U.S. patent application number 09/846903 was filed with the patent office on 2003-10-23 for plant regulatory sequences for selective control of gene expression.
Invention is credited to Conner, Timothy W., Dubois, Patrice, Malven, Marianne, Masucci, James D..
Application Number | 20030200565 09/846903 |
Document ID | / |
Family ID | 22745121 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030200565 |
Kind Code |
A1 |
Conner, Timothy W. ; et
al. |
October 23, 2003 |
Plant regulatory sequences for selective control of gene
expression
Abstract
Promoters from male reproductive tissues were isolated from corn
(Zea mays). These promoters can be used in plants to regulate
transcription of target genes including genes for control of
fertility, insect or pathogen tolerance, herbicide tolerance or any
gene of interest.
Inventors: |
Conner, Timothy W.;
(Wildwood, MO) ; Dubois, Patrice; (Richmond
Heights, MO) ; Malven, Marianne; (Ellisville, MO)
; Masucci, James D.; (Manchester, MO) |
Correspondence
Address: |
MONSANTO COMPANY
800 N. LINDBERGH BLVD.
ATTENTION: G.P. WUELLNER, IP PARALEGAL, (E2NA)
ST. LOUIS
MO
63167
US
|
Family ID: |
22745121 |
Appl. No.: |
09/846903 |
Filed: |
May 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60201255 |
May 1, 2000 |
|
|
|
Current U.S.
Class: |
800/287 ;
536/23.2; 536/23.6; 536/24.1; 800/274; 800/320.3 |
Current CPC
Class: |
C12N 15/8231 20130101;
C12N 15/8222 20130101 |
Class at
Publication: |
800/287 ;
536/23.2; 536/23.6; 800/320.3; 536/24.1; 800/274 |
International
Class: |
C12N 015/82; C07H
021/04; A01H 001/02; A01H 005/00 |
Claims
What is claimed is:
1. An isolated nucleic acid comprising a sequence selected from the
group consisting of SEQ ID NOS:79-98, or a fragment, region, or cis
element of said sequence thereof, said isolated nucleic acid being
capable of regulating transcription of an operably linked DNA
sequence.
2. The isolated nucleic acid of claim 1 wherein the isolated
nucleic acid is a promoter.
3. The isolated nucleic acid of claim 2 wherein the promoter is a
hybrid promoter.
4. The isolated nucleic acid of claim 3 wherein said isolated
nucleic acid confers enhanced expression of operably linked genes
in male reproductive tissues.
5. The isolated nucleic acid of claim 4 wherein said isolated
nucleic acid confers enhanced expression of operably linked genes
in anthers.
6. The isolated nucleic acid of claim 5 wherein said isolated
nucleic acid confers enhanced expression of operably linked genes
in wheat anthers.
7. The isolated nucleic acid of claim 4 further comprising a
minimal promoter.
8. The isolated nucleic acid of claim 7 wherein the minimal
promoter is selected from the group consisting of a minimal CaMV
and a rice actin promoter.
9. The isolated nucleic acid of claim 8 wherein the minimal
promoter is a minimal CaMV 35S promoter.
10. A promoter comprising a nucleic acid sequence selected from the
group consisting of SEQ ID NOS: 79-98 and fragments thereof.
11. The promoter of claim 10 wherein said promoter confers enhanced
expression of operably linked genes in male reproductive
tissues.
12. The promoter of claim 11 wherein said promoter confers enhanced
expression of operably linked genes in anthers.
13. The promoter of claim 12 wherein said promoter confers enhanced
expression of operably linked genes in wheat anthers.
14. A cell comprising a DNA construct comprising an isolated
nucleic acid sequence selected from the group consisting of SEQ ID
NOS:79-98 or a fragment, region, or cis element of said sequence
thereof, and operably linked to said nucleic acid sequence, a
transcribable DNA sequence and a 3' non-translated region.
15. A transgenic plant comprising a DNA construct comprising an
isolated nucleic acid sequence selected from the group consisting
of SEQ ID NOS:79-98 or a fragment, region, or cis element of said
sequence thereof, and operably linked to said nucleic acid
sequence, a transcribable DNA sequence and a 3' non-translated
region.
16. A method of regulating transcription of a DNA sequence
comprising operably linking the DNA sequence to a promoter
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOS:79-98.
17. The method of claim 16 comprising operably linking the DNA
sequence to a hybrid promoter comprising the nucleic acid sequence
selected from the group consisting of SEQ ID NOS:79-98.
18. The method of claim 16 wherein operably linking the nucleic
acid sequence selected from the group consisting of SEQ ID
NOS:79-98 or fragment thereof to the promoter confers enhanced
expression of operably linked genes in male reproductive
tissues.
19. The method of claim 18 wherein said male reproductive tissues
comprise monocot or dicot male reproductive tissues.
20. The method of claim 19 wherein said male reproductive tissues
comprise anthers.
21. The method of claim 20 wherein said male reproductive tissues
comprise wheat anthers.
22. The method of claim 16 comprising operably linking a minimal
promoter to the nucleic acid sequences selected from the group
consisting of SEQ ID NOS:79-98 or fragment, region, or cis element
thereof.
23. A method of making a transgenic plant comprising introducing
into a cell of a plant a DNA construct comprising: (i) a promoter
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOS:79-98 or a fragment, region or cis element
thereof, and, operably linked to the promoter, (ii) a transcribable
DNA sequence and (iii) a 3' non-translated region.
24. A method of isolating at least two 5' regulatory sequences that
confer enhanced expression of operably linked genes in male
reproductive tissues from a plant comprising: (i) evaluating a
collection of nucleic acid sequences of ESTs derived from at least
one cDNA library prepared from a plant cell type of interest; (ii)
comparing EST sequences from at least one target plant cDNA library
and at least one non-target cDNA libraries of ESTs from a different
plant cell type; (iii) subtracting common EST sequences found in
both target and non-target libraries; (iv) designing gene specific
primers from the remaining EST sequences after said subtraction;
and (v) isolating the corresponding 5' flanking and regulatory
sequences from a genomic library prepared from the target plant
comprising the use of said primers.
25. The method of claim 24 wherein said male reproductive tissues
are from monocot or dicot plants.
26. The method of claim 25 wherein said male reproductive tissues
comprise anthers.
27. The method of claim 26 wherein said male reproductive tissues
comprise wheat anthers.
Description
RELATED APPLICATION DATA
[0001] This application claims priority to U.S. Provisional
Application 60/201,255, filed on May 1, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates to the isolation and use of
nucleic acid molecules for control of gene expression in plants,
specifically novel plant promoters.
BACKGROUND OF THE INVENTION
[0003] One of the goals of plant genetic engineering is to produce
plants with agronomically important characteristics or traits.
Recent advances in genetic engineering have provided the requisite
tools to transform plants to contain and express foreign genes
(Kahl et al. , 1995, World Journal of Microbiology and
Biotechnology 11:449-460). Particularly desirable traits or
qualities of interest for plant genetic engineering would include,
but are not limited to, resistance to insects, fungal diseases, and
other pests and disease-causing agents, tolerances to herbicides,
enhanced stability, yield, or shelf-life, environmental tolerances,
and nutritional enhancements. The technological advances in plant
transformation and regeneration have enabled researchers to take
pieces of DNA, such as a gene or genes from a heterologous source,
or a native source, but modified to have different or improved
qualities, and incorporate the exogenous DNA into the plant's
genome. The gene or gene(s) can then be expressed in the plant cell
to exhibit the added characteristic(s) or trait(s). In one
approach, expression of a novel gene that is not normally expressed
in a particular plant or plant tissue may confer a desired
phenotypic effect. In another approach, transcription of a gene or
part of a gene in an antisense orientation may produce a desirable
effect by preventing or inhibiting expression of an endogenous
gene.
[0004] Isolated plant promoters are useful for modifying plants
through genetic engineering to have desired phenotypic
characteristics. In order to produce such a transgenic plant, a
vector that includes a heterologous gene sequence that confers the
desired phenotype when expressed in the plant is introduced into
the plant cell. The vector also includes a plant promoter that is
operably linked to the heterologous gene sequence, often a promoter
not normally associated with the heterologous gene. The vector is
then introduced into a plant cell to produce a transformed plant
cell, and the transformed plant cell is regenerated into a
transgenic plant. The promoter controls expression of the
introduced DNA sequence to which the promoter is operably linked
and thus affects the desired characteristic conferred by the DNA
sequence.
[0005] Because the promoter is a regulatory element that plays an
integral part in the overall expression of a gene or gene(s), it
would be advantageous to have a variety of promoters to tailor gene
expression such that a gene or gene(s) is transcribed efficiently
at the right time during plant growth and development, in the
optimal location in the plant, and in the amount necessary to
produce the desired effect. In one case, for example, constitutive
expression of a gene product may be beneficial in one location of
the plant, but less beneficial in another part of the plant. In
other cases, it may be beneficial to have a gene product produced
at a certain developmental stage of the plant, or in response to
certain environmental or chemical stimuli. The commercial
development of genetically improved germplasm has also advanced to
the stage of introducing multiple traits into crop plants, often
referred to as a gene stacking approach. In this approach, multiple
genes conferring different characteristics of interest can be
introduced into a plant. It is important when introducing multiple
genes into a plant, that each gene is modulated or controlled for
optimal expression and that the regulatory elements are diverse, to
reduce the potential of gene silencing that can be caused by
recombination of homologous sequences. In light of these and other
considerations, it is apparent that optimal control of gene
expression and regulatory element diversity are important in plant
biotechnology.
[0006] The proper regulatory sequences must be present and in the
proper location with respect to the DNA sequence of interest for
the newly inserted DNA to be transcribed and thereby, if desired,
translated into a protein in the plant cell. These regulatory
sequences include, but are not limited to, a promoter, a 5'
untranslated leader, and a 3' polyadenylation sequence. The ability
to select the tissues in which to transcribe such foreign DNA and
the time during plant growth in which to obtain transcription of
such foreign DNA is also possible through the choice of appropriate
promoter sequences that control transcription of these genes.
[0007] A variety of different types or classes of promoters can be
used for plant genetic engineering. Promoters can be classified on
the basis of range or tissue specificity. For example, promoters
referred to as constitutive promoters are capable of transcribing
operatively linked DNA sequences efficiently and expressing said
DNA sequences in multiple tissues. Tissue-enhanced or
tissue-specific promoters can be found upstream and operatively
linked to DNA sequences normally transcribed in higher levels in
certain plant tissues or specifically in certain plant tissues.
Other classes of promoters would include, but are not limited to,
inducible promoters that can be triggered by external stimuli such
as chemical agents, developmental stimuli, or environmental
stimuli. Thus, the different types of promoters desired can be
obtained by isolating the regulatory regions of DNA sequences that
are transcribed and expressed in a constitutive, tissue-enhanced,
or inducible manner.
[0008] The technological advances of high-throughput sequencing and
bioinformatics has provided additional molecular tools for promoter
discovery. Particular target plant cells, tissues, or organs at a
specific stage of development, or under particular chemical,
environmental, or physiological conditions can be used as source
material to isolate the mRNA and construct cDNA libraries. The cDNA
libraries are quickly sequenced, and the expressed sequences can be
catalogued electronically. Using sequence analysis software,
thousands of sequences can be analyzed in a short period, and
sequences from selected cDNA libraries can be compared. The
combination of laboratory and computer-based subtraction methods
allows researchers to scan and compare cDNA libraries and identify
sequences with a desired expression profile. For example, sequences
expressed preferentially in one tissue can be identified by
comparing a cDNA library from one tissue to cDNA libraries of other
tissues and electronically "subtracting" common sequences to find
sequences only expressed in the target tissue of interest. The
tissue enhanced sequence can then be used as a probe or primer to
clone the corresponding full-length cDNA. A genomic library of the
target plant can then be used to isolate the corresponding gene and
the associated regulatory elements, including but not limited to
promoter sequences.
[0009] Multiple genes that have a desired expression profile such
as in male reproductive tissues can be isolated by selectively
comparing cDNA libraries of target tissues of interest with
non-target or background cDNA libraries to find the 5' regulatory
regions associated with the expressed sequences in those target
libraries. The promoter sequences can be isolated from the genomic
DNA flanking the desired genes. The isolated promoter sequences can
be used for selectively modulating expression of any operatively
linked gene and provide additional regulatory element diversity in
a plant expression vector in gene stacking approaches.
SUMMARY OF THE INVENTION
[0010] The present invention provides isolated plant promoter
sequences that comprise nucleic acid regions located upstream of
the 5' end of plant DNA structural coding sequences that are
transcribed in male reproductive tissues. The plant promoter
sequences are capable of modulating or initiating transcription of
DNA sequences to which they are operably linked.
[0011] The present invention provides nucleic acid sequences
comprising regulatory sequences as shown in SEQ ID NOS: 79-98 that
are located upstream of the 5' end of plant DNA structural coding
sequences and transcribed in male reproductive tissues.
[0012] In one aspect, the present invention provides nucleic acid
sequences comprising a sequence selected from the group consisting
of SEQ ID NOS: 79-98 or any fragments or regions of the sequence or
cis elements of the sequence that are capable of regulating
transcription of operably linked DNA sequences.
[0013] The present invention also provides nucleic acid sequences
comprising a sequence selected from the group consisting of SEQ ID
NOS: 79-98 that are promoters.
[0014] Another aspect of the present invention relates to the use
of one or more cis elements, or fragments thereof of the disclosed
5' promoter sequences that can be combined to create novel
promoters or used in a novel combination with another heterologous
regulatory sequence to create a chimeric promoter capable of
modulating transcription of an operably linked DNA sequence.
[0015] Hence, the present invention relates to the use of nucleic
acid sequences disclosed in SEQ ID NOS: 79-98 or any fragment,
region, or cis element of the disclosed sequences that are capable
of regulating transcription of a DNA sequence when operably linked
to the DNA sequence. Therefore, the invention not only encompasses
the sequences as disclosed in SEQ ID NOS: 79-98, but also includes
any truncated or deletion derivatives, or fragments or regions
thereof that are capable of functioning independently as a promoter
including cis elements that are capable of functioning as
regulatory sequences in conjuction with one or more regulatory
sequences when operably linked to a transcribable sequence.
[0016] The present invention thus encompasses a novel promoter or
chimeric or hybrid promoter comprising a nucleic acid of SEQ ID
NOS: 79-98. The chimeric or hybrid promoters can consist of any
length fragments, regions, or cis elements of the disclosed
sequences of SEQ ID NOS: 79-98 combined with any other
transcriptionally active minimal or full-length promoter. For
example, a promoter sequence selected from SEQ ID NOS: 79-98 may be
combined with a CaMV 35S or other promoter to construct a novel
chimeric promoter. A minimal promoter can also be used in
combination with the nucleic acid sequences of the present
invention. A novel promoter also comprises any promoter constructed
by engineering the nucleic acid sequences disclosed in SEQ ID NOS:
79-98 or any fragment, region, or cis element of the disclosed
sequences in any manner sufficient to transcribe an operably linked
DNA sequence.
[0017] Another aspect of the present invention relates to the
ability of the promoter sequences of SEQ ID NOS: 79-98, or
fragments, regions, or cis elements thereof to regulate
transcription of operably linked transcribable sequences in male
reproductive tissues. Fragments, regions, or cis elements of SEQ ID
NOS: 79-98 that are capable of regulating transcription of operably
linked DNA sequences in certain tissues may be isolated from the
disclosed nucleic acid sequences of SEQ ID NOS: 79-98 and used to
engineer novel promoters.
[0018] The present invention also encompasses DNA constructs
comprising the disclosed sequences as shown in SEQ ID NOS: 79-98 or
any fragments, regions, or cis elements thereof, including novel
promoters generated using the disclosed sequences or any fragment,
region, or cis element of the disclosed sequences.
[0019] The present invention also includes any transgenic cells and
plants containing the DNA disclosed in the sequences as shown in
SEQ ID NOS: 79-98, or any fragments, regions, or cis elements
thereof.
[0020] The present invention also provides a method of regulating
transcription of a DNA sequence comprising operably linking the DNA
sequence to any promoter comprising a nucleic acid comprising all
or any fragment, region or cis element of a sequence selected from
the group consisting of SEQ ID NOS: 79-98.
[0021] In another embodiment the present invention provides a
method of regulating expression of DNA sequences in male
reproductive tissues by operably linking a sequence selected from
the group consisting of SEQ ID NOS: 79-98, or any fragment, region,
or cis element of the disclosed sequences to any transcribable DNA
sequence. The fragments, regions, or cis elements of the disclosed
promoters as shown in SEQ ID NOS: 79-98 can be engineered and used
independently in novel combinations including multimers, or
truncated derivatives and the novel promoters can be operably
linked with a transcribable DNA sequence. Alternatively the
disclosed fragments, regions, or cis elements of the disclosed
sequences can be used in combination with a heterologous promoter
including a minimal promoter to create a novel chimeric or hybrid
promoter and the novel chimeric promoter can be operably linked to
a transcribable DNA sequence.
[0022] The present invention also provides a method of making a
transgenic plant by introducing into a cell of a plant a DNA
construct comprising: (i) a promoter comprising a nucleic acid
comprising a sequence selected from the group consisting of SEQ ID
NOS: 79-98, or fragment, region, or cis element thereof, and
operably linked to the promoter, (ii) a transcribable DNA sequence
and (iii) a 3' untranslated region.
[0023] The present invention also provides a method of isolating at
least one 5' regulatory sequence of a desired expression profile
from a target plant of interest by evaluating a collection of
nucleic acid sequences of ESTs derived from one or more cDNA
libraries prepared from a plant cell type of interest, comparing
EST sequences from at least one target plant cDNA library and one
or more non-target cDNA libraries of ESTs from a different plant
cell type, subtracting common EST sequences found in both target
and non-target libraries, designing gene-specific primers from the
remaining ESTs after the subtraction that are representative of the
targeted expressed sequences, and isolating the corresponding 5'
flanking and regulatory sequences, that includes promoter sequences
from a genomic library prepared from the target plant using the
gene specific primers.
[0024] The foregoing and other aspects of the invention will become
more apparent from the following detailed description and
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a plasmid map of pMON19469.
[0026] FIG. 2 is a plasmid map of pMON51850.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0027] Seq ID NOs: 1-3 are adaptor sequences.
[0028] Seq ID NOs: 4-78 are fully synthesized primers derived from
known Zea mays sequences.
[0029] Seq ID NOs: 79-98 are promoter sequences isolated from Zea
mays.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Definitions and Methods
[0031] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant art.
The nomenclature for DNA bases as set forth at 37 CFR .sctn.1.822
is used. The standard one- and three-letter nomenclature for amino
acid residues is used.
[0032] "Nucleic acid (sequence)" or "polynucleotide (sequence)"
refers to single- or double-stranded DNA or RNA of genomic or
synthetic origin, i.e., a polymer of deoxyribonucleotide or
ribonucleotide bases, respectively, read from the 5' (upstream) end
to the 3' (downstream) end. The nucleic acid can represent the
sense or complementary (antisense) strand.
[0033] "Native" refers to a naturally occurring ("wild-type")
nucleic acid sequence.
[0034] "Heterologous" sequence refers to a sequence that originates
from a foreign source or species or, if from the same source, is
modified from its original form.
[0035] An "isolated" nucleic acid sequence is substantially
separated or purified away from other nucleic acid sequences that
the nucleic acid is normally associated with in the cell of the
organism in which the nucleic acid naturally occurs, i.e., other
chromosomal or extrachromosomal DNA. The term embraces nucleic
acids that are biochemically purified so as to substantially remove
contaminating nucleic acids and other cellular components. The term
also embraces recombinant nucleic acids and chemically synthesized
nucleic acids.
[0036] The term "substantially purified", as used herein, refers to
a molecule separated from substantially all other molecules
normally associated with it in its native state. More preferably, a
substantially purified molecule is the predominant species present
in a preparation. A substantially purified molecule may be greater
than 60% free, preferably 75% free, more preferably 90% free from
the other molecules (exclusive of solvent) present in the natural
mixture. The term "substantially purified" is not intended to
encompass molecules present in their native state.
[0037] A first nucleic acid sequence displays "substantial
identity" to a reference nucleic acid sequence if, when optimally
aligned (with appropriate nucleotide insertions or deletions
totaling less than 20 percent of the reference sequence over the
window of comparison) with the other nucleic acid (or its
complementary strand), there is at least about 75% nucleotide
sequence identity, preferably at least about 80% identity, more
preferably at least about 85% identity, and most preferably at
least about 90% identity over a comparison window of at least 20
nucleotide positions, preferably at least 50 nucleotide positions,
more preferably at least 100 nucleotide positions, and most
preferably over the entire length of the first nucleic acid.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith and Waterman
(Adv. Appl. Math. 2: 482, 1981); by the homology alignment
algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970); by
the search for similarity method of Pearson and Lipman (Proc. Natl.
Acad. Sci. USA 85:2444, 1988); preferably by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA) in the Wisconsin Genetics Software Package Release 7.0
(Genetics Computer Group, 575 Science Dr., Madison, Wis.). The
reference nucleic acid may be a full-length molecule or a portion
of a longer molecule. Alternatively, two nucleic acids have
substantial identity if one hybridizes to the other under stringent
conditions, as defined below.
[0038] A first nucleic acid sequence is "operably linked" with a
second nucleic acid sequence when the sequences are so arranged
that the first nucleic acid sequence affects the function of the
second nucleic acid sequence. Preferably, the two sequences are
part of a single contiguous nucleic acid molecule and more
preferably are adjacent. For example, a promoter is operably linked
to a gene if the promoter regulates or mediates transcription of
the gene in a cell.
[0039] A "recombinant" nucleic acid is made by an artificial
combination of two otherwise separated segments of sequence, e.g.,
by chemical synthesis or by the manipulation of isolated segments
of nucleic acids by genetic engineering techniques. Techniques for
nucleic-acid manipulation are well-known (see, e.g., Molecular
Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et
al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989, Sambrook et al., 1989; Current Protocols in Molecular
Biology, ed. Ausubel et al., Greene Publishing and
Wiley-Interscience, New York, 1992, with periodic updates, Ausubel
et al., 1992; and PCR Protocols: A Guide to Methods and
Applications, Academic Press: San Diego, Innis et al., 1990).
Methods for chemical synthesis of nucleic acids are discussed, for
example, in Beaucage and Carruthers (Tetra. Letts. 22:1859-1862,
1981), and Matteucci et al. (J. Am. Chem. Soc. 103:3185, 1981).
Chemical synthesis of nucleic acids can be performed, for example,
on commercial automated oligonucleotide synthesizers.
[0040] A "synthetic nucleic acid sequence" can be designed and
chemically synthesized for enhanced expression in particular host
cells and for the purposes of cloning into appropriate vectors.
Host cells often display a preferred pattern of codon usage (Murray
et al., 1989). Synthetic DNAs designed to enhance expression in a
particular host should therefore reflect the pattern of codon usage
in the host cell. Computer programs are available for these
purposes including but not limited to the "BestFit" or "Gap"
programs of the Sequence Analysis Software Package, Genetics
Computer Group, Inc. (University of Wisconsin Biotechnology Center,
Madison, Wis. 53711).
[0041] "Amplification" of nucleic acids or "nucleic acid
reproduction" refers to the production of additional copies of a
nucleic acid sequence and is carried out using polymerase chain
reaction (PCR) technologies. A variety of amplification methods are
known in the art and are described, inter alia, in U.S. Pat. Nos.
4,683,195 and 4,683,202 and by Innis et al. (PCR Protocols: A Guide
to Methods and Applications, Academic Press, San Diego, 1990). In
PCR, a primer refers to a short oligonucleotide of defined sequence
that is annealed to a DNA template to initiate the polymerase chain
reaction.
[0042] "Transformed", "transfected", or "transgenic" refers to a
cell, tissue, organ, or organism into which has been introduced a
foreign nucleic acid, such as a recombinant vector. Preferably, the
introduced nucleic acid is integrated into the genomic DNA of the
recipient cell, tissue, organ or organism such that the introduced
nucleic acid is inherited by subsequent progeny. A "transgenic" or
"transformed" cell or organism also includes progeny of the cell or
organism and progeny produced from a breeding program employing
such a "transgenic" plant as a parent in a cross and exhibiting an
altered phenotype resulting from the presence of a recombinant
construct or vector.
[0043] The term "gene" refers to chromosomal DNA, plasmid DNA,
cDNA, synthetic DNA, or other DNA that encodes a peptide,
polypeptide, protein, or RNA molecule, and regions flanking the
coding sequence involved in the regulation of expression. Some
genes can be transcribed into mRNA and translated into polypeptides
(structural genes); other genes can be transcribed into RNA (e.g.,
rRNA, tRNA); and other types of genes function as regulators of
expression (regulator genes).
[0044] "Expression" of a gene refers to the transcription of a gene
to produce the corresponding mRNA and translation of this mRNA to
produce the corresponding gene product, i.e., a peptide,
polypeptide, or protein. Gene expression is controlled or modulated
by regulatory elements including 5' regulatory elements such as
promoters.
[0045] "Genetic component" refers to any nucleic acid sequence or
genetic element that may also be a component or part of an
expression vector. Examples of genetic components include, but are
not limited to, promoter regions, 5' untranslated leaders, introns,
genes, 3' untranslated regions, and other regulatory sequences or
sequences that affect transcription or translation of one or more
nucleic acid sequences.
[0046] The terms "recombinant DNA construct", "recombinant vector",
"expression vector" or "expression cassette" refer to any agent
such as a plasmid, cosmid, virus, BAC (bacterial artificial
chromosome), autonomously replicating sequence, phage, or linear or
circular single-stranded or double-stranded DNA or RNA nucleotide
sequence, derived from any source, capable of genomic integration
or autonomous replication, comprising a DNA molecule in which one
or more DNA sequences have been linked in a functionally operative
manner.
[0047] "Complementary" refers to the natural association of nucleic
acid sequences by base-pairing (A-G-T pairs with the complementary
sequence A-C-T). Complementarity between two single-stranded
molecules may be partial, if only some of the nucleic acids pair
are complementary, or complete, if all bases pair are
complementary. The degree of complementarity affects the efficiency
and strength of hybridization and amplification reactions.
[0048] "Homology" refers to the level of similarity between nucleic
acid or amino acid sequences in terms of percent nucleotide or
amino acid positional identity, respectively, i.e., sequence
similarity or identity. Homology also refers to the concept of
similar functional properties among different nucleic acids or
proteins.
[0049] "ESTs" or Expressed Sequence Tags are short sequences of
randomly selected clones from a cDNA (or complementary DNA) library
that are representative of the cDNA inserts of these randomly
selected clones (McCombie et al., Nature Genetics, 1:124, 1992;
Kurata et al., Nature Genetics, 8: 365,1994; Okubo et al., Nature
Genetics, 2: 173, 1992).
[0050] The term "electronic Northern" refers to a computer-based
sequence analysis that allows sequences from multiple cDNA
libraries to be compared electronically based on parameters the
researcher identifies including abundance in EST populations in
multiple cDNA libraries, or exclusively to EST sets from one or
combinations of libraries.
[0051] "Subsetting" refers to a method of comparing nucleic acid
sequences from different or multiple sources that can be used to
assess the expression profile of the nucleic acid sequences that
reflects gene transcription activity and message stability in a
particular tissue, at a particular time, or under particular
conditions.
[0052] "Promoter" refers to a nucleic acid sequence located
upstream or 5' to a translational start codon of an open reading
frame (or protein-coding region) of a gene and that is involved in
recognition and binding of RNA polymerase II and other proteins
(trans-acting transcription factors) to initiate transcription. A
"plant promoter" is a native or non-native promoter that is
functional in plant cells. Constitutive promoters are functional in
most or all tissues of a plant throughout plant development.
Tissue-, organ- or cell-specific promoters are expressed only or
predominantly in a particular tissue, organ, or cell type,
respectively. Rather than being expressed "specifically" in a given
tissue, organ, or cell type, a promoter may display "enhanced"
expression, i.e., a higher level of expression, in one part (e.g.,
cell type, tissue, or organ) of the plant compared to other parts
of the plant. Temporally regulated promoters are functional only or
predominantly during certain periods of plant development or at
certain times of day, as in the case of genes associated with
circadian rhythm, for example. Inducible promoters selectively
express an operably linked DNA sequence in response to the presence
of an endogenous or exogenous stimulus, for example, by chemical
compounds (chemical inducers) or in response to environmental,
hormonal, chemical, or developmental signals. Inducible or
regulated promoters include, for example, promoters regulated by
light, heat, stress, flooding or drought, phytohormones, wounding,
or chemicals such as ethanol, jasmonate, salicylic acid, or
safeners.
