U.S. patent application number 13/335795 was filed with the patent office on 2012-04-19 for nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement.
This patent application is currently assigned to MONSANTO TECHNOLOGY LLC. Invention is credited to Scott E. Andersen, Yongwei Cao, Michael D. Edgerton, David K. Kovalic, Jingdong Liu, Yihua Zhou.
Application Number | 20120096599 13/335795 |
Document ID | / |
Family ID | 46331816 |
Filed Date | 2012-04-19 |
United States Patent
Application |
20120096599 |
Kind Code |
A1 |
Kovalic; David K. ; et
al. |
April 19, 2012 |
Nucleic Acid Molecules and Other Molecules Associated with Plants
and Uses Thereof for Plant Improvement
Abstract
Recombinant polynucleotides and recombinant polypeptides useful
for improvement of plants are provided. The disclosed recombinant
polynucleotides and recombinant polypeptides find use in production
of transgenic plants to produce plants having improved
properties.
Inventors: |
Kovalic; David K.; (Clayton,
MO) ; Zhou; Yihua; (Ballwin, MO) ; Cao;
Yongwei; (Lexington, MA) ; Andersen; Scott E.;
(St. Louis, MO) ; Edgerton; Michael D.; (St.
Louis, MO) ; Liu; Jingdong; (Chesterfield,
MO) |
Assignee: |
MONSANTO TECHNOLOGY LLC
St. Louis
MO
|
Family ID: |
46331816 |
Appl. No.: |
13/335795 |
Filed: |
December 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11978197 |
Oct 29, 2007 |
8106174 |
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13335795 |
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10767701 |
Jan 29, 2004 |
7834146 |
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11978197 |
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09684016 |
Oct 10, 2000 |
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10767701 |
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09654617 |
Sep 5, 2000 |
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09684016 |
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09850147 |
May 8, 2001 |
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10767701 |
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60202213 |
May 8, 2000 |
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Current U.S.
Class: |
800/298 ;
111/100; 47/58.1SE; 536/23.6 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8247 20130101; C12N 15/8218 20130101; C12N 15/8251
20130101; C12N 15/8273 20130101; C12N 15/8216 20130101; C07H 21/04
20130101; C12N 15/8274 20130101; C12N 15/8275 20130101; C12N 15/82
20130101; C12N 15/8261 20130101 |
Class at
Publication: |
800/298 ;
536/23.6; 47/58.1SE; 111/100 |
International
Class: |
A01H 5/00 20060101
A01H005/00; A01C 7/00 20060101 A01C007/00; A01G 1/00 20060101
A01G001/00; A01H 5/10 20060101 A01H005/10; C12N 15/29 20060101
C12N015/29 |
Claims
1-3. (canceled)
4. A transformed plant comprising a nucleic acid molecule which
comprises: (a) an exogenous promoter region which functions in a
plant cell to cause the production of an mRNA molecule; which is
linked to; (b) a structural nucleic acid molecule, wherein said
structural nucleic acid molecule comprises a nucleic acid sequence,
wherein said nucleic acid sequence shares between 100% and 90%
sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 1 through SEQ ID NO: 31,564, or the
complement of SEQ ID NO: 1 through SEQ ID NO: 31,564, which is
operably linked to (c) a 3' non-translated sequence that functions
in said plant cell to cause the termination of transcription and
the addition of polyadenylated ribonucleotides to said 3' end of
said mRNA molecule.
5. The transformed plant according to claim 4, wherein said nucleic
acid sequence is the complement of a nucleic acid sequence selected
from the group consisting of SEQ ID NO: 1 through SEQ ID NO:
31,564.
6. The transformed plant according to claim 4, wherein said nucleic
acid sequence is in the antisense orientation of a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 31,564.
7. The transformed plant according to claim 4, wherein said nucleic
acid sequence shares between 100% and 95% sequence identity with a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1 through SEQ ID NO: 31,564 or the complement of SEQ ID NO: 1
through SEQ ID NO: 31,564.
8. The transformed plant according to claim 7, wherein said nucleic
acid sequence shares between 100% and 98% sequence identity with a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1 through SEQ ID NO: 31,564 or the complement of SEQ ID NO: 1
through SEQ ID NO: 31,564.
9. The transformed plant according to claim 8, wherein said nucleic
acid sequence shares between 100% and 99% sequence identity with a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1 through SEQ ID NO: 31,564 or the complement of SEQ ID NO: 1
through SEQ ID NO: 31,564.
10. The transformed plant according to claim 9, wherein said
nucleic acid sequence shares 100% sequence identity with a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1
through SEQ ID NO: 31,564 and the complement of SEQ ID NO: 1
through SEQ ID NO: 31,564.
11. A transformed seed comprising a transformed plant cell
comprising a nucleic acid molecule which comprises: (a) an
exogenous promoter region which functions in said plant cell to
cause the production of an mRNA molecule; which is linked to; (b) a
structural nucleic acid molecule, wherein said structural nucleic
acid molecule comprises a nucleic acid sequence, wherein said
nucleic acid sequence shares between 100% and 90% sequence identity
to a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 31,564, or the complement of SEQ ID
NO: 1 through SEQ ID NO: 31,564, which is linked to (c) a 3'
non-translated sequence that functions in said plant cell to cause
the termination of transcription and the addition of polyadenylated
ribonucleotides to said 3' end of said mRNA molecule.
12. The transformed seed according to claim 11, wherein said
nucleic acid sequence is the complement of a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 31,564.
13. The transformed seed according to claim 11, wherein said
exogenous promoter region functions in a seed cell.
14. The transformed seed according to claim 11, wherein said
nucleic acid sequence shares between 100% and 95% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 31,564 or the complement of SEQ ID
NO: 1 through SEQ ID NO: 31,564.
15. The transformed seed according to claim 14, wherein said
nucleic acid sequence shares between 100% and 98% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 31,564 or the complement of SEQ ID
NO: 1 through SEQ ID NO: 31,564.
16. The transformed seed according to claim 15, wherein said
nucleic acid sequence shares between 100% and 99% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 31,564 or the complement of SEQ ID
NO: 1 through SEQ ID NO: 31,564.
17. The transformed seed according to claim 16, wherein said
nucleic acid sequence shares 100% sequence identity with a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1
through SEQ ID NO: 31,564 and the complement of SEQ ID NO: 1
through SEQ ID NO: 31,564.
18. A method of growing a transgenic plant comprising (a) planting
a transformed seed comprising a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 1 through SEQ ID NO: 31,564, or
the complement of SEQ ID NO: 1 through SEQ ID NO: 31,564, and (b)
growing a plant from said seed.
19. A substantially purified nucleic acid molecule comprising a
nucleic acid sequence, wherein said nucleic acid sequence shares
between 100% and 90% sequence identity to a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 31,564, or the complement of SEQ ID NO: 1 through SEQ ID NO:
31,564.
20. The substantially purified nucleic acid molecule of claim 19,
wherein said nucleic acid molecule encodes a sorghum protein or
fragment thereof.
Description
[0001] This application claims the benefit of and is a continuation
in part of prior U.S. application Ser. No. 09/684,016 filed Oct.
10, 2000, and prior U.S. application Ser. No. 09/850,147 filed May
7, 2001, both of which are hereby incorporated by reference in
their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] Two copies of the sequence listing (Seq. Listing Copy 1 and
Seq. Listing Copy 2) and a computer-readable form of the sequence
listing, all on CD-ROMs, each containing the file named
pa.sub.--00620.rpt, which is 74,251,352 bytes (measured in MS-DOS)
and was created on Jan. 27, 2004, are herein incorporated by
reference.
FIELD OF THE INVENTION
[0003] Disclosed herein are inventions in the field of plant
biochemistry and genetics. More specifically recombinant
polynucleotides and recombinant polypeptides from Sorghum for use
in plant improvement are provided. Methods of using the recombinant
polynucleotides and recombinant polypeptides for production of
transgenic plants with improved biological characteristics are
disclosed.
BACKGROUND OF THE INVENTION
[0004] The ability to develop transgenic plants with improved
traits depends in part on the identification of polynucleotides
that are useful for the production of transformed plants having
desirable qualities. In this regard, the discovery of
polynucleotide sequences of genes, and the polypeptides encoded by
such genes, is needed. Molecules comprising such polynucleotides
may be used, for example, in recombinant DNA constructs useful for
imparting unique genetic properties into transgenic plants.
SUMMARY OF THE INVENTION
[0005] The present invention provides a recombinant polynucleotide
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 31,564. The present invention also provides a recombinant
polypeptide selected from the group consisting of SEQ ID NO: 31,565
through SEQ ID NO: 63,128.
[0006] The present invention also provides a method of producing a
plant having an improved property, wherein said method comprises
transforming a plant with a recombinant construct comprising a
promoter region functional in a plant cell operably joined to a
polynucleotide comprising a coding sequence for a polypeptide
associated with said property, and growing said transformed
plant.
