U.S. patent application number 10/343477 was filed with the patent office on 2005-03-24 for floral development genes.
Invention is credited to Ananiev, Evgueni V., Bruggemann, Edward, Cahoon, Edgar B., Cahoon, Rebecca E., Danilevskaya, Olga, Hermon, Pedro, Klein, Theodore M., Rafalski, J. Antoni, Sakai, Hajime, Shirbroun, David M..
Application Number | 20050066394 10/343477 |
Document ID | / |
Family ID | 32823675 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050066394 |
Kind Code |
A1 |
Danilevskaya, Olga ; et
al. |
March 24, 2005 |
Floral development genes
Abstract
This invention relates to an isolated nucleic acid fragment
encoding floral development proteins, more specifically FT, TFL or
Ap3 homologs. The invention also relates to the construction of a
recombinant DNA construct encoding all or a portion of the floral
development proteins, in sense or antisense orientation, wherein
expression of the recombinant DNA construct results in production
of altered levels of the FT, TFL or Ap3 homologs in a transformed
host cell.
Inventors: |
Danilevskaya, Olga;
(Johnston, IA) ; Hermon, Pedro; (Johnston, IA)
; Shirbroun, David M.; (West Des Moines, IA) ;
Bruggemann, Edward; (West Des Moines, IA) ; Ananiev,
Evgueni V.; (Johnston, IA) ; Cahoon, Edgar B.;
(Webster Groves, MO) ; Cahoon, Rebecca E.;
(Webster Groves, MO) ; Klein, Theodore M.;
(Wilmington, DE) ; Rafalski, J. Antoni;
(Wilmington, DE) ; Sakai, Hajime; (Newark,
DE) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL INC.
7100 N.W. 62ND AVENUE
P.O. BOX 1000
JOHNSTON
IA
50131
US
|
Family ID: |
32823675 |
Appl. No.: |
10/343477 |
Filed: |
June 24, 2003 |
PCT Filed: |
November 21, 2001 |
PCT NO: |
PCT/US01/43750 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60253415 |
Nov 28, 2000 |
|
|
|
Current U.S.
Class: |
800/287 ;
435/200; 435/419; 435/468; 435/6.13; 435/69.1; 536/23.2;
800/278 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8287 20130101; C12N 15/8214 20130101; C12N 15/8289
20130101; Y02A 40/146 20180101; C12N 15/8261 20130101 |
Class at
Publication: |
800/287 ;
435/006; 435/069.1; 435/200; 435/419; 435/468; 536/023.2 |
International
Class: |
A01H 001/00; C12N
015/82; C12Q 001/68; C07H 021/04; C12N 009/24; C12N 005/04 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising: (a) a first nucleotide
sequence encoding a polypeptide having FT ot TFL homolog activity,
wherein the amino acid sequence of the polypeptide and the amino
acid sequence of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 26, 28, 30,
34, 36, 40, 42, 44, 54, 56, 58, or 60, have at least 80% sequence
identity based on the Clustal alignment method, or (b) a second
nucleotide sequence encoding a polypeptide having FT or TFL homolog
activity, wherein the amino acid sequence of the polypeptide and
the amino acid sequence of SEQ ID NO:2, 22, 24, 32, or 38, have at
least 85% sequence identity based on the Clustal alignment method,
or (c) a third nucleotide sequence encoding a polypeptide having
Ap3 homolog activity, wherein the amino acid sequence of the
polypeptide and the amino acid sequence of SEQ ID NO:50, has at
least 90% sequence identity based on the Clustal alignment method,
or (d) a fourth nucleotide sequence encoding a polypeptide having
FT or TFL homolog activity, wherein the amino acid sequence of the
polypeptide and the amino acid sequence of SEQ ID NO:20, has at
least 95% sequence identity based on the Clustal alignment method,
or (e) a fifth nucleotide sequence encoding a polypeptide having
Ap3 homolog activity, wherein the amino acid sequence of the
polypeptide and the amino acid sequence of SEQ ID NO:46, or 48,
have at least 95% sequence identity based on the Clustal alignment
method, or (f) the complement of the first, second, third, fourth,
or fifth, nucleotide sequence, wherein the complement and the
nucleotide sequence contain the same number of nucleotides and are
100% complementary.
2. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO:4, 6,
8, 10, 12, 14, 16, 18, 26, 28, 30, 34, 36, 40, 42, 44, 54, 56, 58,
or 60, have at least 85% identity based on the Clustal alignment
method.
3. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO:2, 4,
6, 8, 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 54, 56, 58, or 60, have at least 90% identity based on the
Clustal alignment method.
4. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO:2, 4,
6, 8, 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 50, 54, 56, 58, or 60 have at least 95% identity based on
the Clustal alignment method.
5. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide comprises the amino acid sequence of SEQ ID
NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, or 60.
6. The polynucleotide of claim 1 wherein the nucleotide sequence
comprises the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47,
49, 53, 55, 57, 59, 63, 64, 65, 66 or 67.
7. A vector comprising the polynucleotide of claim 1.
8. A recombinant DNA construct comprising the polynucleotide of
claim 1 operably linked to a regulatory sequence.
9. A method for transforming a cell, comprising transforming a cell
with the polynucleotide of claim 1.
10. A cell comprising the recombinant DNA construct of claim 8.
11. A method for producing a plant comprising transforming a plant
cell with the polynucleotide of claim 1 and regenerating a plant
from the transformed plant cell.
12. A plant comprising the recombinant DNA construct of claim
8.
13. A seed comprising the recombinant DNA construct of claim 8.
14. An isolated polynucleotide comprising a first nucleotide
sequence, wherein the first nucleotide sequence contains at least
30 nucleotides, and wherein the first nucleotide sequence is
comprised by another polynucleotide, wherein the other
polynucleotide includes: (a) a second nucleotide sequence, wherein
the second nucleotide sequence encodes a polypeptide having FT
homolog activity, wherein the amino acid sequence of the
polypeptide and the amino acid sequence of SEQ ID NO:4, 6, 8, 10,
12, 14, 16, 18, 26, 28, 30, 34, 36, 40, 42, 44, 54, 56, 58, or 60,
having at least 80% sequence identity based on the Clustal
alignment method, or (b) a third nucleotide sequence, wherein the
third nucleotide sequence encodes a polypeptide having FT homolog
activity, wherein the amino acid sequence of the polypeptide and
the amino acid sequence of SEQ ID NO:2, 22, 24, 32, or 38, having
at least 85% sequence identity based on the Clustal alignment
method, or (c) a fourth nucleotide sequence, wherein the fourth
nucleotide sequence encodes a polypeptide having Ap3 homolog
activity, wherein the amino acid sequence of the polypeptide and
the amino acid sequence of SEQ ID NO:50 has at least 90% sequence
identity based on the Clustal alignment method, or (d) a fifth
nucleotide sequence, wherein the fifth nucleotide sequence encodes
a polypeptide having FT homolog activity, wherein the amino acid
sequence of the polypeptide and the amino acid sequence of SEQ ID
NO:20 has at least 95% sequence identity based on the Clustal
alignment method, or (e) a sixth nucleotide sequence, wherein the
sixth nucleotide sequence encodes a polypeptide having Ap3 homolog
activity, wherein the amino acid sequence of the polypeptide and
the amino acid sequence of SEQ ID NO:46, or 48, has at least 95%
sequence identity based on the Clustal alignment method, or (f) the
complement of the second, third, fourth, fifth, or sixth nucleotide
sequence, wherein the complement and the second, third, fourth,
fifth, or sixth nucleotide sequence contain the came number of
nucleotides and are 100% complementary.
15. An isolated polypeptide having FT or Ap3 homolog activity,
wherein the amino acid sequence of the polypeptide and the amino
acid sequence of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 26, 28, 30,
34, 36, 40, 42, 44, 54, 56, 58, or 60, have at least 80% identity
based on the Clustal alignment method.
16. The polypeptide of claim 15, wherein the amino acid sequence of
the polypeptide and the amino acid sequence of SEQ ID NO:2, 4, 6,
8, 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 54, 56, 58, or 60, have at least 85% identity based on the
Clustal alignment method.
17. The polypeptide of claim 15, wherein the amino acid sequence of
the polypeptide and the amino acid sequence of SEQ ID NO:2, 4, 6,
8, 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 50, 54, 56, 58, or 60 have at least 90% identity based on the
Clustal alignment method.
18. The polypeptide of claim 15, wherein the amino acid sequence of
the polypeptide and the amino acid sequence of SEQ ID NO:2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48, 50, 54, 56, 58, or 60 have at least 95% identity
based on the Clustal alignment method.
19. The polypeptide of claim 15, wherein the amino acid sequence of
the polypeptide comprises the amino acid sequence of SEQ ID NO:2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 54, 56, 58, or 60.
20. A method for isolating a polypeptide encoded by the
polynucleotide of claim 1 comprising isolating the polypeptide from
a cell containing a recombinant DNA construct comprising the
polynucleotide operably linked to a regulatory sequence.
Description
APPLICATION PRIORITY INFORMATION
[0001] This continuation-in-part application claims the benefit of
International Patent Application No. PCT/US01/43750, filed Nov. 21,
2001, and U.S. Provisional Application No. 60/253,415, filed Nov.
28, 2000, the entire contents of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to nucleic acid
fragments encoding floral development proteins in plants and
seeds.
BACKGROUND OF THE INVENTION
[0003] Flowering in plants is a consequence of the transition of
the shoot apex from vegetative to reproductive growth in response
to environmental and internal signals. Currently, there is little
information about how plants coordinate the activities of the cells
that give rise to reproductive plant tissues, however, research has
focused on identifying the genes that control this developmental
process. Floral homeotic genes that control the specification of
meristem and organ identity in developing flowers have been
identified in Arabidopsis thaliana and Antirrhinum majus. Most of
these genes belong to a large family of regulatory genes that
possess a characteristic DNA binding domain known as the MADS-box.
Members of this gene family display primarily floral-specific
expression and are homologous to transcription factors found in
several animal and fungal species. Molecular evolutionary analysis
reveal that there are appreciable differences in the substitution
rates between different domains of these plant MADS-box genes.
Phylogenetic analysis also demonstrate that members of the plant
MADS-box gene family are organized into several distinct gene
groups: the AGAMOUS, APETALA3 (Ap3)/PISTILLATA and APETALA1/AGL9
groups. Several genes that belong to the APETALA3 (Ap3) group have
been identified in Arabidopsis thaliana (Jack et al., (1992) Cell
68:683-697). Genes of this group have been shown to play a role in
the control of organ identity of petals and stamens during floral
development (Bowman et al., (1989) Plant Cell 1:37-52 and Bowman et
al., (1991) Development 112:1-20; Weigel and Meyerowitz (1994) Cell
78:203-209; Coen and Meyerowitz (1991) Nature 353:31-37; WO
93/21322). Thus, the shared evolutionary history of members of a
gene group appear to reflect the distinct functional roles these
MAD-box genes play in flower development.
[0004] The flowering locus T gene (FT) encodes a protein that
appears to be involved in the regulating plant growth by
controlling the rate at which maturation occurs. For example, an
increase in FT function has been shown to produce early flowering
(Kardailsky et al., (1999) Science 286:1962-1965). Thus the FT gene
may be useful to accelerate flowering in various crops.
[0005] The deduced sequence of the FT protein is similar to the
sequence of TERMINAL FLOWER 1 (TFL1) and shares sequence similarity
with membrane-associated mammalian proteins (Kardailsky et al.,
(1999) Science 286:1962-1965). TFL1 in Arabidopsis, and the
homologous Antirrhinum gene CENTRORADIALIS (CEN) play a key role in
determining inflorescence architecture (Bradley et al. (1997)
Science 275:80-83; WO 97/10339; WO 99/53070).
[0006] FT protein belongs to a family of membrane-associated
phosphatidylethanolamine-bind proteins (PEBP), which may function
as kinase inhibitors to regulate the signal transudation pathways
(Kardailsky et al., Activation tagging of the floral inducer FT,
Science, Dec 3;286 (5446), 1962-5,1999); Kobayashi et al., A pair
of related genes with antagonistic roles in mediating flower
signals, Science, December 3;286 (5446), 1960-2,1999). The
Arabidopsis gene TFL encodes a protein related to FT. Genes play
antagonistic roles in the floral transition such as TFL is a
repressor of flowering whereas FT is an activator (Kardailsky et
al., 1999; Kobayashi et al., 1999). International patent
application PCT/US01/43750 claims 9 corn homologs of the
Arabidopsis FT. For continuity, maize genes were named ZmFT or
ZmTFL (Table 1) accordingly to the degree of the translational
homology to Arabidopsis FT-TFL proteins (GenBank accession numbers
FT AB027504, TFL U77674).
[0007] There is a great deal of interest in identifying the genes
that encode proteins involved in cellular differentiation in
plants. These genes may be used in plant cells to control
development. Accordingly, the availability of nucleic acid
sequences encoding all or a portion of an Ap3 or FT or TFL1 gene
homolog would facilitate studies to better understanding
development in plants and provide genetic tools to enhance or
otherwise alter plant developmental processes. Nucleic acid
fragments encoding Ap3 homologs may be useful for engineering plant
sterility/fertility, and flower development and morphology. Nucleic
acid fragments encoding FT or TFL1 homologs may be useful for
engineering flowering time, plant growth rate, inflorescence
architecture, and tissue culture morphology and rate of cell
division to enhance transformation.
SUMMARY OF THE INVENTION
[0008] The present invention concerns isolated polynucleotides
comprising a nucleotide sequence encoding a polypeptide having FT,
TFL or Ap3 homolog activity wherein the amino acid sequence of the
polypeptide and the amino acid sequence of SEQ ID NO:2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 54, 56, 58, or 60 have at least 80% sequence
identity. It is preferred that the identity be at least 85%, it is
preferable if the identity is at least 90%, it is more preferred
that the identity be at least 95%. The present invention also
relates to isolated polynucleotides comprising the complement of
the nucleotide sequence, wherein the complement and the nucleotide
sequence contain the same number of nucleotides and are 100%
complementary. More specifically, the present invention concerns
isolated polynucleotides encoding the polypeptide sequence of SEQ
ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, or 60 or nucleotide
sequences comprising the nucleotide sequence of SEQ ID NO:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41,
43, 45, 47, 49, 53, 55, 57, 59, 63, 64, 65, 66 or 67.
[0009] In a first embodiment, the present invention concerns an
isolated polynucleotide comprising: (a) a nucleotide sequence
encoding a polypeptide comprising at least 50, 100, 150, 160, 170,
175, or 200, amino acids, wherein the amino acid sequence of the
polypeptide and the amino acid sequence of SEQ ID NO:2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 54, 56, 58, or 60 have at least 80%, 85%, 90%, or
95% identity based on the Clustal alignment method, or (b) the
complement of the nucleotide sequence, wherein the complement and
the nucleotide sequence contain the same number of nucleotides and
are 100% complementary. The polypeptide preferably comprises the
amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54,
56, 58, or 60. The nucleotide sequence preferably comprises the
nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57,
59, 63, 64, 65, 66, or 67. The polypeptide preferably is a FT, TFL
or Ap3 homolog.
[0010] In a second embodiment, the present invention relates to a
recombinant DNA construct comprising any of the isolated
polynucleotides of the present invention operably linked to a
regulatory sequence, and a cell, a plant, and a seed comprising the
recombinant DNA construct.
[0011] In a third embodiment, the present invention relates to a
vector comprising any of the isolated polynucleotides of the
present invention.
[0012] In a fourth embodiment, the present invention relates to an
isolated polynucleotide comprising a nucleotide sequence comprised
by any of the polynucleotides of the first embodiment, wherein the
nucleotide sequence contains at least 30, 40, 50, 60, 100, 150,
160, 170, 175, or 200 nucleotides.
[0013] In a fifth embodiment, the present invention relates to a
method for transforming a cell comprising transforming a cell with
any of the isolated polynucleotides of the present invention, and
the cell transformed by this method. Advantageously, the cell is
eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a
bacterium.
[0014] In a sixth embodiment, the present invention relates to a
method for producing a transgenic plant comprising transforming a
plant cell with any of the isolated polynucleotides of the present
invention and regenerating a plant from the transformed plant cell.
The invention also concerns the transgenic plant produced by this
method, and the seed obtained from this transgenic plant.
[0015] In a seventh embodiment, the present invention concerns an
isolated polypeptide comprising an amino acid sequence comprising
at least 50, 100, 150, 160, 170, 175, or 200, amino acids, wherein
the amino acid sequence and the amino acid sequence of SEQ ID NO:2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 54, 56, 58, or 60 have at least 80%,
85%, 90%, or 95% identity based on the Clustal alignment method.
The amino acid sequence preferably comprises the amino acid
sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, or
60. The polypeptide preferably is a FT, TFL or Ap3 homolog.
[0016] In an eighth embodiment, the invention concerns a method for
isolating a polypeptide encoded by the polynucleotide of the
present invention comprising isolating the polypeptide from a cell
containing a recombinant DNA construct comprising the
polynucleotide operably linked to a regulatory sequence.
[0017] In a ninth embodiment, the present invention relates to a
virus, preferably a baculovirus, comprising any of the isolated
polynucleotides of the present invention or any of the recombinant
DNA constructs of the present invention.
[0018] In a tenth embodiment, the invention relates to a method of
selecting an isolated polynucleotide that affects the level of
expression of a gene encoding a FT, TFL or Ap3 homolog protein or
activity in a host cell, preferably a plant cell, the method
comprising the steps of: (a) constructing an isolated
polynucleotide of the present invention or an isolated recombinant
DNA construct of the present invention; (b) introducing the
isolated polynucleotide or the isolated recombinant DNA construct
into a host cell; (c) measuring the level of the FT, TFL or Ap3
homolog protein or activity in the host cell containing the
isolated polynucleotide; and (d) comparing the level of the FT, TFL
or Ap3 homolog protein or activity in the host cell containing the
isolated polynucleotide with the level of the FT, TFL or Ap3
homolog protein or activity in the host cell that does not contain
the isolated polynucleotide.
[0019] In an eleventh embodiment, the invention concerns a method
of obtaining a nucleic acid fragment encoding a substantial portion
of a FT, TFL or Ap3 homolog protein, preferably a plant FT, TFL or
Ap3 homolog protein comprising the steps of: synthesizing an
oligonucleotide primer comprising a nucleotide sequence of at least
30 (preferably at least 40, most preferably at least 60) contiguous
nucleotides derived from a nucleotide sequence selected from the
group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57,
or 59, and the complement of such nucleotide sequences; and
amplifying a nucleic acid fragment (preferably a cDNA inserted in a
cloning vector) using the oligonucleotide primer. The amplified
nucleic acid fragment preferably will encode a substantial portion
of a FT, TFL or Ap3 homolog amino acid sequence.
[0020] In a twelfth embodiment, this invention relates to a method
of obtaining a nucleic acid fragment encoding all or a substantial
portion of the amino acid sequence encoding a FT, TFL or Ap3
homolog protein comprising the steps of: probing a cDNA or genomic
library with an isolated polynucleotide of the present invention;
identifying a DNA clone that hybridizes with an isolated
polynucleotide of the present invention; isolating the identified
DNA clone; and sequencing the cDNA or genomic fragment that
comprises the isolated DNA clone.
[0021] In a thirteenth embodiment, this invention concerns a method
for positive selection of a transformed cell comprising: (a)
transforming a host cell with the recombinant DNA construct of the
present invention or an expression cassette of the present
invention; and (b) growing the transformed host cell, preferably a
plant cell, such as a monocot or a dicot, under conditions which
allow expression of the FT, TFL or Ap3 homolog polynucleotide in an
amount sufficient to complement a null mutant, or a conditional
null mutant, to provide a positive selection means.
[0022] In a fourteenth embodiment, this invention relates to a
method of altering the level of expression of a FT, TFL or Ap3
homolog protein in a host cell comprising: (a) transforming a host
cell with a recombinant DNA construct of the present invention; and
(b) growing the transformed host cell under conditions that are
suitable for expression of the recombinant DNA construct wherein
expression of the recombinant DNA construct results in production
of altered levels of the FT, TFL or Ap3 homolog protein in the
transformed host cell.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0023] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing which form a part of this application.
[0024] FIG. 1 depicts an alignment of amino acid sequences of FT
homologs encoded by nucleotide sequences derived from a contig
assembled from balsam pear clones fds.pk0003.h2, fds.pk0026.d10,
and fds1n.pk001.p18 (SEQ ID NO:4), garden balsam clone
ids.pk0031.a5 (SEQ ID NO:6), contig assembled from corn clones
cbn10.pk0052.f5, cbn2.pk0035.f12, cco1n.pk0010.h3, p0095.cwsas14f,
p0119.cmtmg45rb, and p0128.cpicl42r (SEQ ID NO:8), corn clone
cc71se-b.pk0003.h10 (SEQ ID NO:10), corn clone cco1n.pk0037.d10
(SEQ ID NO:12), contig assembled from corn clones cen3n.pk0004.e9,
cen3n.pk0047.h7, cen3n.pk0093.f1, cen3n.pk0165.f1, and
p0120.cdeae63r (SEQ ID NO:14), corn clone p0014.ctush42r (SEQ ID
NO:16), corn clone p0081.chcad07r (SEQ ID NO:18), corn clone
p0104.cabak14rb (SEQ ID NO:20), corn clone p0118.chsaq04rb (SEQ ID
NO:22), rice clone rls24.pk0017.c7 (SEQ ID NO:30), rice clone
rr1.pk0043.f9 (SEQ ID NO:32), contig assembled from soybean clones
se3.pk0036.g4 and se6.pk0039.h6 (SEQ ID NO:36), soybean clone
srr2c.pk002.o7 (SEQ ID NO:38), contig assembled from soybean clone
ssl.pk0007.a9 and a PCR fragment sequence (SEQ ID NO:40), wheat
clone wdk2c.pk012.o17 (SEQ ID NO:42), and wheat clone
wdk9n1.pk001.o20 (SEQ ID NO:44) and Oryza sativa (NCBI GI No.
5360178; SEQ ID NO:51). Amino acids which are conserved among all
and at least two sequences with an amino acid at that position are
indicated with an asterisk (*). Dashes are used by the program to
maximize alignment of the sequences.
[0025] FIG. 2 depicts an alignment of amino acid sequences of Ap3
homologs encoded by nucleotide sequences derived from corn clone
cta1n.pk0050.f8 (SEQ ID NO:46), corn clone ctnlc.pk002.j23 (SEQ ID
NO:48), soybean clone sf1n.pk001.I16 (SEQ ID NO:50), and Oryza
sativa (NCBI GI No. 5295980; SEQ ID NO:52). Amino acids which are
conserved among all and at least two sequences with an amino acid
at that position are indicated with an asterisk (*). Dashes are
used by the program to maximize alignment of the sequences.
[0026] FIG. 3 delineated a phylogenetic analysis of the family of
membrane-associated phosphatidylethanolamine-binding proteins. The
phylogenetic tree was constructed by the maximum parsimony methods.
The phylogram clearly delimits two major clades that correspond to
FT and TFL proteins.
[0027] FIG. 4 diagrams the genomic structures of the FT/TFL
sequences.
[0028] The genomic region of ZmFT1 (SEQ ID NO: 63) is composed of a
promoter (1-2211 nt), 5'UTR (2212-2385 nt), exon 1 (2386-3580 nt),
exon 2 (2741-2802 nt), exon 3 (9718-9760 nt), exon 4 (9845-10067),
3'UTR (10067-10316).
[0029] The genomic region of ZmFT2 (SEQ ID NO: 64) is composed of
5'UTR (1-93), exon1 (94-293), exon2 (468-525), exon3 (765-806),
exon 4 (1411-1651), 3'UTR (1652-1840).
[0030] The genomic region of ZmFT3 (SEQ ID NO: 65) is composed of a
promoter (286-4375 nt), 5'UTR (4376-4542 nt), exon 1 (4543-4743
nt), exon 2 (4894-4953 nt), exon 3 (5688-5728 nt), exon 4
(6166-6396 nt), 3'UTR (6397-6860).
[0031] The genomic region of ZmTFL1 (SEQ ID NO: 66) is composed of
two copies of ZmTFL1 gene arranged in a perfect tandem. The first
copy has a partial promoter (1-562 nt), 5'UTR (486-563 nt), exon 1
(564-763 nt), exon 2 (846-9907 nt), exon 3 (1056-1096 nt), exon 4
(1176-1364 nt), 3'UTR (1395-1611 nt), 3' downstream segment
(1612-2435 nt). The second copy begins from 2436 nt and shows the
identical structure as the first one. Genomic organization of
ZmTFL1 gene is an example of an unusual configuration of a tandem
array of two gene copies. The unit length in tandem is 2292 nt,
which include a 5'upstream sequence (364 nt), exon/intron genic
segment (1116 nt) and 3'downstream sequence (812 nt). A promoter
for the second ZmTFL1 copy may be defined between nucleotides 1611
and 2435 (824 nt total length). Almost identical nucleotide
sequences of both units suggest a very recent duplication of ZmTFL1
gene in Mol 7 genome.
[0032] Genomic region of ZmTFL2 (SEQ ID NO: 67) is composed of a
promoter (1-1450 nt), 5'UTR (1451-1518 nt), exon 1 (1519-1780 nt),
exon 2 (2097-2137 nt), exon 3 (2309-2595 nt), 3'UTR (2596-2881
nt).
[0033] Table 1 lists the polypeptides that are described herein,
the designation of the cDNA clones that comprise the nucleic acid
fragments encoding polypeptides representing all or a substantial
portion of these polypeptides, and the corresponding identifier
(SEQ ID NO:) as used in the attached Sequence Listing. The sequence
descriptions and Sequence Listing attached hereto comply with the
rules governing nucleotide and/or amino acid sequence disclosures
in patent applications as set forth in 37 C.F.R.
.sctn.1.821-1.825.
1TABLE 1 Floral Development Proteins SEQ ID NO: (Amino Protein
(Plant Source) Clone Designation (Nucleotide) Acid) FT Homolog
eal1c.pk006.e6 1 2 (Peruvian Lily) FT Homolog Contig of 3 4 (Balsam
Pear) fds.pk0003.h2 fds.pk0026.d10 fds1n.pk001.p18 FT Homolog
(Garden ids.pk0031.a5 5 6 Balsam) FT Homolog (Corn) Contig of 7 8
cbn10.pk0052.f5 cbn2.pk0035.f12 cco1n.pk0010.h3 p0095.cwsas14f
p0119.cmtmg45rb p0128.cpicl42r FT Homolog (Corn)
cc71se-b.pk0003.h10 9 10 FT Homolog (Corn) cco1n.pk0037.d10 11 12
FT Homolog (Corn) Contig of 13 14 cen3n.pk0004.e9 cen3n.pk0047.h7
cen3n.pk0093.f1 cen3n.pk0165.f1 p0120.cdeae63r FT Homolog (Corn)
p0014.ctush42r 15 16 FT Homolog (Corn) p0081.chcad07r 17 18 FT
Homolog (Corn) p0104.cabak14rb 19 20 FT Homolog (Corn)
p0118.chsaq04rb 21 22 FT Homolog (Rice) rbm1c.pk001.a6 23 24 FT
Homolog (Rice) Contig of 25 26 rl0n.pk0022.h10 rl0n.pk0022.h11 FT
Homolog (Rice) rlr48.pk0001.b1 27 28 FT Homolog (Rice)
rls24.pk0017.c7 29 30 FT Homolog (Rice) rr1.pk0043.f9 31 32 FT
Homolog (Rice) rsr9n.pk001.d1 33 34 FT Homolog (Soybean) Contig of
35 36 se3.pk0036.g4 se6.pk0039.h6 FT Homolog (Soybean)
srr2c.pk002.o7 37 38 FT Homolog (Soybean) Contig of 39 40
ssl.pk0007.a9 PCR fragment sequence FT Homolog (Wheat)
wdk2c.pk012.o17 41 42 FT Homolog (Wheat) wdk9n1.pk001.o20 43 44 Ap3
Homolog (Corn) cta1n.pk0050.f8 45 46 Ap3 Homolog (Corn)
ctn1c.pk002.j23 47 48 Ap3 Homolog (Soybean) sfl1n.pk001.l16 49 50
FT Homolog (Corn) cta1n.pk0058.d11b 53 54 FT Homolog (Rice)
rbm1c.pk001.a6:fis 55 56 FT Homolog (Rice) rl0n.pk0022.h10:fis 57
58 FT Homolog (Rice) rsr9n.pk001.d1:fis 59 60 FT Homolog 61
(Arabiopsis) FT Homolog (Rice) 62 FT Homolog (Corn) 63 FT Homolog
(Corn 64 FT Homolog (Corn) 65 TFL Homolog (Corn) 66 TFL Homolog
(Corn) 67
[0034] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Res. 13:3021-3030 (1985) and in the
Biochemical J. 219 (No. 2):345-373 (1984) which are herein
incorporated by reference. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn.1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0035] In the context of this disclosure, a number of terms shall
be utilized. The terms "polynucleotide", "polynucleotide sequence",
"nucleic acid sequence", and "nucleic acid fragment"/"isolated
nucleic acid fragment" are used interchangeably herein. These terms
encompass nucleotide sequences and the like. A polynucleotide may
be a polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic
DNA, or mixtures thereof. An isolated polynucleotide of the present
invention may include at least 30 contiguous nucleotides,
preferably at least 40 contiguous nucleotides, most preferably at
least 60 contiguous nucleotides derived from SEQ ID NO:1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43,
45, 47, 49, 53, 55, 57, 59, 63, 64, 65, 66 or 67 or the complement
of such sequences.
[0036] The term "isolated" refers to materials, such as nucleic
acid molecules and/or proteins, which are substantially free or
otherwise removed from components that normally accompany or
interact with the materials in a naturally occurring environment.
Isolated polynucleotides may be purified from a host cell in which
they naturally occur. Conventional nucleic acid purification
methods known to skilled artisans may be used to obtain isolated
polynucleotides. The term also embraces recombinant polynucleotides
and chemically synthesized polynucleotides.
[0037] The term "recombinant" means, for example, that a nucleic
acid sequence is made by an artificial combination of two otherwise
separated segments of sequence, e.g., by chemical synthesis or by
the manipulation of isolated nucleic acids by genetic engineering
techniques.
[0038] As used herein, "contig" refers to a nucleotide sequence
that is assembled from two or more constituent nucleotide sequences
that share common or overlapping regions of sequence homology. For
example, the nucleotide sequences of two or more nucleic acid
fragments can be compared and aligned in order to identify common
or overlapping sequences. Where common or overlapping sequences
exist between two or more nucleic acid fragments, the sequences
(and thus their corresponding nucleic acid fragments) can be
assembled into a single contiguous nucleotide sequence.
[0039] As used herein, "substantially similar" refers to nucleic
acid fragments wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the polypeptide encoded by the
nucleotide sequence. "Substantially similar" also refers to nucleic
acid fragments wherein changes in one or more nucleotide bases does
not affect the ability of the nucleic acid fragment to mediate
alteration of gene expression by gene silencing through for example
antisense or co-suppression technology. "Substantially similar"
also refers to modifications of the nucleic acid fragments of the
instant invention such as deletion or insertion of one or more
nucleotides that do not substantially affect the functional
properties of the resulting transcript vis--vis the ability to
mediate gene silencing or alteration of the functional properties
of the resulting protein molecule. It is therefore understood that
the invention encompasses more than the specific exemplary
nucleotide or amino acid sequences and includes functional
equivalents thereof. The terms "substantially similar" and
"corresponding substantially" are used interchangeably herein.
[0040] Substantially similar nucleic acid fragments may be selected
by screening nucleic acid fragments representing subfragments or
modifications of the nucleic acid fragments of the instant
invention, wherein one or more nucleotides are substituted, deleted
and/or inserted, for their ability to affect the level of the
polypeptide encoded by the unmodified nucleic acid fragment in a
plant or plant cell. For example, a substantially similar nucleic
acid fragment representing at least 30 contiguous nucleotides,
preferably at least 40 contiguous nucleotides, most preferably at
least 60 contiguous nucleotides derived from the instant nucleic
acid fragment can be constructed and introduced into a plant or
plant cell. The level of the polypeptide encoded by the unmodified
nucleic acid fragment present in a plant or plant cell exposed to
the substantially similar nucleic fragment can then be compared to
the level of the polypeptide in a plant or plant cell that is not
exposed to the substantially similar nucleic acid fragment.
