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
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
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.
(1995a,b).
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
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
(Schmulling, 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., 1995a,b). The success
of genetic ablation
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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.
Details and applications
of antisense (Kooter, 1993; Mol et al., 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 et al., 1993; Matzke et al., 1993) technologies in plants have been
described in the
scientific literature. 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., 1995a). Genes that have been
characterized as acting
early in the development of floral structures include LEAFY (LFY) from
Arabidopsis (Weigel et
al., 1992), APETALA1 (AP1) from Arabidopsis (Mandel et al, 1992a,b), 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.,
1993). DEFICIENS
(DEF) is a floral homeotic gene from Antirrhinum that is expressed throughout
flower development
(Schwarz-Sommer et al. 1992).
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
(MCM1), the
floral homeotic genes AG and DEF, and human serum response 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 AP1 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
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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: AP1 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. ( 1996) 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, 1994; Purugganan et al., 1995; and Yanofsky, 1995. AG and DEF
have been
characterized as MADS box genes; while FLO and LFY 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 MADS 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 have 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 Torr. and Gray, also classified as P.
balsamifera subsp.
trichocarpa; Brayshaw, 1965) 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
3 5 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
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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 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
carpels in the center), the Populus flower apparently has only two whorls (a
reduced perianth cup
surrounding either stamens or carpels). 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
(Boes 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 DISCLOSURE
The present invention provides four floral homeotic genes from Populus
trichocarpa. The
four genes are herein termed PTLF, PTD, PTAG-1 and PTAG-2. These genes are
homologs of
floral homeotic genes isolated from other plant species. Specifically, PTLF 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, PTLF 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 carpel 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 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 PTLF, 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
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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 PTLF, PTD, PTAG-1 or PTAG-2 nucleic acid
sequences, and may be
longer, comprising at least 20, 25, 50, or 100 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 transgenic plants. Thus, in one aspect, such molecules are useful to
monitor the expression
of transgenes comprising some portion of the PTD, PTLF, 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, PTLF,
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 stringent conditions 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.
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
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of PTD, PTLF, PTAG-I 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 promoter
sequence is a promoter sequence from PTD, PTLF, 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. The sequence
comprises the following regions:
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Nucleotide numbers Feature
1-1872 5' regulatory region
1752-1756 probable CAAT box
1782-1786 probable CAAT box
1845-1851 probable TATA box
1873-2188 Exon 1 (including inferred
5' UTR)
2189-2327 Intron 1
2328-2394 Exon 2
2395-2484 Intron 2
2485-2546 Exon 3
2547-2652 Intron 3
2653-2752 Exon 4
2753-3309 Intron 4
3310-3351 Exon 5
3352-3432 Intron 5
3433-3477 Exon 6
3478-3584 Intron 6
3585-4000 Exon 7
3765-4285 3' regulatory region (including
3' UTR)
3765-4000 3' UTR
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. The
sequence
comprises the following regions:
Amino Acid numbers Feature
1-57 MADS domain
87-154 K-domain
Seq. LD. No. 5 shows the nucleic acid sequence of the PTLF gene. The sequence
comprises the following regions:
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Nucleotide numbers Feature
1-2638 5' regulatory region
2477-2481 probable CAAT box
2536-2542 probable TATA box
2568-2574 probable TATA box
2628-3074 Exon 1
3075-3655 Intron 1
3656-3990 Exon 2
3991-4679 Intron 2
4680-5197 Exon 3
5043-5197 3' UTR
5043-5656 3' regulatory region (including
3' UTR)
Seq. LD. No. 6 shows the nucleic acid sequence of the PTLF cDNA
Seq. LD. No. 7 shows the nucleic acid sequence of the PTLF ORF.
Seq. LD. No. 8 shows the amino acid sequence of the PTLF polypeptide.
Seq. LD. No. 9 shows the nucleic acid sequence of the PTAG-1 gene. The
sequence
comprises the following regions:
Nucleotide numbers Feature
1-2410 5' regulatory region
2411-2588 Exon 1
2589-3056 Intron 1
3057-3296 Exon 2
3297-8161 Intron 2
8162-8243 Exon 3
8244-8894 Intron 3
8895-8956 Exon 4
8957-9041 Intron 4
9042-9141 Exon 5
9142-9284 Intron 5
9285-9326 Exon 6
9327-9529 Intron 6
9530-9571 Exon 7
9572-971 I Intron 7
9712-9878 Exon 8
9879-10930 Intron 8
10931-11215 Exon 9
10935-I 1485 3' regulatory region (including
3' UTR)
10935-11215 3' UTR
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. The
sequence comprises the following regions:
Amino Acid numbers Feature
17-72 MADS domain
106-172 K-domain
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Seq. LD. No. 13 shows the nucleic acid sequence of the PTAG-2 gene. The
sequence
comprises the following regions:
Nucleotide numbers Feature
I-2336 5' regulatory region
21 I 8-2122 probable CAAT box
2256-2262 probable TATA box
2337-2421 Exon 1
2422-2913 Intron 1
2914-3153 Exon 2
3154-7035 Intron 2
7036-7117 Exon 3
7118-7946 Intron 3
7947-8008 Exon 4
8009-8094 Intron 4
8095-8194 Exon 5
8195-8331 Intron 5
8332-8373 Exon 6
8374-8529 Intron 6
8530-8571 Exon 7
8572-8700 Intron 7
8701-8863 Exon 8
8864-9396 Intron 8
9397-9691 Exon 9
8863-10007 3' regulatory region (including
3' UTR)
8863-8863 joined to 9397-96913' UTR
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. The
sequence comprises the following regions:
Amino Acid numbers Feature
16-72 MADS domain
3 5 106-172 K-domain
Seq. LD. Nos. 17-24 show oligonucleotide primers that may be used to amplify
portions
of the disclosed floral homeotic nucleic acid sequences.
DETAILED DESCRIPTION
I. Definitions and Abbreviations
Unless otherwise noted, technical terms are used according to conventional
usage.
Definitions of common terms in molecular biology may be found in Benjamin
Lewin, Genes V,
published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et
al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell 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).
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In order to facilitate review of the various embodiments of the invention, the
following
definitions of terms are provided:
Isolated: An "isolated" biological component (such as a nucleic acid or
protein or
organelle) has been substantially separated or purified away from other
biological components in
the cell of the organism in which the component naturally occurs, i.e., other
chromosomal and
extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and
proteins that have
been "isolated" include nucleic acids and proteins purified by standard
purification methods. The
term also embraces nucleic acids and proteins 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). cDNA is synthesized in the laboratory by reverse transcription from
messenger RNA
extracted from cells.
Oligonucleotide: A linear polynucleotide sequence of up to about 100
nucleotide bases in
length.
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 coding sequence.
Generally, operably
linked DNA sequences are contiguous and, where necessary to join two protein-
coding regions, in
the same reading frame.
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.
Ortbolog: Two nucleotide or amino acid 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. Orthologous sequences are also homologous sequences.
Probes and primers: Molecules useful as nucleic acid probes and primers may
readily be
prepared based on the nucleic acids provided by this invention. Typically, but
not necessarily, such
molecules are oligonucleotides, i.e., linear nucleic acid molecules of up to
about 100 nucleotides
bases in length. However, longer nucleic acid molecules, up to and including
the full length of a
particular floral homeotic gene may also be employed for such purposes.
A nucleic acid probe comprises at least one copy (and typically many copies)
of an isolated
nucleic acid molecule of known sequence that is used in a nucleic acid
hybridization protocol.
Generally (but not always) the nucleic acid molecule is 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).
Primers are short nucleic acids, usually DNA oligonucleotides 8-10 nucleotides
or more in
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length, and more typically 15-25 nucleotides 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.
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, O 1991, Whitehead Institute for Biomedical
Research, Cambridge, MA).
One of skill in the art will appreciate that the specificity of a particular
probe or primer increases
with its length. Thus, for example, a primer comprising 20 consecutive
nucleotides of the cDNA
disclosed in Seq. LD. No. 2 will anneal to a target sequence such as a
homologous sequence in
Eucalyptus contained within a Eucalyptus cDNA library with a higher
specificity than a
corresponding primer of only 15 nucleotides. Thus, in order to obtain greater
specificity, probes and
primers may be selected that comprise 20, 25, 30, 35, 40, 50, 75, 100 or more
consecutive nucleotides
of the disclosed nucleic acid sequences.
The invention thus includes isolated nucleic acid molecules that comprise
specified
lengths of the disclosed floral homeotic sequences. Such molecules may
comprise at least 8-10, 15,
20, 25, 30, 35, 40, 50, 75, or 100 consecutive nucleotides of these sequences
and may be obtained
from any region of the disclosed sequences. By way of example, the floral
homeotic genes shown
in the Sequence Listing may be apportioned into halves or quarters based on
sequence length, and
the isolated nucleic acid molecules may be derived from the first or second
halves of the molecules,
or any of the four quarters. The PTD cDNA, shown in Seq. LD. No. 2 may be used
to illustrate
this. This cDNA is 924 nucleotides in length and so may be hypothetically
divided into halves
(nucleotides 1-462 and 463-924) or quarters (nucleotides 1-231, 232-462, 463-
693 and 694-924).
Nucleic acid molecules may be selected that comprise at least 8-10, 15, 20,
25, 30, 35, 40, 50, 75 or
100 consecutive nucleotides of any of these portions of the floral homeotic
genes. Thus, one such
nucleic acid molecule might comprise at least 25 consecutive nucleotides of
the region comprising
nucleotides 1-924 of the disclosed floral homeotic genes.
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. Generally, a
preparation of a floral homeotic protein is purified such that the floral
homeotic protein represents
at least 5% of the total protein content of the preparation. For particular
applications, higher purity
may be desired, such that preparations in which the floral homeotic protein
represents at least 50%
or at least 75% of the total protein content may be employed.
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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.
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 naked DNA 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 that contain the
introduced transgene (whether
produced sexually or asexually).
Vector: A nucleic acid molecule as introduced into a host cell, thereby
producing a
transformed host cell. A vector may include nucleic acid sequences that permit
it to replicate in a
host cell, such as an origin of replication. A vector may also include one or
more selectable marker
genes and other genetic elements known in the art.
Sequence identity: the relatedness of two nucleic acid sequences, or two amino
acid
sequences is typically expressed in terms of the identity between the
sequences (in the case of
amino acid sequences, similarity is an alternative assessment). Sequence
identity is frequently
measured in terms of percentage identity; the higher the percentage, the more
similar the two
sequences are. Homologs of a disclosed floral homeotic protein or nucleic acid
sequence will
possess a relatively high degree of sequence identity when aligned using
standard methods.
Methods of alignment of sequences for comparison are well known in the art.
Various
programs and alignment algorithms are described in: Smith and Waterman (1981);
Needleman and
Wunsch (1970); Pearson and Lipman (1988); Higgins and Sharp (1988); Higgins
and Sharp (1989);
Corpet et al. ( 1988); Huang et al. ( 1992); and Pearson et al. ( 1994).
Altschul et al. ( 1994) presents
a detailed consideration of sequence alignment methods and homology
calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) is
available from several sources, including the National Center for
Biotechnology Information
(NCBI, Bethesda, MD) and on the Internet, for use in connection with the
sequence analysis
programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at
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http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence
identity using
this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast
help.html.
Homologs of the disclosed floral homeotic proteins are typically characterized
by possession
of at least 50% sequence identity counted over the full length alignment with
the amino acid sequence
of a selected floral homeotic protein using the NCBI Blast 2.0, gapped blastp
set to default
parameters. Proteins with even greater similarity to the reference sequences
will show increasing
percentage identities when assessed by this method, such as at least 60%, at
least 65%, at least 70%,
at least 75%, at least 80%, at least 90% or at least 95% sequence identity.
When less than the entire
sequence is being compared for sequence identity, homologs will typically
possess at least 75%
sequence identity over short windows of 10-20 amino acids, and may possess
sequence identities of at
least 85% or at least 90% or 95% depending on their similarity to the
reference sequence. Methods
for determining sequence identity over such short windows are described at
http://www.ncbi.nlm.nih.gov/BLAST/blast FAQs.html. One of skill in the art
will appreciate that
these sequence identity ranges are provided for guidance only; it is entirely
possible that strongly
significant homologs could be obtained that fall outside of the ranges
provided. The present
invention provides not only the peptide homologs as described above, but also
nucleic acid molecules
that encode such homologs.
Homologs of the disclosed floral homeotic nucleic acids are typically
characterized by
possession of at least 50% sequence identity counted over the full length
alignment with the nucleic
acid sequence of a selected floral homeotic gene using the NCBI Blast 2.0,
blastn set to default
parameters. Homologs with even greater similarity to the reference sequences
will show increasing
percentage identities when assessed by this method, such as at least 60%, at
least 65%, at least
70%, at least 75%, at least 80%, at least 90% or at least 95% sequence
identity.
An alternative indication that two nucleic acid molecules are closely related
is that the two
molecules hybridize to each other under stringent conditions. Stringent
conditions are sequence
dependent and are different under different environmental parameters.
Generally, stringent
conditions are selected to be about 5°C to 20°C lower than the
thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined
ionic strength and pH) at which 50% of the target sequence hybridizes to a
perfectly matched
probe. Conditions for nucleic acid hybridization and calculation of
stringencies can be found in
Sambrook et al. (1989) and Tijssen (1993). Nucleic acid molecules that
hybridize under stringent
conditions to a disclosed nucleic acid sequences will typically hybridize to a
probe corresponding
to either the entire cDNA or selected portions of the cDNA under wash
conditions of 0.2x SSC,
0.1% SDS at 65°C.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless
encode similar amino acid sequences, due to the degeneracy of the genetic
code. It is understood
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that changes in nucleic acid sequence can be made using this degeneracy to
produce multiple
nucleic acid sequence that all encode substantially the same protein.
