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POLY ADP-RIBOSE POLYMERASE GENE AND ITS USES
FIELD OF THE INVENTION
The invention is drawn to the genetic manipulation of plants.
BACKGROUND OF THE INVENTION
The physiological and metabolic state of plant cells directly influences the
plant response to external stimuli. The plant response to disease includes a
host of
cellular processes to enable plants to defend themselves from pathogenic
agents.
These processes apparently form an integrated set of resistance mechanisms
that is
activated by initial infection and then limits further spread of the invading
pathogenic
microorganism.
The transformation of plants is a complex process. The process involves
1 ~ contacting cells with a DNA to be integrated into the plant cell genome.
Generally,
genetic transformation of eukaryotic cells is a random event. That is, the
foreign
DNA is integrated into the genome at random positions. Often several copies,
or parts
of copies, of the transforming DNA are integrated in a single position, and/or
at
different positions, resulting in a transformed cell containing multiple
copies of the
foreign DNA.
Because the metabolic state of the plant cell is instrumental in various
processes, it would be beneficial to be able to influence the stale of the
cells.
Accordingly, there is a need for genes and methods for altering the metabolic
state of
plant cells.
CA 02312591 2003-07-25
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CA 02312591 2005-O1-11
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In another aspect, there is described a method for
modulating the metabolic state of a plant cell, said method
comprising transforming said plant cell with a DNA
construct, said construct comprising a promoter that drives
expression in a plant cell operably linked with a nucleotide
sequence selected from the group consisting of: (a) a
nucleotide sequence encoding a poly ADP-ribose polymerase
having the amino acid sequence set forth in SEQ ID N0:2; (b)
the nucleotide sequence set forth in SEQ ID N0:1; and (c) a
nucleotide sequence fully complementary to a nucleotide
sequence of (a) or (b).
In another aspect, there is described a method for
generating a transformed plant, said method comprising the
steps of: (a) transforming a plant cell with the chimeric
DNA molecule or vector of the invention; and (b) growing the
transformed plant cell of (a) into a plant.
In another aspect, there is described an isolated
polypeptide comprising an amino acid sequence having at
least 70o identity to SEQ ID N0:2, wherein the polypeptide
has poly ADP-ribose polymerase activity and wherein the
amino acid sequence having at least 70o identity to SEQ ID
N0:2 comprises at least two functional zinc fingers.
In another aspect, there is described an isolated
DNA molecule which encodes the isolated polypeptide of the
invention, or which is fully complementary to the DNA
molecule encoding the isolated polypeptide of the invention.
In another aspect, there is described an isolated
DNA molecule comprising a nucleotide sequence selected from
the group consisting of: (a) a nucleotide sequence having at
least 90o sequence identity to SEQ ID NO: l, wherein said
nucleotide sequence encodes a polypeptide having poly ADP-
2a
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62451-853(S)
ribose polymerase activity, said polypeptide comprising at
least two functional zinc fingers; and (b) a nucleotide
sequence fully complementary to the nucleotide sequence of
(a) .
In another aspect, there is described a chimeric
DNA molecule comprising a promoter capable of driving
expression of a nucleic acid sequence in a plant cell,
operably linked to the DNA molecule of the invention.
In another aspect, there is described a vector
comprising the chimeric DNA molecule of the invention.
In another aspect, there is described a method for
modulating the metabolic state of a plant cell, the method
comprising transforming the plant cell with a DNA construct,
the construct comprising a promoter that drives expression
in a plant cell operably linked to the DNA molecule of the
invention.
In another aspect, there is described a method for
generating a transformed plant, said method comprising the
steps of: (a) transforming a plant cell with the chimeric
DNA molecule of the invention; and (b) growing the
transformed plant cell of (a) into a plant.
In another aspect, there is described a method for
generating a transformed plant, said method comprising the
steps of: (a) transforming a plant cell with the vector of
the invention; and (b) growing the transformed plant cell of
(a) into a plant.
In another aspect, there is described a
transcription cassette and an expression cassette
comprising the DNA molecule of the invention.
2b
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In another aspect, there is described use of the
transcription cassette or the expression cassette of the
invention to suppress endogenous poly ADP-ribose polymerase
in a plant.
In another aspect, there is described a process
for propagating a transgenic plant, the process comprising
crossing a first parent transgenic plant comprising the
transcription cassette or the expression cassette of the
invention with a second parent plant, to produce progeny
plant comprising the transcription cassette or the
expression cassette.
In another aspect, there is described a process
for propagating a transgenic plant, the process comprising
obtaining a seed from a transgenic plant comprising the
transcription cassette or the expression cassette of the
invention and germinating the seed into a plant comprising
the transcription cassette or the expression cassette.
In another aspect, there is described a process
for obtaining transgenic seeds, the process comprising
pollinating a plant comprising the transcription cassette or
the expression cassette of the invention and harvesting
seeds from the plant.
DETAILED DESCRIPTION OF THE INVENTION
Poly ADP-Ribose Polymerase genes and methods for
their use are provided. In particular, the amino acid and
nucleotide sequences for the maize poly ADP-ribose
polymerase (PARP) are provided as SEQ ID NOs: 2 and 1,
respectively. Also of interest are portions of the
sequences of the invention. The nucleotide and amino acid
sequences of the C-terminal domain of the maize poly
2c
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ADP-ribose polymerase is provided in SEQ ID NOs: 3 and 4,
respectively. The nucleotide sequence of the Zinc fingers
is provided in SEQ ID N0:5.
PARP is generally described as a nuclear enzyme
found in most eukaryotes. Structure-function studies have
shown that animal PARPS may be divided into at least three
subdomains. The N-terminal part contains two zinc fingers
and has a high affinity for nicked V-shaped DNA.
Interaction of PARP with nicked DNA strongly enhances the
activity of the catalytic domain, which is very well
conserved among PARPs and located in the carboxyl-terminus
of the protein. (Veda et al. (1985) Ann. Rev. Biochem.
54:73-100; Sdhah et al. (1995) Anal. Biochem. 227:1-13). A
putative plant PARP has recently been cloned (Babiychuk
et al. (1997), EMBL Accession No. AJ222589).
PARP catalyzes both the transfer of ADP-ribose
from NAD+, mainly to the carboxyl group of a glutamic acid
residue on target proteins, and subsequent ADP-ribose
polymerization (Ueda et al. (1985) Ann. Rev. Biochem.
54:73-100; Sdhah et a1. (1995) Anal. Biochem. 227:1-13).
Inhibition of PARP reportedly facilitates genetic
transformation in plants (see WO 97/06267).
2d
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PARP is generally required in most cases where DNA is cleaved and rejoined,
such as in DNA repair, DNA, recombination, gene rearrangements and
transposition.