[0041] For example, it is well known in the art that antisense
suppression and co-suppression of gene expression may be
accomplished using nucleic acid fragments representing less than
the entire coding region of a gene, and by using nucleic acid
fragments that do not share 100% sequence identity with the gene to
be suppressed. Moreover, alterations in a nucleic acid fragment
which result in the production of a chemically equivalent amino
acid at a given site, but do not effect the functional properties
of the encoded polypeptide, are well known in the art. Thus, a
codon for the amino acid alanine, a hydrophobic amino acid, may be
substituted by a codon encoding another less hydrophobic residue,
such as glycine, or a more hydrophobic residue, such as valine,
leucine, or isoleucine. Similarly, changes which result in
substitution of one negatively charged residue for another, such as
aspartic acid for glutamic acid, or one positively charged residue
for another, such as lysine for arginine, can also be expected to
produce a functionally equivalent product. Nucleotide changes which
result in alteration of the N-terminal and C-terminal portions of
the polypeptide molecule would also not be expected to alter the
activity of the polypeptide. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products.
Consequently, an isolated polynucleotide comprising a nucleotide
sequence of at least 30 (preferably at least 40, most preferably at
least 60) contiguous nucleotides derived from a nucleotide sequence
selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47,
49, 53, 55, 57, 59, 63, 64, 65, 66 or 67, and the complement of
such nucleotide sequences may be used to affect the expression
and/or function of a FT, TFL or Ap3 homolog in a host cell. A
method of using an isolated polynucleotide to affect the level of
expression of a polypeptide in a host cell (eukaryotic, such as
plant or yeast, prokaryotic such as bacterial) may comprise the
steps of: constructing an isolated polynucleotide of the present
invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric
gene into a host cell; measuring the level of a polypeptide or
enzyme activity in the host cell containing the isolated
polynucleotide; and comparing the level of a polypeptide or enzyme
activity in the host cell containing the isolated polynucleotide
with the level of a polypeptide or enzyme activity in a host cell
that does not contain the isolated polynucleotide.
[0042] Moreover, substantially similar nucleic acid fragments may
also be characterized by their ability to hybridize. Estimates of
such homology are provided by either DNA-DNA or DNA-RNA
hybridization under conditions of stringency as is well understood
by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic
Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions
can be adjusted to screen for moderately similar fragments, such as
homologous sequences from distantly related organisms, to highly
similar fragments, such as genes that duplicate functional enzymes
from closely related organisms. Post-hybridization washes determine
stringency conditions. One set of preferred conditions uses a
series of washes starting with 6.times.SSC, 0.5% SDS at room
temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at
45.degree. C. for 30 min, and then repeated twice with
0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 min. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times.SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times.SSC, 0.1%
SDS at 65.degree. C.
[0043] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Suitable nucleic acid fragments
(isolated polynucleotides of the present invention) encode
polypeptides that are at least about 70% identical, preferably at
least about 80% identical to the amino acid sequences reported
herein. Preferred nucleic acid fragments encode amino acid
sequences that are at least about 85% identical to the amino acid
sequences reported herein. More preferred nucleic acid fragments
encode amino acid sequences that are at least about 90% identical
to the amino acid sequences reported herein. Most preferred are
nucleic acid fragments that encode amino acid sequences that are at
least about 95% identical to the amino acid sequences reported
herein. Suitable nucleic acid fragments not only have the above
identities but typically encode a polypeptide having at least 50
amino acids, preferably at least 100 amino acids, more preferably
at least 150 amino acids, still more preferably at least 200 amino
acids, and most preferably at least 250 amino acids. Sequence
alignments and percent identity calculations were performed using
the Megalign program of the LASERGENE bioinformatics computing
suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the
sequences was performed using the Clustal method of alignment
(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default
parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default
parameters for pairwise alignments using the Clustal method were
KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
[0044] A "substantial portion" of an amino acid or nucleotide
sequence comprises an amino acid or a nucleotide sequence that is
sufficient to afford putative identification of the protein or gene
that the amino acid or nucleotide sequence comprises. Amino acid
and nucleotide sequences can be evaluated either manually by one
skilled in the art, or by using computer-based sequence comparison
and identification tools that employ algorithms such as BLAST
(Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.
Biol. 215:403-410; see also the explanation of the BLAST alogarithm
on the world wide web site for the National Center for
Biotechnology Information at the National Library of Medicine of
the National Institutes of Health). In general, a sequence of ten
or more contiguous amino acids or thirty or more contiguous
nucleotides is necessary in order to putatively identify a
polypeptide or nucleic acid sequence as homologous to a known
protein or gene. Moreover, with respect to nucleotide sequences,
gene-specific oligonucleotide probes comprising 30 or more
contiguous nucleotides may be used in sequence-dependent methods of
gene identification (e.g., Southern hybridization) and isolation
(e.g., in situ hybridization of bacterial colonies or bacteriophage
plaques). In addition, short oligonucleotides of 12 or more
nucleotides may be used as amplification primers in PCR in order to
obtain a particular nucleic acid fragment comprising the primers.
Accordingly, a "substantial portion" of a nucleotide sequence
comprises a nucleotide sequence that will afford specific
identification and/or isolation of a nucleic acid fragment
comprising the sequence. The instant specification teaches amino
acid and nucleotide sequences encoding polypeptides that comprise
one or more particular plant proteins. The skilled artisan, having
the benefit of the sequences as reported herein, may now use all or
a substantial portion of the disclosed sequences for purposes known
to those skilled in this art. Accordingly, the instant invention
comprises the complete sequences as reported in the accompanying
Sequence Listing, as well as substantial portions of those
sequences as defined-above.
[0045] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid fragment comprising a
nucleotide sequence that encodes all or a substantial portion of
the amino acid sequences set forth herein. The skilled artisan is
well aware of the "codon-bias" exhibited by a specific host cell in
usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a nucleic acid fragment for improved
expression in a host cell, it is desirable to design the nucleic
acid fragment such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
[0046] "Synthetic nucleic acid fragments" can be assembled from
oligonucleotide building blocks that are chemically synthesized
using procedures known to those skilled in the art. These building
blocks are ligated and annealed to form larger nucleic acid
fragments which may then be enzymatically assembled to construct
the entire desired nucleic acid fragment. "Chemically synthesized",
as related to a nucleic acid fragment, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of
nucleic acid fragments may be accomplished using well established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the nucleic acid fragments can be tailored for optimal gene
expression based on optimization of the nucleotide sequence to
reflect the codon bias of the host cell. The skilled artisan
appreciates the likelihood of successful gene expression if codon
usage is biased towards those codons favored by the host.
Determination of preferred codons can be based on a survey of genes
derived from the host cell where sequence information is
available.
[0047] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Chimeric gene" refers any gene
that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature.
"Endogenous gene" refers to a native gene in its natural location
in the genome of an organism. A "foreign-gene" refers to a gene not
normally found in the host organism, but that is introduced into
the host organism by gene transfer. Foreign genes can comprise
native genes inserted into a non-native organism, or chimeric
genes. A "transgene" is a gene that has been introduced into the
genome by a transformation procedure.
[0048] "Coding sequence" refers to a nucleotide sequence that codes
for a specific amino acid sequence. "Regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, and polyadenylation recognition sequences.
[0049] "Promoter" refers to a nucleotide sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
The promoter sequence consists of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a nucleotide sequence which can
stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or may be composed of different
elements derived from different promoters found in nature, or may
even comprise synthetic nucleotide segments. It is understood by
those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental conditions. Promoters which cause a nucleic acid
fragment to be expressed in most cell types at most times are
commonly referred to as "constitutive promoters". New promoters of
various types useful in plant cells are constantly being
discovered; numerous examples may be found in the compilation by
Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is
further recognized that since in most cases the exact boundaries of
regulatory sequences have not been completely defined, nucleic acid
fragments of different lengths may have identical promoter
activity.
[0050] "Translation leader sequence" refers to a nucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner and Foster (1995) Mol. Biotechnol.
3:225-236).
[0051] "3' non-coding sequences" refer to nucleotide sequences
located downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.
[0052] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from post-transcriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger
RNA (mRNA)" refers to the RNA that is without introns and that can
be translated into polypeptides by the cell. "cDNA" refers to DNA
that is complementary to and derived from an mRNA template. The
cDNA can be single-stranded or converted to double stranded form
using, for example, the Klenow fragment of DNA polymerase 1.
"Sense-RNA" refers to an RNA transcript that includes the mRNA and
so can be translated into a polypeptide by the cell. "Antisense
RNA" refers to an RNA transcript that is complementary to all or
part of a target primary transcript or mRNA and that blocks the
expression of a target gene (see U.S. Pat. No. 5,107,065,
incorporated herein by reference). The complementarity of an
antisense RNA may be with any part of the specific nucleotide
sequence, i.e., at the 5' non-coding sequence, 3' non-coding
sequence, introns, or the coding sequence. "Functional RNA" refers
to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may
not be translated but yet has an effect on cellular processes.
[0053] The term "operably linked" refers to the association of two
or more nucleic acid fragments on a single polynucleotide so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0054] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide. "Antisense inhibition" refers to the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein. "Overexpression" refers to the production of a
gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms. "Co-suppression"
refers to the production of sense RNA transcripts capable of
suppressing the expression of identical or substantially similar
foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated
herein by reference).
[0055] A "protein" or "polypeptide" is a chain of amino acids
arranged in a specific order determined by the coding sequence in a
polynucleotide encoding the polypeptide. Each protein or
polypeptide has a unique function.
[0056] "Altered levels" or "altered expression" refers to the
production of gene product(s) in transgenic organisms in amounts or
proportions that differ from that of normal or non-transformed
organisms.
[0057] "Mature protein" or the term "mature" when used in
describing a protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or propeptides present
in the primary translation product have been removed. "Precursor
protein" or the term "precursor" when used in describing a protein
refers to the primary product of translation of mRNA; i.e., with
pre- and propeptides still present. Pre- and propeptides may be but
are not limited to intracellular localization signals.
[0058] A "chloroplast transit peptide" is an amino acid sequence
which is translated in conjunction with a protein and directs the
protein to the chloroplast or other plastid types present in the
cell in which the protein is made. "Chloroplast transit sequence"
refers to a nucleotide sequence that encodes a chloroplast transit
peptide. A "signal peptide" is an amino acid sequence which is
translated in conjunction with a protein and directs the protein to
the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant
Mol. Biol. 42:21-53). If the protein is to be directed to a
vacuole, a vacuolar targeting signal (supra) can further be added,
or if to the endoplasmic reticulum, an endoplasmic reticulum
retention signal (supra) may be added. If the protein is to be
directed to the nucleus, any signal peptide present should be
removed and instead a nuclear localization signal included (Raikhel
(1992) Plant Phys. 100:1627-1632).
[0059] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol. 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference). Thus, isolated polynucleotides of the present invention
can be incorporated into recombinant constructs, typically DNA
constructs, capable of introduction into and replication in a host
cell. Such a construct can be a vector that includes a replication
system and sequences that are capable of transcription and
translation of a polypeptide-encoding sequence in a given host
cell. A number of vectors suitable for stable transfection of plant
cells or for the establishment of transgenic plants have been
described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory
Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for
Plant Molecular Biology, Academic Press, 1989; and Flevin et al.,
Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990.
Typically, plant expression vectors include, for example, one or
more cloned plant genes under the transcriptional control of 5' and
3' regulatory sequences and a dominant selectable marker. Such
plant expression vectors also can contain a promoter regulatory
region (e.g., a regulatory region controlling inducible or
constitutive, environmentally- or developmentally-regulated, or
cell- or tissue-specific expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
[0060] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, 1989
(hereinafter "Maniatis").
[0061] "PCR" or "polymerase chain reaction" is well known by those
skilled in the art as a technique used for the amplification of
specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).
[0062] The present invention concerns an isolated polynucleotide
comprising a nucleotide sequence encoding a FT, TFL or Ap3 homolog
polypeptide having at least 80%, 85%, 90%, 95%, or 100% identity,
based on the Clustal method of alignment, when compared to a
polypeptide selected from the group consisting of SEQ ID NO:2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 54, 56, 58, or 60.
[0063] This invention also relates to the isolated complement of
such polynucleotides, wherein the complement and the polynucleotide
consist of the same number of nucleotides, and the nucleotide
sequences of the complement and the polynucleotide have 100%
complementarity.
[0064] Nucleic acid fragments encoding at least a portion of
several floral development proteins have been isolated and
identified by comparison of random plant cDNA sequences to public
databases containing nucleotide and protein sequences using the
BLAST algorithms well known to those skilled in the art. The
nucleic acid fragments of the instant invention may be used to
isolate cDNAs and genes encoding homologous proteins from the same
or other plant species. Isolation of homologous genes using
sequence-dependent protocols is well known in the art. Examples of
sequence-dependent protocols include, but are not limited to,
methods of nucleic acid hybridization, and methods of DNA and RNA
amplification as exemplified by various uses of nucleic acid
amplification technologies (e.g., polymerase chain reaction, ligase
chain reaction).
[0065] For example, genes encoding other FT, TFL or Ap3 homolog,
either as cDNAs or genomic DNAs, could be isolated directly by
using all or a portion of the instant nucleic acid fragments as DNA
hybridization probes to screen libraries from any desired plant
employing methodology well known to those skilled in the art.
Specific oligonucleotide probes based upon the instant nucleic acid
sequences can be designed and synthesized by methods known in the
art (Maniatis). Moreover, an entire sequence can be used directly
to synthesize DNA probes by methods known to the skilled artisan
such as random primer DNA labeling, nick translation, end-labeling
techniques, or RNA probes using available in vitro transcription
systems. In addition, specific primers can be designed and used to
amplify a part or all of the instant sequences. The resulting
amplification products can be labeled directly during amplification
reactions or labeled after amplification reactions, and used as
probes to isolate full length cDNA or genomic fragments under
conditions of appropriate stringency.
[0066] In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to
amplify longer nucleic acid fragments encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA
85:8998-9002) to generate cDNAs by using PCR to amplify copies of
the region between a single point in the transcript and the 3' or
5' end. Primers oriented in the 3' and 5' directions can be
designed from the instant sequences. Using commercially available
3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments
can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA
86:5673-5677; Loh et al. (1989) Science 243:217-220). Products
generated by the 3' and 5' RACE procedures can be combined to
generate full-length cDNAs (Frohman and Martin (1989) Techniques
1:165). Consequently, a polynucleotide comprising a nucleotide
sequence of at least 30 (preferably at least 40, most preferably at
least 60) contiguous nucleotides derived from a nucleotide sequence
selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 39, 41, 43, 45, 47,
49, 53, 55, 57, 59, 63, 64, 65, 66 or 67 and the complement of such
nucleotide sequences may be used in such methods to obtain a
nucleic acid fragment encoding a substantial portion of an amino
acid sequence of a polypeptide.
[0067] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).
[0068] In another embodiment, this invention concerns viruses and
host cells comprising either the chimeric genes of the invention as
described herein or an isolated polynucleotide of the invention as
described herein. Examples of host cells which can be used to
practice the invention include, but are not limited to, yeast,
bacteria, and plants.
[0069] As was noted above, the nucleic acid fragments of the
instant invention may be used to create transgenic plants in which
the disclosed polypeptides are present at higher or lower levels
than normal or in cell types or developmental stages in which they
are not normally found. This would have the effect of altering
floral development and/or axillary meristems in transgenic plants.
For instance, FT is an activator of flowering, while FT-like
proteins (TFL) are repressor of flowering. Inhibition of TFL, or
over-expression of FT, by chemical treatment, co-suppression, or
mutation leads to a proliferation of flower formation which is
useful for seed yield in crops such as corn, soybean, rice, and
wheat. Over-expression of TFL, or inhibition of FT, suppresses
flower formation which is useful for crops such as spinach or
lettuce where leaves are desired and seed formation is not. The use
of conditional promoters to control FT or TFL expression allows one
to control the timing of flower formation, to delay flowering when
vegetative growth is advantageous, or accelerate flowering in
breeding where reduced generation time is desired. AP3 is required
for the determination of the second and third whorls of the floral
meristem which give rise to the petals and stamen. Suppression of
AP3 has the effect of creating male-sterile flowers, which is
advantageous in crops such as corn where outcrossing can lead to
hybrid vigor. Induction of male-sterility in self-pollinating
plants such as tomato has great commercial value in terms of
breeding.
[0070] Overexpression of the proteins of the instant invention may
be accomplished by first constructing a chimeric gene in which the
coding region is operably linked to a promoter capable of directing
expression of a gene in the desired tissues at the desired stage of
development. The chimeric gene may comprise promoter sequences and
translation leader sequences derived from the same genes. 3'
Non-coding sequences encoding transcription termination signals may
also be provided. The instant chimeric gene may also comprise one
or more introns in order to facilitate gene expression.
[0071] Plasmid vectors comprising the instant isolated
polynucleotide (or chimeric gene) may be constructed. The choice of
plasmid vector is dependent upon the method that will be used to
transform host plants. The skilled artisan is well aware of the
genetic elements that must be present on the plasmid vector in
order to successfully transform, select and propagate host cells
containing the chimeric gene. The skilled artisan will also
recognize that different independent transformation events will
result in different levels and patterns of expression (Jones et al.
(1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.
Genetics 218:78-86), and thus that multiple events must be screened
in order to obtain lines displaying the desired expression level
and pattern. Such screening may be accomplished by Southern
analysis of DNA, Northern analysis of mRNA expression, Western
analysis of protein expression, or phenotypic analysis.
[0072] For some applications it may be useful to direct the instant
polypeptides to different cellular compartments, or to facilitate
its secretion from the cell. It is thus envisioned that the
chimeric gene described above may be further supplemented by
directing the coding sequence to encode the instant polypeptides
with appropriate intracellular targeting sequences such as transit
sequences (Keegstra (1989) Cell 56:247-253), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear
localization signals (Raikhel (1992) Plant Phys.100:1627-1632) with
or without removing targeting sequences that are already present.
While the references cited give examples of each of these, the list
is not exhaustive and more targeting signals of use may be
discovered in the future.
[0073] Gene or Trait Stacking
[0074] In certain embodiments the nucleic acid sequences of the
present invention can be stacked with any combination of
polynucleotide sequences of interest in order to create plants with
a desired phenotype. For example, the polynucleotides of the
present invention may be stacked with any other polynucleotides of
the present invention, such as any combination of ZmFT1, ZmFT2, and
ZmFT3 (SEQ ID NOS: 63, 64, and 65), or with other genes implicated
in flower development pathways such as ZmTFL1 or ZmTFL2 (SEQ ID
NOS: 66 and 67). The combinations generated can also include
multiple copies of any one of the polynucleotides of interest. The
polynucleotides of the present invention can also be stacked with
any other gene or combination of genes to produce plants with a
variety of desired trait combinations including but not limited to
traits desirable for animal feed such as high oil genes (e.g., U.S.
Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S.
Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley
high lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106;
and WO 98/20122); and high methionine proteins (Pedersen et al.
(1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359;
and Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased
digestibility (e.g., modified storage proteins (U.S. Provisional
Application Serial No. 60,246,455, filed Nov. 11, 2000); and
thioredoxins (U.S. Provisional Application Ser. No. 60/250,705,
filed Dec. 12, 2000)), the disclosures of which are herein
incorporated by reference. The polynucleotides of the present
invention can also be stacked with traits desirable for insect,
disease or herbicide resistance (e.g. Bacillus thuringiensis toxic
proteins (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5723,756;
5,593,881; Geiser et al (1986) Gene 48:109); lectins (Van Damme et
al. (1994) Plant Mol. Biol. 24:825); fumonisin detoxification genes
(U.S. Pat. No. 5,792,931); avirulence and disease resistance genes
(Jones et al. (1994) Science 266:789; Martin et al. (1993) Science
262:1432; Mindrinos et al. (1994) Cell 78:1089); acetolactate
synthase (ALS) mutants that lead to herbicide resistance such as
the S4 and/or Hra mutations; inhibitors of glutamine synthase such
as phosphinothricin or basta (e.g., bar gene); and glyphosate
resistance (EPSPS gene)); and traits desirable for processing or
process products such as high oil (e.g., U.S. Pat. No. 6,232,529);
modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
5,952,544; WO 94/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE) and starch debranching enzymes (SDBE)); and
polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.
170:5837-5847) facilitate expression of polyhydroxyalkanoates
(PHAs)), the disclosures of which are herein incorporated by
reference. One could also combine the polynucleotides of the
present invention with polynucleotides providing agronomic traits
such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk
strength, flowering time, or transformation technology traits such
as cell cycle regulation or gene targeting (e.g. WO 99/61619; WO
00/17364; WO 99/25821), the disclosures of which are herein
incorporated by reference.
[0075] These stacked combinations can be created by any method
including but not limited to cross breeding plants by any
conventional or TopCross methodology, or genetic transformation. If
the traits are stacked by genetically transforming the plants, the
polynucleotide sequences of interest can be combined at any time
and in any order. For example, a transgenic plant comprising one or
more desired traits can be used as the target to introduce further
traits by subsequent transformation. The traits can be introduced
simultaneously in a co-transformation protocol with the
polynucleotides of interest provided by any combination of
transformation cassettes. For example, if two sequences will be
introduced, the two sequences can be contained in separate
transformation cassettes (trans) or contained on the same
transformation cassette (cis). Expression of the sequences can be
driven by the same promoter or by different promoters. In certain
cases, it may be desirable to introduce a transformation cassette
that will suppress the expression of the polynucleotide of
interest. This may be combine with any combination of other
suppression cassettes or overexpression cassettes to generate the
desired combination of traits in the plant.
[0076] It may also be desirable to reduce or eliminate expression
of genes encoding the instant polypeptides in plants for some
applications. In order to accomplish this, a chimeric gene designed
for co-suppression of the instant polypeptide can be constructed by
linking a gene or gene fragment encoding that polypeptide to plant
promoter sequences. Alternatively, a chimeric gene designed to
express antisense RNA for all or part of the instant nucleic acid
fragment can be constructed by linking the gene or gene fragment in
reverse orientation to plant promoter sequences. Either the
co-suppression or antisense chimeric genes could be introduced into
plants via transformation wherein expression of the corresponding
endogenous genes are reduced or eliminated.
[0077] Molecular genetic solutions to the generation of plants with
altered gene expression have a decided advantage over more
traditional plant breeding approaches. Changes in plant phenotypes
can be produced by specifically inhibiting expression of one or
more genes by antisense inhibition or cosuppression (U.S. Pat. Nos.
5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression
construct would act as a dominant negative regulator of gene
activity. While conventional mutations can yield negative
regulation of gene activity these effects are most likely
recessive. The dominant negative regulation available with a
transgenic approach may be advantageous from a breeding
perspective. In addition, the ability to restrict the expression of
a specific phenotype to the reproductive tissues of the plant by
the use of tissue specific promoters may confer agronomic
advantages relative to conventional mutations which may have an
effect in all tissues in which a mutant gene is ordinarily
expressed.
[0078] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppression technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
sense or antisense genes may require the use of different chimeric
genes utilizing different regulatory elements known to the skilled
artisan. Once transgenic plants are obtained by one of the methods
described above, it will be necessary to screen individual
transgenics for those that most effectively display the desired
phenotype. Accordingly, the skilled artisan will develop methods
for screening large numbers of transformants. The nature of these
screens will generally be chosen on practical grounds. For example,
one can screen by looking for changes in gene expression by using
antibodies specific for the protein encoded by the gene being
suppressed, or one could establish assays that specifically measure
enzyme activity. A preferred method will be one which allows large
numbers of samples to be processed rapidly, since it will be
expected that a large number of transformants will be negative for
the desired phenotype.
[0079] In another embodiment, the present invention relates to an
isolated polypeptide comprising: (a) a first amino acid sequence
comprising at least 50 or 100 amino acids, wherein the first amino
acid sequence and the amino acid sequence of SEQ ID NO:6, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:28, SEQ
ID NO:30, SEQ ID NO:34, or SEQ ID NO:42 have at least 80%, 85%,
90%, or 95% identity based on the Clustal alignment method, (b) a
second amino acid sequence comprising at least 50 or 100 amino
acids, wherein the second amino acid sequence and the amino acid
sequence of SEQ ID NO:2 have at least 85%, 90%, or 95% identity
based on the Clustal alignment method, (c) a third amino acid
sequence comprising at least 100 amino acids, wherein the third
amino acid sequence and the amino acid sequence of SEQ ID NO:4, SEQ
ID NO:16, or SEQ ID NO:40 have at least 80%, 85%, 90%, or 95%
identity based on the Clustal alignment method, (d) a fourth amino
acid sequence comprising at least 100 amino acids, wherein the
fourth amino acid sequence and the amino acid sequence of SEQ ID
NO:24 have at least 85%, 90%, or 95% identity based on the Clustal
alignment method, (e) a fifth amino acid sequence comprising at
least 150 amino acids, wherein the fifth amino acid sequence and
the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:44 have at
least 80%, 85%, 90%, or 95% identity based on the Clustal alignment
method, (f) a sixth amino acid sequence comprising at least 150
amino acids, wherein the sixth amino acid sequence and the amino
acid sequence of SEQ ID NO:38 have at least 85%, 90%, or 95%
identity based on the Clustal alignment method, (g) a seventh amino
acid sequence comprising at least 150 amino acids, wherein the
seventh amino acid sequence and the amino acid sequence of SEQ ID
NO:50 have at least 90% or 95% identity based on the Clustal
alignment method, (h) an eighth amino acid sequence comprising at
least 160 amino acids, wherein the eighth amino acid sequence and
the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:32 have at
least 85%, 90%, or 95% identity based on the Clustal alignment
method, (i) a ninth amino acid sequence comprising at least 170
amino acids, wherein the ninth amino acid sequence and the amino
acid sequence of SEQ ID NO:20 have at least 95% identity based on
the Clustal alignment method, (j) a tenth amino acid sequence
comprising at least 175 amino acids, wherein the tenth amino acid
sequence and the amino acid sequence of SEQ ID NO:18 have at least
80%, 85%, 90%, or 95% identity based on the Clustal alignment
method, or (k) an eleventh amino acid sequence comprising at least
200 amino acids, wherein the eleventh amino acid sequence and the
amino acid sequence of SEQ ID NO:46 or SEQ ID NO:48 have at least
95% identity based on the Clustal alignment method. The first amino
acid sequence preferably comprises the amino acid sequence of SEQ
ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:26,
SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:34, or SEQ ID NO:42, the
second amino acid sequence preferably comprises the amino acid
sequence of SEQ ID NO:2, the third amino acid sequence preferably
comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:16, or
SEQ ID NO:40, the fourth amino acid sequence preferably comprises
the amino acid sequence of SEQ ID NO:24, the fifth amino acid
sequence preferably comprises the amino acid sequence of SEQ ID
NO:8 or SEQ ID NO:44, the sixth amino acid sequence preferably
comprises the amino acid sequence of SEQ ID NO:38, the seventh
amino acid sequence preferably comprises the amino acid sequence of
SEQ ID NO:50, the eighth amino acid sequence preferably comprises
the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:32, the ninth
amino acid sequence preferably comprises the amino acid sequence of
SEQ ID NO:20, the tenth amino acid sequence preferably comprises
the amino acid sequence of SEQ ID NO:18, and the eleventh amino
acid sequence preferably comprises the amino acid sequence of SEQ
ID NO:46 or SEQ ID NO:48. The polypeptide preferably is a FT or Ap3
homolog.
[0080] The instant polypeptides (or portions thereof) may be
produced in heterologous host cells, particularly in the cells of
microbial hosts, and can be used to prepare antibodies to these
proteins by methods well known to those skilled in the art. The
antibodies are useful for detecting the polypeptides of the instant
invention in situ in cells or in vitro in cell extracts. Preferred
heterologous host cells for production of the instant polypeptides
are microbial hosts. Microbial expression systems and expression
vectors containing regulatory sequences that direct high level
expression of foreign proteins are well known to those skilled in
the art. Any of these could be used to construct a chimeric gene
for production of the instant polypeptides. This chimeric gene
could then be introduced into appropriate microorganisms via
transformation to provide high level expression of the encoded
floral development protein. An example of a vector for high level
expression of the instant polypeptides in a bacterial host is
provided (Example 7).
[0081] All or a substantial portion of the polynucleotides of the
instant invention may also be used as probes for genetically and
physically mapping the genes that they are a part of, and used as
markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. For example, the instant nucleic acid fragments may be
used as restriction fragment length polymorphism (RFLP) markers.
Southern blots (Maniatis) of restriction-digested plant genomic DNA
may be probed with the nucleic acid fragments of the instant
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et al. (1987) Genomics 1:174-181) in orderto construct a genetic
map. In addition, the nucleic acid fragments of the instant
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the instant nucleic acid sequence in the
genetic map previously obtained using this population (Botstein et
al. (1980) Am. J. Hum. Genet. 32:314-331).
[0082] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bernatzky and Tanksley (1986)
Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe
genetic mapping of specific cDNA clones using the methodology
outlined above or variations thereof. For example, F2 intercross
populations, backcross populations, randomly mated populations,
near isogenic lines, and other sets of individuals may be used for
mapping. Such methodologies are well known to those skilled in the
art.
[0083] Nucleic acid probes derived from the instant nucleic acid
sequences may also be used for physical mapping (i.e., placement of
sequences on physical maps; see Hoheisel et al. In: Nonmammalian
Genomic Analysis: A Practical Guide, Academic press 1996, pp.
319-346, and references cited therein).
[0084] Nucleic acid probes derived from the instant nucleic acid
sequences may be used in direct fluorescence in situ hybridization
(FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although
current methods of FISH mapping favor use of large clones (several
to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20),
improvements in sensitivity may allow performance of FISH mapping
using shorter probes.
[0085] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96),
polymorphism of PCR-amplified fragments (CAPS; Sheffield et al.
(1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy
Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0086] Loss of function mutant phenotypes may be identified for the
instant cDNA clones either by targeted gene disruption protocols or
by identifying specific mutants for these genes contained in a
maize population carrying mutations in all possible genes
(Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA
86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA
92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter
approach may be accomplished in two ways. First, short segments of
the instant nucleic acid fragments may be used in polymerase chain
reaction protocols in conjunction with a mutation tag sequence
primer on DNAs prepared from a population of plants in which
Mutator transposons or some other mutation-causing DNA element has
been introduced (see Bensen, supra). The amplification of a
specific DNA fragment with these primers indicates the insertion of
the mutation tag element in or near the plant gene encoding the
instant polypeptides. Alternatively, the instant nucleic acid
fragment may be used as a hybridization probe against PCR
amplification products generated from the mutation population using
the mutation tag sequence primer in conjunction with an arbitrary
genomic site primer, such as that for a restriction enzyme
site-anchored synthetic adaptor. With either method, a plant
containing a mutation in the endogenous gene encoding the instant
polypeptides can be identified and obtained. This mutant plant can
then be used to determine or confirm the natural function of the
instant polypeptides disclosed herein.
EXAMPLES
[0087] The present invention is further defined in the following
Examples, in which parts and percentages are by weight and degrees
are Celsius, unless otherwise stated. It should be understood that
these Examples, while indicating preferred embodiments of the
invention, are given by way of illustration only. From the above
discussion and these Examples, one skilled in the art can ascertain
the essential characteristics of this invention, and without
departing from the spirit and scope thereof, can make various
changes and modifications of the invention to adapt it to various
usages and conditions. Thus, various modifications of the invention
in addition to those shown and described herein will be apparent to
those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
[0088] The disclosure of each reference set forth herein is
incorporated herein by reference in its entirety.
Example 1
Composition of cDNA Libraries; Isolation and Sequencing of cDNA
Clones
[0089] cDNA libraries representing mRNAs from various Peruvian lily
(Alstroemeria caryophylla), balsam pear (Momordica charantia),
garden balsam (Impatiens balsamia), corn (Zea mays), rice (Oryza
sativa), soybean (Glycine max), and wheat (Triticum aestivum)
tissues were prepared. The characteristics of the libraries are
described below. Corn developmental stages are explained in the
publication "How a corn plant develops" from the Iowa State
University Coop. Ext. Service Special Report No. 48 reprinted June
1993.
