U.S. patent application number 14/639636 was filed with the patent office on 2015-06-25 for novel eto1 genes and use of same for reduced ethylene and improved stress tolerance in plants.
The applicant listed for this patent is Pioneer Hi-Bred International Inc.. Invention is credited to XIAOMING BAO, NICHOLAS J. Bate.
Application Number | 20150176018 14/639636 |
Document ID | / |
Family ID | 42990277 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150176018 |
Kind Code |
A1 |
Bate; NICHOLAS J. ; et
al. |
June 25, 2015 |
Novel ETO1 genes and use of same for reduced ethylene and improved
stress tolerance in plants
Abstract
The invention provides isolated ethylene over-producer 1 (ETO1)
nucleic acid molecules which are associated with ethylene
production in plants and their encoded proteins. The present
invention provides methods and compositions relating to altering
ethylene production and abiotic stress response in plants. The
invention further provides recombinant expression cassettes, host
cells, transgenic plants and antibody compositions.
Inventors: |
Bate; NICHOLAS J.; (RALEIGH,
NC) ; BAO; XIAOMING; (Beijing, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pioneer Hi-Bred International Inc. |
Johnston |
IA |
US |
|
|
Family ID: |
42990277 |
Appl. No.: |
14/639636 |
Filed: |
March 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14071206 |
Nov 4, 2013 |
9000262 |
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14639636 |
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12850717 |
Aug 5, 2010 |
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14071206 |
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61231379 |
Aug 5, 2009 |
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Current U.S.
Class: |
800/283 ;
435/320.1 |
Current CPC
Class: |
C12N 15/8249 20130101;
C07K 14/415 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415 |
Claims
1. A method for reducing ethylene biosynthesis in a plant,
comprising: (a) introducing into a plant cell a recombinant
expression cassette comprising a first polynucleotide operably
linked to a heterologous promoter, wherein said expression cassette
directs expression of said first polynucleotide, wherein the first
polynucleotide is selected from the group consisting of: i. a
polynucleotide comprising the full length nucleotide sequence of
SEQ ID NO: 1, 3, 5, or 7; ii. a polynucleotide having at least 95%
sequence identity to the full length of the sequence set forth in
SEQ ID NO: 1, 3, 5, or 7, wherein the polynucleotide encodes a
polypeptide having ethylene over-producer 1 (ETO1) activity and
wherein the polypeptide comprises the N-terminal domain provided as
SEQ ID NO: 11 and the C-terminal domain provided as SEQ ID NO: 12;
and iii. a polynucleotide encoding a polypeptide comprising the
full length amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or
8; and (b) culturing the plant cell under plant cell growing
conditions and regenerating a plant therefrom, wherein the level of
ethylene biosynthesis in said plant is reduced, relative to a
control.
2. The method of claim 1, wherein the heterologous promoter is a
tissue-preferred promoter.
3. The method of claim 1, wherein the plant cell is from a plant
selected from the group consisting of maize, soybean, sunflower,
sorghum, canola, wheat, alfalfa, cotton, rice, barley and
millet.
4. The method of claim 3, wherein the plant is maize.
5. The method of claim 1, wherein the heterologous promoter is a
stress-inducible promoter.
6. A transgenic plant produced by the method of claim 1.
7. The transgenic plant of claim 6, wherein the plant has decreased
ethylene production when compared to a control plant.
8. A recombinant expression cassette, comprising a polynucleotide
operably linked to a heterologous promoter, wherein said
polynucleotide is selected from the group consisting of: (a) a
polynucleotide comprising the full length nucleotide sequence of
SEQ ID NO: 1, 3, 5, or 7; (b) a polynucleotide having at least 95%
sequence identity to the full length of the sequence set forth in
SEQ ID NO: 1, 3, 5, or 7, wherein the polynucleotide encodes a
polypeptide having ETO1 activity; and (c) a polynucleotide encoding
a polypeptide comprising the full length amino acid sequence set
forth in SEQ ID NO: 2, 4, 6, or 8.
9. The recombinant expression cassette of claim 8, wherein the
heterologous promoter is a stress-inducible promoter.
10. The recombination expression cassette of claim 8, wherein the
heterologous promoter is a tissue-preferred promoter.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/850,717 filed Aug. 5, 2010 and claims the
benefit of U.S. Provisional Patent Application No. 61/231,379,
filed Aug. 5, 2009, both of which are hereby incorporated herein in
their entirety by reference.
TECHNICAL FIELD
[0002] The present invention relates generally to plant molecular
biology. More specifically, it relates to nucleic acids and methods
for modulating their expression in plants.
BACKGROUND OF THE INVENTION
[0003] Plant hormones have been intensively studied for decades for
their diverse and complex effects on plant life. Of the five main
hormones-auxins, ethylene, abscisic acid, cytokinins and
gibberellins--the molecular signaling and mode of action of
ethylene has been the most fully researched.
[0004] Ethylene (C.sub.2H.sub.4) is a gaseous plant hormone that
affects myriad developmental processes and fitness responses in
plants, such as germination, flower and leaf senescence, fruit
ripening, leaf abscission, root nodulation, programmed cell death
and responsiveness to stress and pathogen attack. Over the past
decade, genetic screens have identified more than a dozen genes
involved in the ethylene response in plants.
[0005] Ethylene and the ethylene response pathways govern diverse
processes in plants, and these effects are sometimes affected by
the action of other plant hormones, other physiological signals and
the environment, both biotic and abiotic. For example, it is known
that cytokinin can cause ethylene like effects through the action
of ethylene. In addition, abscisic acid can inhibit ethylene
production and signaling. Auxin and ethylene are also known to
cooperate in various physiological phenomena. Such physiological
activities of ethylene include, but are not limited to, promotion
of food ripening, abscission of leaves and fruit of dicotyledonous
species, flower senescence, stem extension of aquatic plants, gas
space (aerenchyma) development in roots, leaf epinastic curvatures,
stem and shoot swelling (in association with stunting), femaleness
in cucurbits, fruit growth in certain species, apical hook closure
in etiolated shoots, root hair formation, flowering in the
Bromeliaceae, diageotropism of etiolated shoots, and increased gene
expression (e.g., of polygalacturonase, cellulase, chitinases,
.beta.1,3-glucanases, etc.). Ethylene is released naturally by
ripening fruit and is also produced by most plant tissues, e.g., in
response to stress (e.g., drought, crowding, disease or pathogen
attack, temperature (cold or heat) stress, wounding, etc.) and in
maturing and senescing organs.
[0006] Ethylene is generated from methionine by a well-defined
pathway involving the conversion of S-adenosyl-L-methionine (SAM or
Ado Met) to the cyclic amino acid 1-aminocyclopropane-1-carboxylic
acid (ACC) which is facilitated by ACC synthase. ACC synthase is an
aminotransferase which catalyzes the rate limiting step in the
formation of ethylene by converting S-adenosylmethionine to
ACC.
[0007] Ethylene is then produced from the oxidation of ACC through
the action of ACC oxidase (also known as the ethylene forming
enzyme) with hydrogen cyanide as a secondary product that is
detoxified by .beta.-cyanoalanine synthase. Finally, ethylene can
be metabolized by oxidation to CO.sub.2 or to ethylene oxide and
ethylene glycol.
[0008] There is a continuing need for modulation of ethylene
production and its response pathways in plants for manipulating
plant development or stress responses. This invention relates to
novel ethylene over-producer 1 (ETO1) sequences and their use in
plants to inhibit ethylene production by removal of a critical
component on the ethylene synthesis pathway. The invention includes
novel polynucleotide sequences, expression constructs, vectors,
plant cells and resultant plants. These and other features of the
invention will become apparent upon review of the following
materials.
SUMMARY OF THE INVENTION
[0009] This invention involves the identification and
characterization of novel ETO1 genes from maize and soybean which
may be introduced into plants to modulate ethylene production and
improve stress tolerance in plants. ETO1 is a protein that
negatively regulates ACS activity and concomitant ethylene
production. ACS refers to ACC synthase, where ACC is
1-aminocyclopropane-1-carboxylic acid.
[0010] The invention comprises polynucleotides, related
polypeptides and all conservatively modified variants of the maize
and soybean ETO1 sequences presented herein.
[0011] The invention also includes methods to alter the genetic
composition of crop plants, especially maize and soybean, so that
such crops can be more tolerant to abiotic stress conditions and to
modulate other ethylene mediated responses. The utility of this
class of invention is then both yield enhancement and stress
tolerance.
[0012] Ethylene-mediated responses include but are not limited to
those involving: crowding tolerance, seed set and development,
growth in compacted soils, flooding tolerance, maturation and
senescence, drought tolerance and disease resistance. This
invention provides methods and compositions to effect various
alterations in the ethylene-mediated response in a plant that would
result in improved agronomic performance, particularly under
stress.
[0013] Therefore, in one aspect, the present invention relates to
an isolated nucleic acid molecule comprising an isolated
polynucleotide sequence encoding an ETO1 protein which will bind to
the C-terminus of ACS6 and target the molecule for degradation. One
embodiment of the invention is an isolated polynucleotide
comprising a nucleotide sequence selected from the group consisting
of: (a) the nucleotide sequence comprising SEQ ID NO: 1, 3, 5, 7 or
9; (b) the nucleotide sequence encoding an amino acid sequence
comprising SEQ ID NO: 2, 4, 6, 8 or 10; (c) a polynucleotide having
a specified sequence identity to a polynucleotide encoding a
polypeptide of the present invention; (d) a polynucleotide which is
complementary to the polynucleotide of (a) and (e) a polynucleotide
comprising a specified number of contiguous nucleotides from a
polynucleotide of (a) or (b). The isolated nucleic acid molecule
can be DNA.
[0014] Compositions of the invention include an isolated
polypeptide comprising an amino acid sequence selected from the
group consisting of: (a) SEQ ID NO: 2, 4, 6, 8 or 10 and (b) the
amino acid sequence comprising a specified sequence identity to SEQ
ID NO: 2, 4, 6, 8 or 10, wherein said polypeptide has ETO1
activity.
[0015] In another aspect, the present invention relates to a
recombinant expression cassette comprising a nucleic acid molecule
as described. Additionally, the present invention relates to a
vector containing the recombinant expression cassette. Further, the
vector containing the recombinant expression cassette can
facilitate the transcription and translation of the nucleic acid
molecule in a host cell. The present invention also relates to the
host cells able to express the polynucleotide of the present
invention. A number of host cells could be used, such as but not
limited to, microbial, mammalian, plant or insect. Preferably, the
host cells are non-human host cells.
[0016] In yet another embodiment, the present invention is directed
to a transgenic plant or plant cell, containing the nucleic acid
molecules of the present invention. Preferred plants containing the
polynucleotides of the present invention include but are not
limited to maize, soybean, sunflower, sorghum, canola, wheat,
alfalfa, cotton, rice, barley, tomato and millet. In another
embodiment, the transgenic plant is a maize plant or plant cell.
Another embodiment is the transgenic seeds from the transgenic
plant.
[0017] The plants of the invention can have altered ethylene
production/response as compared to a control plant. In some plants,
the altered ethylene production/response is directed to a
vegetative tissue, a reproductive tissue or a vegetative tissue and
a reproductive tissue. Plants of the invention can have at least
one of the following phenotypes including but not limited to:
differences in crowding tolerance, seed set and development, growth
in compacted soils, flooding tolerance, drought tolerance,
maturation and senescence and disease resistance, compared to non
transformed plants.
[0018] Methods for decreasing ethylene synthesis in a plant are
provided by introducing to the same an ETO1 protein, thereby
targeting ACS6 for degradation and removing it from the ethylene
synthesis pathway. The method can comprise introducing into the
plant an ETO1 polynucleotide of the invention.
[0019] In a further aspect, the present invention relates to a
polynucleotide amplified from a Zea mays or Glycine max nucleic
acid library using primers which selectively hybridize, under
stringent hybridization conditions, to loci within polynucleotides
of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0020] Units, prefixes and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. Numeric ranges recited within the specification are
inclusive of the numbers defining the range and include each
integer within the defined range. Amino acids may be referred to
herein by either their commonly known three letter symbols or by
the one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to
by their commonly accepted single-letter codes. Unless otherwise
provided for, software, electrical, and electronics terms as used
herein are as defined in The New IEEE Standard Dictionary of
Electrical and Electronics Terms (5th edition, 1993). The terms
defined below are more fully defined by reference to the
specification as a whole.
[0021] By "amplified" is meant the construction of multiple copies
of a nucleic acid sequence or multiple copies complementary to the
nucleic acid sequence using at least one of the nucleic acid
sequences as a template. Amplification systems include the
polymerase chain reaction (PCR) system, ligase chain reaction (LCR)
system, nucleic acid sequence based amplification (NASBA, Cangene,
Mississauga, Ontario), Q-Beta Replicase systems,
transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., Diagnostic Molecular
Microbiology: Principles and Applications, Persing, et al., Ed.,
American Society for Microbiology, Washington, D. C. (1993). The
product of amplification is termed an amplicon.
[0022] The term "antibody" includes reference to antigen binding
forms of antibodies (e.g., Faba, F (ab) 2). The term "antibody"
frequently refers to a polypeptide substantially encoded by an
immunoglobulin gene or immunoglobulin genes or fragments thereof
which specifically bind and recognize an analyte (antigen).
However, while various antibody fragments can be defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that such fragments may be synthesized de novo either
chemically or by utilizing recombinant DNA methodology. Thus, the
term antibody, as used herein, also includes antibody fragments
such as single chain FV, chimeric antibodies (i.e., comprising
constant and variable regions from different species), humanized
antibodies (i.e., comprising a complementarity determining region
(CDR) from a non-human source) and heteroconjugate antibodies
(e.g., bispecific antibodies).
[0023] The term "antigen" includes reference to a substance to
which an antibody can be generated and/or to which the antibody is
specifically immunoreactive. The specific immunoreactive sites
within the antigen are known as epitopes or antigenic determinants.
These epitopes can be a linear array of monomers in a polymeric
composition-such as amino acids in a protein- or consist of or
comprise a more complex secondary or tertiary structure. Those of
skill will recognize that all immunogens (i.e., substances capable
of eliciting an immune response) are antigens; however some
antigens, such as haptens, are not immunogens but may be made
immunogenic by coupling to a carrier molecule. An antibody
immunologically reactive with a particular antigen can be generated
in vivo or by recombinant methods such as selection of libraries of
recombinant antibodies in phage or similar vectors. See, e.g.,
Huse, et al., (1989) Science 246:1275-1281 and Ward, et al., (1989)
Nature 341:544-546 and Vaughan, et al., (1996) Nature Biotech.
14:309-314.
[0024] As used herein, "antisense orientation" includes reference
to a duplex polynucleotide sequence that is operably linked to a
promoter in an orientation where the antisense strand is
transcribed. The antisense strand is sufficiently complementary to
an endogenous transcription product such that translation of the
endogenous transcription product is often inhibited.
[0025] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refers to
those nucleic acids which encode identical or conservatively
modified variants of the amino acid sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations" and represent one species
of conservatively modified variation. Every nucleic acid sequence
herein that encodes a polypeptide also, by reference to the genetic
code, describes every possible silent variation of the nucleic
acid.
[0026] One of ordinary skill will recognize that each codon in a
nucleic acid (except AUG, which is ordinarily the only codon for
methionine and UGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, each silent variation of a nucleic acid
which encodes a polypeptide of the present invention is implicit in
each described polypeptide sequence and is within the scope of the
present invention.
[0027] As to amino acid sequences, one of skill will recognize that
individual substitution, deletion or addition to a nucleic acid,
peptide, polypeptide or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence results in a "conservatively modified variant"
where the alteration results in the substitution of an amino acid
with a chemically similar amino acid. Thus, any number of amino
acid residues selected from the group of integers consisting of
from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7
or 10 alterations can be made.
[0028] Conservatively modified variants typically provide similar
biological activity as the unmodified polypeptide sequence from
which they are derived. For example, substrate specificity, enzyme
activity, or ligand/receptor binding is generally at least 30%,
40%, 50%, 60%, 70%, 80% or 90% of the native protein for its native
substrate. Conservative substitution tables providing functionally
similar amino acids are well known in the art.
[0029] The following six groups each contain amino acids that are
conservative substitutions for one another: [0030] 1) Alanine (A),
Serine (S), Threonine (T); [0031] 2) Aspartic acid (D), Glutamic
acid (E); [0032] 3) Asparagine(N), Glutamine (Q); [0033] 4)
Arginine (R), Lysine (K); [0034] 5) Isoleucine(I), Leucine (L),
Methionine (M), Valine (V); and [0035] 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins
W. H. Freeman and Company.
[0036] By "encoding" or "encoded", with respect to a specified
nucleic acid molecule, is meant comprising the information for
translation into the specified protein. A nucleic acid molecule
encoding a protein may comprise intervening sequences (e.g.,
introns) within translated regions of the nucleic acid molecule, or
may lack such intervening non-translated sequences (e.g., as in
cDNA). The information by which a protein is encoded is specified
by the use of codons. Typically, the amino acid sequence is encoded
by the nucleic acid molecule using the "universal" genetic code.
However, variants of the universal code, such as are present in
some plant, animal and fungal mitochondria, the bacterium
Mycoplasma capricolum or the ciliate Macronucleus, may be used when
the nucleic acid is expressed therein. When the nucleic acid is
prepared or altered synthetically, advantage can be taken of known
codon preferences of the intended host where the nucleic acid is to
be expressed.
[0037] For example, although nucleic acid sequences of the present
invention may be expressed in both monocotyledonous and
dicotyledonous plant species, sequences can be modified to account
for the specific codon preferences and GC content preferences of
monocotyledons or dicotyledons as these preferences have been shown
to differ (Murray, et al., (1989) Nucl. Acids Res. 17:477-498).
Thus, the maize preferred codon for a particular amino acid may be
derived from known gene sequences from maize. Maize codon usage for
28 genes from maize plants is listed in Table 4 of Murray, et al.,
supra.
[0038] As used herein "full-length sequence" in reference to a
specified polynucleotide or its encoded protein means having the
entire amino acid sequence of, a native (nonsynthetic), endogenous,
biologically active form of the specified protein. Methods to
determine whether a sequence is full-length are well known in the
art including such exemplary techniques as northern or western
blots, primer extension, S 1 protection, and ribonuclease
protection. See, e.g., Plant Molecular Biology:A Laboratory Manual,
Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to known
full-length homologous (orthologous and/or paralogous) sequences
can also be used to identify full-length sequences of the present
invention. Additionally, consensus sequences typically present at
the 5' and 3' untranslated regions of mRNA aid in the
identification of a polynucleotide as full-length. For example, the
consensus sequence ANNNNAUGG, where the AUG codon therein
represents the N-terminal methionine, aids in determining whether
the polynucleotide has a complete 5' end. Consensus sequences at
the 3' end, such as polyadenylation sequences, aid in determining
whether the polynucleotide has a complete 3' end.
[0039] As used herein, "heterologous" in reference to a nucleic
acid is a nucleic acid that originates from a foreign species, or,
if from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous structural gene is from a species different from that
from which the structural gene was derived, or, if from the same
species, one or both are substantially modified from their original
form. A heterologous protein may originate from a foreign species
or, if from the same species, is substantially modified from its
original form by deliberate human intervention.
[0040] By "host cell" is meant a cell which contains a vector and
supports the replication and/or expression of the vector. Host
cells may be prokaryotic cells such as E. coli or eukaryotic cells
such as yeast, insect, amphibian or mammalian cells. Preferably,
host cells are monocotyledonous or dicotyledonous plant cells. A
particularly preferred monocotyledonous host cell is a maize host
cell.
[0041] The term "hybridization complex" includes reference to a
duplex nucleic acid structure formed by two single-stranded nucleic
acid sequences selectively hybridized with each other.
[0042] By "immunologically reactive conditions" or "immunoreactive
conditions" is meant conditions which allow an antibody, reactive
to a particular epitope, to bind to that epitope to a detectably
greater degree (e.g., at least 2-fold over background) than the
antibody binds to substantially any other epitopes in a reaction
mixture comprising the particular epitope. Immunologically reactive
conditions are dependent upon the format of the antibody binding
reaction and typically are those utilized in immunoassay protocols.
See Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York (1988), for a description of
immunoassay formats and conditions.
[0043] The term "introduced" in the context of inserting a nucleic
acid molecule into a cell, means "transfection" or "transformation"
or "transduction" and includes reference to the incorporation of a
nucleic acid molecule into a eukaryotic or prokaryotic cell where
the nucleic acid molecule may be incorporated into the genome of
the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA),
converted into an autonomous replicon or transiently expressed
(e.g., transfected mRNA).
[0044] The term "isolated" refers to material, such as a nucleic
acid molecule or a protein, which is: (1) substantially or
essentially free from components that normally accompany or
interact with it as found in its naturally occurring environment.
The isolated material optionally comprises material not found with
the material in its natural environment; or (2) if the material is
in its natural environment, the material has been synthetically
(non-naturally) altered by deliberate human intervention to a
composition and/or placed at a location in the cell (e.g., genome
or subcellular organelle) not native to a material found in that
environment. The alteration to yield the synthetic material can be
performed on the material within or removed from its natural state.
For example, a naturally occurring nucleic acid molecule becomes an
isolated nucleic acid molecule if it is altered, or if it is
transcribed from DNA which has been altered, by means of human
intervention performed within the cell from which it originates.
See, e.g., Compounds and Methods for Site Directed Mutagenesis in
Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo
Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al.,
PCT/US93/03868. Likewise, a naturally occurring nucleic acid
molecule (e.g., a promoter) becomes isolated if it is introduced by
nonnaturally occurring means to a locus of the genome not native to
that nucleic acid molecule. Nucleic acid molecules which are
"isolated" as defined herein are also referred to as "heterologous"
nucleic acid molecules.
[0045] Unless otherwise stated, the term "ETO1 nucleic acid" (also
referred to herein as "ETO1 nucleic acid molecule") is a nucleic
acid (also referred to herein as a "nucleic acid molecule") of the
present invention and means a nucleic acid comprising a
polynucleotide of the present invention (an "ETO1 polynucleotide")
encoding an ETO1 polypeptide with ETO1 activity. An "ETO1 gene" is
a gene of the present invention and refers to a heterologous
genomic form of a full-length ETO1 polynucleotide.
[0046] A "subject plant or plant cell" is one in which genetic
alteration, such as transformation, has been affected as to a gene
of interest or is a plant or plant cell which is descended from a
plant or cell so altered and which comprises the alteration. A
"control" or "control plant" or "control plant cell" provides a
reference point for measuring changes in phenotype of the subject
plant or plant cell.
[0047] A control plant or plant cell may comprise, for example: (a)
a wild-type plant or cell, i.e., of the same genotype as the
starting material for the genetic alteration which resulted in the
subject plant or cell; (b) a plant or plant cell of the same
genotype as the starting material but which has been transformed
with a null construct (i.e., with a construct which has no known
effect on the trait of interest, such as a construct comprising a
marker gene); (c) a plant or plant cell which is a non-transformed
segregant among progeny of a subject plant or plant cell; (d) a
plant or plant cell genetically identical to the subject plant or
plant cell but which is not exposed to conditions or stimuli that
would induce expression of the gene of interest; or (e) the subject
plant or plant cell itself, under conditions in which the gene of
interest is not expressed.
[0048] As used herein, "localized within the chromosomal region
defined by and including" with respect to particular markers
includes reference to a contiguous length of a chromosome delimited
by and including the stated markers.
[0049] As used herein, "marker" includes reference to a locus on a
chromosome that serves to identify a unique position on the
chromosome. A "polymorphic marker" includes reference to a marker
which appears in multiple forms (alleles) such that different forms
of the marker, when they are present in a homologous pair, allow
transmission of each of the chromosomes of that pair to be
followed. A genotype may be defined by use of one or a plurality of
markers.
[0050] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides (e.g., peptide nucleic
acids).
[0051] By "nucleic acid library" is meant a collection of isolated
DNA or RNA molecules which comprise and substantially represent the
entire transcribed fraction of a genome of a specified organism.
Construction of exemplary nucleic acid libraries, such as genomic
and cDNA libraries, is taught in standard molecular biology
references such as Berger and Kimmel, Guide to Molecular Cloning
Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.,
San Diego, Calif. (Berger); Sambrook, et al., Molecular Cloning-A
Laboratory Manual, 2nded., Vol. 1-3 (1989); and Current Protocols
in Molecular Biology, Ausubel, et al., Eds., Current Protocols, a
joint venture between Greene Publishing Associates, Inc. and John
Wiley & Sons, Inc. (1994).
[0052] As used herein "operably linked" includes reference to a
functional linkage between a promoter and a second sequence,
wherein the promoter sequence initiates and mediates transcription
of the DNA sequence corresponding to the second sequence.
Generally, operably linked means that the nucleic acid sequences
being linked are contiguous and where necessary to join two protein
coding regions, contiguous and in the same reading frame.
[0053] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and
progeny of same. Plant cell, as used herein includes, without
limitation, a cell derived from a seed, suspension culture, embryo,
meristematic region, callus tissue, leaf, root, shoot, gametophyte,
sporophyte, pollen or microspore. The class of plants which can be
used in the methods of the invention is generally as broad as the
class of higher plants amenable to transformation techniques,
including both monocotyledonous and dicotyledonous plants. A
particularly preferred plant is Zea mays.
[0054] As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that
have the essential nature of a natural ribonucleotide in that they
hybridize, under stringent hybridization conditions, to
substantially the same nucleotide sequence as naturally occurring
nucleotides and/or allow translation into the same amino acid(s) as
the naturally occurring nucleotide(s). A polynucleotide can be
full-length or a subsequence of a native or heterologous structural
or regulatory gene. Unless otherwise indicated, the term includes
reference to the specified sequence as well as the complementary
sequence thereof. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucleotides" as that term
is intended herein. Moreover, DNAs or RNAs comprising unusual
bases, such as inosine, or modified bases, such as tritylated
bases, to name just two examples, are polynucleotides as the term
is used herein. It will be appreciated that a great variety of
modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art.
[0055] The term polynucleotide as it is employed herein embraces
such chemically, enzymatically or metabolically modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including among other things,
simple and complex cells.
[0056] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. The essential nature of
such analogues of naturally occurring amino acids is that, when
incorporated into a protein, that protein is specifically reactive
to antibodies elicited to the same protein but consisting entirely
of naturally occurring amino acids. The terms "polypeptide",
"peptide" and "protein" are also inclusive of modifications
including, but not limited to, glycosylation, lipid attachment,
sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation. It will be appreciated, as is
well known and as noted above, that polypeptides are not always
entirely linear. For instance, polypeptides may be branched as a
result of ubiquitization, and they may be circular, with or without
branching, generally as a result of post translation events,
including natural processing event and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translation natural process and by entirely synthetic methods,
as well. Further, this invention contemplates the use of both the
methionine-containing and the methionine-less amino terminal
variants of the protein of the invention.
[0057] As used herein "promoter" includes reference to a region of
DNA upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A "plant promoter" is a promoter capable of
initiating transcription in plant cells whether or not its origin
is a plant cell. Exemplary plant promoters include, but are not
limited to, those that are obtained from plants, plant viruses, and
bacteria which comprise genes expressed in plant cells such as
Agrobacterium or Rhizobium. Examples of promoters under
developmental control include promoters that preferentially
initiate transcription in certain tissues, such as leaves, roots,
or seeds. Such promoters are referred to as "tissue preferred".
Promoters which initiate transcription only in certain tissue are
referred to as "tissue specific". A "cell type" preferred promoter
primarily drives expression in certain cell types in one or more
organs, for example, vascular cells in roots or leaves. An
"inducible" or "repressible" promoter is a promoter which is under
environmental control. Examples of environmental conditions that
may effect transcription by inducible promoters include anaerobic
conditions or the presence of light. Tissue specific, tissue
preferred, cell type preferred, and inducible promoters are members
of the class of "non-constitutive" promoters. A "constitutive"
promoter is a promoter which is active under most environmental
conditions and/or in most tissues of a plant and/or at most
developmental stages.
[0058] The term "ETO1 polypeptide" refers to a polypeptide of the
present invention which has ETO1 activity and refers to one or more
amino acid sequences, in glycosylated or non-glycosylated form. The
term is also inclusive of fragments, variants, homologs, alleles or
precursors (e.g., preproproteins or proproteins) thereof which
retain activity. An "ETO1 protein" is a protein of the present
invention and comprises an ETO1 polypeptide. "ETO1 activity" means
that the polypeptide is capable of binding to the C-terminus of ACS
Class II enzymes and ushering them to the 26S proteasome for
degradation resulting in a decrease in ethylene production as
measurable by any of a number of available protocols.
[0059] As used herein "recombinant" includes reference to a cell or
vector, that has been modified by the introduction of a
heterologous nucleic acid or that the cell is derived from a cell
so modified. Thus, for example, recombinant cells express genes
that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under-expressed or not expressed at
all, as a result of deliberate human intervention. The term
"recombinant" as used herein does not encompass the alteration of
the cell or vector by naturally occurring events (e.g., spontaneous
mutation, natural transformation/transduction/transposition) such
as those occurring without deliberate human intervention.
[0060] As used herein, a "recombinant expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically,
with a series of specified nucleic acid elements which permit
transcription of a particular nucleic acid in a host cell. The
recombinant expression cassette can be incorporated into a plasmid,
chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid
fragment. Typically, the recombinant expression cassette portion of
an expression vector includes, among other sequences, a nucleic
acid to be transcribed and a promoter.
[0061] As used herein, a chimeric gene comprises a coding sequence
operably linked to a transcription initiation region that is
heterologous to the coding sequence.
[0062] The terms "residue" and "amino acid residue" and "amino
acid" are used interchangeably herein to refer to an amino acid
that is incorporated into a protein, polypeptide, or peptide
(collectively "protein"). The amino acid may be a naturally
occurring amino acid and unless otherwise limited, may encompass
non-natural analogs of natural amino acids that can function in a
similar manner as naturally occurring amino acids.
[0063] The term "selectively hybridizes" includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to as other biologics. Thus, under designated
immunoassay conditions, the specified antibodies bind to an analyte
having the recognized epitope to a substantially greater degree
(e.g., at least 2-fold over background) than to substantially all
analytes lacking the epitope which are present in the sample.
Specific binding to an antibody under such conditions may require
an antibody that is selected for its specificity for a particular
protein. For example, antibodies raised to the polypeptides of the
present invention can be selected from to obtain antibodies
specifically reactive with polypeptides of the present invention.
The proteins used as immunogens can be in native conformation or
denatured so as to provide a linear epitope.
[0064] A variety of immunoassay formats may be used to select
antibodies specifically reactive with a particular protein (or
other analyte). For example, solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immunoreactive with a protein. See, Harlow and Lane, Antibodies, A
Laboratory Manual, Cold Spring Harbor Publications, New York
(1988), for a description of immunoassay formats and conditions
that can be used to determine selective reactivity.
[0065] The term "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences can be identified which are 100% complementary to the
probe (homologous probing).
[0066] Alternatively, stringency conditions can be adjusted to
allow some mismatching in sequences so that lower degrees of
similarity are detected (heterologous probing). Generally, a probe
is less than about 1000 nucleotides in length, optionally less than
500 nucleotides in length.
[0067] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree.
C. and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0
MNaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1 MNaCl, 1% SDS at 37.degree. C., and a wash in <RTI
0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1
MNaCl, 1% SDS at 37.degree. C. and a wash in 0.1.times.SSC at 60 to
65.degree. C. Specificity is typically the function of
post-hybridization washes, the critical factors being the ionic
strength and temperature of the final wash solution. For DNA/DNA
hybrids, the Tm can be approximated from the equation of Meinkoth
and Wahl, (1984) Anal. Biochem., 138:267-284:Tm=81.5.degree.
C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the
molarity of monovalent cations, % GC is the percentage of guanosine
and cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The Tm is the temperature (under defined
ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. Tm is reduced by
about 1.degree. C. for each 1% of mismatching; thus, Tm,
hybridization and/or wash conditions can be adjusted to hybridize
to sequences of the desired identity. For example, if sequences
with >90% identity are sought, the Tm can be decreased
10.degree. C. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point(Tm) for the
specific sequence and its complement at a defined ionic strength
and pH. However, severely stringent conditions can utilize a
hybridization and/or wash at 1, 2, 3 or 4.degree. C. lower than the
thermal melting point (Tm); moderately stringent conditions can
utilize a hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C.
lower than the thermal melting point(Tm); low stringency conditions
can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or
20.degree. C. lower than the thermal melting point (Tm). Using the
equation, hybridization and wash compositions, and desired Tm,
those of ordinary skill will understand that variations in the
stringency of hybridization and/or wash solutions are inherently
described. If the desired degree of mismatching results in a Tm of
less than 45.degree. C. (aqueous solution) or 32.degree. C.
(formamide solution) it is preferred to increase the SSC
concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays", Elsevier, New York (1993); and Current
Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds.,
Greene Publishing and Wiley-Interscience, New York (1995).
[0068] As used herein, "transgenic plant" includes reference to a
plant which comprises within its genome a heterologous
polynucleotide. Generally, the heterologous polynucleotide is
stably integrated within the genome such that the polynucleotide is
passed on to successive generations. The heterologous
polynucleotide may be integrated into the genome alone or as part
of a recombinant expression cassette. "Transgenic" is used herein
to include any cell, cell line, callus, tissue, plant part or
plant, the genotype of which has been altered by the presence of
heterologous nucleic acid including those transgenics initially so
altered as well as those created by sexual crosses or asexual
propagation from the initial transgenic. The term "transgenic" as
used herein does not encompass the alteration of the genome
(chromosomal or extra-chromosomal) by conventional plant breeding
methods or by naturally occurring events such as random
cross-fertilization, non recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition or spontaneous mutation.
[0069] As used herein, "vector" includes reference to a nucleic
acid used in transfection of a host cell and into which can be
inserted a polynucleotide. Vectors are often replicons. Expression
vectors permit transcription of a nucleic acid inserted
therein.
[0070] The following terms are used to describe the sequence
relationships between a polynucleotide/polypeptide of the present
invention with a reference polynucleotide/polypeptide: (a)
"reference sequence", (b) "comparison window", (c) "sequence
identity" and (d) "percentage of sequence identity".
[0071] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison with a
polynucleotide/polypeptide of the present invention. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence or
the complete cDNA or gene sequence.
[0072] (b) As used herein, "comparison window" includes reference
to a contiguous and specified segment of a
polynucleotide/polypeptide sequence, wherein the
polynucleotide/polypeptide sequence may be compared to a reference
sequence and wherein the portion of the polynucleotide/polypeptide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. Generally, the comparison window is at least 20
contiguous nucleotides/amino acids residues in length and
optionally can be 30, 40, 50, 100 or longer. Those of skill in the
art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide/polypeptide
sequence, a gap penalty is typically introduced and is subtracted
from the number of matches.
[0073] Methods of alignment of sequences for comparison are
well-known in the art. Optimal alignment of sequences for
comparison may be conducted by the local homology algorithm of
Smith and Waterman, (1981) Adv. Appl. Math. 2:482; by the homology
alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.