PARP has been shown to modify PARP itself, histones, high mobility group
chromosomal proteiins, topoi.somerase, endonucleases and DNA polyrnerases.
(Ueda
et al. (1985) Ann. Rev. Biochem. 54:73-100; Sdhah et al. (1995) Anal.
Biochein.
227:1-13)
Initially, the enzyme synthesizes an ester linkage preferentially between the
glutamyl( -) or sometimes the C-ten~ninal( -)carboxyl group on the acceptor
protein:
and the 1'-OH of the ribosyly group of ADP-ribose. Subsequently, up to 45-SO
ADP
units are added via a 2'-1'phosphodiester bond. Branching of the poly (ADP)-
ribosyl
chains via the 2'-1'phosphodiester linkages is also observed. See, for
example, Ueda
et al. (1985) Ann. Rev. Biocl:em. 5~: 73-100; and Shah et al. {1995) Anal.
Biochem.
227:1-13.
Compositions of the invention include isolated nucleic acid molecules
1 S encoding the PARP proteins of the invention, as well as fragments and
variants
thereof. The term "isolated" refers to material, such as a nucleic acid or a
protein,
which is: (1) substantially or essentially free from components that normally
accompany or interact with it as found in its naturally occurring environment.
Thus,
for a nucleic acid, the sequence is lacking a flanking sequence either 3' or
S' or both.
The isolated material optionaly comprises material not found with the material
in its
natural environment; or (2) if the material is in its natural environment, the
material
has been syntheticallly (non-naturally) altered by deliberate human
intervention to a
composition and/or placed at a locus in the cell (e.g., genome or subcellular
organelle)
not native to a material found in that environment. The alteration to yield
the
2S synthetic material c,an be performed on the material within or removed from
its natural
state. For example, a naturally occurring nucleic acid becomes an isolated
nucleic
acid if it is altered, or if it is transcribed from DNA which has been
altered, by non-
natural, synthetic (i..e., "man-made") methods performed within the cell from
which it
originates. See, e.g., Compounds and Methods for Site Directed Mutagenesis in
Eukaryotic Cells, Kmiec, U.S. Patent No. S,56S,3S0; In Vivo Homologous
Sequence
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Targeting in Eukaryotic Cells; Zarling et al., PCT/LJS93/03868. Likewise, a
naturally
occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced
by non-
naturally occurring means to a locus of the genome not native to that nucleic
acid.
Nucleic acids which are "isolated" as defined herein, are also referred to as
"heterologous" nucleic acids.
As used herein, "localized within the chromosomal region defined by and
including" with respect to particular markers includes reference to a
contiguous length
of a chromosome dc;limited by and including the stated markers.
As used herein, "marker" includes reference to a locus on a chromosome that
serves to identify a nznique position on the chromosome. A "polymorphic
marker"
includes reference to a marker which appears in multiple forms (alleles) such
that
different forms of the marker, when they are present in a homologous pair,
allow
transmission of each of the chromosomes in that pair to be followed. A
genotype may
be defined by use oi" one or a plurality of markers.
As used herein "oper,ably linked" includes reference to a functional linkage
between a promoter and a second sequence, wherein the promoter sequence
initiates
and mediates transcription of the DNA sequence corresponding to the second
sequence. Generally, operably linked means that the nucleic acid sequences
being
linked are contiguous and, where necessary to join two protein coding regions,
contiguous and in tree same reading frame.
The nucleotide sequences of the invention can be used to isolate other
homologous sequences in other plant species. Methods are readily available in
the art
for the hybridization of nucleic acid sequences. Coding sequences from other
plants
may be isolated according to well known techniques based on their sequence
homology to the coding sequences set forth herein. In these techniques all or
part of
the maize coding sequence is used as a probe which selectively hybridizes to
other
PARP coding sequences present in a population of cloned genomic DNA fragments
or
cDNA fragments (i.e. genornic or cDNA libraries) from a chosen organism. For
example, the entire maize PARP sequence or portions thereof may be used as
probes
capable of specifically hybridizing to corresponding coding sequences and
messenger
4
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RNAs. To achieve specific hybridization under a variety of conditions, such
probes
include sequences that are unique and are preferably at least about 10
nucleotides in
length, and most preferably at least about 20 nucleotides in length. Such
probes may
be used to amplify the PART coding sequences of interest from a chosen
organism by
the well-known process of polymerase chain reaction (PCR). This technique may
be
used to isolate additional coding sequences from a desired organism or as a
diagnostic
assay to determine the presence of coding sequences in an organism.
Such techniques include hybridization screening of plated DNA libraries
(either plaques or colonies; see, e.g.. Sambrook et al., Molecular Cloning,
eds., Cold
Spring Harbor Laboratory Press ( 1989)) and amplification by PCR using
oligonucleotide primers corresponding to sequence domains conserved among the
amino acid sequences (see, e.g. Innis et al., PCR Protocols, a Guide to
Methods and
Applications, eds., Academic; Press (1990)). For example, hybridization of
such
sequences may be carried out under conditions of reduced stringency, medium
stringency or even stringent conditions (e.g., conditions represented by a
wash
stringency of 35-40~% Formamide with Sx Denhardt's solution, 0.5% SDS and lx
SSPE at 37 C; conditions represented by a wash stringency of 40-45% Formamide
with Sx Denhardt's solution, 0.5% SDS, and lx SSPE at 42 C; and conditions
represented by a wash stringency of 50% Formamide with Sx Denhardt's solution,
0.5% SDS and lx SSPE at 4:~ C, respectively}, to DNA encoding the PARP genes
disclosed herein in a standard hybridization assay. See J. Sambrook et al.,
Molecular
Cloning, A Laboratory Manual 2d ed. (1989) Cold Spring Harbor Laboratory. In
general, sequences which code for the defense activators and other activator
proteins
of the invention and hybridize to the sequences disclosed herein will be at
least SO%
homologous, 70% homologous, and even 85% homologous or more with the
disclosed sequence. That is, the sequence similarity of sequences may range,
sharing
at least about 50%, about 70'%, and even about 85% sequence similarity.
The following terms are used to describe the sequence relationships between
two or more nucleic acids or polynucleotides: (a) "reference sequence", (b)
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"comparison window", (c) "sequence identity", (d) "percentage of sequence
identity",
and (e) "substantial identity""
(a) As used herein, "reference sequence" is a defined sequence used as a
basis for sequence comparison. A reference sequence may be a subset or the
entirety
of a specified sequence; for example, as a segment of a full-length cDNA or
gene
sequence, or the complete cDNA or gene sequence.