2TABLE 2 cDNA Libraries from Peruvian Lily, Balsam Pear, Garden
Balsam, Corn, Rice, Soybean, and Wheat Library Tissue Clone cbn10
Corn Developing Kernel (Embryo and Endosperm); cbn10.pk0052.f5 10
Days After Pollination cbn2 Corn Developing Kernel Two Days After
Pollination cbn2.pk0035.f12 cc71se-b Corn Callus Type II Tissue,
Somatic Embryo Formed cc71se- b.pk0003.h10 cco1n Corn Cob of 67 Day
Old Plants Grown in Green cco1n.pk0010.h3 House* cco1n.pk0037.d10
cen3n Corn Endosperm 20 Days After Pollination* cen3n.pk0004.e9
cen3n.pk0047.h7 cen3n.pk0093.f1 cen3n.pk0165.f1 cta1n Corn Tassel*
cta1n.pk0050.f8 cta1n.pk0058.d11b ctn1c Corn Tassel, Night
Harvested ctn1c.pk002.j23 eal1c Peruvian Lily Mature Leaf from
Mature Stem eal1c.pk006.e6 fds Balsam Pear Developing Seed
fds.pk0003.h2 fds.pk0026.d10 fds1n Balsam Pear Developing Seed
fds1n.pk001.p18 ids Garden Balsam Developing Seed ids.pk0031.a5
p0014 Corn Leaf p0014.ctush42r p0081 Corn Pedicel 10 Days After
Pollination p0081.chcad07r p0095 Ear Leaf Sheath*; Growth
Conditions: Field; Control p0095.cwsas14f or Untreated Tissues;
Growth Stage: 2-3 weeks After Pollen Shed p0104 Corn V5-Stage Root
Infested With Corn Root Worm* p0104.cabak14rb p0118 Corn Stem
Tissue Pooled From the 4-5 Internodes p0118.chsaq04rb Subtending
The Tassel At Stages V8-V12, Night Harvested* p0119 Corn V12-Stage
Ear Shoot With Husk, Night p0119.cmtmg45rb Harvested* p0120 Pooled
Endosperm: 18, 21, 24, 27 and 29 Days After p0120.cdeae63r
Pollination* p0128 Corn Primary and Secondary Immature Ear
p0128.cpicl42r rbm1c Rice Bran 0 Hrs After Milling rbm1c.pk001.a6
rl0n Rice 15 Day Old Leaf* rl0n.pk0022.h10 rl0n.pk0022.h11 rlr48
Resistant Rice Leaf 15 Days After Germination, 48 rlr48.pk0001.b1
Hours After Infection of Strain Magnaporthe grisea 4360-R-62
(AVR2-YAMO) rls24 Susceptible Rice Leaf 15 Days After Germination,
24 rls24.pk0017.c7 Hours After Infection of Strain Magnaporthe
grisea 4360-R-67 (AVR2-YAMO) rr1 Rice Root of Two Week Old
Developing Seedling rr1.pk0043.f9 rsr9n Rice Leaf 15 Days After
Germination, Harvested 2-72 rsr9n.pk001.d1 Hours Following
Infection With Magnaporthe grisea (4360-R-62 and 4360-R-67)* se3
Soybean Embryo, 17 Days After Flowering se3.pk0036.g4 se6 Soybean
Embryo, 26 Days After Flowering se6.pk0039.h6 sfl1n Soybean
Immature Flower* sfl1n.pk001.l16 srr2c Soybean 8-Day-Old Root
srr2c.pk002.o7 ssl Soybean Seedling 5-10 Days After Germination
ssl.pk0007.a9 wdk2c Wheat Developing Kernel, 7 Days After Anthesis
wdk2c.pk012.o17 wdk2c.pk017.p2l wdk2c.pk008.n3 wdk9n1 Wheat Kernels
3, 7, 14 and 21 Days After Anthesis* wdk9n1.pk001.o20 *These
libraries were normalized essentially as described in U.S. Pat. No.
5,482,845, incorporated herein by reference.
[0090] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries
are converted into plasmid libraries according to the protocol
provided by Stratagene. Upon conversion, cDNA inserts will be
contained in the plasmid vector pBluescript. In addition, the cDNAs
may be introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991)
Science 252:1651-1656). The resulting ESTs are analyzed using a
Perkin Elmer Model 377 fluorescent sequencer.
[0091] Full-insert sequence (FIS) data is generated utilizing a
modified transposition protocol. Clones identified for FIS are
recovered from archived glycerol stocks as single colonies, and
plasmid DNAs are isolated via alkaline lysis. Isolated DNA
templates are reacted with vector primed M13 forward and reverse
oligonucleotides in a PCR-based sequencing reaction and loaded onto
automated sequencers. Confirmation of clone identification is
performed by sequence alignment to the original EST sequence from
which the FIS request is made.
[0092] Confirmed templates are transposed via the Primer Island
transposition kit (PE Applied Biosystems, Foster City, Calif.)
which is based upon the Saccharomyces cerevisiae Tyl transposable
element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772).
The in vitro transposition system places unique binding sites
randomly throughout a population of large DNA molecules. The
transposed DNA is then used to transform DH10B electro-competent
cells (Gibco BRL/Life Technologies, Rockville, Md.) via
electroporation. The transposable element contains an additional
selectable marker (named DHFR; Fling and Richards (1983) Nucleic
Acids Res. 11:5147-5158), allowing for dual selection on agar
plates of only those subclones containing the integrated
transposon. Multiple subclones are randomly selected from each
transposition reaction, plasmid DNAs are prepared via alkaline
lysis, and templates are sequenced (ABI Prism dye-terminator
ReadyReaction mix) outward from the transposition event site,
utilizing unique primers specific to the binding sites within the
transposon.
[0093] Sequence data is collected (ABI Prism Collections) and
assembled using Phred/Phrap (P. Green, University of Washington,
Seattle). Phred/Phrap is a public domain software program which
re-reads the ABI sequence data, re-calls the bases, assigns quality
values, and writes the base calls and quality values into editable
output files. The Phrap sequence assembly program uses these
quality values to increase the accuracy of the assembled sequence
contigs. Assemblies are viewed by the Consed sequence editor (D.
Gordon, University of Washington, Seattle).
[0094] In some of the clones the cDNA fragment corresponds to a
portion of the 3'-terminus of the gene and does not cover the
entire open reading frame. In order to obtain the upstream
information one of two different protocols are used. The first of
these methods results in the production of a fragment of DNA
containing a portion of the desired gene sequence while the second
method results in the production of a fragment containing the
entire open reading frame. Both of these methods use two rounds of
PCR amplification to obtain fragments from one or more libraries.
The libraries some times are chosen based on previous knowledge
that the specific gene should be found in a certain tissue and some
times are randomly-chosen. Reactions to obtain the same gene may be
performed on several libraries in parallel or on a pool of
libraries. Library pools are normally prepared using from 3 to 5
different libraries and normalized to a uniform dilution. In the
first round of amplification both methods use a vector-specific
(forward) primer corresponding to a portion of the vector located
at the 5'-terminus of the clone coupled with a gene-specific
(reverse) primer. The first method uses a sequence that is
complementary to a portion of the already known gene sequence while
the second method uses a gene-specific primer complementary to a
portion of the 3'-untranslated region (also referred to as UTR). In
the second round of amplification a nested set of primers is used
for both methods. The resulting DNA fragment is ligated into a
pBluescript vector using a commercial kit and following the
manufacturer's protocol. This kit is selected from many available
from several vendors including Invitrogen (Carlsbad, Calif.),
Promega Biotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.).
The plasmid DNA is isolated by alkaline lysis method and submitted
for sequencing and assembly using Phred/Phrap, as above.
Example 2
Identification of cDNA Clones
[0095] cDNA clones encoding floral development proteins were
identified by conducting BLAST (Basic Local Alignment Search Tool;
Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also the
explanation of the BLAST alogarithm on the world wide web site for
the National Center for Biotechnology Information at the National
Library of Medicine of the National Institutes of Health) searches
for similarity to sequences contained in the BLAST "nr" database
(comprising all non-redundant GenBank CDS translations, sequences
derived from the 3-dimensional structure Brookhaven Protein Data
Bank, the last major release of the SWISS-PROT protein sequence
database, EMBL, and DDBJ databases). The cDNA sequences obtained in
Example 1 were analyzed for similarity to all publicly available
DNA sequences contained in the "nr" database using the BLASTN
algorithm provided by the National Center for Biotechnology
Information (NCBI). The DNA sequences were translated in all
reading frames and compared for similarity to all publicly
available protein sequences contained in the "nr" database using
the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272)
provided by the NCBI. For convenience, the P-value (probability) of
observing a match of a cDNA sequence to a sequence contained in the
searched databases merely by chance as calculated by BLAST are
reported herein as "pLog" values, which represent the negative of
the logarithm of the reported P-value. Accordingly, the greater the
pLog value, the greater the likelihood that the cDNA sequence and
the BLAST "hit" represent homologous proteins.
[0096] ESTs submitted for analysis are compared to the genbank
database as described above. ESTs that contain sequences more 5- or
3-prime can be found by using the BLASTn algorithm (Altschul et al
(1997) Nucleic Acids Res. 25:3389-3402.) against the DuPont
proprietary database comparing nucleotide sequences that share
common or overlapping regions of sequence homology. Where common or
overlapping sequences exist between two or more nucleic acid
fragments, the sequences can be assembled into a single contiguous
nucleotide sequence, thus extending the original fragment in either
the 5 or 3 prime direction. Once the most 5-prime EST is
identified, its complete sequence can be determined by Full Insert
Sequencing as described in Example 1. Homologous genes belonging to
different species can be found by comparing the amino acid sequence
of a known gene (from either a proprietary source or a public
database) against an EST database using the tBLASTn algorithm. The
tBLASTn algorithm searches an amino acid query against a nucleotide
database that is translated in all 6 reading frames. This search
allows for differences in nucleotide codon usage between different
species, and for codon degeneracy.
Example 3
Characterization of cDNA Clones Encoding Flowering Locus T (FT)
Homologs
[0097] The BLASTX search using the EST sequences from clones listed
in Table 3 revealed similarity of the polypeptides encoded by the
cDNAs to FT and its homologs from Citrus unshiu (NCBI GenBank
Identifier (GI) No. 4903139), Arabidopsis thaliana (NCBI GI Nos.
5002246 and 2190540), Oryza sativa (NCBI GI Nos. 5360178 and
5360180) and Nicotiana tabacum (NCBI GI No. 5453314). Shown in
Table 3 are the BLAST results for individual ESTs ("EST"), the
sequences of the entire cDNA inserts comprising the indicated cDNA
clones ("FIS"), the sequences of contigs assembled from two or more
ESTs ("Contig"), sequences of contigs assembled from an FIS and one
or more ESTs ("Contig*"), or sequences encoding an entire protein
derived from an FIS, a contig, or an FIS and PCR fragment sequence
("CGS"):
3TABLE 3 BLAST Results for Sequences Encoding Polypeptides
Homologous to Flowering Locus T (FT) Protein BLAST Results Clone
Status NCBI GI No. pLog Score eal1c.pk006.e6 EST 4903139 65.70
Contig of CGS 5002246 75.00 fds.pk0003.h2 fds.pk0026.d10
fds1n.pk001.p18 ids.pk0031.a5 (FIS) CGS 5002246 61.70 Contig of CGS
4903139 77.00 cbn10.pk0052.f5 cbn2.pk0035.f12 cco1n.pk0010.h3
p0095.cwsas14f p0119.cmtmg45rb p0128.cpicl42r cc71se-b.pk0003.h10
(FIS) CGS 5002246 59.15 cco1n.pk0037.d10 (FIS) CGS 2190540 68.52
Contig of CGS 5002246 57.30 cen3n.pk0004.e9 cen3n.pk0047.h7
cen3n.pk0093.f1 cen3n.pk0165.f1 p0120.cdeae63r p0014.ctush42r (FIS)
CGS 4903139 59.00 p0081.chcad07r (FIS) CGS 4903139 81.00
p0104.cabak14rb (FIS) CGS 5360178 93.70 p0118.chsaq04rb (FIS) CGS
5360180 82.10 rbm1c.pk001.a6 EST 5002246 46.04 Contig of Contig
4903139 43.30 rl0n.pk0022.h10 rl0n.pk0022.h11 rlr48.pk0001.b1 EST
5002246 14.52 rls24.pk0017.c7 (FIS) CGS 5002246 64.70 rr1.pk0043.f9
(FIS) CGS 5360178 82.10 rsr9n.pk001.d1 EST 2190540 35.70 Contig of
CGS 5002246 76.00 se3.pk0036.g4 se6.pk0039.h6 (FIS) srr2c.pk002.o7
(FIS) CGS 5360180 77.52 Contig of CGS 5453314 73.70 ssl.pk0007.a9
PCR fragment sequence wdk2c.pk012.o17 (FIS) CGS 5002246 62.30
wdk9n1.pk001.o20 (FIS) CGS 4903139 75.70 cta1n.pk0058.d11b FIS
15218709 53.40 rbm1c.pk001.a6:fis CGS 5002246 56.10
rl0n.pk0022.h10:fis FIS 14517620 36.70 rsr9n.pk001.d1:fis CGS
15218709 70.39
[0098] The PCR fragment that was used to extend the nucleotide
sequence obtained from clone ssl.pk0007.a9 was obtained via methods
(e.g., RACE techniques) well-known to those skilled in the art.
[0099] The amino acid sequence of the polypeptide encoded by the
insert in clone wdk2c.pk012.o17 is identical to the amino acid
sequence of the polypeptide encoded by the nucleotide sequence of a
contig assembled from ESTs derived from clones wdk2c.pk008.n3,
wdk2c.pk012.o17, and wdk2c.pk017.p21.
[0100] FIG. 1 presents an alignment of the amino acid sequences set
forth in SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 30, 32,
36, 38, 40, 42, and 44 and the Oryza sativa sequence (NCBI GI No.
5360178; SEQ ID NO:51).
[0101] The data in Table 4 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs:4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 30, 32, 36, 38, 40, 42, and 44 and
the Oryza sativa sequence (NCBI GI No. 5360178; SEQ ID NO:51).
4TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to FT Protein Percent Identity to SEQ ID NO. NCBI GI No.
2 69.3 [gi 4903139] 4 72.3 [gi 5002246] 6 61.3 [gi 5002246] 8 74.6
[gi 4903139] 10 60.5 [gi 5002246] 12 67.2 [gi 2190540] 14 60.1 [gi
5002246] 16 59.9 [gi 4903139] 18 78.0 [gi 4903139] 20 94.8 [gi
5360178] 22 83.8 [gi 5360180] 24 56.2 [gi 5002246] 26 64.8 [gi
4903139] 28 52.9 [gi 5002246] 30 65.9 [gi 5002246] 32 83.2 [gi
5360178] 36 74.4 [gi 5002246] 38 76.3 [gi 5360180] 40 74.6 [gi
5453314] 42 64.2 [gi 5002246] 44 72.9 [gi 4903139] 54 60.8 [gi
15218709] 56 57.2 [gi 5002246] 58 66.3 [gi 14517620] 60 69.0 [gi
15218709]
[0102] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. It
will be recognized by one skilled in the art that conserved
sequence elements within the encoded polypeptide are useful in
identifying homologous enzymes. Two such elements, although not
necessarily the only elements, are the sequences
Asp-Pro-Asp-Xaa-Pro-Xaa-- Pro-Ser-Xaa-Pro found, for example, at
positions 70-79 of SEQ ID NO:4, and Gly-Ile-His-Arg found, for
example at positions 115-118 of SEQ ID NO:4. Sequence alignments
and BLAST scores and probabilities indicate that the nucleic acid
fragments comprising the instant cDNA clones encode a substantial
portion of a polypeptide encoded by a member of TFL1/FT gene
family. These sequences represent the first Peruvian lily, balsam
pear, garden balsam, corn, soybean, wheat sequences and new rice
sequences encoding flowering locus T (FT or TFL) homologs known to
Applicant.
Example 4
Characterization of cDNA Clones Encoding Ap3 Homologs
[0103] The BLASTX search using the EST sequences from clones listed
in Table 5 revealed similarity of the polypeptides encoded by the
cDNAs to MADS box proteins (Ap3 homologs) from Oryza sativa (NCBI
GI Nos. 5295980 and 7446534) and Medicago sativa (NCBI GI No.
2827300). Shown in Table 5 are the BLAST results for individual
ESTs ("EST"), the sequences of the entire cDNA inserts comprising
the indicated cDNA clones ("FIS"), the sequences of contigs
assembled from two or more ESTs ("Contig"), sequences of contigs
assembled from an FIS and one or more ESTs ("Contig*"), or
sequences encoding an entire protein derived from an FIS, a contig,
or an FIS and PCR ("CGS"):
5TABLE 5 BLAST Results for Sequences Encoding Polypeptides
Homologous to Ap3 Protein BLAST Results Clone Status NCBI GI No.
pLog Score cta1n.pk0050.f8 (FIS) CGS 5295980 113.00 ctn1c.pk002.j23
(FIS) CGS 7446534 109.00 sfl1n.pk001.l16 (FIS) CGS 2827300
114.00
[0104] FIG. 2 presents an alignment of the amino acid sequences set
forth in SEQ ID NOs:46, 48, and 50 and the Oryza sativa sequence
(NCBI GI No. 5295980; SEQ ID NO:52). The data in Table 6 represents
a calculation of the percent identity of the amino acid sequences
set forth in SEQ ID NOs:46, 48, and 50 and the Oryza sativa
sequence (NCBI GI No. 5295980; SEQ ID NO:52).
6TABLE 6 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Ap3 Protein Percent Identity to SEQ ID NO. [NCBI GI
No.] 46 86.6 [gi 5295980] 48 91.4 [gi 7446534] 50 85.9 [gi
2827300]
[0105] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. It will be recognized by one skilled in the art that
conserved sequence elements within the encoded polypeptide are
useful in identifying homologous enzymes. One such element,
although not necessarily the only element, is the sequence
Arg-Gly-Lys-Ile-Xaa-Ile-
-Lys-Arg-Ile-Glu-Asn-Xaa-Thr-Asn-Arg-Gln-Val-Thr-Xaa-Ser-Lys-Arg-Arg-Xaa-G-
ly-Xaa-Xaa-Lys-Lys-Ala found, for example, at positions 3-32 of SEQ
ID NO:46. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of an Ap3 homolog. These
sequences represent the first soybean sequence and new corn
sequences encoding Ap3 homologs known to Applicant.
Example 5
Expression of Chimeric Genes in Monocot Cells
[0106] A chimeric gene comprising a cDNA encoding the instant
polypeptides in sense orientation with respect to the maize 27 kD
zein promoter that is located 5' to the cDNA fragment, and the 10
kD zein 3' end that is located 3' to the cDNA fragment, can be
constructed. The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites (NcoI or SmaI) can be
incorporated into the oligonucleotides to provide proper
orientation of the DNA fragment when inserted into the digested
vector pML 103 as described below. Amplification is then performed
in a standard PCR. The amplified DNA is then digested with
restriction enzymes NcoI and SmaI and fractionated on an agarose
gel. The appropriate band can be isolated from the gel and combined
with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid
pML103 has been deposited under the terms of the Budapest Treaty at
ATCC (American Type Culture Collection, 10801 University Blvd.,
Manassas, Va. 20110-2209), and bears accession number ATCC 97366.
The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter
fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI
fragment from the 3' end of the maize 10 kD zein gene in the vector
pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at
15.degree. C. overnight, essentially as described (Maniatis). The
ligated DNA may then be used to transform E. coli XL1-Blue
(Epicurian Coli XL-1 Blue.TM.; Stratagene). Bacterial transformants
can be screened by restriction enzyme digestion of plasmid DNA and
limited nucleotide sequence analysis using the dideoxy chain
termination method (Sequenase.TM. DNA Sequencing Kit; U.S.
Biochemical). The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD
zein promoter, a cDNA fragment encoding the instant polypeptides,
and the 10 kD zein 3' region.
[0107] The chimeric gene described above can then be introduced
into corn cells by the following procedure. Immature corn embryos
can be dissected from developing caryopses derived from crosses of
the inbred corn lines H99 and LH132. The embryos are isolated 10 to
11 days after pollination when they are 1.0 to 1.5 mm long. The
embryos are then placed with the axis-side facing down and in
contact with agarose-solidified N6 medium (Chu et al. (1975) Sci.
Sin. Peking 18:659-668). The embryos are kept in the dark at
27.degree. C. Friable embryogenic callus consisting of
undifferentiated masses of cells with somatic proembryoids and
embryoids borne on suspensor structures proliferates from the
scutellum of these immature embryos. The embryogenic callus
isolated from the primary explant can be cultured on N6 medium and
sub-cultured on this medium every 2 to 3 weeks.
[0108] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst
Ag, Frankfurt, Germany) may be used in transformation experiments
in order to provide for a selectable marker. This plasmid contains
the Pat gene (see European Patent Publication 0 242 236) which
encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT
confers resistance to herbicidal glutamine synthetase inhibitors
such as phosphinothricin. The pat gene in p35S/Ac is under the
control of the 35S promoter from Cauliflower Mosaic Virus (Odell et
al. (1985) Nature 313:810-812) and the 3' region of the nopaline
synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens.
[0109] The particle bombardment method (Klein et al. (1987) Nature
327:70-73) may be used to transfer genes to the callus culture
cells. According to this method, gold particles (1 .mu.m in
diameter) are coated with DNA using the following technique. Ten
.mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold
particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5 M
solution) and spermidine free base (20 .mu.L of a 1.0 M solution)
are added to the particles. The suspension is vortexed during the
addition of these solutions. After 10 minutes, the tubes are
briefly centrifuged (5 sec at 15,000 rpm) and the supernatant
removed. The particles are resuspended in 200 .mu.L of absolute
ethanol, centrifuged again and the supernatant removed. The ethanol
rinse is performed again and the particles resuspended in a final
volume of 30 .mu.L of ethanol. An aliquot (5 .mu.L) of the
DNA-coated gold particles can be placed in the center of a
Kapton.TM. flying disc (Bio-Rad Labs). The particles are then
accelerated into the corn tissue with a Biolistic.TM. PDS-1000/He
(Bio-Rad Instruments, Hercules Calif.), using a helium pressure of
1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0
cm.
[0110] For bombardment, the embryogenic tissue is placed on filter
paper over agarose-solidified N6 medium. The tissue is arranged as
a thin lawn and covered a circular area of about 5 cm in diameter.
The petri dish containing the tissue can be placed in the chamber
of the PDS-1000/He approximately 8 cm from the stopping screen. The
air in the chamber is then evacuated to a vacuum of 28 inches of
Hg. The macrocarrier is accelerated with a helium shock wave using
a rupture membrane that bursts when the He pressure in the shock
tube reaches 1000 psi.
[0111] Seven days after bombardment the tissue can be transferred
to N6 medium that contains bialophos (5 mg per liter) and lacks
casein or proline. The tissue continues to grow slowly on this
medium. After an additional 2 weeks the tissue can be transferred
to fresh N6 medium containing bialophos. After 6 weeks, areas of
about 1 cm in diameter of actively growing callus can be identified
on some of the plates containing the bialophos-supplemented medium.
These calli may continue to grow when sub-cultured on the selective
medium.
[0112] Plants can be regenerated from the transgenic callus by
first transferring clusters of tissue to N6 medium supplemented
with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be
transferred to regeneration medium (Fromm et al. (1990)
Bio/Technology 8:833-839).
Example 6
Expression of Chimeric Genes in Dicot Cells
[0113] A seed-specific expression cassette composed of the promoter
and transcription terminator from the gene encoding the p subunit
of the seed storage protein phaseolin from the bean Phaseolus
vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be
used for expression of the instant polypeptides in transformed
soybean. The phaseolin cassette includes about 500 nucleotides
upstream (5') from the translation initiation codon and about 1650
nucleotides downstream (3') from the translation stop codon of
phaseolin. Between the 5' and 3' regions are the unique restriction
endonuclease sites NcoI (which includes the ATG translation
initiation codon), SmaI, KpnI and XbaI. The entire cassette is
flanked by HindIII sites.
[0114] The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the expression vector. Amplification is then
performed as described above, and the isolated fragment is inserted
into a pUC18 vector carrying the seed expression cassette.
[0115] Soybean embryos may then be transformed with the expression
vector comprising sequences encoding the instant polypeptides. To
induce somatic embryos, cotyledons, 3-5 mm in length dissected from
surface sterilized, immature seeds of the soybean cultivar A2872,
can be cultured in the light or dark at 26.degree. C. on an
appropriate agar medium for 6-10 weeks. Somatic embryos which
produce secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos which multiplied as early, globular staged embryos,
the suspensions are maintained as described below.
[0116] Soybean embryogenic suspension cultures can be maintained in
35 mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C.
with florescent lights on a 16:8 hour day/night schedule. Cultures
are subcultured every two weeks by inoculating approximately 35 mg
of tissue into 35 mL of liquid medium.
[0117] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0118] A selectable marker gene which can be used to facilitate
soybean transformation is a chimeric gene composed of the .sup.35S
promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the
3' region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The seed expression cassette
comprising the phaseolin 5' region, the fragment encoding the
instant polypeptides and the phaseolin 3' region can be isolated as
a restriction fragment. This fragment can then be inserted into a
unique restriction site of the vector carrying the marker gene.
[0119] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.L
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
.mu.L of the DNA-coated gold particles are then loaded on each
macro carrier disk.
[0120] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.1 5 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0121] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post
bombardment with fresh media containing 50 mg/mL hygromycin. This
selective media can be refreshed weekly. Seven to eight weeks post
bombardment, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated green tissue
is removed and inoculated into individual flasks to generate new,
clonally propagated, transformed embryogenic suspension cultures.
Each new line may be treated as an independent transformation
event. These suspensions can then be subcultured and maintained as
clusters of immature embryos or regenerated into whole plants by
maturation and germination of individual somatic embryos.
Example 7
Expression of Chimeric Genes in Microbial Cells
[0122] The cDNAs encoding the instant polypeptides can be inserted
into the T7 E. coli expression vector pBT430. This vector is a
derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135)
which employs the bacteriophage T7 RNA polymerase/T7 promoter
system. Plasmid pBT430 was constructed by first destroying the
EcoRI and HindIII sites in pET-3a at their original positions. An
oligonucleotide adaptor containing EcoRI and Hind III sites was
inserted at the BamHI site of pET-3a. This created pET-3aM with
additional unique cloning sites for insertion of genes into the
expression vector. Then, the NdeI site at the position of
translation initiation was converted to an NcoI site using
oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM
in this region, 5'-CATATGG, was converted to 5'-CCCATGG in
pBT430.
[0123] Plasmid DNA containing a cDNA may be appropriately digested
to release a nucleic acid fragment encoding the protein. This
fragment may then be purified on a 1% low melting agarose gel.
Buffer and agarose contain 10 .mu.g/ml ethidium bromide for
visualization of the DNA fragment. The fragment can then be
purified from the agarose gel by digestion with GELase.TM.
(Epicentre Technologies, Madison, Wis.) according to the
manufacturer's instructions, ethanol precipitated, dried and
resuspended in 20 .mu.L of water. Appropriate oligonucleotide
adapters may be ligated to the fragment using T4 DNA ligase (New
England Biolabs (NEB), Beverly, Mass.). The fragment containing the
ligated adapters can be purified from the excess adapters using low
melting agarose as described above. The vector pBT430 is digested,
dephosphorylated with alkaline phosphatase (NEB) and deproteinized
with phenol/chloroform as described above. The prepared vector
pBT430 and fragment can then be ligated at 16.degree. C. for 15
hours followed by transformation into DH5 electrocompetent cells
(GIBCO BRL). Transformants can be selected on agar plates
containing LB media and 100 .mu.g/mL ampicillin. Transformants
containing the gene encoding the instant polypeptides are then
screened for the correct orientation with respect to the T7
promoter by restriction enzyme analysis.
[0124] For high level expression, a plasmid clone with the cDNA
insert in the correct orientation relative to the T7 promoter can
be transformed into E. coli strain BL21 (DE3) (Studier et al.
(1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium
containing ampicillin (100 mg/L) at 25.degree. C. At an optical
density at 600 nm of approximately 1, IPTG
(isopropylthio-.beta.-galactoside, the inducer) can be added to a
final concentration of 0.4 mM and incubation can be continued for 3
h at 25.degree.. Cells are then harvested by centrifugation and
re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1
mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of
1 mm glass beads can be added and the mixture sonicated 3 times for
about 5 seconds each time with a microprobe sonicator. The mixture
is centrifuged and the protein concentration of the supernatant
determined. One .mu.g of protein from the soluble fraction of the
culture can be separated by SDS-polyacrylamide gel electrophoresis.
Gels can be observed for protein bands migrating at the expected
molecular weight.
Example 8
Phylogenetic Analysis of Corn Translational Homologs of the
Arabidopsis Flowering proteins FT (Flowering Locus T) and TFL
(Terminal Flower)
[0125] The relationship of maize and Arabidopsis FT-TFL proteins
was revealed by phylogenetic analysis. Seven maize FT-TFL putative
proteins were included in the phylogenetic analysis as well:
Arabidopsis FT (GenBank accession number AB027504) and TFL (GenBank
accession number U77674), rice Hd3a, heading QTL3 (GenBank
accession number AB052942), rice RCN1 (GenBank accession number
AF159882), snapdragon CEN, centroradialis (GenBank accession number
S81193), tomato SP, self-pruning (GenBank accession number U84140).
The mice PEBP protein (GenBank accession number AF300422) was used
as an outgroup to root the tree. The phylogenetic tree was
constructed by the maximum parsimony methods (FIG. 3). The
phylogram clearly delimits two major clades that corresponded to
the FT and TFL proteins. Maize proteins ZmFT4 and ZmFT5 form an
outgroup with the mice protein mPEBP, which suggests that their
functions are not related to the flowering time.
[0126] The FT lade consists of five maize proteins with different
degrees of amino acid identity. ZmFT1 is closely related to the
Arabidopsis FT sharing 71% identity and 83% similarity. ZmFT3 is
closely related to the rice Hd3a QTL for photoperiod sensitivity
(GenBank accession number AB052942) sharing 88% identity and 94%
similarity. ZmFT3 shows less than 60% identity to both proteins
from rice and Arabidopsis.
[0127] The TFL lade consists of six members including the
snapdragon CEN and tomato SP proteins. This lade shows higher
conservation of amino acid sequences than the FT lade. The maize
proteins ZmFTL1 and ZmFTL2 are tightly associated with the rice
RCN1 (Nakagawa et al., 2002). ZmTFL1 is highly homologous to the
rice RCN1 sharing 94% identity and 98% similarity. ZmFTL2 is also
close to RCN1 sharing 85% identity and 91% similarity. The high
level of the amino acid conservation between species indicates that
TFL protein structures are under a strong evolution pressure for
function. Thus ZmTFL proteins may play roles as repressors of
flowering similar to the Arabidopsis TFL and rice RCN1.
Example 9
Genomic Sequences of Maize FT-TFL Homologs
[0128] To get genomic sequences of ZmFT and ZmTFL genes, BAC
(bacterial artificial chromosome) libraries were screened with
inserts of EST's SEQ ID NO: 7, 9, 15, 17, 19, 21 (Table 1). HindIII
fragments of BAC DNAs were sub-cloned into a plasmid
BluescriptIISK(+) and sequenced at the DuPont sequencing
facilities. The genomic sequences of ZmFT1, ZmFT2, ZmFT3, ZmTFL1
and ZmTFL2 are listed in the sequence listing for this
specification (Sequence ID NOS: 63, 64, 65, 66 and 67,
respectively).
[0129] The genomic structures of the ZmFT and ZmTFL sequences are
shown in FIG. 4, and are described in detail as follows:
[0130] The genomic region of ZmFT1 (SEQ ID NO: 63) is composed of a
promoter (1-2211 nt), 5'UTR (2212-2385 nt), exon 1 (2386-3580 nt),
exon 2 (2741-2802 nt), exon 3 (9718-9760 nt), exon 4 (9845-10067),
3'UTR (10067-10316).
[0131] The genomic region of ZmFT2 (SEQ ID NO: 64) is composed of
5'UTR (1-93), exon1 (94-293), exon2 (468-525), exon3 (765-806),
exon 4 (1411-1651), 3'UTR (1652-1840).
[0132] The genomic region of ZmFT3 (SEQ ID NO: 65) is composed of a
promoter (286-4375 nt), 5'UTR (4376-4542 nt), exon 1 (4543-4743
nt), exon 2 (4894-4953 nt), exon 3 (5688-5728 nt), exon 4
(6166-6396 nt), 3'UTR (6397-6860).
[0133] The genomic region of ZmTFL1 (SEQ ID NO: 66) is composed of
two copies of ZmTFL1 gene arranged in a perfect tandem. The first
copy has a partial promoter (1-562 nt), 5'UTR (486-563 nt), exon 1
(564-763 nt), exon 2 (846-9907 nt), exon 3 (1056-1096 nt), exon 4
(1176-1364 nt), 3'UTR (1395-1611 nt), 3' downstream segment
(1612-2435 nt). The second copy begins from 2436 nt and shows the
identical structure as the first one. The genomic organization of
ZmTFL1 gene exhibits an unusual configuration of a tandem array of
two gene copies. The unit length in tandem is 2292 nt, which
include a 5'upstream sequence (364 nt), exon/intron genic segment
(1116 nt) and 3'downstream sequence (812 nt). A promoter for the
second ZmTFL1 copy may be defined between nucleotides 1611 and 2435
(824 nt total length). Almost identical nucleotide sequences of
both units suggest a very recent duplication of ZmTFL1 gene in Mo17
genome.