Floral Specific Promoter: As used herein, the term "floral specific promoter"
refers to a
regulatory sequence which confers gene expression only in, or predominantly
in, floral tissues.
The complete sequences of four floral specific promoters are disclosed herein:
the promoter of
PTD, located within the 5' regulatory region comprising nucleotides 1-1872 of
Seq. LD. No. 1; the
promoter of PTFL, located within the 5' regulatory region comprising
nucleotides 1-2638 of Seq.
LD. No. 5; the promoter of PTAG-1, located within the 5' regulatory region
comprising 1-2410 of
Seq. LD. No. 9; and the promoter of PTAG-2, located within the 5' regulatory
region comprising
nucleotides 1-2336 of Seq. LD. No. 13). Accordingly, these promoter sequences
may be used to
produce transgene constructs that are specifically or predominantly expressed
in floral tissues. One
of skill in the art will recognize that effective floral-specific expression
may be achieved with less
than the entire promoter sequences noted above. Thus, by way of example,
floral-specific
expression may be obtained by employing sequences comprising 500 nucleotides
or fewer (e.g.,
250, 200, 150, or 100 nucleotides) upstream of the start codon, AUG, of the
disclosed gene
sequences.
The determination of whether a particular sub-region of the disclosed
sequences operates
to confer floral specific expression in a particular system (taking into
account the plant species into
which the construct is being introduced, the level of expression required,
etc.), is preformed using
known methods, such as operably linking the promoter sub-region to a marker
gene (e.g. GUS),
introducing such constructs into plants and then determining the level of
expression of the marker
gene in floral and other plant tissues. Sub-regions which confer only or
predominantly floral
expression, are considered to contain the necessary elements to confer floral
specific expression.
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.
(1987) and Innis et al. (1990).
III. Isolation and Characterization of PTLF
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
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(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 MgCl2, 100 ItM each dNTP, 4 Itg primer XT, 10 ltCi
[a32P]-dCTP, and
200 U M-MLV reverse transcriptase (Gibco BRL) in 50 ltL. Incubations were
performed at 37°C
for I hr, then the cDNA was purified with GeneClean (BIO101 ) silica matrix.
Typical yields were
10-40 ng of cDNA, as determined by'ZP incorporation. The size ranges of the
cDNA samples were
characterized 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/1tL
in 10 mM
TrisHCl, I mM EDTA (pH 8.0) and stored at -20°C.
cDNA libraries were prepared using the Lambda-ZAP CDNA cloning kit
(Stratagene).
From 5 ltg 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
FLOlLFYhomolog was obtained by touchdown PCR (Don et al., 1991) ofthe 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, 30 sec; 58°C, 30 sec; 71°C, 1 min) x 2,
(94°C, 30 sec; 56°C, 30 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 PTLF genomic clone was isolated by screening the genomic library using
probes
derived from the PTLF 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, 1984). 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 PTLF 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/Entrez/nucleotide.html)
under accession
number U93196 and is shown in Seq. LD. No. 5. The positions of the two introns
found in both
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FLO and LFY are conserved in PTLF. 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 deduced PTLF amino acid sequence
with several
FLO/LFY homologs revealed conserved amino- and carboxyl-terminal domains (133
and 175
residues, respectively, in PTLF) linked by a poorly conserved, highly charged
domain (69
residues). The overall sequence identity between PTLF and FLO (Coen et al.,
1990) is 79%, with
88% amino acid sequence similarity.
Due to the limited seasonal availability of inflorescence and flower tissue,
and the
difficulty of obtaining large amounts of developing meristems, the levels of
PTLF expression were
compared using RT-PCR. PTLF was detected most strongly in developing
inflorescences, with no
significant differences between samples from male and female trees.
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 Itm in thickness. Probes were generated from a
plasmid
consisting of the PTLF cDNA inserted between the EcoRI and Kpn I sites of the
vector
pBluescriptII SK (-), and were not alkaline hydrolyzed. A PTLF antisense probe
hybridized
strongly to the floral meristems and developing flowers of both male and
female plants. PTLF 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. PTLF 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 PTLF cDNA were produced for
analysis in
transgenic trees. The insert from the cDNA clone of PTLF 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 (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).
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Although overexpression of LFY in aspens was reported to result in short,
bushy plants
that flower within a year (Weigel and Nilsson, 1995), no such obvious
phenotypes were seen with
PTLF. During more than one year of growth in soil in a greenhouse, and an
additional year at a
field site in Corvallis, OR, few differences were noted for any of the
transgenics relative to control
plants.
IV. 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 AGL 1. 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. The MADS domain extends over amino acids 1-57, while the K-domain
extends over
amino acids 87-154. The MADS domain is 93% conserved with respect to DEF,
whereas the K
domain is 85% conserved at the amino acid level.
To determine if the promoter of PTD would confer the floral-specific
expression, 1.9 kb of
its promotor and 5' untranslated region were fused to a GUS-intron reporter
gene, and introduced
into Arabidopsis, tobacco and poplar. GUS expression was observed in floral
tissues including
petals and stamens. This expression pattern is characteristic of a "B
function" gene like
APETALA3, suggesting that PTD has retained the regulatory motifs (i.e.
sequence patterns) that
direct it to stamens and petals (though poplar has no true petals). No
vegetative GUS expression
was observed, except in poplar, where vegetative expression was confined to
leaf like structures
subtending induced floral structures.
V. Isolation and Characterization of PTAG-1 and PTAG-2
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-1 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 17-72 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
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acids in length (Seq. LD. No. 16). The PTAG-2 polypeptide contains both a MADS
domain and a
K-domain. The MADS domain extends from amino acids 16-72 and the K-domain from
amino
acids 106-172. The PTAG-2 nucleotide and amino acid sequences are available on
GenBank under
accession number AF052571.
Like AG (Yanofsky et al., 1990), both PTAG 1 and PTAG2 contain 8 introns at
conserved
positions. All introns have canonical donor (GT) and acceptor (AG) sites.
At the amino acid level, PTAG-1 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. Northern analysis of PTAG gene expression in populus revealed
that transcripts are
present in immature and mature flowers from both male and female trees. In
addition, low levels of
PTAG gene expression are present in all vegetative tissues tested.
Interestingly, the size of the
transcripts from the vegetative tissues are shorter (~--150-200 bp) than the
floral transcripts. This
size difference is not due to alternate intron/exon splicing.
EXAMPLES
The following examples are provided to illustrate the scope of the invention.
Example 1
Preferred Method of Making the Populus Genes and cDNAs
With the provision of the four Populus floral homeotic nucleic acid sequences
PTD,
PTLF, 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 cDNA or gene,
or the entire cDNA or genes. Variations in amplification conditions may be
required to
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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.
( 1987). 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 primers:
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)
1 O 5' AGGAAGGCGAAGTTCATGGGATCCAAA 3' (Seq. LD. No. 19)
A derivative version of the PTD ORF that lacks the MADS 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
PTLF 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, PTLF, 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
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, PTLF, PTAG-1 or PTAG-2 gene
or cDNA
sequences may be used.
Example 2
Use of the Populus Genes and cDNAs to Modify Fertility 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
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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 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);
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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);
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, PTLF, 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, PTLF, 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 one
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, Seguoia,
Abies, Thuja,
Libocedrus, Chamaecyparis and Larix. In particular, members of the genera
Eucalyptus, Acacia
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and Gmelina, which are becoming 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 transfection 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 (CaMV) 35S
promoter, which confers
constitutive, high-level expression in most plant tissues (see, e.g., Odell et
al., 1994, Dekeyser et
al., 1990, Terada and 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, Schaffner 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 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, P'TLF, 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'-
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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 transfotmants. 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.
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
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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. 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.
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 function in the same manner as the
non-mutated form of the
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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 PTLF, 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:
RNases, such as
barnase from Bacillus amyloliquefaciens and RNase-T1 from Aspergillus (Mariani
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); DTA (diphtheria toxin A) (Pappenheimer,
1977) and
glucanase (Worrall et al., 1992).
d. 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 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.
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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 ofA.
tumefaciens (Settler, 1993; Strauss et al., 1995a,b), 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 polymerise chain reaction (RT-PCR).
Example 3
Introduction of antisense PTD cDNA into hybrid aspens
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 PTAG-1 sequences in plants
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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 MADS box domain is deleted is amplified using the
following primer
combination:
S 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 pDW151 (Weigel and Nilsson,
1995) and the
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 Sequence Variants
As noted above, modification of the activity of floral homeotic polypeptides
such as PTD,
PTLF, PTAG-1 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 art and are described in Innis et al. (1990).
As noted, variant DNA molecules also include those created by DNA genetic
engineering
techniques, for example, M 13 primer mutagenesis. Details of these techniques
are provided in
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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.
Nucleic acid molecules and proteins that are variants of those disclosed
herein may be
identified by the degree of sequence identity that they share with a nucleic
acid molecule or protein
disclosed herein. Typically, such variants share at least 50% sequence
identity with a disclosed
nucleic acid or protein, as determined by the methods described above for
homologs.
Alternatively, for nucleic acid molecules, variants may be identified by their
ability to hybridize to
a disclosed sequence under stringent conditions, as described above.
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 32nd amino acid
residue of the
Poplar PTD protein shown in Seq. LD. No. 4 is alanine. This is encoded in the
Poplar PTD open
reading frame by the nucleotide codon triplet GCC. Because of the degeneracy
of the genetic code,
three other nucleotide codon triplets: GCT, GCA and GCG, also code for
alanine. Thus, the
nucleotide sequence of the Poplar PTD 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. Thus, this
invention also
encompasses nucleic acid sequences which encode a floral homeotic protein but
which vary from
the disclosed nucleic acid sequences by virtue of the degeneracy of the
genetic code.
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 Poplar floral homeotic proteins,
yet which proteins are
clearly derivative of these proteins. Newly derived proteins may also be
selected in order to obtain
variations on the characteristic of the Poplar floral homeotic proteins. Such
derivatives include
those with variations in amino acid sequence including minor 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.
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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 30
residues. Deletions or insertions preferably are made in adjacent pairs, i.e.,
a deletion of two
residues or insertion of two residues. Substitutions, deletions, insertions or
any combination
thereof may be combined to arrive at a final construct. Obviously, 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 typically regarded as conservative
substitutions.
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Table 1
Original Residue Conservative Substitutions
Ala ser
Arg lys
Asn gln; his
Asp glu
Cys ser
G In asn
G lu asp
Gly pro
His asn; gln
Ile leu, val
Leu ile; val
Lys arg; gln; glu
Met leu; ile
Phe met; leu; tyr
Ser thr
Thr ser
Trp tyr
Tyr trp; phe
Val ile; leu
Substantial changes in transcription factor 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., Beryl 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 aspartyl; 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.
Homologous polypeptides that share at least 50% amino acid sequence identity
to the
disclosed PTD, PTLF, PTAG-1 or PTAG-2 amino acid sequences as determined using
BLAST 2.0,
gapped blastp, with default parameters, are encompassed by this invention.
Homologs with even
greater similarity to the reference sequences will show increasing percentage
identities when
assessed by this method, such as at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%,
at least 90% or at least 95% sequence identity. Such homologous peptides are
preferably at least 10
amino acids in length, and more preferably at least 25 or 50 amino acids in
length. When less than
the entire sequence is being compared for sequence identity, homologs will
typically possess at
least 75% sequence identity over short windows of 10-20 amino acids, and may
possess sequence
identities of at least 85% or at least 90% or 95% depending on their
similarity to the reference
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sequence. Also encompassed by the present invention are the nucleic acid
sequences that encode
these homologous peptides.
Similarly, homologous nucleic acids that share at least 50% nucleotide
identity to the
disclosed PTD, PTLF, PTAG-1 or PTAG-2 nucleic acid sequences as determined
using BLAST
2.0, gapped blastn, with default parameters, are encompassed by this
invention. Homologs with
even greater similarity to the reference sequences will show increasing
percentage identities when
assessed by this method, such as at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%,
at least 90% or at least 95% sequence identity. 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 Sambrook 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 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)
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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 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 floral 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 (MHC) 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
32
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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, I5, 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 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.
