienti~ic Publications Fi~th Edition p39~.
In a second aspect o~ the present invention there is provided recombinant nucleic acid, particularly in the ~orm o~ DNA or cRNA ~mRNA), encoding ~or expression o~ Rb protein that is characteristic o~ plants This nucleic acid is characterised by one or more characteristic regions that di~er ~rom known animal Rb protein nucleic acid and is exempli~ied 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 o~ the codons in SEQ ID No. 1, and in where the RNA the T is U. The ~ most pre~erred DNA or RNA are capable of hybridate with the polynucleotide o~ the SEQ ID No. 1 in conditions o~
low stringency, pre~erably being the hybridization CA 022~7828 1998-12-11 produced in conditions o~ high stringency.
The expressions "conditions o~ low stringency" and "conditions of high stringency" are understood by those skilled, but are conveniently exempli~ied in US 5202257, Col-9-Col 10. I~ some modi~ications were made to lead to the expression of a protein with dif~erent amino acids, preferably of the same ]~ind o~ the corresponding amino acids to the SEQ ID No 1; that is, are conservative substitutions. Such substitutions are known by those s]cilled, for example, see US 5380712, and it is only contemplated when the protein has activity with retinoblastoma protein.
Pre~erred DNA or ~RNA encodes ~or 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 pre~erably ~rom 50~ to 64~
homology. Particularly the plant Rb protein so encoded has the ~706 amino acid o~ human Rb conserved. Pre~erably the spacer sequence between the A and B pockets is not conserved with respect to animal Rb proteins, pre~erably being less than 50~ homologous to the same region as ~ound in such animal proteins. Most pre~erably the protein so encoded has 80~ or more homology with that of SEQ NO 2 o~ the sequence listing attached hereto, still more pre~erably 90~ or more and most pre~erably 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 o~ SQ ID No 2 In a third aspect of the present invention there is provided the protein expressed by the recombinant DNA or RNA o~ the second aspect, novel proteins derived ~rom such DNA or RNA, and protein derived ~rom naturally occurring DNA or RNA by mutagenic means such as use of mutayenic PCR primers.