(b) As u~:ed herein, "comparison window" means includes reference to a
contiguous and specified segment of a polynucleotide sequence, wherein the
polynucleotide sequence in the comparison window may comprise additions or
deletions (i.e., gaps) compared to the reference sequence (which does not
comprise
additions or deletions) for optimal alignment of the two sequences. Generally,
the
comparison window is at least 20 contiguous nucleotides in length, and
optionally can
be 30, 40, 50, 100, o~r longer. Those of skill in the art understand that to
avoid a high
similarity to a reference sequence due to inclusion of gaps in the
polynucleotide
sequence a gap penalty is typically introduced and is subtracted from the
number of
matches.
Methods of alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison may be conducted by the local
homology algorithm. of Smith et al. (1981) Adv. Appl. Math. 2:482; by the
homology
alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; by the
search for
similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; by
computerized implementations of these algorithms, including, but not limited
to:
CLUSTAL in the P(:/Gene program by Intelligenetics, Mountain View, California,
GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin,
USA; the CLUSTAL program is well described by Higgins et al. (1988) Gene
73:237-
244; Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic
Acids
Research 16:10881-90; Huarig et al. (1992) Computer Applications in the
Biosciences
8:155-65, and Person et al. (11994) Methods ofMolecular Biology 24:307-331;
preferred computer ~tlignmen.t methods also include the BLASTP, BLASTN, and
6
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WO 99/37789 PCTNS99/01591
BLASTX algorithms. Altschul et al. (1990) J. Mol. Biol. 215:403-410. Alignment
is
also often performed by inspection and manual alignment.
(c) As used herein, "sequence identity" or "identity" in the context of two
nucleic acid or polypeptide sequences includes reference to the residues in
the two
sequences which are; the same when aligned for maximum correspondence over a
specified comparison window. When percentage of sequence identity is used in
reference to proteins. it is recognized that residue positions which are not
identical
often differ by conservative ~unino acid substitutions, where amino acid
residues are
substituted for other amino acid residues with similar chemical properties
(e.g. charge
or hydrophobicity) and therefore do not change the functional properties of
the
molecule. When sequences differ in conservative substitutions, the percent
sequence
identity may be adjusted upwards to correct for the conservative nature of the
substitution. Sequences which differ by such conservative substitutions are
said to
have "sequence similarity" or "similarity". Means for making this adjustment
are
well-known to those of skill in the art. Typically this involves scoring a
conservative
substitution as a pari;ial rather than a full mismatch, thereby increasing the
percentage
sequence identity. Thus, for example, where an identical amino acid is given a
score
of 1 and a non-conservative substitution is given a score of zero, a
conservative
substitution is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program PC/GENE
(Intelligenetics, Mountain View, California, USA).
(d) As used herein, "percentage of sequence identity" means the value
determined by comparing two optimally aligned sequences over a comparison
window, wherein the portion of the polynucleotide sequence in the comparison
window may compriise additions or deletions (i.e., gaps) as compared to the
reference
sequence (which doc;s not comprise additions or deletions) for optimal
alignment of
the two sequences. 7.'he percentage is calculated by determining the number of
positions at which the identical nucleic acid base or amino acid residue
occurs in both
sequences to yield the number of matched positions, dividing the number of
matched
7
CA 02312591 2000-07-26
WO 99/37789 PCT/US99/01591
positions by the total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence identity.
(e) (i) The term "substantial identity" of polynucleotide sequences means
that a polynucleotide comprises a sequence that has at least 70% sequence
identity,
preferably at least 80%, more; preferably at least 90% and most preferably at
least
95%, compared to a reference sequence using one of the alignment programs
described using standard par<uneters. One of skill will recognize that these
values can
be appropriately adjusted to determine corresponding identity of proteins
encoded by
two nucleotide sequences by taking into account codon degeneracy, amino acid
sequences for these purposes normally means sequence identity of at least 60%,
more
preferably at least 70%, 80%, 90%, and most preferably at least 95%.
Polypeptides
which are "substantially similar" share sequences as noted above except that
residue
positions which are not identical may differ by conservative amino acid
changes.
Another indication that nucleotide sequences are substantially identical is if
two molecules hybridize to each other under stringent conditions. Generally,
stringent
conditions are selected to be about 5 C to about 20 C lower than the thermal
melting
point (Tm) for the specific sequence at a defined ionic strength and pH. The
Tm is the
temperature (under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Typically, stringent wash
conditions are those in which. the salt concentration is about 0.02 molar at
pH 7 and
the temperature is at least about 50, 55, or 60 C. However, nucleic acids
which do not
hybridize to each other under stringent conditions are still substantially
identical if the
polypeptides which they encode are substantially identical. This may occur,
e.g.,
when a copy of a nucleic acid is created using the maximum codon degeneracy
permitted by the genetic code. One indication that two nucleic acid sequences
are
substantially identic,~l is that the polypeptide which the first nucleic acid
encodes is
immunologically cross reactive with the polypeptide encoded by the second
nucleic
acid.
(e) (ii) The terms "substantial identity" in the context of peptide indicates
that a peptide comprises a sequence with at least 70% sequence identity to a
reference
8
CA 02312591 2000-07-26
WO 99/37789 PCT/US99/01591
sequence, preferably 80%, more preferably 85%, most preferably at least 90% or
95%
sequence identity to the reference sequence over a specified comparison
window.
Preferably, optimal alignment is conducted using the homology alignment
algorithm
of Needleman et al. (1970) J, Mol. Biol. 48:443. An indication that two
peptide
sequences are substantially identical is that one peptide is immunologically
reactive
with antibodies raised against the second peptide. 'Thus, a peptide is
substantially
identical to a second peptide.,, for example, where the two peptides differ
only by a
conservative substifiution.
Fragments annd variants of the disclosed nucleotide sequences and proteins
encoded thereby are also encompassed by the present invention. By "fragment"
is
intended a portion of the nucleotide sequence or a portion of the amino acid
sequence
and hence protein encoded thereby. Fragments of a nucleotide sequence may
encode
protein fragments that retain the biological activity of the native protein
and hence
confer resistance to nematodes. Alternatively, fragments of a nucleotide
sequence that
are useful as hybridization probes generally do not encode fragment proteins
retaining
biological activity. ~Chus, fragments of a nucleotide sequence may range from
at least
about 20 nucleotides, about '_>0 nucleotides, about 100 nucleotides, and up to
the entire
nucleotide sequence: encoding the proteins of the invention.
By "variants" is intended substantially similar sequences. For nucleotide
sequences, conservative variants include those sequences that, because of the
degeneracy of the g~.netic code, encode the amino acid sequence of one of the
proteins
conferring resistance to nematodes. Generally, nucleotide sequence variants of
the
invention will have at least 70%, generally, 80%, preferably up to 90%
sequence
identity to its respective native nucleotide sequence.