[0053] Any plant promoter can be used as a 5' regulatory sequence
for modulating expression of a particular gene or genes. One
preferred promoter would be a plant RNA polymerase II promoter.
Plant RNA polymerase II promoters, like those of other higher
eukaryotes, have complex structures and are comprised of several
distinct elements. One such element is the TATA box or
Goldberg-Hogness box, which is required for correct expression of
eukaryotic genes in vitro and accurate, efficient initiation of
transcription in vivo. The TATA box is typically positioned at
approximately -25 to -35, that is, at 25 to 35 basepairs (bp)
upstream (5') of the transcription initiation site, or cap site,
which is defined as position +1 (Breathnach and Chambon, Ann. Rev.
Biochem. 50:349-383, 1981; Messing et al., In: Genetic Engineering
of Plants, Kosuge et al., eds., pp. 211-227, 1983). Another common
element, the CCAAT box, is located between -70 and -100 bp. In
plants, the CCAAT box may have a different consensus sequence than
the functionally analogous sequence of mammalian promoters (the
plant analogue has been termed the "AGGA box" to differentiate it
from its animal counterpart; Messing et al., In: Genetic
Engineering of Plants, Kosuge et al., eds., pp. 211-227, 1983). In
addition, virtually all promoters include additional upstream
activating sequences or enhancers (Benoist and Chambon, Nature 290:
304-310, 1981; Gruss et al., Proc. Natl. Acad. Sci. USA 78:943-947,
1981; and Khoury and Gruss, Cell 27:313-314, 1983) extending from
around -100 bp to -1,000 bp or more upstream of the transcription
initiation site. Enhancers have also been found 3' to the
transcriptional start site.
[0054] When fused to heterologous DNA sequences, such promoters
typically cause the fused sequence to be transcribed in a manner
that is similar to that of the gene sequence that the promoter is
normally associated with. Promoter fragments that include
regulatory sequences can be added (for example, fused to the 5' end
of, or inserted within, an active promoter having its own partial
or complete regulatory sequences (Fluhr et al., Science
232:1106-1112, 1986; Ellis et al., EMBO J. 6:11-16, 1987;
Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990,
1987; Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988; Comai et
al., Plant Mol. Biol. 15:373-381, 1991). Alternatively,
heterologous regulatory sequences can be added to the 5' upstream
region of an inactive, truncated promoter, e.g., a promoter
including only the core TATA and, sometimes, the CCAAT elements
(Fluhr et al., Science 232:1106-1112, 1986; Strittmatter and Chua,
Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987; Aryan et al., Mol.
Gen. Genet. 225:65-71, 1991).
[0055] Promoters are typically comprised of multiple distinct
"cis-acting transcriptional regulatory elements," or simply
"cis-elements," each of which appears to confer a different aspect
of the overall control of gene expression (Strittmatter and Chua,
Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987; Ellis et al., EMBO J.
6:11-16, 1987; Benfey et al., EMBO J. 9:1677-1684, 1990). Cis
elements bind trans-acting protein factors that regulate
transcription. Some cis elements bind more than one factor, and
trans-acting transcription factors may interact with different
affinities with more than one cis element (Johnson and McKnight,
Ann. Rev. Biochem. 58:799-839, 1989). Plant transcription factors,
corresponding cis elements, and analysis of their interaction are
discussed, for example, in Martin (Curr. Opinions Biotech.
7:130-138, 1996), Murai (Methods in Plant Biochemistry and
Molecular Biology, Dashek, ed., CRC Press, 1997, pp. 397-422), and
Maliga et al. (Methods in Plant Molecular Biology, Cold Spring
Harbor Press, 1995, pp. 233-300). The promoter sequences of the
present invention can contain "cis elements" that can confer or
modulate gene expression.
[0056] Cis elements can be identified by a number of techniques,
including deletion analysis, i.e., deleting one or more nucleotides
from the 5' end or internal to a promoter; DNA binding protein
analysis using Dnase I footprinting; methylation interference;
electrophoresis mobility-shift assays, in vivo genomic footprinting
by ligation-mediated PCR; and other conventional assays; or by
sequence similarity with known cis element motifs by conventional
sequence comparison methods. The fine structure of a cis element
can be further studied by mutagenesis (or substitution) of one or
more nucleotides or by other conventional methods (see for example,
Methods in Plant Biochemistry and Molecular Biology, Dashek, ed.,
CRC Press, 1997, pp. 397-422; and Methods in Plant Molecular
Biology, Maliga et al., eds., Cold Spring Harbor Press, 1995, pp.
233-300).
[0057] Cis elements can be obtained by chemical synthesis or by
cloning from promoters that include such elements, and they can be
synthesized with additional flanking sequences that contain useful
restriction enzyme sites to facilitate subsequent manipulation. In
one embodiment, the promoters are comprised of multiple distinct
"cis-acting transcriptional regulatory elements," or simply
"cis-elements," each of which appears to confer a different aspect
of the overall control of gene expression (Strittmatter and Chua,
Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987; Ellis et al., EMBO J.
6:11-16, 1987; Benfey et al., EMBO J. 9:1677-1684, 1990). In a
preferred embodiment, sequence regions comprising "cis elements" of
the nucleic acid sequences of SEQ ID NOS: 79-98 are identified
using computer programs designed specifically to identify cis
elements, or domains or motifs within sequences.
[0058] The present invention includes cis elements of SEQ ID NOS:
79-98, or homologues of cis elements known to affect gene
regulation that show homology with the nucleic acid sequences of
the present invention. A number of such elements are known in the
literature, such as elements that are regulated by numerous factors
such as light, heat, or stress; elements that are regulated or
induced by pathogens or chemicals, and the like. Such elements may
either positively or negatively regulated gene expression,
depending on the conditions. Examples of cis elements would
include, but are not limited to, oxygen responsive elements (Cowen
et al., J. Biol. Chem. 268(36):26904, 1993), light regulatory
elements (see for example, Bruce and Quaill, Plant Cell 2(11):
1081, 1990; and Bruce et al., EMBO J. 10:3015, 1991), a cis element
reponsive to methyl jasmonate treatment (Beaudoin and Rothstein,
Plant Mol. Biol. 33:835, 1997), salicylic acid responsive elements
(Strange et al., Plant J. 11:1315, 1997), heat shock response
elements (Pelham et al., Trends Genet. 1:31, 1985), elements
responsive to wounding and abiotic stress (Loace et al., Proc.
Natl. Acad. Sci. U.S.A. 89:9230, 1992; Mhiri et al., Plant Mol.
Biol. 33:257, 1997), low temperature elements (Baker et al., Plant
Mol. Biol. 24:701, 1994; Jiang et al., Plant Mol. Biol. 30:679,
1996; Nordin et al., Plant Mol. Biol. 21:641, 1993; Zhou et al., J.
Biol. Chem. 267:23515, 1992), and drought responsive elements,
(Yamaguchi et al., Plant Cell 6:251-264, 1994; Wang et al., Plant
Mol. Biol. 28:605, 1995; Bray, Trends in Plant Science 2:48,
1997).
[0059] The present invention therefore encompasses fragments or cis
elements of the disclosed nucleic acid molecules, and such nucleic
acid fragments can include any region of the disclosed sequences.
The promoter regions or partial promoter regions of the present
invention as shown in SEQ ID NOS: 79-98 can contain one or more
regulatory elements including but not limited to cis elements or
domains that are capable of regulating expression of operably
linked DNA sequences, preferably in male reproductive tissues.
[0060] Plant promoters can include promoters produced through the
manipulation of known promoters to produce synthetic, chimeric, or
hybrid promoters. Such promoters can also combine cis elements from
one or more promoters, for example, by adding a heterologous
regulatory sequence to an active promoter with its own partial or
complete regulatory sequences (Ellis et al., EMBO J. 6:11-16, 1987;
Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990,
1987; Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988; Comai et
al., Plant. Mol. Biol. 15:373-381, 1991). Chimeric promoters have
also been developed by adding a heterologous regulatory sequence to
the 5' upstream region of an inactive, truncated promoter, i.e., a
promoter that includes only the core TATA and, optionally, the
CCAAT elements (Fluhr et al., Science 232:1106-1112, 1986;
Strittmatter and Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990,
1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991).
[0061] The design, construction, and use of chimeric or hybrid
promoters comprising one or more of cis elements of SEQ ID NOS:
79-98 for modulating or regulating the expression of operably
linked nucleic acid sequences is also encompassed by the present
invention.
[0062] The promoter sequences, fragments, regions or cis elements
thereof of SEQ ID NOS: 79-98 are capable of transcribing operably
linked DNA sequences in male reproductive tissues and therefore can
selectively regulate expression of genes in these tissues.
[0063] The promoter sequences of the present invention are useful
for regulating gene expression in male reproductive tissues such as
tassels, anthers, and pollen. For a number of agronomic traits,
transcription of a gene or genes of interest is desirable in
multiple tissues in order to confer the desired characteristic(s).
The availability of suitable promoters that regulate transcription
of operably linked genes in selected target tissues of interest is
important because it may not be desirable to have expression of a
gene in every tissue, but only in certain tissues. For example, if
one desires to selectively express a target gene for controlling
fertility in corn, it would be advantageous to have a promoter that
confers enhanced expression in reproductive tissues. The promoter
sequences of the present invention are capable of regulating
operably linked DNA sequence particularly in male reproductive
tissues and have utility for regulating transcription of any target
gene including, but not limited to, genes for control of fertility,
insect or pathogen tolerance, herbicide tolerance or any gene of
interest. Consequently, it is important to have a wide variety of
choices of 5' regulatory elements for any plant biotechnology
strategy.
[0064] The advent of genomics, which comprises molecular and
bioinformatics techniques, has resulted in rapid sequencing and
analyses of a large number of DNA samples from a vast number of
targets, including but not limited to plant species of agronomic
importance. To identify the nucleic acid sequences of the present
invention from a database or collection of cDNA sequences, the
first step involves constructing cDNA libraries from specific plant
tissue targets of interest. Briefly, the cDNA libraries are first
constructed from these tissues that are harvested at a particular
developmental stage or under particular environmental conditions.
By identifying differentially expressed genes in plant tissues at
different developmental stages or under different conditions, the
corresponding regulatory sequences of those genes can be identified
and isolated. Transcript imaging enables the identification of
tissue-preferred sequences based on specific imaging of nucleic
acid sequences from a cDNA library. By transcript imaging as used
herein is meant an analysis that compares the abundance of
expressed genes in one or more libraries. The clones contained
within a cDNA library are sequenced and the sequences compared with
sequences from publicly available databases. Computer-based methods
allow the researcher to provide queries that compare sequences from
multiple libraries. The process enables quick identification of
clones of interest compared with conventional hybridization
subtraction methods known to those of skill in the art.
[0065] Using conventional methodologies, cDNA libraries can be
constructed from the mRNA (messenger RNA) of a given tissue or
organism using poly dT primers and reverse transcriptase
(Efstratiadis et al., Cell 7:279, 1976; Higuchi et al., Proc. Natl.
Acad. Sci. U.S.A. 73:3146, 1976; Maniatis et al., Cell 8:163, 1976;
Land et al., Nucleic Acids Res. 9:2251, 1981; Okayama et al., Mol.
Cell. Biol. 2:161, 1982; Gubler et al., Gene 25:263, 1983).
[0066] Several methods can be employed to obtain full-length cDNA
constructs. For example, terminal transferase can be used to add
homopolymeric tails of dC residues to the free 3' hydroxyl groups
(Land et al., Nucleic Acids Res. 9:2251, 1981). This tail can then
be hybridized by a poly dG oligo that can act as a primer for the
synthesis of full length second strand cDNA. Okayama and Berg,
reported a method for obtaining full-length cDNA constructs (Mol.
Cell Biol. 2:161, 1982). This method has been simplified by using
synthetic primer adapters that have both homopolymeric tails for
priming the synthesis of the first and second strands and
restriction sites for cloning into plasmids (Coleclough et al.,
Gene 34:305, 1985) and bacteriophage vectors (Krawinkel et al.,
Nucleic Acids Res. 14:1913, 1986; Han et al., Nucleic Acids Res.
15:6304, 1987).
[0067] These strategies can be coupled with additional strategies
for isolating rare mRNA populations. For example, a typical
mammalian cell contains between 10,000 and 30,000 different mRNA
sequences (Davidson, Gene Activity in Early Development, 2nd ed.,
Academic Press, New York, 1976). The number of clones required to
achieve a given probability that a low-abundance mRNA will be
present in a cDNA library is N=(1n(1-P))/(1n(1-1/n)) where N is the
number of clones required, P is the probability desired, and 1/n is
the fractional proportion of the total mRNA that is represented by
a single rare mRNA (Sambrook et al., 1989).
[0068] One method to enrich preparations of mRNA for sequences of
interest is to fractionate by size. One such method is to
fractionate by electrophoresis through an agarose gel (Pennica et
al., Nature 301:214, 1983). Another method employs sucrose gradient
centrifugation in the presence of an agent, such as methylmercuric
hydroxide, that denatures secondary structure in RNA (Schweinfest
et al., Proc. Natl. Acad. Sci. U.S.A. 79:4997-5000, 1982).
[0069] A frequently adopted method is to construct equalized or
normalized cDNA libraries (Ko, Nucleic Acids Res. 18:5705, 1990;
Patanjali et al., Proc. Natl. Acad. Sci. U.S.A. 88:1943, 1991).
Typically, the cDNA population is normalized by subtractive
hybridization (Schmid et al., J. Neurochem. 48:307, 1987; Fargnoli
et al., Anal. Biochem. 187:364, 1990; Travis et al., Proc. Natl.
Acad. Sci. U.S.A. 85:1696, 1988; Kato, Eur. J. Neurosci. 2:704,
1990; Schweinfest et al., Genet. Anal. Tech. Appl. 7:64, 1990).
Subtraction represents another method for reducing the population
of certain sequences in the cDNA library (Swaroop et al., Nucleic
Acids Res. 19:1954, 1991). Normalized libraries can be constructed
using the Soares procedure (Soares et al., Proc. Natl. Acad. Sci.
U.S.A. 91:9228, 1994). This approach is designed to reduce the
initial 10,000-fold variation in individual cDNA frequencies to
achieve abundance within one order of magnitude while maintaining
the overall sequence complexity of the library. In the
normalization process, the prevalence of high-abundance cDNA clones
decreases dramatically, clones with mid-level abundance are
relatively unaffected, and clones for rare transcripts are
effectively increased in abundance.
[0070] ESTs can be sequenced by a number of methods. Two basic
methods can be used for DNA sequencing, the chain termination
method (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74: 5463,
1977) and the chemical degradation method (Maxam and Gilbert, Proc.
Nat. Acad. Sci. U.S.A. 74: 560, 1977). Automation and advances in
technology, such as the replacement of radioisotopes with
fluorescence-based sequencing, have reduced the effort required to
sequence DNA (Craxton, Methods, 2: 20, 1991; Ju et al., Proc. Natl.
Acad. Sci. U.S.A. 92: 4347, 1995; Tabor and Richardson, Proc. Natl.
Acad. Sci. U.S.A. 92: 6339, 1995). Automated sequencers are
available from a number of manufacturers including Pharmacia
Biotech, Inc., Piscataway, N.J. (Pharmacia ALF); LI-COR, Inc.,
Lincoln, Nebr. (LI-COR 4,000); and Millipore, Bedford, Mass.
(Millipore BaseStation).
[0071] ESTs longer than 150 bp have been found to be useful for
similarity searches and mapping (Adams et al., Science 252:1651,
1991). EST sequences normally range from 150-450 bases. This is the
length of sequence information that is routinely and reliably
generated using single run sequence data. Typically, only single
run sequence data is obtained from the cDNA library (Adams et al.,
Science 252:1651, 1991). Automated single run sequencing typically
results in an approximately 2-3% error or base ambiguity rate
(Boguski et al., Nature Genetics, 4:332, 1993).
[0072] EST databases have been constructed or partially constructed
from, for example, C. elegans (McCombrie et al., Nature Genetics
1:124, 1992); human liver cell line HepG2 (Okubo et al., Nature
Genetics 2:173, 1992); human brain RNA (Adams et al., Science
252:1651, 1991; Adams et al., Nature 355:632, 1992); Arabidopsis,
(Newman et al., Plant Physiol. 106:1241, 1994); and rice (Kurata et
al., Nature Genetics 8:365, 1994). The present invention uses ESTs
from a number of cDNA libraries prepared from male reproductive
tissues of corn as a tool for the identification of genes expressed
in these target tissues, which then facilitates the isolation of 5'
regulatory sequences such as promoters that regulate the genes.
[0073] Computer-based sequence analyses can be used to identify
differentially expressed sequences including, but not limited to,
those sequences expressed in one tissue compared with another
tissue. For example, a different set of sequences can be found from
cDNA isolated from root tissue versus leaf tissue. Accordingly,
sequences can be compared from cDNA libraries prepared from plants
grown under different environmental or physiological conditions.
Once the preferred sequences are identified from the cDNA library
of interest, the genomic clones can be isolated from a genomic
library prepared from the plant tissue, and corresponding
regulatory sequences including but not limited to 5' regulatory
sequences can be identified and isolated.
[0074] In one preferred embodiment, expressed sequence tags (EST)
sequences from a variety of cDNA libraries are catalogued in a
sequence database. This database is used to identify promoter
targets from a particular tissue of interest. The selection of
expressed sequence tags for subsequent promoter isolation is
reflective of the presence of one or more sequences among the
representative ESTs from a random sampling of an individual cDNA
library or a collection of cDNA libraries. For example, the
identification of regulatory sequences that direct the expression
of transcripts in male reproductive tissues is conducted by
identifying ESTs found in tissues such as tassel and anther, and
absent or in lower abundance in other cDNA libraries in the
database. The identified EST leads are then evaluated for relative
abundance within the library and the expression profile for a given
EST is assessed. By abundance as used herein is meant the number of
times a clone or cluster of clones appears in a library. The
sequences that are enhanced or in high abundance in a specific
tissue or organ that represent a target expression profile are
identified in this manner and primers can be designed from the
identified EST sequences. A PCR-based approach can be used to
amplify flanking regions from a genomic library of the target plant
of interest. A number of methods are known to those of skill in the
art to amplify unknown DNA sequences adjacent to a core region of
known sequence. Methods include but are not limited to inverse PCR
(IPCR), vectorette PCR, Y-shaped PCR, and genome walking
approaches.
[0075] In a preferred embodiment, genomic DNA ligated to an adaptor
is subjected to a primary round of PCR amplification with a
gene-specific primer and a primer that anneals to the adaptor
sequence. The PCR product is next used as the template for a nested
round of PCR amplification with a second gene-specific primer and
second adaptor. The resulting fragments from the nested PCR
reaction are then isolated, purified and subcloned into an
appropriate vector. The fragments are sequenced, and the
translational start sites can be identified when the EST is derived
from a truncated cDNA. The fragments can be cloned into plant
expression vectors as transcriptional or translational fusions with
a reporter gene such as .beta.-glucuronidase (GUS). The constructs
can be tested in transient analyses, and subsequently the 5'
regulatory regions are operably linked to other genes and
regulatory sequences of interest in a suitable plant transformation
vector and the transformed plants are analyzed for the expression
of the gene(s) of interest, by any number of methods known to those
of skill in the art.
[0076] Any plant can be selected for the identification of genes
and regulatory sequences. Examples of suitable plant targets for
the isolation of genes and regulatory sequences would include but
are not limited to Acadia, alfalfa, apple, apricot, Arabidopsis,
artichoke, arugula, asparagus, avocado, banana, barley, beans,
beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage,
canola, cantaloupe, carrot, cassava, castorbean, cauliflower,
celery, cherry, chicory, cilantro, citrus, clementines, clover,
coconut, coffee, corn, cotton, cucumber, Douglas fir, eggplant,
endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape,
grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon,
lily, lime, Loblolly pine, linseed, mango, melon, mushroom,
nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion,
orange, an ornamental plant, palm, papaya, parsley, parsnip, pea,
peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain,
plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine,
radiscchio, radish, rapeseed, raspberry, rice, rye, sorghum,
Southern pine, soybean, spinach, squash, strawberry, sugarbeet,
sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea,
tobacco, tomato, triticale, turf, turnip, a vine, watermelon,
wheat, yams, and zucchini. Particularly preferred plant targets
would include corn, cotton, rice, soybean, and wheat.
[0077] The nucleic acid molecules of the present invention are
isolated from corn (Zea mays). The corn plant develops about 20-21
leaves, silks about 65 days post-emergence, and matures about 125
days post-emergence. Normal corn plants follow a general pattern of
development, but the time interval between different stages and
morphology varies between different hybrids, growth and
environmental conditions.
[0078] There are a number of identifiable stages in corn plant
development. The stages are defined as vegetative (V) and
reproductive (R) stages. Subdivisions of the V stages are
numerically designated as V1, V2,V3, etc., through V(n) where (n)
represents the last leaf stage before tasseling (VT) and the first
V stage is the emergence (VE) stage. For example, VE is the
emergence from the soil of a seedling leaf, V1 represents the first
true leaf, V2 represents the second leaf, etc. The reproductive
stages include the first appearance of silk to the mature seed and
are represented as follows: R1 is silking, R2 is blistering, R3 is
the milk stage, R4 is the dough stage, R5 is the dent stage, and R6
is physiological maturity (see for example, Ritchie et al. (1986)
How a Corn Plant Develops, Iowa State University of Science and
Technology Cooperative Exension Service, Ames, Iowa 48: 1-21).
[0079] Any type of plant tissue can be used as a target tissue for
the identification of genes and associated regulatory sequences.
For the present invention, corn male reproductive tissue is used.
More preferably corn tassel tissues are the target tissues for
identification of promoter sequences. Corn cDNA libraries can be
constructed from several different plant developmental stages. More
preferably corn plants at stages V6-V9 are used. Background or
non-target libraries can include but are not limited to libraries
such as leaf, root, embryo, callus, shoot, seedling, endosperm,
culm, ear, and silks.
[0080] Any method that allows a differential comparison between
different types or classes of sequences can be used to isolate
genes or regulatory sequences of interest. For example, in one
differential screening approach, a cDNA library from mRNA isolated
from a particular tissue can be prepared in a bacteriophage host
using a commercially available cloning kit. The plaques are spread
onto plates containing lawns of a bacterial host such as E. coli to
generate bacteriophage plaques. About 10.sup.5-10.sup.6 plaques can
be lifted onto DNA-binding membranes. Duplicate membranes are
probed using probes generated from mRNA from the target and
non-target or background tissue. The probes are labeled to
facilitate detection after hybridization and development. Plaques
that hybridize to target tissue-derived probes but not to
non-target tissue derived probes that display a desired
differential pattern of expression can be selected for further
analysis. Genomic DNA libraries can also be prepared from a chosen
species by partial digestion with a restriction enzyme and size
selecting the DNA fragments within a particular size range. The
genomic DNA can be cloned into a suitable vector including but not
limited to a bacteriophage and prepared using a suitable vector
such as a bacteriophage using a suitable cloning kit from any
number of vendors (see for example Stratagene, La Jolla Calif. or
Gibco BRL, Gaithersburg, Md.).
[0081] Differential hybridization techniques as described are well
known to those of skill in the art and can be used to isolate a
desired class of sequences. By classes of sequences as used herein
is meant sequences that can be grouped based on a common identifier
including but not limited to sequences isolated from a common
target plant, a common library, or a common plant tissue type. In a
preferred embodiment, sequences of interest are identified based on
sequence analyses and querying of a collection of diverse cDNA
sequences from libraries of different tissue types. The disclosed
method provides an example of a differential screening approach
based on electronic sequence analyses of plant ESTs derived from
diverse cDNA libraries.
[0082] A number of methods used to assess gene expression are based
on measuring the mRNA level in an organ, tissue, or cell sample.
Typical methods include but are not limited to RNA blots,
ribonuclease protection assays and RT-PCR. In another preferred
embodiment, a high-throughput method is used whereby regulatory
sequences are identified from a transcript profiling approach. The
development of cDNA microarray technology enables the systematic
monitoring of gene expression profiles for thousands of genes
(Schena et al, Science, 270: 467, 1995). This DNA chip-based
technology arrays thousands of cDNA sequences on a support surface.
These arrays are simultaneously hybridized to multiple labeled cDNA
probes prepared from RNA samples of different cell or tissue types,
allowing direct comparative analysis of expression. This technology
was first demonstrated by analyzing 48 Arabidopsis genes for
differential expression in roots and shoots (Schena et al, Science,
270:467, 1995). More recently, the expression profiles of over 1400
genes were monitored using cDNA microarrays (Ruan et al, The Plant
Journal 15:821, 1998). Microarrays provide a high-throughput,
quantitative and reproducible method to analyze gene expression and
characterize gene function. The transcript profiling approach using
microarrays thus provides another valuable tool for the isolation
of regulatory sequences such as promoters associated with those
genes.
[0083] The present invention uses high throughput sequence analyses
to form the foundation of rapid computer-based identification of
sequences of interest. Those of skill in the art are aware of the
resources available for sequence analyses. Sequence comparisons can
be done by determining the similarity of the test or query sequence
with sequences in publicly available or proprietary databases
("similarity analysis") or by searching for certain motifs
("intrinsic sequence analysis") (e.g., cis elements) (Coulson,
Trends in Biotechnology, 12:76, 1994; Birren et al., Genome
Analysis, 1:543, 1997).
[0084] The nucleotide sequences provided in SEQ ID NOS: 79-98 or
fragments thereof, or complements thereof, or a nucleotide sequence
at least 90% identical, preferably 95% identical even more
preferably 99% or 100% identical to the sequence provided in SEQ ID
NOS: 79-98 or fragment thereof, or complement thereof, can be
"provided" in a variety of mediums to facilitate use. Such a medium
can also provide a subset thereof in a form that allows one of
skill in the art to examine the sequences.
[0085] In one application of this embodiment, a nucleotide sequence
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. One of skill in the art can readily
appreciate how any of the presently known computer readable media
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide sequence of the present
invention.
[0086] By providing one or more of nucleotide sequences of the
present invention, those of skill in the art can routinely access
the sequence information for a variety of purposes. Computer
software is publicly available that allows one of skill in the art
to access sequence information provided in a computer readable
medium. Examples of public databases would include but are not
limited to the DNA Database of Japan (DDBJ)
(http://www.ddbj.nig.ac.jp/);Genbank (http://www.ncbi.nlm.nih.gov/-
web/Genbank/Index.html); and the European Molecular Biology
Laboratory Nucleic Acid Sequence Database (EMBL)
(http://www.ebi.ac.uk/ebi_docs/embl- _db.html) or versions thereof.
A number of different search algorithms have been developed,
including but not limited to the suite of programs referred to as
BLAST programs. There are five implementations of BLAST, three
designed for nucleotide sequence queries (BLASTN, BLASTX, and
TBLASTX) and two designed for protein sequence queries (BLASTP and
TBLASTN) (Coulson, Trends in Biotechnology, 12:76-80, 1994; Birren
et al., Genome Analysis, 1:543, 1997).
[0087] Any program designed for motif searching also has utility in
the present invention. Sequence analysis programs designed for
motif searching can be used for identification of cis elements.
Preferred computer programs would include but are not limited to
MEME, SIGNAL SCAN, and GENESCAN. MEME is a program that identifies
conserved motifs (either nucleic acid or peptide) in a group of
unaligned sequences. MEME saves these motifs as a set of profiles.
These profiles can be used to search a database of sequences. A
MEME algorithm (version 2.2) can be found in version 10.0 of the
GCG package; MEME (Bailey and Elkan, Machine Learning,
21(1-2):51-80,1995 and the location of the website is
http://www.sdsc.edu/MEME/meme/website/COPYRIGHT.html. SIGNALSCAN is
a program that identifies known motifs in the test sequences using
information from other motif databases (Prestridge, CABIOS 7,
203-206, 1991). SIGNALSCAN version 4.0 information is available at
the following website:
http://biosci.cbs.umn.edu/software/sigscan.html. The ftp site for
SIGNALSCAN is ftp://biosci.cbs.umn.edu/software/sigscan.html.
Databases used with SIGNALSCAN include PLACE
(http://www.dna.affrc.go.ip/- htdocs/PLACE; Higo et al., Nucleic
Acids Research 27(1):297-300, 1999) and TRANSFAC (Heinemeye, X. et
al., Nucleic Acid Research 27(1):318-322) that can be found at the
following website: http://transfac.gbf.de/.GENESCAN is another
suitable program for motif searching (Burge and Karlin, J. Mol.