CA 022~7828 1998-12-11 WO 97/47745 PCT~P97/03070 In a ~ourth aspect there are provided vectors, cells and plants and animals comprising the recombinant DNA or RNA o~ correct sense or anti-sense, o~ the invention In a particularly pre~erred use of the ~irst aspect there is provided a method o~ controlling cell or viral growth comprising administering the DNA, RNA or protein o~ 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 o~ the invention or ~ragments thereof that are capable o~ 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 o~ the speci~ic characteristics o~ the plants protein and o~ 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 o~ the SEQ
ID No l 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 o~ more bases.
The present invention will now be illustrated further by reference to the ~ollowing non-limiting Examples.
Further embodiments falling within the scope of the claims attached here~o will occur to those skilled in the light o~ these Figures.
Fig. 1. The sub-~igure A shows the relative lengths of the present Zm~bl protein and the human retinoblastoma proteins. The sub-figure B shows the alignment o~ the CA 022~7828 1998-12-11 W O 97/47745 PCT/~P97/03070 amino acids sequences o~ the Pocket A and Pocket B o~ the ZmRbl with that o~ the Xenopus, chicken, rat and three human protein (Rb, plO7 and pl30).
Fig. 2. This ~igure is a map o~ the main characteristics o~ the WDV virus and the pWori vector derived ~rom WDV
and the positions o~ the deletions and mutations used in order to establish that the LXCXE moti~ is required ~or its replication in plants cells.
EXAMPLE 1.
IsolatiQn Q:E :DNA and protein expressinq clones.
Total RNA was isolated ~rom maize root and mature leaves by grinding the material previously ~rozen in liquid nitrogen essentially as described in $oni et al (1995). The major and minor p75ZmRbl mRNAs were identi~ied by hybridization to a random-primed 32P-labelled PstI internal ~ragment (1.4 kb).
A portion o~ a maize cDNA library (106 p~u) in lZAPII
(Stratagene) was screened by subsequent hybridization to 5'-labelled oligonucleotides designed to be complementary to a known EST sequence o~ homologue maize o~ pl30. These oligonucleotides were 5'-AATAGACACATCGATCAA/G (M.5m, nt positions 1~11-1438) and 5'-GTAATGATACCA~CATGG (M.3c, nt positions 1606-1590)(Isogen Biosciences).
A~ter the second round o~ screeniny, pBluescript SK-25:~ (pBS) phagemids ~rom positive clones were isolated by invivo excision with ExAssist helper phage (Stratagene) according to protocols recommended by the manu~acturer.
DNA sequencing was carried out using a SequenaseTM Kit (USB).
30:~ The 5'-end o~ the mRNAs encoding p75ZmRbl was determined by RACE-PCR. Poly-A~mRNA was puri~ied by chromatography on oligo-dT-cellulose (Amersham). The ~irst strand was synthesized using oligonucleotide DraI35 (5'-GATTTAAAATCAAGCTCC, nt positions 113-96). After denaturation at 90~C ~or 3 min, RNA was eliminated by CA 022~7828 1998-12-11 W O 97/47745 PCT~EP97/03070 RNase treatment, the cDNA recovered and 5'-tailed with terminal trans~erase and dATP. Then a PCR ~ragment was ampli~ied using primer DraI35 and the linker-primer (50 ~ bp) o~ the Stratagene cDNA synthesis kit.
One o~ the positive clones so produced contained a ~4 kb insert that, according to restriction analysis, extended both 5' and 3' o~ 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 ~rame capable o~ encoding a protein o~ 683 amino acids (predicted Mr 75247, p75ZmRbl) ~ollowed by a 1646 bp 3'-untranslated region. Untranslated regions o~
similar length have been also ~ound in m~mm~ ian 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 ~rom 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 o~ the sequences encoding WDV proteins (Sanz and Gutierrez, unpublished). Plasmid p35S.Rbl was constructed by inserting the CaMV 35S promoter (obtained ~rom pWDV3:35SGUS) upstream o~ the ZmRbl cDNA in the pBS
vector. Plasmid p35S.130 ~as constructed by introducing the complete coding sequence o~ human pl30 instead o~
ZmRbl sequences into p35S.Rbl. Plasmid p35.AtB was constructed by substituting sequences encoding the WDV
RepA and RepB ORFs instead o~ 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 022~7828 1998-12-11 position 31 contains a consensus translation start (Kozak, M. J. Mol. Biol. 196, 9~7 (1987)). To determine whether the cDNA contained the ~ull-length ZmRbl coding region, the 5'-end o~ the mRNAs was ampli~ied by RACE-PCR
using an oligonucleotide derived from a region close to the putative initiator AUG, which would produce a fragment o~ ~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 o~ the ~Ipocket~ 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. ~. Cell 66, 1155 (l991))(Lee W. L. et al, Science 235, 1394 (1987);
Bernards et al, Proc. Matl. Acad. Sci USA 86, 6974 (1989)), with the A and B subdomains exhibitlng the highest homology (~50-64~). Interestingly, amino acid C706 in human Rb, critical ~or 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 o~ the sub-domain A and several in the C-terminal area which are potential sites of phosphorilation. A nucleic acid pre~erred group which encodes proteins in which one or more of these sites are CA 022~7828 1998-12-11 W 097/47745 PCT/~P97/03~70 changed or deleted, making the protein more resistant to the phosphorilation and thus, to its ~unctionality, ~or example li~king to E2F or similar. This can be easily carried out by means o~ mutagenesis conducted by means o~
PCR.

