WO 01/64022 1 PCT/USO1/05454
LEAFY COTYLEDON I GENES AND THEIR USES
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a Continuation-In-Part ("CIP") of United States
Patent Application Serial Number (USSN) 09/193,931, filed November 17, 1998,
which is a
CIP of USSN 09/103,478, filed June 24, 1998, which is a CIP of USSN
09/026,221, filed
February 19, 1998, which is a CIP of USSN 08/804,534, filed February 21, 1997.
Each of
the aforementioned applications is explicitly incorporated herein by reference
in their entirety
and for all purposes.
FIELD OF THE INVENTION
The present invention is directed to plant genetic engineering. In particular,
it
relates to new embryo-specific genes useful in improving agronomically
important plants.
BACKGROUND OF THE INVENTION
Embryogenesis in higher plants is a critical stage of the plant life cycle in
which the primary organs are established. Embryo development can be separated
into two
main phases: the early phase in which the primary body organization of the
embryo is laid
down and the late phase which involves maturation, desiccation and dormancy.
In the early
phase, the symmetry of the embryo changes from radial to bilateral, giving
rise to a hypocotyl
with a shoot meristem surrounded by the two cotyledonary primordia at the
apical pole and a
root meristem at the basal pole. In the late phase, during maturation the
embryo achieves its
maximum size and the seed accumulates storage proteins and lipids. Maturation
is ended by
the desiccation stage in which the seed water content decreases rapidly and
the embryo passes
into metabolic quiescent state. Dormancy ends with seed germination, and
development
continues from the shoot and the root meristem regions.
The precise regulatory mechanisms which control cell and organ
differentiation during the initial phase of embryogenesis are largely unknown.
The plant
hormone abscisic acid (ABA) is thought to play a role during late
embryogenesis, mainly in
the maturation stage by inhibiting germination during embryogenesis (Black, M.
(1991 ). In
Abscisic Acid: Physiology and Biochemistry, W. J. Davies and H. G. Jones, eds.
(Oxford:
Bios Scientific Publishers Ltd.), pp. 99-124) Koornneef, M., and Karssen, C.
M. (1994). In
Arabidopsis, E. M. Meyerowitz and C. R. Sommerville, eds. (Cold Spring Harbor:
Cold
Spring Harbor Laboratory Press), pp. 313-334). Mutations which effect seed
development
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and are ABA insensitive have been identified in Arabidopsis and maize. The ABA
insensitive (abi3) mutant of Arabidopsis and the viviparousl (vpl ) mutant of
maize are
detected mainly during late embryogenesis (McCarty, et al., ( 1989) Plant Cell
1, 523-532 and
Parcy et al., (1994) Plant Cell6, 1567-1582). Both the VPl gene and the ABI3
genes have
been isolated and were found to share conserved regions (Giraudat, J. ( 1995)
C'ur~r~ent
Opinion in Cell Biology 7:232-238 and McCarty, D. R. (1995). Annu. Rev. Plant
Physiol.
Plant Mol. Biol. 46:71-93). The VP1 gene has been shown to function as a
transcription
activator (McCarty, et al., ( 1991 ) Cell 66:895-906). It has been suggested
that ABI3 has a
similar function.
Another class of embryo defective mutants involves three genes: LEAFY
COTYLEDON1 and 2 (LEC1, LEC2) and FUSCA3 (FUS3). These genes are thought to
play a central role in late embryogenesis (Baumlein, et crl. (1994) Plant J.
6:379-387; Meinke.
D. W. (1992) Science 258:1647-1650; Meinke c~t crl.. Plant Cell 6:1049-1064.
West et crl.,
(1994) PlcrrZt Cell 6:1731-1745). Lil:e the abi3 mutant, leafy cotyledon-type
mutants are
defective in late embryogenesis. In these mutants, seed morphology is altered.
the shoot
meristem is activated early, storage proteins are lacking and developing
cotyledons
accumulate anthocyanin. As with abi3 mutants, they are desiccation intolerant
and therefore
die during late embryogenesis. Nevertheless, the immature mutants embryos can
be rescued
to give rise to mature and fertile plants. However, unlike abi3 when the
immature mutants
germinate they exhibit trichomes on the adaxial surface of the cotyledon.
Trichomes are
normally present only on leaves, stems and sepals, not cotyledons. Therefore,
it is thought
that the leafy cotyledon type genes have a role in specifying cotyledon
identity during
embryo development.
Among the above mutants, the lec 1 mutant exhibits the most extreme
phenotype during embryogenesis. For example. the maturation and
postgermination
programs are active simultaneously in the lecl mutant (West et al., 1994),
suggesting a
critical role for LEC 1 in gene regulation during late embryogenesis.
In spite of the recent progress in defining the genetic control of embryo
development, further progress is reduired in the identification and analysis
of genes expressed
specifically in the embryo and seed. Characterization ol~ such scenes would
allow For the
genetic engineering plants with a variety of desirable traits. For instance.
modulation of th c
expression of genes which control embryo development may be used to alter
traits such as
accumulation of storage proteins in leaves and cotyledons. Alternatively,
promoters from
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embryo or seed-specific genes can be used to direct expression of desirable
heterologous
genes to the embryo or seed. The present invention addresses these and other
needs.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the isolation and characterization
of
LEC 1 genes. The invention provides isolated nucleic acid molecules comprising
a LEC 1
polynucleotide sequence which is at least 68% identical to the B domain of SEQ
ID N0:2.
The invention also provides expression cassettes comprising a promoter
operably linked to a heterologous polynucleotide sequence or complement
thereof, encoding
a LEC1 polypeptide comprising a sequence which is at least 68% identical to
the B domain of
SEQ ID N0:2. In some embodiments, the polynucleotide sequence is heterologous
to any
element in the expression cassette. In a preferred embodiment, the B domain
comprises a
polypeptide between about amino acid residue 28 and amino acid residue 117 of
SEQ ID
N0:2. In a more preferred embodiment, the B domain comprises a polypeptide
sequence
with an amino terminus at amino acid residues 28-35 and a carboxy terminus at
amino acid
residues 103-1 17 of SEQ ID N0:2.
In particularly preferred embodiments, the LECI polypeptide is shown in SEQ
ID N0:20 or 22. Such LEC 1 polypeptides can be encoded by the polynucleotide
sequences
shown in SEQ ID N0:19 or SEQ ID N0:21, respectively. In another embodiment,
the LECI
polypeptide is a fusion between two or more LEC 1 polypeptides of polypeptide
subsequences.
The expression cassette comprises a promoter operably linked to the LEC 1
polynucleotide or its complement. For example, the promoter can be a
constitutive promoter.
Alternatively, the promoter can be a promoter from a LEC 1 gene. For instance,
the LEC I
promoter can be fi-om about nucleotide 1 to about nucleotide 1998 of SEQ ID
N0:3. In one
embodiment, the heterologous polynucleotide can be linked to the promoter in
the antisense
orientation. In another embodiment, the promoter is SEQ ID N0:23. The promoter
can
further compromise SEQ ID N0:24.
In another embodiment, the invention provides an expression cassette
comprising a promoter operably linked to a heterologous polvnucleotide
sequence, or
complement thereof, encoding a LEC 1 polypeptide comprising a subsequence at
least 90°~~
identical to the A or C domain of a LEC1 polypeptide. The polynucleotide
sequence can be
heterologous to any element in the expression cassette. Such expression
cassettes can encode
fusions of two or more LEC 1 polypeptides or polypeptide subsequences.
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The invention also provides for an expression cassette for the expression of
heterologous polypeptides in a plant. The expression cassette comprises a LEC
1 promoter
operably linked to a heterologous polynucleotide. In some embodiments, the LEC
1 promoter
is at least 70% identical to SEQ ID N0:23. In some embodiments, the expression
cassette
promoter comprises a promoter at least 70% identical to SEQ ID N0:24.
Preferably, the
promoter comprises the sequence displayed in SEQ ID N0:24.
The invention also provides an isolated nucleic acid or complement thereof;
encoding a LECI polypeptide comprising a subsequence at least 68% identical to
the B
domain of SEQ ID N0:2, with the proviso that the nucleic acid is not clone
MNJ7. In a
preferred embodiment, the B domain comprises a polypeptide sequence with an
amino
terminus at amino acids 28-35 and a carboxy terminus at amino acids 103-I 17
of SEQ ID
N0:2. In another embodiment, the LEC1 polypeptide is shown in SEQ ID NO: 20 or
SEQ
ID N0:22. Such LECl polypeptides can be encoded by the polynucleotide
sequences shown
in SEQ ID N0:19 or SEQ ID N0:21, respectively. In another embodiment, the LEC1
polypeptide is a fusion between two or more LEC 1 polypeptides of polypeptide
subsequences.
The isolated nucleic acid can further compromise a promoter operably linked
to the LEC1-encoding nucleic acid. The promoter can be a constitutive
promoter.
Alternatively, the promoter can be a promoter from a LEC 1 gene. For instance,
the LEC 1
promoter can be from about nucleotide 1 to about nucleotide 1998 of SEQ ID
N0:3. In one
embodiment, the heterologous polynucleotide can be linked to the promoter in
the antisense
orientation.
The invention provides a host cell comprising expression cassettes or nucleic
acids of the invention. Thus, in one embodiment, the host cells of the
invention comprise an
expression cassette comprising a promoter operably linked to a heterologous a
polynucleotide
sequence, or complement thereof, encoding a LEC 1 polypeptide with a
subsequence at least
68% identical to the B domain of SEQ ID N0:2. In other embodiments, the host
cell of the
invention comprises an expression cassette comprising a promoter operable
linked to a
heterologous polynucleotide sequence or complement thereof, encoding a LEC 1
polypeptide
with a subsequence at least 90% identical to the A or C domain of a LEC1
polypeptide.
Other embodiments include hosts cells comprising an expression cassette
comprising a
promoter at least 70% identical to SEQ ID N0:23 or an isolated nucleic acid
comprising a
subsequence at least 68% identical to the B domain of SEQ ID N0:2, so long as
the nucleic
acid is not clone MNJ7.
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The invention also provides isolated polypeptides comprising amino acid
sequences at least 68% identical to the B domain of SEQ ID N0:2 and capable of
exhibiting
at least one of the biological activities of the polypeptides encoded in SEQ
ID NO:1. SEQ ID
N0:19 or SEQ ID N0:21, or a fragment thereof. Antibodies capable of binding
the above-
described polypeptide are also provided.
Also provided are methods of introducing an isolated nucleic acid into a host
cell. The method comprises providing an expression cassette of nucleic acid of
the invention
as described herein and contacting the expression cassette or nucleic acid
with the host cell
under conditions that permit insertion of the nucleic acid into the host cell.
The invention also provides transgenic plant cells or plants comprising an
expression cassette comprising a promoter operably linked to a heterologous
polynucleotide
sequence, or complement thereof, encoding a LEC 1 polypeptide comprising a
sequence
which is at least 68% identical to the B domain of SEQ ID N0:2. In a preferred
embodiment,
the LEC1 polypeptide is shown in SEQ ID NO: 20 or SEQ ID N0:22. Such LEC1
polypeptides can be encoded by the polynucleotide sequences shown in SEQ ID
NO:19 or
SEQ ID N0:21, respectively. The invention also provides plants that are
regenerated from
the plant cells discussed above.
The expression cassette promoter can be a constitutive promoter.
Alternatively, the promoter can be a promoter from a LEC I gene. For instance,
the LEC 1
promoter can be from about nucleotide 1 to about nucleotide 1998 of SEQ ID
N0:3. In one
embodiment, the heterologous polynucleotide can be linked to the promoter in
the antisense
orientation. In another embodiment. the promoter is SEQ ID N0:23. The promoter
can also
further comprise SEQ ID N0:24.
The invention also provides an expression cassette for the expression of a
heterologous polynucleotide in a plant cell, comprising a promoter
polynucleotide at least
70% identical to SEQ ID N0:23, wherein the promoter polynucleotide is operably
linked to a
heterologous polynucleotide. In one embodiment, the promoter polynucleutide is
SEQ ID
N0:23. The promoter can also further comprise a polynucleotide at least 70%
identical to
SEQ ID N0:24. In a preferred embodiment, the promoter comprises SEQ ID N0:24.
The invention also provides methods of modulating transcription comprising,
introducing into the plant an expression cassette containing a plant promoter
operably linked
to a heterologous LECl polynucleotide, the heterologous LEC'I polynucleotide
encoding a
LEC 1 polypeptide comprising a subsequence at least 68% identical to the B
domain of SEQ
ID N0:2 and detecting a plant with modulated transcription. Embodiments of
these methods
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include where the LEC1 polynucleotide is SEQ ID N0:2, SEQ ID N0:20 or SEQ ID
N0:22.
In other embodiments, the LEC1 polypeptides are encoded by SEQ ID NO:1. SEQ ID
N0:19
or SEQ ID N0:21. Preferred embodiments of the invention include the method
where
transcription modulation results in induction of embyonic characteristics in a
plant. In an
alternative embodiment, transcription modulation results in induction of seed
development.
The invention also provides a method of detecting a nucleic acid in a sample.
The
method comprises providing an isolated LEC1 nucleic acid molecule comprising a
polynucleotide sequence, or complement thereof, encoding a LEC 1 polypeptide
with a
subsequence at least 68% identical to the B domain of SEQ ID N0:2., contacting
the isolated
nucleic acid molecule with a sample under conditions which permit a comparison
of the
sequence of the isolated nucleic acid molecule with the sequence of DNA in the
sample; and
analyzing the result of the comparison. In some embodiments, the isolated
nucleic acid
molecule and the sample are contacted under conditions which permit the
formation of a
duplex between complementary nucleic acid sequences.
Definitions
The phrase "nucleic acid" refers to a single or double-stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
Nucleic acids may
also include modified nucleotides that permit correct read through by a
polymerise and do
not alter expression of a polypeptide encoded by that nucleic acid.
The phrase "polynucleotide sequence" or "nucleic acid sequence" includes
both the sense and antisense strands of a nucleic acid as either individual
single strands or in
the duplex. It includes, but is not limited to, self replicating plasmids,
chromosomal
sequences, and infectious polymers of DNA or RNA.
The phrase "nucleic acid sequence encoding" refers to a nucleic acid which
directs the expression of a specific protein or peptide. The nucleic acid
sequences include
both the DNA strand sequence that is transcribed into RNA and the RNA sequence
that is
translated into protein. The nucleic acid sequences include both the full
length nucleic acid
sequences as well as non-full length sequences derived from the full length
sequences. It
should be further understood that the sequence includes the degenerate colons
of the native
sequence or sequences which may be introduced to provide colon preference in a
specific
host cell.
The term "promoter" refers to a region or sequence determinants located
upstream or downstream from the start of transcription and which are involved
in recognition
and binding of RNA polymerise and other proteins to initiate transcription. A
"plant
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promoter" is a promoter capable of initiating transcription in plant cells.
Such promoters
need not be of plant origin, for example, promoters derived from plant
viruses, such as the
CaMV35S promoter, can be used in the present invention.
The term "plant" includes whole plants, shoot vegetative organs/structures
(e.g. leaves, stems and tubers), roots, flowers and floral organs/structures
(e.~>. bracts, sepals,
petals, stamens. carpets, anthers and ovules), seed (including embryo,
endosperm, and seed
coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground
tissue, and the
like) and cells (e.g. guard cells, egg cells, trichomes and the like), and
progeny of same. The
class of plants that can be used in the method of the invention is generally
as broad as the
class of higher and lower plants amenable to transformation techniques,
including
angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms. ferns,
and
multicellular algae. It includes plants of a variety of ploidy levels,
including aneuploid-
polyploid. diploid, haploid and hemizygous.
A polynucleotide sequence is "heterologous to" an organism or a second
polynucleotide sequence if it originates from a foreign species, or. if from
the same species, is
modified from its original form. For example, a promoter operably linked to a
heterologous
coding sequence refers to a coding sequence from a species different from that
from which
the promoter was derived, or, if from the same species, a coding sequence
which is different
from any naturally occurring allelic variants. As defined here, a modif ed LEC
I coding
sequence which is heterologous to an operably linked LEC 1 promoter does not
include the ~I~-
DNA insertional mutants as described in West et al.. The Plcrnt C.'ell 6:1731-
174 ( 1994).
A polynucleotide "exogenous to" an individual plant is a polynucleotide which
is introduced into the plant by any means other than by a sexual cross.
Examples of means
by which this can be accomplished are described below, and include
Agrobacterium-
mediated transformation, biolistic methods, electroporation, in planta
techniques, and the
like. Such a plant containing the exogenous nucleic acid is referred to here
as an R,
generation transgenic plant. Transgenic plants which arise from sexual cross
or by selfin~a arc
descendants of such a plant.
As used herein an "embryo-specific gene" or "seed specific gene" is a ~lene
that is preferentially expressed during embryo development in a plant. For
purposes of this
disclosure, embryo development begins with the first cell divisions in the
zygote and
continues through the late phase of embryo development (characterized by
maturation-
desiccation, dormancy), and ends with the production of a mature and
desiccated seed.
Embryo-specific genes can be further classified as "early phase-specific" and
"late phase-
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specific". Early phase-specific genes are those expressed in embryos up to the
end of embryo
morphogenesis. Late phase-specific genes are those expressed from maturation
through to
production of a mature and desiccated seed.
A "LEC1 polynucleotide" is a nucleic acid sequence comprising (or consisting
of) a coding region of about 100 to about 900 nucleotides, sometimes from
about 300 to
about 630 nucleotides, which hybridizes to SEQ ID NO:I under stringent
conditions (as
defined below), or which encodes a LEC1 polypeptide. LEC1 polynucleotides can
also be
identified by their ability to hybridize under low stringency conditions
(e.g., Tm ~40°C) to
nucleic acid probes having a sequence from position 1 to 81 in SEQ ID NO:1 or
from
position 355 to 627 in SEQ ID NO:1.
A "promoter from a LEC 1 gene" or ''LEC 1 promoter" wil l typically be about
500 to about 2000 nucleotides in length. usually from about 750 to 1500.
Exemplary
promoter sequences are shown as nucleotides 1-1998 of SEQ ID N0:3 or as SEQ ID
N0:23.
A LECI promoter can also be identified by its ability to direct expression in
all, or essentially
all, proglobular embryonic cells, as well as cotyledons and axes of a late
embryo.