48:443; by the search for similarity method of Pearson and Lipman,
(1988) Proc. Natl. Acad. Sci. 85:2444; by computerized
implementations of these algorithms, including, but not limited to:
CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,
Calif.; GAP, BESTFIT, BLAST, FASTA and TFASTA in the GCG Wisconsin
Genetics Software Package, Version 10 (available from Accelrys
Inc., 9685 Scranton Road, San Diego, Calif., USA). The CLUSTAL
program is well described by Higgins and Sharp, (1988) Gene
73:237-244; Higgins and Sharp, (1989) CABIOS 5:151-153; Corpet, et
al., (1988) Nucleic Acids Research 16:10881-90; Huang, et al.,
(1992) Computer Applications in the Biosciences 8:155-65 and
Pearson, et al., (1994) Methods in Molecular Biology
24:307-331.
[0074] The BLAST family of programs which can be used for database
similarity searches includes: BLASTN for nucleotide query sequences
against nucleotide database sequences; BLASTX for nucleotide query
sequences against protein database sequences; BLASTP for protein
query sequences against protein database sequences; TBLASTN for
protein query sequences against nucleotide database sequences and
TBLASTX for nucleotide query sequences against nucleotide database
sequences. See, Current Protocols in Molecular Biology, Chapter 19,
Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience,
New York (1995); Altschul et al., (1990) J. Mol. Biol., 215:403-410
and Altschul, et al., (1997) Nucleic Acids Res. 25:3389-3402.
[0075] Software for performing BLAST analyses is publicly
available, e.g., through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold. These initial neighborhood word hits act as seeds for
initiating searches to find longer HSPs containing them. The word
hits are then extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Cumulative scores are calculated using, for nucleotide sequences,
the parameters M (reward score for a pair of matching residues;
always >0) and N (penalty score for mismatching residues; always
<0). For amino acid sequences, a scoring matrix is used to
calculate the cumulative score.
[0076] Extension of the word hits in each direction are halted
when: the cumulative alignment score falls off by the quantity X
from its maximum achieved value; the cumulative score goes to zero
or below, due to the accumulation of one or more negative-scoring
residue alignments; or the end of either sequence is reached. The
BLAST algorithm parameters W, T and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a word length (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as
defaults a word length (W) of 3, an expectation (E) of 10 and the
BLOSUM62 scoring matrix (see, Henikoff and Henikoff, (1989) Proc.
Natl. Acad. Sci. USA 89:10915).
[0077] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul,
(1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5877). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. BLAST searches assume that proteins can be
modeled as random sequences. However, many real proteins comprise
regions of nonrandom sequences which may be homopolymeric tracts,
short-period repeats or regions enriched in one or more amino
acids. Such low-complexity regions may be aligned between unrelated
proteins even though other regions of the protein are entirely
dissimilar. A number of low-complexity filter programs can be
employed to reduce such low-complexity alignments. For example, the
SEG (Wooten and Federhen, (1993) Comput. Chem., 17:149-163) and XNU
(Claverie and States, (1993) Comput. Chem., 17:191-201)
low-complexity filters can be employed alone or in combination.
[0078] Unless otherwise stated, nucleotide and protein
identity/similarity values provided herein are calculated using GAP
(GCG Version 10) under default values. GAP (Global Alignment
Program) can also be used to compare a polynucleotide or
polypeptide of the present invention with a reference sequence. GAP
uses the algorithm of Needleman and Wunsch (J. Mol. Biol.
48:443-453 (1970)) to find the alignment of two complete sequences
that maximizes the number of matches and minimizes the number of
gaps. GAP considers all possible alignments and gap positions and
creates the alignment with the largest number of matched bases and
the fewest gaps. It allows for the provision of a gap creation
penalty and a gap extension penalty in units of matched bases. GAP
must make a profit of gap creation penalty number of matches for
each gap it inserts. If a gap extension penalty greater than zero
is chosen, GAP must, in addition, make a profit for each gap
inserted of the length of the gap times the gap extension penalty.
Default gap creation penalty values and gap extension penalty
values in Version 10 of the Wisconsin Genetics Software Package for
protein sequences are 8 and 2, respectively. For nucleotide
sequences the default gap creation penalty is 50 while the default
gap extension penalty is 3. The gap creation and gap extension
penalties can be expressed as an integer selected from the group of
integers consisting of from 0 to 100. Thus, for example, the gap
creation and gap extension penalties can each independently be: 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.
[0079] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the Wisconsin Genetics Software Package is BLOSUM62
(see, Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA
89:10915).
[0080] Multiple alignment of the sequences can be performed using
the CLUSTAL method of alignment (Higgins and Sharp, (1989) CABIOS
5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
CLUSTAL method are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5.
[0081] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes
reference to the residues in the two sequences which are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences which differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well-known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, (1988) Computer Applic. Biol. Sci., 4:11-17,
e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Calif., USA).
[0082] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
Overview
[0083] The present invention provides, among other things,
compositions and methods for modulating (i.e., increasing or
decreasing) the level of polynucleotides and polypeptides of the
present invention in plants. In particular, the polynucleotides and
polypeptides of the present invention can be expressed temporally
or spatially, e.g., at developmental stages, in tissues, and/or in
quantities, which are uncharacteristic of non-recombinantly
engineered plants. Thus, the present invention provides utility in
such exemplary applications as provided below.
[0084] Applicants have isolated a novel ETO1 protein that may be
used in the modulation of ethylene activity and production in
plants. The novel protein, its nucleotide sequences encoding the
same and resultant constructs, vectors and modified plant cells,
tissues, seeds and organs form the basis of the invention. The
enzyme thus finds utility in a number of stress response
applications such as the following.
Crowding Tolerance
[0085] The agronomic performance of crop plants is often a function
of how well they tolerate planting density. Overcrowded plants grow
poorly. The stress of overcrowding can be due to simple limitations
of nutrients, water and sunlight. Crowding stress may also be due
to enhanced contact between plants. Plants often respond to
physical contact by slowing growth and thickening their
tissues.
[0086] Ethylene has been implicated in plant crowding response. For
example, ethylene insensitive tobacco plants did not slow growth
when contacting neighboring plants (Knoester, et al., (1998) PNAS
USA 95:1933-1937). There is also evidence that ethylene, and the
plant's response to it is involved in water deficit stress and that
ethylene may be causing changes in the plant that limit its growth
and aggravate the symptoms of drought stress beyond the loss of
water itself.
[0087] The present invention provides for decreasing ethylene
production in a plant, in particular cereals such as maize, by
providing one or more novel ETO1 polynucleotides or their protein
products to promote tolerance of close spacing with reduced stress
and yield loss.
Seed Set and Development in Maize
[0088] Ethylene plays a number of roles in seed development. For
example, in maize ethylene is linked to programmed cell death of
developing endosperm cells (Young, et al., (1997) Plant Physiol.,
115:737-751). In addition, ethylene is linked to kernel abortion,
such as occurs at the tips of ears, especially in plants grown
under stressful conditions (Cheng and Lur, (1996) Physiol. Plant
98:245-252). Reduced kernel seed set is of course a contributor to
reduced yields. Consequently, the present invention provides
plants, in particular maize plants, that have reduced ethylene
action by providing for and/or modulating the expression/activity
of the novel ETO1 polynucleotides of the invention.
Growth in Compacted Soils
[0089] Plant growth is affected by the density and compaction of
soils. Denser, more compacted soils typically result in poorer
plant growth. The trend in agriculture towards more minimal till
planting and cultivation practices, with the goal of soil and
energy conservation, is increasing the need for crop plants that
can perform well under these conditions.
[0090] Ethylene is well-known to affect plant growth and
development and one effect of ethylene is to promote tissue
thickening and growth retardation when encountering mechanical
stress, such as compacted soils. This can affect both the roots and
shoots. This effect is presumably adaptive in some circumstances in
that it results in stronger, more compact tissues that can force
their way through or around, obstacles such as compacted soils.
However, in such conditions, the production of ethylene and the
activation of the ethylene pathway may exceed what is needed for
adaptive accommodation to the mechanical stress of the compacted
soils. And, of course, any resulting unnecessary growth inhibition
would be an undesired agronomic result.
[0091] The present invention provides for decreasing ethylene
production in a plant, in particular cereals such as maize, by
providing for and/or modulating the expression/activity of one or
more novel ETO1 polynucleotides or their protein products. Such
modulated plants grow and germinate better in compacted soils,
resulting in higher stand counts, the herald of higher yields.
Flooding Tolerance
[0092] Flooding and water-logged soils cause substantial losses in
crop yield each year around the world. Flooding can be both
widespread or local, transitory or prolonged. Ethylene has been
implicated in flooding mediated damage. In fact, in flooded
conditions ethylene production can rise. There are two main reasons
for this rise: 1) under such flooded conditions, which creates
hypoxia, plants produce more ethylene and 2) under flooded
conditions the diffusion of ethylene away from the plant is slowed,
because ethylene is minimally soluble in water, resulting in a rise
of intra-plant ethylene levels.
[0093] Ethylene in flooded maize roots can also inhibit
gravitropism, which is normally adaptive during germination in that
it orients the roots down and the shoots up. Gravitropism is a
factor in determining root architecture, which in turn plays an
important role in soil resource acquisition. Manipulation of
ethylene levels could be used to impact root angle for drought
tolerance, flood tolerance, greater standability and/or improved
nutrient uptake. For example, a root growing at a more erect angle
(steeper) would likely grow more deeply in soil and thus obtain
water at greater depths, improving drought tolerance. In the
absence of drought stress a converse argument could be made for
more efficient root uptake of nutrients and water in the upper
layers of the soil profile, by roots which are more parallel to the
soil surface. In general, roots that have a angle nearer that of
vertical (steep) are also more susceptible to root lodging than
roots with a shallow angle (parallel to the surface) that can be
more root lodging resistant.
[0094] In addition to inhibition of gravitropism, it is likely that
ethylene evolution in flooded conditions inhibits growth,
especially of roots. Such inhibition will likely contribute to poor
plant growth overall, and consequently is a disadvantageous
agronomic trait.
[0095] The present invention provides for decreasing ethylene
production in a plant, in particular cereals such as maize, by
providing for and/or modulating the expression/activity of one or
more novel ETO1 polynucleotides or their protein products. Such
plants should grow and germinate better in flooded conditions or
water-logged soils, resulting in higher stand counts.
Plant Maturation and Senescence
[0096] Ethylene is known to be involved in controlling senescence,
fruit ripening and abscission. The role of ethylene in fruit
ripening is well-established and industrially applied. The
prediction based on precedent would be that ethylene
underproduction/insensitivity would result in slower seed ripening
and the converse would result in more rapid seed ripening.
Abscission is primarily studied for dicot plants and apparently has
little application to monocots such as cereals. Ethylene mediated
senescence also is mostly studied in dicots, but control of
senescence is agronomically important for both dicot and monocot
crop species. Ethylene insensitivity can cause a delay of, but not
arrest, senescence. The senescence process mediated by ethylene
bears some similarities to the cell death process in disease
symptoms and in abscission zones.
[0097] Controlling ethylene production, as through the control of
one or more novel ETO1 genes, could result in modulation of
maturity rates for crop plants such as maize.
[0098] The present invention provides for decreasing ethylene
production in a plant, in particular cereals such as maize, by
providing for and/or modulating the expression/activity of one or
more novel ETO1 polynucleotides or their protein products which may
contribute to a later maturing plant, which is desirable for
placing crop varieties in different maturity zones.
Tolerance to Other Abiotic Stresses
[0099] Many stresses on plants induce the production of ethylene
(see, Morgan and Drew, (1997) Physiol. Plant 100:620-630). These
stresses can be cold, heat, wounding, pollution, drought and
hypersalinity. Mechanical impedance (soil compaction) and flooding
stresses were addressed above. It appears that several of these
stresses operate through common mechanisms, such as water deficit.
Clearly drought causes water deficit; crowding stress may also
cause water deficit. Additionally, in maize chilling can cause an
elevation in ethylene production and activity, and this induction
is apparently due to chilling causing water deficit in cells
(Janowaik and Dorffling, (1995) J. Plant Physiol. 147:257-262).
[0100] Some of the ethylene production following stresses may serve
an adaptive purpose by regulating ethylene-mediated processes in
the plant that result in a plant reorganized in such manner to
better acclimate to the stress encountered. However, there is also
evidence that ethylene production during stress can result in an
aggravation of negative symptoms resulting from the stress, such as
yellowing, tissue death and senescence.
[0101] To the extent that ethylene production during stress causes
or augments negative stress-related symptoms, it would be desirable
to create a crop plant with reduced ethylene production. Towards
that end, the present invention provides for decreasing ethylene
production in a plant, in particular cereals such as maize, by
providing for and/or modulating the expression/activity of one or
more novel ETO1 polynucleotides or their protein products to create
plants that avoid certain ethylene-mediated effects.
Disease Resistance
[0102] Crop plants can be susceptible to a wide variety of
pathogens, whether viruses, bacteria, fungi or insects. This
susceptibility results in large crop yield losses annually
worldwide. Crop breeders have endeavored to breed more resistant or
tolerant varieties which can withstand pathogen attack. Additional
genetic engineering strategies seek the same end. In many
plant-pathogen interactions the symptoms of disease, most often
tissue necrosis and resulting poor plant growth, are known to be
the result of an active plant defense response to the pathogen.
That is, the symptoms are caused directly by the plant and not
simply by the pathogen. From among the list of all crop plants and
their potential list of pathogens, resistance is the rule, and
susceptibility the exception. Susceptible interactions are often
thought to result from an improper or insufficient activation
defense by the plant that results in a runaway symptom development
and an inability to contain the pathogen.
[0103] Ethylene has long been known to be associated with plant
pathogen defense systems. Many pathogenesis related genes are
induced in expression at the level of mRNA by ethylene. The trend
in our understanding of the role of ethylene in plant pathogen
defense is towards ethylene and ethylene mediated effects being
viewed as principally part of the downstream reactions to pathogen
attack, as in symptom development. Ethylene seems to be involved in
the plant's response to the stress of pathogen attack and in tissue
damage inflicted by the pathogen. In a susceptible interaction
ethylene may actually promote tissue damage. Consequently, in such
situations, blocking ethylene production or action may actually
result in less tissue damage, that is, more apparent resistance,
even though the pathogen is compatible with the plant. Blocking
ethylene action is known to result in either more susceptibility
(e.g., Knoester, et al., (1988)) or more resistance (e.g., Lund, et
al., (1998) Plant Cell 10:371-382), which indicates that the role
of ethylene action is complex, as is to be expected, for it depends
upon the interactions of diverse plants and pathogens.
[0104] The present invention provides for the use of one or more
novel ETO1 polynucleotides or their protein products to affect
enhanced resistance to plant stresses, in particular for monocots
such as maize.
[0105] For most applications this will involve the reduction in
ethylene production by providing for and/or modulating the
expression/activity of novel ETO1 polynucleotides or their
proteins, with the goal of causing plants that produce less
ethylene in response to stress and thereby plants that are less
prone to tissue damage following exposure to abiotic stressors.
ETO1 is a potent negative regulator of ACS and ethylene production,
see, for example, Chae and Faure, et al., (2003) Plant Cell
15(2):545-559; Christians, et al., (2009) The Plant Journal
57(2):332-345; Wang, et al., (2004) Nature 428(6986):945-950;
Yoshida, et al., (2005) BMC Plant Biology 5:14 and Yoshida, et al.,
(2006) Plant Molecular Biology 62(3):427-437.
Plant Transformation
[0106] The generation of transgenic plants is central to crop plant
genetic engineering strategies. Transgenesis typically involves the
introduction of exogenous DNA into the plants cells via a variety
of methods, such as particle bombardment or Agrobacterium
infection, which is usually followed by tissue culture and plant
regeneration. Transgenic plant production remains a costly and rate
limiting step in genetic engineering, especially for many of the
most economically important crop plants, such as the cereals, like
maize.
Improving the Efficiency of this Process is Therefore of Great
Importance.
[0107] It has been accepted for a long time that ethylene action
has negative consequences for plant transformation. As a result
various approaches to bind, trap or otherwise block the
accumulation of ethylene are employed in transformation and tissue
culture (see, Songstad, et al., (1991) Plant Cell Reports
9:694-702). The particle bombardment method causes substantial
tissue/cell damage and such damage is known to elicit ethylene
accumulation. Moreover, in most tissue culture methods, some tissue
grows better than others, as is designed in chemical selection of
transformants. Such dying tissue can emit ethylene and cause
inhibition of positive transformants. Aggravating these effects is
the confinement of plant tissues in containers for the purpose of
tissue regeneration that can result in the accumulation of
ethylene, also causing growth retardation. As ethylene is known to
promote slower tissue growth and even cell/tissue death, having a
means to block or minimize ethylene action during transformation is
desired.
[0108] Consequently, the present invention also provides for use of
the ETO1 sequences herein to create transient or stable reductions
in ethylene action by increasing the expression/activity of ETO1
polynucleotides or polypeptides.
Other Utilities
[0109] The present invention also provides isolated nucleic acids
comprising polynucleotides of sufficient length and complementarity
to a gene of the present invention to use as probes or
amplification primers in the detection, quantitation or isolation
of gene transcripts. For example, isolated nucleic acids of the
present invention can be used as probes in detecting deficiencies
in the level of mRNA in screenings for desired transgenic plants,
for detecting mutations in the gene (e.g., substitutions, deletions
or additions), for monitoring upregulation of expression or changes
in enzyme activity in screening assays of compounds, for detection
of any number of allelic variants (polymorphisms), orthologs or
paralogs of the gene, or for site directed mutagenesis in
eukaryotic cells (see, e.g., U.S. Pat. No. 5,565,350). The isolated
nucleic acids of the present invention can also be used for
recombinant expression of their encoded polypeptides, or for use as
immunogens in the preparation and/or screening of antibodies. The
isolated nucleic acids of the present invention can also be
employed for use in sense or antisense suppression of one or more
genes of the present invention in a host cell, tissue or plant.
Attachment of chemical agents which bind, intercalate, cleave
and/or cross-link to the isolated nucleic acids of the present
invention can also be used to modulate transcription or
translation.
[0110] The present invention also provides isolated proteins
comprising a polypeptide of the present invention (e.g.,
preproenzyme, proenzyme or enzymes). The present invention also
provides proteins comprising at least one epitope from a
polypeptide of the present invention. The proteins of the present
invention can be employed in assays for enzyme agonists or
antagonists of enzyme function, or for use as immunogens or
antigens to obtain antibodies specifically immunoreactive with a
protein of the present invention. Such antibodies can be used in
assays for expression levels, for identifying and/or isolating
nucleic acids of the present invention from expression libraries,
for identification of homologous polypeptides from other species or
for purification of polypeptides of the present invention.
[0111] The isolated nucleic acids and polypeptides of the present
invention can be used over a broad range of plant types,
particularly monocots such as the species of the family Gramineae
including Hordeum, Secale, Tritium, Sorghum (e.g., S. bicolor) and
Zea (e.g., Z. mays). The isolated nucleic acid and proteins of the
present invention can also be used in species from the genera:
Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago,
Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium,
Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,
Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum,
Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca,
Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium,
Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis,
Browallia, Glycine, Pisum, Phaseolus, Lolium, Oryza and Avena.
Nucleic Acids
[0112] The present invention provides, among other things, isolated
nucleic acids of RNA, DNA and analogs and/or chimeras thereof,
comprising a polynucleotide of the present invention.
[0113] A polynucleotide of the present invention is inclusive of:
[0114] (a) a polynucleotide encoding a polypeptide of SEQ ID NO: 2,
4, 6, 8 or 10 and conservatively modified and polymorphic variants
thereof, including exemplary polynucleotides of SEQ ID NO: 1, 3, 5,
7 or 9, (ETO1); [0115] (b) an isolated polynucleotide which is the
product of amplification from a plant nucleic acid library using
primer pairs which selectively hybridize under stringent conditions
to loci within a polynucleotide of the present invention; [0116]
(c) an isolated polynucleotide which selectively hybridizes to a
polynucleotide of (a) or (b); [0117] (d) an isolated polynucleotide
having a specified sequence identity with polynucleotides of (a),
(b) or (c); [0118] (e) an isolated polynucleotide encoding a
protein having a specified number of contiguous amino acids from a
prototype polypeptide, wherein the protein is specifically
recognized by antisera elicited by presentation of the protein and
wherein the protein does not detectably immunoreact to antisera
which has been fully immunosorbed with the protein; [0119] (f)
complementary sequences of polynucleotides of (a), (b), (c), (d) or
(e); and [0120] (g) an isolated polynucleotide comprising at least
a specific number of contiguous nucleotides from a polynucleotide
of (a), (b), (c), (d), (e) or (f); [0121] (h) an isolated
polynucleotide from a full-length enriched cDNA library having the
physico-chemical property of selectively hybridizing to a
polynucleotide of (a), (b), (c), (d), (e), (f) or (g); [0122] (i)
an isolated polynucleotide made by the process of: 1) providing a
full-length enriched nucleic acid library, 2) selectively
hybridizing the polynucleotide to a polynucleotide of (a), (b),
(c), (d), (e), (f), (g) or (h), thereby isolating the
polynucleotide from the nucleic acid library.
A. Polynucleotides Encoding A Polypeptide of the Present
Invention
[0123] As indicated in (a), above, the present invention provides
isolated nucleic acids comprising a polynucleotide of the present
invention, wherein the polynucleotide encodes a polypeptide of the
present invention. Every nucleic acid sequence herein that encodes
a polypeptide also, by reference to the genetic code, describes
every possible silent variation of the nucleic acid. One of
ordinary skill will recognize that each codon in a nucleic acid
(except AUG, which is ordinarily the only codon for methionine and
UGG, which is ordinarily the only codon for tryptophan) can be
modified to yield a functionally identical molecule. Thus, each
silent variation of a nucleic acid which encodes a polypeptide of
the present invention is implicit in each described polypeptide
sequence and is within the scope of the present invention.
Accordingly, the present invention includes polynucleotides of the
present invention and polynucleotides encoding a polypeptide of the
present invention.
B. Polynucleotides Amplified from a Plant Nucleic Acid Library
[0124] As indicated in (b), above, the present invention provides
an isolated nucleic acid comprising a polynucleotide of the present
invention, wherein the polynucleotides are amplified, under nucleic
acid amplification conditions, from a plant nucleic acid
library.
[0125] Nucleic acid amplification conditions for each of the
variety of amplification methods are well known to those of
ordinary skill in the art. The plant nucleic acid library can be
constructed from a monocot such as a cereal crop. Exemplary cereals
include corn, sorghum, alfalfa, canola, wheat or rice. The plant
nucleic acid library can also be constructed from a dicot such as
soybean. Zea mays lines B73, PHRE1, A632, BMP2#10, W23 and Mol7 are
known and publicly available. Other publicly known and available
maize lines can be obtained from the Maize Genetics Cooperation
(Urbana, Ill.).
[0126] Wheat lines are available from the Wheat Genetics Resource
Center (Manhattan, Kans.). The nucleic acid library may be a cDNA
library, a genomic library or a library generally constructed from
nuclear transcripts at any stage of intron processing. cDNA
libraries can be normalized to increase the representation of
relatively rare cDNAs. In optional embodiments, the cDNA library is
constructed using an enriched full-length cDNA synthesis method.
Examples of such methods include Oligo-Capping (Maruyama and
Sugano, (1994) Gene 138:171-174), Biotinylated CAP Trapper
(Carninci, et al., (1996) Genomics 37:327-336) and CAP Retention
Procedure (Edery, et al., (1995) Molecular and Cellular Biology
15:3363-3371). Rapidly growing tissues or rapidly dividing cells
are preferred for use as an mRNA source for construction of a cDNA
library. Growth stages of corn are described in "How a Corn Plant
Develops, "Special Report No. 48, Iowa State University of Science
and Technology Cooperative Extension Service, Ames, Iowa, Reprinted
February 1993.
[0127] A polynucleotide of this embodiment (or subsequences
thereof) can be obtained, for example, by using amplification
primers which are selectively hybridized and primer extended, under
nucleic acid amplification conditions, to at least two sites within
a polynucleotide of the present invention or to two sites within
the nucleic acid which flank and comprise a polynucleotide of the
present invention or to a site within a polynucleotide of the
present invention and a site within the nucleic acid which
comprises it. Methods for obtaining 5' and/or 3' ends of a vector
insert are well known in the art. See, e.g., RACE (Rapid
Amplification of Complementary Ends) as described in Frohman, in
PCR Protocols: A Guide to Methods and Applications, Innis, et al.,
Eds. (Academic Press, Inc., San Diego), pp. 28-38 (1990)), see
also, U.S. Pat. No. 5,470,722, and Current Protocols in Molecular
Biology, Unit 15.6, Ausubel, et al., Eds., Greene Publishing and
Wiley-Interscience, New York (1995); Frohman and Martin, (1989)
Techniques 1:165.
[0128] Optionally, the primers are complementary to a subsequence
of the target nucleic acid which they amplify but may have a
sequence identity ranging from about 85% to 99% relative to the
polynucleotide sequence which they are designed to anneal to. As
those skilled in the art will appreciate, the sites to which the
primer pairs will selectively hybridize are chosen such that a
single contiguous nucleic acid can be formed under the desired
nucleic acid amplification conditions. The primer length in
nucleotides is selected from the group of integers consisting of
from at least 15 to 50. Thus, the primers can be at least 15, 18,
20, 25, 30, 40 or 50 nucleotides in length. Those of skill will
recognize that a lengthened primer sequence can be employed to
increase specificity of binding (i.e., annealing) to a target
sequence. A non-annealing sequence at the 5' end of a primer (a
"tail") can be added, for example, to introduce a cloning site at
the terminal ends of the amplicon.
[0129] The amplification products can be translated using
expression systems well known to those of skill in the art. The
resulting translation products can be confirmed as polypeptides of
the present invention by, for example, assaying for the appropriate
catalytic activity (e.g., specific activity and/or substrate
specificity) or verifying the presence of one or more epitopes
which are specific to a polypeptide of the present invention.
Methods for protein synthesis from PCR derived templates are known
in the art and available commercially. See, e.g., Amersham Life
Sciences, Inc, Catalog '97, p. 354.
C. Polynucleotides which Selectively Hybridize to a Polynucleotide
of (A) or (B)
[0130] As indicated in (c), above, the present invention provides
isolated nucleic acids comprising polynucleotides of the present
invention, wherein the polynucleotides selectively hybridize, under
selective hybridization conditions, to a polynucleotide of sections
(A) or (B) as discussed above. Thus, the polynucleotides of this
embodiment can be used for isolating, detecting, and/or quantifying
nucleic acids comprising the polynucleotides of (A) or (B). For
example, polynucleotides of the present invention can be used to
identify, isolate or amplify partial or full-length clones in a
deposited library.
[0131] In some embodiments, the polynucleotides are genomic or cDNA
sequences isolated or otherwise complementary to a cDNA from a
dicot or monocot nucleic acid library.
[0132] Exemplary species of monocots and dicots include, but are
not limited to: maize, canola, soybean, cotton, wheat, sorghum,
sunflower, alfalfa, oats, sugar cane, millet, barley and rice. The
cDNA library comprises at least 50% to 95% full-length sequences
(for example, at least 50%, 60%, 70%, 80%, 90% or 95% full-length
sequences). The cDNA libraries can be normalized to increase the
representation of rare sequences. See, e.g., U.S. Pat. No.
5,482,845. Low stringency hybridization conditions are typically,
but not exclusively, employed with sequences having a reduced
sequence identity relative to complementary sequences. Moderate and
high stringency conditions can optionally be employed for sequences
of greater identity. Low stringency conditions allow selective
hybridization of sequences having about 70% to 80% sequence
identity and can be employed to identify orthologous or paralogous
sequences.
D. Polynucleotides Having a Specific Sequence Identity with the
Polynucleotides of (A), (B) or (C)
[0133] As indicated in (d), above, the present invention provides
isolated nucleic acids comprising polynucleotides of the present
invention, wherein the polynucleotides have a specified identity at
the nucleotide level to a polynucleotide as disclosed above in
sections (A), (B), or (C), above. Identity can be calculated using,
for example, the BLAST, CLUSTALW or GAP algorithms under default
conditions. The percentage of identity to a reference sequence is
at least 60% and rounded upwards to the nearest integer, can be
expressed as an integer selected from the group of integers
consisting of from 60 to 99. Thus, for example, the percentage of
identity to a reference sequence can be at least 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% to a
full-length sequence of the invention.
[0134] Optionally, the polynucleotides of this embodiment will
encode a polypeptide that will share an epitope with a polypeptide
encoded by the polynucleotides of sections (A), (B) or (C). Thus,
these polynucleotides encode a first polypeptide which elicits
production of antisera comprising antibodies which are specifically
reactive to a second polypeptide encoded by a polynucleotide of
(A), (B) or (C). However, the first polypeptide does not bind to
antisera raised against itself when the antisera has been fully
immunosorbed with the first polypeptide. Hence, the polynucleotides
of this embodiment can be used to generate antibodies for use in,
for example, the screening of expression libraries for nucleic
acids comprising polynucleotides of (A), (B) or (C), or for
purification of, or in immunoassays for, polypeptides encoded by
the polynucleotides of (A), (B) or (C). The polynucleotides of this
embodiment comprise nucleic acid sequences which can be employed
for selective hybridization to a polynucleotide encoding a
polypeptide of the present invention.
[0135] Screening polypeptides for specific binding to antisera can
be conveniently achieved using peptide display libraries. This
method involves the screening of large collections of peptides for
individual members having the desired function or structure.
[0136] Antibody screening of peptide display libraries is well
known in the art. The displayed peptide sequences can be from 3 to
5000 or more amino acids in length, frequently from 5100 amino
acids long, and often from about 8 to 15 amino acids long. In
addition to direct chemical synthetic methods for generating
peptide libraries, several recombinant DNA methods have been
described. One type involves the display of a peptide sequence on
the surface of a bacteriophage or cell. Each bacteriophage or cell
contains the nucleotide sequence encoding the particular displayed
peptide sequence. Such methods are described in PCT Patent
Application Publication Numbers 1991/17271, 1991/18980, 1991/19818
and 1993/08278. Other systems for generating libraries of peptides
have aspects of both in vitro chemical synthesis and recombinant
methods. See, PCT Patent Application Publication Numbers
1992/05258, 1992/14843 and 1997/20078. See also, U.S. Pat. Nos.
5,658,754 and 5,643,768. Peptide display libraries, vectors and
screening kits are commercially available from such suppliers as
Invitrogen (Carlsbad, Calif.).
E. Polynucleotides Encoding a Protein Having a Subsequence from a
Prototype Polypeptide and Cross-Reactive to the Prototype
Polypeptide
[0137] As indicated in (e), above, the present invention provides
isolated nucleic acids comprising polynucleotides of the present
invention, wherein the polynucleotides encode a protein having a
subsequence of contiguous amino acids from a prototype polypeptide
of the present invention such as are provided in (a), above. The
length of contiguous amino acids from the prototype polypeptide is
selected from the group of integers consisting of from at least 10
to the number of amino acids within the prototype sequence. Thus,
for example, the polynucleotide can encode a polypeptide having a
subsequence having at least 10, 15, 20, 25, 30, 35, 40, 45 or 50,
contiguous amino acids from the prototype polypeptide. Further, the
number of such subsequences encoded by a polynucleotide of the
instant embodiment can be any integer selected from the group
consisting of from 1 to 20, such as 2, 3, 4 or 5. The subsequences
can be separated by any integer of nucleotides from 1 to the number
of nucleotides in the sequence such as at least 5, 10, 15, 25, 50,
100 or 200 nucleotides.
[0138] The proteins encoded by polynucleotides of this embodiment,
when presented as an immunogen, elicit the production of polyclonal
antibodies which specifically bind to a prototype polypeptide such
as but not limited to, a polypeptide encoded by the polynucleotide
of (a) or (b), above. Generally, however, a protein encoded by a
polynucleotide of this embodiment does not bind to antisera raised
against the prototype polypeptide when the antisera has been fully
immunosorbed with the prototype polypeptide. Methods of making and
assaying for antibody binding specificity/affinity are well known
in the art. Exemplary immunoassay formats include ELISA,
competitive immunoassays, radioimmunoassays, Western blots,
indirect immunofluorescent assays and the like.
[0139] In a preferred assay method, fully immunosorbed and pooled
antisera which is elicited to the prototype polypeptide can be used
in a competitive binding assay to test the protein. The
concentration of the prototype polypeptide required to inhibit 50%
of the binding of the antisera to the prototype polypeptide is
determined. If the amount of the protein required to inhibit
binding is less than twice the amount of the prototype protein,
then the protein is said to specifically bind to the antisera
elicited to the immunogen.
[0140] Accordingly, the proteins of the present invention embrace
allelic variants, conservatively modified variants and minor
recombinant modifications to a prototype polypeptide.
[0141] A polynucleotide of the present invention optionally encodes
a protein having a molecular weight as the non-glycosylated protein
within 20% of the molecular weight of the full-length
non-glycosylated polypeptides of the present invention. Molecular
weight can be readily determined by SDS-PAGE under reducing
conditions. Optionally, the molecular weight is within 15% of a
full length polypeptide of the present invention, more preferably
within 10% or 5% and most preferably within 3%, 2% or 1% of a full
length polypeptide of the present invention. Optionally, the
polynucleotides of this embodiment will encode a protein having a
specific enzymatic activity at least 50%, 60%, 80% or 90% of a
cellular extract comprising the native, endogenous full-length
polypeptide of the present invention.
[0142] Further, the proteins encoded by polynucleotides of this
embodiment will optionally have a substantially similar affinity
constant (Km) and/or catalytic activity (i.e., the microscopic rate
constant, kcat) as the native endogenous, full-length protein.
Those of skill in the art will recognize that kcat/Km value
determines the specificity for competing substrates and is often
referred to as the specificity constant. Proteins of this
embodiment can have akcat/Km value at least 10% of a full-length
polypeptide of the present invention as determined using the
endogenous substrate of that polypeptide. Optionally, the kcat/Km
value will be at least 20%, 30%, 40%, 50% and most preferably at
least 60%, 70%, 80%, 90% or 95% the kcat/Km value of the
full-length polypeptide of the present invention.
[0143] Determination of kcat, Km and kcat/Km can be determined by
any number of means well known to those of skill in the art. For
example, the initial rates (i.e., the first 5% or less of the
reaction) can be determined using rapid mixing and sampling
techniques (e.g., continuous-flow, stopped-flow or rapid quenching
techniques), flash photolysis or relaxation methods (e.g.,
temperature jumps) in conjunction with such exemplary methods of
measuring as spectrophotometry, spectrofluorimetry, nuclear
magnetic resonance or radioactive procedures. Kinetic values are
conveniently obtained using a Lineweaver Burk or Eadie-Hofstee
plot.
F. Polynucleotides Complementary to the Polynucleotides of
(A)-(E)
[0144] As indicated in (f), above, the present invention provides
isolated nucleic acids comprising polynucleotides complementary to
the polynucleotides of paragraphs A-E, above. As those of skill in
the art will recognize, complementary sequences base-pair
throughout the entirety of their length with the polynucleotides of
sections (A)-(E) (i.e., have 100% sequence identity over their
entire length). Complementary bases associate through hydrogen
bonding in double stranded nucleic acids. For example, the
following base pairs are complementary: guanine and cytosine;
adenine and thymine and adenine and uracil.