DETAILED DESCRIPTION OF THE INVENTION
[0007] The present invention provides recombinant polynucleotides
and recombinant polypeptides from Sorghum. The recombinant
polynucleotides and recombinant polypeptides of the present
invention find a number of uses, for example in recombinant DNA
constructs, in physical arrays of molecules, for use as plant
breeding markers, and for use in computer based storage and
analysis systems.
[0008] The recombinant polynucleotides of the present invention
also find use in generation of transgenic plants to provide for
increased or decreased expression of the polypeptides encoded by
the recombinant polynucleotides provided herein. As used herein a
"transgenic" organism is one whose genome has been altered by the
incorporation of foreign genetic material or additional copies of
native genetic material, e.g. by transformation or recombination.
As a result of such biotechnological applications, plants,
particularly crop plants, having improved properties are obtained.
Crop plants of interest in the present invention include, but are
not limited to soy, cotton, canola, maize, wheat, sunflower,
sorghum, alfalfa, barley, millet, rice, tobacco, fruit and
vegetable crops, and turf grass. In one embodiment the disclosed
recombinant polynucleotides provide plants having improved yield
resulting from improved utilization of key biochemical compounds,
such as nitrogen, phosphorous and carbohydrate, or resulting from
improved responses to environmental stresses, such as cold, heat,
drought, salt, and attack by pests or pathogens. Recombinant
polynucleotides of the present invention may be used to provide
plants having improved growth and development, and ultimately
increased yield, as the result of modified expression of plant
growth regulators or modification of cell cycle or photosynthesis
pathways. Other traits of interest that may be modified in plants
using polynucleotides of the present invention include flavonoid
content, seed oil and protein quantity and quality, herbicide
tolerance, and rate of homologous recombination.
Polynucleotides
[0009] Depending on the intended use, the recombinant
polynucleotides of the present invention may be present in the form
of DNA, such as cDNA or genomic DNA, or as RNA, for example mRNA.
The polynucleotides of the present invention may be single or
double stranded and may represent the coding, or sense strand of a
gene, or the non-coding, antisense, strand. In one embodiment, the
recombinant polynucleotides of this invention represent cDNA
sequences from Sorghum. DNA sequences representing the recombinant
polynucleotides are provided herein as SEQ ID NO: 1 through SEQ ID
NO: 31,564.
[0010] The term "recombinant polynucleotide" as used herein refers
to a polynucleotide produced by recombinant DNA technology. In one
embodiment a recombinant polynucleotide may be produced by
separation from substantially all other molecules normally
associated with it in its native state. A recombinant
polynucleotide may be greater than 60% free, greater than 75% free,
greater than 90% free, or greater than 95% free from the other
molecules (exclusive of solvent) present in the natural mixture. In
another embodiment, a recombinant polynucleotide may be separated
from nucleic acids which normally flank the polynucleotide in
nature. Thus, polynucleotides fused to regulatory or coding
sequences with which they are not normally associated, for example
as the result of recombinant techniques, are considered recombinant
polynucleotides herein. Such molecules are considered recombinant
polynucleotides even when present, for example in the chromosome of
a host cell, or in a nucleic acid solution. The term recombinant
polynucleotide as used herein is not intended to encompass
molecules present in their native state.
[0011] It is understood that the molecules of the invention may be
labeled with reagents that facilitate detection of the molecule. As
used herein, a label can be any reagent that facilitates detection,
including fluorescent labels, chemical labels, or modified bases,
including nucleotides with radioactive elements, e.g. .sup.32P,
.sup.33P, .sup.35S or .sup.125I such as .sup.32P
deoxycytidine-5'-triphosphate (.sup.32PdCTP).
[0012] Recombinant polynucleotides of the present invention are
capable of specifically hybridizing to other polynucleotides under
certain circumstances. As used herein, two polynucleotides are said
to be capable of specifically hybridizing to one another if the two
molecules are capable of forming an anti-parallel, double-stranded
nucleic acid structure. A polynucleotide is said to be the
"complement" of another polynucleotide if the molecules exhibit
complete complementarity. As used herein, molecules are said to
exhibit "complete complementarity" when every nucleotide in each of
the polynucleotides is complementary to the corresponding
nucleotide of the other. Two polynucleotides are said to be
"minimally complementary" if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one
another under at least conventional "low-stringency" conditions.
Similarly, the polynucleotides are said to be "complementary" if
they can hybridize to one another with sufficient stability to
permit them to remain annealed to one another under conventional
"high-stringency" conditions. Conventional stringency conditions
are known to those skilled in the art and can be found, for example
in Molecular Cloning: A Laboratory Manual, 3.sup.rd edition Volumes
1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold
Spring Harbor Laboratory Press, 2000.
[0013] Departures from complete complementarity are therefore
permissible, as long as such departures do not completely preclude
the capacity of the polynucleotides to form a double-stranded
structure. Thus, in order for a polynucleotide to serve as a primer
or probe it need only be sufficiently complementary in sequence to
be able to form a stable double-stranded structure under the
particular solvent and salt concentrations employed. Appropriate
stringency conditions which promote DNA hybridization are, for
example, 6.0.times. sodium chloride/sodium citrate (SSC) at about
45.degree. C., followed by a wash of 2.0.times.SSC at 50.degree. C.
Such conditions are known to those skilled in the art and can be
found, for example in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989). Salt concentration and temperature
in the wash step can be adjusted to alter hybridization stringency.
For example, conditions may vary from low stringency of about
2.0.times.SSC at 40.degree. C. to moderately stringent conditions
of about 2.0.times.SSC at 50.degree. C. to high stringency
conditions of about 0.2.times.SSC at 50.degree. C.
[0014] As used herein "sequence identity" refers to the extent to
which two optimally aligned polynucleotides or polypeptide
sequences are invariant throughout a window of alignment of
components, e.g. nucleotides or amino acids. An "identity fraction"
for aligned segments of a test sequence and a reference sequence is
the number of identical components which are shared by the two
aligned sequences divided by the total number of components in the
reference sequence segment, i.e. the entire reference sequence or a
smaller defined part of the reference sequence. "Percent identity"
is the identity fraction times 100. Comparison of sequences to
determine percent identity can be accomplished by a number of
well-known methods, including for example by using mathematical
algorithms, such as those in the BLAST suite of sequence analysis
programs.
[0015] In one embodiment this invention provides recombinant
polynucleotides comprising regions that encode polypeptides. The
encoded polypeptides may be the complete protein encoded by the
gene represented by the polynucleotide, or may be fragments of the
encoded protein. In one embodiment, polynucleotides provided herein
encode polypeptides constituting a substantial portion of the
complete protein. In another embodiment polynucleotides provided
herein encode polypeptides constituting a sufficient portion of the
complete protein to provide the relevant biological activity.
[0016] In one embodiment recombinant polynucleotides of the present
invention encode polypeptides involved in one or more important
biological function in plants. Such recombinant polynucleotides may
be expressed in transgenic plants to produce plants having improved
phenotypic properties and/or improved response to stressful
environmental conditions. See, for example, Table 1 of U.S.
application Ser. No. 10/767,701 for a list of SEQ ID numbers
representing the recombinant polynucleotides that may be expressed
in transgenic plants to impart an improved plant property where
improved plant properties are provided for each sequence in the
PRODUCT_CAT_DESC column.
[0017] Recombinant polynucleotides of the present invention are
generally used to impart such improved properties by providing for
enhanced protein activity in a transgenic organism, such as a
transgenic plant, although in some cases, improved properties are
obtained by providing for reduced protein activity in a transgenic
plant. Reduced protein activity and enhanced protein activity are
measured by reference to a wild type cell or organism and can be
determined by direct or indirect measurement. Direct measurement of
protein activity might include an analytical assay for the protein,
per se, or enzymatic product of protein activity. Indirect assay
might include measurement of a property affected by the protein.
Enhanced protein activity can be achieved in a number of ways, for
example by overproduction of mRNA encoding the protein or by gene
shuffling. One skilled in the art will know methods to achieve
overproduction of mRNA, for example by providing increased
recombinant copies of a gene or by introducing a recombinant
construct having a heterologous promoter operably linked to a
recombinant polynucleotide encoding a polypeptide into a target
cell or organism. Reduced protein activity can be achieved by a
variety of mechanisms including antisense, mutation, or knockout.
Antisense RNA will reduce the level of expressed protein resulting
in reduced protein activity as compared to wild type activity
levels. A mutation in the gene encoding a protein may reduce the
level of expressed protein and/or interfere with the function of
expressed protein to cause reduced protein activity.
