U.S. patent application number 09/770657 was filed with the patent office on 2002-04-18 for plant proteins.
This patent application is currently assigned to Consejo Superior de Investigaciones Cientificas. Invention is credited to Gutierrez-Armenta, Crisanto, Sanz-Burgos, Andres Pelayo.
Application Number | 20020046416 09/770657 |
Document ID | / |
Family ID | 8293501 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020046416 |
Kind Code |
A1 |
Gutierrez-Armenta, Crisanto ;
et al. |
April 18, 2002 |
Plant proteins
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.
Inventors: |
Gutierrez-Armenta, Crisanto;
(Cantoblanco, ES) ; Sanz-Burgos, Andres Pelayo;
(Cantoblanco, ES) |
Correspondence
Address: |
Nixon & Vanderhye P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201
US
|
Assignee: |
Consejo Superior de Investigaciones
Cientificas
|
Family ID: |
8293501 |
Appl. No.: |
09/770657 |
Filed: |
January 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09770657 |
Jan 29, 2001 |
|
|
|
09213293 |
Dec 14, 1998 |
|
|
|
Current U.S.
Class: |
800/279 ;
536/23.6; 800/278; 800/301 |
Current CPC
Class: |
C07K 14/415 20130101;
Y02A 40/146 20180101; C12N 15/8261 20130101; C12N 15/8283
20130101 |
Class at
Publication: |
800/279 ;
800/278; 800/301; 536/23.6 |
International
Class: |
C12N 015/82; A01H
005/00; C12N 015/29 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 1996 |
ES |
PCT/ES96/00130 |
Claims
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 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 DWA 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 in 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.
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.
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.
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 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.
Description
DESCRIPTION
[0001] 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.
[0002] 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)).
[0003] 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., Surez-Lpez,
P. and Gutirrez, 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. and Murray, J. A. H. Plant Cell
7, 85-103 (1995)).
[0004] The present inventors have now identified characteristic
sequences of plant Rb proteins and corresponding encoding
polynucleotides for the first time, isolated such a protein and
polynucleotide, and particularly have identified sequences that
distinguish it from known animal Rb protein sequences. The
inventors have determined that a known DNA sequence from the maize
encoding a vegetable Rb plant protein and is hereinafter called
ZmRb1. ZmRb1 has been demonstrated by the inventors to interact in
yeasts with RepA, a plant geminivirus protein containing LXCXE
motif essential for its function. The inventors have further
determined that geminivirus DNA replication is reduced in plant
cells transfected with plasmids encoding either ZmRb1 or human
p130, a member of the human Rb family.
[0005] 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 ZmRb1
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.
[0006] 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.
[0007] A preferred method of control using such proteins involves
applying these to the plant cell, either directly or by
introduction of DNA or RNA encoding for their expression into the
plant cell which it is desired to treat. 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.
[0008] 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.
[0009] 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.
[0010] The DNA or RNA can have a sequence that contains the
degenerated substitution in the nucleotides of the codons in SEQ ID
No. 1, and in where the RNA the T is U. The most preferred DNA or
RNA are capable of hybridate with the polynucleotide of the SEQ ID
No. 1 in conditions of low stringency, preferably being the
hybridization produced in conditions of high stringency.
[0011] The expressions "conditions of low stringency" and
"conditions of high stringency" are understood by those skilled,
but are conveniently exemplified in U.S. Pat. No. 5,202,257,
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 U.S. Pat. No. 5,380,712,
and it is only contemplated when the protein has activity with
retinoblastoma protein.
[0012] Preferred DNA or cRNA encodes for a plant Rb protein having
A and B pocket sub-domains having between 30% and 75% homology with
human Rb protein, particularly as compared with p130, more
preferably from 50% to 64% homology. 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 NO 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 ZmRb1 of SQ ID No 2.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] Alternatively, the method uses an expression vector capable
of producing anti-sense RNA of the cDNA of the invention.
[0017] 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.
[0018] 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.
[0019] Figures.
[0020] FIG. 1. The sub-figure A shows the relative lengths of the
present ZmRb1 protein and the human retinoblastoma proteins. The
sub-figure B shows the alignment of the amino acids sequences of
the Pocket A and Pocket B of the ZmRb1 with that of the Xenopus,
chicken, rat and three human protein (Rb, p107 and p130).