By "variant" protein is intended a protein derived from the native protein by
deletion (so-called truncation) or addition of one or more amino acids to the
N-
terminal and/or C-terminal end of the native protein; deletion or addition of
one or
more amino acids apt one or snore sites in the native protein; or substitution
of one or
more amino acids apt one or snore sites in the native protein. Such variants
may result
from, for example, genetic polymorphism or from human manipulation. Methods
for
9
CA 02312591 2001-05-08
62451-853(5)
such manipulations are generally known in the art.
The proteins of the invention may be altered in various ways including amino
acid substitutions, deletions, truncations, and insertions. Methods for such
manipulations are generally known in the art. For example, amino acid sequence
variants of the activator proteins can be prepared by mutations in the DNA.
Methods
for mutagenesis and nucleotide sequence alterations are well known in the art.
See,
for example, Kunkel et al. (1985) Proc. Natl. Acad. Sci. USA 82:488-492;
Kunkel et
al. ( 1987) Methods in Enzymol. I ~-1:367-382; U.S. Patent No. 4,873,192;
Walker and
Gaastra (eds.) Techniques in ~Llolecular Biology, MacMillan Publishing
Company,
NY (1983) and the references cited therein. Thus, the genes and nucleotide
sequences
of the invention include both the naturally occurring sequences as well as
variant and
mutant forms. Likewise, the proteins of the invention encompass both naturally
occurring proteins as well as variants and modified forms thereof. Such
variants wilt
continue to possess the desired PARP activity. Obviously, the mutations that
will be
1 ~ made in the DNA encoding the variant must not place the sequence out of
reading
frame and preferably will not create complementary regions that could produce
secondary mRNA structure. See, EP Patent Application Publication No. 7,444.
PARP is present in all higher eukaryotes. Therefore, it is recognized that the
nucleotide sequence encoding the PARP may be utilized from any eukaryotic
source,
including vertebrates, arthropods, mollusks, slime moulds, dinoflagellates,
fungi,
mammals, chicken, Xenopus and insects. See, for example, Heller et al. ( 1995)
J.
Biol. Chem. 270:11178-11180; Schreiber et al. (1995) Proc. Natl. .Acad. Sci.
USA
92:4753-4757; Ueda et al. (1985) Ann. Rev. Biochem. 54:73-100; Brightwell et
al.
(1975) Biochem. J. 147:119-129; Kofler et al. (1993) ibid 293:275-281;
Collinge et
al. ( 1994) Mol. Gen. Genet. 2=h:686-693; Scovassi et al. ( 1986) Eur. J.
Biochem.
1.59:77-84; Simonin et al. (1991) Anal. Blochem. 195:226-231; Masutani et al.
(1994)
Eur. J. Biochem. 220:607-614,
It is recognized that the plant cell can be transformed with a nucleotide
sequence encoding PARP, a nucleotide sequence encoding a portion of PARP,
preferably the C-terminal portion of PARP, as well as with a nucleotide
sequence
CA 02312591 2000-07-26
WO 99/37789 PCT/(JS99/Oi591
encoding the antisense sequence for the PARP gene, or portions thereof. In
this
manner, the level of expression of the PARP in the plant cell can be
modulated, i.e.,
increased or decreased, respectively. Levels of expression of the sense or
antisense
sequence can be regulated by the promoter utilized to express the gene.
S Promoters for the expression of genes in plant cells are known in the art.
Promoters are available for constitutive, tissue specific, inducible, etc.
Such
promoters include, for example, 3SS promoter, Meyer et al. (1997) J. Gen.
Virol.
78:3147-3151; biotin carbox;ylase, Bas et al. (1997) Plant Mol. Biol. 35:539-
SSO;
oxidase, Lasserre et al. (1997) Mol. Gen. Genet 256:211-222; cab, Shiina et
al. (1997)
Plant Physiol. 115:477-483; phospholipase, Xu et al. (1997) Plant Physiol.
115:387-
395; farnesyltransferase, Zhou et al. (1997) Plant J. 12:921-930;
plastocyanin,
Helliwell et al. (1997) Plant.J. 12:499-506; CVMV promoter, Verdaquer et al.
(1996)
Plant Mol. Biol. 31:1129-1139; actin, An et al. (1996) Plant J. 10:107-121;
heat
shock, Prandl et al. x;1996) Plant Mol. Biol. 31:157-162; ubiquitin, thionin,
3SS,
1 S Holtorf et al. ( 1995) Plant ~l'ol. Biol. 29:637-646; Callis et al. (
1990) J. Biol. Chem.
265:12486-12493; histone, Atanossova et al. {1992) Plant J. 2:291-300; rol C,
Fladung et al. (1993) Plant Mol. Biol. 23:749-757; histone, Brignon et al.
(1993)
Plant J. 4:445-457; Lepetit et al. ( 1992) Mol. Gen. Genet. 231:276-285.
As indicated, recent studies on the mechanism of PARP suggests involvement
of the enzyme in regulation of DNA repair, recombination and replication. The
enzyme is rapidly activated by DNA and exhibits a high affinity for naked
single-
stranded or double-stranded DNA. Any perturbation in the cellular morphology
and/or physiology that causes a change in chromatin conformation generally
results in
a rapid increase in PARP activity. PARP is an important modulator of the fate
of
2S DNA introduced into a plant cell. Accordingly, plants transformed with
either a sense
or antisense PARP nucleotide sequence may be utilized to increase
transformation
frequency in plant cells. Therefore, the present invention provides for the
regulation
of the levels of PARP in the plant cell to determine its effect on plant
transformation
and gene targeting.
11
CA 02312591 2001-05-08
62451-853(S)
It is further recognized that because the enzyme plays a role in cellular
stress,
it may be beneficial to increase the levels of the enzyme to prevent plant
disease or
pathogen attack. In this manner, constitutive or inducible promoters may be
utilized.
Constitutive promoters include, for example, those disclosed in U.S. Patent
Nos.
S 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5.268,463;
5,608,142. Inducible promoters are known in the art
and include, for example, pathogen inducible promoters, such as promoters from
pathogenesis-related proteins (PR proteins) which are induced following
infection by
a pathogen; e.g., PR proteins. SAR proteins, beta-1,3-glucanase, chitinase,
etc. See,
for example, Redolfi et crl. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes
et crl.
(1992) The Plant Cell -1:645-656; and Van Loon (1985) Plant wlol. ~'irol. -I:1
11-116.
Of interest are promoters that are expressed locally at or near the site of
pathogen
infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-
342;
Matton et al. ( 1989) Molecular Plant-~t~licrobe Interactions 2:325-331;
Somsisch et
1 ~ al. ( 1986) Proc. Natl. Acad Sci. USA 83:2427-2430; Somsisch et al. (
1988)
~~lolecular and General Genetics ?:93-98; and Yang, Y ( 1996) Proc. ~~'atl.