[0018] In one embodiment, the invention is a fragment of a
disclosed recombinant polynucleotide consisting of oligonucleotides
of at least 15, at least 16 or 17, at least 18 or 19, or at least
20 or more consecutive nucleotides. Such oligonucleotides are
fragments of the larger recombinant polynucleotides having a
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 31,564, and find use, for example as probes and primers
for detection of the polynucleotides of the present invention.
[0019] In one embodiment the present invention is a functional
variant of a recombinant polynucleotide provided herein. As used
herein, a "functional variant" refers to any second polynucleotide
varying from a first polynucleotide sequence in such a way so as
not to significantly affect the function when compared to the
function of the first polynucleotide. Such functional variants may
be naturally occurring, including homologous polynucleotides from
the same or a different species, or may be non-natural functional
variants, for example polynucleotides synthesized using chemical
synthesis methods, or generated using recombinant DNA techniques.
With respect to nucleotide sequences, degeneracy of the genetic
code provides the possibility to substitute at least one base of
the protein encoding sequence of a gene with a different base
without causing the amino acid sequence of the polypeptide produced
from the gene to be changed. Hence, in one embodiment, a
recombinant polynucleotide of the present invention may have any
base sequence that has been changed from SEQ ID NO: 1 through SEQ
ID NO: 31,564 by substitution in accordance with degeneracy of the
genetic code. See for example, U.S. Pat. No. 5,500,365, which is
hereby incorporated by reference.
[0020] Polynucleotides of the present invention that are functional
variants of the polynucleotides provided herein will generally
demonstrate significant identity with the polynucleotides provided
herein. Of particular interest are polynucleotide homologs having
at least about 60% sequence identity, at least about 70% sequence
identity, at least about 80% sequence identity, at least about 85%
sequence identity, and at least about 90%, 95% or even greater,
such as 98% or 99% sequence identity with polynucleotide sequences
described herein.
Polypeptides
[0021] In one embodiment this invention also provides recombinant
polypeptides. Amino acid sequences of the recombinant polypeptides
of the present invention are provided herein as SEQ ID NO: 31,565
through SEQ ID NO: 63,128.
[0022] As used herein, the term "polypeptide" refers to an
unbranched chain of amino acid residues that are covalently linked
by an amide linkage between the carboxyl group of one amino acid
and the amino group of another. The term polypeptide can encompass
whole proteins (i.e. a functional protein encoded by a particular
gene), as well as fragments of proteins. In one embodiment the
invention is a recombinant polypeptide which represents a whole
protein. In another embodiment the invention is a recombinant
polypeptide which represents a sufficient portion of an entire
protein to impart the relevant biological activity of the protein.
The term "protein" also includes molecules consisting of one or
more polypeptide chains. Thus, a recombinant polypeptide of the
present invention may also constitute an entire gene product, but
only a portion of a functional oligomeric protein having multiple
polypeptide chains.
[0023] As used herein, the term "recombinant polypeptide" refers to
a polypeptide produced as a result of recombinant DNA technology.
The term recombinant polypeptide as used herein is not intended to
encompass molecules present in their native state.
[0024] In one embodiment the invention is a recombinant polypeptide
involved in one or more important biological properties in a plant.
Such recombinant polypeptide may be produced in transgenic plants
to provide plants having improved phenotypic properties and/or
improved response to stressful environmental conditions. In some
cases, decreased expression of such polypeptide may be desired,
such decreased expression being obtained by use of the
polynucleotide sequences provided herein, for example in antisense
or cosuppression methods. See, Table 1 of U.S. application Ser. No.
10/767,701 for a list of improved plant properties and PROTEIN_NUM
for the recombinant polypeptide whose expression may be altered in
transgenic plants to impart such improvements. A summary of such
improved properties and polypeptides of interest for increased or
decreased expression is provided below.
[0025] Yield/Nitrogen: Yield improvement by improved nitrogen flow,
sensing, uptake, storage and/or transport. Polypeptides useful for
imparting such properties include those involved in aspartate and
glutamate biosynthesis, polypeptides involved in aspartate and
glutamate transport, polypeptides associated with the TOR (Target
of Rapamycin) pathway, nitrate transporters, ammonium transporters,
chlorate transporters and polypeptides involved in tetrapyrrole
biosynthesis.
[0026] Yield/Carbohydrate: Yield improvement by effects on
carbohydrate metabolism, for example by increased sucrose
production and/or transport. Polypeptides useful for improved yield
by effects on carbohydrate metabolism include polypeptides involved
in sucrose or starch metabolism, carbon assimilation or
carbohydrate transport, including, for example sucrose transporters
or glucose/hexose transporters, enzymes involved in
glycolysis/gluconeogenesis, the pentose phosphate cycle, or
raffinose biosynthesis, and polypeptides involved in glucose
signaling, such as SNF1 complex proteins.
[0027] Yield/Photosynthesis: Yield improvement resulting from
increased photosynthesis. Polypeptides useful for increasing the
rate of photosynthesis include phytochrome, photosystem I and II
proteins, electron carriers, ATP synthase, NADH dehydrogenase and
cytochrome oxidase.
[0028] Yield/Phosphorus: Yield improvement resulting from increased
phosphorus uptake, transport or utilization. Polypeptides useful
for improving yield in this manner include phosphatases and
phosphate transporters.
[0029] Yield/Stress tolerance: Yield improvement resulting from
improved plant growth and development by helping plants to tolerate
stressful growth conditions. Polypeptides useful for improved
stress tolerance under a variety of stress conditions include
polypeptides involved in gene regulation, such as
serine/threonine-protein kinases, MAP kinases, MAP kinase kinases,
and MAP kinase kinase kinases; polypeptides that act as receptors
for signal transduction and regulation, such as receptor protein
kinases; intracellular signaling proteins, such as protein
phosphatases, GTP binding proteins, and phospholipid signaling
proteins; polypeptides involved in arginine biosynthesis;
polypeptides involved in ATP metabolism, including for example
ATPase, adenylate transporters, and polypeptides involved in ATP
synthesis and transport; polypeptides involved in glycine betaine,
jasmonic acid, flavonoid or steroid biosynthesis; and hemoglobin.
Enhanced or reduced activity of such polypeptides in transgenic
plants will provide changes in the ability of a plant to respond to
a variety of environmental stresses, such as chemical stress,
drought stress and pest stress.
[0030] Cold tolerance: Polypeptides of interest for improving plant
tolerance to cold or freezing temperatures include polypeptides
involved in biosynthesis of trehalose or raffinose, polypeptides
encoded by cold induced genes, fatty acyl desaturases and other
polypeptides involved in glycerolipid or membrane lipid
biosynthesis, which find use in modification of membrane fatty acid
composition, alternative oxidase, calcium-dependent protein
kinases, LEA proteins and uncoupling protein.
[0031] Heat tolerance: Polypeptides of interest for improving plant
tolerance to heat include polypeptides involved in biosynthesis of
trehalose, polypeptides involved in glycerolipid biosynthesis or
membrane lipid metabolism (for altering membrane fatty acid
composition), heat shock proteins and mitochondrial NDK.
[0032] Osmotic tolerance: Polypeptides of interest for improving
plant tolerance to extreme osmotic conditions include polypeptides
involved in proline biosynthesis.
[0033] Drought tolerance: Polypeptides of interest for improving
plant tolerance to drought conditions include aquaporins,
polypeptides involved in biosynthesis of trehalose or wax, LEA
proteins and invertase.
[0034] Pathogen or pest tolerance: Polypeptides of interest for
improving plant tolerance to effects of plant pests or pathogens
include proteases, polypeptides involved in anthocyanin
biosynthesis, polypeptides involved in cell wall metabolism,
including cellulases, glucosidases, pectin methylesterase,
pectinase, polygalacturonase, chitinase, chitosanase, and cellulose
synthase, and polypeptides involved in biosynthesis of terpenoids
or indole for production of bioactive metabolites to provide
defense against herbivorous insects.
[0035] Cell cycle modification: Polypeptides encoding cell cycle
enzymes and regulators of the cell cycle pathway are useful for
manipulating growth rate in plants to provide early vigor and
accelerated maturation leading to improved yield. Improvements in
quality traits, such as seed oil content, may also be obtained by
expression of cell cycle enzymes and cell cycle regulators.
Polypeptides of interest for modification of cell cycle pathway
include cyclins and EIF5alpha pathway proteins, polypeptides
involved in polyamine metabolism, polypeptides which act as
regulators of the cell cycle pathway, including cyclin-dependent
kinases (CDKs), CDK-activating kinases, CDK-inhibitors, Rb and
Rb-binding proteins, and transcription factors that activate genes
involved in cell proliferation and division, such as the E2F family
of transcription factors, proteins involved in degradation of
cyclins, such as cullins, and plant homologs of tumor suppressor
polypeptides.
[0036] Seed protein yield/content: Polypeptides useful for
providing increased seed protein quantity and/or quality include
polypeptides involved in the metabolism of amino acids in plants,
particularly polypeptides involved in biosynthesis of
methionine/cysteine and lysine, amino acid transporters, amino acid
efflux carriers, seed storage proteins, proteases, and polypeptides
involved in phytic acid metabolism.