G. Polynucleotides which are Subsequences of the Polynucleotides of
(A)-(F)
[0145] As indicated in (g), above, the present invention provides
isolated nucleic acids comprising polynucleotides which comprise at
least 15 contiguous bases from the polynucleotides of sections (A)
through (F) as discussed above. The length of the polynucleotide is
given as an integer selected from the group consisting of from at
least 15 to the length of the nucleic acid sequence from which the
polynucleotide is a subsequence of. Thus, for example,
polynucleotides of the present invention are inclusive of
polynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75
or 100 contiguous nucleotides in length from the polynucleotides of
(A)-(F). Optionally, the number of such subsequences encoded by a
polynucleotide of the instant embodiment can be any integer
selected from the group consisting of from 1 to 20, such as 2, 3, 4
or 5. The subsequences can be separated by any integer of
nucleotides from 1 to the number of nucleotides in the sequence
such as at least 5, 10, 15, 25, 50, 100 or 200 nucleotides.
[0146] Subsequences can be made by in vitro synthetic, in vitro
biosynthetic or in vivo recombinant methods. In optional
embodiments, subsequences can be made by nucleic acid
amplification. For example, nucleic acid primers will be
constructed to selectively hybridize to a sequence (or its
complement) within, or co-extensive with, the coding region.
[0147] The subsequences of the present invention can comprise
structural libraries as are known in the art and discussed briefly
below. The cDNA library comprises at least 50% to 95% full-length
sequences (for example, at least 50%, 60%, 70%, 80%, 90% or 95%
full-length sequences). The cDNA library can be constructed from a
variety of tissues from a monocot or dicot at a variety of
developmental stages. Exemplary species include maize, wheat, rice,
canola, soybean, cotton, sorghum, sunflower, alfalfa, oats, sugar
cane, millet, barley and rice. Methods of selectively hybridizing,
under selective hybridization conditions, a polynucleotide from a
full-length enriched library to a polynucleotide of the present
invention are known to those of ordinary skill in the art. Any
number of stringency conditions can be employed to allow for
selective hybridization. In optional embodiments, the stringency
allows for selective hybridization of sequences having at least
70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity over the
length of the hybridized region. Full-length enriched cDNA
libraries can be normalized to increase the representation of rare
sequences.
H. Polynucleotide Products Made by a cDNA Isolation Process
[0148] As indicated in (I), above, the present invention provides
an isolated polynucleotide made by the process of: 1) providing a
full-length enriched nucleic acid library, 2) selectively
hybridizing the polynucleotide to a polynucleotide of paragraphs
(A), (B), (C), (D), (E), (F), (G) or (H) as discussed above and
thereby isolating the polynucleotide from the nucleic acid library.
Full-length enriched nucleic acid libraries are constructed as
discussed in paragraph (G) and below. Selective hybridization
conditions are as discussed in paragraph (G). Nucleic acid
purification procedures are well known in the art.
[0149] Purification can be conveniently accomplished using
solid-phase methods; such methods are well known to those of skill
in the art and kits are available from commercial suppliers such as
Advanced Biotechnologies (Surrey, UK). For example, a
polynucleotide of paragraphs (A)-(H) can be immobilized to a solid
support such as a membrane, bead or particle. See, e.g., U.S. Pat.
No. 5,667,976. The polynucleotide product of the present process is
selectively hybridized to an immobilized polynucleotide and the
solid support is subsequently isolated from non-hybridized
polynucleotides by methods including, but not limited to,
centrifugation, magnetic separation, filtration, electrophoresis,
and the like.
Construction of Nucleic Acids
[0150] The isolated nucleic acids of the present invention can be
made using (a) standard recombinant methods, (b) synthetic
techniques or combinations thereof. In some embodiments, the
polynucleotides of the present invention will be cloned, amplified
or otherwise constructed from a monocot such as corn, rice or wheat
or a dicot such as soybean.
[0151] The nucleic acids may conveniently comprise sequences in
addition to a polynucleotide of the present invention. For example,
a multi-cloning site comprising one or more endonuclease
restriction sites may be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences may
be inserted to aid in the isolation of the translated
polynucleotide of the present invention. For example, a
hexahistidine marker sequence provides a convenient means to purify
the proteins of the present invention. A polynucleotide of the
present invention can be attached to a vector, adapter or linker
for cloning and/or expression of a polynucleotide of the present
invention. Additional sequences may be added to such cloning and/or
expression sequences to optimize their function in cloning and/or
expression, to aid in isolation of the polynucleotide or to improve
the introduction of the polynucleotide into a cell. Typically, the
length of a nucleic acid of the present invention less the length
of its polynucleotide of the present invention is less than 20
kilobase pairs, often less than 15 kb, and frequently less than 10
kb. Use of cloning vectors, expression vectors, adapters, and
linkers is well known and extensively described in the art. For a
description of various nucleic acids see, for example, Stratagene
Cloning Systems, Catalogs 1999 (La Jolla, Calif.); and Amersham
Life Sciences, Inc, Catalog '99 (Arlington Heights, Ill.).
A. Recombinant Methods for Constructing Nucleic Acids
[0152] The isolated nucleic acid compositions of this invention,
such as RNA, cDNA, genomic DNA or a hybrid thereof, can be obtained
from plant biological sources using any number of cloning
methodologies known to those of skill in the art. In some
embodiments, oligonucleotide probes which selectively hybridize,
under stringent conditions, to the polynucleotides of the present
invention are used to identify the desired sequence in a cDNA or
genomic DNA library. Isolation of RNA and construction of cDNA and
genomic libraries is well known to those of ordinary skill in the
art. See, e.g., Plant Molecular Biology: A Laboratory Manual,
Clark, Ed., Springer-Verlag, Berlin (1997); and Current Protocols
in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and
Wiley-Interscience, New York (1995).
[0153] A1. Full-Length Enriched cDNA Libraries
[0154] A number of cDNA synthesis protocols have been described
which provide enriched full-length cDNA libraries. Enriched
full-length cDNA libraries are constructed to comprise at least
60%, and more preferably at least 70%, 80%, 90% or 95% full-length
inserts amongst clones containing inserts. The length of insert in
such libraries can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
kilobase pairs. Vectors to accommodate inserts of these sizes are
known in the art and available commercially. See, e.g.,
Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12
kb cloning capacity). An exemplary method of constructing a greater
than 95% pure full-length cDNA library is described by Carninci, et
al., (1996) Genomics 37:327-336. Other methods for producing
full-length libraries are known in the art. See, e.g., Edery, et
al., (1995) Mol. Cell Biol. 15(6):3363-3371 and PCT Application
Publication Number WO 1996/34981.
[0155] A2. Normalized or Subtracted cDNA Libraries
[0156] A non-normalized cDNA library represents the mRNA population
of the tissue it was made from. Since unique clones are
out-numbered by clones derived from highly expressed genes their
isolation can be laborious. Normalization of a cDNA library is the
process of creating a library in which each clone is more equally
represented. Construction of normalized libraries is described in
Ko, (1990) Nucl. Acids. Res. 18(19):5705-5711; Patanjali, et al.,
(1991) Proc. Natl. Acad. USA 88:1943-1947; U.S. Pat. Nos.
5,482,685, 5,482,845 and 5,637,685. In an exemplary method
described by Soares, et al., normalization resulted in reduction of
the abundance of clones from a range of four orders of magnitude to
a narrow range of only 1 order of magnitude. Proc. Natl. Acad. Sci.
USA 91:9228-9232 (1994).
[0157] Subtracted cDNA libraries are another means to increase the
proportion of less abundant cDNA species. In this procedure, cDNA
prepared from one pool of mRNA is depleted of sequences present in
a second pool of mRNA by hybridization. The cDNA: mRNA hybrids are
removed and the remaining un-hybridized cDNA pool is enriched for
sequences unique to that pool. See, Foote, et al. in, Plant
Molecular Biology: A Laboratory Manual, Clark, Ed.,
Springer-Verlag, Berlin (1997); Kho and Zarbl, (1991) Technique
3(2):58-63; Sive and St. John, (1988) Nucl. Acids Res.
16(22):10937; Current Protocols in Molecular Biology, Ausubel, et
al., Eds., Greene Publishing and Wiley-Interscience, New York
(1995) and Swaroop, et al., (1991) Nucl. Acids Res. 19(8):1954.
cDNA subtraction kits are commercially available. See, e.g.,
PCR-Select (Clontech, Palo Alto, Calif.).
[0158] To construct genomic libraries, large segments of genomic
DNA are generated by fragmentation, e.g., using restriction
endonucleases, and are ligated with vector DNA to form concatemers
that can be packaged into the appropriate vector. Methodologies to
accomplish these ends and sequencing methods to verify the sequence
of nucleic acids are well known in the art. Examples of appropriate
molecular biological techniques and instructions sufficient to
direct persons of skill through many construction, cloning, and
screening methodologies are found in Sambrook, et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide
to Molecular Cloning Techniques, Berger and Kimmel, Eds., San
Diego: Academic Press, Inc. (1987), Current Protocols in Molecular
Biology, Ausubel, et al., Eds., Greene Publishing and
Wiley-Interscience, New York (1995); Plant Molecular Biology: A
Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits
for construction of genomic libraries are also commercially
available.
[0159] The cDNA or genomic library can be screened using a probe
based upon the sequence of a polynucleotide of the present
invention such as those disclosed herein. Probes may be used to
hybridize with genomic DNA or cDNA sequences to isolate homologous
genes in the same or different plant species. Those of skill in the
art will appreciate that various degrees of stringency of
hybridization can be employed in the assay and either the
hybridization or the wash medium can be stringent.
[0160] The nucleic acids of interest can also be amplified from
nucleic acid samples using amplification techniques. For instance,
polymerase chain reaction (PCR) technology can be used to amplify
the sequences of polynucleotides of the present invention and
related genes directly from genomic DNA or cDNA libraries. PCR and
other in vitro amplification methods may also be useful, for
example, to clone nucleic acid sequences that code for proteins to
be expressed, to make nucleic acids to use as probes for detecting
the presence of the desired mRNA in samples, for nucleic acid
sequencing, or for other purposes. The T4 gene 32 protein
(Boehringer Mannheim) can be used to improve yield of long PCR
products.
[0161] PCR-based screening methods have been described. Wilfinger,
et al., describe a PCR-based method in which the longest cDNA is
identified in the first step so that incomplete clones can be
eliminated from study. Bio Techniques 22(3):481-486 (1997). Such
methods are particularly effective in combination with a
full-length cDNA construction methodology, above.
B. Synthetic Methods for Constructing Nucleic Acids
[0162] The isolated nucleic acids of the present invention can also
be prepared by direct chemical synthesis by methods such as the
phosphotriester method of Narang, et al., (1979) Meth. Enzymol.
68:90-99; the phosphodiester method of Brown, et al., (1979) Meth.
Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage,
et al., (1981) Tetra. Lett. 22:1859-1862; the solid phase
phosphoramidite triester method described by Beaucage and
Caruthers, (1981) Tetra. Letts. 22(20):1859-1862, e.g., using an
automated synthesizer, e.g., as described in Needham-VanDevanter,
et al., (1984) Nucleic Acids Res. 12:6159-6168 and the solid
support method of U.S. Pat. No. 4,458,066. Chemical synthesis
generally produces a single stranded oligonucleotide. This may be
converted into double stranded DNA by hybridization with a
complementary sequence or by polymerization with a DNA polymerase
using the single strand as a template. One of skill will recognize
that while chemical synthesis of DNA is best employed for sequences
of about 100 bases or less, longer sequences may be obtained by the
ligation of shorter sequences.
Recombinant Expression Cassettes
[0163] The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention. A nucleic acid sequence coding for the desired
polypeptide of the present invention, for example a cDNA or a
genomic sequence encoding a full length polypeptide of the present
invention, can be used to construct a recombinant expression
cassette which can be introduced into the desired host cell. A
recombinant expression cassette will typically comprise a
polynucleotide of the present invention operably linked to
transcriptional initiation regulatory sequences which will direct
the transcription of the polynucleotide in the intended host cell,
such as tissues of a transformed plant.
[0164] For example, plant expression vectors may include (1) a
cloned plant gene under the transcriptional control of 5' and 3'
regulatory sequences and (2) a dominant selectable marker. Such
plant expression vectors may also contain, if desired, a promoter
regulatory region (e.g., one conferring inducible or constitutive,
environmentally- or developmentally-regulated or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site and/or a polyadenylation signal.
[0165] A plant promoter fragment can be employed which will direct
expression of a polynucleotide of the present invention in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Examples of constitutive promoters include the
cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the
cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),
the Nos promoter, the pEmu promoter, the rubisco promoter, the
GRP1-8 promoter and other transcription initiation regions from
various plant genes known to those of skill. Constitutive promoters
of particular interest for use in soybean include SCP1 and
At-UBQ10.
[0166] Examples of promoters under developmental control include
promoters that initiate transcription only, or preferentially, in
certain tissues, such as leaves, roots, fruit, seeds, or flowers.
Exemplary promoters include the anther specific promoter 5126 (U.S.
Pat. Nos. 5,689,049 and 5,689,051), glob-1 promoter and gamma-zein
promoter.
[0167] Promoters of interest in constructs designed to drive
expression preferentially in female reproductive tissues include
the maize Zag2.1 promoter (GenBank Number X80206; Schmidt, et al.,
(1993) Plant Cell 5(7):729-737); maize Zap promoter (U.S. Pat. No.
7,560,612); maize ckx1-2 promoter (US Patent Application
Publication Number 2002/0152500 A1); ZM-ADF4 (US Patent Application
Publication Number 2009/0094713); maize eep1 promoter (US Patent
Application Publication Number 2004/0237147); maize end2 promoter,
(U.S. Pat. Nos. 6,528,704 and 6,903,205); maize lec1 promoter (U.S.
Pat. No. 7,122,658); maize F3.7 promoter (Baszczynski, et al.,
(1997) Maydica 42:189-201); maize tb1 promoter (Hubbarda, et al.,
(2002) Genetics 162:1927-1935); maize eep2 promoter (US Patent
Application Publication Number 2004/0237147); maize thioredoxinH
promoter, U.S. Provisional Patent Application Ser. No. 60/514,123);
maize Zm40 promoter (U.S. Pat. No. 6,403,862) maize mLIP15 promoter
(U.S. Pat. No. 6,479,734); maize ESR promoter (U.S. Pat. No.
7,276,596); maize PCNA2 promoter (US Patent Application Publication
Number 2005/0120404).
[0168] Root-preferred promoters include Zm-NAS2 (U.S. patent
application Ser. No. 12/030,455, filed Feb. 13, 2008), Zm-Cyclol
promoter (U.S. Pat. No. 7,268,226), Zm-Metallothionein promoters
(U.S. Pat. Nos. 6,774,282; 7,214,854 and 7,214,855 (also known as
RootMET2)), Zm-MSY promoter (SEQ ID NO: 64; U.S. Patent Application
Ser. No. 60/971,310 filed Sep. 11, 2007) or MsZRP promoter (SEQ ID
NO: 65; see, U.S. Pat. No. 5,633,363). Additional root-preferred
promoters include the VfENOD-GRP3 gene promoter (Kuster, et al.,
(1995) Plant Mol. Biol. 29(4):759-772); rolB promoter (Capana, et
al., (1994) Plant Mol. Biol. 25(4):681-691; and the CRWAQ81
root-preferred promoter with the ADH first intron (US Patent
Application Publication Number 2005/0097633). See also, U.S. Pat.
Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;
5,110,732 and 5,023,179.
[0169] Alternatively, the plant promoter may be under more precise
environmental control. Such promoters are referred to here as
"inducible" promoters. Environmental conditions that may effect
transcription by inducible promoters include pathogen attack,
anaerobic conditions, or the presence of light. Examples of
inducible promoters are the Adhl promoter which is inducible by
hypoxia or cold stress, the Hsp70 promoter which is inducible by
heat stress and the PPDK promoter which is inducible by light.
[0170] The operation of a promoter may also vary depending on its
location in the genome. Thus, an inducible promoter may become
fully or partially constitutive in certain locations.
[0171] Both heterologous and non-heterologous (i.e., endogenous)
promoters can be employed to direct expression of the nucleic acids
of the present invention. These promoters can also be used, for
example, in recombinant expression cassettes to drive expression of
antisense nucleic acids to reduce, increase, or alter concentration
and/or composition of the proteins of the present invention in a
desired tissue. Thus, in some embodiments, the nucleic acid
construct will comprise a promoter functional in a plant cell, such
as in Zea mays, operably linked to a polynucleotide of the present
invention. Promoters useful in these embodiments include the
endogenous promoters driving expression of a polypeptide of the
present invention.
[0172] In some embodiments, isolated nucleic acids which serve as
promoter or enhancer elements can be introduced in the appropriate
position (generally upstream) of a non-heterologous form of a
polynucleotide of the present invention so as to up or down
regulate expression of a polynucleotide of the present invention.
For example, endogenous promoters can be altered in vivo by
mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.
5,565,350; Zarling, et al., PCT Patent Application Number
PCT/US93/03868) or isolated promoters can be introduced into a
plant cell in the proper orientation and distance from a gene of
the present invention so as to control the expression of the gene.
Gene expression can be modulated under conditions suitable for
plant growth so as to alter the total concentration and/or alter
the composition of the polypeptides of the present invention in
plant cell.
[0173] Thus, the present invention provides compositions, and
methods for making, heterologous promoters and/or enhancers
operably linked to a native, endogenous (i.e., nonheterologous)
form of a polynucleotide of the present invention.
[0174] Methods for identifying promoters with a particular
expression pattern, in terms of, e.g., tissue type, cell type,
stage of development and/or environmental conditions, are well
known in the art. See, e.g., The Maize Handbook, Chapters 114-115,
Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn
Improvement, 3rd edition, Chapter 6, Sprague and Dudley, Eds.,
American Society of Agronomy, Madison, Wis. (1988).
[0175] A typical step in promoter isolation methods is
identification of gene products that are expressed with some degree
of specificity in the target tissue. Amongst the range of
methodologies are: differential hybridization to cDNA libraries;
subtractive hybridization; differential display; differential 2-D
protein gel electrophoresis; DNA probe arrays and isolation of
proteins known to be expressed with some specificity in the target
tissue. Such methods are well known to those of skill in the art.
Commercially available products for identifying promoters are known
in the art such as Clontech's (Palo Alto, Calif.) Universal Genome
Walker Kit.
[0176] For the protein-based methods, it is helpful to obtain the
amino acid sequence for at least a portion of the identified
protein, and then to use the protein sequence as the basis for
preparing a nucleic acid that can be used as a probe to identify
either genomic DNA directly, or preferably, to identify a cDNA
clone from a library prepared from the target tissue. Once such a
cDNA clone has been identified, that sequence can be used to
identify the sequence at the 5' end of the transcript of the
indicated gene. For differential hybridization, subtractive
hybridization and differential display, the nucleic acid sequence
identified as enriched in the target tissue is used to identify the
sequence at the 5' end of the transcript of the indicated gene.
Once such sequences are identified, starting either from protein
sequences or nucleic acid sequences, any of these sequences
identified as being from the gene transcript can be used to screen
a genomic library prepared from the target organism. Methods for
identifying and confirming the transcriptional start site are well
known in the art.
[0177] In the process of isolating promoters expressed under
particular environmental conditions or stresses, or in specific
tissues, or at particular developmental stages, a number of genes
are identified that are expressed under the desired circumstances,
in the desired tissue, or at the desired stage. Further analysis
will reveal expression of each particular gene in one or more other
tissues of the plant. One can identify a promoter with activity in
the desired tissue or condition but that does not have activity in
any other common tissue.
[0178] To identify the promoter sequence, the 5' portions of the
clones described here are analyzed for sequences characteristic of
promoter sequences. For instance, promoter sequence elements
include the TATA box consensus sequence (TATAAT), which is usually
an AT-rich stretch of 5-10 bp located approximately 20 to 40 base
pairs upstream of the transcription start site. Identification of
the TATA box is well known in the art. For example, one way to
predict the location of this element is to identify the
transcription start site using standard RNA-mapping techniques such
as primer extension, S 1 analysis, and/or RNase protection. To
confirm the presence of the AT-rich sequence, a structure-function
analysis can be performed involving mutagenesis of the putative
region and quantification of the mutation's effect on expression of
a linked downstream reporter gene. See, e.g., The Maize Handbook,
Chapter 114, Freeling and Walbot, Eds., Springer, New York,
(1994).
[0179] In plants, further upstream from the TATA box, at
positions-80 to-100, there is typically a promoter element (i.e.,
the CAAT box) with a series of adenines surrounding the
trinucleotide G (or T) N G. Messing, et al., in Genetic Engineering
in Plants, Kosage, Meredith and Hollaender, Eds., pp. 221-227 1983.
In maize, there is no well conserved CAAT box but there are several
short, conserved protein-binding motifs upstream of the TATA box.
These include motifs for the trans-acting transcription factors
involved in light regulation, anaerobic induction, hormonal
regulation or anthocyanin biosynthesis, as appropriate for each
gene.
[0180] Once promoter and/or gene sequences are known, a region of
suitable size is selected from the genomic DNA that is 5' to the
transcriptional start, or the translational start site and such
sequences are then linked to a coding sequence. If the
transcriptional start site is used as the point of fusion, any of a
number of possible 5' untranslated regions can be used in between
the transcriptional start site and the partial coding sequence. If
the translational start site at the 3' end of the specific promoter
is used, then it is linked directly to the methionine start codon
of a coding sequence.
[0181] If polypeptide expression is desired, it is generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region. The polyadenylation region can be
derived from the natural gene, from a variety of other plant genes,
or from T-DNA. The 3' end sequence to be added can be derived from,
for example, the nopaline synthase or octopine synthase genes or
alternatively from another plant gene or less preferably from any
other eukaryotic gene.
[0182] An intron sequence can be added to the 5' untranslated
region or the coding sequence of the partial coding sequence to
increase the amount of the mature message that accumulates in the
cytosol. Inclusion of a spliceable intron in the transcription unit
in both plant and animal expression constructs has been shown to
increase gene expression at both the mRNA and protein levels up to
1000-fold. Buchman and Berg, (1988) Mol. Cell Biol. 8:4395-4405;
Callis, et al., (1987) Genes Dev. 1:1183-1200. Such intron
enhancement of gene expression is typically greatest when placed
near the 5' end of the transcription unit. Use of maize introns
Adhl-S intron 1, 2 and 6, the Bronze-1 intron are known in the art.
See generally, The Maize Handbook, Chapter 116, Freeling and
Walbot, Eds., Springer, New York (1994).
[0183] The vector comprising the sequences from a polynucleotide of
the present invention will typically comprise a marker gene which
confers a selectable phenotype on plant cells. Usually, the
selectable marker gene will encode antibiotic resistance, with
suitable genes including genes coding for resistance to the
antibiotic spectinomycin (e.g., the aada gene), the streptomycin
phosphotransferase (SPT) gene coding for streptomycin resistance,
the neomycin phosphotransferase (NPTII) gene encoding kanamycin or
genetic in resistance, the hygromycin phosphotransferase (HPT) gene
coding for hygromycin resistance, genes coding for resistance to
herbicides which act to inhibit the action of acetolactate synthase
(ALS), in particular the sulfonylurea-type herbicides (e.g., the
acetolactate synthase (ALS) gene containing mutations leading to
such resistance in particular the S4 and/or Hra mutations), genes
coding for resistance to herbicides which act to inhibit action of
glutamine synthase, such as phosphinothricin or basta (e.g., the
bar gene) or other such genes known in the art. The bar gene
encodes resistance to the herbicide basta, the nptII gene encodes
resistance to the antibiotic kanamycin, and the ALS gene encodes
resistance to the herbicide chlorsulfuron.
[0184] Typical vectors useful for expression of genes in higher
plants are well known in the art and include vectors derived from
the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
described by Rogers, et al., (1987) Meth. in Enzymol. 153:253-277.
These vectors are plant integrating vectors in that on
transformation, the vectors integrate a portion of vector DNA into
the genome of the host plant. Exemplary A. tumefaciens vectors
useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al.,
(1987) Gene 61:1-11 and Berger, et al., (1989) Proc. Natl. Acad.
Sci. USA 86:8402-8406. Another useful vector herein is plasmid
pBI101.2 that is available from Clontech Laboratories, Inc. (Palo
Alto, Calif.).
[0185] A polynucleotide of the present invention can be expressed
in either sense or antisense orientation as desired. It will be
appreciated that control of gene expression in either sense or
anti-sense orientation can have a direct impact on the observable
plant characteristics. Antisense technology can be conveniently
used to inhibit gene expression in plants. To accomplish this, a
nucleic acid segment from the desired gene is cloned and operably
linked to a promoter such that the anti-sense strand of RNA will be
transcribed. The construct is then transformed into plants and the
antisense strand of RNA is produced.
[0186] In plant cells, it has been shown that antisense RNA
inhibits gene expression by preventing the accumulation of mRNA
which encodes the enzyme of interest, see, e.g., Sheehy, et al.,
(1988) Proc. Nat'l. Acad. Sci. (USA) 85:8805-8809 and Hiatt, et
al., U.S. Pat. No. 4,801,340.
[0187] Another method of suppression is sense suppression.
Introduction of nucleic acid configured in the sense orientation
has been shown to be an effective means by which to block the
transcription of target genes. For an example of the use of this
method to modulate expression of endogenous genes see, Napoli, et
al., (1990) The Plant Cell 2:279-289 and U.S. Pat. No.
5,034,323.
[0188] Catalytic RNA molecules or ribozymes can also be used to
inhibit expression of plant genes. It is possible to design
ribozymes that specifically pair with virtually any target RNA and
cleave the phosphodiester backbone at a specific location, thereby
functionally inactivating the target RNA. In carrying out this
cleavage, the ribozyme is not itself altered, and is thus capable
of recycling and cleaving other molecules, making it a true enzyme.
The inclusion of ribozyme sequences within antisense RNAs confers
RNA cleaving activity upon them, thereby increasing the activity of
the constructs. The design and use of target RNA-specific ribozymes
is described in Haseloff, et al., (1988) Nature 334:585-591. A
variety of cross-linking agents, alkylating agents and radical
generating species as pendant groups on polynucleotides of the
present invention can be used to bind, label, detect, and/or cleave
nucleic acids. For example, Vlassov, et al., (1986) Nucleic Acids
Res 14:4065-4076, describe covalent bonding of a single-stranded
DNA fragment with alkylating derivatives of nucleotides
complementary to target sequences. A report of similar work by the
same group is that by Knorre, et al., (1985) Biochimie 67:785 789.
Iverson and Dervan.
[0189] The present invention further provides a protein comprising
a polypeptide having a specified sequence identity with a
polypeptide of the present invention. The percentage of sequence
identity is an integer selected from the group consisting of from
60 to 99. Exemplary sequence identity values include 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%
to a full-length sequence of the invention.
[0190] As those of skill will appreciate, the present invention
includes catalytically active polypeptides of the present invention
(i.e., enzymes). Catalytically active polypeptides have a specific
activity of at least 20%, 30% or 40%, and preferably at least 50%,
60% or 70% and most preferably at least 80%, 90% or 95% that of the
native (non-synthetic), endogenous polypeptide. Further, the
substrate specificity (kcat/Km) is optionally substantially similar
to the native (non-synthetic), endogenous polypeptide. Typically,
the Km will be at least 30%, 40% or 50%, that of the native
(non-synthetic), endogenous polypeptide and more preferably at
least 60%, 70%, 80% or 90%. Methods of assaying and quantifying
measures of enzymatic activity and substrate specificity (heat/Km),
are well known to those of skill in the art.
[0191] Generally, the proteins of the present invention will, when
presented as an immunogen, elicit production of an antibody
specifically reactive to a polypeptide of the present invention.
Further, the proteins of the present invention will not bind to
antisera raised against a polypeptide of the present invention
which has been fully immunosorbed with the same polypeptide.
Immunoassays for determining binding are well known to those of
skill in the art. A preferred immunoassay is a competitive
immunoassay as discussed, infra. Thus, the proteins of the present
invention can be employed as immunogens for constructing antibodies
immunoreactive to a protein of the present invention for such
exemplary utilities as immunoassays or protein purification
techniques.
Expression of Proteins in Host Cells
[0192] Using the nucleic acids of the present invention, one may
express a protein of the present invention in a recombinantly
engineered cell such as bacteria, yeast, insect, mammalian or
preferably plant cells. The cells produce the protein in a
non-natural condition (e.g., in quantity, composition, location
and/or time), because they have been genetically altered through
human intervention to do so.
[0193] It is expected that those of skill in the art are
knowledgeable in the numerous expression systems available for
expression of a nucleic acid encoding a protein of the present
invention. No attempt to describe in detail the various methods
known for the expression of proteins in prokaryotes or eukaryotes
will be made.
[0194] In brief summary, the expression of isolated nucleic acids
encoding a protein of the present invention will typically be
achieved by operably linking, for example, the DNA or cDNA to a
promoter (which is either constitutive or regulatable), followed by
incorporation into an expression vector. The vectors can be
suitable for replication and integration in either prokaryotes or
eukaryotes. Typical expression vectors contain transcription and
translation terminators, initiation sequences, and promoters useful
for regulation of the expression of the DNA encoding a protein of
the present invention. To obtain high level expression of a cloned
gene, it is desirable to construct expression vectors which
contain, at the minimum, a strong promoter to direct transcription,
a ribosome binding site for translational initiation and a
transcription/translation terminator. One of skill would recognize
that modifications can be made to a protein of the present
invention without diminishing its biological activity. Some
modifications may be made to facilitate the cloning, expression, or
incorporation of the targeting molecule into a fusion protein.
[0195] Such modifications are well known to those of skill in the
art and include, for example, a methionine added at the amino
terminus to provide an initiation site, or additional amino acids
(e.g., poly His) placed on either terminus to create conveniently
located purification sequences. Restriction sites or termination
codons can also be introduced.
A. Expression in Prokaryotes
[0196] Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used.
Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site
sequences, include such commonly used promoters as the beta
lactamase (penicillinase) and lactose (lac) promoter systems
(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp)
promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057)
and the lambda derived P L promoter and N-gene ribosome binding
site (Shimatake, et al., (1981) Nature 292:128). The inclusion of
selection markers in DNA vectors transfected in E. coli is also
useful. Examples of such markers include genes specifying
resistance to ampicillin, tetracycline, or chloramphenicol.
[0197] The vector is selected to allow introduction into the
appropriate host cell. Bacterial vectors are typically of plasmid
or phage origin. Appropriate bacterial cells are infected with
phage vector particles or transfected with naked phage vector DNA.
If a plasmid vector is used, the bacterial cells are transfected
with the plasmid vector DNA. Expression systems for expressing a
protein of the present invention are available using Bacillus sp.
and Salmonella (Palva, et al., (1983) Gene 22:229-235; Mosbach, et
al., (1983) Nature 302:543-545).
B. Expression in Eukaryotes
[0198] A variety of eukaryotic expression systems such as yeast,
insect cell lines, plant and mammalian cells, are known to those of
skill in the art. As explained briefly below, a polynucleotide of
the present invention can be expressed in these eukaryotic systems.
In some embodiments, transformed/transfected plant cells, as
discussed infra, are employed as expression systems for production
of the proteins of the instant invention.
[0199] Synthesis of heterologous proteins in yeast is well known.
Sherman, et al., Methods in Yeast Genetics, Cold Spring Harbor
Laboratory (1982) is a well recognized work describing the various
methods available to produce the protein in yeast. Two widely
utilized yeast for production of eukaryotic proteins are
Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and
protocols for expression in Saccharomyces and Pichia are known in
the art and available from commercial suppliers (e.g.,
Invitrogen).
[0200] Suitable vectors usually have expression control sequences,
such as promoters, including 3-phosphoglycerate kinase or alcohol
oxidase and an origin of replication, termination sequences and the
like as desired.
[0201] A protein of the present invention, once expressed, can be
isolated from yeast by lysing the cells and applying standard
protein isolation techniques to the lysate. The monitoring of the
purification process can be accomplished by using Western blot
techniques or radioimmunoassay or other standard immunoassay
techniques.
[0202] The sequences encoding proteins of the present invention can
also be ligated to various expression vectors for use in
transfecting cell cultures of, for instance, mammalian, insect or
plant origin. Illustrative of cell cultures useful for the
production of the peptides are mammalian cells. Mammalian cell
systems often will be in the form of monolayers of cells although
mammalian cell suspensions may also be used. A number of suitable
host cell lines capable of expressing intact proteins have been
developed in the art, and include the HEK293, BHK21 and CHO cell
lines. Expression vectors for these cells can include expression
control sequences, such as an origin of replication, a promoter
(e.g., the CMV promoter, a HSVtk promoter or pgk (phosphoglycerate
kinase) promoter), an enhancer (Queen, et al., (1986) Immunol. Rev.
89:49) and necessary processing information sites, such as ribosome
binding sites, RNA splice sites, polyadenylation sites (e.g., an
SV40 large T Ag poly A addition site) and transcriptional
terminator sequences. Other animal cells useful for production of
proteins of the present invention are available, for instance, from
the American Type Culture Collection.
[0203] Appropriate vectors for expressing proteins of the present
invention in insect cells are usually derived from the SF9
baculovirus. Suitable insect cell lines include mosquito larvae,
silkworm, army worm, moth and Drosophila cell lines such as a
Schneider cell line (see, Schneider, (1987) Embryol. Exp. Morphol.
27:353-365.
[0204] As with yeast, when higher animal or plant host cells are
employed, polyadenylation or transcription terminator sequences are
typically incorporated into the vector. An example of a terminator
sequence is the polyadenylation sequence from the bovine growth
hormone gene. Sequences for accurate splicing of the transcript may
also be included. An example of a splicing sequence is the VP1
intron from SV40 (Sprague, et al., (1983) J. Virol. 45:773-781).
Additionally, gene sequences to control replication in the host
cell may be incorporated into the vector such as those found in
bovine papilloma virus type-vectors. Saveria-Campo, Bovine
Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning Vol.
II a Practical Approach, Glover, Ed., IRL Press, Arlington, Va. pp.
213-238 (1985).
Increasing the Activity and/or Level of an ETO1 Polypeptide
[0205] Methods are provided to increase the activity and/or level
of the ETO1 polypeptide of the invention. An increase in the level
and/or activity of the ETO1 polypeptide of the invention can be
achieved by providing to the plant an ETO1 polypeptide. The ETO1
polypeptide can be provided by introducing the amino acid sequence
encoding the ETO1 polypeptide into the plant, introducing into the
plant a nucleotide sequence encoding an ETO1 polypeptide or
alternatively by modifying a genomic locus encoding the ETO1
polypeptide of the invention.
[0206] As discussed elsewhere herein, many methods are known in the
art for providing a polypeptide to a plant including, but not
limited to, direct introduction of the polypeptide into the plant,
introducing into the plant (transiently or stably) a polynucleotide
construct encoding a polypeptide having enhanced activity. It is
also recognized that the methods of the invention may employ a
polynucleotide that is not capable of directing, in the transformed
plant, the expression of a protein or an RNA. Thus, the level
and/or activity of an ETO1 polypeptide may be increased by altering
the gene encoding the ETO1 polypeptide or its promoter. See, e.g.,
Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT Application
Serial Number PCT/US93/03868. Therefore mutagenized plants that
carry mutations in ETO1 genes, where the mutations increase
expression of the ETO1 or increase the activity of the encoded ETO1
polypeptide, are provided.