Biol. 268, 78-94, 1997), and version 1.0 information is available
at the following website:
http://gnomic.stanford.edu/GENESCANW.html. As used herein, "a
target structural motif" or "target motif" refers any rationally
selected sequence or combination of sequences in which the
sequence(s) are chosen based on a three-dimensional configuration
that is formed upon the folding of the target motif. There are a
variety of target motifs known to those of skill in the art.
Protein target motifs include, but are not limited to, enzymatic
active sites and signal sequences. Preferred target motifs of the
present invention would include but are not limited to promoter
sequences, cis elements, hairpin structures and other expression
elements such as protein binding sequences.
[0088] As used herein, "search means" refers to one or more
programs that are implemented on the computer-based system to
compare a target sequence or target structural motif with the
sequence information stored within the data storage means. Search
means are used to identify fragments or regions of the sequences of
the present invention that match a particular target sequence or
target motif. Multiple sequences can also be compared in order to
identify common regions or motifs that may be responsible for
specific functions. For example, cis elements or sequence domains
that confer a specific expression profile can be identified when
multiple promoter regions of similar classes of promoters are
aligned and analyzed by certain software packages.
[0089] The present invention further provides systems, particularly
computer-based systems, that contain the sequence information
described herein. As used herein, a "computer-based system" refers
to the hardware means, software means, and data storage means used
to analyze the nucleotide sequence information of the present
invention. The minimum hardware means of the computer-based systems
of the present invention comprises a central processing unit (CPU),
input means, output means, and data storage means. Those of skill
in the art can appreciate that any one of the available
computer-based systems are suitable for use in the present
invention.
[0090] SEQ ID NOS: 4-76 are primers designed from the cDNA
sequences identified from the computer-based sequence comparisons.
These sequences are used to extend the nucleic acid sequence using
polymerase chain reaction (PCR) amplification techniques (see for
example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol.
51:263, 1986; Erlich et al., European Patent Appln. 50,424;
European Patent Appln. 84,796, European Patent Appln. 258,017,
European Patent Appln. 237,362; Mullis, European Patent Appln.
201,184; Mullis et al., U.S. Pat. No. 4,683,202; Erlich, U.S. Pat.
No. 4,582,788; and Saiki et al., U.S. Pat. No. 4,683,194). A number
of PCR amplification methods are known to those of skill in the art
and are used to identify nucleic acid sequences adjacent to a known
sequence. For example, inverse PCR (IPCR) methods to amplify
unknown DNA sequences adjacent to a core region of known sequence
have been described. Other methods are also available such as
capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111, 1991),
and walking PCR (Parker et al., Nucleic Acids Res 19:3055, 1991). A
number of manufacturers have also developed kits based on
modifications of these methods for the purposes of identifying
sequences of interest. Technical advances including improvements in
primer and adaptor design, improvements in the polymerase enzyme,
and thermocycler capabilies have facilitated quicker, more
efficient methods for isolating sequences of interest.
[0091] In a preferred embodiment, the flanking sequences containing
the 5' regulatory elements of the present invention are isolated
using a genome-walking approach (Universal GenomeWalker.TM. Kit,
CLONTECH Laboratories, Inc., Palo Alto, Calif.). In brief, the
purified genomic DNA is subjected to a restriction enzyme digest
that produces genomic DNA fragments with ends that are ligated with
GenomeWalker.TM. adaptors. GenomeWalker.TM. primers are used along
with gene specific primers in two consecutive PCR reactions
(primary and nested PCR reactions) to produce PCR products
containing the 5' regulatory sequences that are subsequently cloned
and sequenced.
[0092] In addition to their use in modulating gene expression, the
promoter sequences of the present invention also have utility as
probes or primers in nucleic acid hybridization experiments. The
nucleic acid probes and primers of the present invention can
hybridize under stringent conditions to a target DNA sequence. The
term "stringent hybridization conditions" is defined as conditions
under which a probe or primer hybridizes specifically with a target
sequence(s) and not with non-target sequences, as can be determined
empirically. The term "stringent conditions" is functionally
defined with regard to the hybridization of a nucleic-acid probe to
a target nucleic acid (i.e., to a particular nucleic-acid sequence
of interest) by the specific hybridization procedure (see for
example Sambrook et al., 1989, at 9.52-9.55 and 9.47-9.52,
9.56-9.58; Kanehisa, Nucl. Acids Res. 12:203-213, 1984; Wetmur and
Davidson, J. Mol. Biol. 31:349-370, 1968). Appropriate stringency
conditions that 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., and they
are known to those skilled in the art or can be found in laboratory
manuals including but not limited to Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6. For
example, the salt concentration in the wash step can be selected
from a low stringency of about 2.0.times.SSC at 50.degree. C. to a
high stringency of about 0.2.times.SSC at 50.degree. C. In
addition, the temperature in the wash step can be increased from
low stringency conditions at room temperature, about 22.degree. C.,
to high stringency conditions at about 65.degree. C. Both
temperature and salt may be varied, or either the temperature or
the salt concentration may be held constant while the other
variable is changed. For example, hybridization using DNA or RNA
probes or primers can be performed at 65.degree. C. in 6.times.SSC,
0.5% SDS, 5.times. Denhardt's, 100 .mu.g/mL nonspecific DNA (e.g.,
sonicated salmon sperm DNA) with washing at 0.5.times.SSC, 0.5% SDS
at 65.degree. C., for high stringency.
[0093] It is contemplated that lower stringency hybridization
conditions such as lower hybridization and/or washing temperatures
can be used to identify related sequences having a lower degree of
sequence similarity if specificity of binding of the probe or
primer to target sequence(s) is preserved. Accordingly, the
nucleotide sequences of the present invention can be used for their
ability to selectively form duplex molecules with complementary
stretches of DNA fragments. Detection of DNA segments via
hybridization is well-known to those of skill in the art. Thus
depending on the application envisioned, one will desire to employ
varying hybridization conditions to achieve varying degrees of
selectivity of probe towards target sequence and the method of
choice will depend on the desired results.
[0094] The nucleic acid sequences in SEQ ID NOS: 79-98, and any
variants thereof, are capable of hybridizing to other nucleic acid
sequences under appropriately selected conditions of stringency. As
used herein, two nucleic acid molecules 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 nucleic acid molecule is said to be the "complement"
of another nucleic acid molecule if they exhibit complementarity.
As used herein, molecules are said to exhibit "complete
complementarity" when every nucleotide of one of the molecules is
complementary to a nucleotide of the other. Two molecules 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 molecules 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 described by Sambrook et al. (Molecular Cloning, A Laboratory
Manual, 2.sup.nd Ed., Cold Spring Harbor Press, Cold Spring Harbor,
N.Y., 1989), and by Haymes et al. (Nucleic Acid Hybridization, A
Practical Approach, IRL Press, Washington, D.C., 1985).
[0095] In a preferred embodiment, the nucleic acid sequences SEQ ID
NOS: 79-98 or a fragment, region, cis element, or oligomer of these
sequences may be used in hybridization assays of other plant
tissues to identify closely related or homologous genes and
associated regulatory sequences. These include but are not limited
to Southern or northern hybridization assays on any substrate
including but not limited to an appropriately prepared plant
tissue, cellulose, nylon, or combination filter, chip, or glass
slide. Such methodologies are well known in the art and are
available in a kit or preparation that can be supplied by
commercial vendors.
[0096] Of course, nucleic acid fragments can also be obtained by
other techniques such as by directly synthesizing the fragment by
chemical means, as is commonly practiced by using an automated
oligonucleotide synthesizer. Fragments can also be obtained by
application of nucleic acid reproduction technology, such as the
PCR.TM. (polymerase chain reaction) technology or by recombinant
DNA techniques generally known to those of skill in the art of
molecular biology. Regarding the amplification of a target
nucleic-acid sequence (e.g., by PCR) using a particular
amplification primer pair, "stringent PCR conditions" refer to
conditions that permit the primer pair to hybridize only to the
target nucleic-acid sequence to which a primer having the
corresponding wild-type sequence (or its complement) would bind and
preferably to produce a unique amplification product.
[0097] A fragment of a nucleic acid as used herein is a portion of
the nucleic acid that is less than full-length. For example, for
the present invention any length of nucleotide sequence that is
less than the disclosed nucleotide sequences of SEQ ID NOS: 79-98
is considered to be a fragment. A fragment can also comprise at
least a minimum length capable of hybridizing specifically with a
native nucleic acid under stringent hybridization conditions as
defined above. The length of such a minimal fragment is preferably
at least 8 nucleotides, more preferably 15 nucleotides, even more
preferably at least 20 nucleotides, and most preferably at least 30
nucleotides of a native nucleic acid sequence.
[0098] The nucleic acid sequences of the present invention can also
be used as probes and primers. Nucleic acid probes and primers can
be prepared based on a native gene sequence. A "probe" is an
isolated nucleic acid to which is attached a conventional
detectable label or reporter molecule, e.g., a radioactive isotope,
ligand, chemiluminescent agent, or enzyme. "Primers" are isolated
nucleic acids that are annealed to a complementary target DNA
strand by nucleic acid hybridization to form a hybrid between the
primer and the target DNA strand, then extended along the target
DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs
can be used for amplification of a nucleic acid sequence, e.g., by
the polymerase chain reaction (PCR) or other conventional
nucleic-acid amplification methods.
[0099] Probes and primers are generally 15 nucleotides or more in
length, preferably 20 nucleotides or more, more preferably 25
nucleotides, and most preferably 30 nucleotides or more. Such
probes and primers hybridize specifically to a target DNA or RNA
sequence under high stringency hybridization conditions and
hybridize specifically to a target native sequence of another
species under lower stringency conditions. Preferably, probes and
primers according to the present invention have complete sequence
similarity with the native sequence, although probes differing from
the native sequence and that retain the ability to hybridize to
target native sequences may be designed by conventional methods.
Methods for preparing and using probes and primers are described
(see Sambrook et al., 1989; Ausubel et al., 1992, and Innis et al.,
1990). PCR-primer pairs can be derived from a known sequence, for
example, by using computer programs intended for that purpose such
as Primer (Version 0.5, .COPYRGT. 1991, Whitehead Institute for
Biomedical Research, Cambridge, Mass.). Primers and probes based on
the native promoter sequences disclosed herein can be used to
confirm and, if necessary, to modify the disclosed sequences by
conventional methods, e.g., by re-cloning and re-sequencing.
[0100] In another embodiment, the nucleotide sequences of the
promoters disclosed herein can be modified. Those skilled in the
art can create DNA molecules that have variations in the nucleotide
sequence. The nucleotide sequences of the present invention as
shown in SEQ ID NOS: 79-98 may be modified or altered to enhance
their control characteristics. One preferred method of alteration
of a nucleic acid sequence is to use PCR to modify selected
nucleotides or regions of sequences. These methods are known to
those of skill in the art. Sequences can be modified, for example
by insertion, deletion or replacement of template sequences in a
PCR-based DNA modification approach. "Variant" DNA molecules are
DNA molecules containing changes in which one or more nucleotides
of a native sequence is deleted, added, and/or substituted,
preferably while substantially maintaining promoter function. In
the case of a promoter fragment, "variant" DNA can include changes
affecting the transcription of a minimal promoter to which it is
operably linked. Variant DNA molecules can be produced, for
example, by standard DNA mutagenesis techniques or by chemically
synthesizing the variant DNA molecule or a portion thereof.
[0101] In another embodiment, the nucleotide sequences as shown in
SEQ ID NOS: 79-98 include any length of said nucleotide sequences
that are capable of regulating an operably linked DNA sequence. For
example, the sequences as disclosed in SEQ ID NOS: 79-98 may be
truncated or portions deleted and still be capable of regulating
transcription of an operably linked DNA sequence. In a related
embodiment, a cis element of the disclosed sequences may confer a
particular specificity such as conferring enhanced expression of
operably linked DNA sequences in certain tissues. Consequently, any
sequence fragments, portions, or regions of the disclosed sequences
of SEQ ID NOS: 79-98 can be used as regulatory sequences including
but not limited to cis elements or motifs of the disclosed
sequences. For example, one or more base pairs may be deleted from
the 5' or 3' end of a promoter sequence to produce a "truncated"
promoter. One or more base pairs can also be inserted, deleted, or
substituted internally to a promoter sequence. Promoters can be
constructed such that promoter fragments or elements are operably
linked for example, by placing such a fragment upstream of a
minimal promoter. A minimal or basal promoter is a piece of DNA
that is capable of recruiting and binding the basal transcription
machinery. One example of basal transcription machinery in
eukaryotic cells is the RNA polymerase II complex and its accessory
proteins. The enzymatic components of the basal transcription
machinery are capable of initiating and elongating transcription of
a given gene, utilizing a minimal or basal promoter. That is, there
are not added cis-acting sequences in the promoter region that are
capable of recruiting and binding transcription factors that
modulate transcription, e.g., enhance, repress, render
transcription hormone-dependent, etc. Substitutions, deletions,
insertions or any combination thereof can be combined to produce a
final construct.
[0102] Native or synthetic nucleic acids according to the present
invention can be incorporated into recombinant nucleic acid
constructs, typically DNA constructs, capable of introduction into
and replication in a host cell. In one preferred embodiment, the
nucleotide sequences of the present invention as shown in SEQ ID
NOS: 79-98 or fragments, variants or derivatives thereof are
incorporated into an expression vector cassette that includes the
promoter regions of the present invention operably linked to a
genetic component such as a selectable, screenable, or scorable
marker gene. The disclosed nucleic acid sequences of the present
invention are preferably operably linked to a genetic component
such as a nucleic acid that confers a desirable characteristic
associated with plant morphology, physiology, growth and
development, yield, nutritional enhancement, disease or pest
resistance, or environmental or chemical tolerance. These genetic
components such as marker genes or agronomic genes of interest can
function in the identification of a transformed plant cell or
plant, or a produce a product of agronomic utility.
[0103] In a preferred embodiment, one genetic component produces a
product that serves as a selection device and functions in a
regenerable plant tissue to produce a compound that would confer
upon the plant tissue resistance to an otherwise toxic compound.
Genes of interest for use as a selectable, screenable, or scorable
marker would include but are not limited to GUS (coding sequence
for beta-glucuronidase), GFP (coding sequence for green fluorescent
protein), LUX (coding gene for luciferase), antibiotic resistance
marker genes, or herbicide tolerance genes. Examples of transposons
and associated antibiotic resistance genes include the transposons
Tns (bla), Tn5 (nptII), Tn7 (dhfr), penicillins, kanamycin (and
neomycin, G418, bleomycin); methotrexate (and trimethoprim);
chloramphenicol; and tetracycline.
[0104] Characteristics useful for selectable markers in plants have
been outlined in a report on the use of microorganisms (Advisory
Committee on Novel Foods and Processes, July 1994). These include
stringent selection with minimum number of nontransformed tissues,
large numbers of independent transformation events with no
significant interference with the regeneration, application to a
large number of species, and availability of an assay to score the
tissues for presence of the marker.
[0105] A number of selectable marker genes are known in the art and
several antibiotic resistance markers satisfy these criteria,
including those resistant to kanamycin (nptII), hygromycin B (aph
IV) and gentamycin (aac3 and aacC4). Useful dominant selectable
marker genes include genes encoding antibiotic resistance genes
(e.g., resistance to hygromycin, kanamycin, bleomycin, G418,
streptomycin or spectinomycin); and herbicide resistance genes
(e.g., phosphinothricin acetyltransferase). A useful strategy for
selection of transformants for herbicide resistance is described,
e.g., in Vasil (Cell Culture and Somatic Cell Genetics of Plants,
Vols. I-III, Laboratory Procedures and Their Applications Academic
Press, New York, 1984). Particularly preferred selectable marker
genes for use in the present invention would include genes that
confer resistance to compounds such as antibiotics like kanamycin
and herbicides like glyphosate (Della-Cioppa et al., Bio/Technology
5(6), 1987; U.S. Pat. No. 5,463,175; U.S. Pat. No. 5,633,435).
Other selection devices can also be implemented and would still
fall within the scope of the present invention.
[0106] For the practice of the present invention, conventional
compositions and methods for preparing and using vectors and host
cells are employed, as discussed, inter alia, in Sambrook et al.,
1989. In a preferred embodiment, the host cell is a plant cell. A
number of vectors suitable for stable transfection of plant cells
or for the establishment of transgenic plants have been described
in, e.g., Pouwels et al. (Cloning Vectors: A Laboratory Manual,
1985, supp. 1987); Weissbach and Weissbach (Methods for Plant
Molecular Biology, Academic Press, 1989); Gelvin et al. (Plant
Molecular Biology Manual, Kluwer Academic Publishers, 1990); and
Croy (Plant Molecular Biology LabFax, BIOS Scientific Publishers,
1993). Plant expression vectors can include, for example, one or
more cloned plant genes under the transcriptional control of 5' and
3' regulatory sequences. They can also include a selectable marker
as described to select for host cells containing the expression
vector. Such plant expression vectors also contain a promoter
regulatory region (e.g., a regulatory region controlling inducible
or constitutive, environmentally or developmentally regulated, or
cell- or tissue-specific expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and a polyadenylation signal. Other
sequences of bacterial origin are also included to allow the vector
to be cloned in a bacterial host. The vector will also typically
contain a broad host range prokaryotic origin of replication. In a
particularly preferred embodiment, the host cell is a plant cell
and the plant expression vector comprises a promoter region as
disclosed in SEQ ID NOS: 79-98, an operably linked transcribable
sequence, and a transcription termination sequence. Other
regulatory sequences envisioned as genetic components in an
expression vector include, but is not limited to, non-translated
leader sequence that can be coupled with the promoter. Plant
expression vectors also can comprise additional sequences including
but not limited to restriction enzyme sites that are useful for
cloning purposes.
[0107] A number of promoters have utility for plant gene expression
for any gene of interest including but not limited to selectable
markers, scorable markers, genes for pest tolerance, disease
tolerance, nutritional enhancements and any other gene that confers
a desirable trait or characteristic. Examples of constitutive
promoters useful for plant gene expression include, but are not
limited to, the cauliflower mosaic virus (CaMV) 35S promoter, which
confers constitutive, high-level expression in most plant tissues
(see, e.g., Odel et al., Nature 313:810, 1985), including monocots
(see, e.g., Dekeyser et al., Plant Cell 2:591, 1990; Terada and
Shimamoto, Mol. Gen. Genet. 220:389, 1990); the nopaline synthase
promoter (An et al., Plant Physiol. 88:547, 1988); the octopine
synthase promoter (Fromm et al., Plant Cell 1:977, 1989); and the
figwort mosaic virus (FMV) promoter as described in U.S. Pat. No.
5,378,619.
[0108] A variety of plant gene promoters that are regulated in
response to environmental, hormonal, chemical, and/or developmental
signals can be used for expression of an operably linked gene in
plant cells, including promoters regulated by (1) heat (Callis et
al., Plant Physiol. 88:965, 1988), (2) light (e.g., pea rbcS-3A
promoter, Kuhlemeier et al., Plant Cell 1:471, 1989; maize rbcS
promoter, Schaffner and Sheen, Plant Cell 3:997, 1991; or
chlorophyll a/b-binding protein promoter, Simpson et al., EMBO J.
4:2723, 1985), (3) hormones, such as abscisic acid (Marcotte et
al., Plant Cell 1:969, 1989), (4) wounding (e.g., wunI, Siebertz et
al., Plant Cell 1:961, 1989); or (5) chemicals such as methyl
jasmonate, salicylic acid, or safener. It may also be advantageous
to employ (6) organ-specific promoters (e.g., Roshal et al., EMBO
J. 6:1155, 1987; Schernthaner et al., EMBO J. 7:1249, 1988; Bustos
et al., Plant Cell 1:839, 1989). The promoters of the present
invention are plant promoters that are capable of transcribing
operably linked DNA sequences in male reproductive tissues and can
be operably linked to any gene of interest in an expression
vector.
[0109] Plant expression vectors can include RNA processing signals,
e.g., introns, which may be positioned upstream or downstream of a
polypeptide-encoding sequence in the transgene. In addition, the
expression vectors may include additional regulatory sequences from
the 3'-untranslated region of plant genes (Thornburg et al., Proc.
Natl. Acad. Sci. USA 84:744, 1987; An et al., Plant Cell 1:115,
1989), e.g., a 3' terminator region to increase mRNA stability of
the mRNA, such as the PI-II terminator region of potato or the
octopine or nopaline synthase 3' terminator regions. Five-end
non-translated regions of a mRNA can play an important role in
translation initiation and can also be a genetic component in a
plant expression vector. For example, non-translated 5' leader
sequences derived from heat shock protein genes have been
demonstrated to enhance gene expression in plants (see, for example
U.S. Pat. No. 5,362,865). These additional upstream and downstream
regulatory sequences may be derived from a source that is native or
heterologous with respect to the other elements present on the
expression vector.
[0110] The promoter sequences of the present invention are used to
control gene expression in plant cells. The disclosed promoter
sequences are genetic components that are part of vectors used in
plant transformation. The promoter sequences of the present
invention can be used with any suitable plant transformation
plasmid or vector containing a selectable or screenable marker and
associated regulatory elements, as described, along with one or
more nucleic acids expressed in a manner sufficient to confer a
particular desirable trait. Examples of suitable structural genes
of agronomic interest envisioned by the present invention would
include but are not limited to one or more genes for insect
tolerance, such as a B.t., pest tolerance such as genes for fungal
disease control, herbicide tolerance such as genes conferring
glyphosate tolerance, and genes for quality improvements such as
yield, nutritional enhancements, environmental or stress
tolerances, or any desirable changes in plant physiology,
fertilizer, growth, development, morphology or plant
product(s).
[0111] Alternatively, the DNA coding sequences can effect these
phenotypes by encoding a non-translatable RNA molecule that causes
the targeted inhibition of expression of an endogenous gene, for
example via antisense- or cosuppression-mediated mechanisms (see,
for example, Bird et al., Biotech. Gen. Engin. Rev. 9:207,1991).
The RNA could also be a catalytic RNA molecule (i.e., a ribozyme)
engineered to cleave a desired endogenous mRNA product (see for
example, Gibson and Shillitoe, Mol. Biotech. 7:125,1997). Thus, any
gene that produces a protein or mRNA that expresses a phenotype or
morphology change of interest is useful for the practice of the
present invention.
[0112] In addition to regulatory elements or sequences located
upstream (5') or within a DNA sequence, there are downstream (3')
sequences that affect gene expression and thus the term regulatory
sequence as used herein refers to any nucleotide sequence located
upstream, within, or downstream to a DNA sequence that controls,
mediates, or affects expression of a gene product in conjunction
with the protein synthetic apparatus of the cell.
[0113] The promoter sequences of the present invention may be
modified, for example for expression in other plant systems. In
another approach, novel hybrid promoters can be designed or
engineered by a number of methods. Many promoters contain upstream
sequences that activate, enhance or define the strength and/or
specificity of the promoter (Atchison, Ann. Rev. Cell Biol. 4:127,
1988). T-DNA genes, for example, contain "TATA" boxes defining the
site of transcription initiation and other upstream elements
located upstream of the transcription initiation site modulate
transcription levels (Gelvin, In: Transgenic Plants, Kung and Us,
eds, San Diego: Academic Press, pp.49-87, 1988). Ni and colleagues
combined a trimer of the octopine synthase (ocs) activator to the
mannopine synthase (mas) activator plus promoter and reported an
increase in expression of a reporter gene (Ni et al., The Plant
Journal 7:661, 1995). The upstream regulatory sequences of the
present invention can be used for the construction of such chimeric
or hybrid promoters. Methods for construction of variant promoters
of the present invention include but are not limited to combining
control elements of different promoters or duplicating portions or
regions of a promoter (see for example U.S. Pat. No. 5,110,732 and
U.S. Pat. No. 5,097,025). Those of skill in the art are familiar
with the standard resource materials that 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 isolation
of genes, (see for example Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, 1989; Maliga et al.,
Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995;
Birren et al., Genome Analysis: volume 1, Analyzing DNA, (1997),
volume 2, Detecting Genes, (1998), volume 3, Cloning Systems,
(1999) volume 4, Mapping Genomes, (1999), Cold Spring Harbor,
N.Y.).
[0114] The promoter sequences of the present invention may be
incorporated into an expression vector using screenable or scorable
markers as described and tested in transient analyses that provide
an indication of gene expression in stable plant systems. Methods
of testing gene expression in transient assays are known to those
of skill in the art. Transient expression of marker genes has been
reported using a variety of plants, tissues and DNA delivery
systems. For example, types of transient analyses can include but
are not limited to direct gene delivery via electroporation or
particle bombardment of tissues in any transient plant assay using
any plant species of interest. Such transient systems would include
but are not limited to protoplasts from suspension cultures in
wheat (Zhou et al., Plant Cell Reports 12:612. 1993,
electroporation of leaf protoplasts of wheat (Sethi et al., J. Crop
Sci. 52: 152, 1983; electroporation of protoplast prepared from
corn tissue (Sheen, The Plant Cell 3: 225, 1991), or particle
bombardment of specific tissues of interest. The present invention
encompasses the use of any transient expression system to evaluate
regulatory sequences operatively linked to selected reporter genes,
marker genes or agronomic genes of interest. Examples of plant
tissues envisioned to test in transients via an appropriate
delivery system would include, but are not limited to, leaf base
tissues, callus, cotyledons, roots, endosperm, embryos, floral
tissue, pollen, and epidermal tissue.
[0115] Any scorable or screenable marker can be used in a transient
assay. Preferred marker genes for transient analyses of the
promoters or 5' regulatory sequences of the present invention
include a GUS gene or a GFP gene. The expression vectors containing
the 5' regulatory sequences operably linked to a marker gene are
delivered to the tissues and the tissues are analyzed by the
appropriate mechanism, depending on the marker. The quantitative or
qualitative analyses are used as a tool to evaluate the potential
expression profile of the 5' regulatory sequences when operatively
linked to genes of agronomic interest in stable plants. Ultimately,
the 5' regulatory sequences of the present invention are directly
incorporated into suitable plant transformation expression vectors
comprising the 5' regulatory sequences operatively linked to a
transcribable DNA sequence interest, transformed into plants and
the stably transformed plants and progeny thereof analyzed for the
desired expression profile conferred by the 5' regulatory
sequences.
[0116] Those of skill in the art are aware of the vectors suitable
for plant transformation. Suitable vectors would include but are
not limited to disarmed Ti-plasmids for Agrobacterium-mediated
methods. These vectors can contain a resistance marker, 1-2 T-DNA
borders, and origins of replication for E. coli and Agrobacterium
along with one or more genes of interest and associated regulatory
regions. Those of skill in the art are aware that for
Agrobacterium-mediated approaches a number of strains and methods
are available. Such strains would include but are not limited to
Agrobacterium strains C58, LBA4404, EHA101 and EHA105. Particularly
preferred strains are Agrobacterium tumefaciens strains. Other DNA
delivery systems for plant transformation are also known to those
of skill in the art and include, but are not limited to, particle
bombardment of selected plant tissues.
[0117] Exemplary nucleic acids that may be introduced by the
methods encompassed by the present invention include, for example,
DNA sequences or genes from another species, or even genes or
sequences that originate with or are present in the same species
but are incorporated into recipient cells by genetic engineering
methods rather than classical reproduction or breeding techniques.
However, the term exogenous is also intended to refer to genes that
are not normally present in the cell being transformed, or perhaps
simply not present in the form, structure, etc., as found in the
transforming DNA segment or gene, or genes that are normally
present yet which one desires, e.g., to have over-expressed. Thus,
the term "exogenous" gene or DNA is intended to refer to any gene
or DNA segment that is introduced into a recipient cell, regardless
of whether a similar gene may already be present in such a cell.
The type of DNA included in the exogenous DNA can include DNA that
is already present in the plant cell, DNA from another plant, DNA
from a different organism, or a DNA generated externally, such as a
DNA sequence containing an antisense message of a gene, or a DNA
sequence encoding a synthetic or modified version of a gene.
[0118] The plant transformation vectors containing the promoter
sequences of the present invention may be introduced into plants by
any plant transformation method. Several methods are available for
introducing DNA sequences into plant cells and are well known in
the art. Suitable methods include but are not limited to bacterial
infection, binary bacterial artificial chromosome vectors, direct
delivery of DNA (e.g. via PEG-mediated transformation,
desiccation/inhibition-mediated DNA uptake, electroporation,
agitation with silicon carbide fibers), and acceleration of DNA
coated particles (reviewed in Potrykus, Ann. Rev. Plant Physiol.
Plant Mol. Biol., 42: 205, 1991).