[0021] 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
[0022] Isolation of DNA and Protein Expressing Clones.
[0023] 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
p75ZmRb1 mRNAs were identified by hybridization to a random-primed
32P-labelled PstI internal fragment (1.4 kb).
[0024] A portion of a maize cDNA library (106 pfu) in IZAPII
(Stratagene) was screened by subsequent hybridization to
5'-labelled oligonucleotides designed to be complementary to a
known EST sequence of homologue maize of p130. These
oligonucleotides were 5'-AATAGACACATCGATCAA/G (M.5m, nt positions
1411-1438) and 5'-GTAATGATACCAACATGG (M.3c, nt positions 1606-1590)
(Isogen Biosciences).
[0025] 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).
[0026] The 5'-end of the mRNAs encoding p75ZmRb1 was determined by
RACE-PCR. Poly-A+mRNA was purified by chromatography on
oligo-dT-cellulose (Amersham). The first strand was synthesized
using oligonucleotide DraI35 (5'-GATTTAAAATCAAGCTCC, nt positions
113-96). After denaturation at 90.degree. C. for 3 min, RNA was
eliminated by RNase treatment, the cDNA recovered and 5'-tailed
with terminal transferase and DATP. Then a PCR fragment was
amplified using primer DraI35 and the linker-primer (50 bp) of the
Stratagene cDNA synthesis kit.
[0027] One of the positive clones so produced contained a .about.4
kb insert that, according to restriction analysis, extended both 5'
and 3' of the region contained in the Expressed Sequence Tag used.
The nucleotide sequence corresponding to the longest cDNA insert
(3747 bp) is shown in SEQ ID No. 1. This ZmRb1 cDNA contains a
single open reading frame capable of encoding a protein of 683
amino acids (predicted Mr 75247, p75ZmRb1) followed by a 1646 bp
3'-untranslated region. Untranslated regions of similar length have
been also found in mammalian Rb cDNAs (Lee, W. -L. et al, Science
235, 1394 (1987); Bernards, R. 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, .about.2.7.+-.0.2 kb in length. In addition, a minor
.about.3.7.+-.0.2 kb message also appears. Heterogeneous
transcripts have been detected in other species (Destre, O. H. J.
et al, Dev. Biol. 153, 141 (1992)).
[0028] Plasmid pWori.DELTA..DELTA. was constructed by deleting in
pWori most of the sequences encoding WDV proteins (Sanz and
Gutierrez, unpublished). Plasmid p35S.Rb1 was constructed by
inserting the CaMV 35S promoter (obtained from pWDV3:35SGUS)
upstream of the ZmRb1 cDNA in the pBS vector. Plasmid p35S.130 was
constructed by introducing the complete coding sequence of human
p130 instead of ZmRb1 sequences into p35S.Rb1. Plasmid p35.A+B was
constructed by substituting sequences encoding the WDV RepA and
RepB ORFs instead of ZmRb1 in p35S.Rb1 plasmid. (See Soni, R. and
Murray, J. A. H. Anal. Biochem. 218, 474-476 (1994)).
[0029] The sequence around the methionine codon at nucleotide
position 31 contains a consensus translation start (Kozak, M. J.
Mol. Biol. 196, 947 (1987)). To determine whether the cDNA
contained the full-length Zmb1 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 .about.150 bp. The results are consistent
with the ZmRb1 cDNA clone containing the complete coding
region.
[0030] The ZmRb1 protein contains segments homologous to the A and
B subdomains of the "pocket" that is present in all members of the
Rb family. These subdomains are separated by a non-conserved
spacer. ZmRb1 also contains non-conserved N-terminal and C-terminal
domains. Overall, ZmRb1 shares .about.28-30% amino acid identity
(.about.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 (1991)) (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 (.about.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 p75ZmRb1.
[0031] Note: The 561-577 amino acids encompass a proline-rich
domain.
[0032] ZmRb1 contains 16 consensus sites, SP or TP for
phosphorilation by cyclins dependant kinases (CDKs) with one of the
5'-tail of the sub-domain A and several in the C-terminal area
which are potential sites of phosphorilation. A nucleic acid
preferred group which encodes proteins in which one or more of
these sites are changed or deleted, making the protein more
resistant to the phosphorilation and thus, to its functionality,
for example linking to E2F or similar. This can be easily carried
out by means of mutagenesis conducted by means of PCR.