[0134] The genomic region of ZmTFL2 (SEQ ID NO: 67) is composed of
a promoter (1-1450 nt), 5'UTR (1451-1518 nt), exon 1 (1519-1780
nt), exon 2 (2097-2137 nt), exon 3 (2309-2595 nt), and 3'UTR
(2596-2881 nt).
[0135] The overall genomic structures of ZmFT1, Zm FT2, ZmFT3 and
ZmTFL1 are very similar, each comprising 4 exons and 3 introns.
They are similar to the Arabidopsis FT (GenBank accession number
NC003070) and TFL genes (GenBank accession number NC003076) and the
rice gene Hd3a (GenBank accession number AB052942). The genomic
structure of ZmTFL2 is different as it contains only 2 introns. The
absence of intron 1 results in a fusion of exon1 and exon 2. The
coding segments of exons are nearly identical ranging in sizes
195-204 nt (exon1), 61nt (exon2), 41nt (exon3) and 213-238 nt (exon
4). This is consistent with the very close sizes of encoded
proteins of 173-177 amino acids. Conversely, intron lengths vary
significantly between the ZmFT and ZmTFL genes. Intron1 ranges from
0 to 186 nt, intron 2 ranges from 147 to 6916 nt, and intron 3
ranges from 39 to 604 nt. The ZmFT1 gene possesses the most unusual
intron, with its second intron being 6917 nt, which is 7 times
longer than a coding sequence. Such a long intron size is very
uncommon in plants and raises a possibility of the particular role
of this intron in controlling the ZmFT1 gene expression.
Example 10
Map Position of ZmFT and ZmFTL Genes and Correlation with QTL's
Loci for Flowering Time
[0136] All ZmFT and ZmFTL genes have been mapped to chromosomes
using the maize-oat addition lines (Kynast et al., 2001). Pairs of
gene-specific primers (table 2) were designed to amplify each gene
from the 10 samples of the oat DNA each of which carrying a single
maize chromosome. Genes were mapped to following chromosomes:
ZmFT1--chromosome 8, ZmFT2--chromosome 3, ZmFT3--chromosome 6,
ZmTFL1--chromosome 3, ZmTFL2--chromosome 6. The ZmFT1 gene is
mapped to chromosome 8 where two major QTLs for early flowering,
vgt1 and vgt2 are located (Vladutu et al., 1999). To place ZmFT1
more precisely on chromosome 8, the mapping population SX19 SYN4
derived from B37 and Mo17 was used. PCR primers SEQ ID NO: 6 and
SEQ ID NO: 7 (table 2) were designed. These primers amplify an
insertion/deletion polymorphism between B73 and Mol 7 in the 5'
untranslated region of ZmFT1. The 270 lines from the SX19 SYN4
population were genotyped for the ZmFT1 gene. Co-segregation
analysis was performed using marker data and genetic maps assembled
from both public and Pioneer sources. Linkage between ZmFT1 and
each marker was tested by applying a 2.times.2 X.sup.2 test for
independent segregation (one degree of freedom); the threshold for
declaring linkage was X.sup.2>18. The genome-wide cumulative
type I error probability was 0.05 assuming 1500 marker tests.
[0137] Significant linkage was detected between ZmFT1 and a group
of markers located on chromosome 8; no linkage was detected
elsewhere. The nearest marker to ZmFT1 was UMC32b (14 recombinant
lines out of 239 lines genotyped). UMC32b is located at 199 cM on
the public 2002 IBM map (568 cM total genetic distance for
chromosome 8). ZmFT1 was also tightly linked to UMC120a (16/145
recombinants). UMC120a is located at 55 cM on the public 1998 UMC
map (183 cM total After converting the observed two-point
recombination fractions to single meioisis genetic distances, ZmFT1
lies from 1.1 cM UMC32b (direction not determined) and 2.2 cM below
UMC120a.
[0138] Vgt1 and Vgt2 are linked QTLs for flowering time and leaf
number defined by Vladutu, et al. (1999) on chromosome 8 between
markers UMC236 and UMC89a. UMC236 is located at 54cM of UMC 1998
map. UMC89a is located at 327cM of 2002 IBM map. This places ZmFT1
between UMC236 and UMC89a. Therefore ZmFT1 is a candidate gene for
either or both QTLs.
Example 11
[0139] Temporal and Spatial Expression of ZmFT-TFL Genes During
Plant Development
[0140] ZmFT genes comprise a family of related genes with a
significant homology to each other. To validate their roles in the
transition from the vegetative growth to reproductive, their
expressions have been assayed throughout corn plant development.
The transition to flowering is a complex development event.
Internal and external signals entail irreversible changes in a
seedling growing point, the shoot apical meristem. During switching
to flowering the shoot apical meristem (SAM) stops producing leaves
and commits to form influences. In maize, the floral signals are
generated in immature leaves (Colasanti et al., 1998). Thus two
tissues play central roles in the transition to flowering, immature
leaves and the shoot apical meristem. If candidate genes are
expressed in those tissues, they are very likely to play a role in
timing of flowering. The SAM shows morphological changes during the
transition to flowering such as elongation and branch primordial.
This moment was recorded as the transition point. Seedlings (B73
inbred line) have been grown in a green house under standard
conditions. Every 3 days seedlings were examined for morphological
changes in the SAM and 3-5 SAMs and several immature leaves were
sampled. Samples at 8 time points were collected. Total RNAs were
isolated from sampled tissues and RT-PCR has been performed for
each of ZmFT-TFL genes. Pairs of gene-specific primers were
designed according EST sequences to amplify specifically one cDNA
at time (table 2). Mature leaves, the embryo from 15 days after
pollination, kernels and seedling roots were tested for expression
of ZmFT genes as well. Out of 6 genes examined, ZmFT1, ZmTFL1 and
ZmTFL2 showed expression in the shoot apical meristem in a very
specific pattern for each gene. ZmFT2, ZmFT3 and ZmFT4 are
expressed neither in the SAM nor in immature leaves, which excluded
them from the floral transition genes. ZmFT2 is expressed in mature
leaves that still may play some role in flowering. ZmFT3 is
expressed only in pedicel, and ZmFT 4 is expressed in the embryo.
They may function in other pathways not related to the floral
transition.
[0141] As shown by RT-PCR, ZmFT1 gene is not expressed in the SAM
from seedlings during vegetative growth up 26 days after planting.
The transition from vegetative to reproductive growth occurs in the
SAM around 22-26 days after planting. An RT-PCR band from ZmFT1
mRNA appears in the SAM at 33 days after planting when the floral
transition occurred. Thus ZmFT1 gene is expressed in the young
inflorescence. ZmTFL1 and ZmTFL2 are expressed in the SAM during
vegetative growth at 3-22 days after planting. They cease their
expression before the floral transition around 26 days.
[0142] ZmFT1 demonstrates a pattern of expression consistent with
function as an activator of the flowering. Its transcription is not
detected in the SAM during a vegetative growth, but it is activated
sharply in the SAM very early after the transition. Conversely,
ZmTFL1 and ZmTFL2 are active in the SAM during the vegetative
growth and their expressions decline just before the meristem
transition to the reproductive growth. This pattern of expression
is consistent with their functions as flowering repressors.
Example 12
Expression of ZmTF1 Gene in the Shoot Apical Meristem of the
Flowering Mutant id1 (Indeterminate 1)
[0143] The temporal and spatial patterns of ZmFT1 expression are
consistent with its function as a floral activator in corn. Id1
(indeterminate) gene is the only maize cloned gene with a clear
role in a floral transition (Colasanti et al., 1998). The Id1 gene
encodes transcription factors and regulates the production of a
transmissible signal in the immature leaves that induces the
transition of the SAM to reproductive development. To test the
possibility whether the ZmFT1 expression is related to the Id1
function, the SAM were sampled from wild type and id1 homozygous
siblings after the floral transition. Wild type plants were sampled
at 27, 33, 40 and 48 days after planting. Id mutants were sampled
also at 66 and 70 days because of delayed flowering phenotypes.
RT-PCR analysis has demonstrated that ZmFT1 is expressed in the
inflorescence up to 48 days, but it did not expression in the
inflorescence of the id1 seedling, even at the later stage of 70
days. Thus these data strongly suggest ZmFT1 is in the same pathway
with the Id1 transcription factor. Activation of ZmFT1
transcription requires a floral signal produced in immature leaves
under control of the Id1gene. The absence of this signal in the id1
homozygous mutant prevents ZmFT1 expression. Thus ZmFT1 is
operating down stream from the Id1 gene in the same pathway. The
expression patterns of ZmTFL1 and ZmTFL2 are not significantly
affected in the id1 mutant seedlings. Those genes may be placed in
independent pathways.
Example 13
Inactivation of ZmFT1 and ZmTFL2 Genes by the Mutator Transposon
Insertions.
[0144] Gene inactivation can be used to confirm the function of
ZmFT-TFL genes in flowering. Pioneer proprietary system TUSC (Trait
Utility System for Corn) was used to screen ZmFT1, ZmTFL1, and
ZmTFL2 genes disrupted by the Mutator transposable element
insertion. F.sub.2 families segregating for the Mutator insertions
were screened by PCR with the Mu specific primer (SEQ ID NO: 24)
and gene specific primers SEQ ID NO: 21, 22, 23 (table 2). Positive
signals were found for the Mutator insertions in ZmFT1 and ZmTFL2,
not in ZmTFL1. The Mu insertion sites were sequenced from PCR
products. TUSC plants were grown and crossed to different inbred
lines to segregate away non-related mutations created by
Mu-activity. Flowering phenotypic analysis known to those of skill
in the art will demonstrate which genes exhibit and/or modify
flowering function.
[0145] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0146] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will be thorough and complete, and will fully convey the
scope of the invention to those skilled in the art. Like numbers
refer to like elements throughout.
[0147] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing descriptions and the associated drawings. Therefore, it
is to be understood that the invention is not to be limited to the
specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims. Although specific terms are employed herein, they
are used in a generic and descriptive sense only and not for
purposes of limitation.
Sequence CWU 1
1
67 1 492 DNA Alstroemeria caryophylla 1 attgatttct agctagctcc
tttgcttgca atagatatat aatgagcgga gaaagcgaaa 60 ccctggtgat
tggtagggtg gtgggggacg tgttggaccc ctacactaaa accacggcgc 120
tcaggatcag gtatggatcg aaagaggtga cgtgcgggca cgagctaaag ccatcgcagg
180 tcgtcataca gccaagggtg gaggttggag ggaaggatct caggaccttt
tacacacttg 240 tgatggtaga ccctgatgct ccgagcccaa gcaacccaca
ccttagggcg tatctacatt 300 ggctggtgac tgacctcccg ggaactactg
gagctagctt cgggcaagag gtgatgaggt 360 acgagagccc aaggccaaca
ttagggattc accgcttcgt cttcgtgctg ttccggcagc 420 tcgggcggca
gacggtgcag gtgcccaccc ccgggaggcg ccagaacttc aacacaaggg 480
gctttgcaag ag 492 2 150 PRT Astroemeria caryophylla 2 Met Ser Gly
Glu Ser Glu Thr Leu Val Ile Gly Arg Val Val Gly Asp 1 5 10 15 Val
Leu Asp Pro Tyr Thr Lys Thr Thr Ala Leu Arg Ile Arg Tyr Gly 20 25
30 Ser Lys Glu Val Thr Cys Gly His Glu Leu Lys Pro Ser Gln Val Val
35 40 45 Ile Gln Pro Arg Val Glu Val Gly Gly Lys Asp Leu Arg Thr
Phe Tyr 50 55 60 Thr Leu Val Met Val Asp Pro Asp Ala Pro Ser Pro
Ser Asn Pro His 65 70 75 80 Leu Arg Ala Tyr Leu His Trp Leu Val Thr
Asp Leu Pro Gly Thr Thr 85 90 95 Gly Ala Ser Phe Gly Gln Glu Val
Met Arg Tyr Glu Ser Pro Arg Pro 100 105 110 Thr Leu Gly Ile His Arg
Phe Val Phe Val Leu Phe Arg Gln Leu Gly 115 120 125 Arg Gln Thr Val
Gln Val Pro Thr Pro Gly Arg Arg Gln Asn Phe Asn 130 135 140 Thr Arg
Gly Phe Ala Arg 145 150 3 615 DNA Momordica charantia unsure
(567)...(567) n = A, C, G or T 3 gtttgtggcg ctagcctttg tgatctctca
tggctatgtc cgtggaccct ctggtggtcg 60 gccgagtgat cggagacgtg
gtcgacatgt ttgtgccaac tgctaacctg gcagtctact 120 tcaactccaa
acatgttact aatggttgcg acattaagcc ttctcttgcg gttaacccac 180
caaggctcgt cattccgggc catcctcgcg acctttacac tttggtgatg acagatccag
240 atgctccgag tcctagcgaa cctcatatga gagaatgggt ccattggata
attgtagaca 300 ttcccggagg ctcaacaatg acccaaggga aggagattct
gccgtacacc ggcccacgtc 360 cacccatcgg aatccaccgc tacatccttt
tactgttcaa gcaaaagggt cctgtggggt 420 tgatcgagca accaccgagc
cgcgcaaact tcagcactcg cctgtttgct aagcacctcg 480 acctggacct
gccggtggcg gccacctact tcaactctca gaaggaacca gccaccaaaa 540
agttcgcaat gtaatctgaa ccaagtngtc aacccaaacc aaaaaaaaat ngnagtcatc
600 cacgggcaaa atttc 615 4 174 PRT Momordica charantia 4 Met Ala
Met Ser Val Asp Pro Leu Val Val Gly Arg Val Ile Gly Asp 1 5 10 15
Val Val Asp Met Phe Val Pro Thr Ala Asn Leu Ala Val Tyr Phe Asn 20
25 30 Ser Lys His Val Thr Asn Gly Cys Asp Ile Lys Pro Ser Leu Ala
Val 35 40 45 Asn Pro Pro Arg Leu Val Ile Pro Gly His Pro Arg Asp
Leu Tyr Thr 50 55 60 Leu Val Met Thr Asp Pro Asp Ala Pro Ser Pro
Ser Glu Pro His Met 65 70 75 80 Arg Glu Trp Val His Trp Ile Ile Val
Asp Ile Pro Gly Gly Ser Thr 85 90 95 Met Thr Gln Gly Lys Glu Ile
Leu Pro Tyr Thr Gly Pro Arg Pro Pro 100 105 110 Ile Gly Ile His Arg
Tyr Ile Leu Leu Leu Phe Lys Gln Lys Gly Pro 115 120 125 Val Gly Leu
Ile Glu Gln Pro Pro Ser Arg Ala Asn Phe Ser Thr Arg 130 135 140 Leu
Phe Ala Lys His Leu Asp Leu Asp Leu Pro Val Ala Ala Thr Tyr 145 150
155 160 Phe Asn Ser Gln Lys Glu Pro Ala Thr Lys Lys Phe Ala Met 165
170 5 859 DNA Impatiens balsamia 5 gcacgagagc tcatctttcc cagttttgct
cccccttttg gctaaaatgt ctcagatctc 60 tgcctccatt gaccctctca
ttatgtgcag aatcatagga gatgtggttg atgtgtttgt 120 tcccaccacg
gctatgaatg tctactttgg gaacaagcat gttaccaatg gctgtaacat 180
caagccttcc atggcttatg atgccccaaa tgtcactatt tctgggatgc ctcatgagct
240 ttacactctt gtgatgacag atccagatgc tccaagtcca agtgagccct
ccatgaggga 300 atgggtccac tgggttgtga ccaacattcc cgggggcagc
agtgcggctc aagggaaaga 360 gctggtgtcc tacatgggtc catgcccagc
tattgggatt catcgctaca ttttgatcct 420 gtaccgtcag tccatatatg
tggaccagaa cattgagaag cctaacatca taaccagggc 480 caacttcagc
accagggctt tctctcatca cctttgcctg ggagttcctg tggccactgt 540
ttacttcaat gctcagaagg agcccctgaa ccagcgcaag aatgtgtgaa ggaacggccc
600 tggagcggcg agagaacgtg gagcaagcta cttcgtttgt cttttccttt
tagtataagt 660 aatatcatgc attagcatga ccctaagaat aattgatgtt
gtgggatatg tgtgttttac 720 catctctttg tttggttatg ttatgcattt
ccctttaggc tttaatgttt gtatgcattt 780 ccctttggct taatatttca
atgcatttcc ctcaaaaaaa aaaaaaaaaa aaaaaaaaaa 840 aaaaaaaaaa
aaaaaaaaa 859 6 180 PRT Impatiens balsamia 6 Met Ser Gln Ile Ser
Ala Ser Ile Asp Pro Leu Ile Met Cys Arg Ile 1 5 10 15 Ile Gly Asp
Val Val Asp Val Phe Val Pro Thr Thr Ala Met Asn Val 20 25 30 Tyr
Phe Gly Asn Lys His Val Thr Asn Gly Cys Asn Ile Lys Pro Ser 35 40
45 Met Ala Tyr Asp Ala Pro Asn Val Thr Ile Ser Gly Met Pro His Glu
50 55 60 Leu Tyr Thr Leu Val Met Thr Asp Pro Asp Ala Pro Ser Pro
Ser Glu 65 70 75 80 Pro Ser Met Arg Glu Trp Val His Trp Val Val Thr
Asn Ile Pro Gly 85 90 95 Gly Ser Ser Ala Ala Gln Gly Lys Glu Leu
Val Ser Tyr Met Gly Pro 100 105 110 Cys Pro Ala Ile Gly Ile His Arg
Tyr Ile Leu Ile Leu Tyr Arg Gln 115 120 125 Ser Ile Tyr Val Asp Gln
Asn Ile Glu Lys Pro Asn Ile Ile Thr Arg 130 135 140 Ala Asn Phe Ser
Thr Arg Ala Phe Ser His His Leu Cys Leu Gly Val 145 150 155 160 Pro
Val Ala Thr Val Tyr Phe Asn Ala Gln Lys Glu Pro Leu Asn Gln 165 170
175 Arg Lys Asn Val 180 7 1078 DNA Zea mays 7 ggcgcgccgg ccggccggtc
gattccccca ctccactcgc cgcccgcggc tgggctgcgc 60 tgcgcatcga
cgacggacga cgacacaatc acccccaccc cccgtccaat cagcagcgga 120
cgagggacga ccacggcccc ccgtctgccg cacgcgcgcc cgctctgcca gctgctgcta
180 ctactgctaa acctcgccca ccagtcgcgt gaggaaatag caacctgctg
agctcgctcg 240 ttcgctcgct cgcctgcctt cttccctggg caagctagct
agctaggatc gaggaggagc 300 tctgcccggc catgcagcgt ggggatccgc
tggtggtggg ccgcatcatc ggcgacgtgg 360 tggacccctt cgtgcgccgg
gtgccgctcc gcgtcgccta cgccgcgcgc gaggtctcca 420 acggctgcga
gctcaggccc tccgccatcg ccgaccagcc gcgcgtcgag gtcggcggac 480
ccgacatgcg caccttctac accctcgtga tggtagatcc tgatgcgccg agccccagcg
540 atcccaacct cagggagtac ctgcactggc tggtcactga tattccggcg
acgactggag 600 tatcttttgg gaccgaggtc gtgtgctacg agagcccacg
gccggtgctg gggatccacc 660 gggtcgtgtt tctgctcttc cagcagctcg
gccggcagac ggtgtacgcc ccggggtggc 720 ggcagaactt cagcacccgc
gacttcgccg agctctacaa cctcggcttg ccggtcgccg 780 ccgtctactt
caactgccag agggagtccg gaaccggtgg gagaagaatg tgatctcgac 840
ccggccgggt ggaaattaat aagatgacgg gtaatcgggt atatgtatat atttatatat
900 atatgtatat gtacgtgtat ttgatctggt ggcctttggt tatattgggt
ggggtgtatt 960 tgatatatta tctgtggcag attggcgcat tctctggcgc
atatttgata gctacatgta 1020 tctatttata cagatataaa gcgagcaata
atatgcatat gagagggttc agccaaaa 1078 8 173 PRT Zea mays 8 Met Gln
Arg Gly Asp Pro Leu Val Val Gly Arg Ile Ile Gly Asp Val 1 5 10 15
Val Asp Pro Phe Val Arg Arg Val Pro Leu Arg Val Ala Tyr Ala Ala 20
25 30 Arg Glu Val Ser Asn Gly Cys Glu Leu Arg Pro Ser Ala Ile Ala
Asp 35 40 45 Gln Pro Arg Val Glu Val Gly Gly Pro Asp Met Arg Thr
Phe Tyr Thr 50 55 60 Leu Val Met Val Asp Pro Asp Ala Pro Ser Pro
Ser Asp Pro Asn Leu 65 70 75 80 Arg Glu Tyr Leu His Trp Leu Val Thr
Asp Ile Pro Ala Thr Thr Gly 85 90 95 Val Ser Phe Gly Thr Glu Val
Val Cys Tyr Glu Ser Pro Arg Pro Val 100 105 110 Leu Gly Ile His Arg
Val Val Phe Leu Leu Phe Gln Gln Leu Gly Arg 115 120 125 Gln Thr Val
Tyr Ala Pro Gly Trp Arg Gln Asn Phe Ser Thr Arg Asp 130 135 140 Phe
Ala Glu Leu Tyr Asn Leu Gly Leu Pro Val Ala Ala Val Tyr Phe 145 150
155 160 Asn Cys Gln Arg Glu Ser Gly Thr Gly Gly Arg Arg Met 165 170
9 929 DNA Zea mays 9 gcacgagaag aaaccgaacg agggtttagc tagcaaaata
aacagaagca agcaagctag 60 ctagagctaa ggatcgagat cgagatcgac
cgaccgacga cgatcagcat ggcgcgcttc 120 gtggatccgc tggtggtggg
gcgggtgatc ggcgaggtgg tggacctgtt cgtgccttcc 180 atctccatga
ccgtcgccta tgatggctcc aaggacatca gcaacggctg cctcctcaag 240
ccgtccgcca ccgccgcgcc gccgctcgtc cgcatctccg gccgccgcaa cgacctctac
300 acgctgatca tgacggaccc cgatgcgcct agccccagca acccgaccat
gagagagtac 360 ctccactgga tagtgattaa cataccagga ggaacagatg
ctactaaagg tgaggaggtg 420 gtggagtaca tgggcccgcg gccgccggtg
ggcatccacc gctacgtgct ggtgctgttc 480 gagcagaaga cgcgcgtgca
cgcggaggcc cccggcgacc gcgccaactt caagacgcgc 540 gcgttcgcgg
cggcgcacga gctcggcctc cccactgccg tcgtctactt caacgcgcag 600
aaggagcccg ccagccgccg ccgctagcta gcagctcctc tctgaggcat gccagatgca
660 tgcgtgtgcg tgcaggtgca accaccgcac tgccggcggc tacgtatgac
cggtgaataa 720 aaagttttac tgcaccgtaa gcatgctcgc cctgttgcta
ttggtatatg ttagcagtgt 780 ggcagtctgt atgtagtagc tattcgcttg
catctatgca ctctatgtta gtatgcgtac 840 gtgtggttcc ggaacttttg
gagtcttatc taaatactat tgagtaaaac tccagtagtt 900 cactcttaaa
caaaaaaaaa aaaaaaaaa 929 10 172 PRT Zea mays 10 Met Ala Arg Phe Val
Asp Pro Leu Val Val Gly Arg Val Ile Gly Glu 1 5 10 15 Val Val Asp
Leu Phe Val Pro Ser Ile Ser Met Thr Val Ala Tyr Asp 20 25 30 Gly
Ser Lys Asp Ile Ser Asn Gly Cys Leu Leu Lys Pro Ser Ala Thr 35 40
45 Ala Ala Pro Pro Leu Val Arg Ile Ser Gly Arg Arg Asn Asp Leu Tyr
50 55 60 Thr Leu Ile Met Thr Asp Pro Asp Ala Pro Ser Pro Ser Asn
Pro Thr 65 70 75 80 Met Arg Glu Tyr Leu His Trp Ile Val Ile Asn Ile
Pro Gly Gly Thr 85 90 95 Asp Ala Thr Lys Gly Glu Glu Val Val Glu
Tyr Met Gly Pro Arg Pro 100 105 110 Pro Val Gly Ile His Arg Tyr Val
Leu Val Leu Phe Glu Gln Lys Thr 115 120 125 Arg Val His Ala Glu Ala
Pro Gly Asp Arg Ala Asn Phe Lys Thr Arg 130 135 140 Ala Phe Ala Ala
Ala His Glu Leu Gly Leu Pro Thr Ala Val Val Tyr 145 150 155 160 Phe
Asn Ala Gln Lys Glu Pro Ala Ser Arg Arg Arg 165 170 11 899 DNA Zea
mays 11 ttcaagccaa gttagcttgc ctcgaagatt gccaatcata gctagccatg
tcaagggacc 60 cacttgttgt aggcaacgta gttggagata tcttggaccc
atttatcaaa tcagcatcac 120 tcagagtcct atacaacaat agagaactga
ctaatggatc tgagttcagg ccatcgcaag 180 tagcttatga accaaggatt
gagattgctg gatatgacat gaggaccctt tacactttgg 240 taatggtgga
tcctgactca ccaagtccaa gcaatccaac aaaaagagag taccttcact 300
ggttggtgac agatattcca gaatcaacag atgtgagctt tggaaatgag gtagtaagct
360 atgaaagccc aaagccaagt gctggaatac atcgcttcgt ctttgttctg
gtccgccaat 420 ctgtcaggca aactatttat gcgccaggat ggagacaaaa
tttcaacaca agagacttct 480 cagcactcta taatctagga ccacctgtgg
cctcagtgtt cttcaactgc caaagggaga 540 atgggtgcgg tggcagacga
tatattagat gatactcact ccgttctttt ttatttgtcg 600 cgttttagtt
taaaaataaa ctagcggacg acaaatattc gagaacggag gtagtattag 660
aataacctcc tctacatgag gactgacgga attctgtatg aggccaagca caccgaatgg
720 gtagtaaacg ctggacctta atttctagac tactttccca cctctacaag
atttgactat 780 gctagaaacg aatttcactt accatgtgaa atgtgataaa
tatattccaa ctatatgttc 840 ctgcctcctt gataatgaat actactcagc
attggttttg taaaaaaaaa aaaaaaaaa 899 12 174 PRT Zea mays 12 Met Ser
Arg Asp Pro Leu Val Val Gly Asn Val Val Gly Asp Ile Leu 1 5 10 15
Asp Pro Phe Ile Lys Ser Ala Ser Leu Arg Val Leu Tyr Asn Asn Arg 20
25 30 Glu Leu Thr Asn Gly Ser Glu Phe Arg Pro Ser Gln Val Ala Tyr
Glu 35 40 45 Pro Arg Ile Glu Ile Ala Gly Tyr Asp Met Arg Thr Leu
Tyr Thr Leu 50 55 60 Val Met Val Asp Pro Asp Ser Pro Ser Pro Ser
Asn Pro Thr Lys Arg 65 70 75 80 Glu Tyr Leu His Trp Leu Val Thr Asp
Ile Pro Glu Ser Thr Asp Val 85 90 95 Ser Phe Gly Asn Glu Val Val
Ser Tyr Glu Ser Pro Lys Pro Ser Ala 100 105 110 Gly Ile His Arg Phe
Val Phe Val Leu Val Arg Gln Ser Val Arg Gln 115 120 125 Thr Ile Tyr
Ala Pro Gly Trp Arg Gln Asn Phe Asn Thr Arg Asp Phe 130 135 140 Ser
Ala Leu Tyr Asn Leu Gly Pro Pro Val Ala Ser Val Phe Phe Asn 145 150
155 160 Cys Gln Arg Glu Asn Gly Cys Gly Gly Arg Arg Tyr Ile Arg 165
170 13 893 DNA Zea mays 13 ggccgtagat agtaagtaga tcacgcagcg
cagtagctct ggattaatta ataataattg 60 ctcgtgcgtg tgtccagagc