A "LEC1 polypeptide'' is a sequence of about 50 to about 210, sometimes 100
to 150, amino acid residues encoded by a LEC1 polynucleotide. A full length
LEC1
polypeptide and fragments containing a CCAAT binding factor (CBF) domain can
act as a
subunit of a protein capable of acting as a transcription factor in plant
cells. LEC 1
polypeptides are often distinguished by the presence of a sequence which is
required for
binding the nucleotide sequence: CCAAT. In particular, a short region of seven
residues
(MPIANVI) at residues 34-40 of SEQ ID NO: 3 shows a high degree of similarity
to a region
that has been shown to required for binding the CCAAT box. Similarly. residues
61-72 of
SEQ ID NO: 3 (IQECVSEYISFV) is nearly identical to a region that contains a
subunit
interaction domain (Ring. et al.. ( 1993 ) EMl3() J. 12:4647-4655).
As used herein, a homolog of a particular embryo-specific gene (e.g.. SEQ ID
NO:1 ) is a second gene in the same plant type or in a different plant type.
which has a
polynucleotide sequence of at least 50 contiguous nucleotides which are
substantially
identical (determined as described below) to a sequence in the first gene. It
is believed that,
in general. homologs share a common evolutionary past.
"Increased or enhanced LEC 1 activity or expression of the LEC'1 gene" refers
to an augmented change in LEC1 activity. Examples of such increased activity
or expression
include the following. LEC 1 activity or expression of the LECI gene is
increased above the
level of that in wild-type, non-trans'genic control plants (i.e. the quantity
of LEC 1 activity or
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expression of the LECl gene is increased). LEC1 activity or expression of the
LECI gene is
in an organ, tissue or cell where it is not normally detected in wild-type,
non-transgenic
control plants (i.e. spatial distribution of LEC1 activity or expression of
the LEC'I gene is
increased). LEC 1 activity or expression is increased when LEC 1 activity or
expression of the
LECI gene is present in an organ, tissue or cell for a longer period than in a
wild-type, non-
transgenic controls (i.e. duration of LEC 1 activity or expression of the
LEC'I gene is
increased).
A "polynucleotide sequence from" a particular embryo-specific gene is a
subsequence or full length polynucleotide sequence of an embryo-specific gene
which, when
present in a transgenic plant. has the desired effect, for example. inhibiting
expression of the
endogenous gene driving expression of an heterologous polynucleotide. A full
length
sequence of a particular gene disclosed here may contain about 95%, usually at
least about
98% of an entire sequence shown in the Sequence Listing. below.
The term "reproductive tissues" as used herein includes fiwit, ovules, seeds.
pollen, pistols, flowers, or any embryonic tissue.
In the case of both expression of transgenes and inhibition of endogenous
genes (e.g., by antisense, or sense suppression) one of shill will recognize
that the inserted
polynucleotide sequence need not be identical and may be "substantially
identical" to a
sequence of the gene from which it was derived. As explained below, these
variants are
specifically covered by this term.
In the case where the inserted polynucleotide sequence is transcribed and
translated to produce a functional polypeptide. one of shill will recognize
that because of
codon degeneracy a number of polynucleotide sequences will encode the same
polypeptide.
These variants are specifically covered by the term "polynucleotide sequence
from" a
particular embryo-specific gene, such as LEC 1. In addition, the term
specifically includes
sequences (e.g., full length sequences) substantially identical (determined as
described
below) with a LEC 1 gene sequence and that encode proteins that retain the
function of a
LEC I polypeptide.
In the case of polynucleotides used to inhibit expression of an endogenous
gene, the introduced sequence need not be perfectly identical to a sequence of
the target
endogenous gene. The introduced polynucleotide sequence will typically be at
least
substantially identical (as determined below) to the target endogenous
sequence.
Two nucleic acid sequences or polypeptides are said to be "identical" if the
sequence of nucleotides or amino acid residues. respectively, in the two
seduences is the
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same when aligned for maximum correspondence as described below. The term
"complementary to" is used herein to mean that the sequence is complementary
to all or a
portion of a reference polynucleotide sequence.
Optimal alignment of sequences for comparison may be conducted by the
local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 ( 1981 ),
by the
homology alignment algorithm of Needle man and Wunsch J Nlol. Biol. 48:443 (
1970), by
the search for similarity method of Pearson and Lipman P~°oc. Natl.
Acad Sci. (U.S.A.) 85:
2444 (1988), by computerized implementations of these algorithms (GAP,
BESTFIT,
BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
"Percentage of sequence identity" is determined by comparing two optimally
aligned sequences over a comparison window, wherein the portion of the
polynucleotide
sequence in the comparison window may comprise additions or deletions (i.e.,
gaps) as
compared to the reference sequence (which does not comprise additions or
deletions) for
optimal alignment of the two sequences. The percentage is calculated by
determining the
number of positions at which the identical nucleic acid base or amino acid
residue occurs in
both sequences to yield the number of matched positions, dividing the number
of matched
positions by the total number of positions in the window of comparison and
mllltlplylng the
result by 100 to yield the percentage of sequence identity.
The term "substantial identity" of polynucleotide sequences means that a
polynucleotide comprises a sequence that has at least 25% sequence identity.
Alternatively,
percent identity can be any integer from 25% to 100%. More preferred
embodiments include
at least: 25%, 30%, 35%. 40%, 45%, 50%, _55%. 60%, 65%, 70%, 75%, 80%, 85%.
90%.
95%, or 99%. compared to a reference sequence using the programs described
herein;
preferably BLAST using standard parameters, as described below. Accordingly,
LEC 1
sequences of the invention include nucleic acid sequences that have
substantial identity to
SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:19 and SEQ ID N0:21. LEC1
sequences of the invention include poly peptide sequences having substantial
identify to SEQ
ID N0:2, SEQ ID N0:20 or SEQ ID N0:22. One of skill will recognize that these
values can
be appropriately adjusted to determine corresponding identity of proteins
encoded by two
nucleotide sequences by taking into account codon degeneracy, amino acid
similarity,
reading frame positioning and the like. Substantial identity of amino acid
sequences for these
purposes normally means sequence identity of at least 40%. Preferred percent
identity of
polypeptides can be any integer from 40% to 100%. More preferred embodiments
include at
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least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Most preferred
embodiments
include 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% and 75%. Polypeptides which are
"substantially similar" share sequences as noted above except that residue
positions which are
not identical may differ by conservative amino acid changes. Conservative
amino acid
substitutions refer to the interchangeability of residues having similar side
chains. For
example, a group of amino acids having aliphatic side chins is glycine,
alanine, valine,
leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side
chains is
serine and threonine; a group of amino acids having amide-containing side
chains is
asparagine and glutamine; a group of amino acids having aromatic side chains
is
phenylalanine, tyrosine, and tryptophan; a group of amino acids havin<~ basic
side chains is
lysine, arginine, and histidine; and a group of amino acids having sulfur-
containing side
chains is cysteine and methionine. Preferred conservative amino acids
substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,
alanine-valine-
aspartic acid-glutamic acid, and asparagine-glutamine.
Another indication that nucleotide sequences are substantially identical is if
two molecules hybridize to each other. or a third nucleic acid, under
stringent conditions.
Stringent conditions are sequence dependent and will be different in different
circumstances.
Generally, stringent conditions are selected to be about 5"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
seduence
hybridizes to a perfectly matched probe. Typically- stringent conditions will
be those in
which the salt concentration is about 0.02 molar at pH 7 and the temperature
is at least about
60"C.
In the present invention. mRNA encoded by embryo-specific genes of the
invention can be identified in Northern blots under stringent conditions using
cDNAs of the
invention or fragments of at least about 100 nucleotides. For the purposes of
this disclosure.
stringent conditions for such RNA-DNA hybridizations are those which include
at least one
wash in 0.2X SSC at 63°C for 20 minutes, or equivalent conditions.
Genomic DNA or cDNA
comprising genes of the invention can be identified using the same cDNAs (or
fragments of
at least about 100 nucleotides) under stringent conditions- which for purposes
of this
disclosure, include at least one wash (usually 2) in 0.2X SSC at a temperature
of at least
about 50°C, usually about 55°C, for 20 minutes. or equivalent
conditions.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A shows a schematic representation of the three domains of the LEC 1
polypeptide. Figure 2B shows a comparison of the predicted amino acid sequence
of the B
domain encoded by LEC 1 with HAP3 homologs from maize, chicken, lamprey,
Xenopus
laveis, human, mouse, rat, Emericella nidulans, Schizosaccharomyces pombe,
Saccharomyces cerevisiae, and Kluyveromyces lactis. The DNA-binding region and
the
subunit interaction region are indicated. Numbers indicate amino acid
positions of the B
domains.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention provides new embryo-specific genes useful in
genetically engineering plants. Polynucleotide sequences from the genes of the
invention can
be used, for instance, to direct expression of desired heterologous genes in
embryos (in the
case of promoter sequences) or to modulate development of embryos or embyonic
characteristics on other organs (e.g., by enhancing expression of the gene in
a transgenic
plant). In particular, the invention provides a new gene from Arabidopsis
referred to here as
LEC 1. LEC 1 encodes polypeptides which subunits of a protein which acts as a
transcription
factor. Thus, modulation of the expression of this gene can be used to
manipulate a number
of useful traits, such as increasing or decreasing storage protein content in
cotyledons or
leaves.
Generally, the nomenclature and the laboratory procedures in recombinant
DNA technology described below are those well known and commonly employed in
the art.
Standard techniques are used for cloning, DNA and RNA isolation. amplification
and
purification. Generally enzymatic reactions involving DNA ligase, DNA
polymerase,
restriction endonucleases and the like are performed according to the
manufacturer's
specifications. These techniques and various other techniques are generally
performed
according to Sambrook et al., Molecular Cloning - A Laboratory Manual, 2nd.
ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor. New York, (1989).
CA 02399886 2002-08-22
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Isolation of nucleic acids of the invention
The isolation of sequences from the genes of the invention may be
accomplished by a number of techniques. For instance, oligonucleotide probes
based on the
sequences disclosed here can be used to identify the desired gene in a cDNA or
genomic
DNA library from a desired plant species. To construct genomic libraries,
large segments of
genomic DNA are generated by random fragmentation, e.g. using restriction
endonucleases,
and are ligated with vector DNA to form concatemers that can be packaged into
the
appropriate vector. To prepare a library of embryo-specific cDNAs, mRNA is
isolated from
embryos and a cDNA library that contains the gene transcripts is prepared from
the mRNA.
I 0 The cDNA or genomic library can then be screened using a probe based upon
the sequence of a cloned embryo-specific gene such as the polynucleotides
disclosed here.
Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate
homologous genes in the same or different plant species.
Alternatively, the nucleic acids of interest can be amplified from nucleic
acid
samples using amplification techniques. For instance, polymerase chain
reaction (PCR)
technology to amplify the sequences of the genes directly from mRNA, from
cDNA, from
genomic libraries or cDNA libraries. PCR and other in vitro amplification
methods may also
be useful, for example, to clone nucleic acid sequences that code for proteins
to be expressed,
to make nucleic acids to use as probes for detecting the presence of the
desired mRNA in
samples, for nucleic acid sequencing, or for other purposes.
Appropriate primers and probes for identifying embryo-specific genes from
plant tissues are generated from comparisons of the sequences provided herein.
For a general
overview of PCR see PCR Protocols: A Guide to Methods and Applications.
(Innis, M.
Gelfand, D., Sninsl<y, J. and White, T., eds.), Academic Press, San Diego (
1990).
Appropriate primers for this purpose include, for instance: UP primer - 5' GGA
ATT CAG
CAA CAA CCC AAC CCC A 3" and LP primer - 5' LP primer - 5' GCT CTA GAC ATA
CAA CAC TTT TCC TTA 3'. Alternatively, the following primer pairs can be used:
ATG ACC AGC TCA GTC ATA GTA GC 3' and 5' GCC ACA CAT GGT GGT TGC TGC
TG 3' or 5' GAG ATA GAG ACC GAT CGT GGT TC 3' and 5' TCA CTT ATA CTG ACC
ATA ATG GTC 3'. A third set of primers include: ~'-AGG ATC CAT GGA ACG TGG
AGG CTT CCA T-3' and ~"-ATC TAG ATC AGT ACT TAT GTT GTT GAG TCG-3'. The
amplifications conditions are typically as follows. Reaction components: 10 mM
Tris-HC1.
pH 8.3, 50 mM potassium chloride, 1.~ mM magnesium chloride, 0.001 % gelatin,
200
microM (uM) dATP, 200 microM dCTP, 200 microM dGTP. 200 microM dTTP, 0.4
microM
CA 02399886 2002-08-22
WO 01/64022 14 PCT/USO1/05454
primers, and 100 units per ml Taq polymerise. Program: 96 C for 3 min., 30
cycles of 96 C
for 45 sec., 50 C for 60 sec., 72 for 60 sec, followed by 72 C for 5 min.
Polynucleotides may also be synthesized by well-known techniques as
described in the technical literature. See, e.g., Carruthers et u1., Cold
Spring Harbor Symp.
Quint. l3iol. 47:411-418 (1982), and Adams et al., J. Am. Chem. S'oc. 105:661
(1983).
Double stranded DNA fragments may then be obtained either by synthesizing the
complementary strand and annealing the strands together under appropriate
conditions, or by
adding the complementary strand lISlIlg DNA polymerise with an appropriate
primer
sequence.
Analysis of LEC1 Gene Sequences
The genus of LECl nucleic acid sequences of the invention includes genes and
gene products identified and characterized by analysis using the sequences
nucleic acid
sequences, including SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:19 and
SEQ
ID N0:21, and protein sequences, including SEQ ID N0:2. SEQ ID N0:20 and SEQ
ID
N0:22. LEC1 sequences of the invention include nucleic acid sequences having
substantial
identity to SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:19 and SEQ ID
N0:21.
LEC 1 sequences of the invention include polypeptide sequences having
substantial identify
to SEQ ID N0:2, SEQ ID N0:20 and SEQ ID N0:22.
LEC1 nucleic acid sequences also include Iusions between two or more LECI
genes. Different domains of different genes can be fused. LECI gene fusions
can be linked
directly or can be attached by additional amino acids that link the two of
more fusion
partners.
Gene fusions can be generated by basic recombinant DNA techniques as
described below. Selection of gene Iusions will depend on the desired
phenotype caused by
the gene fusion. For instance, if phenotypes associated with the A domain of
one LEC I
protein are desired with phenotypes associated with the B domain of a second
LEC 1 protein.
the a fusion of the first LEC I protein's A domain to the second LEC 1's B
domain would be
created. The fusion can subsequently be tested in vitro or in vioo for the
desired phenotypes.
Use of nucleic acids of the invention to inhibit gene expression
The isolated sequences prepared as described herein, can be used to prepare
expression cassettes useful in a number of techniques. For example, expression
cassettes of
the invention cam be used to suppress endogenous LECl gene expression.
IWibiting
expression can be useful, for instance, in weed control (by transferring an
inhibitory sequence
CA 02399886 2002-08-22
WO 01/64022 15 PCT/USO1/05454
to a weedy species and allowing it to be transmitted through sexual crosses)
or to produce
fruit with small and non-viable seed.
A number of methods can be used to inhibit gene expression in plants. For
instance, antisense technology can be conveniently used. To accomplish this, a
nucleic acid
segment from the desired gene is cloned and operably linked to a promoter such
that the
antisense strand of RNA will be transcribed. The expression cassette is then
transformed into
plants and the antisense strand of RNA is produced. In plant cells, it has
been suggested that
antisense RNA inhibits gene expression by preventing the accumulation of mRNA
which
encodes the enzyme of interest, see, e.g., Sheehy et ul., Pr°oc. lVcrt.
Accrd. Sci. USA,
85:8805-8809 (1988), and Hiatt et crl., U.S. Patent No. 4,801,340.
The antisense nucleic acid sequence transformed into plants will be
substantially identical to at least a portion of the endogenous embryo-
specil7c gene or genes
to be repressed. The sequence, however, does not have to be perfectly
identical to inhibit
expression. The vectors of the present invention can be designed such that the
inhibitory
effect applies to other proteins within a family of genes exhibiting homology
or substantial
homology to the target gene.
For antisense suppression, the introduced sequence also need not be full
length
relative to either the primary transcription product or fully processed mRNA.
Generally,
higher homology can be used to compensate for the use of a shorter sequence.
Furthermore.
the introduced sequence need not have the same intron or exon pattern, and
homology of non-
coding segments may be equally effective. Normally. a seduence of between
about 30 or 40
nucleotides and about full length nucleotides should be used. though a
sequence of at least
about 100 nucleotides is preferred, a sequence of at least about 200
nucleotides is more
preferred, and a sequence of at least about 500 nucleotides is especially
preferred.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression
of embryo-specific genes. It is possible to design ribozymes that specifically
pair with
virtually any target RNA and cleave the phosphodiester backbone at a specific
location,
thereby functionally inactivating the target RNA. In carrying out this
cleavage, the ribozyme
is not itself altered, and is thus capable of recycling and cleaving other
molecules, making it a
true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers
RNA-cleaving activity upon them, thereby increasing the activity of the
constructs.
A number of classes of ribozymes have been identified. One class of
ribozymes is derived from a number of small circular RNAs that are capable of
self-cleavage
and replication in plants. The RNAs replicate either alone (viroid RNAs) or
with a helper
CA 02399886 2002-08-22
WO 01/64022 16 PCT/USO1/05454
virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid
and the
satellite RNAs from tobacco ringspot virus, lueerne transient streak virus,
velvet tobacco
mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle
virus. The
design and use of target RNA-specific ribozymes is described in Haseloff et
al. Ncrtur°e,
334:585-591 (1988).
Another method of suppression is sense suppression. Introduction of
expression cassettes in which a nucleic acid is configured in the sense
orientation with respect
to the promoter has been shown to be an effective means by which to block the
transcription
of target genes. For an example of the use of this method to modulate
expression of
endogenous genes see, Napoli et al., The Plcrnt Cell 2:279-289 (1990). and
U.S. Patents Nos.
5,034,323, 5,231,020, and 5,283,184.
Generally, where inhibition of expression is desired, some transcription of
the
introduced sequence occurs. The effect may occur where the introduced sequence
contains
no coding sequence per se, but only intron or untranslated sequences
homologous to
sequences present in the primary transcript of the endogenous sequence. The
introduced
sequence generally will be substantially identical to the endogenous sequence
intended to be
repressed. This minimal identity will typically be greater than about GS%, but
a higher
identity might exert a more effective repression of expression of the
endogenous seduences.