In vivo activity.
Replication o~ wheat dwarf geminivirus (WD~) is dep~n~nt upon an intact LXCXE moti~ o~ the viral RepA
protein. This motif can mediate interaction with a member o~ the human Rb ~amily, pl30, in yeasts. There~ore, 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-trans~ormed with a plasmid encoding the ~usion GAL4BD-RepA protein and with plasmids encoding di~erent GAL4AD ~usion protein. The GAL4AD-p75ZmRbl ~usion could also complex with GAL4BD-Rep~ to allow growth o~ the recipient yeast cells in the absence o~
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 ~orm o~
p75ZmRbl. The role o~ the LXCXE moti~ in RepA-p75ZmRbl interaction was assessed using a point mutation in WDV
RepA (E198K) which we previously showed to destroy interaction with human pl30. ~o-trans~ormation o~ ZmRbl with a plasmid encoding the ~usion GAL4BD-RepA(E198K) indicated that the interaction between RepA and p75ZmRbl occurred through the LXCXE moti~.
In this respect, the E198K mutant of WDV RepA behaves similarly to analogous point mutants o~ 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 5~, =: :
CA 022~7828 1998-12-11 1091 (1987); DeCarpio, J. A. et al., ibid., p. 275 (1988)).
Speci~ic interaction between maize p75ZmRbl and WD~
RepA in the yeast two-hybrid system (Fields et al) relied on the ability to reconstitute a ~unctional GAL4 activity ~rom two separated GAL4 ~usion proteins containing the DNA binding domain (GAL4BD) and the activation domain (GAL4AD). Yeast HF7c cells were co-trans~ormed with a plasmid expressing the GAL4BD-RepA or the GAL4BD-RepA(E198K) ~usions and the plasmids expressing the GAL4AD alone (Vec) or fused to human pl30, maize p75 (p75ZmRbl) or a 69 amino acids N-terminal d~letion o~ p75 (p75ZmRbl-DN). Cells were strea~ed on plates with or without histidine according to the distribution shown in the upper le~t corner. The ability to grow in the absence o~ histidine depends on the ~unctional reconstitution o~
a GAL4 activity upon interaction o~ the ~usion proteins, since this triggers expression o~ the HIS3 gene which is under the control o~ a GAL4 responsive element. The growth characteristics o~ these yeast co-trans~ormants correlate with the levels o~ b-galactosidase activity.
Procedures ~or two-hybrid analysis are described in Xie et al (1995). The GAL4AD-ZmRbl ~usions were construed in the pGAD424 vector.

In vivo activity.
Geminivirus DNA replication requires the cellular DNA
replication machinery as well as other S-phase speci~ic ~actors (Davies, J. W. and Stanley, J. Trends Genet. 5, 77 (1989); Lazarowitz, S. Crit. Rev. Plant Sci. 11, 3~7 (1992)). ~onsistent with this requirement, geminivirus in~ection appears to drive non-proli~erating cells into S-phase, as indicated by the accumulation o~ the proli~erating cell nuclear antigen (PCNA), a protein which is not normall~- present in the nuclei o~