cgccatggct gcccatgtgg acccgctggt tgtggggagg 120 gtgatcggcg
acgtggtgga cttgttcgtg ccgacggtgg ccgtgtcggc gcgcttcggc 180
gccaaggacc tcaccaacgg ctgcgagatc aagccatccg tcgccgcggc cgctcccgcc
240 gtcctcatcg ccggcagggc caacgacctc ttcaccctgg ttatgactga
cccagatgct 300 ccgagcccta gcgagccaac gatgagggag ttgctccact
ggctggtggt taacatacca 360 ggtggagcag atgcttctca aggcggtgag
acggtggtgc cgtacgtggg cccgcgcccg 420 ccggtgggta tccaccgcta
cgtgctggtg gtgtaccagc agaaggcccg cgtcacggct 480 ccgccgtcgc
tggcgccggc gacggaggcg acgcgcgcac ggttcagcaa ccgcgccttc 540
gccgaccgcc atgacctagg cctccctgtc gccgccatgt tcttcaacgc gcagaaggag
600 acagctagtc gccgccgcca ctactgagac aggctgatcg tcgtccaacg
gcaattacgt 660 acccagcaaa gcttaagcca gccgctgcag tcactcatct
catcgagaag aagacaatct 720 tcctagtcgc tgttcttgcc aagtactagt
accttgttaa ttattatgta agctaaaccc 780 gtgtgcctgt gattatattg
ggacgtgtct cgctttaata caaccgctca acttgtggcg 840 tttaattatt
ttatttatta gatataccaa ggtgtcatca agtcacttgc ctt 893 14 180 PRT Zea
mays 14 Met Ala Ala His Val Asp Pro Leu Val Val Gly Arg Val Ile Gly
Asp 1 5 10 15 Val Val Asp Leu Phe Val Pro Thr Val Ala Val Ser Ala
Arg Phe Gly 20 25 30 Ala Lys Asp Leu Thr Asn Gly Cys Glu Ile Lys
Pro Ser Val Ala Ala 35 40 45 Ala Ala Pro Ala Val Leu Ile Ala Gly
Arg Ala Asn Asp Leu Phe Thr 50 55 60 Leu Val Met Thr Asp Pro Asp
Ala Pro Ser Pro Ser Glu Pro Thr Met 65 70 75 80 Arg Glu Leu Leu His
Trp Leu Val Val Asn Ile Pro Gly Gly Ala Asp 85 90 95 Ala Ser Gln
Gly Gly Glu Thr Val Val Pro Tyr Val Gly Pro Arg Pro 100 105 110 Pro
Val Gly Ile His Arg Tyr Val Leu Val Val Tyr Gln Gln Lys Ala 115 120
125 Arg Val Thr Ala Pro Pro Ser Leu Ala Pro Ala Thr Glu Ala Thr Arg
130 135 140 Ala Arg Phe Ser Asn Arg Ala Phe Ala Asp Arg His Asp Leu
Gly Leu 145 150 155 160 Pro Val Ala Ala Met Phe Phe Asn Ala Gln Lys
Glu Thr Ala Ser Arg 165 170 175 Arg Arg His Tyr 180 15 837 DNA Zea
mays 15 ccacgcgtcc ggtactgtga gagtaaggct aaagtcgccg gataatataa
gaccagcaat 60 aacaagctag tttgccctcg ttctccaaca aaatgtctga
tgtggagccg ctggttctgg 120 ctcatgtcat acgagatgtg ttggattcat
ttgcaccaag tatcgggctc agaataacct 180 acaacagcag gttacttcta
tcaggtgttg agctgaaacc atccgcggtt gtgaataagc 240 caagagttga
tgttgggggc accgacctca gggtgttcta cacattggta ttagtggatc 300
cagatgcccc aagcccaagc aatccatcac tgagggagta tctgcactgg atggtgatag
360 acattcctgg aacaactgga gccagctttg gtcaggagct catgttttac
gagaggccag 420 agccgaggtc cggcatacac cgcatggtgt tcgtgctgtt
ccggcagctc ggcaggggga 480 cggtgtttgc accagacatg cggcacaact
tcaactgcaa gagcttcgcc cgtcagtacc 540 acctggacgt cgtggctgcc
acgtatttca actgccaaag ggaggcagga tccgggggca 600 gaaggttcag
gccggagagc tcgtaaggaa tgaagcatgc acagaagaag actgcagcgc 660
tttcgcatgc atatgatcta tcgtcgtcct gcggaatata tatatagtaa ccgttgttat
720 atggaataat gtgcatgaaa ttggtatcag atgcaccgac ccgtacgtac
gtaattaatg 780 tttgttatta cacgcagaca tataatatac atactcattc
acaaaaaaaa aaaaaaa 837 16 177 PRT Zea mays 16 Met Ser Asp Val Glu
Pro Leu Val Leu Ala His Val Ile Arg Asp Val 1 5 10 15 Leu Asp Ser
Phe Ala Pro Ser Ile Gly Leu Arg Ile Thr Tyr Asn Ser 20 25 30 Arg
Leu Leu Leu Ser Gly Val Glu Leu Lys Pro Ser Ala Val Val Asn 35 40
45 Lys Pro Arg Val Asp Val Gly Gly Thr Asp Leu Arg Val Phe Tyr Thr
50 55 60 Leu Val Leu Val Asp Pro Asp Ala Pro Ser Pro Ser Asn Pro
Ser Leu 65 70 75 80 Arg Glu Tyr Leu His Trp Met Val Ile Asp Ile
Pro
Gly Thr Thr Gly 85 90 95 Ala Ser Phe Gly Gln Glu Leu Met Phe Tyr
Glu Arg Pro Glu Pro Arg 100 105 110 Ser Gly Ile His Arg Met Val Phe
Val Leu Phe Arg Gln Leu Gly Arg 115 120 125 Gly Thr Val Phe Ala Pro
Asp Met Arg His Asn Phe Asn Cys Lys Ser 130 135 140 Phe Ala Arg Gln
Tyr His Leu Asp Val Val Ala Ala Thr Tyr Phe Asn 145 150 155 160 Cys
Gln Arg Glu Ala Gly Ser Gly Gly Arg Arg Phe Arg Pro Glu Ser 165 170
175 Ser 17 1191 DNA Zea mays 17 ccacgcgtcc ggtagtacct tggccaaacg
acttagctat caagctcgac cgaagctaag 60 ctaccaagct agtagccttc
ttggtcacgt accggccgtt gttgattgca gcggtcaagc 120 acacacaagc
taggcagcta gctagctaga gctagggtcg tcggatagat cgacatggcc 180
ggcagggaca gggagccgct ggtggttggt agggtggtcg gcgacgtgct ggaccccttc
240 gtccggacca ccaacctcag ggtcagctac ggggccagga ccgtgtccaa
cggctgcgag 300 ctcaagccgt ccatggtggt gcaccagccc agggtcgagg
tcgggggacc tgacatgagg 360 accttctaca ccctcgtgat ggtggacccg
gatgctccga gcccaagcga cccgaacctt 420 agggagtacc tacactggct
ggtgacggat attccgggaa ctactggggc agcatttggg 480 caagaggtga
tctgctacga gagccctcgg ccgaccatgg ggatccaccg cttcgtgctg 540
gtgctgttcc agcagctggg gcggcagacg gtgtacgccc cgggctggcg ccagaacttc
600 aacaccaggg acttcgccga gctctacaac ctgggcccgc ccgtcgccgc
cgtctacttc 660 aactgccagc gtgaggccgg ctctgggggc aggaggatgt
actcgtgatc ggatgcatgg 720 ttacatacca tgcacactac tcactccatc
gtctccatac atgtagacgg acgatggtgc 780 atgcatcgat cgtcaactac
tcaacaatta cgaactagaa atacacgcgt atatatacat 840 atataaatat
gcatatatac cggtactgta catgtcgccg tacacgcgca ggtggctgct 900
gctagcttgc tataccggcc ggtggtactg agcaggcagc atgcgctata tacttgcttg
960 gcgacgacgt gcagtgtgtg tatacaataa tgagcggccg gccggctagc
agggcgacga 1020 gccgtggctt tagcaataca tataccatgc aggcatgtgt
gtgtgcagtg cgtgccaagg 1080 tacggtaacg tattaattat tgtgcacata
cacatatgta tacgtacata tgcgtaaata 1140 tgaatgtgta cgtatacata
tgcatgctgg ttaattaaaa aaaaaaaaaa a 1191 18 177 PRT Zea mays 18 Met
Ala Gly Arg Asp Arg Glu Pro Leu Val Val Gly Arg Val Val Gly 1 5 10
15 Asp Val Leu Asp Pro Phe Val Arg Thr Thr Asn Leu Arg Val Ser Tyr
20 25 30 Gly Ala Arg Thr Val Ser Asn Gly Cys Glu Leu Lys Pro Ser
Met Val 35 40 45 Val His Gln Pro Arg Val Glu Val Gly Gly Pro Asp
Met Arg Thr Phe 50 55 60 Tyr Thr Leu Val Met Val Asp Pro Asp Ala
Pro Ser Pro Ser Asp Pro 65 70 75 80 Asn Leu Arg Glu Tyr Leu His Trp
Leu Val Thr Asp Ile Pro Gly Thr 85 90 95 Thr Gly Ala Ala Phe Gly
Gln Glu Val Ile Cys Tyr Glu Ser Pro Arg 100 105 110 Pro Thr Met Gly
Ile His Arg Phe Val Leu Val Leu Phe Gln Gln Leu 115 120 125 Gly Arg
Gln Thr Val Tyr Ala Pro Gly Trp Arg Gln Asn Phe Asn Thr 130 135 140
Arg Asp Phe Ala Glu Leu Tyr Asn Leu Gly Pro Pro Val Ala Ala Val 145
150 155 160 Tyr Phe Asn Cys Gln Arg Glu Ala Gly Ser Gly Gly Arg Arg
Met Tyr 165 170 175 Ser 19 902 DNA Zea mays 19 ccacgcgtcc
ggtattcttg agtgcattcg cttgctccat tcagtcagag cattccttgt 60
gcaaaattca aatacctgtc acaccaacca tgtctaggtc tgtggagcct ctcatagtcg
120 ggcgggtgat tggagaagtt ctcgactcct ttaacccatg tgtcaagatg
atagtaacct 180 acaactcaaa caaacttgta ttcaatggcc atgagatcta
cccatcagca attgtatcta 240 aacctagggt agaggttcaa gggggtgatt
tgcggtcttt cttcacattg gttatgacag 300 acccagatgt tccaggacca
agtgatccat atctaaggga gcaccttcat tggatcgtga 360 ctgatatacc
tgggacaaca gatgcctcct ttgggcgaga ggtcataagc tatgagagcc 420
caagacctaa catcggtatc cacaggttca tttttgtgct cttcaagcag aagggtaggc
480 aaactgtaac cgtgccatcc ttcagagatc atttcaacac ccggcagttt
gctgaggaaa 540 atgaccttgg cctcccagta gctgctgtct acttcaatgc
acagagagaa actgcagcta 600 ggagacgttg aaaattccag ctcttattgt
ccacctgatg ataataaagg ccttctgatc 660 ttctttctag gaagccaatg
aacttattct acattaaatt ctcctgagcc ctaccgtata 720 aataaaccag
atgcgttttg ctgattgtat tagtattaga atgctttgta cgtggcaaga 780
atgagaatta caaatggtca atgcttgtgg taaaatttga tgtgtaaaaa aaaaaaaaaa
840 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 900 gg 902 20 173 PRT Zea mays 20 Met Ser Arg Ser Val
Glu Pro Leu Ile Val Gly Arg Val Ile Gly Glu 1 5 10 15 Val Leu Asp
Ser Phe Asn Pro Cys Val Lys Met Ile Val Thr Tyr Asn 20 25 30 Ser
Asn Lys Leu Val Phe Asn Gly His Glu Ile Tyr Pro Ser Ala Ile 35 40
45 Val Ser Lys Pro Arg Val Glu Val Gln Gly Gly Asp Leu Arg Ser Phe
50 55 60 Phe Thr Leu Val Met Thr Asp Pro Asp Val Pro Gly Pro Ser
Asp Pro 65 70 75 80 Tyr Leu Arg Glu His Leu His Trp Ile Val Thr Asp
Ile Pro Gly Thr 85 90 95 Thr Asp Ala Ser Phe Gly Arg Glu Val Ile
Ser Tyr Glu Ser Pro Arg 100 105 110 Pro Asn Ile Gly Ile His Arg Phe
Ile Phe Val Leu Phe Lys Gln Lys 115 120 125 Gly Arg Gln Thr Val Thr
Val Pro Ser Phe Arg Asp His Phe Asn Thr 130 135 140 Arg Gln Phe Ala
Glu Glu Asn Asp Leu Gly Leu Pro Val Ala Ala Val 145 150 155 160 Tyr
Phe Asn Ala Gln Arg Glu Thr Ala Ala Arg Arg Arg 165 170 21 980 DNA
Zea mays 21 ccacgcgtcc gcgcacatag ggaacagaag ctactagctc cagcacaaaa
cacctactgc 60 ttcaactgta ccgttagaca tgtcaagggt gttggagcct
ctcattgtgg ggaaagtgat 120 tggtgaggtc ctggaccatt tcaaccccac
ggtgaagatg gtggtcacct acaactccaa 180 caagcaggtg ttcaacgggc
acgagttctt cccttcggca gtggccgcca agccgcgtgt 240 tgaggtccaa
gggggcgacc tcaggtcctt cttcacgttg gtgatgaccg accccgatgt 300
tcctggacct agtgatccat acttgaggga gcaccttcac tggattgtca ctgatattcc
360 tgggactacc gatgcttctt ttgggaaaga ggtggtgagc tacgagatcc
caaagccaaa 420 cattggcatc cacaggttca tctttgtgct gttccggcag
aagagccggc aagcggtgaa 480 cccgccgtcg tcgaaggacc gcttcagcac
ccgccagttc gctgaggaga acgacctcgg 540 cctccccgtc gccgccgtct
acttcaacgc gcagcgcgag accgccgccc gccgacgcta 600 accgtacggc
tcaacgtacg aaagaagacc atcctacgac gcttgcaatt agctgggcaa 660
gcaaagcttt ttttttcatc ctgagtcgat ctttacgtat gtatgtttgt ttaaataaaa
720 aggtagctaa tcagctgctt ggctgtgacc ccacgagcta gcagctacaa
cctactggta 780 catgctgcac attttagctg atttatgaag gtgacaatat
gattggtagg gttgcaatgt 840 tgactgggca tagtgtaaca acttaagcaa
tggccatggg cgagtacgtg tcgagtggtg 900 aagttgaagg gaagtttata
ttaaaagcaa ggccatgtct tgtattacct tgcctaaaaa 960 aaaaaaaaaa
aaaaaaaaag 980 22 173 PRT Zea mays 22 Met Ser Arg Val Leu Glu Pro
Leu Ile Val Gly Lys Val Ile Gly Glu 1 5 10 15 Val Leu Asp Asn Phe
Asn Pro Thr Val Lys Met Thr Ala Thr Tyr Gly 20 25 30 Ala Asn Lys
Gln Val Phe Asn Gly His Glu Phe Phe Pro Ser Ala Val 35 40 45 Ala
Gly Lys Pro Arg Val Glu Val Gln Gly Gly Asp Leu Arg Ser Phe 50 55
60 Phe Thr Leu Val Met Thr Asp Pro Asp Val Pro Gly Pro Ser Asp Pro
65 70 75 80 Tyr Leu Arg Glu His Leu His Trp Ile Val Thr Asp Ile Pro
Gly Thr 85 90 95 Thr Asp Ala Ser Phe Gly Arg Glu Val Val Ser Tyr
Glu Ser Pro Arg 100 105 110 Pro Asn Ile Gly Ile His Arg Phe Ile Leu
Val Leu Phe Arg Gln Lys 115 120 125 Arg Arg Gln Ala Val Ser Pro Pro
Pro Ser Arg Asp Arg Phe Ser Thr 130 135 140 Arg Gln Phe Ala Glu Asp
Asn Asp Leu Gly Leu Pro Val Ala Ala Val 145 150 155 160 Tyr Phe Asn
Ala Gln Arg Glu Thr Ala Ala Arg Arg Arg 165 170 23 405 DNA Oryza
sativa unsure (346)...(346) n = A, C, G, or T 23 ggagagatcg
atggcccgtt tcgtggatcc gctggtggtg ggacgggtga tcggggaggt 60
ggtggatttg ttcgttccat ccatctccat gaccgccgcc tacggcgaca gggacatcag
120 caacggctgc ctcgtccgcc catccgccgc cgactaccct cccctcgtcc
gcatctccgg 180 ccgccgcaac gacctctaca ccctgatcat gacggacccg
gacgcaccta gccctagcga 240 cccatccatg agggagtttc tccactggat
cgtggttaac ataccggggg gaacagatgc 300 atctaaaggt gaggagatgg
tggagtacat ggggccacgg gcgacngtgg ggataaacaa 360 gtacnttgct
ggtgctgtac aacaaaaagc gcgctttctg ggacg 405 24 128 PRT Oryza sativa
UNSURE (119)...(119) Xaa = any amino acid 24 Met Ala Arg Phe Val
Asp Pro Leu Val Val Gly Arg Val Ile Gly Glu 1 5 10 15 Val Val Asp
Leu Phe Val Pro Ser Ile Ser Met Thr Ala Ala Tyr Gly 20 25 30 Asp
Arg Asp Ile Ser Asn Gly Cys Leu Val Arg Pro Ser Ala Ala Asp 35 40
45 Tyr Pro Pro Leu Val Arg Ile Ser Gly Arg Arg Asn Asp Leu Tyr Thr
50 55 60 Leu Ile Met Thr Asp Pro Asp Ala Pro Ser Pro Ser Asp Pro
Ser Met 65 70 75 80 Arg Glu Phe Leu His Trp Ile Val Val Asn Ile Pro
Gly Gly Thr Asp 85 90 95 Ala Ser Lys Gly Glu Glu Met Val Glu Tyr
Met Gly Pro Arg Ala Thr 100 105 110 Val Gly Ile Asn Lys Tyr Xaa Ala
Gly Ala Val Gln Gln Lys Ala Arg 115 120 125 25 419 DNA Oryza sativa
unsure (221)...(221) n = A, C, G or T 25 cttacaccta atcccagcaa
cccaaccttg agggaatacc tgcactggat ggtgactgat 60 atcccatcat
cgacggacga tagctttggg cgggagatcg taacatacga aagcccaagc 120
cccaccatgg gcatccaccg catcgtgatg gtgttgtatc agcagcttgg gcgcggcacg
180 gtgttcgcgc cgcagtgggt ccagaacttc aacctgcgca ntttcgcgcg
ccgtttcaac 240 ctcggcaagc cggtggccgc catgtacttc aactgcnagc
gcccgacagg cacaggtggg 300 aggaggccaa ctgattgatc aatatcgtcg
atttcgtctt ctagctcttg tacatgttga 360 gtgttganca atataatggc
cactcatgca tatatatata tatatatata tatatatat 419 26 105 PRT Oryza
sativa UNSURE (74)...(74) Xaa = any amino acid 26 Leu Thr Pro Asn
Pro Ser Asn Pro Thr Leu Arg Glu Tyr Leu His Trp 1 5 10 15 Met Val
Thr Asp Ile Pro Ser Ser Thr Asp Asp Ser Phe Gly Arg Glu 20 25 30
Ile Val Thr Tyr Glu Ser Pro Ser Pro Thr Met Gly Ile His Arg Ile 35
40 45 Val Met Val Leu Tyr Gln Gln Leu Gly Arg Gly Thr Val Phe Ala
Pro 50 55 60 Gln Trp Val Gln Asn Phe Asn Leu Arg Xaa Phe Ala Arg
Arg Phe Asn 65 70 75 80 Leu Gly Lys Pro Val Ala Ala Met Tyr Phe Asn
Cys Xaa Arg Pro Thr 85 90 95 Gly Thr Gly Gly Arg Arg Pro Thr Asp
100 105 27 400 DNA Oryza sativa unsure (3)...(3) n = A, C, T or G
27 aancacagtc acacacacac agcagaagaa gaagaaaccg aacgagggtt
tagctagcaa 60 aataaacaga agcaagcaag ctagctagag ctaaggatcg
agatcgagat cgaccgaccg 120 acgacgatca actagcatgg cgcgcttcgt
ggatccgctg gtggtggggc nngtgatcgg 180 cgaggtggtg gacctgttcn
tgccttccat ctccatgacc gtcgcctatn atngccccaa 240 ggacatcanc
aacggctgcc tcctcaagcn gtccgccacc gccgcgccgc cggtcgtccn 300
catctccggc cgccgcnacg acctctacac nctgatgcat gacggacccc natnngccta
360 nccccagcaa cccgaccatg agggantacc nncactggat 400 28 87 PRT Oryza
sativa UNSURE (12)...(12) Xaa = any amino acid 28 Met Ala Arg Phe
Val Asp Pro Leu Val Val Gly Xaa Val Ile Gly Glu 1 5 10 15 Val Val
Asp Leu Phe Xaa Pro Ser Ile Ser Met Thr Val Ala Tyr Xaa 20 25 30
Xaa Pro Lys Asp Ile Xaa Asn Gly Cys Leu Leu Lys Xaa Ser Ala Thr 35
40 45 Ala Ala Pro Pro Val Val Xaa Ile Ser Gly Arg Arg Xaa Asp Leu
Tyr 50 55 60 Thr Leu Met Met Thr Asp Pro Xaa Xaa Pro Xaa Pro Ser
Asn Pro Thr 65 70 75 80 Met Arg Xaa Tyr Xaa His Trp 85 29 1226 DNA
Oryza sativa 29 ggcataagta tatatctgac aaattcagag aaattcagag
agtcaccgcg agagcttaag 60 ctagctagct agccggccat ggcatcgcat
gtggacccgc tggtggtggg gagggtgatc 120 ggcgacgtgg tggacctgtt
cgtgccgacg acggccatgt cggtgcggtt cgggaccaag 180 gacctcacca
acggctgcga gatcaagccg tccgtcgccg ccgcgccgcc cgccgtgcag 240
atcgccggca gggtcaacga gctcttcgct ctggtcatga ctgatccaga tgctcctagc
300 cccagcgagc cgactatgag agagtggctt cactggctgg tggttaacat
accaggtgga 360 acagatcctt ctcaagggga tgtggtggtg ccgtacatgg
ggccacggcc gccggtgggg 420 atccaccgct acgtgatggt gctgttccag
cagaaggcgc gcgtggcggc gccgccgccc 480 gacgaggacg ccgcgcgcgc
caggttcagc acgcgcgcct tcgccgaccg ccacgacctc 540 ggcctccccg
tcgccgccct ctacttcaac gcccagaagg agcccgccaa ccgccgccgc 600
cgctactagc ctccctcccc tcgctcggcg tcgcccatcc atccatccat ggacggcgac
660 ggcgacctag ctagctaata agccatcggt cggccatgct cgccgtccaa
actatcatgc 720 accatatcat gtcgtcgttt atgtggttaa ttaattattt
ccggcgtttt attactgtgt 780 ggtgccgtac atggggccac ggccgccggt
ggggatccac cgctacgtga tggtgctgtt 840 ccagcagaag gcgcgcgtgg
cggcgccgcc gcccgacgag gacgccgcgc gcgccaggtt 900 cagcacgcgc
gccttcgccg accgccacga cctcggcctc cccgtcgccg ccctctactt 960
caacgcccag aaggagcccg ccaaccgccg ccgccgctac tagcctccct cccctcgctc
1020 ggcgtcgccc atccatccat ccatggacgg cgacggcgac ctagctagct
aataagccat 1080 cggtcggcca tgctcgccgt ccaaactatc atgcaccata
tcatgtcgtc gtttatgtgg 1140 ttaattaatt atttccggcg ttttattact
gtgtggtgat taaaaaaaaa aaaaaaaaaa 1200 aaaaaaaaaa aaaaaaaaaa aaaaaa
1226 30 176 PRT Oryza sativa 30 Met Ala Ser His Val Asp Pro Leu Val
Val Gly Arg Val Ile Gly Asp 1 5 10 15 Val Val Asp Leu Phe Val Pro
Thr Thr Ala Met Ser Val Arg Phe Gly 20 25 30 Thr Lys Asp Leu Thr
Asn Gly Cys Glu Ile Lys Pro Ser Val Ala Ala 35 40 45 Ala Pro Pro
Ala Val Gln Ile Ala Gly Arg Val Asn Glu Leu Phe Ala 50 55 60 Leu
Val Met Thr Asp Pro Asp Ala Pro Ser Pro Ser Glu Pro Thr Met 65 70
75 80 Arg Glu Trp Leu His Trp Leu Val Val Asn Ile Pro Gly Gly Thr
Asp 85 90 95 Pro Ser Gln Gly Asp Val Val Val Pro Tyr Met Gly Pro
Arg Pro Pro 100 105 110 Val Gly Ile His Arg Tyr Val Met Val Leu Phe
Gln Gln Lys Ala Arg 115 120 125 Val Ala Ala Pro Pro Pro Asp Glu Asp
Ala Ala Arg Ala Arg Phe Ser 130 135 140 Thr Arg Ala Phe Ala Asp Arg
His Asp Leu Gly Leu Pro Val Ala Ala 145 150 155 160 Leu Tyr Phe Asn
Ala Gln Lys Glu Pro Ala Asn Arg Arg Arg Arg Tyr 165 170 175 31 1295
DNA Oryza sativa 31 gcacgagatt gcctgcacct agccacatca tatattcaga
gagagagctg agagagcagt 60 acaagagtgt atactacact tagcagctca
tcagttatta gttcactagt tcagccactg 120 accatcgaat caattcaggt
gagataatct tgagatagat atacggccat gtcgagggtg 180 ctggagcctc
tcattgtggg gaaggtgatc ggcgaggtgc tggacaactt caaccccacg 240
gtgaagatga cggccaccta cggcgccaac aagcaggtgt tcaacggcca cgagttcttc
300 ccctccgccg tcgccggcaa gccgcgcgtc gaggtccagg gcggcgacct
caggtccttc 360 ttcacattgg tgatgactga ccctgatgtg ccagggccta
gtgatccata cctgagggag 420 catcttcact ggattgttac tgatattcct
gggactactg atgcctcttt tgggagggag 480 gtggtgagct acgagagccc
gcggccaaac atcggcatcc acaggttcat cctggtgctg 540 ttccggcaga
agcgccggca ggcggtgagc ccgccgccgt cgagggaccg cttcagcacc 600
cgccagttcg ccgaggacaa cgacctcggc ctccccgtcg ccgccgtcta cttcaacgcg
660 cagcgcgaga ccgccgctcg ccgccgctaa tggctaccga cgacggcgac
gacgacgacg 720 accctgacac cgcgacgacc gatcttgcat ggacaaaaca
atataatcga gcttaattaa 780 ttactactac ttctactggc attttctatt
agttttccta tttcccctac attaatttca 840 ctgtcaaata aggcacactg
tgattagctg cagctagcta gctttgctcg tgtgtgtgag 900 ctagctagct
cgtagctaca gggcaggcct acagactacc agttcgtgct tgtttgcatg 960
cacattatca gattatccta gttgatttgt gaattaatca aggtgatcat aggattgtga
1020 gtgagcaatc gcaatcgcaa tgtgcaagct atggttgact agcagcagtg
agagatctct 1080 agctagctac actagttgaa gcaatggcca tccatggcag
gagagtccta gcatcccccc 1140 tgcatatatg cctacctact attcaactgc
tgttcttcga ttcaattcgc tggtgcttgc 1200 agtgtacttt gtttgatcct
gtgatcacta cttcttgcca cttgtttttg taatccgatc 1260 ggtgtcactt
tttctgtaaa aaaaaaaaaa aaaaa 1295 32 173 PRT Oryza sativa 32 Met Ser
Arg Val Leu Glu Pro Leu Ile Val Gly Lys Val Ile Gly Glu 1 5 10 15
Val Leu Asp Asn Phe Asn Pro Thr Val Lys Met Thr Ala Thr Tyr Gly 20
25 30 Ala Asn Lys Gln Val Phe Asn Gly His Glu Phe Phe Pro Ser Ala
Val 35 40 45 Ala Gly Lys Pro Arg Val Glu Val Gln Gly Gly Asp Leu
Arg Ser Phe 50 55 60 Phe Thr Leu Val Met Thr Asp Pro Asp Val Pro
Gly Pro Ser Asp Pro 65 70 75 80 Tyr Leu Arg Glu His Leu His Trp Ile
Val Thr Asp Ile Pro Gly Thr 85 90 95 Thr Asp Ala Ser Phe Gly Arg
Glu Val Val Ser Tyr Glu Ser Pro Arg
100 105 110 Pro Asn Ile Gly Ile His Arg Phe Ile Leu Val Leu Phe Arg
Gln Lys 115 120 125 Arg Arg Gln Ala Val Ser Pro Pro Pro Ser Arg Asp
Arg Phe Ser Thr 130 135 140 Arg Gln Phe Ala Glu Asp Asn Asp Leu Gly
Leu Pro Val Ala Ala Val 145 150 155 160 Tyr Phe Asn Ala Gln Arg Glu
Thr Ala Ala Arg Arg Arg 165 170 33 567 DNA Oryza sativa unsure
(401)...(401) n = A, C, G or T 33 ctcaagttag cttcttagca cagcctcttc
ttgctcaact cctgaagatc atcaatcttc 60 actagccatg tcaagggacc
cacttgtcgt aggacatgtt gttggggata tcttagaccc 120 attcaacaaa
tcagcatcac tcaaggtcct atacaacaac aaggaattaa caaatgggtc 180
tgagctcaaa ccgtcacagg tagcaaatga accaaggatt gaaattgctg gccgcgacat
240 aaggaacctt tacactctgg tgatggtgga tcctgactcg ccaagtccaa
gcaacccaac 300 aaaaagagaa taccttcatt gggttgggtg acaagacatt
ccaagaatcg gcaaatgcta 360 attatggaaa tgaagtttgt cagttatgaa
aagcccaaaa ncaaactgca nggatacatc 420 cgttttgncc ttaanantaa
ttccgccaat atgtncaaca agactaatta tgcancaaga 480 tgggggaaca
aaatttcaat acaaagagaa tttttccgca acgntaaaac cttggncctc 540
ccngtgggaa caatggttct caaattg 567 34 87 PRT Oryza sativa 34 Met Ser
Arg Asp Pro Leu Val Val Gly His Val Val Gly Asp Ile Leu 1 5 10 15
Asp Pro Phe Asn Lys Ser Ala Ser Leu Lys Val Leu Tyr Asn Asn Lys 20
25 30 Glu Leu Thr Asn Gly Ser Glu Leu Lys Pro Ser Gln Val Ala Asn
Glu 35 40 45 Pro Arg Ile Glu Ile Ala Gly Arg Asp Ile Arg Asn Leu
Tyr Thr Leu 50 55 60 Val Met Val Asp Pro Asp Ser Pro Ser Pro Ser
Asn Pro Thr Lys Arg 65 70 75 80 Glu Tyr Leu His Trp Val Gly 85 35
850 DNA Glycine max 35 atggcagcct ccgtggatcc cctagtggtt ggtcgcgtga
tcggcgatgt ggtagacatg 60 ttcattcctt cagtcaacat gtccgtttac
tttgggtcga agcacgtcac aaatggctgt 120 gacatcaagc catccattgc
catcagccct cctaagctca ccctcaccgg caacatggat 180 aacctctaca
cactggttat gactgatcct gacgcaccta gccccagtga accaagcatg 240
cgcgagtgga tacattggat cttagttgac atacctggag gaacaaaccc atttcgcgga
300 aaagagattg tttcatatgt gggaccaaga ccacctattg gaatacatcg
ctatatcttt 360 gtgttgtttc aacagaaagg acctttaggt cttgtggagc
aaccaccaac tcgagcaagc 420 ttcaacactc gttattttgc caggcaattg
gacttgggac ttccagtggc cactgtctac 480 ttcaactctc aaaaagaacc
tgctgttaag aggcgctgaa tctagctata ttgtaaccat 540 cagtgtctct
cttgagatat gcatggttgg aatatacttt taagatatca gaactatata 600
aaatctatga ctagtgcgta aataatgcag ggagagggtg tgtgtaaagt aatatagttg
660 tccaagacat gagtggtgcc gaccatttcc ccggctttga taactttttt
tctctatata 720 tatacctctc tttcactcta tcaaatatat aaagttaatc
tttattaaaa aaaaaaaaaa 780 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840 aaaaaaaaaa 850 36 172 PRT
Glycine max 36 Met Ala Ala Ser Val Asp Pro Leu Val Val Gly Arg Val
Ile Gly Asp 1 5 10 15 Val Val Asp Met Phe Ile Pro Ser Val Asn Met
Ser Val Tyr Phe Gly 20 25 30 Ser Lys His Val Thr Asn Gly Cys Asp
Ile Lys Pro Ser Ile Ala Ile 35 40 45 Ser Pro Pro Lys Leu Thr Leu
Thr Gly Asn Met Asp Asn Leu Tyr Thr 50 55 60 Leu Val Met Thr Asp
Pro Asp Ala Pro Ser Pro Ser Glu Pro Ser Met 65 70 75 80 Arg Glu Trp
Ile His Trp Ile Leu Val Asp Ile Pro Gly Gly Thr Asn 85 90 95 Pro
Phe Arg Gly Lys Glu Ile Val Ser Tyr Val Gly Pro Arg Pro Pro 100 105
110 Ile Gly Ile His Arg Tyr Ile Phe Val Leu Phe Gln Gln Lys Gly Pro
115 120 125 Leu Gly Leu Val Glu Gln Pro Pro Thr Arg Ala Ser Phe Asn
Thr Arg 130 135 140 Tyr Phe Ala Arg Gln Leu Asp Leu Gly Leu Pro Val
Ala Thr Val Tyr 145 150 155 160 Phe Asn Ser Gln Lys Glu Pro Ala Val
Lys Arg Arg 165 170 37 969 DNA Glycine max 37 gcacgagcat aacaattgta
ttcctccctt ccttagctcc actacctctt ctctcttcct 60 ccttgttcct
tcctcttaca atggcaagaa tgcctttaga gcctctaata gtggggagag 120
tcataggaga agttcttgat tcttttacca caagcacaaa aatgattgtg agttacaaca
180 agaatcaagt ctacaatggc catgaactct tcccttccac tgtcaacacc
aagcccaagg 240 ttgagattga gggtggtgat atgaggtcct tctttacact
gatcatgact gaccctgatg 300 ttcctggccc tagtgaccct tatctgagag
agcacttgca ctggatagtg acagatattc 360 caggcacaac agatgccaca
tttgggaaag agttggtgag ctatgagatc ccaaagccta 420 atattgggat
ccataggttt gtgtttgtcc tgttcaagca aaagcgtaga cagtgtgtta 480