Substantially greater identity of more than about 80% is preferred, though
about 95% to
absolute identity would be most preferred. As with antisense regulation, the
effect should
apply to any other proteins within a similar family of genes exhibiting
homology or
substantial homology.
For sense suppression, the introduced sequence in the expression cassette,
needing less than absolute identity, also need not be full length, relative to
either the primary
transcription product or fully processed mRNA. This may be preferred to avoid
concurrent
production of some plants which are overexpressers. A higher identity in a
shorter than full
length sequence compensates for a longer, less identical sequence.
Furthermore, the
introduced sequence need not have the same intron or exon pattern, and
identity of non-
coding segments will be equally effective. Normally, a sequence of the size
ranges noted
above for antisense regulation is used.
One of skill in the a.rt will recognize that using technology based on
specific
nucleotide sequences (e.~J.. antisense or sense suppression technology),
families of
homologous genes can be suppressed with a sin<~le sense or antisense
transcript. For
instance, if a sense or antisense transcript is designed to have a sequence
that is conserved
CA 02399886 2002-08-22
WO 01/64022 1 ~ PCT/USO1/05454
among a family of genes (e.g., the B domain of LEC1), then multiple members of
a gene
family can be suppressed. Conversely, if the goal is to only suppress one
member of a
homologous gene family, then the sense or antisense transcript should be
targeted to
sequences with the most vairance between family members. For instance, an
antisense
transcript identical to the A and C domains of LEC 1 can be used to suppress
LEC 1 without
suppressing related genes such as described in SEQ ID N0:19 or SEQ ID N0:21.
Another means of inhibiting LEC 1 function in a plant is by creation of
dominant negative mutations. In this approach, non-functional, mutant LEC 1
polypeptides,
which retain the ability to interact with wild-type subunits are introduced
into a plant.
Identification of residues that can be changed to create a dominant negative
can be
determined by published work examining interaction of different subunits of
CBF homologs
from different species (see, e.g., Sinha et al., ( 1995). Proc. Nall. Accrcl
Sci. USA
92:1624-1628. j
Use of nucleic acids of the invention to enhance gene expression
Isolated sequences prepared as described herein can also be used to prepare
expression cassettes which enhance or increase endogenous LEC1 gene
expression. Where
overexpression of a gene is desired, the desired gene from a different species
may be used to
decrease potential sense suppression effects. Enhanced expression of LEC 1
polynucleotides
is useful, for example, to increase storage protein content in plant tissues.
Such techniques
may be particularly useful for improving the nutritional value of plant
tissues.
Any of a number of means well known in the art can be used to increase LEC 1
activity in plants. Enhanced expression is useful, for example, to induce
embyonic
characteristics in plants or plant organs. Any organ can be targeted, such as
shoot ve<~etative
organs/structures (e.g. leaves, stems and tubers), roots, l7owers and floral
organs/structures
(e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed
(including embryo,
endosperm, and seed coat) and fruit. Alternatively, one or several LEC 1 genes
can be
expressed constitutively (e.g., using the CaMV 35S promoter).
One of skill will recognize that the polypeptides encoded by the genes of the
invention, like other proteins, have different domains which perform different
functions.
Thus, the gene sequences need not be full len~~th, so long as the desired
functional domain of
the protein is expressed. As explained above, LEC 1 polypeptides are related
to CCAAT
box-binding factor (CBF) proteins. CBFs are highly conserved family of
transcription factors
that regulate gene activity in ettl<aryotic organisms (see, e.g" Mantvani (7
992) Narcl. Acido
CA 02399886 2002-08-22
WO 01/64022 18 PCT/USO1/05454
Res. 20:1087-1091; Li (1992) Nucleic Acids Res. 20:1087-1091). LEC1 was found
to have
high similarity to a portion of the HAP3 subunit of CBF. Thus, without being
bound to any
particular theory or mechanism, LEC 1 is likely to act as a transcriptional
modulator. HAP3
is divided into three domains, an amino terminal A domain, a central B domain,
and a
carboxyl terminal C domain, as shown diagrammatically in Figure 2A.
Specifically, LEC 1.
has between about 75% and 85% sequence similarity, which is equivalent to 55%
to 63%
sequence identity, with the B domains of the other HAP3 homologs shown in
Figure 2B; see
also, Example 1, below. Figure 2B shows the amino acid sequence homology
between LEC1
and other CBF homologs.
The LEC 1 polypeptide also has an amino terminal A domain, a central B
domain, and a carboxyl terminal C domain. The three domains of the LEC 1
polypeptide are
defined as follows: in SEQ ID N0:2, the A domain is located between about
amino acid
position 1 to about position 27; the B domain is located between about amino
acid position 28
to about position 117; and, the C domain is located between about position 1
18 to about
position 208. The B domain of LEC 1, L 1 L and Phcr.seolus L 1 L are all close
1y related,
whereas the A and C domains display almost no homology to each other.
The nucleotide sequence for LECI corresponding to each domain is displayed
in SEQ ID NO 1, e.g., the A domain is located between about nucleotide
position I to about
nucleotide position 82; the B domain is located between about nucleotide
position 83 to about
nucleotide position 351; the C domain is located between about nucleotide
position 352 to
about nucleotide position 624.
One of skill in the art will recognize that the domain boundaries are
approximate. The boundaries for the domains of the LEC 1 polypeptides and
nucleotides can
vary from 1 to 20 amino acids residues (1-60 nucleotides) from the boundaries
listed above.
The DNA binding activity, and, therefore, transcription activation function,
ol~
LECI polypeptides is thought to be modulated by a short region of seven
residues, MPIANVI
(found, e.g., at residues 34-40 of SEQ ID NO: 2). Thus, the polypeptides of
the invention
will often retain these sequences.
Modification of endogenous LEC1 genes
Methods for introducing genetic mutations into plant genes and selecting
plants with desired traits are well known. For instance, seeds or other plant
material can be
treated with a mutagenic chemical substance, according to standard techniques.
Such
chemical substances include, but are not limited to, the following: diethyl
sulfate, ethylene
CA 02399886 2002-08-22
WO 01/64022 19 PCT/USO1/05454
imine, ethyl methanesulfonate and N-nitroso-N-ethylurea. Alternatively,
ionizing radiation
from sources such as, X-rays or gamma rays can be used.
Modified protein chains can also be readily designed utilizing
vat°ious
recombinant DNA techniques well known to those skilled in the art and
described for
instance, in Sambrook et al., supra. Hydroxylamine can also be used to
introduce single base
mutations into the coding region of the gene (Sikorski, et u1.. (1991). Meth.
Enzymol. 194:
302-318). For example, the chains can vary from the naturally occurring
sequence at the
primary structure level by amino acid substitutions, additions, deletions, and
the like. These
modifications can be used in a number of combinations to produce the final
modified protein
chain.
Alternatively, homologous recombination can be used to induce targeted gene
modifcations by specifically targeting the LECl gene in vivo (.see,
~renerully, Grewal and
Klar, Genetics 146: 1221-1238 (1997) and Xu W crl., Genes Dev. 10: 241 1-2422
(1996)).
Homologous recombination has been demonstrated in plants (Puchta et crl.,
Experienticr 50:
277-284 (1994), Swoboda et crl., EMBO.I. 13: 484-489 (1994); Ofli-inga et
crl.. Proc. Ncrtl.
Acad Sci. USA 90: 7346-7350 (1993); and Kempin et crl. Nature 389:802-803
(1997)).
In applying homologous recombination technology to the genes of the
invention, mutations in selected portions of an LEC'l gene sequences
(including 5' upstream.
3' downstream, and intragenic regions) such as those disclosed here are made
in vitro and
then introduced into the desired plant using standard techniques. Since the
efficiency of
homologous recombination is known to be dependent on the vectors used, use of
dicistronic
gene targeting vectors as described by Mountford c~i crl., I'roc. Ncrtl.
Ac~crcl. fci. US .9 91: 430 3-
4307 (1994); and Vaulont et crl., Tr~crn.sgenic Re.s. 4: 247-255 (1995) are
conveniently used to
increase the efficiency of selecting for altered LEC'I gene expression in
transgenic plants.
The mutated gene will interact with the target wild-type gene in such a way
that homologous
recombination and targeted replacement of the wild-type gene will occur in
transgenic plant
cells, resulting in suppression of LEC 1 activity.
Alternatively, oligonucleotides composed of a contiguous stretch of RNA and
DNA residues in a duplex conformation with double hairpin caps on the ends can
be used.
The RNA/DNA sequence is designed to align with the sequence of the target LECI
gene and
to contain the desired nucleotide change. Introduction of the chimeric
oligonucleotide on an
extrachromosomal T-DNA plasmid results in efficient and specific LEC 1 gene
conversion
directed by chimeric molecules in a small number of transformed plant cells.
This method is
CA 02399886 2002-08-22
WO 01/64022 20 PCT/USO1/05454
described in Cole-Strauss et al., Science 273:1386-1389 (1996) and Yoon et al.
Proc. Natl.
Acad. Sci. USA 93: 2071-2076 (1996).
Desired modified LEC 1 polypeptides can be identified using assays to screen
for the presence or absence of wild type LEC 1 activity. Such assays can be
based on the
S ability of the LEC 1 protein to functionally complement the hap3 mutation in
yeast. As noted
above, it has been shown that homologs from different species functionally
interact with
yeast subunits ofthe CBF. (Sinha, et al., (1995). Proc. Natl. Acad. Sci. USA
92:1624-1628);
see, also, Becker, et al., (1991). Proc. Ncrtl. Acad. Sci. U.SA 88: 1968-
1972). The reporter for
this screen can be any of a number of standard reporter genes such as the lacZ
gene encoding
beta-galactosidase that is fused with the regulatory DNA sequences and
promoter of the yeast
CYC 1 gene. This promoter is regulated by the yeast CBF.
A plasmid containing the LEC1 cDNA clone is mutagenized in vitro
according to techniques well known in the art. The cDNA inserts are excised
from the
plasmid and inserted into the cloning site of a yeast expression vector such
as pYES2
1 S (Invitrogen). The plasmid is introduced into hap3- yeast containing a lacZ
reporter that is
regulated by the yeast CBF such as pLG26SUP1-lacZ (Guarente, et crl., (1984)
Cell 36:
317-321). Transformants are then selected and a filter assay is used to test
colonies for
beta-galactosidase activity. After confirming the results of activity assays,
immunochemical
tests using a LEC 1 antibody are performed on yeast lines that lack beta-
galactosidase activity
to identify those that produce stable LEC 1 protein but lack activity. The
mutant LEC 1 genes
are then cloned from the yeast and their nucleotide sequence determined to
identify the nature
of the lesions.
In other embodiments. the promoters derived from the LECI genes of the
invention can be used to drive expression of heterologous genes in an embryo-
specific or
2S seed-specific manner, such that desired gene products are present in the
embryo, seed, or
fruit. Suitable structural genes that could be used for this purpose include
genes encoding
proteins useful in increasing the nutritional value of seed or fruit. Examples
include genes
encoding enzymes involved in the biosynthesis of antioxidants such as vitamin
A, vitamin C.
vitamin E and melatonin. Other suitable genes encoding proteins involved in
modification of
fatty acids, or in the biosynthesis of lipids, proteins. and carbohydrates.
Still other genes can
be those encoding proteins involved in auxin and auxin analog biosynthesis for
increasing
fruit size, genes encoding pharmaceutically useful compounds, and genes
encoding plant
resistance products to combat fungal or other infections of the seed.
CA 02399886 2002-08-22
WO 01/64022 21 PCT/USO1/05454
Typically, desired promoters are identified by analyzing the 5' sequences of a
genomic clone corresponding to the embryo-specific genes described here.
Sequences
characteristic of promoter sequences can be used to identify the promoter.
Sequences
controlling eukaryotic gene expression have been extensively studied. For
instance, promoter
sequence elements include the TATA box consensus sequence (TATAAT), which is
usually
20 to 30 base pairs upstream of the transcription start site. In most
instances the TATA box
is required for accurate transcription initiation. In plants, further upstream
from the TATA
box, at positions -80 to -100, there is typically a promoter element with a
series of adenines
surrounding the trinucleotide G (or T) N G. J. Messing et al., in Genetic
Engineering in
Plants, pp. 221-227 (Kosage, Meredith and Hollaender, eds. (1983)).
A number of methods are known to those of skill in the art for identifying and
characterizing promoter regions in plant genomic DNA (see, e.g.. Jordano, et
crl., Plcrnt C'c~ll,
1: 855-866 (1989); Bustos, et u1., Plcrnt Cell, 1:839-854 (1989); Green, et
u1., EMBOJ. 7,
4035-4044 (1988); Meier, et crl., Plcrnt C'c~ll, 3, 309-316 (1991); and Zhang,
et u1., Plunt
P>zysiology 110: 1069-1079 (1996)).
Preparation of recombinant vectors
To use isolated sequences in the above techniques, recombinant DNA vectors
suitable for transformation of plant cells are prepared. Techniques for
transforming a wide
variety of higher plant species are well known and described in the technical
and scientific
literature. See, for example. Weisin~~ et crl. .~nri. Rev. Genet. 22:421-477
(1988). A DNA
sequence coding for the desired polypeptide, for example a cDNA sequence
encoding a lull
length protein, will preferably be combined with transcriptional and
translational initiation
regulatory sequences which will direct the transcription of the sequence ti-om
the gene in the
2~ intended tissues of the transformed plant.
For example, for overexpression, a plant promoter fragment may be employed
which will direct expression of the gene in all tissues of a regenerated
plant. Such promoters
are referred to herein as "constitutive" promoters and are active under most
environmental
conditions and states of development or cell differentiation. Examples of
constitutive
promoters include the cauliflower mosaic virus (CaMV) 35S transcription
initiation region.
the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, and
other
transcription initiation regions from various plant genes known to those of
skill.
Alternatively. the plant promoter may direct expression of the polynucleotide
of the invention in a specific tissue (tissue-specific promoters) or may be
otherwise under
CA 02399886 2002-08-22
WO 01/64022 22 PCT/USO1/05454
more precise environmental control (inducible promoters). Examples of tissue-
specific
promoters under developmental control include promoters that initiate
transcription only in
certain tissues, such as fruit, seeds, or flowers. As noted above, the
promoters from the LEC 1
genes described here are particularly useful for directing gene expression so
that a desired
gene product is located in embryos or seeds. Other suitable promoters include
those from
genes encoding storage proteins or the lipid body membrane protein, oleosin.
Examples of
environmental conditions that may affect transcription by inducible promoters
include
anaerobic conditions, elevated temperature, or the presence of light.
If proper polypeptide expression is desired, a polyadenylation region at the
3'-
end of the coding region should be included. The polyadenylation region can be
derived
from the natural gene, from a variety of other plant genes, or from T-DNA.
The vector comprising the sequences (e.g., promoters or coding regions) from
genes of the invention will typically comprise a marker gene which confers a
selectable
phenotype on plant cells. For example, the marker may encode biocide
resistance,
I S particularly antibiotic resistance, such as resistance to kanamycin, 6418,
bleomycin,
hygromycin, or herbicide resistance, such as resistance to chlorosluforon or
Basta.
LEC 1 nucleic acid sequences of the invention are expressed recombinantly in
plant cells to enhance and increase levels of endo<~enous LEC1 polypeptides.
Alternatively,
antisense or other LEC I constructs (described above) are used to suppress LEC
1 levels of
expression. A variety of different expression constructs, such as expression
cassettes and
vectors suitable for transformation of plant cells can be prepared. Technidues
for
transforming a wide variety of higher plant species are well known and
described in the
technical and scientific literature. See, e.g., Weising et ol. Ann. Reo.
Genet. 22:421-477
(1988). A DNA sequence coding for a LEC1 polypeptide, e.g., a cDNA sequence
encoding
a full length protein, can be combined with cis-acting (promoter) and traps-
acting (enhances)
transcriptional regulatory sequences to direct the timing, tissue type and
levels of
transcription in the intended tissues of the transformed plant. Translational
control elements
can also be used.
The invention provides a LEC1 nucleic acid operably linked to a promoter
which, in a preferred embodiment, is capable of driving the transcription of
the LEC 1 coding
sequence in plants. The promoter can be, e.g., derived from plant or viral
sources. The
promoter can be, e.g., constitutively active, inducible. or tissue specific.
In construction of
recombinant expression cassettes, vectors, transgenics, of the invention. a
different promoters
CA 02399886 2002-08-22
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can be chosen and employed to differentially direct gene expression, e.g., in
some or all
tissues of a plant or animal.
Typically, desired promoters are identified by analyzing the 5' sequences of a
genomic clone corresponding to the embryo-specific genes described here.
Sequences
characteristic of promoter sequences can be used to identify the promoter.
Sequences
controlling eukaryotic gene expression have been extensively studied. For
instance, promoter
sequence elements include the TATA box consensus sequence (TATAAT), which is
usually
20 to 30 base pairs upstream of the transcription start site. In most
instances the TATA box
is required for accurate transcription initiation. In plants, further upstream
from the TATA
box, at positions -80 to -100, there is typically a promoter element with a
series of adenines
surrounding the trinucleotide G (or T) N G. J. Messing et al., in Genetic
Engineering in
Plants, pp. 221-227 (Kosage, Meredith and Hollaender, eds. (1983)). A number
of methods
are known to those of skill in the art for identifying and characterizing
promoter regions in
plant genomie DNA (see, e.g.. Jordano. ct al., Plcrr~t C.'c~ll. 1: 855-866 ( I
989): Bustos. et al..
Plant Cell, 1:839-854 (1989); Green, et al.. EMBOJ. 7, 4035-4044 (1988).
Meier, et crl..
Plant C.'ell, 3, 309-316 (1991); and Zhang (1996) Plcrnt Phv.siolu~ry 110:1069-
1079).
Constitutive Promoters
A promoter fragment can be employed which will direct expression of LEC 1
nucleic acid in all transformed cells or tissues, e.g. as those of a
regenerated plant. Such
promoters are referred to herein as "constitutive" promoters and are active
under most
environmental conditions and states of development or cell differentiation.