CA 022~7828 1998-12-11 W 097/47745 PCT~EP97/03070 di~erentiated cells (Nagar, S., Pedersen, T. J., - Carric~{, K. M., Hanley-Bowdoin, L. and Robertson, D.
Plant Cell 7, 705 (1995)). The inventors ~inding that e~icient WDV DNA replication requires an intact LXCXE
moti~ in RepA coupled with the discovery o~ a plant homolog o~ Rb supports the model that, as in animal cells, sequestration o~ plant Rb by viral Rep~ protein promotes inappropriate entry o~ in~ected cells into S-phase. There~ore, one way to investigate the ~unction o~
p75z-m-Rbl was to measure geminivirus DNA replication in cells trans~ected with a plasmid bearing the ZmRbl sequences under a promoter ~unctional in plant cells, an approach analogous to that previously used in human cells (Uzvolgi, E. et al., Cell Growth Dif~ 2, 297 (1991)).
Accumulation o~ newly replicated viral plasmid DNA was impaired in wheat cells trans~ected with plasmids expressing p75ZmRbl or human pl30, when expression o~ 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, inter~erence with viral DNA replication by p75ZmRbl and human pl30 strongly evidences a role ~or retinoblastoma protein in the control o~ the G1/S
transition in plants. The existence o~ a plant Rb homolog implies that despite their ancient divergence, plant and animal cells use, at least in part, similar regulatory proteins and pathways ~or cell cycle control.
Two lines o~ evidences rein~orce this model. First, a gene encoding a protein that complements speci~ically the G1/S, but not the G2/M transition o~ the budding yeast cdc28 mutant has been identi~ied in al~al~a cells (Hirt, H., Pay, A., Bogre, L., Meskiene, I. and Heberle-Bors, E.
Plant J. 4, 61 (1993)). Second, plant homologs o~ D-type cyclins have been isolated ~rom Arabidopsis and these, like their m~m~1ian relatives, contain LXCXE moti~s. In CA 022~7828 1998-12-11 W O 97/47745 PCTrEP97/03070 concert with plant versions o~ CDK4 and CDK6, plant D-type cyclins may regulate passage through G1 phase by controllin~ the phosphorylation state o~ Rb-like proteins.
In animal cells, the Rb ~amily has been implicated in tumor suppression and in the control o~ differentiation and development. Thus, p75ZmRbl could also play key regulatory roles at other levels during the plant cell li~e One key question that is raised by the existence o~
Rb homologs in plant cells in whether, as in animals disruption o~ 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 o~ both Agrobacterium tume~aciens and A. rhyzogenes contains an LXCXE moti~. Although the VirB4 protein is required ~or tumor induction (Hooykas, P. J. J. and Beijersbergen, A.
G. M. Annu. Rev. Phytopathol. 32, 157 (1994), the ~unction o~ its LXCXE moti~ in this context remains to be examined. Geminivirus in~ection is not accompanied by tumor development in the in~ected plant, but in some cases an abnormal growth of enactions has been observed (G. Da~alla and B. Gronenborn, personal communication).
Inhibition o~ wheat dwarf geminivirus (WDV) DNA
replication by ZmRbl or human pl30 in cultured wheat cells was carried out as ~ollows. A. Wheat cells were trans~ected, as indicated, with pWori (Xie et al. lg95) alone (0.5g), a replicating WDV-based plasmid which encodes WDV proteins required ~or viral DNA replication, and with control plasmid pBS (10 g) or p35S.Rbl (10 g), which encodes ZmRbl sequences under the control o~ the CaMV 35S promoter. Total DNA was puri~ied one and two days a~ter trans~ection, e~ual amounts fractionated in agarose gels and ethidium bromide staining and viral pWori DNA identi~ied by Southern hybridization. Plasmid DNA represents exclusively newly-replicated plasmid DNA

CA 022~7828 l998-l2-ll WO 97/47745 PCT~P97/03070 since it is fully resistant to DpnI digestion and - sensitive to Mbol. Note that the MboI-digested samples were run for about hal:E oE the length than the undigested samples. B. To test the efi~ect of hllm~n 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 e~ect oE ZmRbl or human pl30 on WDV DNA replication when expression o~ viral proteins was directed by the CaMV 35S
promoter, the test plasmid pWori~ (which does not encode Eunctional WDV replication proteins but replicates when they are provided by a di:E~erent plasmid, i. e. pWori) was used Wheat cells were co-transf~ected, as indicated, with pWoriA~ (0.25 g), pWori (0.25 g), p35S.A+B (6 g), p35S.Rbl (10 g) and/or p35S.130 (10 g). Replication o~
the test plasmid (pWori~) was analyzed 36 hours after transi~ection and was detected as described in part A
using ethidium bromide staining; Southern hybridization.
Plasmids pWori (M1) and pWori~ (M2; Sanz and Gutiérrez, unpublished), 100 pg in each case, were used as mar]~ers.
Suspension cultures oE wheat cells, trans~ection by particle bombardment and analysis of viral DNA
replication were carried out as described in (Xie et al.
1995), except that DNA extraction was modi~ied as in ~Soni and Murray. Arnal. Biochem. 218, 474-476 (1995).

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) COM~Ul~ 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

CA 022~7828 l998-l2-ll W O 97147745 PCT~EP97/03070 (B) LOC~TION: 31..2079 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Met Glu Cy6 Phe Glll Ser Asn Leu GAA A~A ATG GAG AAA CTA TGT AAT TCT AAT AGC TGT A~A GGG GAG CTT 102 Glu Lys Met Glu Lys Leu Cys 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 A~:l Leu Gly His Ser Ly~ Cys Ala Phe Glu Thr Leu Ala Ser Pro Thr Lys Thr Ile Lys Asn Met Leu Thr Val Pro Ser S~r Pro Leu 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 Leu Arg Glu Val Ile Ser Ser Leu Pro Asp Lys Pro Ser Ser Lys Leu Gln Glll 105 110 115 120.