[0037] Seed oil yield/content: Polypeptides useful for providing
increased seed oil quantity and/or quality include polypeptides
involved in fatty acid and glycerolipid biosynthesis,
beta-oxidation enzymes, enzymes involved in biosynthesis of
nutritional compounds, such as carotenoids and tocopherols, and
polypeptides that increase embryo size or number or thickness of
aleurone.
[0038] Disease response in plants: Polypeptides useful for
imparting improved disease responses to plants include polypeptides
encoded by cercosporin induced genes, antifungal proteins and
proteins encoded by R-genes or SAR genes. Expression of such
polypeptides in transgenic plants will provide an increase in
disease resistance ability of plants.
[0039] Galactomannanan biosynthesis: Polypeptides involved in
production of galactomannans are of interest for providing plants
having increased and/or modified reserve polysaccharides for use in
food, pharmaceutical, cosmetic, paper and paint industries.
[0040] Flavonoid/isoflavonoid metabolism in plants: Polypeptides of
interest for modification of flavonoid/isoflavonoid metabolism in
plants include cinnamate-4-hydroxylase, chalcone synthase and
flavonol synthase. Enhanced or reduced activity of such
polypeptides in transgenic plants will provide changes in the
quantity and/or speed of flavonoid metabolism in plants and may
improve disease resistance by enhancing synthesis of protective
secondary metabolites or improving signaling pathways governing
disease resistance.
[0041] Plant growth regulators: Polypeptides involved in production
of substances that regulate the growth of various plant tissues are
of interest in the present invention and may be used to provide
transgenic plants having altered morphologies and improved plant
growth and development profiles leading to improvements in yield
and stress response. Of particular interest are polypeptides
involved in the biosynthesis of plant growth hormones, such as
gibberellins, cytokinins, auxins, ethylene and abscisic acid, and
other proteins involved in the activity and/or transport of such
polypeptides, including for example, cytokinin oxidase,
cytokinin/purine permeases, F-box proteins, G-proteins and
phytosulfokines.
[0042] Herbicide tolerance: Polypeptides of interest for producing
plants having tolerance to plant herbicides include polypeptides
involved in the shikimate pathway, which are of interest for
providing glyphosate tolerant plants. Such polypeptides include
polypeptides involved in biosynthesis of chorismate, phenylalanine,
tyrosine and tryptophan.
[0043] Transcription factors in plants: Transcription factors play
a key role in plant growth and development by controlling the
expression of one or more genes in temporal, spatial and
physiological specific patterns. Enhanced or reduced activity of
such polypeptides in transgenic plants will provide significant
changes in gene transcription patterns and provide a variety of
beneficial effects in plant growth, development and response to
environmental conditions. Transcription factors of interest
include, but are not limited to myb transcription factors,
including helix-turn-helix proteins, homeodomain transcription
factors, leucine zipper transcription factors, MADS transcription
factors, transcription factors having AP2 domains, zinc finger
transcription factors, CCAAT binding transcription factors,
ethylene responsive transcription factors, transcription initiation
factors and UV damaged DNA binding proteins.
[0044] Homologous recombination: Increasing the rate of homologous
recombination in plants is useful for accelerating the
introgression of transgenes into breeding varieties by
backcrossing, and to enhance the conventional breeding process by
allowing rare recombinants between closely linked genes in phase
repulsion to be identified more easily. Polypeptides useful for
expression in plants to provide increased homologous recombination
include polypeptides involved in mitosis and/or meiosis, including
for example, resolvases and polypeptide members of the RAD52
epistasis group.
[0045] Lignin biosynthesis: Polypeptides involved in lignin
biosynthesis are of interest for increasing plants' resistance to
lodging and for increasing the usefulness of plant materials as
biofuels.
[0046] In one embodiment of the invention, the function of a
recombinant polypeptide is determined by comparison of the amino
acid sequence of the recombinant polypeptide to amino acid
sequences of known polypeptides. A variety of homology based search
algorithms are available to compare a query sequence to a protein
database, including for example, BLAST, FASTA, and Smith-Waterman.
In the present application, BLASTX and BLASTP algorithms are used
to provide protein function information. A number of values are
examined in order to assess the confidence of the function
assignment. Useful measurements include "E-value" (also shown as
"hit_p"), "percent identity", "percent query coverage", and
"percent hit coverage".
[0047] In BLAST, E-value, or expectation value, represents the
number of different alignments with scores equivalent to or better
than the raw alignment score, S, that are expected to occur in a
database search by chance. The lower the E value, the more
significant the match. Because database size is an element in
E-value calculations, E-values obtained by BLASTing against public
databases, such as GenBank, have generally increased over time for
any given query/entry match. In setting criteria for confidence of
polypeptide function prediction, a "high" BLAST match is considered
herein as having an E-value for the top BLAST hit provided in Table
1 of U.S. application Ser. No. 10/767,701 of less than 1E-30; a
medium BLASTX E-value is 1E-30 to 1E-8; and a low BLASTX E-value is
greater than 1E-8. The top BLAST hit and corresponding E values are
provided in Table 1 of U.S. application Ser. No. 10/767,701.
[0048] Percent identity refers to the percentage of identically
matched amino acid residues that exist along the length of that
portion of the sequences which is aligned by the BLAST algorithm.
In setting criteria for confidence of polypeptide function
prediction, a "high" BLAST match is considered herein as having
percent identity for the top BLAST hit provided in Table 1 of U.S.
application Ser. No. 10/767,701 of at least 70%; a medium percent
identity value is 35% to 70%; and a low percent identity is less
than 35%.
[0049] In one embodiment of the invention, the protein function
assignment in the present invention is determined using
combinations of E-values, percent identity, query coverage and hit
coverage. Query coverage refers to the percent of the query
sequence that is represented in the BLAST alignment. Hit coverage
refers to the percent of the database entry that is represented in
the BLAST alignment. In one embodiment of the invention, function
of a query polypeptide is inferred from function of a protein
homolog where either (1) hit_p<1e-30 or % identity>35% AND
query_coverage>50% AND hit_coverage>50%, or (2) hit_p<1e-8
AND query_coverage>70% AND hit_coverage>70%.
[0050] Another aspect of the invention comprises a functional
variant which differs in one or more amino acids from those of a
recombinant polypeptide provided herein as the result of one or
more conservative amino acid substitutions. It is well known in the
art that one or more amino acids in a reference sequence can be
substituted with at least one other amino acid, the charge and
polarity of which are similar to that of the native amino acid,
resulting in a silent change. For instance, valine is a
conservative substitute for alanine and threonine is a conservative
substitute for serine. Conservative substitutions for an amino acid
within a polypeptide sequence can be selected from other members of
the class to which the naturally occurring amino acid belongs.
Amino acids can be divided into the following four groups: (1)
acidic amino acids, (2) basic amino acids, (3) neutral polar amino
acids, and (4) neutral nonpolar amino acids. Representative amino
acids within these various groups include, but are not limited to:
(1) acidic (negatively charged) amino acids such as aspartic acid
and glutamic acid; (2) basic (positively charged) amino acids such
as arginine, histidine, and lysine; (3) neutral polar amino acids
such as glycine, serine, threonine, cysteine, tyrosine, asparagine,
and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids
such as alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine. Conserved substitutes
for an amino acid within a polypeptide sequence can be selected
from other members of the group to which the naturally occurring
amino acid belongs. For example, a group of amino acids having
aliphatic side chains is glycine, alanine, valine, leucine, and
isoleucine; a group of amino acids having aliphatic-hydroxyl side
chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Examples of conservative amino acid substitution groups
are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and
asparagine-glutamine. In one embodiment a recombinant polypeptide
of the invention may differ in one or more amino acids as the
result of deletion or insertion of one or more amino acids in a
native sequence. See for example, U.S. Pat. No. 5,500,365, which is
hereby incorporated by reference.
[0051] One embodiment of the present invention is a variant which
has the same function as a recombinant polypeptide provided herein,
but with increased or decreased activity or altered specificity.
Such variations in protein activity can be achieved by mutagenesis
or may exist naturally in polypeptides encoded by related genes,
for example in a related polypeptide encoded by a different allele
or in a different species. Variant polypeptides may be obtained by
well known nucleic acid or protein screening methods using DNA or
antibody probes, for example by screening libraries for genes
encoding related polypeptides, or in the case of expression
libraries, by screening directly for variant polypeptides.
Screening methods for obtaining a modified protein or enzymatic
activity of interest by mutagenesis are disclosed in U.S. Pat. No.