Promoters that
drive expression continuously under physiological conditions are referred to
as "constitutive"
promoters and are active under most environmental conditions and states of
development or
cell differentiation. Examples of constitutive promoters include those Ii-om
viruses which
infect plants, such as the cauliflower mosaic virus (CaMV) 3~S transcription
initiation region
(see, e.g., Dagless (1997) Arch. I~irol. 142:183-191 ); the 1'- or 2'-
promoter derived from T-
DNA of Agrobacterium tumafaciens (see, e.g., Mengiste (1997) supra; O'Grady
(1990 Plcrnt
Mol. Biol. 29:99-108); the promoter of the tobacco mosaic virus: the promoter
of Fi'wvort
mosaic virus (see, e.g., Maiti (1997) Trcrns~Tenic Res. 6:143-156); actin
promoters, such as the
Arabidopsis actin gene promoter (see, e.g., Huan<~ ( 1997) Plcrrn ll~lul.
l3iol. 1997 33:12-I 39);
alcohol dehydrogenase (Adh) gene promoters (see, e.g., Millar (1996) Plant
Mol. Biol.
31:897-904); ACTH from Arabidopsis (Huang et ccl. Plant Mol. Biol. 33:12-139
(1996)),
Cat3 from Anabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 2~
1:196-203
(1996)), the gene encoding stearoyl-acyl carrier protein desaturase from
Br°crssicu napes
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(Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176 (1994)),
GPcI from
maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565 (1989)),
Gpc? from
maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112
(1997)), other
transcription initiation regions from various plant genes known to those of
skill. See also
Holtorf (1995) "Comparison of different constitutive and inducible promoters
for the
overexpression of transgenes in Arabidopsis thaliana," Plcrnt Mol. Biol.
29:637-646.
Inducible Promoters
Alternatively, a plant promoter may direct expression of the LEC 1 nucleic
acid of the invention under the influence of changing environmental conditions
or
developmental conditions. Examples of environmental conditions that may effect
transcription by inducible promoters include anaerobic conditions, elevated
temperature,
drought, or the presence of light. Such promoters are referred to herein as
"inducible"
promoters. For example, the invention incorporates the drought-inducible
promoter of maize
(Busk (1997) supra); the cold, drought, and high salt inducible promoter from
potato (Kirch
(1997) Plant Mol. Biol. 33:897-909).
Alternatively, plant promoters which are inducible upon exposure to plant
hormones, such as auxins, are used to express the nucleic acids of the
invention. For
example, the invention can use the auxin-response elements E1 promoter
fragment (AuxREs)
in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the
auxin-
responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and
hydrogen
peroxide) (Chen (1996) Plunt .l. 10: 9~5-966); the auxin-inducible parC
promoter from
tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (
1997) ~Llol. Plant
Microbe Interact. 10:933-937); and, the promoter responsive to the stress
hormone abscisic
acid (Sheen (1996) Science 274:1900-1902).
2~ Plant promoters which are inducible upon exposure to chemicals reagents
which can be applied to the plant, such as herbicides or antibiotics, are also
used to express
the nucleic acids of the invention. For example, the maize In2-2 promoter,
activated by
benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plcrnt
Cell Physiol.
38:568-577); application of different herbicide safeners induces distinct gene
expression
patterns, including expression in the root, hydathodes, and the shoot apical
meristem. LEC I
coding sequence can also be under the control of, e.g., a tetracycline-
inducible promoter, e.g.,
as described with transgenic tobacco plants containing the Avena sativa L.
(oat) arginine
decarboxylase gene (Masgrau ( 1997) Plant J. 11:465-473); or, a salicylic acid-
responsive
element (Stange (1997) Plant J. 11:1315-1324.
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Tissue-Specific Promoters
Alternatively, the plant promoter may direct expression of the polynucleotide
of the invention in a specific tissue (tissue-specific promoters). Tissue
specific promoters are
transcriptional control elements that are only active in particular cells or
tissues at specific
times during plant development, such as in vegetative tissues or reproductive
tissues.
Promoters from the LEC1 genes of the invention are particularly useful for
tissue-specific
direction of gene expression so that a desired gene product is generated only
or preferentially
in embryos or seeds, as described below.
Examples of tissue-specific promoters under developmental control include
promoters that initiate transcription only (or primarily only) in certain
tissues, such as
vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as
fruit, ovules, seeds,
pollen, pistols, flowers, or any embryonic tissue. Reproductive tissue-
specific promoters
may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-
specific. seed
and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or
some combination
thereof.
Suitable seed-specific promoters are derived from the following genes: MAC 1
from maize, Sheridan (1996) Genetics 142:1009-1020; Cat3 from maize, GenBank
No.
L05934, Abler (1993) Plant Mol. Biol. 22:10131-1038; vivparous-1 li-om
Arabidopsis,
Genbank No. U93215; atmycl from Arabidopsis, Urao (1996) Plcrrzt Mol. Biol.
32:571-57;
Conceicao (1994) Plunt 5:493-505; napA from Brassica napes, GenBank No.
J02798,
Josefsson (1987) JBL 26:12196-1301, the napin gene family from Brassica napes.
Sjodahl
(1995) Pluntcr 197:264-271.
The ovule-specific BEL1 gene described in Reiser (1995) Cell 83:735-742,
GenBanlc No. U39944, can also be used. See also Ray ( 1994) Pros. Nutl. Aced.
Sci. USA
91:5761-5765. The egg and central cell specific FIE1 promoter is also a useful
reproductive
tissue-specific promoter.
Sepal and petal specific promoters are also used to express LEC 1 nucleic
acids
in a reproductive tissue-specific manner°. For example, the Arabidopsis
floral homeotic gene
APETALA1 (AP1) encodes a putative transcription factor that is expressed in
young flower
primordia, and later becomes localized to sepals and petals (see, e.g.,
Gustafson- Brown
(1994) Cell 76:131-143; Mandel (1992) Nature 360:273-277). A related promoter,
for AP2.
a floral homeotic gene that is necessary for the normal development of sepals
and petals in
floral whorls, is also useful (see, e.g.. Drews ( 1991 ) Cell 65:991-1002:
Bowman ( 1991 ) Plain
Cell 3:749-758). Another useful promoter is that controlling the expression of
the unusual
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floral organs (ufo) gene of Arabidopsis, whose expression is restricted to the
junction
between sepal and petal primordia (Bossinger (1996) Development 122:1093-
1102).
A maize pollen-specific promoter has been identified in maize (Guerrero
(1990) Mol. Gen. Genet. 224:161-168). Other genes specifically expressed in
pollen are
described, e.g., by Wakeley (1998) PlantMol. Biol. 37:187-192; Ficker (1998)
Mol. Gen.
Genet. 257:132-142; Kulikauskas (1997) Plant Mol. Biol. 34:809-814; Treacy
(1997) Plant
Mol. Biol. 34:603-611.
Other suitable promoters include those from genes encoding embryonic
storage proteins. For example, the gene encoding the 2S storage protein from
Brassica napes.
Dasgupta (1993) Gene 133:301-302; the 2s seed storage protein gene family from
Arabidopsis; the gene encoding oleosin 20kD from Brassica napes, GenBank No.
M63985;
the genes encoding oleosin A, Genbanl: No. 009118, and, oleosin B, Genbank No.
0091 19,
from soybean; the gene encoding oleosin from Arabidopsis, Genbank No. 217657:
the gene
encoding oleosin 181cD from maize, GenBank No. J05212, Lee (1994) Plcrnt Mol.
Biol.
26:1981-1987; and, the gene encoding low molecular weight sulphur rich protein
from
soybean, Choi (1995) Mol Gen, Genet. 246:266-268, can be used. The tissue
specific E8
promoter from tomato is particularly useful for directing gene expression so
that a desired
gene product is located in fruits.
A tomato promoter active during fruit ripening, senescence and abscission of
leaves and, to a lesser extent, of flowers can be used (Blame ( 1997) Plant J
12:731-746).
Other exemplary promoters include the pistol specific promoter in the potato
(Solarium
tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase (Ficker
(1997) Plcrnt
Mol. Biol. 35:425-431 ); the Blec4 gene from pea (Pisum sativum cv. Alaska),
active in
epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa.
This makes it a
useful tool to target the expression of foreign genes to the epidermal layer
of actively
growing shoots.
A variety of promoters specifically active in vegetative tissues, such as
leaves.
stems, roots and tubers, can also be used to express the LEC 1 nucleic acids
of the invention.
For example, promoters controlling patatin, the major storage protein of the
potato tuber. can
be used, see, e.g., Kim (1994) Plant Mol. Biol. 26:603-615; Martin (1997)
Plant J. 11:53-62.
The ORF13 promoter from Agrobacterium rhizogenes which exhibits high activity
in roots
can also be used (Hansen (1997) Mol. Gen. Genet. 254:337-343. Other useful
vegetative
tissue-specific promoters include: the tarin promoter of the gene encoding a
globulin from a
major taro (Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra
(1995) Plant
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Mol. Biol. 28:137-144); the curculin promoter active during taro corm
development (de
Castro (1992) Plaht Cell 4:1549-1559) and the promoter for the tobacco root-
specific gene
TobRB7, whose expression is localized to root meristem and immature central
cylinder
regions (Yamamoto ( 1991 ) Plant Cell 3:371-382).
Leaf specific promoters, such as the ribulose biphosphate carboxylase (RBCS)
promoters can be used. For example, the tomato RBCSI, RBCS2 and RBCS3A genes
are
expressed in leaves and light-grown seedlings, only RBCS 1 and RBCS2 are
expressed in
developing tomato fruits (Meier (1997) FEBS Lett. 415:91-95). A ribulose
bisphosphate
carboxylase promoters expressed almost exclusively in mesophyll cells in leaf
blades and leal
sheaths at high levels, described by Matsuolca ( 1994) Plcrnt .l. 6:31 1-319,
can be used.
Another leaf specific promoter is the light harvesting chlorophyll a/b binding
protein gene
promoter. see, e.g., Shiina (1997) Plant Physiol. 115:477-483; Casal (1998)
Plant Physiol.
116:1533-1538. The Arabidopsis thaliana myb-related gene promoter (AtmybS)
described by
Li (1996) FEBS Lett. 379:117-121. is leaf specific. The AtmybS promoter is
expressed in
developing leaf trichomes, stipules, and epidermal cells on the margins of
young rosette and
cauline leaves, and in immature seeds. AtmybS mRNA appears between
fertilization and the
16 cell stage of embryo development and persists beyond the heart stage. A
leaf promoter
identified in maize by Busk (1997) Plant J. 11:1285-1295, can also be used.
Another class of useful vegetative tissue-specific promoters are meristematic
(root tip and shoot apex) promoters. For example, the ''SHOOTMERISTEMLESS" and
"SCARECROW" promoters, which are active in the developing shoot or root apical
meristems, described by Di Laurenzio ( 1996) C'c.~ll 86:423-433; and, Long (
1996) Ncriarre
379:66-69; can be used. Another useful promoter is that which controls the
expression of
3-hydroxy-3- methylglutaryl coenzyme A reductase HMG2 gene. whose expression
is
restricted to meristematic and floral (secretory zone of the stigma, mature
pollen grains,
gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto (
1995) Plain C.'ell.
7:517-527). Also useful are knl-related genes from maize and other species
which show
meristem-specific expression, see, e.g., Granger (1996) Plcr>7t Mol. Biol.
31:373-378;
Kerstetter (1994) Plant C.'ell 6:1877-1887; Hale (1995) Philos. Tr~arrs. R.
Soc. Loud. B. Biul.
Sci. 350:45-51. For example, the Arabidopsis thaliana KNAT1 promoter. In the
shoot apex.
KNAT1 transcript is localized primarily to the shoot apical meristem; the
expression of
KNAT1 in the shoot meristem decreases during tile floral transition and is
restricted to the
cortex of the inflorescence stem (see, e.g., Lincoln ( 1994) Plan/ Cell 6:1859-
1876).
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One of skill will recognize that a tissue-specific promoter may drive
expression of operably linked sequences in tissues other than the target
tissue. Thus, as used
herein a tissue-specific promoter is one that drives expression preferentially
in the target
tissue, but may also lead to some expression in other tissues as well.
In another embodiment, a LEC 1 nucleic acid is expressed through a
transposable element. This allows for constitutive, yet periodic and
infrequent expression of
the constitutively active polypeptide. The invention also provides for use of
tissue-specific
promoters derived from viruses which can include, e.g., the tobamovirus
sub~enomic
promoter (Kumagai (1995) Proc. Natl. Acucl. Sci. USA 92:1679-1683; the rice
tungro
bacilliform virus (RTBV), which replicates only in phloem cells in infected
rice plants. with
its promoter which drives strong phloem-specific reporter gene expression: the
cassava vein
mosaic virus (CVMV) promoter, with highest activity in vascular elements. in
leaf mesophyll
cells, and in root tips (Verdaguer (1996) Plarr~ iLlol. Biol. 31:1 129-1139).
Production of trans~~enic plants
DNA constructs of the invention may be introduced into the genome of the
desired plant host by a variety of conventional techniques. For example, the
DNA construct
may be introduced directly into the genomic DNA of the plant cell using
techniques such as
electroporation and microinjection of plant cell protoplasts, or the DNA
constructs can be
introduced directly to plant tissue using ballistic methods, such as DNA
particle
bombardment. Alternatively, the DNA constructs may be combined with suitable T-
DNA
flanking regions and introduced into a conventional Agrobacterium tumefaciens
host vector.
The virulence functions of tile Agrobacterium tumefaciens host will direct the
insertion of the
construct and adjacent marker into the plant cell DNA when the cell is
infected by the
bacteria.
Microinjection techniques are known in the art and well described in the
scientific and patent literature. The introduction of DNA constmcts using
polyethylene
glycol precipitation is described in Paszkowski et al. EmOo .l 3:2717-2722 (
1984).
Electroporation techniques are described in Fromm et crl. Pr°oc. Null.
Acacl. Sci. US.~ 82:5824
(1985). Ballistic transformation techniques are described in Klein et al.
.Vuturc 327:70-73
( 1987).
Agrobacterium tumefaciens-mediated transformation techniques, including
disarming and use of binary vectors, are well described in the scientific
literature. See. for
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WO 01/64022 29 PCTNS01/05454
example Horsch et al. Science 233:496-498 (1984), and Fraley et al.
Pr°oc. Natl. Acad. Sci.
USA 80:4803 (1983).
Transformed plant cells which are derived by any of the above transformation
techniques can be cultured to regenerate a whole plant which possesses the
transformed
genotype and thus the desired phenotype such as seedlessness. Such
regeneration techniques
rely on manipulation of certain phytohormones in a tissue culture growth
medium, typically
relying on a biocide and/or herbicide marker which has been introduced
together with the
desired nucleotide sequences. Plant regeneration from cultured protoplasts is
described in
Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell
Culture, pp. 124-176,
MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of
Plants,
Plant Protoplasts, pp. 21-73, CRC Press. Boca Raton, 198. Regeneration can
also be
obtained from plant callus, explants, organs, or parts thereof. Such
regeneration techniques
are described generally in Klee et crl. Arm. Rev. of~Plcrnt Pl~y.s. 38:467-486
(1987).
The nucleic acids of the invention can be used to confer desired traits on
essentially any plant. Thus, the invention has use over a broad range of
plants, including
species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus,
Capsicum,
Cucumis, Cucurbita, Daucus. Fragaria, Glycine, Gossypium, Helianthus,
Heterocallis,
Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot,
Majorana,
Medicago, Nicotiana. Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus, Prunus,
Raphanus,
Secale, Senecio, Sinapis, Solanum, Sorghum, Trigonella, Triticum, Vitis,
Vigna, and, Zea.
The LEC 1 genes of the invention are particularly useful in the production of
transgenic plants
in the genus Brassica. Examples include broccoli, cauliflower, brussel
sprouts, canola, and
the like.
Use and Recombinant Expression of LEC 1 in Combination with other Genes
The LEC 1 nucleic acids of the invention can be expressed together with other
structural or regulatory genes to achieve a desired effect. A cell or plant,
such as a
transformed cell or a transgenic plant, can be transformed, engineered or bred
to co-express
both LEC 1 nucleotide and/or LEC 1 polypeptide, and another gene or gene
product.
Alternatively, two or more LEC1 nucleic acids can be co-expressed together in
the same
plant or cell.
The LEC 1 nucleic acids of the invention, when expressed in plant
reproductive or vegetative tissue, can induce ectopic embryo morphogenesis.
Thus, in one
embodiment, a LEC1 nucleic acid of the invention is expressed in a sense
conformation in a
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WO 01/64022 30 PCTNS01/05454
transgenic plant to induce the expression of ectopic embryo-like structures.
as discussed
above. In another embodiment, LEC I is co-expressed with a gene or nucleic
acid that
increases reproductive tissue mass, e.g., increases fruit size, seed mass,
seed protein or seed
oils. For example, co-expression of antisense nucleic acid to ADC genes, such
as AP2 and
RAP2 genes of Arabidopsis, will dramatically increase seed mass, seed protein
and seed oils;
see, e.g., Jofulcu, et al., WO 98/07842; Okamuro (1997) Proc. Natl. Acucl.
S'ci. USA
94:7076-7081; Okamuro (1997) Plum C'c~ll 9:37-47; Jofuku (1994) Plan/ Cell
6:121 I-1225.
Thus, co-expression of a LEC 1 of the invention, to induce ectopic expression
oi~ embronic
cells and tissues, together with another plant nucleic acid and/or protein,
such as the seed-
mass enhancing antisense AP2 nucleic acid, generates a cell, tissue, or plant
(e.g., a
transgenic plant) with increased ti-uit and seed mass, greater yields of
embryonic storage
proteins, and the like.
In another embodiment. the LEC 1 nucleic acids of the invention are expressed
in plant reproductive or vegetative cells and tissues which lack the ability
to produce
I 5 functional ADC genes. such as AP2 and RAP2 genes. The LEC 1 nucleic acid
can be
expressed in an ADC "knockout" transgenic plant. Alternatively, the LEC 1
nucleic acid can
be expressed in a cell, tissue or plant expressing a mutant ADC nucleic acid
or gene product.
Expression of LEC1 nucleic acid in any of these non-functioning ADC models
will also
produce a cell, tissue or plant with increased ti-uit and seed mass, greater
yields of embryonic
storage proteins, and the like.
One of skill will recognize that after the expression cassette is stably
incorporated in transgenic plants and confirmed to be operable, it can be
introduced into other
plants by sexual crossing. Any of a number of standard breeding techniques can
be used,
depending upon the species to be crossed.