Phe Leu Ser Ser Cys Asp Arg Asp Leu Thr Asn Ala Val Thr Glu Arg Val Ser Ile Val Leu Glu Ala lle Phe Pro Thr LyE; Ser Ser Ala As Arg Gly Val Ser Leu Gly Leu A6n Cys Ala Asn Ala Phe Asp Ile Pro 155 160 _ 165 Trp Ala Glu Ala Arg Lys Val Glu Ala Ser Lys Leu Tyr Tyr Arg Val CA 022~7828 l998-l2-ll W 097/47745 PCT~EP97/03070 Leu Glu Ala IIe Cys Arg Ala Glu Leu Glll Asll Ser Asn Val Asn Asn 185 l9Q 195 200 Leu Thr Pro Leu Leu Ser As~l Glu Arg Phe ~is Arg Cys Leu Ile A1R

Cys Ser Ala Asp Leu Val Leu Ala Thr Hi~ Lys Thr Val Ile Met Met Phe Pro Ala Val Leu Glu Ser Thr Gly Leu Thr Ala Phe Asp Leu Ser 23~ 240 245 AAA ATA ATT GAG ~AC TTT GTG AGA CAT GAA GAG ACC CTC CCA AGA GAA 822 Lys Ile Ile Glu Asll Phe Val Arg His G1u Glu Thr Leu Pro Arg Glu ~50 255 26C' TTG AAA AGG cAr CTA AAT TCC TTA GAA GAA CAG CTT TTG GAA AGC ATG 870 Leu Lys Arg Hls _eu Asll Ser Leu Glu Glu Glll Leu Leu Glu Ser Met Ala Trp Glu Lys 5 y Ser Ser Leu Tyr Asn Ser Leu Ile Val Ala Arg ~~5 290 295 Pro Ser Val ALa Ser Glu lle A611 Arg Leu Gly Leu Leu Ala Glu Pro ATG CCA TCT CTT ~~T GAC TTA GTG TCA AGG CAG AAT GTT CGT ATC GAG 1014 Met Pro Ser Leu Asp Asp Leu Val Ser Arg Glll A611 Val Al-g Ile Glu Gly Leu Pro Ala ~hr Pro Ser Ly6 Lys Arg Ala Ala Gly Pro Asp Asp AAC GCT GAT CCT _~A TCA CCA AAG AGA TCG TGC AAT GAA TCT AGG AAC 1110 Asn Ala Asp Pro Arg Ser Pro Lys Arg Ser Cys Asn Glu Ser Arg As 145 35Q 355 36n ACA GTA GTA GAG CGC AAT TTG C~G ACA CCT CCA CCC AAG CAA AGC CAC 1158 Thr Val Val Glu A~-g A6n Leu Gln Thr Pro Pro Pro Lys Glll Ser Hi6 M~t Val Ser Thr '~r Leu Lys Ala Lys Cys His Pro Leu Glll Ser Thr 38C~ 385 390 CA 022~7828 1998-12-11 W 097/47745 PCT~EP97/03070 Phe Ala Ser Pro Thr Val Cys Asn Pro Val Gly Gly Asll Glu Lys Cy~

