Note: Descriptions are shown in the official language in which they were submitted.
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FLORAL HOMEOTIC GENES FOR MANIPULATION OF FLOWERING
IN POPLAR AND OTHER PLANT SPECIES
Field of the Invention
This invention relates to nucleic acid molecules isolated from Populus
species, and
methods of using these molecules and derivatives thereof to produce plants,
particularly trees
such as Populus species, that have modified fertility characteristics.
Background of the Invention
The increasing demand for pulp and paper products and the diminishing
availability
of productive forest lands are being addressed in part by efforts to develop
trees that produce
increased yields in shorter growth periods. Many such efforts are focused on
the production
of transgenic trees having modified growth characteristics, such as reduced
lignin content
(see for example, U. S. Patent No. 5,451,514, "Modification of Lignin
Synthesis in Plants"),
and resistance to insect, viruses and herbicides. A major concern with the
production of
transgenic trees is the possibility that the transgenic traits might be
introduced into
indigenous tree populations by cross-fertilization. Thus, for example, the
introduction of
genes for insect resistance into indigenous tree populations could accelerate
the evolution of
resistant insects, adversely affect endangered insect species and interfere
with normal food
chains. Because of these concerns, the U.S. and other governments have
instituted
regulatory review processes to assess the risks associated with proposed
environmental
releases of transgenic plants (both for field trials and commercial
production).
Genetic engineering of sterility into trees offers the possibility of securing
introduced
genes in the engineered tree; trees that produce neither pollen nor seeds will
not be able to
transmit introduced genes by normal routes of reproduction. Additional
potential benefits of
engineering sterility into trees include increased wood yields and reduced
production of
allergens such as pollen. For a review of engineering reproductive sterility
in forest trees,
see Strauss et al: (1995).
Two primary methods for engineering sterility have been described. In the
first
method, termed genetic ablation, a cytotoxic gene is expressed under the
control of a
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reproductive tissue-specific promoter. Cytotoxic genes employed in this method
to date
include RNase (Mariani et al., 1990; Mariani et al. 1992; Reynarts et al.
1993; Goldman et al.
1994), ADP-ribosyl transferase (Thorsness et al., 1991; Kandasamy, 1993;
Thorseness et al.,
1993), the Agrobacterium RoIC gene (Schmiilling, 1993), and glucanase (Worrall
et al.
1992, Paul et al., 1992). The expression of the cytotoxic gene results
(ideally) in the death of
all cells in which the reproductive tissue-specific promoter is active. It is
therefore critical
that the promoter be highly specific to the reproductive tissue to avoid
pleiotropic effects on
vegetative tissue. For this reason, genome position effects on the transgene
need to be
monitored (see Strauss et al., 1995). The success of genetic ablation methods
in trees will
thus depend on the availability of a suitable reproductive tissue-specific
promoter for the tree
species in question.
The second method for engineering sterility involves inhibiting the expression
of
genes that are essential for reproduction. This can be accomplished in a
number of ways,
including the use of antisense RNA, sense suppression and promoter-based
suppression.
Effective antisense (Kooter, 1993; Mol et~.l., 1994; Van der Meer et al. 1992;
Pnueli et al.,
1994), sense suppression (Flavell, 1994; Jorgensen, 1992; Taylor et al., 1992)
and promoter-
based suppression (Brusslan, 1993; Matzke, 1993) in plants have been
described. The key
to the use of any of these methods in the production of sterile trees is the
identification of
appropriate indigenous genes, i.e, disruption of the expression of such genes
must result in
the abolition of correct reproductive tissue development.
Genes specifically expressed in reproductive tissues have been isolated from a
number of plant species (for a review, see Strauss et al., 1995). Genes that
have been .
characterized as acting early in the development of floral structures include
LEAFY (LFY)
from Arabidopsis (Weigel et al. 1992), APETALA1 (AP 1) from Arabidopsis
(Mandel et al,
1992), and FLORICAULA (FLO) from Antirrhinum (Coen et al., 1990), which
regulate the
transition from inflorescence to floral meristems. APETALA2 (AP2) appears to
regulate the
AGAMOUS gene (AG) which plays a role in differentiation of male and female
floral tissues
(see Okamuro et al., 1992). DEFICIENS (DEF) is a floral homeotic gene from
Antirrhinum
that is expressed throughout flower development (Schwarz-Sommer et al. 1992).
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The majority of floral homeotic genes are members of the MADS-box family of
transcription factors (Yanofsky et al., 1990). The MADS-box is a conserved
region of
approximately 60 amino acid residues. MADS is an acronym for the first four
known genes
in which the MADS-box was identified: yeast minichromosomal maintenance factor
(MCM 1 ), the floral homeotic genes AG and DEF, and human serum respons a
factor (SRF).
Plant MADS-box genes contain four domains: the highly conserved MADS-box
region
located near or at the 5' end of the translated region in plant genes; the L
or linker region
between the MADS and K domains; the K domain, a moderately conserved keratin-
like
region predicted to form amphipathic a-helices; and a highly variable carboxy-
terminal
region. The K-box is only present in plant MADS-box genes. It is thought to be
involved
in protein-protein interactions (Pnueli et al., 1991).
Studies have shown that the organization of the MADS domain in plants is
similar to
that in SRF; the basic N-terminal portion of the domain is required for DNA-
binding and the
C-terminal half of the box is required for dimerization. Because MADS proteins
bind DNA
as dimers, the MADS box as well as a C-terminal extension that is involved in
dimerization
are required for DNA-binding. The C-terminal extension varies throughout the
gene family.
C-terminal deletions indicate that the minimal DNA-binding domain of AP 1 and
AG
includes the MADS-box and part of the L region, whereas AP3 and PI require a
portion of
the K box in addition to the MADS and L regions (Riechmann et al., 1996). The
difference
in the sizes of the minimal binding domains is thought to reflect the
dimerization
characteristics of the respective proteins: AP 1 and AG bind DNA as homodimers
whereas
AP3/PI and their Antirrhinum homologs DEF/GLO bind as heterodimers.
MADS-box proteins have been found to bind to a motif found in target gene
promoters referred to as the CArG-box. CArG-box motifs are also found in the
promoters of
MADS-box genes, where they are thought to be targets for auto-regulation.
Riechmann et al
used circular permutation and phasing analysis to detect conformational
changes in DNA
that resulted from MADS-box protein binding (Reichmann et al., 1996). They
found that
bound API, AP3/Pl, and AG all induce DNA bending oriented toward the minor
groove. For
a review of MADS box biology, see Ma et al., 1994; Puruggana et al., 1995; and
Yanofsky,
1995. AG and DEF have been characterized as MARS box genes; while FLO and LFY
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appear to encode transcription factors and have proline-rich and acidic
domains, they are not
MADS box genes.
Following a functional analyses of MADS box genes, Mizukami et al. (1996)
created
deletion mutants of AG in which various domains of the gene, including the
MADS and K
boxes were deleted. Based on their results, they proposed that dominant
negative mutations
of MADS box genes could be created by deleting the all or part of the MARS
domain, or by
deleting all or part of the K domain or by deleting various portions of the 3'
region of the
AG open reading frame. It was proposed that the proteins encoded by these
deletion mutants
would be able to bind either the target DNA (i.e., the nucleotide sequence to
which the
transcription factor binds) or the protein co-factors required for
transcription, but not both.
Thus, it was proposed that such mutant proteins would interfere with the
functioning of the
coexisting corresponding endogenous gene. The studies of floral homeotic genes
discussed
in the preceding paragraphs havc been primarily undertaken in model plants
such as
Arabidopsis and Antirrhinum; few, if any, studies have addressed the genetics
of flowering
in tree. species at the molecular level. '
Species of the genus Populus are becoming increasingly important in the
forestry
industry, particularly for pulp and paper production, in part because of their
fast growth
characteristics. This group includes aspens (species of Populus section Leuce
and their
hybrids), and hybrids between black cottonwood (P. trichocarpa Ton. and Gray,
also
classified as P. balsamifera subsp. trichocarpa; Brayshaw, 1960 and eastern
cottonwood
(P. deltoides L.). These species are also well suited to manipulation by
genetic engineering
because they are fast-growing, have relatively small genomes, are easy to
regenerate in vitro,
and are susceptible to transformation with Agrobacterium. To date however,
relatively few
genes have been cloned from these species. Notably, the genetic basis
underlying floral
development in these species is almost completely uncharacterized.
Floral development in the genus Populus is significantly different from what
is seen
in a typical hermaphroditic annual (Nagaraj, 1952; Boes and Strauss, 1994).
The apices of
the branches do not become inflorescences. The flowers are borne on axillary
inflorescences, or catkins, with male and female flowers found on separate
trees, although
occasionally mixed inflorescences or hermaphroditic flowers are seen. The
inflorescences
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appear from dormant buds in the spring, usually occurring from about five
years of age.
Instead of the usual structure of four concentric whorls of organs (sepals
outermost, followed
by petals, then stamens surrounding one or more carpets in the center), the
Populus flower
apparently has only two whorls (a reduced perianth cup surrounding either
stamens or
carpets). Unlike several other species that produce unisexual flowers through
developmental
arrest or degeneration of one set of organs (Cheng et al., 1983; Grant et al.,
1994), Populus
does not initiate male organs in female flowers or vice versa (Goes and
Strauss, 1994;
Sheppard, 1997). After releasing pollen or seeds, the entire inflorescences
are shed (Kaul,
1995). By late spring, the inflorescence buds for the next year's flowers have
already been
initiated in the axils of the current year's leaves, and will develop for
several more months
before going dormant.
The availability of genes that control floral development in Populus species
would
permit the production of genetically engineered sterile trees. In turn, the
ability to control
fertility of Populus trees in this way would be of great value in
environmental and biosafety
of Populus trees engineered for improved agronomic characteristics. It is to
such genes that
the present invention is directed.
Summary of the Invention
The present invention provides four floral homeotic genes from Populus
trichocarpa.
The four genes are herein termed PTFL, PTD, PTAG-1 and PTAG-2. These genes are
homologs of floral homeotic genes isolated from other plant species.
Specifically, PTFL is a
homolog of LEAFY (LFY) and FLORICAULA (FLO), PTD is a homolog of DEFICIENS
(DEF) and PTAG-1 and PTAG-2 are homologs of AGAMOUS (AG). The Populus genes
are shown to be expressed in floral tissues; for example, PTFL is expressed in
immature
inflorescences on which floral promordia are developing, whereas PTD is
expressed strongly
in stamen primordia from the onset of organogenesis. PTD is also expressed at
low levels in
carpet primordia.
The invention provides the nucleic acid sequences of these four Populus genes,
the
corresponding cDNA sequences and the deduced amino acid sequences of the
encoded
polypeptides. Along with these sequences, the present invention also provides
methods of
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using the gene and cDNA sequences to produce genetically engineered Populus
species and
other trees having modified fertility characteristics, including sterility.
Genetic constructs useful in producing genetically engineered Populus and
other
trees include antisense versions of PTFL, PTD, PTAG-1 and PTAG-2, dominant
negative
mutants of these genes, and constructs useful for sense suppression. In
addition, the
promoter sequences of these genes may be used to obtain floral-specific
expression of genes
such as cytotoxins that may be employed in genetic ablation strategies to
produce trees
having modified fertility characteristics, including sterility.
In one aspect, the invention provides isolated nucleic acid molecules
comprising
portions of the disclosed nucleic acid sequences. Such molecules comprise at
least 15
consecutive nucleotides of the disclosed PTFL, PTD, PTAG-1 or PTAG-2 nucleic
acid
sequences, and may be longer, comprising at least 20 or 25 or 50 consecutive
nucleotides of
these sequences. Such molecules are useful, among other things, as primers and
probes for
amplifying all or parts of the disclosed sequences and for detecting the
expression of the
nucleic acid molecules in cells, such as cells of tran'sgenic plants. Thus, in
one aspect, such
molecules are useful to monitor the expression of transgenes comprising some
portion of the
PTD, PTFL, PTAG-1 or PTAG-2 molecules.
Modification of the fertility traits of plants, such as Populus species may
also be
obtained by introducing genetic constructs containing variants of all or
portions of the
disclosed PTD, PTFL, PTAG-1 or PTAG-2 sequences. Such variants are provided by
the
invention and may comprise a nucleotide sequence of at least 50 (or, for
example, at least
100) nucleotides in length which sequence hybridizes under conditions of
defined
stringency (such as at least 75% or 90% stringency) to the disclosed nucleic
acid sequences.
Alternatively, such variants may share a specified percentage of sequence
identity with the
disclosed nucleic acid sequences (e.g., at least 75% or at least 90% sequence
identity) as
determined using a specified sequence alignment program.
The disclosed nucleic acid molecules and variant forms of these molecules may
be
assembled in nucleic acid vectors for introduction into cells, such as plant
cells. Thus,
another aspect of the invention comprises the disclosed nucleic acid molecules
and variants
thereof, and vectors comprising these molecules.
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In another embodiment, the invention provides transgenic plants comprising the
vectors. Such transgenic plants may have altered phenotypes (compared to non-
transgenic
plants of the same species) including modified fertility characteristics.
Modified fertility
characteristics include modifications in the timing of flowering, for example,
advancing the
timing of flowering relative to non-transgenic plants of the same species, and
sterility.
Sterility may be complete sterility, or may be male only or female only
sterility. Examples of
transgenic plants provided by the present invention include genetically
engineered sterile
Populus and Eucalyptus species.
In another embodiment, the invention provides transgenic plants that comprise
a
recombinant expression cassette, wherein the recombinant expression cassette
comprises a
promoter sequence operably linked to a first nucleic acid sequence, and
wherein the first
nucleic acid sequence comprises all or part of one of the disclosed nucleic
acid molecules, or
a variant of one of the disclosed nucleic acid molecules. By way of example,
such transgenic
plants include plants in which the first nucleic acid is arranged in reverse
orientation to the
promoter sequence in the recombinant expression cassette, such that an
antisense RNA is
produced. In another example, such transgenic plants include plants in which
the first
nucleic acid is a dominant negative mutant of PTD, PTFL, PTAG-1 or PTAG-2,
produced by
deletion of part of the coding region, such as the 3' portion of the open
reading frame, or all
or part of a MADS or K-box region of the coding region. In other embodiments,
the
promoter sequence driving expression of the first nucleic acid may be a
promoter that
confers enhanced expression of the first nucleic acid molecule in floral
tissues of the plant
relative to non-floral tissues.
In other embodiments, the expression of at least one endogenous gene in
transgenic
plants containing such a recombinant expression cassette will be modified as a
result of the
cassette. In particular embodiments, that modified expression will affect the
fertility of the
plant, and will render the plant sterile.
In yet other embodiments, the invention provides transgenic plants comprising
a
recombinant expression cassette, wherein the recombinant expression cassette
comprises a
promoter sequence operably linked to a first nucleic acid sequence, and
wherein the
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promoter sequence is a promoter sequence from PTD, PTFL, PTAG-1 or PTAG-2. In
particular embodiments, the first nucleic acid sequence encodes a cytotoxic
polypeptide.
These and other aspects of the invention are described in more detail below.
Sequence Listing
The nucleic and amino acid sequences listed in the accompanying Sequence
Listing
are showed using standard letter abbreviations for nucleotide bases, and three
letter code for
amino acids. Only one strand of each nucleic acid sequence is shown, but the
complementary strand is understood to be included by any reference to the
displayed strand.
Seq. LD. No. 1 shows the nucleic acid sequence of the PTD gene.
Seq. LD. No. 2 shows the nucleic acid sequence of the PTD cDNA.
Seq. LD. No. 3 shows the nucleic acid sequence of the PTD ORF.
Seq. LD. No. 4 shows the amino acid sequence of the
PTD polypeptide.
Seq. LD. No. 5 shows the nucleic acid sequence of
the PTFL gene.
~. Seq. LD. No. 6 shows the nucleic acid sequence of
the PTFL cDNA.
Seq. LD. No. 7 shows the nucleic acid sequence of
the PTFL ORF.
Seq. LD. No. 8 shows the amino acid sequence of the
PTFL polypeptide.
Seq. LD. No. 9 shows the nucleic acid sequence of
the PTAG-1 gene.
Seq. LD. No. 10 shows the nucleic acid sequence of
the PTAG-1 cDNA.
Seq. LD. No. 11 shows the nucleic acid sequence of the PTAG-1 ORF.
Seq. LD. No. 12 shows the amino acid sequence of the PTAG-1 polypeptide.
Seq. LD. No. 13 shows the nucleic acid sequence of the PTAG-2 gene.
Seq. LD. No. 14 shows the nucleic acid sequence of the PTAG-2 cDNA.
Seq. LD. No. 15 shows the nucleic acid sequence of the PTAG-2 ORF.
Seq. LD. No. 16 shows the amino acid sequence of the PTAG-2 polypeptide.
Seq. LD. Nos. 17-23 show oligonucleotide primers that may be used to amplify
portions of the disclosed floral homeotic nucleic acid sequences.
Brief Description of the Drawings
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Fig. 1 shows the sequence of the PTD gene. Exons are capitalized, the cDNA and
all
motifs are underlined. The MARS box is indicated by a solid line above the
sequence, the
K-domain by a dashed line. The TATA motif is double-underlined.
Fig. 2 shows an alignment of MADS and K-domains in various plant polypeptides.
Fig. 2A shows the alignment of MADS domains, Fig. 2B shows the alignment of K
domains.
Detailed Description of the Invention
I. Definitions and Abbreviations
To facilitate review of the various embodiments of the present invention, the
following definitions of terms and explanations of abbreviations are provided.
Isolated: An "isolated" nucleic acid has been substantially separated or
purified
away from other nucleic acid sequences in the cell of the organism in which
the nucleic acid
naturally occurs, i. e., other chromosomal and extrachremosomal DNA and RNA.
The term
"isolated" thus encompasses nucleic acids purified by standard nucleic acid
purification
methods. The term also embraces nucleic acids prepared by recombinant
expression in a
host cell as well as chemically synthesized nucleic acids.
cDNA (complementary DNA): A piece of DNA lacking internal, non-coding
segments (introns) and regulatory sequences which determine transcription.
cDNA is
synthesized in the laboratory by reverse transcription from messenger RNA
extracted from
cells.
ORF (open reading frame): A series of nucleotide triplets (codons) coding for
amino acids without any termination codons. These sequences are usually
translatable into a
peptide.
Ortholog: Two nucleotide sequences are orthologs of each other if they share a
common ancestral sequence and diverged when a species carrying that ancestral
sequence
split into two species. Ortholgous sequences are also homologous sequences.
Probes and primers: Nucleic acid probes and primers may readily be prepared
based on the nucleic acids provided by this invention. A probe comprises an
isolated
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nucleic acid attached to a detectable label or reporter molecule. Typical
labels include
radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods
for labeling
and guidance in the choice of labels appropriate for various purposes are
discussed, e.g., in
Sambrook et al. (1989) and Ausubel et al. (1987). Probes are useful in nucleic
acid
hybridization methods such as Southern blotting, to detect the presence of
particular target
nucleic acid sequences. Probes are typically 20 or more nucleotides in length.
Primers are short nucleic acids, preferably DNA oligonucleotides 15
nucleotides or
more in length. Primers may be annealed to a complementary target DNA strand
by nucleic
acid hybridization to form a hybrid between the primer and the target DNA
strand, and then
extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs
can be
used for amplification of a nucleic acid sequence, e.g., by the polymerase
chain reaction
(PCR) or other nucleic-acid amplification methods known in the art.
One of skill in the art will appreciate that the specificity of probes and
primers
generally increases with length. At a minimum, probes and primers typically
comprise at
least 15 or 20 consecutive nucleotides of the nucleic acid on which they are
based.
However, to enhance specificity and reduce background hybridization, probes
and primers
of increasing length may be used. Thus, probes and primers included in the
present
invention may comprise at least 25, 30, 3~, 50 or 100 consecutive nucleotides
of a nucleic
acid molecule provided by the invention. Methods for preparing and using
probes and
primers are described, for example, in Sambrook et al. (1989), Ausubel et al.
(1987), and
Innis et al., (1990). PCR primer pairs can be derived from a known sequence,
for example,
by using computer programs intended for that purpose such as Primer (Version
0.5, D 1991,
Whitehead Institute for Biomedical Research, Cambridge, MA).
Purified: The term purified does not require absolute purity; rather, it is
intended as
a relative term. Thus, for example, a purified PTAG-1 protein preparation is
one in which
the PTAG-1 protein is more pure than the protein in its natural environment
within a cell.
Preferably, a preparation of a purified protein according to the present
invention is purified
such that the subject protein represents at least 50% of the total protein
content of the
preparation.
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Operably linked: A first nucleic acid sequence is operably linked with a
second
nucleic acid sequence when the first nucleic acid sequence is placed in a
functional
relationship with the second nucleic acid sequence. For instance, a promoter
is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the
S coding sequence. Generally, operably linked DNA sequences are contiguous
and, where
necessary to join two protein coding regions, in the same reading frame.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not
naturally occurring or has a sequence that is made by an artificial
combination of two
otherwise separated segments of sequence. This artificial combination is often
accomplished
by chemical synthesis or, more commonly, by the artificial manipulation of
isolated
segments of nucleic acids, e.g., by genetic engineering techniques.
Vector: A nucleic acid molecule as introduced into a host cell, thereby
producing a
transformed host cell. A vector may include an origin of replication, one or
more selectable
marker genes and other genetic elements known in the art.
. Transformed: A transformed cell is a cell into which has been introduced a
nucleic
acid molecule by molecular biology techniques. As used herein, the term
transformation
encompasses all techniques by which a nucleic acid molecule might be
introduced into such
a cell, including Agrobacterium-mediated transformation, transfection with
viral vectors,
transformation with plasmid vectors and introduction of nucleic acids by
electroporation,
lipofection, and particle gun acceleration.
Transgenic plant: As used herein, this term refers to a plant that contains
recombinant genetic material not normally found in plants of this type and
which has been
introduced into the plant in question (or into progenitors of the plant) by
human
manipulation. Thus, a plant that is grown from a plant cell into which
recombinant DNA is
introduced by transformation is a transgenic plant, as are all offspring of
that plant which
contain the introduced DNA (whether produced sexually or asexually).
Additional definitions of terms commonly used in molecular genetics can be
found in
Benjamin Lewin, Genes V published by Oxford University Press, 1994 (ISBN 0-19-
854287-
9); Kendrew et al (eds.), The Encyclopedia ofMolecular Biology, published by
Blackwell
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Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular
Biology
and Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc.,
1995 (ISBN 1-56081-569-8).
II. Methods
The four floral homeotic genes were obtained, and the present invention can be
practiced, using standard molecular biology and plant transformation
procedures, unless
otherwise noted. Standard molecular biology procedures are described in
Sambrook et al
(1989), Ausubel et al. (1988) and Innis et al. (1990).
III. Isolation and Characterization of PTFL
Genomic DNA was purified from dormant vegetative buds of a single Populus
trichocarpa tree using a modified CTAB extraction technique (Wagner et al.,
1987). After
centrifugation to pellet nuclei, a large gummy pellet of resin was evident.
This was left intact
during the resuspension of nuclei, and then discarded. Normal yield of DNA was
approximately 1 mg per 40 g of tissue. A genomic library was constructed from
DNA
partially digested with Sau3A, filled in with DNA Pol I and dATP and dGTP, and
ligated
into LambdaGem-12 vector (Stratagene) having partially filled-in Xho I sites.
Packaging of
the DNA into phage particles was performed with GigaPack Gold II (Stratagene).
RNA was extracted using the lithium dodecyl sulfate method of Baker et al.
(1990),
and purified by centrifugation through a 5.7 M CsCI pad. After redissolving
the RNA pellet
in TE, pH 8.0, NaCI was added to 400 mM and the RNA was precipitated with EtOH
to
remove excess CsCI. PolyA+ RNA was selected using oligo dT-cellulose columns
(mRNA
Separation Kit, Clontech). RNA was stored at -80 C until use. Ten-microgram
samples of
total RNA were used as templates for single-stranded cDNA synthesis. Reactions
included
50 mM TrisHCl (pH 8.3), 75 mM KCI, 10 mM dithiothreitol, 3 mM MgClz, 100 ~M
each
dNTP, 4 ~g primer XT, 10 ~lCi [a3zP]-dCTP, and 200 U M-MLV reverse
transcriptase (Gibco
BRL) in 50 ~1L. Incubations were performed at 37 C for 1 hr, then the cDNA was
purified
with GeneClean (BIO101) silica matrix. Typical yields were 10 - 40 ng of cDNA,
as
determined by 3zP incorporation. The size ranges of the cDNA samples were
characterized
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by alkaline gel electrophoresis. cDNA products were between 500 to 4000 bases
in length,
with an average size of 1000 bases. The DNA was diluted to 0.25 ng/11L in 10
mM TrisHCl,
1 mM EDTA (pH 8.0) and stored at -20 C.
cDNA libraries were prepared using the Lambda-ZAP CDNA cloning kit
(Stratagene). From 5 ~lg of polyA+ RNA, approximately 106 clones were
recovered per
preparation, with an average size of 1 kb and a size range of 500 by to 3 kb.