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Example I
This example describes the isolation and characterization of an exemplary
LEC 1 gene.
Experimental Procedures
Plant Material
A lecl-2 mutant was identified from a population of Arabidopsis thaliana
ecotype Wassilewskija (Ws-O) lines mutagenized with T-DNA insertions as
described before
(West et al., 1994). The abi3-3, fus3-3 and lecl-1 mutants were generously
provided by
Peter McCourt, University of Toronto and David Meinl:e, Oklahoma State
University. Wild
type plants and mutants were grown under constant light at 22°C.
Double mutants were constructed by intercrossin g the mutant lines lecl-1.
lecl-2, abi3-3, fus3-3, and lec2. The genotype of the double mutants was
verified through
backcrosses with each parental line. Double mutants were those who failed to
complement
both parent lines. Homozygous single and double mutants were generated by
germinatin~~
intact seeds or dissected mature embryos before desiccation on basal media.
Isolation and Sequence analysis of Genomic and cDNA Clones
Genomic libraries of Ws-O wild type plants. lecl-1 and lecl-2 mutants were
made in GEM 11 vector according to the instructions of the manufacturer
(Promega). Two
silique-specific cDNA libraries (stages globular to heart and heart to young
torpedo) were
made in ZAPII vector (Stratagene).
The genomic library of lecl-2 was screened using right and left T-DNA
specific probes according to standard techniques. About 12 clones that
cosegregate ~~ith the
mutation, were isolated and purified and the entire DNAs were further labeled
and used as
probes to screen a southern blot containing wild type and lec 1-1 genomic DNA.
One clone
hybridized with plant DNA and was further analyzed. A 7.1 1:b XhoI fragment
containin<~
the left border and the plant sequence flanking the T-DNA was subcloned into
pBluescript-KS plasmid (Stratagene) to form ML7 and sequenced using a left
border specific
primer (5' GCATAGATGCACTCGAAATCAGCC 3'). The T-DNA organization was
partially verified using southern analysis with T-DNA left and right borders
and PBR322
probes. The results suggested that the other end of the T-DNA is also composed
of left
border. This was confirmed by generating a fCR fragment using a genomic plant
DNA
primer (LP primer 5' GCT CTA GAC ATA CAA CAC TTT TCC TTA 3') and a T-DNA left
border specific primer (5' GCTTGGTAATAATTGTCATTAG 3') and sequencing.
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The EcoRI insert of ML7 was used to screen a wild type genomic library.
Two overlapping clones were purified and a 7.4 EcoRI genomic fragment from the
wild type
DNA region was subcloned into pBluescript-KS plasmid making WT74. This
fragment was
sequenced (SEQ ID NO: 4) and was used to screen lecl-1 genomic library and
wild type
silique-specific cDNA libraries. 8 clones from the lecl-1 genomic library were
identified
and analyzed by restriction mapping.
From these clones the exact site of the deletion in lecl-1 was mapped and
sequenced by amplifying a Xbp PCR fragment using primers (H21 - 5' H21 - 5'
CTA AAA
ACA TCT ACG GTT CA 3'; H 17 - ~' TTT GTG GTT GAC CGT TTG GC 3') flanking the
deletion region in lecl-1 genomic DNA. Clones were isolated from both cDNA
libraries
and partially sequenced. The sequence of the cDNA clones and the wild type
genomic clone
matched exactly, confirming that both derived from the same locus. All
hybridizations were
performed under stringent conditions with 32P random prime probes
(Strata<~ene).
Sequencing was done using the automated dideoxy chain termination method
(Applied Biosystems, Foster City, CA). Data base searches were performed at
the National
Center for Biotechnology Information by using the BLAST network service.
Alignment of
protein sequences was done using PILEUP program (Genetics Computer Group.
Madison,
WI)
DNA and RNA blot analysis
Genomic DNA was isolated from leaves by using the CTAB-containing buffer
Dellaporta, et al., (1983). Plant Mol. Biol. Reporter 1: 19-21. Two micrograms
of DNA was
digested with different restriction endonucleases, electrophoretically
separated in 1 % agarose
gel, and transferred to a nylon membrane (I-lybond N; Amersham).
Total RNA was prepared from siliques, two days old seedlings, stems. leaves,
?5 buds and roots. Poly(A)+ RNA was purified from total RNA by oligo(dT)
cellulose
chromatography, and two micrograms of each Poly(A)+ RNA samples were separated
in 1 °/>
denatured formaldehyde-agarose gel. Hybridizations were done under stringent
conditions
unless it specifies otherwise. Radioactive probes were prepared as described
above.
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Complementation of lecl mutants
A 3.4 kb Bstyl fragment of genomic DNA (SEQ ID NO: 3) containing
sequences from 1.992 kb upstream of the ORF to a region 579 by downstream from
the poly
A site was subcloned into the hygromycin resistant binary vector pBIB-Hyg. The
LECI
cDNA was placed under the control of the 35S promoter and the ocs
polyadenylation signals
by inserting a PCR fragment spanning the entire coding region into the plasmid
pART7. The
entire regulatory fragment was then removed by digestion with NotI and
transferred into the
hygromycin resistant binary vector BJ49. The binary vectors were introduced
into the
Agrobacterium strain GV3101, and constructions were checked by re-isolation of
the
plasmids and restriction enzyme mapping, or by PCR. Transformation to
homozygous lecl-1
and lecl-2 mutants were done using the in planta transformation procedure
(Bechtold, et al..
(1993). Comptes Rendus de 1'Academie des Sciences Serie III Sciences de la
Vie, 316:
1194-1199. Dry seeds from lecl mutants were selected for transformants by
their ability to
germinate after desiccation on plates containing ~ghnl hygromycin. The
transformed plants
were tested for the present of the transgene by PCR and by screening the
siliques for the
present of viable seeds.
In Situ Hybridization
Experiments were performed as described previously by Dietrich et al. (1989)
Plant Cell 1: 73-80. Sections were hybridized with LEC 1 antisense probe. As a
negative
control, the LEC 1 antisense probe was hybridized to seed sections of lec I
mutants. In
addition, a sense probe was prepared and reacted with the wild type seed
sections.
Results
Genetic Interaction Between Leafy Cotyledon-Type Mutants and abi3
In order to understand the genetic pathways which regulate late embryogenesis
we took advantage of three Arabidopsis mutants lec2, fus3-3 and abi3-3 that
cause similar
defects in late embryogenesis to those of lecl-1 or lecl-2. These mutants are
desiccation
intolerant, sometimes viviparous and have activated shoot apical meristems.
The lec2 and
fus3-3 mutants are sensitive to ABA and possess trichomes on their cotyledons
and therefore
can be categorized as leafy cotyledon-type mutants (Meinke et al.. 1994). l,he
abi3-3
mutants belong to a different class of late embryo defective mutations that is
insensitive to
ABA and does not have trichomes on the cotyledons.
The two classes of mutants were crossed to lec 1-1 and lec 1-2 mutants to
construct plants homozygous to both mutations. The lec 1 and lec2 mutations
interact
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synergistically, resulting in a double mutant which is arrested in a stage
similar to the late
heart stage, the double mutant embryo, however. is larger. The lec 1 or lec2
and fus3-3
double mutants did not display any epistasis and the resulting embryo had an
intermediate
phenotype. The lecl/abi3-3 double mutants and lec2/abi3-3 double mutants were
ABA
insensitive and had a lec-like phenotype. There was no different between
double mutants that
consist of either lecl-1 or lecl-2.
No epistasis was seen between the double mutants indicating that each of the
above genes, the LEC-type and ABI3 genes, operate in different genetic
pathways.
LEC1 Functions Early in Embryo~enesis
The effects of lecl is not limited to late embryogenesis, it also has a role
in
early embryogenesis. The embryos of the lecl/lec2 double mutants were arrested
in the early
stages of development, while the single mutants developed into mature embryos,
suggesting
that these genes act early during development.
Further examination of the early stages of the single and double mutations
showed defects in the shape, size and cell division pattern of the mutants
suspensors. The
suspensor of wild type embryo consists of a single file of six to eight cells,
whereas the
suspensors of the mutants are often enlarged and undergo periclinal divisions.
Leafy
cotyledon mutants exhibit suspensor anomalies at the globular or transition
stage whereas
wild type and abi3 mutant do not show any abnormalities.
The number of anomalous suspensors increases as the embryos continue to
develop. At the torpedo stage, the wild type suspensor cells undergo
programmed cell death,
but in the mutants secondary embryos often develop from the abnormal
suspensors and, when
rescued, give rise to twins.
The Or#~anization of the LEC1 Locus in Wild Type Plants and lecl Mutants
Two mutant alleles of the LEC 1 gene have been reported, lec 1-1 and lec 1-2
(Meinke, 1992; West et al., 1994). Both mutants were derived from a population
of plants
mutagenized insertionally with T-DNA (Feldmann and Marla, 1987), although lecl-
1 is not
tagged. The lecl-2 mutant contains multiple T-DNA insertions. A specific
subset of T-DNA
fragments were found to be closely linked with the mutation. A genomic library
of lecl-?
was screened using right and left borders T-DNA as probes. Genomic clones
containin~~
T-DNA fragments that cosegregate with the mutation were isolated and tested on
Southern
blots of both wild type and lecl-1 plants. Only one clone hybridized with
Arabidopsis DNA
and also gave polymorphic restriction fragment in lecl-1.
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The lecl-1 polymorphism resulted from a small deletion, approximately 2 lcb
in length. Using sequences from the plant fragment flanking the T-DNA, the
genomic wild
type DNA clones and the lecl-1 genomic clones were isolated. An EcoRI fragment
of 7.4 Icb
of the genomic wild type DNA that corresponded to the polymorphic restriction
fragment in
lecl-1 was further analyzed and sequenced. The exact site of the deletion in
lecl-1 was
identified using a PCR fragment that was generated by primers, within the
expected borders
of the deleted fragment, and sequencing.
In the wild type genomic DNA that corresponded to the lec 1-1 deletion. a 626
by ORF was identified. Southern analysis of wild type DNA and the two mutants
DNA
probed with the short DNA fragment of the ORF revealed that both the wild type
and lecl-2
DNA contain the ORF while the lecl-1 genomic DNA did not hybridize. The exact
insertion
site of the 'T-DNA in lec 1-2 mutant was determined by PCR and sequencin~~ and
it was found
that the T-DNA was inserted 115 by upstream of the ORF's translational
lllltlat1011 COdOIl 111
the 5' region of the gene.
At the site of the T-DNA insertion a small deletion of 21 plant nucleic acids
and addition of 20 unknown nucleic acids occurred. These results suggest that
in lecl-2 the
T-DNA interferes with the regulation of the ORF while in lec 1-1 the whole
gene is deleted.
Thus, both lecl alleles contain DNA disruptions at the same locus, confirming
the identity oC
the LEC 1 locus.
The lecl Mutants Can Be Complement by Transformation
To prove that the 7.4 kb genomic wild type fragment indeed contained the
ORF of the LEC 1 gene, we used a genomic fragment of 3395 by (SEQ ID NO: 3)
within that
fragment to transform homozygous lecl-1 and lecl-2 plants. The clone consists
of a 3395
by BstYI restriction fragment containing the gene and the promoter region. The
translation
start codon (ATG) of the polypeptide is at 1999 and the stop codon is at 2625
(TGA). There
are no introns in the gene.
The tl-ansformed plants were selected on hygromycin plates and were tested to
contain the wild type DNA fiagment by PCR analysis. Both transgenic mutants
were able to
produce viable progeny, that were desiccation tolerant and did not posses
trichomes on their
cotyledons. We concluded that the 3.4 1b fragment can complement the lec 1
mutation and
since there is only one ORF in the deleted 2 1<b fra~~ment in lecl-1 we
suggest that this ORF
corresponds to the LEC 1 gene.
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The LECI Gene is a Member of Gene Family
In order to isolate the LEC 1 gene two cDNA libraries of young siliques were
screened using the 7.4 lcb DNA fragment as a probe. Seventeen clones were
isolated and after
further analysis and partial sequencing they were all found to be identical to
the genomic
ORF. The cDNA contains 626 by ORF specifying 208 amino acid protein (SEQ ID
NO:I
and SEQ ID N0:2).
The LECI cDNA was used to hybridize a DNA gel blot containing Ws-O
genomic DNA digested with three different restriction enzymes. Using low
stringency
hybridization we found that there is at least one more gene. This confirmed
our finding of
two more Arabidopsis ESTs that show homology to the LEC 1 gene.
The LEC 1 gene is Embryo Specific
The lecl mutants are affected mostly during embryogenesis. Rescued mutants
can give rise to homozygous plants that have no obvious abnormalities other
than the
presence of trichomes on their cotyledons and their production of defective
progeny.
Therefore, we expected the LEC 1 gene to have a role mainly during
embryo'~enesis and not
during vegetative growth. To test this assumption poly (A)+ RNA was isolated
from siliques.
seedling, roots, leaves, stems and buds of wild type plants and li-om siliques
of lecl plants.
Only one band was detected on northern blots using either the LEC 1 gene as a
probe or the
7.41<b genomic DNA fragment suggesting that there is only one gene in the
genomic DNA
fragment which is active transcriptionally. The transcript was detected only
in siliques
containing young and mature embryos and was not detected in seedlings, roots.
leaves, stems
and buds indicating that the LEC 1 gene is indeed embryo specific. In
addition, no RNA was
detected in siliques of both alleles of lecl mutants confirming that this ORF
corresponds to
the LEC 1 gene.
Expression Pattern of the LEC 1 Gene
To study how the LEC 1 gene specifies cotyledons identity, we analyzed its
expression by in situ hybridization. We specifically focused on young
developing embryos
since the mutants abnormal suspensors phenotype indicates that the LEC 1 gene
should be
active very early during development.
During embryogenesis. the LEC 1 transcript was first detected in proglobular
embryos. The transcript was found in all cells of the proembryo and was also
found in the
suspensor and the endosperm. However, from the globular stage and on it
accumulates more
in the outer layer of the embryo, namely the protodenn and in the outer part
of the ground
meristem leaving the procambium without a signal. At the torpedo stage the
signal was
CA 02399886 2002-08-22
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stronger in the cotyledons and the root meristem, and was more limited to the
protoderm
layer. At the bent cotyledon stage the signal was present throughout the
embryo and at the
last stage of development when the embryo is mature and filling the whole seed
we could not
detect the LEC 1 transcript. This might be due to sensitivity limitation and
may imply that if
the LEC1 transcript is expressed at that stage it is not localized in the
mature embryo, but
rather spread throughout the embryo.
The LEC 1 gene encodes a Homology of CCAAT binding factor.
Comparison of the deduced amino acid sequence of LEC 1 to the GenBanl:
reveals significant similarity to a subunit of a transcription factor, the
CCAAT box binding
factor (CBF). CBFs are highly conserved family of transcription factors that
regulate gene
activity in eukaryotic organisms Mantvani, et al., . (1992). Nucl. Acids Res.
20: 1087-1091.
They are hetero-oligomeric proteins that consist of between three to four non-
homologous
subunits. LEC1 was found to have high similarity to CBF-A subunit. This
subunit has three
domains; A and C which show no conservation between l:in~~doms and a central
domain, B-
which is highly conserved evolutionary. Similarly the LEC 1 gene is composed
of three
domains. The LEC1 B domain shares between 75%-8~% similarity alld SJ%-63%
identity
with different B domains that are found in organisms ranging from yeast to
human. Within
this central domain, two highly conserved amino acid segments are present.
Deletion and
mutagenesis analysis in the CBF-A yeast homolog hap3 protein demonstrated that
a short
region of seven residues (42-48) (LPIANVA) is required for binding the CCAAT
box, while
the subunit interaction domain lies in the region between residues 69-80
(MQECVSEFISFV)
(Ring et al., supra). LEC1 protein shares h lgh homology to those regions.
DISCUSS10N
The lecl mutant belongs to the leafy cotyledon class that interferes mainly
with the embryo program and therefore is thought to play a central regulatory
role during
embryo development. It was shown before that LEC1 gene activity is required to
suppress
germination during the maturation stage. Therefore, we analyzed the genetic
interaction of
homozygous double mutants of the different members of the leafy cotyledon
class and the
abi3 mutant that has an important role during embryo maturation. All the five
different
combinations of the double mutants showed either an intermediate phenotype or
an additive
effect. No epistatic relationship among the four genes was found. These
findings suggest
that the different genes act in parallel genetic pathways. Of special interest
was the double
mutant lecl/lec2 that was arrested morphologically at the heart stage, but
continued to grow
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in that shape. This double mutant phenotype indicates that both genes LEC 1
and LEC2 are
essential for early morphogenesis and their products may interact directly or
indirectly in the
young developing embryo.
The Role of LEC 1 in Embryo~enesis
One of the proteins that mediate CCAAT box function, is an heteromeric
protein called CBF (also called NFY or CPI). CBF is a transcription activator
that regulates
constitutively expressed genes, but also participates in differential
activation of
developmental genes Wingender, E. ( 1993). Gene Regulation in Eulcaryotes (New
York:
VCH Publishers). In mammalian cells, three subunits have been identified CBF-
A, CBF-B
and CBF-C and all of which are required for DNA binding. In yeast, the CBF
homolog HAl'
activates the CYC 1 and other genes involved in the mitochondrial electron
transport Johnson,
et al., Proteins. Annu. Rev. Biochem. 58, 799-840. ( 1989). HAP consists of
four subunits
hap2, hap3, hap4 and hap5. Only hap2, 3 and 5 are required for DNA binding.
CBF-A- B
and C show high similarity to the yeast hap3- 2 and 5, respectively. It was
also reported that
I5 mammalian CBF-A and B can be functionally interchangeable with the
corresponding yeast
subunits (Sinha et al., supra.).
The LECI gene encodes a protein that shows more then 75% similarity to the
conserved region of CBF-A. CCAAT motifs are not common in plants' promoters
and their
role in transcription regulation is not clear. However, maize and Brassica
homologs have
been identified. A search of the GenBank revealed several Arabidopsis ES'l~s
that show high
similarity to CBF-A, B and C. Accession numbers of CBF-A (HAP3) homologs:
H37368,
H76589; CBF-B (HAP2) homologs: T20769; CBF-C CHAPS) homologs: T43909, T44300.
These findings and the pleiotropic affects of LEC 1 suggest that LEC 1 is a
member of a
heteromeric complex that functions as a transcription factor.