[0119] Methods for specifically transforming dicots primarily use
Agrobacterium tumefaciens. For example, transgenic plants reported
include but are not limited to cotton (U.S. Pat. No. 5,004,863;
U.S. Pat. No. 5,159,135; U.S. Pat. No. 5,518,908, WO 97/43430),
soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011; McCabe
et al., Bio/Technology, 6:923, 1988; Christou et al., Plant
Physiol., 87:671, 1988); Brassica (U.S. Pat. No. 5,463,174), and
peanut (Cheng et al., Plant Cell Rep., 15: 653, 1996).
[0120] Similar methods have been reported in the transformation of
monocots. Transformation and plant regeneration using these methods
have been described for a number of crops including but not limited
to asparagus (Asparagus officinalis; Bytebier et al., Proc. Natl.
Acad. Sci. U.S.A., 84: 5345, 1987); barley (Hordeum vulgarae; Wan
and Lemaux, Plant Physiol., 104: 37, 1994); maize (Zea mays; Rhodes
et al., Science, 240: 204, 1988; Gordon-Kamm et al., Plant Cell, 2:
603, 1990; Fromm et al., Bio/Technology, 8: 833, 1990; Koziel et
al., Bio/Technology, 11: 194, 1993); oats (Avena sativa; Somers et
al., Bio/Technology, 10: 1589, 1992); orchardgrass (Dactylis
glomerata; Horn et al., Plant Cell Rep., 7: 469, 1988); rice (Oryza
sativa, including indica and japonica varieties, Toriyama et al.,
Bio/Technology, 6: 10, 1988; Zhang et al., Plant Cell Rep., 7: 379,
1988; Luo and Wu, Plant Mol. Biol. Rep., 6: 165, 1988; Zhang and
Wu, Theor. Appl. Genet., 76: 835, 1988; Christou et al.,
Bio/Technology, 9: 957, 1991); sorghum (Sorghum bicolor; Casas et
al., Proc. Natl. Acad. Sci. U.S.A., 90: 11212, 1993); sugar cane
(Saccharum spp.; Bower and Birch, Plant J., 2: 409, 1992); tall
fescue (Festuca arundinacea; Wang et al., Bio/Technology, 10: 691,
1992); turfgrass (Agrostis palustris; Zhong et al., Plant Cell
Rep., 13: 1, 1993); wheat (Triticum aestivum; Vasil et al.,
Bio/Technology, 10: 667, 1992; Weeks et al., Plant Physiol., 102:
1077, 1993; Becker et al., Plant, J. 5: 299, 1994), and alfalfa
(Masoud et al., Transgen. Res., 5: 313, 1996). It is apparent to
those of skill in the art that a number of transformation
methodologies can be used and modified for production of stable
transgenic plants from any number of target crops of interest.
[0121] The transformed plants are analyzed for the presence of the
genes of interest and the expression level and/or profile conferred
by the promoter sequences of the present invention. Those of skill
in the art are aware of the numerous methods available for the
analysis of transformed plants. A variety of methods are used to
assess gene expression and determine if the introduced gene(s) is
integrated, functioning properly, and inherited as expected. For
the present invention the promoters can be evaluated by determining
the expression levels of genes to which the promoters are
operatively linked. A preliminary assessment of promoter function
can be determined by a transient assay method using reporter genes,
but a more definitive promoter assessment can be determined from
the analysis of stable plants. Methods for plant analysis include
but are not limited to Southern blots or northern blots, PCR-based
approaches, biochemical analyses, phenotypic screening methods,
field evaluations, and immunodiagnostic assays.
[0122] The methods of the present invention including but not
limited to cDNA library preparation, genomic library preparation,
sequencing, sequence analyses, PCR technologies, vector
construction, transient assays, and plant transformation methods
are well known to those of skill in the art and are carried out
using standard techniques or modifications thereof.
[0123] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention. However, those of skill in
the art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention, therefore all
matter set forth or shown in the accompanying drawings is to be
interpreted as illustrative and not in a limiting sense.
EXAMPLES
Example 1
[0124] Plant Material, DNA Isolation and Library Construction
[0125] The target cDNA libraries included three tassel libraries.
The background cDNA libraries included cDNA libraries prepared from
leaf, root, embryo, callus, shoot, seedling, endosperm, culm, ear,
and silks.
[0126] Plant Growth Conditions
[0127] Seeds are planted at a depth of about 3 cm in soil into
2"-3" pots containing Metro 200 growing medium and transplanted
into larger 10" pots containing the same soil after 2-3 weeks.
Plants were fertilized as needed. A total of about 900 mg Fe is
added to each pot. Corn plants are grown in the greenhouse in 15 hr
day/9 hr night cycles. The daytime temperature is 26.7.degree. C.
and the night temperature is 21.1.degree. C. Lighting is provided
by 1000 W sodium vapor lamps.
[0128] Tissue Isolation
[0129] The corn immature tassel cDNA library (SATMON001) is
generated using maize (B73, Illinois Foundation Seeds, Champaign,
Ill. U.S.A.). The corn plant is at the V6 plant developmental
stage. The tassel is an immature tassel 2-3 cm in length. The
immature tassel is frozen in liquid nitrogen and the harvested
tissue is stored at -80.degree. C. until the RNA is prepared.
[0130] The corn immature tassel cDNA library (SATMON021) is
generated using maize (DK604, Dekalb Genetics, Dekalb Ill.,
U.S.A.). The corn plant is at the V8 plant developmental stage. The
tassels, which are about 15-20 cm in length, are collected and
frozen in liquid nitrogen. The harvested tissue is stored at
-80.degree. C. until the RNA is prepared.
[0131] The corn immature tassel cDNA library (SATMON024) containing
tassel with glume, anthers, and pollen is generated using maize
(DK604, Dekalb Genetics, Dekalb, Ill. U.S.A.). The corn plant is at
the V9 plant developmental stage. The tassel is at the rapidly
developing stage, and a tassel about 37 cm along with the glume,
anthers, and pollen are collected and frozen in liquid nitrogen.
The harvested tissue is stored at -80.degree. C. until the RNA is
prepared.
[0132] The RNA is purified using Trizol reagent available from Life
Technologies (Gaithersburg, Md.) 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 Corporation, Lake Success, N.Y.). Two modifications to the
Trizol protocol include centrifuging the ground tissue samples at
12,000.times.g for 10 minutes at 4.degree. C. after the addition of
Trizol to remove insoluble material, and precipitating the RNA with
0.25 mL isopropanol and 0.25 mL 0.8M NaCl per 1.0 mL Trizol used.
All the samples are precipitated with 0.1 volume of 3M NaOAc and
3.0 volumes of ethanol. RNAs are resuspended in distilled water at
a concentration of 2 .mu.g/.mu.L. The RNAs are DNase treated for 10
minutes at room temperature with RNase free DNase (BMB,
Indianapolis, Ind.) and samples are extracted with
phenol/chloroform and isopropanol precipitated as described, and
resuspended in distilled water at a concentration of 1
.mu.g/.mu.L.
[0133] Construction of 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. The Superscript.TM.
Plasmid System for cDNA synthesis and Plasmid Cloning (Gibco BRL,
Life Technologies, Gaithersburg, Md.) is used, following the
conditions suggested by the manufacturer.
[0134] The cDNA libraries are plated on LB agar containing the
appropriate antibiotics for selection and incubated at 37.degree.
C. for a sufficient time to allow the growth of individual
colonies. Single colonies are individually placed in each well of a
96-well microtiter plate containing LB liquid including 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., Santa Clara, Calif.).
[0135] Template plasmid DNA clones are used for subsequent
sequencing. For sequencing, the ABI PRISM dRhodamine Terminator
Cycle Sequencing Ready Reaction Kit with AmpliTaq.RTM. DNA
Polymerase, FS, is used (PE Applied Biosystems, Foster City,
Calif.).
[0136] After cDNA synthesis, the samples are diluted with one
volume of water, and one microliter is used for each
tissue-specificity PCR assay. Corn cDNAs were synthesized for
tissue specificity testing methods well known in the art include
leaf, root, rachus, early anther, kernals from 6 cm ear, kernals
from 4 cm ears, silk, glumme/lemma/palea, mature anther and pollen,
meristem, microspores, culm, tassel, ear, and husk.
Example 2
[0137] Promoter Lead Identification
[0138] The database of EST sequences derived from the cDNA
libraries prepared from various corn tissues is used to identify
the genes with the correct expression profile from which promoter
candidates can be isolated for expression of operably linked DNA
sequences in male reproductive tissues. The sequences are also used
as query sequences against GenBank databases that contain
previously identified and annotated sequences and searched for
regions of homology using BLAST programs. The selection of
expressed sequence tags (ESTs) for subsequent promoter isolation is
reflective of the presence of one or more sequences among the
representative ESTs from a random sampling of an individual cDNA
library or collection of cDNA libraries. To identify regulatory
sequences that regulate the expression of transcripts in the target
tissues of interest from EST sequences in the database, a
subsetting function is done, requesting ESTs found in target
libraries such as the three tassel libraries and EST clones in all
other libraries were subtracted. The background or non-target
libraries included the following: SATMON013 (corn meristem),
SATMON020 (corn callus), SATMON022 (corn immature ear), SATMON023
(corn ear, growing silks), SATMON025 (corn regenerating callus),
SATMON004 (corn leaf), SATMON009 (corn leaf, V8 stage), SATMON011
(corn leaf, undeveloped), SATMON016 (corn sheath), SATMON026 (corn
leaf), SATMON027 (corn leaf, water stressed 6 days), SATMON031
(corn leaf V4 stage), SATMONN01 (corn leaf normalize), SATMON003
(corn root), SATMON007 (corn primary root), SATMON010 (corn root V8
stage), SATMON028 (corn root, water stressed 6 days), SATMON030
(corn root V4 stage), SATMONN05 (corn root, normalize), SATMON014
(corn endosperm, 14 days after pollination), SATMON017 (corn
embryo, 21 days after pollination), SATMON033 (corn embryo, 13 days
after pollination), SATMON036 (corn endosperm 22 days after
pollination), SATMONN04 (corn embryo, 21-DAP, normalized),
SATMONN06 (corn embryo, 21-DAP, normalized), SATMON008 (corn
primary shoot), SATMON012 (corn seedling, 2 days post-germination),
SATMON019 (corn culm), SATMON029 (corn seedling, etiolated 4 days),
and SATMON034 (corn seedling, cold stressed). The cDNA clones
identified from the subsetting are candidates for
tissue-enhanced/specific genes and are further pursued. Rt-PCR
reactions are performed for each of the leads to determine if a
male-specific/enhanced pattern of expression is observed. The EST
sequences that were detectable predominantly in male tissues are
used to isolate the tissue enhanced/specific promoters.
[0139] Table 1 provides background clone ID (EST) information and
GenBank identifier (gi) information for the ESTs used for
subsequent isolation of the promoter sequences of SEQ ID NOS:
79-98. The promoter leads were all obtained from the SATMON024
library source as described above. Sequence annotation is listed
for the clone IDs based on a GenBank BLAST search with a p-value
cut-off of 10.sup.-8. The information is subject to change as new
sequences are submitted to the sequence databases. The annotations
for the ESTs are listed as follows with the annotation information
in parentheses: Clone ID 700352826); Clone ID 700353038 (Cynodon
dactyloncalcium-binding pollen allergen gene, partial cds; p value
7e-53); Clone ID 700354918 (Phleum pratense mRNA for
polygalacturonase, partial: p-value 1e-08); Clone ID 700353844 (Z.
mays pollen specific mRNA C-terminal (clone 4H7) p-value 6e-26);
Clone ID 700355306 (Holcus lanatus mRNA for major group I allergen
Hol 1 1; p-value le-21)); Clone ID 700353142); Clone ID 700282503
); Clone ID 700282409 (Z. mays ZmPRO1 mRNA for profilin 1; p value
2e-34); Clone ID 700352616); Clone ID 700354681 (Z. mays ZmPRO2
mRNA for profilin 2; p-value e-137); 700353007); 700352625); and
Clone ID 700382630 (Z. mays mRNA for pectin methylesterase-like
protein; p value 1e-08).
1TABLE 1 Promoter Summary Information Clone ID SEQ ID NO. GenBank
Identifier (gi) 700352826 79 none 700353038 80, 81 g1864023
700354918 82 aj238848 700353844 83 x57275 700355306 84 aj012714
700353142 85 none 700282503 86 none 700282409 87 x73279 700282409
88 x73279 700352616 89 none 700354681 90 x73280 700353007 91 none
700352625 92 none 700382630 93 y13285 700352826 94 none 700353038
95, 96, 97, 98 g1864023
Example 3
[0140] Genomic Library Construction, PCR Amplification and Promoter
Isolation
[0141] A number of methods are known to those of skill in the art
for genomic library preparation. For genomic libraries of the
present invention, corn DNA (Maize hybrid Fr27.times.FrMol17) is
isolated by a CsCl purification protocol according to Ausubel et
al. (1992) or by a CTAB purification method (Rogers and Bendich,
Plant Mol. Biol., 5:69, 1988). Reagents are available commercially
(see, for example Sigma Chemical Co., St. Louis, Mo.). The
libraries are prepared according to manufacturer instructions
(GENOME WALKER, a trademark of CLONTECH Laboratories, Inc, Palo
Alto, Calif.). In separate reactions, genomic DNA is subjected to
restriction enzyme digestion overnight at 37.degree. C. with the
following blunt-end endonucleases: EcoRV, ScaI, DraI, PvuII, or
StuI (CLONTECH Laboratories, Inc., Palo Alto, Calif.). The reaction
mixtures are extracted with phenol:chloroform, ethanol
precipitated, and resuspended in Tris-EDTA buffer. The purified
blunt-ended genomic DNA fragments are then ligated to the
GenomeWalker.TM. adaptors and ligation of the resulting DNA
fragments to adaptors were done according to manufacturer protocol.
The GenomeWalker.TM. sublibraries are aliquoted and stored at
-20.degree. C.
[0142] Genomic DNA ligated to the GenomeWalker.TM. adaptor (above)
is subjected to a primary round of PCR amplification with
gene-specific primer 1 (GSP1) and a primer that anneals to the
Adaptor sequence, adaptor primer 1 (AP1) (SEQ ID NO:1). A diluted
(1:50) aliquot of the primary PCR reaction is used as the input DNA
for a nested round of PCR amplification with gene-specific primer 2
(GSP2) and adaptor primer 2 (AP2) (SEQ ID NO:2), or adaptor primer
3 (AP3) (SEQ ID NO:3). The annealing temperatures of the
GenomeWalker.TM. primary primer (AP1) and nested primer (AP2) are
59.degree. C. and 71.degree. C., respectively. Generally, gene
specific primers are designed to have the following
characteristics: 26-30 nucleotides in length, GC content of 40-60%
with resulting temperatures for most of the gene specific primers
in the high 60.degree. C. range or about 70.degree. C. The Taq
polymerase used is Amplitaq Gold.TM. or Expand HiFidelity
(Boehringer Mannheim) available through Perkin-Elmer Biosystems
(Branchbury, N.J.). A number of temperature cycling instruments and
reagent kits are commercially available for performing PCR
experiments and include those available from PE Biosystems (Foster
City, Calif.), Stratagene (La Jolla, Calif.), and MJ Research Inc.
(Watertown, Mass.). Following the primary PCR reaction, an aliquot
is taken (10-15 .mu.L) for agarose gel analysis. Isolation of each
unknown sequence required amplification from 5 sub-genomic
libraries and a negative control (without DNA).
[0143] The PCR components and conditions generally used are
outlined below:
2 PRIMARY PCR Component Amount/Volume required Sub-library aliquot
1 .mu.L Gene-specific primer 1 1 .mu.L (100 pmol) GenomeWalker .TM.
Adaptor primer 1 (AP1) 1 .mu.L dNTP mix (10 mM of each dNTP) 1
.mu.L DMSO 2.5 .mu.L (or 2-5% final concentration) 10X PCR buffer
(containing MgCl.sub.2) 5 .mu.L (final concentration of 1X)
Amplitaq Gold .TM. or Expand HiFidelity 0.5 .mu.L Distilled Water
For final reaction volume of 50 .mu.L Reaction Conditions for
Primary PCR: A. 1 minutes at 95.degree. C. B. 94.degree. C. for 2
seconds, 70.degree. C. for 3 minutes; repeat 94.degree.
C./70.degree. C. cycling for total of 7 times C. 94.degree. C. for
2 seconds, 65.degree. C. for 3 minutes; repeat 94.degree.
C./65.degree. C. cycling for total of 36 times D. 65.degree. C. for
4 minutes as a final extension E. 10.degree. C. for an extended
incubation NESTED PCR (secondary PCR reaction) Component
Amount/Volume Required 1:50 dilution of the primary PCR reaction 1
.mu.L Gene-specific primer 2 1 .mu.L (100 pmol) GenomeWalker .TM.
Adaptor primer 2 or 3 1 .mu.L (AP2 or AP3)) dNTP mix (10 mM of each
dNTP) 1 .mu.L DMSO 2.5 .mu.L 10X PCR buffer (containing MgCl.sub.2)
5 .mu.L (final concentration of 1X) Amplitaq Gold .TM. 0.5 .mu.L
Distilled water to final reaction volume of 50 .mu.L Reaction
Conditions for Nested PCR: A. 1 minutes at 95.degree. C. B.
94.degree. C. for 2 seconds, 70.degree. C. for 3 minutes; repeat
94.degree. C./70.degree. C. cycling for total of 5 times C.
94.degree. C. for 2 seconds, 65.degree. C. for 3 minutes; repeat
94.degree. C./65.degree. C. cycling for total of 24 times D.
65.degree. C. for 4 minutes as a final extension E. 10.degree. C.
for an extended incubation For RT-PCR the corn inbred line H99 is
used and the generic PCR reaction conditions are outlined below: 1
uL cDNA 5 uL 10x BMB PCR reaction buffer (supplied with taq DNA
polymerase) 1 uL 10 mM dNTPs 1 uL 10 uM primer 1 1 uL 10 uM primer
2 0.5 uL taq DNA polymerase (BMB, Indianapolis, IN) 40.5 uL H2O 1
uL DMSO (optional)
[0144] 3a. 700353038 Clone ID Analysis and Promoter Isolation
[0145] To determine the distribution of the clone ID 700353038
transcripts in corn, RT-PCR is performed using the SEQ ID NO:4 and
SEQ ID NO:5 primers following a standard RT-PCR protocol using cDNA
derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel from
4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, or silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 94.degree. C. 1 minute and 35
cycles of 94.degree. C. 5 seconds, 52.degree. C. 30 seconds and
72.degree. C. 30 seconds. PCR products are amplified for this clone
using cDNA derived from anther, glume/lemma/palea, microspores, and
pollen but not with cDNA derived from ear, husk, kernel, meristem,
rachis, leaf, root, or silk.
[0146] For the isolation of the clone ID 700353038 promoter, SEQ ID
NO:14 is used in combination with SEQ ID NO:1 in a standard
GenomeWalker.TM. PCR reaction with the following conditions: Expand
Hi Fidelity DNA Polymerase (BMB Indianapolis, Ind.) is used in
conjunction with the supplied buffer #2, 2 .mu.L of a 1:2 dilution
of GenomeWalker.TM. libraries made according to the manufacturer's
protocol (Clontech, Palo Alto, Calif.) and made with maize genomic
DNA. The following cycling parameters are used: 94.degree. C. 1
minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3 min,
and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3 minutes. For
the nested secondary PCR reaction, 1 .mu.L of the primary reaction
was used with SEQ ID No: 15 and SEQ ID NO: 3 (AP3) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB Indianapolis, Ind.) with the supplied buffer #2.
The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4
seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4
seconds and 67.degree. C. 3 minutes.
[0147] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis, and bands of approximately
900 bp and 800 bp are cut out, purified using the Qiaquick gel
extraction kit (Qiagen, Valencia, Calif.) and eluted with 30 .mu.L
10 mM Tris pH. 8.5. Five microliters of the purified band is
ligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.).
DNA from individual clones are isolated using the Qiagen Plasmid
Mini kit (Qiagen, Valencia, Calif.) and sequenced using the M13
forward primers and M13 reverse primers. Sequence analysis
indicated the two fragments shared 3' homology but diverged at
their 5' end. The promoter sequences are shown in SEQ ID NO:80 and
SEQ ID NO: 81.
[0148] To determine if the large open reading frames identified in
the isolated genomic fragments are transcribed, RT-PCR is performed
using the following primer pairs: 1. SEQ ID NOS:6 and 7; 2. SEQ ID
NOS:8 and 9; 3. SEQ ID NOS:10 and 11; and 4. SEQ ID NOS:12 and 13.
RT-PCR is performed using a standard RT-PCR protocol using cDNA
derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel from
4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, or silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 94.degree. C. 1minute and 35
cycles of 94.degree. C. 5 seconds, 52.degree. C. 30 seconds and
72.degree. C. 30 seconds.
[0149] Two "ATG" sequences are identified, in frame with an ORF in
the EST sequence. Primers are designed to the sequences 5' to each
of these putative start codons (SEQ ID NO:16 and SEQ ID NO:17). To
isolate the promoter fragment and 5' leader sequence by PCR, the
SEQ ID NO:16 primer and the SEQ ID NO:17 primer are separately used
with the AP3 primer (SEQ ID NO:3) in a standard PCR reaction
containing 1 .mu.L of DNA from either the 800 bp or 900 bp clone ID
700353038 promoter fragment, using 0.5 uL BMB taq polymerase and
the supplied buffer. The cycling parameters are as follows:
94.degree. C. 1 minute, 20 cycles of 94.degree. C. 5 seconds
55.degree. C. 15 seconds and 72.degree. C. 30 seconds.
[0150] The amplified promoter fragments are analyzed by agarose gel
electrophoresis, and bands of approximately 850 bp and 750 bp are
cut out and purified using the Qiaquick gel extraction kit (Qiagen,
Valencia, Calif.) following the conditions recommended by the
manufacturer. The resulting bands are designated SEQ ID NOS: 95 and
96 (amplified from SEQ ID NO: 81), and SEQ ID NOS: 97 and 98
(amplified from SEQ ID NO: 80). The fragments are eluted with 30
.mu.L 10 mM Tris pH. 8.5. Five microliter of the purified band is
ligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.).
DNA from individual clones is isolated using the Qiagen Plasmid
Mini kit (Qiagen, Valencia, Calif.). The DNA is digested with Hind
III and Bgl II, and the resulting promoter fragments are analyzed
by agarose gel electrophoresis and bands of approximately 850 bp
and 750 bp are cut out, purified using the Qiaquick gel extraction
kit (Qiagen, Valencia, Calif.) and eluted with 30 .mu.L 10 mM Tris
pH. 8.5. Five microliter of the purified band is ligated to 1 .mu.L
of an expression vector such as pMON19469 shown in FIG. 1, that is
prepared by digesting 10 .mu.g with BglII and HindIII, separating
by agarose gel electrophoresis and purified using the Qiaquick gel
extraction kit (Qiagen, Valencia, Calif.) and eluted with 30 .mu.L
10 mM Tris pH. 8.5. The resulting plasmids contain the promoter
fragments, hsp70 intron, and GUS gene and are used to assay
promoter activity of the promoter fragments.
[0151] 3b. 700352826 Clone ID Analysis and Promoter Isolation
[0152] To determine the distribution of the clone ID 700352826
transcripts in corn, RT-PCR was performed using the SEQ ID NO:18
and SEQ ID NO:19 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 94.degree. C. 1 minute and 35
cycles of 94.degree. C. 5 seconds, 52.degree. C. 30 seconds and
72.degree. C. 30 seconds. PCR products are obtained with cDNA
derived from anther, glume/lemma/palea, micropsores, and pollen but
not with cDNA derived from ear, husk, kernel, meristem, rachis,
leaf, root, or silk.
[0153] For the isolation of the clone ID 700352826 promoter, SEQ ID
NO:20 is used in combination with SEQ ID NO:1 AP1 in a standard
GenomeWalker.TM. PCR reaction with the following conditions: Expand
Hi Fidelity DNA Polymerase (BMB Indianapolis, Ind.) is used in
conjunction with the supplied buffer #2, 2 .mu.L of a 1:2 dilution
of GenomeWalker.TM. libraries made according to the manufacturer's
protocol (Clontech, Palo Alto, Calif.) and made with maize genomic
DNA. The following cycling parameters are used: 94.degree. C. 1
minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3 min,
and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3 minutes. For
the nested secondary PCR reaction, 1 .mu.L of the primary reaction
is used with SEQ ID NO:21 and SEQ ID NO:3 (AP3) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB Indianapolis, Ind.) with the supplied buffer #2.
The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4
seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4
seconds and 67.degree. C. 3 minutes.
[0154] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis and bands of approximately
500 bp and 3000 bp are cut out, purified using the Qiaquick gel
extraction kit (Qiagen, Valencia, Calif.) and eluted with 30 .mu.L
10 mM Tris pH. 8.5. Five microliters of the purified bands is
ligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.).
DNA from individual clones is isolated using the Qiagen Plasmid
Mini kit (Qiagen, Valencia, Calif.) and is sequenced using M13
forward primers and M13 reverse primers.
[0155] The sequence for clone ID 700352826 is derived from a
truncated cDNA. A BLAST comparison of the promoter sequence against
the GenBank database identified polygalacturase clones that allowed
the determination of the translational initiation codon. SEQ ID
NO:22 is designed just upstream of the predicted ATG. To isolate
the functional promoter fragment and 5' leader sequence by PCR of
clone ID 700352826, the SEQ ID NO:22 primer and SEQ ID NO: 3
(AP3)primer are used in a standard PCR reaction containing 1 .mu.L
of cloned DNA containing clone ID 700352826 promoter fragment,
using BMB taq polymerase and the supplied buffer. The cycling
parameters are as follows: 94.degree. C. 1 minute, 20 cycles of
94.degree. C. 5 seconds 55.degree. C. 15 seconds and 72.degree. C.
30 seconds. The sequence of the promoter is SEQ ID NO:79.
[0156] The amplified promoter fragments are analyzed by agarose gel
electrophoresis and purified using the Qiaquick gel extraction kit
(Qiagen, Valencia, Calif.) and eluted with 30 .mu.L 10 mM Tris pH.
8.5 and subsequently cloned into a plasmid as shown in FIG. 1
(pMON19469).
[0157] 3c. 700354918 Clone Analysis and Promoter Isolation
[0158] To determine the distribution of the clone ID 700354918
transcripts in corn, RT-PCR is performed using the SEQ ID NO:23 and
SEQ ID NO:24 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 94.degree. C. 1 minute and 35
cycles of 94.degree. C. 5 seconds, 52.degree. C. 30 seconds and
72.degree. C. 30 seconds. PCR products are produced with cDNA
derived from anther, glume/lemma/palea, micropsores, and pollen but
not with cDNA derived from ear, husk, kernel, meristem, rachis,
root, leaf, or silk.
[0159] For the isolation of the clone ID 700354918 promoter, SEQ ID
NO:25 is used in combination with SEQ ID NO:1 (AP1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 2 .mu.L of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.) using maize
genomic DNA. The following cycling parameters are used: 94.degree.
C. 1 minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3
min, and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3
minutes. For the nested, secondary PCR reaction, 1 .mu.L of the
primary reaction is used with SEQ ID NO:26 and SEQ ID NO:3 (AP3) in
a standard GenomeWalker.TM. PCR reaction using Expand Hi Fidelity
DNA Polymerase (BMB, Indianapolis, Ind.) with the supplied buffer
#2 . The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4
seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4
seconds and 67.degree. C. 3 minutes.
[0160] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis and a 700 bp band is
isolated, purified using the Qiaquick gel extraction kit (Qiagen,
Valencia, Calif.), and eluted with 30 .mu.L 10 mM Tris pH. 8.5.
Five microliters of the purified band is ligated to 50 ng of
pGEM-T-Easy vector (Promega, Madison, Wis.). DNA from individual
clones is isolated using the Qiagen Plasmid Mini kit (Qiagen,
Valencia, Calif.) and sequenced using the M13 forward primers and
M13 reverse primers.
[0161] The clone ID 700354918 sequence is derived from a truncated
cDNA and the translation initiation codon was unknown. A BLAST of
the 5' end of the promoter against the GenBank database produced
polygalacturase clones that allowed the prediction of the
translational initiation codon 67 nucleotides from the 5' end of
promoter. To isolate the functional promoter fragment and 5' leader
sequence of clone ID 700354918 by PCR, SEQ ID NO:27 is used in
combination with SEQ ID NO: 1 (AP1) in a standard GenomeWalker.TM.
PCR reaction using Expand Hi Fidelity DNA Polymerase (BMB,
Indianapolis, Ind.) in conjunction with the supplied buffer #2.