EXAMPLE 2
[0033] In vivo Activity.
[0034] 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, p130,
in yeasts. Therefore, the inventors investigated whether p75ZmRb1
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-p75ZmRb1 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 p130 protein. RepA could also bind to some extent to
a N-terminally truncated form of p75ZmRb1. The role of the LXCXE
motif in RepA-p75ZmRb1 interaction was assessed using a point
mutation in WDV RepA (E198K) which we previously showed to destroy
interaction with human p130. Co-transformation of ZmRb1 with a
plasmid encoding the fusion GAL4BD-RepA(E198K) indicated that the
interaction between RepA and p75ZmRb1 occurred through the LXCXE
motif.
[0035] 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, 1091 (1987); DeCarpio, J. A. et al., ibid., p. 275
(1988)).
[0036] Specific interaction between maize p75ZmRb1 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 p130, maize p75 (p75ZmRb1) or a 69 amino acids N-terminal
deletion of p75 (p75ZmRb1-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.
[0037] Procedures for two-hybrid analysis are described in Xie et
al (1995). The GAL4AD-ZmRb1 fusions were construed in the pGAD424
vector.
EXAMPLE 3
[0038] In vivo Activity.
[0039] 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
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 p75ZmRb1 was to measure
geminivirus DNA replication in cells transfected with a plasmid
bearing the ZmRb1 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 p75ZmRb1 or human p130, when
expression of WDV replication protein(s) is directed wither by the
WDV promoter or by the CaMV 35S promoter.
[0040] Since WDV DNA replication requires an S-phase cellular
environment, interference with viral DNA replication by p75ZmRb1
and human p130 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.
[0041] 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., Py, 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
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.
[0042] In animal cells, the Rb family has been implicated in tumor
suppression and in the control of differentiation and development.
Thus, p75ZmRb1 could also play key regulatory roles at other levels
during the plant cell life. One key question that is raised by the
existence of Rb 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 LXCXE motif. Although the VirB4 protein is
required for tumor induction (Hooykas, P. J. J. and Beijersbergen,
A. G. M. Annu. Rev. Phytopathol. 32, 157 (1994), the function of
its LXCXE motif in this context remains to be examined. Geminivirus
infection is not accompanied by tumor development in the infected
plant, but in some cases an abnormal growth of enactions has been
observed (G. Dafalla and B. Gronenborn, personal
communication).
[0043] Inhibition of wheat dwarf geminivirus (WDV) DNA replication
by ZmRb1 or human p130 in cultured wheat cells was carried out as
follows. A. Wheat cells were transfected, as indicated, with pWori
(Xie et al. 1995) alone (0.5 g), a replicating WDV-based plasmid
which encodes WDV proteins required for viral DNA replication, and
with control plasmid pBS (10 g) or p35S.Rb1 (10 g), which encodes
ZmRb1 sequences under the control of the CaMV 35S 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 DNA
represents exclusively newly-replicated plasmid DNA 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 p130 on
WDV DNA replication, wheat cells were co-transfected with pWori
(0.5 g) and plasmids pBS (control), p35S.Rb1 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 ZmRb1 or human p130 on WDV DNA replication when
expression of viral proteins was directed by the CaMV 35S promoter,
the test plasmid pWori.DELTA..DELTA. (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 pWori.DELTA..DELTA. (0.25
g), pWori (0.25 g), p35S.A+B (6 g), a p35S.Rb1 (10 g) and/or
p35S.130 (10 g). Replication of the test plasmid
(pWori.DELTA..DELTA.) was analyzed 36 hours after transfection and
was detected as described in part A using ethidium bromide
staining; Southern hybridization. Plasmids pWori (M1) and
pWori.DELTA..DELTA. (M2; Sanz and Gutirrez, 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).