ctccacccac ttcaagggac cacttcaaca cacgcaaatt cgcagcagag aacgaccttg
540 ccctccctgt ggctgctgtc tacttcaatg cacagaggga aacggctgca
agaagacgct 600 agctatagct gctgattttg ccactgcttc aaccaaacta
gtattgtatt gtattgaata 660 aagcgataaa aaaaggtaca agtacaagga
gtttcagtag tggaattaag ttgatcctca 720 catgtggctt caaataactt
gcaggaaggg aagataatta atcattttct agtttgaccc 780 gtgtgtatgc
tacgttttat tttactttcc atctgttgtg taaacattat actactacgt 840
gtattattat tacctcgtgg gactactatg aggtgtgatc ttatatatag aaataagagg
900 tttggtacgc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 960 aaaaaaaaa 969 38 173 PRT Glycine max 38 Met Ala Arg
Met Pro Leu Glu Pro Leu Ile Val Gly Arg Val Ile Gly 1 5 10 15 Glu
Val Leu Asp Ser Phe Thr Thr Ser Thr Lys Met Ile Val Ser Tyr 20 25
30 Asn Lys Asn Gln Val Tyr Asn Gly His Glu Leu Phe Pro Ser Thr Val
35 40 45 Asn Thr Lys Pro Lys Val Glu Ile Glu Gly Gly Asp Met Arg
Ser Phe 50 55 60 Phe Thr Leu Ile Met Thr Asp Pro Asp Val Pro Gly
Pro Ser Asp Pro 65 70 75 80 Tyr Leu Arg Glu His Leu His Trp Ile Val
Thr Asp Ile Pro Gly Thr 85 90 95 Thr Asp Ala Thr Phe Gly Lys Glu
Leu Val Ser Tyr Glu Ile Pro Lys 100 105 110 Pro Asn Ile Gly Ile His
Arg Phe Val Phe Val Leu Phe Lys Gln Lys 115 120 125 Arg Arg Gln Cys
Val Thr Pro Pro Thr Ser Arg Asp His Phe Asn Thr 130 135 140 Arg Lys
Phe Ala Ala Glu Asn Asp Leu Ala Leu Pro Val Ala Ala Val 145 150 155
160 Tyr Phe Asn Ala Gln Arg Glu Thr Ala Ala Arg Arg Arg 165 170 39
836 DNA Glycine max unsure (622)...(622) n = A, C, G or T 39
gttttagagc atattttctt catttttctt gcattccttc tctttgcaat tgatgtctag
60 gctaatggaa caaccacttg ttgtgggaag agtgatagga gaagtggttg
acattttcag 120 cccaagtgta agaatgaatg ttacatattc cactaagcaa
gttgctaatg gtcatgagtt 180 aatgccttct actattatgg ccaagccacg
cgttgagatt ggtggtgatg acatgaggac 240 tgcttatacc ttgatcatga
cagacccaga tgctccaagt cctagtgatc cacatctgag 300 ggaacatctc
cactggacgg ttacagatat ccctggcacc acagatgtct cttttggtaa 360
agagatagtg ggctatgaga gtccaaaacc agtaatagga atccacaggt atgtgttcat
420 tttgttcaag cagagaggaa gacagacagt caggcctcct tcttcaagag
accatttcaa 480 cacaaggagg ttctcagaag agaatggcct tggcctacca
gttgctgtag tttacttcaa 540 tgctcaaaga gagactgccg caagaaggag
gtgattcctg aagaagaaga agaagaagaa 600 gaaaggttgc agcagtaaat
anaattaatt ttgtttcaac cttaatcatc tcataatgag 660 atttgtttcc
tttggttttc ttaggggttg gcatggttga gtaaggaaga taggtgtgtt 720
gatgaatctc tcacacatca atgtttcttg tccatttctt tgggtcacaa cgaggaactg
780 taggtagtgt gtcaacagag tgtatctgat gacttaacgt cactggaaag gtgagg
836 40 173 PRT Glycine max 40 Met Ser Arg Leu Met Glu Gln Pro Leu
Val Val Gly Arg Val Ile Gly 1 5 10 15 Glu Val Val Asp Ile Phe Ser
Pro Ser Val Arg Met Asn Val Thr Tyr 20 25 30 Ser Thr Lys Gln Val
Ala Asn Gly His Glu Leu Met Pro Ser Thr Ile 35 40 45 Met Ala Lys
Pro Arg Val Glu Ile Gly Gly Asp Asp Met Arg Thr Ala 50 55 60 Tyr
Thr Leu Ile Met Thr Asp Pro Asp Ala Pro Ser Pro Ser Asp Pro 65 70
75 80 His Leu Arg Glu His Leu His Trp Thr Val Thr Asp Ile Pro Gly
Thr 85 90 95 Thr Asp Val Ser Phe Gly Lys Glu Ile Val Gly Tyr Glu
Ser Pro Lys 100 105 110 Pro Val Ile Gly Ile His Arg Tyr Val Phe Ile
Leu Phe Lys Gln Arg 115 120 125 Gly Arg Gln Thr Val Arg Pro Pro Ser
Ser Arg Asp His Phe Asn Thr 130 135 140 Arg Arg Phe Ser Glu Glu Asn
Gly Leu Gly Leu Pro Val Ala Val Val 145 150 155 160 Tyr Phe Asn Ala
Gln Arg Glu Thr Ala Ala Arg Arg Arg 165 170 41 893 DNA Triticum
aestivum 41 ttcggcacga ggggagatcc agctagctag ctggctagtt ttgctgttgc
tgctcgacct 60 catcgccatc ctccggctat ggcagcccat gtggatcccc
ttgtggttgg gagggtgatc 120 ggtgacgtgg tggacatgtt cgtgcccacc
atgccggtga ccgtgcgctt cgggacgaag 180 gacctgacga acggctgcga
gatcaagccg tccatcgccg acgcggcgcc ctcgatccag 240 atagccggcc
gggccggcga tctcttcacc ctggttatga ctgatccgga cgcaccgagc 300
cccagcgagc caaccatgaa ggagtggctt cactggctgg tggttaacat acctggtgga
360 tcagatcctt ctcaagggga ggaggtggtg ccctacatgg gtccgaagcc
gccgttgggc 420 atccaccgct acgtgctggt gctgttccag cagaaggcgc
gtgtgctggc gccggctccc 480 ggcggcgaca cagcagcgtc ggccatgcgc
gcgcggttca gcacccgtgc cttcgcagag 540 cgccatgacc tggggctccc
cgtcgccgcc atgtacttca acgcgcagaa ggagccggcc 600 aaccgccgcc
gccgctacta gctcgtcgcc gccggccgat caaacccgct gctgcctgct 660
ggtgctgcct gctggtccgt ctgtgtgtgc gtgcatgcgc gcgggcccaa taaattaacc
720 atatcgatct tgtcgttctc atgaacaatc tgggcttgta ttgtgtggta
ctctttgttt 780 gttttttctt gcgggtgcgt gtggtactct ttggaccata
tacatattta ccgctttctc 840 tcttcgttgt attcattgat tatgtgtgag
atccaaaaaa aaaaaaaaaa aac 893 42 180 PRT Triticum aestivum 42 Met
Ala Ala His Val Asp Pro Leu Val Val Gly Arg Val Ile Gly Asp 1 5 10
15 Val Val Asp Met Phe Val Pro Thr Met Pro Val Thr Val Arg Phe Gly
20 25 30 Thr Lys Asp Leu Thr Asn Gly Cys Glu Ile Lys Pro Ser Ile
Ala Asp 35 40 45 Ala Ala Pro Ser Ile Gln Ile Ala Gly Arg Ala Gly
Asp Leu Phe Thr 50 55 60 Leu Val Met Thr Asp Pro Asp Ala Pro Ser
Pro Ser Glu Pro Thr Met 65 70 75 80 Lys Glu Trp Leu His Trp Leu Val
Val Asn Ile Pro Gly Gly Ser Asp 85 90 95 Pro Ser Gln Gly Glu Glu
Val Val Pro Tyr Met Gly Pro Lys Pro Pro 100 105 110 Leu Gly Ile His
Arg Tyr Val Leu Val Leu Phe Gln Gln Lys Ala Arg 115 120 125 Val Leu
Ala Pro Ala Pro Gly Gly Asp Thr Ala Ala Ser Ala Met Arg 130 135 140
Ala Arg Phe Ser Thr Arg Ala Phe Ala Glu Arg His Asp Leu Gly Leu 145
150 155 160 Pro Val Ala Ala Met Tyr Phe Asn Ala Gln Lys Glu Pro Ala
Asn Arg 165 170 175 Arg Arg Arg Tyr 180 43 886 DNA Triticum
aestivum 43 gcacgaggcc gccttcatta acatatcgcc acttgcgccg gcggccggcg
gagaagggcg 60 ccagtggtga caggaggaag aagatggtgg ggagcggcat
gcatgcccag cgcggggacc 120 cgctggtggt ggggcgcgtg atcggcgacg
tggtggaccc gttcgtgcgg cgggtggcgc 180 tgcgggtcgg ctacgcgtcc
agggacgtgg ccaacggctg cgagctgagg ccgtccgcca 240 tcgccgaccc
gccgcgcgtc gaggtcggcg gcccggacat gcgcaccttc tacacgctgg 300
tgatggtgga tccggatgct ccaagtccca gcgatcccag ccttagggag tacttgcact
360 ggctggtcac cgacatcccg gcgacgacag gagtgtcttt tgggaccgag
gtggtgtgct 420 acgagggccc gcggccggtg ctcgggatcc accggctggt
gttcctgctc ttccagcagc 480 tgggccgcca gacggtgtac gccccggggt
ggcggcagaa cttcagcacc cgcgacttcg 540 ccgagctcta caacctcggc
ctgcccgtcg ccgccgtcta cttcaactgc cagagggaga 600 ccggaaccgg
cgggagaagg atgtgatgat caactccttg tataatacca gtacttaagt 660
agtataagtg acgacacaag atgatgatga tgatgaggtc gtatgggtgg tggtttatac
720 agggcgaaat ggagaaagaa ttgtaatgtt gaagaaataa taactatgcg
tgcgactttt 780 ttgatccgat gccggtgcga tactacaaag attaaaaaga
tgttaggatc caaaaaaaaa 840 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaa 886 44 180 PRT Triticum aestivum 44 Met Val Gly
Ser Gly Met His Ala Gln Arg Gly Asp Pro Leu Val Val 1 5 10 15 Gly
Arg Val Ile Gly Asp Val Val Asp Pro Phe Val Arg Arg Val Ala 20 25
30 Leu Arg Val Gly Tyr Ala Ser Arg Asp Val Ala Asn Gly Cys Glu Leu
35 40 45 Arg Pro Ser Ala Ile Ala Asp Pro Pro Arg Val Glu Val Gly
Gly Pro 50 55 60 Asp Met Arg Thr Phe Tyr Thr Leu Val Met Val Asp
Pro Asp Ala Pro 65 70 75 80 Ser Pro Ser Asp Pro Ser Leu Arg Glu Tyr
Leu His Trp Leu Val Thr 85 90 95 Asp Ile Pro Ala Thr Thr Gly Val
Ser Phe Gly Thr Glu Val Val Cys 100 105 110 Tyr Glu Gly Pro Arg Pro
Val Leu Gly Ile His Arg Leu Val Phe Leu 115 120 125 Leu Phe Gln Gln
Leu Gly Arg Gln Thr Val Tyr Ala Pro Gly Trp Arg 130 135 140 Gln Asn
Phe Ser Thr Arg Asp Phe Ala Glu Leu Tyr Asn Leu Gly Leu 145 150 155
160 Pro Val Ala Ala Val Tyr Phe Asn Cys Gln Arg Glu Thr Gly Thr Gly
165 170 175 Gly Arg Arg Met 180 45 1257 DNA Zea mays 45 ctcgctcaga
cagctctgct agctgcatcc tcctaactct ccaggtctct ctctcctctc 60
ccaactccca agtcccatcc ggatcgagac gctggaggcg gagcgccccc ccgggacggc
120 ggcggcgacg atggggcgcg gcaagatcga gatcaagcgg atcgagaacg
ccaccaaccg 180 ccaggtgacc tactccaagc gccggacggg gatcatgaag
aaggcgcgcg agctcaccgt 240 gctctgcgac gcccaggtcg ccatcatcat
gttctcctcc accggcaagt accacgagtt 300 ctgcagcccc ggaaccgaca
tcaagaccat ctttgaccgg taccagcagg ccatcgggac 360 cagcctatgg
atcgagcagt atgagaatat gcagcgcacg ctgagccatc tcaaggacat 420
caatcgtggt ctgcgcacag agattaggca aaggatgggc gaggatctgg acagtctgga
480 cttcgacgag ctgcgcggcc tcgagcaaaa cgtcgacgcg gctctcaagg
aggttcgcca 540 taggaagtac catgtgatca gcacgcagac tgatacctac
aagaaaaagg tgaagcactc 600 gcacgaggcg tacaagaacc tgcagcagga
gctaggcatg cgggaggacc cggcgttcgg 660 gtacgtggac aacacgggcg
ccggcgtcgc ctgggacggc gcggcggcgg cgctgggcgg 720 cgccccgccg
gacatgtacg ccttccgcgt ggtgcccagc cagcccaacc tgcacggcat 780
ggcctacggc ttccacgacc tccgcctggg ctagcgcatc catcaccatg ctgggtggtg
840 ctgctcgatc ctactgcatg gcaatgcaag ctggttggtt agttcgctca
tgcatcgtcc 900 gtcaacaaag caagtaagca atgcaatgca accgaggtac
tgtaatagcc aataaaatct 960 actgcatact gcaaacccaa ttactggtag
cttagctacc gcgtgtgtac gaatcaaccg 1020 attaattacc gcgcccttag
cttgcatgtc gtcgtcgtct gtgcttttgg cgttcgtaga 1080 catgtgtgta
ttgtatgcat gggtcctgtt catctgcatc catgcatgtt gtttatgatt 1140
gtaattgttg tgtgaaatgg ctgtactttg ttatgatcac gtgaaattat atctacattc
1200 gtgggaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa
1257 46 227 PRT Zea mays 46 Met Gly Arg Gly Lys Ile Glu Ile Lys Arg
Ile Glu Asn Ala Thr Asn 1 5 10 15 Arg Gln Val Thr Tyr Ser Lys Arg
Arg Thr Gly Ile Met Lys Lys Ala 20 25 30 Arg Glu Leu Thr Val Leu
Cys Asp Ala Gln Val Ala Ile Ile Met Phe 35 40 45 Ser Ser Thr Gly
Lys Tyr His Glu Phe Cys Ser Pro Gly Thr Asp Ile 50 55 60 Lys Thr
Ile Phe Asp Arg Tyr Gln Gln Ala Ile Gly Thr Ser Leu Trp 65 70 75 80
Ile Glu Gln Tyr Glu Asn Met Gln Arg Thr Leu Ser His Leu Lys Asp 85
90 95 Ile Asn Arg Gly Leu Arg Thr Glu Ile Arg Gln Arg Met Gly Glu
Asp 100 105 110 Leu Asp Ser Leu Asp Phe Asp Glu Leu Arg Gly Leu Glu
Gln Asn Val 115 120 125 Asp Ala Ala Leu Lys Glu Val Arg His Arg Lys
Tyr His Val Ile Ser 130 135 140 Thr Gln Thr Asp Thr Tyr Lys Lys Lys
Val Lys His Ser His Glu Ala 145 150 155 160 Tyr Lys Asn Leu Gln Gln
Glu Leu Gly Met Arg Glu Asp Pro Ala Phe 165 170 175 Gly Tyr Val Asp
Asn Thr Gly Ala Gly Val Ala Trp Asp Gly Ala Ala 180 185 190 Ala Ala
Leu Gly Gly Ala Pro Pro Asp Met Tyr Ala Phe Arg Val Val 195 200 205
Pro Ser Gln Pro Asn Leu His Gly Met Ala Tyr Gly Phe His Asp Leu 210
215 220 Arg Leu Gly 225 47 1089 DNA Zea mays 47 ccacgcgtcc
gaccgcaccg gcaccaccac caaccgagcg gctccaggct cctgctcagg 60
aaggggagaa gaggcgagcc ttccttggga agtcgcagga ggagagaagg ggaacaaaga
120 tggggcgcgg caagatcgag atcaagcgga tcgagaactc caccaaccgc
caggtgacct 180 tctccaagcg ccgcaacggg atcctcaaga aggcgcggga
gatcagcgtg ctctgcgacg 240 ccgaggtcgg cgtcgtcgtc ttctccagcg
ccggcaagct ttacgactac tgctccccga 300 agacatcgct atcaaaaatc
ctggagaagt accagaccaa ctctggaaag atactgtggg 360 gtgagaagca
caagagcctt agtgcagaga ttgaccgtat aaagaaagag aacgacacca 420
tgcagatcga gctcaggcac ctgaaaggtg aagatctaaa ctcgctgcaa cccaaagacc
480 tgatcatgat cgaggaggca cttgataatg
gactgacgaa cctgaatgag aaactgatgg 540 agcactggga aaggcgtgtg
acaaacacta agatgatgga agacgagaac aaattgctgg 600 ccttcaaact
ccaccagcaa gatatcgcgc tgagcggcag catgagagag cttgagctgg 660
gttaccatcc tgaccgggac ctggcggccc agatgccaat caccttccgc gtgcagccca
720 gccatcccaa cttgcaggag aacaattaga ctgctggatg ccctcgttcc
actcgccgag 780 gatttcaccc agccaccacc gctggcttgt atgccctcgt
gcgctggcaa ctgtatcttt 840 atctttccgg tatgtttgga tgaacgtata
atgtgtgtca gtgtcggtcg catgacgtgc 900 cgatgtcgtg catctctctc
tctctctgcc agagcagcgg aactctgcac cgtgagtaac 960 ttaattggta
ccgtatgatc tgccggacag tgaatagttt atgtgagtgg gtcaaaccat 1020
aatgtgtagt atttgtgtcg aactgtcaat ggcacgtatt tggattttca ctaaaaaaaa
1080 aaaaaaaag 1089 48 209 PRT Zea mays 48 Met Gly Arg Gly Lys Ile
Glu Ile Lys Arg Ile Glu Asn Ser Thr Asn 1 5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Asn Gly Ile Leu Lys Lys Ala 20 25 30 Arg Glu
Ile Ser Val Leu Cys Asp Ala Glu Val Gly Val Val Val Phe 35 40 45
Ser Ser Ala Gly Lys Leu Tyr Asp Tyr Cys Ser Pro Lys Thr Ser Leu 50
55 60 Ser Lys Ile Leu Glu Lys Tyr Gln Thr Asn Ser Gly Lys Ile Leu
Trp 65 70 75 80 Gly Glu Lys His Lys Ser Leu Ser Ala Glu Ile Asp Arg
Ile Lys Lys 85 90 95 Glu Asn Asp Thr Met Gln Ile Glu Leu Arg His
Leu Lys Gly Glu Asp 100 105 110 Leu Asn Ser Leu Gln Pro Lys Asp Leu
Ile Met Ile Glu Glu Ala Leu 115 120 125 Asp Asn Gly Leu Thr Asn Leu
Asn Glu Lys Leu Met Glu His Trp Glu 130 135 140 Arg Arg Val Thr Asn
Thr Lys Met Met Glu Asp Glu Asn Lys Leu Leu 145 150 155 160 Ala Phe
Lys Leu His Gln Gln Asp Ile Ala Leu Ser Gly Ser Met Arg 165 170 175
Glu Leu Glu Leu Gly Tyr His Pro Asp Arg Asp Leu Ala Ala Gln Met 180
185 190 Pro Ile Thr Phe Arg Val Gln Pro Ser His Pro Asn Leu Gln Glu
Asn 195 200 205 Asn 49 926 DNA Glycine max 49 gcacgaggct atggctagag
gaaagatcca gatcaagagg atagagaaca acaccaaccg 60 ccaggtcact
tactctaaac gacggaatgg ccttttcaag aaggccaacg agcttaccgt 120
tctctgcgat gccaaggttt ctattattat gttctccagc actggaaaac tccaccagta
180 catcagcccc tccacctcaa caaagcagtt cttcgatcaa taccagatga
ctctgggagt 240 tgatctctgg aactctcatt acgagaatat gcaagagaac
ttgaagaaac tgaaagaggt 300 gaataggaat cttcgtaagg agattaggca
gagaatggga gattgtctga acgagctggg 360 catggaagat ctcaagctcc
ttgaggaaga aatggacaag gccgccaagg ttgttcgtga 420 gcgtaagtat
aaggtgataa caaatcagat tgacacccag aggaaaaagt ttaataacga 480
gaaagaagtg cacaacaggc tcctgcatga cttggatgca aaagcagaag atccacgttt
540 tgcattgata gataatggag gggagtatga gtctgtgatc ggattctcaa
atttaggtcc 600 acgcatgttc gcattgagca tacaaccaag ccatcctagt
gcccatagcg gaggagcagg 660 ctctgatctt accacttacc ctttactttt
ctagtacgca attgcttaag ctctctccat 720 cagaaatacc atattcactc
aaatttcaat aagaatgact tgttgcagtt tgtacttaac 780 cacaaaacaa
tctcacgaat cttctccgtg gaacgcatgt gtgaattatt caattgcaac 840
tactgttatc tgtattttct ttttgcctaa tcatatacca taaacatgaa gttgtgcttc
900 cttttaaaaa aaaaaaaaaa aaaaaa 926 50 227 PRT Glycine max 50 Met
Ala Arg Gly Lys Ile Gln Ile Lys Arg Ile Glu Asn Asn Thr Asn 1 5 10
15 Arg Gln Val Thr Tyr Ser Lys Arg Arg Asn Gly Leu Phe Lys Lys Ala
20 25 30 Asn Glu Leu Thr Val Leu Cys Asp Ala Lys Val Ser Ile Ile
Met Phe 35 40 45 Ser Ser Thr Gly Lys Leu His Gln Tyr Ile Ser Pro
Ser Thr Ser Thr 50 55 60 Lys Gln Phe Phe Asp Gln Tyr Gln Met Thr
Leu Gly Val Asp Leu Trp 65 70 75 80 Asn Ser His Tyr Glu Asn Met Gln
Glu Asn Leu Lys Lys Leu Lys Glu 85 90 95 Val Asn Arg Asn Leu Arg
Lys Glu Ile Arg Gln Arg Met Gly Asp Cys 100 105 110 Leu Asn Glu Leu
Gly Met Glu Asp Leu Lys Leu Leu Glu Glu Glu Met 115 120 125 Asp Lys
Ala Ala Lys Val Val Arg Glu Arg Lys Tyr Lys Val Ile Thr 130 135 140
Asn Gln Ile Asp Thr Gln Arg Lys Lys Phe Asn Asn Glu Lys Glu Val 145
150 155 160 His Asn Arg Leu Leu His Asp Leu Asp Ala Lys Ala Glu Asp
Pro Arg 165 170 175 Phe Ala Leu Ile Asp Asn Gly Gly Glu Tyr Glu Ser
Val Ile Gly Phe 180 185 190 Ser Asn Leu Gly Pro Arg Met Phe Ala Leu
Ser Ile Gln Pro Ser His 195 200 205 Pro Ser Ala His Ser Gly Gly Ala
Gly Ser Asp Leu Thr Thr Tyr Pro 210 215 220 Leu Leu Phe 225 51 173
PRT Oryza sativa 51 Met Ser Arg Ser Val Glu Pro Leu Val Val Gly Arg
Val Ile Gly Glu 1 5 10 15 Val Leu Asp Thr Phe Asn Pro Cys Met Lys
Met Ile Val Thr Tyr Asn 20 25 30 Ser Asn Lys Leu Val Phe Asn Gly
His Glu Leu Tyr Pro Ser Ala Val 35 40 45 Val Ser Lys Pro Arg Val
Glu Val Gln Gly Gly Asp Leu Arg Ser Phe 50 55 60 Phe Thr Leu Val
Met Thr Asp Pro Asp Val Pro Gly Pro Ser Asp Pro 65 70 75 80 Tyr Leu
Arg Glu His Leu His Trp Ile Val Thr Asp Ile Pro Gly Thr 85 90 95
Thr Asp Ala Ser Phe Gly Arg Glu Val Ile Ser Tyr Glu Ser Pro Lys 100
105 110 Pro Asn Ile Gly Ile His Arg Phe Ile Phe Val Leu Phe Lys Gln
Lys 115 120 125 Arg Arg Gln Thr Val Ile Val Pro Ser Phe Arg Asp His
Phe Asn Thr 130 135 140 Arg Arg Phe Ala Glu Glu Asn Asp Leu Gly Leu
Pro Val Ala Ala Val 145 150 155 160 Tyr Phe Asn Ala Gln Arg Glu Thr
Ala Ala Arg Arg Arg 165 170 52 224 PRT Oryza sativa 52 Met Gly Arg
Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Ala Thr Asn 1 5 10 15 Arg
Gln Val Thr Tyr Ser Lys Arg Arg Thr Gly Ile Met Lys Lys Ala 20 25
30 Arg Glu Leu Thr Val Leu Cys Asp Ala Gln Val Ala Ile Ile Met Phe
35 40 45 Ser Ser Thr Gly Lys Tyr His Glu Phe Cys Ser Pro Ser Thr
Asp Ile 50 55 60 Lys Gly Ile Phe Asp Arg Tyr Gln Gln Ala Ile Gly
Thr Ser Leu Trp 65 70 75 80 Ile Glu Gln Tyr Glu Asn Met Gln Arg Thr
Leu Ser His Leu Lys Asp 85 90 95 Ile Asn Arg Asn Leu Arg Thr Glu
Ile Arg Gln Arg Met Gly Glu Asp 100 105 110 Leu Asp Gly Leu Glu Phe
Asp Glu Leu Arg Gly Leu Glu Gln Asn Val 115 120 125 Asp Ala Ala Leu
Lys Glu Val Arg His Arg Lys Tyr His Val Ile Thr 130 135 140 Thr Gln
Thr Glu Thr Tyr Lys Lys Lys Val Lys His Ser Tyr Glu Ala 145 150 155
160 Tyr Glu Thr Leu Gln Gln Glu Leu Gly Leu Arg Glu Glu Pro Ala Phe
165 170 175 Gly Phe Val Asp Asn Thr Gly Gly Gly Trp Asp Gly Gly Ala
Gly Ala 180 185 190 Gly Ala Ala Ala Asp Met Phe Ala Phe Arg Val Val
Pro Ser Gln Pro 195 200 205 Asn Leu His Gly Met Ala Tyr Gly Gly Asn
His Asp Leu Arg Leu Gly 210 215 220 53 613 DNA Zea mays unsure
(613)...(613) n = A, C, G or T 53 tacttctcgg cgtcggcgct gctccgagtg
atgtacggcg ggcgcgagat gacctgcggg 60 tcggagctca ggccgtcgca
ggtggcgagc gagccgacgg tgcacatcac ggggggccgc 120 gacgggaggc
cggtgctcta cacactggtg atgctggacc ccgatgcgcc cagcccaagc 180
aacccctcca agcgggagta tctccattgg ttggtgactg acataccaga aggagctggt
240 gccaatcatg ggaacgaggt ggtggcgtac gagagccccc ggccatcggc
ggggatccac 300 cgattcgtgt tcatcgtgtt ccggcaggcg gtccggcagg
cgatctacgc gcctgggtgg 360 cgcgccaact tcaacaccag ggacttcgcc
gcctgctaca gcctcggacc gcctgtcgcc 420 gccacctact tcaactgcca
gagggagggc ggctgcggtg gtcggaggta caggtgatga 480 atcgagagag
agcatgcatc ccaacaaggc ggtgatgaca cgtgacccat cctatgacaa 540
gttatatata ttagcacata ccacaaaaat aaacaataca tatatatatg tctccatctc
600 tatctgcaat atn 613 54 158 PRT Zea mays 54 Tyr Phe Ser Ala Ser
Ala Leu Leu Arg Val Met Tyr Gly Gly Arg Glu 1 5 10 15 Met Thr Cys
Gly Ser Glu Leu Arg Pro Ser Gln Val Ala Ser Glu Pro 20 25 30 Thr
Val His Ile Thr Gly Gly Arg Asp Gly Arg Pro Val Leu Tyr Thr 35 40
45 Leu Val Met Leu Asp Pro Asp Ala Pro Ser Pro Ser Asn Pro Ser Lys
50 55 60 Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro Glu Gly
Ala Gly 65 70 75 80 Ala Asn His Gly Asn Glu Val Val Ala Tyr Glu Ser
Pro Arg Pro Ser 85 90 95 Ala Gly Ile His Arg Phe Val Phe Ile Val
Phe Arg Gln Ala Val Arg 100 105 110 Gln Ala Ile Tyr Ala Pro Gly Trp
Arg Ala Asn Phe Asn Thr Arg Asp 115 120 125 Phe Ala Ala Cys Tyr Ser
Leu Gly Pro Pro Val Ala Ala Thr Tyr Phe 130 135 140 Asn Cys Gln Arg
Glu Gly Gly Cys Gly Gly Arg Arg Tyr Arg 145 150 155 55 945 DNA
Oryza sativa 55 gcacgaggga gagatcgatg gcccgtttcg tggatccgct
ggtggtggga cgggtgatcg 60 gggaggtggt ggatttgttc gttccatcca
tctccatgac cgccgcctac ggcgacaggg 120 acatcagcaa cggctgcctc
gtccgcccat ccgccgccga ctaccctccc ctcgtccgca 180 tctccggccg
ccgcaacgac ctctacaccc tgatcatgac ggacccggac gcacctagcc 240
ctagcgaccc atccatgagg gagtttctcc actggatcgt ggttaacata ccggggggaa
300 cagatgcatc taaaggtgag gagatggtgg agtacatggg gccacggccg
acggtgggga 360 tacacaggta cgtgctggtg ctgtacgagc agaaggcgcg
cttcgtggac ggcgcgctga 420 tgccgccggc ggacaggccc aacttcaaca
caagagcatt cgcggcgtac catcagctcg 480 gcctccccac cgccgtcgtc
cacttcaact cccagaggga gcccgccaac cgccgccgct 540 aatagtaata
gcctactatc tctatctatc tatccataat gaagaaagca agcacgcctg 600
cggatgcggc cggccggccc tactatatta ttacaataat atagtttttg aataattaag
660 ctagctagct ctcaactcaa gtatacttac tggaactcga ctgcgttgcg
tacgcatgtc 720 ctcatcatac gtacgaacgt gcgtgtccac gtactgtgta
ctagctagcg agtactctct 780 ccatatatat cttcctccac cgtcgtgtgg
tacgttttaa caacgtacat gcatgcatgg 840 ataatgcagg ctctatatat
atatatataa tactactgta ctgtactgta tgctttaatt 900 aattttgtgg
tttgctctca aaaaaaaaaa aaaaaaaaaa aaaaa 945 56 174 PRT Oryza sativa
56 Met Ala Arg Phe Val Asp Pro Leu Val Val Gly Arg Val Ile Gly Glu
1 5 10 15 Val Val Asp Leu Phe Val Pro Ser Ile Ser Met Thr Ala Ala
Tyr Gly 20 25 30 Asp Arg Asp Ile Ser Asn Gly Cys Leu Val Arg Pro
Ser Ala Ala Asp 35 40 45 Tyr Pro Pro Leu Val Arg Ile Ser Gly Arg
Arg Asn Asp Leu Tyr Thr 50 55 60 Leu Ile Met Thr Asp Pro Asp Ala
Pro Ser Pro Ser Asp Pro Ser Met 65 70 75 80 Arg Glu Phe Leu His Trp
Ile Val Val Asn Ile Pro Gly Gly Thr Asp 85 90 95 Ala Ser Lys Gly
Glu Glu Met Val Glu Tyr Met Gly Pro Arg Pro Thr 100 105 110 Val Gly
Ile His Arg Tyr Val Leu Val Leu Tyr Glu Gln Lys Ala Arg 115 120 125
Phe Val Asp Gly Ala Leu Met Pro Pro Ala Asp Arg Pro Asn Phe Asn 130
135 140 Thr Arg Ala Phe Ala Ala Tyr His Gln Leu Gly Leu Pro Thr Ala
Val 145 150 155 160 Val His Phe Asn Ser Gln Arg Glu Pro Ala Asn Arg
Arg Arg 165 170 57 639 DNA Oryza sativa 57 gcacgagctt acacctaatc
ccagcaaccc aaccttgagg gaatacctgc actggatggt 60 gactgatatc
ccatcatcga cggacgatag ctttgggcgg gagatcgtaa catacgaaag 120
cccaagcccc accatgggca tccaccgcat cgtgatggtg ttgtatcagc agcttgggcg
180 cggcacggtg ttcgcgccgc aggtgcgtca gaacttcaac ctgcgcagct
tcgcgcgccg 240 tttcaacctc ggcaagccgg tggccgccat gtacttcaac
tgccagcgcc cgacaggcac 300 aggtgggagg aggccaacct gatctgatca
atatcgatcg atcttcgatc ttctagctct 360 tgtacatgtt gagtgttgac
caatataatg gccactcatg catatatata tatatgcagt 420 gtgtctagcc
agctgcatgc aactttgtct acgtgcttat ataattaaac aaatgcatat 480
atagccggcc gtatcataaa gttcctagct ataaaagcta tagaataaat gtcgccccac
540 ttggtcagtt ggtgtacatg acggctccta agtgtgctat catgaatatg
ctaataatag 600 cagtttagta tatcatcccc gcaaaaaaaa aaaaaaaaa 639 58
104 PRT Oryza sativa 58 Leu Thr Pro Asn Pro Ser Asn Pro Thr Leu Arg
Glu Tyr Leu His Trp 1 5 10 15 Met Val Thr Asp Ile Pro Ser Ser Thr
Asp Asp Ser Phe Gly Arg Glu 20 25 30 Ile Val Thr Tyr Glu Ser Pro
Ser Pro Thr Met Gly Ile His Arg Ile 35 40 45 Val Met Val Leu Tyr
Gln Gln Leu Gly Arg Gly Thr Val Phe Ala Pro 50 55 60 Gln Val Arg
Gln Asn Phe Asn Leu Arg Ser Phe Ala Arg Arg Phe Asn 65 70 75 80 Leu
Gly Lys Pro Val Ala Ala Met Tyr Phe Asn Cys Gln Arg Pro Thr 85 90
95 Gly Thr Gly Gly Arg Arg Pro Thr 100 59 1004 DNA Oryza sativa 59
gcacgagctc aagttagctt cttagcacag cctcttcttg ctcaactcct gaagatcatc
60 aatcttcact agccatgtca agggacccac ttgtcgtagg acatgttgtt
ggggatatct 120 tagacccatt caacaaatca gcatcactca aggtcctata
caacaacaag gaattaacaa 180 atgggtctga gctcaaaccg tcacaggtag
caaatgaacc aaggattgaa attgctggcc 240 gcgacataag gaacctttac