A
hybridization probe for the Populus FLOlLFY homolog was obtained by touchdown
PCR
(Don et al., 1991) of the cDNA library with a degenerate primer specific to a
highly
conserved region of the FLO and LFY genes and a primer specific for the vector
plus 3'-end
of polyadenylated cDNAs. The PCR protocol was as follows: (94 C, 30 sec; 60 C,
30 sec;
71 C, 1 min) X 2, (94 C, 3 0 sec; 5 8 C, 3 0 sec; 71 C, 1 min) X 2, (94 C, 3 0
sec; 56 C, 3 0
sec; 71 C, 1 min) X 2, (94 C, 30 sec; 54 C, 30 sec; 71 C, 1 min) X 2, (94 C,
30 sec; 52 C,
30 sec; 71 C, 1 min) X 2, (94 C, 30 sec; 50 C, 30 sec; 71 C, 1 min) X 8, (94
C, 30 sec; 52
C, 30 sec; 71 C, 1 min) X 25. The approximately 480 by fragment obtained was
gel-purified
and subcloned into pBluescript SK(-) for further characterization.
The PTFL genomic clone was isolated by screening the genomic library using
probes
derived from the PTFL cDNA sequence. Sequencing of the cDNA was performed
using the
dideoxy- terminator-based Sequenase 2.0 kit (Unites States Biochemical Corp.),
according to
the methods described by the manufacturer. Most sequencing of the cDNA and
subclones of
the gene was done using universal primers on nested deletions created with
ExoIII
(Henikoff, 1982). Gaps were filled in by sequencing from specific primers
synthesized at
Oregon State University. Sequence analysis was performed using PCGENE
(Intelligenetics).
A total of 5,656 by of the PTFL gene locus was sequenced, including 2,638 by
upstream of the initiation codon and 457 by downstream of the polyA addition
site. This
sequence is available on GenBank
(http://www.ncbi.nlm.nih.gov/web/search/index.html)
under accession number U93196 and is shown in Seq. LD. No. 5. The positions of
the two
introns found in both FLO and LFY are conserved in PTFL. A probable TATA box
and a
CART box upstream of the coding sequence are underlined. The longest cDNA
obtained
(Seq. LD. No. 6) includes an open reading frame (Seq. LD. No. 7) that encodes
for a
predicted polypeptide of 377 amino acid residues (Seq. LD. No. 8). Comparison
of the
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deduced PTFL amino acid sequence with several FLO/LFY homologs revealed
conserved
amino- and carboxyl-terminal domains (133 and 175 residues, respectively, in
PTFL) linked
by a poorly conserved, highly charged domain (69 residues). The overall
sequence identity
between PTFL and FLO (Coen et al., 1990) is 79%, with 88% amino acid sequence
similarity.
For in situ hybridization analysis, tissue samples from various sources were
fixed,
embedded, sectioned, and hybridized as described by Kelly et al. (1995), with
the following
modifications. Sections were 10 elm in thickness. Probes were generated from a
plasmid
consisting of the PTFL cDNA inserted between the EcoRI and Kpn I sites of the
vector
pBluescriptII SK (-), and were not alkaline hydrolysed. A PTFL antisense probe
hybridized
strongly to the floral meristems and developing flowers of both male and
female plants.
PTFL was not detected in the apical inflorescence meristem, but was seen in
the flanking
nascent floral meristems. Developing flowers showed expression in the immature
carpels
and anthers. Both male and female flowers exhibited some hybridization on the
inner
(adaxial) rim of the perianth cup during the middle stages of development.
PTFL also
showed marked hybridization to bracts. Hybridization was observed with
vegetative buds
from mature branches. The pattern of hybridization showed that there was RNA
in the axils
of the newly formed leaves, but not in the center of the vegetative meristem.
There was also
significant expression in the tips of the leaf primordia, and in some portions
of the
surrounding developing leaves.
Overexpression and antisense constructs of PTFL cDNA were produced for
analysis
in transgenic trees. The insert from the cDNA clone of PTFL was cut out using
EcoR I and
Kpn I, and the ends were polished with T4 DNA polymerase. The insert was then
ligated
into the Sma I site of pBI121 (Jefferson et al., 1987). Clones with each
orientation were
identified by PCR, and the structures of the junction sites near the promoters
of both were
verified by sequencing of the PCR fragments. Hybrid aspens were used for
transformation,
in part because of the relative ease of transformation, and in part because of
concern that
transgenic cottonwoods might interact with native cottonwoods in the vicinity
of the
experimental site. The P. tremula X alba hybrid aspen female clone 717-1B4 and
the P.
tremula X tremuloides hybrid aspen male clone 353-38 were transformed with
pDW151
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(Weigel and Nilsson, 1995) and the above binary vectors using Agrobacterium
tumefasciens
strain C58 (Leple et al., 1992) with modifications as described by Han et al.
(1996).
III. Isolation and Characterization of PTD
The PTD cDNA and gene were isolated by probing the Populus cDNA library
described above at low stringency using an Eco RI fragment of pCIT2241 (Ma et
al., 1991)
which contains the MADS box region of AGLl. The PTD cDNA (Seq. LD. No. 2)
comprises an open reading frame (Seq. LD. No. 3) encoding a 227 amino acid
polypeptide
(Seq. LD. No. 4). The PTD gene (Seq. LD. No. 1) consists of seven exons.
The PTD polypeptide is 81 % conserved overall with respect to DEF. PTD has
MADS and K domains (see Fig. 1). The MARS domain extends over amino acids 1-
60,
while the K-domain extends over amino acids 88-143. The MARS domain is 93%
conserved with respect to DEF, whereas the K domain is 85% conserved at the
amino acid
level (Fig. 2).
IV. Isolation and Characterization of PTAG1 and PTAG2
Two cDNAs and their corresponding genes were isolated from Populus using the
methodologies described above and a probe derived from the 3' region of the AG
cDNA.
Denoted PTAG-l and PTAG-2, these two sequences are the orthologs of AG.
The genomic, cDNA and open reading frame sequences of PTAG-1 are shown in
Seq. LD. Nos. 9, 10 and 1 l, respectively. The open reading frame encodes a
polypeptide of
241 amino acids in length (Seq. LD. No. 12). The PTAG-1 polypeptide contains
both a
MADS domain and a K-domain. The MADS domain extends from amino acids 17-33 and
the K-domain from amino acids 106-172. The PTAG-1 nucleotide and amino acid
sequences are available on GenBank under accession number AF052570.
The genomic, cDNA and open reading frame sequences of PTAG-2 are shown in
Seq. LD. Nos. 13, 14 and 15, respectively. The open reading frame encodes a
polypeptide of
238 amino acids in length (Seq. LD. No. 16). The PTAG-1 polypeptide contains
both a
MADS domain and a K-domain. The MADS domain extends from amino acids 17-33 and
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the K-domain from amino acids 106-172. The PTAG-2 nucleotide and amino acid
sequences
are available on GenBank under accession number AF052571.
At the amino acid level, PTAG-l and PTAG 2 are 89% identical, and show 72-75%
sequence similarity with AG.
Because AG is only expressed in floral tissues and is essential for the
development of
both male and female reproductive organs, it is ideally suited for use in
modifying fertility
through genetic engineering approaches. In situ hybridization studies show
that the PTAG
genes in Populus are expressed in the central zone of both male and female
floral meristems,
and, as with AG, expression begins before reproductive primordia emerge and
continues in
developing stamens and carpets.
EXAMPLES
The following examples are provided to illustrate the scope of the invention.
. Ezample 1
Preferred Method of Makinis the Populus Genes and cDNAs
With the provision of the four Populus floral homeotic nucleic acid sequences
PTD,
PTFL, PTAG-1 and PTAG-2, the polymerase chain reaction (PCR) may now be
utilized in a
preferred method for producing the cDNAs and genes, as well as derivatives of
these
sequences. PCR amplification of the sequence may be accomplished either by
direct PCR
from an appropriate cDNA or genomic library. Alternatively, the cDNAs may be
amplified
by Reverse-Transcription PCR (RT-PCR) using RNA extracted from Populus cells
as a
template. Similarly, the gene sequences may be directly amplified using
Populus genomic
DNA as a template. Methods and conditions for both direct PCR and RT-PCR are
known in
the art and are described in Innis et al. (1990). Suitable plant cDNA and
genomic libraries
for direct PCR include Populus libraries made by methods described above.
Other tree
cDNA and genomic libraries may be used in order to amplify orthologous cDNAs
of tree
species, such as Pinus and Eucalyptus.
The selection of PCR primers will be made according to the portions of the
cDNA or
gene that are to be amplified. Primers may be chosen to amplify small segments
of the
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cDNA or gene, or the entire cDNA or genes. Variations in amplification
conditions may be
required to accommodate primers of differing lengths; such considerations are
well known in
the art and are discussed in Innis et al. (1990), Sambrook et al. (1989), and
Ausubel et al
(1992). By way of example only, the PTD cDNA molecule as shown in Seq. LD. No.
2 (with
the exception of the 5' poly-A tail) may be amplified using the following
combination of
pnmers:
5' ATGGGTCGTGGAAAGATTGAAATCAAG 3' (Seq. LD. No. 17)
5' ATTTGTGAAAAAGAGCTTTTATATTTA 3' (Seq. LD. No. 18)
The open reading frame portion of the PTD cDNA may be amplified using the
following
primer pair:
5' ATGGGTCGTGGAAAGATTGAAATCAAG 3' (Seq. LD. No. 17)
5' AGGAAGGCGAAGTTCATGGGATCCAAA 3' (Seq. LD. No. 19)
A derivative version of the PTD ORF that lacks the MARS box domain may be
amplified
using the following primers:
5' TCCACATCGACAAAGAAGATCTACGAT 3' (Seq. LD. No. 20)
5' AGGAAGGCGAAGTTCATGGGATCCAAA 3' (Seq. LD. No. 19)
These primers are illustrative only; it will be appreciated by one skilled in
the art that
many different primers may be derived from the provided cDNA and gene
sequences in
order to amplify particular regions of the provided nucleic acid molecules.
Suitable
amplification conditions include those described above for the original
isolation of the PTFL
cDNA. As is well known in the art, amplification conditions may need be varied
in order to
amplify orthologous genes where the sequence identity is not 100%; in such
cases, the use
of nested primers, as described above may be beneficial. Resequencing of PCR
products
obtained by these amplification procedures is recommended; this will
facilitate confirmation
of the amplified cDNA sequence and will also provide information on natural
variation on
this sequence in different ecotypes, cultivars and plant populations.
Oligonucleotides that are derived from the PTD, PTFL, PTAG-1 and PTAG-2 cDNA
and gene sequences and which are suitable for use as PCR primers to amplify
corresponding
nucleic acid sequences are encompassed within the scope of the present
invention.
Preferably, such oligonucleotide primers will comprise a sequence of 15-20
consecutive
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nucleotides of the selected cDNA or gene sequence. To enhance amplification
specificity,
primers comprising at least 25, 30, 35, 50 or 100 consecutive nucleotides of
the PTD, PTFL,
PTAG-1 or PTAG-2 gene or cDNA sequences may be used.
Ezample 2
Use of the Ponulus Genes and cDNAs to Modifv Fertilit3~ Characteristics
Once a nucleic acid encoding a protein involved in the determination of a
particular
plant characteristic, such as flowering, has been isolated, standard
techniques may be used to
express the nucleic acid in transgenic plants in order to modify that
particular plant
characteristic. One approach is to clone the nucleic acid into a vector, such
that it is operably
linked to control sequences (e.g., a promoter) which direct expression of the
nucleic acid in
plant cells. The transformation vector is then introduced into plant cells by
one of a number
of techniques (e.g., electroporation and Agrobacterium-mediated
transformation) and
progeny plants containing the introduced nucleic acid are selected. Preferably
all or part of
the transformation vector will stably integrate into the genome of the plant
cell. That part of
the vector which integrates into the plant cell and which contains the
introduced nucleic acid
and associated sequences for controlling expression (the introduced
"transgene") may be
referred to as the recombinant expression cassette.
Selection of progeny plants containing the introduced transgene may be made
based
upon the detection of an altered phenotype. Such a phenotype may result
directly from the
nucleic acid cloned into the transformation vector or may be manifested as
enhanced
resistance to a chemical agent (such as an antibiotic) as a result of the
inclusion of a
dominant selectable marker gene incorporated into the transformation vector.
The choice of (a) control sequences and (b) how the nucleic acid (or selected
portions
of the nucleic acid) are arranged in the transformation vector relative to the
control sequences
determine, in part, how the plant characteristic affected by the introduced
nucleic acid is
modified. For example, the control sequences may be tissue specific, such that
the nucleic
acid is only expressed in particular tissues of the plant (e.g., reproductive
tissues) and so the
affected characteristic will be modified only in those tissues. The nucleic
acid sequence may
be arranged relative to the control sequence such that the nucleic acid
transcript is expressed
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normally, or in an antisense orientation. Expression of an antisense RNA that
is the reverse
complement of the cloned nucleic acid will result in a reduction of the
targeted gene product
(the targeted gene product being the protein encoded by the plant gene from
which the
introduced nucleic acid was derived). Over-expression of the introduced
nucleic acid,
resulting from a plus-sense orientation of the nucleic acid relative to the
control sequences in
the vector, may lead to an increase in the level of the gene product, or may
result in a
reduction in the level of the gene product due to co-suppression (also termed
"sense
suppression") of that gene product. In another approach, the nucleic acid
sequence may be
modified such that certain domains of the encoded peptide are deleted.
Depending on the
domain deleted, such modified nucleic acid may act as dominant negative
mutations,
suppressing the phenotypic effects of the corresponding endogenous gene.
Successful examples of the modification of plant characteristics by
transformation
with cloned nucleic acid sequences are replete in the technical and scientific
literature.
Selected examples, which serve to illustrate the level of knowledge in this
field of
technology include:
U.S. Patent No. 5,432,068 to Albertson (control of male fertility using
externally
inducible promoter sequences);
U.S. Patent No. 5,686,649 to Chua (suppression of plant gene expression using
processing-defective RNA constructs);
U. S. Patent No. 5,659,124 to Crossland (transgenic male sterile plants);
U. S. Patent No. 5,451,514 to Boudet (modification of lignin synthesis using
antisense RNA and co-suppression);
U.S. Patent No. 5,443,974 to Hitz (modification of saturated and unsaturated
fatty
acid levels using antisense RNA and co-suppression);
U.S. Patent No. 5,530,192 to Murase (modification of amino acid and fatty acid
composition using antisense RNA);
U. S. Patent No. 5,455,167 to Voelker (modification of medium chain fatty
acids)
U.S. Patent No. 5,231,020 to Jorgensen (modification of flavonoids using co-
suppression);
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U.S. Patent No. 5,583,021 to Dougherty (modification of virus resistance by
expression of plus-sense RNA); and
Mizukami et al. (1996) (dominant negative mutations in floral development
using
partial deletions of AG).
These examples include descriptions of transformation vector selection,
transformation techniques and the production of constructs designed to over-
express an
introduced nucleic acid, dominant negative mutant forms, untranslatable RNA
forms or
antisense RNA. In light of the foregoing and the provision herein of the PTD,
PTFL, PTAG-
1 and PTAG-2 cDNA and gene sequences, it is apparent that one of skill in the
art will be
able to introduce these cDNAs or genes, or derivative forms of these sequences
(e.g.,
antisense forms), into plants in order to produce plants having modified
fertility
characteristics, particularly sterility. This Example provides a description
of the approaches
that may be used to achieve this goal. For convenience the PTD, PTFL, PTAG-1
and PTAG-
2 cDNAs and genes disclosed herein will be generically referred to as the
"floral homeotic
nucleic acids," and the encoded polypeptides as the "floral homeotic
polypeptides". Example
3 provides an exemplary illustration of how an antisense form of ane of these
floral homeotic
nucleic acids, specifically the PTD cDNA, may be introduced into poplar
species using
Agrobacterium transformation, in order to produce genetically engineered
sterile poplars.
Example 4 provides an exemplary illustration of how mutant forms of PTAG-1 may
be
produced and introduced into poplar species to produce modified fertility
characteristics.
a. Plant Types
The floral homeotic nucleic acids disclosed herein may be used to produce
transgenic
plants having modified fertility characteristics. In particular, the amenable
plant species
include, but are not limited to, members of the genus Populus, including
Populus
trichocarpa (commonly known as black cottonwood, California poplar and western
balsam
poplar) and poplar hybrid species. Other woody species that are amenable to
fertility
modification by the methods disclosed herein include members of the genera
Picea, Pinus
Pseudotsuga, Tsuga, Sequoia, Abies, Thuja, Libocedrus, Chamaecyparis and
Larix. In
particular, members of the genera Eucalyptus, Acacia and Gmelina, which are
becoming
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increasingly important for pulp production, may be engineered for sterility
using the nucleic
acid sequences and methods disclosed here.
b. Vector construction, choice of promoters
A number of recombinant vectors suitable for stable tra.nsfection of plant
cells or for
the establishment of transgenic plants have been described including those
described in
Pouwels et al., (1987), Weissbach and Weissbach, (1989), and Gelvin et al.,
(1990).
Typically, plant transformation vectors include one or more cloned plant genes
(or cDNAs)
under the transcriptional control of 5' and 3' regulatory sequences and a
dominant selectable
marker. Such plant transformation vectors typically also contain a promoter
regulatory
region (e.g., a regulatory region controlling inducible or constitutive,
environmentally or
developmentally regulated, or cell- or tissue-specific expression), a
transcription initiation
start site, a ribosome binding site, an RNA processing signal, a transcription
termination site,
and/or a polyadenylation signal.
Examples of constitutive plant promoters which may be useful for expressing
the
floral homeotic nucleic acids include: the cauliflower mosaic virus (CaNl~ 35S
promoter,
which confers constitutive, high-level expression in most plant tissues (see,
e.g., Odel et al.,
1985, Dekeyser et al., 1990, Terada a.nd Shimamoto, 1990; Benfey and Chua.
1990); the
nopaline synthase promoter (An et al., 1988); and the octopine synthase
promoter (Fromm et
al., 1989).
A variety of plant gene promoters that are regulated in response to
environmental,
hormonal, chemical, and/or developmental signals, also can be used for
expression of the
floral homeotic nucleic acids in plant cells, including promoters regulated
by: (a) heat (Callis
et al., 1988; Ainley, et al. 1993; Gilmartin et al. 1992); (b) light (e.g.,
the pea rbcS-3A
promoter, Kuhlemeier et al., 1989, and the maize rbcS promoter, Scha.ffner and
Sheen, 1991;
(c) hormones, such as abscisic acid (Marcotte et al., 1989); (d) wounding
(e.g., wunl,
Siebertz et al., 1989); and (e) chemicals such as methyl jasminate or
salicylic acid (see also
Gatz 1997) can also be used to regulate gene expression.
Alternatively, tissue specific (root, leaf, flower, and seed for example)
promoters
(Carpenter et al. 1992, Denis et al. 1993, Opperman et al. 1993, Stockhause et
al. 1997;
Roshal et al., 1987; Schernthaner et al., 1988; and Bustos et al., 1989) can
be fused to the
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coding sequence to obtained particular expression in respective organs. In
addition, the
timing of the expression can be controlled by using promoters such as those
acting at
senescencing (Gan and Amasino 1995) or late seed development (Odell et al.
1994).
The promoter regions of the PTD, PTFL, PTAG-1 or PTAG-2 gene sequences confer
floral-specific (or floral-enriched) expression in Populus. Accordingly, these
native
promoters may be used to obtain floral-specific (or floral-enriched)
expression of the
introduced transgene.
Plant transformation vectors may also include RNA processing signals, for
example,
introns, which may be positioned upstream or downstream of the ORF sequence in
the
transgene. In addition, the expression vectors may also include additional
regulatory
sequences from the 3'-untranslated region of plant genes, e.g., a 3'
terminator region to
increase mRNA stability of the mRNA, such as the PI-II terminator region of
potato or the
octopine or nopaline synthase 3' terminator regions.
Finally, as noted above, plant transformation vectors may also include
dominant
selectable marker genes to allow for the ready selection of transformants.
Such genes
include those encoding antibiotic resistance genes (e.g., resistance to
hygromycin,
kanamycin, bleomycin, 6418, streptomycin or spectinomycin) and herbicide
resistance
genes (e.g., phosphinothricin acetyltransferase).
c. Arrangement of floral homeotic nucleic acid sequence in vector
Modified fertility characteristics in plants may be obtained using the floral
homeotic
nucleic acid sequences disclosed herein in a variety of forms. Over-
expression, sense-
suppression, antisense RNA and dominant negative mutant forms of the disclosed
floral
homeotic nucleic acid sequences may be constructed in order to modulate or
supplement the
expression of the corresponding endogenous floral homeotic genes, and thereby
to produce
plants having modified fertility characteristics. Alternatively, the floral-
specific (or floral-
enriched) expression conferred by the promoters of the disclosed floral
homeotic genes may
be employed to obtain corresponding expression of cytotoxic products. Such
constructs will
comprise the appropriate floral homeotic promoter sequence operably linked to
a suitable
open reading frame (discussed further below) and will be useful in genetic
ablation
approaches to engineering sterility in plants.
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i. Modulation/supplementation of floral homeotic
nucleic acid expression
The particular arrangement of the floral homeotic nucleic acid sequence in the
transformation vector will be selected according to the type of expression of
the sequence
that is desired.
Enhanced expression of a floral homeotic nucleic acid may be achieved by
operably
linking the floral homeotic nucleic acid to a constitutive high-level promoter
such as the
CaMV 35S promoter. As noted below, modified activity of a floral homeotic
polypeptide in
planta may also be achieved by introducing into a plant a transformation
vector containing a
variant form of a floral homeotic nucleic acid, for example a form which
varies from the
exact nucleotide sequence of the disclosed floral homeotic nucleic acid.
A reduction in the activity of a floral homeotic polypeptide in the transgenic
plant
may be obtained by introducing into plants antisense constructs based on the
floral homeotic
nucleic acid sequence. For expression of antisense RNA, the floral homeotic
nucleic acid is
arranged in reverse orientation relative to the promoter sequence in the
transformation
vector. The introduced sequence need not be the full length floral homeotic
nucleic acid,
and need not be exactly homologous to the floral homeotic nucleic acid found
in the plant
type to be transformed. Generally, however, where the introduced sequence is
of shorter
length, a higher degree of homology to the native floral homeotic nucleic acid
sequence will
be needed for effective antisense suppression. Preferably, the introduced
antisense sequence
in the vector will be at least 30 nucleotides in length, and improved
antisense suppression
will typically be observed as the length of the antisense sequence increases.
Preferably, the
length of the antisense sequence in the vector will be greater than 100
nucleotides.
2~ Transcription of an antisense construct as described results in the
production of RNA
molecules that are the reverse complement of mRNA molecules transcribed from
the
endogenous floral homeotic gene in the plant cell. Although the exact
mechanism by which
antisense RNA molecules interfere with gene expression has not been
elucidated, it is
believed that antisense RNA molecules bind to the endogenous mRNA molecules
and
thereby inhibit translation of the endogenous mRNA.
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Suppression of endogenous floral homeotic polypeptide activity can also be
achieved
using ribozymes. Ribozymes are synthetic RNA molecules that possess highly
specific
endoribonuclease activity. The production and use of ribozymes are disclosed
in U.S. Patent
No. 4,987,071 to Cech and U.S. Patent No. 5,543,508 to Haselhoff. The
inclusion of
ribozyme sequences within antisense RNAs may be used to confer RNA cleaving
activity on
the antisense RNA, such that endogenous mRNA molecules that bind to the
antisense RNA
are cleaved, which in turn leads to an enhanced antisense inhibition of
endogenous gene
expression.
Constructs in which the floral homeotic nucleic acid (or variants thereon) are
over-
expressed may also be used to obtain co-suppression of the endogenous floral
homeotic
nucleic acid gene in the manner described in U.S. Patent No. 5,231,021 to
Jorgensen. Such
co-suppression (also termed sense suppression) does not require that the
entire floral
homeotic nucleic acid cDNA or gene be introduced into the plant cells, nor
does it require
that the introduced sequence be exactly identical to the endogenous floral
homeotic nucleic
acid gene. However, as with antisense suppression, the suppressive efficiency
will be
enhanced as ( 1 ) the introduced sequence is lengthened and (2) the sequence
similarity
between the introduced sequence and the endogenous floral homeotic nucleic
acid gene is
increased.
Constructs expressing an untranslatable form of the floral homeotic nucleic
acid
mRNA may also be used to suppress the expression of endogenous floral homeotic
genes.
Methods for producing such constructs are described in U.S. Patent No.
5,583,021 to
Dougherty et al. Preferably, such constructs are made by introducing a
premature stop
codon into the floral homeotic nucleic acid ORF.