Sequence CWU 1
1
2 1 3747 DNA Zea mays 1 gaattcggca cgagcaaagg tctgattgat atggaatgtt
tccagtcaaa tttggaaaaa 60 atggagaaac tatgtaattc taatagctgt
aaaggggagc ttgattttaa atcaattttg 120 atcaataatg attatattcc
ctatgatgag aactcgacgg gggattccac caatttagga 180 cattcaaagt
gtgcctttga aacattggca tctcccacaa agacaataaa gaacatgctg 240
actgttccta gttctccttt gtcaccagcc accggtggtt cagtcaagat tgtgcaaatg
300 acaccagtaa cttctgccat gacgacagct aagtggcttc gtgaggtgat
atcttcattg 360 ccagataagc cttcatctaa gcttcagcag tttctgtcat
catgcgatag ggatttgaca 420 aatgctgtca cagaaagggt cagcatagtt
ttggaagcaa tttttccaac caaatcttct 480 gccaatcggg gtgtatcgtt
aggtctcaat tgtgcaaatg cctttgacat tccgtgggca 540 gaagccagaa
aagtggaggc ttccaagttg tactataggg tattagaggc aatctgcaga 600
gcggagttac aaaacagcaa tgtaaataat ctaactccat tgctgtcaaa tgagcgtttc
660 caccgatgtt tgattgcatg ttcagcggac ttagtattgg cgacacataa
gacagtcatc 720 atgatgtttc ctgctgttct tgagagtacc ggtctaactg
catttgattt gagcaaaata 780 attgagaact ttgtgagaca tgaagagacc
ctcccaagag aattgaaaag gcacctaaat 840 tccttagaag aacagctttt
ggaaagcatg gcatgggaga aaggttcatc attgtataac 900 tcactgattg
ttgccaggcc atctgttgct tcagaaataa accgccttgg tcttttggct 960
gaaccaatgc catctcttga tgacttagtg tcaaggcaga atgttcgtat cgagggcttg
1020 cctgctacac catctaaaaa acgtgctgct ggtccagatg acaacgctga
tcctcgatca 1080 ccaaagagat cgtgcaatga atctaggaac acagtagtag
agcgcaattt gcagacacct 1140 ccacccaagc aaagccacat ggtgtcaact
agtttgaaag caaaatgcca tccactccag 1200 tccacatttg caagtccaac
tgtctgtaat cctgttggtg ggaatgaaaa atgtgctgac 1260 gtgacaattc
atatattctt ttccaagatt ctgaagttgg ctgctattag aataagaaac 1320
ttgtgcgaaa gggttcaatg tgtggaacag acagagcgtg tctataatgt cttcaagcag
1380 attcttgagc aacagacaac attatttttt aatagacaca tcgatcaact
tatcctttgc 1440 tgtctttatg gtgttgcaaa ggtttgtcaa ttagaactca
cattcaggga gatactcaac 1500 aattacaaaa gagaagcaca atgcaagcca
gaagtttttt caagtatcta tattgggagt 1560 acgaaccgta atggggtatt
agtatcgcgc catgttggta tcattacttt ttacaatgag 1620 gtatttgttc
cagcagcgaa gcctttcctg gtgtcactaa tatcatctgg tactcatcca 1680
gaagacaaga agaatgctag tggccaaatt cctggatcac ccaagccatc tcctttccca
1740 aatttaccag atatgtcccc gaagaaagtt tcagcatctc ataatgtata
tgtgtctcct 1800 ttgcggcaaa ccaagttgga tctactgctg tcaccaagtt
ccaggagttt ttatgcatgc 1860 attggtgaag gcacccatgc ttatcagagc
ccatctaagg atttggctgc tataaatagc 1920 cgcctaaatt ataatggcag
gaaagtaaac agtcgattaa atttcgacat ggtgagtgac 1980 tcagtggtag
ccggcagtct gggccagata aatggtggtt ctacctcgga tcctgcagct 2040
gcatttagcc ccctttcaaa gaagagagag acagatactt gatcaattat aaatggtggc
2100 ctctctcgta tatagctcac agatccgtgc tccgtagcag tctattcttc
tgaataagtg 2160 gattaactgg agcgatttaa ctgtacatgt atgtgttagt