5,939,250, which is hereby incorporated by reference. An
alternative approach to the generation of variants uses random
recombination techniques such as "DNA shuffling" as disclosed in
U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721 and 5,837,458; and
International Applications WO 98/31837 and WO 99/65927, all of
which are hereby incorporated by reference. An alternative method
of molecular evolution involves a staggered extension process
(StEP) for in vitro mutagenesis and recombination of nucleic acid
molecule sequences, as disclosed in U.S. Pat. No. 5,965,408 and
International Application WO 98/42832, both of which are hereby
incorporated by reference.
[0052] Polypeptides of the present invention that are functional
variants of the polypeptides provided herein will generally
demonstrate significant identity with the polypeptides provided
herein. One embodiment of the invention is a polypeptide having at
least about 35% sequence identity, at least about 50% sequence
identity, at least about 60% sequence identity, at least about 70%
sequence identity, at least about 80% sequence identity, and at
least about 85%, 90%, 95% or even greater sequence identity with a
recombinant polypeptide sequence described herein. One embodiment
of the invention is a polypeptide having an amino acid sequence
provided herein (reference polypeptides) and functional variants of
such reference polypeptide, wherein such functional variant
comprises at least about 50 consecutive amino acids having at least
about 90% identity to about a 50 amino acid polypeptide fragment of
said reference polypeptide.
Recombinant DNA Constructs
[0053] In one embodiment the invention encompasses the use of
recombinant polynucleotides in recombinant constructs, i.e.
constructs comprising recombinant polynucleotides that are
constructed or modified outside of cells and that join nucleic
acids that are not found joined in nature. Using methods known to
those of ordinary skill in the art, recombinant polynucleotides of
the invention can be inserted into recombinant DNA constructs that
can then be introduced into a host cell of choice for expression of
the encoded polypeptide or to provide for reduction of expression
of the encoded polypeptide, for example by antisense or
cosuppression methods. Potential host cells include both
prokaryotic and eukaryotic cells. One embodiment of the invention
uses a recombinant polynucleotide of the present invention for
preparation of recombinant constructs for use in plant
transformation.
[0054] In plant transformation, exogenous genetic material is
transferred into a plant cell. As used herein "exogenous" refers to
a nucleic acid molecule, for example a recombinant DNA construct
comprising a recombinant polynucleotide of the present invention,
produced outside the organism, e.g. plant, into which it is
introduced. An exogenous nucleic acid molecule can have a naturally
occurring or non-naturally occurring nucleic acid sequence. One
skilled in the art recognizes that an exogenous nucleic acid
molecule can be derived from the same species into which it is
introduced or from a different species. Such exogenous genetic
material may be transferred into either monocot or dicot plants
including, but not limited to, soy, cotton, canola, maize,
teosinte, wheat, rice, and Arabidopsis plants. Transformed plant
cells comprising such exogenous genetic material may be regenerated
to produce whole transformed plants.
[0055] Exogenous genetic material may be transferred into a plant
cell by the use of a recombinant construct, also known as a vector,
designed for such a purpose. A recombinant construct can comprise a
number of sequence elements, including promoters, encoding regions,
and selectable markers. Recombinant constructs are available which
have been designed to replicate in both E. coli and A. tumefaciens
and have all of the features required for transferring large
inserts of DNA into plant chromosomes. Design of such vectors is
generally within the skill of the art.
[0056] A recombinant construct will generally include a plant
promoter to direct transcription of the protein-encoding region or
the antisense sequence of choice. Numerous promoters, which are
active in plant cells, have been described in the literature. These
include the nopaline synthase (NOS) promoter and octopine synthase
(OCS) promoters carried on tumor-inducing plasmids of Agrobacterium
tumefaciens or caulimovirus promoters such as the Cauliflower
Mosaic Virus (CaMV) 19S or 35S promoter (U.S. Pat. No. 5,352,605),
and the Figwort Mosaic Virus (FMV) 35S-promoter (U.S. Pat. No.
5,378,619). These promoters and numerous others have been used to
create recombinant vectors for expression in plants. Any promoter
known or found to cause transcription of DNA in plant cells can be
used in the present invention. Other useful promoters are
described, for example, in U.S. Pat. Nos. 5,378,619; 5,391,725;
5,428,147; 5,447,858; 5,608,144; 5,614,399; 5,633,441; and
5,633,435, all of which are hereby incorporated by reference.
[0057] In addition, promoter enhancers, such as the CaMV 35S
enhancer or a tissue specific enhancer, may be used to enhance gene
transcription levels. Enhancers often are found 5' to the start of
transcription in a promoter that functions in eukaryotic cells, but
can often be inserted in the forward or reverse orientation 5' or
3' to the coding sequence. In some instances, these 5' enhancing
elements are introns. Deemed to be particularly useful as enhancers
are the 5' introns of the rice actin 1 and rice actin 2 genes.
Examples of other enhancers which could be used in accordance with
the invention include elements from octopine synthase genes, the
maize alcohol dehydrogenase gene intron 1, elements from the maize
shrunken 1 gene, the sucrose synthase intron, the TMV omega
element, and promoters from non-plant eukaryotes.
[0058] Recombinant constructs can also contain one or more 5'
non-translated leader sequences which serve to enhance polypeptide
production from the resulting mRNA transcripts. Such sequences may
be derived from the promoter selected to express the gene or can be
specifically modified to increase translation of the mRNA. Such
regions may also be obtained from viral RNAs, from suitable
eukaryotic genes, or from a synthetic gene sequence. For a review
of optimizing expression of transgenes, see Koziel et al. (1996)
Plant Mol. Biol. 32:393-405).
[0059] Recombinant constructs may also include, with the coding
region of interest, a nucleic acid sequence that acts, in whole or
in part, to terminate transcription of that region. One type of 3'
untranslated sequence which may be used is a 3' UTR from the
nopaline synthase gene (nos 3') of Agrobacterium tumefaciens. Other
3' termination regions of interest include those from a gene
encoding the small subunit of a ribulose-1,5-bisphosphate
carboxylase-oxygenase (rbcS), and more specifically, from a rice
rbcS gene (U.S. Pat. No. 6,426,446), the 3' UTR for the T7
transcript of Agrobacterium tumefaciens, the 3' end of the protease
inhibitor I or II genes from potato or tomato, and the 3' region
isolated from Cauliflower Mosaic Virus. Alternatively, one also
could use a gamma coixin, oleosin 3 or other 3' UTRs from the genus
Coix (PCT Publication WO 99/58659).
[0060] Recombinant constructs may also include a selectable marker.
Selectable markers may be used to select for plants or plant cells
that contain the exogenous genetic material. Useful selectable
marker genes include those conferring resistance to antibiotics
such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin
(aac3 and aacC4) or resistance to herbicides such as glufosinate
(bar or pat) and glyphosate (EPSPS). Examples of such selectable
markers are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435;
5,780,708 and 6,118,047, all of which are hereby incorporated by
reference.
[0061] Recombinant constructs may also include a screenable marker.
Screenable markers may be used to monitor transformation. Exemplary
screenable markers include genes expressing a colored or
fluorescent protein such as a luciferase or green fluorescent
protein (GFP), a .beta.-glucuronidase or uidA gene (GUS) which
encodes an enzyme for which various chromogenic substrates are
known or an R-locus gene, which encodes a product that regulates
the production of anthocyanin pigments (red color) in plant
tissues. Other possible selectable and/or screenable marker genes
will be apparent to those of skill in the art.
[0062] Recombinant constructs may also include a transit peptide
for targeting of a gene target to a plant organelle, particularly
to a chloroplast, leucoplast or other plastid organelle, see for
example U.S. Pat. No. 5,188,642, which is hereby incorporated by
reference.
[0063] For use in Agrobacterium mediated transformation methods,
recombinant constructs of the present invention may also include
T-DNA border regions flanking the DNA to be inserted into the plant
genome to provide for transfer of the DNA into the plant host
chromosome as discussed in more detail below. An exemplary plasmid
that finds use in such transformation methods is pMON18365, a T-DNA
vector that can be used to clone exogenous genes and transfer them
into plants using Agrobacterium-mediated transformation. See
published U.S. Patent Application 20030024014, which is hereby
incorporated by reference. This vector contains the left border and
right border sequences necessary for Agrobacterium transformation.
The plasmid also has origins of replication for maintaining the
plasmid in both E. coli and Agrobacterium tumefaciens strains.
[0064] A candidate gene is prepared for insertion into the T-DNA
vector, for example using well-known gene cloning techniques such
as PCR. Restriction sites may be introduced onto each end of the
gene to facilitate cloning. For example, candidate genes may be
amplified by PCR techniques using a set of primers. Both the
amplified DNA and the cloning vector are cut with the same
restriction enzymes, for example, NotI and PstI. The resulting
fragments are gel-purified, ligated together, and transformed into
E. coli. Plasmid DNA containing the vector with inserted gene may
be isolated from E. coli cells selected for spectinomycin
resistance, and the presence of the desired insert verified by
digestion with the appropriate restriction enzymes. Undigested
plasmid may then be transformed into Agrobacterium tumefaciens
using techniques well known to those in the art, and transformed
Agrobacterium cells containing the vector of interest selected
based on spectinomycin resistance. These and other similar
recombinant constructs useful for plant transformation may be
readily prepared by one skilled in the art.