Acad. Sci.
C~SA 93:14972-14977. See also, Chen et al. ( 1996) Plant J. 10:955-966; Zhang
et al.
( 1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. ( 1993 ) Plant
J. 3:191-
201; Siebertz et al. (1989) Plant Cel! 1:961-968; and the references cited
therein. Of
particular interest is the inducible promoter for the maize PRms gene, whose
expression is induced by the pathogen Fusarirrm moniliforme (see, for example,
Cordero et al. (1992) Physiological and Moleculur Plant Pathology -II: I 89-
200).
The PARP genes or antisense nucleotides of the invention can be introduced
into any plant. The genes or nucleotide sequences to be introduced will be
used in
expression cassettes for expression in any plant of interest.
Such expression cassettes will comprise a transcriptional initiation region
linked to the gene encoding the PARP gene or antisense nucleotide of interest.
Such
an expression cassette is provided with a plurality of restriction sites for
insertion of
the gene of interest to be under the transcriptional regulation of the
regulatory regions.
The expression cassette may additionally contain selectable marker genes.
12
CA 02312591 2001-05-08
62451-853(S)
The transcriptional initiation region, the promoter, may be native or
analogous
or foreign or heterologous to the plant host. Additionally, the promoter may
be the
natural sequence or alternatively a synthetic sequence. By foreign is intended
that the
transcriptional initiation region is not found in the native plant into which
the
transcriptional initiation region is introduced. As used herein a chicneric
gene
comprises a coding sequence operably linked to a transcription initiation
region that is
heterologous to the coding sequence.
The transcriptional cassette will include in the 5'-3' direction of
transcription, a
transcriptional and translational initiation region, a DNA sequence of
interest, and a
transcriptional and translational termination region functional in plants. The
termination region may be native with the transcriptional initiation region,
may be
native with the DNA sequence of interest, or may be derived from another
source.
Convenient termination regions are available from the Ti-plasmid of A.
tumejaciens,
such as the octopine synthase and nopaline synthase termination regions. See
also,
1 p Guerineau et al. ( I 99 I ) Mol. Gen. Genet. 262:141-144; Proudfoot ( 1991
) Cell 6-1:671-
674; Sanfacon et crl. (1991) Genes Dev. x:141-149; Mogen et al. (1990) Pla~~t
Cell
2:1261-1272; Munroe et al. (1990) Gene 91:151-1~8; Ballas et al. (1989)
Nucleic
Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 1:9627-9639.
The genes of the invention are provided in expression cassettes for expression
in the plant of interest. The cassette will include 5' and 3' regulatory
sequences
operably linked to the gene of interest. The cassette may additionally contain
at least
one additional gene to be cotransformed into the organism. Alternatively, the
additional genes) can be provided on another expression cassette. Where
appropriate,
the genes) may be optimized for increased expression in the transformed plant.
That
is, the genes can be synthesized using plant preferred codons for improved
expression.
Methods are available in the art for synthesizing plant preferred genes. See,
for
example, U.S. Patent Nos. 5,380,831, 5,436, 391, and Murray eyl. (1989)
Nucleic
Acids Res. 17:477-498,
Additional sequence modifications are known to enhance gene expression in a
cellular host. These include elimination of sequences encoding spurious
13
CA 02312591 2000-07-26
WO 99/37789 PCT/US99/01591
polyadenylation signals, exon-intron splice site signals, transposon-like
repeats, and
other such well-characterized sequences which may be deleterious to gene
expression.
The G-C content ofthe sequence may be adjusted to levels average for a given
cellular host, as calculated by reference to known genes expressed in the host
cell.
When possible, the ;sequence is modified to avoid predicted hairpin secondary
mRNA
structures.
The expression cassettes may additionally contain 5' leader sequences in the
expression cassette construct. Such leader sequences can act to enhance
translation.
Translation leaders are known in the art and include: picornavirus leaders,
for
example, EMCV leader (Encephalomyocarditis S' noncoding region) (Elroy-Stein
et
al. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic
Virus); Virology 15.x:9-20), and human immunoglobulin heavy-chain binding
protein
{BiP), (Macejak et al. (1991) Nature 353:90-94; untranslated leader from the
coat
protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling et al. (1987)
Nature
325:622-625; tobacco mosaic virus leader (TMV), (Gallie, D.R. et al. (1989)
Molecular Biology of RNA, pages 237-256; and maize chlorotic mottle virus
leader
(MCMV) (Lommel, S.A. et al. (1991) Virology 81:382-385). See also, Della-
Cioppa
et al. (1987) Plant ~'hysiology 84:965-968. Other methods known to enhance
translation can also be utilized, for example, introns, and the like.
In preparing the expression cassette, the various DNA fragments may be
manipulated, so as to provide; for the DNA sequences in the proper orientation
and, as
appropriate, in the proper reading frame. Towards this end, adapters or
linkers may be
employed to join the DNA fragments or other manipulations may be involved to
provide for convenient restriction sites, removal of'superfluous DNA, removal
of
restriction sites, or the like. :For this purpose, in vitro mutagenesis,
primer repair,
restriction, annealing, resubstitutions, e.g. transitions and transversions,
may be
involved.
The genes o:f the present invention can be used to transform any plant. In
this
manner, genetically modified plants, plant cells, plant tissue, seed, and the
like can be
14
CA 02312591 2001-05-08
62451-853(5)
obtained. Transformation protocols may vary depending on the type of plant or
plant
cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of
transforming plant cells include microinjection (Crossway et al. ( 1986)
Biotechniques
-1:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad Sci. USA
83:5602-5606, Agrobacterium mediated transformation (Hinchee et al. ( 1988)
Biotechnology 6:915-921 ), direct gene transfer (Paszkowski et al. ( 1984)
EMBO J.
3:2717-2722), and ballistic particle acceleration (see, for example, Sanford
et al., U.S.
Patent 4,945,050; Tomes et al. Direct DNA Transfer into Intact Plant Cells via
Microprojectile Bombardment In Gamborg and Phillips (eds.) Plant Cell, Tissue
and
Organ Cultzrre: Fundamental Methods, Springer-Verlag, Berlin ( 1995); and
McCabe
et al. (1988) Biotechnology 6:923-926). Also see, Weissinger et al. (1988)
Ann. Rev.
Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology
5:27-37
(onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et
al.
(1988) BiolTechnology 6:923-926 (soybean); Datta et al. (1990) Biotechnology
8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 8:4305-4309
(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes m al.