The model suggests that LEC 1 acts as transcription activator to several sets
of
genes, which keep the embryonic program on and repress the germination
process.
Defective LEC 1 expression partially shuts down the embryonic program and as a
result the
cotyledons lose their embryonic characteristics and the germination program is
active in the
embryo.
Example 2
This example demonstrates that LEC I is sulticient to induce embryonic
pathways in transgenic plants.
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The phenotype of lecl mutants and the gene's expression pattern indicated
that LEC 1 functions specifically during embryogenesis. A LEC I cDNA clone
under the
control of the cauliflower mosaic virus 3 5 S promoter was transferred into
lee 1-1 mutant
plants in planta using standard methods as described above.
Viable dry seeds were obtained from lecl-I mutants transformed with the
35S/LEC1 construct. However, the transformation efficiency was only
approximately 0.6%
of that obtained normally. In several experiments, half the seeds that
germinated ( 12/23 )
produced seedlings with an abnormal morphology. Unlike wild type seedlings,
these
35S/LEC1 seedlings possessed cotyledons that remained fleshy and that failed
to expand.
Roots often did not extend or extended abnormally and sometimes greened. These
seedlings
occasionally produced a single pair of organs on the shoot apex at the
position normally
occupied by leaves. Unlike wild type leaves, these organs did not expand and
did not possess
trichomes. Morphologically, these leaf like structures more closely resembled
embryonic
cotyledons than leaves.
I S The other 35S/LEC I seeds that remained viable after drying produced
plants
that grow vegetatively. The majority of these plants (7) flowered and produced
I 00°r~ lee 1
mutant seeds. Amplification experiments confirmed that the seedlings contained
the
transgene, suggesting that the 35S/LEC1 gene was inactive in these T2 seeds.
No vegetative
abnormalities were observed in these plants with the exception that a few
displayed defects in
apical dominance. A few plants (2) were male sterile and did not produce
progeny. One
plant that produced progeny segregated 25% mutant Lecl- seeds that, when
germinated
before desiccation and grown to maturity, gave rise to 100% mutant seed, as
expected for a
single transgene locus. The other 75% of seeds contained embryos with either a
wild type
phenotype or a phenotype intermediate between lee 1 mutants and wild type.
Only 25°ro of the
dry seed from this plant germinated, and all seedlings resembled the embryo-
like seedlings
described above. Some seedlings continued to ~n-ow and displayed a strikin;~
phenotype.
These 35S/LEC1 plants developed two types of structures on leaves. One type
resembled
embryonic cotyledons while the other looked like intact torpedo stage embryos.
Thus.
eetopic expression of LEC 1 induces the morphogenesis phase of embryo
development in
vegetative cells.
Because many 35S/I_ECI seedlings exhibited embryonic characteristics. the
seedlings were analyzed for expression of genes specifically active in
embryos. Cruciferin A
storage protein mRNA accumulated tluoughout the 3~S/LECl seedlings, including
the leaf=
like structures. Proteins with sizes characteristic of 12S storage protein
cruciferin
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accumulated in these transgenic seedlings. Thus, 35S/LECI seedings displaying
an embryo-
like phenotype accumulated embryo-specific mRNAs and proteins. LEC 1 mRNA
accumulated to a high level in these 35S/LECl seedlings in a pattern similar
to early stage
embryos but not in wild type seedlings. LEC I is therefore sufficient to alter
the fate of
vegetative cells by inducing embryonic programs of development.
The ability of LEC1 to induce embryonic programs of development in
vegetative cells establishes the gene as a central regulator of embryogenesis.
LEC I is
sufficient to induce both the seed maturation pathway as indicated by the
induction of storage
protein genes in the 35S/LECI seedlings. The presence of ectopic embryos on
leaf surfaces
and cotyledons at the position of leaves also shows that LEC 1 can activate
the embryo
morphogenesis pathway. Thus, LEC1 regulates both early and late embryonic
processes.
Example 3
This example shows that LEC 1 is expressed in zygotes and that the promoters
I 5 of the invention can therefore be used to target expression in zygotes.
To determine precisely when the LEC1 gene becomes activated, LEC1 RNA
levels were analyzed in the egg apparatus of mature female gametophytes before
fertilization.
in zygotes after fertilization, and in very early stage embryos containing an
apical cell and
two to three suspensor cells. In situ hybridization experiments showed that
LEC 1 RNA was
present in zygotes and early stage embryos but was not detected in female
gametophytes.
These results show that the LEC 1 promoter becomes active in the zygote. The
LEC 1 is
therefore useful to target the expression of sense or antisense versions of
regulatory genes or
cytotoxic genes to zygotes and early stage embryos.
Example 4
This example shows the identification of a LECI homolog from Arabidopsis
designated the LEAFY COTJ'LEDONI-LIKE gene.
A Blast search was conducted through the Arabidopsis Database
(http://genome-www.stanford.edu/Arabidopsis/) usin g the LEC'I cDNA nucleotide
sequence
as a probe to identify homologs of the HAP3 subunit of CCAAT box binding
transcription
factor from Arabidopsis. The Arabidopsis BAC clone, MNJ7 (Accession Number
AB025628), contains a gene, designated LECI-Like (LIL), that displays the
highest amino
acid sequence identity with the LECI protein of any known Arabidopsis H IP3
gene. The
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nucleotide and amino acid sequences of LIL are shown in SEQ ID N0:19 and SEQ
ID
N0:20, respectively.
The Polymerase Chain Reaction (PCR) was used to amplify the LIL gene,
which lacks introns. Primers designed to amplify the LIL open reading frame
contained
BamHI and XbaI restriction enzyme sites for cloning purposes. The forward
primer,
BAMMNJ7-~ sequence is 5'-AGGATCCATGGAACGTGGAGGCTTCCAT-3' with the
BamHI site underlined. The reverse primer, 3-MNJ7XBA sequence is 5'-
ATCTAGATCAGTACTTATGTTGTTGAGTCG-3' with the XbaI site underlined. The
PCR conditions were as follows: 30 cycles of 4~ seconds at 94°C, 45
seconds at 53°C, and 3
minutes at 72°C. AmphiTaq DNA polymerase (Perkin Elmer Cetus, 761 Main
Ave.,
Norwalk, CT 06859) was used. PCR products were cloned using the TOPO TA
Cloning Kit
(Invitrogen, Carlsbad. CA 92008). The nucleotide sequence ofthe cloned LIL
gene was
determined to confirm its identity.
Accumulation of LEC'I-LIKE RNA
The LIL clone was hybridized with gel blots containing 20 pg of total RNA
from leaves, stems, roots, seedlings, and siliques containing either proembryo
to heart stage
(early) embryos, heart to torpedo stage (middle) embryos, or torpedo to mature
(late)
embryos. LIL RNA was detected only in siliques containing all three stages of
embryos.
Detection of the LIL RNA in siliques from Ic~cl-I mutants showed that the RNA
detected
was not LECI. Thus, like LEC'I, LIL accumulates specifically during
embryogenesis.
Complementation of lecl-I Mutation by LIL
The LIL clone was inserted into the LEC'I promoter/terminator cassette within
the plant transformation vector BJ49. The LEC'I promoter/terminator cassette
consists of
1992 by of DNA 5' of the LECI translation start codon and 770 by 3' of the
LEC'I cDNA
translation stop codon (H.S. Lee, R.W. Kwong. and J.J. Harada. unpublished
results). The
promoter and terminator are separated by a short polylinker with BgIII and
Avrll restriction
endonuclease sites in which the LIL gene was inserted.
This construct was transferred into homozygous lecl-I null mutants using in
planta transformation procedures with Agr°obactenizun tun2efucier~s
strain GV3101. Unlike
lecl-I mutant plants whose progeny die following desiccation, plants
transformed with the
LIL construct produced viable seedlings. PCR amplification experiments
confirmed that the
viable seedlings have the lecl-1 mutation and the transgene. Seedlings
morphologically
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WO 01/64022 42 PCT/USO1/05454
resembled wild type rather than lecl mutant plants. These results show that
the LlL gene
complements the lecl mutation, suggesting overlapping functions for the two
genes.
Example 5
This example shows the identification of a LEC'1 ortholog from scarlet runner
bean.
Constructing an Embryo-Proper cDNA Library from the Globular Embrvo of Scarlet
Runner
Bean
A cDNA library was constructed with 150 ng of total RNA isolated from
embryo propers (EP) of the scarlet runner bean (SRB; Pha.s~eolus coccineus)
that were
dissected from globular-stage embryo. The SMART PCR cDNA Library Construction
Kit
(Clontech, cat # K1051-1) was used according to a manufacturer's protocol.
Briefly, first
strand cDNA was synthesized from EP total RNA using Superscript II RNase H-
reverse
transcriptase (Gibco/BRL, cat # l 8064-014) in the presence of an Sf7 IB-site
containing
oligo-dT primer (CDSIII/3' PCR primer. Clontech) and a SMART Ill containing an
Sli IA-
site primer (Clontech). Second strand was generated by polymerase chain
reaction using ~'-
and 3' PCR primers (Clontech). Double-stranded cDNA was digested with Sfi I
restriction
enzyme (New England BioLabs, cat # 123S) and then size-fractionated over a
CHROMA S-
400 sepharose column (Clontech). After analyzing collected fractions on a 1.1
% agarose gel.
four fractions containing high amount of cDNAs in a range of 0.~ 1:b to 4 kb
~~ere pooled and
precipitated in an ethanol/salt solution at -20°C overnight. A cDNA
pellet was recovered by
centrifugation and resuspended in 7 uL of sterile water. cDNA inserts were
ligated to Sfi I-
digested lambda arms (lTriplEx2, Clontech). Ligation mixtures were packaged
into phage
heads using Gigapack III Gold Packaging Extract (Stratagene).
Isolation of the Scarlet Runner Bean LEC.'1 ortholo~~ cDNA.
The cDNA library was converted from a lambda form to a plasmid form via
Cre-Lox system (in vivo excision, Clontech). Colonies were picked randomly for
plasmid
DNA isolation. The nucleotide sequences of cDNA clones were determined using
BigDye
terminator, a ~'-TriplEx sequencing primer (Clontech), and the ABI Prism 377
DNA
sequencer (Perkin-Ehner Applied Biosystems). The identity of the cDNA clone
was
determined by BlastX and BlastN analyses.
A BlastX search revealed that a cDNA clone pPCEP 112 encoded a protein
(SEQ ID N0:22) with high amino acid sequence identity to the Arabidopsis LEC1.
especially
in the conserved B domain. However. a BlastN result indicated that this SRB
cDNA
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WO 01/64022 43 PCT/USO1/05454
sequence is more similar to the Arabidopsis LIL gene at the nucleotide level.
The entire
sequence of the pPCEP112 insert was determined to be 988 by (SEQ ID N0:21).
Spatial Expression Pattern of the LECl-Like Gene in SRB Seeds
To examine the spatial expression pattern of scarlet runner bean LIL gene in
embryos, we carried out in situ hybridization analyses. The full length cDNA
insert of
pBSEP112 was used as the template for sense and antisense RNA probe synthesis.
The LIL
mRNA accumulated in both the embryo proper (EP) and suspensor (S) of a 5 days
after
pollination embryo. In the 7 days after pollination seeds, the RNA is
localized intensively in
the epidermal layer of the embryo proper and moderately in every cell in both
the embryo
proper and suspensor. Only background signal was detected using the sense
probe. In
conclusion, the spatial expression pattern of SRB LEC1-like gene in the
globular embryo is
similar to that of Arabidopsis LEC I .
The above examples are provided to illustrate the invention but not to limit
its
scope. Other variants of the invention will be readily apparent to one of
ordinary skill in the
art and are encompassed by the appended claims. All publications, databases,
Genbank
sequences, patents, and patent applications cited herein are hereby
incorporated by reference.
CA 02399886 2002-08-22
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Harada, John
Lotan, Tamar
Ohto, Masa-aki
Goldberg, Robert B.
Fischer, Robert L.
Bui, Anhthu
Kwong, Raymond
(ii) TITLE OF INVENTION: Leafy Cotyledonl Genes and Their Uses