Each reaction contains 2 .mu.L of a 1:2 dilution of
GenomeWalker.TM. libraries made according to the manufacturer's
protocol (Clontech, Palo Alto, Calif.) and made with maize genomic
DNA. The following cycling parameters are used: 94.degree. C. 1
minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3 min,
and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3 minutes. For
the nested secondary PCR reaction, 1 .mu.L of the primary reaction
is used with SEQ ID NO:28 and SEQ ID NO:3 (AP3) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2.
The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4
seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4
seconds and 67.degree. C. 3 minutes.
[0162] The PCR products are purified by gel electrophoresis as
described and cloned into a vector such as shown in FIG. 1
(pMON19469). The promoter sequence is shown in SEQ ID NO:82.
[0163] 3d. 700353844 Clone Analysis and Promoter Isolation
[0164] To determine the distribution of the clone ID 700353844
transcripts in corn, RT-PCR is performed using the SEQ ID NO:29 and
SEQ ID NO:30 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 94.degree. C. 1 minute and 35
cycles of 94.degree. C. 5 seconds, 52.degree. C. 30 seconds and
72.degree. C. 30 seconds. PCR products are obtained with cDNA
derived from anther, glume/lemma/palea, micropsores, and pollen but
not with cDNA derived from ear, husk, kernel, meristem, rachis,
leaf, root, or silk.
[0165] For the isolation of the clone ID 700353844 promoter, SEQ ID
NO:31 is used in combination with SEQ ID NO:1 (AP1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 2 .mu.L of a 1:2
dilution of the Genome Walker libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.) using maize
genomic DNA. The following cycling parameters are used: 94.degree.
C. 1 minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3
min, and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3
minutes. For the nested, secondary PCR reaction, 1 .mu.L of the
primary reaction is used with SEQ ID NO:32 and SEQ ID NO:3 (AP3) in
a standard GenomeWalker.TM. PCR reaction using Expand Hi Fidelity
DNA Polymerase (BMB Indianapolis, Ind.) with the supplied buffer #2
. The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4
seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4
seconds and 67.degree. C. 3 minutes.
[0166] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis and a 500 bp band is
isolated, purified using the Qiaquick gel extraction kit (Qiagen,
Valencia, Calif.) and eluted with 30 .mu.L 10 mM Tris pH. 8.5. Five
microliters of the purified band is ligated to 50 ng of pGEM-T-Easy
vector (Promega, Madison, Wis.). DNA from 2 individual clones is
isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,
Calif.) and sequenced using the M13 forward primers and M13 reverse
primers. The sequence of the promoter fragment is shown in SEQ ID
NO:83. The promoter fragment is subsequently cloned into a plasmid
as shown in FIG. 1 (pMON19469).
[0167] 3e. 700355306b Clone Analysis and Promoter Isolation
[0168] To determine the distribution of the clone ID 700355306b
transcripts in corn, RT-PCR is performed using the SEQ ID NO:33 and
SEQ ID NO:34 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 95.degree. C. 1 minute and 35
cycles of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by a 10-minute incubation at 72
C. Bands are amplified with cDNA derived from anther, micropsores,
and pollen but not with cDNA derived from ear, husk,
glume/lemma/palea, kernel, meristem, rachis, leaf, root, or
silk.
[0169] For the isolation of the clone ID 700355306b promoter, SEQ
ID NO:35 is used in combination with SEQ ID NO:1 (AP1) in a
standard GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 2 .mu.L of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.) using maize
genomic DNA. The following cycling parameters are used: 94.degree.
C. 1 minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3
min, and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3
minutes. For the nested, secondary PCR reaction, 1 .mu.L of the
primary reaction is used with SEQ ID NO: 36 and SEQ ID NO:2 (AP2)
in a standard GenomeWalker.TM. PCR reaction using Expand Hi
Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with the supplied
buffer #2 . The reactions are carried out under the following
cycling conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree.
C. 4 seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree.
C. 4 seconds and 67.degree. C. 3 minutes followed by 7 minutes at
67.degree. C.
[0170] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis bands are isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.), and eluted with 30 .mu.L 10 mM Tris pH. 8.5. To add a
HindIII restriction site to the 5' end of the 700355306b promoter
fragment, 1 .mu.L of the isolated DNA is amplified under standard
GenomeWalker.TM. PCR conditions using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2 in
combination with primers SEQ ID NO:37 and SEQ ID NO:3 (AP3). The
reactions are carried out under the following cycling conditions:
94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4 seconds,
72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4 seconds
and 67.degree. C. 3 minutes followed by 7 minutes at 67.degree.
C.
[0171] Twenty five microliters of the tertiary PCR reaction is
analyzed by agarose gel electrophoresis, the promoter band is
isolated, purified using the Qiaquick gel extraction kit (Qiagen,
Valencia, Calif.) and eluted with 30 .mu.L 10 mM Tris pH. 8.5. Five
microliters of the purified band is ligated to 50 ng of pGEM-T-Easy
vector (Promega, Madison, Wis.). DNA from individual clones is
isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,
Calif.) and sequenced by the using the M13 forward primers and M13
reverse primers. The sequence of the promoter fragment is SEQ ID
NO:84. The promoter fragment is subsequently cloned into a plasmid
as shown in FIG. 1 (pMON19469).
[0172] 3f. 700353142 Clone Analysis and Promoter Isolation
[0173] To determine the distribution of the clone ID 700353142
transcripts in corn, RT-PCR is performed using the SEQ ID NO:38 and
SEQ ID NO:39 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 95.degree. C. 1 minute and 35
cycles of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by a 10-minute incubation at
72.degree. C. Bands are amplified with cDNA derived from anther,
micropsores, glume/lemma/palea, husk, meristem, and pollen but not
with cDNA derived from ear, kernel, rachis, leaf, root, or
silk.
[0174] For the isolation of the clone ID 700353142 promoter, SEQ ID
NO:40 is used in combination with SEQ ID NO:1 (AP1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contained 2 .mu.L of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.) using maize
genomic DNA. The following cycling parameters are used: 94.degree.
C. 1 minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3
min, and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3
minutes. For the nested, secondary PCR reaction, 1 .mu.L of the
primary reaction is used with SEQ ID NO:41 and SEQ ID NO:2 (AP2) in
a standard GenomeWalker.TM. PCR reaction using Expand Hi Fidelity
DNA Polymerase (BMB Indianapolis, Ind.) with the supplied buffer #2
. The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4
seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4
seconds and 67.degree. C. 3 minutes followed by 7 minutes at
67.degree. C.
[0175] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis, and PCR products are
isolated, purified using the Qiaquick gel extraction kit (Qiagen,
Valencia, Calif. cat # 28704) and eluted with 30 .mu.L 10 mM Tris
pH. 8.5. To add a HindIII restriction site to the 5' end of the
700353142 promoter fragment, 1 .mu.L of the isolated DNA is
amplified under standard GenomeWalker.TM. PCR conditions using
Expand Hi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with
the supplied buffer #2 in combination with primers SEQ ID NO:42 and
SEQ ID NO:3 (AP3). The reactions are carried out under the
following cycling conditions: 94.degree. C. 1 minute, 5 cycles of
94.degree. C. 4 seconds, 72.degree. C. 3 minutes, and 20 cycles of
94.degree. C. 4 seconds and 67.degree. C. 3 minutes followed by 7
minutes at 67.degree. C.
[0176] Twenty five microliters of the tertiary PCR reaction is
analyzed by agarose gel electrophoresis. The promoter band is
isolated, purified using the Qiaquick gel extraction kit (Qiagen,
Valencia, Calif.), and eluted with 30 .mu.L 10 mM Tris pH. 8.5.
Five microliters of the purified band is ligated to 50 ng of
pGEM-T-Easy vector (Promega, Madison, Wis.). DNA from individual
clones is isolated using the Qiagen Plasmid Mini kit (Qiagen,
Valencia, Calif.) and sequenced using the M13 forward primers and
M13 reverse primers. The sequence of the promoter is SEQ ID
NO:85
[0177] 3g. 700282503 Clone Analysis and Promoter Isolation
[0178] To determine the distribution of the clone ID 700282503
transcripts in corn, 2 RT-PCR experiments are performed. In one
experiment SEQ ID NO: 43 and SEQ ID NO:44 primers are used
following a standard RT-PCR protocol using cDNA derived from
anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm ears,
leaf, meristem, mature anther/pollen, microspores, rachis, root,
and silk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is used
in combination with the supplied reaction buffer. Cycling
parameters are as follows: 95.degree. C. for 1 minute and 35 cycles
of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by a 10-minute incubation at
72.degree. C. Bands are amplified with cDNA derived from anther,
micropsores, glume/lemma/palea, husk, kernal, and pollen but not
with cDNA derived from ear, rachis, leaf, root, meristem, or silk.
In the second RT-PCR reaction SEQ ID NO:43 and SEQ ID NO:44 primers
are used following a standard RT-PCR protocol using cDNA derived
from anther, 6 cm ear, glume/lemma/palea, husk, kernel from 4 cm
ears, leaf, meristem, mature anther/pollen, microspores, rachis,
root, and silk. Taq DNA polymerase from BMB (Indianapolis, Ind.) is
used in combination with the supplied reaction buffer. Cycling
parameters, are as follows: 95.degree. C. 1 minute and 35 cycles of
95.degree. C. 15 seconds, 50.degree. C. 30 seconds and 72.degree.
C. 30 seconds followed by a 10-minute incubation at 72.degree. C.
Bands are amplified with cDNA derived from anther, kernal, and
pollen but not with cDNA derived from ear, rachis, leaf, root,
glume/lemma/palea, microspore, husk, meristem, or silk.
[0179] For the isolation of the clone ID 700282503 promoter, SEQ ID
NO:46 is used in combination with SEQ ID NO: 1 (AP 1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 2 .mu.l of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.) using maize
genomic DNA. The following cycling parameters are used: 94.degree.
C. 1 minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3
min, and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3
minutes. For the nested, secondary PCR reaction, 1 .mu.L of the
primary reaction was used with SEQ ID NO:47 and SEQ ID NO:2 (AP2)
in a standard GenomeWalker.TM. PCR reaction using Expand Hi
Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with the supplied
buffer #2. The reactions are carried out under the following
cycling conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree.
C. 4 seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree.
C. 4 seconds and 67.degree. C. 3 minutes followed by 7 minutes at
67.degree. C.
[0180] Twenty five microliters of the secondary PCR reaction was
analyzed by agarose gel electrophoresis and PCR products are
isolated, purified using the Qiaquick gel extraction kit (Qiagen,
Valencia,Calif.) and eluted with 30 .mu.L 10 mM Tris pH. 8.5. To
add a HindIII restriction site to the 5' end of the 700282503
promoter fragment, 1 .mu.L of the isolated DNA is amplified under
standard Genome Walker.TM.PCR conditions using Expand Hi Fidelity
DNA Polymerase (BMB Indianapolis, Ind.) with the supplied buffer #2
in combination with primers SEQ ID NO:48 and SEQ ID No:3 (AP3). The
reactions are carried out under the following cycling conditions:
94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4 seconds,
72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4 seconds
and 67.degree. C. 3 minutes followed by 7 minutes at 67.degree.
C.
[0181] Twenty five microliters of the tertiary PCR reaction is
analyzed by agarose gel electrophoresis. A band is isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.) and eluted with 30 .mu.L 10 mM Tris pH. 8.5. 5 .mu.L of the
purified band is ligated to 50 ng of pGEM-T-Easy vector (Promega,
Madison, Wis., Cat. #A1360). DNA from individual clones was
isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,
Calif. Cat. #12125) and sequenced using the M13 forward primers and
M13 reverse primers. The sequence of the promoter is SEQ ID
NO:86.
[0182] 3h. 700282409 Clone Analysis and Promoter Isolation
(Profilin 2)
[0183] To determine the distribution of the clone ID 700282409
transcripts in corn, RT-PCR is performed using the SEQ ID NO:49 and
SEQ ID NO:50 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 95.degree. C. for 1 minute and
35 cycles of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by a 10 minute incubation at
72.degree. C. Bands are amplified with cDNA derived from anther,
glume/lemma/palea, meristem, microspore, silk, and pollen but not
with cDNA derived from ear, husk, kernel, rachis, leaf, or
root.
[0184] For the isolation of the clone ID 700282409 promoter, SEQ ID
NO:51 is used in combination with SEQ ID NO:1 (AP1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 2 .mu.L of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.) using maize
genomic DNA. The following cycling parameters are used: 94.degree.
C. 1 minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3
min, and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3
minutes. For the nested, secondary PCR reaction, 1 .mu.L of the
primary reaction is used with SEQ ID NO:52 and SEQ ID No. 2 (AP2)
in a standard GenomeWalker.TM. PCR reaction using Expand Hi
Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with the supplied
buffer #2. The reactions are carried out under the following
cycling conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree.
C. 4 seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree.
C. 4 seconds and 67.degree. C. 3 minutes followed by 7 minutes at
67.degree. C.
[0185] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis. The bands are isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.) and eluted with 30 .mu.L 10 mM Tris pH. 8.5. To add a
HindIII restriction site to the 5' end of the 700282409 promoter
fragment, 1 .mu.L of the isolated DNA is amplified under standard
Genome Walker.TM. PCR conditions using Expand Hi Fidelity DNA
Polymerase (BMB Indianapolis, Ind.) with the supplied buffer #2 in
combination with primers SEQ ID NO:53 and SEQ ID NO:3 (AP3). The
reactions are carried out under the following cycling conditions:
94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4 seconds,
72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4 seconds
and 67.degree. C. 3 minutes followed by 7 minutes at 67.degree.
C.
[0186] Twenty five microliters of the tertiary PCR reaction is
analyzed by agarose gel electrophoresis. A band is isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.) and eluted with 30 .mu.L 10 mM Tris pH. 8.5. Five
microliters of the purified band is ligated to 50 ng of pGEM-T-Easy
vector (Promega, Madison, Wis.). DNA from individual clones is
isolated using the Qiagen Plasmid Mini kit (Qiagen, Valencia,
Calif.) and sequenced using the M13 forward primers and M13 reverse
primers. The sequence designated profilin 2 is shown in SEQ ID
NO:87.
[0187] 3i. 700282409 Clone Analysis and Promoter Isolation
(Profilin 1)
[0188] All RT-PCR data for 700282409--profilin 1 is the same as the
information for profilin 2 as described in Example 3h.
[0189] For the isolation of the clone ID 700282409 promoter, SEQ ID
NO:51 is used in combination with SEQ ID NO:1 (AP1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 1.5 .mu.L of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.) using maize
genomic DNA. The following cycling parameters are used: 94.degree.
C. 1 minute, 7 cycles of 94.degree. C. 4 seconds, 70.degree. C. 3
min, and 33 cycles 94.degree. C. 4 seconds, 68.degree. C. 3
minutes. For the nested, secondary PCR reaction, 1 .mu.L of the
primary reaction is used with SEQ ID NO:54 with SEQ ID No:3 (AP3).
The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 4
seconds, 72.degree. C. 3 minutes, and 20 cycles of 94.degree. C. 4
seconds and 67.degree. C. 3 minutes followed by 7 minutes at
67.degree. C.
[0190] Twenty five microliters of the tertiary PCR reaction is
analyzed by agarose gel electrophoresis. A is isolated, purified
using the Qiaquick gel extraction kit (Qiagen, Valencia, Calif.)
and eluted with 30 .mu.L 10 mM Tris pH. 8.5. Five microliters of
the purified band is ligated to 50 ng of pGEM-T-Easy vector
(Promega, Madison, Wis.). DNA from individual clones is isolated
using the Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.) and
sequenced using the M13 forward primers and M13 reverse primers.
The fall length promoter sequence is shown in SEQ ID NO:88.
[0191] 3j. 700352616 Clone Analysis and Promoter Isolation
[0192] To determine the distribution of the clone ID 700352616
transcripts in corn, RT-PCR is performed using the SEQ ID NO:55 and
SEQ ID NO:56 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 95.degree. C. 1 minute and 35
cycles of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by 10 minutes at 72.degree. C.
Bands are amplified with cDNA derived from anther,
glume/lemma/palea, and microspore but not with cDNA derived from
ear, husk, kernel, rachis, leaf, pollen, silk, or root.
[0193] For the isolation of the clone ID 700352616 promoter, SEQ ID
NO:58 is used in combination with SEQ ID NO:1 (AP1) in a standard
Genome Walker PCR reaction using Expand Hi Fidelity DNA Polymerase
(BMB, Indianapolis, Ind.) in conjunction with the supplied buffer
#2. Each reaction contains 2 .mu.L of a 1:2 dilution of the
GenomeWalker.TM. libraries made according to the manufacturer's
protocol (Clontech, Palo Alto, Calif.). The following cycling
parameters are used: 94.degree. C. 1 minute, 7 cycles of 94.degree.
C. 2 seconds, 72.degree. C. 3 min, and 36 cycles 94.degree. C. 2
seconds, 66.degree. C. 3 minutes followed by a 4-minute incubation
at 66.degree. C. For the nested, secondary PCR reaction, 1 .mu.L of
a 1:50 dilution of the primary reaction is used with SEQ ID NO:59
and SEQ ID NO:2 (AP2) in a standard GenomeWalker.TM. PCR reaction
using Expand Hi Fidelity DNA Polymerase (BMB Indianapolis, Ind.)
with the supplied buffer #2. The reactions are carried out under
the following cycling conditions: 94.degree. C. 1 minute, 5 cycles
of 94.degree. C. 2 seconds, 72.degree. C. 3 minutes, and 25 cycles
of 94.degree. C. 2 seconds and 67.degree. C. 3 minutes followed by
4 minutes at 67.degree. C.
[0194] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis. The bands are isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.), and eluted with 30 .mu.L ddH2O. Five microliters of the
purified band is ligated to 50 ng of pGEM-T-Easy vector (Promega,
Madison, Wis.). DNA from individual clones is isolated using the
Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.) and sequenced
using the M13 forward primers and M13 reverse primers.
[0195] To add a HindIII restriction site to the 5' end and a BamH1
site at the 3' end of the 700352616 promoter fragment, 1 .mu.L of
the isolated DNA is amplified under standard Genome Walker.TM. PCR
conditions using Expand Hi Fidelity DNA Polymerase (BMB,
Indianapolis, Ind.) with the supplied buffer #2 in combination with
primers SEQ ID NO:57 and SEQ ID NO.3 (AP3). The reactions are
carried out under the following cycling conditions: 94.degree. C. 1
minute, 3 cycles of 94.degree. C. 2 seconds,70.degree. C. 3 minutes
and 12 cycles 94.degree. C. 2 seconds 67.degree. C. 3 minutes
followed by a 4-minute incubation at 67.degree. C. Twenty five
microliters of this PCR reaction is analyzed by agarose gel
electrophoresis. The bands are isolated, purified using the
Qiaquick gel extraction kit (Qiagen, Valencia, Calif.), and eluted
with 30 .mu.L ddH2O. Five microliters of the purified band is
ligated to 50 ng of pGEM-T-Easy vector (Promega, Madison, Wis.).
DNA from individual clones is isolated using the Qiagen Plasmid
Mini kit (Qiagen, Valencia, Calif.). The promoter sequence is shown
in SEQ ID NO:89.
[0196] The promoter is cloned into a plasmid vector such as shown
in FIG. 1 (pMON19469).
[0197] 3k. 700354681 Clone Analysis and Promoter Isolation
[0198] To determine the distribution of the clone ID 700354681
transcripts in corn, RT-PCR is performed using the SEQ ID NO:60 and
SEQ ID NO:61 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 95.degree. C. 1 minute and 35
cycles of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by 10 minutes at 72.degree. C.
Bands are amplified with cDNA derived from anther, pollen, and
microspore but not with cDNA derived from ear, glume/lemma/palea,
husk, kernel, rachis, leaf, pollen, silk, or root.
[0199] For the isolation of the clone ID 700354681 promoter, SEQ ID
NO:62 is used in combination with SEQ ID NO:1 (AP1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 2 .mu.L of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.). The
following cycling parameters are used: 94.degree. C. 1 minute, 7
cycles of 94.degree. C. 2 seconds, 72.degree. C. 3 min, and 36
cycles 94.degree. C. 2 seconds, 66.degree. C. 3 minutes followed by
a 4-minute incubation at 66.degree. C. For the nested, secondary
PCR reaction, 1 .mu.L of a 1:50 dilution of the primary reaction is
used with SEQ ID NO:63 and SEQ ID NO:2 (AP2) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2.
The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 2
seconds, 72.degree. C. 3 minutes, and 25 cycles of 94.degree. C. 2
seconds and 67.degree. C 3 minutes followed by 4 minutes at
67.degree. C.
[0200] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis. The bands are isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.) and eluted with 30 .mu.L ddH2O. Five microliters of the
purified band is ligated to 50 ng of pGEM-T-Easy vector (Promega,
Madison, Wis.). DNA from individual clones is isolated using the
Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.) and sequenced
using the M13 forward primers and M13 reverse primers.
[0201] To add a HindIII restriction site to the 5' end and Bgl II,
BamH1 sites at the 3' end of the 700354681 promoter fragment, 1
.mu.L of the clone harboring the promoter sequence is amplified
under standard GenomeWalker.TM. PCR conditions using Expand Hi
Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with the supplied
buffer #2 in combination with primers SEQ ID NO:64 and SEQ ID NO:3
(AP3). The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 3 cycles of 94.degree. C. 2
seconds, 70.degree. C. 3 minutes and 12 cycles of 94.degree. C. 2
seconds 67.degree. C. 3 minutes followed by a 4-minute incubation
at 67.degree. C. A 25 .mu.L aliquot of this PCR reaction is
analyzed by agarose gel electrophoresis. The bands are isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.), and eluted with 30 .mu.L ddH2O. Five microliters of the
purified band is ligated to 50 ng of pGEM-T-Easy vector (Promega,
Madison, Wis.). DNA from individual clones is isolated using the
Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.). SEQ ID NO:90 is
the promoter sequence for clone ID 700354681 Sequence analysis
shows SEQ ID NO:90 to be a fragment of SEQ ID NO: 87.
[0202] The promoter band is purified using the Qiaquick gel
extraction kit (Qiagen, Valencia, Calif.), and eluted with 30 .mu.L
ddH.sub.2O. An aliquot of the purified band is ligated to an
expression vector as shown in FIG. 1 (pMON19469) that is prepared
for example by digesting 10 .mu.g with BglII and HindIII,
separating by agarose gel electrophoresis and isolating the vector
band using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.), and eluted with 30 .mu.L 10 mM Tris pH 8.5.
[0203] 31. 700353007 Clone Analysis and Promoter Isolation
[0204] To determine the distribution of the clone ID 700353007
transcripts in corn, RT-PCR is performed using the SEQ ID NO:65 and
SEQ ID NO:66 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 95.degree. C. for 1 minute and
35 cycles of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by 10 minutes at 72.degree. C.
Bands are amplified with cDNA derived from anther,
glume/lemma/palea, pollen, and microspore but not with cDNA derived
from ear, husk, kernel, rachis, leaf, silk, or root.
[0205] For the isolation of the clone ID 700353007 promoter, SEQ ID
NO:67 is used in combination with SEQ ID NO:1 AP1 in a standard
Genome Walker PCR reaction using Expand Hi Fidelity DNA Polymerase
(BMB, Indianapolis, Ind.) in conjunction with the supplied buffer
#2. Each reaction contains 2 .mu.L of a 1:2 dilution of the
GenomeWalker.TM. libraries made according to the manufacturer's
protocol (Clontech, Palo Alto, Calif.). The following cycling
parameters are used: 94.degree. C. 1 minute, 7 cycles of 94.degree.
C. 2 seconds, 72.degree. C. 3 min, and 36 cycles 94.degree. C. 2
seconds, 66.degree. C. 3 minutes followed by 4 minute incubation at
66.degree. C. For the nested, secondary PCR reaction, 1 .mu.L of a
1:50 dilution of the primary reaction was used with SEQ ID NO:68
and SEQ ID NO:2 (AP2) in a standard GenomeWalker.TM.PCR reaction
using Expand Hi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.)
with the supplied buffer #2. The reactions are carried out under
the following cycling conditions: 94.degree. C. for 1 minute, 5
cycles of 94.degree. C. 2 seconds, 72.degree. C. 3 minutes, and 25
cycles of 94.degree. C. 2 seconds and 67.degree. C. 3 minutes
followed by 4 minutes at 67.degree. C.
[0206] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis. The bands are isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.) and eluted with 30 .mu.l ddH2O. Five microliters of the
purified band is ligated to 50 ng of pGEM-T-Easy vector (Promega,
Madison, Wis.). DNA from individual clones is isolated using the
Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.), and sequenced
using the M13 forward primers and M13 reverse primers.
[0207] To add a HindIII restriction site to the 5' end and BglII,
BamH1 sites at the 3' end of the 700353007 promoter fragment, 1
.mu.L of the isolated DNA is amplified under standard Genome
Walker.TM. PCR conditions using Expand Hi Fidelity DNA Polymerase
(BMB, Indianapolis, Ind.) with the supplied buffer #2 in
combination with primers SEQ ID NO:69 and SEQ ID NO:3 (AP3). The
reactions are carried out under the following cycling conditions:
94.degree. C. 1 minute, 3 cycles of 94.degree. C. 2
seconds,70.degree. C. 3 minutes and 12 cycles 94.degree. C. 2
seconds 67.degree. C. 3 minutes followed by 4-minute incubation at
67.degree. C. A 25 .mu.L aliquot of this PCR reaction is analyzed
by agarose gel electrophoresis. The bands are isolated, purified
using the Qiaquick gel extraction kit (Qiagen, Valencia, Calif.),
eluted and ligated to pGEM-T-Easy vector (Promega, Madison, Wis.).
DNA from individual clones is isolated using the Qiagen Plasmid
Mini kit (Qiagen, Valencia, Calif.). The sequence of the promoter
fragment is SEQ ID NO:91.
[0208] The promoter fragment is cloned into a vector such as shown
in FIG. 1 (pMON19469) containing the hsp70 intron/GUS cassette.
[0209] 3m. 700352625 Clone Analysis and Promoter Isolation
[0210] To determine the distribution of the clone ID 700352625
transcripts in corn, RT-PCR is performed using the SEQ ID NO:70 and
SEQ ID NO:71 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 95.degree. C. for 1 minute and
35 cycles of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by 10 minutes at 72.degree. C.
Bands are amplified with cDNA derived from anther,
glume/lemma/palea, pollen, and microspore but not with cDNA derived
from ear, husk, kernel, rachis, leaf, silk, or root.
[0211] For the isolation of the clone ID 700352625 promoter, SEQ ID
NO:72 is used in combination with SEQ ID NO:1 (AP1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 2 .mu.L of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.). The
following cycling parameters are used: 94.degree. C. 1 minute, 7
cycles of 94.degree. C. 2 seconds, 72.degree. C. 3 min, and 36
cycles 94.degree. C. 2 seconds, 66.degree. C. 3 minutes followed by
4 minute incubation at 66.degree. C. For the nested, secondary PCR
reaction, 1 .mu.L of a 1:50 dilution of the primary reaction was
used with SEQ ID NO:73 and SEQ ID NO. 2 (AP2) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2.
The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 2
seconds, 72.degree. C. 3 minutes, and 25 cycles of 94.degree. C. 2
seconds and 67.degree. C. 3 minutes followed by 4 minutes at
67.degree. C.
[0212] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis. The bands are isolated,
purified using the Qiaquick gel extraction kit (Qiagen, Valencia,
Calif.) and eluted with 30 .mu.L ddH2O. Five microliters of the
purified band is ligated to 50 ng of pGEM-T-Easy vector (Promega,
Madison, Wis.). DNA from individual clones is isolated using the
Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.), and sequenced
using the M13 forward primers and M13 reverse primers. To add a
HindIII restriction site to the 5' end and a BamH1 site at the 3'
end of the 700352625 promoter fragment, 1 .mu.L of the isolated DNA
was amplified under standard Genome Walker.TM. PCR conditions using
Expand Hi Fidelity DNA Polymerase (BMB, Indianapolis, Ind.) with
the supplied buffer #2 in combination with primers SEQ ID NO:74 and
SEQ ID NO:3 (AP3). The reactions are carried out under the
following cycling conditions: 94.degree. C. 1 minute, 3 cycles of
94.degree. C. 2 seconds,70.degree. C. 3 minutes and 12 cycles
94.degree. C. 2 seconds 67.degree. C. 3 minutes followed by
4-minute incubation at 67.degree. C. Twenty five microliters of
this PCR reaction is analyzed by agarose gel electrophoresis. The
bands are isolated, purified and ligated to pGEM-T-Easy vector DNA
(Promega, Madison, Wis.). DNA from individual clones is isolated
using the Qiagen Plasmid Mini kit (Qiagen, Valencia, Calif.). The
promoter sequence is shown in SEQ ID NO:92. The promoter fragment
is cloned in an expression vector as shown in FIG. 1
(pMON19469).