Direct
DNA Transfer into Intact Plant Cells via Microprojectile Bombardment in
Gamborg
and Phillips (eds.) Plant Cell, Tissue and Organ Culture: Fundamental
Nlethods,
Springer-Verlag, Berlin (1995) (maize); Klein et al. (1988) Plant Physiol.
91:440-444
(maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooydaas-Van
Slogteren & Hooykaas ( 1984) Nature (London) 311:763-764; Bytebier et al. (
1987)
Proc. Natl. Acad Sci. USA 8.1:5345-5349 (Liliaceae); De Wet et al. .(1985) in
The
Experimental Manipulation ojOvule Tissues ed. G.P. Chapman et al., pp. 197-
209.
Longman, NY (pollen); Kaepp(er et al. (1990) Plant Cell Reports 9:415-418; and
Kaeppler et al. ( 1992) T heor. Appl. Genet. 84:560-566 (whisker-mediated
transformation); D Halluin et al. (1992) Plant Cell =1:1495-1505
(electroporation); Li
et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995)
Annals of
Botarry 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology I=1:745-
750
(maize via Agrohacterium tumefcrciens),
CA 02312591 2000-07-26
WO 99/37789 PCT/US99/01591
The cells which have been transformed may be grown into plants in
accordance with conventional ways. See, for example, McCormick et al. (1986)
Plant
Cell Reports 5:81-84. These. plants may then be grown, and either pollinated
with the
same transformed strain or different strains, and the resulting hybrid having
the
desired phenotypic characteristic identified. Two or more generations may be
grown
to ensure that the subject phenotypic characteristic is stably maintained and
inherited
and then seeds harvested to ensure the desired phenotype or other property has
been
achieved.
The present invention also provides isolated nucleic acid comprising
polynucleotides of sufficient length and complementarity to a gene to use as
probes or
amplification primers in the detection, quantitation, or isolation of gene
transcripts.
For example, isolated nucleic acids of the present invention can be used as
probes in
detecting deficiencies in the level of mRNA in screenings for desired
transgenic
plants, for detecting mutations in the gene (e.g., substitutions, deletions,
or additions),
1 S for monitoring upre;gulation of expression or changes in enzyme activity
in screening
assays of compounds, for detection of any number of allelic variants
(polymorphisms)
of the gene, or for use as molecular markers in plant breeding programs. The
isolated
nucleic acids of the present invention can also be employed for use in sense
or
antisense suppression of a PARP gene in a host cell, tissue, or plant. See,
Tools to
Determine the Function of Genes, 1995 Proceedings of the Fiftieth Annual Corn
and
Sorghum Industry Research Conference, American Seed Trade Association,
Washington, D.C., l'.995. Additionally, non-translated 5 or 3 regions of the
polynucleotides of the present invention can be used to modulate turnover of
heterologous mRNAs and/or protein synthesis.
The present invention provides a method of genotyping a plant comprising a
polynucleotide of th,e present invention. Preferably, the plant is a monocot,
such as
maize or sorghum. Genotyping provides a means of distinguishing homologs of a
chromosome pair and can be used to differentiate segregants in a plant
population.
Molecular marker methods can be used for phylogenetic studies, characterizing
genetic relationships among crop varieties, identifying crosses or somatic
hybrids,
16
CA 02312591 2001-05-08
62451-853(5)
localizing chromosomal segments affecting monogenic traits, map based cloning,
and
the study of quantitative inheritance. See, e.g., Plant Molecular Biology: A
Laboratory Manual, Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1997). For
molecular marker methods, see generally, The DNA Revolution by Andrew H.
Paterson 1996 (Chapter 2) in: Genome Mapping in Plants led. Andrew H.
Paterson)
by Academic Press/R. G. Landis Company, Austin, Texas, pp.7-21.
The particular method of genotyping in the present invention may employ any
number of molecular marker analytic techniques such as, but not limited to,
restriction
fragment length polymorphisms (RFLPs). RFLPs are the product ofallelic
differences between DNA restriction fragments caused by nucleotide sequence
variability. As is well known to those of skill in the art, RFLPs are
typically detected
by extraction of genomic DNA and digestion with a restriction enzyme.
Generally,
the resulting fragments are separated according to size and hybridized with a
probe;
single copy probes are preferred. Restriction fragments from homologous
1 ~ chromosomes are revealed. Differences in fragment size among alleles
represent an
RFLP. Thus, the present invention further provides a means to follow
segregation of
a PARP gene or nucleic acid of the present invention as well as chromosomal
sequences genetically linked to these genes or nucleic acids using such
techniques as
RFLP analysis. Linked chromosomal sequences are within 50 centiMorgans (cM),
often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably
within 5, 3,
2, or I cM of a PARP gene.
In the present invention, the nucleic acid probes employed for molecular
marker mapping of plant nuclear genomes selectively hybridize, under selective
hybridization conditions, to a gene encoding a polynucleotide of the present
invention.
In preferred embodiments, the probes are selected from polynucleotides of the
present
invention. Typically, these probes are cDNA probes or Pst I genomic clones.
The
length of the probes is discussed in greater detail in the references
noted above but are typically at least 15 bases in length, more
preferably at least 20, 25, 30, 35, 40, or 50 bases in length.
Generally, however, the probes are less than about 1 kilobase in
length. Preferably, the probes are single copy probes that hybridize
to a unique locus in a haploid
17
CA 02312591 2000-07-26
WO 99/37789 PC'TNS99/01591
chromosome complement.
The method of detecting an RFLP comprises the steps of (a) digesting
genomic DNA of a plant with a restriction enzyme; (b) hybridizing a nucleic
acid
probe, under selective hybridization conditions, to a sequence of a
polynucleotide of
the present of said genomic DNA; (c) detecting therefrom a RFLP. Other methods
of
differentiating polymorphic (;allelic) variants of polynucleotides of the
present
invention can be had by utilizing molecular marker techniques well known to
those of
skill in the art including such. techniques as: 1) single stranded
conformation analysis
(SSCP); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection
assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins
which
recognize nucleotide mismatches, such as the E coli mutS protein; and 6)
allele-
specific PCR. Other approaches based on the detection of mismatches between
the
two complementary DNA strands include clamped denaturing gel electrophoresis
(CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage (CMC).
Thus, the present invention further provides a method of genotyping comprising
the
steps of contacting, under stringent hybridization conditions, a sample
suspected of
comprising a polynucleotide of the present invention with a nucleic acid
probe.
Generally, the sample is a plant sample; preferably, a sample suspected of
comprising
a maize polynucleotide of the present invention (e.g., gene, mRNA). The
nucleic acid
probe selectively hybridizes, under stringent conditions, to a subsequence of
a
polynucleotide of tl;~e present invention comprising a polymorphic marker.