Ala Asp Val Thl- Ile His Ile Phe Phe Ser Lys Ilc Leu Lys Leu Ala Ala Ile Arg Ile Arg Asn Leu Cys Glu Arg Val Glll Cys Val Glu Gl Thr Glu Arg Val Tyr Asll Val Phe Lys Gln Ile Leu Glu Gln Gln Thr Thr Leu Phe Phc Asn Arg Pis Il~ Asp Glll Leu Ile Leu Cys Cys Leu Tyr Gly Val Ala Lys Val Cys Glll Leu Glu Leu Thr Phe Arg Glu Ile Leu Asll Asn Tyr Lys Arg Glu Ala Glll Cys Lys Pro Glu Val Phe Ser Ser Ile Tyl Ile Gly Ser Thr Asll Arg Asn Gly Val Leu Val Ser Arg 505 510 515 . 520 Hls Val Gly Ile Ile Thr Phe Tyr Asn Glu Val Phe Val Pro Ala Ala Lys Pro Phe Leu Val Ser Leu Ile Ser Ser Gly Thr Hls Pro Glu Asp Lys Lys Asll Ala Ser Gly Gll~ Ile Pro Gly S~r Pro Lys Pro Ser Pro Phe Pro Asll Leu Pro Asp Met Ser Pro Lys Lys Val Ser Ala S~r His 57~ 575 580 Asll Val Tyr Val Ser Pro Leu Arg G11l Thr Lys Leu Asp Leu Leu Leu CA 022~7828 l998-l2-ll W 097/47745 PCT~EP97/03070 Ser Pro Ser Ser Arg Scr Phe Tyr Ala Cy6 Ile Gly Glu Gly Thr Hi6 GCT TAT CAG AGC CCA TCT AAG GAT TTG GCT GCT ATA AAT AGC CGC CTA l926 Ala Tyr Gln Ser Pro S~r Lys A6p Leu Ala Ala Ile A6n Ser Arg Leu As~1 Tyr A611 Gly Arg Ly6 Val A611 Ser Arg Leu A611 Phe Asp Met Val 635 6~0 645 Ser A6p Ser Val Val Ala Gly Ser Leu Gly Gl11 Ile A611 Gly Gly Ser hCC TCG GAT CCT GCA GCT GCA TTT AGC CCC rTT TCA AAG AAG AGA GAG 2070 Thr Ser Asp P1-o Ala Ala Ala Ph~ Ser Pro Leu Ser Ly6 Lys Arg Glu ~'' 670 675 680 ACA GAT ACT TGATCAATTA TAAATGGTGG CCTCTCTCGT ATATAGCTC~ 2119 Thr A6p Thr CAGATCCGTG CTCCGTAGCA GTCTATTCTT CTGAATAAGT GGATTAACTG GAGCGATTTA 217 g .h~ iAG CTATCACCAT TTCTCTGCTG ATTGAACATA TCCGCTGTGT AGAGTGCTAA 2299 TGhATCTTTA GTTTTCATTG GGCTGACATA ACAAATCTTT ATCCTAGTTG G~l~i~ll~ill 2359 GGGAGGCATT CATCAGGGTT ATATTTGGTT GTCAAAAAGT ACTGTACTTA ATTCACATCT 24:L9 TTACAGGTAT AAGCATGCCA ACTCTAAACT ATATGAATCC TTTTTATATT CTCATTTTTA '>539 AGTACTTCTC I~ ACTTTTGTAC TGTATATTTC CAIi~ll~l~C ~TCAGACTGA 2599 55TTACA~TC AGCCTTTACT GCTTTATATT GTTCTACTAA l'lll'~iliL~L~i CACAGCCAGG 2779 ,'.CGATTGGTT TTCTGCATCA ATCAATCTTT TTTAGGACAA GATATTTTTG TATGCTACAC 2839 TTCCCAAATT GCAATTAATC CAGAAGTCTA ~ LA TTCTATTAGT TCTCAGCAAC 2899 AGTGAATGAA TATG~ATCAG TCATGCTGAT AGATGTTCAT CTGGTTATTC t~AAAcAATcT 2959 CA 022~7828 1998-12-11 WO 97/47745 PCT~P97/03070 TGTACGGTAG TGATGGCAGG AGACGTTCTT A~ACAGCAGC TGCTCCTTCA GCTTGTAATG 3139 TTCCAGCTGC ACCGTGTCAA ATGTGAAGGC CCTGCAAGGC TTTCCAGGCC GCACCAATCC 33l9 TGCTTGCTTC TTGAAGATAC ATATGGTGCC ACCTAAATAA AAG~~ . TGGTTATGTC 3379 TGAGAGCTAC ~ "lG~AG CAAACGGAGG GCGATATAGG TTGATAGCCA ATTTCAGTTC 3559 TCTATATACA AGCAGCGGAT lll~lllAGA GTTAGCTTTT GAGATGCATC Alll~lll~A 3619 CATCTGATTC T,l~l~ll~l AACTCGGAGT CGCGTAGAAG TTAGAATGCT AACTGACCTT 3679 AATTTTCACC GAATAATTTG CTAGCGTTTT TCAGTATGAA AT~ll~l~l TAAAAAAAAA 3739 (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 Gl1~ Ser Asn Leu Glu Lys Met Glu Ly6 Leu Cy6 A
l 5 lO 15 -Ser AB1~ Ser Cys Lys Gly Glu Leu A6p Phe Lys Ser Ile Le~l Ile Asn A611 Asp Tyr Ile Pro Tyr Asp Glu Asn Ser Thr Gly Asp Ser Thr A