actctggtga tggtggatcc tgactcgcca agtccaagca 300 acccaacaaa
aagagaatac cttcattggt tggtgacaga cattccagaa tcggcaaatg 360
ctagttatgg aaatgaagtt gtcagttatg aaagcccaaa accaactgca gggatacatc
420 gttttgtctt tatattattt cgccaatatg tacaacagac tatttatgca
ccaggatgga 480 gaccaaattt caatacaaga gatttttccg cactgtataa
tcttggacct cctgtggcag 540 cagtgttctt caattgccag agggagaacg
gatgtggagg cagacggtac attagataaa 600 agtcaggatc attcatagcc
ctctacaaga agaggtgata ttcatgtgag aagatgaatg 660 gggtcaggca
catcgcaacg tgctggtcaa tggtggacct tttaatgtat cttcatttaa 720
gaactactac ctttgatacg tatccaggca ctaaacaagg tgctttacga atgaatttag
780 cttcagatct catcttggag aacactttat ctggttcttc aggaacgaaa
tcctactgat 840 tctgcaccca acaactgttg tccatgtcat gttcaaaagc
gactatcaaa gcaacaaatt 900 gagtgcatca ttgaagaatg caactgataa
cacgtcatgt tctttaaaaa agaaagcatc 960 ctaggcttac tgagaacttt
gcataaaaaa aaaaaaaaaa aaaa 1004 60 174 PRT Oryza sativa 60 Met Ser
Arg Asp Pro Leu Val Val Gly His Val Val Gly Asp Ile Leu 1 5 10 15
Asp Pro Phe Asn Lys Ser Ala Ser Leu Lys Val Leu Tyr Asn Asn Lys 20
25 30 Glu Leu Thr Asn Gly Ser Glu Leu Lys Pro Ser Gln Val Ala Asn
Glu 35 40 45 Pro Arg Ile Glu Ile Ala Gly Arg Asp Ile Arg Asn Leu
Tyr Thr Leu 50 55 60 Val Met Val Asp Pro Asp Ser Pro Ser Pro Ser
Asn Pro Thr Lys Arg 65 70 75 80 Glu Tyr Leu His Trp Leu Val Thr Asp
Ile Pro Glu Ser Ala Asn Ala 85 90 95 Ser Tyr Gly Asn Glu Val Val
Ser Tyr Glu Ser Pro Lys Pro Thr Ala 100 105 110 Gly Ile His Arg Phe
Val Phe Ile Leu Phe Arg Gln Tyr Val Gln Gln 115 120 125 Thr Ile Tyr
Ala Pro Gly Trp Arg Pro Asn Phe Asn Thr Arg Asp Phe 130 135 140 Ser
Ala Leu Tyr Asn Leu Gly Pro Pro Val Ala Ala Val Phe Phe Asn 145 150
155 160 Cys Gln Arg Glu Asn Gly Cys Gly Gly Arg Arg Tyr Ile Arg 165
170 61 175 PRT Arabidopsis thaliana 61 Met Ser Ile Asn Ile Arg Asp
Pro Leu Ile Val Ser Arg Val Val Gly 1 5 10 15 Asp Val Leu Asp Pro
Phe Asn Arg Ser Ile Thr Leu Lys Val Thr Tyr 20 25 30 Gly Gln Arg
Glu Val Thr Asn Gly Leu Asp Leu Arg Pro Ser Gln Val 35 40 45 Gln
Asn Lys Pro Arg Val Glu Ile Gly Gly Glu Asp Leu Arg Asn Phe 50 55
60 Tyr Thr Leu Val Met Val Asp Pro Asp Val Pro Ser Pro Ser Asn Pro
65 70 75 80 His Leu Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro
Ala Thr 85 90 95 Thr Gly Thr Thr Phe Gly Asn Glu Ile Val Cys Tyr
Glu Asn Pro Ser 100 105 110 Pro Thr Ala Gly Ile His Arg Val Val Phe
Ile Leu Phe Arg Gln Leu 115 120 125 Gly Arg Gln Thr Val Tyr Ala Pro
Gly Trp Arg Gln Asn Phe Asn Thr 130 135 140 Arg Glu Phe Ala Glu Ile
Tyr Asn Leu Gly Leu Pro Val Ala Ala Val 145 150 155 160 Phe Tyr Asn
Cys Gln Arg
Glu Ser Gly Cys Gly Gly Arg Arg Leu 165 170 175 62 179 PRT Oryza
sativa 62 Met Ala Gly Ser Gly Arg Asp Arg Asp Pro Leu Val Val Gly
Arg Val 1 5 10 15 Val Gly Asp Val Leu Asp Ala Phe Val Arg Ser Thr
Asn Leu Lys Val 20 25 30 Thr Tyr Gly Ser Lys Thr Val Ser Asn Gly
Cys Glu Leu Lys Pro Ser 35 40 45 Met Val Thr His Gln Pro Arg Val
Glu Val Gly Gly Asn Asp Met Arg 50 55 60 Thr Phe Tyr Thr Leu Val
Met Val Asp Pro Asp Ala Pro Ser Pro Ser 65 70 75 80 Asp Pro Asn Leu
Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro 85 90 95 Gly Thr
Thr Ala Ala Ser Phe Gly Gln Glu Val Met Cys Tyr Glu Ser 100 105 110
Pro Arg Pro Thr Met Gly Ile His Arg Leu Val Phe Val Leu Phe Gln 115
120 125 Gln Leu Gly Arg Gln Thr Val Tyr Ala Pro Gly Trp Arg Gln Asn
Phe 130 135 140 Asn Thr Lys Asp Phe Ala Glu Leu Tyr Asn Leu Gly Ser
Pro Val Ala 145 150 155 160 Ala Val Tyr Phe Asn Cys Gln Arg Glu Ala
Gly Ser Gly Gly Arg Arg 165 170 175 Val Tyr Pro 63 13400 DNA Zea
mays 63 atattatttt cactcgcgag ctgctaaaga aaagcgccag tgataataat
gtttccacag 60 gcgttttctt aagcagcccg ccagtgaaaa ttcagttttg
tttgaaaatt tgaaaaaagg 120 caggaaactt atactggcgg gcagctaaag
aaaaccgcca gtgataataa tctttccact 180 ggcggtgtga taacgaatgc
ggacacaata tttattctta ggcggggctg cttaaaacac 240 cgccagtgat
aataatattt ccacatgcgg tttcttaagc aaaccgccag tgctaatgat 300
atttacactg gcgggctgct aaataaaacc gtcagtgcta aagatattta cactgacggc
360 tgacgaacaa ccgcctgtga aaaagcccga tttctactgg cacctagcac
tggcggcact 420 gaaaaacgcc agtgcaaata gctttaggac cgccactata
gagcttctgt gtactagtgc 480 tgccttattc cctccaccac ccggccttgt
cacgctcgtt gtcgagtact agaaaatctt 540 gtgtgttgtt gacgttattt
ggctcattct tgagcttttc tgaacgaacc agtggactgc 600 tcgtccacgg
ctcgatcggc gtaattagag atgttgtaac gggtcacgtc tacaacctag 660
tctaattagg ataaaagtgc acactgaaca catgattagc gcatgtcacc caataataac
720 ctcgatcgtt cgccctccac cggaatcgaa gccaggacag gacaggcacc
gcaggagatc 780 gacacgtcaa cccatcttgt tttttctacg accgctatta
ttatttacca cttgcttact 840 tagtacttac tgtgcggtgc ggggtaattg
gtaaacgggt gtcgacgtcc cgggcgcgta 900 cgtcgtgttg cggttactgt
taccgcgccg tggtcgtggt gggccagcaa agtctggcgg 960 gcggccaatg
ggaactcgac agagcggggg agacatgacg gggctgatag ctgtacgtcc 1020
tccaatccca tgcacccggc cacccatccg ctccgctgtc tcgtcagccg tcagctacct
1080 ctctagctgc tcgctcctca agcagtcaag cttctcaatg gaacagtatc
tatctatccc 1140 tacagtgggc agtggtgcca tacatggcca aaagttttag
cactatatga caccaaccag 1200 caagtttaat aatatagctc aggacaggtt
ttaggatggt aatatgtcgt atatagttaa 1260 ctaaaaacct actcatataa
taactctcca tagagacaaa ttattgtaga aaccgtagcc 1320 ggattaaagc
tgcagcaaac cgcaacgaac atggaagctg cctatttttt ctccctccac 1380
ttctcttatt tttatgtcac cataaatctg acgtgatatc tcttatacta ctccgtctat
1440 atcaacttat tatacttgct ctaacaccta gtattgtcgg tgcctatcta
taccgtcaaa 1500 aaatttgaaa tagtgtaaag attacaaata taatgtaaaa
atatgaatat ttataccaat 1560 atagatcgga tctatcatat ttaaaattat
aagaaatcat aattcataat cacatgtaca 1620 caacataaat cacctttaca
taccatcggt tagtctaagc actttgtcgt gttgtgttcg 1680 tgctaggcca
ttgtgatggg gtgacaacat cggtacgaca ttggatctcg tactgtattg 1740
acacatgtac tgtgtgaatc gtgttgtatc atgctgcaac actatgaatc gtgttgtatt
1800 tagtgtcaac ttatttagca cgactcactc gaccatctgg atcatgtatc
taaaaaatta 1860 gctggctaaa aactaacaga tattagctac tccttactaa
tcctgggcag ggcaggagga 1920 ggggcagggg cccccctccc tttcctagac
gagctagcaa acagcggtca gcagcaggga 1980 cagtccattt ccgtctgacc
cgcccgcggc cgttccccca ccccgccttt ccttgcaccc 2040 acccagcaag
cagagcccac cggatggacg gacgccccgc gcgccggccg gccggtcgat 2100
tcccccactc cactcgccgc ccgcggctgg gctgcgctgc gcatcgacga cggacgacga
2160 cacaatcacc cccacccccc gtccaatcag cagcggacga gggacgacca
cggccccccg 2220 tctgccgcac gcgcgcccgc tctgccagct gctgctacta
ctgctaaacc tcgcccacca 2280 gtcgcgtgag gaaatagcaa cctgcttgag
ctcgttcgct cgctcgcctg ccttcttccc 2340 tgggcaagct agctagctag
gatcgaggag gagctctgcc cggccatgca gcgtggggat 2400 ccgctggtgg
tgggccgcat catcggcgac gtggtggacc ccttcgtgcg ccgggttccg 2460
ctccgcgtcg cctacgccgc gcgcgaggtc tccaacggct gcgagctcag gccctccgcc
2520 atcgccgacc agccgcgcgt cgaggtcggc ggacccgaca tgcgcacctt
ctacaccctc 2580 gtacgtaaca gtccgttcac tcgatcttct cattatatcc
atccactgca tccctactat 2640 agacagacag tacatgcgtg cgtcggtgcg
tgttgtacac gattccaagc catcagccag 2700 cttaacccgc atgcctgaat
ctgactaatt ctgatctgca ggtgatggta gatcctgatg 2760 cgccgagccc
cagcgatccc aacctcaggg agtacctgca ctggtaatct actactagta 2820
gctagctagc cgcatatccg tttccatcca tccacgtact agtctacaaa acaaactgca
2880 tgcttcgttg ctttgcttct ctgcagcaag catggcctta gaataaacga
tctcggtaca 2940 tatatattta tctatttatc agtctaagat attttaaatc
ttactacata ttttttttaa 3000 atgaacctat attcttaatc gtatataaag
taagtataat tacatactca tatactttat 3060 atgcacttgc atatttttta
tcatactatt gtagataatt tagtgcgacc atatacttta 3120 tttatatgta
cttcatggag acatatatat atcataaatg tacatatata agaaactatt 3180
tcttaaaaat gcaaaataat tttcatatat acatacatat atacattgca tatggtaaca
3240 cacgtactgt ccacttcccg tctctttcag catgctacaa cacacgtact
gtcaggatct 3300 tgtctacatt ctccacctcg tacggtaggt agtactgtgt
actagcatga tataaataaa 3360 ctaatccaga gtccaccagc ctccctttcc
agttctagag atcaataata tatgcaagca 3420 aactactatt tagttgtgaa
tgacattaga tatctacctg ggcgcatttg tagaaacatt 3480 atcccgatta
agagtaaaga tgttactaac tgccctgatc acagatacaa tatcaaatat 3540
ctattcgaca tgttctttac gctatatatg tgtagttagt tcgttctcga atatttgtcg
3600 ctggctggtt catttttcaa ctaaaacgtg acaaataaat tagaacgaag
agagtactgt 3660 attagaatgt tagtttatgc aatttcaagt atttgtacag
aactgtggaa gatatcgacg 3720 catgttgctg ccacatgatc tgatgtatta
ctagctagct tttaaggcac acaatagcaa 3780 ccatgccttt tattttccca
ggcaaattat ttgagatggt aactcattcc agacatcttt 3840 taggctagtg
gtatgcatta gatttcgtta acaccaatac ccatatacca tctgtgtaga 3900
aaaaatgaag tagataatct gtagatgcac caatatcata acctacgtgt ctgccgtcca
3960 taattctttg gaaacttcaa attcctaaac catgagtaat cgtataagag
catcaccata 4020 tcatatatct atccggtctc ctatactaaa aaaatactac
ctccgttctc gaatatttgt 4080 cgcccgctag ttcatttttg aactaaaacg
tgacaaataa aaaagaacgg agggagtaac 4140 attttaaaag aggatgtgca
ttttagaaca tgaggtgcta caaaagatct tctatctcat 4200 ttcctatatc
tataatttta agagatattc taagagatgc attatttctc ctaaataaaa 4260
gaaacaaccc tcccttatct atccctaaag aaatatagga agcaaatttt aggtaatcta
4320 ctacaatagt gccctcttaa ctcttatatc ctaaattaat tttaagatgc
tcttttaaaa 4380 taacgtattt gagatgcaat ataagtgtgt gtttggttta
ggagccacat gggatagtta 4440 acctctatcc taattttttt agttgggtgg
acctcatctc acacgtttgg ttagagggat 4500 ggggtcattc taggttttct
gtttggttca ttggaaaata gatgtagatt gagaatctaa 4560 cacatggatc
ccacgtgtca ttctctttct cttcccttct ctaccttctt ccctaatcat 4620
gctcatcata ccccccatca tgtcacgtgg ttggaacgct tgtaccacac cgcccaattg
4680 ccatgactcc tacatcgtgc cctgcatcca tagtttttaa gctacactac
tcatcgtgtc 4740 gtccatgttt gccacaacca aatcgtcacc cctcactagc
accaacgggt gctcgagctt 4800 ggcttgcacc accggagttg ggtctgtgtg
ccaccaccac ccctcatcga tcatcatcgt 4860 gctcaccctc gagctcgacc
catcatcgct ctgctacgtc cggacatgcc cctgatgtgg 4920 gttcatttgc
tccacttgaa tcgaatgaat tggatgaatc catatctatg aggaatattc 4980
tttttgcggg atgaacccaa ccccctagtc tctcaaaaat ggattcgacc ctacttgacc
5040 taatgatacc tttgaaccaa acacacacta aaatatttgg tggagatgct
ttaaccattg 5100 gttttatatt tgtactcaag gcagactagt ggatgcaatt
actttatact cctactcgaa 5160 acatgagcaa ttgccttaat ggcatggcac
caagaaacat agaatacatc tttgagctag 5220 tcccaagaaa gaataggtcg
gttgcctgtg agttcaatgg tgggaattgg atcaggtctt 5280 tgagaagcaa
gatcacattg ttggttcaaa ttgaggaatt catctccctt tggattagac 5340
tccaagattt ttatcttcaa ccgcaggttc cgaactccat tacatggaaa tggagccttg
5400 acaatgtctt catagttaat ttagcctaca aagtgcaatc catcaaatct
tgcagccaca 5460 tcaaatctag tcttgcaatg gaaggcaagg gttgatgacc
caacatgtaa ggtttttcct 5520 tggattcttc tacaggacaa actcctcact
gtgaacaatc ttgccactag gggttggcca 5580 caccattcga gtcgctccct
ctacaatctt gccactaggg gatggccaca ccatccgagt 5640 cacaccctct
acaattggtc cctagagact aagggtgggc attttaaaac caaaaaccaa 5700
acccgaactc gaacccgaat aaaccaaatt atcgatctat ttgggttttg aggttcaggt
5760 ttggttccta catgtactat attttgaggt atgagtttgg gttcgttttc
tagcctttaa 5820 aacctaaata gaccgaataa cccgaaattt aaaaaactat
tagtatgtga tgatattatt 5880 atgtgattta tgaacttatt agctaaaaat
tatgatgaca tcttaatgat ggtatatata 5940 tctctatatg ctatttttta
tagtaacatg ttgtaataat agtacttcaa attaactatt 6000 tattatattt
atatttatat taacaaaaga cactagtctc tctactattt gatctatacg 6060
gtggatgaat agaccgaacc aaaattgtgt gtctattcag gttcgattcc caaaattatt
6120 tttgaaaatt ttggttctca tttttttaga actcgaaatt ttaaaaacta
aatagactga 6180 atcaaatcac ccttatagac tgaatgccca accctactag
agattggcct tctctctatt 6240 tccattgccc tttcgcctgt tgtgcaggat
tatattctag cttgggaagg tatgaccctc 6300 cctgcctagg cagacttggc
tcactcctct aacattaagg actggtggga agcagcgtcc 6360 tccccactcc
caaaaaccca aaaccgtgag ttcaacgaca tcgtggtata cactctatgg 6420
aacctatgga aggagagaaa ccagaggatc ttcaaaaata gctctctaaa ccctatccag
6480 gtagccttga ggatcaaaga ggatgtttta agttttaggt gggctatatc
taccctataa 6540 ttgggttgtt gcagtctctt agcaactacc tgctctatgt
gggtctgctg gaaatgttcc 6600 tcccaccgtg tgtgttaggc gatgttgtat
ccggtcttcg gaccttgttt cttgttttct 6660 aaactctttc cccattaatg
gatatgcaga gttcctgcat ttcattcaaa acaatatcga 6720 aacatgggac
atgagtgtat tttaaacatt gaactttatt atctttgatc aataattggc 6780
ccaattgtat aaaaagttta tagtacaaaa cttgtaacat tatatttgtg tacataaata
6840 ccttcttcag catctggtcc accaaagaat caaccttaat ctacctagga
ggcaataaat 6900 gaacacaaca tccacaaaac ttggacaaga atgcataggg
atctacttgt taaaacatgt 6960 agtagaacac acatatagta aaacacacat
gagatggaaa tgtgttgcgt gttgtcctct 7020 tcttgatcac atgagcatct
ccgacaaggt gcatcaaaat agtgtaccaa aaatcaaaat 7080 actacattta
aagtgtttag gacactaaaa acaaattaag ctacaacagt taagccccac 7140
attatatatt ttagaaaata attacattat taggaaagaa aaggttgaag atgatgtaga
7200 aaacaagaat aggacactaa tctagggtgt agtatgtttg gatcacttac
tgatgtgccc 7260 tatatttttt gataaaaatt gtaaatagga tattggtgtt
gttttttatg agttaaaccc 7320 tatatttcaa agtaggaaat tgttttaagg
cactaggtat gttctaagtg tggaatccct 7380 ctccacctaa ctaggttgtc
ctttttcagc atctattggt ggatagccaa ccacaaaaaa 7440 ataaaattga
cattttttag aggcctaagg gcaaagaaaa actaaaacaa cgaaggagta 7500
acgagctgat tttgtgaagc actaactaga agatgacaat ttcggtcagt tgctcgttcc
7560 gagcgagtca ccgaaaaatt tgtctcgaaa gtccccttta cgactagttt
gaaaacttaa 7620 atcccttctg ggattttcgg gaattaagga gaaaattaag
gctaatttcc atccctcaaa 7680 tccccaggaa tcccgaaggg atttaagttt
ccaaactagc ccttagatag aaggtctcct 7740 gtatatatat gtcaaggtat
ttggacaaag caccattaaa agagcacctt gtatatcttt 7800 tttctgaata
cgcaggagag ttgtgtatta gtatattaag aagagtaaag gtctgggaac 7860
agatcaaaat acaaggacac tactcataga ggacgaaaac acaaaaacat aaagagactc
7920 gtttatggcc caaaacacga cagcaacgat ctagccgtta gagagtaggc
gcaggccaca 7980 agctagagcc ccttgtatat ccatgagcca acttaagggt
gtgttcggaa gcaaagtatt 8040 ttaaaactat gattttagaa tactggtttt
aaattgttat gacacaacta tgatattttt 8100 tacatctctt taagcgatac
ttatttgtag acaaagtatc tcaaaccatg atatctagta 8160 tgcatatgtt
agaagctagc tataaacaaa aactttggtt tagagtggag tttcaagtac 8220
tctaaaaata ctataaatac tataggaact actaaatcat gttatttaaa atagaatttt
8280 ttacagagta gttaaacgcc tcttagcaac aaaaaaaccc atagtactta
ctccttgtca 8340 tgacacttca gtgccgcagg gctagagccc aatggagtca
aagttgggct ccccaacgaa 8400 gccatatcac tatatacatt ttccgttcat
caactagcta gctagctcaa cgaccatttt 8460 tctaatgggt ttttcttttt
ctaaagaaaa ttagttcatt ttcttttggc agaagttcac 8520 atctcgataa
aatttatatt tatggttagc ttggcaacac cattttattt tcaagaggtt 8580
tttattttat caagaaaaat tagttcattt tctcttggaa aaataaaaat ccattagaaa
8640 aatggggttg tcaaactagt ccttatttag ttttccattg ttaatttact
accggagtaa 8700 tttgagcgat gtggagaatt gaacttgaat ggcatttgac
tgtaattagt tagtactgtc 8760 tcataaggaa aatatttttc tttagaaaaa
gatactgaag caggctatat ttcttcaaca 8820 aaagaaaaga ataaaactga
aagactcaaa gcgctaacat acaataatga cataaagctg 8880 attgatagga
cagattatta gaagatccgt gcacacttgt tttggtgttg acaccgcgcg 8940
cctagcttta agtagctgct agttaatttg attttgtaac tgttcaaggc gagaattcaa
9000 atattatcta atgaattaat gatgatacgc cgcatcacct gaaacacaag
agttctttga 9060 acagtggcga ccaatatctt ggattcttct gctgtatttt
gtttctatag attcacctgc 9120 aaatatcatc tttatcacaa tcgtacctgt
aactcaaatg catctatagt tacaaagtca 9180 ccggccagcc tatacacata
tgctgatgtg caaaacggca agaccttgct agcattatat 9240 tttggggaca
tactaaagat ttcctgggtc agtccagtcc aattacagca acttctctta 9300
gaatcttgaa ctcgcgatat accacactga caaacaaaca aatcttatgt acaagcatgc
9360 aagcgagcct cttggtctgt aagatcacga tctaactgta gataagatcg
tgattcgtta 9420 agaggtcatc tataaatcta cgtgagctag tggtggaaat
gtttaagtgt taaatgtaat 9480 taatggttag atcataatct aacgaaccag
caacctcgct tgcacataag accttacagt 9540 actaggttcg tacgtgacct
ttcatgtata tagcacacat acatgtacga gtctactata 9600 cgtgttacag
cacatatata tacatctaac agatttctcg aacctgagcc atgatctgat 9660
gatagcatcc catatacaaa gatgtgccaa ctcaaagtcg ctttgatctt ctgttgcagg
9720 ctggtcactg atattccggc gacgactgga gtatcttttg gtatgtacta
gtacacagta 9780 ggaaccatga acgagcttaa ccaaagtgct agctaatgcc
atgcttggac atccatgaac 9840 gcgcagggac cgaggtcgtg tgctacgaga
gcccacggcc ggtgctgggg atccaccggg 9900 tcgtgtttct gctcttccag
cagctcggcc ggcagacggt gtacgccccg gggtggcggc 9960 agaacttcag
cacccgcgac ttcgccgagc tctacaacct cggcttgccg gtcgccgccg 10020
tctacttcaa ctgccagagg gagtccggaa ccggtgggag aagaatgtga tctcgacccg
10080 gctgggtgga aattaataag atgacgggta atcgggtata tgtatatatt
tatatatata 10140 tatatatgta tatgtacgtg tatttgatct ggtggccttt
tgttacattt gggtgaggtg 10200 tatttgatat tatctgtgga agattggcgc
attctctggc gcatatttga tagctacatg 10260 tatctattta tacagatata
aagcgagcaa taatatgcat atgagagggt tcagccacat 10320 cagtcaatac
tctctccttt atactgtgat gatgactact tatctgaatc atctgattac 10380
aactacatgc ttgcttgcta gctcactggt cgagctgctg gatgcagcca tgcatgcaga
10440 gaaatgaacc cacacatgcc gagggagacg agcagctgct tcctctcgcc
atttaatggt 10500 ttggccaaca cacagataaa tcatttgggg gacaagggca
aggatcaaga tgatggattg 10560 gggaagaaca cgggtgtgcc ttcacttccc
ttccttcctt ccttcctttc tttctttctt 10620 tcttttttct ttgagagaga
acgagaagaa tgcgggccgt agttgcactc gagaccccgc 10680 ccaacggcct
gctacgggcc attaaatcga acattccatg cgaacgcaaa aaaaaaagga 10740
cccaagttag cacaactcgc gggggggttc ggcccagcac actcctagag tcagaactga
10800 aattcctagc acatgcttca tcacccaaca aaagaatcag agcattaaga
gcgtagtaca 10860 gatcaacgat tattagcaac accaagacca taagtacttg
catccgagtt atgtacattc 10920 tatactgttc tctaaggtct gagtgcacat
ggaaagatga gagctatcct gtccagcttc 10980 agcttgcagc actatggcgg
tacctggcag ctgagccata cttctgataa gaggccaaat 11040 accggacgaa
aaatgaggaa gcggcaacca gggggtacaa atgctgagat tatcatgaag 11100
ggaacggcga tggctcctca gctggaagct tgtccgcggg ctttagctcc cagttgcgtg
11160 gattggtgtc cgtcgaatca gacttggaaa tgtacagcgc cttgttgtgg
aacttgtgaa 11220 gtgttttccg gtgtccgatg cttacgtagg taatgccggc
agcttcaatc tgactgtata 11280 gatgagcctg tgacacaact aaagctagtc
agagttgaca tcaaagtagg tgcgggtaca 11340 ctggttcttg gtccatgcaa
aaaggatgtc ctacagcaac accaaccctt ttcttttctt 11400 ctactctgtg
atggggtgaa agcatttgtg cgactgtgtg gactatgaat aacaagcagg 11460
caaaaaaaca agtatagcag tgtaactaga gttcagttac ctcgtttgct tcatcgagtg
11520 cgcttgtcga ctcgtccagc aggaccaaag tgggtgtagc aagtaatagc
cgggcgaacg 11580 cgagacgctg ctgctccccc agcgaaagaa cactggccca
gtcatgggta gagtccaagc 11640 cgttgaaacg aggcaatatg taacccagct
tgacgtcctc aaggacttcg atcagttcag 11700 ctgtcgaggg cacctcggat
ttggcaccga ccagatctga tgtggagact tcggacagaa 11760 aaggaagagg
atctgctgat cattttgaaa ggaaaaaaaa ggatttagga taaatgcata 11820
tgcatgtttc aagaaaacgc tgccacgagc actttcacac gtacacttgg tagttttttt
11880 cccctgaaca cactccaaac agatttaagt agttttgttc gacatcgtac
aactaacgag 11940 aagggctacc tgagttttta tcatcattat ctggtgagtg
ttgaacctct tcagtccacg 12000 taggatagag caactgttga cgaagtgtcc
ccagaaccat atacggcctt tgtgggacga 12060 agaatatgcc attatctctt
ctttgcttgg aactttgcaa cgattcttct tcgcccttta 12120 aattcatgct
ggatggttta tcagaacttg gatttggttt ttgaagctgc gtagaacctc 12180
tcacgtggta tatgatctcc ccagttccac tagtccagag gccggccaaa gcacgtaaca
12240 gcgacgtctt cccacttcca ctaggtccca tcacctaaag tgaaaaaaaa
agcatagcac 12300 ctcagcgatg agtcaagtgt acagagataa catttgtttt
gccgaatgcg caagtagtta 12360 aagaaatgga ataccagtag gtggtccttg
tcttttagtt ccatagtgag gccagtaata 12420 agaacatttc cacttcttgg
tgttaacaat gtcaagttac gaatttccag aaccatgcat 12480 ggatcagact
gtgttagtga tccattagaa ctaggaacac aaggaccgct gcttctgaaa 12540
acaatgttga tcccatcaat gctatcacgt tgagatgata gagaggactc gtttccatcc
12600 aatagatcat caaactcacc tatattacaa agaatttaca gttaaccacg
ttaatagagg 12660 gggtatagag gcaggaaggg aggaagggag ggaggggaga
agtcacctag acgatcgata 12720 actgctgaga atgcactaat tgactgaaat
tggaaaacaa tgagagagaa atcactaaga 12780 atatgattga aagcagacac
cgattggttt attactccaa actcaatttt ccctgagaag 12840 tacattggag
caacaactgc agctggcaga atctgaatca aatatcgata accattggtg 12900
aaaaactcca gattccgaga agctatcagc aattcctgat acataataaa ataaaatata
12960 tgaactggcc aacctggaaa aatatttaac agtaggaatt tatggctaaa
tatgaagata 13020 cagagcagct gcattgtgat aagtcagtag tagagcacat
aaagagccag ttactcttac 13080 agtaaaaaga actcaaccaa cctacaaaga
attgtggcgt gtaaataaga ataaaatagg 13140 cacccaaatt tgagttgaag
aggactcgaa tctgggtggt agggatgtag acccacacct 13200 ccccaggctc
agttcctcac ctgccacagt atcaccatta ttagcagttt gttggagtat 13260
atcagaatca acaacttgta tttgattatt acaaaagaaa atttattgcc caagaggtat
13320 gatttacatg attatcagat cttagtcgca gagaaaacca gggtatagaa
atacaagtga 13380 tctgctttac taatggcaaa 13400 64 1840 DNA Zea mays
64 ccacgcgtcc ggtactgtga gagtaaggct aaagtcgccg gataatataa
gaccagcaat 60 aacaagctag tttgccctcg ttctccaaca aaatgtctga
tgtggagccg ctggttctgg 120 ctcatgtcat acgagatgtg ttggattcat
ttgcaccaag tatcgggctc agaataacct 180 acaacagcag gttacttcta
tcaggtgttg agctgaaacc atccgcggtt gtgaataagc 240 caagagttga
tgttgggggc accgacctca gggtgttcta cacattggta agcgaataaa 300
atacgatgtg tgcttgtgtt ctgtcatgtg tggagccatg gcagcatcca ctaacaatta
360 acctcttata
tattgtcaat gtgactacac aacacctgaa aatttaaaca gctcaatatc 420
tcttgtgctc tagctaactt attggttctt catttacata aggtattagt ggatccagat
480 gccccaagcc caagcaatcc atcactgagg gagtatctgc actggtaagt
ttgttcatta 540 ttagttcttt attagtcaaa tggcaggttt gtttgaaatg
tatttagctt tcccagtgaa 600 ctaaatagct ttgagtgtgt tgtcctagca
cgacacatgc acatgttata tatatatata 660 tgttctcacc ttttccttgt
tttaatgtat catagacatt tcttccttgt tctcaccttt 720 ttagacattt
cccagtgaat ctaaatttgt gctgcatatg taggatggtg atagacattc 780
ctggaacaac tggagccagc tttggtatga tcccatgaaa tcatattttc cgtctggatt
840 ccttcgcttt tagcatatat catctatgat cgaaaaagat cctcggctgt
gctaactgac 900 tcacaatcaa caaaagtgat ctatcacctt ttagcggttc
caatgaccct aactcagaaa 960 aacgtttttt ttagaagtca tacatatata
tagagagaaa aatctagatt cataccaaac 1020 aaggctaaag tttttcttag
cattgtgaac agaattggag ttcatatata attaaacata 1080 aatgctaatg
ctaatgggag atgtttggtt cgaggaatca cttcatccaa aatgagatgt 1140
tacatcatag gtccgatcca tttctcaaat ttggtgtgat aacctcattc cttatatttg
1200 tactaattaa atatgagaaa tgaggtgatg atagatcaac tcattccatt
tcacaaacca 1260 aacaaaaaaa ataaaaagtg gaagatgatg aactagcttg
ttccttaaat caaacaccct 1320 aaacaaaaaa agagaagtgg aagatgatag
actagctcgt tctttaaaca aacacactaa 1380 atatatacta gaccatctct
tgctgcaggt caggagctca tgttttacga gaggccagag 1440 ccgaggtccg
gcatacaccg catggtgttc gtgctgttcc ggcagctcgg cagggggacg 1500
gtgtttgcac cagacatgcg gcacaacttc aactgcaaga gcttcgcccg tcagtaccac
1560 ctggacgtcg tggctgccac gtatttcaac tgccaaaggg aggcaggatc
cgggggcaga 1620 aggttcaggc cggagagctc gtaaggaatg aagcatgcac
agaagaagac tgcagcgctt 1680 tcgcatgcat atgatctatc gtcgtcctgc