Reducing the Activity and/or Level of an ETO1 Polypeptide
[0207] In certain embodiments, methods are provided to reduce or
eliminate the activity of an ETO1 polypeptide of the invention by
transforming a plant cell with an expression cassette that
expresses a polynucleotide that inhibits the expression of the ETO1
polypeptide. The polynucleotide may inhibit the expression of the
ETO1 polypeptide directly, by preventing transcription or
translation of the ETO1 associated messenger RNA or indirectly, by
encoding a polypeptide that inhibits the transcription or
translation of an ETO1 gene encoding ETO1 polypeptide. Methods for
inhibiting or eliminating the expression of a gene in a plant are
well known in the art and any such method may be used in the
present invention to inhibit the expression of ETO1
polypeptide.
[0208] In accordance with the present invention, the expression of
an ETO1 polypeptide is inhibited if the protein level of the ETO1
polypeptide is less than 70% of the protein level of the same ETO1
polypeptide in a plant that has not been genetically modified or
mutagenized to inhibit the expression of that ETO1 polypeptide. In
particular embodiments of the invention, the protein level of the
ETO1 polypeptide in a modified plant according to the invention is
less than 60%, less than 50%, less than 40%, less than 30%, less
than 20%, less than 10%, less than 5% or less than 2% of the
protein level of the same ETO1 polypeptide in a plant that is not a
mutant or that has not been genetically modified to inhibit the
expression of that ETO1 polypeptide. The expression level of the
ETO1 polypeptide may be measured directly, for example, by assaying
for the level of ETO1 polypeptide expressed in the plant cell or
plant, or indirectly, for example, by measuring the ethylene
response in the plant cell or plant, or by measuring the is
phenotypic changes in the plant. Methods for performing such assays
are described elsewhere herein.
[0209] In other embodiments of the invention, the activity of the
ETO1 polypeptide is reduced or eliminated by transforming a plant
cell with an expression cassette comprising a polynucleotide
encoding a polypeptide that inhibits the activity of an ETO1
polypeptide. The activity of an ETO1 polypeptide is inhibited
according to the present invention if the activity of the ETO1
polypeptide is less than 70% of the activity of the same ETO1
polypeptide in a plant that has not been modified to inhibit the
activity of that polypeptide. In particular embodiments of the
invention, the activity of the ETO1 polypeptide in a modified plant
according to the invention is less than 60%, less than 50%, less
than 40%, less than 30%, less than 20%, less than 10% or less than
5% of the activity of the same polypeptide in a plant that that has
not been modified to inhibit the expression of that ETO1
polypeptide. The activity of an ETO1 polypeptide is "eliminated"
according to the invention when it is not detectable by the assay
methods described elsewhere herein. Methods of determining the
alteration of activity of an ETO1 polypeptide are described
elsewhere herein.
[0210] In other embodiments, the activity of an ETO1 polypeptide
may be reduced or eliminated by disrupting the gene encoding the
ETO1 polypeptide. The invention encompasses mutagenized plants that
carry mutations in ETO1 genes, where the mutations reduce
expression of the associated gene or inhibit the activity of the
encoded ETO1 polypeptide.
[0211] Thus, many methods may be used to reduce or eliminate the
activity of an ETO1 polypeptide. In addition, more than one method
may be used to reduce the activity of a single ETO1
polypeptide.
[0212] 1. Polynucleotide-Based Methods:
[0213] In some embodiments of the present invention, a plant is
transformed with an expression cassette that is capable of
expressing a polynucleotide that inhibits the expression of an ETO1
polypeptide of the invention. The term "expression" as used herein
refers to the biosynthesis of a gene product, including the
transcription and/or translation of said gene product. For example,
for the purposes of the present invention, an expression cassette
capable of expressing a polynucleotide that inhibits the expression
of at least one ETO1 polypeptide is an expression cassette capable
of producing an RNA molecule that inhibits the transcription and/or
translation of at least one ETO1 polypeptide of the invention. The
"expression" or "production" of a protein or polypeptide from a DNA
molecule refers to the transcription and translation of the coding
sequence to produce the protein or polypeptide, while the
"expression" or "production" of a protein or polypeptide from an
RNA molecule refers to the translation of the RNA coding sequence
to produce the protein or polypeptide.
[0214] Examples of polynucleotides and methodology that inhibit the
expression of an ETO1 polypeptide include, sense suppression,
cosuppression, antisense suppression, double stranded RNA
interference, hairpin RNA Interference, intron-containing hairpin
RNA interference, amplicon-mediated interference, ribozymes, small
interfering RNA or micro RNA. Other methods of inhibition can
include polypeptide-based inhibition of gene expression, or of
protein activity as well as gene disruption.
[0215] 2. Mutant Plants with Reduced Activity:
[0216] Additional methods for decreasing or eliminating the
expression of endogenous genes in plants are also known in the art
and can be similarly applied to the instant invention. These
methods include other forms of mutagenesis, such as ethyl
methanesulfonate-induced mutagenesis, deletion mutagenesis, and
fast neutron deletion mutagenesis used in a reverse genetics sense
(with PCR) to identify plant lines in which the endogenous gene has
been deleted. For examples of these methods see, Ohshima, et al.,
(1998) Virology 243:472-481; Okubara, et al., (1994) Genetics
137:867-874 and Quesada, et al., (2000) Genetics 154:421-436; each
of which is herein incorporated by reference. In addition, a fast
and automatable method for screening for chemically induced
mutations, TILLING (Targeting Induced Local Lesions In Genomes),
using denaturing HPLC or selective endonuclease digestion of
selected PCR products is also applicable to the instant invention.
See, McCallum, et al., (2000) Nat. Biotechnol. 18:455-457, herein
incorporated by reference.
[0217] Mutations that impact gene expression or that interfere with
the function (enhanced activity) of the encoded protein are well
known in the art. Insertional mutations in gene exons usually
result in null-mutants. Mutations in conserved residues are
particularly effective in inhibiting the activity of the encoded
protein. Conserved residues of plant ETO1 polypeptides suitable for
mutagenesis with the goal to eliminate activity have been
described. Such mutants can be isolated according to well-known
procedures, and mutations in different ETO1 associated loci can be
stacked by genetic crossing. See, for example, Gruis, et al.,
(2002) Plant Cell 14:2863-2882.
[0218] The invention encompasses additional methods for reducing or
eliminating the activity of one or more ETO1 polypeptide. Examples
of other methods for altering or mutating a genomic nucleotide
sequence in a plant are known in the art and include, but are not
limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors,
RNA:DNA repair vectors, mixed-duplex oligonucleotides,
self-complementary RNA:DNA oligonucleotides and recombinogenic
oligonucleobases. Such vectors and methods of use are known in the
art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181;
5,756,325; 5,760,012; 5,795,972 and 5,871,984; each of which are
herein incorporated by reference. See also, WO 1998/49350, WO
1999/07865, WO 1999/25821 and Beetham, et al., (1999) Proc. Natl.
Acad. Sci. USA 96:8774-8778, each of which is herein incorporated
by reference.
Transfection/Transformation of Cells
[0219] The method of transformation/transfection is not critical to
the instant invention; various methods of transformation or
transfection are currently available. As newer methods are
available to transform crops or other host cells they may be
directly applied.
[0220] Accordingly, a wide variety of methods have been developed
to insert a DNA sequence into the genome of a host cell to obtain
the transcription and/or translation of the sequence to effect
phenotypic changes in the organism. Thus, any method which provides
for effective transformation/transfection may be employed.
A. Plant Transformation
[0221] A DNA sequence coding for the desired polypeptide of the
present invention, for example a cDNA or a genomic sequence
encoding a full length protein, will be used to construct a
recombinant expression cassette which can be introduced into the
desired plant.
[0222] Isolated nucleic acid acids of the present invention can be
introduced into plants according to techniques known in the art.
Generally, recombinant expression cassettes as described above and
suitable for transformation of plant cells are prepared. Techniques
for transforming a wide variety of higher plant species are well
known and described in the technical, scientific and patent
literature. See, for example, Weising et al., (1988) Ann. Rev.
Genet. 22:421-477. For example, the DNA construct may be introduced
directly into the genomic DNA of the plant cell using techniques
such as electroporation, polyethylene glycol (PEG), poration,
particle bombardment, silicon fiber delivery or microinjection of
plant cell protoplasts or embryogenic callus. See, e.g., Tomes, et
al., Direct DNA Transfer into Intact Plant Cells Via
Microprojectile Bombardment. pp. 197213 in Plant Cell, Tissue and
Organ Culture, Fundamental Methods. eds. Gamborg and Phillips.
Springer-Verlag Berlin Heidelberg New York, 1995. Alternatively,
the DNA constructs may be combined with suitable T-DNA flanking
regions and introduced into a conventional Agrobacterium
tumefaciens host vector. The virulence functions of the
Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria. See, U.S. Pat. No. 5,591,616.
[0223] The introduction of DNA constructs using PEG precipitation
is described in Paszkowski, et al., (1984) Embo J. 3:2717-2722.
Electroporation techniques are described in Fromm, et al., (1985)
Proc. Natl. Acad. Sci. (USA) 82:5824. Ballistic transformation
techniques are described in Klein et al., (1987) Nature
327:70-73.
[0224] Agrobacterium tumefaciens-mediated transformation techniques
are well described in the scientific literature. See, for example
Horsch, et al., (1984) Science 233:496-498 and Fraley et al.,
(1983) Proc. Natl. Acad. Sci. (USA) 80:4803. Although Agrobacterium
is useful primarily in dicots, certain monocots can be transformed
by Agrobacterium. For instance, Agrobacterium transformation of
maize is described in U.S. Pat. No. 5,550,318.
[0225] Other methods of transfection or transformation include (1)
Agrobacterium rhizogenes-mediated transformation (see, e.g.,
Lichtenstein and Fuller In: Genetic Engineering, vol. 6, PWJ Rigby,
Ed., London, Academic Press, 1987; and Lichtenstein and Draper, In:
DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985),
PCT Application Number PCT/US87/02512 (WO 1988/02405 published Apr.
7, 1988) describes the use of A. rhizogenes strain A4 and its Ri
plasmid along with A. tumefaciens vectors pARC8 orpARC16 (2)
liposome-mediated DNA uptake (see, e.g., Freeman, et al., (1984)
Plant Cell Physiol. 25:1353), (3) the vortexing method (see, e.g.,
Kindle, (1990) Proc. Natl. Acad. Sci., (USA) 87:1228).
[0226] DNA can also be introduced into plants by direct DNA
transfer into pollen as described by Zhou, et al., (1983) Methods
in Enzymology 101:433; Hess, (1987) Intern Rev. Cytol. 107:367;
Luo, et al., (1988) Plant Mol. Biol. Reporter 6:165. Expression of
polypeptide coding genes can be obtained by injection of the DNA
into reproductive organs of a plant as described by Pena, et al.,
(1987) Nature 325:274.
[0227] DNA can also be injected directly into the cells of immature
embryos and the rehydration of desiccated embryos as described by
Neuhaus, et al., (1987) Theor. Appl. Genet. 75:30 and Benbrook, et
al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass.,
pp. 27-54 (1986). A variety of plant viruses that can be employed
as vectors are known in the art and include cauliflower mosaic
virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic
virus.
B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal
Cells
[0228] Animal and lower eukaryotic (e.g., yeast) host cells are
competent or rendered competent for transfection by various means.
There are several well-known methods of introducing DNA into animal
cells. These include: calcium phosphate precipitation, fusion of
the recipient cells with bacterial protoplasts containing the DNA,
treatment of the recipient cells with liposomes containing the DNA,
DEAE dextran, electroporation, biolistics and micro-injection of
the DNA directly into the cells. The transfected cells are cultured
by means well known in the art. Kuchler, Biochemical Methods in
Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc.
(1977).
Synthesis of Proteins
[0229] The proteins of the present invention can be constructed
using non-cellular synthetic methods. Solid phase synthesis of
proteins of less than about 50 amino acids in length may be
accomplished by attaching the C-terminal amino acid of the sequence
to an insoluble support followed by sequential addition of the
remaining amino acids in the sequence. Techniques for solid phase
synthesis are described by Barany and Merrifield, Solid-Phase
Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis,
Biology, Vol. 2: Special Methods in Peptide Synthesis, Part A.;
Merrifield, et al., (1963) J. Am. Chem. Soc. 85:2149-2156 and
Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce
Chem. Co., Rockford, Ill. (1984). Proteins of greater length may be
synthesized by condensation of the amino and carboxy termini of
shorter fragments. Methods of forming peptide bonds by activation
of a carboxy terminal end (e.g., by the use of the coupling reagent
N,N'-dicycylohexylcarbodiimide) are known to those of skill.
Purification of Proteins
[0230] The proteins of the present invention may be purified by
standard techniques well known to those of skill in the art.
Recombinantly produced proteins of the present invention can be
directly expressed or expressed as a fusion protein. The
recombinant protein is purified by a combination of cell lysis
(e.g., sonication, French press) and affinity chromatography. For
fusion products, subsequent digestion of the fusion protein with an
appropriate proteolytic enzyme releases the desired recombinant
protein.
[0231] The proteins of this invention, recombinant or synthetic,
may be purified to substantial purity by standard techniques well
known in the art, including detergent solubilization, selective
precipitation with such substances as ammonium sulfate, column
chromatography, immunopurification methods and others. See, for
instance, Scopes, Protein Purification: Principles and Practice,
Springer-Verlag: New York (1982); Deutscher, Guide to Protein
Purification, Academic Press (1990). For example, antibodies may be
raised to the proteins as described herein. Purification from E.
coli can be achieved following procedures described in U.S. Pat.
No. 4,511,503. The protein may then be isolated from cells
expressing the protein and further purified by standard protein
chemistry techniques as described herein. Detection of the
expressed protein is achieved by methods known in the art and
include, for example, radioimmunoassays, Western blotting
techniques or immunoprecipitation.
Transgenic Plant Regeneration
[0232] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant which possesses the transformed genotype. Such
regeneration techniques often rely on manipulation of certain
phytohormones in a tissue culture growth medium. For transformation
and regeneration of maize see, Gordon-Kamm, et al., (1990) The
Plant Cell 2:603-618.
[0233] Plants cells transformed with a plant expression vector can
be regenerated, e.g., from single cells, callus tissue or leaf
discs according to standard plant tissue culture techniques. It is
well known in the art that various cells, tissues and organs from
almost any plant can be successfully cultured to regenerate an
entire plant. Plant regeneration from cultured protoplasts is
described in Evans, et al., Protoplasts Isolation and Culture,
Handbook of Plant Cell Culture, Macmillan Publishing Company, New
York, pp. 124-176 (1983) and Binding, Regeneration of Plants, Plant
Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).
[0234] The regeneration of plants containing the foreign gene
introduced by Agrobacterium from leaf explants can be achieved as
described by Horsch, et al., (1985) Science 227:1229-1231. In this
procedure, transformants are grown in the presence of a selection
agent and in a medium that induces the regeneration of shoots in
the plant species being transformed as described by Fraley, et al.,
(1983) Proc. Natl. Acad. Sci. USA 80:4803. This procedure typically
produces shoots within two to four weeks and these transformant
shoots are then transferred to an appropriate root-inducing medium
containing the selective agent and an antibiotic to prevent
bacterial growth. Transgenic plants of the present invention may be
fertile or sterile.
[0235] Regeneration can also be obtained from plant callus,
explants, organs, or parts thereof. Such regeneration techniques
are described generally in Kleen, et al., (1987) Ann. Rev. of Plant
Phys. 38:467-486. The regeneration of plants from either single
plant protoplasts or various explants is well known in the art.
See, for example, Methods for Plant Molecular Biology, A. Weissbach
and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif.
(1988). This regeneration and growth process includes the steps of
selection of transformant cells and shoots, rooting the
transformant shoots and growth of the plantlets in soil. For maize
cell culture and regeneration see generally, The Maize Handbook,
Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn
Improvement, 3rd edition, Sprague and Dudley Eds., American Society
of Agronomy, Madison, Wis. (1988).
[0236] One of skill will recognize that after the recombinant
expression cassette is stably incorporated in transgenic plants and
confirmed to be operable, it can be introduced into other plants by
sexual crossing. Any of a number of standard breeding techniques
can be used, depending upon the species to be crossed. In
vegetatively propagated crops, mature transgenic plants can be
propagated by the taking of cuttings or by tissue culture
techniques to produce multiple identical plants.
[0237] Selection of desirable transgenics is made and new varieties
are obtained and propagated vegetatively for commercial use. In
seed propagated crops, mature transgenic plants can be self crossed
to produce a homozygous inbred plant. The inbred plant produces
seed containing the newly introduced heterologous nucleic acid.
These seeds can be grown to produce plants that would produce the
selected phenotype.
[0238] Parts obtained from the regenerated plant, such as flowers,
seeds, leaves, branches, fruit, and the like are included in the
invention, provided that these parts comprise cells comprising the
isolated nucleic acid of the present invention. Progeny and
variants, and mutants of the regenerated plants are also included
within the scope of the invention, provided that these parts
comprise the introduced nucleic acid sequences. Transgenic plants
expressing the selectable marker can be screened for transmission
of the nucleic acid of the present invention by, for example,
standard immunoblot and DNA detection techniques. Transgenic lines
are also typically evaluated on levels of expression of the
heterologous nucleic acid. Expression at the RNA level can be
determined initially to identify and quantitate expression-positive
plants. Standard techniques for RNA analysis can be employed and
include PCR amplification assays using oligonucleotide primers
designed to amplify only the heterologous RNA templates and
solution hybridization assays using heterologous nucleic
acid-specific probes. The RNA-positive plants can then analyzed for
protein expression by Western immunoblot analysis using the
specifically reactive antibodies of the present invention. In
addition, in situ hybridization and immunocytochemistry according
to standard protocols can be done using heterologous nucleic acid
specific polynucleotide probes and antibodies, respectively, to
localize sites of expression within transgenic tissue. Generally, a
number of transgenic lines are usually screened for the
incorporated nucleic acid to identify and select plants with the
most appropriate expression profiles.
[0239] A preferred embodiment is a transgenic plant that is
homozygous for the added heterologous nucleic acid; i.e., a
transgenic plant that contains two added nucleic acid sequences,
one gene at the same locus on each chromosome of a chromosome pair.
A homozygous transgenic plant can be obtained by sexually mating
(selfing) a heterozygous transgenic plant that contains a single
added heterologous nucleic acid, germinating some of the seed
produced and analyzing the resulting plants produced for altered
expression of a polynucleotide of the present invention relative to
a control plant (i.e., native, nontransgenic). Back-crossing to a
parental plant and out-crossing with a non-transgenic plant are
also contemplated.
Modulation of Polypeptide Levels and/or Composition
[0240] The present invention further provides a method for
modulating (i.e., increasing or decreasing) the concentration or
ratio of the polypeptides of the present invention in a plant or
part thereof. Modulation can be effected by increasing or
decreasing the concentration and/or the ratio of the polypeptides
of the present invention in a plant.
[0241] The method comprises introducing into a plant cell a
recombinant expression cassette comprising a polynucleotide of the
present invention as described above to obtain a transformed plant
cell, culturing the transformed plant cell under plant cell growing
conditions and inducing or repressing expression of a
polynucleotide of the present invention in the plant for a time
sufficient to modulate concentration and/or the ratios of the
polypeptides in the plant or plant part.
[0242] In some embodiments, the concentration and/or ratios of
polypeptides of the present invention in a plant may be modulated
by altering, in vivo or in vitro, the promoter of a gene to up- or
down-regulate gene expression. In some embodiments, the coding
regions of native genes of the present invention can be altered via
substitution, addition, insertion, or deletion to decrease activity
of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350;
Zarling, et al., PCT Patent Application Number PCT/US93/03868. And
in some embodiments, an isolated nucleic acid (e.g., a vector)
comprising a promoter sequence is transfected into a plant
cell.
[0243] Subsequently, a plant cell comprising the promoter operably
linked to a polynucleotide of the present invention is selected for
by means known to those of skill in the art, such as, but not
limited to, Southern blot, DNA sequencing or PCR analysis using
primers specific to the promoter and to the gene and detecting
amplicons produced therefrom. A plant or plant part altered or
modified by the foregoing embodiments is grown under plant forming
conditions for a time sufficient to modulate the concentration
and/or ratios of polypeptides of the present invention in the
plant. Plant forming conditions are well known in the art and
discussed briefly, supra.
[0244] In general, concentration or the ratios of the polypeptides
is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80% or 90% relative to a native control plant, plant
part, or cell lacking the aforementioned recombinant expression
cassette. Modulation in the present invention may occur during
and/or subsequent to growth of the plant to the desired stage of
development. Modulating nucleic acid expression temporally and/or
in particular tissues can be controlled by employing the
appropriate promoter operably linked to a polynucleotide of the
present invention in, for example, sense or antisense orientation
as discussed in greater detail, supra. Induction of expression of a
polynucleotide of the present invention can also be controlled by
exogenous administration of an effective amount of inducing
compound. Inducible promoters and inducing compounds which activate
expression from these promoters are well known in the art. In
preferred embodiments, the polypeptides of the present invention
are modulated in monocots, particularly maize.
Molecular Markers
[0245] The present invention provides a method of genotyping a
plant comprising a polynucleotide of the present invention.
Optionally, the plant is a monocot, such as maize or sorghum.
Genotyping provides a means of distinguishing homologs of a
chromosome pair and can be used to differentiate segregants in a
plant population. Molecular marker methods can be used for
phylogenetic studies, characterizing genetic relationships among
crop varieties, identifying crosses or somatic hybrids, localizing
chromosomal segments affecting monogenic traits, map based cloning
and the study of quantitative inheritance. See, e.g., Clark, Ed.,
Plant Molecular Biology: A Laboratory Manual. Berlin, Springer
Verlag, 1997, Chapter 7. For molecular marker methods, see
generally, "The DNA Revolution" in: Paterson, Genome Mapping in
Plants (Austin, Tex., Academic Press/R. G. Landis Company, 1996)
pp. 7-21.
[0246] The particular method of genotyping in the present invention
may employ any number of molecular marker analytic techniques such
as, but not limited to, restriction fragment length polymorphisms
(RFLPs). RFLPs are the product of allelic differences between DNA
restriction fragments resulting from nucleotide sequence
variability. As is well known to those of skill in the art, RFLPs
are typically detected by extraction of genomic DNA and digestion
with a restriction enzyme. Generally, the resulting fragments are
separated according to size and hybridized with a probe; single
copy probes are preferred. Restriction fragments from homologous
chromosomes are revealed.
[0247] Differences in fragment size among alleles represent an
RFLP. Thus, the present invention further provides a means to
follow segregation of a gene or nucleic acid of the present
invention as well as chromosomal sequences genetically linked to
these genes or nucleic acids using such techniques as RFLP
analysis. Linked chromosomal sequences are within 50 centiMorgans
(cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more
preferably within 5, 3, 2 or 1 cM of a gene of the present
invention.
[0248] In the present invention, the nucleic acid probes employed
for molecular marker mapping of plant nuclear genomes selectively
hybridize, under selective hybridization conditions, to a gene
encoding a polynucleotide of the present invention. In preferred
embodiments, the probes are selected from polynucleotides of the
present invention.
[0249] Typically, these probes are cDNA probes or
restriction-enzyme treated (e.g., Pst I) genomic clones. The length
of the probes is discussed in greater detail, supra, but are
typically at least 15 bases in length, more preferably at least 20,
25, 30, 35, 40 or 50 bases in length. Generally, however, the
probes are less than about 1 kilobase in length. Preferably, the
probes are single copy probes that hybridize to a unique locus in a
haploid chromosome complement. Some exemplary restriction enzymes
employed in RFLP mapping are EcoRl, EcoRv and Sstl. As used herein
the term "restriction enzyme" includes reference to a composition
that recognizes and alone or in conjunction with another
composition, cleaves at a specific nucleotide sequence.
[0250] The method of detecting an RFLP comprises the steps of (a)
digesting genomic DNA of a plant with a restriction enzyme; (b)
hybridizing a nucleic acid probe, under selective hybridization
conditions, to a sequence of a polynucleotide of the present of
said genomic DNA; (c) detecting therefrom a RFLP. Other methods of
differentiating polymorphic (allelic) variants of polynucleotides
of the present invention can be had by utilizing molecular marker
techniques well known to those of skill in the art including such
techniques as: 1) single stranded conformation analysis (SSCA); 2)
denaturing gradient gel electrophoresis (DGGE); 3) RNase protection
assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of
proteins which recognize nucleotide mismatches, such as the E. coli
mutS protein and 6) allele-specific PCR. Other approaches based on
the detection of mismatches between the two complementary DNA
strands include clamped denaturing gel electrophoresis (CDGE);
heteroduplex analysis (HA); and chemical mismatch cleavage (CMC).
Thus, the present invention further provides a method of genotyping
comprising the steps of contacting, under stringent hybridization
conditions, a sample suspected of comprising a polynucleotide of
the present invention with a nucleic acid probe. Generally, the
sample is a plant sample; preferably, a sample suspected of
comprising a maize polynucleotide of the present invention (e.g.,
gene, mRNA). The nucleic acid probe selectively hybridizes, under
stringent conditions, to a subsequence of a polynucleotide of the
present invention comprising a polymorphic marker. Selective
hybridization of the nucleic acid probe to the polymorphic marker
nucleic acid sequence yields a hybridization complex. Detection of
the hybridization complex indicates the presence of that
polymorphic marker in the sample. In preferred embodiments, the
nucleic acid probe comprises a polynucleotide of the present
invention.
UTRs and Codon Preference
[0251] In general, translational efficiency has been found to be
regulated by specific sequence elements in the 5' non-coding or
untranslated region (5' UTR) of the RNA. Positive sequence motifs
include translational initiation consensus sequences (Kozak, (1987)
Nucleic Acids Res. 15:8125) and the 7-methylguanosine cap structure
(Drummond, et al., (1985) Nucleic Acids Res. 13:7375). Negative
elements include stable intramolecular 5' UTR stem-loop structures
(Muesing, et al., (1987) Cell 48:691) and AUG sequences or short
open reading frames preceded by an appropriate AUG in the 5' UTR
(Kozak, supra, Rao, et al., (1988) Mol. and Cell. Biol. 8:284).
Accordingly, the present invention provides 5' and/or 3'
untranslated regions for modulation of translation of heterologous
coding sequences.
[0252] Further, the polypeptide-encoding segments of the
polynucleotides of the present invention can be modified to alter
codon usage. Altered codon usage can be employed to alter
translational efficiency and/or to optimize the coding sequence for
expression in a desired host such as to optimize the codon usage in
a heterologous sequence for expression in maize. Codon usage in the
coding regions of the polynucleotides of the present invention can
be analyzed statistically using commercially available software
packages such as "Codon Preference" available from the University
of Wisconsin Genetics Computer Group (see, Devereaux, et al.,
(1984) Nucleic Acids Res. 12:387-395) or MacVector 4.1 (Eastman
Kodak Co., New Haven, Conn.). Thus, the present invention provides
a codon usage frequency characteristic of the coding region of at
least one of the polynucleotides of the present invention. The
number of polynucleotides that can be used to determine a codon
usage frequency can be any integer from 1 to the number of
polynucleotides of the present invention as provided herein.
Optionally, the polynucleotides will be full-length sequences. An
exemplary number of sequences for statistical analysis can be at
least 1, 5, 10, 20, 50 or 100.
Sequence Shuffling
[0253] The present invention provides methods for sequence
shuffling using polynucleotides of the present invention, and
compositions resulting therefrom. Sequence shuffling is described
in PCT Application Publication Number WO 1997/20078. See also,
Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509.
Generally, sequence shuffling provides a means for generating
libraries of polynucleotides having a desired characteristic which
can be selected or screened for. Libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides which comprise sequence regions which have
substantial sequence identity and can be homologously recombined in
vitro or in vivo. The population of sequence-recombined
polynucleotides comprises a subpopulation of polynucleotides which
possess desired or advantageous characteristics and which can be
selected by a suitable selection or screening method. The
characteristics can be any property or attribute capable of being
selected for or detected in a screening system, and may include
properties of: an encoded protein, a transcriptional element, a
sequence controlling transcription, RNA processing, RNA stability,
chromatin conformation, translation or other expression property of
a gene or transgene, a replicative element, a protein-binding
element, or the like, such as any feature which confers a
selectable or detectable property. In some embodiments, the
selected characteristic will be a decreased Km and/or increased
KCat over the wild-type protein as provided herein. In other
embodiments, a protein or polynucleotide generated from sequence
shuffling will have a ligand binding affinity greater than the
non-shuffled wild-type polynucleotide. The increase in such
properties can be at least 110%, 120%, 130%, 140% or at least 150%
of the wild-type value.
Generic and Consensus Sequences
[0254] Polynucleotides and polypeptides of the present invention
further include those having: (a) a generic sequence of at least
two homologous polynucleotides or polypeptides, respectively, of
the present invention and (b) a consensus sequence of at least
three homologous polynucleotides or polypeptides, respectively, of
the present invention. The generic sequence of the present
invention comprises each species of polypeptide or polynucleotide
embraced by the generic polypeptide or polynucleotide sequence,
respectively. The individual species encompassed by a
polynucleotide having an amino acid or nucleic acid consensus
sequence can be used to generate antibodies or produce nucleic acid
probes or primers to screen for homologs in other species, genera,
families, orders, classes, phyla, or kingdoms. For example, a
polynucleotide having a consensus sequence from a gene family of
Zea mays can be used to generate antibody or nucleic acid probes or
primers to other Gramineae species such as wheat, rice, or
sorghum.
[0255] Alternatively, a polynucleotide having a consensus sequence
generated from orthologous genes can be used to identify or isolate
orthologs of other taxa. Typically, a polynucleotide having a
consensus sequence will be at least 25, 30, or 40 amino acids in
length, or 20, 30, 40, 50, 100 or 150 nucleotides in length. As
those of skill in the art are aware, a conservative amino acid
substitution can be used for amino acids which differ amongst
aligned sequence but are from the same conservative substitution
group as discussed above. Optionally, no more than 1 or 2
conservative amino acids are substituted for each 10 amino acid
length of consensus sequence.
[0256] Similar sequences used for generation of a consensus or
generic sequence include any number and combination of allelic
variants of the same gene, orthologous or paralogous sequences as
provided herein. Optionally, similar sequences used in generating a
consensus or generic sequence are identified using the BLAST
algorithm's smallest sum probability (P(N)). Various suppliers of
sequence-analysis software are listed in chapter 7 of Current
Protocols in Molecular Biology, Ausubel, et al., Eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc. (Supplement 30).
[0257] A polynucleotide sequence is considered similar to a
reference sequence if the smallest sum probability in a comparison
of the test nucleic acid to the reference nucleic acid is less than
about 0.1, more preferably less than about 0.01, or 0.001 and most
preferably less than about 0.0001 or 0.00001. Similar
polynucleotides can be aligned and a consensus or generic sequence
generated using multiple sequence alignment software available from
a number of commercial suppliers, such as the Genetics Computer
Group's (Madison, Wis.) PILEUP software, Vector NTI's (North
Bethesda, Md.) ALIGNX or Genecode's (Ann Arbor, Mich.) SEQUENCHER.
Conveniently, default parameters of such software can be used to
generate consensus or generic sequences.
Machine Applications
[0258] The present invention provides machines, articles of
manufacture, and processes for identifying, modeling or analyzing
the polynucleotides and polypeptides of the present invention.
Identification methods permit identification of homologues of the
polynucleotides or polypeptides of the present invention while
modeling and analysis methods permit recognition of structural or
functional features of interest.
A. Machines: Data Processing Systems
[0259] In one embodiment, the present invention provides a machine
having: 1) a memory comprising data representing at least one
genetic sequence, 2) a genetic identification, analysis, or
modeling program with access to the data, 3) a data processor which
executes instructions according to the program using the genetic
sequence or a subsequence thereof and 4) an output for storing or
displaying the results of the data processing.
[0260] The machine of the present invention is a data processing
system, typically a digital computer. The term "computer" includes
one or several desktop or portable computers, computer
workstations, servers (including intranet or internet servers),
mainframes and any integrated system comprising any of the above
irrespective of whether the processing, memory, input or output of
the computer is remote or local, as well as any networking
interconnecting the modules of the computer. Data processing can
thus be remote or distributed amongst several processors at one or
multiple sites. The data processing system comprises a data
processor, such as a central processing unit (CPU), which executes
instructions according to an application program. As used herein,
machines, articles of manufacture and processes are exclusive of
the machines, manufactures, and processes employed by the United
States Patent and Trademark Office or the European Patent Office
when data representing the sequence of a polypeptide or
polynucleotide of the present invention is used for patentability
searches.
[0261] The machine of the present invention includes a memory
comprising data representing at least one genetic sequence. As used
herein, "genetic sequence" refers to the primary sequence (i.e.,
amino acid or nucleotide sequence) of a polynucleotide or
polypeptide of the present invention. The genetic sequence can
represent a partial sequence from a full-length protein, genomic
DNA or full-length cDNA/mRNA. Nucleic acids or proteins comprising
a genetic sequence that is identified, analyzed or modeled
according to the present invention can be cloned or
synthesized.
[0262] As those of skill in the art will be aware, the form of
memory of a machine of the present invention, or the particular
embodiment of the computer readable medium, are not critical
elements of the invention and can take a variety of forms. The
memory of such a machine includes, but is not limited to, ROM or
RAM or computer readable media such as, but not limited to,
magnetic media such as computer disks or hard drives or media such
as CD-ROMs, DVDs, and the like. The memory comprising the data
representing the genetic sequence includes main memory, a register
and a cache. In some embodiments the data processing system stores
the data representing the genetic sequence in memory while
processing the data and wherein successive portions of the data are
copied sequentially into at least one register of the data
processor for processing. Thus, the genetic sequence stored in
memory can be a genetic sequence created during computer runtime or
stored beforehand. The machine of the present invention includes a
genetic identification, analysis or modeling program (discussed
below) with access to the data representing the genetic sequence.
The program can be implemented in software or hardware.
[0263] The present invention further contemplates that the machine
of the present invention will reference, directly or indirectly, a
utility or function for the polynucleotide or polypeptide of the
present invention. For example, the utility/function can be
directly referenced as a data element in the machine and accessible
by the program. Alternatively, the utility/function of the genetic
can be indirectly referenced to an electronic or written record.
The function or utility of the genetic sequence can be a function
or utility for the genetic sequence, or the data representing the
sequence (i.e., the genetic sequence data).
[0264] Exemplary function or utilities for the genetic sequence
include: 1) its name (per International Union of Biochemistry and
Molecular Biology rules of nomenclature) or the function of the
enzyme or protein represented by the genetic sequence, 2) the
metabolic pathway that the protein represented by the genetic
sequence participates in, 3) the substrate or product or structural
role of the protein represented by the genetic sequence or 4) the
phenotype (e.g., an agronomic or pharmacological trait) affected by
modulating expression or activity of the protein represented by the
genetic sequence.
[0265] The machine of the present invention also includes an output
for displaying, printing or recording the results of the
identification, analysis or modeling performed using a genetic
sequence of the present invention. Exemplary outputs include
monitors, printers or various electronic storage mechanisms (e.g.,
floppy disks, hard drives, main memory) which can be used to
display the results or employed as a means to input the stored data
into a subsequent application or device.
[0266] In some embodiments, data representing a genetic sequence of
the present invention is a data element within a data structure.