Leu Gly ~i6 Ser Lys Cy6 Ala Phe Glu Thr Leu Ala Ser Pro Thr Ly6 ~ ' CA 022~7828 1998-12-11 W O 97/47745 PCT~P97/03070 Thr Ile Ly6 Asn Met Leu Thr Val Pro S~r Ser Pro Leu Ser Pro Ala ~hr Gly Gly Ser Val Lys Ile Val Gln Met Thr Pro Val Thr Ser Ala ~~t Thî Thr Ala LYB Trp Leu Arg Glu Val Ilc Ser Ser Leu Pro Asp Ly6 Pro Ser Ser Ly6 Leu Gln Gln Phe Leu Ser Ser Cys A6p Arg A6p Leu Thr Asn Ala Val Thr Glu Arg Val Ser Ile Val Leu Glu Ala Ile 13(J 135 140 Phe~ E'ro Thl- Ly6 Ser S~r Ala Asn Arg Gly Val Ser Leu Gly Leu A

~yF Ala Asll Ala Phe Asp Ile Pro T1-p Ala Glu Ala A~-g Lys Val Glu ~la Ser Lys Leu Tyr Tyr Arg Val Leu Glu Ala Ile Cys Arg Ala Glu Leu Glll Asn Ser Asll Val Asll Asn Leu Thr Pro Leu Leu Ser Asll Glu 1~5 200 205 Arg Phe H1s Arg Cy6 Leu Ile Ala Cy~ Ser Ala Asp Leu Val Leu Ala Th~ i6 Lys Thr Val Ile Met Met Phe Pro Ala Val Leu Glu Ser Thl-' '5 230 235 Z40 ~ly Le~l T}lr Ala Plle Asp Leu Ser Lys Ile Ile Glu Asn Phe Val Arg245 250 255 ~is Glu Glu Thr L~u Pro Arg Glu Leu Lys Arg Hi6 Leu Asll Ser Leu Glu Glu Glll Leu Leu Glu Ser Met Ala Trp Glu Ly6 Gly Ser Ser Leu Tyr Asll Ser Leu Ile Val Ala Arg Pro Ser Val Ala Ser Glu Ile As ~0 295 300 Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser Leu Asp Asp Leu Val ~er Arg Glll Asll Val Arg Ile Glu Gly Leu Plo Ala Thr Pro Ser Lys ~y6 Arg Ala Ala Gly Pro A6p A6p AB1l Ala A6p Pro Arg Ser Pro Lys CA 022~7828 1998-12-11 W 097/47745 PCT~EP97/03070 Arg Ser Cys Asn Glu Ser Arg A611 Thr Val Val Glu Arg A6n Leu Gl Thr Pro Pro Pro Ly6 Gl11 Ser Hi6 Met. Val Ser Thr Ser Leu Ly6 Ala Ly6 Cy6 Hi6 Pro Leu Gl11 Ser Thr Phe Ala Ser Pro Thr Val Cy6 A6n Pro Val Gly Gly Asr~ Glu Ly6 CYB Ala ABP Val Thr Ile His Ilc Phe Phe Ser LYB ~le Leu Ly6 Leu Ala Ala Ile Arg Ile Arg A611 Leu Cy6 Glu Arg Val Gl11 CYB Val Glu Gl11 Thr Glu Arg Val Tyr A611 Val Phe Ly~ Gl1~ Ile Leu Glu Gl11 Gl11 Thr Thr Leu P1~e Phe A611 Arg Hi6 Ile A6p Gl1~ L~u Ile Leu Cy6 Cy6 Leu Tyr Gly Val Ala Ly6 Val Cy6 Gl Leu Glu Leu Thl- Phe Arg Glu Ile Leu A611 A611 Tyr Lys Arg Glu Ala Gln CYB LYB Pro Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr Acn Arg AB11 Gly ial Leu Val Ser Arg His Val Gly Ile Ile Thr Phe Tyr 51~ 520 525 AB11 Glu Val Phe Val Pro Ala Ala Ly6 Pro Phe Leu Val Ser Leu Ile Ser Ser Gly Thr Hi6 Pro Glu A6p Ly6 Ly6 A611 Ala Ser Gly Gl11 Ile 545 550 555 . 560 Pro Gly Ser Pro Ly6 Pro Ser Pro Phe Pro A611 Leu Pro A6p Met Ser Pro LYB Ly6 Val Ser Ala Ser Hi6 A611 Val Tyr Val Ser Pro Leu Arg Gl~ Thr LYB Leu ABP Leu Leu Leu Ser Pro Ser Ser Arg S~r Phe Tyr 59~ 600 605 - Ala Cy6 Ile Gly Glu Gly Thr Hi6 Ala Tyr Gl11 Ser Pro Ser Ly6 A6p Leu Ala Ala _le AB11 Ser Arg Leu A~.11 Tyr AB11 Gly Arg Lys Val AB11 W O 97/47745 PCT~EP97/03070 Ser Arg Leu A311 Phe A~p Met Val Ser A~p Ser Val Val Ala Gly Ser Leu Gly Gll1 Ile ~11 Gly Gly Ser Thr Ser A~p Pro Ala Ala Ala Phe Ser Pro Leu S~r LYG Ly~ Arg Glu Thr A~p Thr W O 97147745 PCT~EP97/03070 INFORMATION RELATIVE TO THE DEPOSIT O~ A MICRO-ORGANISM
The micro-organism to which re~erence is made in page 6 o~ the disclosure has been deposited in the ~ollowing institution:
5 COLECCION ESPANOLA DE CULTIVOS TIPO (CECT) Departamento de Microbiologla Facultad de Ciencias Biologicas 46100 BURJASOT (Valencia) Spain Deposit identi~ication: pBS.Rbl Deposit date: June 12, 1996 Order No.: 4699 This in~ormation appears re~lected in the ~orm PCE/RO/134 enclosed to the request.