gagaagcagc agtttttagg 2220 cagcaaactg tttcaagtta gcttttgagc
tatcaccatt tctctgctga ttgaacatat 2280 ccgctgtgta gagtgctaat
gaatctttag ttttcattgg gctgacataa caaatcttta 2340 tcctagttgg
ctggttgttg ggaggcattc atcagggtta tatttggttg tcaaaaagta 2400
ctgtacttaa ttcacatctt tcacattttt cactagcaat agcagcccca aattgctttc
2460 ctgactagga acatattctt tacaggtata agcatgccaa ctctaaacta
tatgaatcct 2520 ttttatattc tcatttttaa gtacttctct gtttctgcta
cttttgtact gtatatttcc 2580 agcttctcca tcagactgat gatcccatat
tcagtgtgct gcaagtgatt tgaccatatg 2640 tggcttatcc ttcaggtatg
tctcatgttg tgacttcatt gctgattgct tttgtaatgg 2700 tactgttgag
ttcatttctg gttacaatca gcctttactg ctttatattg ttctactaat 2760
tttggcttgc acagccagga cgattggttt tctgcatcaa tcaatctttt ttaggacaag
2820 atatttttgt atgctacact tcccaaattg caattaatcc agaagtctac
cttgttttat 2880 tctattagtt ctcagcaaca gtgaatgaat atgaatcagt
catgctgata gatgttcatc 2940 tggttattcc aaacaatctg acatcgcatc
tctttctgca agtgagatga agaaaacctg 3000 aaatgctatc accatttaaa
acattggctt ctggaagttc aggtgattag caggagacgt 3060 tctgacattg
ccattgacat gtacggtagt gatggcagga gacgttctta aacagcagct 3120
gctccttcag cttgtaatgt ctgattgtat tgaccaagag catccacctt gccttatggt
3180 actaactgaa tgagctggtg acgctgactc atctgcataa tggcagatgc
ttaaccatct 3240 ttaggagctc atgtcatgat tccagctgca ccgtgtcaaa
tgtgaaggcc ctgcaaggct 3300 ttccaggccg caccaatcct gcttgcttct
tgaagataca tatggtgcca cctaaataaa 3360 agctgtttct ggttatgtct
gtccttgaca tgtcaacaga ttagtgttgg gttgcagtca 3420 tgtggtgttt
aagtcttgga gaaggcgaga agtcattgct gccagcattg tgatcgtcag 3480
gcacagaagt actcaaaagt gagagctact tgttgcgagc aaacggaggg cgatataggt
3540 tgatagccaa tttcagttct ctatatacaa gcagcggatt ttgtttagag
ttagcttttg 3600 agatgcatca tttctttcac atctgattct gtgtgttgta
actcggagtc gcgtagaagt 3660 tagaatgcta actgacctta attttcaccg
aataatttgc tagcgttttt cagtatgaaa 3720 tccttgtctt aaaaaaaaaa aaaaaaa
3747 2 683 PRT Unknown Description of Unknown Organism protein 2
Met Glu Cys Phe Gln Ser Asn Leu Glu Lys Met Glu Lys Leu Cys Asn 1 5
10 15 Ser Asn Ser Cys Lys Gly Glu Leu Asp Phe Lys Ser Ile Leu Ile
Asn 20 25 30 Asn Asp Tyr Ile Pro Tyr Asp Glu Asn Ser Thr Gly Asp
Ser Thr Asn 35 40 45 Leu Gly His Ser Lys Cys Ala Phe Glu Thr Leu
Ala Ser Pro Thr Lys 50 55 60 Thr Ile Lys Asn Met Leu Thr Val Pro
Ser Ser Pro Leu Ser Pro Ala 65 70 75 80 Thr Gly Gly Ser Val Lys Ile
Val Gln Met Thr Pro Val Thr Ser Ala 85 90 95 Met Thr Thr Ala Lys
Trp Leu Arg Glu Val Ile Ser Ser Leu Pro Asp 100 105 110 Lys Pro Ser
Ser Lys Leu Gln Gln Phe Leu Ser Ser Cys Asp Arg Asp 115 120 125 Leu
Thr Asn Ala Val Thr Glu Arg Val Ser Ile Val Leu Glu Ala Ile 130 135