Transformation Methods and Transgenic Plants
[0065] Methods and compositions for transforming bacteria and other
microorganisms are known in the art. See for example Molecular
Cloning: A Laboratory Manual, 3.sup.rd edition Volumes 1, 2, and 3.
J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor
Laboratory Press, 2000.
[0066] Technology for introduction of DNA into cells is well known
to those of skill in the art. Methods and materials for
transforming plants by introducing a transgenic DNA construct into
a plant genome in the practice of this invention can include any of
the well-known and demonstrated methods including electroporation
as illustrated in U.S. Pat. No. 5,384,253, microprojectile
bombardment as illustrated in U.S. Pat. Nos. 5,015,580; 5,550,318;
5,538,880; 6,160,208; 6,399,861 and 6,403,865,
Agrobacterium-mediated transformation as illustrated in U.S. Pat.
Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840 and 6,384,301, and
protoplast transformation as illustrated in U.S. Pat. No.
5,508,184, all of which are hereby incorporated by reference.
[0067] Any of the recombinant polynucleotides of the present
invention may be introduced into a plant cell in a permanent or
transient manner in combination with other genetic elements such as
vectors, promoters enhancers etc. Further any of the recombinant
polynucleotides of the present invention may be introduced into a
plant cell in a manner that allows for production of the
polypeptide or fragment thereof encoded by the recombinant
polynucleotide in the plant cell, or in a manner that provides for
decreased expression of an endogenous gene and concomitant
decreased production of protein.
[0068] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating added, exogenous genes. Selfing of
appropriate progeny can produce plants that are homozygous for both
added, exogenous genes that encode a polypeptide of interest.
Back-crossing to a parental plant and out-crossing with a
non-transgenic plant are also contemplated, as is vegetative
propagation.
[0069] Expression of the recombinant polynucleotides of the present
invention and the concomitant production of polypeptides encoded by
the recombinant polynucleotides is of interest for production of
transgenic plants having improved properties, particularly,
improved properties which result in crop plant yield improvement.
Expression of recombinant polypeptides of the present invention in
plant cells may be evaluated by specifically identifying the
protein products of the introduced genes or evaluating the
phenotypic changes brought about by their expression. It is noted
that when the polypeptide being produced in a transgenic plant is
native to the target plant species, quantitative analyses comparing
the transformed plant to wild type plants may be required to
demonstrate increased expression of the polypeptide of this
invention.
[0070] Assays for the production and identification of specific
proteins make use of various physical-chemical, structural,
functional, or other properties of the proteins. Unique
physical-chemical or structural properties allow the proteins to be
separated and identified by electrophoretic procedures, such as
native or denaturing gel electrophoresis or isoelectric focusing,
or by chromatographic techniques such as ion exchange or gel
exclusion chromatography. The unique structures of individual
proteins offer opportunities for use of specific antibodies to
detect their presence in formats such as an ELISA assay.
Combinations of approaches may be employed with even greater
specificity such as western blotting in which antibodies are used
to locate individual gene products that have been separated by
electrophoretic techniques. Additional techniques may be employed
to absolutely confirm the identity of the product of interest such
as evaluation by amino acid sequencing following purification.
Although these are among the most commonly employed, other
procedures may be additionally used.
[0071] Assay procedures may also be used to identify the expression
of proteins by their functionality, particularly where the
expressed protein is an enzyme capable of catalyzing chemical
reactions involving specific substrates and products. These
reactions may be measured, for example in plant extracts, by
providing and quantifying the loss of substrates or the generation
of products of the reactions by physical and/or chemical
procedures.
[0072] In many cases, the expression of a gene product is
determined by evaluating the phenotypic results of its expression.
Such evaluations may be simply as visual observations, or may
involve assays. Such assays may take many forms including but not
limited to analyzing changes in the chemical composition,
morphology, or physiological properties of the plant. Chemical
composition may be altered by expression of genes encoding enzymes
or storage proteins which change amino acid composition and may be
detected by amino acid analysis, or by enzymes which change starch
quantity which may be analyzed by near infrared reflectance
spectrometry. Morphological changes may include greater stature or
thicker stalks.
[0073] Plants with decreased expression of a gene of interest can
also be achieved through the use of polynucleotides of the present
invention, for example by expression of antisense nucleic acids, or
by identification of plants transformed with sense expression
constructs that exhibit cosuppression effects.
[0074] Antisense approaches are a way of preventing or reducing
gene function by targeting the genetic material as disclosed in
U.S. Pat. Nos. 4,801,540; 5,107,065; 5,759,829; 5,910,444;
6,184,439; and 6,198,026, all of which are hereby incorporated by
reference. The objective of the antisense approach is to use a
sequence complementary to the target gene to block its expression
and create a mutant cell line or organism in which the level of a
single chosen protein is selectively reduced or abolished.
Antisense techniques have several advantages over other `reverse
genetic` approaches. The site of inactivation and its developmental
effect can be manipulated by the choice of promoter for antisense
genes or by the timing of external application or microinjection.
Antisense can manipulate its specificity by selecting either unique
regions of the target gene or regions where it shares homology to
other related genes.
[0075] The principle of regulation by antisense RNA is that RNA
that is complementary to the target mRNA is introduced into cells,
resulting in specific RNA:RNA duplexes being formed by base pairing
between the antisense substrate and the target. Under one
embodiment, the process involves the introduction and expression of
an antisense gene sequence. Such a sequence is one in which part or
all of the normal gene sequences are placed under a promoter in
inverted orientation so that the `wrong` or complementary strand is
transcribed into a noncoding antisense RNA that hybridizes with the
target mRNA and interferes with its expression. An antisense vector
is constructed by standard procedures and introduced into cells by
transformation, transfection, electroporation, microinjection,
infection, etc. The type of transformation and choice of vector
will determine whether expression is transient or stable. The
promoter used for the antisense gene may influence the level,
timing, tissue, specificity, or inducibility of the antisense
inhibition.
[0076] As used herein "gene suppression" means any of the
well-known methods for suppressing expression of protein from a
gene including sense suppression, anti-sense suppression and RNAi
suppression. In suppressing genes to provide plants with a
desirable phenotype, anti-sense and RNAi gene suppression methods
are preferred. For a description of anti-sense regulation of gene
expression in plant cells see U.S. Pat. No. 5,107,065. For a
description of RNAi gene suppression in plants by transcription of
a dsRNA see U.S. Pat. No. 6,506,559, U.S. Patent Application
Publication No. 2002/0168707 A1, and U.S. patent application Ser.
Nos. 09/423,143 (see WO 98/53083), 09/127,735 (see WO 99/53050),
and 09/084,942 (see WO 99/61631), all of which are hereby
incorporated by reference. Suppression of an gene by RNAi can be
achieved using a recombinant DNA construct having a promoter
operably linked to a DNA element comprising a sense and anti-sense
element of a segment of genomic DNA of the gene, e.g., a segment of
at least about 23 nucleotides, more preferably about 50 to 200
nucleotides where the sense and anti-sense DNA components can be
directly linked or joined by an intron or artificial DNA segment
that can form a loop when the transcribed RNA hybridizes to form a
hairpin structure. For example, genomic DNA from a polymorphic
locus of SEQ ID NO: 1 through SEQ ID NO: 31,564 can be used in a
recombinant construct for suppression of a cognate gene by RNAi
suppression.
[0077] Insertion mutations created by transposable elements may
also prevent gene function. For example, in many dicot plants,
transformation with the T-DNA of Agrobacterium may be readily
achieved and large numbers of transformants can be rapidly
obtained. Also, some species have lines with active transposable
elements that can efficiently be used for the generation of large
numbers of insertion mutations, while some other species lack such
options. Mutant plants produced by Agrobacterium or transposon
mutagenesis and having altered expression of a polypeptide of
interest can be identified using the polynucleotides of the present
invention. For example, a large population of mutated plants may be
screened with polynucleotides encoding the polypeptide of interest
to detect mutated plants having an insertion in the gene encoding
the polypeptide of interest.
[0078] In one embodiment of the invention, polynucleotides of the
present invention may be used in site-directed mutagenesis.
Site-directed mutagenesis may be utilized to modify nucleic acid
sequences, particularly as it is a technique that allows one or
more of the amino acids encoded by a nucleic acid molecule to be
altered (e.g., a threonine to be replaced by a methionine). Three
basic methods for site-directed mutagenesis are often employed.
These are cassette mutagenesis, primer extension, and methods based
upon PCR.