(iii) NUMBER OF SEQUENCES: 18
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Townsend and Townsend and Crew LLP
(B) STREET: Two Embarcadero Center, Eighth Floor
(C) CITY: San Francisco
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94111-3834
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/804.534
(B) FILING DATE: 21-FEB-1997
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bastian, Kevin L.
(B) REGISTRATION NUMBER: 34,774
(C) REFERENCE/DOCKET NUMBER: 023070-077600US
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 576-0200
(B) TELEFAX: (415) 576-0300
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 627 base pairs
1
WO 01/64022 CA 02399886 2002-08-22 pCT~S01/05454
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..627
(D) OTHER INFORMATION: /product= "LEC 1 "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATG ACC AGC TCA GTC ATA GTA GCC GGC GCC GGT GAC AAG AAC AAT GG~r
48
Met Thr Ser Ser Val Ile Val Ala Gly Ala Gly Asp Lys Asn Asn Gly
1 5 10 15
ATC GTG GTC CAG CAG CAA CCA CCA TGT GTG GCT CGT GAG CAA GAC CAA
96
Ile Val Val Gln Gln Gln Pro Pro Cys Val Ala Arg Glu Gln Asp Gln
20 25 30
TAC ATG CCA ATC GCA AAC GTC ATA AGA ATC ATG CGT AAA ACC TTA CCG
144
Tyr Met Pro Ile Ala Asn Val Ile Arg Ile Met Arg Lys Thr Leu Pro
35 40 45
TCT CAC GCC AAA ATC TCT GAC GAC GCC AAA GAA ACG ATT CAA GAA TGT
192
Ser His Ala Lys Ile Ser Asp Asp Ala Lys Glu Thr Ile Gln Glu Cys
50 55 60
GTC TCC GAG TAC ATC AGC TTC GTG ACC GGT GAA GCC AAC GAG CGT TGC
240
Val Ser Glu Tyr Ile Ser Phe Val Thr Gly Glu Ala Asn Glu Arg Cys
65 70 75 80
CAA CGT GAG CAA CGT AAG ACC .ATA ACT GCT GAA GAT ATC CTT TGG GCT
288
Gln Arg Glu Gln Arg Lys Thr Ile Thr Ala Glu Asp Ile Leu Trp Ala
85 90 95
ATG AGC AAG CTT GGG TTC GAT AAC TAC GTG GAC CCC CTC ACC GTG TTC
336
Met Ser Lys Leu Gly Phe Asp Asn Tyr Val Asp Pro Leu Thr Val Phe
100 105 110
2
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ATT AAC CGG TAC CGT GAG ATA GAG ACC GAT CGT GGT TCT GCA CTT AGA
384
Ile Asn Arg Tyr Arg Glu Ile Glu Thr Asp Arg Gly Ser Ala Leu Arg
115 120 125
GGT GAG CCA CCG TCG TTG AGA CAA ACC TAT GGA GGA AAT GGT .ATT GGG
432
Gly Glu Pro Pro Ser Leu Arg Gln Thr Tyr Gly Gly Asn Gly Ile Gly
130 135 140
TTT CAC GGC CCA TCT CAT GGC CTA CCT CCT CCG GGT CCT TAT GGT TAT
480
Phe His Gly Pro Ser His C=ly Leu Pro Pro Pro Gly Pro Tyr Gly Tyr
145 150 155 160
GGT ATG TTG GAC CAA TCC ATG GTT ATG GGA GGT GGT CGG TAC TAC CAA
528
Gly Met Leu Asp Gln Ser Met Val Met Gly Gly Gly Arg Tyr Tyr Gln
165 170 175
AAC GGG TCG TCG GGT CAA GAT GAA TCC AGT GTT GGT GGT GGC TCT TCG
576
Asn Gly Ser Ser Gly Gln Asp Glu Ser Ser Val Gly Gly Gly Ser Ser
180 185 190
TCT TCC ATT AAC GGA ATG CCG GCT TT1' GAC CAT TAT GGT CAG TAT AAG
624
Ser Ser Ile Asn Gly Met Pro Ala Phe Asp His Tyr Gly Gln Tyr Lys
195 200 205
TGA 627
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Thr Ser Ser Val Ile Val Ala Gly Ala Gly Asp Lys Asn Asn Gly
1 5 10 1~
Ile Val Val Gln Gln Gln Pro Pro Cys Val Ala Arg Glu Gln Asp Gln
20 25 30
3
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Tyr Met Pro Ile Ala Asn Val Ile Arg Ile Met Arg Lys Thr Leu Pro
35 40 45
Ser His Ala Lys Ile Ser Asp Asp Ala Lys Glu Thr Ile Gln Glu Cys
50 55 60
Val Ser Glu Tyr Ile Ser Phe Val Thr Gly Glu Ala ASI1 Glu Arg Cys
65 70 75 80
Gln Arg Glu Gln Arg Lys Thr Ile Thr Ala Glu Asp Ile Leu Trp Ala
85 90 95
Met Ser Lys Leu Gly Phe .Asp Asn Tyr Val Asp Pro Leu Thr Val Phe
100 105 110
Ile Asn Arg Tyr Arg Glu Ile Glu Thr Asp Arg Gly Ser Ala Leu Arg
115 120 125
Gly Glu Pro Pro Ser Leu Arg Gln Thr Tyr Gly Gly Asn Gly Ile Gly
130 135 140
Phe His Gly Pro Ser His Gly Leu Pro Pro Pro Gly Pro Tyr Gly Tyr
145 150 155 160
Gly Met Leu Asp Gln Ser Met Val Met Gly Gly Gly Arg Tyr Tyr Gln
165 170 175
Asn Gly Ser Ser Gly Gln Asp Glu Ser Ser Val Gly Gly Gly Ser Ser
180 185 190
Ser Ser Ile Asn Gly Met Pro Ala Phe Asp His Tyr Gly Gln Tyr Lys
195 200 205
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3395 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
AGATCCAAAA CAGGTCA'rGG ACTGGGCCGT AAACTCTATC CAAAATTCTT CATGTTTTTC 60
CATCTTTCAA AAATCTTTAT CCACCATTCC ATTACTAGGG TGTTGGTTTT ATTTTATTTG 120
TTGATTAATT ATGTATTAGA AAATGTAAAG CAATATTCAA TTGTAACATG CATCATCTAA 1~0
4
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CACCAATATC TTGTACTAAC CTTTTGTAAT TTTCCTATAA ACATTTTAAA 240
AGGCTAATTT
AAATAAAAAT TACAATAAAC GTGATAACTC ACTTTCGTAA CGCATATTTA 300
TTCAAATATA
CCAAAATTTA CCATTTTAAG TAAGAGAATC TTTTTAAAAT TAATTTTCAA 360
TTTCATTAAT
TAAGAAACAA AGAATTTACT GAAACCTATA TTTTATTAAA TTTTAATAAA 420
ATATATGACT
AAAATAACGT CACGTGAATC TTTCTCAGCC GTTCGATAAT CGAATACTTT 480
ATTGACTAAG
TATTTATTTA G.AAAATTTTA AACAACACTT AATTTCTAGA AACAAAGAGA 540
GCCTCATATG
TATAAAAATC TTCTTCTTAT CTTTCTTTCT TTCTTAATAG TCTTTATTTT
TACTTAATTA 600
CTTTGGTAAT TTGTGAAAAA CACAACCAAT GAGAGAAGAG CAGTTTGACT
GGCCACATAG 660
CCAA'rGAGAC AAGCCAATGG GAAAGAGATA TAGAGACCTC GTAAGAACCG C
CTCCTTTGC 720
ATTTGTATCA TCTCTCTATA AAACCACTCA ACCATCAACC TNTCTTTGCA 780
TGCAACAAAT
CACTCAAATA ATTATTTTAT AAAGAACAAA AAAAAAAAGA CGGCAGAGAA
ACAATGGAAC84U
GTGGAGCTCC CTTCTCTCAC TATCAGCTAC CCAAATCCAT CTCTGGTAAT 900
CTAAGTGGCT
ATTTGTATAC AGTATATACT TGCCTCCATG TATATTTATA TTCTCGTGAA 960
AAATTGGAGA
CATGCTTTAT GAATTTTATG AGACTTTGCA ACAACGAACG AGATGCTTTC 1020
TCTCTAGAAA
TTTAAATTTA GATTTGTGAA GGTTTTGGGA ATGGCCCGGA GAAGACGATT 1080
TTATATA'1'AC
ATGCATGCAA GAGTTTGATA TGTATATTGT TTCATCATGG CTGAGTCAAA I
GTTTTATCCA 140
AATATTTCCA TGGTGTGGTA TTAGTTAAAC AAATCTCTCG TATGTGTCAT 1200
TGAATATACC
CGTGCATGTA CCAGGAATGT TTTTGATTCT AAAAACGTTT TTTTCTTTGT 1260
TGTAACGGTT
GAGTTTTTTT CTTCGTTTCA AAACGAGATT CTCGTTTGTC TCTTCCCTTG 1320
TCTAAAAACA
TCTACGGTTC ATGTGATTCA AAAACACTAA AAAAATATAA ACTCATTTTT 1380
TTTTAATACT
TAACATTTAA ACTATATATA TATATATATA TATATATATC TTATACTAGT 1440
CCCAAGTTTT
AGTGTGAGGT TTTTTTATTC AAAATCTATC AG'1'ACATTTT TTGGAAAAGA 1500
ACTAAGTGAA
ATTTTCTCCA AATTTTCCTT TTACTATTGA TTTTTTAATT ACTGGATGTC 560
,A'I'TAACTTTA 1
ATCTTTTGAT TCTTTCAACG TTTACCATTG GGAACCTTCA CATGAAATAA 1620
ATGTCTACTT
TATTGAGTCA TACCTTCGTC AACATAAATT AATTGATGTT CTTCTCCAAA 1680
TTTTGAGTTT
TTGGTTTTTC TAATAATCTT AACGAAAGCT TTTTGGTATA CATGTAAAAC 1740
GTAACGGCAA
GAATCTGAAC AGTCTACTCA ACGGGGTCCA TAAGTCTAGA ATGTAGACCC
CACAAACTTA 1800
CTCTTATCTT ATTGGTCCGT AACTAAGAAC GTGTCCCTCT GATTCTCTTG 860
TTTTCTTCTA 1
ATTAATTCGT ATCCTACAAA TTTAATTATC ATTTCTACTT CAACTAATCT 920
TTTTTTATTT 1
CCTAAAGATT TCAATTTCTC TCTGTATTTT CTATGAACAG AATTGAACTT 1980
GGACCAGCAC
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AGCAACAACC CAACCCCAAT GACCAGCTCA GTCATAGTAG CCGGCGCCGG TGACAAGAAC2040
AATGGTATCG TGGTCCAGCA GCAACCACCA TGTGTGGCTC GTGAGCAAGA CCAATACATG 2100
CCAATCGCAA ACGTCATAAG AATCATGCGT AAAACCTTAC CGTCTCACGC CAAAATCTCT 21 GO
GACGACGCCA AAGAAACGAT TCAAGAATGT GTCTCCGAGT ACATCAGCTT CGTGACCGGT 2220
GAAGCCAACG AGCGTTGCCA ACGTGAGCAA CGTAAGACCA TAACTGCTGA AGATATCCTT 2280
TGGGCTATGA GCAAGCTTGG GTTCGATAAC TACGTGGACC CCCTCACCGT GTTCATTAAC 2340
CGGTACCGTG AGATAGAGAC CGATCGTGGT TCTGCACTTA GAGGTGAGCC ACCGTCGTTG 2400
AGACAAACCT ATGGAGGAAA TGGTATTGGG TTTCACGGCC CATCTCATGG CCTACCTCCT 2460
CCGGGTCCTT ATGGTTATGG TATGTTGGAC CAATCCATGG TTATGGGAGG TGGTCGGTAC 2520
TACCAAAACG GGTCGTCGGG TCAAGATGAA TCCAGTGTTG GTGGTGGCTC TTCGTCTTCC 280
ATTAACGGAA TGCCGGCTTT TGACCATTAT GGTCAGTATA AGTGAAGAAG GAGTTATTC'1~ 2640
TCATTTT'1'AT ATCTATTCAA AACATGTGTT TCGATAGATA TTTTATTTTT ATGTCTTATC 2700
AATAACATTT CTATATAATG TTGCTTCTTT AAGGAAAAGT GTTGTATGTC AATACTTTAT 2760
GAGAAACTGA TTTATATATG CAAATGATTG AATCCAAACT GTTTTGTGGA TTAAACTCTA 2820
TGCAACATTA TATATTTACA TGATCTAAAG GTTTTGTAAT TCAAAAGCTG TCATAGTTAG 2880
AAGATAACTA AACATTGTAG TAACCAAGTT TAATTTACTT TTTTGAGTTT ACATAACTAA 2940
CCAAGCCAAA AGGTTATAAA ATCTAAATTC GTTGAGTTGT CAAACTTCTG AAGATTGCTA 3000
TCCTCTTTGA GTTGCTTTCT TTTGGGTGCT TGAGTTTCAT TAGGCTGAGC TGACTCGTTG 3060
CTCTCTAGTC TTTCATCTCT GTCTTTTCCA AGGATTCATA ACGTTGGTCG CTCTCTGTTT 3 I20
CTGCCTACAC TTCTTCAAGG GATCATTACT GAGGCTAAGA GTTAAAGACC TGAACCATGG 3180
TTTTCTGTAA CTGGTTCAAG TTCATTCTCC GGTTATTGTG TGGTTATCTT TCGGTTAGAT 3240
TGAAACCCAT ATGTTTGCTC TGTTTCTTCT AGTTCCAAGT TTAATTTCCG GTTATTGTTT 3300
GGCTTTTTAA AAGTTTTTAA GGTCTATTCT ATGTAAAGAC TATTCTACGT ACGTACATTT 3360
ATCGCAAAAT TGAAAGATTA TAAAAAAAAT TGAAA 339
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7560 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
AATTNACCCT CACTAAAGGG AACAAAAGCT GGGTACCGGG CCCCCCCTCG
AGGTCGACGG 60
TATCGATAAG CTTGATATCG AATTCGTGGC CATTAGACCC ATAACTATAT
GACGATGTTA 120
AAGAGAAAAT AAATCATAAA TAAAATAAGA GTCCTTATC.A ATAAACCTAA
TTGGCTAATT 180
TCAACCTCAA AGAGTAGTAG GAACAGGTAA GGTGAAGCCA AACAGCTCCT
TTTACAGTTG 240
GACCACTAGA GCTGATCTGG CATACAAAGT ATGCTTATTG GGCTGTCACG
GCCCATCCGC 300
AAAATGTCGT TGGTTACGAA GCATCCACGA CATAGACGGT GCCACATGTT
AGAAAAGTGT 360
TTCGGCGATC AAGATTGTGT CCACATCATT AGACGTCTGA ACTGTCCACG
TGTCTATCAA 420
AGCTGGCGTC AAACATTACG TTTTCGTCGT TTGCGCCTCC TAGTTCACAC
GTGCAACGAA 480
CGCGTGCGAC GTATCAAAAT TGTTAATTTT AGCCATGTAT AAAGAATATC
TACAAAATTA 540
ACCTCAGGAA TATTTTTGTT TTTTCAATTG AGGCCATAAT ATACNTNCCG
ATNGAAAAAT 600
TTTNCANCAT ATCNCTAATA TCAAAAAATT ATGATGTTAG TAAACGTAAA
AAATTTACAC 660
AAAATAANTT TCACAAAACT TANNGGGGAA ATTGGAACAA ANAAAAGACT
GGTGAGTGAT 720
AAGCGATGAT GGCCGGTGAA TCAGGTAGCC GTCCTACAAC GTGGTTGATT
TTGAGCAAAC 780
TCCTATCTAC TCTTCACACT ATTGGAAATC CCAAAATGTC GTCACACCA1,
AATAATGTGA 840
ATTTTGTTAT GGAATTTGAG GGAAACAGTA GATATATGTT TCAACCAGTG
AAAGTTACCC 900
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TCCTTTGGAC ATATCTACGA NAGTAGAAAG TAGAAACATT CACTAAACGT
GACAACTTTA 960
TAAATTTTCT TTTTGTAACT TTTCTTTAGA TTTATTTACG ANAAGAGAAA
TATAAACGTC 1020
ATGCTAATAA AAAATGCATT ATTTTCTACC ATCTAGCTAG AATATTGATC
AAGTCTTCAC 1080
GTTTTTTGTT TATCTCTTCT CTCATAGGCA TGTCCACAAA AGGGTAAGTT
TTACTGGTTC 1140
AAAATATTGC ATGAGTACTA CTAAGCTCGT ATAGTTTGAT CTTACTATCA
TTGCGATGAG 1200
GGTTGTTAGT TTGGAAGAAA TAAGGATTTA TGCAAATGGT AATCATTATG
TCTGCTATTT 1260
AAGAAGTAAA TTATGATGCT TGTTGCGTGA ACATATTAAA TTTGC:GAAAA
ATAAGCAAGG 1320
ATACACGAGA GAAGCTCAGA TATTCACGTA ACGATGTTTC ATCTCTTCTC
ATTGAGGAAA 1380
CATATGGCCA TGATATAGCT AATAAGCCTA CGGGATTGTC NTTTCAACGC
CGAATCTACC 1440
AAACTGTTCC ATCTCTTATT ATATATAGTT TGGTTATTTA AGTAATTAGA
TGCATCATAA 1500
TCTTTTTTTC TGCCAGTTGT AATGCAGATA AAAATATATT GGTTGTTCTA
AGGATTGTTC 1560
AAACGTGCAT GTGTACAAGT TATTATTTAT ATACTTTCAT CTACATGCGA
TGCGTTATTT 1620
ATAATGATAA AACTAAGATT TTTAGTTAAA TTTAATAAAG AGC TTACGAG
CTACAATTAA 1680
TTAGAAATGG TTGCTCAGAA ATCAGAATAC TATATATGAA AAAAGAAGTT
GGTATACTTG 1740
AAAAAAGAAA AAACTACTTG AAAAGATGGT AAAAGATATA GAACGAGTAT
ATATCTTACT 1800
CAAGCACGAT AGAAGTTTGT ATCAAAACAT TGCGTTCCAA ACCAATGTTT
GAAGATGGTC 1860
AAAGGTGCTA CTCATGATGT GGTGCGAAGA AGCTTACGAA AAATTCTGCA
ATGAGAGATA 1920
8
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ACTTTATGGG CTGCTTGTTC AATATATTGA AAATCATGGT AGACAACACC
AAACTCTCCT 1980
TTACCAGAAG TCATATTTCC TTAACCTCAG AATAAGTAAA TCTTCTAGTT
TATTATTTGA 2040
AAGTTGAGCG TATAATTGCA ATGAAACTTT TACCAATTCA CCGCCTCCTA
ACTGAGTTGT 2100
TGTATTATCC TATCTCTTTA GCTATCCTTT CCTTGCTCTT GCTCCACCTG
CATGTGGCCT 2160
CTTTATTTAT AATCTCTCTA GATTCTGCTA AAGATGTNTG TTCAAAATGG
TTTATCTTTA 2220
AGGGAAGCAA AGTGAATGGA AACATTTAAA GAAAAAAAAA ACTTTTAGCA
GAGTTCCATG 2280
AGATTTCATA CTGATGATAA CTAAAATAAT CTTATATGCG TAAGATTATT
TTAGTTCTAA 2340
ACTTCATTTT GAAATGAGAG GTCATTGGCC AGGAAAGATT CAATATTGGT
TCTTTGTTAA 2400
TTCTCGTTGG TTTGTTTTTA GTATGGGCTA GATCCAAAAC AGGTCATGGA
CTGGGCCGTA 2460
AACTCTATCC AAAATTCTTC ATGTTTTTCC ATCTTTCAAA AATCTTTATC
CACCATTCCA 2520
TTACTAGGGT GTTGGTTTTA TTTTATTTGT TGATTAATTA TGTATTAGAA
AATGTAAAGC 2580
AATATTCAAT TGTAACATGC ATCATCTAAC ACCAATATCT TGTACTAACC
TTTTGTAATT 2640
TTCCTATAAA CATTTTAAAA GGCTAATTTA AATAAAAATT ACAATAAACG
TGATAACTCA 2700
CTTTCGTAAC GCATATTTAT TCAAATATAC CAAAATTTAC CATTTTAAGT
AAGAGAATCT 2760
TTTTAAAATT AATTTTCAAT TTCATTAATT AAGAAACAAA GAATTTACTG
AAACCTATAT 2820
TTTATTAAAT TTTAATAAAA TATATGACTA AAATAACGTC ACGTGAATCT
TTCTCAGCCG 2880
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TTCGATAATC GAATACTTTA TTGACTAAGT ATTTATTTAG AAAATTTTAA
ACAACACTTA 2940
ATTTCTAGAA ACAAAGAGAG CCTCATATGT ATAAAAATCT TCTTCTTATC
TTTCTTTCTT 3000
TCTTAATAGT CTTTATTTTT ACTTAATTAC TTTGGTAATT TGTGAAAAAC
ACAACCAATG 3060
AGAGAAGAGC AGTTTGACTG GCCACATAGC CAATGAGACA AGCCAATGGG
AAAGAGATAT 3120
AGAGACCTCG TAAGAACCGC TCCTTTGCCA TTTGTATCAT CTCTCTATAA
AACCACTCAA 3180
CCATCAACCT NTCTTTGCAT GCAACAAATC ACTCAAATAA TTATTTTATA
AAGAACAAAA 3240
AAAAAAAGAC GGCAGAGAAA CAATGGAACG TGGAGCTCCC TTCTCTCACT
ATCAGCTACC 3300
CAAATCCATC TCTGGTAATC TAAGTGGCTA TTTGTATACA GTATATACTT
GCCTCCATGT 3360