Selective
hybridization of the nucleic acid probe to the polymorphic marker nucleic acid
sequence yields a h;,~bridization complex. Detection of the hybridization
complex
indicates the presence of that polymorphic marker in the sample. In preferred
embodiments, the nucleic acid probe comprises a polynucleotide of the present
invention.
18
CA 02312591 2000-07-26
WO 99/37789 PCT/US99/01591
EXPERIMENTAL
Materials and Methods
Chemicals and Reagents
All chemicals used in this study were of molecular biology grade. Trizma
base (Tris {hydroxymethyl) aminomethane; abbreviated hereafter as Tris), N-2
hydroxy-ethyl-pipcrazine-N''-2-ethane sulfonic acid (Hepes),
Ethylenediaminetetraacetic acid, sodium salt (EDTA), Magnesium chloride, Urea,
were procured from Sigma Chemical Co. Analytical grade glycerol was obtained
from Baxter. Dithiothreitol (DTT), PefablocSC, Pepstatin, Bestatin, all
restriction
enzymes, DNA and RNA purification kits as well as markers were purchased from
Boehringer Mannheim. Immunodetection kits for Western blots, silver staining
and
Colloidal Coomassie Blue staining were from Novex. All radioactive chemicals
were
purchased from NE~f~-Dupont and NEN. Chromotopographic resins were purchased
either from Sigma, lBioRad or Pharmacia.
Cell Culture
The enzyme is isolated from a Hi II embryogenic callus cell line.
Exponentially growing cultures of 612B4 cells were maintained in dark at
28°C.
(Armstrong et al. (1992) Theor. Appl. Genet. 84:755-762}. The cell suspensions
are
in MS medium supplemented with 2-4-dichlorophenoxyaxcetic acid (2.5 mg/1).
Cultures are grown for a week on a gyroratatory shaker at 150 rpm and
harvested by
decantation. Routinely, 60-80 g of cells are obtained from 800-900 ml cultures
grown
in 12-14 flasks.
Cells are hawested by filtration and used to prepare whole cell extracts (WCE)
form 612B4 cells u;>ing the Bionebulizer (Glas-Col, Terre Haute, Indiana). The
process for WCE preparation is outlined in Schema 1. All operations were
earned out
at 4°C or on ice unless mentioned otherwise.
Chromatography on Heparin-agarose: About 300 ml of Heparin -agarose
(Sigma) was washed extensively with 20 mM Hepes-KOH pH 7.9, 0.1 mM EDTA,
19
CA 02312591 2001-05-08
62451-853(S)
20% glycerol (HGED buffer), packed into a 5.0 x 30 cm Econo column(Biorad) and
connected to the Econo System (Biorad). The matrix was equilibrated with HGED
+
100 mM KCI. Three batches of crude WCE extract (approx. 1.8-2.0 a of total
pooled
protein in 60-80 ml) were loaded on the column at a flow rate of 15-20 ml/hr.
The
column was washed extensively with equilibration buffer till the A,s°
of the effluent
was < 0.1 unit (approx. 900 ml).
Small aliquots of peak fractions were saved for PAR.P assays and all fractions
(7-8 ml each) showing A,$° > 0.1 unit were pooled. Protein was
precipitated by
adding solid ammonium sulfate (0.4 g/ml). The mixture was centrifuged at
40,000 x g
for 30 min., dissolved in minimum amount HGED and dialyzed against HGED+100
mM KCI containing Pefabloc and DTT. This fraction is designated HA-1. The
column was further washed with 900 ml each HGED + 400 mM KCI followed by
HGED + 1 M KCI. Fractions from both washes were processed as above and
designated as HA-2 and HA-3. PARP assays were performed on HA-1, 2 and 3 and
1 ~ the active fraction (HA-2) was used for further purification.
Chromatography on DNA-cellulose: DNA-cellulose (Sigma) was washed
extensively with HGED and packed in the Econo column~(2.5 x 30 cm). The column
was connected to the Econo System and equilibrated with HGED+100mM KCI.
2U Partially pure PARP from three Heparin-agarose column was loaded on the DNA-
cellulose column. Unbound protein was removed by washing with HGED + 100 mM
KCI (200 ml; designated as DC-1 ). The bound protein was eluted with HGED + 1
M
KCI (designated DC-2). All fractions was processed as described above for
activity
and protein.
Chromatography on Histone-agarose: Histone-agarose (Sigma) was washed
extensively with HGED and packed into an Econo column(1.5 x 15 cm). The column
was equilibrated in I-IGED + 100 mM KCI. Active fraction from DNA-cellulose
(DC-
2) was further fractionated on I-Iistone-agarose by washing the column
successively
with I-IGED containing 100 mM, 400 mM and 1 M KCI. All fractions were
processed
*Trademark 20
CA 02312591 2001-05-08
62451-853(S)
as above and dialyzed against 20 mM Tris-HCI buffer pH 7.9 containing 100 111M
KCI.
Chromatography on Mini-Q column: Mini-Q column (Pharmacia) was
connected to Smart-LC (Pharmacia) and equilibrated by washing with five bed
volumes of HGED followed by five bed volumes of Tris-HCI buffer + 100 mM KCI.
Active PARP from the Histone-agarose step was loaded on the column. The column
was washed with three bed volumes of equilibration buffer and 400 p l
fractions were
collected. Column was further developed using a step gradient of KCI at 400
mM,
600 mM and 1 M in Tris-HCI, pH 8Ø All fractions were tested for PARP
activity as
described below.
Enzyme Assays
Catalytic activity of PARP is assayed following published protocols (Shah et
al. (1995) Anal. l3iochem 227:1-13) with modifications suitable for the plant
enzyme.
Briefly, the enzyme (in a total volume of 25 pl of 20 mM Hepes pH 7.9, 100 mM
KCI) is incubated with 2.5-5 pCi of oo-''-P-NAD+, 2 111/ml final concentration
of
bovine histone (fraction IV), 2 pg of activated calf thymus DNA and 0.5 mM
DTT.
The reactions are carried out at 6°C unless otherwise mentioned. At the
end of the
appropriate time intervals, the labeled protein is precipitated with 25% TCA.
The
precipitate is collected by centrifugation at 16,000 x g for 10 min., washed 2
x with
5% TCA and counted in a LSC. Protein heated at 65°C for 5 min. is used
as a
negative control.