[0079] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant organisms and the
screening and isolating of clones.
Arrays
[0080] In one embodiment of the invention, the recombinant
polynucleotides or recombinant polypeptides of this invention may
be used to prepare arrays of target molecules arranged on a surface
of a substrate. The target molecules may be known molecules, e.g.
polynucleotides (including oligonucleotides) or polypeptides, which
are capable of binding to specific probes, such as complementary
nucleic acids or specific antibodies. The target molecules may be
immobilized, e.g. by covalent or non-covalent bonding, to the
surface in small amounts of substantially purified and isolated
molecules in a grid pattern. By immobilized it is meant that the
target molecules maintain their position relative to the solid
support under hybridization and washing conditions. Target
molecules are deposited in small footprint, isolated quantities of
"spotted elements" of preferably single-stranded polynucleotide
preferably arranged in rectangular grids in a density of about 30
to 100 or more, e.g. up to about 1000, spotted elements per square
centimeter. In one embodiment of the invention, the arrays comprise
at least about 100 or more, e.g. at least about 1000 to 5000,
distinct target polynucleotides per unit substrate. Where detection
of transcription for a large number of genes is desired, the
economics of arrays favors a high density design criteria provided
that the target molecules are sufficiently separated so that the
intensity of the indicia of a binding event associated with highly
expressed probe molecules does not overwhelm and mask the indicia
of neighboring binding events. For high-density microarrays each
spotted element may contain up to about 10.sup.7 or more copies of
the target molecule, e.g. single stranded cDNA, on glass substrates
or nylon substrates.
[0081] Arrays of this invention may be prepared with molecules from
a single species, preferably a plant species, or with molecules
from other species, particularly other plant species. Arrays with
target molecules from a single species can be used with probe
molecules from the same species or a different species due to the
ability of cross species homologous genes to hybridize. It is
generally preferred for high stringency hybridization that the
target and probe molecules are from the same species.
[0082] In one embodiment of the invention, the organism of interest
is a plant and the target molecules are polynucleotides or
oligonucleotides with nucleic acid sequences having at least about
80 percent sequence identity to a corresponding sequence of the
same length in a recombinant polynucleotide having a sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 31,564 or complements thereof. In another embodiment of the
invention, at least about 10% of the target molecules on an array
have at least about 15 consecutive nucleotides of sequence having
at least about 80% and up to about 100% identity with a
corresponding sequence of the same length in a recombinant
polynucleotide having a sequence selected from the group consisting
of SEQ ID NO: 1 through SEQ ID NO: 31,564 or complements or
fragments thereof.
[0083] Such arrays are useful in a variety of applications,
including gene discovery, genomic research, molecular breeding and
bioactive compound screening. One use of arrays is in the analysis
of differential gene transcription, e.g. transcription profiling
where the production of mRNA in different cells, normally a cell of
interest and a control, is compared and discrepancies in gene
expression are identified. In such assays, the presence of
discrepancies indicates a difference in gene expression levels in
the cells being compared. Such information is useful for the
identification of the types of genes expressed in a particular cell
or tissue type in a known environment. Such applications generally
involve the following steps: (a) preparation of probe, e.g.
attaching a label to a plurality of expressed molecules; (b)
contact of probe with the array under conditions sufficient for
probe to bind with corresponding target, e.g. by hybridization or
specific binding; (c) removal of unbound probe from the array; and
(d) detection of bound probe.
[0084] A probe may be prepared with RNA extracted from a given cell
line or tissue. The probe may be produced by reverse transcription
of mRNA or total RNA and labeled with radioactive or fluorescent
labeling. A probe is typically a mixture containing many different
sequences in various amounts, corresponding to the numbers of
copies of the original mRNA species extracted from the sample.
[0085] The initial RNA sample for probe preparation will typically
be derived from a physiological source. The physiological source
may be selected from a variety of organisms, with physiological
sources of interest including single celled organisms such as yeast
and multicellular organisms, including plants and animals,
particularly plants, where the physiological sources from
multicellular organisms may be derived from particular organs or
tissues of the multicellular organism, or from isolated cells
derived from an organ, or tissue of the organism. The physiological
sources may also be multicellular organisms at different
developmental stages (e.g., 10-day-old seedlings), or organisms
grown under different environmental conditions (e.g.,
drought-stressed plants) or treated with chemicals.
[0086] In preparing the RNA probe, the physiological source may be
subjected to a number of different processing steps, where such
processing steps might include tissue homogenation, cell isolation
and cytoplasmic extraction, nucleic acid extraction and the like,
where such processing steps are known to the those of skill in the
art. Methods of isolating RNA from cells, tissues, organs or whole
organisms are well known to those skilled in the art.
Computer Based Systems and Methods
[0087] In one embodiment of the invention, the sequence of the
molecules of this invention can be provided in a variety of media
to facilitate use thereof. Such media may provide a subset thereof
in a form that allows a skilled artisan to examine the sequences.
In a one embodiment, about 20, about 50, about 100, and about 200
or more of the polynucleotide and/or the polypeptide sequences of
the present invention can be recorded on computer readable media.
As used herein, "computer readable media" refers to any medium that
can be read and accessed directly by a computer. Such media
include, but are not limited to: magnetic storage media, such as
floppy discs, hard disc, storage medium, and magnetic tape; optical
storage media such as CD-ROM; electrical storage media such as RAM
and ROM; and hybrids of these categories such as magnetic/optical
storage media. A skilled artisan can readily appreciate how any of
the presently known computer readable media can be used to create a
manufacture comprising a computer readable medium having recorded
thereon a nucleotide sequence of the present invention.
[0088] As used herein, "recorded" refers to a process for storing
information on computer readable media. A skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable media to generate media comprising
the nucleotide sequence information of the present invention. A
variety of data storage structures are available to a skilled
artisan for creating a computer readable medium having recorded
thereon a nucleotide sequence of the present invention. The choice
of the data storage structure will generally be based on the means
chosen to access the stored information. In addition, a variety of
data processor programs and formats can be used to store the
nucleotide sequence information of the present invention on
computer readable media. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
skilled artisan can readily adapt any number of data processor
structuring formats (e.g., text file or database) in order to
obtain a computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0089] By providing one or more of polynucleotide or polypeptide
sequences of the present invention in a computer readable medium, a
skilled artisan can routinely access the sequence information for a
variety of purposes. The examples which follow demonstrate how
software which implements the BLAST and BLAZE search algorithms on
a Sybase system can be used to identify open reading frames (ORFs)
within the genome that contain homology to ORFs or polypeptides
from other organisms. Such ORFs are polypeptide encoding fragments
within the sequences of the present invention and are useful in
producing commercially important polypeptides such as enzymes used
in amino acid biosynthesis, metabolism, transcription, translation,
RNA processing, nucleic acid and a protein degradation, protein
modification, and DNA replication, restriction, modification,
recombination, and repair.
[0090] One embodiment of the invention provides systems,
particularly computer-based systems, which contain the sequence
information described herein. Such systems are designed to identify
commercially important fragments of the nucleic acid molecule of
the present invention. As used herein, "a computer-based system"
refers to the hardware, software, and memory used to analyze the
sequence information of the present invention. A skilled artisan
can readily appreciate that any one of the currently available
computer-based systems are suitable for use in the present
invention.
[0091] As indicated above, the computer-based systems of the
present invention comprise a database having stored therein a
polynucleotide sequence, polypeptide sequence, or both of the
present invention and the necessary hardware and software for
supporting and implementing a homology search. As used herein,
"database" refers to memory system that can store searchable
nucleotide sequence information. As used herein "query sequence" is
a polynucleotide sequence, or a polypeptide sequence, or a
polynucleotide sequence corresponding to a polypeptide sequence, or
a polypeptide sequence corresponding to a polynucleotide sequence,
that is used to query a collection of polynucleotide or polypeptide
sequences. As used herein, "homology search" refers to one or more
programs which are implemented on the computer-based system to
compare a query sequence, i.e., gene or peptide or a conserved
region (motif), with the sequence information stored within the
database. Homology searches are used to identify segments and/or
regions of the sequence of the present invention that match a
particular query sequence. A variety of known searching algorithms
are incorporated into commercially available software for
conducting homology searches of databases and computer readable
media comprising sequences of molecules of the present
invention.
[0092] Sequence length of a query sequence may be from about 10 to
about 100 or more amino acid residues or from about 20 to about 300
or more nucleotide residues. There are a variety of motifs known in
the art. Protein motifs include, but are not limited to, enzymatic
active sites and signal sequences. An amino acid query is converted
to all of the nucleic acid sequences that encode that amino acid
sequence by a software program, such as TBLASTN, which is then used
to search the database. Nucleic acid query sequences that are
motifs include, but are not limited to, promoter sequences, cis
elements, hairpin structures and inducible expression elements
(protein binding sequences).