33
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Date of Deposit: October 1, 1999
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38
CA 02319853 2000-12-22
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
2 (B) COMPUTER: IBM PC compatible
0
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,319,853
(B) FILING DATE: 02-OCT-2000
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
3 (viii) ATTORNEY/AGENT INFORMATION:
O
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 63198-1293
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID NO.: 1:
4 O (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 4285
(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:
AAGCTTGTCA GACCCAACAA ATATGGACCT GATATGCTTG TCATACCAAC TTAAACTCGA 60
50 GTCAGATATA TTTAATATTA TTATTCATAT TATTAATATA ATTATTAATA AATTTGAAAA 120
AATATTATTA CTATCGAAAA AAACTATAAA TTTGATTTGA ATGATAAAAT TAAAAATTAA 180
AAATATTTTT ATTATCATTA TTGTTAATAT AATTATTAAA AAATTTAAAA AATATATTAT 240
TACTATTGAG AAAAACCATA ACTGTTTATG CGACATTTTA TGTCATGGAA AATGAGCTGA 300
AAAAAACCAA TAAAAAGAAA AAAACTAATG AAAAAAAAGA AAAAAAAATA TGAATTAACT 360
GGGTTAACCC TTGAAACCAG GTTACCCCGT CAAACCTTGG ATTCGTGTCG TGAAAGTTTG 420
TTAACTAAAT AGAAAAAAAA AATTGACGGG TTACCCAGAA TTAACTGGGC TAACCCGTCA 480
AACCAGGTTA CCTATCAAAC CCGGGATCCG TGTCATGAAA GTTTGATAAC TAAATAGAAA 540
ACAATTGAAC ATTAACAACC TAAATTAAAC GAAAAAAATT AATTAAAAAC AAGAAAACAA 600
AAACAAACAA AAAACATAAG CATGTTAGTA ATGAGGAAAA AGAAAAAAAA TTTGATTCAA 660
60 CTGAGTTAAC CCGTCAAACC CGGGATTCGC GTCATGAAAG TTTGATAACT AAATAGAAAA 720
AAAAATCGAC GGGTTAAACG AAAAAAAATT AACAAACTAA ACTAAACAAA AAAAAATTGA 780
TTAAAAAGGA AAAAAGCAAA AAAAATAATT TCGGGTTAAC TCATCAAACC AGGTTAACCC 840
GTCAAACCCG AGATCCGTGT CATGAAAGTC TGATAACTAA ATAATTTTTT TTTTCACATT 900
AACAAACTAA ATTAAACAAA AAAAATTCAT TAAAAGAAAA AAAAACACAA AGAAAAAAGC 960
39
CA 02319853 2000-12-22
AAAAAAAAACCTATAATAGCATAAATAAATAAATAAAAACAGGAAAAAAATTTTTTAAAA1020
AAAACCTTTCAATCACTAATACATAGAAGGTGTGGGGAAAGCCACAGTGATTTCCCCGTA1080
CCTTTTAAAGTATTACTTAATATATAGGTGAATTTAATTGACCGTCACGAAAAAGACTAT1140
TCTGGCTTCCTCTTACAATGGACGCTATCTAAATTCAAATACTTTGAAAAAAGATTTAAT1200
CCTGTAACCTTCTTTCGTTTTTTTATGCCTTCAATCCATCTATTTATTGTTTTTATGATT1260
TTTCTTAGATACAAAAGAGCATATTTTAAAGAAGAAAAAAATAAGCTAAGCACCTCAAGT1320
TTTGATTTTTTTTTTATTTTGCAGCCAATTTTTTAAATATTAAAATTTTCATAATAGATC1380
AAAGGATAATTCAAAATTGCATCCAAATAACAACATTAGTAATGGAAGGACTTATGGTAT1440
GAATGGATCAATAATATAAGGGCTGAATTAACAACATTTTTTTTATTTAGATCCTGTTTA1500
TTTTTACGTTTTAAAAATATTTTTGAAATTATTTTATTTTTTATTATAAATTAATATTTT1560
TAGATCATTTTAATACGTTAATATAAAAAATAATTTTTTTAAAAAAATTTATTTTAATAT1620
ATTTTTTAAAAATAATATTTAAAAAAACAATCATAACAATATTCTCATTACCTAACACAG1680
TCATGGAACAGGAATGAGAAAAGGTCTTATCAGTAAATTGCTTGCATGTCATGTCAAGGT1740
GTATGAACCTCCCAATACTTCTCACGCTACCCTTCAGAAATCCAATCTCAGAAGCCACAG1800
ACAATCTAAGTTACGCTACAATCAACTTTCCATCACCCTTTCCTTATTTAGAAACTCCAC1860
TTAATCACATTTCACCCTTTTTCATCATCTTCTCTTTCCCTTCAAGAAGCCTAGGTACTG1920
TGCAAGAAACCCTTATCTCTCCCCCTCAGTATTTACTTTTGTTTAGTGCTACAGCTTTCA1980
CAAAGAAGTAAGGAAAAAATATGGGTCGTGGAAAGATTGAAATCAAGAAGATCGAAAACC2040
CCACAAACAGGCAAGTCACCTACTCGAAGAGAAGAAATGGTATTTTCAAGAAAGCCCAAG2100
2 AACTCACTGTACTTTGTGATGCTAAGGTCTCTCTTATCATGTTCTCCAACACTAACAAAC2160
O
TCAATGAGTACATTAGCCCCTCCACATCGTACGTATACTCGTATCATGTTTCTGGCTAAG2220
TATTTCTTCCGTGCTTTCTCTTCTTTCTTTCTTTTCTTGTCTTTTATGTTGCAGTTTTAT2280
GAAACCTTGGTAATGGAACCGTAGTTTTTATTGTTAATTATGACCAGGACAAAGAAGATC2340
TACGATCAATATCAGAACGCTTTAGGCATAGATCTGTGGGGCACTCAATACGAGGTTAAC2400
CTTTCTTTTCTGTCTTTCTTCTAATGTTTGATCTATAGGACGAATATGAGATTCTTCAAA2460
GGATTTTGTTTGTGAGGTTTGCAGAAAATGCAAGAGCACTTGAGGAAGCTGAATGATATC2520
AATCATAAGCTGAGACAAGAAATCAGGTAACTTCAAAAGAAATAACCTTCGCATATATGC2580
ATGTGGTTATGGTTTTTATGGGAATATCTGTAAATTTGTGGAGCTACTAATTAAGGTATT2640
TGTTTTTAACAGGCAGAGGAGAGGAGAGGGCCTGAATGATCTGAGCATTGATCATCTGCG2700
3 CGGTCTTGAGCAACATATGACTGAAGCCTTGAATGGTGTGCGTGGCAGGAAGGTCAGATG2760
O
TTTTCAAGTGAACATCTTTATATAATTATCAAGTTCTAATTCCTAAAATTTGAGCTTACT2820
AGTAATTTGAGTTCGGTCCGGTGTATCAAGCAGGTTAATCTAGATCTAGTTTTTTTTCCT2880
TACCAAATCAAAGTCATTTTGAGGATTTTTTAATAAAAAATATTGAATTTTGAATCAACT2940
TATACAAATTCATCAATCTACAACTCGAATCTTACATTTAATCAAACTTTCAAATTAGAT3000
CTTATAAATATGATATTAACCGGTCGGTGTTTTATGTACATTAATATTATGTTTTAGTTG3060
AACTCTTTTATCATTTTTTTTTTTTAAATTTGAGTTATTTTAATCCTTATCAATTTTTAT3120
CATTTTGGGATTCTTGGAAACCCTGGTTAGAAAGAAAATACACACCCTTGAACTTGTGCT3180
TCTTTACCTTTGCATTATGGATTTTCATGAACTGGATTTTGGGTAACCCTTAACCTCATC3240
TATAGAAGGGATATGCCTTGTAATTAACACTTTACACTTACAAGTTCAACATTCTTTGAT3300
4 TATTTACAGTACCATGTGATCAAAACACAAAACGAAACCTACAGGAAGAAGGTTAGTGAT3360
O
AAAAAGAACATTTTACCTCTTCAATTTCATGCATGTAGCTTTTGGAACAAATTCTCTGGC3420
GATTAATTGCAGGTGAAGAATTTAGAGGAGAGACATGGAAACCTCTTGATGGAATATGTA3480
AGAATCTAAATTTTCATGTGCTTGTTTTCGCTAATTTTCCAACTTGGAAAAACACATGGA3540
TTAAACCTGAGATTTTTTTTTTCTTTTGTGCTTTGGGATTTAAGGAAGCAAAACTAGAGG3600
ATCGACAGTATGGTTTAGTGGACAATGAAGCTGCTGTTGCACTTGCAAATGGGGCTTCCA3660
ACCTCTATGCATTCCGCCTGCATCACGGGCACAACCACCACCACCATCTCCCTAATCTTC3720
ACCTTGGAGATGGATTTGGAGCCCATGAACTTCGCCTTCCTTGAGTGGTGCTTGAGGTCG3780
ACCTTCCAGCTCTTCAGACATCTTATCTAAATGCGTGTGCTAACTAGAGATGCTATCTAA3840
TATTATTTAATAATTAATTAAGAGCCCGGAAGTAAAAAATACTTTCATAGATTGTAATTT3900
50 ACCTCAGGGTAATGTGTATGGCAGCATATTAGATTGTGATTTGAGCAAGGAATGTCATTC3960
CTTATGGATTAATTAAATATAAAAGCTCTTTTTCACAAATATAATTCCACTTGGAGTAGC4020
ATTCTGCAATATCCCATATGATCTGCAGGCTTAATAATTATATGATTGAAATGTGTTGGA4080
TCAACCGTCATATGTATGTATGTATGTATGTATGTATACGTATGTGTATACTAGGGAGTC4140
AACAACACAGGGGGTGTAAGCACCAAATGCATTATCCACTGTTTTTGCCCAAACCCCATT4200
TGGCATAGGTCGACAATACCATACCAATGCCTCCGAAGCCATCCTTCCCCGCCGCCCTAC4260
ACAAACCAAAACCGCTGAATTCCTG 4285
(2) INFORMATION FOR SEQ ID NO.: 2:
60 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 946
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
CA 02319853 2000-12-22
(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:
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 ATG TTC 144
Gln Glu Leu Thr Val Leu Cys Asp Ala Lys Val Ser Leu Ile Met Phe
35 40 45
2 O TCC AAC ACT AAC AAA CTC AAT GAG TAC ATT AGC CCC TCC ACA TCG ACA 192
Ser 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
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 A.sn Asp Leu Ser Ile Asp His Leu Arg Gly Leu Glu Gln His Met
115 120 125
4 O 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
50 165 170 175
TAT GGT TTA GTG GAC AAT GAA GCT GCT GTT GCA CTT GCA AAT GGG GCT 576
Tyr Gly Leu Val Asp Asn Glu Ala Ala Val Ala Leu Ala Asn Gly Ala
180 185 190
TCC AAC CTC TAT GCA TTC CGC CTG CAT CAC GGG CAC AAC CAC CAC CAC 624
Ser Asn Leu Tyr Ala Phe Arg Leu His His Gly His Asn His His His
195 200 205
60 CAT CTC CCT AAT CTT CAC CTT GGA GAT GGA TTT GGA GCC CAT GAA CTT 672
His Leu Pro Asn Leu His Leu Gly Asp Gly Phe Gly Ala His Glu Leu
210 215 220
41
CA 02319853 2000-12-22
CGC CTT CCT TGA GTGGTGCTTG AGGTCGACCT TCCAGCTCTT CAGACATCTT 724
Arg Leu Pro
225
ATCTAAATGC GTGTGCTAAC TAGAGATGCT ATCTAATATT ATTTAATAAT TAATTAAGAG 784
CCCGGAAGTA AAAAATACTT CCATAGATTG TAATTTACCT CAGGGTAATG TGTATGGCAG 844
CATATTAGAT TGTGATTTGA GCAAGGAATG TCATTCCTTA TGGATTAATT AAATATAAAA 904
GCTCTTTTTC ACAAATAAAA P~~~1AAAAAAA P~AAAAAAAAA AA 946
(2) INFORMATION FOR SEQ ID NO.: 3:
(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
2 O (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
30 20 25 30
CAA GAA CTC ACT GTA CTT TGT GAT GCT AAG GTC TCT CTT ATC ATG TTC 144
Gln Glu Leu Thr Val Leu Cys Asp Ala Lys Val Ser Leu Ile Met Phe
35 40 45
TCC AAC ACT AAC AAA CTC AAT GAG TAC ATT AGC CCC TCC ACA TCG ACA 192
Ser Asn Thr Asn Lys Leu Asn Glu Tyr Ile Ser Pro Ser Thr Ser Thr
50 55 60
4 O AAG AAG ATC TAC GAT CAA TAT CAG AAC GCT TTA GGC ATA GAT CTG TGG 240
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
50 loo 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
60 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
42
CA 02319853 2000-12-22
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
TAT GGT TTA GTG GAC AAT GAA GCT GCT GTT GCA CTT GCA AAT GGG GCT 576
Tyr Gly Leu Val Asp Asn Glu Ala Ala Val Ala Leu Ala Asn Gly Ala
180 185 190
TCC AAC CTC TAT GCA TTC CGC CTG CAT CAC GGG CAC AAC CAC CAC CAC 624
Ser Asn Leu Tyr Ala Phe Arg Leu His His Gly His Asn His His His
195 200 205
CAT CTC CCT AAT CTT CAC CTT GGA GAT GGA TTT GGA GCC CAT GAA CTT 672
His Leu Pro Asn Leu His Leu Gly Asp Gly Phe Gly Ala His Glu Leu
210 215 220
CGC CTT CCT 681
Arg Leu Pro
225
(2) INFORMATION FOR SEQ ID NO.: 4:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 227
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
3 0 (A) ORGANISM: Populus balsamifera subsp. trichocarpa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 4:
Met Gly Arg Gly Lys Ile Glu Ile Lys Lys Ile Glu Asn Pro Thr Asn
1 5 10 15
Arg Gln Val Thr Tyr Ser Lys Arg Arg Asn Gly Ile Phe Lys Lys Ala
20 25 30
Gln Glu Leu Thr Val Leu Cys Asp Ala Lys Val Ser Leu Ile Met Phe
35 40 45
Ser Asn Thr Asn Lys Leu Asn Glu Tyr Ile Ser Pro Ser Thr Ser Thr
55 60
Lys Lys Ile Tyr Asp Gln Tyr Gln Asn Ala Leu Gly Ile Asp Leu Trp
65 70 75 80
Gly Thr Gln Tyr Glu Lys Met Gln Glu His Leu Arg Lys Leu Asn Asp
85 90 95
5 0 Ile Asn His Lys Leu Arg Gln Glu Ile Arg Gln Arg Arg Gly Glu Gly
100 105 110
Leu Asn Asp Leu Ser Ile Asp His Leu Arg Gly Leu Glu Gln His Met
115 120 125
Thr Glu Ala Leu Asn Gly Val Arg Gly Arg Lys Tyr His Val Ile Lys
130 135 140
Thr Gln Asn Glu Thr Tyr Arg Lys Lys Val Lys Asn Leu Glu Glu Arg
145 150 155 160
His Gly Asn Leu Leu Met Glu Tyr Glu Ala Lys Leu Glu Asp Arg Gln
165 170 175
43
CA 02319853 2000-12-22
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
1 0 Arg Leu Pro
225
(2) INFORMATION 5:
FOR SEQ
ID NO.:
(i) SEQUENCE
CHARACTERISTICS
(A) LENGTH:
5656
(B) TYPE: nucleic
acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
2 (ii) MOLECULE DNA
O TYPE:
(vi) ORIGINAL
SOURCE:
(A) ORGANISM: us balsamifera trichocarpa
Popul subsp.