The data structure may be defined by the computer programs that
define the processes of identification, modeling or analysis (see
below) or it may be defined by the programming of separate data
storage and retrieval programs subroutines or systems. Thus, the
present invention provides a memory for storing a data structure
that can be accessed by a computer programmed to implement a
process for identification, analysis or modeling of a genetic
sequence. The data structure, stored within memory, is associated
with the data representing the genetic sequence and reflects the
underlying organization and structure of the genetic sequence to
facilitate program access to data elements corresponding to logical
sub-components of the genetic sequence. The data structure enables
the genetic sequence to be identified, analyzed or modeled. The
underlying order and structure of a genetic sequence is data
representing the higher order organization of the primary sequence.
Such higher order structures affect transcription, translation,
enzyme kinetics or reflects structural domains or motifs.
[0267] Exemplary logical sub-components which constitute the higher
order organization of the genetic sequence include but are not
limited to: restriction enzyme sites, endopeptidase sites, major
grooves, minor grooves, beta-sheets, alpha helices, open reading
frames (ORFs), 5' untranslated regions (UTRs), 3' UTRs, ribosome
binding sites, glycosylation sites, signal peptide domains,
intron-exon junctions, poly-A tails, transcription initiation
sites, translation start sites, translation termination sites,
methylation sites, zinc finger domains, modified amino acid sites,
preproprotein-proprotein junctions, proprotein-protein junctions,
transit peptide domains, single nucleotide polymorphisms (SNPs),
simple sequence repeats (SSRs), restriction fragment length
polymorphisms(RFLPs), insertion elements, transmembrane spanning
regions and stem-loop structures.
[0268] In another embodiment, the present invention provides a data
processing system comprising at least one data structure in memory
where the data structure supports the accession of data
representing a genetic sequence of the present invention. The
system also comprises at least one genetic identification, analysis
or modeling program which directs the execution of instructions by
the system using the genetic sequence data to identify, analyze or
model at least one data element which is a logical sub-component of
the genetic sequence. An output for the processing results is also
provided.
B. Articles of Manufacture: Computer Readable Media
[0269] In one embodiment, the present invention provides a data
structure in a computer readable medium that contains data
representing a genetic sequence of the present invention. The data
structure is organized to reflect the logical structuring of the
genetic sequence, so that the sequence can be analyzed by software
programs capable of accessing the data structure. In particular,
the data structures of the present invention organize the genetic
sequences of the present invention in a manner which allows
software tools to perform an identification, analysis or modeling
using logical elements of each genetic sequence.
[0270] In a further embodiment, the present invention provides a
machine-readable media containing a computer program and genetic
sequence data. The program provides instructions sufficient to
implement a process for effecting the identification, analysis or
modeling of the genetic sequence data. The media also includes a
data structure reflecting the underlying organization and structure
of the data to facilitate program access to data elements
corresponding to logical sub-components of the genetic sequence,
the data structure being inherent in the program and in the way in
which the program organizes and accesses the data.
[0271] An example of a data structure resembles a layered hash
table, where in one dimension the base content of the sequence is
represented by a string of elements A, T, C, G and N. The direction
from the 5' end to the 3' end is reflected by the order from the
position 0 to the position of the length of the string minus one.
Such a string, corresponding to a nucleotide sequence of interest,
has a certain number of substrings, each of which is delimited by
the string position of its 5' end and the string position of its 3'
end within the parent string. In a second dimension, each substring
is associated with or pointed to one or multiple attribute fields.
Such attribute fields contain annotations to the region on the
nucleotide sequence represented by the substring.
[0272] For example, a sequence under investigation is 520 bases
long and represented by a string named SeqTarget. There is a minor
groove in the 5' upstream non-coding region from position 12 to 38,
which is identified as a binding site for an enhancer protein HM-A,
which in turn will increase the transcription of the gene
represented by SeqTarget. Here, the substring is represented as
(12, 38) and has the following attributes: [upstream uncoded],
[minor groove], [HM-A binding] and [increase transcription upon
binding by HM-A]. Similarly, other types of information can be
stored and structured in this manner, such as information related
to the whole sequence, e.g., whether the sequence is a full length
viral gene, a mammalian house keeping gene or an EST from clone X,
information related to the 3' down stream non-coding region, e.g.,
hair pin structure and information related to various domains of
the coding region, e.g., Zinc finger.
[0273] This data structure is an open structure and is robust
enough to accommodate newly generated data and acquired knowledge.
Such a structure is also a flexible structure. It can be trimmed
down to a1-D string to facilitate data mining and analysis steps,
such as clustering, repeat-masking and HMM analysis. Meanwhile,
such a data structure also can extend the associated attributes
into multiple dimensions. Pointers can be established among the
dimensioned attributes when needed to facilitate data management
and processing in a comprehensive genomics knowledge base.
Furthermore, such a data structure is object-oriented. Polymorphism
can be represented by a family or class of sequence objects, each
of which has an internal structure as discussed above. The common
traits are abstracted and assigned to the parent object, whereas
each child object represents a specific variant of the family or
class. Such a data structure allows data to be efficiently
retrieved, updated and integrated by the software applications
associated with the sequence database and/or knowledge base.
C. Processes: Identification, Analysis, or Modeling
[0274] The present invention also provides a process of
identifying, analyzing, or modeling data representing a genetic
sequence of the present invention. The process comprises: 1)
providing a machine having a hardware or software implemented
genetic sequence identification, modeling, or analysis program with
data representing a genetic sequence, 2) executing the program
while granting it access to the genetic sequence data and 3)
displaying or outputting the results of the identification,
analysis, or modeling. Data structures made by the processes of the
present invention and embodied within a computer readable medium
are also provided herein.
[0275] A further process of the present invention comprises
providing a memory embodied with data representing a genetic
sequence and developing within the memory a data structure
associated with the data and reflecting the underlying organization
and structure of the data to facilitate program access to data
elements corresponding to logical subcomponents of the sequence. A
computer is programmed with a program containing instructions
sufficient to implement the process for effecting the
identification, analysis or modeling of the genetic sequence and
the program is executed on the computer while granting the program
access to the data and to the data structure within the memory. The
program results are outputted.
[0276] Identification, analysis, and modeling programs are well
known in the art and available commercially. The program typically
has at least one application to: 1) identify the structural role or
enzymatic function of the gene which the genetic sequence encodes
or is translated from, 2) analyzes and identifies higher order
structures within the genetic sequence or 3) model the
physico-chemical properties of a genetic sequence of the present
invention in a particular environment.
[0277] Included amongst the modeling/analysis tools are methods to:
1) recognize overlapping sequences (e.g., from a sequencing
project) with a polynucleotide of the present invention and create
an alignment called a "contig"; 2) identify restriction enzyme
sites of a polynucleotide of the present invention; 3) identify the
products of a TI ribonuclease digestion of a polynucleotide of the
present invention; 4) identify PCR primers with minimal
self-complementarity; 5) compute pairwise distances between
sequences in an alignment, reconstruct phylogentic trees using
distance methods, and calculate the degree of divergence of two
protein coding regions; 6) identify patterns such as coding
regions, terminators, repeats, and other consensus patterns in
polynucleotides of the present invention; 7) identify RNA secondary
structure; 8) identify sequence motifs, isoelectric point,
secondary structure, hydrophobicity and antigenicity in
polypeptides of the present invention; 9) translate polynucleotides
of the present invention and backtranslate polypeptides of the
present invention and 10) compare two protein or nucleic acid
sequences and identifying points of similarity or dissimilarity
between them.
[0278] Identification of the function/utility of a genetic sequence
is typically achieved by comparative analysis to a gene/protein
database and establishing the genetic sequence as a candidate
homologue (i.e., ortholog or paralog) of a gene/protein of known
function/utility.
[0279] A candidate homologue has statistically significant
probability of having the same biological function (e.g., catalyzes
the same reaction, binds to homologous proteins/nucleic acids, has
a similar structural role) as the reference sequence to which it is
compared. Sequence identity/similarity is frequently employed as a
criterion to identify candidate homologues. In the same vein,
genetic sequences of the present invention have utility in
identifying homologs in animals or other plant species,
particularly those in the family Gramineae such as, but not limited
to, sorghum, wheat or rice. Function is frequently established on
the basis of sequence identity/similarity. Exemplary sequence
comparison systems are provided for in sequence analysis software
such as those provided by the Genetics Computer Group (Madison,
Wis.) or InforMax</RTI
[0280] The present invention further provides methods for detecting
a polynucleotide of the present invention in a nucleic acid sample
suspected of containing a polynucleotide of the present invention,
such as a plant cell lysate, particularly a lysate of maize. In
some embodiments, a gene of the present invention or portion
thereof can be amplified prior to the step of contacting the
nucleic acid sample with a polynucleotide of the present invention.
The nucleic acid sample is contacted with the polynucleotide to
form a hybridization complex. The polynucleotide hybridizes under
stringent conditions to a gene encoding a polypeptide of the
present invention. Formation of the hybridization complex is used
to detect a gene encoding a polypeptide of the present invention in
the nucleic acid sample. Those of skill will appreciate that an
isolated nucleic acid comprising a polynucleotide of the present
invention should lack cross-hybridizing sequences in common with
non-target genes that would yield a false positive result.
[0281] Detection of the hybridization complex can be achieved using
any number of well known methods. For example, the nucleic acid
sample, or a portion thereof, may be assayed by hybridization
formats including but not limited to, solution phase, solid phase,
mixed phase, or in situ hybridization assays. Briefly, in solution
(or liquid) phase hybridizations, both the target nucleic acid and
the probe or primer are free to interact in the reaction mixture.
In solid phase hybridization assays, probes or primers are
typically linked to a solid support where they are available for
hybridization with target nucleic in solution. In mixed phase,
nucleic acid intermediates in solution hybridize to target nucleic
acids in solution as well as to a nucleic acid linked to a solid
support. In in situ hybridization, the target nucleic acid is
liberated from its cellular surroundings in such as to be available
for hybridization within the cell while preserving the cellular
morphology for subsequent interpretation and analysis. The
following articles provide an overview of the various hybridization
assay formats: Singer, et al., (1986) Biotechniques 4(3):230-250;
Haase, et al., Methods in Virology, Vol. VII, pp. 189-226 (1984);
Wilkinson, The theory and practice of in situ hybridization in: In
situ Hybridization, Wilkinson, Ed., IRL Press, Oxford University
Press, Oxford; and Nucleic Acid Hybridization: A Practical
Approach, Hames and Higgins, Eds., IRL Press (1987).
Nucleic Acid Labels and Detection Methods
[0282] The means by which nucleic acids of the present invention
are labeled is not a critical aspect of the present invention and
can be accomplished by any number of methods currently known or
later developed. Detectable labels suitable for use in the present
invention include any composition detectable by spectroscopic,
radioisotopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means.
[0283] Useful labels in the present invention include biotin for
staining with labeled streptavidin conjugate, magnetic beads,
fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green
fluorescent protein and the like), radiolabels (e.g., 3H, 125I,
35S, I4C or 32 p), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.,
polystyrene, polypropylene, latex, etc.) beads.
[0284] Nucleic acids of the present invention can be labeled by any
one of several methods typically used to detect the presence of
hybridized nucleic acids. One common method of detection is the use
of autoradiography using probes labeled with 3H, 125I, 35S, I4C or
32 p, or the like. The choice of radioactive isotope depends on
research preferences due to ease of synthesis, stability, and half
lives of the selected isotopes. Other labels include ligands which
bind to antibodies labeled with fluorophores, chemiluminescent
agents and enzymes. Alternatively, probes can be conjugated
directly with labels such as fluorophores, chemiluminescent agents
or enzymes. The choice of label depends on sensitivity required,
ease of conjugation with the probe, stability requirements and
available instrumentation. Labeling the nucleic acids of the
present invention is readily achieved such as by the use of labeled
PCR primers.
[0285] In some embodiments, the label is simultaneously
incorporated during the amplification step in the preparation of
the nucleic acids. Thus, for example, polymerase chain reaction
(PCR) with labeled primers or labeled nucleotides will provide a
labeled amplification product. In another embodiment, transcription
amplification using a labeled nucleotide (e.g., fluorescein-labeled
UTP and/or CTP) incorporates a label into the transcribed nucleic
acids.
[0286] Non-radioactive probes are often labeled by indirect means.
For example, a ligand molecule is covalently bound to the probe.
The ligand then binds to an anti-ligand molecule which is either
inherently detectable or covalently bound to a detectable signal
system, such as an enzyme, a fluorophore or a chemiluminescent
compound. Enzymes of interest as labels will primarily be
hydrolases, such as phosphatases, esterases and glycosidases or
oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, etc. Chemiluminescers include
luciferin and 2,3-dihydrophthalazinediones, e.g., luminol.
[0287] Ligands and anti-ligands may be varied widely. Where a
ligand has a natural anti-ligand, namely ligands such as biotin,
thyroxine and cortisol, it can be used in conjunction with its
labeled, naturally occurring anti-ligands. Alternatively, any
haptenic or antigenic compound can be used in combination with an
antibody. Probes can also be labeled by direct conjugation with a
label. For example, cloned DNA probes have been coupled directly to
horseradish peroxidase or alkaline phosphatase.
[0288] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with a substrate and detecting the reaction product produced
by the action of the enzyme on the substrate and colorimetric
labels are detected by simply visualizing the colored label.
Antibodies to Proteins
[0289] Antibodies can be raised to a protein of the present
invention, including individual, allelic, strain, or species
variants, and fragments thereof, both in their naturally occurring
(full-length) forms and in recombinant forms. Additionally,
antibodies are raised to these proteins in either their native
configurations or in non-native configurations. Many methods of
making antibodies are known to persons of skill. A variety of
analytic methods are available to generate a hydrophilicity profile
of a protein of the present invention. Such methods can be used to
guide the artisan in the selection of peptides of the present
invention for use in the generation or selection of antibodies
which are specifically reactive, under immunogenic conditions, to a
protein of the present invention. See, e.g., Janin, (1979) Nature
277:491-492; Wolfenden, et al., (1981) Biochemistry 20:849-855;
Kyte and Doolite, (1982) J. Mol Biol. 157:105-132; Rose, et al.,
(1985) Science 229:834838. The following discussion is presented as
a general overview of the techniques available; however, one of
skill will recognize that many variations upon the following
methods are known.
[0290] A number of immunogens are used to produce antibodies
specifically reactive with a protein of the present invention. An
isolated recombinant, synthetic or native polynucleotide of the
present invention are the preferred antigens for the production of
monoclonal or polyclonal antibodies. Polypeptides of the present
invention are optionally denatured, and optionally reduced, prior
to formation of antibodies for screening expression libraries or
other assays in which a putative protein of the present invention
is expressed or denatured in a non-native secondary, tertiary, or
quartenary structure.
[0291] The protein of the present invention is then injected into
an animal capable of producing antibodies. Either monoclonal or
polyclonal antibodies can be generated for subsequent use in
immunoassays to measure the presence and quantity of the protein of
the present invention. Methods of producing polyclonal antibodies
are known to those of skill in the art. In brief, an antigen,
preferably a purified protein, a protein coupled to an appropriate
carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a
protein incorporated into an immunization vector such as a
recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) is mixed
with an adjuvant and animals are immunized with the mixture. The
animal's immune response to the immunogen preparation is monitored
by taking test bleeds and determining the titer of reactivity to
the protein of interest. When appropriately high titers of antibody
to the immunogen are obtained, blood is collected from the animal
and antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the protein is performed where
desired (See, e.g., Coligan, Current Protocols in Immunology,
Wiley/Greene, NY (1991) and Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press, NY (1989)).
[0292] Antibodies, including binding fragments and single chain
recombinant versions thereof, against predetermined fragments of a
protein of the present invention are raised by immunizing animals,
e.g., with conjugates of the fragments with carrier proteins as
described above. Typically, the immunogen of interest is a protein
of at least about 5 amino acids, more typically the protein is 10
amino acids in length, preferably, 15 amino acids in length and
more preferably the protein is 20 amino acids in length or greater.
The peptides are typically coupled to a carrier protein (e.g., as a
fusion protein) or are recombinantly expressed in an immunization
vector. Antigenic determinants on peptides to which antibodies bind
are typically 3 to 10 amino acids in length.
[0293] Monoclonal antibodies are prepared from hybrid cells
secreting the desired antibody. Monoclonals antibodies are screened
for binding to a protein from which the antigen was derived.
Specific monoclonal and polyclonal antibodies will usually have an
antibody binding site with an affinity constant for its cognate
monovalent antigen at least between 106-107, usually at least 108,
preferably at least 109, more preferably at least 101 and most
preferably at least 101 liters/mole.
[0294] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies are found in, e.g., Basic and Clinical
Immunology, 4th ed., Stites et al., Eds., Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane, Supra; Goding, Monoclonal Antibodies: Principles
and Practice, 2nd ed., Academic Press, New York, N.Y. (1986); and
Kohler and Milstein, (1975) Nature 256:495-497. Summarized briefly,
this method proceeds by injecting an animal with an antigen
comprising a protein of the present invention. The animal is then
sacrificed and cells taken from its spleen, which are fused with
myeloma cells. The result is a hybrid cell or "hybridoma" that is
capable of reproducing in vitro.
[0295] The population of hybridomas is then screened to isolate
individual clones, each of which secrete a single antibody species
to the antigen. In this manner, the individual antibody species
obtained are the products of immortalized and cloned single B cells
from the immune animal generated in response to a specific site
recognized on the antigenic substance.
[0296] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors (see, e.g.,
Huse, et al., (1989) Science 246:1275-1281 and Ward, et al., (1989)
Nature 341:544-546 and Vaughan, et al., (1996) Nature Biotechnology
14:309-314). Alternatively, high avidity human monoclonal
antibodies can be obtained from transgenic mice comprising
fragments of the unrearranged human heavy and light chain Ig loci
(i.e., mini locus transgenic mice). Fishwild, et al., (1996) Nature
Bio Tech. 14:845-851. Also, recombinant immunoglobulins may be
produced. See, Cabilly, U.S. Pat. No. 4,816,567 and Queen, et al.,
(1989) Proc. Nat'l Acad. Sci. 86:10029-10033.
[0297] The antibodies of this invention are also used for affinity
chromatography in isolating proteins of the present invention.
Columns are prepared, e.g., with the antibodies linked to a solid
support, e.g., particles, such as agarose, SEPHADEX, or the like,
where a cell lysate is passed through the column, washed and
treated with increasing concentrations of a mild denaturant,
whereby purified protein are released.
[0298] The antibodies can be used to screen expression libraries
for particular expression products such as normal or abnormal
protein. Usually the antibodies in such a procedure are labeled
with a moiety allowing easy detection of presence of antigen by
antibody binding. Antibodies raised against a protein of the
present invention can also be used to raise anti-idiotypic
antibodies. These are useful for detecting or diagnosing various
pathological conditions related to the presence of the respective
antigens.
[0299] Frequently, the proteins and antibodies of the present
invention will be labeled by joining, either covalently or
non-covalently, a substance which provides for a detectable signal.
A wide variety of labels and conjugation techniques are known and
are reported extensively in both the scientific and patent
literature. Suitable labels include radionucleotides, enzymes,
substrates, cofactors, inhibitors, fluorescent moieties,
chemiluminescent moieties, magnetic particles, and the like.
[0300] Plants exhibiting an altered ethylene-dependent phenotype as
compared with wild-type plants can be selected among other methods,
by visual observation. For example, an altered ethylene-dependent
phenotype may be detected by utilization of the "triple response."
The "triple response" consists of three distinct morphological
changes in dark-grown seedlings upon exposure to ethylene:
inhibition of hypocotyl and root elongation, radial swelling of the
stem and exaggeration of the apical hook. Thus, a triple response
displayed in the presence of ethylene inhibitors would indicate one
type of altered ethylene-dependent phenotype. Ethylene affects a
vast array of agriculturally important plant processes, including
fruit ripening, flower and leaf senescence and leaf abscission. The
ability to control the sensitivity of plants to ethylene could thus
significantly improve the quality and longevity of many crops. The
invention includes plants produced by the method of the invention,
as well as plant tissue and seeds.
"Stacking" of Constructs and Traits
[0301] In certain embodiments, the nucleic acid sequences of the
present invention can be used in combination ("stacked") with other
polynucleotide sequences of interest in order to create plants with
a desired phenotype. The polynucleotides of the present invention
may be stacked with any gene or combination of genes, and the
combinations generated can include multiple copies of any one or
more of the polynucleotides of interest. The desired combination
may affect one or more traits; that is, certain combinations may be
created for modulation of gene expression affecting ACC synthase
activity and/or ethylene production. Other combinations may be
designed to produce plants with a variety of desired traits,
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 1998/20122) 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. patent application Ser. No.
10/053,410, filed Nov. 7, 2001) and thioredoxins (U.S. patent
application Ser. No. 10/005,429, filed Dec. 3, 2001)), 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; 5,723,756; 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 1994/11516)); 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 affecting 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 1999/61619; WO
2000/17364; WO 1999/25821), the disclosures of which are herein
incorporated by reference.
[0302] 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 of
interest 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 a
polynucleotide of interest. This may be accompanied by any
combination of other suppression cassettes or over-expression
cassettes to generate the desired combination of traits in the
plant.
Use in Breeding Methods
[0303] The transformed plants of the invention may be used in a
plant breeding program. The goal of plant breeding is to combine,
in a single variety or hybrid, various desirable traits. For field
crops, these traits may include, for example, resistance to
diseases and insects, tolerance to heat and drought, reduced time
to crop maturity, greater yield and better agronomic quality. With
mechanical harvesting of many crops, uniformity of plant
characteristics such as germination and stand establishment, growth
rate, maturity and plant and ear height is desirable. Traditional
plant breeding is an important tool in developing new and improved
commercial crops. This invention encompasses methods for producing
a maize plant by crossing a first parent maize plant with a second
parent maize plant wherein one or both of the parent maize plants
is a transformed plant displaying a staygreen phenotype, a
sterility phenotype, a crowding resistance phenotype, or the like,
as described herein.
[0304] Plant breeding techniques known in the art and used in a
maize plant breeding program include, but are not limited to,
recurrent selection, bulk selection, mass selection, backcrossing,
pedigree breeding, open pollination breeding, restriction fragment
length polymorphism enhanced selection, genetic marker enhanced
selection, doubled haploids and transformation. Often combinations
of these techniques are used.
[0305] The development of maize hybrids in a maize plant breeding
program requires, in general, the development of homozygous inbred
lines, the crossing of these lines and the evaluation of the
crosses. There are many analytical methods available to evaluate
the result of a cross. The oldest and most traditional method of
analysis is the observation of phenotypic traits. Alternatively,
the genotype of a plant can be examined.
[0306] A genetic trait which has been engineered into a particular
maize plant using transformation techniques can be moved into
another line using traditional breeding techniques that are well
known in the plant breeding arts. For example, a backcrossing
approach is commonly used to move a transgene from a transformed
maize plant to an elite inbred line, and the resulting progeny
would then comprise the transgene(s). Also, if an inbred line was
used for the transformation, then the transgenic plants could be
crossed to a different inbred in order to produce a transgenic
hybrid maize plant. As used herein, "crossing" can refer to a
simple X by Y cross, or the process of backcrossing, depending on
the context.
[0307] The development of a maize hybrid in a maize plant breeding
program involves three steps: (1) the selection of plants from
various germplasm pools for initial breeding crosses; (2) the self
ing of the selected plants from the breeding crosses for several
generations to produce a series of inbred lines, which, while
different from each other, breed true and are highly uniform and
(3) crossing the selected inbred lines with different inbred lines
to produce the hybrids. During the inbreeding process in maize, the
vigor of the lines decreases. Vigor is restored when two different
inbred lines are crossed to produce the hybrid. An important
consequence of the homozygosity and homogeneity of the inbred lines
is that the hybrid created by crossing a defined pair of inbreds
will always be the same. Once the inbreds that give a superior
hybrid have been identified, the hybrid seed can be reproduced
indefinitely as long as the homogeneity of the inbred parents is
maintained.
[0308] Transgenic plants of the present invention may be used to
produce, e.g., a single cross hybrid, a three-way hybrid or a
double cross hybrid. A single cross hybrid is produced when two
inbred lines are crossed to produce the F1 progeny. A double cross
hybrid is produced from four inbred lines crossed in pairs
(A.times.B and C.times.D) and then the two F1 hybrids are crossed
again (A.times.B).times.(C.times.D). A three-way cross hybrid is
produced from three inbred lines where two of the inbred lines are
crossed (A.times.B) and then the resulting F1 hybrid is crossed
with the third inbred (A.times.B).times.C. Much of the hybrid vigor
and uniformity exhibited by F1 hybrids is lost in the next
generation (F2). Consequently, seed produced by hybrids is consumed
rather than planted.
Antibodies
[0309] The polypeptides of the invention can be used to produce
antibodies specific for the polypeptides of SEQ ID NO: 2, 4, 6, 8
or 10 and conservative variants thereof. Antibodies specific for,
e.g., SEQ ID NO: 2, 4, 6, 8 or 10 and related variant polypeptides
are useful, e.g., for screening and identification purposes, e.g.,
related to the activity, distribution and expression of ACC
synthase.
[0310] Antibodies specific for the polypeptides of the invention
can be generated by methods well known in the art. Such antibodies
can include, but are not limited to, polyclonal, monoclonal,
chimeric, humanized, single chain, Fab fragments and fragments
produced by a Fab expression library.
[0311] Polypeptides do not require biological activity for antibody
production. The full length polypeptide, subsequences, fragments or
oligopeptides can be antigenic. Peptides used to induce specific
antibodies typically have an amino acid sequence of at least about
10 amino acids and often at least 15 or 20 amino acids. Short
stretches of a polypeptide, e.g., selected from among SEQ ID NO: 2,
4, 6, 8 or 10, can be fused with another protein, such as keyhole
limpet hemocyanin and antibody produced against the chimeric
molecule.
[0312] Numerous methods for producing polyclonal and monoclonal
antibodies are known to those of skill in the art and can be
adapted to produce antibodies specific for the polypeptides of the
invention, e.g., corresponding to SEQ ID NO: 2, 4, 6, 8 or 10. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene,
NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold
Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical
Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif.,
and references cited therein; Goding (1986) Monoclonal Antibodies:
Principles and Practice (2d ed.) Academic Press, New York, N.Y.;
Fundamental Immunology, e.g., 4th Edition (or later), W. E. Paul
(ed.), Raven Press, N.Y. (1998) and Kohler and Milstein, (1975)
Nature 256:495-497. Other suitable techniques for antibody
preparation include selection of libraries of recombinant
antibodies in phage or similar vectors. See, Huse, et al., (1989)
Science 246:1275-1281 and Ward, et al., (1989) Nature 341:544-546.
Specific monoclonal and polyclonal antibodies and antisera will
usually bind with a K.sub.D of at least about 0.1 .mu.M, preferably
at least about 0.01 .mu.M or better and most typically and
preferably, 0.001 .mu.M or better.
Kits for Modulating Plant Stress Response
[0313] Certain embodiments of the invention can optionally be
provided to a user as a kit. For example, a kit of the invention
can contain one or more nucleic acid, polypeptide, antibody,
diagnostic nucleic acid or polypeptide, e.g., antibody, probe set,
e.g., as a cDNA microarray, one or more vector and/or cell line
described herein. Most often, the kit is packaged in a suitable
container. The kit typically further comprises one or more
additional reagents, e.g., substrates, labels, primers, or the like
for labeling expression products, tubes and/or other accessories,
reagents for collecting samples, buffers, hybridization chambers,
cover slips, etc. The kit optionally further comprises an
instruction set or user manual detailing preferred methods of using
the kit components for discovery or application of gene sets. When
used according to the instructions, the kit can be used, e.g., for
evaluating expression or polymorphisms in a plant sample, e.g., for
evaluating ACC synthase, ethylene production, stress response
potential, crowding resistance potential, sterility, etc.
Alternatively, the kit can be used according to instructions for
using at least one ACC synthase polynucleotide sequence to control
ethylene production in a plant.
Other Nucleic Acid and Protein Assays
[0314] In the context of the invention, nucleic acids and/or
proteins are manipulated according to well known molecular biology
methods. Detailed protocols for numerous such procedures are
described in, e.g., in Ausubel, et al., Current Protocols in
Molecular Biology (supplemented through 2004) John Wiley &
Sons, New York ("Ausubel"); Sambrook, et al., Molecular Cloning-A
Laboratory Manual (2nd Ed.), Vol. 1 3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and Berger
and Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
("Berger").
[0315] In addition to the above references, protocols for in vitro
amplification techniques, such as the polymerase chain reaction
(PCR), the ligase chain reaction (LCR), Q.beta.-replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA), useful, e.g., for amplifying polynucleotides of the
invention, are found in Mullis, et al., (1987) U.S. Pat. No.
4,683,202; PCR Protocols A Guide to Methods and Applications (Innis
et al. eds) Academic Press Inc. San Diego, Calif. (1990) ("Innis");
Arnheim and Levinson, (1990) C&EN 36; The Journal Of NIH
Research (1991) 3:81; Kwoh, et al., (1989) Proc Natl Acad Sci USA
86:1173; Guatelli, et al., (1990) Proc Natl Acad Sci USA 87:1874;
Lomell, et al., (1989) J Clin. Chem 35:1826; Landegren, et al.,
(1988) Science 241:1077; Van Brunt, (1990) Biotechnology 8:291; Wu
and Wallace, (1989) Gene 4:560; Barringer, et al., (1990) Gene
89:117 and Sooknanan and Malek, (1995) Biotechnology 13:563.
Additional methods, useful for cloning nucleic acids in the context
of the invention, include Wallace, et al., U.S. Pat. No. 5,426,039.
Improved methods of amplifying large nucleic acids by PCR are
summarized in Cheng, et al., (1994) Nature 369:684 and the
references therein.
[0316] Certain polynucleotides of the invention can be synthesized
utilizing various solid-phase strategies involving mononucleotide-
and/or trinucleotide-based phosphoramidite coupling chemistry. For
example, nucleic acid sequences can be synthesized by the
sequential addition of activated monomers and/or trimers to an
elongating polynucleotide chain. See, e.g., Caruthers, et al.,
(1992) Meth Enzymol 211:3. In lieu of synthesizing the desired
sequences, essentially any nucleic acid can be custom ordered from
any of a variety of commercial sources, such as The Midland
Certified Reagent Company (mcrc@oligos.com) (Midland, Tex.), The
Great American Gene Company (available on the World Wide Web at
genco.com) (Ramona, Calif.), ExpressGen, Inc. (available on the
World Wide Web at expressgen.com) (Chicago III.), Operon
Technologies, Inc. (available on the World Wide Web at operon.com)
(Alameda Calif.), and many others.
[0317] Although the present invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
TABLE-US-00001 TABLE 1 Sequences in Sequence Listing SEQ ID NO
PP/NT DESCRIPTION 1 nucleotide ZM-ETO1-1 cfp3n.pk009.o19.f.FIS 2
polypeptide ZM-ETO1-1 cfp3n.pk009.o19.f.FIS 3 nucleotide ZM-ETO1-2
cfp6n.pk073.o21.FIS 4 polypeptide ZM-ETO1-2cfp6n.pk073.o21.FIS 5
nucleotide ZM-ETO1-3 cta1.pk0036.f.FIS 6 polypeptide ZM-ETO1-3
cta1.pk0036.f + cfp1n.pk047.e9a.FIS 7 nucleotide ZM-ETO1-4
cfp7n.pk074.p17.FIS 8 polypeptide ZM-ETO1-4 cfp7n.pk074.p17.FIS 9
nucleotide GM-ETO1-1 PSO415110 genomic 10 polypeptide GM-ETO1-1
PSO415110 11 polypeptide N-Terminal Domain of ETO1 (Consensus) 12
polypeptide C-Terminal Domain of ETO1 (Consensus) 13 nucleotide
ASal-A20 oligonucleotide
EXAMPLES
Example 1
Construction of cDNA Libraries
Total RNA Isolation
[0318] Total RNA for SEQ ID NO: 1, 3, 5 and 7 was obtained from
maize genotype Hill (Armstrong and Phillips, (1988) Crop Sci.
28:363-369) and from night harvested leaf tissue at the V8-V10
stage of maize genotype B75. Total RNA for SEQ ID NO: 9 was
obtained from soybean. The total RNA was isolated from the maize
and soybean tissues with TRIzol Reagent (Life Technology Inc.
Gaithersburg, Md.) using a modification of the guanidine
isothiocyanate/acid-phenol procedure described by Chomczynski and
Sacchi (Chomczynski and Sacchi, (1987) Anal. Biochem. 162:156). In
brief, plant tissue samples were pulverized in liquid nitrogen
before the addition of the TRIzol Reagent and then were further
homogenized with a mortar and pestle. Addition of chloroform
followed by centrifugation was conducted for separation of an
aqueous phase and an organic phase. The total RNA was recovered by
precipitation with isopropyl alcohol from the aqueous phase.
Poly (A)+RNA Isolation
[0319] The selection of poly (A)+RNA from total RNA was performed
using PolyATact system (Promega Corporation, Madison, Wis.). In
brief, biotinylated oligo(dT) primers were used to hybridize to the
3' poly (A) tails on mRNA. The hybrids were captured using
streptavidin coupled to paramagnetic particles and a magnetic
separation stand. The mRNA was washed at high stringency conditions
and eluted by RNase-free deionized water. cDNA Library Construction
cDNA synthesis was performed and unidirectional cDNA libraries were
constructed using the SuperScript Plasmid System (Life Technology
Inc. Gaithersburg, Md.). The first strand of cDNA was synthesized
by priming an oligo(dT) primer containing a Not I site.
[0320] The reaction was catalyzed by SuperScript Reverse
Transcriptase II at 45.degree. C. The second strand of cDNA was
labeled with alpha-32P-dCTP and a portion of the reaction was
analyzed by agarose gel electrophoresis to determine cDNA sizes.
cDNA molecules smaller than 500 base pairs and unligated adapters
were removed by Sephacryl-5400 chromatography. The selected cDNA
molecules were ligated into pSPORTI vector in between of Not I and
Sal I sites.
TABLE-US-00002 TABLE 2 cDNAs, Corresponding Sequence Identifiers,
and Source cfp3n.pk009.o19.f SEQ ID 1 & 2 Maize Ear, pooled
V10-V14-v16- VT, Full-length enriched normalized cfp6n.pk073.o21.f
SEQ ID 3 & 4 Maize Leaf and Seed pooled, Full-length enriched
normalized cfp6n.pk003.j6 SEQ ID 5 & 6 Maize Leaf and Seed
pooled, Full-length enriched normalized cfp7n.pk074.p17 SEQ ID 7
& 8 Maize Root, Pooled stages, Full-length enriched, normalized
sfl1.pk0066.b1 SEQ ID 9 & 10 Soybean (Glycine max L.) immature
flower
Based on the sequence comparison of the soybean and maize sequences
two domains that are highly conserved across all of the sequences
were identified: one N-terminal and one C-terminal.