[0093] One embodiment of the invention, provides an input device
for receiving a query sequence, a memory for storing sequences (the
query sequences of the present invention and sequences identified
using a homology search as described above), and an output device
for outputting the identified homologous sequences. A variety of
structural formats for the input and output presentations can be
used to input and output information in the computer-based systems
of the present invention. One format for an output presentation
ranks fragments of the sequence of the present invention by varying
degrees of homology to the query sequence. Such presentation
provides a skilled artisan with a ranking of sequences that contain
various amounts of the query sequence and identifies the degree of
homology contained in the identified fragment.
[0094] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
Example 1
[0095] A cDNA library is generated from Sorghum tissue. Tissue is
harvested and immediately frozen in liquid nitrogen. The harvested
tissue is stored at -80.degree. C. until preparation of total RNA.
The total RNA is purified using Trizol reagent from Invitrogen
Corporation (Invitrogen Corporation, Carlsbad, Calif., U.S.A.),
essentially as recommended by the manufacturer. Poly A+ RNA (mRNA)
is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Biotech, Oslow,
Norway).
[0096] Construction of plant cDNA libraries is well known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. cDNA
libraries are prepared using the Superscript.TM. Plasmid System for
cDNA synthesis and Plasmid Cloning (Invitrogen Corporation,
Carlsbad, Calif., U.S.A.), as described in the Superscript II cDNA
library synthesis protocol. The cDNA libraries are quality
controlled for a good insert:vector ratio.
[0097] The cDNA libraries are plated on LB agar containing the
appropriate antibiotics for selection and incubated at 37.degree.
for a sufficient time to allow the growth of individual colonies.
Single colonies are individually placed in each well of a 96-well
microtiter plates containing LB liquid including the selective
antibiotics. The plates are incubated overnight at approximately
37.degree. C. with gentle shaking to promote growth of the
cultures. The plasmid DNA is isolated from each clone using Qiaprep
plasmid isolation kits, using the conditions recommended by the
manufacturer (Qiagen Inc., Valencia, Calif. U.S.A.).
[0098] The template plasmid DNA clones are used for subsequent
sequencing. Sequences of recombinant polynucleotides may be
obtained by a number of sequencing techniques known in the art,
including fluorescence-based sequencing methodologies. These
methods have the detection, automation, and instrumentation
capability necessary for the analysis of large volumes of sequence
data. With these types of automated systems, fluorescent
dye-labeled sequence reaction products are detected and data
entered directly into the computer, producing a chromatogram that
is subsequently viewed, stored, and analyzed using the
corresponding software programs. These methods are known to those
of skill in the art and have been described and reviewed.
Example 2
[0099] The open reading frame in each recombinant polynucleotide
sequence is identified by a combination of predictive and homology
based methods. The longest open reading frame (ORF) is determined,
and the top BLAST match is identified by BLASTX against NCBI. The
top BLAST hit is then compared to the predicted ORF, with the BLAST
hit given precedence in the case of discrepancies.
[0100] Functions of polypeptides encoded by the polynucleotide
sequences of the present invention are determined using a
hierarchical classification tool, termed FunCAT, for Functional
Categories Annotation Tool. Most categories collected in FunCAT are
classified by function, although other criteria are used, for
example, cellular localization or temporal process. The assignment
of a functional category to a query sequence is based on BLASTX
sequence search results, which compare two protein sequences.
FunCAT assigns categories by iteratively scanning through all blast
hits, starting with the most significant match, and reporting the
first category assignment for each FunCAT source classification
scheme. In the present invention, function of a query polypeptide
is inferred from the function of a protein homolog where either (1)
hit_p<1e-30 or % identity>35% AND query_coverage>50% AND
hit_coverage>50%, or (2) hit_p<1e-8 AND query_coverage>70%
AND hit_coverage>70%.
[0101] Functional assignments from five public classification
schemes, GO_BP, GO_CC, GO_MF, KEGG, and EC, and one internal
Monsanto classification scheme, POI, are provided in Table 1 of
U.S. application Ser. No. 10/767,701. The column under the heading
"CAT_TYPE" indicates the source of the classification. GO_BP=Gene
Ontology Consortium-biological process; GO_CC=Gene Ontology
Consortium-cellular component; GO_MF=Gene Ontology
Consortium-molecular function; KEGG=KEGG functional hierarchy;
EC=Enzyme Classification from ENZYME data bank release 25.0;
POI=Pathways of Interest. The column under the heading "CAT_DESC"
provides the name of the subcategory into which the query sequence
was classified. The column under the heading "PRODUCT_HIT_DESC"
provides a description of the BLAST hit to the query sequences that
led to the specific classification. The column under the heading
"HIT_E" provides the e-value for the BLAST hit. It is noted that
the e-value in the HIT_E column may differ from the e-value based
on the top BLAST hit provided in the E_VALUE column since these
calculations were done on different days, and database size is an
element in E-value calculations. E-values obtained by BLASTing
against public databases, such as GenBank, will generally increase
over time for any given query/entry match.
[0102] Sequences useful for producing transgenic plants having
improved biological properties are identified from their FunCAT
annotations and are also provided in Table 1 of U.S. application
Ser. No. 10/767,701. A biological property of particular interest
is plant yield. Plant yield may be improved by alteration of a
variety of plant pathways, including those involving nitrogen,
carbohydrate, or phosphorus utilization and/or uptake. Plant yield
may also be improved by alteration of a plant's photosynthetic
capacity or by improving a plant's ability to tolerate a variety of
environmental stresses, including cold, heat, drought and osmotic
stresses. Other biological properties of interest that may be
improved using sequences of the present invention include pathogen
or pest tolerance, herbicide tolerance, disease resistance, growth
rate (for example by modification of cell cycle, by expression of
transcription factors, or expression of growth regulators), seed
oil and/or protein yield and quality, rate and control of
recombination, and lignin content.
[0103] Sequences of recombinant polynucleotides are provided herein
as SEQ ID NO: 1 through SEQ ID NO: 31,564 and sequences of
recombinant polypeptides are provided as SEQ ID NO: 31,565 through
SEQ ID NO: 63,128. Descriptions of each of these recombinant
polynucleotide and recombinant polypeptide sequences are provided
in Table 1 of U.S. application Ser. No. 10/767,701.
TABLE-US-00001 TABLE 1 (of U.S. Application No. 10/767,701) Column
Descriptions SEQ_NUM provides the SEQ ID NO for the listed
recombinant polynucleotide sequences. CONTIG_ID provides an
arbitrary sequence name taken from the name of the clone from which
the cDNA sequence was obtained. PROTEIN_NUM provides the SEQ ID NO
for the recombinant polypeptide sequence NCBI_GI provides the
GenBank ID number for the top BLAST hit for the sequence. The top
BLAST hit is indicated by the National Center for Biotechnology
Information GenBank Identifier number. NCBI_GI_DESCRIPTION refers
to the description of the GenBank top BLAST hit for the sequence.
E_VALUE provides the expectation value for the top BLAST match.
MATCH_LENGTH provides the length of the sequence which is aligned
in the top BLAST match TOP_HIT_PCT_IDENT refers to the percentage
of identically matched nucleotides (or residues) that exist along
the length of that portion of the sequences which is aligned in the
top BLAST match. CAT_TYPE indicates the classification scheme used
to classify the sequence. GO_BP = Gene Ontology
Consortium--biological process; GO_CC = Gene Ontology Consortium--
cellular component; GO_MF = Gene Ontology Consortium--molecular
function; KEGG = KEGG functional hierarchy (KEGG = Kyoto
Encyclopedia of Genes and Genomes); EC = Enzyme Classification from
ENZYME data bank release 25.0; POI = Pathways of Interest. CAT_DESC
provides the classification scheme subcategory to which the query
sequence was assigned. PRODUCT_CAT_DESC provides the FunCAT
annotation category to which the query sequence was assigned.
PRODUCT_HIT_DESC provides the description of the BLAST hit which
resulted in assignment of the sequence to the function category
provided in the cat_desc column. HIT_E provides the E value for the
BLAST hit in the hit_desc column. PCT_IDENT refers to the
percentage of identically matched nucleotides (orr residues) that
exist along the length of that portion of the sequences which is
aligned in the BLAST match provided in hit_desc. QRY_RANGE lists
the range of the query sequence aligned with the hit. HIT_RANGE
lists the range of the hit sequence aligned with the query.
QRY_CVRG provides the percent of query sequence length that matches
to the hit (NCBI) sequence in the BLAST match (% qry cvrg = (match
length / query total length) .times. 100). HIT_CVRG provides the
percent of hit sequence length that matches to the query sequence
in the match generated using BLAST (% hit cvrg = (match length /
hit total length) .times. 100).
[0104] All publications and patent applications cited herein are
hereby incorporated by reference in their entirely to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0105] 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 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120096599A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120096599A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References