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO.: 5:
AGTATATATACTAAATAAATATATAAACTTGTAAAAAATAAAAGAAAAATAATCATTGCA60
TGCAAACTAAACAAACATTAAAATTATACTTAAACAAAACTAATCTAAAATGAAGTTTTT120
AAAAGGTAATTATGACATAGCCACGAGCCACTCAATAAACCTTTATAAGATTTAAATTGA180
TGCTAAAATATATTTTTTTTTATTTTTTGCATCATTAAAGAAATAACTCAAAAGCATCTT240
TTATTTTTTAAATATTAATTTATTAGAACAATACTTGATATCTATTGAAATAATACTCAA300
TATCTATCTATAAATCAAAAAACCTAAACTCTAGATTGTAAAAAATAATAATAATAGAAG360
3 AGCCACCCATCCAAAACTTCTATATTATTTGACTTGAAAGCAAAAACATTAATACACATA420
O
ATTCATGAAAAATACTCATGAAAGTCTATAATTCACAAAAGAATTGATGAATATTCATAT480
ATAGTTCACTAATAACATTCATTTTCATCATATAATTAACGTATTAATTCAAGTACTAAA540
ATATTTTATGAACTAAAAGAAATTATTGATCAAAGAAAGACTCAATAACAAATATTTTTT600
TATTAATCAAACTCAAATTCAAATTCATGAACCCTCAAATCCATTATCAAATCCATAAAC660
CTATTTGGGGTTTGAGATTTTTGTTATCCAAGGGTTTTATGGAGACAATTTATCATTCCC720
TTTTTATTAGTCTTTTTTATTATATATTAATATTTTATATTAAAATACTAATTACAAAAT780
TCAATATGATTTTAATCTTGGACCTCATATATAATTCCGCTTTAAAACTCCGACTCATAT840
TCTAAACCCAATTCCAACATGGACTAAACAATTAATCCCAATATTAGAGGGAACAAATTA900
TTTATTTCTTAACAACACGAAAACTAAAGTATATCACTCTGCAAAATGTAATTACAAGTC960
4 CTTCGTGTTTAGGCTAGTTTGAAGATGCCTGTGGTTGGAGACCAGAGACATCAAATTAAT1020
O
GTTTTTTTATAGTAACATGTGCTCAAGTTGCATGCATTTTTCGTACCAACAAAATACATG1080
TAAAATCATCATCCATTAATCAAATTGCAATGATTCATAGCATATGCATAACGCATGTGT1140
CTGTGCATGTTTTAGCTGGTTCAATTCTTGCAGATTGTACTGCTAAATGTACGTACTAGC1200
ACCTCAAATCACAGTGACCTCCCAAATATTGCACAGACCTCTTTGTTTACAAATTTCAAG1260
CATCCTAATTAATCTCCCAAGTGACATCTGGTGGCCATGTTGCGGCCCTGACAAGCAGCT1320
GAGAAATTCTCCAACATTAGAGGGATTCAATGTTCTGTTCAATGTTTGGATACATTGATT1380
CTGCATTGCAACGCTAATCACGGTCTGTTCTCCGGCAAGGGGGGGAAAAACAATGATCAG1440
GGATAAGGCAGCGAATGTCTGGTGAAAACAAGGGTATTTTCATACTTTTCTCAGGTTCGT1500
GTAGTCAGCAATGAACGAAACGAGGCAAATCCAACCAAGTAGAAAAACCTCATGAGTAAC1560
GAGAAAGTCGAGGAGACAGTATCTGGCACCCTCAGATGCATCATACCTTGCGATGAGCCA1620
O
GAAACTAAGATGATTCTAGTGACGTCTAAATCATCAATCCCACGGTTAAAAGGACACCAT1680
AACCCAAGCCACTAGAATATCTGCTTACGCAGCAACCACACTGCAAAGCCACGACGAAGA1740
ACTACAAAGATACGGATATAACATGATATAAATATATTAATACTTAATTCTTCAAGGTCT1800
TGGATTATGAACTTTTTTGTTCATATTTATTTTATTATATTGAAAAACTCGAAATAAATA1860
AGACGATTATTATAAGAATTCTTAAATCATGTTTATCAAATTTTGTCCTATCTAGAGACC1920
ATTAATAATTGTGTGTGGATTAATTCACCAAAAACTTAAATGAAAAGTAACTTTATCTAT1980
CTAGAGATGGAAAAGGAACTCAATTACCCTCAATAATAAAATTGGATGGAAATCATCTAG2040
ATGGTGGTCCAGTAGTAAGATTTTGGGACTAAAAGGTTTGTTCTCTTTGTGGTCTCAGGT2100
TCGAGCCATGTGGTTGCTTATATGATGACCACTGAAAATTTACATGGTCGTTAACTTCAG2160
60 GGCCCGTGGGATTAGTCGAGGTGCGTCAAGTTAGTCTGGACACCCATATTAATCTAAAAA2220
AAAAAATTAAATGGCAAAAAATATTTTGAATGTTGAAGTAAAAAAAGTGAAAGGGAGGTA2280
GTAAAACAATATACGACCTAACAGGAGAGGAGTCCAATCAAGTAGATCATGTGTCAAGAG2340
ATGAGTGGATAGAAGAACTTCAAGTGAAGAATGTATGCAGGGAACCAAATGTGTGAATGA2400
CACAAAGATCTGACTAGTTCGATTTCAACTGTCCAGTTCCGAAGAAACATCAAAACCCTT2460
44
CA 02319853 2000-12-22
TAATTCTGTTAGCTTCCCAATACATACAAA CTCGTCCTGT2520
AAAGAAAAAA
AGACAAAAAA
TAAGGGCAGTTTTGGTATATAAATAAAACAAGAAGCTCACTTGTCTTTATATATCTACCA2580
AATCCAAGACATGCACCTGTGAAAGATCACAGAGAGAGAGACAAGGGGGCAGATAGATAT2640
GGATCCGGAGGCTTTCACGGCGAGTTTGTTCAAATGGGATACGAGAGCAATGGTGCCACA2700
TCCTAACCGTCTGCTTGAAATGGTGCCCCCGCCTCAGCAGCCACCGGCTGCGGCGTTTGC2760
TGTAAGGCCAAGGGAGCTATGTGGGCTAGAGGAGTTGTTTCAAGCTTATGGTATTAGGTA2820
CTACACGGCAGCAAAAATAGCTGAACTCGGGTTCACAGTGAACACCCTTTTGGACATGAA2880
AGACGAGGAGCTTGATGAAATGATGAATAGTTTGTCTCAGATCTTTAGGTGGGATCTTCT2940
TGTTGGTGAGAGGTATGGTATTAAAGCTGCTGTTAGAGCTGAAAGAAGAAGGCTTGATGA3000
GGAGGATCCTAGGCGTAGGCAATTGCTCTCTGGTGATAATAATACAAATACTCTTGATGC3060
TCTCTCCCAAGAAGGTTTGGTTAGCATTGATTCTACCTTTTAGTGTAATTAAGCTAAGCT3120
CATACTATTACTAGCTATAGGAGTCCATGGCCAATTTGTTGTAGTTTTGTAGAGTAAATT3180
AATTCTATGTATACTTGGATAAGATAATTAGCTTATTATAAGATGTTACTTGCCAGCTTA3240
TAATTTCCATATACAACAATCATTTTCATTCCCTTTTCCTTTTCTTATATATGAAATTTA3300
GTTCAAGTATAAGTGCTTGTACACCAATGTATGTTTACTCTAGTCATATCAATTCTACTT3360
TGCAGGGTTGGTTTCTTGCTAATTAATCACCATGCTCAATATTAGAGTAGTAATTCTCTT3420
AACTAAGTCCAGGTTAGCTAGCTTTTGGTTTCTTGTTAATTGCCGCACATACTTAGCTTA3480
AATTAGTTCTCAAGGTAATAGTTAGCTTAATAGCTTTGAGCTCATACTGGTTTCTATAAA3540
ATAAATGAACAAAATCTGATTGTTTCGAAAAATTAAATAACATTAACTTATTAAACTTAT3600
2 TTTCCTTTCCTTAATTTTTAATTTTTGCTTGTTTCTTGGGTGGTTGTGTGTTCAGGTTTC3660
0
TCTGAGGAGCCAGTACAGCAAGACAAGGAGGCAGCAGGGAGCGGTGGAAGAGGGACATGG3720
GAGGCAGTGGCAGCGGGGGAGAGGAAGAAACAGTCAGGGCGGAAGAAAGGCCAAAGAAAG3780
GTGGTGGACCTTGATGGAGATGATGAACATGGTGGTGCTATCTGTGAGAGACAGCGGGAG3840
CACCCATTCATTGTAACAGAGCCTGGTGAAGTGGCACGTGGCAAAAAGAACGGTCTTGAT3900
TACCTCTTCCATTTATATGAACAGTGTCGTGATTTCTTGATCCAAGTCCAAAGCATTGCG3960
AAGGAGAGGGGAGAAAAATGCCCCACTAAGGTACGAAGAGTCAGCTTCGCGAGGGATTGA4020
TTTTTATTTAGAAATATATTAAAATAATATTTTTTATATTTTAAAATTTATTTTTAATAT4080
TAATATATTAAAATAATATAAAAATACTGAAAAATAATTTTTTAAAAAATAATTTTTTTT4140
CAAAAATATTTACAAAACAAACTGTGTCTAAAGAACACATTTAGACCGTTAATTTCTGCA4200
3 AGTCTCAACATTTCAATGGTTCTTGTCTTGGACCCACATAGACCAGCCATTGTATTCTGG4260
O
ACTGGACTGGAGTTATGCCCCCACCTGAATTTGCCTTTCACAGCTGTCCCGATAAAAACG4320
TGACAACTCATGTACTGGTTTCTGGTCCCTGTCATTTTAGACCTGCTATTTGCAGTGGGA4380
TACTTATTGGTTACTCTTACTAGTCGATCATCGTTATTTGAATATTTCAAATATTCTGAT4440
TTTGGAAGTTTGTACGATGTCGTGTCACGTGGATCTTGTGAAACCTGGTTGATGTCAACT4500
ATTGTCGAACTGGACCAAAATCCATTACATTCTGAGTTTCTCTAGTGTTTTCCTGCCATG4560
GAACCTGAAAGCCCATGTTGATGGTTAGGACTTAGAATTTGATTAGCCCTAAATGGAACA4620
GTGAGTAATTATGCTAAGAAAAATGGTTTTTTTTTGTTTTGTTTTGTGTTTGGTTATAGG4680
TGACAAATCAGGTGTTTAGGTATGCCAAGAAGGCAGGAGCAAGCTACATCAACAAGCCCA4740
AAATGAGACACTACGTGCATTGCTATGCTTTACATTGCCTCGATGAGGACGCATCCAATG4800
4 CACTTAGGAGAGCGTTCAAGGAGAGAGGAGAAAATGTTGGAGCATGGAGACAGGCTTGTT4860
O
ACAAGCCCCTTGTAGCCATCGCATCACGCCAAGGCTGGGACATAGATTCCATTTTCAATG4920
CTCATCCTCGGCTTGCCATTTGGTATGTGCCGACCAAGCTCCGTCAACTTTGTTATGCAG4980
AGCGCAATAGTGCCACTTCTTCAAGCTCTGTCTCTGGTACTGGAGGTCACCTGCCGTTTT5040
GAGTTCTTAATTATGCCAAGATAAATACTCCTATCTCTATAAAATTGTCAAAATGTATGT5100
TGTAGCGAGGTCAGGACAAAGTATTGGTTGATGGAGGATGGTTCATTAAATTTCACATCC5160
TTGACTATTTATATATCATGATATGCTTAAAGGCTCTAATCATTGTTTACGTCGATGGAA5220
CTATTATATTTCTAATTTAGTTTTCAGGGAAGTCTAGGCTGCTGGTGCCTACAGTGTCCA5280
TAAATTTGAGCAAAATGGCCAAAAGGGGCCAATTGGGACCCACTAAATTAATTTGGTGGT5340
GCAGTCCCCCTTACAATACGACTGCATGTAATACTTGTCCAAAATTTGAGTGCAGTTCAT5400
5 AGGCTGTTACTTTAAACAGACAAACACATGATGACAAGATAAAAGGCATGGATAATTCTT5460
O
GTCTTCTTGAGGTGCCAACATGCAAAATGCCATGTCAGGTTGTTGATTTGATTTCTAATT5520
GTTAACCATTACTGTTTTTTTTGCCATAACCATGCAATGGTGCTAAAGTTAGATGCCATA5580
AAAGATGTATCATGGCAGCCTGCAATGCAAATAAAAACGGGGAAACAATGGAAAGTTGCC5640
AGAAATTTCAATTACT 5656
(2) INFORMATION 6:
FOR SEQ
ID NO.:
(i) SEQUENCE
CHARACTERISTICS
(A) LENGTH:
1308
60 (B) TYPE: nucleic
acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE DNA
TYPE:
(vi) ORIGINAL
SOURCE:
CA 02319853 2000-12-22
(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
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
Glu Leu Cys Gly Leu Glu Glu Leu Phe Gln Ala Tyr Gly Ile Arg Tyr
50 55 60
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
3 O 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
40 130 135 140
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
5 O 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
60 210 215 220
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
46
CA 02319853 2000-12-22
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 AGA 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
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