TABLE-US-00003 N-TERMINAL DOMAIN (SEQ ID NO: 11)
F-X8-C-X-R-X3-A-X-L-S-X-P-X4-L-X-G-X-F-X-E-X17-M-
X6-S-X16-L-X2-A-X2-F-C-C-X2-L-K-X2-C-X3-L-X8-A-X8- E-X5-L-X3-CLQ
C-TERMINAL DOMAIN (SEQ ID NO: 12)
W-S-X-V-D-D-X2-S-L-X-V-X3-M-L-X8-L-X-F-R-Q-S-L-L-
L-L-R-L-N-C-X3-A-M-R-X-L-X2-A-X8-E-R-L-V-Y-E-G-W-
X-L-Y-D-X-G-X3-E-X-L-X-K-A-X3-I-X3-R-S-F-E-A-X-F-
L-X-A-Y-X-L-X5-D-X6-V-X3-L-X2-A-X2-C-X2-D-X-L-R-K-
G-Q-A-X-N-N-X-G-X2-Y-X5-L-D-X-A-X3-Y-X2-A-X4-H-X-
R-A-X-Q-G-L-A-R-V-X2-L-X-N-X4-A-X2-E-M-T-X-L-X-E-
X5-A-X-A-Y-E-K-R-S-E-Y-X2-R-X5-D-L-X5-L-D-P-X-R-X-
Y-P-Y-R-Y-R-A-A-V-L-M-D
Example 2
cDNA Sequencing and Library Subtraction
Sequencing Template Preparation
[0321] Individual colonies were picked and DNA was prepared either
by PCR with M13 forward primers and M13 reverse primers, or by
plasmid isolation. All the cDNA clones were sequenced using M13
reverse primers.
[0322] Q-bot Subtraction Procedure cDNA libraries subjected to the
subtraction procedure were plated out on 22.times.22 cm.sup.2 agar
plate at density of about 3,000 colonies per plate. The plates were
incubated in a 37.degree. C. incubator for 12-24 hours. Colonies
were picked into 384-well plates by a robot colony picker, Q-bot
(GENETIX Limited). These plates were incubated overnight at
37.degree. C.
[0323] Once sufficient colonies were picked, they were pinned onto
22.times.22 cm.sup.2 nylon membranes using Q-bot. Each membrane
contained 9,216 colonies or 36,864 colonies. These membranes were
placed onto agar plate with appropriate antibiotic. The plates were
incubated at 37.degree. C. for overnight.
[0324] After colonies were recovered on the second day, these
filters were placed on filter paper pre-wetted with denaturing
solution for four minutes, then were incubated on top of a boiling
water bath for additional four minutes. The filters were then
placed on filter paper pre-wetted with neutralizing solution for
four minutes. After excess solution was removed by placing the
filters on dry filter papers for one minute, the colony side of the
filters were place into Proteinase K solution, incubated at
37.degree. C. for 40-50 minutes. The filters were placed on dry
filter papers to dry overnight. DNA was then cross-linked to nylon
membrane by UV light treatment.
[0325] Colony hybridization was conducted as described by Sambrook,
et al., (in Molecular Cloning: A laboratory Manual, 2nd Edition).
The following probes were used in colony hybridization: [0326] 1.
First strand cDNA from the same tissue as the library was made from
to remove the most redundant clones. [0327] 2. 48-192 most
redundant cDNA clones from the same library based on previous
sequencing data. [0328] 3. 192 most redundant cDNA clones in the
entire maize sequence database. [0329] 4. ASaI-A20 oligonucleotide:
TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA AAA (SEQ ID NO: 13),
removes clones containing a poly A tail but no cDNA. [0330] 5. cDNA
clones derived from rRNA.
[0331] The image of the autoradiography was scanned into computer
and the signal intensity and cold colony addresses of each colony
was analyzed. Re-arraying of cold colonies from 384 well plates to
96 well plates was conducted using Q-bot.
Example 3
Homology Search
[0332] This example describes identification of the gene from a
computer homology search. Gene identities were determined by
conducting BLAST (Basic Local Alignment Search Tool; Altschul, et
al., (1993) J. Mol. Biol. 215:403-410) searches under default
parameters 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
were analyzed for similarity to all publicly available DNA
sequences contained in the "nr" database using the BLASTN
algorithm.
[0333] 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) Nature Genetics 3:266-272) provided by the NCBI. In
some cases, the sequencing data from two or more clones containing
overlapping segments of DNA were used to construct contiguous DNA
sequences.
Example 4
Transformation and Regeneration of Transgenic Plants
[0334] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing the ETO1 sequence operably
linked to the a promoter such as a drought-inducible promoter RAB17
promoter (Vilardell, et al., (1990) Plant Mol Biol 14:423-432), a
constitutive promoter, a female preferred promoter, such as ZM-ADF4
(US Patent Application Publication Number 2009/0094713) or EEP1 (US
Patent Application Publication Number 2004/0237147) or a root
specific promoter and the selectable marker gene MO-PAT, which
confers resistance to the herbicide Bialaphos or the BAR selectable
marker. Alternatively, the selectable marker gene is provided on a
separate plasmid. Transformation is performed as follows. Media
recipes follow below.
Preparation of Target Tissue
[0335] The ears are husked and surface sterilized in 30% Clorox@
bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two
times with sterile water. The immature embryos are excised and
placed embryo axis side down (scutellum side up), 25 embryos per
plate, on 560Y medium for 4 hours and then aligned within the
2.5-cm target zone in preparation for bombardment.
Preparation of DNA
[0336] A plasmid vector comprising the ETO1 sequence operably
linked to an ubiquitin promoter is made. This plasmid DNA plus
plasmid DNA containing a PAT selectable marker is precipitated onto
1.1 .mu.m (average diameter) tungsten pellets using a CaCl.sub.2
precipitation procedure as follows:
[0337] 100 .mu.l prepared tungsten particles in water
[0338] 10 .mu.l (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g total
DNA)
[0339] 100 .mu.l 2.5 M CaCl.sub.2
[0340] 10 .mu.l 0.1 M spermidine
[0341] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol and centrifuged for 30 seconds. Again the liquid is removed
and 105 .mu.l 100% ethanol is added to the final tungsten particle
pellet. For particle gun bombardment, the tungsten/DNA particles
are briefly sonicated and 10 .mu.l spotted onto the center of each
macrocarrier and allowed to dry about 2 minutes before
bombardment.
Particle Gun Treatment
[0342] The sample plates are bombarded at level #4 in a particle
gun. All samples receive a single shot at 650 PSI, with a total of
ten aliquots taken from each tube of prepared particles/DNA.
Subsequent Treatment
[0343] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos, and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288 J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5'' pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for increased drought tolerance. Assays to measure improved drought
tolerance are routine in the art and include, for example,
increased kernel set under drought conditions when compared to
control maize plants under identical environmental conditions.
Alternatively, the transformed plants can be monitored for a
modulation in meristem development (e.g., a decrease in spikelet
formation on the ear). See, for example, Bruce, et al., (2002)
Journal of Experimental Botany 53:13-25.
Bombardment and Culture Media
[0344] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts
(SIGMA C-1416), 1.0 ml/I Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose,
1.0 mg/l 2,4-D and 2.88 g/l L-proline (brought to volume with D-I
H.sub.2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite
(added after bringing to volume with D-I H.sub.2O) and 8.5 mg/l
silver nitrate (added after sterilizing the medium and cooling to
room temperature). Selection medium (560R) comprises 4.0 g/l N6
basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose
and 2.0 mg/l 2,4-D (brought to volume with D-I H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after
bringing to volume with D-I H.sub.2O) and 0.85 mg/l silver nitrate
and 3.0 mg/l bialaphos (both added after sterilizing the medium and
cooling to room temperature).
[0345] Plant regeneration medium (288 J) comprises 4.3 g/l MS salts
(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and
0.40 g/l glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog, (1962) Physiol. Plant 15:473), 100 mg/l
myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose and 1.0 ml/I of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.2O
after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing
to volume with D-I H.sub.2O) and 1.0 mg/l indoleacetic acid and 3.0
mg/l bialaphos (added after sterilizing the medium and cooling to
60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/l MS
salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100
g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL
and 0.40 g/l glycine brought to volume with polished D-I H.sub.2O),
0.1 g/l myo-inositol and 40.0 g/l sucrose (brought to volume with
polished D-I H.sub.2O after adjusting pH to 5.6) and 6 g/l
bacto-agar (added after bringing to volume with polished D-I
H.sub.2O), sterilized and cooled to 60.degree. C.
Example 5
Agrobacterium-Mediated Transformation
[0346] For Agrobacterium-mediated transformation of maize with an
expression construct with the ETO1 sequence of the present
invention, preferably the method of Zhao is employed (U.S. Pat. No.
5,981,840 and PCT Patent Application Publication WO 1998/32326; the
contents of which are hereby incorporated by reference). Briefly,
immature embryos are isolated from maize and the embryos contacted
with a suspension of Agrobacterium, where the bacteria are capable
of transferring the ETO1 sequences to at least one cell of at least
one of the immature embryos (step 1: the infection step). In this
step the immature embryos are preferably immersed in an
Agrobacterium suspension for the initiation of inoculation. The
embryos are co-cultured for a time with the Agrobacterium (step 2:
the co-cultivation step). Preferably the immature embryos are
cultured on solid medium following the infection step. Following
this co-cultivation period an optional "resting" step is
contemplated. In this resting step, the embryos are incubated in
the presence of at least one antibiotic known to inhibit the growth
of Agrobacterium without the addition of a selective agent for
plant transformants (step 3: resting step). Preferably the immature
embryos are cultured on solid medium with antibiotic, but without a
selecting agent, for elimination of Agrobacterium and for a resting
phase for the infected cells. Next, inoculated embryos are cultured
on medium containing a selective agent and growing transformed
callus is recovered (step 4: the selection step). Preferably, the
immature embryos are cultured on solid medium with a selective
agent resulting in the selective growth of transformed cells. The
callus is then regenerated into plants (step 5: the regeneration
step) and preferably calli grown on selective medium are cultured
on solid medium to regenerate the plants. Plants are monitored and
scored for a modulation in meristem development: for instance,
alterations of size and appearance of the shoot and floral
meristems and/or increased yields of leaves, flowers and/or
fruits.
Example 6
Soybean Embryo Transformation
[0347] Soybean embryos are bombarded with a plasmid containing an
ETO1 sequence operably linked to an ubiquitin or other constitutive
promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm
in length dissected from surface-sterilized, immature seeds of the
soybean cultivar A2872, are cultured in the light or dark at
26.degree. C. on an appropriate agar medium for six to ten weeks.
Somatic embryos producing secondary embryos are then excised and
placed into a suitable liquid medium. After repeated selection for
clusters of somatic embryos that multiplied as early,
globular-staged embryos, the suspensions are maintained as
described below.
[0348] 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.
[0349] 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 PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0350] A selectable marker gene that can be used to facilitate
soybean transformation is a transgene composed of the 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 expression cassette
comprising an ETO1 encoding sequence operably linked to the
ubiquitin promoter 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.
[0351] 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
microliters of the DNA-coated gold particles are then loaded on
each macro carrier disk.
[0352] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 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.
[0353] 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
Sunflower Meristem Tissue Transformation
[0354] Sunflower meristem tissues are transformed with an
expression cassette containing an ETO1 sequence operably linked to
a ubiquitin promoter as follows (see also, European Patent Number
EP 0 486233, herein incorporated by reference and
Malone-Schoneberg, et al., (1994) Plant Science 103:199-207).
Mature sunflower seed (Helianthus annuus L.) are dehulled using a
single wheat-head thresher. Seeds are surface sterilized for 30
minutes in a 20% Clorox.RTM. bleach solution with the addition of
two drops of Tween.RTM. 20 per 50 ml of solution. The seeds are
rinsed twice with sterile distilled water.
[0355] Split embryonic axis explants are prepared by a modification
of procedures described by Schrammeijer, et al. (Schrammeijer, et
al., (1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in
distilled water for 60 minutes following the surface sterilization
procedure. The cotyledons of each seed are then broken off,
producing a clean fracture at the plane of the embryonic axis.
Following excision of the root tip, the explants are bisected
longitudinally between the primordial leaves. The two halves are
placed, cut surface up, on GBA medium consisting of Murashige and
Skoog mineral elements (Murashige, et al., (1962) Physiol. Plant,
15:473-497), Shepard's vitamin additions (Shepard, (1980) in
Emergent Techniques for the Genetic Improvement of Crops
(University of Minnesota Press, St. Paul, Minn.) 0, 40 mg/l adenine
sulfate, 30 g/I sucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25
mg/l indole-3-acetic acid (IAA), 0.1 mg/I gibberellic acid (GA3),
pH 5.6 and 8 g/l Phytagar.
[0356] The explants are subjected to microprojectile bombardment
prior to Agrobacterium treatment (Bidney, et al., (1992) Plant Mol.
Biol. 18:301-313). Thirty to forty explants are placed in a circle
at the center of a 60.times.20 mm plate for this treatment.
Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are
resuspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM
EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each
plate is bombarded twice through a 150 mm nytex screen placed 2 cm
above the samples in a PDS 1000.RTM. particle acceleration
device.
[0357] Disarmed Agrobacterium tumefaciens strain EHA105 is used in
all transformation experiments. A binary plasmid vector comprising
the expression cassette that contains the ETO1 gene operably linked
to the ubiquitin promoter is introduced into Agrobacterium strain
EHA105 via freeze-thawing as described by Holsters, et al., (1978)
Mol. Gen. Genet. 163:181-187. This plasmid further comprises a
kanamycin selectable marker gene (i.e., nptII). Bacteria for plant
transformation experiments are grown overnight (28.degree. C. and
100 RPM continuous agitation) in liquid YEP medium (10 gm/l yeast
extract, 10 gm/l Bactopeptone, and 5 gm/l NaCl, pH 7.0) with the
appropriate antibiotics required for bacterial strain and binary
plasmid maintenance. The suspension is used when it reaches an
OD.sub.600 of about 0.4 to 0.8. The Agrobacterium cells are
pelleted and resuspended at a final OD.sub.600 of 0.5 in an
inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l
NH.sub.4Cl, and 0.3 gm/l MgSO.sub.4.
[0358] Freshly bombarded explants are placed in an Agrobacterium
suspension, mixed, and left undisturbed for 30 minutes. The
explants are then transferred to GBA medium and co-cultivated, cut
surface down, at 26.degree. C. and 18-hour days. After three days
of co-cultivation, the explants are transferred to 374B (GBA medium
lacking growth regulators and a reduced sucrose level of 1%)
supplemented with 250 mg/l cefotaxime and 50 mg/l kanamycin
sulfate. The explants are cultured for two to five weeks on
selection and then transferred to fresh 374B medium lacking
kanamycin for one to two weeks of continued development. Explants
with differentiating, antibiotic-resistant areas of growth that
have not produced shoots suitable for excision are transferred to
GBA medium containing 250 mg/l cefotaxime for a second 3-day
phytohormone treatment. Leaf samples from green,
kanamycin-resistant shoots are assayed for the presence of NPTII by
ELISA and for the presence of transgene expression by assaying for
a modulation in meristem development (i.e., an alteration of size
and appearance of shoot and floral meristems).
[0359] NPTII-positive shoots are grafted to Pioneer.RTM. hybrid
6440 in vitro-grown sunflower seedling rootstock. Surface
sterilized seeds are germinated in 48-0 medium (half-strength
Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH 5.6) and
grown under conditions described for explant culture. The upper
portion of the seedling is removed, a 1 cm vertical slice is made
in the hypocotyl and the transformed shoot inserted into the cut.
The entire area is wrapped with parafilm to secure the shoot.
Grafted plants can be transferred to soil following one week of in
vitro culture. Grafts in soil are maintained under high humidity
conditions followed by a slow acclimatization to the greenhouse
environment. Transformed sectors of T.sub.0 plants (parental
generation) maturing in the greenhouse are identified by NPTII
ELISA and/or by ETO1 activity analysis of leaf extracts while
transgenic seeds harvested from NPTII-positive T.sub.0 plants are
identified by ETO1 analysis of small portions of dry seed
cotyledon.
[0360] An alternative sunflower transformation protocol allows the
recovery of transgenic progeny without the use of chemical
selection pressure. Seeds are dehulled and surface-sterilized for
20 minutes in a 20% Clorox bleach solution with the addition of two
to three drops of Tween 20 per 100 ml of solution, then rinsed
three times with distilled water. Sterilized seeds are imbibed in
the dark at 26.degree. C. for 20 hours on filter paper moistened
with water. The cotyledons and root radical are removed, and the
meristem explants are cultured on 374E (GBA medium consisting of MS
salts, Shepard vitamins, 40 mg/l adenine sulfate, 3% sucrose, 0.5
mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA and 0.8% Phytagar at pH 5.6)
for 24 hours under the dark. The primary leaves are removed to
expose the apical meristem, around 40 explants are placed with the
apical dome facing upward in a 2 cm circle in the center of 374M
(GBA medium with 1.2% Phytagar) and then cultured on the medium for
24 hours in the dark.
[0361] Approximately 18.8 mg of 1.8 .mu.m tungsten particles are
resuspended in 150 .mu.l absolute ethanol. After sonication, 8
.mu.l of it is dropped on the center of the surface of
macrocarrier. Each plate is bombarded twice with 650 psi rupture
discs in the first shelf at 26 mm of Hg helium gun vacuum.
[0362] The plasmid of interest is introduced into Agrobacterium
tumefaciens strain EHA105 via freeze thawing as described
previously. The pellet of overnight-grown bacteria at 28.degree. C.
in a liquid YEP medium (10 g/l yeast extract, 10 g/l Bactopeptone,
and 5 g/l NaCl, pH 7.0) in the presence of 50 .mu.g/l kanamycin is
resuspended in an inoculation medium (12.5 mM 2-mM 2-(N-morpholino)
ethanesulfonic acid, MES, 1 g/l NH.sub.4Cl and 0.3 g/l MgSO.sub.4
at pH 5.7) to reach a final concentration of 4.0 at OD.sub.600.
Particle-bombarded explants are transferred to GBA medium (374E),
and a droplet of bacteria suspension is placed directly onto the
top of the meristem. The explants are co-cultivated on the medium
for 4 days, after which the explants are transferred to 374C medium
(GBA with 1% sucrose and no BAP, IAA, GA3 and supplemented with 250
.mu.g/ml cefotaxime). The plantlets are cultured on the medium for
about two weeks under 16-hour day and 26.degree. C. incubation
conditions.
[0363] Explants (around 2 cm long) from two weeks of culture in
374C medium are screened for a modulation in meristem development
(i.e., an alteration of size and appearance of shoot and floral
meristems). After positive explants are identified, those shoots
that fail to exhibit modified ETO1 activity are discarded, and
every positive explant is subdivided into nodal explants. One nodal
explant contains at least one potential node. The nodal segments
are cultured on GBA medium for three to four days to promote the
formation of auxiliary buds from each node. Then they are
transferred to 374C medium and allowed to develop for an additional
four weeks. Developing buds are separated and cultured for an
additional four weeks on 374C medium. Pooled leaf samples from each
newly recovered shoot are screened again by the appropriate protein
activity assay. At this time, the positive shoots recovered from a
single node will generally have been enriched in the transgenic
sector detected in the initial assay prior to nodal culture.
[0364] Recovered shoots positive for modified ETO1 expression are
grafted to Pioneer hybrid 6440 in vitro-grown sunflower seedling
rootstock. The rootstocks are prepared in the following manner.
Seeds are dehulled and surface-sterilized for 20 minutes in a 20%
Clorox.RTM. bleach solution with the addition of two to three drops
of Tween 20 per 100 ml of solution, and are rinsed three times with
distilled water. The sterilized seeds are germinated on the filter
moistened with water for three days, then they are transferred into
48 medium (half-strength MS salt, 0.5% sucrose, 0.3% gelrite pH
5.0) and grown at 26.degree. C. under the dark for three days, then
incubated at 16-hour-day culture conditions. The upper portion of
selected seedling is removed, a vertical slice is made in each
hypocotyl, and a transformed shoot is inserted into a V-cut. The
cut area is wrapped with parafilm. After one week of culture on the
medium, grafted plants are transferred to soil. In the first two
weeks, they are maintained under high humidity conditions to
acclimatize to a greenhouse environment.
Example 8
Rice Tissue Transformation
[0365] One method for transforming DNA into cells of higher plants
that is available to those skilled in the art is high-velocity
ballistic bombardment using metal particles coated with the nucleic
acid constructs of interest (see, Klein, et al., (1987) Nature
(London) 327:70-73 and see, U.S. Pat. No. 4,945,050). A Biolistic
PDS-1000/He (BioRAD Laboratories, Hercules, Calif.) is used for
these complementation experiments. The particle bombardment
technique is used to transform the ETO1 mutants and wild type rice
with DNA fragments
[0366] The bacterial hygromycin B phosphotransferase (Hpt II) gene
from Streptomyces hygroscopicus that confers resistance to the
antibiotic is used as the selectable marker for rice
transformation. In the vector, pML18, the Hpt II gene was
engineered with the 35S promoter from Cauliflower Mosaic Virus and
the termination and polyadenylation signals from the octopine
synthase gene of Agrobacterium tumefaciens. pML18 was described in
WO 1997/47731, which was published on Dec. 18, 1997, the disclosure
of which is hereby incorporated by reference.
[0367] Embryogenic callus cultures derived from the scutellum of
germinating rice seeds serve as source material for transformation
experiments. This material is generated by germinating sterile rice
seeds on a callus initiation media (MS salts, Nitsch and Nitsch
vitamins, 1.0 mg/l 2,4-D and 10 .mu.M AgNO.sub.3) in the dark at
27-28.degree. C. Embryogenic callus proliferating from the
scutellum of the embryos is the transferred to CM media (N6 salts,
Nitsch and Nitsch vitamins, 1 mg/l 2,4-D, Chu, et al., (1985) Sci.
Sinica 18:659-668). Callus cultures are maintained on CM by routine
sub-culture at two week intervals and used for transformation
within 10 weeks of initiation.
[0368] Callus is prepared for transformation by subculturing
0.5-1.0 mm pieces approximately 1 mm apart, arranged in a circular
area of about 4 cm in diameter, in the center of a circle of
Whatman #541 paper placed on CM media. The plates with callus are
incubated in the dark at 27-28.degree. C. for 3-5 days. Prior to
bombardment, the filters with callus are transferred to CM
supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr in
the dark. The petri dish lids are then left ajar for 20-45 minutes
in a sterile hood to allow moisture on tissue to dissipate.
[0369] Each genomic DNA fragment is co-precipitated with pML18
containing the selectable marker for rice transformation onto the
surface of gold particles. To accomplish this, a total of 10 .mu.g
of DNA at a 2:1 ratio of trait:selectable marker DNAs are added to
50 .mu.l aliquot of gold particles that have been resuspended at a
concentration of 60 mg ml.sup.-1. Calcium chloride (50 .mu.l of a
2.5 M solution) and spermidine (20 .mu.l of a 0.1 M solution) are
then added to the gold-DNA suspension as the tube is vortexing for
3 min. The gold particles are centrifuged in a microfuge for 1 sec
and the supernatant removed. The gold particles are then washed
twice with 1 ml of absolute ethanol and then resuspended in 50
.mu.l of absolute ethanol and sonicated (bath sonicator) for one
second to disperse the gold particles. The gold suspension is
incubated at -70.degree. C. for five minutes and sonicated (bath
sonicator) if needed to disperse the particles. Six .mu.l of the
DNA-coated gold particles are then loaded onto mylar macrocarrier
disks and the ethanol is allowed to evaporate.
[0370] At the end of the drying period, a petri dish containing the
tissue is placed in the chamber of the PDS-1000/He. The air in the
chamber is then evacuated to a vacuum of 28-29 inches 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 1080-1100 psi. The tissue is placed approximately 8 cm from
the stopping screen and the callus is bombarded two times. Two to
four plates of tissue are bombarded in this way with the DNA-coated
gold particles. Following bombardment, the callus tissue is
transferred to CM media without supplemental sorbitol or
mannitol.
[0371] Within 3-5 days after bombardment the callus tissue is
transferred to SM media (CM medium containing 50 mg/l hygromycin).
To accomplish this, callus tissue is transferred from plates to
sterile 50 ml conical tubes and weighed. Molten top-agar at
40.degree. C. is added using 2.5 ml of top agar/100 mg of callus.
Callus clumps are broken into fragments of less than 2 mm diameter
by repeated dispensing through a 10 ml pipet. Three ml aliquots of
the callus suspension are plated onto fresh SM media and the plates
are incubated in the dark for 4 weeks at 27-28.degree. C. After 4
weeks, transgenic callus events are identified, transferred to
fresh SM plates and grown for an additional 2 weeks in the dark at
27-28.degree. C.
[0372] Growing callus is transferred to RM1 media (MS salts, Nitsch
and Nitsch vitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite+50 ppm
hyg B) for 2 weeks in the dark at 25.degree. C. After 2 weeks the
callus is transferred to RM2 media (MS salts, Nitsch and Nitsch
vitamins, 3% sucrose, 0.4% gelrite+50 ppm hyg B) and placed under
cool white light (.about.40 .mu.Em.sup.-2s.sup.-1) with a 12 hr
photo period at 25.degree. C. and 30-40% humidity. After 2-4 weeks
in the light, callus begin to organize, and form shoots. Shoots are
removed from surrounding callus/media and gently transferred to RM3
media (1/2.times.MS salts, Nitsch and Nitsch vitamins, 1%
sucrose+50 ppm hygromycin B) in phytatrays (Sigma Chemical Co., St.
Louis, Mo.) and incubation is continued using the same conditions
as described in the previous step.
[0373] Plants are transferred from RM3 to 4'' pots containing Metro
mix 350 after 2-3 weeks, when sufficient root and shoot growth have
occurred. The seed obtained from the transgenic plants is examined
for genetic complementation of the ETO1 mutation with the wild-type
genomic DNA containing the ETO1 gene.
Example 9
Variants of ETO1 Sequences
[0374] A. Variant Nucleotide Sequences of ETO1 Proteins that do not
Alter the Encoded Amino Acid Sequence
[0375] The ETO1 sequences having the nucleotide sequence of the
open reading frame with about 70%, 75%, 80%, 85%, 90% and 95%
nucleotide sequence identity when compared to the starting
unaltered ORF nucleotide sequence of the corresponding SEQ ID NO:
1, 3, 5, 7 or 9. These functional variants are generated using a
standard codon table. While the nucleotide sequence of the variants
are altered, the amino acid sequence encoded by the open reading
frames do not change.
[0376] B. Variant Amino Acid Sequences of ETO1 Polypeptides
[0377] Variant amino acid sequences of the ETO1 polypeptides are
generated. In this example, one amino acid is altered.
Specifically, the open reading frames are reviewed to determine the
appropriate amino acid alteration. The selection of the amino acid
to change is made by consulting the protein alignment (with the
other orthologs and other gene family members from various
species). An amino acid is selected that is deemed not to be under
high selection pressure (not highly conserved) and which is rather
easily substituted by an amino acid with similar chemical
characteristics (i.e., similar functional side-chain). Using the
protein alignment, an appropriate amino acid can be changed. Once
the targeted amino acid is identified, the procedure outlined in
the following section C is followed. Variants having about 70%,
75%, 80%, 85%, 90% and 95% nucleic acid sequence identity are
generated using this method.
[0378] C. Additional Variant Amino Acid Sequences of ETO1
Polypeptides
[0379] In this example, artificial protein sequences are created
having 80%, 85%, 90% and 95% identity relative to the reference
protein sequence. This latter effort requires identifying conserved
and variable regions from the alignment and then the judicious
application of an amino acid substitutions table. These parts will
be discussed in more detail below.
[0380] Largely, the determination of which amino acid sequences are
altered is made based on the conserved regions among ETO1 protein
or among the other ETO1 polypeptides. Based on the sequence
alignment, the various regions of the ETO1 polypeptide that can
likely be altered are represented in lower case letters, while the
conserved regions are represented by capital letters. It is
recognized that conservative substitutions can be made in the
conserved regions below without altering function. In addition, one
of skill will understand that functional variants of the ETO1
sequence of the invention can have minor non-conserved amino acid
alterations in the conserved domain.
[0381] Artificial protein sequences are then created that are
different from the original in the intervals of 80-85%, 85-90%,
90-95% and 95-100% identity. Midpoints of these intervals are
targeted, with liberal latitude of plus or minus 1%, for example.
The amino acids substitutions will be effected by a custom Perl
script. The substitution table is provided below in Table 3.
TABLE-US-00004 TABLE 3 Substitution Table Rank of Amino Strongly
Similar and Order to Acid Optimal Substitution Change Comment I L,
V 1 50:50 substitution L I, V 2 50:50 substitution V I, L 3 50:50
substitution A G 4 G A 5 D E 6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R
12 R K 13 N Q 14 Q N 15 F Y 16 M L 17 First methionine cannot
change H Na No good substitutes C Na No good substitutes P Na No
good substitutes
[0382] First, any conserved amino acids in the protein that should
not be changed is identified and "marked off" for insulation from
the substitution. The start methionine will of course be added to
this list automatically. Next, the changes are made.
[0383] H, C and P are not changed in any circumstance. The changes
will occur with isoleucine first, sweeping N-terminal to
C-terminal. Then leucine, and so on down the list until the desired
target it reached. Interim number substitutions can be made so as
not to cause reversal of changes. The list is ordered 1-17, so
start with as many isoleucine changes as needed before leucine, and
so on down to methionine. Clearly many amino acids will in this
manner not need to be changed. L, I and V will involve a 50:50
substitution of the two alternate optimal substitutions.
[0384] The variant amino acid sequences are written as output. Perl
script is used to calculate the percent identities. Using this
procedure, variants of the ETO1 polypeptides are generating having
about 80%, 85%, 90% and 95% amino acid identity to the starting
unaltered ORF nucleotide sequence of SEQ ID NO: 1, 3, 5, 7 or
9.
Example 10
Transgenic Maize Plants
[0385] T.sub.0 transgenic maize plants containing the ETO1
construct under the control of a promoter are generated. These
plants are grown in greenhouse conditions, under the FASTCORN
system, as detailed in US Patent Application Publication Number
2003/0221212, U.S. patent application Ser. No. 10/367,417.
[0386] Each of the plants is then analyzed for measurable
alteration in one or more of the following characteristics in the
following manner:
[0387] T.sub.1 progeny derived from self fertilization of each
T.sub.0 plant containing a single copy of each construct that were
found to segregate 1:1 for the transgenic event were analyzed for
improved growth rate in low KNO.sub.3. Growth is monitored up to
anthesis when cumulative plant growth, growth rate and ear weight
were determined for transgene positive, transgene null, and
non-transformed controls events. The distribution of the phenotype
of individual plants was compared to the distribution of a control
set and to the distribution of all the remaining treatments.
Variances for each set were calculated and compared using an F
test, comparing the event variance to a non-transgenic control set
variance and to the pooled variance of the remaining events in the
experiment. The greater the response to KNO.sub.3, the greater the
variance within an event set and the greater the F value. Positive
results will be compared to the distribution of the transgene
within the event to make sure the response segregates with the
transgene.
[0388] The article "a" and "an" are used herein to refer to one or
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one or more
element.
[0389] 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.