[0213] 3n. 700382630 Clone Analysis and Promoter Isolation
[0214] To determine the distribution of the clone ID 700382630
transcripts in corn, RT-PCR is performed using the SEQ ID NO:75 and
SEQ ID NO:76 primers following a standard RT-PCR protocol using
cDNA derived from anther, 6 cm ear, glume/lemma/palea, husk, kernel
from 4 cm ears, leaf, meristem, mature anther/pollen, microspores,
rachis, root, and silk. Taq DNA polymerase from BMB (Indianapolis,
Ind.) is used in combination with the supplied reaction buffer.
Cycling parameters are as follows: 95.degree. C. for 1 minute and
35 cycles of 95.degree. C. 15 seconds, 50.degree. C. 30 seconds and
72.degree. C. 30 seconds followed by 10 minutes at 72.degree. C.
Bands are amplified with cDNA derived from anther,
glume/lemma/palea, pollen, and microspore but not with cDNA derived
from ear, husk, kernel, rachis, leaf, silk, or root.
[0215] For the isolation of the clone ID 700382630 promoter, SEQ ID
NO:77 is used in combination with SEQ ID NO:1 (AP1) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) in conjunction with the
supplied buffer #2. Each reaction contains 2 .mu.L of a 1:2
dilution of the GenomeWalker.TM. libraries made according to the
manufacturer's protocol (Clontech, Palo Alto, Calif.). The
following cycling parameters are used: 94.degree. C. 1 minute, 7
cycles of 94.degree. C. 2 seconds, 72.degree. C. 3 min, and 36
cycles 94.degree. C. 2 seconds, 66.degree. C. 3 minutes followed by
4 minute incubation at 66.degree. C. For the nested, secondary PCR
reaction, 1 .mu.L of a 1:50 dilution of the primary reaction is
used with SEQ ID NO:78 and SEQ ID NO:3 (AP3) in a standard
GenomeWalker.TM. PCR reaction using Expand Hi Fidelity DNA
Polymerase (BMB, Indianapolis, Ind.) with the supplied buffer #2.
The reactions are carried out under the following cycling
conditions: 94.degree. C. 1 minute, 5 cycles of 94.degree. C. 2
seconds, 72.degree. C. 3 minutes, and 25 cycles of 94.degree. C. 2
seconds and 67.degree. C. 3 minutes followed by 4 minutes at
67.degree. C.
[0216] Twenty five microliters of the secondary PCR reaction is
analyzed by agarose gel electrophoresis. The bands are isolated,
purified, and ligated to pGEM-T-Easy vector (Promega, Madison,
Wis.) DNA. DNA from individual clones is isolated using the Qiagen
Plasmid Mini kit (Qiagen, Valencia, Calif.), and sequenced using
the M13 forward primers and M13 reverse primers. The promoter
sequence is shown in SEQ ID NO:93.
Example 4
[0217] Promoter Isolation and Cloning
[0218] The DNA fragments resulting from the nested PCR
amplification described above are isolated and gel purified. A 25
.mu.L aliquot of the secondary PCR is run on an agarose gel. The
DNA fragment of the secondary PCR product is purified from the
agarose gel using the Qiagen Kit following the conditions suggested
by the manufacturer. The purified DNA is ligated to pGEM-T Easy
vector (pGEM-T Easy Vector System I, Promega Corp., Madison, Wis.)
following the conditions recommended by the manufacturer. An
aliquot of the ligation reaction is transformed into a suitable E.
coli host such as DH10B and the cells plated on selection medium
(for DH10B, 100 g/mL carbenicillin). Bacterial transformants are
selected, grown in liquid culture, and the plasmid DNA isolated
using a commercially available kit such as the Qiaprep Spin
Microprep Kit (Qiagen Corp., Valencia, Calif.). Purified plasmid
containing the predicted insert size based on restriction enzyme
analysis are sequenced using the dye terminator method in both
directions using the M13 forward and reverse primers shown in SEQ
ID NO:4 (M13 forward primer) and SEQ ID NO:5 (M13 reverse primer).
Restriction enzymes are available from a number of manufacturers
(see, for example, Boehringer Mannheim (Indianapolis, Ind.). The 5'
flanking regions containing the promoter sequences are determined
and shown in SEQ ID NOS:79-98. Engineering restriction sites for
cloning into suitable vectors is done using standard molecular
biology techniques known to those skilled in the art.
Example 5
[0219] Transient Analysis of Promoter Activity in Protoplasts and
Microspores
[0220] For transient expression, promoter fragments are cloned into
expression vectors such as pMON19469 shown in FIG. 1. Plasmid
pMON19469 is an expression vector consisting of the following
genetic components: P-e35S is the promoter for the 35S RNA from
CaMV containing a duplication of the -90 to -300 region; HSP70
intron is the intervening sequence of the maize heat shock protein
as described in U.S. Pat. Nos. 5,593,874 (herein incorporated by
reference in its entirety) and 5,859,347 (herein incorporated by
reference in its entirety); GUS:1 is the coding region for
beta-glucuronidase; nos 3' is the termination signal from the
nopaline synthase gene; ori-M13 and ori-pUC are origins of
replication; AMP is the coding region for ampicillin selection. If
a translational start codon of a target promoter is identified, the
fragment is cloned into pMON19469 in place of the P-e35S genetic
element. If an AUG is not identified, the promoter fragment is
cloned into an expression vector modified to enable translational
fusions with a reporter gene such as .beta.-glucuronidase (GUS)
(Jefferson et al., EMBO J., 6:3901, 1987) or green fluorescent
protein (GFP) as described in Pang et al. (Plant Physiol. 112:893,
1996).
[0221] The expression constructs are tested in a transient plant
assay. A number of assays are available and known to those skilled
in the art. Analysis of reporter genes in a protoplast system can
be used to assess the activity of a regulatory element, such as a
promoter operably linked to the reporter gene. A leaf protoplast
isolation and electroporation protocol is followed essentially as
described by Sheen (The Plant Cell 3:225-245, 1991) with the
following modifications: the seed used is FR27RHM.times.FRMol7RHM
from Illinois Foundation Seeds. The seed is surface sterilized for
2 minutes in 95% ethanol, rinsed twice with sterile water, 30
minutes in 50% bleach (Clorox) plus 2 drops of Tween-20, three
rinses in sterile water followed by a 5-minute soak in
benlate/captan solution to prevent fungal growth. The seeds are
germinated in phytotrays containing 100 ml 1/2 MS media (2.2 g/L MS
salts, 0.25% gelrite), 7 seeds per phytotray. The seeds are grown 5
days at 26.degree. C. in 16/8 hour day/night photoperiod and 7 days
in the dark at 28.degree. C. The second leaf from each plant is
sliced longitudinally using Feather No. 11 surgical blades.
Digestion time is two hours and 10 minutes in the light at
26.degree. C. After digestion, the plates are swirled two times at
80-100 rpm for 20 seconds each and the protoplast/enzyme solution
is pipetted through a 190 .mu.m tissue collector. Protoplasts are
counted using a hemacytometer counting only protoplasts that are
intact and circular. Ten to fifty micrograms of DNA containing the
vector of interest is added per cuvette. Final protoplast densities
at electroporation range from 3.times.10.sup.6/mL to
4.5.times.10.sup.6/mL. Electroporations are performed in the light
using Bio-Rad Gene pulser cuvettes (Bio-Rad Hercules, Calif.) with
a 0.4 cm gap and a maximum volume of 0.8 mL at 125 .mu.Farads
capacitance and 260 volts. The protoplasts are incubated on ice
after resuspension in electroporation buffer and are kept on ice in
cuvettes until 10 minutes after electroporation. The protoplasts
are kept at room temperature for ten minutes before adding 7 mL of
protoplast growth medium. The protoplast culture medium has been
described (Fromm et al., Methods in Enzymology 153, 351-366, 1987).
Culture plates are layered with the growth medium and 1.5%
SeaPlaque agarose (FMC BioProducts, Rockland, Me.) to prevent
protoplast loss. Samples are cultured in the light at 26.degree.
C., 16/8 day/night cycle, until harvested for the assay (typically
18-22 hours after electroporation). Samples are pipetted from the
petri plates to 15 mL centrifuge tubes and harvested by
centrifugation at 800-1000 rpm. The supernatant is removed and
samples are assayed immediately for the gene of interest. Samples
can also be frozen for later analysis.
[0222] For analysis of promoter activity in a wheat protoplast
system, the method for isolation and preparation of wheat
protoplasts is performed as described by Zhou et al. (Plant Cell
Reports 12:612, 1993). The electroporation buffer used has been
described (Li et al., 1995). The culture medium used is MS1 MSM
(4.4 g Gibco MS salts/L, 1.25 ml Thiamine HCL (0.4 mg/mL), 1 mL
2,4-D (1 mg/mL), 20 g/L sucrose, 0.15 mL asparagine (15 mg/mL),
0.75 g MgCl.sub.2. 109 g/L 0.6M mannitol, pH5.5. Mustang
protoplasts are used for protoplast isolation about four days after
subculture. Briefly, 8 g of wheat cell suspension is poured into a
culture tube, the cells are allowed to settle. The medium is
removed and remaining cells are resuspended with 40 mL enzyme
solution, transferred to a petri plate, wrapped in foil, and
incubated at 26.degree. C. for 2 hours on a rotator at 40 rpm. The
suspension is centrifuged at 200 g for 8 min., washed twice with
centrifugation between each wash, resuspended in 10 mL wash
solution and stored on ice. The number of protoplasts is determined
and the volume adjusted to a final concentration of
4.times.10.sup.6 protoplasts/ml. About 0.75 mL of protoplasts is
added to each electroporation cuvette and up to about 50 .mu.g
plasmid DNA of the vector in 50 .mu.L solution is added to the
protoplasts. The electroporation conditions are 960 .mu.Farads and
160 volts using a Bio-Rad Gene Pulser (Bio-Rad Laboratories,
Hercules, Calif.). The samples remain on ice for 10 minutes prior
to and during electroporation. After electroporation, the samples
are left on ice for about 10 minutes and removed and allowed to
warm to room temperature for 10 minutes. The electroporated cells
are pipetted into MS1 WSM medium and incubated in the dark for
18-22 hours at 24.degree. C. The cells are harvested by
centrifugation at 200-250 g for 8 minutes and frozen on dry ice for
subsequent analysis of expression of the gene of interest.
[0223] In another transient assay system, barley microspores are
used. For this assay shoots are collected and spikes are removed
from the sheath and placed in 15.times.100 mm plates. Fifteen
microliters of 0.3 M ice cold mannitol is added into each plate
containing 10 spikes. The plates are sealed with parafilm and kept
at 4.degree. C. for 3-4 days. Pre-treated spikes are cut about 1-2
cm into a chilled blender cup (about 10 two-rowed spikes
needed/plate). The spikes are covered with enough cold mannitol to
create a slurry and blended at low speed in a Waring blender for
6-10 seconds. The slurry is filtered through cheesecloth or a nylon
membrane and the filtrate is filtered through a 100.mu. mesh nylon
membrane into a 50 mL centrifuge tube. The mixture is spun for 5
minutes at 900 rpm and the liquid is decanted and microspore pellet
resuspended in 2 mL liquid FHG medium. The microspores are filtered
through three layers of Whatman #2 filter paper into a filtering
flask under vacuum. About 2 mL microspores are dropped on each
filter set and the uppermost filter paper is transferred to solid
medium (FHG+0.25 M mannitol+0.25 M sorbitol on a 15.times.100 mm
plate). The plates are sealed with parafilm and incubated in the
dark at 25.degree. C. for 3-4 hours prior to particle bombardment.
A number of methods of particle bombardment can be used (see, for
example, Klein et al., Bio/Technology 6:559, 1988; Christou,
Particle Bombardment for Genetic Engineering of Plants, Academic
Press, 1996). After bombardment, the plates are sealed and kept at
25.degree. C. for 20-24 hours. 0.3 M mannitol solution is used to
wash microspores from the filter paper and the microspores are
collected by centrifugation and analyzed for expression of the gene
of interest. The FHG medium recipe is as follows: Macroelements
(mg/L) include, 1900 mg KNO, 165 mg NH.sub.4NO.sub.3, 170 mg
KH.sub.2PO.sub.4, 370 mg MgSO.sub.4 7 H.sub.2O, 440 mg CaCl.sub.2
2H.sub.2O; Microelements (mg/L) include 40 mg FeNa.EDTA, 22.3 mg
MnSO.sub.4.5H.sub.2O, 6.2 mg H.sub.3B0.sub.3, 8.6 mg ZnSO.sub.4,
0.025 CuSO.sub.4.5H.sub.2O, 0.25 mg NaM004.2H.sub.2O.
Example 6
[0224] Transient Analysis of Promoter Activity in Wheat
Reproductive Tissues
[0225] For analysis of promoter activity in wheat reproductive
tissues such as wheat anthers and ovaries, constructs containing
the potential promoter, the HSP70 intron and the GUS gene are
bombarded into wheat anthers and ovaries from wheat spikes in which
the boot is just beginning to open. One spike of anthers and
ovaries is dissected per plate (1 liter plate medium containing 4.4
g MS salt, 40 g maltose, 40 g raffinose, 22.78 g mannitol, 1.95 g
MES and 4 g phytagel at pH 5.8). Two and a half micrograms of each
DNA sample (1 .mu.g/.mu.L) to be tested is precipitated with 12.5
.mu.L tungsten, 5.0 .mu.L 0.1M spermidine and 12.5 .mu.L 1.0M
calcium chloride for 40 minutes at room temperature. For gunpowder
bombardments, 12.5 .mu.L of the supernatant is removed and
remainder of the sample is sonicated before each shot. Two and a
half microliters of the DNA precipitant is bombarded per shot. For
the helium gun bombardments, the precipitated DNA is spun down,
washed with 70% EtOH and with 100% EtOH and resuspended in 40 .mu.L
100% EtOH. Five microliters of the DNA is bombarded per plate. For
either method, each plate is shot twice, and two plates are assayed
per DNA sample. After bombardment, the plates are incubated
overnight at 24.degree. C. in the dark. The next day the anther and
ovaries are transferred to a GUS staining solution. To increase the
penetration of the staining solution, the samples are put in a
vacuum chamber for 10 minutes. Anthers and ovaries are incubated in
the staining solution at 37.degree. C. for 16-24 hours. The
staining solution is replaced with 70% Ethanol, and the tissues are
stored at 4.degree. C. Staining is strictly qualitative, either
there is expression or not. The staining indicates nothing of the
tissue specificity of the potential promoters in stable plates,
because the wheat ovary is a very promiscuous tissue that allows
many active promoters to be expressed in this transient system.
Example 7
[0226] Promoter Activity from Transient Assay Analysis
[0227] In general, transcriptional regulatory elements necessary
for promoter activity are located within a few hundred bases of the
transcriptional start site. In many plant promoters, regulatory
elements sufficient for driving heterologous gene expression in a
spatial and temporal pattern that mimics the expression of the
endogenous gene are located within 1000 base pairs 5' of the
transcriptional start site. There are some genes, however, where
transcriptional regulatory elements can be located kilobases away,
5' or 3' to the transcriptional start site.
[0228] The transient assay is a system well suited to determining
if sufficient regulatory elements are present for transcription
initiation. As described herein, a DNA fragment is operationally
linked to a reporter gene of interest and that construct is
"placed" (through particle bombardments, electroporation, etc) into
cells, protoplasts or tissues. Expression of the reporter gene in
the recipient cells indicates that enough regulatory sequences
reside in the DNA fragment to initiate transcription. Thereby, the
DNA fragment can be considered a promoter. The transient assay does
not provide any data regarding the pattern of gene expression the
promoter fragment would provide in vivo. A prediction of the
promoter's activity can be made based on the pattern of the
endogenous gene's activity, but the accuracy of this prediction is
dependent on whether the promoter fragment contains all the
necessary regulatory elements responsible for the proper expression
of the endogenous gene.
[0229] A negative result in a transient assay does not necessarily
indicate that a tested DNA fragment has no promoter activity. In
addition to experimental error, some of the conditions which could
result in a negative result are: 1). A translational start codon is
located within the DNA fragment thereby blocking expression of the
reporter gene; 2). The DNA fragment contains a transcriptional
start site and a splice donor site, but lacks a splice acceptor
site. Therefore, the reporter gene is not expressed because the
message is not properly spliced; 3). Transcription factors specific
to the function of the promoter region may not be present in the
tissues used for the transient assay; or 4). The level of
transcription is below the limits of detection of the assay.
[0230] To test the DNA fragments contained in Example 3 for
promoter activity, SEQ ID NOS: 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97 and 98 are assayed by particle
bombardment of barley microspores (see Example 5) or particle
bombardment of wheat reproductive tissues (see Example 6). As
described herein, the experimental cassette used to test each
putative promoter fragment contains the fragment operationally
linked to the hsp70 intron and the GUS gene. The experiment for
each construct is carried out at least 2 times. A construct
containing the e35S promoter operationally linked to the hsp70
intron and GUS gene is used as a positive control and a no-DNA
bombardment is used as a negative control. The data are summarized
in Table 2. Table 2. Summary of transient assay data testing for
promoter activity of the DNA fragments containing SEQ ID NOS:
82-98. In the first column are the Clone IDs of the EST sequences
used to isolate the promoter fragments (see Example 3). The second
column are the SEQ ID numbers of the fragments tested in the
transient assay. The construct names are listed in the third
column. Each construct contains a fragment operably linked to the
hsp70 intron and GUS gene. The Fourth column indicates the
transient assay used. The fifth column is the level of GUS activity
detected based on a qualitative evaluation of the number and
intensity of positively staining cells in the assay. Low indicates
much lower than 35S, medium indicates slightly lower than 35S, and
high indicates greater than or equal to 35S.
3 SEQ ID Level Clone ID # Construct Transient Assay Detected
700353038 96 pMON48133 wheat reproductive low tissue 98 pMON48132
wheat reproductive low tissue 95 pMON48131 wheat reproductive low
tissue 97 pMON48130 wheat reproductive low tissue 700352826 94
pMON48136 wheat reproductive medium tissue 700354918 82 pMON48134
wheat reproductive not tissue detected 700353844 83 pMON48137 wheat
reproductive medium tissue 700355306 84 pMON53312 wheat
reproductive not tissue detected 700353142 85 pMON53306 wheat
reproductive low tissue 700282503 86 pMON53320 wheat reproductive
not tissue detected 700282409 88 pMON53310 wheat reproductive high
tissue 700282409 87 pMON53308 wheat reproductive medium tissue
700352616 89 pMON49324 wheat reproductive low tissue 89 pMON49323
wheat reproductive not tissue detected 89 pMON49322 wheat
reproductive not tissue detected 700354681 90 pMON49300 wheat
reproductive high tissue 700353007 91 pMON49332 wheat reproductive
low tissue 91 pMON49327 wheat reproductive not tissue detected 91
pMON49326 wheat reproductive low tissue 91 pMON49325 wheat
reproductive low tissue 700352625 92 pMON49337 wheat reproductive
low tissue 92 pMON49330 wheat reproductive low tissue 92 pMON49329
wheat reproductive not tissue detected 92 pMON49328 wheat
reproductive low tissue 700382630 93 pMON49336 barley microspore
low
[0231] 7a: 700353038
[0232] For clone ID 700353038, two promoter fragments are isolated
that comprise SEQ ID NOS:80 and 81. Two potential translational
start codons (ATG) are identified at the 3' end of each fragment.
Therefore, two new fragments are generated from each fragment which
has 3' ends just upstream of one of the ATG's (see Example 3A).
These new promoter fragments comprise SEQ ID NOS:95 and 96 (derived
from SEQ ID NO:81) and SEQ ID NOS:97 and 98 (derived from SEQ ID
NO:80). Each DNA fragment comprising each of SEQ ID NOS: 95, 96,
97, and 98 operably linked to the hsp70 intron and the GUS gene to
generate constructs pMON48131, pMON48133, pMON48130 and pMON48132,
respectively. These constructs are tested in the wheat reproductive
tissue transient assay. GUS activity is detected for each
construct. The level of expression detected is much lower than seen
with e35S but greater than that seen with the no DNA bombardments.
These data indicate that SEQ ID NOS:95, 96, 97, and 98 contain
promotor activity and translation initiated within the GUS
gene.
[0233] 7b: 700352826
[0234] The fragment comprising SEQ ID NO:79 has an internal HindIII
site at position 416. SEQ ID NO: 94 contains the sequences 417-2213
of SEQ ID NO:79 (all sequences 3' of the HindIII site, inclusive).
The fragment comprising SEQ ID NO:94 is operably linked to the
hsp70 intron and the GUS gene to generate the construct pMON48136.
This construct is tested for promotor activity using the wheat
reproductive tissue transient assay. GUS activity is detected at a
level slightly below that seen with the e35S positive control
indicating that SEQ ID NO:94 has promotor activity.
[0235] 7c: 700354918
[0236] The fragment comprising SEQ ID NO:82 is operably linked to
the hsp70 intron and the GUS gene to generate the construct
pMON48134. This construct is tested for promoter activity using the
wheat reproductive tissue transient assay. GUS activity is not
detected which could be due to any of the reasons described
above.
[0237] 7d: 700353844
[0238] The fragment comprising SEQ ID NO:83 is operably linked to
the hsp70 intron and the GUS gene to generate the construct
pMON48137. This construct is tested for promoter activity using the
wheat reproductive tissue transient assay. GUS activity is detected
at a level slightly below that seen with the e35S positive control
indicating that SEQ ID NO:83 has promoter activity.
[0239] 7e: 700355306
[0240] The fragment comprising SEQ ID NO:84 is operably linked to
the hsp70 intron and the GUS gene to generate the construct
pMON53312. This construct is tested for promoter activity using the
wheat reproductive tissue transient assay. GUS activity is not
detected which could be due to any of the reasons described
above.
[0241] 7f: 700353142
[0242] The fragment comprising SEQ ID NO:85 is operably linked to
the hsp70 intron and the GUS gene to generate the construct
pMON53306. This construct is tested in the wheat reproductive
tissue transient assay. GUS activity is detected at a level of
expression much lower than seen with e35S but greater than that
seen with the no DNA bombardments indicating that SEQ ID NO:85 has
promoter activity.
[0243] 7g: 700282503
[0244] The fragment comprising SEQ ID NO:86 is operably linked to
the hsp70 intron and the GUS gene to generate the construct
pMON53320. This construct is tested for promoter activity using the
wheat reproductive tissue transient assay. GUS activity is not
detected which could be due to any of the reasons described
above.
[0245] 7h: 700282409 Profilin 2 and 700354681
[0246] The fragment comprising SEQ ID NO:90 is a smaller version of
the putative promoter comprising SEQ ID NO:87. The fragment
comprising SEQ ID NO:90 is operably linked to the hsp70 intron and
the GUS gene to generate the construct pMON449300. The fragment
comprising SEQ ID NO:87 is operably linked to the hsp70 intron and
the GUS gene to generate the construct pMON53308. Both constructs
are tested in the wheat reproductive tissue transient assay. GUS
activity is detected with each fragment tested. GUS activity is
detected at a level slightly below that seen with the e35S positive
control with SEQ ID NO:87. Gus activity is detected at a level
equal to that seen with the e35S positive control with SEQ ID
NO:90. These data indicate that both SEQ ID NOS:87 and 90 contain
promoter activity.
[0247] 7i: 700282409 Profilin 1
[0248] The fragment comprising SEQ ID NO:88 is operably linked to
the hsp70 intron and the GUS gene to generate the construct
pMON53310. This construct is tested in the wheat reproductive
tissue transient assay.. Gus activity is detected at a level equal
to that seen with the e35S positive control indicating that SEQ ID
NO:88 has promoter activity.
[0249] 7j: 700352616
[0250] The translational start codon is not readily identifiable in
the EST sequence for clone ID 7003542616. Therefore, the fragment
comprising SEQ ID NO:89 is placed in vectors designed to generated
GUS fusions (see Example 5). Three constructs are generated,
pMON49322, pMON49323, and pMON49324 each representing a different
reading frame for a putative translational fusion between the
sequences in SEQ ID NO:89 and GUS. All three constructs are tested
in the wheat reproductive tissue transient assay. GUS activity is
detected with only one construct, at a level of expression much
lower than seen with e35S but greater than that seen with the no
DNA bombardments. These data indicate that a translational start
codon is located within SEQ ID NO:89 and that SEQ ID NO:89 has
promoter activity.
[0251] 7k: 700353007
[0252] The translational start codon is not readily identifiable in
the EST sequence for clone ID 700353007. Therefore, the fragment
comprising SEQ NO: ID 92 is placed in vectors designed to generate
GUS fusions (see Example 5). Three constructs are generated,
pMON49325, pMON49326, and pMON49327, each representing a different
reading frame for a putative translational fusion between the
sequences in SEQ ID NO:91 and GUS. All three constructs are tested
in the wheat reproductive tissue transient assay. GUS activity is
detected with two constructs, at a level of expression much lower
than seen with e35S but greater than that seen with the no DNA
bombardments. This indicates that translation does initiate off the
translational start codon located within the GUS gene. To test
that, SEQ ID NO:91 is tested using the conventional vector
described in Example 5, which has an inframe STOP codon upstream of
the GUS translational start codon. With this construct, pMON 49332,
GUS activity is detected at a level of expression much lower than
seen with e35S but greater than that seen with the no DNA
bombardments. These data indicate that SEQ ID NO:91 has promoter
activity.
[0253] 7l: 700352625
[0254] The translational start codon is not readily identifiable in
the EST sequence for clone ID 700352625. Therefore, the fragment
comprising SEQ ID NO:92 is placed in vectors designed to generate
GUS fusions (see Example 5). Three constructs are generated,
pMON49328, pMON49329, and pMON49330, each representing a different
reading frame for a putative translational fusion between the
sequences in SEQ ID NO:92 and GUS. All three constructs are tested
in the wheat reproductive tissue transient assay. GUS activity is
detected with two constructs, at a level of expression much lower
than seen with e35S but greater than that seen with the no DNA
bombardments. This indicates translation is initiating at the
translational start codon within the GUS gene. To test that, SEQ ID
NO:92 is tested using the conventional vector described in Example
5, which has an in frame STOP codon upstream of the GUS
translational start codon. With this construct, pMON49337, GUS
activity is detected at a level of expression much lower than seen
with e35S but greater than that seen with the no DNA bombardments
indicating that SEQ ID NO:92 has promoter activity.
[0255] 7m: 700382630
[0256] The fragment comprising SEQ ID NO:93 is operably linked to
the hsp70 intron and the GUS gene to generate the construct
pMON49336. This is tested in the barley microspore transient assay.
GUS activity is detected at a level of expression much lower than
seen with e35S but greater than that seen with the no DNA
bombardments indicating that SEQ ID NO:93 has promoter
activity.
Example 8
[0257] Promoter Activity Analysis in Plants
[0258] For stable plant transformation the 5' regulatory sequences
are cloned into a plant transformation vector such as shown in FIG.
2. Plasmid pMON51850 is a double border (right and left T-DNA
borders) plant transformation vector and contains the following
genetic components: NOS 3' is the termination signal from the
nopaline synthase gene; ori-322 and ori-V are origins of
replication; kan is the coding region for kanomycin selection.
[0259] The promoter is operably linked to any gene of interest such
as a glyphosate tolerance gene along with other regulatory
sequences including, but not limited to, non-translated leaders and
terminators as described above, and transformed into a target crop
of interest via an appropriate delivery system such as
Agrobacterium-mediated transformation (see, for example, U.S. Pat.
No. 5,569,834, herein incorporated by reference in its entirety,
U.S. Pat. No. 5,416,011, herein incorporated by reference in its
entirety, U.S. Pat. No. 5,631,152, herein incorporated by reference
in its entirety, U.S. Pat. No. 5,159,135, herein incorporated by
reference in its entirety and U.S. Pat. No. 5,004,863, herein
incorporated by reference in its entirety) or particle bombardment
methods (see, for example, Patent Applns. WO 92/15675. WO 97/48814
and European Patent Appln. 586,355, and U.S. Pat. Nos. 5,120,657,
5,503,998, 5,830,728 and 5,015,580, all of which are herein
incorporated by reference in their entirety). A large number of
transformation and regeneration systems and methods are available
and well-known to those skilled in the art. The stably transformed
plants and progeny are subsequently analyzed for expression of the
gene in tissues of interest by any number of molecular,
immunodiagnostic, biochemical, and/or field evaluation methods
known to those skilled in the art.