140 Phe Pro Thr Lys Ser Ser Ala Asn Arg Gly Val Ser Leu Gly Leu Asn
145 150 155 160 Cys Ala Asn Ala Phe Asp Ile Pro Trp Ala Glu Ala Arg
Lys Val Glu 165 170 175 Ala Ser Lys Leu Tyr Tyr Arg Val Leu Glu Ala
Ile Cys Arg Ala Glu 180 185 190 Leu Gln Asn Ser Asn Val Asn Asn Leu
Thr Pro Leu Leu Ser Asn Glu 195 200 205 Arg Phe His Arg Cys Leu Ile
Ala Cys Ser Ala Asp Leu Val Leu Ala 210 215 220 Thr His Lys Thr Val
Ile Met Met Phe Pro Ala Val Leu Glu Ser Thr 225 230 235 240 Gly Leu
Thr Ala Phe Asp Leu Ser Lys Ile Ile Glu Asn Phe Val Arg 245 250 255
His Glu Glu Thr Leu Pro Arg Glu Leu Lys Arg His Leu Asn Ser Leu 260
265 270 Glu Glu Gln Leu Leu Glu Ser Met Ala Trp Glu Lys Gly Ser Ser
Leu 275 280 285 Tyr Asn Ser Leu Ile Val Ala Arg Pro Ser Val Ala Ser
Glu Ile Asn 290 295 300 Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser
Leu Asp Asp Leu Val 305 310 315 320 Ser Arg Gln Asn Val Arg Ile Glu
Gly Leu Pro Ala Thr Pro Ser Lys 325 330 335 Lys Arg Ala Ala Gly Pro
Asp Asp Asn Ala Asp Pro Arg Ser Pro Lys 340 345 350 Arg Ser Cys Asn
Glu Ser Arg Asn Thr Val Val Glu Arg Asn Leu Gln 355 360 365 Thr Pro
Pro Pro Lys Gln Ser His Met Val Ser Thr Ser Leu Lys Ala 370 375 380
Lys Cys His Pro Leu Gln Ser Thr Phe Ala Ser Pro Thr Val Cys Asn 385
390 395 400 Pro Val Gly Gly Asn Glu Lys Cys Ala Asp Val Thr Ile His
Ile Phe 405 410 415 Phe Ser Lys Ile Leu Lys Leu Ala Ala Ile Arg Ile
Arg Asn Leu Cys 420 425 430 Glu Arg Val Gln Cys Val Glu Gln Thr Glu
Arg Val Tyr Asn Val Phe 435 440 445 Lys Gln Ile Leu Glu Gln Gln Thr
Thr Leu Phe Phe Asn Arg His Ile 450 455 460 Asp Gln Leu Ile Leu Cys
Cys Leu Tyr Gly Val Ala Lys Val Cys Gln 465 470 475 480 Leu Glu Leu
Thr Phe Arg Glu Ile Leu Asn Asn Tyr Lys Arg Glu Ala 485 490 495 Gln
Cys Lys Pro Glu Val Phe Ser Ser Ile Tyr Ile Gly Ser Thr Asn 500 505
510 Arg Asn Gly Val Leu Val Ser Arg His Val Gly Ile Ile Thr Phe Tyr
515 520 525 Asn Glu Val Phe Val Pro Ala Ala Lys Pro Phe Leu Val Ser
Leu Ile 530 535 540 Ser Ser Gly Thr His Pro Glu Asp Lys Lys Asn Ala
Ser Gly Gln Ile 545 550 555 560 Pro Gly Ser Pro Lys Pro Ser Pro Phe
Pro Asn Leu Pro Asp Met Ser 565 570 575 Pro Lys Lys Val Ser Ala Ser
His Asn Val Tyr Val Ser Pro Leu Arg 580 585 590 Gln Thr Lys Leu Asp
Leu Leu Leu Ser Pro Ser Ser Arg Ser Phe Tyr 595 600 605 Ala Cys Ile
Gly Glu Gly Thr His Ala Tyr Gln Ser Pro Ser Lys Asp 610 615 620 Leu
Ala Ala Ile Asn Ser Arg Leu Asn Tyr Asn Gly Arg Lys Val Asn 625 630
635 640 Ser Arg Leu Asn Phe Asp Met Val Ser Asp Ser Val Val Ala Gly
Ser 645 650 655 Leu Gly Gln Ile Asn Gly Gly Ser Thr Ser Asp Pro Ala
Ala Ala Phe 660 665 670 Ser Pro Leu Ser Lys Lys Arg Glu Thr Asp Thr
675 680
* * * * *