W 097/47745 PCTrEP97/03070 I~nUICAl~O~S RUELATIYG TO A ~Epo~srrE~ MICROORGh~MlSM
~T ~el3~) 6 . 24 snd o ow~ng CKS p~gC . ~ne ~ C ~ON O~ DEPOSrr pBS . Rbl ~u~t~dq~it~lrei~?ifi~io~sl~-t~ cat COLECCION ESPA~OLA DE CULTIVOS TIPO (CECT) A~Yqf~ ~ y - ~d~p~o~) Departamento de MicrobioLogia FacuLtad de Ciencias Bio~ogicas 46100 BURJASOT (VaLencia~
Spain Dalc Rf d~posit ~ N~
12 June 1996 1 4699 C J~l~DlI~OlYAI. ~NV~ NS r~ ~ f~ ~ u ~t a D. DESIG~.~TfiD S~rEs FoR W~C~ I.NDIC~ TlONS ~RE h~hDE t~ ~ror~ ' 'S' ~ SJ~ATE FllRNIS~N~ OF ~nI~ ol~5 rZ~ ~l~y~ J
n . . ~ 1. w~ ~ ~ ~c - ~ , SL ~UW~ s ~Q~S~

r~,.. ~,.. ,~Of~ n~nly ~o~ ~n~n~ Bu~uU5Co~y ~et~~v~rccci~ed~ hc .~ " ~ n ~ r~ edbyt~e r - ~ B~re~

YI cfJice~ _ A ~ ' of fic~:r .~.A.PASCHE
.~m PCT~~O~34(~y1992~

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 of 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 inter with viral LXCXE motif without effecting 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 characterized 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 in claim 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.

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.
a 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 is 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 charged 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 SP or TP sites round in the SEQ ID No. 2 have been changed or deleted.

17. A recombinant vector characterised in that in 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 nuleic 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 retincoblastoma 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.