[0260] The results from the transient assay analysis described in
Examples 7 are qualitative regarding the promoter activity of the
DNA fragments tested. Although these promoters are predicted to be
active in male reproductive tissues, the transient assay analysis
does not demonstrate any support for this. To determine the
promoter activity in male reproductive tissues, the promoter
fragments are cloned upstream of a gene of interest (either GUS or
the MS2 coat protein), placed in a plant transformation vector and
transformed into plants. Tissues from R0 plants are harvested and
assayed for the gene of interest.
[0261] Detection of GUS activity in male reproductive tissues is
described in Example 5. For the detection of the MS2 coat protein,
anther extracts are analyzed by immunodetection on Western Blots or
ELISA analysis. For western blots, the T7 tag monoclonal and horse
radish peroxidase conjugated antibody (Novagen, Madison, Wis.) is
used to detect MS2 protein expression in anther tissues. Total
protein is extracted (extraction buffer containing 1.times. PBS and
.01% Tween-20) from anther, 10 .mu.g of total protein sample is
separated on a 10-20% polyacrylamide gradient gel (BioRAD,
Hercules, Calif.) and transferred onto ECL nitrocellulose membrane
(Amersham, Arlington, Ill.). A 1:5000 dilution of primary antisera
is used to detect ACOX protein using the ECL detection system
(Amersham, Arlington, Ill.). A 15 Kd protein is detected.
[0262] For ELISA quantification of MS2 coat protein levels in
anthers, crude anther extracts containing 1 ug total protein is
added to a 96-well Nunc-Immuno MaxiSorb plate coated with 100 .mu.l
of purified polyclonal anti-MS2 coat protein IgG antibody (0.1
ng/.mu.L) in coating buffer (15 mM Na.sub.2CO.sub.3, 35 mM
NaHCO.sub.3, pH 9.6). The plate is sealed and incubated at
37.degree. C. for one hour. The plate is then rinsed three times
with washing buffer (1.times. PBS, 0.05% Tween 20, pH7.4). Fifty
microliters of the anther extract containing 1 .mu.g of total
protein is added to a well, followed by addition of 50 .mu.l of a
1:10,000 dilution of anti-T7 tag monoclonal and HSP conjugated
antibody (Novagen). The loaded plate is incubated at 37.degree. C.
for one hour then rinsed three times with washing buffer. To
develop the plate, 100 .mu.l of substrate (a 1:1 mixture of
H.sub.2O.sub.2 and TMB (3,3'-5,5'-tetra methyl benzidine),
Kirgeggard and Perry, #50-76-03, Gaithersburg, Md.) is added to
each well and the plate is incubated at room temperature for 3-5
min. One hundred microliters of stop solution (3M H.sub.3PO.sub.4)
is added to terminate the reaction. The plate is read on a Spectra
Max 340 (Molecular Devices, Sunnyvail, Calif.) at 450 nm.
[0263] Plasmids can be transformed into either monocot or dicot
plants. The promoter fragments are derived from corn (Zea mays).
Therefore, activity in a dicot plant would indicate a broad
spectrum of plants in which the promoter is active. The dicot plant
Arabidopsis thaliana offers several advantages as a model system to
study promoter systems: ease of transformation, quick life cycle,
and multiple stages of floral development on each plant. Monocot
promoters that are active in Arabidopsis anthers are likely to be
active in many monocot and dicot species. To test this, some
promoters which are active in Arabidopsis are also tested in
monocot plants. As shown below and summarized in Table 3, all
promoter fragments tested that are active in Arabidopsis are also
active in monocots. Table 3. Summary of promoter activity in stably
transformed plants. In the first column are the Clone IDs of the
EST sequences used to isolate the promoter fragments (see Example
3). The second column shows the SEQ ID numbers of the fragments
tested in the transient assay. The third column lists the introns
used in the constructs. No introns are used in constructs for dicot
transformation. The fourth column lists the reporter genes used in
the constructs. The fifth column show the construct names. In the
sixth column are the organisms transformed. The seventh column
shows the type of assay used to detect the reporter gene. The
eighth column shows the number of plants assayed. The ninth column
shows the number of plants showing male expression and the last
column describes any other tissues where the reporter protein is
detected.
4 Num Number of Sho Plants M Clone ID SEQ ID Intron Gene Assayed
Construct Organism Assay Type Assayed Expr 700353038 98 none GUS
pMON48183 Arabidopsis GUS activity 4 98 hsp70 intron MS2 coat
protein pMON42438 rice Western 5 700352826 94 none GUS pMON48185
Arabidopsis GUS activity 4 700353844 83 none GUS 48186 Arabidopsis
GUS activity 1 83 hsp70 intron MS2 coat protein 42439 rice Western
3 700282409 88 hsp70 intron MS2 coat protein 42938 rice Western 5
88 hsp70 intron MS2 coat protein 42938 wheat Elisa 4 88 hsp70
intron MS2 coat protein 42938 wheat Western 4 88 none GUS 48194
Arabidopsis GUS activity 4 88 hsp70 intron MS2 coat protein 52006
wheat Western 6 88 hsp70 intron GUS 53322 wheat GUS activity 11
700354681 90 hsp70 intron MS2 coat protein 42914 rice Western 5 90
hsp70 intron MS2 coat protein 42914 wheat Western 19 90 hsp70
intron MS2 coat protein 42936 wheat Elisa 1 90 hsp70 intron MS2
coat protein 42936 wheat Western 3 90 none GUS 51818 Arabidopsis
GUS activity 7 700353007 91 hsp70 intron MS2 coat protein 52003
wheat Elisa 2 91 hsp70 intron MS2 coat protein 52003 wheat Western
2 700352625 92 hsp70 intron M52 coat protein 52021 wheat Elisa
1
[0264] 8a. 700353038
[0265] To test for anther activity in dicot plants the fragment
comprising SEQ ID NO: 98 is places upstream of the GUS gene and put
into a plant transformation vector resulting in the construct
pMON48183. This construct is used to transform Arabidopsis
thaliana. Two of four show expression in the male reproductive
tissues specifically. No GUS expression is detected in other floral
tissues, stem or leaf. To test for anther activity in monocot
plants, the fragment comprising SEQ ID NO:98 is placed upstream of
the hsp70 intron/MS2 coat protein gene cassette and put into a
plant transformation vector resulting in the construct pMON42438.
This construct is used to transform Oryza sativum (rice). Anthers
from five independent R0 rice plants are assayed by Western Blot
for MS2 coat protein. All 5 plants are positive for MS2 coat
protein. These data indicate that SEQ ID NO:98 can act as an anther
enhanced promoter in both monocots and dicots.
[0266] 8b. 700352826
[0267] To test for anther activity in dicot plants the fragment
comprising SEQ ID NO:94 is placed upstream of the GUS gene and put
into a plant transformation vector resulting in the construct
pMON48185. This construct is used to transform Arabidopsis
thaliana. Four of four independent events show expression in the
male reproductive tissues. GUS expression is also detected in
immature seed, seedlings and cut stems but not detected leaves,
roots or other floral organs. These data indicate that SEQ ID NO:94
can act as an anther enhanced promoter in dicots. Because it is
monocot-derived promoter, it is likely to be active in monocot
anthers as well.
[0268] 8c. 700353844
[0269] To test for anther activity in dicot plants the fragment
comprising SEQ ID NO:83 is placed upstream of the GUS gene and put
into a plant transformation vector resulting in the construct
pMON48186. This construct is used to transform Arabidopsis
thaliana. One event is obtained and it shows expression in the male
reproductive tissues specifically. No GUS expression is detected in
other floral tissues, leaves, or stems. To test for anther activity
in monocots, the fragment comprising SEQ ID NO:83 is placed
upstream of the hsp70 intron/MS2 coat protein gene cassette and put
into a plant transformation vector resulting in the construct
pMON42439. This construct is used to transform Oryza sativum.
Anthers from three independent R0 rice plants are assayed by
Western Blot for MS2 coat protein. Two of three plants are positive
for MS2 coat protein. These data indicate that SEQ ID NO:83 can act
as an anther enhanced promoter in both monocots and dicots.
[0270] 8d: 700282409
[0271] To test for anther activity in dicot plants, the fragment
comprising SEQ ID NO:88 is placed upstream of the GUS gene and put
into a plant transformation vector resulting in the construct
pMON48194. This construct is used to transform Arabidopsis
thaliana. Two of four events show GUS expression in the anthers.
One of four events has detectable GUS expression in roots but no
other GUS expression is detected in other tissues.
[0272] To test for anther activity in monocot plants, the fragment
comprising SEQ ID NO:88 is placed upstream of the hsp70 intron/MS2
coat protein gene cassette and put into a plant transformation
vector resulting in the constructs pMON42938 and pMON52006. The
construct pMON42938 is used to transform Oryza sativum (rice) and
Triticum aesitivum (wheat). Anthers from five independent R0 rice
plants are assayed by Western Blot for MS2 coat protein. Two of
five plants are positive for MS2 coat protein. Anthers from four
independent R0 wheat plants were assayed by ELISA for MS2 coat
protein. Two of four plants are positive for MS2 coat protein.
Anthers from four independent R0 wheat plants are assayed by
Western Blot for MS2 coat protein but expression is below the
limits of detection for each event. The construct pMON52006 is used
to transform wheat. Anthers from six independent R0 wheat plants
are assayed by Western Blot for MS2 coat protein. Three of six
plants are positive for MS2 coat protein. These data indicate that
SEQ ID NOS: 88 can act as an anther enhanced promoter in both
monocots and dicots.
[0273] 8e: 700354681
[0274] Because SEQ ID NO: 90 is a subfragment of SEQ ID NO: 87,
only SEQ ID NO: 90 was tested. The fragment comprising SEQ ID NO:90
is placed upstream of the GUS gene and put into a plant
transformation vector resulting in the construct pMON51818. This
construct is used to transform Arabidopsis thaliana. Five of seven
events show GUS expression in the anthers. GUS expression is not
detected in other floral tissues.
[0275] The fragment comprising SEQ ID NO:90 is placed upstream of
the hsp70 intron/MS2 coat protein gene cassette and put into a
plant transformation vector resulting in the constructs pMON42914
and pMON42936. The construct pMON42914 is used to transform Oryza
sativum (rice) and Triticum aesitivum (wheat). Anthers from five
independent R0 rice plants are assayed by Western Blot for MS2 coat
protein. Four of five plants are positive for MS2 coat protein.
Anthers from nineteen independent R0 wheat plants are assayed by
Western for MS2 coat protein. Fourteen of nineteen plants were
positive for MS2 coat protein. The construct pMON42936 is used to
transform wheat. Anthers from three independent R0 wheat plants are
assayed by Western Blot for MS2 coat protein. One of three plants
is positive for MS2 coat protein. These data indicate that SEQ ID
NO: 90 can act as an anther enhanced promoter in both monocots and
dicots. Because SEQ ID NO: 90 acts as an anther enhanced promoter,
it is probable that SEQ ID NO: 87 also acts as an anther enhanced
promtoer.
[0276] 8f. 700353007
[0277] To test for anther activity in monocot plants, the fragment
comprising SEQ ID NO:91 is placed upstream of the hsp70 intron/MS2
coat protein gene cassette and put into a plant transformation
vector resulting in the construct pMON52003. This construct is used
to transform Triticum aesitivum (wheat). Anthers from two
independent R0 wheat plants are assayed by Western Blot for MS2
coat protein. MS2 coat protein is not detected in either event.
Anthers from two independent R0 wheat plants are assayed by ELISA
for MS2 coat protein. MS2 coat protein is not detected in either
event. At this time the data obtained have not been able to
demonstrate if this particular construct containing SEQ ID NO:91 is
active in anther in transgenic wheat as the number of the events
evaluated is very low. Further evaluations are needed.
[0278] 8g: 700352625
[0279] To test for anther activity in monocot plants, the fragment
comprising SEQ ID NO:92 is placed upstream of the hsp70 intron/MS2
coat protein gene cassette and put into a plant transformation
vector resulting in the construct pMON52021. This construct is used
to transform Triticum aesitivum (wheat). Anthers from one R0 wheat
plant is assayed by Western Blot for MS2 coat protein. MS2 coat
protein is not detected. At this time the data obtained have not
been able to demonstrate if this particular construct containing
SEQ ID NO:92 is active in anther in transgenic wheat as the number
of the events evaluated is very low. Further evaluations are
needed.
Example 9
[0280] Identification of Cis Elements and Engineering Novel
Promoters
[0281] Cis acting regulatory elements necessary for proper promoter
regulation can be identified by a number of means. In one method,
deletion analysis is carried out to remove regions of the promoter
and the resulting promoter fragments are assayed for promoter
activity. DNA fragments are considered necessary for promoter
regulation if the activity of the truncated promoter is altered
compared to the original promoter fragment. Through this deletion
analysis, small regions of DNA can be identified which are
necessary for positive or negative regulation of transcription.
Promoter sequence motifs can also be identified and novel promoters
engineered to contain these cis elements for modulating expression
of operably linked transcribable sequences. See for example U.S.
Pat. No. 5,223,419, herein incorporated by reference in its
entirety, U.S. Pat. No. 4,990,607, herein incorporated by reference
in its entirety, and U.S. Pat. No. 5,097,025, herein incorporated
by reference in its entirety.
[0282] An alternative approach is to look for similar sequences
between promoters with similar expression profiles. Promoters with
overlapping patterns of activity can have common regulatory
mechanisms. Several computer programs can be used to identify
conserved, sequence motifs between promoters, including, but not
limited to, MEME, SIGNAL SCAN, or GENE SCAN. These motifs can
represent binding sites for transcription factors which act to
regulate the promoters. Once the sequence motifs are identified,
their function can be assayed. For example, the motif sequences can
be deleted from the promoter to determine if the motif is necessary
for proper promoter function. Alternatively, the motif can be added
to a minimal promoter to test whether it is sufficient to activate
transcription. Suspected negative regulatory elements can be tested
for sufficiency by adding to an active promoter and testing for a
reduction in promoter activity. Some cis acting regulatory elements
may require other elements to function. Therefore, multiple
elements can be tested in various combinations by any number of
methods known to those skilled in the art.
[0283] Once functional promoter elements have been identified,
promoter elements can be modified at the nucleotide level to affect
protein binding. The modifications can cause either higher or lower
affinity binding which would affect the level of transcription from
that promoter.
[0284] Promoter elements can act additively or synergistically to
affect promoter activity. In this regard, promoter elements from
different 5' regulatory regions can be placed in tandem to obtain a
promoter with a different spectrum of activity or different
expression profile. Accordingly, combinations of promoter elements
from heterologous sources or duplication of similar elements or the
same element can confer a higher level of expression of operably
linked transcribable sequences. For example, a promoter element can
be multimerized to increase levels of expression specifically in
the pattern affected by that promoter element.
[0285] The technical methods needed for constructing expression
vectors containing the novel engineered 5' regulatory elements are
known to those of skill in the art. The engineered promoters are
tested in expression vectors and tested transiently by operably
linking the novel promoters to a suitable reporter gene such as GUS
and testing in a transient plant assay. The novel promoters are
operably linked to one or more genes of interest and incorporated
into a plant transformation vector along with one or more
additional regulatory elements and transformed into a target plant
of interest by a suitable DNA delivery system. The stably
transformed plants and subsequent progeny are evaluated by any
number of molecular, immunodiagnostic, biochemical, phenotypic, or
field methods suitable for assessing the desired agronomic
characteristic(s).
Sequence CWU 1
1
98 1 22 DNA Artificial Sequence Description of Artificial Sequence
adaptor sequence 1 gtaatacgac tcactatagg gc 22 2 19 DNA Artificial
Sequence Description of Artificial Sequence adaptor sequence 2
actatagggc acgcgtggt 19 3 30 DNA Artificial Sequence Description of
Artificial Sequence adaptor sequence 3 agggcaagct tggtcgacgg
cccgggctgg 30 4 17 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 4 ctgacggagg cgctacg
17 5 17 DNA Artificial Sequence Description of Artificial Sequence
fully synthesized primer 5 gttgaagtcg atgcagc 17 6 17 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 6 cgtcgggtat agattta 17 7 17 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 7 ccatgactca cttcctg 17 8 17 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 8
cgaatctgct acggatc 17 9 17 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 9 acacggtatc tctgagc
17 10 17 DNA Artificial Sequence Description of Artificial Sequence
fully synthesized primer 10 cacacgtaat cgtaatg 17 11 17 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 11 ccatgcacca gctgcag 17 12 17 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 12 cgaatctgct acggatc 17 13 17 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 13
atgcgcagac gttgagg 17 14 24 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 14 ggaccccagc
gtccgtagcg cctc 24 15 33 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 15 ggatcccagc
tccgacagcg agatcttacc gtc 33 16 35 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 16
ggatccagat ctgtccgccg tctccgacat tagcg 35 17 35 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 17 ggatccagat ctagcgaatc tgctacggat caata 35 18 17 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 18 ggacatcacc atccagc 17 19 17 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 19 catcgagcgt gccggag 17 20 27 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 20
ggatgccatc gaagctggat ggtgatg 27 21 38 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 21
ggatccagat ctaagtagag agggcccacc aggtagtc 38 22 37 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 22 ggatccagat ctcccctttg ctagttctct cctcgcc 37 23 17 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 23 acgacctgga caagtac 17 24 17 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 24 tcgccttcac gttgtcg 17 25 27 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 25
cagctcgccg tgtacttgtc caggtcg 27 26 39 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 26
ggatccagat ctaggttgcc atccagctgg atggcgatg 39 27 27 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 27 cacccggaga gcgttgtgtg cggaagc 27 28 37 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 28 ggatccagat ctccttcctg tggccgccgg ctctcct 37 29 17 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 29 gtggcatcat cgtcagc 17 30 20 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 30 cctcgcacag cgtcgagcag 20 31 27 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 31
ctgccctcgc acagcgtcga gcagaag 27 32 39 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 32
ggatccagat cttcggcgat tgttgatgga tcggagaag 39 33 17 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 33 ccatggccaa gaagggt 17 34 21 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 34
cccttctcct tgatgtccac c 21 35 26 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 35
ccttcttggc catggcgccg aacgcc 26 36 39 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 36
ggatccagat ctcgaagtgg tacgccgcga taggctcat 39 37 34 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 37 ggatccagat ctcatgtccg tagatgtgca ccac 34 38 21 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 38 gccggcgaga gcatggcgat g 21 39 19 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 39 cgttgctgtg gtctgcttg 19 40 28 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 40 gccgacacca gggtgacctc caggacac 28 41 40 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 41 ggatccagat ctcatgctct cgccggcgaa ggtgttttgc 40 42 34 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 42 ggatccagat ctctcgccgg cgaaggtgtt ttgc 34 43
19 DNA Artificial Sequence Description of Artificial Sequence fully
synthesized primer 43 ctacgactag ctagattcc 19 44 18 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 44 gcggattctg ttcttgcc 18 45 18 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 45
gcggattctg ttcttccc 18 46 27 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 46 acgcggatcc
tggtgggcgc catcgcg 27 47 39 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 47 ggatccagat
cttgggcgcc atcgcgcgtg gaatctagc 39 48 32 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 48
ggatccagat ctcgtttgcg gtgttcgcgt tg 32 49 15 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 49 ccgctttagt tcagt 15 50 15 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 50
cccgcatttc atttc 15 51 27 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 51 gtctgttgtc
catgcgattc acgctac 27 52 43 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 52 ggatccagat
ctcatgcgat tcacgctaca gccaaatgat cga 43 53 32 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 53 ggatccagat ctgcccggtc agacatgttt ac 32 54 33 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 54 ggatccagat ctgctcggcg cgttggtcgg tcg 33 55 18
DNA Artificial Sequence Description of Artificial Sequence fully
synthesized primer 55 atgagggttc ttgtagag 18 56 18 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 56 cccatcagtc cgctgttg 18 57 40 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 57
ggatcctaga tctaaacaca gagactaaca gcttctctac 40 58 27 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 58 tgccgtgtga tcatattcta gagacac 27 59 27 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 59 aaacacagag actaacagct tctctac 27 60 18 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 60 attctccagc gcaggtag 18 61 18 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 61
tgccctggct cgtcgaag 18 62 27 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 62 cctgccacga
catctttgcc cggtcag 27 63 27 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 63 tcgcacatca
ggtgctcgtc cacgtac 27 64 38 DNA Artificial Sequence Description of
Artificial Sequence fully synthesized primer 64 ggatcctaga
tctctgcgct ggagaatgga tcggagag 38 65 19 DNA Artificial Sequence
Description of Artificial Sequence fully synthesized primer 65
gaatcatcgg aataatggc 19 66 16 DNA Artificial Sequence Description
of Artificial Sequence fully synthesized primer 66 taggagcggg
agcatc 16 67 27 DNA Artificial Sequence Description of Artificial
Sequence fully synthesized primer 67 catcggcgga tgccatggac ctaccct
27 68 27 DNA Artificial Sequence Description of Artificial Sequence
fully synthesized primer 68 gccaggagga gcacgacgag gaacacg 27 69 40
DNA Artificial Sequence Description of Artificial Sequence fully
synthesized primer 69 ggatcctaga tctgccagga ggagcacgac gaggaacacg
40 70 19 DNA Artificial Sequence Description of Artificial Sequence