3 0 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 AAA 1308
(2) INFORMATION FOR SEQ ID NO.: 7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1131
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii.) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
5 0 (A) ORGANISM: Populus balsamifera subsp. trichocarpa
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (1)..(1131)
(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
60 Ala Met Val Pro His Pro Asn Arg Leu Leu Glu Met Val Pro Pro Pro
20 25 30
47
CA 02319853 2000-12-22
CAG CAG CCA CCG GCT GCG GCG TTT GCT GTA AGG CCA AGG GAG CTA TGT 144
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
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
3 0 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
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
5 0 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
48
CA 02319853 2000-12-22
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 AGA 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
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
3 O (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(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
40 20 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
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
145 150 155 160
49
CA 02319853 2000-12-22
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
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
4 0 Ser Gly Thr Gly Gly His Leu Pro Phe
370 375
(2) INFORMATION FOR SEQ ID NO.: 9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 11485
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
5 O (ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Populus balsamifera subsp. trichocarpa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 9:
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
TGTTGTTGGT TGTCATTGGG TGTATAAGAT CAAGACTAAT TCTGATGGGT CTATTGAGCA 300
ATACAAAGCT AGGCTGGTTG CAAAAGGATA CTCTCAACAT TATGGTATGG ACTATGAGGA 360
6 O 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
CA 02319853 2000-12-22
TTAAGTGCACTGATGCAGGTCGTATCATTCTGTCTTTATATGTTGATAACATGATTATTA720
TTGGTGATGACATTGATGGTATTTCAGTCTTGAAGACAAAGTTGGCTAGACGATTTGAAA780
TGAAAGATTTGGGTTATCTTCAATATTTCCTGGGTATTGAGGTAGCATACTCACCTAGAG840
GTTACCTTCTTTCTCAGTCGAAATATGTTGCAGATATTCTTGAGCAGACTAGACTTACTG900
ATAACAAAACTGTAGATACTCCTATTGAGGTCAACGTGAGGTACTCTTCTTCTGATGGTT960
TACCTTTGATAGATCTTACTTTATACCACACTATTGTTAGGAGTTTGGTATATCTCACCA1020
TTACTCGTCCAGATATTGCATATGCTGTTCATGTTGTTAGTCAGTTTGTTGCTTCTCTTA1080
CTACTGTTCACTGGGCAGCTGTTATTCGTATTTTGCGATATCTTCGGGGTACAGTTTTTC1140
AGAGTCTTTTACTTTCATCCACCTCTTTCTTGGAGTTGCGTGCATACTCTGATGCTGATC1200
ATGGTAGTGATCCCACAGATCGCAAGTCTGTTACCGGGTTCTGTATCTTTTTAGGTGATT1260
CTCTTATTTCTTGGAAGAGCAAGAAACAATCTATTGTTTCTCAATCATCCATCGAAGCAG1320
AATATCGTGCCATGACATCTACTACCAAAGAGATTGTTTGGTTATGTTGGTTACTTGCTG1380
ATATGAGAGTTTCATTTTCTCATCCTACTCCTATGTATTGTGACAACCAGAGTTCTATTC1440
AGATTGCTCACAACTCGGTTTTTCATGAGCGAACTAAGCACATTGAGATCGATTGTCATC1500
TTACTCATCATCATCTCAAGCATGGCACCATTGCTTTACCTTTTGTTCCTTCTTCCTTAC1560
AGATTGCAGATTTCTTTATCAAGGCGCATTCCATCTCTCGTTTTTGTTTTCAGGTTGGCA1620
AACTCTCGATGCTTGTAGCTGCCGCATTGTGAGTTTGAGGGGAGATGTTAAATAATATTT1680
ATGTAGTCTTATTTATTAAGGGTAGAATAGTACTTTCAGTTTAACCTATATATACTTTAT1740
TTGTATTTAGGTTAAGACTAAGCATTCATAATAAATGTATCATTAAGAATTCTAGCCTCC1800
2 TTTTCGTGTTTCATTTTAATTATTTTTAACAATCTTGTATTAATATATGTGAATTGATTG1860
O
AATTAAATCTTGTAATACAATTTAATTGATTCTAAGTTAAACAATCTGCTGGGGAACATT1920
CATACAACTATCTTTCTTTTCGTTTCAAGTAGGCAGGAAATAAAACGTTTTTAGTTTAGG1980
TGACTAAACAATGGAATTTAATGAAATAAGGGTAGAGATGAGGTCTGAGGTTATCTTGTT2040
AAGCACCTTCCCATTTGAACCATGATTTTGTCGTTAAGCACTGAGAGTGTAACTTAGCCC2100
TAAAACGTCTCACTCACCCCATTATAATTCATTTTCAGAAAGTCCCTTGCTTTTCTCTCT2160
AATGACCTAAATCATTTCCTTGAAAGCCAAAAATAAAAAATAAAAACGAATATAGTGGAG2220
AGTTATTGAGGTCTGAATCTGACGACAGATTCCCACCTTTAGCCTCTTCTTTTTAATTCC2280
TCTTCAATGCTCACCACTCATCAATACCAAGATAAGAAAAAGAAAAAAAAATGGAAAAAT2340
TATTGAAGAAGAGAAATTACAAAGACAGTAGTTAGACTTGGTAGAAGTATTGTTATATAT2400
3 AAAGATTGGATGAGAGGTTGTTTTTCACTTTATAAATACCCACCTCTTAGCCCAAACTTG2460
O
CTTCCATTTTCTTCATCTCTCTACTAGTTAGATTTGTAGGAGAAATCCCAAAGGAAAAGA2520
TCCTCACTTTCTCTACACATTAACTGCTATCTACAGCCCCTAGCTACTTTGTTTTATTTC2580
CTCCCAAGGTTAGTTACTAAAACATGGAGTCATAAATCTCGTTGTATTCTTCAGTGCTTC2640
ATCACTTGTTTTGGGCTAATTAATCAATCTTTTCACGTTTCAAAACCCACCTCTTCTTTT2700
TCTGTTTTGATCACTCAGAAACCCCAAAAAATACAACTTTCAAACATTTCTGTCTCCCTT2760
TCCCATTTCAATCTCCAGATTGAAGCACCAGTGATTTATTTTTGTTTTGTTGATTGATTA2820
TTTTGACCATAACCAATAAACCATAACAATCGCAATTCAGAAGCTCCAGACGTTCATCGA2880
CCCCTTTTTCTTATGTTTATTTTATATTACTTCCATCCTGGACTACTCATTTGGACAAAA2940
AAAGTATTGCTAAATATGCTATGAGTTGTGCATATATTATTCTTGAATTAGTAGTATTTT3000
4 TTTCATTTTATTACATTTTTTGTGTTGTCACTCAGTTTGTGTTTTGGATCAGCTAGCTAG3060
O
GCTGCAGCTATGGAATATCAAAATGAATCCCTTGAGAGCTCCCCCCTGAGGAAGCTGGGA3120
AGGGGAAAGGTGGAGATCAAGCGGATCGAGAACACCACCAATCGCCAAGTCACTTTCTGC3180
AAAAGGCGCAGTGGTTTGCTCAAGAAAGCCTACGAATTATCTGTTCTTTGCGATGCTGAG3240
GTTGCACTCATCGTCTTCTCTAGCCGCGGTCGCCTTTATGAGTACTCTAACGATAGGTAA3300
ATAAATCTAATTTTAGATATTTGCTTCTCTGGATCTTAAATTCTCCATGTTACAAGCCCT3360
CTATCTTCATGTGGTCACTTTTTTTTTTTTTTTATCTTCCTTTCTGCCCCAAAGAGATTT3420
TTTTATCCTCTCTATTTTGCTTATGTTAGTGTTAATTTTTAGCTTTAATTGGTTTCTTTC3480
ATTTTCATTTTCTTTCTTTCATGAATGATCATTAAATGGTTTTCAATTTCTAAGGTGGGA3540
AATTTATTATTATTATTATTATTTTGTGTTTAATCTCTGGGTAAAGGATTTAAAGCAAAA3600
50 GAGACACAATCATTCCTTATGCTGCAGTTTAGATTGAGTTTCTTATCTAACTGAGATTCA3660
CTTGTCTTTCTTTCTTTCTTTCTCTTCTCTTACCCTTTAGACGATGCTGATGCACACGTT3720
ATTTTGAGTTCTTGGTTTGGTAAAAACATAGATCTGGTATAATAAACAGACATAGAAGCA3780
CTATATGAGTGTAGTATGGTAGCAGAAATAAGTATAGGTCTGTGAGATCAGCCTCTTTAT3840
CTCCTCCCTTGTTGTTAATTTTGTTGTTTCCGTTTTTCTTTCTCTTCCATTATTCCTCTT3900
GCACTCTCTATCTCTCGCTTTTTTTTTGCACATACTTGTTTGTTTGTGTCATCTACGAGG3960
CTAAAGAGATTGCCTATAGCCAAAGCTGTCATCTTCTCATTAGTCCAAACCCTCCATCTC4020
TTTTCACTTCCTAGTTAAATAGCACGTCAATTAGACATCAAGAAAGCAAAAGTACCATGT4080
CAAATAACCGTGAAAAAGAAGAAGAACAAAGAAAGGTTTTTTTAATTTGTCATGTCACTC4140
AAACATATATTATTAGGGTTTCAAATCCCAAATCCCCAGATGGGTTTTTCATCTTATTTT4200
60 ATTTTTCCAAACCAATCCAGGGTTTTTCCCCTAATCACACGAAATTTCCCAAAATCTCAG4260
TTTGAACCCACGAGGGGATAGTGAAAACCTTTCTGTTAGTCAATGCATAACCCCAGTTAG4320
GGTTCATAGTTAGGGTTCATATTCAAGTAACCACATGAAATCATCGAAATCGTACATTAA4380
CATTCAAGGAAAACTGTTAAATCAAGCAAGTGGACCCTTCCACAACCAATCAAAACTCAG4440
TTAGATTTCACCTAGATTTTTACCCCTTTTTTAACCTGGGTAAGTATGGTACAGTAATCG4500
51
CA 02319853 2000-12-22
GTTAGGGTTTAGTAGCCAGTCAAATAGATCAGATTGTTGTTCGGGTTTATGAACAGAATC4560
TTTGGTAACGTCACACACGATTTTTCAGTTCTTGCCTACTGACAAAAGGCTTTATGTCAT4620
GATTCCTTAAACTGAACCCAAGATTTTTAACTTCCGATCCCCCTGGAAAAAATATGAAAT4680
TCCAAAAATTGTCCATTTCTTCTCCTTAGATCTCTCTCTATCTCTCTCCCGGTTAAATTG4740
TTTCCATGGTGAAAGCAGAGAGATGGATCAATGAGAATGGGTTAACCAAGGCCATAATGA4800
TGGCACTGTTTAAGATCTTGTATAGATATATTTATATAAGTTTTTTTTTTTTTTAATTTA4860
AAGAGAGATTTAGCCCCATTTGTATTTTTACGGTGAGAAAACACTTTTATAAAAAATTGA4920
TATTTTTTTAAAAATTATTTTTTATATTTTTTAGATTATTTTTATGTGTTAATATTAAAA4980
ATAAATTTTTTAAAATATAAAAAATATTATATTAATATATTTTAAATAAAAAATTAACCG5040
TTGATGACAATATTGAGAGAAAGAGAGTCGTGAAGAGAGAATGAACGACAACTGTTAACC5100
AGTGGAAGAGTTCTGTCAATTTTGGTTTCTTCTATGTAATAGAAAGCCTACAACTCTAGC5160
TGGTATTGTACGGCTCTGCTTCTCTCAGAGTTTCAGTCTGAGACTAATAAAATGTCCGAT5220
TAGTACAATATTTTATTACAATGAAATAGAATATCGAGGTGGGTAATAGAGTGAGTTTAA5280