Finally, dominant negative mutant forms of the disclosed sequences may be used
to
block endogenous floral homeotic polypeptide activity using approaches similar
to that
described by Mizukami et al. (1996). Such mutants require the production of
mutated forms
of the floral homeotic polypeptide that bind either to an endogenous binding
target (for
example, a nucleic acid sequence in the case of floral homeotic polypeptides,
such as PTD,
that function as transcription factors) or to a second polypeptide sequence
(such as
transcription co-factors), but do not function normally after such binding
(i.e. do not
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function in the same manner as the non-mutated form of the polypeptide). By
way of
example, such dominant mutants can be constructed by deleting all or part of
the C-terminal
domain of a floral homeotic polypeptide, leaving an intact MADS domain.
Polypeptides
lacking all or part of the C-terminal region may bind to the appropriate DNA
target, but are
unable to interact with protein co-factors, thereby blocking transcription.
Alternatively,
dominant negative mutants may be produced by deleting all or part of the MADS
domain, or
all or part of the K-domain.
ii. Genetic ablation
An alternative approach to modulating floral development is to specifically
target a
cytotoxic gene product to the floral tissues. This may be achieved by
producing transgenic
plants that express a cytotoxic gene product under the control of a floral-
specific promoter,
such as the promoter regions of PTFL, PTD, PTAG-1 and PTAG-2 as disclosed
herein. The
promoter regions of these gene sequences are generally contained within the
first 150 base
pairs of sequence upstream of the open reading frame, although floral-specific
expression
may be conferred by using smaller regions of this sequence. Thus, regions as
small as the
first 50 base pairs of sequence upstream of the open reading frame may be
effective in
conferring floral-specific expression. However, longer regions, such as at
least 100, 150,
200 or 250 base pairs of the upstream sequences are preferred.
A number of known cytotoxic gene products may be expressed under the control
of
the disclosed promoter sequences of the floral homeotic genes. These include:
Rl~lases, such
as barnase from Bacillus amyloliquefaciens and RNase-T1 from Aspergillus
(Ma.riani et al.,
1990; Mariani et al., 1992; Reynaerts et al., 1993); ADP-ribosyl-transferase
(Diphtheria
toxin A chain) (Pappenheimer, 1977; Thorness et al., 1991; Kandasamy et al.,
1993); RoIC
from Agrobacterium rhizogenes (Schmulling et al., 1993); and glucanase
(Worrall et al.,
1992).
c. Transformation and regeneration techniques
Constructs designed as discussed above to modulate or supplement expression of
native floral homeotic genes in plants, or to express cytotoxins in a tissue-
specific manner
can be introduced into plants by a variety of means. Transformation and
regeneration of
both monocotyledonous and dicotyledonous plant cells is now routine, and the
selection of
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the most appropriate transformation technique will be determined by the
practitioner. The
choice of method will vary with the type of plant to be transformed; those
skilled in the art
will recognize the suitability of particular methods for given plant types.
Suitable methods
may include, but are not limited to: electroporation of plant protoplasts;
liposome-mediated
transformation; polyethylene mediated transformation; transformation using
viruses; micro-
injection of plant cells; micro-projectile bombardment of plant cells; vacuum
infiltration; and
Agrobacterium tumefaciens (AT) mediated transformation. Typical procedures for
transforming and regenerating plants are described in the patent documents
listed at the
beginning of this section.
Methods that are particularly suited to the transformation of woody species
include
(for Picea species) methods described in Ellis et al. (1991, 1993) and (for
Populus species)
the use of A. tumefaciens (Settler, 1993; Strauss et al., 1995), A. rhizogenes
(Han et al., 1996)
and biolistics (McCown et al., 1991).
e. Selection of transformed plants
Following transformation and regeneration of plants with the transformation
vector,
transformed plants are preferably selected using a dominant selectable marker
incorporated
into the transformation vector. Typically, such a marker will confer
antibiotic resistance on
the seedlings of transformed plants, and selection of transformants can be
accomplished by
exposing the seedlings to appropriate concentrations of the antibiotic.
After transformed plants are selected and grown to maturity, the effect on
fertility can
be determined by visual inspection of floral morphology, including the
determination of the
production of pollen or ova. In addition, the effect on the activity of the
endogenous floral
homeotic gene may be directly determined by nucleic acid analysis
(hybridization or PCR
methodologies) or immunoassay of the expressed protein. Antisense or sense
suppression of
the endogenous floral homeotic gene may be detected by analyzing mRNA
expression on
. Northern blots or by reverse transcription polymerase chain reaction (RT-
PCR).
Example 3
Introduction of antisense PTD cDNA into hybrid aspens
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By way of example, the following methodology may be used to produce poplar
trees
with modified expression of PTD. The PTD cDNA (Seq. LD. No. 2) is excised from
the
cloning vector and blunt ended using T4 DNA polymerise. The cDNA is then
ligated into
the Sma I site of pBI121 (Jefferson et al., 1987), and clones containing the
cDNA in reverse
orientation with respect to the promoter are identified by sequence analysis.
Hybrid aspens, such as the P. tremula X alba hybrid aspen and the P. tremula X
tremuloides hybrid aspen are transformed with pDW 151 (Weigel and Nilsson,
1995) and the
above binary vectors using Agrobacterium tumefasciens strain C58 (Leple et
al., 1992) with
modifications as described by Han et al. (1996).
Expression of the antisense transgene is assessed in immature plants by
extraction of
mRNA and northern blotting using the PTD cDNA as a probe, or by RT-PCR. Levels
of
PTD protein are analyzed by extraction and concentration of cellular proteins
followed by
western blotting, or by in situ hybridization.
Example 4
Expression of mutant PTAGl sequences in plants
PTAG-1 mutants are constructed by PCR amplification using standard PCR
methodologies as described above and a Populus cDNA library as a template. A
mutant
form of PTAG-1 in which the MARS box domain is deleted is amplified using the
following
primer combination:
5' GTCACTTTCTGCAAAAGGCGCAGTGGT 3' (Seq. LD. No. 21)
5' AACTAACTGAAGGGCCATCTGATCTTG 3' (Seq. LD. No. 22)
A mutant form of PTAG-1 in which a portion of the 3' region of the encoded
polypeptide is deleted is amplified using the following primer combination:
5' ATGGAATATCAAAATGAATCCCTTGAG 3' (Seq. LD. No. 23)
5'ATTCATGCTCTGTCGCTTTCTTTCATTCT 3' (Seq. LD. No. 24)
The amplified products are cloned using standard cloning vectors and then
ligated
into a transformation vector such as pBI121 (Jefferson et al., 1987).
Hybrid aspens, such as the P.-tremula X alba hybrid aspen and the P. tremula X
tremuloides hybrid aspen are transformed with pDW 151 (Weigel and Nilsson,
1995) and the
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pBI121 binary vector containing the mutant PTAG-1 construct using
Agrobacterium
tumefasciens strain C58 (Leple et al., 1992) with modifications as described
by Han et al.
( 1996).
Expression of the mutant PTAG-1 transgenes is assessed in immature plants by
extraction of mRNA and northern blotting using the PTAG-1 cDNA as a probe or
by RT-
PCR. Levels of mutant protein are analyzed by extraction and concentration of
cellular
proteins followed by western blotting, or by in situ hybridization.
Example 5
Production of Sec~nence Variants
As noted above, modification of the activity of floral homeotic polypeptides
such as
PTD, PTFL, PTAG-l and PTAG-2 in plant cells can be achieved by transforming
plants with
a selected floral homeotic nucleic acid (cDNA or gene, or parts therof),
antisense constructs
based on the disclosed floral homeotic nucleic acid sequences or other
variants on the
disclosed sequences. Sequence variants include not only genetically engineered
sequence
variants, but also naturally occurring variants that arise within Populus
populations,
including allelic variants and polymorphisms, as well as variants that occur
in different
genotypes and species of Populus. These naturally occurring variants may be
obtained by
PCR amplification from genomic or cDNA libraries made from genetic material of
Populus
species, or by RT-PCR from mRNA from such species, or by other methods known
in the
art, including using the disclosed nucleic acids as probes to hybridize with
genetic libraries.
Methods and conditions for both direct PCR and RT-PCR are known in the a.rt
and are
described in Innis et al. (1990).
As noted, variant DNA molecules also include those created by DNA genetic
engineering techniques, for example, M13 primer mutagenesis. Details of these
techniques
are provided in Sambrook et al. (1989), Ch. 15. By the use of such techniques,
variants may
be created which differ in minor ways from the floral homeotic cDNA or gene
sequences
disclosed. DNA molecules and nucleotide sequences which are derived from the
floral
homeotic nucleic acids disclosed include DNA sequences which hybridize under
stringent
conditions to the DNA sequences disclosed, or fragments thereof.
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Hybridization conditions resulting in particular degrees of stringency will
vary
depending upon the nature of the hybridization method of choice and the
composition and
length of the hybridizing DNA used. Generally, the temperature of
hybridization and the
ionic strength (especially the Na+ concentration) of the hybridization buffer
will determine
the stringency of hybridization. Calculations regarding hybridization
conditions required for
attaining particular degrees of stringency are discussed by Sambrook et al.
(1989), chapters 9
and 11, herein incorporated by reference. By way of illustration only, a
hybridization
experiment may be performed by hybridization of a DNA molecule (for example, a
variant of
the PTAG-1 cDNA sequence) to a target DNA molecule (for example, the PTAG-1
cDNA
sequence) which has been electrophoresed in an agarose gel and transferred to
a
nitrocellulose membrane by Southern blotting (Southern, 1975), a technique
well known in
the art and described in (Sambrook et al., 1989). Hybridization with a target
probe labeled
with [3zP]-dCTP is generally carried out in a solution of high ionic strength
such as 6xSSC at
a temperature that is 20-25 C below the melting temperature, Tm, described
below. For such
Southern hybridization experiments where the target DNA molecule on the
Southern blot
contains 10 ng of DNA or more, hybridization is typically carried out for 6-8
hours using
1-2 ng/ml radiolabeled probe (of specific activity equal to 10 9 CPM/~g or
greater).
Following hybridization, the nitrocellulose filter is washed to remove
background
hybridization. The washing conditions should be as stringent as possible to
remove
background hybridization but to retain a specific hybridization signal. The
term Tm
represents the temperature above which, under the prevailing ionic conditions,
the
radiolabeled probe molecule will not hybridize to its target DNA molecule. The
Tm of such a
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hybrid molecule may be estimated from the following equation (Bolton and
McCa.rthy,
1962):
Tm = 81.5 C - 16.6(log lo[Na+]) + 0.41 (%G+C) - 0.63 (% formamide) - (600/
Where 1= the length of the hybrid in base pairs.
This equation is valid for concentrations of Na+ in the range of 0.01 1~ to
0.4 M, and it
is less accurate for calculations of Tm in solutions of higher [Na+]. The
equation is also
primarily valid for DNAs whose G+C content is in the range of 30% to 75%, and
it applies
to hybrids greater than 100 nucleotides in length (the behavior of
oligonucleotide probes is
described in detail in Ch. 11 of Sambrook et al., 1989).
Thus, by way of example, for a 150 base pair DNA probe derived from the first
150
base pairs of the open reading frame of the PTAG-1 cDNA (with a hypothetical
%GC =
45%), a calculation of hybridization conditions required to give particular
stringencies may
be made as follows:
For this example, it is assumed that the filter will be washed in 0.3 xSSC
solution
following hybridization, thereby [Na'] = 0.045M; %GC = 45%; Formamide
concentration = 0; l = 150 base pairs; and
Tm = 81.5 - 16(loglo[Na+]) + (0.41 x 45) - (600/150)
and so Tm = 74.4 C.
The Tm of double-stranded DNA decreases by 1-1.5 C with every 1% decrease in
homology (Bonner et al., 1973). Therefore, for this given example, washing the
filter in
0.3 xSSC at 59.4-64.4 C will produce a stringency of hybridization equivalent
to 90%.
Alternatively, washing the hybridized filter in 0.3 xSSC at a temperature of
65.4-68.4 C will
yield a hybridization stringency of 94%. The above example is given entirely
by way of
theoretical illustration. One skilled in the art will appreciate that other
hybridization
techniques may be utilized and that variations in experimental conditions will
necessitate
alternative calculations for stringency.
DNA sequences which hybridize under hybridization conditions of at least 75%,
more preferably at least 80%, more preferably at least 85%, more preferably at
least 90% and
most preferably at least 95% stringency to the PTD, PTFL, PTAG-l and PTAG-2
nucleic
acid sequences are encompassed within the present invention. Such nucleotide
sequences
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are preferably at least 50 nucleotides on length, and more preferably at least
100 or 250
nucleotides in length. Such sequences are useful in generating constructs for
modifying the
activity of the corresponding polypeptides in plants, and thereby the
fertility characteristics
of plants.
The degeneracy of the genetic code further widens the scope of the present
invention
as it enables major variations in the nucleotide sequence of a DNA molecule
while
maintaining the amino acid sequence of the encoded protein. For example, the
fifth amino
acid residue of the PTFL protein is alanine. This is encoded in the PTFL open
reading frame
by the nucleotide codon triplet GCT. Because of the degeneracy of the genetic
code, three
other nucleotide codon triplets--GCA, GCC and GCG--also code for alanine.
Thus, the
nucleotide sequence of the PTFL ORF could be changed at this position to any
of these three
codons without affecting the amino acid composition of the encoded protein or
the
characteristics of the protein. Based upon the degeneracy of the genetic code,
variant DNA
molecules may be derived from the cDNA and gene sequences disclosed herein
using
standard DNA mutagenesis techniques as described above, or by synthesis of DNA
sequences. Such molecules may be employed to over-express the floral homeotic
polypeptides in plants and thereby produce plants having modified fertility
characteristics.
One skilled in the art will recognize that DNA mutagenesis techniques may be
used
not only to produce variant DNA molecules, but will also facilitate the
production of
proteins which differ in certain structural aspects from the disclosed floral
homeotic
polypeptides. Newly derived proteins may also be selected in order to obtain
variations on
the characteristic of the floral homeotic polypeptides, as will be more fully
described below.
Such derivatives include those with variations in amino acid sequence
including deletions,
additions and substitutions.
While the site for introducing an amino acid sequence variation is
predetermined, the
mutation per se need not be predetermined. For example, in order to optimize
the
performance of a mutation at a given site, random mutagenesis may be conducted
at the
target codon or region and the expressed protein variants screened for the
optimal
combination of desired activity. Techniques for making substitution mutations
at
predetermined sites in DNA having a known sequence as described above are well
known.
31
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Amino acid substitutions are typically of single residues; insertions usually
will be
on the order of about from 1 to 10 amino acid residues; and deletions will
range about from 1
to 60 residues. Substitutions, deletions, insertions or any combination
thereof may be
combined to arnve at a final construct. The mutations that are made in the DNA
encoding
the protein must not place the sequence out of reading frame and preferably
will not create
complementary regions that could produce secondary mRNA structure.
Substitutional variants are those in which at least one residue in the amino
acid
sequence has been removed and a different residue inserted in its place. Such
substitutions
generally are made in accordance with the following Table 1 when it is desired
to finely
modulate the characteristics of the protein. Table 1 shows amino acids which
may be
substituted for an original amino acid in a protein and which are regarded as
conservative
substitutions.
Table 1.
_ Orisinal Residue Conservative Substitutions
Ala s er
~g lys
Asn gln; his
~p glu
Cys ser
Gln asn
Glu asp
Gly pro
His asn; gln
Ile leu, val
Leu ile; val
Lys ~g~ g~~ glu
Met leu; ile
Phe met; leu; tyr
3 0 Ser thr
Thr ser
Trp tyr
Tyr trp; phe
Val ile~ leu
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Substantial changes in enzymatic function or other features are made by
selecting
substitutions that are less conservative than those in Table 1, i.e.,
selecting residues that
differ more significantly in their effect on maintaining (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a sheet or helical
conformation,
(b) the charge or hydrophobicity of the molecule at the target site, or (c)
the bulk of the side
chain. The substitutions which in general are expected to produce the greatest
changes in
protein properties will be those in which (a) a hydrophilic residue, e.g.,
seryl or threonyl, is
substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,
phenylalanyl, valyl or
alanyl; (b) a cysteine or proline is substituted for (or by) any other
residue; (c) a residue
having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is
substituted for (or by)
an electronegative residue, e.g., glutamyl or asparrtyl; or (d) a residue
having a bulky side
chain, e.g., phenylalanine, is substituted for (or by) one not having a side
chain, e.g., glycine.
Sequence variants may alternatively be defined by the degree of sequence
identity.
Methods of determining the level of sequence identity between two given
protein or
nucleotide sequences are well known and include the BLAST algorithm and search
program
that is available at http://www.ncbi.nlm.nih.govBLAST (Altschul et al., 1990;
Altschul et
al., 1994). A description of how to determine sequence identity using this
program is
available at http://www.ncbi.nlm.nih.govBLAST/blast help.html.
Homologous polypeptides that share at least 70% amino acid sequence identity
to the
disclosed PTD, PTFL, PTAG-1 or PTAG-2 amino acid sequences as determined using
BLAST 2.0, gapped blastp, with default parameters, are encompassed by this
invention.
Such homologous peptides will more preferably share at least 75%, more
preferably at least
80% and still more preferably at least 90% or 95% sequence identity to one of
the disclosed
sequences as determined using the specified program. Such homologous peptides
are
preferably at least 10 amino acids in length, and more preferably at least 25
or 50 amino
acids in length. Also encompassed by the present invention are the nucleic
acid sequences
that encode these homologous peptides.
Similarly, homologous nucleic acids that share at least 70% nucleotide
identity to the
disclosed PTD, PTFL, PTAG-1 or PTAG-2 nucleic acid sequences as determined
using
BLAST 2.0, gapped blastn, with default parameters, are encompassed by this
invention.
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Such homologous nucleic acid sequences will more preferably share at least
75%, more
preferably at least 80% and still more preferably at least 90% or 95% sequence
identity to
one of the disclosed sequences as determined using the specified program. Such
homologous nucleic acids are preferably at least 50 nucleotides on length, and
more
preferably at least 100 or 250 nucleotides in length.
Example 6
Other Applications of the Disclosed Sequences
The disclosed floral homeotic nucleic acids and polypeptide are useful as
laboratory
reagents to study and analyze floral gene expression in plants, including
plants engineered
for modified fertility characteristics. For example, probes and primers
derived from the PTD
sequence, as well as monoclonal antibodies specific for the PTD polypeptide
may be used to
detect and quantify expression of PTD in seedlings transformed with an
antisense PTD
construct as described above. Such analyses would facilitate detection of
those
transformants that display modified PTD expression and which may therefore be
good
candidates for having modified fertility characteristics.
The production of probes and primers derived from the disclosed sequences is
described in detail above. Production of monoclonal antibodies requires that
all or part of
the protein against which the antibodies to be raised be purified. With the
provision herein
of the floral homeotic nucleic acid sequences, as well as the sequences of the
encoded
polypeptides, this may be achieved by expression in heterologous expression
systems, or
chemical synthesis of peptide fragments.
Many different expression systems are available for expressing cloned nucleic
acid
molecules. Examples of prokaryotic and eukaryotic expression systems that are
routinely
used in laboratories are described in Chapters 16-17 of Sa.mbrook et al.
(1989). Such
systems may be used to express the floral homeotic polypeptides at high levels
to faciliate
purification.
By way of example only, high level expression of a floral homeotic polypeptide
may
be achieved by cloning and expressing the selected cDNA in yeast cells using
the pYES2
yeast expression vector (Invitrogen, San Diego, CA). Secretion of the
recombinant floral
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homeotic polypeptide from the yeast cells may be achieved by placing a yeast
signal
sequence adjacent to the floral homeotic nucleic acid coding region. A number
of yeast
signal sequences have been characterized, including the signal sequence for
yeast invertase.
This sequence has been successfully used to direct the secretion of
heterologous proteins
from yeast cells, including such proteins as human interferon (Chang et al.,
1986), human
lactoferrin (Liang and Richardson, 1993) and prochymosin (Smith et al., 1985).
Alternatively, the enzyme may be expressed at high level in prokaryotic
expression systems,
such as E. coli.
Monoclonal or polyclonal antibodies may be produced to the selected floral
homeotic
polypeptide or portions thereof. Optimally, antibodies raised against a
specified floral
homeotic polypeptide will specifically detect that polypeptide. That is, for
example,
antibodies raised against the PTD polypeptide would recognize and bind the PTD
polypeptide and would not substantially recognize or bind to other proteins
found in poplar
cells. The determination that an antibody specifically detects PTD is made by
any one of a
number of standard immunoassay methods; for instance, the Western blotting
technique
(Sambrook et al., 1989). To determine that a given antibody preparation (such
as one
produced in a mouse against PTD) specifically detects PTD by Western blotting,
total
cellular protein is extracted from poplar cells and electrophoresed on a
sodium dodecyl
sulfate-polyacrylamide gel. The proteins are then transferred to a membrane
(for example,
nitrocellulose) by Western blotting, and the antibody preparation is incubated
with the
membrane. After washing the membrane to remove non-specifically bound
antibodies, the
presence of specifically bound antibodies is detected by the use of an anti-
mouse antibody
conjugated to an enzyme such as alkaline phosphatase; application of the
substrate 5-bromo-
4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production
of a dense blue
compound by immuno-localized alkaline phosphatase. Antibodies which
specifically detect
PTD will, by this technique, be shown to bind to substantially only the PTD
band (which
will be localized at a given position on the gel determined by its molecular
weight). Non-
specific binding of the antibody to other proteins may occur and may be
detectable as a weak
signal on the Western blot. The non-specific nature of this binding will be
recognized by
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one skilled in the art by the weak signal obtained on the Western blot
relative to the strong
primary signal arising from the specific antibody-PTD binding.
Substantially pure floral homeotic polypeptides suitable for use as an
immunogen
may be isolated from transformed cells as described above. Concentration of
protein in the
final preparation is adjusted, for example, by concentration on an Amicon
filter device, to the
level of a few micrograms per milliliter. Alternatively, peptide fragments of
the specified
Moral homeotic polypeptide may be utilized as immunogens. Such fragments may
be
chemically synthesized using standard methods, or may be obtained by cleavage
of the
whole floral homeotic polypeptide followed by purification of the desired
peptide fragments.
Peptides as short as 3 or 4 amino acids in length are immunogenic when
presented to the
immune system in the context of a Major Histocompatibility Complex (NIPIC)
molecule,
such as MHC class I or MHC class II. Accordingly, peptides comprising at least
3 and
pereferably at least 4, 5, 6 or 10 or more consecutive amino acids of the
disclosed floral
homeotic polypeptide amino acid sequences may be employed as immuogens to
raise
antibodies. Because naturally occurring epitopes on proteins are frequently
comprised of
amino acid residues that are not adjacently arranged in the peptide when the
peptide
sequence is viewed as a linear molecule, it may be advantageous to utilize
longer peptide
fragments from the floral homeotic polypeptide amino acid sequences in order
to raise
antibodies. Thus, for example, peptides that comprise at least 10, 15, 20, 25
or 30
consecutive amino acid residues of the floral homeotic polypeptide amino acid
sequence
may be employed. Monoclonal or polyclonal antibodies to the intact floral
homeotic
polypeptide or peptide fragments of this protein may be prepared as described
below.
Monoclonal antibody to epitopes of the selected floral homeotic polypeptide
can be
prepared from murine hybridomas according to the classical method of Kohler
and Milstein
(1975) or derivative methods thereof. Briefly, a mouse is repetitively
inoculated with a few
micrograms of the selected protein over a period of a few weeks. The mouse is
then
sacrificed, and the antibody-producing cells of the spleen isolated. The
spleen cells are fused
by means of polyethylene glycol with mouse myeloma cells, and the excess
unfused cells
destroyed by growth of the system on selective media comprising aminopterin
(HAT media).
The successfully fused cells are diluted and aliquots of the dilution placed
in wells of a
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microtiter plate where growth of the culture is continued. Antibody-producing
clones are
identified by detection of antibody in the supernatant fluid of the wells by
immunoassay
procedures, such as ELISA, as originally described by Engvall (1980), and
derivative
methods thereof. Selected positive clones can be expanded and their monoclonal
antibody
product harvested for use. Detailed procedures for monoclonal antibody
production are
described in Harlow and Lane (1988).
Having illustrated and described the principles of isolating the Populus
floral
homeotic genes, the proteins encoded by these genes and modes of use of these
biological
molecules, it should be apparent to one skilled in the art that the invention
can be modified
in arrangement and detail without departing from such principles. We claim all
modifications coming within the spirit and scope of the claims presented
herein.