Microseqc~en cing
Protein samples obtained from the Mini-Q column purification step was
electrophoresed in duplicate on a 10% polyacrylamide gels using 0.1% SDS in
the
running buffer (Shah et al. (1995) Anal. Biochem 227:1-13). One half of the
gel was
used to detect protein hands with a Colloidal Coormassie staining kit (Novex)
following manufacturer s instructions. The other half was used in the activity
blot
assay to confirm position of the active PARP on the gel. Stained protein band
*Trademark 21
CA 02312591 2000-07-26
WO 99/377$9 PCT/US99/01591
corresponding to active PARP was cut out from the gel and used microsequencing
earned out at the W.M. Keck Foundation Biotechnology Resource Laboratory if
Yale
University. In gel t:ryptic digestion of the protein, Matrix Assisted Laser
Desorption
Mass Spectrometry (MALD',f-MS) of the isolated peptides, and amino sequencing
of
representative peptides was carried out following protocols detailed elsewhere
(Stone
et al. (1990) In: Methods in .~nzyomology 193:389-412); (Stone et al. (1991)
In:
5,..
Methods in Protein Sequence Anal ysis 133-141); Williams et al. (1995) In:
Techniques in Protein Chemistry 6:143-152); Williams et al. (1995) In: Protein
Protocol Handbook 365-378).
Ant~entide; Antibodies
Synthesis of the peptide antigens and antibody generation was carried out at
Research Genetics, Inc. Two peptides (P-1 and P-2) were used as for antibody
generation using two different protocols. In the first protocol, peptide P-1
was
synthesized as a multiple antigenic peptide (MAP) following published
protocols
(Tam, J.P. (1988) Proc. Nat. Acad Sci. USA 85:5409-5413). Antiserum was
collected
and analyzed for cross-reactivity to PARP using Western blots (Shah et al.
(1995)
Anal. Biochem 227:1-13). In the second protocol, P-2 was synthesized as MAP
(Tam,
J.P. (1988) Proc. Ncrtl. Aead. Sci. USA 85:5409-5413) as well as a linear
peptide
(Barany et al. (1980) In: The Peptides 2:1-284). The linear peptide was
conjugated to
hemocyanin using published methods (Walter et al. (1980) Proc. Natl. Acad.
Sci. USA
77:5 I 97-5200) and 'used for ;immunization. The immunization protocol for
both types
of antigens was essentially the same and is detailed below.
Two New Zealand rabbits (4-6 months old) were used for immunization with
each type of antigen.. The antigens were prepared by dissolving 500 ~g MAP
peptide
in 500 ~l of saline and mixed with equal 500 pl of complete Freund's adjuvant
and
injected subcutaneously at three to four dorsal sites. Same concentration of
each
antigen (in saline) was mixed with equal volume of incomplete Freund's
adjuvant and
injected as before at: two, foL~r and six weeks after the first immunization.
Animals
were bled from the ;auricular artery to collect 30-SU ml blood on days 0, 27,
57 and 69.
22
62451-853(S)
CA 02312591 2001-05-08
Blood samples were allowed to clot at room temperature for 15 min. and serum
was
isolated from each sample by centrifugation at 5,000 x g for 10 min. Cell-free
serum
was decanted gently into a clean tube and stored at -20°C till further
use.
cDNA Cloning
Total RNA was isolated from corn tissues with TRIzoI Reagent (Life
Technology, Inc. Gaithersburg, MD) using a modification of the guanidine
isothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi
(Chomczynski et al. (1987) Anal. Biochem 162:156). In brief, plant tissue
samples
I 0 were pulverized in liquid nitrogen before the addition of the
TRIzolReagent, and then
were further homogenized with a mortar and pestle. Addition of chloroform by
centrifugation was conducted for separation of an aqueous phase and an organic
phase. The total RNA was recovered by precipitation with isopropyl alcohol
from the
aqueous phase.
I ~ The selection of poly(A) + RNA from total RNA was performed using
PolyATract system (Promega Corporation, Madison, WI). In brief, biotinylated
oligo
(dT) primers were used to hybridize to the 3' poly(A) tails on mRNA. The
hybrids
were captured using streptavidin coupled to paramagnetic particles and a
magnetic
separation stand. The mRNA was washed at high stringent condition and eluted
by
20 RNase-free deionized water.
Synthesis of the cDNA was performed and unidirectional cDNA libraries were
constructed using the Superscript Plasmid System (Life Technology, Inc.,
Gaithersburg, MD). First strand of cDNA was synthesized by priming an
oligo(dT)
primer containing a Not I site. The reaction was catalyzed by Superscript
Reverse
2~ Transcriptase II at 45°C. The second strand of cDNA was labeled with
alpha-'ZY-
dCTP and portions of the molecules smaller than 500 base pairs and unligated
adapters were removed by Sephacryl-5400 chromatography. The selected cDNA
molecules were ligated into pSPORTI~reference vector between the Not I and Sal
I
si tes.
*Trademark
23
CA 02312591 2001-05-08
62451-853(S)
Individual colonies were picked and DNA was prepared either by PCR with
M 13 forward primers and M 13 reverse primers, or by plasmid miniprep
isolation. All
the cDNA clones were sequenced using M13 reverse primers.
Arral ty ica!
Protein was estimated by the Bradford method (Bradford, M. (1976) ibid
72:248-254) using bovine y-globulin as standard. Activity blots, Western blots
and
product analysis were performed essentially following published protocols (10,
20-
22), except that all essays were carried out at 6°C.
Identification of Zinc Fingers
Two PCR primers were designed to encompass both the Zinc fingers of the
maize PARP sequence. These primers were used for reverse transcriptase
assisted
PCR using the Titan 1 tube RT-PCR kit from Boeheringer Mannheim. Maize callus
1 ~ and leaf mRNA was used as template. The PCR product was purified using Qia
Quick*PCR product purification columns (Qiagen) and sequenced using an ABI
sequencer. Sequenced data is shown in SEQ ID NO. S.
Isolation of PARP from maize cells
Cells
(60-80 g)
Suspend in Nebulization Buffer ( 4 ml/g)
(20 mM Hepes pH 7.9, 20% glycerol, 0.4 molal sorbitol, 0.l mM EDTA)
(+DTT and PI cocktail)
Bionebulization at 100 psi x 4
Filter through cheesecloth
Filtrate
Add 0.1 volume of SASS
*Trademark 24
CA 02312591 2001-05-08
62451-853(S)
Mix gently for 1 hr.
Centrifuge at 100,000 x g
Add 0.4 of solid (NH4),SO, per ml/S 100
Mix for 30 min.
Centrifuge at 40,000 x g for 30 min.
Dissolve precipitate in HGED buffer and dialyze overnight
Centrifuge at 40,000 x g for 30 min.
Store WCE at -80°C
Scheme I
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention
pertains.
Although the foregoing invention has been described
in some detail by way of illustration and example for purposes
of clarity of understanding, it will be obvious that certain
changes and modifications may be practiced within the scope of
the appended claims.
CA 02312591 2003-07-25
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