GGAGATTATCCACTATGTAATGGGTTATTGACACGTGGAGAATATTTGACCGCTGATCTA5340
CCTTGGCCAATCATATTGTAGGATTCAGTGACAGCTTGGCAGAGACAGCCAATCAATGTC5400
TCGACGAAGTTAAGGTATAAGGAAATCTAGAAAAGCGGTTCTTGTCTGAATTGACAAGAT5460
GTGTTCACATTTTACTGAGATTATTATGGCAAAATTTTAGGATTTCCTTCGCATTGTGTC5520
GAGGAAAGACTGGATAATCAGACTGACTCGGAGAGCTGTGGTTTTGTCATTCATCTTCTT5580
TTTAGGGTTTTCTACGAGTTAACTTAATGGAGTTATTCGTTGATTTGACTGTTTAATTGC5640
2 CTTACCGTCAAGCTTTGTTATAATAAGGATTTTTTAAATTGTTTTTTTTATTTATAAATA5700
O
TATTAAAATAATATTTTTTAATTTTTAAGATGGCATATCAAAAATATTTTAAAAAATAAA5760
AAAATAATTTGAAATAAAACAAAAATTAATTTTTTTAAAACAATATTTTTAACGCAATAA5820
CAAATTCTTAATCTTTTACTCATATATCTTAAATTTACGAGAGTTTTTTCCAAAAAGATA5880
AAGAGATATATGTAAGCGATAAAGTATTAGTAACCTCACATAAAATAATGTACAATAATA5940
GATAAAAACTAAATTTTATATAAAAATTGAATTTCAATCCACTTTCTTTTTTCGTGGATC6000
ATAAGGAGTTGGACTTGCTTTTTTCACGGTAATTTGACCAAAGAAAGAGTTAATACAAAT6060
AATATTAATTAAGATATTATCTCTTGTTGTTTGTTCTTGTTTTGAAATAATTTAGTTTTT6120
TTTTTAAGAAAAAAAAGTTTTTCCAATACATAAGCAATACAAAAGTGTTTGAACATGGTA6180
ATTCTTCTTCTTCTTAGTTGACCAAATTACATTTGGTAGACTAAAGTTGTTCATATATAT6240
3 GCTACCATTGATAGAGTCATTGGCCAATTATATGTTTTTACGTCATTATATTTGAATTCT6300
O
TTTGTTAATAGTAATTATTAATCACTGAAGTTATTGCATTCTTGTCAGCTGATAAACTCC6360
AAGTTGTAATTTTATGTTTGATCTTGTAATTAAGAGCAAGCCAGGAGGACATCTCTAGTG6420
TTCGAGGAAATTGACAAAATTTGCTTCCTCAAATATATTTTTGTTTTTCATTGGACAAAA6480
ATACATGTTATATATATATATATATATATATATATATATATATATATATATAATGCCTAT6540
ATTTTGTGAGTAGTTCCATAAGTTTAGGATATGTTTGAGGTAGTTTAACATAAGCATTTG6600
ATTTTTTTTTTCAATCCTTATATCAAAATTATCATAAAACAATTAAAAAATCATTAATTT6660
ATTTTATTTTTTTAATTAAAAAAAACACTTATAAACACAGTATTACCCAAATACAGATTT6720
ATGAAGCCGCCATGTGGTAAAAAAATACATGTTAGAGATATCAGAAGTTTACAAGCATGT6780
TTATATGCGTTAATGTGGCATATGAAATGTCATATCAATTGCGTTACAAAGCTTTTCTTG6840
4 TGCTAAGTGTGGCGTTAGTAATAAGCAAGTGTTTGTAAGAATTGTCAACACGTGTGTTTA6900
O
CTTACTTGAAAGAACATTAATTGCTAATTTTATTAAATAATTAATCCTTCCTATTACTAT6960
CTTGGGATAGGTTGAAGAGCATAAGGAAAAGGGTTACCATGATAAATACAAAAAATAAAA7020
AAGGAGGAAGGAGTAGTTTTCAATTTTATTTTAATTGTCAATACTATGTGCTTGGTGAAA7080
AGTTATCTGTCCTCATTTTTATTTATTGTTTTTTACAAAAAGCATAGAATAATGTGTGTT7140
TCATGTGTTTGGTTAGAGGTTATAGATGAAAAGCTTTAATAATAAATAGTAGCTAAATAT7200
ACTTCATTGTTTGAGTGGTAGAGGAGATTTTTAAAATTTATGAAGACTACAATTCTCTTT7260
CATTTCAAATAACATCCCTATTTTAGTGGTGAGATTAATGTATTTGTTTCTCTTTTTCTA7320
TTTTCTTTTATCAATATTATATATAAAACTAAAATGCATCAGTGTTTTACTATGGATTGA7380
TCATAATGCAATTCACTATAAAATAATTGATGCTTCCCTTAAAAAACCAAATAATTAAAC7440
5 AAACACTCAGGGTTAATTTTGTATTTTCATATCTTTATTGCATAGTGTAATTATTTCTAT7500
O
GTCCTTGAAAAAAGAAAAAAAACACTAGGGTTTTTTTAAAAAAGTTTCATATTTTTTTGT7560
ATAGTGTAATTATCCCACTTTTGGGGCCAACTTTTTTTTACCTAAGGTAAAGGGGTATTT7620
TTGGTTTTTTTATGTTTGTTTTTTTGCAATTATTATATGGGATCAAGAGTGTTATGATCT7680
TTTTATATAAAAAAAAAATGGTTGACACGTGATCTACAATTCCCCCTCCCTTTTCATTCC7740
TAACCTTGAAAGTCTTAGTGAAACATATAGTTATAATAAAGAAATATTATCTCTAGTTTT7800
GCAAATTAATTTCATAACATCAATTAAATATTCTGATAAGGTAAAGTTATTTAGGATGGA7860
GAAATTTACATAATGAAGCCTCCTTCTGCCTGAGTAGTGCATTTCTATGGTATTTATGAG7920
CATCAATTCTACAATCCATTGAAGCAAAAGAACTAACCTTCTTGAAACCCTCTTGCAGAT7980
AATTGTGAGTGAATGTAAGTCCACTACGAAATATTCACACGATTACGCACTTAGTTATCA8040
60 TTAAACTTTGTTTTTGGTGCTTTGCATTTTCTTAATTAGATTCTTCCACAGCTTTCCAAT8100
GCACATTTTGATGACTTTTTTTATTTTATTTTTCTTGATGGAAATGTTGACATGATTGCA8160
GTGTCAAATCAACAATTGAGAGGTACAAAAAGGCATCTGCAGATTCTTCAAACACTGGGT8220
CTGTTTCTGAAGCCAATGCTCAGGTACCATATATCAGCTCTAACTAACAATTTGTACTCA8280
TAATATCTATTAGATGGAGTTCAAGCATAATATTCCTCCCAATAATTTATTGCCAATATA8340
52
CA 02319853 2000-12-22
GTGCTATGCTACCACTTCATTCACTCTTTCTTGATAACCCCAGCTTGTATAAAATCTATT8400
AGATACCTCTAAGTTTTTGCCTTACCTTTCTCACTAGTGTCTGACATGACACTAGTGTTC8460
ACATGGATTAGCATCTCGGAGTTGAAGGTTGTCTGGCTTCTTCGAANATCCAGGGTTTTC8520
AAGAAGGTTTGTACATTGGGAGGCCCGTGGTTATAAACCTACTGTGTAAATGGTTTGATA8580
AATAATGATTCATCAGATTTGAGTAATAGTCTTTTAATTTCTTTGTAAATGTTGTCTATG8640
TTTTTTCCAGTCCTCCCTACACACACTCTGATAATTATAACCAATTTTGTTTCGCTTCCT8700
CCTTTCGCTATGCTCCTACTGAATTTATTTCCAGTTTGATTCAGTATTATATGCATGTTT8760
ACAAGAAAATAGAAGGGGGGAATCTACATCACTGAGATTTTCTACCTGTATTTTATCAAC8820
TGATCTAATATGAACTTGAGGCTCTTAATTTTGTTATATATAATGTTTTATTGCCTTTTG8880
TTCTTGCATCTCAGTACTACCAGCAAGAAGCTGCCAAGCTGCGTTCCCAAATTGGTAATT8940
TGCAGAATTCAAACAGGTCAGAGCCTGTTTGATATTGATCTATTTGTCAGATGATATCGT9000
TTTCTCTTCCAAACTCCGCTTAAGTATAAATTATATTTCAGGCATATGCTGGGTGAAGCG9060
CTTAGTTCATTGAGTGTGAAGGAACTTAAGAGTTTGGAAATACGACTTGAGAAAGGAATA9120
AGCAGAATTCGTTCCAAAAAGGTTTTGATACTAGTACCGAATTGATACTATCACATTTTT9180
TTGTTTTACTTGGATATCACATTTCCATGTATGGCCATTAACAAGTTTTGTGTTCATACT9240
TTCCTGCTATGTTTCTAAAAAATTCCTCCCGCAAACCTTGCCAGAATGAGCTGTTGTTTG9300
CAGAAATCGAGTATATGCAGAAGAGGGTAATGCTTCTTATGTTATCACATTTCCCATTTA9360
TTTAATATTTATTGTTTTCTGGTGGAGTATATTCTATATGATTGTTATATATTCTGAGGT9420
AAAAGTCATCTAGTGTTTATTAACATAATGATTCTATGGTCAACTTATTCCTTCCTGTTT9480
2 TCACTCCGAGATTTTCCTTTGATTCCTTGAATGAAAATGCACATTACAGGAGGTTGACTT9540
O
GCACAACAATAACCAGCTTCTCCGAGCAAAGGTCTTTCTTCTATCTATCTATTTATCCAT9600
CTCGAGTGAGGGCAAGGATGCGTGCGTGTGCATGAATGAAGATCTCTATGTCTTATATCG9660
TTAGTGAGCTGTTTATAATTTAGAAATATGAGGCTTATCTTGATAGTGCAGATTTCAGAG9720
AATGAAAGAAAGCGACAGAGCATGAATTTGATGCCAGGAGGAGCAGACTTTGAGATCGTG9780
CAGTCTCAACCATATGACTCTCGGAACTATTCTCAAGTGAATGGATTACAGCCTGCAAGT9840
CATTACTCACATCAAGATCAGATGGCCCTTCAGTTAGTGTAAGTATCTCCTTTGTAACGA9900
ATAATAGGTTTTCATTAACCGGACAACCAGATTTAGTGTTGTGCATTCATAAAATACAAT9960
TAATTACTTTAATTTGGAGATGTTCCAAAAGTTGCAACTGCATGGTTCATGGGCTCTAAT10020
TTCTTGGAAGTATATAACCGATGCTATGTCTTTTCATTCTCATAATTACTGATCAGTCCC10080
3 TTATAGATGATTATTTGCAGATTCTTATGACCATTTTCCCATTGAGATTATAAGATTTTG10140
O
ACATCGAATAGTTGGACTAGGAGTAAAGAGCTGTTGCTGTTATTTAGCACCCCAAAGGAA10200
ATATTATATACCTCTGAACCAATTGAATGGCCGACCTAGGTTTACTGAAATGTTTAGCTG10260
TAAGAAGGTTAAGTGTTATCAGATTCCCCAAGTGAGAAGTACATGTTTCTTAGCATACTT10320
TATGTTTCACGCACCTTGATTTTTCAAACTTTGTTTATCGATTTCTGAACTAAAGTGACT10380
ACATTATAGAACTTGAACCTAAAATTACTCTCCTCACTATAGGTGAAATCAGATTACTTG10440
AAAATACTACTAAAAAAAATTATGGCGTTTGCTGGTATTTCTAACATCTTTTCTGCTAAT10500
CTTGTATTAATTTTCTCCTAGATGAACTTGTTATTATGTAAAAAGGTTTCATTACTCATG10560
CAATGGTGCACTAATGCTTGAGGAGTTCCAAGTAACTTTGCTGTCTCATGTAAAGAAGAG10620
TGCTGAAGTTCACTATGGTTTAACTTCTACTGCACTGCTTGATATTGCCATGAACTCTGA10680
4 CATCATTTGGCTTGATCTTGTTCTAAAATCTAAATGAAATAATTCTCTCTTACTATATAT10740
O
CTTCTTAACCCTTTGCATATGATTAAGTGGTCTTTGATAGGATATCATTAAAACCTCGCA10800
TAAAAGCTACCATTTTATAAATTTCAAACTCCACGACGCATTTTCTGGTGATTCCATTGC10860
TGATTATTGTTTAAAGACATCATTATTCCAATTAGTACATGTATAATAATTTCCTCTGTT10920
GTTGGTGCAGTTAATAATCTCCAAGTGCAGCAGTTTCTCGCATTTCCATATTCCATGGAG10980
AGTACCTGGGTTTCCATTGAGCGCAAAAGCTACATGTATGCTAAAAAACCTGAAGTAGCG11040
TAAATCATATTTGTCTGGGTGGGAGGGCCTAGTACTCTTCCTCTATGTATTAACTATCCT11100
GTCCCAGTTAAGACATAAGAAATGTCAGAGAAGGATTTCTTTTCTGTATGTTTCATGAAG11160
GCATTAAGATGCTGTTACAGTTGTGACTAACTTATTATATATGTCTTACTGCTTCATCTT11220
GTGATATTTTCTTGCATGTTAATCTGATTAAAGTGTAGCTTAGACCATTCACCATGTTAA11280
5 TGGTGACTTGTTGGTGACTACTAGTAGCTGTAGCTCTCCGTAGTACTGCTATGCCTTCAA11340
O
AAAATGATGGGTCGGAAATTACTAGCTAGCTAGTATTGCTGTTTCATTCAATCTCTGCTT11400
TAACCCAAAAATCAGGACTAGTGGATTAGCATACCTCTCACCAGGACAATGCACTAGAGC11460
ACATTTTCATCTTCTTCTCATATTT 11485
(2) INFORMATION 10:
FOR SEQ
ID NO.:
(i) SEQUENCE
CHARACTERISTICS
(A) LENGTH:
1219
(B) TYPE: nucleic
acid
60 (C) STRAN DEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE DNA
TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: trichocarpa
Populus
balsamifera
subsp.