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42
CA 02227940 1999-OS-20
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: THE STATE OF OREGON ACTING BY AND THROUGH THE STATE
BOARD OF HIGHER EDUCATION ON BEHALF OF OREGON STATE
UNIVERSITY
(ii) TITLE OF INVENTION: FLORAL HOMEOTIC GENES FOR MANIPULATION OF
FLOWERING IN POPLAR TREES AND OTHER PLANT
SPECIES
(iii) NUMBER OF SEQUENCES: 24
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
2 O (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,227,940
(B) FILING DATE: 07-APR-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/080,851
(B) FILING DATE: 06-APR-1998
(viii) ATTORNEY/AGENT INFORMATION:
3 O (A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 63198-1218
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486
- 43 -
63198-1218
CA 02227940 1999-OS-20
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID NO.: 1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 4192
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 1:
TAATATAATT ATTAATAAAT TTGAAAAAAT ATTATTACTA TCGAAAAAAA CTATAAATTT60
GATTTGAATG ATAAAATTAA AAATTAAAAA TATTTTTATT ATCATTATTG TTAATATAAT120
TATTAAAAAA TTTAAAAAAT ATATTATTAC TATTGAGAAA AACCATAACT GTTTATGCGA180
CATTTTATGT CATGGAAAAT GAGCTGAAAA AAACCAATAA AAAGAAAAAA ACTAATGAAA240
AAAAAGAAAA AAAAATATGA ATTAACTGGG TTAACCCTTG AAACCAGGTT ACCCCGTCAA300
ACCTTGGATT CGTGTCGTGA AAGTTTGTTA ACTAAATAGA F~~AAAAAAAT TGACGGGTTA360
CCCAGAATTA ACTGGGCTAA CCCGTCAAAC CAGGTTACCT ATCAAACCCG GGATCCGTGT420
2 O CATGAAAGTT TGATAACTAA ATAGAAAACA ATTGAACATT AACAACCTAA 480
ATTAAACGAA
AAAAATTAAT TAAAAACAAG AAAACAAAAA CAAACAAAAA ACATAAGCAT GTTAGTAATG540
AGGAAAAAGA AAAAAAATTT GATTCAACTG AGTTAACCCG TCAAACCCGG GATTCGCGTC600
ATGAAAGTTT GATAACTAAA TAGAAAAAAA AATCGACGGG TTAAACGAAA AAAAATTAAC660
AAACTAAACT AAACAAAAAA AAATTGATTA AAAAGGAAAA AAGCAAAAAA AATAATTTCG720
GGTTAACTCA TCAAACCAGG TTAACCCGTC AAACCCGAGA TCCGTGTCAT GAAAGTCTGA780
TAACTAAATA ATTTTTTTTT TCACATTAAC AAACTAAATT AAACAAAAAA AATTCATTAA840
AAGAAAAAAA AACACAAAGA AAAAAGCAAA AAAAAACCTA TAATAGCATA AATAAATAAA900
TAAAAACAGG AAAAAAATTT TTTAAAAAAA ACCTTTCAAT CACTAATACA TAGAAGGTGT960
GGGGAAAGCC ACAGTGATTT CCCCGTACCT TTTAAAGTAT TACTTAATAT ATAGGTGAAT1020
3 O TTAATTGACC GTCACGAAAA AGACTATTCT GGCTTCCTCT TACAATGGAC 1080
GCTATCTAAA
TTCAAATACT TTGAAAAAAG ATTTAATCCT GTAACCTTCT TTCGTTTTTT TATGCCTTCA1140
ATCCATCTAT TTATTGTTTT TATGATTTTT CTTAGATACA AAAGAGCATA TTTTAAAGAA1200
GAAAAAAATA AGCTAAGCAC CTCAAGTTTT GATTTTTTTT TTATTTTGCA GCCAATTTTT1260
TAAATATTAA AATTTTCATA ATAGATCAAA GGATAATTCA AAATTGCATC CAAATAACAA1320
- 44 -
63198-1218
CA 02227940 1999-OS-20
CATTAGTAAT GGAAGGACTT ATGGTATGAA TGGATCAATA ATATAAGGGC TGAATTAACA 1380
ACATTTTTTT TATTTAGATC CTGTTTATTT TTACGTTTTA AAAATATTTT TGAAATTATT 1440
TTATTTTTTA TTATAAATTA ATATTTTTAG ATCATTTTAA TACGTTAATA TAAAAAATAA 1500
TTTTTTTAAA AAAATTTATT TTAATATATT TTTTAAAAAT AATATTTAAA AAAACAATCA 1560
TAACAATATT CTCATTACCT AACACAGTCA TGGAACAGGA ATGAGAAAAG GTCTTATCAG 1620
TAAATTGCTT GCATGTCATG TCAAGGTGTA TGAACCTCCC AATACTTCTC ACGCTACCCT 1680
TCAGAAATCC AATCTCAGAA GCCACAGACA ATCTAAGTTA CGCTACAATC AACTTTCCAT 1740
CACCCTTTCC TTATTTAGAA ACTCCACTTA ATCACATTTC ACCCTTTTTC ATCATCTTCT 1800
CTTTCCCTTC AAGAAGCCTA GGTACTGTGC AAGAAACCCT TATCTCTCCC CCTCAGTATT 1860
TACTTTTGTT TAGTGCTACA GCTTTCACAA AGAAGTAAGG AAAAAATATG GGTCGTGGAA 1920
AGATTGAAAT CAAGAAGATC GAAAACCCCA CAAACAGGCA AGTCACCTAC TCGAAGAGAA 1980
GAAATGGTAT TTTCAAGAAA GCCCAAGAAC TCACTGTACT TTGTGATGCT AAGGTCTCTC 2040
TTATCATTGT CCCCAACACT AACAAACTCA ATGAGTACAT TAGCCCCTCC ACATCGTACG 2100
TATACTCGTA TCATGTTTCT GGCTAAGTAT TTCTTCCGTG CTTTCTCTTC TTTCTTTCTT 2160
TTCTTGTCTT TTATGTTGCA GTTTTATGAA ACCTTGGTAA TGGAACCGTA GTTTTTATTG 2220
TTAATTATGA CCAGGACAAA GAAGATCTAC GATCAATATC AGAACGCTTT AGGCATAGAT 2280
CTGTGGGGCA CTCAATACGA GGTTAACCTT TCTTTTCTGT CTTTCTTCTA ATGTTTGATC 2340
TATAGGACGA ATATGAGATT CTTCAAAGGA TTTTGTTTGT GAGGTTTGCA GAAAATGCAA 2400
GAGCACTTGA GGAAGCTGAA TGATATCAAT CATAAGCTGA GACAAGAAAT CAGGTAACTT 2460
CAAAAGAAAT AACCTTCGCA TATATGCATG TGGTTATGGT TTTTATGGGA ATATCTGTAA 2520
ATTTGTGGAG CTACTAATTA AGGTATTTGT TTTTAACAGG CAGAGGAGAG GAGAGGGCCT 2580
GAATGATCTG AGCATTGATC ATCTGCGCGG TCTTGAGCAA CATATGACTG AAGCCTTGAA 2640
TGGTGTGCGT GGCAGGAAGG TCAGATGTTT TCAAGTGAAC ATCTTTATAT AATTATCAAG 2700
TTCTAATTCC TAAAATTTGA GCTTACTAGT AATTTGAGTT CGGTCCGGTG TATCAAGCAG 2760
GTTAATCTAG ATCTAGTTTT TTTTCCTTAC CAAATCAAAG TCATTTTGAG GATTTTTTAA 2820
TAAAAAATAT TGAATTTTGA ATCAACTTAT ACAAATTCAT CAATCTACAA CTCGAATCTT 2880
ACATTTAATC AAACTTTCAA ATTAGATCTT ATAAATATGA TATTAACCGG TCGGTGTTTT 2940
ATGTACATTA ATATTATGTT TTAGTTGAAC TCTTTTATCA TTTTTTTTTT TTAAATTTGA 3000
GTTATTTTAA TCCTTATCAA TTTTTATCAT TTTGGGATTC TTGGAAACCC TGGTTAGAAA 3060
3 O GAAAATACAC ACCCTTGAAC TTGTGCTTCT TTACCTTTGC ATTATGGATT TTCATGAACT 3120
GGATTTTGGG TAACCCTTAA CCTCATCTAT AGAAGGGATA TGCCTTGTAA TTAACACTTT 3180
ACACTTACAA GTTCAACATT CTTTGATTAT TTACAGTACC ATGTGATCAA AACACAAAAC 3240
GAAACCTACA GGAAGAAGGT TAGTGATAAA AAGAACATTT TACCTCTTCA ATTTCATGCA 3300
TGTAGCTTTT GGAACAAATT CTCTGGCGAT TAATTGCAGG TGAAGAATTT AGAGGAGAGA 3360
- 45 -
63198-1218
CA 02227940 1999-OS-20
CATGGAAACC TCTTGATGGA ATATGTAAGA ATCTAAATTT TCATGTGCTT GTTTTCGCTA 3420
ATTTTCCAAC TTGGAAAAAC ACATGGATTA AACCTGAGAT TTTTTTTTTC TTTTGTGCTT 3480
TGGGATTTAA GGAAGCAAAA CTAGAGGATC GACAGTATGG TTTAGTGGAC AATGAAGCTG 3540
CTGTTGCACT TGCAAATGGG GCTTCCAACC TCTATGCATT CCGCCTGCAT CACGGGCACA 3600
ACCACCACCA CCATCTCCCT AATCTTCACC TTGGAGATGG ATTTGGAGCC CATGAACTTC 3660
GCCTTCCTTG AGTGGTGCTT GAGGTCGACC TTCCAGCTCT TCAGACATCT TATCTAAATG 3720
CGTGTGCTAA CTAGAGATGC TATCTAATAT TATTTAATAA TTAATTAAGA GCCCGGAAGT 3780
AAAAAATACT TTCATAGATT GTAATTTACC TCAGGGTAAT GTGTATGGCA GCATATTAGA 3840
TTGTGATTTG AGCAAGGAAT GTCATTCCTT ATGGATTAAT TAAATATAAA AGCTCTTTTT 3900
CACAAATATA ATTCCACTTG GAGTAGCATT CTGCAATATC CCATATGATC TGCAGGCTTA 3960
ATAATTATAT GATTGAAATG TGTTGGATCA ACCGTCATAT GTATGTATGT ATGTATGTAT 4020
GTATACGTAT GTGTATACTA GGGAGTCAAC AACACAGGGG GTGTAAGCAC CAAATGCATT 4080
ATCCACTGTT TTTGCCCAAA CCCCATTTGG CATAGGTCGA CAATACCATA CCAATGCCTC 4140
CGAAGCCATC CTTCCCCGCC GCCCTACACA AACCAAAACC GCTGAATTCC TG 4192
(2) INFORMATION FOR SEQ ID NO.: 2:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 924
2 0 (B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (1)..(684)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2:
3 O ATG GGT CGT GGA AAG ATT GAA ATC AAG AAG ATC GAA AAC CCC ACA AAC 48
Met Gly Arg Gly Lys Ile Glu Ile Lys Lys Ile Glu Asn Pro Thr Asn
1 5 10 15
AGG CAA GTC ACC TAC TCG AAG AGA AGA AAT GGT ATT TTC AAG AAA GCC 96
Arg Gln Val Thr Tyr Ser Lys Arg Arg Asn Gly Ile Phe Lys Lys Ala
25 30
- 46 -
63198-1218
~
' CA 02227940 1999-OS-20
CAA GAA CTC ACT GTA CTT TGT GAT GCT AAG GTC TCT CTT ATC ATT GTC 144
Gln Glu LeuThrVal LeuCys AspAlaLys ValSerLeuIle IleVal
35 40 45
CCC AAC ACTAACAAA CTCAAT GAGTACATT AGCCCCTCCACA TCGACA 192
Pro Asn ThrAsnLys LeuAsn GluTyrIle SerProSerThr SerThr
50 55 60
AAG AAG ATCTACGAT CAATAT CAGAACGCT TTAGGCATAGAT CTGTGG 240
Lys Lys IleTyrAsp GlnTyr GlnAsnAla LeuGlyIleAsp LeuTrp
65 70 75 gp
GGC ACT CAATACGAG AAAATG CAAGAGCAC TTGAGGAAGCTG AATGAT 288
Gly Thr GlnTyrGlu LysMet GlnGluHis LeuArgLysLeu AsnAsp
85 90 95
ATC AAT CATAAGCTG AGACAA GAAATCAGG CAGAGGAGAGGA GAGGGC 336
Ile Asn HisLysLeu ArgGln GluIleArg GlnArgArgGly GluGly
2 100 105 110
0
CTG AAT GATCTGAGC ATTGAT CATCTGCGC GGTCTTGAGCAA CATATG 384
Leu Asn AspLeuSer IleAsp HisLeuArg GlyLeuGluGln HisMet
115 120 125
ACT GAA GCCTTGAAT GGTGTG CGTGGCAGG AAGTACCATGTG ATCAAA 432
Thr Glu AlaLeuAsn GlyVal ArgGlyArg LysTyrHisVal IleLys
130 135 140
3 ACA CAA AACGAAACC TACAGG AAGAAGGTG AAGAATTTAGAG GAGAGA 480
O
Thr Gln AsnGluThr TyrArg LysLysVal LysAsnLeuGlu GluArg
145 150 155 160
CAT GGA AACCTCTTG ATGGAA TATGAAGCA AAACTAGAGGAT CGACAG 528
His Gly AsnLeuLeu MetGlu TyrGluAla LysLeuGluAsp ArgGln
165 170 175
TAT GGT TTAGTGGAC AATGAA GCTGCTGTT GCACTTGCAAAT GGGGCT 576
Tyr Gly LeuValAsp AsnGlu AlaAlaVal AlaLeuAlaAsn GlyAla
40 180 185 190
TCC AAC CTCTATGCA TTCCGC CTGCATCAC GGGCACAACCAC CACCAC 624
Ser Asn LeuTyrAla PheArg LeuHisHis GlyHisAsnHis HisHis
195 200 205
CAT CTC CCTAATCTT CACCTT GGAGATGGA TTTGGAGCCCAT GAACTT 672
His Leu ProAsnLeu HisLeu GlyAspGly PheGlyAlaHis GluLeu
210 215 220
50 CGC CTT CCTTGAGTGGTGCTTG GACCT 724
AGGTC TCCAGCTCTT
CAGACATCTT
Arg Leu Pro
225
ATCTAAATGC ATCTAATATT ATTTAATAAT
784
GTGTGCTAAC TAATTAAGAG
TAGAGATGCT
CCCGGAAGTA TAATTTACCT CAGGGTAATG
844
AAAAATACTT TGTATGGCAG
CCATAGATTG
CATATTAGAT TCATTCCTTA TGGATTAATT
904
TGTGATTTGA AAATATAAAA
GCAAGGAATG
GCTCTTTT TC 924
ACAAATAAAA
60 (2) INFORMATION FOR SEQ ID NO.: 3:
- 47 -
63198-1218
CA 02227940 1999-OS-20
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 681
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (1)..(681)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 3:
ATG GGT CGT GGA AAG ATT GAA ATC AAG AAG ATC GAA AAC CCC ACA AAC 48
Met Gly Arg Gly Lys Ile Glu Ile Lys Lys Ile Glu Asn Pro Thr Asn
1 5 10 15
AGG CAA GTC ACC TAC TCG AAG AGA AGA AAT GGT ATT TTC AAG AAA GCC 96
Arg Gln Val Thr Tyr Ser Lys Arg Arg Asn Gly Ile Phe Lys Lys Ala
25 30
CAA GAA CTC ACT GTA CTT TGT GAT GCT AAG GTC TCT CTT ATC ATT GTC 144
Gln Glu Leu Thr Val Leu Cys Asp Ala Lys Val Ser Leu Ile Ile Val
35 40 45
CCC AAC ACT AAC AAA CTC AAT GAG TAC ATT AGC CCC TCC ACA TCG ACA 192
Pro Asn Thr Asn Lys Leu Asn Glu Tyr Ile Ser Pro Ser Thr Ser Thr
50 55 60
AAG AAG ATC TAC GAT CAA TAT CAG AAC GCT TTA GGC ATA GAT CTG TGG 240
3 0 Lys Lys Ile Tyr Asp Gln Tyr Gln Asn Ala Leu Gly Ile Asp Leu Trp
65 70 75 80
GGC ACT CAA TAC GAG AAA ATG CAA GAG CAC TTG AGG AAG CTG AAT GAT 288
Gly Thr Gln Tyr Glu Lys Met Gln Glu His Leu Arg Lys Leu Asn Asp
85 90 95
ATC AAT CAT AAG CTG AGA CAA GAA ATC AGG CAG AGG AGA GGA GAG GGC 336
Ile Asn His Lys Leu Arg Gln Glu Ile Arg Gln Arg Arg Gly Glu Gly
100 105 110
CTG AAT GAT CTG AGC ATT GAT CAT CTG CGC GGT CTT GAG CAA CAT ATG 384
Leu Asn Asp Leu Ser Ile Asp His Leu Arg Gly Leu Glu Gln His Met
115 120 125
ACT GAA GCC TTG AAT GGT GTG CGT GGC AGG AAG TAC CAT GTG ATC AAA 432
Thr Glu Ala Leu Asn Gly Val Arg Gly Arg Lys Tyr His Val Ile Lys
130 135 140
ACA CAA AAC GAA ACC TAC AGG AAG AAG GTG AAG AAT TTA GAG GAG AGA 480
Thr Gln Asn Glu Thr Tyr Arg Lys Lys Val Lys Asn Leu Glu Glu Arg
145 150 155 160
CAT GGA AAC CTC TTG ATG GAA TAT GAA GCA AAA CTA GAG GAT CGA CAG 528
His Gly Asn Leu Leu Met Glu Tyr Glu Ala Lys Leu Glu Asp Arg Gln
165 170 175
- 48 -
63198-1218
CA 02227940 1999-OS-20
TAT GGT TTA GTG GAC AAT GAA GCT 576
GCT GTT GCA CTT GCA AAT GGG GCT
Tyr Gly Leu Val Asp Asn Glu Ala Ala Asn
Ala Val Leu Gly
Ala Ala
180 185 190
TCC AAC CTC TAT GCA TTC CGC CTG GGG CACCAC CAC 624
CAT CAC CAC
AAC
Ser Asn Leu Tyr Ala Phe Arg Leu Gly HisHis His
His His His
Asn
195 200 205
CAT CTC CCT AAT CTT CAC CTT GGA TTTGGA CATGAA CTT 672
GAT GGA GCC
His Leu Pro Asn Leu His Leu Gly PheGly HisGlu Leu
Asp Gly Ala
210 215 220
CGC CTT CCT 681
Arg Leu Pro
225
(2) INFORMATION FOR SEQ ID NO.:
4:
2 (i) SEQUENCE CHARACTERISTICS
0
(A) LENGTH: 227
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera trichocarpa
subsp.