ATATTTATAT TCTCGTGAAA AATTGGAGAC ATGCTTTATG AATTTTATGA
GACTTTGCAA 3420
CAACGAACGA GATGCTTTCT CTCTAGAAAT TTAAATTTAG ATTTGTGAAG
GTTTTGGGAA 3480
TGGCCCGGAG AAGACGATTT TATATATACA TGCATGCAAG AGTTTGATAT
GTATATTGTT 3540
TCATCATGGC TGAGTCAAAG TTTTATCCAA ATATTTCCAT GGTGTGGTAT
TAGTTAAACA 3600
AATCTCTCGT ATGTGTCATT GAATATACCC GTGCATGTAC CAGGAATGTT
TTTGATTCTA 3660
AAAACGTTTT TTTCTTTGTT GTAACGGTTG AGTTTTTTTC TTCGTTTCAA
AACGAGATTC 3720
TCGTTTGTCT CTTCCCTTGT CTAAAAACAT CTACGGTTCA TGTGATTCAA
AAACACTAAA 3780
AAAATATAAA CTCATTTTTT TTTAATACTT AACATTTAAA CTATATATAT
ATATATATAT 3840
ATATATATCT TATACTAGTC CCAAGTTTTA GTGTGAGGTT TTTTTATTCA
AAATCTATCA 3900
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GTACATTTTT TGGAAAAGAA CTAAGTGAAA TTTTCTCCAA ATTTTCCTTT
TACTATTGAT 3960
TTTTTAATTA CTGGATGTCA TTAACTTTAA TCTTTTGATT CTTTCAACGT
TTACC.ATTGG 4020
GAACCTTCAC ATGAAATAAA TGTCTACTTT ATTGAGTCAT ACCTTCGTCA
ACATAAATTA 4080
ATTGATGTTC TTCTCCAAAT TTTGAGTTTT TGGTTTTTCT AATAATCTTA
ACGAAAGCTT 4140
TTTGGTATAC ATGTAAAACG TAACGGCAAG AATCTGAACA GTCTACTCAA
CGGGGTCCAT 4200
AAGTCTAGAA TGTAGACCCC ACAAACTTAC TCTTATCTTA TTGGTCCGTA
ACTAAGAACG 4260
TGTCCCTCTG ATTCTCTTGT TTTCTTCTAA TTAATTCGTA TCCTACAAAT
TTAATTATCA 4320
TTTCTACTTC AACTAATCTT TTTTTATTTC CTAAAGATTT CAATTTCTCT
CTGTATTTTC 4380
TATGAACAGA ATTGAACTTG GACCAGCACA GCAACAACCC AACCCCAATG
ACCAGCTCAG 4440
TCATAGTAGC CGGCGCCGGT GACAAGAACA ATGGTATCGT GGTCCAGCAG
CAACCACCAT 4500
GTGTGGCTCG TGAGCAAGAC CAATACATGC CAATCGCAAA CGTCATAAGA
ATCATGCGTA 4560
AAACCTTACC GTCTCACGCC AAAATCTCTG ACGACGCCAA AGAAACGATT
CAAGAATGTG 4620
TCTCCGAGTA CATCAGCTTC GTGACCGGTG AAGCCAACGA GCGTTGCCAA
CGTGAGCAAC 4680
GTAAGACCAT AACTGCTGAA GATATCCTTT GGGCTATGAG CAAGCTTGGG
TTCGATAACT 4740
ACGTGGACCC CCTCACCGTG TTCATTAACC GGTACCGTGA GATAGAGACC
GATCGTGGTT 4800
CTGCACTTAG AGGTGAGCCA CCGTCGTTGA GACAAACCTA TGGAGGAAAT
GGTATTGGGT 4860
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TTCACGGCCC ATCTCATGGC CTACCTCCTC CGGGTCCTTA TGGTTATGGT
ATGTTGGACC 4920
AATCCATGGT TATGGGAGGT GGTCGGTACT ACCAAAACGG GTCGTCGGGT
CAAGATGAAT 4980
CCAGTGTTGG TGGTGGCTCT TCGTCTTCCA TTAACGGAAT GCCGGCTTTT
GACCATTATG 5040
GTCAGTATAA GTGAAGAAGG AGTTATTCTT CATTTTTATA TCTATTCAAA
ACATGTGTTT S 100
CGATAGATAT TTTATTTTTA TGTCTTATCA ATAACATTTC TATATAATGT
TGCTTCTTTA 5160
AGGAAAAGTG TTGTATGTCA ATACTTTATG AGAAACTGAT TTATA1'ATGC
AAATGATTGA 5220
ATCCAAACTG TTTTGTGGAT TAAACTCTAT GCAACATTAT ATATTTACAh
GATCTAAAGG 5280
TTTTGTAATT CAAAAGCTGT CATAGTTAGA AGATAACTAA ACATTGTAGT
AACCAAGTTT 5340
AATTTACTTT TTTGAGTTTA CATAACTAAC CAAGCCAAAA GGTTATAAAA
TCTAAATTCG 5400
TTGAGTTGTC AAACTTCTGA AGATTGCTAT CCTCTTTGAG TTGCTT1'CTT
TTGGGTGCTT 5460
GAGTTTCATT AGGCTG.AGCT GACTCGTTGC TCTCTAGTCT TTCATCTCTG
TCTTTTCCAA 5520
GGATTCATAA CGTTGGTCGC TCTCTGTTTC TGCCTACACT TCTTCAAGGG
ATCATTACTG 5580
AGGCTAAGAG TTAAAGACCT GAACCATGGT TTTCTGTAAC TGGTTCAAGT
TCATTCTCCG 5640
GTTATTGTGT GGTTATCTTT CGGTTAGATT GAAACCCATA ~I~GTTTGCTCT
GTTTCTTCTA 5700
GTTCCAAGTT TAATTTCCGG TTATTGTTTG GCTTTTTAAA AGTTTTTAAG
GTCTATTCTA 5760
TGTAAAGACT ATTCTACGTA CGTACATTTA TCGCAAAATT GAAAGATTAT
AAAAAAAATT 5820
GAAAGATCCA AAGGAAACCA ATAGATTAAA CTAAAATGTA GTATCCTTTT
TATCATTTTA 5880
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GGCTATGTTT TCTTTTAAGA AAGCTTTGGT AGTTAACTCT GTTTAAAAGA
AAAAAAAGAG 5940
ATGCATAAAT TAAATTTAAG TTTCTAGAAC TTTTGGATAA ACATATT.AAG
CTAAAGAAAT 6000
TAAACTAAAG GGCGTAAATG CAAGCTTGTT ATGCGTTATT GAAAACATTA
CCTCTAAATT 6060
AAATAGCCCA ATATTGAAAA CCTTAAGCTT CTTTGATCCC CTTAACTTGT
TTGTCCACCA 6120
AGTATTAGTT CATCTCTTAA CACGGCAACT CGAAACGGCA CAATGGACAA
ACATGGTCTT 6180
TCAAAAACCA CTTCCCAATA CATCCATCGT CAAACTCGTG GCCACATGGT
AAGGTCACCA 6240
CTATTTCTCC CTTTTCAAAC TCCTCCAAAC AAATTGTGCA CACACTGGCG
TCAGAGTTGG 6300
ATTTCTTCTT ATTATTATAT ACTTTCCTTG CCAAACGGTC AACCACAAAC
TTATTTGCCG 6360
GTCTAATTAA CTCGATATTA TTGGTGGTCT CATCAAAC'GA GTCAATCCGA
GGAGGAGGTG 6420
GAACAATGAC TTTACAG'rAC ATGTAAACTA ACGTAGCACA AACTGAAGAG
TCTACCATAG 6480
AAATCGACTT ACAGATTCGT TCAGTGAGTT GAGAGTTAGC AATGTCAACA
TATTGTTCGG 6540
AGAGCCCTGC TGAGTACAAC CATTCATTCA GTTTTTTCGA GTCATTAGGG
TAGGAGGATA 6600
TGACACCTTC GTAGTCATTG TACGAGAGAA CGAAATTTGG TGGAAGAC'TA
ATTGATGTGT 6660
CCGATCTTCG GGCACTTACG CAGATTTTGA ATGATCCAGC ATCTTGTGAT
TTCGGTTTGA 6720
GGTCTATTTC GCCGCCAAAG GATATT'rCCG CTTCCATAGC TATCAAAGAG
AAAGAAAAAT 6780
AGTGAATCCA AGGTTTAGGG TT1'CTTTTCT TTGTCTTNC T TA~I'ATATAGA
GGCGCTAGAT 6840
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TGTATTAAGG ATTATACATA TATATAAGTA ATTGCAATTT GTGAGTTTAT
CCTTATTCAT 6900
TTTTAATTTT ATTTACCTTT ATTTAGTTGA TATTGTGTCC TTTTCCTAGG
TAGCATTTCC 6960
TTCCATCTGT GTTAATTATT AGCATTTCCT TTCCTTTGTC TTATTTGCCT
TTATTTCGTA 7020
GGAAGAAATC CTTTATGNAC CCCATCTTGG CTGAGAACTT GAGATGATTT
TAAATCCTCA 7080
AAAATTATTC AATTTATGAT TTCGAAATTG ATATACACTT TATATTTTCT
CCTAAAAAAC 7140
CATATTGTAC TAAGAAAAGT AGAAAACCAG ACTTTTTAAT ATGTTAGATT
TTAATTGGGT 7200
TCTTAAAGTG TTTTAGCGTT TNACACCGGT TATTCTCCAA AATCCAAACT
CTATAATTAT 7260
AGTTTTTAAG TATAAATTAA TCCGGTTGGC CCAATTAGTG GACCGTTTAA
AGAGTAGACA 7320
CTTTTTTTTT TATATATCGA C TACCATAAA ACTTTAACGA TTAATATTTT
TGGATAATAA 7380
GCGATCGTTT TGAGGCGTCC CAATTTTTTT TGTTTCTTTT TATATGAGAA
ATGGGTTTAA 7440
GAAAAACTGC AATTTTGTCC ATAAAGCTAG TCAGAATTCC TGCAGCCCGG
GGGATCCACT 7500
AGTTCTAGAG CGGCCGCCAC CGCGGTGGAG CTCCAATTCG CCCTATAGTG
AGTCGTATTA 7560
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
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Met Pro Ile Ala Asn Val Ile
1 5
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Ile Gln Glu Cys Val Ser Glu Tyr Ile Ser Phe Val
1 5 10
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GGAATTCAGC AACAACCCAA CCCCA 25
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GCTCTAGACA TACAACACTT TTCCTTA 27
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(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ATGACCAGCT CAGTCATAGT AGC 23
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GCCACACATG GTGGTTGCTG CTG ?3
(2) INFORMATION FOR SEQ ID NO:I I
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l 1:
GAGATAGAGA CCGATCGTGG TTC ?3
(2) INFORMATION FOR SEQ ID N0:12:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
TCACTTATAC TGACCA_TAAT GGTC 24
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GCATAGATGC ACTCGAAATC AGCC 24
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
GCTTGGTAAT AATTGTCATT AG 22
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CTAAAAACAT CTACGGTTCA 20
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
TTTGTGGTTG ACCGTTTGGC 20
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Leu Pro Ile Ala Asn Val Ala
1 5
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Gln Glu Cys Val Ser Glu Phe Ile Ser Phe Val
1 5 10
SEQ ID N0:19: Arabidopsis L1L gene (pMNJ7 sequence)
ATGGCAGAGG GCAGTATGCG TCCTCCAGAA TTCAACCAGC CTAACAAAAC
CAGTAATGGT GGTGAGGAGG AGTGCACGGT GAGGGAGCAA GACAGGTTCA
TGCCTATTGC CAACGTGATA CGGATCATGC GGAGGATCTT ACCTGCTCAC
GCCAAGATCT CAGATGACTC CAAGGAGACG ATCCAAGAGT GTGTTTCGGA
GTACATCAGC TTCATAACAG GGGAGGCTAA TGAGCGGTGC CAGCGGGAAC
AGCGCAAGAC CATCACTGCT GAGGACGTCT TGTGGGCAAT GAGCAAGCTC
GGTTTTGATG ACTACATCGA ACCCCTCACG TTGTACCTCC ACCGCTACAG
AGAGTTGGAA GGTGAAAGAG GGGTTAGCTG CAGTGCTGGG TCCGTTAGTA
TGACCAACGG CTTGGTGGTC AAGAGGCCTA ATGGGACCAT GACCGAGTAT
GGAGCCTACG GGCCTGTGCC AGGGATTCAC ATGGCGCAGT ACCATTATCG
TCATCAGAAC GGGTTTGTTT TCAGTGGTAA CGAACCTAAT TCTAAGATGA
GTGGTTCATC TTCAGGAGCA AGTGGCGCCA GAGTTGAAGT ATTTCCGACT
CAACAACATA AGTACTGA
SEQ ID N0:20: Arabidopsis L1L protein
MAEGSMRPPE FNQPNKTSNG GEEECTVREQ DRFMPIANVI RIMRRILPAH
AKISDDSKET IQECVSEYIS FITGEANERC QREQRKTITA EDVLWAMSKL
GFDDYIEPLT LYLHRYRELE GERGVSCSAG SVSMTNGLVV KRPNGTMTEY
GAYGPVPGIH MAQYHYRHQN GFVFSGNEPN SKMSGSSSGA SGARVEVFPT
QQHKY
SEQ ID N0:21: Phaseolua~ gene
GATCTCTCAACCCAACCCTTTCATTTTCATTTTCATTTTCATTTTTCCATCACTTCACTGTC'
ACCATGGAAAG
TGGAGGCTTTCATGGCTACCGCAAGCTCCCCAACACCACCTCTCCTGGGTTGAAGCTGTCAG
TGTCAGACATG
AACAACGTGAACACGAGTAGGCAGGTAGCAGGAGACAACAACCACACAGCGGATGAGAGCAA
CGAATGCACTG
TGAGGGAGCAAGACCGTTTCATGCCAATTGCAAATGTGATCAGGATCATGCGAAAGATTCTT
CCTCCACATGC
CAAGATCTCAGGTGATGCCAAAGAAACAATTCAAGAGTGTGTGTCTGAGTACATCAGCTTTA
TCACCGGAGAG
GCAAACGAGCGTTGCCAGAGGGAACAACGCAAGACCATAACTGCTGAGGACGTGCTTTGGGC
CATGAGCAAGC
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TTGGATTTGATGATTACATGGAGCCACTGACCATGTACCTTCACAGGTATCGTGAGCTTGAG
GGTGACCGAAC
CTCCATGAGAGGTGAATCATTGGGGAAGAGGACTATTGAATACGCCCCTATGGGTGTTGGCG
TTGCTACTGCT
TTTGTGCCACCACAGTTTCACCCAAATGGATACTATGGTCCTGCCATGGGAGCTTACGTTGC
GCCACCAAATG
CTGCGTCCTCTCATCACCATGGAATGCCAAATACTGAACCGAATGCTCGCTCCATGTGAATT
GATGATGATGA
GGAGGAGGAGGAGGAAGACGACGAGTGTTGAGTTAGTAGAAGAAGAATACTTTAATTAATTA
GCTTAACTCTC
GGTAATTAGAGTACTGTTGTTGAGGGTACGTAGTAAACTTTATAATTAAGGGGATGGATGGG
ATTAAGGAGTT
CTGATATTCCTAATCCTAATCAGGCCTATGTTAATTTATGTAATAACTCTGCTTATGTTTTT
GGATTTTCTGA
TGTTGTTCCAAAAAAAAAAAAAA~1AAAAAAAAAAAAAA
SEQ ID N0:22: Phaseolu.r protein
MESGGFHGYRKLPNTTSPGLKLSVSDMNNVNTSRQVAGDNNHTADESNECTVREQDRFMPIA
NVIRIMRKILP
PHAKISGDAKETIQECVSEYISFITGEANERCQREQRKTITAEDVLWAMSKLGFDDYMEPLT
MYLHRYRELEG
DRTSMRGESLGKRTIEYAPMGVGVATAFVPPQFHPNGYYGPAMGAYVAPPNAASSHHHGMPN
TEPNARSM
SEQ ID N0:23: 5' untranslated region
tgggttttca aaggaagagg
atgattctct tcctcctctt caaatggagt ttcaagctc~a aaatcgcatc tcttgggatg
gtctctctct caggtataaa tctcaccatt aaaaatgtga gctttttgtt ~~aactttgga
tctgttactg tgaaaagttg ttactttttt tctgtattat taagagt~ta attttttttc
acgtttatta gaagcttgtt tggtagagac ctcctaaaca cattctcttc ctcttgatat
atttgagctt tgcggtatca tttgattcta gattggttga ctggtgcatc actgaacact
ctcagcttaa agcattaaac tttgcagata tcaatcagat tggtgtgccg tcattacaag
cttttacagt gttggtttat accacttcta agcagtgttt gtctatatat tctgcggaac
ttttggatta ttagttctta gatagtgtaa ccatgttgga agctttgagt ttttgataag
tactttccaa tttttgattt tgcagctcct ctgttgatag cagcgatagt gactcatctc
cagacgttcg caagaccgtc acgggtaaaa gaaagcggga aacaagggta aagctggagc
atttcttgga gaagcttgtg gggagtatga tgaagcggca ggagaagatg cataatcagt
tgattaatgt gatggagaag atggaagtgg agagaatacg ccgtgaggaa gcttggaggc
aacaggaaac cgagaggatg acacagaatg aagaagcacg gaagcaagag atggcacgca
acttgtctct catctctttc atcagaagtg ttactggtga cgagatcgag atccctaaac
agtgtgaatt cccgcaacca ctccagcaga ttcttccgga acaatgtaaa gacgagaaat
gtgaatccgc tcagagagaa agagauataa agtttagata ctcaagcggc agtggcagca
gtggtagaag gtggccgcaa gaggaagtgc aggcattgat aagttcgaga agcgatgtgg
aagagaagac ggggatcaac aagggagcga tttgggatga gatatcagca agaatgaaag
aaagagggta cgaaagatct gcgaaaaagt gtaaggagaa gtgggagaac atgaacaagt
actataggag agtgacggaa ggtgggcaga aacagcctga gcacagcaag actcgctcat
actttgagaa acttggaaat ttttacaaga ccatttcctc gggagagagg gaaaaatgag
tgaaagattt taaatttagg tgtttttggc acgcaaaacg ggagaacttg tagatgatta
cctcgagttt aatttttata tctttggtgt agtttataat ttaaaactct acggctctgt
atttgtagaa ggttcgaata aaaaagacaa atacgttggg gtgattggga ttttgtaacg
gctaagggag acgaggagaa ggatcctcgg tcacatcgat tatggctgcc acgttgttga
acttgtgagg tctgaaatta caaat~ctga cacttgccaa cactattaq~ tttattccaa
ttactctttc ttctctctca ttcca~tctc ttcttcaaat g~ttcttaat t_cgggcatt
ggttattatt atttataggg atattcacaa acacaaaagt cgtgtattta gaacaagaaa
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gatatggaac gtggaggctt ccatggctac cgcaagctgt ccgtgaacaa caccactcct
tctccaccag gtagtgccat tctctatacc ccctcttttc acaggctctc ttcatttcag
ttgcatgcga aaccattctc tgcaatccct ccattgtcat gtctgtactc ttttcatgac
gaacagttaa tgaaatagct tttcaatctt ataaaccgcg catgcagacg tcatcgaagc
cattatgcac taaaacttcc atttttctta tttttgttag gattagcagc gaattttctg
SEQ ID N0:24: 3' untranslated region
ga acaatggcta ataacataga cagctgacag agtcataact
gttagtaggt gcaagctgta gcttatgaat tcaagtttaa gcgaaaacaa tgctgctttt
tctttgttta ttatctatct agttgaaaga acattgtgtt tttcatctga tctgtcttgt
ggtaaagtat gtcaataaag cattagtttt gcaaaccgca tgcatgtgat attacaaaat
tcacggtgaa ttcgtaatgc gtcttggttc aaaatagaaa gagactaaac attccagatt
tcaattctca gctacagaaa tgagtgttta acggatacag aaacaactct cacaatcttc
attcatttca tttagctact actttccaaa ggaacttcaa cgcatacctt tttcctctcc
agaagatcat gtttgtctgc actctcgttt gcctcagtat ctttctcctg atgctcttca
gatatatgtt ccaatttcga acaatcaaca ggatcaagtc cggttctttt cctctgagga
atcacagtga agaaggctgt tttccagtcc ctagtctcca gaaacttgac gagtatctcc
aaaacttggt tcacagtgag aacctaaatc aataaaaacc acaaatctta cattaacaaa
gtacataaag tagaggtttt ttgtgttgtg cccaatgaga caagaattga agtggccatt
tagttacctg agaacttgac attttcatat actctcctat gggaagctta gctgttttaa
tgccttgttc ttgagccttg gtcatggtga tccctttgaa ccggtttcga tccactaagc
caccgataat gtagatatgc ttagggtcaa gatcatccaa aacagtttca gaatcagccg
taagatacac caaattatct ttctgatcag ccatggcttc aatgtaacac ctactttcct
tttcaatgaa ccatttctca aaaccaggaa gcttgtcaag ctcagtactc atcttccc
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