[0390] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
1313395DNAZea mays 1cggctctctc tctagctgca gattggtgcc tcacccctag
cggtcggtgg ttggggccgg 60gttccgacgg ctgctcggga gctcgactgg ggcttggaag
tccaaagccg ccgcctcctc 120gtggcgcgcc gctccggaat tcttgccttc
ttgggctccg aatctcgccg cccggtgttt 180gatttgcggc gttcctagcg
aattcggggc tgatttgccc ccctatggcg aggatttgag 240tggactcgag
aggaacaagt agaggggagt cctgatttgg gcccctgccg tggtatgtgc
300catgaggaag ctcttcttct ccgagtcggc ctgcaaggag accaagctcc
actgcgcgcc 360ccactcatgg ctgcccctcg agagggggaa gctctccaag
ttctccggcc atgccgccgc 420cggctcctcc atagaggcat tgatgaagat
gccggagccg gcagtgcttc cgtacttcaa 480gcccgcgaac tatgtcgaca
tactggctca gatacacgag gagctggagt cctgccctcc 540tgacgagaag
tcctgcctgt acctgctcca gttccaggtc ttccgcggcc ttggggaggc
600caagctgtca cggaggagcc tccagtctgc gtgggagaag gcgagcacca
tacacgagaa 660gctcatcttt ggggcgtggc tcaagtacga gaagaaaggg
gaggaggcaa tctccgacct 720gctcagctcg tgctgcaagt gctcacagga
gttcaggctg ctggattttg tgtcgcaagt 780ctccactggg tcacacatga
tgaactatga tgatgatgat gatgagtctg atgagtttcg 840gggttctgcg
gtggttcatt tccggataag agatgatatg attgcatgcg atcgacggaa
900acttgcagct ctgtcaactc cactgtatgc aatgcttaac ggtggattta
gggaatccta 960tctggaggtc attgacatgt ctagaaatgg tatctcccct
attggcatga gggcaatcag 1020taaattcagc ctatcaggaa gactacctta
tttgtcagca gatgctatct tggagatgct 1080tgattttgcc aataagtttt
gctgcaaggg cctcaaggat gcctgtgagc gaaagcttgc 1140ttctttcatc
tcttcaaggc aagatgctat agatttcatg gagtgtgctc ttgagctggg
1200ctgttccatt cttgctgctt catgcttaca agtgctcttg aatgagcttc
cagagtgctt 1260gaatgatgaa caagtggtta ggatattctc ctctgcaaat
aaggcacaga gattgacaat 1320ggttggcaat gcatctttct ccctatattg
ccttctcagt gaagtctccg tgagtaccaa 1380cccaacatcg gatgtcactg
tgagtttctt ggaaaaactg gtagagtcgg catcagattc 1440taggcagaag
cagctggcct tacatcagct ggcatgcacc agatttttaa ggaaagatta
1500ccctgaatct gagtgcttgt tcaatgctgc cttttctgct ggccatcttt
attcgttagt 1560gggtttggct agattggcct ctctgagggg taataagcat
tttgctctca agttgctaga 1620ctctgtgatg tcatctcggt ggcctcttgg
atggatgtat caagagagag cactctattt 1680ggatggtgat aacaagttag
aaaatcttaa caaggctact gagttggacc ctacccttac 1740atatccctat
atgtttcgag ctgcatcttt gatgaaaagg caaagtgttg aagctgcatt
1800gatggagata aaccggatcc ttggatttaa actggtgctg gagtgcttag
aactaaggtt 1860ctgttgctac cttgcccttg aggattatag ggctgcctta
tgtgacgtgc aggcaatcct 1920cactcttgcc ccagattatc gtatgattgg
tggccgggtt gctgccaagc agctgcgaat 1980gctagtgcta gagaatgtag
agcagtggac acctgctgac tgttggatgc aactttatga 2040tcgctggtcg
tctgtggatg atatagggtc cctctctgtt atatatcaaa tgctggagtc
2100agagactgcc aaaggagttt tgtactttag acaatctttg cttcttctca
gattaaactg 2160tcctgaggcg gcaatgagga gtttgcagct tgctcgtgag
catgctgcaa gtgatcatga 2220aaggcttgtc tatgaaggat ggatattgta
tgatactggc cactgcgagg aaggactgca 2280gaaagcggaa gcatcaattg
caatacaacg gtcatttgag gcattttttc tgaaagctta 2340tgctttggct
gattcgagtc ttgatccttc gaccacagca acagttgtat cacttctaga
2400agatgcattg cggtgtccct cagatagact tcggaagggt caagctctaa
acaaccttgg 2460aagtgtttat gtggattgtg ggaagcttga cctggcagct
gaatgctaca ttaacgccct 2520aaagatcggc cacaccagag cgcaccaagg
ccttgcgagg gttcatttcc ttcggaacaa 2580cagagtcggt gcgtatgatg
aaatgaccaa gctgatagag aaggccagga acaacgcgtc 2640ggcatacgag
aagagatctg agtactgcga gcgggagctg acgaaaacgg acttgcagat
2700ggtcaccaaa ctcgaccctc tgcgagtcta cccctacaga taccgtgctg
ctgtgctgat 2760ggacaaccac aaggagaaag aggccgtcgc ggagctgacc
agggcgatcg ccttcaaggc 2820ggacctgaac ctgctccacc tgcgcgcggc
cttccacgag cacatcggcg acatctcgag 2880cgccctccgg gactgccgcg
cggccctcct ggtggacccc aaccaccagg agatgctgga 2940gctgcaccac
cgggtgaaca gccaggaacc atgagcggag cgccgccatg gtgtacatac
3000aggacgtgat aggaagcccc tcatagccaa ccccccgcca taccagtgta
tgttttgtac 3060catacacagc atgtcaatgt aaggatagta gaaaagccac
tttaggtccc tccctggctc 3120cctccccttg caaaccaaac acctacattc
cttgtgtgcc tagttagata tgttgtttgc 3180catatagcct ttcccttagt
aaattattgt tgtcgactgt gattaagcct cctaattgta 3240cccgccacgt
ggcccgtgct gccagaatca agaagttttg actgtacctt ctgtatgtaa
3300atgcaatggg ggaaaaatga tgaatggaag cttttgtcaa gcctgcaggc
aacttgctgg 3360ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa
33952890PRTZea mays 2Met Arg Lys Leu Phe Phe Ser Glu Ser Ala Cys
Lys Glu Thr Lys Leu 1 5 10 15 His Cys Ala Pro His Ser Trp Leu Pro
Leu Glu Arg Gly Lys Leu Ser 20 25 30 Lys Phe Ser Gly His Ala Ala
Ala Gly Ser Ser Ile Glu Ala Leu Met 35 40 45 Lys Met Pro Glu Pro
Ala Val Leu Pro Tyr Phe Lys Pro Ala Asn Tyr 50 55 60 Val Asp Ile
Leu Ala Gln Ile His Glu Glu Leu Glu Ser Cys Pro Pro 65 70 75 80 Asp
Glu Lys Ser Cys Leu Tyr Leu Leu Gln Phe Gln Val Phe Arg Gly 85 90
95 Leu Gly Glu Ala Lys Leu Ser Arg Arg Ser Leu Gln Ser Ala Trp Glu
100 105 110 Lys Ala Ser Thr Ile His Glu Lys Leu Ile Phe Gly Ala Trp
Leu Lys 115 120 125 Tyr Glu Lys Lys Gly Glu Glu Ala Ile Ser Asp Leu
Leu Ser Ser Cys 130 135 140 Cys Lys Cys Ser Gln Glu Phe Arg Leu Leu
Asp Phe Val Ser Gln Val 145 150 155 160 Ser Thr Gly Ser His Met Met
Asn Tyr Asp Asp Asp Asp Asp Glu Ser 165 170 175 Asp Glu Phe Arg Gly
Ser Ala Val Val His Phe Arg Ile Arg Asp Asp 180 185 190 Met Ile Ala
Cys Asp Arg Arg Lys Leu Ala Ala Leu Ser Thr Pro Leu 195 200 205 Tyr
Ala Met Leu Asn Gly Gly Phe Arg Glu Ser Tyr Leu Glu Val Ile 210 215
220 Asp Met Ser Arg Asn Gly Ile Ser Pro Ile Gly Met Arg Ala Ile Ser
225 230 235 240 Lys Phe Ser Leu Ser Gly Arg Leu Pro Tyr Leu Ser Ala
Asp Ala Ile 245 250 255 Leu Glu Met Leu Asp Phe Ala Asn Lys Phe Cys
Cys Lys Gly Leu Lys 260 265 270 Asp Ala Cys Glu Arg Lys Leu Ala Ser
Phe Ile Ser Ser Arg Gln Asp 275 280 285 Ala Ile Asp Phe Met Glu Cys
Ala Leu Glu Leu Gly Cys Ser Ile Leu 290 295 300 Ala Ala Ser Cys Leu
Gln Val Leu Leu Asn Glu Leu Pro Glu Cys Leu 305 310 315 320 Asn Asp
Glu Gln Val Val Arg Ile Phe Ser Ser Ala Asn Lys Ala Gln 325 330 335
Arg Leu Thr Met Val Gly Asn Ala Ser Phe Ser Leu Tyr Cys Leu Leu 340
345 350 Ser Glu Val Ser Val Ser Thr Asn Pro Thr Ser Asp Val Thr Val
Ser 355 360 365 Phe Leu Glu Lys Leu Val Glu Ser Ala Ser Asp Ser Arg
Gln Lys Gln 370 375 380 Leu Ala Leu His Gln Leu Ala Cys Thr Arg Phe
Leu Arg Lys Asp Tyr 385 390 395 400 Pro Glu Ser Glu Cys Leu Phe Asn
Ala Ala Phe Ser Ala Gly His Leu 405 410 415 Tyr Ser Leu Val Gly Leu
Ala Arg Leu Ala Ser Leu Arg Gly Asn Lys 420 425 430 His Phe Ala Leu
Lys Leu Leu Asp Ser Val Met Ser Ser Arg Trp Pro 435 440 445 Leu Gly
Trp Met Tyr Gln Glu Arg Ala Leu Tyr Leu Asp Gly Asp Asn 450 455 460
Lys Leu Glu Asn Leu Asn Lys Ala Thr Glu Leu Asp Pro Thr Leu Thr 465
470 475 480 Tyr Pro Tyr Met Phe Arg Ala Ala Ser Leu Met Lys Arg Gln
Ser Val 485 490 495 Glu Ala Ala Leu Met Glu Ile Asn Arg Ile Leu Gly
Phe Lys Leu Val 500 505 510 Leu Glu Cys Leu Glu Leu Arg Phe Cys Cys
Tyr Leu Ala Leu Glu Asp 515 520 525 Tyr Arg Ala Ala Leu Cys Asp Val
Gln Ala Ile Leu Thr Leu Ala Pro 530 535 540 Asp Tyr Arg Met Ile Gly
Gly Arg Val Ala Ala Lys Gln Leu Arg Met 545 550 555 560 Leu Val Leu
Glu Asn Val Glu Gln Trp Thr Pro Ala Asp Cys Trp Met 565 570 575 Gln
Leu Tyr Asp Arg Trp Ser Ser Val Asp Asp Ile Gly Ser Leu Ser 580 585
590 Val Ile Tyr Gln Met Leu Glu Ser Glu Thr Ala Lys Gly Val Leu Tyr
595 600 605 Phe Arg Gln Ser Leu Leu Leu Leu Arg Leu Asn Cys Pro Glu
Ala Ala 610 615 620 Met Arg Ser Leu Gln Leu Ala Arg Glu His Ala Ala
Ser Asp His Glu 625 630 635 640 Arg Leu Val Tyr Glu Gly Trp Ile Leu
Tyr Asp Thr Gly His Cys Glu 645 650 655 Glu Gly Leu Gln Lys Ala Glu
Ala Ser Ile Ala Ile Gln Arg Ser Phe 660 665 670 Glu Ala Phe Phe Leu
Lys Ala Tyr Ala Leu Ala Asp Ser Ser Leu Asp 675 680 685 Pro Ser Thr
Thr Ala Thr Val Val Ser Leu Leu Glu Asp Ala Leu Arg 690 695 700 Cys
Pro Ser Asp Arg Leu Arg Lys Gly Gln Ala Leu Asn Asn Leu Gly 705 710
715 720 Ser Val Tyr Val Asp Cys Gly Lys Leu Asp Leu Ala Ala Glu Cys
Tyr 725 730 735 Ile Asn Ala Leu Lys Ile Gly His Thr Arg Ala His Gln
Gly Leu Ala 740 745 750 Arg Val His Phe Leu Arg Asn Asn Arg Val Gly
Ala Tyr Asp Glu Met 755 760 765 Thr Lys Leu Ile Glu Lys Ala Arg Asn
Asn Ala Ser Ala Tyr Glu Lys 770 775 780 Arg Ser Glu Tyr Cys Glu Arg
Glu Leu Thr Lys Thr Asp Leu Gln Met 785 790 795 800 Val Thr Lys Leu
Asp Pro Leu Arg Val Tyr Pro Tyr Arg Tyr Arg Ala 805 810 815 Ala Val
Leu Met Asp Asn His Lys Glu Lys Glu Ala Val Ala Glu Leu 820 825 830
Thr Arg Ala Ile Ala Phe Lys Ala Asp Leu Asn Leu Leu His Leu Arg 835
840 845 Ala Ala Phe His Glu His Ile Gly Asp Ile Ser Ser Ala Leu Arg
Asp 850 855 860 Cys Arg Ala Ala Leu Leu Val Asp Pro Asn His Gln Glu
Met Leu Glu 865 870 875 880 Leu His His Arg Val Asn Ser Gln Glu Pro
885 890 33486DNAZea mays 3ccgcccccca ccgcgctctc gccctccctc
tccctctcta gcggtagatt ggtgctgttc 60ccccctaatc ggtcggcggt tgggggccgg
gttccgacgt ctgcccggga gctcgaccgg 120gcctccgatc cgggctccca
agccgccgcc tcccggggcg cccctccgga attcttgctt 180tctcgggctc
cgaatctcgc cgcctgtcgt cgccagcggc tggcgcgggg gcttggttcc
240cggcgtttca ttgcgaattc gggggcgatc tggggcccct ggtctggatg
gcgaggattt 300gagtggattc gagacgaaca agtggaggtg gagccctgat
ctgggtggcg cccccgccgt 360ggtatgtgcc atgaggaagc tcttcttctc
cgagtcggcc tgcaaagaga ccaagcttca 420ctccgcgccc cactcatggc
tgcccctcga gagggggaag ctctccaagt tctccggcca 480tgccgccgcc
ggctcctcca tagagtcctt gatgaagatg ccagagccgg ctgtgcttcc
540gcacttcaag cccgcggact atgtcgacgt actggctcag atacacgagg
agctggagtc 600ctgcccccct gacgacaagt cctccctgta cctcctccag
tatcaggtct tccgtggcct 660cggcgaggcc aagctgtctc ggaggagcct
ccagtctgcg tgggagaagg ggagcaccat 720acacgagaag ctcatcttcg
gggcatggct caagtacgag aagaaagggg aggaggccat 780ctccgacctg
ctcagctcgt gcagcaagtg cttgcaggag ttcaggctgc tggatttcgt
840gttgcaggtc tccactgggt cacatgtgat aaactacgat ggtgatgatg
atgagtttcg 900gggttctgcg gtggttcatt tccggataag agatgatatg
gttgcgtgcg atcgtcggaa 960gctcgcggcg ctgtcaactc cactgtatgc
aatgcttaac ggtggattta gggaatcata 1020tctagaggtc attgacatgt
ctagaaatgg tatctcccct attggcatga gggcaatcag 1080taaattcagc
ctatcaggaa gactaccgta tttgtcagct gatgctatct tggagattct
1140tgattttgcc aataagtttt gctgcaaggg cctcaaggat gcctgtgagc
gaaagcttgc 1200ttctttcgtc tcttcaaggc aagatgctat agacttcatg
gagtgcgctc ttgagctggg 1260ctgttccatc cttgctgctt catgcttgca
agtgctcttg aatgagcttc cagagtgctt 1320gaatgatgaa caagtggtta
ggatattctc ttctgcaaat aaggcacaga gattgacaat 1380ggttggcaat
gcatctttct ccctatattg tcttcttagt gaagtctcca tgagtaccaa
1440cccaacatcg gatgtcactg taagtttctt ggaaaagctg gtagagtcgg
catcagattc 1500taggcaaaat cagctggcct tacatcagct ggcatgcacc
aaatttctaa ggaaagatta 1560ccctgaatct gagcgcctgt tcaatgctgc
attttctgcc ggccatctct attcgatagt 1620gggtttagct agattggcct
ctctgagggg taataagcat tttgctctca agttgctaga 1680ctctgtcatg
tcatctcggt ggcctcttgg gtggatgtat caagagagag ctctatattt
1740ggatggtgat aacaagttag aaaatcttaa caaggctact gagttggacc
ctactcttac 1800atatccctat atgttccgag ctgcatcttt gatgaaaagg
caaagtgttg aagctgcatt 1860gatggagatc aaccggatac ttggattcaa
gctggtgctg gagtgcttag aactaaggtt 1920ctgttgctac cttgcccttg
aggattatag ggctgcctta tgtgacgtgc aggcaatact 1980cactcttgcc
ccagattatc gtatgattgg tggccgggtt gctgccaagc agctgagaat
2040gctagtgcta gagaatgtag agcagtggac agctgctgac tgttggatgc
agctttatga 2100tcgctggtca tctgtggatg atatagggtc cctctctgtt
atatatcaaa tgttggagtc 2160agataccgcc aaaggagttt tgtactttag
gcaatctttg cttcttctca gattaaactg 2220tcctgaggcg gcaatgagga
gtttgcagct tgctcgtgag catgctgcga gtgatcatga 2280aaggcttgtc
tatgaaggat ggatattgta tgatactggc cactgcgagg aaggattgca
2340gaaggcagaa gcatccattg caatacaaag gtcatttgag gcattttttc
tgaaagctta 2400tgctttggct gattcgagtc ttgatccttc tacctcagca
acagttgtat cacttctaga 2460agatgcattg cggtgtccct cagatagact
tcggaagggt caggctctaa acaacctcgg 2520aagtgtttat gtggattgtg
ggaagctaga cctggcagct gaatgctaca ttaatgcact 2580aaagatcggt
cacaccagag cgcatcaagg ccttgcaagg gttcatttcc tacggaacaa
2640cagagctggt gcatacgacg aaatgaccaa gctgatagag aaggccagga
acaacgcttc 2700ggcatatgag aagagatccg agtactgtga ccgggagctg
acgaaaacgg acctgcagat 2760ggtcaccaaa ctcgaccctc tgcgagttta
tccttacaga taccgtgctg ctgtgctgat 2820ggacaaccac aaggagaaag
aggcgatcgc ggagctgacc aaggccatcg ccttcaaggc 2880ggacctgaac
ctgctccacc tgcgcgcggc cttccacgag cacgtgggcg acgtctcgag
2940cgccctccag gactgccgcg cggccctctc ggtggacccc aaccaccagg
agatgctgga 3000gcttcaccac cgggtgaaca gccaggaacc ctgagcgcgc
tcccacggtg tacatacagg 3060acaggaagcc cctcatcata gccaaccggc
cataccggtg tatgttttgt accatacaca 3120gcagatcaga tcaatgtaag
gacacagtag aaagccacat tagatccctc cccttgaaaa 3180ccaaacaccc
cattccttgt gtccctaatt attagatata tatatgtgtt gtttgctata
3240gcctcccctt agtaagttgt tgctgccgat tgtgattaag cctcctaatt
gtacccgcca 3300tgtgccccag cggcccgtgc ttccagaatc aagaagtttt
gactgtacca tgtgtatgta 3360agtgaaatgg gggaacaaag gatggtggaa
gcttttgtcc gcgccaaact gtcaagcatg 3420caggcacctg ttcttgccga
gcacttgatt gattgcaaaa aaaaaaaaaa aaaaaaaaaa 3480aaaaaa
34864887PRTZea mays 4Met Arg Lys Leu Phe Phe Ser Glu Ser Ala Cys
Lys Glu Thr Lys Leu 1 5 10 15 His Ser Ala Pro His Ser Trp Leu Pro
Leu Glu Arg Gly Lys Leu Ser 20 25 30 Lys Phe Ser Gly His Ala Ala
Ala Gly Ser Ser Ile Glu Ser Leu Met 35 40 45 Lys Met Pro Glu Pro
Ala Val Leu Pro His Phe Lys Pro Ala Asp Tyr 50 55 60 Val Asp Val
Leu Ala Gln Ile His Glu Glu Leu Glu Ser Cys Pro Pro 65 70 75 80 Asp
Asp Lys Ser Ser Leu Tyr Leu Leu Gln Tyr Gln Val Phe Arg Gly 85 90
95 Leu Gly Glu Ala Lys Leu Ser Arg Arg Ser Leu Gln Ser Ala Trp Glu
100 105 110 Lys Gly Ser Thr Ile His Glu Lys Leu Ile Phe Gly Ala Trp
Leu Lys 115 120 125 Tyr Glu Lys Lys Gly Glu Glu Ala Ile Ser Asp Leu
Leu Ser Ser Cys 130 135 140 Ser Lys Cys Leu Gln Glu Phe Arg Leu Leu
Asp Phe Val Leu Gln Val 145 150 155 160 Ser Thr Gly Ser His Val Ile
Asn Tyr Asp Gly Asp Asp Asp Glu Phe 165 170 175 Arg Gly Ser Ala Val
Val His Phe Arg Ile Arg Asp Asp Met Val Ala 180 185 190 Cys Asp Arg
Arg Lys Leu Ala Ala Leu Ser Thr Pro Leu Tyr Ala Met 195 200 205 Leu
Asn Gly Gly Phe Arg Glu Ser Tyr Leu Glu Val Ile Asp Met Ser 210 215
220 Arg Asn Gly Ile Ser Pro Ile Gly Met Arg Ala Ile Ser Lys Phe Ser
225 230 235 240 Leu Ser Gly Arg Leu Pro Tyr Leu Ser Ala Asp Ala Ile
Leu Glu Ile 245 250 255 Leu Asp Phe Ala Asn Lys Phe Cys Cys Lys Gly
Leu Lys Asp Ala Cys 260 265 270 Glu Arg Lys Leu Ala Ser Phe Val Ser
Ser Arg Gln Asp Ala Ile Asp 275 280 285 Phe Met Glu Cys Ala Leu Glu
Leu Gly Cys Ser Ile Leu Ala Ala Ser 290 295 300 Cys Leu Gln Val Leu
Leu Asn Glu Leu Pro Glu Cys Leu Asn Asp Glu 305 310 315 320 Gln Val
Val Arg Ile Phe Ser Ser Ala Asn Lys Ala Gln Arg Leu Thr 325 330 335
Met Val Gly Asn Ala Ser Phe Ser Leu Tyr Cys Leu Leu Ser Glu
Val 340 345 350 Ser Met Ser Thr Asn Pro Thr Ser Asp Val Thr Val Ser
Phe Leu Glu 355 360 365 Lys Leu Val Glu Ser Ala Ser Asp Ser Arg Gln
Asn Gln Leu Ala Leu 370 375 380 His Gln Leu Ala Cys Thr Lys Phe Leu
Arg Lys Asp Tyr Pro Glu Ser 385 390 395 400 Glu Arg Leu Phe Asn Ala
Ala Phe Ser Ala Gly His Leu Tyr Ser Ile 405 410 415 Val Gly Leu Ala
Arg Leu Ala Ser Leu Arg Gly Asn Lys His Phe Ala 420 425 430 Leu Lys
Leu Leu Asp Ser Val Met Ser Ser Arg Trp Pro Leu Gly Trp 435 440 445
Met Tyr Gln Glu Arg Ala Leu Tyr Leu Asp Gly Asp Asn Lys Leu Glu 450
455 460 Asn Leu Asn Lys Ala Thr Glu Leu Asp Pro Thr Leu Thr Tyr Pro
Tyr 465 470 475 480 Met Phe Arg Ala Ala Ser Leu Met Lys Arg Gln Ser
Val Glu Ala Ala 485 490 495 Leu Met Glu Ile Asn Arg Ile Leu Gly Phe
Lys Leu Val Leu Glu Cys 500 505 510 Leu Glu Leu Arg Phe Cys Cys Tyr
Leu Ala Leu Glu Asp Tyr Arg Ala 515 520 525 Ala Leu Cys Asp Val Gln
Ala Ile Leu Thr Leu Ala Pro Asp Tyr Arg 530 535 540 Met Ile Gly Gly
Arg Val Ala Ala Lys Gln Leu Arg Met Leu Val Leu 545 550 555 560 Glu
Asn Val Glu Gln Trp Thr Ala Ala Asp Cys Trp Met Gln Leu Tyr 565 570
575 Asp Arg Trp Ser Ser Val Asp Asp Ile Gly Ser Leu Ser Val Ile Tyr
580 585 590 Gln Met Leu Glu Ser Asp Thr Ala Lys Gly Val Leu Tyr Phe
Arg Gln 595 600 605 Ser Leu Leu Leu Leu Arg Leu Asn Cys Pro Glu Ala
Ala Met Arg Ser 610 615 620 Leu Gln Leu Ala Arg Glu His Ala Ala Ser
Asp His Glu Arg Leu Val 625 630 635 640 Tyr Glu Gly Trp Ile Leu Tyr
Asp Thr Gly His Cys Glu Glu Gly Leu 645 650 655 Gln Lys Ala Glu Ala
Ser Ile Ala Ile Gln Arg Ser Phe Glu Ala Phe 660 665 670 Phe Leu Lys
Ala Tyr Ala Leu Ala Asp Ser Ser Leu Asp Pro Ser Thr 675 680 685 Ser
Ala Thr Val Val Ser Leu Leu Glu Asp Ala Leu Arg Cys Pro Ser 690 695
700 Asp Arg Leu Arg Lys Gly Gln Ala Leu Asn Asn Leu Gly Ser Val Tyr
705 710 715 720 Val Asp Cys Gly Lys Leu Asp Leu Ala Ala Glu Cys Tyr
Ile Asn Ala 725 730 735 Leu Lys Ile Gly His Thr Arg Ala His Gln Gly
Leu Ala Arg Val His 740 745 750 Phe Leu Arg Asn Asn Arg Ala Gly Ala
Tyr Asp Glu Met Thr Lys Leu 755 760 765 Ile Glu Lys Ala Arg Asn Asn
Ala Ser Ala Tyr Glu Lys Arg Ser Glu 770 775 780 Tyr Cys Asp Arg Glu
Leu Thr Lys Thr Asp Leu Gln Met Val Thr Lys 785 790 795 800 Leu Asp
Pro Leu Arg Val Tyr Pro Tyr Arg Tyr Arg Ala Ala Val Leu 805 810 815
Met Asp Asn His Lys Glu Lys Glu Ala Ile Ala Glu Leu Thr Lys Ala 820
825 830 Ile Ala Phe Lys Ala Asp Leu Asn Leu Leu His Leu Arg Ala Ala
Phe 835 840 845 His Glu His Val Gly Asp Val Ser Ser Ala Leu Gln Asp
Cys Arg Ala 850 855 860 Ala Leu Ser Val Asp Pro Asn His Gln Glu Met
Leu Glu Leu His His 865 870 875 880 Arg Val Asn Ser Gln Glu Pro 885
53763DNAZea mays 5ggcgggaggg cgggggcgac gcgcgcgcac cagccgttcc
gccggagaac tcccgctctg 60gcacgatcgt cgccgtgcga caggctgctg gccaaacgcg
cgcgcgatag ccgaggggag 120gaggacgtag aggaggggta agccggctgc
ggaattcacc atgaccaata acttcctcac 180gacgataaag agcctgaagc
tgatcgaggg ttgcaaagcc gcacaattat acgccttaag 240ctccgttggg
gcagcctcca cgtccggctc gggggatgcc ggagggagca gcaacggcaa
300gccccagcct cctccgccgc caaagaccat ctcgatgcgg tccggatcgc
tgtactaccc 360gcacgcggcg ccgtccacgt cgggcgcctt cgtgcccgag
ccgcacctgc cgtgcggcct 420cccggtggcc gacgccctcg agccggccct
ggacgcgtgc ctgcgccccg tcgaccacgt 480cggcgtgctc gccgcgtcgt
accggcgggt ctcggccgcc acggcggggg gcgacgacga 540cctctgcgac
gcgtacctgg agcagcacgc gctgttccag tcgatcggcg acgcgaggct
600gatccggcgg gcgctgcggg ccgcgcgcgt ccacgcggac aacccgcacc
ggcgcgccgt 660gctcgccgcg tggctccggt acgagcgccg cgaggacgag
ctcgacccgg cgccgccgcc 720gctcgcgccc tgcaccgcga cgacgccgct
gctcgagtgc ccccgcgccg ctgtcttcgc 780cagcgtgtcc cactcccaca
gcgtggaccc ggtctgcccg tgccgccgcc caccgcttcc 840tccagtcacc
cctccaccgc accgcctgag gcgcaacacg tcgggcgccg cctccgagat
900gagcgaggag gaggagccgg agaccaatga cctgtggttc atcatcggcg
aggaggaggt 960agcgtgcgag cggtcgtgca tcgcggcgct ctcaaagccg
ctcaacaccc tcctctacgg 1020cgggttcgcc gaggcgcacc gcgaccggat
cgacttctcc cgcgacggca tcacgccgcg 1080cggcatgcgc gcggtctccg
cctacagccg ccacggccgc gtggacgact tcccgcccga 1140cgtcatctcc
cagctcctcg cattcgccaa caagttctgc tgcgagggcc tgaaggcagc
1200ttgcgacaac cagctcgcgg ccatggtgcg gggtctcgac gacgcccggt
ccctcatcga 1260catcggcctc gaggaggcct cccacctcct cgtcgcctcc
tgcctccagg cgttcctgcg 1320ggagctcccc aagtcgctca cgtgcccgga
catcgcgcgc ctgctctgca gcccagaggg 1380gcgagagcgc cttgacatct
ccggtaacgc gtccttcgcg ctctaccact tcctctctta 1440cgtcgccatg
gagcaggaca tgaggtcgaa caccacggtg atgctgctgg agaggctgaa
1500tgaattcgcg gagcagccat ggcagaagca gctggcactg caccagctcg
ggtgcgtgat 1560gctccagcgc ggcgagttcg aggaagcgca ggagtggttc
gaggccgccg tcggcgaggg 1620ccatgtgtac tcggtcgccg gagaggcacg
tgccaagtac aagcgcgggc acaagtacgc 1680cgcgtacaag ctaatgaaca
gtattctcgg cgagtacgac gaacccgccg ggtggatgta 1740ccaagagcgc
tccctgtact gtgtcggcaa ggagaagttg gctgatctgc aggcggcgac
1800ggagctggac cctacgatga cattcccgta caaatatcgt gcgtgcgcgc
tgctggagga 1860ggacaatgct gcgtccgcga tcgcagagat cagcagggtc
gtcggtttca agatggcgac 1920cgattgcctt gagctccggg cgtggttcta
ccttgcgctt gagcagtgcg agctggctgt 1980gcaggacgtg agggcgatat
tgacgttgga tccaacctac atgatgttcc acgggagaat 2040gcacggggag
cagctgattg agctcctccg aggacaggtg cagcagtggg atatggcgga
2100ttgctggatg cagctgtacg gtcggtggtc ggcggtggat gacatcggct
ctctggcggt 2160tgtccagcag atgctctcca gggaacccgg aaacagcagc
ttgcggtttc gacagtcact 2220tctccttcta aggctaaact gtcagaaagc
tgccatgcgc agtttgcgat atgctcggaa 2280cagcacgctc catgagcatg
agaggctcgt atacgaaggg tggattctgt atgacagtgg 2340gcatcgcgac
gaagcgttag ccaaggccga gcagtcgatc ggactccaga gatcattcga
2400agccttcttc ctcaaggcgt acgccttagg agattctagc cttgacacgg
aatcctcgct 2460ctccgtggtc cagcttctgg aacatgccaa cagctgtgct
tccgacaacc ttcgcaaggg 2520gcaggcatac aacaacatgg ggagcatcta
cgtggactgt gacatgctgg acgaggctgc 2580cgagtgctac ggcatcgcgc
tgaacataaa gcacacacgg gcgcatcagg gcctagctcg 2640agtccactac
ttgaaaaaca ggaaaaaggt tgcgtttgag gagatgacga agctcgtgga
2700gattgccagc aactgcgcgt cggcgtatga aaagcggtcg gaatacggtg
agcgcgaagc 2760tgcgaggagc gacctgaaca tggcgacgct tcttgatcct
accaggactt atccttacag 2820atacagagca gctgtactga tggacgaggg
caaggaggag gaggcgatcg cggagctgtc 2880aggagccata gctttcaagc
cggacctcca gctgctgcac ctccgcgcgg cgttcttcga 2940ctccatgggc
gagcgcgaga gcgccctgcg ggactgcgag gccgcgctct gcctggaccc
3000gacccacggc gacacattgg agctgtacag caaagcctcc accaccaagg
ccgaacccca 3060gagctaggca gccagccggc cggccggccg gcaggccgcc
gctctcctcg tcgtcgattc 3120agctgcggtt tttgcgaggc aggatgatga
gacgatctct tctctactct catggggtgg 3180aagctgcaga tcagtgaggc
aggagcaccg gaacatgcac atatctcttc taagagtata 3240tacaagagcc
ttagttctgt tactgttaga gttggacatg gggaggcagc accgcaggag
3300attgagtgcg tgttgcctta agggtagact gcgcaggtga ggtgacaaag
agcatgcact 3360gcactgcact gcaccacata tgtgcatcca aggttgaaga
cgaccagcac ctccggtcag 3420aagagaggaa ggagaggcgg ctggagaatg
agagccaggt cagcagggtg tgcaaaccgc 3480cggcggtacc aacgaatctt
cctctttttc ttcttttgct tgaatttatg ccttgtgacg 3540tgcatctgga
ggcacgactg attacaaaag aatacgagtt tttttaaagt aacgcagcgc
3600gaaagggaag attcttcctg ctgccgactg cacgctgtat tatgtatgag
tcgtggctcc 3660gtcgtgcctc cagctaacga ggccctgaca tgcatctgct
gcattgctac acgttcgttc 3720gtgttcacaa ctacgctttg ttctttcgtt
ccaattccaa atc 37636968PRTZea mays 6Met Thr Asn Asn Phe Leu Thr Thr
Ile Lys Ser Leu Lys Leu Ile Glu 1 5 10 15 Gly Cys Lys Ala Ala Gln
Leu Tyr Ala Leu Ser Ser Val Gly Ala Ala 20 25 30 Ser Thr Ser Gly
Ser Gly Asp Ala Gly Gly Ser Ser Asn Gly Lys Pro 35 40 45 Gln Pro
Pro Pro Pro Pro Lys Thr Ile Ser Met Arg Ser Gly Ser Leu 50 55 60
Tyr Tyr Pro His Ala Ala Pro Ser Thr Ser Gly Ala Phe Val Pro Glu 65
70 75 80 Pro His Leu Pro Cys Gly Leu Pro Val Ala Asp Ala Leu Glu
Pro Ala 85 90 95 Leu Asp Ala Cys Leu Arg Pro Val Asp His Val Gly
Val Leu Ala Ala 100 105 110 Ser Tyr Arg Arg Val Ser Ala Ala Thr Ala
Gly Gly Asp Asp Asp Leu 115 120 125 Cys Asp Ala Tyr Leu Glu Gln His
Ala Leu Phe Gln Ser Ile Gly Asp 130 135 140 Ala Arg Leu Ile Arg Arg
Ala Leu Arg Ala Ala Arg Val His Ala Asp 145 150 155 160 Asn Pro His
Arg Arg Ala Val Leu Ala Ala Trp Leu Arg Tyr Glu Arg 165 170 175 Arg
Glu Asp Glu Leu Asp Pro Ala Pro Pro Pro Leu Ala Pro Cys Thr 180 185
190 Ala Thr Thr Pro Leu Leu Glu Cys Pro Arg Ala Ala Val Phe Ala Ser
195 200 205 Val Ser His Ser His Ser Val Asp Pro Val Cys Pro Cys Arg
Arg Pro 210 215 220 Pro Leu Pro Pro Val Thr Pro Pro Pro His Arg Leu
Arg Arg Asn Thr 225 230 235 240 Ser Gly Ala Ala Ser Glu Met Ser Glu
Glu Glu Glu Pro Glu Thr Asn 245 250 255 Asp Leu Trp Phe Ile Ile Gly
Glu Glu Glu Val Ala Cys Glu Arg Ser 260 265 270 Cys Ile Ala Ala Leu
Ser Lys Pro Leu Asn Thr Leu Leu Tyr Gly Gly 275 280 285 Phe Ala Glu
Ala His Arg Asp Arg Ile Asp Phe Ser Arg Asp Gly Ile 290 295 300 Thr
Pro Arg Gly Met Arg Ala Val Ser Ala Tyr Ser Arg His Gly Arg 305 310
315 320 Val Asp Asp Phe Pro Pro Asp Val Ile Ser Gln Leu Leu Ala Phe
Ala 325 330 335 Asn Lys Phe Cys Cys Glu Gly Leu Lys Ala Ala Cys Asp
Asn Gln Leu 340 345 350 Ala Ala Met Val Arg Gly Leu Asp Asp Ala Arg
Ser Leu Ile Asp Ile 355 360 365 Gly Leu Glu Glu Ala Ser His Leu Leu
Val Ala Ser Cys Leu Gln Ala 370 375 380 Phe Leu Arg Glu Leu Pro Lys