(xi) SEQUENCE DESCRIPTION: SEQ ID 4:
NO.:
Met Gly Arg Gly Lys Ile Glu Ile IleGlu ProThr Asn
Lys Lys Asn
3 1 5 10 15
0
Arg Gln Val Thr Tyr Ser Lys Arg GlyIle LysLys Ala
Arg Asn Phe
25 30
Gln Glu Leu Thr Val Leu Cys Asp ValSer IleIle Val
Ala Lys Leu
35 40 45
Pro Asn Thr Asn Lys Leu Asn Glu SerPro ThrSer Thr
Tyr Ile Ser
50 55 60
40
Lys Lys Ile Tyr Asp Gln Tyr Gln LeuGly AspLeu Trp
Asn Ala Ile
65 70 75 80
Gly Thr Gln Tyr Glu Lys Met Gln LeuArg LeuAsn Asp
Glu His Lys
85 90 95
Ile Asn His Lys Leu Arg Gln Glu GlnArg GlyGlu Gly
Ile Arg Arg
100 105 110
50 Leu Asn Asp Leu Ser Ile Asp His GlyLeu GlnHis Met
Leu Arg Glu
115 120 125
Thr Glu Ala Leu Asn Gly Val Arg LysTyr ValIle Lys
Gly Arg His
130 135 140
Thr Gln Asn Glu Thr Tyr Arg Lys LysAsn GluGlu Arg
Lys Val Leu
145 150 155 160
- 49 -
63198-1218
CA 02227940 1999-OS-20
His Gly Asn Leu Leu Met Glu Tyr Glu Ala Lys Leu Glu Asp Arg Gln
165 170 175
Tyr Gly Leu Val Asp Asn Glu Ala Ala Val Ala Leu Ala Asn Gly Ala
180 185 190
Ser Asn Leu Tyr Ala Phe Arg Leu His His Gly His Asn His His His
195 200 205
His Leu Pro Asn Leu His Leu Gly Asp Gly Phe Gly Ala His Glu Leu
210 215 220
Arg Leu Pro
225
(2) INFORMATION FOR SEQ ID NO.: 5:
2 O (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 5656
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 5:
AGTATATATA CTAAATAAAT ATATAAACTT GTAAAAAATA AAAGAAAAAT AATCATTGCA 60
3 O TGCAAACTAA ACAAACATTA AAATTATACT TAAACAAAAC TAATCTAAAA TGAAGTTTTT 120
AAAAGGTAAT TATGACATAG CCACGAGCCA CTCAATAAAC CTTTATAAGA TTTAAATTGA 180
TGCTAAAATA TATTTTTTTT TATTTTTTGC ATCATTAAAG AAATAACTCA AAAGCATCTT 240
TTATTTTTTA AATATTAATT TATTAGAACA ATACTTGATA TCTATTGAAA TAATACTCAA 300
TATCTATCTA TAAATCAAAA AACCTAAACT CTAGATTGTA AAAAATAATA ATAATAGAAG 360
AGCCACCCAT CCAAAACTTC TATATTATTT GACTTGAAAG CAAAAACATT AATACACATA 420
ATTCATGAAA AATACTCATG AAAGTCTATA ATTCACAAAA GAATTGATGA ATATTCATAT 480
ATAGTTCACT AATAACATTC ATTTTCATCA TATAATTAAC GTATTAATTC AAGTACTAAA 540
ATATTTTATG AACTAAAAGA AATTATTGAT CAAAGAAAGA CTCAATAACA AATATTTTTT 600
TATTAATCAA ACTCAAATTC AAATTCATGA ACCCTCAAAT CCATTATCAA ATCCATAAAC 660
4 O CTATTTGGGG TTTGAGATTT TTGTTATCCA AGGGTTTTAT GGAGACAATT TATCATTCCC 720
TTTTTATTAG TCTTTTTTAT TATATATTAA TATTTTATAT TAAAATACTA ATTACAAAAT 780
TCAATATGAT TTTAATCTTG GACCTCATAT ATAATTCCGC TTTAAAACTC CGACTCATAT 840
TCTAAACCCA ATTCCAACAT GGACTAAACA ATTAATCCCA ATATTAGAGG GAACAAATTA 900
- 50 -
63198-1218
CA 02227940 1999-OS-20
TTTATTTCTT AACAACACGA AAACTAAAGT ATATCACTCT GCAAAATGTA ATTACAAGTC 960
CTTCGTGTTT AGGCTAGTTT GAAGATGCCT GTGGTTGGAG ACCAGAGACA TCAAATTAAT 1020
GTTTTTTTAT AGTAACATGT GCTCAAGTTG CATGCATTTT TCGTACCAAC AAAATACATG 1080
TAAAATCATC ATCCATTAAT CAAATTGCAA TGATTCATAG CATATGCATA ACGCATGTGT 1140
CTGTGCATGT TTTAGCTGGT TCAATTCTTG CAGATTGTAC TGCTAAATGT ACGTACTAGC 1200
ACCTCAAATC ACAGTGACCT CCCAAATATT GCACAGACCT CTTTGTTTAC AAATTTCAAG 1260
CATCCTAATT AATCTCCCAA GTGACATCTG GTGGCCATGT TGCGGCCCTG ACAAGCAGCT 1320
GAGAAATTCT CCAACATTAG AGGGATTCAA TGTTCTGTTC AATGTTTGGA TACATTGATT 1380
CTGCATTGCA ACGCTAATCA CGGTCTGTTC TCCGGCAAGG GGGGGAAAAA CAATGATCAG 1440
GGATAAGGCA GCGAATGTCT GGTGAAAACA AGGGTATTTT CATACTTTTC TCAGGTTCGT 1500
GTAGTCAGCA ATGAACGAAA CGAGGCAAAT CCAACCAAGT AGAAAAACCT CATGAGTAAC 1560
GAGAAAGTCG AGGAGACAGT ATCTGGCACC CTCAGATGCA TCATACCTTG CGATGAGCCA 1620
GAAACTAAGA TGATTCTAGT GACGTCTAAA TCATCAATCC CACGGTTAAA AGGACACCAT 1680
AACCCAAGCC ACTAGAATAT CTGCTTACGC AGCAACCACA CTGCAAAGCC ACGACGAAGA 1740
ACTACAAAGA TACGGATATA ACATGATATA AATATATTAA TACTTAATTC TTCAAGGTCT 1800
TGGATTATGA ACTTTTTTGT TCATATTTAT TTTATTATAT TGAAAAACTC GAAATAAATA 1860
AGACGATTAT TATAAGAATT CTTAAATCAT GTTTATCAAA TTTTGTCCTA TCTAGAGACC 1920
ATTAATAATT GTGTGTGGAT TAATTCACCA AAAACTTAAA TGAAAAGTAA CTTTATCTAT 1980
CTAGAGATGG AAAAGGAACT CAATTACCCT CAATAATAAA ATTGGATGGA AATCATCTAG 2040
2 0 ATGGTGGTCC AGTAGTAAGA TTTTGGGACT AAAAGGTTTG TTCTCTTTGT GGTCTCAGGT 2100
TCGAGCCATG TGGTTGCTTA TATGATGACC ACTGAAAATT TACATGGTCG TTAACTTCAG 2160
GGCCCGTGGG ATTAGTCGAG GTGCGTCAAG TTAGTCTGGA CACCCATATT AATCTAAAAA 2220
AAAAAATTAA ATGGCAAAAA ATATTTTGAA TGTTGAAGTA AAAAAAGTGA AAGGGAGGTA 2280
GTAAAACAAT ATACGACCTA ACAGGAGAGG AGTCCAATCA AGTAGATCAT GTGTCAAGAG 2340
ATGAGTGGAT AGAAGAACTT CAAGTGAAGA ATGTATGCAG GGAACCAAAT GTGTGAATGA 2400
CACAAAGATC TGACTAGTTC GATTTCAACT GTCCAGTTCC GAAGAAACAT CAAAACCCTT 2460
TAATTCTGTT AGCTTCCCAA TACATACAAA AAAGAAAAAA AGACAAAAAA CTCGTCCTGT 2520
TAAGGGCAGT TTTGGTATAT AAATAAAACA AGAAGCTCAC TTGTCTTTAT ATATCTACCA 2580
AATCCAAGAC ATGCACCTGT GAAAGATCAC AGAGAGAGAG ACAAGGGGGC AGATAGATAT 2640
3 0 GGATCCGGAG GCTTTCACGG CGAGTTTGTT CAAATGGGAT ACGAGAGCAA TGGTGCCACA 2700
TCCTAACCGT CTGCTTGAAA TGGTGCCCCC GCCTCAGCAG CCACCGGCTG CGGCGTTTGC 2760
TGTAAGGCCA AGGGAGCTAT GTGGGCTAGA GGAGTTGTTT CAAGCTTATG GTATTAGGTA 2820
CTACACGGCA GCAAAAATAG CTGAACTCGG GTTCACAGTG AACACCCTTT TGGACATGAA 2880
AGACGAGGAG CTTGATGAAA TGATGAATAG TTTGTCTCAG ATCTTTAGGT GGGATCTTCT 2940
- 51 -
63198-1218
CA 02227940 1999-OS-20
TGTTGGTGAG AGGTATGGTA TTAAAGCTGC TGTTAGAGCT GAAAGAAGAA GGCTTGATGA 3000
GGAGGATCCT AGGCGTAGGC AATTGCTCTC TGGTGATAAT AATACAAATA CTCTTGATGC 3060
TCTCTCCCAA GAAGGTTTGG TTAGCATTGA TTCTACCTTT TAGTGTAATT AAGCTAAGCT 3120
CATACTATTA CTAGCTATAG GAGTCCATGG CCAATTTGTT GTAGTTTTGT AGAGTAAATT 3180
AATTCTATGT ATACTTGGAT AAGATAATTA GCTTATTATA AGATGTTACT TGCCAGCTTA 3240
TAATTTCCAT ATACAACAAT CATTTTCATT CCCTTTTCCT TTTCTTATAT ATGAAATTTA 3300
GTTCAAGTAT AAGTGCTTGT ACACCAATGT ATGTTTACTC TAGTCATATC AATTCTACTT 3360
TGCAGGGTTG GTTTCTTGCT AATTAATCAC CATGCTCAAT ATTAGAGTAG TAATTCTCTT 3420
AACTAAGTCC AGGTTAGCTA GCTTTTGGTT TCTTGTTAAT TGCCGCACAT ACTTAGCTTA 3480
AATTAGTTCT CAAGGTAATA GTTAGCTTAA TAGCTTTGAG CTCATACTGG TTTCTATAAA 3540
ATAAATGAAC AAAATCTGAT TGTTTCGAAA AATTAAATAA CATTAACTTA TTAAACTTAT 3600
TTTCCTTTCC TTAATTTTTA ATTTTTGCTT GTTTCTTGGG TGGTTGTGTG TTCAGGTTTC 3660
TCTGAGGAGC CAGTACAGCA AGACAAGGAG GCAGCAGGGA GCGGTGGAAG AGGGACATGG 3720
GAGGCAGTGG CAGCGGGGGA GAGGAAGAAA CAGTCAGGGC GGAAGAAAGG CCAAAGAAAG 3780
GTGGTGGACC TTGATGGAGA TGATGAACAT GGTGGTGCTA TCTGTGAGAG ACAGCGGGAG 3840
CACCCATTCA TTGTAACAGA GCCTGGTGAA GTGGCACGTG GCAAAAAGAA CGGTCTTGAT 3900
TACCTCTTCC ATTTATATGA ACAGTGTCGT GATTTCTTGA TCCAAGTCCA AAGCATTGCG 3960
AAGGAGAGGG GAGAAAAATG CCCCACTAAG GTACGAAGAG TCAGCTTCGC GAGGGATTGA 4020
TTTTTATTTA GAAATATATT AAAATAATAT TTTTTATATT TTAAAATTTA TTTTTAATAT 4080
2 O TAATATATTA AAATAATATA AAAATACTGA AAAATAATTT TTTAAAAAAT AATTTTTTTT 4140
CAAAAATATT TACAAAACAA ACTGTGTCTA AAGAACACAT TTAGACCGTT AATTTCTGCA 4200
AGTCTCAACA TTTCAATGGT TCTTGTCTTG GACCCACATA GACCAGCCAT TGTATTCTGG 4260
ACTGGACTGG AGTTATGCCC CCACCTGAAT TTGCCTTTCA CAGCTGTCCC GATAAAAACG 4320
TGACAACTCA TGTACTGGTT TCTGGTCCCT GTCATTTTAG ACCTGCTATT TGCAGTGGGA 4380
TACTTATTGG TTACTCTTAC TAGTCGATCA TCGTTATTTG AATATTTCAA ATATTCTGAT 4440
TTTGGAAGTT TGTACGATGT CGTGTCACGT GGATCTTGTG AAACCTGGTT GATGTCAACT 4500
ATTGTCGAAC TGGACCAAAA TCCATTACAT TCTGAGTTTC TCTAGTGTTT TCCTGCCATG 4560
GAACCTGAAA GCCCATGTTG ATGGTTAGGA CTTAGAATTT GATTAGCCCT AAATGGAACA 4620
GTGAGTAATT ATGCTAAGAA AAATGGTTTT TTTTTGTTTT GTTTTGTGTT TGGTTATAGG 4680
3 O TGACAAATCA GGTGTTTAGG TATGCCAAGA AGGCAGGAGC AAGCTACATC AACAAGCCCA 4740
AAATGAGACA CTACGTGCAT TGCTATGCTT TACATTGCCT CGATGAGGAC GCATCCAATG 4800
CACTTAGGAG AGCGTTCAAG GAGAGAGGAG AAAATGTTGG AGCATGGAGA CAGGCTTGTT 4860
ACAAGCCCCT TGTAGCCATC GCATCACGCC AAGGCTGGGA CATAGATTCC ATTTTCAATG 4920
CTCATCCTCG GCTTGCCATT TGGTATGTGC CGACCAAGCT CCGTCAACTT TGTTATGCAG 4980
- 52 -
63198-1218
CA 02227940 1999-OS-20
AGCGCAATAG TGCCACTTCT TCAAGCTCTG TCTCTGGTAC TGGAGGTCAC CTGCCGTTTT 5040
GAGTTCTTAA TTATGCCAAG ATAAATACTC CTATCTCTAT AAAATTGTCA AAATGTATGT 5100
TGTAGCGAGG TCAGGACAAA GTATTGGTTG ATGGAGGATG GTTCATTAAA TTTCACATCC 5160
TTGACTATTT ATATATCATG ATATGCTTAA AGGCTCTAAT CATTGTTTAC GTCGATGGAA 5220
CTATTATATT TCTAATTTAG TTTTCAGGGA AGTCTAGGCT GCTGGTGCCT ACAGTGTCCA 5280
TAAATTTGAG CAAAATGGCC AAAAGGGGCC AATTGGGACC CACTAAATTA ATTTGGTGGT 5340
GCAGTCCCCC TTACAATACG ACTGCATGTA ATACTTGTCC AAAATTTGAG TGCAGTTCAT 5400
AGGCTGTTAC TTTAAACAGA CAAACACATG ATGACAAGAT AAAAGGCATG GATAATTCTT 5460
GTCTTCTTGA GGTGCCAACA TGCAAAATGC CATGTCAGGT TGTTGATTTG ATTTCTAATT 5520
GTTAACCATT ACTGTTTTTT TTGCCATAAC CATGCAATGG TGCTAAAGTT AGATGCCATA 5580
AAAGATGTAT CATGGCAGCC TGCAATGCAA ATAAAAACGG GGAAACAATG GAAAGTTGCC 5640
AGAAATTTCA ATTACT 5656
(2) INFORMATION FOR SEQ ID NO.: 6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1305
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
2 O (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (12)..(1145)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 6:
GGCAGATAGA T ATG GAT CCG GAG GCT TTC ACG GCG AGT TTG TTC AAA TGG 50
Met Asp Pro Glu Ala Phe Thr Ala Ser Leu Phe Lys Trp
1 5 10
GAT ACG AGA GCA ATG GTG CCA CAT CCT AAC CGT CTG CTT GAA ATG GTG 98
Asp Thr Arg Ala Met Val Pro His Pro Asn Arg Leu Leu Glu Met Val
15 20 25
CCC CCG CCT CAG CAG CCA CCG GCT GCG GCG TTT GCT GTA AGG CCA AGG 146
Pro Pro Pro Gln Gln Pro Pro Ala Ala Ala Phe Ala Val Arg Pro Arg
30 35 40 45
GAG CTA TGT GGG CTA GAG GAG TTG TTT CAA GCT TAT GGT ATT AGG TAC 194
4 0 Glu Leu Cys Gly Leu Glu Glu Leu Phe Gln Ala Tyr Gly Ile Arg Tyr
50 55 60
- 53 -
63198-1218
CA 02227940 1999-OS-20
TAC ACG GCA GCA AAA ATA GCT GAA CTC GGG TTC ACA GTG AAC ACC CTT 242
Tyr Thr Ala Ala Lys Ile Ala Glu Leu Gly Phe Thr Val Asn Thr Leu
65 70 75
TTG GAC ATG AAA GAC GAG GAG CTT GAT GAA ATG ATG AAT AGT TTG TCT 290
Leu Asp Met Lys Asp Glu Glu Leu Asp Glu Met Met Asn Ser Leu Ser
80 85 90
CAG ATC TTT AGG TGG GAT CTT CTT GTT GGT GAG AGG TAT GGT ATT AAA 338
Gln Ile Phe Arg Trp Asp Leu Leu Val Gly Glu Arg Tyr Gly Ile Lys
95 100 105
GCT GCT GTT AGA GCT GAA AGA AGA AGG CTT GAT GAG GAG GAT CCT AGG 386
Ala Ala Val Arg Ala Glu Arg Arg Arg Leu Asp Glu Glu Asp Pro Arg
110 115 120 125
CGT AGG CAA TTG CTC TCT GGT GAT AAT AAT ACA AAT ACT CTT GAT GCT 434
Arg Arg Gln Leu Leu Ser Gly Asp Asn Asn Thr Asn Thr Leu Asp Ala
130 135 140
CTC TCC CAA GAA GGT TTC TCT GAG GAG CCA GTA CAG CAA GAC AAG GAG 482
Leu Ser Gln Glu Gly Phe Ser Glu Glu Pro Val Gln Gln Asp Lys Glu
145 150 155
GCA GCA GGG AGC GGT GGA AGA GGG ACA TGG GAA GCA GTG GCA GCG GGG 530
Ala Ala Gly Ser Gly Gly Arg Gly Thr Trp Glu Ala Val Ala Ala Gly
160 165 170
GAG AGG AAG AAA CAG TCA GGG CGG AAG AAA GGC CAA AGA AAG GTG GTG 578
Glu Arg Lys Lys Gln Ser Gly Arg Lys Lys Gly Gln Arg Lys Val Val
175 180 185
GAC CTT GAT GGA GAT GAT GAA CAT GGT GGT GCT ATC TGT GAG AGA CAG 626
Asp Leu Asp Gly Asp Asp Glu His Gly Gly Ala Ile Cys Glu Arg Gln
190 195 200 205
CGG GAG CAC CCA TTC ATT GTA ACA GAG CCT GGT GAA GTG GCA CGT GGC 674
Arg Glu His Pro Phe Ile Val Thr Glu Pro Gly Glu Val Ala Arg Gly
210 215 220
AAA AAG AAC GGT CTT GAT TAC CTC TTC CAT TTA TAT GAA CAG TGT CGT 722
Lys Lys Asn Gly Leu Asp Tyr Leu Phe His Leu Tyr Glu Gln Cys Arg
225 230 235
GAT TTC TTG ATC CAA GTC CAA AGC ATT GCG AAG GAG AGG GGA GAA AAA 770
Asp Phe Leu Ile Gln Val Gln Ser Ile Ala Lys Glu Arg Gly Glu Lys
240 245 250
TGC CCC ACT AAG GTG ACA AAT CAG GTG TTT AGG TAT GCC AAG AAG GCA 818
Cys Pro Thr Lys Val Thr Asn Gln Val Phe Arg Tyr Ala Lys Lys Ala
255 260 265
GGA GCA AGC TAC ATC AAC AAG CCC AAA ATG AGA CAC TAC GTG CAT TGC 866
Gly Ala Ser Tyr Ile Asn Lys Pro Lys Met Arg His Tyr Val His Cys
270 275 280 285
TAT GCT TTA CAT TGC CTC GAT GAG GAC GCA TCC AAT GCA CTT AGG AGA 914
Tyr Ala Leu His Cys Leu Asp Glu Asp Ala Ser Asn Ala Leu Arg Arg
290 295 300
GCG TTC AAG GAG AGG GGA GAA AAT GTT GGA GCA TGG AGA CAG GCT TGT 962
Ala Phe Lys Glu Arg Gly Glu Asn Val Gly Ala Trp Arg Gln Ala Cys
305 310 315
TAC AAG CCC CTT GTA GCC ATC GCA TCA CGC CAA GGC TGG GAC ATA GAT 1010
Tyr Lys Pro Leu Val Ala Ile Ala Ser Arg Gln Gly Trp Asp Ile Asp
320 325 330
- 54 -
63198-1218
CA 02227940 1999-OS-20
TCC ATT TTC AAT GCT CAT CCT CGG CTT GCC ATT TGG TAT GTG CCG ACC 1058
Ser Ile Phe Asn Ala His Pro Arg Leu Ala Ile Trp Tyr Val Pro Thr
335 340 345
AAG CTC CGT CAA CTT TGT TAT GCA GAG CGC AAT AGT GCC ACT TCT TCA 1106
Lys Leu Arg Gln Leu Cys Tyr Ala Glu Arg Asn Ser Ala Thr Ser Ser
350 355 360 365
AGC TCT GTC TCT GGT ACT GGA GGT CAC CTG CCG TTT TGA GTTCTTAATT 1155
Ser Ser Val Ser Gly Thr Gly Gly His Leu Pro Phe
370 375
ATGCCAAGAT AAATACTCCT ATCTCTATAA AATTGTCAAA ATGTATGTTG TAGCGAGGTC 1215
AGGACAAAGT ATTGGTTGAT GGAGGATGGT TCATTAAATT TCACATCCTT GACTATTTAT 1275
ATATCATGAT ATGCTTAAAG GCTCTAAAAA 1305
(2) INFORMATION FOR SEQ ID NO.: 7:
(i) SEQUENCE CHARACTERISTICS
2 O (A) LENGTH: 1131
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (1)..(1131)
3 O (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 7:
ATG GAT CCG GAG GCT TTC ACG GCG AGT TTG TTC AAA TGG GAT ACG AGA 48
Met Asp Pro Glu Ala Phe Thr Ala Ser Leu Phe Lys Trp Asp Thr Arg
1 5 10 15
GCA ATG GTG CCA CAT CCT AAC CGT CTG CTT GAA ATG GTG CCC CCG CCT 96
Ala Met Val Pro His Pro Asn Arg Leu Leu Glu Met Val Pro Pro Pro
25 30
CAG CAG CCA CCG GCT GCG GCG TTT GCT GTA AGG CCA AGG GAG CTA TGT 144
4 0 Gln Gln Pro Pro Ala Ala Ala Phe Ala Val Arg Pro Arg Glu Leu Cys
35 40 45
GGG CTA GAG GAG TTG TTT CAA GCT TAT GGT ATT AGG TAC TAC ACG GCA 192
Gly Leu Glu Glu Leu Phe Gln Ala Tyr Gly Ile Arg Tyr Tyr Thr Ala
50 55 60
GCA AAA ATA GCT GAA CTC GGG TTC ACA GTG AAC ACC CTT TTG GAC ATG 240
Ala Lys Ile Ala Glu Leu Gly Phe Thr Val Asn Thr Leu Leu Asp Met
65 70 75 80
- 55 -
63198-1218
CA 02227940 1999-OS-20
AAA GAC GAG GAG CTT GAT GAA ATG ATG AAT AGT TTG TCT CAG ATC TTT 288
Lys Asp Glu Glu Leu Asp Glu Met Met Asn Ser Leu Ser Gln Ile Phe
85 90 95
AGG TGG GAT CTT CTT GTT GGT GAG AGG TAT GGT ATT AAA GCT GCT GTT 336
Arg Trp Asp Leu Leu Val Gly Glu Arg Tyr Gly Ile Lys Ala Ala Val
100 105 110 .