53
CA 02319853 2000-12-22
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (196)..(921)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 10:
TGAGAGGTTG TTTTTCACTT TATAAATACC CACCTCTTAG CCCAAACTTG CTTCCATTTT 60
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
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
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
3 0 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
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
5 0 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
54
CA 02319853 2000-12-22
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
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 CAAAAAAAAA AAAAAAAA 1219
(2) INFORMATION FOR SEQ ID NO.: 11:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 723
(B) TYPE: nucleic acid
2 O (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)..(723)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 11:
ATG GAA TAT CAA AAT GAA TCC CTT GAG AGC TCC CCC CTG AGG AAG CTG 48
3 0 Met Glu Tyr Gln Asn Glu Ser Leu Glu Ser Ser Pro Leu Arg Lys Leu
1 5 10 15
GGA 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
20 25 30
CAA GTC ACT TTC TGC AAA AGG CGC AGT GGT TTG CTC AAG AAA GCC TAC 144
Gln Val Thr Phe Cys Lys Arg Arg Ser 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 TCT 192
Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe Ser
55 60
AGC CGC GGT CGC CTT TAT GAG TAC TCT AAC GAT AGT GTC AAA TCA ACA 240
Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn Asp Ser Val Lys Ser Thr
65 70 75 80
ATT GAG AGG TAC AAA AAG GCA TCT GCA GAT TCT TCA AAC ACT GGG TCT 288
5 0 Ile Glu Arg Tyr Lys Lys Ala Ser Ala Asp Ser Ser Asn Thr Gly Ser
85 90 95
GTT TCT GAA GCC AAT GCT CAG TAC TAC CAG CAA GAA GCT GCC AAG CTG 336
Val Ser Glu Ala Asn Ala Gln Tyr Tyr Gln Gln Glu Ala Ala Lys Leu
100 105 110
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
CA 02319853 2000-12-22
CGA CTT GAG AAA GGA ATA AGC AGA ATT CGT TCC AAA AAG AAT GAG CTG 480
Arg Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys Lys Asn Glu Leu
145 150 155 160
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
Gln Pro Ala Ser His Tyr Ser His Gln Asp Gln Met Ala Leu Gln Leu
225 230 235 240
GTT 723
Val
(2) INFORMATION FOR SEQ ID NO.: 12:
3 O (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
40 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
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
56
CA 02319853 2000-12-22
Arg Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys Lys Asn Glu Leu
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 13:
FOR SEQ
ID NO.:
(i) SEQUENCE
CHARACTERISTICS
(A) LENGTH:
10007
(B) TYPE: nucleic
acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE DNA
TYPE:
(vi) ORIGINAL
SOURCE:
3 (A) ORGANISM: trichocarpa
0 Populus
balsamifera
subsp.
(xi) SEQUENCE ID NO.:
DESCRIPTION: 13:
SEQ
TCCATTATTT CAACAATAGATTCATTTACACTAGCATGGATACTTCAATGAATAAGAAGT60
GTGTTATTGT TTGGAGTAATAGACACCTATAATTCTCAAACCTTTTACTTTATTTTTATT120
TCCTTTGTTA TATTACATTTTTCATTTCTTTATTGGGTTTTCATTGACAGGATGGCTAGA180
TTAATATAGT TTCTTGACTTTAATAAATAAAAAAAAGATCAAGACTCTCTTCACAAACCT240
TTACAAAATT GGGCGCTATATCTAAACTAAAAAACTTAAGATTATATACTATCTAAGGAG300
TAGCACACTA TAAATAACATTATAAAGGTAGTTTGTTGAGCGGAACTAGACTTTGCAAAA360
TAACTTTCCA ATATAGCTTTTCTTGTTGATGTTGACCTTTTAATTTAGGATCAAACACTT420
GTAAATTACA ATTAAAAGGCTTATTTTTGTTTGCCATTTTTACCAAGCAATGTTAGGATT480
4 GCTAGAGATT AGTTTTTCCATGGAATAAGAAGTTATCTTTAAAGGGCTTAAAAGACCTAG540
O
TAGCTTGACA AGGCTATGACTTGTGTTGTTTTGGATCGTATGGTTATTGTTATAGAGGTG600
CTAGTGGTTA AAGACATCCATCATGGAGGTGGTGATGACTTAAAAGAGTTAGATGTAAAT660
TGGAGACTTA TGTTATTCTCACATAAAAAATGTTAGCCTCCGACATTGTTTTTGGATGTG720
TAAAATCAAT GTACCATTTTATTCTTCATTGTTTGTTTTCCTTATTATGACTTTTACAAA780
TTTATCCTTT AGGTGATGAAATTCCTTCAATCTTGTTCTATTTTTTTTTTTAATTCTTGG840
TACGTAGTTC TGTACTTAATCAAGCAACATAAAATAGTGATGCCATCTTCATCACTCTAT900
AAACGTGGAA ACCCAAATCTCTGGCTTTTATTCATGATTAAAGTCATTTCTAGATTTTTT960
TAGACGTTCA AGTGAGATTTAGGGTTCAATAAGAGAGGATCAATGGTGAAAATAGAAGAA1020
CAAAGTTGTT GTGGTTAAGTTGACTCGGTGGTTGTTGAGTTGGGATATGAAGGAATAGAT1080
5 GGTAGACTAA TCTAGTGTTTTTGTCCACTTGAGTTCTTAATTATTATTCCATCTCCATGA1140
O
CTATTTCCAT CTTCTTCTTCAGTGATATTGTTTATACTCTGTGATTTGGGTTTATTGGAA1200
CTTATTATTG AGGCAGCTCATCCATAGAAATTTGGTACTTGCTTCAACAAACCACTAAAA1260
TGTTGTGTGG TTAATATTTGAGAATGCGCGAAAAAAGCATCGTACTAAATTTGGGTTCCC1320
GACTGGATGA AGAGAGATGTGATTACTTAATTTATTTGGATTTTCGGGGTTTATTAGATT1380
TTTGGAAAGG TAATACGATATCATTGGTTTTGAGAGGAAATAACATTGGGATTTTGATGA1440
TTTTTGAATA ATAAAATTAAGTTTTTTCTTGATTCATTTGTTAATAGAAAGAGAAGAGGG1500
ATAGCTCTCT TATTCTAGCAGAAGTACGTATATGAGCTATGGGATTTAATTCTTAATTTT1560
GTATGAGTTA TTGATCAAAGAAAAAGCAATGATGTGAGAAGTCTATATATATAATTTCTC1620
CTACGTACTC CGTTGAACCTTTTTTCCTAATAAAAATTGATAGAAAATCTACAACATATA1680
60 CAGAGAAATG TGAAGTTCTTCAATTGAGAATAAATCGTTTCAAAAGGACGTAGGAATCTC1740
CTTGTAGTGA GTGAAACTCCAAGAAAATTAAACAACCTGCTGGGGAACATCCATACAACT1800
ATCCTCCGAT CCCTTCTTTTCTTTTCAAGTAGGCAGGCAATAAAACGTATTTAGCATAGC1860
CAAGTTCAAA AAAAAAACAAGAAGAAGAAGAAGCAATGAAATAAGGGAAAAGATGAGGTT1920
CTCTTGTTAA GCACCTTTCATTTGTACCATAATTTTGTCCTTGGAATGATTAGAGAGCCC1980
57
CA 02319853 2000-12-22
AAAAACGTGTTATTCACCCCAGAAAAATCCATTTTCAAAA TCTTGATGAC2040
AGTCCCTTTC
CTAAATCATTCACATGGAAGCCAAGGAAGAAAATGAAAAAAACGAATATAGTGGATGGTT2100
ATTGAGGTCTCAGTCTTCCTATAGCGTATTCTCTAATTAATTCCAAGATAP~~;~1AAAAAAA2160
AAAATTACAAGGATGGTGTAGATAAACTTAGTAGAAAGTATTGTTATATATATATATATA2220
TATATGGGAATGGATGAAAGGTCGTTTATCACTTTTATAAATGCCCACCTCTTAGCCCCA2280
ACTTGCTTCCATTTTCTGCATCTCTCCTACTCAGATTCGTAGGAACAAAGAAGAGAGAAA2340
CCCCAGAGCAAAAGATCCTTACTTTCTCTCCTTAATAACTACTATCTCTACAACCCCTAC2400
TTTGGTTTATTTCCTCCCAAGGTTAGTTACCAAAACACTGAGACATATATCTCGTTGTAT2460
TCTTGAGTGCTTCACTTGTTTGGGGCTTATCAATCTTCTGATCTTCTTATCTCTTCTTCA2520
TCATAGTGACTGAGGAACCCCATCAGATGAAACTTTTAATTTTCTAAAAAAGATTTACTT2580
ACAAACGTTTCTGTCACTCTCTGCCGTTTCAATCTCCAGATTGAAGCATTACTAGTTCAT2640
CCCTTTGTTTTGTTTCTCAATTATTTTCATATCCATGAAACCATAACAAGGGCTAATTCA2700
AGAGCTAGCTGCAGGCGTTCATGGAACCCCTTTCTTCTGTTTATTTTGTCTTCCATCATG2760
AGCTATTCAGTGCTCAAGAGTATTCCTGCTAAATATGCTATGAATTATCCTTATATATAA2820
ATCATTCTTGAATTAATTACTAGCTAGTAGTTCAGTAATTTTATTACTCTCTTTTCTGCT2880
GTCTTCACCCAGTTTGTGTTTTGGATCAGCTAGCTAGGCAGCAGCTATGGCATACCAAAA2940
TGAACCCCAAGAGAGCTCTCCCCTGAGGAAGCTGGGGAGGGGAAAGGTGGAGATCAAGCG3000
GATCGAGAACACCACCAATCGCCAAGTCACTTTCTGCAAAAGGCGGAATGGTTTGCTCAA3060
GAAAGCCTATGAATTATCTGTTCTTTGCGATGCTGAGGTTGCACTCATCGTCTTCTCCAG3120
2 CCGTGGACGCCTTTATGAGTACTCTAACAATAGGTATATACTTAGTTCCTCGGCTCATGA3180
O
ATTCTCCATGTTGCAAACCCTCTTCAAGTGCTCAAAGTTGGTTTTTCTTGCTTTCTCATC3240
CAAAGGGATTTGTTTTTTCTTTTTGCTTATGTCAGTGTTAATTTTTATTGCTTTGGTTTT3300
GAGCTGTTTCTTTAATTGGTTTTCTTCCATCATCATTTTCTTTCTTCAATTGGTTTTCAA3360
CGTTTGTTGTGGGGAAAAAAAATAGGAGCCTGGTGTCAAGGTTTTTAGCTTCTGAGCTAG3420
ATCTTCGGGTGTCTTTAAAGTAAAAGAACACAATCATTCTTTATGCTGCAGTTTGGATTG3480
AATTTCTTCTCAAAATACAATTCACTTGTCTTTCTTTCTTCTATTTCTTTTCTTTTCCTT3540
GTATAAGCATAATTAATGTTTTGTTTTTCCTTTTCTTTATTTCACCCTTAGATGATTGTG3600
ATGCATACATGATTTTGAGTTCTTGGTACATAGATCTGGTGTATTAGATAGACATAGAAG3660
CACAATTATAAGTGTAATAAGGTAGTAGAAACAAGTAGAGGGCTGGGAAAATGTATGCAG3720
3 GCATGTGATATCAGCCTCTTTATCTCCTCCCTTGATGTTAAGTTTGCTGTTTCCTTTTTC3780
O
TTTCTTTTCCATCATTCCTCTTGAACTCTGCCTCTCTCCTTTACTCTTTTCTTGCACATA3840
CATGCATGTTTGAGTCATCTCTAGGGCTAAAGAGATTACCTATAGCTAAAGCTGTCATCT3900
TCTCATTAGTCCAAACCCTCCCATCTCTTCTCACTTCCAAAATAGCACGTCAGTCGGACA3960
TAAGAAGAAAAGAGTACAAAGTCAAATTAAATGTGAAAAAAAAAGAAAGGGTTTTTTTAT4020
ATGTCATGTCACCAAACACACAAACATATATTACTAGGGTTTCAAAATCCAAATCCCCAA4080
ATGGGTTTCTTCATCTTATTTTATTTTTCCAAAACAATCACTAGGATCTCTCAATTTAGG4140
ATTCTTTTTCCTCTAATTCACACGAATTTCACAAAATCTCTGTTCGAACCCACGTGGGGA4200
AAGTGAAAAGCTTTTTGTTTTTCAAGCATAGCCCTAGTTAGGGTTCATATTTAAGTAACC4260
ACTTGAAGTCATCAAAATTGAACCGAAACTTTAGTGCAAACTATTCAATCAACCATGTGG4320
4 ATTCTTCCATAACCAGTCAAAAATTAAGTTAGATTTCACCTAGATTTTTACCCTTTTTAA4380
O
CCTCGGTAAGAAGGGTACAGTAACTGGTTAGGGTTTAATAGCCAGTTCAATATATCAGAT4440
TGTTGTTTTGGTTTATGAAAAGAATCTTTGGTCACGTCACACACGATTTTTCAGTTCTTG4500
ACTACTGACAAAAGGGTTCAAGTCATGATTCATGAAAATGAACCATAAATTTTGAACTCC4560
CAATCTGCAAAAAAAAAGAAGAAGAAGCAATACCACACAGAATATTGTCCATTTCTTCTC4620
CTTAGATCCCTCCCTCTCTCTGTTATTTTCTTTCCCATAGTGAAAGAGAGATGGAACAAC4680
GAGAAAGGGTTAGCTAAGGTCATGATGATGCCATTGTTGGTCATTGTTGAGTGTGGTTTG4740
CGTTTGTTCAAGATCTTGAATATATGTATGTATGTATGTGTATGTATGTACGCAAGTTCT4800
TTTAGGAAGAGAGAGTTAATACAGAGAGAGAAAGAGAAGAGACGATGTACGACAAGTGCT4860
AGCCAATGGGAGACTTCTGTCAATTTTGGCTTTTTTAATGTAAATAGAAGCGTAAAACTC4920
5 TAGCAGCTGCTGCTGCTGCTCCTCTCTGAGAGTTTCAGTCACATTCAAGAAAACAAAAAA4980
O
AAATAATTTTTTATCTTATTACAAATGAAATAGAATATCGAGGTGGGTAATAGATGGTGG5040
CTCAAGAGATTATCCACCATAGAAGAAAAAAAGAAAAATAGATATTGCCCACCATGTAAT5100
GAGGAATTGACAAGTGGGATAGATGTGACCGTTGATTTAGCTTGGCCAATCATGTTATAG5160
GACTCAGTGACAGCTTGGCAGAGACAGCCAATCACTGGCTCGACGAAGTTAAGGTATCAG5220
AAAATCTAGATATCTGGGTCTTGTCTTAATTGACAAAATGTGTTCACATCTTACTGTTAT5280
TATTATGGCAAAATTTTAGGATGCACAAAGAACTGGGTGGAGGTTTTCCCATCCATCTTC5340
TTCTTTAGGGTTTTAAATGAGTTAACTTAATGGAGTTATTAGTTGATTTGACTCTTTGAT5400
TTGAATGTTCTTACCTTAAAATCATGGCTTAACATCATCCGGTGTTGGTACAGGGGCATG5460
ATTTTGCTCTCTCCCTCTTCAGTCAATTTCATCATTTATTTTGATATATAATTTTCTTTT5520