ggaatatata tatagtaacc gttgttatat 1740 ggaataatgt gcatgaaatt
ggtatcagat gcaccgaccc gtacgtacgt aattaatgtt 1800 tgttattaca
cgcagacata taatatacat actcattcac 1840 65 7160 DNA Zea mays 65
ttcgatatca agcttatgaa attatctaca actcctttta ccaacctaaa gtttaattct
60 gttgataaaa ttgcctaaaa cttaagagaa cagattatgc atagaattat
gagactaaaa 120 aactgctata atcaaactgc aataactttc tgatctgatg
tctgattgag ggcccgttca 180 tttctactgg aagagaccag gattagttcc
aaatctccaa aacttatagt aattattgaa 240 ggaatccaag ccagatttat
tccacccctt cattccatgc gaaatgaata ggccctgagg 300 tgttcttcgc
ggccacagaa agatctataa attgtctata actcatagat agactttttg 360
tttttttgag gccactaaga ccacaaaaaa ctgccaagaa cagaaattgt caaatctgtc
420 attctgtaga aactaaattc gaggtcaaaa cctaacccca catctaatcc
aacctctaat 480 cctttaatcc ccatgatcga caacctccaa aagcacataa
ttatagggag gaacaaggat 540 cccatcctta cctagggttt ccccaagaaa
atctacaaga agagatgggg tagaacatct 600 ccttgatcct ctgttcctct
tcaattcaac gtgctcctcc tcttctcgat ccttgctaca 660 taggggaaga
tcaagggagg aggaagcaat ggagggcggc tgcaatcgga ggagaggggc 720
ggccagccaa gggggaaaag ggaggggcgg ccaagccaag ggagaaaaag ggggggtggc
780 ggccaagaga gggaaaaagg gaaaaggggt ggccacctag ggttttgtgg
gagaaagaaa 840 agacgcctca tatgaattat gactttgcat ccaacggccc
acaacccttt ccctcatcca 900 cgcaccgaaa atcaactttt gggcaacatt
tttttgggat attacacaat tctggcagat 960 gcttcgggat ttctgaatag
gtctcctaga cctcggtcag tggtgaccct tactctgtgc 1020 gcctctaagt
agtgccttag tttgcgcgaa gccatgatga ccgagtaggc tatcttctct 1080
agctctgtca tactgcactt ggaactggtg agtacctcgg aaacagaata tatcgggcat
1140 tgttgggtct taccgtgttt gcacctttct tgtaccagag ttgcgctgac
cacgctgggt 1200 gacgttgtga tgtgaggtag cgcggggtcc gggctagtga
gtgtcgctag atcagattta 1260 atattgtttt aaggactcaa aggctgcagt
ctcctctggg ccctaggcaa agtccttggc 1320 tccgcgcagc gttttcagga
aaatggaagg ttttgctcga tggaagtagc accctaaaat 1380 tctttttagg
gaaactactg taatatttaa ataaatgggt ttcctaatat gggattgact 1440
aaaaaggtta tcatcacatg aattaaattg atattttgtg ggactatata gcagggataa
1500 ggagtcatat tgcatagtga gggacttgat agactgattt cacaaaattg
ctcttggtgt 1560 caggatcacc tagatggttt gctggtgatt ggagtgtggt
gatcacctag agagattgtg 1620 gatgactcgg ctcgggatgt gtatatgatt
gtgagctatt gaccatatgc ccgagcagca 1680 aaaaaccacc tcgtagagag
cacttggtcc ttgcacggac taaggaggaa tgacacccat 1740 gtgcaggtta
tccaacaagg attaatgggg agtgttaact ctctaagacc tcgcgaaaaa 1800
tcttgagtct tctaaaccct tgttttactt ttcgcaaata aatttgtgca atttacttta
1860 ataatttaca ttcttataat tgttacaata gtatagggtt agaatttggg
cgcaaaatct 1920 tttgtataca cattaggcac tcagtttaag tttggaatgc
aattacaata ttattatcga 1980 cagacatttt agagaaattc aattcacccc
atcttaggta tcatgatcct ttcacaagcc 2040 attattgcta atgtggaggc
cataataggg cgacaagatt tggatgccaa ttgagaggct 2100 ggtgatggcc
aatttgaggc tataatggag ttccgatgaa cttgaccatg tccttggcaa 2160
acaccatgct tgactaatga ggatggttta ttttccatac tattgatcct tgattcatag
2220 ttgttcggtg gtggtgggtg tgcttgtttg aggagctcat cgctcgtgtc
ctatcgacca 2280 tcacattgat ctaatgctct ccactccact cgtaatcaca
gtcaagaaag agcacataga 2340 catgttcgtc gaatgatgga tcttggtagc
tactattaca agggcggtgg actggtggtg 2400 tggttagcca ccaaggtagg
gagtgttcta gtatagtgcg aagatgaaag taaaagggag 2460 gcattaggga
gacaatatga ttattgtggt ggtacgacct tgtgatcgat gaccataagg 2520
caccaactcc tccaaagcac gtgagtcttg actatttaag gtttctttat ttaatcaact
2580 ttgctctttc taggttgtgt gggctgacca actaattcat ttgggcagtg
ggccaactat 2640 agtggtcttg ggctactgga atctaggttt gatgtggctc
gtgagctggc tcattggcga 2700 ttcaagctaa gatgttacct caactcgtta
cgaaataaga tcaagccaaa ccattaaggg 2760 tccatttgat tggagagcca
catgagcaga gcagttccat tccatatttg aagagcatat 2820 atagtggctc
cattccaaat tatgagactg gagtggaatc attatatttt ttgtttgggt 2880
ccatatagca aacagaggga gaagtgaaac gatgagattc caccaatttt tttagagcga
2940 gatcatcccg aatctaatgg gaatattctc gatttagagt ggctccaccc
caccctctct 3000 attcgttccc caaccaaaca cataattgag cggctccatc
gcacctcgct ctccaaccaa 3060 acagaccgta acaaatcgaa ccgagcgagt
tatcgagttt tttatccaac ccttattagt 3120 accatgatct taacctaata
caagtgtgtg tcgtgtgtaa gttataccct ttatttatgg 3180 caagagatct
gacatggttg atgcttgcgt tgccatcttt catcctttca gaccacacac 3240
gggggcaggt ggggctcgag cctcctatcg ctcccgagcc aatagtgcct ccatccccat
3300 atagcttccc gtaaaattac atcctatatt tctcattata tattttatga
ttttcatcgt 3360 tcatcgatgt ttcaatatat aactacttta aactatgata
tttgtatgtt gtgagcggtt 3420 tagtgagatt atgtctctgt tgatcatagt
tcagcctctc ctaaaaattt tcctggctcc 3480 gtcttcgttg agcgcgcatc
ctttgcatcg ccagcgtacc gtgcagcaaa caaaagggcg 3540 gcgaagctag
ctttagttta tgcggcagat ggatcggaaa tcggataggg tgaccccagc 3600
acagcaccag cagatagcgg caatgaagaa taataccagc aggcggacat tattgtagca
3660 gtatatgcac aagaaacacc ctacacatac atatgctgcc cggccgggca
aatcagtgtt 3720 ttgcacaggc actgtgaaaa aaaaacacta aaaaaggtga
ccatgaaact tgcgcctggt 3780 tggcagacat actctcgaca gattagttgg
ggctagcgct acggaactga tcgtcgagaa 3840 gctagcatgc agcagctgcg
cgcttaagat tcagattgag atggacagat agacgtgtac 3900 gtacgtgcgg
tctgcaggag ctagctgagg agaagctagc tagctagcta gctgctatgg 3960
tagccactgc ttttgggccg cgcgggggtc aaaagcgcgc gcgcgatgcg gatgccccgg
4020 ccccgcgcgc atccagcata ctagttctgc atataccgag tgtgtatagc
tagtgtcatc 4080 aggtccacac aagaagaatt ggagatagag aaaatgactg
gagattctat gggggacatg 4140 tgcagatgtg ctactagcag actcagacag
cagcagcagc agcaacaaaa actattagga 4200 acagcacagc agctagccac
acagtagtaa accttaactc atatcagcca atagcatgca 4260 gaagcagcaa
gttggtgacg gtaccatgca tcatatatac tcctgatgag cttgacgtac 4320
ccgccaccgg ccactatata aactggccat cgatcatcag ctagctagcc agcagcaggt
4380 agtaccttgg ccaaaacgac ttagctatca agctcgaccg acgaccgaag
ctaagctacc 4440 aagctactag tagtcttctt ggtcacgttg ttgattgcag
cggtcgagca cacacaagct 4500 agctagctag ccgctagagc tagggtcgtc
ggatagatcg acatggccgg cagggacagg 4560 gagccgctgg tggttggtag
ggtggtcggc gacgtgctgg accccttcgt ccggaccacc 4620 aacctcaggg
tcagctacgg ggccaggacc gtgtccaacg gctgcgagct caagccgtcc 4680
atggtggtgc accagccaag ggtcgaggtc gggggacctg acatgaggac cttctacacc
4740 ctcgtacgtg tatatatata tatatatata cacgtcgtcg ttttacttct
cttcattggc 4800 tagctactta gctagctagc tagcttataa tgatagcccg
ttgatccatc gagatatgat 4860 cgtacgtgat gccatgcatg cttcgttctt
caggtgatgg tggacccaga tgctccgagc 4920 ccaagcgacc cgaaccttag
ggagtaccta cactggtaaa atctatatac gtactcgtcc 4980 tgcacacact
cgatcgatcc cttcactatc tatatatatg tatacataca tatgttatat 5040
attacaagta aaatctcata ctccctccct ccgttcttaa atatatgacg ttgtcgttga
5100 tttttttttc aaaaaatttt tgaccactca ttatatttaa aataaatcat
gagttattat 5160 ttgttttgct gtgatttgtt ttatcactta cgcaccgttt
cgatccttgg aatgcatttt 5220 aataatagta atttggcttt atgcaaaata
tatttgtata ttattattag caagatgtcg 5280 gatatattta tgtgctacat
ttttaatata aaggagtgag tcgaagagcg tcctataatt 5340 tggagagtag
aaacaaattc tactgacaca taaaataatt tctcatcctc caccaaatga 5400
atttgagata gatttatatc tgaactttag aaacaggtgg aatgttaaat ttgaagctaa
5460 atatgttact ttattaaata aattttaatt tctctaaaat gaatggatcc
aaacggttcg 5520 ttaagttaat tacttaaaat tttacatttt taaatacata
tttttaatta gacgagtggt 5580 caattttttt gaaaaaagtc aacaacctcg
tatatattta agaatggaca tcgtatgccc 5640 gggttggttg ggcatgtcct
ctctgatcta ccgtcttctt tcatgcaggc tggtgacgga 5700 tattccggga
actactgggg cagcatttgg tatatatctg tcactccgtc cggtcataaa 5760
ctgtctgttt gttgtctgtc tatacaactg tgtccagaca aactttttac tacacgtacg
5820 tggccggatt tcctatatat atattgtgca tgcagatcaa acgcatatat
acggcgtcac 5880 acttgtcgat cggccaccgg ttatatggag acgtacaaaa
gaattctatc tatagatata 5940 tatgtatatg ataattaatt aagctacaag
ctactataga tatatatgat aatttattat 6000 gttcttgata gccaagttaa
taaaaccata atgacaaaat aacaagaaat aaaaaaatca 6060 ggcgtaggaa
ttataaatag atatgcatgg tgctgtatta tatgtatgtg atgatgatga 6120
tacaaaaaaa ataaactact actggtgtgt ggcacccgtg ctgcagggca agaggtgatc
6180 tgctacgaga gccctcggcc gaccatgggg atccaccgct tcgtgctggt
gctgttccag 6240 cagctggggc ggcagacggt gtacgccccg ggctggcgcc
agaacttcaa caccagggac 6300 ttcgccgagc tctacaacct gggcccgccc
gtcgccgccg tctacttcaa ctgccagcgt 6360 gaggccggct ctgggggcag
gaggatgtac tcgtgatcgg atgcatggtt acataccatg 6420 cacacactac
tcactccatc gtctccatgc atgtagacgg acgatggtgc ctgcatcgat 6480
cgtcaactac tcaacaatta cgaactagaa atacacgcgt atatatacat atataaatat
6540 gcatatatac cggtactgta catgtcgccg tacacgcgca ggtggctgct
agctgcttgc 6600 tataccggcc ggtactgagc aggcagcatg cgctatatac
ttgcttggcg acgacgtgca 6660 gtgtgtgtat acaataatga gcggccggct
agcagggcga cgagccgtgg ctttataaag 6720 caatacatat atactgcagg
gatgtgtgtg tgcagtgcat gccaaggtac ggtaatcgta 6780 ttattgtgca
catacacata tgtatacgta catatgcgta aatatgaatc tgtacgtata 6840
catatgcatg ctggttaatt aaggcgctat cttgtacgta cgtgcttggt gtaataataa
6900 tataagtcct cagtggagaa tatatataat aataaattaa gaactatagg
cttttgtgtg 6960 gacaaaccag cagggttggt atatacatta atttcatgca
tgattttctc cttaatatat 7020 acacaaatta tcgtcataag ggtctcctct
gtttttgatc gtgcaagcgt gctttgaact 7080 tgaacgtacg tctacctgcc
ggggcacgcg gaataatgat acagtggcgt gtagctctca 7140 ccagtacaca
gaagcttgat 7160 66 4711 DNA Zea mays unsure (2376)...(2376) n = A,
C, G or T 66 taagcttctg caaagtaact gtaatgcaga ccatacatgt agatagtgga
gaatgattcc 60 caataataca agatttttac tgtcccactt tcctagagat
gtgttatctg ttatgttttc 120 ttgtggactc cacagtgacc tgaaatactt
catttcagtc atgaaagaac aattccgtgc 180 atacgtagtc atcaaagagg
ggaaaaatgc tttgtcccaa gaaattcaac aaagaaacat 240 gatgcattgc
ttgcatagat tttatctgta actgttacag aagtatgata tcctttgttg 300
gctttcttgg aaaaagtggt acaaccttcc tgttgccatt gcagctgcat ccacaccata
360 cccaactaaa agacacacat gacacatgcc tcacatcctc tttttatatc
ctacactcag 420 cagcagcctc ataaccgcta taaatagagg cttgtcctct
accaatgccc aataagcagc 480 tcacagtatt cttgagtgca ttcgcttgct
ccattcagtc agagcatttc ttgtgcaaaa 540 ttcaaatacc tgtcacacca
accatgtcta ggtctgtgga gcctctcata gtcgggcggg 600 tgattggaga
agttctcgac tcctttaacc catgtgtcaa gatgatagta acctacaact 660
caaacaaact tgtattcaat ggccatgaga tctacccatc agcaattgta tctaaaccta
720 gggtagaggt tcaagggggt gatttgcggt ctttcttcac attggttagc
aatcaaatgt 780 tccatctttg acaaatattc tacaaatact actacttctc
tgatggccat gctcatcaaa 840 tttcaggtta tgacagaccc agatgttcca
ggaccaagtg atccatatct aagggagcac 900 cttcattggt aatattcagc
atttcttacc ttagtaggga tgttaaatag catatgttgc 960 tcaggaagat
agaaccatat cttgcataca aaattgcatg ctttttagca cttaaatggg 1020
agtagtgtga ctaaatggca tccttcacat ttgcaggatc gtgactgata tacctgggac
1080 aacagatgcc tcctttggta ggttctgctg aatcaggctt agtctattcc
atgttttagt 1140 tgttctttga attaataaca attcatctga caattgcagg
gcgagaggtc ataagctatg 1200 agagcccaag acctaacatc ggtatccaca
ggttcatttt tgtgctcttc aagcagaagg 1260 gtaggcaaac tgtaaccgtg
ccatccttca gagatcattt caacacccgg cagtttgctg 1320 aggaaaatga
ccttggcctc ccagtagctg ctgtctactt caatgcacag agagaaactg 1380
cagctaggag acgttgaaaa ttccagctct tattgtccac ctgatgataa taaaggcctt
1440 ctgatcttct ttctaggaag ccaatgaact tattctacat taaattctcc
tgagccctac 1500 cgtataaata aaccagatgc gttttgctga ttgtattagt
attagaatgc tttgtacgtg 1560 gcaagaatga gaattacaaa tggtcaatgc
ttgtggtaaa atttgatgtg taagacatct 1620 atcactgaaa gtcagaaacc
aggcttagtg gacacccctc acagggggaa aatttcatct 1680 tctgtttgca
gcttcgcact ttgtgcactt tttctaacat gcaaactcct ggaaacaaaa 1740
ttcgttggac attttgcacc aacttataga actataaaca ccctaactgc attcgactac
1800 tacattgtta agcaaggcat gtatagaata tggagatgtc taatttacca
acaggaatat 1860 aggattgccc actggaagca tgaaaagtat ggagacatca
atttttgtgc ccctattttt 1920 ccttcagctt gtccttaagc caaatgacca
gaaatccata tcatgtatgc ttgaacatgc 1980 aagatagctg catatctttg
aattaaagga cagacctagt gttggctgct cccacatgtg 2040 tgggttctag
ggaaggagta accaaggcaa gccttatctc tgcatttttg cagagaggtt 2100
gaggaaaagg gaagagaatc ctgtacaaag caacctcctt accagccaga gggtctgagg
2160 atgtcacgag ctaacactaa aaaatcacct ggacacctgg catacaccaa
gaaatgatcc 2220 aaccagaata caaaagactc acacctgacc ctctacatga
ccttctatgc aaccaaatat 2280 gaatgcaaac tgaatgctct ctttcccatt
ggcttctgta tgtgtttatt ggctacatac 2340 aaacaaacta taagatgtaa
caatgcaatg tgtacnattg tatcagttta cctctgttga 2400 tgaagactgg
ggagtggaat ccccattaat acttcatttc agtcatgaaa gaacaattcc 2460
gtgcatacgt agtcatcaaa gaggggaaaa atgctttgtc ccaagaaatt caacaaagaa
2520 acatgatgca ttgcttgcat agattttatc tgtaactgtt acagaagtat
gatatccttt 2580 gttggctttc ttggaaaaag tggtacaacc ttcctgttgc
cattgcagct gcatccacac 2640 catacccaac taaaagacac acatgacaca
tgcctcacat cctcttttta tatcctacac 2700 tcagcagcag cctcataacc
gctataaata gaggcttgtc ctctaccaat gcccaataag 2760 cagctcacag
tattcttgag tgcattcgct tgctccattc agtcagagca tttcttgtgc 2820
aaaattcaaa tacctgtcac accaaccatg tctaggtctg tggagcctct catagtcggg
2880 cgggtgattg gagaagttct cgactccttt aacccatgtg tcaagatgat
agtaacctac 2940 aactcaaaca aacttgtatt caatggccat gagatctacc
catcagcaat tgtatctaaa 3000 cctagggtag aggttcaagg gggtgatttg
cggtctttct tcacattggt tagcaatcaa 3060 atgttccatc tttgacaaat
attctacaaa tactactact tctctgatgg ccatgctcat 3120 caaatttcag
gttatgacag acccagatgt tccaggacca agtgatccat atctaaggga 3180
gcaccttcat tggtaatatt cagcatttct taccttagta gggatgttaa atagcatatg
3240 ttgctcagga agatagaacc atatcttgca tacaaaattg catgcttttt
agcacttaaa 3300 tgggagtagt gtgactaaat ggcatccttc acatttgcag
gatcgtgact gatatacctg 3360 ggacaacaga tgcctccttt ggtaggttct
gctgaatcag gcttagtcta ttccatgttt 3420 tagttgttct ttgaattaat
aacaattcat ctgacaattg cagggcgaga ggtcataagc 3480 tatgagagcc
caagacctaa catcggtatc cacaggttca tttttgtgct cttcaagcag 3540
aagggtaggc aaactgtaac cgtgccatcc ttcagagatc atttcaacac ccggcagttt
3600 gctgaggaaa atgaccttgg cctcccagta gctgctgtct acttcaatgc
acagagagaa 3660 actgcagcta ggagacgttg aaaattccag ctcttattgt
ccacctgatg ataataaagg 3720 ccttctgatc ttctttctag gaagccaatg
aacttattct acattaaatt ctcctgagcc 3780 ctaccgtata aataaaccag
atgcgttttg ctgattgtat tagtattaga atgctttgta 3840 cgtggcaaga
atgagaatta caaatggtca atgcttgtgg taaaatttga tgtgtaagac 3900
atctatcact gaaagtcaga aaccaggctt agtggacacc cctcacaggg ggaaaatttc
3960 atcttctgtt tgcagcttcg cactttgtgc actttttcta acatgcaaac
tcctggaaac 4020 aaaattcgtt ggacattttg caccaactta tagaactata
aacaccctaa ctgcattcga 4080 ctactacatt gttaagcaag gcatgtatag
aatatggaga tgtctaattt accaacagga 4140 atataggatt gcccactgga
agcatgaaaa gtatggagac atcaattttt gtgcccctat 4200 ttttccttca
gcttgtcctt aagccaaatg accagaaatc catatcatgt atgcttgaac 4260
atgcaagata gctgcatatc tttgaattaa aggacagacc tagtgttggc tgctcccaca
4320 tgtgtgggtt ctagggaagg agtaaccaag gcaagcctta tctctgcatt
tttgcagaga 4380 ggttgaggaa aagggaagag aatcctgtac aaagcaacct
ccttaccagc cagagggtct 4440 gaggatgtca cgagctaaca ctaaaaaatc
acctggacac ctggcataca ccaagaaatg 4500 atccaaccag aatacaaaag
actcacacct gaccctctac atgaccttct atgcaaccaa 4560 atatgaatgc
aaactgaatg ctctctttcc cattggcttc tgtatgtgtt tattggctac 4620
atacaaacaa actataagat gtaacaatgc aatgtgtacn attgtatcag tttacctctg
4680 ttgatgaaga ctggggagtg gaatccccat t 4711 67 5966 DNA Zea mays
67 acttgcgtga caggaataat caccagaagt agcaagtccg ctcaagtgca
atgctgttta 60 gggcatgttc ttttgcaccc catagctcag cagagtggtg
cttcagcgtg cctcgctttt 120 ttacctcaag agaaaaagat gctagctcct
gggtgcagtt gtgctccagc tttgagaata 180 cccaagctaa gctcactctc
cagctgcaaa aagaaaaact aaaatatgtg ttgaaacgca 240 aaataaaata
catgtaaaaa taaagggata atcaatcaat ggagagagaa ttcacattaa 300
cgttgtatgc ttctttatat aaagggacag gcaaagaata caagctaaag aactagcata
360 atcaagctgc catctttgac accgttccta cattcactac cacacattat
gtctctaaga 420 atttctgaag aaaatgaaat aattaatgca cgtttagtga
agattctcta tggtacacta 480 ctaataatgt cttcaattaa ctcctcagtg
ctaaatatat acaaattcat actatcattt 540 ttgtacacaa aacataaatt
gtgatgtgat ttgtttcagt aaatcacaat caatacgtcg 600 attagataca
aaaattgtca cctcacttct ggcttctgcg ctcacatagc atgaacaaaa 660
aaaaaacctg gcacactaat aataatcaaa cctaagacta gatcatcacc catgttccag
720 agaaaaatac tgtgaccgcc tctataggga ataaaataat ccatgtggga
agccagtgca 780 aaaccgaatg tactaggcga aaaaaaaata ctaaaccatt
tctgcattac caagtcaacc 840 aacaagtagc tgcagctccg aacggaagtc
aagattttac atgcatccct tataaaccac 900 gaagctaact cccaaaacac
ctgagaaatg ctacgatctt ttatatttca atgtgctcgc 960 ttggcttgat
cgatgtctga aggaagtacg ttatcagtgg atctctgtcg tcgttcaccg 1020
gagaaacttt aatgatatgg atgtaacaaa gagctttttc ctattttgcc aaatgtggat
1080 tctaggcact taactatttc ggcctgaacg cccatgcata tataataata
taaatattgt 1140 gaagggaata aatatctttg agatagatgt ggaaattgga
atatatcgga taataaggaa 1200 cttgtatatc tgctgcaagt gcccctattc
tatatattgt gaagaaacaa tctgacatgt 1260 taagctatat atgatagtag
ccaccgtacg agccgataga gacgctcaaa cctgaacttt 1320 ttaagagctg
taatatatga tgatgatcgg tccatcacac tcagctcgat cgatcggaat 1380
atatagctgg ccttgacgtc tataaataga ggccaaacgc cctggtctct tggcaacaca
1440 cacaagcaca cgcacatagg gaacagaagc tactagctcc agcacaaaac
acctactgct 1500 tcaactgtac cgttagacat
gtcaagggtg ttggagcctc tcattgtggg gaaagtgatt 1560 ggtgaggtcc
tggaccattt caaccccacg gtgaagatgg tggtcaccta caactccaac 1620
aagcaggtgt tcaacgggca cgagttcttc ccttcggcag tggccgccaa gccgcgtgtt
1680 gaggtccaag ggggcgacct caggtccttc ttcacgttgg tgatgaccga
ccccgatgtt 1740 cctggaccta gtgatccata cttgagggag caccttcact
ggtaataatt tttatcatca 1800 atgcgagaat tcataataat atagtctata
tatatcttgc tagataaagt aatcgtataa 1860 gtaaatactg tactaaatag
gggggtatca cctatatatc tactatagtg tagcgcttga 1920 ctcggccatg
catgattagc tagaatttat tacgtatata tttttacaca gctgggttat 1980
gcatgcatgg tcttataaat gcatgttcat atatattatc tcaaggcatt tatatgtata
2040 tatagctaat caggggcgtg tttttttgtt tgctggacca cacactggca
ctggcaggat 2100 tgtcactgat attcctggga ctaccgatgc ttcttttggt
aagtcttttt tcttgtacat 2160 gtcgctttag ttcttgcttg ttttgaagcc
gcagaagagg ggaagagtgg gggcgacgaa 2220 tggataaaaa gggtttttat
gcatgctgta caaatcagtt cgttctttgt cactccatct 2280 aattaatgct
tgtgcttgta tgcatgcagg gaaagaggtg gtgagctacg agatcccaaa 2340
gccaaacatt ggcatccaca ggttcatctt tgtgctgttc cggcagaaga gccggcaagc
2400 ggtgaacccg ccgtcgtcga aggaccgctt cagcacccgc cagttcgctg
aggagaacga 2460 cctcggcctc cccgtcgccg ccgtctactt caacgcgcag
cgcgagaccg ccgcccgccg 2520 acgctaaccg tacggctcaa cgtacgaaag
aagaccatcc tacgacgctt gcaattagct 2580 gggcaagcaa agcttttttt
ttcatcctga gtcgatcttt acgtatgtat gtttgtttaa 2640 ataaaaaggt
agctaatcag ctgcttggct gtgaccccac gagctagcag ctacaaccta 2700
ctggtacatg ctgcacattt tagctgattt atgaaggtga caatatgatt ggtagggttg
2760 caatgttgac tgggcatagt gtaacaactt aagcaatggc catgggcgag
tacgtgtcga 2820 gtggtgaagt tgaagggaag tttatattaa aagcaaggcc
atgtcttgta ttaccttgcc 2880 tattattctg ccatatatac atactggtgt
ggttctgtga agattgcttt gtttgtttga 2940 tgatactgtc actacacagc
tggctgctta ttcttgtcgt gccatcgctt actttttcca 3000 attctcttta
tactatccca cttttcaatg acaagacgtc catatataca tatcaagctg 3060
aaaacaagaa tgcagcagca tctatttcta ggccacctta gctttttcac gtcatttcag
3120 tcgttttata atacacgatc ttgcgatgct agtttctaag tgcatctgtg
catatatgct 3180 agatgggccg tctagccact aggatcacaa ttcacaagtg
catttatctg aataagttta 3240 tgaacgaata tgcctttgaa gaaatatatt
gattacctca gaagatatat cactcaatgt 3300 caggcaacta gccatttcag
ccgtttcaac tgttctcttg aacaggtgta tttatcttga 3360 ttaaattgaa
catgtaggtc tagagactgt atgaaaacca aatatacttt accaaatgtt 3420
aggagaagac acttggcaaa cgagagtttt gctgagcacc tgaggacatc actcagcaaa
3480 gacataactg tgtcgacata aatacataac gacggtcgtc ggttgttgac
ggcgctttaa 3540 cgagttgagt gttgatgttg gccgagagtt taactatcga
caaataagtc tcggcataat 3600 atccttacaa taccatatat aatatgcatg
gtctttggca tgcctcttat tatagctata 3660 tatgttaggt gtgtgttctt
ataccaaagc aagaaactga caggtgacaa gagaagacaa 3720 ctgttgcaac
acgctaagac aactgttgca acttatgtct ggccggatgg ttgtgttaca 3780
attacaacac gctaagacaa ctgttgcatg taaccttctt ttaattgggg gttagttata
3840 gcctaaccct aacacttcca tcggaatgta agtgttcatt taccacgtac
cctctccatt 3900 ccaaaatcta aatttacgct tcacccaata ggaagaatga
tataattaag cagcaaaggc 3960 aatcctaata gtgtatcacg ctagaatgat
attgacacct cagctgctgg atctattgca 4020 gcattaattc aaaatagaaa
gtgataataa tactgtccca aattaaaatt cgttttagct 4080 aatcaatgag
ctcatacaat atttgtatat gttttatatg tgtataggtt catccttatt 4140
tatttaaata gagactaaaa tgactaatat tttaggacga ccgaggtagt accggccaat
4200 gcaagtcaca tatgagtgga agtgtacgca tcaaattcca ggacaaatat
gcgtgttgtg 4260 gtaaaaccgt acctgagctt gatcatttcg ttgcctgtat
taaacagttc tagaaaattc 4320 agataaatac atccttatcc tcatatacta
atataaaata cgagtttcgt aaccctctcc 4380 acagtagtga gggtttttcg
cacgccaacg aaatataaaa aaatgcagct gtcagaattc 4440 aaattgtggc
ctacaagaga gcacctgccg atcatgacta taggttatat tggtttttgt 4500
tatataaaca aactatatat atatatatat atatatatat atatatataa atagtagtga
4560 tgtacgggta actaacgagt actcttgata cgtctggtcg gatgccaggc
tatcacatgc 4620 atatatagta cgacctatcg tcaggtaata cagatgcaga
gataatgcaa acatatatag 4680 catatctgct ccaaaaaaga aagacgatgt
gataaataca agtgaaaaaa tgaacaggca 4740 gctaggccac gctgaggcag
agctgggcaa cctatcacgg cacttgtctt cctgctgtgt 4800 gtggtaccat
accaagtaca gagcattgca gagtgcacag gcgctggggc caaggtggcc 4860
gcaagacaaa catggcgtgg agcgggacag ggcacggatt tgatagggta agttggtcct
4920 tgccacagag cctagctcca cgagaaaccc gctggcctgt cgacgtgtcg
atcctgcgat 4980 cgtctcggca gcccccacac ccccgtcttg ttgggtctcg
actctcgaca gggtggtggc 5040 aaatccatga attatctaca cctgtgagta
cgtacgaggt ggatgcgatc tgcctcctcc 5100 gggccctctg taatatttat
aaaggcattg ccaattgcag ctgcagacat cctggactcc 5160 gatgtgtgga
ccaaagaaag acgcgtagct agggtagagc tgcatgcgtc cagtctcctg 5220
ttatggccgc cggcccgcca agcacactgc acacgcgcac cttctagcta gctactccgc
5280 ttctgtctgg aatggctgga atatataata tgctatgcta ccgtggagcc
gagctctagc 5340 atcagatgtg cttgctgctc cggttaacac ttgtgtctgc
acgtacaaat gaatgccggc 5400 acaggacaca acagttggca gtacaagctg
catcgagatg aacacaatgg tgtgtacaag 5460 cccatgtgtc attgtacaaa
gttgccctgc agtgcgagta ccagaaccgg agcctttgtt 5520 ctcctacaac
aggccctaga acagcatgca gcacgtagat acgctcctgg cgcatggccc 5580
acgtccgatg tccattgcac cagcacaaca tgtgtcgtcc atcatccacg actcgtgccc
5640 tcaggggtcc agtccagcag gtgaacaatc aagacactct gcatcttgtc
aaagacaaca 5700 gcgtgcagtg cttgaatcct ctcatacatg cataccacct
cccagctttt gcccaaagat 5760 cgaattgtca aaggcggcaa cagcatgcca
gcttttgccc ggagaaagat gtaccgttgc 5820 caatgtagct ggatgttatg
tagtaccgga aaacaattaa gcaattttgg gtactgtacc 5880 atcccacagt
tatgtgctcc agtcacctac taaacacaat agtatatatt gctgctctag 5940
aggccacctg ttatattgcc atggct 5966
* * * * *