AGA GCT GAA AGA AGA AGG CTT GAT GAG GAG GAT CCT AGG CGT AGG CAA 384
Arg Ala Glu Arg Arg Arg Leu Asp Glu Glu Asp Pro Arg Arg Arg Gln
115 120 125
TTG CTC TCT GGT GAT AAT AAT ACA AAT ACT CTT GAT GCT CTC TCC CAA 432
Leu Leu Ser Gly Asp Asn Asn Thr Asn Thr Leu Asp Ala Leu Ser Gln
130 135 140
GAA GGT TTC TCT GAG GAG CCA GTA CAG CAA GAC AAG GAG GCA GCA GGG 480
Glu Gly Phe Ser Glu Glu Pro Val Gln Gln Asp Lys Glu Ala Ala Gly
145 150 155 160
AGC GGT GGA AGA GGG ACA TGG GAA GCA GTG GCA GCG GGG GAG AGG AAG 528
Ser Gly Gly Arg Gly Thr Trp Glu Ala Val Ala Ala Gly Glu Arg Lys
165 170 175
AAA CAG TCA GGG CGG AAG AAA GGC CAA AGA AAG GTG GTG GAC CTT GAT 576
Lys Gln Ser Gly Arg Lys Lys Gly Gln Arg Lys Val Val Asp Leu Asp
180 185 190
GGA GAT GAT GAA CAT GGT GGT GCT ATC TGT GAG AGA CAG CGG GAG CAC 624
3 0 Gly Asp Asp Glu His Gly Gly Ala Ile Cys Glu Arg Gln Arg Glu His
195 200 205
CCA TTC ATT GTA ACA GAG CCT GGT GAA GTG GCA CGT GGC AAA AAG AAC 672
Pro Phe Ile Val Thr Glu Pro Gly Glu Val Ala Arg Gly Lys Lys Asn
210 215 220
GGT CTT GAT TAC CTC TTC CAT TTA TAT GAA CAG TGT CGT GAT TTC TTG 720
Gly Leu Asp Tyr Leu Phe His Leu Tyr Glu Gln Cys Arg Asp Phe Leu
225 230 235 240
ATC CAA GTC CAA AGC ATT GCG AAG GAG AGG GGA GAA AAA TGC CCC ACT 768
Ile Gln Val Gln Ser Ile Ala Lys Glu Arg Gly Glu Lys Cys Pro Thr
245 250 255
AAG GTG ACA AAT CAG GTG TTT AGG TAT GCC AAG AAG GCA GGA GCA AGC 816
Lys Val Thr Asn Gln Val Phe Arg Tyr Ala Lys Lys Ala Gly Ala Ser
260 265 270
TAC ATC AAC AAG CCC AAA ATG AGA CAC TAC GTG CAT TGC TAT GCT TTA 864
Tyr Ile Asn Lys Pro Lys Met Arg His Tyr Val His Cys Tyr Ala Leu
275 280 285
CAT TGC CTC GAT GAG GAC GCA TCC AAT GCA CTT AGG AGA GCG TTC AAG 912
His Cys Leu Asp Glu Asp Ala Ser Asn Ala Leu Arg Arg Ala Phe Lys
290 295 300
GAG AGG GGA GAA AAT GTT GGA GCA TGG AGA CAG GCT TGT TAC AAG CCC 960
Glu Arg Gly Glu Asn Val Gly Ala Trp Arg Gln Ala Cys Tyr Lys Pro
305 310 315 320
CTT GTA GCC ATC GCA TCA CGC CAA GGC TGG GAC ATA GAT TCC ATT TTC 1008
Leu Val Ala Ile Ala Ser Arg Gln Gly Trp Asp Ile Asp Ser Ile Phe
325 330 335
AAT GCT CAT CCT CGG CTT GCC ATT TGG TAT GTG CCG ACC AAG CTC CGT 1056
Asn Ala His Pro Arg Leu Ala Ile Trp Tyr Val Pro Thr Lys Leu Arg
340 345 350
- 56 -
63198-1218
CA 02227940 1999-OS-20
CAA CTT TGT TAT GCA GAG CGC AAT AGT GCC ACT TCT TCA AGC TCT GTC 1104
Gln Leu Cys Tyr Ala Glu Arg Asn Ser Ala Thr Ser Ser Ser Ser Val
355 360 365
TCT GGT ACT GGA GGT CAC CTG CCG TTT 1131
Ser Gly Thr Gly Gly His Leu Pro Phe
370 375
(2) INFORMATION FOR SEQ ID NO.: 8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 377
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
2 0 (A) ORGANISM: Populus balsamifera subsp. trichocarpa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 8:
Met Asp Pro Glu Ala Phe Thr Ala Ser Leu Phe Lys Trp Asp Thr Arg
1 5 10 15
Ala Met Val Pro His Pro Asn Arg Leu Leu Glu Met Val Pro Pro Pro
25 30
Gln Gln Pro Pro Ala Ala Ala Phe Ala Val Arg Pro Arg Glu Leu Cys
35 40 45
Gly Leu Glu Glu Leu Phe Gln Ala Tyr Gly Ile Arg Tyr Tyr Thr Ala
50 55 60
Ala Lys Ile Ala Glu Leu Gly Phe Thr Val Asn Thr Leu Leu Asp Met
65 70 75 80
Lys Asp Glu Glu Leu Asp Glu Met Met Asn Ser Leu Ser Gln Ile Phe
85 90 95
4 0 Arg Trp Asp Leu Leu Val Gly Glu Arg Tyr Gly Ile Lys Ala Ala Val
100 105 110
Arg Ala Glu Arg Arg Arg Leu Asp Glu Glu Asp Pro Arg Arg Arg Gln
115 120 125
Leu Leu Ser Gly Asp Asn Asn Thr Asn Thr Leu Asp Ala Leu Ser Gln
130 135 140
Glu Gly Phe Ser Glu Glu Pro Val Gln Gln Asp Lys Glu Ala Ala Gly
50 145 150 155 160
Ser Gly Gly Arg Gly Thr Trp Glu Ala Val Ala Ala Gly Glu Arg Lys
165 170 175
Lys Gln Ser Gly Arg Lys Lys Gly Gln Arg Lys Val Val Asp Leu Asp
180 185 190
- 57 -
63198-1218
CA 02227940 1999-OS-20
Gly Asp Asp Glu His Gly Gly Ala Ile Cys Glu Arg Gln Arg Glu His
195 200 205
Pro Phe Ile Val Thr Glu Pro Gly Glu Val Ala Arg Gly Lys Lys Asn
210 215 220
Gly Leu Asp Tyr Leu Phe His Leu Tyr Glu Gln Cys Arg Asp Phe Leu
225 230 235 240
Ile Gln Val Gln Ser Ile Ala Lys Glu Arg Gly Glu Lys Cys Pro Thr
245 250 255
Lys Val Thr Asn Gln Val Phe Arg Tyr Ala Lys Lys Ala Gly Ala Ser
260 265 270
Tyr Ile Asn Lys Pro Lys Met Arg His Tyr Val His Cys Tyr Ala Leu
275 280 285
His Cys Leu Asp Glu Asp Ala Ser Asn Ala Leu Arg Arg Ala Phe Lys
290 295 300
Glu Arg Gly Glu Asn Val Gly Ala Trp Arg Gln Ala Cys Tyr Lys Pro
305 310 315 320
Leu Val Ala Ile Ala Ser Arg Gln Gly Trp Asp Ile Asp Ser Ile Phe
325 330 335
Asn Ala His Pro Arg Leu Ala Ile Trp Tyr Val Pro Thr Lys Leu Arg
340 345 350
Gln Leu Cys Tyr Ala Glu Arg Asn Ser Ala Thr Ser Ser Ser Ser Val
355 360 365
Ser Gly Thr Gly Gly His Leu Pro Phe
370 375
4 O (2) INFORMATION FOR SEQ ID NO.: 9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 11484
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 9:
50 GGATCCACCT CCACGTCAGT CCATCCGCAT TCGTAAGTCC ATAAAACTAC CAGATTTTGC 60
TTATTCTTGT TATTCTTCAT CATTTACTTC CTTTTTAGCT TCTATTCATT GCCTTTTTGA 120
GCCCTCTTCC TATAAAGAGG CAATTCTTGA TCCGCTTCGG CAACAAGCTA TGAATGAAGA 180
ATTTTCTGCT TTGCATAAGA CAGATACTTG GGATCTGGTT CCTCTACCTC CCGGTAAGAG 240
- 58 -
63198-1218
TCT GGT ACT GGA GGT CAC CTG CCG TTT 1
CA 02227940 1999-OS-20
TGTTGTTGGT TGTCATTGGG TGTATAAGAT CAAGACTAAT TCTGATGGGT CTATTGAGCA 300
ATACAAAGCT AGGCTGGTTG CAAAAGGATA CTCTCAACAT TATGGTATGG ACTATGAGGA 360
AACATTTGCC CCGGTTGCAA AAATGACTAC TATTCGTACT CTTATTGTCG TAGCTTCGAT 420
TCGTCAGTGG CATATTTCTC AGCTTGATGT TAAAAATGCC TTCTTGAATG GAGATCTTCA 480
AGAAGAAGTT TATGTGGCAC TCCCTCCTGG TATTTCATAT GACTCTGGAT ATGTTTGTAA 540
GCTTAAGAAA GCATTAATTA TATGGTCTCA AACAAGCACC CCGTGCTTGG TTTGAGAAAT 600
TCTCTATTGT GATCTCGTCT CTTGGCATTG TTTCTAGCAG TCATGATTCT GCTCTTTTTA 660
TTAAGTGCAC TGATGCAGGT CGTATCATTC TGTCTTTATA TGTTGATAAC ATGATTATTA 720
TTGGTGATGA CATTGATGGT ATTTCAGTCT TGAAGACAAA GTTGGCTAGA CGATTTGAAA 780
TGAAAGATTT GGGTTATCTT CAATATTTCC TGGGTATTGA GGTAGCATAC TCACCTAGAG 840
GTTACCTTCT TTCTCAGTCG AAATATGTTG CAGATATTCT TGAGCAGACT AGACTTACTG 900
ATAACAAAAC TGTAGATACT CCTATTGAGG TCAACGTGAG GTACTCTTCT TCTGATGGTT 960
TACCTTTGAT AGATCTTACT TTATACCACA CTATTGTTAG GAGTTTGGTA TATCTCACCA 1020
TTACTCGTCC AGATATTGCA TATGCTGTTC ATGTTGTTAG TCAGTTTGTT GCTTCTCTTA 1080
CTACTGTTCA CTGGGCAGCT GTTATTCGTA TTTTGCGATA TCTTCGGGGT ACAGTTTTTC 1140
AGAGTCTTTT ACTTTCATCC ACCTCTTTCT TGGAGTTGCG TGCATACTCT GATGCTGATC 1200
ATGGTAGTGA TCCCACAGAT CGCAAGTCTG TTACCGGGTT CTGTATCTTT TTAGGTGATT 1260
CTCTTATTTC TTGGAAGAGC AAGAAACAAT CTATTGTTTC TCAATCATCC ATCGAAGCAG 1320
AATATCGTGC CATGACATCT ACTACCAAAG AGATTGTTTG GTTATGTTGG TTACTTGCTG 1380
2 O ATATGAGAGT TTCATTTTCT CATCCTACTC CTATGTATTG TGACAACCAG AGTTCTATTC 1440
AGATTGCTCA CAACTCGGTT TTTCATGAGC GAACTAAGCA CATTGAGATC GATTGTCATC 1500
TTACTCATCA TCATCTCAAG CATGGCACCA TTGCTTTACC TTTTGTTCCT TCTTCCTTAC 1560
AGATTGCAGA TTTCTTTATC AAGGCGCATT CCATCTCTCG TTTTTGTTTT CAGGTTGGCA 1620
AACTCTCGAT GCTTGTAGCT GCCGCATTGT GAGTTTGAGG GGAGATGTTA AATAATATTT 1680
ATGTAGTCTT ATTTATTAAG GGTAGAATAG TACTTTCAGT TTAACCTATA TATACTTTAT 1740
TTGTATTTAG GTTAAGACTA AGCATTCATA ATAAATGTAT CATTAAGAAT TCTAGCCTCC 1800
TTTTCGTGTT TCATTTTAAT TATTTTTAAC AATCTTGTAT TAATATATGT GAATTGATTG 1860
AATTAAATCT TGTAATACAA TTTAATTGAT TCTAAGTTAA ACAATCTGCT GGGGAACATT 1920
CATACAACTA TCTTTCTTTT CGTTTCAAGT AGGCAGGAAA TAAAACGTTT TTAGTTTAGG 1980
3 O TGACTAAACA ATGGAATTTA ATGAAATAAG GGTAGAGATG AGGTCTGAGG TTATCTTGTT 2040
AAGCACCTTC CCATTTGAAC CATGATTTTG TCGTTAAGCA CTGAGAGTGT AACTTAGCCC 2100
TAAAACGTCT CACTCACCCC ATTATAATTC ATTTTCAGAA AGTCCCTTGC TTTTCTCTCT 2160
AATGACCTAA ATCATTTCCT TGAAAGCCAA AAATAAAAAA TAAAAACGAA TATAGTGGAG 2220
AGTTATTGAG GTCTGAATCT GACGACAGAT TCCCACCTTT AGCCTCTTCT TTTTAATTCC 2280
- 59 -
63198-1218
CA 02227940 1999-OS-20
TCTTCAATGC TCACCACTCA TCAATACCAA GATAAGAAAA AGF~AAAAAAA ATGGAAAAAT 2340
TATTGAAGAA GAGAAATTAC AAAGACAGTA GTTAGACTTG GTAGAAGTAT TGTTATATAA 2400
AAGATTGGAT GAGAGGTTGT TTTTCACTTT ATAAATACCC ACCTCTTAGC CCAAACTTGC 2460
TTCCATTTTC TTCATCTCTC TACTAGTTAG ATTTGTAGGA GAAATCCCAA AGGAAAAGAT 2520
CCTCACTTTC TCTACACATT AACTGCTATC TACAGCCCCT AGCTACTTTG TTTTATTTCC 2580
TCCCAAGGTT AGTTACTAAA ACATGGAGTC ATAAATCTCG TTGTATTCTT CAGTGCTTCA 2640
TCACTTGTTT TGGGCTAATT AATCAATCTT TTCACGTTTC AAAACCCACC TCTTCTTTTT 2700
CTGTTTTGAT CACTCAGAAA CCCCAAAAAA TACAACTTTC AAACATTTCT GTCTCCCTTT 2760
CCCATTTCAA TCTCCAGATT GAAGCACCAG TGATTTATTT TTGTTTTGTT GATTGATTAT 2820
TTTGACCATA ACCAATAAAC CATAACAATC GCAATTCAGA AGCTCCAGAC GTTCATCGAC 2880
CCCTTTTTCT TATGTTTATT TTATATTACT TCCATCCTGG ACTACTCATT TGGACAAAAA 2940
AAGTATTGCT AAATATGCTA TGAGTTGTGC ATATATTATT CTTGAATTAG TAGTATTTTT 3000
TTCATTTTAT TACATTTTTT GTGTTGTCAC TCAGTTTGTG TTTTGGATCA GCTAGCTAGG 3060
CTGCAGCTAT GGAATATCAA AATGAATCCC TTGAGAGCTC CCCCCTGAGG AAGCTGGGAA 3120
GGGGAAAGGT GGAGATCAAG CGGATCGAGA ACACCACCAA TCGCCAAGTC ACTTTCTGCA 3180
AAAGGCGCAG TGGTTTGCTC AAGAAAGCCT ACGAATTATC TGTTCTTTGC GATGCTGAGG 3240
TTGCACTCAT CGTCTTCTCT AGCCGCGGTC GCCTTTATGA GTACTCTAAC GATAGGTAAA 3300
TAAATCTAAT TTTAGATATT TGCTTCTCTG GATCTTAAAT TCTCCATGTT ACAAGCCCTC 3360
TATCTTCATG TGGTCACTTT TTTTTTTTTT TTATCTTCCT TTCTGCCCCA AAGAGATTTT 3420
2 0 TTTATCCTCT CTATTTTGCT TATGTTAGTG TTAATTTTTA GCTTTAATTG GTTTCTTTCA 3480
TTTTCATTTT CTTTCTTTCA TGAATGATCA TTAAATGGTT TTCAATTTCT AAGGTGGGAA 3540
ATTTATTATT ATTATTATTA TTTTGTGTTT AATCTCTGGG TAAAGGATTT AAAGCAAAAG 3600
AGACACAATC ATTCCTTATG CTGCAGTTTA GATTGAGTTT CTTATCTAAC TGAGATTCAC 3660
TTGTCTTTCT TTCTTTCTTT CTCTTCTCTT ACCCTTTAGA CGATGCTGAT GCACACGTTA 3720
TTTTGAGTTC TTGGTTTGGT AAAAACATAG ATCTGGTATA ATAAACAGAC ATAGAAGCAC 3780
TATATGAGTG TAGTATGGTA GCAGAAATAA GTATAGGTCT GTGAGATCAG CCTCTTTATC 3840
TCCTCCCTTG TTGTTAATTT TGTTGTTTCC GTTTTTCTTT CTCTTCCATT ATTCCTCTTG 3900
CACTCTCTAT CTCTCGCTTT TTTTTTGCAC ATACTTGTTT GTTTGTGTCA TCTACGAGGC 3960
TAAAGAGATT GCCTATAGCC AAAGCTGTCA TCTTCTCATT AGTCCAAACC CTCCATCTCT 4020
30 TTTCACTTCC TAGTTAAATA GCACGTCAAT TAGACATCAA GAAAGCAAAA GTACCATGTC 4080
AAATAACCGT GAAAAAGAAG AAGAACAAAG AAAGGTTTTT TTAATTTGTC ATGTCACTCA 4140
AACATATATT ATTAGGGTTT CAAATCCCAA ATCCCCAGAT GGGTTTTTCA TCTTATTTTA 4200
TTTTTCCAAA CCAATCCAGG GTTTTTCCCC TAATCACACG AAATTTCCCA AAATCTCAGT 4260
TTGAACCCAC GAGGGGATAG TGAAAACCTT TCTGTTAGTC AATGCATAAC CCCAGTTAGG 4320
- 60 -
63198-1218
CA 02227940 1999-OS-20
GTTCATAGTT AGGGTTCATA TTCAAGTAAC CACATGAAAT CATCGAAATC GTACATTAAC 4380
ATTCAAGGAA AACTGTTAAA TCAAGCAAGT GGACCCTTCC ACAACCAATC AAAACTCAGT 4440
TAGATTTCAC CTAGATTTTT ACCCCTTTTT TAACCTGGGT AAGTATGGTA CAGTAATCGG 4500
TTAGGGTTTA GTAGCCAGTC AAATAGATCA GATTGTTGTT CGGGTTTATG AACAGAATCT 4560
TTGGTAACGT CACACACGAT TTTTCAGTTC TTGCCTACTG ACAAAAGGCT TTATGTCATG 4620
ATTCCTTAAA CTGAACCCAA GATTTTTAAC TTCCGATCCC CCTGGAAAAA ATATGAAATT 4680
CCAAAAATTG TCCATTTCTT CTCCTTAGAT CTCTCTCTAT CTCTCTCCCG GTTAAATTGT 4740
TTCCATGGTG AAAGCAGAGA GATGGATCAA TGAGAATGGG TTAACCAAGG CCATAATGAT 4800
GGCACTGTTT AAGATCTTGT ATAGATATAT TTATATAAGT TTTTTTTTTT TTTAATTTAA 4860
AGAGAGATTT AGCCCCATTT GTATTTTTAC GGTGAGAAAA CACTTTTATA AAAAATTGAT 4920
ATTTTTTTAA AAATTATTTT TTATATTTTT TAGATTATTT TTATGTGTTA ATATTAAAAA 4980
TAAATTTTTT AAAATATAAA AAATATTATA TTAATATATT TTAAATAAAA AATTAACCGT 5040
TGATGACAAT ATTGAGAGAA AGAGAGTCGT GAAGAGAGAA TGAACGACAA CTGTTAACCA 5100
GTGGAAGAGT TCTGTCAATT TTGGTTTCTT CTATGTAATA GAAAGCCTAC AACTCTAGCT 5160
GGTATTGTAC GGCTCTGCTT CTCTCAGAGT TTCAGTCTGA GACTAATAAA ATGTCCGATT 5220
AGTACAATAT TTTATTACAA TGAAATAGAA TATCGAGGTG GGTAATAGAG TGAGTTTAAG 5280
GAGATTATCC ACTATGTAAT GGGTTATTGA CACGTGGAGA ATATTTGACC GCTGATCTAC 5340
CTTGGCCAAT CATATTGTAG GATTCAGTGA CAGCTTGGCA GAGACAGCCA ATCAATGTCT 5400
CGACGAAGTT AAGGTATAAG GAAATCTAGA AAAGCGGTTC TTGTCTGAAT TGACAAGATG 5460
2 O TGTTCACATT TTACTGAGAT TATTATGGCA AAATTTTAGG ATTTCCTTCG CATTGTGTCG 5520
AGGAAAGACT GGATAATCAG ACTGACTCGG AGAGCTGTGG TTTTGTCATT CATCTTCTTT 5580
TTAGGGTTTT CTACGAGTTA ACTTAATGGA GTTATTCGTT GATTTGACTG TTTAATTGCC 5640
TTACCGTCAA GCTTTGTTAT AATAAGGATT TTTTAAATTG TTTTTTTTAT TTATAAATAT 5700
ATTAAAATAA TATTTTTTAA TTTTTAAGAT GGCATATCAA AAATATTTTA AAAAATAAAA 5760
AAATAATTTG AAATAAAACA AAAATTAATT TTTTTAAAAC AATATTTTTA ACGCAATAAC 5820
AAATTCTTAA TCTTTTACTC ATATATCTTA AATTTACGAG AGTTTTTTCC AAAAAGATAA 5880
AGAGATATAT GTAAGCGATA AAGTATTAGT AACCTCACAT AAAATAATGT ACAATAATAG 5940
ATAAAAACTA AATTTTATAT AAAAATTGAA TTTCAATCCA CTTTCTTTTT TCGTGGATCA 6000
TAAGGAGTTG GACTTGCTTT TTTCACGGTA ATTTGACCAA AGAAAGAGTT AATACAAATA 6060
3 0 ATATTAATTA AGATATTATC TCTTGTTGTT TGTTCTTGTT TTGAAATAAT TTAGTTTTTT 6120
TTTTAAGAAA AAAAAGTTTT TCCAATACAT AAGCAATACA AAAGTGTTTG AACATGGTAA 6180
TTCTTCTTCT TCTTAGTTGA CCAAATTACA TTTGGTAGAC TAAAGTTGTT CATATATATG 6240
CTACCATTGA TAGAGTCATT GGCCAATTAT ATGTTTTTAC GTCATTATAT TTGAATTCTT 6300
TTGTTAATAG TAATTATTAA TCACTGAAGT TATTGCATTC TTGTCAGCTG ATAAACTCCA 6360
- 61 -
63198-1218
CA 02227940 1999-OS-20
AGTTGTAATT TTATGTTTGA TCTTGTAATT AAGAGCAAGC CAGGAGGACA TCTCTAGTGT 6420
TCGAGGAAAT TGACAAAATT TGCTTCCTCA AATATATTTT TGTTTTTCAT TGGACAAAAA 6480
TACATGTTAT ATATATATAT ATATATATAT ATATATATAT ATATATATAT AATGCCTATA 6540
TTTTGTGAGT AGTTCCATAA GTTTAGGATA TGTTTGAGGT AGTTTAACAT AAGCATTTGA 6600
TTTTTTTTTT CAATCCTTAT ATCAAAATTA TCATAAAACA ATTAAAAAAT CATTAATTTA 6660
TTTTATTTTT TTAATTAAAA AAAACACTTA TAAACACAGT ATTACCCAAA TACAGATTTA 6720
TGAAGCCGCC ATGTGGTAAA AAAATACATG TTAGAGATAT CAGAAGTTTA CAAGCATGTT 6780
TATATGCGTT AATGTGGCAT ATGAAATGTC ATATCAATTG CGTTACAAAG CTTTTCTTGT 6840
GCTAAGTGTG GCGTTAGTAA TAAGCAAGTG TTTGTAAGAA TTGTCAACAC GTGTGTTTAC 6900
TTACTTGAAA GAACATTAAT TGCTAATTTT ATTAAATAAT TAATCCTTCC TATTACTATC 6960
TTGGGATAGG TTGAAGAGCA TAAGGAAAAG GGTTACCATG ATAAATACAA AAAATAAAAA 7020
AGGAGGAAGG AGTAGTTTTC AATTTTATTT TAATTGTCAA TACTATGTGC TTGGTGAAAA 7080
GTTATCTGTC CTCATTTTTA TTTATTGTTT TTTACAAAAA GCATAGAATA ATGTGTGTTT 7140
CATGTGTTTG GTTAGAGGTT ATAGATGAAA AGCTTTAATA ATAAATAGTA GCTAAATATA 7200
CTTCATTGTT TGAGTGGTAG AGGAGATTTT TAAAATTTAT GAAGACTACA ATTCTCTTTC 7260
ATTTCAAATA ACATCCCTAT TTTAGTGGTG AGATTAATGT ATTTGTTTCT CTTTTTCTAT 7320
TTTCTTTTAT CAATATTATA TATAAAACTA AAATGCATCA GTGTTTTACT ATGGATTGAT 7380
CATAATGCAA TTCACTATAA AATAATTGAT GCTTCCCTTA AAAAACCAAA TAATTAAACA 7440
AACACTCAGG GTTAATTTTG TATTTTCATA TCTTTATTGC ATAGTGTAAT TATTTCTATG 7500
2 O TCCTTGAAAA AAGAAAAAAA ACACTAGGGT TTTTTTAAAA AAGTTTCATA TTTTTTTGTA 7560
TAGTGTAATT ATCCCACTTT TGGGGCCAAC TTTTTTTTAC CTAAGGTAAA GGGGTATTTT 7620
TGGTTTTTTT ATGTTTGTTT TTTTGCAATT ATTATATGGG ATCAAGAGTG TTATGATCTT 7680
TTTATATAAA AAAAAAATGG TTGACACGTG ATCTACAATT CCCCCTCCCT TTTCATTCCT 7740
AACCTTGAAA GTCTTAGTGA AACATATAGT TATAATAAAG AAATATTATC TCTAGTTTTG 7800
CAAATTAATT TCATAACATC AATTAAATAT TCTGATAAGG TAAAGTTATT TAGGATGGAG 7860
AAATTTACAT AATGAAGCCT CCTTCTGCCT GAGTAGTGCA TTTCTATGGT ATTTATGAGC 7920
ATCAATTCTA CAATCCATTG AAGCAAAAGA ACTAACCTTC TTGAAACCCT CTTGCAGATA 7980
ATTGTGAGTG AATGTAAGTC CACTACGAAA TATTCACACG ATTACGCACT TAGTTATCAT 8040
TAAACTTTGT TTTTGGTGCT TTGCATTTTC TTAATTAGAT TCTTCCACAG CTTTCCAATG 8100
3 O CACATTTTGA TGACTTTTTT TATTTTATTT TTCTTGATGG AAATGTTGAC ATGATTGCAG 8160
TGTCAAATCA ACAATTGAGA GGTACAAAAA GGCATCTGCA GATTCTTCAA ACACTGGGTC 8220
TGTTTCTGAA GCCAATGCTC AGGTACCATA TATCAGCTCT AACTAACAAT TTGTACTCAT 8280
AATATCTATT AGATGGAGTT CAAGCATAAT ATTCCTCCCA ATAATTTATT GCCAATATAG 8340
TGCTATGCTA CCACTTCATT CACTCTTTCT TGATAACCCC AGCTTGTATA AAATCTATTA 8400
- 62 -
63198-1218
CA 02227940 1999-OS-20
GATACCTCTA AGTTTTTGCC TTACCTTTCT CACTAGTGTC TGACATGACA CTAGTGTTCA 8460
CATGGATTAG CATCTCGGAG TTGAAGGTTG TCTGGCTTCT TCGAANATCC AGGGTTTTCA 8520
AGAAGGTTTG TACATTGGGA GGCCCGTGGT TATAAACCTA CTGTGTAAAT GGTTTGATAA 8580
ATAATGATTC ATCAGATTTG AGTAATAGTC TTTTAATTTC TTTGTAAATG TTGTCTATGT 8640
TTTTTCCAGT CCTCCCTACA CACACTCTGA TAATTATAAC CAATTTTGTT TCGCTTCCTC 8700
CTTTCGCTAT GCTCCTACTG AATTTATTTC CAGTTTGATT CAGTATTATA TGCATGTTTA 8760
CAAGAAAATA GAAGGGGGGA ATCTACATCA CTGAGATTTT CTACCTGTAT TTTATCAACT 8820
GATCTAATAT GAACTTGAGG CTCTTAATTT TGTTATATAT AATGTTTTAT TGCCTTTTGT 8880
TCTTGCATCT CAGTACTACC AGCAAGAAGC TGCCAAGCTG CGTTCCCAAA TTGGTAATTT 8940