60 TCCCTAATGTTAAGCCTCTACTGTCACATCTTTAAATTACTAGAGGGATATGTAACTGAT5580
AACTAGTAACTTCACATAAAATACTGTACAATATAGATTTAAAAAAGGAATTTTATATAA5640
AATTTGAACTCTCAATTTTATTTTATTTTTGTTATTTGGATGAGTAGTTTGTCTAAAGCA5700
ATAGCTAATATGGAGGGTATTAAGAAACTGCCTCTAGTTGTTGACACAAAAGCCTTGAAG5760
CACGTATTTTACTCGCTAATTCACACTTCTTGGCTGTGCTCTTACCATCTTGGAAAATAA5820
58
CA 02319853 2000-12-22
ATGGATTTCA TGGTTTCTTAATCTATTTGA TAATGAAAAA5880
AAAAAGTACA AATGATTTAA
TAAAATCAAATGTAAAGTCTTAAATGTAATAAAAAATAATTTTCGATGTTTTTTGAGTGT5940
TTTTTTTTTTATAATTTTGAAATCTTCATATTTATATGTCCATATAAAACATAGGAAAAA6000
TCATCAATTCTCAAAATTATTGGAGAAAAACACCTACATATGCATTATCGATCAACTACA6060
CAAATAGGAAGTAACCATTCGAAGAAAATTAAAGACTAGAGACATCAAAAGTTGACAAAC6120
ATGTGTACATGTGTTAATGTAATCGTGTGCAAGTGTCATGTCAGTTGAGTTACCAGTGCT6180
AAGTGTTGCTTCCGTTAATGTTATAACAAATTATCAATATATGTAAGTACTAATTTTAAA6240
GAATATTGCTATTAAATAGTAATTAAGCTATCTTTGGATACATAGAAGACCATGGGAAGT6300
GAAAAGTTTCCTTGATAAAAGGAGTTGTGGTTTTCAATATATATATATATTCAAGAATGA6360
CATGAGAAGTCTCTTAATACAGGCCTCCAATGAAAAGGAAATGAAGAATAATTTTCCAAT6420
TACTTTCGAGTAAAAAGTTATCTATGTTCAATATTTTCTTTTCTTTAAAAAAAAGAGAGA6480
ACAGAATAGAAGAATGTATAGATCTGTCTGTTTTTTGTTCTTTGATAACTAGAATAGTGT6540
ATGTTTCAGTTTCTTGGTAAGAGGTTAGATGTGAAGTTCTTATAATACTATAACAAAATA6600
TACTTCATCATTTGAGGGGTGGAGGAGATCTTACAAACAAACTACTGAACCCAATTCCCT6660
TTTACTTTTAGTAACATCCCAATTTTGGTGTTGAGATATTGTGATAAGGTAAAGTTATAT6720
ACTTTGGCCAAAGTAATTTACAGGATGAAGCCTTCTTCTCCCTGATTAGTGACTTTCTGA6780
GGTATTAACATCAGAGTTCTGAAGATTATCCAAAAAAACATCAGAGTTCTGAAATTTATT6840
AGGGCCAGAGATTTAATATTCTTGAAATCCTCAATTGCAGATAATTATGAAATTTAAGAT6900
CAAAATGAAATATCCACAGGATCACTTATTTATGACTAAAATTTGTTTTTGGGGTGACAT6960
2 TCTTCCACATTATTAGAATGCACGTTGTGATGACACTTGCTTTTTCTTGATGGAAATGAT7020
O
GACATGAATGCGCAGTGTCAAATCTACAATTGAAAGGTACAAAAAGGCATGTGCAGATTC7080
TTCCAACAACGGGTCAGTTTCTGAAGCCAATGCTCAGGTATCATTTATCAGCTCTAACAA7140
TTGTTTACATGTGCAGATTCTTCCAACACTTTGTTATAATCCTTTGTGTCCTACTGGTTT7200
TTGGTTTTGGATACTGATTAGTTTGTAATGTATGCACTAGGGCTGAAAAAAGGCATACAG7260
AATTATGATATTATAGAACAAAATTACCAATTAACAGTATTTTTCTTTCTTTTTTAATAA7320
ATTACAGTATAGTTTTTCGTGAATTTATGTGCGATCGAGTGTTTACACTGAATTTCAAAA7380
TGTGCATGTACGTTTTGAGGCTAGTGTAGAACCACAGAAAGACAGTATATATGGAACTAC7440
CAGCATATAACAAAATCCTTTTTATGAAATTTTATCGTCGATGTTTTACACTAAATTCTC7500
TCACTATTCATTAACAGCGTAATTAACAACATGCTGTTAAATTATAGAAGGAGTTCAAGC7560
3 AATATTCCTAACAATCATTTATTGCCAATATTTGTCAAATACCCTTTGTGATAACCTCAT7620
O
TTGTGTAAAATCGATTAAATACATACCTATTTAATTTTGCTTCTCAGAGTGGAGGTTTTT7680
TCCTACTGCATTGGGAGTCATGAGTGTAAACCTGCATTATAGCCAGTTTTGTGTACAGAA7740
ACCCTTTTCCTTCCTCTGTTGCTGTGGCCCTATTGTATCAATTTATTTCCAGTTTGATTC7800
GGTATTATATACATGTTTCCAAGAAGTATAAGAGAGAAATGTACATCACTGATATTTTCT7860
ACTTATATTTTGAGTTCTAATCTGAACTCGAGGATCTTAATCTAGTTATTTATAATGTTT7920
TATTGCCTTTTGCTTTTGCATTTCAGTTTTATCAGCAAGAAGCTGCCAAGCTGCGCTCGC7980
AAATTGGTAATTTGCAGAATTCAAACAGGTATGATCATTTGTGATCTTGATCAATTTGTT8040
AGATAAAATTTGTTTTTCCTCTTCCAAACTCCGTTTAAGCAAATTAATTTTCAGGAATAT8100
GCTGGGTGAATCACTTAGTGCATTGAGTGTGAAGGAACTTAAGAGCTTGGAGATAAAACT8160
4 TGAGAAAGGAATTGGTAGAATTCGTTCGAAAAAGGTCTTTATTCTAGTACTCAAATGATT8220
O
CTCTCTTTTTTTAAGTCAAATATCACTTTAATTTTCCTTGTATTGCCACTAACAAGTTTT8280
GTTTTGTCTTGTTTTCCTTTTGTTTTTTAATTCCTCCCTCAAACCTGCCAGAATGAGCTG8340
TTGTTTGCTGAAATTGAGTATATGCAGAAGAGGGTAACAACTTTTGTGCTCATATTCACC8400
ATGACTTCTTCTATTTGAGATAAAAAAATCAAGTTTTTGCCAATTTAATGATCCTATGGT8460
GAACCTCTTCTATTGTATTTTCACTCCAAAAATTTTCTTTGATTCATTGAATGAAAATGC8520
AAATTGCAGGAGATTGACTTGCACAACAATAACCAGCTTCTCCGAGCAAAGGTCTTTCTA8580
CTTATCTATTTATCAATGCCTTGTGTGTGTCTGAACTTGGATCTTAATATCTTAGATCGT8640
TGGTGGGTTGTTTTTATTTAGTAAATATGACACTACGTGGGGCTTATGTTGATGTTGCAG8700
ATTGCAGAGAATGAAAGAAAGCGACAGCACATGAATTTGATGCCGGGAGGTGTCAACTTC8760
50 GAGATCATGCAGTCTCAACCATTTGACTCTCGGAACTATTCTCAAGTTAATGGATTGCCG8820
CCTGCCAATCATTACCCTCATGAAGACCAGCTCTTCAGTTAGTGTAAGTATTTCCTTTGC8880
AATGAGCTGTAGTTTTTCATCAATTAATTACTGATGAGCATATAATTAACTACTTTGATC8940
TGGATGGGTTTCAGTAGCAGCAGCGGCTGAATGGTTCGTGGTCTGTAAAAATTTATTGGA9000
AGGATATAATAACTGATGCTGTGCCTTCTAATTCTCATAATCATTTGATCTTTCAATTAG9060
TTAGATGATGATTTACGCATTCTTATTGAGATTTTTACCATTGGATGATAAGAGGGAATT9120
GCAATATTTAGCTGTTGTACTAAAAGTAGACTGCTGTTATCAGCACCCCATGCTCACTGA9180
AGAACTAGAAGATTACCCAACCTAGTTTTACTTCACTGAACCGTTTGCATGCAAGAACTT9240
AAAGCGTAATCTGATTTCCCAAGTGACAAGTATATGTTTCTAACTCCTTGACGAATCTGC9300
TGCTGATTCCTTTGCTGGTTTATATTATTCTTATGACTACAAACACAATACTTTTCAACT9360
60 AGCTAGTGAATGAATAATCATTTCCTTATGTTGCAGTTAAAAAGCACCAAGTGCAGCAAC9420
TCCTCGCATTTCCATATTCCATGGAGAGTACCTACTATTTCACTGAGCGCAAAAGCTGCA9480
AGTACGCTAAAACAAAAATCTGAAGTAGCATAACTCAAATTTGTGCCGGTGGAGAGCCTA9540
GTACTCTTCCTCCATGTATTGCTTTTCCAGTCCCAGTTAAGACATAACAAATGTCAGATA9600
AGGATTTCTTTTCTGCATGTTTCATGAAGGCACTAAGATGCTGTGACAGTACTTGTGACT9660
59
CA 02319853 2000-12-22
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
(2) INFORMATION FOR SEQ ID NO.: 14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1159
(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
2 0 (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
30 10 15 20
ATC AAG CGG ATC GAG AAC ACC ACC AAT CGC CAA GTC ACT TTC TGC AAA 212
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
4 O 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 85
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
50 90 95 100
CAG TTC TAT CAG CAA GAA GCT GCC AAG CTG CGC TCG CAA ATT GGT AAT 452
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
60 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
CA 02319853 2000-12-22
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
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
GCCTAGTACT CTTCCTCCAT GTATTGCTTT TCCAGTCCCA GTTAAGACAT AACAAATGTC 1015
AGATAAGGAT TTCTTTTCTG CATGTTTCAT GAAGGCACTA AGATGCTGTG ACAGTACTTG 1075
TGACTAACTT ATTATATATT TTGTCTTATA TTTCTTAAAA AAAAAAAAAA F~~AAAAAAAA 1135
3 0 AAAAAAAAAA AAAAAAAAAA AAAA 1159
(2) INFORMATION FOR SEQ ID NO.: 15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 714
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
4 O (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
1 5 10 15
5 O 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
20 25 30
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
60 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
61
CA 02319853 2000-12-22 .
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
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
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
180 185 190
CGA CAG CAC ATG AAT TTG ATG CCG GGA GGT GTC AAC TTC GAG ATC ATG 624
3 0 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
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
(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.: 16:
Met Ala Tyr Gln Asn Glu Pro Gln Glu Ser Ser Pro Leu Arg Lys Leu
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 Asn Gly Leu Leu Lys Lys Ala Tyr
35 40 45
Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe Ser
50 55 60
62
CA 02319853 2000-12-22
Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn Asn Ser Val Lys Ser Thr
65 70 75 80
Ile Glu Arg Tyr Lys Lys Ala Cys Ala Asp Ser Ser Asn Asn Gly Ser
85 90 95
Val Ser Glu Ala Asn Ala Gln Phe Tyr Gln Gln Glu Ala Ala Lys Leu
100 105 110
Arg Ser Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg Asn Met Leu Gly
115 120 125
Glu Ser Leu Ser Ala Leu Ser Val Lys Glu Leu Lys Ser Leu Glu Ile
130 135 140
Lys Leu Glu Lys Gly Ile Gly Arg Ile Arg Ser Lys Lys Asn Glu Leu
145 150 155 160
Leu Phe Ala Glu Ile Glu Tyr Met Gln Lys Arg Glu Ile Asp Leu His
2 0 165 170 175
Asn Asn Asn Gln Leu Leu Arg Ala Lys Ile Ala Glu Asn Glu Arg Lys
180 185 190
Arg Gln His Met Asn Leu Met Pro Gly Gly Val Asn Phe Glu Ile Met
195 200 205
Gln Ser Gln Pro Phe Asp Ser Arg Asn Tyr Ser Gln Val Asn Gly Leu
210 215 220
Pro Pro Ala Asn His Tyr Pro His Glu Asp Gln Leu Phe Ser
225 230 235
(2) INFORMATION FOR SEQ ID NO.: 17:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
4 O (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:
(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
63
CA 02319853 2000-12-22
(2) INFORMATION FOR SEQ ID NO.: 19:
(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.: 19:
AGGAAGGCGA AGTTCATGGG ATCCAAA 27
(2) INFORMATION FOR SEQ ID NO.: 20:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
2 0 (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.: 20:
3 O TCCACATCGA CAAAGAAGAT CTACGAT 27
(2) INFORMATION FOR SEQ ID NO.: 21:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
4 O (vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(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:
S O (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:
60 oligonucleotide primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 22:
AACTAACTGA AGGGCCATCT GATCTTG 27
64
CA 02319853 2000-12-22
(2) INFORMATION FOR SEQ ID NO.: 23:
(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
1 O ( ix) FEATURE
(C) OTHER INFORMATION: Description of Artificial Sequence:
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
2 0 (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.: 24:
3 O ATTCATGCTC TGTCGCTTTC TTTCATTCT 29