Ser Leu Thr Cys Pro Asp Ile Ala Arg 385 390 395 400 Leu Leu Cys Ser
Pro Glu Gly Arg Glu Arg Leu Asp Ile Ser Gly Asn 405 410 415 Ala Ser
Phe Ala Leu Tyr His Phe Leu Ser Tyr Val Ala Met Glu Gln 420 425 430
Asp Met Arg Ser Asn Thr Thr Val Met Leu Leu Glu Arg Leu Asn Glu 435
440 445 Phe Ala Glu Gln Pro Trp Gln Lys Gln Leu Ala Leu His Gln Leu
Gly 450 455 460 Cys Val Met Leu Gln Arg Gly Glu Phe Glu Glu Ala Gln
Glu Trp Phe 465 470 475 480 Glu Ala Ala Val Gly Glu Gly His Val Tyr
Ser Val Ala Gly Glu Ala 485 490 495 Arg Ala Lys Tyr Lys Arg Gly His
Lys Tyr Ala Ala Tyr Lys Leu Met 500 505 510 Asn Ser Ile Leu Gly Glu
Tyr Asp Glu Pro Ala Gly Trp Met Tyr Gln 515 520 525 Glu Arg Ser Leu
Tyr Cys Val Gly Lys Glu Lys Leu Ala Asp Leu Gln 530 535 540 Ala Ala
Thr Glu Leu Asp Pro Thr Met Thr Phe Pro Tyr Lys Tyr Arg 545 550 555
560 Ala Cys Ala Leu Leu Glu Glu Asp Asn Ala Ala Ser Ala Ile Ala Glu
565 570 575 Ile Ser Arg Val Val Gly Phe Lys Met Ala Thr Asp Cys Leu
Glu Leu 580 585 590 Arg Ala Trp Phe Tyr Leu Ala Leu Glu Gln Cys Glu
Leu Ala Val Gln 595 600 605 Asp Val Arg Ala Ile Leu Thr Leu Asp Pro
Thr Tyr Met Met Phe His 610 615 620 Gly Arg Met His Gly Glu Gln Leu
Ile Glu Leu Leu Arg Gly Gln Val 625 630 635 640 Gln Gln Trp Asp Met
Ala Asp Cys Trp Met Gln Leu Tyr Gly Arg Trp 645 650 655 Ser Ala Val
Asp Asp Ile Gly Ser Leu Ala Val Val Gln Gln Met Leu 660 665 670 Ser
Arg Glu Pro Gly Asn Ser Ser Leu Arg Phe Arg Gln Ser Leu Leu 675 680
685 Leu Leu Arg Leu Asn Cys Gln Lys Ala Ala Met Arg Ser Leu Arg Tyr
690 695 700 Ala Arg Asn Ser Thr Leu His Glu His Glu Arg Leu Val Tyr
Glu Gly 705 710 715 720 Trp Ile Leu Tyr Asp Ser Gly His Arg Asp Glu
Ala Leu Ala Lys Ala 725 730 735 Glu Gln Ser Ile Gly Leu Gln Arg Ser
Phe Glu Ala Phe Phe Leu Lys 740 745 750 Ala Tyr Ala Leu Gly Asp Ser
Ser Leu Asp Thr Glu Ser Ser Leu Ser 755 760 765 Val Val Gln Leu Leu
Glu His Ala Asn Ser Cys Ala Ser Asp Asn Leu 770 775 780 Arg Lys Gly
Gln Ala Tyr Asn Asn Met Gly Ser Ile Tyr Val Asp Cys 785 790 795 800
Asp Met Leu Asp Glu Ala Ala Glu Cys Tyr Gly Ile Ala Leu Asn Ile 805
810 815 Lys His Thr Arg Ala His Gln Gly Leu Ala Arg Val His Tyr Leu
Lys 820 825 830 Asn Arg Lys Lys Val Ala Phe Glu Glu Met Thr Lys Leu
Val Glu Ile 835 840 845 Ala Ser Asn Cys Ala Ser Ala Tyr Glu Lys Arg
Ser Glu Tyr Gly Glu 850 855 860 Arg Glu Ala Ala Arg Ser Asp Leu Asn
Met Ala Thr Leu Leu Asp Pro 865 870 875 880 Thr Arg Thr Tyr Pro Tyr
Arg Tyr Arg Ala Ala Val Leu Met Asp Glu 885 890 895 Gly Lys Glu Glu
Glu Ala Ile Ala Glu Leu Ser Gly Ala Ile Ala Phe 900 905 910 Lys Pro
Asp Leu Gln Leu Leu His Leu Arg Ala Ala Phe Phe Asp Ser 915 920 925
Met Gly Glu Arg Glu Ser Ala Leu Arg Asp Cys Glu Ala Ala Leu Cys 930
935 940 Leu Asp Pro Thr His Gly Asp Thr Leu Glu Leu Tyr Ser Lys Ala
Ser 945 950 955 960 Thr Thr Lys Ala Glu Pro Gln Ser 965 73477DNAZea
mays 7cttcctgctc tcgctgagcc tgcaggatct gaatccgagc tcgctcgcat
ccactatctg 60caggccccac gcgccctgtt ccttcctccc gctaacaatc gccctgttcc
cggtttgatc 120cgttgaattc tgcccggcgc gcgggggctt gcgggtgcgc
accggaggct gcatcttttc 180ccggccaaga ttcggtccgg tggggccgtt
cttggcacga tttcacgggc cgtttggcct 240tccctcgccg gatttgttcc
ggctccaggc accaaattcc aatcttttcc tgctgctgct 300gcctctgcga
cactttattc ttctccccca attagcggcg gttagtgtgg attctgattt
360gtaggttcat ttcctggttt cctccgtgag cttctgcggt tggcgtggtt
acgccagtcc 420ctcgcgattt acatctgtga ttcgtttgaa aatctgggag
tgtggagatt tgggaggtct 480tctcgctcct gtgctctatg aggagcagct
tcctgtcgga gtcgccgtgc gacgagcagc 540gcatccatgg atatggtttc
aacccgcagt catggctgca ggtggagcga gggaagctgc 600ccaagtcgtc
ctactcgcct tcctccattg agtcacttat caagattgct gagccacatg
660tagtgccatt gtataagcct ttggattatg ttgaggtgct gtcaaggatc
cacgaggagc 720ttgaacaatg taggccgagc gagctgccag gcctgtactt
ggtccagtcc caggtgtttc 780ggggccttgg agaagcaaaa ttgcgccaga
ggagcctcca ctctgcctgg cgttgtgcaa 840gcagcgtcca tgagaaagtc
atatttgggg catggttgcg gtacgagaag cagggggagg 900agatcatatc
tgacgtcctt gcatcatgtc agaaatgctg tcgagagttt ggtttacttg
960atgttgcctc tgagatgcct gtgcggaatt ttgaggtaat tggttcatgg
gagacaggct 1020cctcgtctca agtttcttcc atggtaacct tccaaataca
ggatggtagg gtgacatgtg 1080ataggtgcaa gattgcgtct ttgtcaatac
cattttgctc catgcttaat ggaccgttca 1140atgagtcaca gcttgagctt
gttgatttgt cagagaatgg tatttcgttg gagggcatga 1200gagctgtttc
tgagtttagt tctacatgta gtttagggga tcttcctgtg gaaatcttat
1260tggagatcct ggtgtttgca aacacatttt gttgtgacag gctaaaagat
gcttgtgata 1320ggaaactggc ttcatttgtt tcaacaaggc aggatgctgt
tgagctcatg
ccgttggcat 1380ttgaagaaaa tgcgccagtt cttgctgctt cttgcttgca
aattttttta caggaacttc 1440ccaattgtct agctgatgat ctagtaatta
gcctcttctt aggtgcaact gcacaacaac 1500aacttatcat ggttggacat
gcatcctttt tgctgtactg cttgcttagt gaagtagcaa 1560tgaacattga
tccgaggaca gaaacaactg tattattgtt agagaagctt gtgcagctag
1620cagttacccc tactcagaag caaatagctt ttcatcaact tgcatgcatt
agacttttga 1680gaaaggaata tagtgaagct gaacaccaat ttgaggttgc
cttctctgcc ggtcatgtgt 1740attcaattgc tggtattgct agagtcgctg
gcattcaagg ccaaaaggct ttggcttatg 1800agaagctcag ttcagtgata
acatcaaatt tgccactggg gtggatgtat ctggagaggt 1860ctttgtattc
tgaaggtgat agaaagctgg cagaccttga caaagcaagc gagctggatc
1920ctactctcac ttacccttac atgtatcgag ctgcatcctt gatgagaaaa
aaagatgcta 1980aacatgcctt agaggaaatt aaccgactct tgggtttcaa
gttagcattg gagtgcctgg 2040agctacggat ctgtctatac ctggctcttg
aagactataa gtctgctatc tgtgatatcc 2100atgcgattct tactctttca
cctgattatc ggatgttgga aggacgtgta gctgcttcca 2160aaataggcac
tcttcttggt gcacatgtcg agcagtggaa tacggctgag tgttggctac
2220aactttatga gcgctggtca tcagttgatg atattggctc cctttcagtg
atctatcgga 2280tgcttgagtc agatgcagca aaaggtgtcc tctactttag
gcaatctttg ctgctcctta 2340ggttgaactg tcccgaggca gcgatgcgca
gtttgcaatt ggcaaggcat catgcagcaa 2400ctgagcatga acgactagta
tatgaggggt ggctcttata tgacacgggg cactatgggg 2460aggccctaca
aaaagcggaa gaatctattt ccattcaaag atcatttgaa gctttctttc
2520tgaaagccta tgttttggct gattcaggag ttgatccttc ttattctgcg
acagttatct 2580cacttcttga agatgcattg aaatgccctt cagaccggct
tcggaagggt caggcattga 2640ataaccttgg tggtgtctat gttgattgtg
gaaagttaga ttcagcagct gattgctata 2700caagtgcatt gaaaattcga
cacactagag cccatcaagg tcttgctcgt gtacattttc 2760tgaggaacaa
cagggaagct gcatatgaag agatgacaaa gttgatagaa aaagctaaaa
2820acaatgcttc ggcttatgag aaacgctcag aatattgtga acgagaacaa
actatgacag 2880atttgcaaac agtgacccaa ttggatcctt tgcgtgttta
tccatacaga tatcgagcag 2940cagtgctgat ggatagccac aaggagaatg
atgcaatagc ggagctcagc cgtgcgatat 3000ccttcaaagc ggacctgcat
ttgctgcatc tccgtgcggc tttccacgag cacattggag 3060atgtacccag
cgctctccgt gattgtagag ccgccctctc cttggacccg aatcaccagg
3120agatgttgga gcttcagaaa cgtgtgaaca gccaagagcc ctgacacatt
gtggtgctgt 3180attgtgtatt gtacaccacg actcatgcca ctttggtttg
cctcgtgaca gacagccttg 3240acctgattca tttttttttc gtttttccgt
taattaatta atcaatcact gtaatacgaa 3300gttttcagga agaaagtaac
agagagcaag agagagaact agttatatga aagcaggcat 3360tgataaagcc
tttttaactc gatgcctgtg gcctctgtag aagtattgtg ctaatccctg
3420aactgtatct tgaaaaagtg attcgtgcat aatgtatttt tcagttctgt tctcttt
34778888PRTZea mays 8Met Arg Ser Ser Phe Leu Ser Glu Ser Pro Cys
Asp Glu Gln Arg Ile 1 5 10 15 His Gly Tyr Gly Phe Asn Pro Gln Ser
Trp Leu Gln Val Glu Arg Gly 20 25 30 Lys Leu Pro Lys Ser Ser Tyr
Ser Pro Ser Ser Ile Glu Ser Leu Ile 35 40 45 Lys Ile Ala Glu Pro
His Val Val Pro Leu Tyr Lys Pro Leu Asp Tyr 50 55 60 Val Glu Val
Leu Ser Arg Ile His Glu Glu Leu Glu Gln Cys Arg Pro 65 70 75 80 Ser
Glu Leu Pro Gly Leu Tyr Leu Val Gln Ser Gln Val Phe Arg Gly 85 90
95 Leu Gly Glu Ala Lys Leu Arg Gln Arg Ser Leu His Ser Ala Trp Arg
100 105 110 Cys Ala Ser Ser Val His Glu Lys Val Ile Phe Gly Ala Trp
Leu Arg 115 120 125 Tyr Glu Lys Gln Gly Glu Glu Ile Ile Ser Asp Val
Leu Ala Ser Cys 130 135 140 Gln Lys Cys Cys Arg Glu Phe Gly Leu Leu
Asp Val Ala Ser Glu Met 145 150 155 160 Pro Val Arg Asn Phe Glu Val
Ile Gly Ser Trp Glu Thr Gly Ser Ser 165 170 175 Ser Gln Val Ser Ser
Met Val Thr Phe Gln Ile Gln Asp Gly Arg Val 180 185 190 Thr Cys Asp
Arg Cys Lys Ile Ala Ser Leu Ser Ile Pro Phe Cys Ser 195 200 205 Met
Leu Asn Gly Pro Phe Asn Glu Ser Gln Leu Glu Leu Val Asp Leu 210 215
220 Ser Glu Asn Gly Ile Ser Leu Glu Gly Met Arg Ala Val Ser Glu Phe
225 230 235 240 Ser Ser Thr Cys Ser Leu Gly Asp Leu Pro Val Glu Ile
Leu Leu Glu 245 250 255 Ile Leu Val Phe Ala Asn Thr Phe Cys Cys Asp
Arg Leu Lys Asp Ala 260 265 270 Cys Asp Arg Lys Leu Ala Ser Phe Val
Ser Thr Arg Gln Asp Ala Val 275 280 285 Glu Leu Met Pro Leu Ala Phe
Glu Glu Asn Ala Pro Val Leu Ala Ala 290 295 300 Ser Cys Leu Gln Ile
Phe Leu Gln Glu Leu Pro Asn Cys Leu Ala Asp 305 310 315 320 Asp Leu
Val Ile Ser Leu Phe Leu Gly Ala Thr Ala Gln Gln Gln Leu 325 330 335
Ile Met Val Gly His Ala Ser Phe Leu Leu Tyr Cys Leu Leu Ser Glu 340
345 350 Val Ala Met Asn Ile Asp Pro Arg Thr Glu Thr Thr Val Leu Leu
Leu 355 360 365 Glu Lys Leu Val Gln Leu Ala Val Thr Pro Thr Gln Lys
Gln Ile Ala 370 375 380 Phe His Gln Leu Ala Cys Ile Arg Leu Leu Arg
Lys Glu Tyr Ser Glu 385 390 395 400 Ala Glu His Gln Phe Glu Val Ala
Phe Ser Ala Gly His Val Tyr Ser 405 410 415 Ile Ala Gly Ile Ala Arg
Val Ala Gly Ile Gln Gly Gln Lys Ala Leu 420 425 430 Ala Tyr Glu Lys
Leu Ser Ser Val Ile Thr Ser Asn Leu Pro Leu Gly 435 440 445 Trp Met
Tyr Leu Glu Arg Ser Leu Tyr Ser Glu Gly Asp Arg Lys Leu 450 455 460
Ala Asp Leu Asp Lys Ala Ser Glu Leu Asp Pro Thr Leu Thr Tyr Pro 465
470 475 480 Tyr Met Tyr Arg Ala Ala Ser Leu Met Arg Lys Lys Asp Ala
Lys His 485 490 495 Ala Leu Glu Glu Ile Asn Arg Leu Leu Gly Phe Lys
Leu Ala Leu Glu 500 505 510 Cys Leu Glu Leu Arg Ile Cys Leu Tyr Leu
Ala Leu Glu Asp Tyr Lys 515 520 525 Ser Ala Ile Cys Asp Ile His Ala
Ile Leu Thr Leu Ser Pro Asp Tyr 530 535 540 Arg Met Leu Glu Gly Arg
Val Ala Ala Ser Lys Ile Gly Thr Leu Leu 545 550 555 560 Gly Ala His
Val Glu Gln Trp Asn Thr Ala Glu Cys Trp Leu Gln Leu 565 570 575 Tyr
Glu Arg Trp Ser Ser Val Asp Asp Ile Gly Ser Leu Ser Val Ile 580 585
590 Tyr Arg Met Leu Glu Ser Asp Ala Ala Lys Gly Val Leu Tyr Phe Arg
595 600 605 Gln Ser Leu Leu Leu Leu Arg Leu Asn Cys Pro Glu Ala Ala
Met Arg 610 615 620 Ser Leu Gln Leu Ala Arg His His Ala Ala Thr Glu
His Glu Arg Leu 625 630 635 640 Val Tyr Glu Gly Trp Leu Leu Tyr Asp
Thr Gly His Tyr Gly Glu Ala 645 650 655 Leu Gln Lys Ala Glu Glu Ser
Ile Ser Ile Gln Arg Ser Phe Glu Ala 660 665 670 Phe Phe Leu Lys Ala
Tyr Val Leu Ala Asp Ser Gly Val Asp Pro Ser 675 680 685 Tyr Ser Ala
Thr Val Ile Ser Leu Leu Glu Asp Ala Leu Lys Cys Pro 690 695 700 Ser
Asp Arg Leu Arg Lys Gly Gln Ala Leu Asn Asn Leu Gly Gly Val 705 710
715 720 Tyr Val Asp Cys Gly Lys Leu Asp Ser Ala Ala Asp Cys Tyr Thr
Ser 725 730 735 Ala Leu Lys Ile Arg His Thr Arg Ala His Gln Gly Leu
Ala Arg Val 740 745 750 His Phe Leu Arg Asn Asn Arg Glu Ala Ala Tyr
Glu Glu Met Thr Lys 755 760 765 Leu Ile Glu Lys Ala Lys Asn Asn Ala
Ser Ala Tyr Glu Lys Arg Ser 770 775 780 Glu Tyr Cys Glu Arg Glu Gln
Thr Met Thr Asp Leu Gln Thr Val Thr 785 790 795 800 Gln Leu Asp Pro
Leu Arg Val Tyr Pro Tyr Arg Tyr Arg Ala Ala Val 805 810 815 Leu Met
Asp Ser His Lys Glu Asn Asp Ala Ile Ala Glu Leu Ser Arg 820 825 830
Ala Ile Ser Phe Lys Ala Asp Leu His Leu Leu His Leu Arg Ala Ala 835
840 845 Phe His Glu His Ile Gly Asp Val Pro Ser Ala Leu Arg Asp Cys
Arg 850 855 860 Ala Ala Leu Ser Leu Asp Pro Asn His Gln Glu Met Leu
Glu Leu Gln 865 870 875 880 Lys Arg Val Asn Ser Gln Glu Pro 885
94548DNAGlycine max 9atgcaacaca gcatcttcgc ctcaatgcgt agcttgaaga
tcatggacgg ttgcaagggc 60actcaggtct acgccatcaa cccctccagc gccaccggcg
gtggaattgg cgagaagctt 120ctccaacagc ttcacgacca catcaaaagc
cacaccctta gaaccaaatc ggttcggaac 180ttacaacctc cgaacatgac
gacgccgtcg gaggttttcg tctccgacgg gtcgctcctt 240ccttacggcc
tccccatgac ggacctccta gagcccaaaa ttgaaccctc cttggtgtcg
300gtggattttg tcgaaaccct cgccggagtc taccgccgca ccgaggaccg
ccaccagttc 360gaccgctccg aggtgtacct cgagcaatgc gcggtattcc
aggggctggc cgacccgaag 420ctcttccgcc gcagcctccg cgccgcccgg
cagcacgcca tcaacgtgca cgcgaaggtc 480gtgctttccg catggcttcg
ctacgagcgc cgcgaggatg agctcatcgg ctcgtccttg 540atggactgca
gcgggaggaa cctcgagtgc ccccgcacca cgctggttcc aggctacgac
600ccggagttgg tgtttgattc ctgcgcgtgc acgggtgcac gcgcaggtaa
tggtgataac 660gataatgatg atgcaatggc aatagtggtt gatgagcaat
gctccacctc ggaagaggag 720gaggaggatg gtgacatgtc tttttgtgtt
ggtgatgatg agattaagtg taataggttc 780aatatagcct cactttcaag
gccctttaag ataatgttgt atggtggatt cattgagtca 840acgagagaga
agataaattt ttcgcggaat tgtttttctg ttgaggcatt gagggctgct
900gaggtgttca gtaggagaaa gaggttgagt catttggagc ccaaggttat
tttggagttg 960ctatctttgg caaaccggtt ttgttgcgag gagatgaaga
atgcttgtga cgcgcatttg 1020gcatcgcttg tttgtgacat agacgatgcc
ttgttgcttg ttgagtatgg actggaggag 1080accgcatacc tgctggtggc
tgcctgcttg caggtgtttc tccgggagct ccctggttcg 1140atgcaaagtt
tgagtgttgt gaagatattt tgtagtccgg agggtaggga taggcttgct
1200ctggcggggc acgcgtcgtt tgtgttgtat tattttttga gtcagattgc
gatggaggaa 1260gagatgaggt cgaacaccac tgtgatgctg ttggagaggt
tagtggagtg cgcaaaggat 1320ggttgggaga agcaagttgc gtttcatcta
ttgggtgttg ttatgcttga gagaaaagaa 1380tacaaagatg cacaatattg
gtttcaggca gcggttgatg cagggcatgc ttattctttg 1440gtgggagttg
caagggcaaa atataagcgt ggtcacacat attcagcata taagttgatg
1500aactcactta tttctgatca taaaccggtt gggtggatgt atcaggaaag
gtctttgtat 1560tgtgttggga aggagaaatt gatggacttg atgtctgcaa
ctgagttaga tccaactctt 1620tcctttccat ataaattccg ggctgtttct
ttcctggagg aaaacaagat tggacctgcc 1680attgcagaaa tcaataaaat
aattggcttc aaggtttctc ctgattgcct tgaattgaga 1740gcttggttct
tgattgccat ggaagattat gaaggagccc tcagggatgt ccgggcaatt
1800ttgacattgg atccaaatta tatgatgttc tatgggcata tgcacggtga
tcagttggta 1860gaacttctcc aaccttttgt tcagcagtgg agtcaggctg
attgctggat tcagttgtat 1920gaccgatggt cctctgttga tgatattggt
tctttggctg ttgtacacca gatgttagca 1980aaagacccgg ggaaaagtct
tttatgcttt cggcaatctc tccttcttct acggtgagtg 2040taatccctac
attaattcag ttatatctta tttcattagt gagttctgat atagattcta
2100gtttgactta ataattttct ccatgcatga attctctgcg aatgaattgc
ctactgtggg 2160ctgttttata cttttcctat tatcagtgac gagtaatatc
aaagtctggc tagaaatatt 2220ttgttgatcc ttttcctttt taactggcca
tatacctgca tgggattcat gtttctattt 2280gctgcaaaac aaactagttt
atagtcacat tttattatat caaaattaga gaaaattgtc 2340acatgttcca
cccatcaaag caaactatgg taaactaatg aaactgcaga tcatttttat
2400tgtgtactgg ccaggatcta cattgaggta tttattatgt aatttgccaa
aagctaccca 2460cattttatgt atagtttcat aaatattagt ccaggaggtg
actttcagtt attgtatctt 2520ctctgctttc agaatgaatg gaaactgata
agtactttga aaaactagtt ggctgtgttt 2580taatacttat tctatctttt
ttagttatgt actttttgct agtttttcct tttagaaagt 2640gttgatactt
gattgttatt aacttagata gctaatttgt cttagtcctg gttcatttta
2700atgccagagc ttgactatta tgggtgttga ttttcctgaa tcttgtaggt
tgaattgtcc 2760aaagtctgcc atgcgtagtt tgcggctggc tagaaatcat
tctacttctg atcatgaaag 2820gcttgtgtat gaaggatgga tactgtatga
cactggttat cgtgaagaag cattagcaaa 2880ggctgaggaa tctatttcta
ttcgaagatc atttgaagct tactttctca aagcttatgc 2940gttagctgac
tcaaatcttg attcagagtc ttcaaagtat gtgatctgtc tcttggagga
3000agctcttaga tgccctttag atggtcttcg gaaaggacaa gtgagtgcta
agttttggat 3060taaacatttt atgctgatat cagtgctgaa gtaatgtatt
tatatttgtt attcttttaa 3120attgtaggca ctgaataatc tagggagtgt
ctatgtagac tgtgataaac tggaccttgc 3180tgctgactgc tacatgaatg
cactcaacat caagcataca cgagcacatc aggggttggc 3240acgtgtatat
catcttaaaa atctccggaa agcagcatat gatgagatga caaagctaat
3300agaaaaggct cggagtaatg catcagctta tgagaaacgt tcagaatatt
gcgaccgtga 3360catggcaaag agtgatctta gtatggcatc acaattggat
cctctaagga cttatcctta 3420ccgatatagg gctgcaggtg agtctcatat
gagtggttct attatcccgt gttttctcct 3480gattattcat gatggtaact
tggtgaggga ggggaattgt agctgtgatt gtaatttgca 3540agccattttt
gtgtttcaaa tattatctgt ggcatttgta tagccatgat ggttttatta
3600ctgtcttaga ttccagggta caatcttcaa tgctgttgtt ttttaccaaa
ttttttccta 3660taaactgatt tgtctgcata acttcataga catattttag
ttttccatga aaaaaatagt 3720agttgatgac tttccttaga tttactatgt
aacatttaaa tgaatgaaaa atcactgtgt 3780ttatctttct gctttctttg
tttacttgtt tatatacccc aactcaaatg ccaaatgttt 3840atgttgacca
cccatgattt atttaagttt cccctctaaa aatgcttttt gtctgaattt
3900gtggcattaa tcatgcagtt actcagaaac attatattgc ataagtattg
caggaaatga 3960cttgtggaat tttcacagtt atgcagtaaa aactgaccat
cgttgctgta aatgttgaca 4020gaaaatcatg gctgttcata ggtttcctgt
tcatagcacg cctatcaaat catacattat 4080tgtagggtac atgttcattt
tgctttgttt tatcatatca agagtgatca gttatgaaga 4140gatcttttga
gtttgtgcct atccgtgtgg ctttgtatgc ttatgttctg acttttggtc
4200ttggttcatg gtgagatact tttgtttgcc atgagcatct tgatagagca
atatgaacac 4260aatatattag aacctctgca acctgttatc aacttctgac
tcaaactctg tttaccagtt 4320ttaatggatg atcataagga agctgaggca
atagaagagc tttcaagagc cattgatttt 4380aagccagatc tgcaactatt
acatcttcga gcggcatttt atgattcaat gggtgatttt 4440gtctctgcag
tccgggactg tgaagcagcc ctttgtcttg atcctaatca taatgagatt
4500cttgatctct gtaataaagc acgggagcat attcgagaac caaagtga
454810954PRTGlycine max 10Met Gln His Ser Ile Phe Ala Ser Met Arg
Ser Leu Lys Ile Met Asp 1 5 10 15 Gly Cys Lys Gly Thr Gln Val Tyr
Ala Ile Asn Pro Ser Ser Ala Thr 20 25 30 Gly Gly Gly Ile Gly Glu
Lys Leu Leu Gln Gln Leu His Asp His Ile 35 40 45 Lys Ser His Thr
Leu Arg Thr Lys Ser Val Arg Asn Leu Gln Pro Pro 50 55 60 Asn Met
Thr Thr Pro Ser Glu Val Phe Val Ser Asp Gly Ser Leu Leu 65 70 75 80
Pro Tyr Gly Leu Pro Met Thr Asp Leu Leu Glu Pro Lys Ile Glu Pro 85
90 95 Ser Leu Val Ser Val Asp Phe Val Glu Thr Leu Ala Gly Val Tyr
Arg 100 105 110 Arg Thr Glu Asp Arg His Gln Phe Asp Arg Ser Glu Val
Tyr Leu Glu 115 120 125 Gln Cys Ala Val Phe Gln Gly Leu Ala Asp Pro
Lys Leu Phe Arg Arg 130 135 140 Ser Leu Arg Ala Ala Arg Gln His Ala
Ile Asn Val His Ala Lys Val 145 150 155 160 Val Leu Ser Ala Trp Leu
Arg Tyr Glu Arg Arg Glu Asp Glu Leu Ile 165 170 175 Gly Ser Ser Leu
Met Asp Cys Ser Gly Arg Asn Leu Glu Cys Pro Arg 180 185 190 Thr Thr
Leu Val Pro Gly Tyr Asp Pro Glu Leu Val Phe Asp Ser Cys 195 200 205
Ala Cys Thr Gly Ala Arg Ala Gly Asn Gly Asp Asn Asp Asn Asp Asp 210
215 220 Ala Met Ala Ile Val Val Asp Glu Gln Cys Ser Thr Ser Glu Glu
Glu 225 230 235 240 Glu Glu Asp Gly Asp Met Ser Phe Cys Val Gly Asp
Asp Glu Ile Lys 245 250 255 Cys Asn Arg Phe Asn Ile Ala Ser Leu Ser
Arg Pro Phe Lys Ile Met 260 265 270 Leu Tyr Gly Gly Phe Ile Glu Ser
Thr Arg Glu Lys Ile Asn Phe Ser 275 280 285 Arg Asn Cys Phe Ser Val
Glu Ala Leu Arg Ala Ala Glu Val Phe Ser 290 295 300 Arg Arg Lys Arg
Leu Ser His Leu Glu Pro Lys Val Ile Leu Glu Leu 305 310 315 320 Leu
Ser Leu Ala Asn Arg Phe Cys Cys Glu Glu Met Lys Asn Ala Cys 325 330
335 Asp Ala His Leu Ala Ser Leu Val Cys Asp Ile Asp Asp Ala Leu Leu
340 345 350 Leu Val Glu Tyr Gly Leu Glu Glu Thr Ala Tyr Leu Leu Val
Ala Ala 355 360 365 Cys Leu Gln Val Phe Leu Arg Glu Leu Pro Gly Ser
Met Gln Ser Leu 370 375 380 Ser Val Val Lys Ile Phe Cys Ser Pro Glu
Gly
Arg Asp Arg Leu Ala 385 390 395 400 Leu Ala Gly His Ala Ser Phe Val
Leu Tyr Tyr Phe Leu Ser Gln Ile 405 410 415 Ala Met Glu Glu Glu Met
Arg Ser Asn Thr Thr Val Met Leu Leu Glu 420 425 430 Arg Leu Val Glu
Cys Ala Lys Asp Gly Trp Glu Lys Gln Val Ala Phe 435 440 445 His Leu
Leu Gly Val Val Met Leu Glu Arg Lys Glu Tyr Lys Asp Ala 450 455 460
Gln Tyr Trp Phe Gln Ala Ala Val Asp Ala Gly His Ala Tyr Ser Leu 465
470 475 480 Val Gly Val Ala Arg Ala Lys Tyr Lys Arg Gly His Thr Tyr
Ser Ala 485 490 495 Tyr Lys Leu Met Asn Ser Leu Ile Ser Asp His Lys
Pro Val Gly Trp 500 505 510 Met Tyr Gln Glu Arg Ser Leu Tyr Cys Val
Gly Lys Glu Lys Leu Met 515 520 525 Asp Leu Met Ser Ala Thr Glu Leu
Asp Pro Thr Leu Ser Phe Pro Tyr 530 535 540 Lys Phe Arg Ala Val Ser
Phe Leu Glu Glu Asn Lys Ile Gly Pro Ala 545 550 555 560 Ile Ala Glu
Ile Asn Lys Ile Ile Gly Phe Lys Val Ser Pro Asp Cys 565 570 575 Leu
Glu Leu Arg Ala Trp Phe Leu Ile Ala Met Glu Asp Tyr Glu Gly 580 585
590 Ala Leu Arg Asp Val Arg Ala Ile Leu Thr Leu Asp Pro Asn Tyr Met
595 600 605 Met Phe Tyr Gly His Met His Gly Asp Gln Leu Val Glu Leu
Leu Gln 610 615 620 Pro Phe Val Gln Gln Trp Ser Gln Ala Asp Cys Trp
Ile Gln Leu Tyr 625 630 635 640 Asp Arg Trp Ser Ser Val Asp Asp Ile
Gly Ser Leu Ala Val Val His 645 650 655 Gln Met Leu Ala Lys Asp Pro
Gly Lys Ser Leu Leu Cys Phe Arg Gln 660 665 670 Ser Leu Leu Leu Leu
Arg Leu Asn Cys Pro Lys Ser Ala Met Arg Ser 675 680 685 Leu Arg Leu
Ala Arg Asn His Ser Thr Ser Asp His Glu Arg Leu Val 690 695 700 Tyr
Glu Gly Trp Ile Leu Tyr Asp Thr Gly Tyr Arg Glu Glu Ala Leu 705 710
715 720 Ala Lys Ala Glu Glu Ser Ile Ser Ile Arg Arg Ser Phe Glu Ala
Tyr 725 730 735 Phe Leu Lys Ala Tyr Ala Leu Ala Asp Ser Asn Leu Asp
Ser Glu Ser 740 745 750 Ser Lys Tyr Val Ile Cys Leu Leu Glu Glu Ala
Leu Arg Cys Pro Leu 755 760 765 Asp Gly Leu Arg Lys Gly Gln Ala Leu
Asn Asn Leu Gly Ser Val Tyr 770 775 780 Val Asp Cys Asp Lys Leu Asp
Leu Ala Ala Asp Cys Tyr Met Asn Ala 785 790 795 800 Leu Asn Ile Lys
His Thr Arg Ala His Gln Gly Leu Ala Arg Val Tyr 805 810 815 His Leu
Lys Asn Leu Arg Lys Ala Ala Tyr Asp Glu Met Thr Lys Leu 820 825 830
Ile Glu Lys Ala Arg Ser Asn Ala Ser Ala Tyr Glu Lys Arg Ser Glu 835
840 845 Tyr Cys Asp Arg Asp Met Ala Lys Ser Asp Leu Ser Met Ala Ser
Gln 850 855 860 Leu Asp Pro Leu Arg Thr Tyr Pro Tyr Arg Tyr Arg Ala
Ala Val Leu 865 870 875 880 Met Asp Asp His Lys Glu Ala Glu Ala Ile
Glu Glu Leu Ser Arg Ala 885 890 895 Ile Asp Phe Lys Pro Asp Leu Gln
Leu Leu His Leu Arg Ala Ala Phe 900 905 910 Tyr Asp Ser Met Gly Asp
Phe Val Ser Ala Val Arg Asp Cys Glu Ala 915 920 925 Ala Leu Cys Leu
Asp Pro Asn His Asn Glu Ile Leu Asp Leu Cys Asn 930 935 940 Lys Ala
Arg Glu His Ile Arg Glu Pro Lys 945 950 11123PRTArtificial
SequenceConsensus Sequence of Conserved N-Terminal Domain 11Phe Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Arg Xaa Xaa Xaa Ala 1 5 10 15
Xaa Leu Ser Xaa Pro Xaa Xaa Xaa Xaa Leu Xaa Gly Xaa Phe Xaa Glu 20
25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 35 40 45 Xaa Met Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa
Xaa Ala Xaa Xaa Phe 65 70 75 80 Cys Cys Xaa Xaa Leu Lys Xaa Xaa Cys
Xaa Xaa Xaa Leu Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Ala Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Xaa 100 105 110 Xaa Xaa Xaa Xaa Leu
Xaa Xaa Xaa Cys Leu Gln 115 120 12240PRTArtificial
SequenceConsensus Sequence of Conserved C-Terminal Domain 12Trp Ser
Xaa Val Asp Asp Xaa Xaa Ser Leu Xaa Val Xaa Xaa Xaa Met 1 5 10 15
Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Phe Arg Gln Ser Leu 20
25 30 Leu Leu Leu Arg Leu Asn Cys Xaa Xaa Xaa Ala Met Arg Xaa Leu
Xaa 35 40 45 Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Arg Leu
Val Tyr Glu 50 55 60 Gly Trp Xaa Leu Tyr Asp Xaa Gly Xaa Xaa Xaa
Glu Xaa Leu Xaa Lys 65 70 75 80 Ala Xaa Xaa Xaa Ile Xaa Xaa Xaa Arg
Ser Phe Glu Ala Xaa Phe Leu 85 90 95 Xaa Ala Tyr Xaa Leu Xaa Xaa
Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Val Xaa Xaa Xaa
Leu Xaa Xaa Ala Xaa Xaa Cys Xaa Xaa Asp Xaa 115 120 125 Leu Arg Lys
Gly Gln Ala Xaa Asn Asn Xaa Gly Xaa Xaa Tyr Xaa Xaa 130 135 140 Xaa
Xaa Xaa Leu Asp Xaa Ala Xaa Xaa Xaa Tyr Xaa Xaa Ala Xaa Xaa 145 150
155 160 Xaa Xaa His Xaa Arg Ala Xaa Gln Gly Leu Ala Arg Val Xaa Xaa
Leu 165 170 175 Xaa Asn Xaa Xaa Xaa Xaa Ala Xaa Xaa Glu Met Thr Xaa
Leu Xaa Glu 180 185 190 Xaa Xaa Xaa Xaa Xaa Ala Xaa Ala Tyr Glu Lys
Arg Ser Glu Tyr Xaa 195 200 205 Xaa Arg Xaa Xaa Xaa Xaa Xaa Asp Leu
Xaa Xaa Xaa Xaa Xaa Leu Asp 210 215 220 Pro Xaa Arg Xaa Tyr Pro Tyr
Arg Tyr Arg Ala Ala Val Leu Met Asp 225 230 235 240
1336DNAArtificial Sequenceprobe 13tcgacccacg cgtccgaaaa aaaaaaaaaa
aaaaaa 36
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