GCAGAATTCA AACAGGTCAG AGCCTGTTTG ATATTGATCT ATTTGTCAGA TGATATCGTT 9000
TTCTCTTCCA AACTCCGCTT AAGTATAAAT TATATTTCAG GCATATGCTG GGTGAAGCGC 9060
TTAGTTCATT GAGTGTGAAG GAACTTAAGA GTTTGGAAAT ACGACTTGAG AAAGGAATAA 9120
GCAGAATTCG TTCCAAAAAG GTTTTGATAC TAGTACCGAA TTGATACTAT CACATTTTTT 9180
TGTTTTACTT GGATATCACA TTTCCATGTA TGGCCATTAA CAAGTTTTGT GTTCATACTT 9240
TCCTGCTATG TTTCTAAAAA ATTCCTCCCG CAAACCTTGC CAGAATGAGC TGTTGTTTGC 9300
AGAAATCGAG TATATGCAGA AGAGGGTAAT GCTTCTTATG TTATCACATT TCCCATTTAT 9360
TTAATATTTA TTGTTTTCTG GTGGAGTATA TTCTATATGA TTGTTATATA TTCTGAGGTA 9420
AAAGTCATCT AGTGTTTATT AACATAATGA TTCTATGGTC AACTTATTCC TTCCTGTTTT 9480
CACTCCGAGA TTTTCCTTTG ATTCCTTGAA TGAAAATGCA CATTACAGGA GGTTGACTTG 9540
2 O CACAACAATA ACCAGCTTCT CCGAGCAAAG GTCTTTCTTC TATCTATCTA TTTATCCATC 9600
TCGAGTGAGG GCAAGGATGC GTGCGTGTGC ATGAATGAAG ATCTCTATGT CTTATATCGT 9660
TAGTGAGCTG TTTATAATTT AGAAATATGA GGCTTATCTT GATAGTGCAG ATTTCAGAGA 9720
ATGAAAGAAA GCGACAGAGC ATGAATTTGA TGCCAGGAGG AGCAGACTTT GAGATCGTGC 9780
AGTCTCAACC ATATGACTCT CGGAACTATT CTCAAGTGAA TGGATTACAG CCTGCAAGTC 9840
ATTACTCACA TCAAGATCAG ATGGCCCTTC AGTTAGTGTA AGTATCTCCT TTGTAACGAA 9900
TAATAGGTTT TCATTAACCG GACAACCAGA TTTAGTGTTG TGCATTCATA AAATACAATT 9960
AATTACTTTA ATTTGGAGAT GTTCCAAAAG TTGCAACTGC ATGGTTCATG GGCTCTAATT 10020
TCTTGGAAGT ATATAACCGA TGCTATGTCT TTTCATTCTC ATAATTACTG ATCAGTCCCT 10080
TATAGATGAT TATTTGCAGA TTCTTATGAC CATTTTCCCA TTGAGATTAT AAGATTTTGA 10140
3 O CATCGAATAG TTGGACTAGG AGTAAAGAGC TGTTGCTGTT ATTTAGCACC CCAAAGGAAA 10200
TATTATATAC CTCTGAACCA ATTGAATGGC CGACCTAGGT TTACTGAAAT GTTTAGCTGT 10260
AAGAAGGTTA AGTGTTATCA GATTCCCCAA GTGAGAAGTA CATGTTTCTT AGCATACTTT 10320
ATGTTTCACG CACCTTGATT TTTCAAACTT TGTTTATCGA TTTCTGAACT AAAGTGACTA 10380
CATTATAGAA CTTGAACCTA AAATTACTCT CCTCACTATA GGTGAAATCA GATTACTTGA 10440
- 63 -
63198-1218
CA 02227940 1999-OS-20
AAATACTACT AAAAAAAATT ATGGCGTTTG CTGGTATTTC TAACATCTTT TCTGCTAATC 10500
TTGTATTAAT TTTCTCCTAG ATGAACTTGT TATTATGTAA AAAGGTTTCA TTACTCATGC 10560
AATGGTGCAC TAATGCTTGA GGAGTTCCAA GTAACTTTGC TGTCTCATGT AAAGAAGAGT 10620
GCTGAAGTTC ACTATGGTTT AACTTCTACT GCACTGCTTG ATATTGCCAT GAACTCTGAC 10680
ATCATTTGGC TTGATCTTGT TCTAAAATCT AAATGAAATA ATTCTCTCTT ACTATATATC 10740
TTCTTAACCC TTTGCATATG ATTAAGTGGT CTTTGATAGG ATATCATTAA AACCTCGCAT 10800
AAAAGCTACC ATTTTATAAA TTTCAAACTC CACGACGCAT TTTCTGGTGA TTCCATTGCT 10860
GATTATTGTT TAAAGACATC ATTATTCCAA TTAGTACATG TATAATAATT TCCTCTGTTG 10920
TTGGTGCAGT TAATAATCTC CAAGTGCAGC AGTTTCTCGC ATTTCCATAT TCCATGGAGA 10980
GTACCTGGGT TTCCATTGAG CGCAAAAGCT ACATGTATGC TAAAAAACCT GAAGTAGCGT 11040
AAATCATATT TGTCTGGGTG GGAGGGCCTA GTACTCTTCC TCTATGTATT AACTATCCTG 11100
TCCCAGTTAA GACATAAGAA ATGTCAGAGA AGGATTTCTT TTCTGTATGT TTCATGAAGG 11160
CATTAAGATG CTGTTACAGT TGTGACTAAC TTATTATATA TGTCTTACTG CTTCATCTTG 11220
TGATATTTTC TTGCATGTTA ATCTGATTAA AGTGTAGCTT AGACCATTCA CCATGTTAAT 11280
GGTGACTTGT TGGTGACTAC TAGTAGCTGT AGCTCTCCGT AGTACTGCTA TGCCTTCAAA 11340
AAATGATGGG TCGGAAATTA CTAGCTAGCT AGTATTGCTG TTTCATTCAA TCTCTGCTTT 11400
AACCCAAAAA TCAGGACTAG TGGATTAGCA TACCTCTCAC CAGGACAATG CACTAGAGCA 11460
CATTTTCATC TTCTTCTCAT ATTT 11484
(2) INFORMATION FOR SEQ ID NO.: 10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1201
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
3 0 ( ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (196)..(921)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 10:
TGAGAGGTTG TTTTTCACTT TATAAATACC CACCTCTTAG CCCAAACTTG CTTCCATTTT 60
- 64 -
63198-1218
CA 02227940 1999-OS-20
CTTCATCTCT CTACTAGTTA GATTTGTAGG AGAAATCCCA AAGGAAAAGA TCCTCACTTT 120
CTCTACACAT TAACTGCTAT CTACAGCCCC TAGCTACTTT GTTTTATTTC CTCCCAAGCT 180
AGCTAGGCTG CAGCT ATG GAA TAT CAA AAT GAA TCC CTT GAG AGC TCC CCC 231
Met Glu Tyr Gln Asn Glu Ser Leu Glu Ser Ser Pro
1 5 10
CTG AGG AAG CTG GGA AGG GGA AAG GTG GAG ATC AAG CGG ATC GAG AAC 279
Leu Arg Lys Leu Gly Arg Gly Lys Val Glu Ile Lys Arg Ile Glu Asn
15 20 25
ACC ACC AAT CGC CAA GTC ACT TTC TGC AAA AGG CGC AGT GGT TTG CTC 327
Thr Thr Asn Arg Gln Val Thr Phe Cys Lys Arg Arg Ser Gly Leu Leu
30 35 40
AAG AAA GCC TAC GAA TTA TCT GTT CTT TGC GAT GCT GAG GTT GCA CTC 375
Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu
45 50 55 60
ATC GTC TTC TCT AGC CGC GGT CGC CTT TAT GAG TAC TCT AAC GAT AGT 423
2 0 Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn Asp Ser
65 70 75
GTC AAA TCA ACA ATT GAG AGG TAC AAA AAG GCA TCT GCA GAT TCT TCA 471
Val Lys Ser Thr Ile Glu Arg Tyr Lys Lys Ala Ser Ala Asp Ser Ser
80 85 90
AAC ACT GGG TCT GTT TCT GAA GCC AAT GCT CAG TAC TAC CAG CAA GAA 519
Asn Thr Gly Ser Val Ser Glu Ala Asn Ala Gln Tyr Tyr Gln Gln Glu
95 100 105
GCT GCC AAG CTG CGT TCC CAA ATT GGT AAT TTG CAG AAT TCA AAC AGG 567
Ala Ala Lys Leu Arg Ser Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg
110 115 120
CAT ATG CTG GGT GAA GCG CTT AGT TCA TTG AGT GTG AAG GAA CTT AAG 615
His Met Leu Gly Glu Ala Leu Ser Ser Leu Ser Val Lys Glu Leu Lys
125 130 135
140
AGT TTG GAA ATA CGA CTT GAG AAA GGA ATA AGC AGA ATT CGT TCC AAA 663
4 0 Ser Leu Glu Ile Arg Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys
145 150 155
AAG AAT GAG CTG TTG TTT GCA GAA ATC GAG TAT ATG CAG AAG AGG GAG 711
Lys Asn Glu Leu Leu Phe Ala Glu Ile Glu Tyr Met Gln Lys Arg Glu
160 165 170
GTT GAC TTG CAC AAC AAT AAC CAG CTT CTC CGA GCA AAG ATT TCA GAG 759
Val Asp Leu His Asn Asn Asn Gln Leu Leu Arg Ala Lys Ile Ser Glu
175 180 185
AAT GAA AGA AAG CGA CAG AGC ATG AAT TTG ATG CCA GGA GGA GCA GAC 807
Asn Glu Arg Lys Arg Gln Ser Met Asn Leu Met Pro Gly Gly Ala Asp
190 195 200
TTT GAG ATC GTG CAG TCT CAA CCA TAT GAC TCT CGG AAC TAT TCT CAA 855
Phe Glu Ile Val Gln Ser Gln Pro Tyr Asp Ser Arg Asn Tyr Ser Gln
205 210 215
220
GTG AAT GGA TTA CAG CCT GCA AGT CAT TAC TCA CAT CAA GAT CAG ATG 903
Val Asn Gly Leu Gln Pro Ala Ser His Tyr Ser His Gln Asp Gln Met
225 230 235
GCC CTT CAG TTA GTT TAA TAATCTCCAA GTGCAGCAGT TTCTCGCATT 951
Ala Leu Gln Leu Val
240
- 65 -
63198-1218
CA 02227940 1999-OS-20
TCCATATTCC ATGGAGAGTA CCTGGGTTTC CATTGAGCGC AAAAGCTACA TGTATGCTAA 1011
AAAACCTGAA GTAGCGTAAA TCATATTTGT CTGGGTGGGA GGGCCTAGTA CTCTTCCTCT 1071
ATGTATTAAC TATCCTGTCC CAGTTAAGAC ATAAGAAATG TCAGAGAAGG ATTTCTTTTC 1131
TGTATGTTTC ATGAAGGCAT TAAGATGCTG TTACAGTTGT GACTAACTTA TTATATATGT 1191
CTTACTGCTT 1201
(2) INFORMATION NO.:11:
FOR SEQ ID
(i) SEQUENCE
CHARACTERISTICS
(A) LENGTH:
723
(B) TYPE: nucleic
acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus ifera bsp.trichocarpa
balsam su
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (1) ..
(723)
2 (xi) SEQUENCE DESCRIPTION:SEQ ID 11:
O NO.:
ATG GAA TAT AAT GAA CTT GAGAGCTCC CCCCTGAGG AAGCTG 48
CAA TCC
Met Glu Tyr Asn Glu Leu GluSerSer ProLeuArg LysLeu
Gln Ser
1 5 10 15
GGA AGG GGA GTG GAG AAG CGGATCGAG AACACCACC AATCGC 96
AAG ATC
Gly Arg Gly Val Glu Lys ArgIleGlu AsnThrThr AsnArg
Lys Ile
25 30
CAA GTC ACT TGC AAA CGC AGTGGTTTG CTCAAGAAA GCCTAC 144
TTC AGG
3 Gln Val Thr Cys Lys Arg SerGlyLeu LeuLysLys AlaTyr
0 Phe Arg
35 40 45
GAA TTA TCT CTT TGC GCT GAGGTTGCA CTCATCGTC TTCTCT 192
GTT GAT
Glu Leu Ser Leu Cys Ala GluValAla LeuIleVal PheSer
Val Asp
50 55 60
AGC CGC GGT CTT TAT TAC TCTAACGAT AGTGTCAAA TCAACA 240
CGC GAG
Ser Arg Gly Leu Tyr Tyr SerAsnAsp SerValLys SerThr
Arg Glu
65 70 75 80
40
ATT GAG AGG AAA AAG TCT GCAGATTCT TCAAACACT GGGTCT 288
TAC GCA
Ile Glu Arg Lys Lys Ser AlaAspSer SerAsnThr GlySer
Tyr Ala
85 90 95
GTT TCT GAA AAT GCT TAC TACCAGCAA GAAGCTGCC AAGCTG 336
GCC CAG
Val Ser Glu Asn Ala Tyr TyrGlnGln GluAlaAla LysLeu
Ala Gln
100 105 110
- 66 -
63198-1218
CA 02227940 1999-OS-20
CGT TCC CAA ATT GGT AAT TTG CAG AAT TCA AAC AGG CAT ATG CTG GGT 384
Arg Ser Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg His Met Leu Gly
115 120 125
GAA GCG CTT AGT TCA TTG AGT GTG AAG GAA CTT AAG AGT TTG GAA ATA 432
Glu Ala Leu Ser Ser Leu Ser Val Lys Glu Leu Lys Ser Leu Glu Ile
130 135 140 .
CGA CTT GAG AAA GGA ATA AGC AGA ATT CGT TCC AAA AAG AAT GAG CTG 480
145 Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys Lys Asn Glu Leu
150 155 160
TTG TTT GCA GAA ATC GAG TAT ATG CAG AAG AGG GAG GTT GAC TTG CAC 528
Leu Phe Ala Glu Ile Glu Tyr Met Gln Lys Arg Glu Val Asp Leu His
165 170 175
AAC AAT AAC CAG CTT CTC CGA GCA AAG ATT TCA GAG AAT GAA AGA AAG 576
Asn Asn Asn Gln Leu Leu Arg Ala Lys Ile Ser Glu Asn Glu Arg Lys
180 185 190
CGA CAG AGC ATG AAT TTG ATG CCA GGA GGA GCA GAC TTT GAG ATC GTG 624
Arg Gln Ser Met Asn Leu Met Pro Gly Gly Ala Asp Phe Glu Ile Val
195 200 205
CAG TCT CAA CCA TAT GAC TCT CGG AAC TAT TCT CAA GTG AAT GGA TTA 672
Gln Ser Gln Pro Tyr Asp Ser Arg Asn Tyr Ser Gln Val Asn Gly Leu
210 215 220
CAG CCT GCA AGT CAT TAC TCA CAT CAA GAT CAG ATG GCC CTT CAG TTA 720
3 0 Gln Pro Ala Ser His Tyr Ser His Gln Asp Gln Met Ala Leu Gln Leu
225 230 235 240
GTT
Val 723
(2) INFORMATION FOR SEQ ID NO.: 12:
4 0 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 241
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 12:
Met Glu Tyr Gln Asn Glu Ser Leu Glu Ser Ser Pro Leu Arg Lys Leu
SO 1 5 10 15
Gly Arg Gly Lys Val Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn Arg
20 25 30
Gln Val Thr Phe Cys Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala Tyr
35 40 45
- 67 -
63198-1218
CA 02227940 1999-OS-20
Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe Ser
50 55 60
Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn Asp Ser Val Lys Ser Thr
65 70 75 80
Ile Glu Arg Tyr Lys Lys Ala Ser Ala Asp Ser Ser Asn Thr Gly Ser
85 90 95
Val Ser Glu Ala Asn Ala Gln Tyr Tyr Gln Gln Glu Ala Ala Lys Leu
100 105 110
Arg Ser Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg His Met Leu Gly
115 120 125
Glu Ala Leu Ser Ser Leu Ser Val Lys Glu Leu Lys Ser Leu Glu Ile
130 135 140
Arg Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys Lys Asn Glu Leu
2 0 145 150 155 160
Leu Phe Ala Glu Ile Glu Tyr Met Gln Lys Arg Glu Val Asp Leu His
165 170 175
Asn Asn Asn Gln Leu Leu Arg Ala Lys Ile Ser Glu Asn Glu Arg Lys
180 185 190
Arg Gln Ser Met Asn Leu Met Pro Gly Gly Ala Asp Phe Glu Ile Val
195 200 205
Gln Ser Gln Pro Tyr Asp Ser Arg Asn Tyr Ser Gln Val Asn Gly Leu
210 215 220
Gln Pro Ala Ser His Tyr Ser His Gln Asp Gln Met Ala Leu Gln Leu
225 230 235 240
Val
(2) INFORMATION FOR SEQ ID NO.: 13:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 10007
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 13:
TCCATTATTT CAACAATAGA TTCATTTACA CTAGCATGGA TACTTCAATG AATAAGAAGT 60
GTGTTATTGT TTGGAGTAAT AGACACCTAT AATTCTCAAA CCTTTTACTT TATTTTTATT 120
TCCTTTGTTA TATTACATTT TTCATTTCTT TATTGGGTTT TCATTGACAG GATGGCTAGA 180
TTAATATAGT TTCTTGACTT TAATAAATAA AAAAAAGATC AAGACTCTCT TCACAAACCT 240
- 68 -
63198-1218
CA 02227940 1999-OS-20
TTACAAAATT GGGCGCTATA TCTAAACTAA AAAACTTAAG ATTATATACT ATCTAAGGAG 300
TAGCACACTA TAAATAACAT TATAAAGGTA GTTTGTTGAG CGGAACTAGA CTTTGCAAAA 360
TAACTTTCCA ATATAGCTTT TCTTGTTGAT GTTGACCTTT TAATTTAGGA TCAAACACTT 420
GTAAATTACA ATTAAAAGGC TTATTTTTGT TTGCCATTTT TACCAAGCAA TGTTAGGATT 480
GCTAGAGATT AGTTTTTCCA TGGAATAAGA AGTTATCTTT AAAGGGCTTA AAAGACCTAG 540
TAGCTTGACA AGGCTATGAC TTGTGTTGTT TTGGATCGTA TGGTTATTGT TATAGAGGTG 600
CTAGTGGTTA AAGACATCCA TCATGGAGGT GGTGATGACT TAAAAGAGTT AGATGTAAAT 660
TGGAGACTTA TGTTATTCTC ACATAAAAAA TGTTAGCCTC CGACATTGTT TTTGGATGTG 720
TAAAATCAAT GTACCATTTT ATTCTTCATT GTTTGTTTTC CTTATTATGA CTTTTACAAA 780
TTTATCCTTT AGGTGATGAA ATTCCTTCAA TCTTGTTCTA TTTTTTTTTT TAATTCTTGG 840
TACGTAGTTC TGTACTTAAT CAAGCAACAT AAAATAGTGA TGCCATCTTC ATCACTCTAT 900
AAACGTGGAA ACCCAAATCT CTGGCTTTTA TTCATGATTA AAGTCATTTC TAGATTTTTT 960
TAGACGTTCA AGTGAGATTT AGGGTTCAAT AAGAGAGGAT CAATGGTGAA AATAGAAGAA 1020
CAAAGTTGTT GTGGTTAAGT TGACTCGGTG GTTGTTGAGT TGGGATATGA AGGAATAGAT 1080
GGTAGACTAA TCTAGTGTTT TTGTCCACTT GAGTTCTTAA TTATTATTCC ATCTCCATGA 1140
CTATTTCCAT CTTCTTCTTC AGTGATATTG TTTATACTCT GTGATTTGGG TTTATTGGAA 1200
CTTATTATTG AGGCAGCTCA TCCATAGAAA TTTGGTACTT GCTTCAACAA ACCACTAAAA 1260
TGTTGTGTGG TTAATATTTG AGAATGCGCG AAAAAAGCAT CGTACTAAAT TTGGGTTCCC 1320
GACTGGATGA AGAGAGATGT GATTACTTAA TTTATTTGGA TTTTCGGGGT TTATTAGATT 1380
2 0 TTTGGAAAGG TAATACGATA TCATTGGTTT TGAGAGGAAA TAACATTGGG ATTTTGATGA 1440
TTTTTGAATA ATAAAATTAA GTTTTTTCTT GATTCATTTG TTAATAGAAA GAGAAGAGGG 1500
ATAGCTCTCT TATTCTAGCA GAAGTACGTA TATGAGCTAT GGGATTTAAT TCTTAATTTT 1560
GTATGAGTTA TTGATCAAAG AAAAAGCAAT GATGTGAGAA GTCTATATAT ATAATTTCTC 1620
CTACGTACTC CGTTGAACCT TTTTTCCTAA TAAAAATTGA TAGAAAATCT ACAACATATA 1680
CAGAGAAATG TGAAGTTCTT CAATTGAGAA TAAATCGTTT CAAAAGGACG TAGGAATCTC 1740
CTTGTAGTGA GTGAAACTCC AAGAAAATTA AACAACCTGC TGGGGAACAT CCATA.CAACT 1800
ATCCTCCGAT CCCTTCTTTT CTTTTCAAGT AGGCAGGCAA TAAAACGTAT TTAGCATAGC 1860
CAAGTTCAAA AAAAAAACAA GAAGAAGAAG AAGCAATGAA ATAAGGGAAA AGATGAGGTT 1920
CTCTTGTTAA GCACCTTTCA TTTGTACCAT AATTTTGTCC TTGGAATGAT TAGAGAGCCC 1980
3 O AAAAACGTGT TATTCACCCC AGAAAAATCC ATTTTCAAAA AGTCCCTTTC TCTTGATGAC 2040
CTAAATCATT CACATGGAAG CCAAGGAAGA AAATGAAAAA AACGAATATA GTGGATGGTT 2100
ATTGAGGTCT CAGTCTTCCT ATAGCGTATT CTCTAATTAA TTCCAAGATA F~~;?~AAAAAAA 2160
AAAATTACAA GGATGGTGTA GATAAACTTA GTAGAAAGTA TTGTTATATA TATATATATA 2220
TATATGGGAA TGGATGAAAG GTCGTTTATC ACTTTTATAA ATGCCCACCT CTTAGCCCCA 2280
- 69 -
63198-1218
CA 02227940 1999-OS-20
ACTTGCTTCC ATTTTCTGCA TCTCTCCTAC TCAGATTCGT AGGAACAAAG AAGAGAGAAA 2340
CCCCAGAGCA AAAGATCCTT ACTTTCTCTC CTTAATAACT ACTATCTCTA CAACCCCTAC 2400
TTTGGTTTAT TTCCTCCCAA GGTTAGTTAC CAAAACACTG AGACATATAT CTCGTTGTAT 2460
TCTTGAGTGC TTCACTTGTT TGGGGCTTAT CAATCTTCTG ATCTTCTTAT CTCTTCTTCA 2520
TCATAGTGAC TGAGGAACCC CATCAGATGA AACTTTTAAT TTTCTAAAAA AGATTTACTT 2580
ACAAACGTTT CTGTCACTCT CTGCCGTTTC AATCTCCAGA TTGAAGCATT ACTAGTTCAT 2640
CCCTTTGTTT TGTTTCTCAA TTATTTTCAT ATCCATGAAA CCATAACAAG GGCTAATTCA 2700
AGAGCTAGCT GCAGGCGTTC ATGGAACCCC TTTCTTCTGT TTATTTTGTC TTCCATCATG 2760
AGCTATTCAG TGCTCAAGAG TATTCCTGCT AAATATGCTA TGAATTATCC TTATATATAA 2820
ATCATTCTTG AATTAATTAC TAGCTAGTAG TTCAGTAATT TTATTACTCT CTTTTCTGCT 2880
GTCTTCACCC AGTTTGTGTT TTGGATCAGC TAGCTAGGCA GCAGCTATGG CATACCAAAA 2940
TGAACCCCAA GAGAGCTCTC CCCTGAGGAA GCTGGGGAGG GGAAAGGTGG AGATCAAGCG 3000
GATCGAGAAC ACCACCAATC GCCAAGTCAC TTTCTGCAAA AGGCGGAATG GTTTGCTCAA 3060
GAAAGCCTAT GAATTATCTG TTCTTTGCGA TGCTGAGGTT GCACTCATCG TCTTCTCCAG 3120
CCGTGGACGC CTTTATGAGT ACTCTAACAA TAGGTATATA CTTAGTTCCT CGGCTCATGA 3180
ATTCTCCATG TTGCAAACCC TCTTCAAGTG CTCAAAGTTG GTTTTTCTTG CTTTCTCATC 3240
CAAAGGGATT TGTTTTTTCT TTTTGCTTAT GTCAGTGTTA ATTTTTATTG CTTTGGTTTT 3300
GAGCTGTTTC TTTAATTGGT TTTCTTCCAT CATCATTTTC TTTCTTCAAT TGGTTTTCAA 3360
CGTTTGTTGT GGGGAAAAAA AATAGGAGCC TGGTGTCAAG GTTTTTAGCT TCTGAGCTAG 3420
ATCTTCGGGT GTCTTTAAAG TAAAAGAACA CAATCATTCT TTATGCTGCA GTTTGGATTG 3480
AATTTCTTCT CAAAATACAA TTCACTTGTC TTTCTTTCTT CTATTTCTTT TCTTTTCCTT 3540
GTATAAGCAT AATTAATGTT TTGTTTTTCC TTTTCTTTAT TTCACCCTTA GATGATTGTG 3600
ATGCATACAT GATTTTGAGT TCTTGGTACA TAGATCTGGT GTATTAGATA GACATAGAAG 3660
CACAATTATA AGTGTAATAA GGTAGTAGAA ACAAGTAGAG GGCTGGGAAA ATGTATGCAG 3720
GCATGTGATA TCAGCCTCTT TATCTCCTCC CTTGATGTTA AGTTTGCTGT TTCCTTTTTC 3780
TTTCTTTTCC ATCATTCCTC TTGAACTCTG CCTCTCTCCT TTACTCTTTT CTTGCACATA 3840
CATGCATGTT TGAGTCATCT CTAGGGCTAA AGAGATTACC TATAGCTAAA GCTGTCATCT 3900
TCTCATTAGT CCAAACCCTC CCATCTCTTC TCACTTCCAA AATAGCACGT CAGTCGGACA 3960
TAAGAAGAAA AGAGTACAAA GTCAAATTAA ATGTGAAAAA AAAAGAAAGG GTTTTTTTAT 4020
3 O ATGTCATGTC ACCAAACACA CAAACATATA TTACTAGGGT TTCAAAATCC AAATCCCCAA 4080
ATGGGTTTCT TCATCTTATT TTATTTTTCC AAAACAATCA CTAGGATCTC TCAATTTAGG 4140
ATTCTTTTTC CTCTAATTCA CACGAATTTC ACAAAATCTC TGTTCGAACC CACGTGGGGA 4200
AAGTGAAAAG CTTTTTGTTT TTCAAGCATA GCCCTAGTTA GGGTTCATAT TTAAGTAACC 4260
ACTTGAAGTC ATCAAAATTG AACCGAAACT TTAGTGCAAA CTATTCAATC AACCATGTGG 4320
- 70 -
63198-1218
CA 02227940 1999-OS-20
r
ATTCTTCCAT AACCAGTCAA AAATTAAGTT AGATTTCACC TAGATTTTTA CCCTTTTTAA 4380
CCTCGGTAAG AAGGGTACAG TAACTGGTTA GGGTTTAATA GCCAGTTCAA TATATCAGAT 4440
TGTTGTTTTG GTTTATGAAA AGAATCTTTG GTCACGTCAC ACACGATTTT TCAGTTCTTG 4500
ACTACTGACA AAAGGGTTCA AGTCATGATT CATGAAAATG AACCATAAAT TTTGAACTCC 4560
CAATCTGCAA AAAAAAAGAA GAAGAAGCAA TACCACACAG AATATTGTCC ATTTCTTCTC 4620
CTTAGATCCC TCCCTCTCTC TGTTATTTTC TTTCCCATAG TGAAAGAGAG ATGGAACAAC 4680
GAGAAAGGGT TAGCTAAGGT CATGATGATG CCATTGTTGG TCATTGTTGA GTGTGGTTTG 4740
CGTTTGTTCA AGATCTTGAA TATATGTATG TATGTATGTG TATGTATGTA CGCAAGTTCT 4800
TTTAGGAAGA GAGAGTTAAT ACAGAGAGAG AAAGAGAAGA GACGATGTAC GACAAGTGCT 4860
AGCCAATGGG AGACTTCTGT CAATTTTGGC TTTTTTAATG TAAATAGAAG CGTAAAACTC 4920
TAGCAGCTGC TGCTGCTGCT CCTCTCTGAG AGTTTCAGTC ACATTCAAGA AAACAAAAA.A 4980
AAATAATTTT TTATCTTATT ACAAATGAAA TAGAATATCG AGGTGGGTAA TAGATGGTGG 5040
CTCAAGAGAT TATCCACCAT AGAAGAAAAA AAGAAAAATA GATATTGCCC ACCATGTAAT 5100
GAGGAATTGA CAAGTGGGAT AGATGTGACC GTTGATTTAG CTTGGCCAAT CATGTTATAG 5160
GACTCAGTGA CAGCTTGGCA GAGACAGCCA ATCACTGGCT CGACGAAGTT AAGGTATCAG 5220
AAAATCTAGA TATCTGGGTC TTGTCTTAAT TGACAAAATG TGTTCACATC TTACTGTTAT 5280
TATTATGGCA AAATTTTAGG ATGCACAAAG AACTGGGTGG AGGTTTTCCC ATCCATCTTC 5340
TTCTTTAGGG TTTTAAATGA GTTAACTTAA TGGAGTTATT AGTTGATTTG ACTCTTTGAT 5400
TTGAATGTTC TTACCTTAAA ATCATGGCTT AACATCATCC GGTGTTGGTA CAGGGGCATG 5460
2 O ATTTTGCTCT CTCCCTCTTC AGTCAATTTC ATCATTTATT TTGATATATA ATTTTCTTTT 5520
TCCCTAATGT TAAGCCTCTA CTGTCACATC TTTAAATTAC TAGAGGGATA TGTAACTGAT 5580
AACTAGTAAC TTCACATAAA ATACTGTACA ATATAGATTT AAAAAAGGAA TTTTATATAA 5640
AATTTGAACT CTCAATTTTA TTTTATTTTT GTTATTTGGA TGAGTAGTTT GTCTAAAGCA 5700
ATAGCTAATA TGGAGGGTAT TAAGAAACTG CCTCTAGTTG TTGACACAAA AGCCTTGAAG 5760
CACGTATTTT ACTCGCTAAT TCACACTTCT TGGCTGTGCT CTTACCATCT TGGAAAATAA 5820
ATGGATTTCA AAAAAGTACA TGGTTTCTTA ATCTATTTGA AATGATTTAA TAATGAAAAA 5880
TAAAATCAAA TGTAAAGTCT TAAATGTAAT AAAAAATAAT TTTCGATGTT TTTTGAGTGT 5940
TTTTTTTTTT ATAATTTTGA AATCTTCATA TTTATATGTC CATATAAAAC ATAGGAAAAA 6000
TCATCAATTC TCAAAATTAT TGGAGAAAAA CACCTACATA TGCATTATCG ATCAACTACA 6060
30 CAAATAGGAA GTAACCATTC GAAGAAAATT AAAGACTAGA GACATCAAAA GTTGACAAAC 6120
ATGTGTACAT GTGTTAATGT AATCGTGTGC AAGTGTCATG TCAGTTGAGT TACCAGTGCT 6180
AAGTGTTGCT TCCGTTAATG TTATAACAAA TTATCAATAT ATGTAAGTAC TAATTTTAAA 6240
GAATATTGCT ATTAAATAGT AATTAAGCTA TCTTTGGATA CATAGAAGAC CATGGGAAGT 6300
GAAAAGTTTC CTTGATAAAA GGAGTTGTGG TTTTCAATAT ATATATATAT TCAAGAATGA 6360
- 71 -
63198-1218
CA 02227940 1999-OS-20
CATGAGAAGT CTCTTAATAC AGGCCTCCAA TGAAAAGGAA ATGAAGAATA ATTTTCCAAT 6420
TACTTTCGAG TAAAAAGTTA TCTATGTTCA ATATTTTCTT TTCTTTAAAA AAAAGAGAGA 6480
ACAGAATAGA AGAATGTATA GATCTGTCTG TTTTTTGTTC TTTGATAACT AGAATAGTGT 6540
ATGTTTCAGT TTCTTGGTAA GAGGTTAGAT GTGAAGTTCT TATAATACTA TAACAAAATA 6600
TACTTCATCA TTTGAGGGGT GGAGGAGATC TTACAAACAA ACTACTGAAC CCAATTCCCT 6660
TTTACTTTTA GTAACATCCC AATTTTGGTG TTGAGATATT GTGATAAGGT AAAGTTATAT 6720
ACTTTGGCCA AAGTAATTTA CAGGATGAAG CCTTCTTCTC CCTGATTAGT GACTTTCTGA 6780
GGTATTAACA TCAGAGTTCT GAAGATTATC CAAAAAAACA TCAGAGTTCT GAAATTTATT 6840
AGGGCCAGAG ATTTAATATT CTTGAAATCC TCAATTGCAG ATAATTATGA AATTTAAGAT 6900
CAAAATGAAA TATCCACAGG ATCACTTATT TATGACTAAA ATTTGTTTTT GGGGTGACAT 6960
TCTTCCACAT TATTAGAATG CACGTTGTGA TGACACTTGC TTTTTCTTGA TGGAAATGAT 7020
GACATGAATG CGCAGTGTCA AATCTACAAT TGAAAGGTAC AAAAAGGCAT GTGCAGATTC 7080
TTCCAACAAC GGGTCAGTTT CTGAAGCCAA TGCTCAGGTA TCATTTATCA GCTCTAACAA 7140
TTGTTTACAT GTGCAGATTC TTCCAACACT TTGTTATAAT CCTTTGTGTC CTACTGGTTT 7200
TTGGTTTTGG ATACTGATTA GTTTGTAATG TATGCACTAG GGCTGAAAAA AGGCATACAG 7260
AATTATGATA TTATAGAACA AAATTACCAA TTAACAGTAT TTTTCTTTCT TTTTTAATAA 7320
ATTACAGTAT AGTTTTTCGT GAATTTATGT GCGATCGAGT GTTTACACTG AATTTCAAAA 7380
TGTGCATGTA CGTTTTGAGG CTAGTGTAGA ACCACAGAAA GACAGTATAT ATGGAACTAC 7440
CAGCATATAA CAAAATCCTT TTTATGAAAT TTTATCGTCG ATGTTTTACA CTAAATTCTC 7500
2 O TCACTATTCA TTAACAGCGT AATTAACAAC ATGCTGTTAA ATTATAGAAG GAGTTCAAGC 7560
AATATTCCTA ACAATCATTT ATTGCCAATA TTTGTCAAAT ACCCTTTGTG ATAACCTCAT 7620
TTGTGTAAAA TCGATTAAAT ACATACCTAT TTAATTTTGC TTCTCAGAGT GGAGGTTTTT 7680
TCCTACTGCA TTGGGAGTCA TGAGTGTAAA CCTGCATTAT AGCCAGTTTT GTGTACAGAA 7740
ACCCTTTTCC TTCCTCTGTT GCTGTGGCCC TATTGTATCA ATTTATTTCC AGTTTGATTC 7800
GGTATTATAT ACATGTTTCC AAGAAGTATA AGAGAGAAAT GTACATCACT GATATTTTCT 7860
ACTTATATTT TGAGTTCTAA TCTGAACTCG AGGATCTTAA TCTAGTTATT TATAATGTTT 7920
TATTGCCTTT TGCTTTTGCA TTTCAGTTTT ATCAGCAAGA AGCTGCCAAG CTGCGCTCGC 7980
AAATTGGTAA TTTGCAGAAT TCAAACAGGT ATGATCATTT GTGATCTTGA TCAATTTGTT 8040
AGATAAAATT TGTTTTTCCT CTTCCAAACT CCGTTTAAGC AAATTAATTT TCAGGAATAT 8100
3 O GCTGGGTGAA TCACTTAGTG CATTGAGTGT GAAGGAACTT AAGAGCTTGG AGATAAAACT 8160
TGAGAAAGGA ATTGGTAGAA TTCGTTCGAA AAAGGTCTTT ATTCTAGTAC TCAAATGATT 8220
CTCTCTTTTT TTAAGTCAAA TATCACTTTA ATTTTCCTTG TATTGCCACT AACAAGTTTT 8280
GTTTTGTCTT GTTTTCCTTT TGTTTTTTAA TTCCTCCCTC AAACCTGCCA GAATGAGCTG 8340
TTGTTTGCTG AAATTGAGTA TATGCAGAAG AGGGTAACAA CTTTTGTGCT CATATTCACC 8400
- 72 -
63198-1218
CA 02227940 1999-OS-20
ATGACTTCTT CTATTTGAGA TAAAAAAATC AAGTTTTTGC CAATTTAATG ATCCTATGGT 8460
GAACCTCTTC TATTGTATTT TCACTCCAAA AATTTTCTTT GATTCATTGA ATGAAAATGC 8520
AAATTGCAGG AGATTGACTT GCACAACAAT AACCAGCTTC TCCGAGCAAA GGTCTTTCTA 8580
CTTATCTATT TATCAATGCC TTGTGTGTGT CTGAACTTGG ATCTTAATAT CTTAGATCGT 8640
TGGTGGGTTG TTTTTATTTA GTAAATATGA CACTACGTGG GGCTTATGTT GATGTTGCAG 8700
ATTGCAGAGA ATGAAAGAAA GCGACAGCAC ATGAATTTGA TGCCGGGAGG TGTCAACTTC 8760
GAGATCATGC AGTCTCAACC ATTTGACTCT CGGAACTATT CTCAAGTTAA TGGATTGCCG 8820
CCTGCCAATC ATTACCCTCA TGAAGACCAG CTCTTCAGTT AGTGTAAGTA TTTCCTTTGC 8880
AATGAGCTGT AGTTTTTCAT CAATTAATTA CTGATGAGCA TATAATTAAC TACTTTGATC 8940
TGGATGGGTT TCAGTAGCAG CAGCGGCTGA ATGGTTCGTG GTCTGTAAAA ATTTATTGGA 9000
AGGATATAAT AACTGATGCT GTGCCTTCTA ATTCTCATAA TCATTTGATC TTTCAATTAG 9060
TTAGATGATG ATTTACGCAT TCTTATTGAG ATTTTTACCA TTGGATGATA AGAGGGAATT 9120
GCAATATTTA GCTGTTGTAC TAAAAGTAGA CTGCTGTTAT CAGCACCCCA TGCTCACTGA 9180
AGAACTAGAA GATTACCCAA CCTAGTTTTA CTTCACTGAA CCGTTTGCAT GCAAGAACTT 9240
AAAGCGTAAT CTGATTTCCC AAGTGACAAG TATATGTTTC TAACTCCTTG ACGAATCTGC 9300
TGCTGATTCC TTTGCTGGTT TATATTATTC TTATGACTAC AAACACAATA CTTTTCAACT 9360
AGCTAGTGAA TGAATAATCA TTTCCTTATG TTGCAGTTAA AAAGCACCAA GTGCAGCAAC 9420
TCCTCGCATT TCCATATTCC ATGGAGAGTA CCTACTATTT CACTGAGCGC AAAAGCTGCA 9480
AGTACGCTAA AACAAAAATC TGAAGTAGCA TAACTCAAAT TTGTGCCGGT GGAGAGCCTA 9540
2 O GTACTCTTCC TCCATGTATT GCTTTTCCAG TCCCAGTTAA GACATAACAA ATGTCAGATA 9600
AGGATTTCTT TTCTGCATGT TTCATGAAGG CACTAAGATG CTGTGACAGT ACTTGTGACT 9660
AACTTATTAT ATATTTTGTC TTATATTTCT TATCTTTCAT CTTGTAATAT TTCTTCGCGT 9720
ATCTAGTATT GCTTTTCATT CAAACCCTTC CGTGACCCAG AATCAGGACC ACTGCCTTAG 9780
CATGCTGTTC ATCAGCGGTA CATGTAATAG AGGCCTCTAT ATTTTGCTGC CAGCTTAATA 9840
TACAGTTTAC ATCTTTCATG TGTGAGTTCA GCACGAGTAA TTAATTTTAT GGTTATTTTC 9900
TTTGTAACAG AGCCTCTTGA TGTCTATTTG TAAGCATTGC GAGGTTTTTA AAGATTAAAT 9960
TAATACGTAA GCTGAATGTC TCGCAAAAGG TACAAATTGC TTCAGCT 10007
3 O (2) INFORMATION FOR SEQ ID NO.: 14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1115
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
- 73 -
63198-1218
CA 02227940 1999-OS-20
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (99)..(815)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 14:
GAAACCCCAG AGCCAAAGAT CCTTACTTTC TCTCCTTAAT AACTACTATC TCTACATCCC 60
CTACTTTGGT TTATTTCCTC CCAAGCTAGG CAGCAGCT ATG GCA TAC CAA AAT GAA 116
Met Ala Tyr Gln Asn Glu
1 5
CCC CAA GAG AGC TCT CCC CTG AGG AAG CTG GGG AGG GGA AAG GTG GAG 164
Pro Gln Glu Ser Ser Pro Leu Arg Lys Leu Gly Arg Gly Lys Val Glu
10 15 20
ATC AAG CGG ATC GAG AAC ACC ACC AAT CGC CAA GTC ACT TTC TGC AAA 212
2 0 Ile Lys Arg Ile Glu Asn Thr Thr Asn Arg Gln Val Thr Phe Cys Lys
25 30 35
AGG CGG AAT GGT TTG CTC AAG AAA GCC TAT GAA TTA TCT GTT CTT TGC 260
Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys
40 45 50
GAT GCT GAG GTT GCA CTC ATC GTC TTC TCC AGC CGT GGA CGC CTT TAT 308
Asp Ala Glu Val Ala Leu Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr
55 60 65 70
GAG TAC TCT AAC AAT AGT GTC AAA TCT ACA ATT GAA AGG TAC AAA AAG 356
Glu Tyr Ser Asn Asn Ser Val Lys Ser Thr Ile Glu Arg Tyr Lys Lys
75 80 g5
GCA TGT GCA GAT TCT TCC AAC AAC GGG TCA GTT TCT GAA GCC AAT GCT 404
Ala Cys Ala Asp Ser Ser Asn Asn Gly Ser Val Ser Glu Ala Asn Ala
90 95 100
CAG TTC TAT CAG CAA GAA GCT GCC AAG CTG CGC TCG CAA ATT GGT AAT 452
4 0 Gln Phe Tyr Gln Gln Glu Ala Ala Lys Leu Arg Ser Gln Ile Gly Asn
105 110 115
TTG CAG AAT TCA AAC AGG AAT ATG CTG GGT GAA TCA CTT AGT GCA TTG 500
Leu Gln Asn Ser Asn Arg Asn Met Leu Gly Glu Ser Leu Ser Ala Leu
120 125 130
AGT GTG AAG GAA CTT AAG AGC TTG GAG ATA AAA CTT GAG AAA GGA ATT 548
Ser Val Lys Glu Leu Lys Ser Leu Glu Ile Lys Leu Glu Lys Gly Ile
135 140 145 150
GGT AGA ATT CGT TCG AAA AAG AAT GAG CTG TTG TTT GCT GAA ATT GAG 596
Gly Arg Ile Arg Ser Lys Lys Asn Glu Leu Leu Phe Ala Glu Ile Glu
155 160 165
TAT ATG CAG AAG AGG GAG ATT GAC TTG CAC AAC AAT AAC CAG CTT CTC 644
Tyr Met Gln Lys Arg Glu Ile Asp Leu His Asn Asn Asn Gln Leu Leu
170 175 180
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CGA GCA AAG ATT GCA GAG AAT GAA AGA AAG CGA CAG CAC ATG AAT TTG 692
Arg Ala Lys Ile Ala Glu Asn Glu Arg Lys Arg Gln His Met Asn Leu
185 190 195
ATG CCG GGA GGT GTC AAC TTC GAG ATC ATG CAG TCT CAA CCA TTT GAC 740
Met Pro Gly Gly Val Asn Phe Glu Ile Met Gln Ser Gln Pro Phe Asp .
200 205 210
TCT CGG AAC TAT TCT CAA GTT AAT GGA TTG CCG CCT GCC AAT CAT TAC 788
Ser Arg Asn Tyr Ser Gln Val Asn Gly Leu Pro Pro Ala Asn His Tyr
215 220 225 230
CCT CAT GAA GAC CAG CTC TTC AGT TAG TTTAAAAAGC ACCAAGTGCA 835
Pro His Glu Asp Gln Leu Phe Ser
235
GCAACTCCTC GCATTTCCAT ATTCCATGGA GAGTACCTAC TATTTCACTG AGCGCAAAAG 895
CTGCAAGTAC GCTAAAACAA AAATCTGAAG TAGCATAACT CAAATTTGTG CCGGTGGAGA 955
2 O GCCTAGTACT CTTCCTCCAT GTATTGCTTT TCCAGTCCCA GTTAAGACAT AACAAATGTC 1015
AGATAAGGAT TTCTTTTCTG CATGTTTCAT GAAGGCACTA AGATGCTGTG ACAGTACTTG 1075
TGACTAACTT ATTATATATT TTGTCTTATA TTTCTTAAAA 1115
(2) INFORMATION FOR SEQ ID NO.: 15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 714
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
30 (D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (1)..(714)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 15:
ATG GCA TAC CAA AAT GAA CCC CAA GAG AGC TCT CCC CTG AGG AAG CTG 48
Met Ala Tyr Gln Asn Glu Pro Gln Glu Ser Ser Pro Leu Arg Lys Leu
40 1 5 10 15
GGG AGG GGA AAG GTG GAG ATC AAG CGG ATC GAG AAC ACC ACC AAT CGC 96
Gly Arg Gly Lys Val Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn Arg
25 30
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CAA GTC ACT TTC TGC AAA AGG CGG AAT GGT TTG CTC AAG AAA GCC TAT 144
Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr
35 40 45
GAA TTA TCT GTT CTT TGC GAT GCT GAG GTT GCA CTC ATC GTC TTC TCC 192
Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe Ser
50 55 60
AGC CGT GGA CGC CTT TAT GAG TAC TCT AAC AAT AGT GTC AAA TCT ACA 240
Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn Asn Ser Val Lys Ser Thr
65 70 75 80
ATT GAA AGG TAC AAA AAG GCA TGT GCA GAT TCT TCC AAC AAC GGG TCA 288
Ile Glu Arg Tyr Lys Lys Ala Cys Ala Asp Ser Ser Asn Asn Gly Ser
85 90 95
GTT TCT GAA GCC AAT GCT CAG TTC TAT CAG CAA GAA GCT GCC AAG CTG 336
Val Ser Glu Ala Asn Ala Gln Phe Tyr Gln Gln Glu Ala Ala Lys Leu
2 0 100 105 110
CGC TCG CAA ATT GGT AAT TTG CAG AAT TCA AAC AGG AAT ATG CTG GGT 384
Arg Ser Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg Asn Met Leu Gly
115 120 125
GAA TCA CTT AGT GCA TTG AGT GTG AAG GAA CTT AAG AGC TTG GAG ATA 432
Glu Ser Leu Ser Ala Leu Ser Val Lys Glu Leu Lys Ser Leu Glu Ile
130 135 140
3 O AAA CTT GAG AAA GGA ATT GGT AGA ATT CGT TCG AAA AAG AAT GAG CTG 480
Lys Leu Glu Lys Gly Ile Gly Arg Ile Arg Ser Lys Lys Asn Glu Leu
145 150 155 160
TTG TTT GCT GAA ATT GAG TAT ATG CAG AAG AGG GAG ATT GAC TTG CAC 528
Leu Phe Ala Glu Ile Glu Tyr Met Gln Lys Arg Glu Ile Asp Leu His
165 170 175
AAC AAT AAC CAG CTT CTC CGA GCA AAG ATT GCA GAG AAT GAA AGA AAG 576
Asn Asn Asn Gln Leu Leu Arg Ala Lys Ile Ala Glu Asn Glu Arg Lys
4 0 180 185 190
CGA CAG CAC ATG AAT TTG ATG CCG GGA GGT GTC AAC TTC GAG ATC ATG 624
Arg Gln His Met Asn Leu Met Pro Gly Gly Val Asn Phe Glu Ile Met
195 200 205
CAG TCT CAA CCA TTT GAC TCT CGG AAC TAT TCT CAA GTT AAT GGA TTG 672
Gln Ser Gln Pro Phe Asp Ser Arg Asn Tyr Ser Gln Val Asn Gly Leu
210 215 220
50 CCG CCT GCC AAT CAT TAC CCT CAT GAA GAC CAG CTC TTC AGT 714
Pro Pro Ala Asn His Tyr Pro His Glu Asp Gln Leu Phe Ser
225 230 235
(2) INFORMATION FOR SEQ ID NO.: 16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 238
60 (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
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(ii) TYPE:
MOLECULE polypeptide
(v i) RIGINAL SOURCE:
O
(A) RGANISM:Po pulus lsamifera bsp.trichocarpa
O ba su
(x i) EQUENCE DESCRIPT ION:SEQID NO.:16:
S
Met AlaTyr GlnAsnGlu ProGlnGlu SerSer ProLeuArg LysLeu
1 5 10 15
Gly ArgGly LysValGlu IleLysArg IleGlu AsnThrThr AsnArg
20 25 30
Gln ValThr PheCysLys ArgArgAsn GlyLeu LeuLysLys AlaTyr
35 40 45
Glu LeuSer ValLeuCys AspAlaGlu ValAla LeuIleVal PheSer
50 55 60
Ser ArgGly ArgLeuTyr GluTyrSer AsnAsn SerValLys SerThr
65 70 75 80
2 Ile GluArg TyrLysLys AlaCysAla AspSer SerAsnAsn GlySer
0
85 90 95
Val SerGlu AlaAsnAla GlnPheTyr GlnGln GluAlaAla LysLeu
100 105 110
Arg SerGln IleGlyAsn LeuGlnAsn SerAsn ArgAsnMet LeuGly
115 120 125
Glu SerLeu SerAlaLeu SerValLys GluLeu LysSerLeu GluIle
30 130 135 140
Lys LeuGlu LysGlyIle GlyArgIle ArgSer LysLysAsn GluLeu
145 150 155 160
Leu PheAla GluIleGlu TyrMetGln LysArg GluIleAsp LeuHis
165 170 175
Asn AsnAsn GlnLeuLeu ArgAlaLys IleAla GluAsnGlu ArgLys
180 185 190
40
Arg GlnHis MetAsnLeu MetProGly GlyVal AsnPheGlu IleMet
195 200 205
Gln SerGln ProPheAsp SerArgAsn TyrSer GlnValAsn GlyLeu
210 215 220
Pro ProAla AsnHisTyr ProHisGlu AspGln LeuPheSer
225 230 235
(2) INFORMATION FOR SEQ ID NO.: 17:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
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(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence .
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 17:
ATGGGTCGTG GAAAGATTGA AATCAAG 27
(2) INFORMATION FOR SEQ ID NO.: 18:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
2 0 (A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 18:
ATTTGTGAAA AAGAGCTTTT ATATTTA 27
(2) INFORMATION FOR SEQ ID NO.: 19:
(i) SEQUENCE CHARACTERISTICS
3 O (A) LENGTH: 27
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
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(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence: .
oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 19:
AGGAAGGCGA AGTTCATGGG ATCCAAA 27
(2) INFORMATION FOR SEQ ID NO.: 20:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
2 0 (C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 20:
TCCACATCGA CAAAGAAGAT CTACGAT 27
(2) INFORMATION FOR SEQ ID NO.: 21:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
(B) TYPE: nucleic acid
3 O (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
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(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 21:
GTCACTTTCT GCAAAAGGCG CAGTGGT 27
(2) INFORMATION FOR SEQ ID NO.: 22:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 22:
AACTAACTGA AGGGCCATCT GATCTTG 27
(2) INFORMATION FOR SEQ ID NO.: 23:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
3 O (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
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oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 23:
ATGGAATATC AAAATGAATC CCTTGAG 27
(2) INFORMATION FOR SEQ ID NO.: 24:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 29
(B) TYPE: nucleic acid
1O (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 24:
ATTCATGCTC TGTCGCTTTC TTTCATTCT 29
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