fully synthesized primer 70 caaacgctgc tgcgctctc 19 71 16 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 71 gagcgtggcg acgacg 16 72 27 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 72 gcgccatttt ctccaggttg ttctctc 27 73 27 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 73 caccgatcaa aacgacaggc tcctctg 27 74 40 DNA Artificial
Sequence Description of Artificial Sequence fully synthesized
primer 74 ggatcctaga tctcaccgat caaaacgaca ggctcctctg 40 75 25 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 75 caaggaggtg ttccacagcg tggcc 25 76 25 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 76 tggcgttgcg cacgacggag tgcat 25 77 27 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 77 accgtgaagt tgtcgccggc cttcttg 27 78 40 DNA
Artificial Sequence Description of Artificial Sequence fully
synthesized primer 78 ggatcctaga tctacgagga tcgccgatag gccacctagg
40 79 2213 DNA Zea mays 79 cctgtccttc ggcatttctg tagctactct
cgacgatgag gtcttcgtcg cctcatcggt 60 aggcaaagtg tcctattgca
cctctaagga ggtaggacta atcaactccc tagtgacacc 120 tatgtaggcc
atctgagcag aggcaatgat ctttcactaa gactgattca gccaaagtta 180
actcttgaag tgctcctaaa caaataactt tggagcaaag tgaattttgg caattgttag
240 gtgaaggcag taaatgtgat ctgccctcac ctcctcagcc acttttatac
tgaaaatgca 300 catgacgcga cacgggtcat ggaaattgtt cccgcgcagc
gtcccaagtc ccccctcgat 360 gatttaactt ctaggtcaac cgtttttact
tcaaatgtat caccttcttc ttaaacaagc 420 tttaggaagt tgtttttcgg
atctttggaa ttgggcctcc aagttataat ttttgcaatc 480 taagctcttt
gcaaagaaaa caaactcata tcgcaatggg ctacttttgg ggaccttcta 540
tgttgaagat cacttgctta aacttttact ttgacacaag ctagtgtgtt tcaggaatat
600 ctctactgat gcacgaagcc aaccttcggt cgggaaggcg tgcagagaag
cattgcacat 660 gaaaggtgaa agggtattaa ccaaagttga tagcttcgat
gacagcaaag gagcttcaga 720 tacaagccaa aggaaaaggc gacgaaggcc
taaatccgag cagccgaaga tggggaaaat 780 acgctactgc cctaacaaca
tttgtaaaca gtgaggggta caattgtaat tatgtactaa 840 gtcggttcgt
ctcccctata aatagatgaa cagtaacccg cataaattac attttgccag 900
gtgctacagc tttgtatagc tcaggctcca aaacacattc gtgctatctt gcactaagaa
960 gtcaatggta tgattgtaaa cttgttttct ataagagaaa tgaaattcta
aggcacatga 1020 gatgagttct catatcttcg tcatgttttt atgtattcta
gtcgattaca tccaaccttc 1080 gtccttgagt agttatccca aagacttaac
acttcaagga tgaaggcttc tactttttaa 1140 cattgtgttg tcttgttttt
tatttcattt agcaattaaa agcaagtgac taacacatgg 1200 ttaaacccaa
gatccgaaaa gaggctaaaa ttgagcaaga atgaacaaaa gttggtaaga 1260
ggaacataaa ccaacctttc ttagcaacat tcttccaaaa aaagaagatc aaaacatgta
1320 cccttgtatt ttgtgaaaac tggatctcca aaattgccta caatggaagg
tggctacgag 1380 aaacggttat aatcgaggag gtagagagaa ttttatgcta
caaccttcac aggcggtttc 1440 cctaagaaac atccactcta aatgtctttg
cacatacggt tcacttaaaa aaccgcaaat 1500 gcaaattgtt cattttcact
ggaggttttt taagcgaacc gctagaggaa atctcatttg 1560 caccggcgat
ccttaagaca tatcatgagc gaggttgcct tggaagccgg aagagttggt 1620
caatgaccta taaaaagcag aggacacagg agtgccctat tcaagcattg cctaaaaata
1680 gcaaaggcca aacgaccatt tcgtgtacat agcaaacggt gctcctctct
ctcaagaaag 1740 gatatcttcg ggaacatcca tccatcccca atccccaaag
gcgaggagag aactagcaaa 1800 ggggaaatgg ctgcttccac aaacaacact
ctcagggtgc tgttcaccct aatggttgta 1860 tgcgccgcag tatgcacagc
gaaaaggact gtagcaaagg caggagactt ggcgccagcc 1920 cctgctccgt
taggagcagg aggcgccacc gcagccccag aaggtgcggc tagagccagc 1980
aggacgttcg acatatcgaa gttcggcgcg accagcgacg gcaagacgga ctcgacacag
2040 gttgcattgc attgcattgc attgcattgc atgttggcac ggtggtgtga
ctgatgcact 2100 ggttcaatga tctatcaggc agtccaggac acgtggacgt
cagcgtgcgg agcgatggga 2160 gacgcaacga tgctcatccc caagggcgac
tacctggtgg gccctctcta ctt 2213 80 809 DNA Zea mays 80 aaaaaaccca
cgggttcacg ggtttgggta ctataggaac aaacccgtac caataaaccc 60
gtcgggtata gatttatgcc cattaacaaa cccatggata tgaaaattga tccaaacccg
120 taccctaata gggtaaaaac ccatcgggtt tcgggtttcg agtacccatt
gtcatcttta 180 acaggaagtg agtcatgggc ctcttgtgcg tttgcgcttc
tcgcttcatg gtccgtgact 240 ttccacgggt acacatatgg gccctaccat
ggctctctta tcaactgggc ctcgaagcct 300 agctagttga tggcttgcat
aattgcattg catggtctcc tctgctccgt ccgactgagc 360 gattcttccg
gtaggggagc tgcagtgcag ctggtgcatg gcgatggatg gctgcgagtg 420
gtccaagaat ttctccccgg catgtcctct cctccagacc tccaccgatg cagcaggctc
480 ctggtagagc taactaaatc ggggacccct tctcaagttt tcatcactat
atatgcagca 540 gatacctaga agagcacgac cgagctagga gaagcgcgaa
cgcgtgcatg cgcagacgtt 600 gaggtcgagg gacacggtat ctctgagctt
catcggagag cgacccgcca ccgccacgct 660 tggccgcaag ccgagaagag
tgccgggccg ggagaccgga cgattattga tccgtagcag 720 attcgctaat
ggcggatacg gcggacatgg agcggatctt caagcggttc gacaccaacg 780
gcgacggtaa gatctcgctg tcggagctg 809 81 910 DNA Zea mays 81
ccgtgacttt ccacgggtac acatatgggc cctaccatgg ctctcttatc aactgggcct
60 cgaagcctag ttagttgatg gcttgcataa ttgcattgca taattgcgct
tctccctacc 120 atgtgcctgt ttgtttcggc ttctgacagc ttctggccac
caaaagctgc tgcggactgc 180 caaacgctct gcttttcagt cagcttctat
aaaattcgtt ggggcaaaaa ccatccaaaa 240 tcaatataaa cacataatcg
gttgagtcgt tgtaatagtt ggaatccgtc actttctaga
300 tattgaaccc tatgaacaac tttatcttcc tccacacgta atcgtaatga
tactcagatt 360 ctttccacag ccaaattccc ccacagccaa attttcagaa
aagctggtca gaaaaaagct 420 gaaccaaaca ggcccatggt ctcctctgct
ccgtccggct gagcgattct tccggtggga 480 gagctgcagc tgttgcatgg
cgatggatgg cagcgaggtg gtccaagaat ttctccccgg 540 catgtcctct
cctccagacc tccaccgatg cagcaggctc ctggtagagc taactaaatc 600
ggggacccct tctcaagttt tcatcactat atatgcagca gatacctaga agagcacgac
660 cgagctagga gaagcgcgaa cgccgtgcat gcgcagacgt tgaggtcgag
ggacacggta 720 tctctgagct tcatcggaga gcgacccgcc accgccacgc
ttggccgcaa gccgagaaga 780 gtgccgggcc gggagaccgg acgattattg
atccgtagca gattcgctaa tggcggagac 840 ggcggacatg gagcggatct
tcaagcggtt cgacaccaac ggcgacggta agatctcgct 900 gtcggagctg 910 82
1511 DNA Zea mays 82 atcgtgtaag gtgatttagt tcatttgttg tgagggaagt
gtggtgcttg gtggtctgtt 60 ccacgtggtt cctttgctcg agttttcatg
ttctcatcat tcttgccttt gtttgagagg 120 cgaggttatt gtcttctcca
ctggagctgg gcagattttg ggttgaacca tcgcgatgta 180 gctagtgaac
ctgtggtctt ttgacattaa ttaaggtagt gttcggttct ggagccagtt 240
gggatggagt ggctccgttc gagagatttt ggggagttgg atggctcggt atttgaatat
300 aatttccctt tttagaacca ctctatcttg taaaggattg gcactcaggg
ttttcttagc 360 tcaaccgtca gtggaaatcg atttctgctc atggttgtct
taactgaacc gccaatgaaa 420 aacgattttc actagcggtt atcgtttacc
tgcctatgaa aataattatt tctactggtc 480 tctaacgctg gaggttctga
aaaacgccag tgcaaataag tttcgaacca tctctataaa 540 acttctttct
actagtgaca tggaaccaat ccgaacaaca ttaaattgag agtgaagtgg 600
cttgatccaa ctatagtcca gaaatcaaac actgcctaga aggctgctgg gcacgaggac
660 ttgaactcta aaaaagatgt ggtgctggtg ctcttcaaaa tatttgattg
ttgttcagca 720 ttttgttttt gtttttgtag gcgtagttcg atttacccat
ttatatgatt cgctgtgata 780 caatatcata tgtaattaaa atcaactgac
cccccgtttg gatcattgga attgaattcc 840 attctaataa tagtaattta
gatatatatc aattaagcta attcagtttt ttgcaaaata 900 tatttgtata
ttattattag caagatgtta gaaatattta tgtgctatat ttttactata 960
gaggggtgag acgaagagtg tcttgtaagt tacagagtag aaacaaattc tactaatgca
1020 taaaatcatt tctcatcctg caccccatga atttgaaccc catgaatttg
agataggctt 1080 atatctgaac tttgaaaagt ggtggaatgt caaatttcaa
attaaataag ttaatttatt 1140 aggtgaattc caattccttt gaaacaaagg
gatctaaacg tcccgtgaga aaatttgcat 1200 gtgcacaaaa gttcacaatt
tgcatgctga cacacgcatc tctgggtccg tacgattggt 1260 aaaacttgat
gaggttgcct ttgtctagca tccgcatcaa taggaccttt gaaacggtaa 1320
gagttggtca tcgagaacct gaaaaaaaac tagaggacag gagttcttta ttcaagcatg
1380 gcctcaaaat agcaaagtcc agacggtcat ttcgtgtaaa tagcagacgg
tgctcctctg 1440 tctcttgcaa tcttccggaa catccatcga tctcccccca
gcggcgagga gagccggcgg 1500 ccacaggaag g 1511 83 459 DNA Zea mays 83
cgtatctagc gactacatgc tacaacatgc tcgatgtcat atacacctat acatgtcact
60 atggcgtatc atactttgtc attaaaatcc acatctaaga catgatccat
gtacaactac 120 gataagatag gagtactagt taaatctctg ttgggcattg
gaccagatca tgctcgtgct 180 gggctttcgg gcctcgtgtt ctctcaccaa
ttatagtggg tagctagtaa atgcatgcat 240 ctatatatgg acatgtatgc
atgctattag agtattagtt agaagcgtac cactgcacga 300 agagagaggt
acgatcggga gggaaactct catggccata cacctatcat ctccttttcg 360
tgacatccta ctgtgtatat ataaccaaca acgatcatgt tagttccaca agcaaattaa
420 acctatcatc atcttctccg atccatcaac aatcgccga 459 84 1503 DNA Zea
mays 84 atcctccaag gtatcagcag tgggtccgga cccccatggg aaagtgctgg
acccctgttt 60 atatggaccg gacctccagg taaggtccag gacctccacg
ggcgcgaact gaaccccttg 120 gatgggtccc ggacccctct gtgtgggatc
cgggccactc acaacaaggt cccgggattc 180 tgggacaaag aatacctgga
ccttgttgaa gaccaagcga gggtccggag ccgacacgtg 240 ttcgggccat
gcggtgtacg cttctgctct ccactcaggt ggagacccga tgctgccacg 300
tggcccacca ccgtgacgta agccagcggg cgaagcctga cgtaaggcct ctgggccaca
360 cggcctctgc atttattaca gataagccgc atcgcgtgtc cactccactg
gcaggcgatg 420 tgccgcctca gaatttaatg agccttgtcc actccactgg
caggcgatgt gccgcctcag 480 catttaatgt gccctgtcca ctctgctgac
aggcaacgac cagccatcct gcaggcggcg 540 tgcctgtcca ttccgttggc
aagcagtacg cctatgctgc ggcatacact gtgctcatca 600 tcactcgcgt
gttaccaagg aggcagcatg ggataccaat actatatgca ctacagacat 660
tatagcgctc ggggttcacc ctggcgttac gggcattagt tgcttccttc catttgtccc
720 tcggcccaca tgtcggggct cagcaccctt gtacgtgccc cccttgagct
ataaaaggga 780 gggcacacga cgttacaagg aagacccaac ttaggctcac
acactcactc aaactcacaa 840 gttcatacaa gctctcaagc tcaatacatc
acacagtgga gtagggtatt acgctctggc 900 ggcccgaacc actctaaacc
cttgtgtgtt cttgtgttct tcccgattcc atctagcagg 960 caaaacgctt
gggcccctcc tcatcttagg atttagggcg ggtgcgttcc gccacccgac 1020
cggagaattc cctctccgac agtactcatg accacaaatt cagaccctgt ttgctagctc
1080 attcatcgta gcatagttcc attcactcat cgaagacaaa acatggttgc
gattgtgagc 1140 accatgtggt cgtcgatgca ggcgcatgtg gcgatggtcg
tggcattggc gttcctagtg 1200 agcggtgact ggtgcggtcc tcccaaggtt
ccccctggca agaacatcac ggccacctac 1260 ggcagcgact ggctggacgc
taaagcgaca tggtatggca agccaacggg tgctggcccc 1320 gacgacaacg
gtggcggctg cgggtacaag gacgtgaaca agcccccctt caatagcatg 1380
ggcgcgtgca gcaacatccc tatcttcaag gatggtctgg gatgtgggtc ctgcttcgag
1440 atcaagtgtg ataagcctgc ggagtgctct ggcaagcccg cggtggtgta
catcacggac 1500 atg 1503 85 658 DNA Zea mays 85 aaattaatac
aaataaaatc atataagtca ctccttctct aaatttatcg tatatacaaa 60
attattttga ttgttattga aaattaatat acaattatat gatgcaagtt tctttaatta
120 gcttattatc tatactatat aaaaatcagt atacacatgt tttatatatc
atatggtact 180 tttttcatat tatagtattg atgaattttt cgcccctcta
tgtcatactc ctggcttcac 240 cctagtctac tacgtcaatt tttttcagta
aatgcatcgc aaaatgattt tgcatttttg 300 gtgtcctaaa tcttaatata
tattcttaga caaatagagt taaacagatg ttaaacatag 360 atttgactta
agataaaaat agattttaga aaatacagaa cagccccagt attctgcatt 420
gctaaaaaac actccgtgaa acaatgtgga ccgcaaaaag ttccttcaaa atcctgccat
480 ctgatgctat ttttggggcc aaactccatc accaaccaaa cacaacctct
tggctttatt 540 taacttgtgc cttgcggatg ttttcgttgt cgagggaata
cgaacgtcgt acgagaacct 600 ttctccctcc tccacctttc tccttttctt
gccacggcaa aacaccttcg ccggcgag 658 86 1173 DNA Zea mays 86
gtcgtcaacc cggtgactgc catgggcccc atgattccgc ccaccatcaa ctgcagcatg
60 accgtgctcc tacgcctgct acaaggtgcg tggagtagtc gttgctttcc
tgcttgctgc 120 tcgatatgca tgccgttcgc gttgccatgc gaatgagacg
aagaagaaac taaaggagga 180 tgccggcctg ttcgtggtcg caggttgcac
ggaggagtac agggacatct ggatgggggc 240 ggtgcatgtg cacgacgccg
ccatggcgca tatcctggtg ttcgagagcc cggcggcgtc 300 cgggaggcac
atctgcgccc agtccatctc ccactggagc gacttcgcgg ccaaggtcgc 360
cgagctgtac cctgagtaca aggtgcccaa gtaagcgacc cgaccatgtt ctgtgaaaat
420 gaaaacctgg atagatagag cattgcttag cttatagttg cgtacgttgc
aggttcccca 480 aggataccca gcctgggctg gtgcgacagg gagccgagga
ggggtccaag aagctcgtcg 540 cgttggggct gcacttcagc cctctggaga
agatcatcag ggacgctgtg gaggccctca 600 agagcagagg ctacatttcg
tagctagccg accgacggca gctatagtgg agtagtatgc 660 ctgtcgaatt
tcgattccca agtggcaaat tctgcaaaac gagtccgcca atatgaacaa 720
taaataaaga acgttgtgat aaaataaagc agattttctg ttgcatttgg cccttcaaag
780 catccgtggt ggtaagattt cctatgatct gtcctggtcg gtcgggcctg
agcacctttt 840 ttctgtagac ggatgcttta tcacctaggg attgttttat
tatattgcta taatgcaaat 900 tggttgatcc aaattaaagc aggatctaaa
atggtcgaca ggctaagctt ataatgaaca 960 cagaaataaa tcaaggtgga
atgtgtccgc aatcgacgct gcgatttcga atgctaaata 1020 aataaatcgg
taacacggac ggacgtagaa gagaagccat tatgcgtggc aggcagcaca 1080
agagctattc aaagccgcgg caacggaggg ctgcaattca caaaccccaa aattaggtca
1140 ccccggccac tttcaacgcg aacaccgcaa acg 1173 87 1587 DNA Zea mays
87 actcttccca tttgcgagga atctccacaa gttggagcct ctcaccctta
caaagttatg 60 atcacaaaga aagcacaaga gtaaggatgg gagagcaaca
cacgcaagac tcaaatccgt 120 agcacaatca cgcacacaag ccaagacttg
agctcgaaac acagcacatg gagtttgcaa 180 ctcaaacaga gctcaaatca
ctaacacagc gaatcaaatg cgtggagacg gagtctggga 240 gtcttagaat
gtttcttgaa agcttggtgt tctgctccat gcgcctaggg gtccctttta 300
aacctccaag acagctagga gtcgttggag atcaacatgg aaggctgatc tttccttctg
360 ccgagtggcg caccggacag tccggtgcgc caccgggcag gtcctgtagc
ttgtccggtg 420 tgcgatctcc ttccatatcg ggcgcatccg accgttgagc
cggcggtctc gttggcgcat 480 cagacactgt acggtgtaca ccggacagtc
tggtgtgccc aaccaaccgt tggcccgtcc 540 acgtgtcacc cgcagatttc
gctgccgacc gttggccgtg agcgccgttg gctcatcgga 600 cagtcattag
gaacgaagca taaacaaaag cgcgtgatgt ggacacacat tagggtattt 660
tgggctaact tgacacactt gagtatttat aagatgtgtc atgggcttga cgactctata
720 ggctagctag cacggcacat aattaggttt gtactttagc gtgccatgct
agcctatatg 780 tgtataaggc cattctcaat tgaagtttta ttggagtttc
attctcatta aatgttgtgc 840 cacataagca aacaagacga catgtcatac
catttaatga agagagatat gagagagttt 900 aatggggaat aaagctctat
ttacactatt tcctagacaa atattgatat ggtatccttg 960 gaattcgtat
gatgaaatcc tcaattgaag aatggcttaa ctggcaccgc tacgtagggg 1020
ctattcaaga accaacaatg tacagttgtt gcaacgtgaa tggttatttg cttcagatta
1080 aagccaattg tttagactga tgcagctgca attcatagag acaaaaacag
tgtagaagcc 1140 gtataagcat taagcaaaca agcgaacatt gcttagctac
aaccaatttg ctgggcttcc 1200 atgggcatcg cagaagtatt gtggctgcat
attgctgaaa ttatagcgag ggcccaaggc 1260 ccatcacttc acttcgaggt
cagcattgta cttttgttaa cgtctcgata aatttgttca 1320 cttaaaatag
accagttcaa ttctggttct agtcaacatg cctggatcca cgggggagcg 1380
aggagacgaa tgtgtggccc gccgcagtga ggccaagccg agcccggtcg tccgtccaac
1440 caccccctcg tttatactat atatacacag acgcacgata cccatatcgt
ggtgctagaa 1500 gcaactgaaa acagccgagc gatctcctct ccctctccct
ctccgatcca ttctccagcg 1560 cagcgaagta aacatgtctg accgggc 1587 88
665 DNA Zea mays 88 aaagaaaatt ggatgaaagt tactcgaccc cgtcaattta
acagtgacat tgaatttgtc 60 gaggctgttg agatgaagac atgccagatt
gagggtattt tactaataag gagattttag 120 gtgtgaatga caaacgcttc
agccgaatac ttaaaagact ggctaagaag aacaaagcta 180 atttggtgga
ggtttcatcc aacagtgatg aggaaagaac accaactctg gtagaagtag 240
agtatgttgc tgcatcaaag agtttttttt ttgtggaaaa cttcagcgtg tagttttatt
300 ggtcagcaat gtttgtttgg cagtagcatg catgagtccg attctctgac
catctccatt 360 taccggtgcc ctggttatta tccccccatc acaagagtgg
ccaacatgca gcccctgaaa 420 cctggcgaag tccaaggggg agcgaggaga
cgaacgtgtg gccacggtga ggtggggatc 480 cggtccttca ccccttcaac
ttgggattcc ctctctattt agccatccgt ccggtgcacg 540 atgctacaag
ctcctcgtca ccagtcagaa aacagtggga tcgagttgtt tcactgcacg 600
agcacatcct ccggcgacca ccggcctccc tctccgtcct ctagcgaccg accaacgcgc
660 cgagc 665 89 833 DNA Zea mays 89 acggaaccta aatatggatg
tcttacaaca gctaattaga tgcgaaaggt tccagcatgc 60 ccattcgtta
ccctgtgaac atgggcagat ctacgggtat tatgttctgg cacaccctac 120
gtatccggta tatcttgccg attatgtttt aatactatga ggttgtttgt ataatcacat
180 ttcacaaatg agagctgaga atttaatccg tgcaaattag tttatttaat
tgtttgggaa 240 tatgttttta agtaggtgaa gataacataa ttaagatatc
gattatgtct cttagtaagg 300 tctcagctaa aaagtcgtat gaactattag
catgactttt cattgattta tattgtaatt 360 tatgaatatt tttaacttac
tttacaaatt taaggattat tatttatatt ttgaacttat 420 cctataattt
aaaatttact atgtaatttc atgtaaaaat ggtttctaat ttgatcgagt 480
atatatatga aaattttaga tgacttatag aaaaattcta gatccgccat tggctgcaga
540 gtgtagagga tgtgcatgca cagatgcact tcattgttgt tatatataca
acaagttttc 600 atgcaataca agcctataaa taaatgtcct gactaagctt
tcgtccacag aatttaccac 660 ttcttccgct gagtactacc gattcaacag
aacagataga ccactcgtta acactgtaca 720 cttctaccta tatattcgct
tctctcctct tgcaaatcat attgtcaata gtaacagtga 780 gaagaacaca
caaaatgagg gttcttgtag agaagctgtt agtctctgtg ttt 833 90 823 DNA Zea
mays 90 ctgcacggta ctccaagtat aagacacagc taaaacacaa cataatgcag
tggtcatgtc 60 taaaacatgt gtcttaccat attcattgta tcaatcagaa
cattcaataa attaaagtga 120 ccaatcagat agtctcctgt cccgaatata
gagctaagac actgtgtctt cgtcaagata 180 catgtcttga gattttttac
attcaccccc ctagacacac tctaagacac aacttaagac 240 acccattgta
catgccctaa ctggcaccgc tacgtagggg ctattcaaga accaaccatg 300
tacagttgtt gcaacgtgaa tggttatttg cttcagatta aagctaatta tttagactga
360 tgcagctgca attcatagag acaaaaacag tgtagaagcc gtataagcat
taagcaaaca 420 agcgaacatt gcttagctac aaccaatttg ctgggcttcc
atgggcatcg cagaagtatt 480 gtggctgcat attgctgaaa ttatagcgag
ggcccaaggc ccatcacttc acttcgaggt 540 cagcattgta cttttgttaa
cgtctcgata aatttgttca cttaaaatag accagttcaa 600 ttctggttct
agtcaacatg cctggatcca cgggggagcg aggagacgaa tgtgtggccc 660
gccgcagtga ggccaagccg agcccggtcg tccgtccaac caccccctcg tttatactat
720 atatacacag acgcacgata cccatatcgt ggtgctagaa gcaactgaaa
acagccgagc 780 gatctcctct ccctctccct ctccgatcca ttctccagcg cag 823
91 1163 DNA Zea mays 91 actacagccc gagggcgcct gtctacgggc ccctcggcgc
agactatctg gttgtcccac 60 cggatagtcc ggtgcacacc agacaattac
tgttcactgt ccggtgtgcc atcaggcgtt 120 ggttgactgc ctttttcttg
gatttcttcg cagtttcttt tgagcttctt ttgttcttga 180 gtcttggact
tctatgcttc tttttatatc ttcttttgag gtgttgcatc ctcattgcct 240
tagtccaatc ctcttcgcat cctgtgaact acaaacataa acactagcaa acacattagt
300 ccacaggttg cgttgttcat caaacaccaa aaccttttga gccaaatggc
acaggtccat 360 tttcattaca gccaccctcc tcagtcgttg gttgttagtt
attttcgacg gctcacgtgt 420 atagccgcca aaatttggca aatttcggca
ccacaatgtc caaccatcga aaattagaca 480 atagggaaaa tcacgggccg
ccctttcatt ttccacggcg aattagggtc accaaaccaa 540 aacaatcgaa
aaccaaacca cgagccttaa tttttgtggc cttgaccgcc aaaaattaca 600
tgttttcttc tagtggtagg gggagttata agcaacaact ctaacaattg tagaaaaata
660 acattgggtg accaagatga gtaagagagg aatttaggat gagattaata
tgtgtttatt 720 gctatctaaa ctttatacat gaggtttcta ggctcgtcat
atgttataga gtcaaaaagt 780 atgacatgtt tttttagtca caacaaagtg
tggctttcca cacttttgtg gtttatcttg 840 tttaactaag attagccatg
acaatttatg agcactcgca tgtttggcca cctatatata 900 gcgagacttg
tgcatccaag acttcttccg tgcgagggta gtgcacgacc ataggacaag 960
aggagcttgc attcgcgcgt ctcaaggcaa caatctcccc taaaaatagc cacacaacat
1020 tcatgttgcc tatatataaa catcgtgcct cgcccgtccc atcatcacag
tcgaaacaaa 1080 gccacaacac atacaggaaa gcaagcaaga atcatcggaa
taatggctcg tgcatgcgtg 1140 ttcctcgtcg tgctcctcct ggc 1163 92 2126
DNA Zea mays 92 cctccgaaat caccgaccac agagatacac ttgcacgggt
gtgcgggcga tcagattttt 60 ggggagcgtc ttcgcgactg ctcgcgtgat
cgtccacagc ttgctgttcg tcgccttccc 120 aagttgacgc gtgctgctgt
tcttcttccc ggcgaccgtt cgagggactg cactgcgtac 180 atcttcctgc
accgacttcg tacggctaca tcgaacaaac acacgagatg tctcgtgtga 240
atggagccac tggtgccttg agcatcggtc cctccgctgg gtacactctg ttcttcgtat
300 ttgtgcatgt ttcattgctg tttactgctt atgcgagtag ttatacacat
atgcacatac 360 atgtcatcac atatatcgca ctgattatct ggattaaatt
aaaactaaaa atgcctaact 420 ttctaacaat atttgcactt gttcttacta
ttcctgtttg atttggtttt ttgattgagt 480 gtgatgagtt gtgaagtaat
gtctataatt gcatctaaat atatatatat gcataaaaat 540 atagaaatac
tcccataaaa acagatccat cttatcttga aagattctat attatcctaa 600
tagatccatc ttgtcctgat aagcatactt attccacctt agagtaacaa tcatgatcta
660 atccaaatta attagatcta atctaatcta atctaattca atataatcta
atttgaccta 720 atttagtcaa aactagtcta atctaatctg aaactcttat
cttattgatc tttcttgcct 780 atatctaaag gttagaacta attaacttat
ctagtccaac ctaggagcaa aacaacaaac 840 atgattctac atattctcat
gaagcttaag ccacctatta agccatatgc tctacctact 900 gagctatttg
gtgtacctac aaacccattt taaccataaa tcatttaatt tgcaattagt 960
gaacattgta attgtcctag ttgcctggtt tgttgtatta tatatttgag taacgagtaa
1020 tgacttcata tttggattga gctagtaact taactaattc caacttggat
atggatgtat 1080 atatggttgt atccgtgggg gtggcctttg atttggttat
atttcacttg agagagtagt 1140 attgtaatgt tctgaagagt tgttcagtat
tttctggaac aaagaagata aatacttccg 1200 taaggttgca caacttatca
tttgaaaggg gttactatgt ttggttgtga tgcatatagc 1260 tcatgctgaa
actaatcatc tttgttgatt agaacggcta ggaggttagc agttgtagaa 1320
gttgcagtaa ttacagaaaa aaaaaggatg gatggaagca atccaatatg tgcatgcaaa
1380 atttgggttt gaacaaggac ttatggtaag agaaacatgc gcttgaacag
tatgtgtagg 1440 agactcaaag gagttctgga gagagaggtt tcttcttttt
tttcttagtt ttgacttccg 1500 gtctataact aacattgtaa gagcaaaaat
gagaaaaaaa catgactaaa gttaaaaata 1560 aggacaccag caaaaccaaa
ggcaccacct taaaaaactg ttttcttttt ctgaacacag 1620 aatcaagggg
atttatttgt tattaatact aacagaaaat gttagcagga gtgacagtct 1680
gttggagcag gattctgcga ggaagccgct tatccataga caggaatttt tttagtgata
1740 agtagggcac actcttaact ttgcatgagg agtttgggta ccaaatacag
gtgaggggtg 1800 aggcttcgtc agtcgttgca caggtaaaga ttgatgattt
gatgtaactt tgaaccgctc 1860 taactaacta aatcgtcctc gagtgtgcgg
cgggtgcgag caacaaggcc gtccgctcct 1920 tgttcgtccc tgcatgtgtt
ccgttcggtt ccatcaattc caccacgaaa taaggctgta 1980 taaatctttc
ctgggcgttc cctctctctc ctgtgcatcg ccggacggaa ccaaacgcca 2040
aacgctgctg cgctctctcc ttctcgtcct gaccccccag agcgagaggg aggggcaccc
2100 agaggagcct gtcgttttga tcggtg 2126 93 2508 DNA Zea mays 93
actgcttgtg aaaagtaaac tgaattctgg ttgtgaacta cttgtttaag taaatgcgtg
60 tttctgtttt ttgttgtcag tgcatttctg ttttcactga tgaaaccacc
atttctgctt 120 tcaatgaatc tattgaactg aactgcaaaa aaagaaattg
ttctattttg tttgagtgca 180 caaacggaac tcaacggaac tagatctgaa
tatgtttgag tgcacaaaca gaaattgttc 240 tgttctggtt tcagtgaact
ccacaaatag aactgaatct attatgtttg agtgtagaaa 300 cagataccga
aatgctacat ctagcactta atctggcaga atcagaaaat tggggcaaat 360
acaagttgtt taagagcaca aacagaaact gaatctgttt gattgcagaa accaataaaa
420 acagaaatat atgttgaaaa taattcataa gtaggcagtg gtggtgctat
ggtgcatacc 480 aggttcttat tcaaatgatt tgctaaagtc aaatatatgc
ttctggtctg attgatttgt 540 gaaactgaaa tggatatttt atttcggcac
tatgaaataa actcactgtg atcctgaaac 600 atatcagttg tgtttgtttt
tgtaaatctt ttataccact aggggagaaa attagcttag 660 ttcaatcgca
tctcatatgt ctaattacca ggggagaaaa ttagcttagt tcattttgtt 720
gctgccatat gggtgaaaaa ataatgagac atctaaatca gtaaattgga aatatagcat
780 cttaaacctg caggtagttt cttaaacctg attctagcta caacttagta
caactaccgg 840 tagtttttta aacctgattc tagctacatg ttttatattg
tggcacaaga acttttaaga 900 acatatgctg atgcccactg tatttagtta
ctacttcaag accaactgta ttttagttac 960 aaatgtgttt tcaagattat
agaaatttgt agctgaaatt atccacacca tatttgtgaa 1020 ctgacatcat
ttctaagaat attactgatt agaatctttc acttttataa tgctttgcag 1080
gagtggcccc tctggagttg aatatgcagt tataaccaaa ttttacccct tttatcctag
1140 aagagttgcc aagacacggt ataagaccat gataatagac taagagagga
tttggctcta 1200 attactatat gttttattta tgcagtccca tgagaacttt
gagtatttgc agattgcttt 1260 attaatttat taaagttaaa gattgtatgt
gttgagtatg
tatccactct tgttggaagt 1320 gtcttgcaat tccaatccaa ggatgtataa
aatactgcat gggctaagta tgtgtttttt 1380 catgtatttg gagtatatat
actttttgtt gcttgagaac atgtatgtac actagaagct 1440 tgtcaattgt
gtgaacttga gttgatccct gtctaacctg agtatatata tatatatata 1500
ttttgttgct tgagaacaag tatgtacaat agaggcttga caattgtgtg aacttgagtt
1560 gaacatgaat tttgataatc acaactcacc atccctttca atatgcttag
aatatagctt 1620 tttataattt ttcaccctac aatacaaaat tgttctatga
aggccatggt acatcatcat 1680 atcctgtatt atcaacctag gatttgtcta
tttcgattaa taatggcatt gagtcaaatt 1740 ttggttgttt caaatgatag
acttcgatat ttgttatgat ttatgagttg attcttgata 1800 gcattactaa
aaaatgacct atgtatatac aagtgtcttc cgttgcaacg cacggacata 1860
tacctagtca atcactaaga ccctaatttt gaagttggga cttagacgtg ttccacgttt
1920 gtaaaggcaa gtatataggt gtatgtatat aagagccggt gtatacaaca
attttttata 1980 agaaaacttg aacaagtagc caggtgttga aatcttcata
tatgtgccga cgccattcaa 2040 catcatattt ggcttctggc gaggatcgta
gtatcaagca acataaaagc aatgacaaac 2100 agcgaagcac aaagatctcc
caggctcgtc ataaactaat cacaatgttg tttgtcctcc 2160 acaattagca
caacccattt tagaaaaaga tgccacgatc gatcgagacg ttggccagct 2220
atcaaacaga taagaactac ccaaatattt cctaaatcca gaacggaaga cccattgact
2280 aggtccttac ctctcaaata gacagactat tcttctccac atcaaaatat
agggactccc 2340 gatgcaacaa acacgggcca ccacacaaca atggtgaaat
gaccatgcat gcatccacgt 2400 ccgtacgcag ccatttcgtc tataaatttg
cttcccatcc gattcaacta caagcttgcg 2460 ggcaaaaatg gcaaaggctc
tcctaggtgg cctatcggcg atcctcgt 2508 94 1797 DNA Zea mays 94
aagctttagg aagttgtttt tcggatcttt ggaattgggc ctccaagtta taatttttgc
60 aatctaagct ctttgcaaag aaaacaaact catatcgcaa tgggctactt
ttggggacct 120 tctatgttga agatcacttg cttaaacttt tactttgaca
caagctagtg tgtttcagga 180 atatctctac tgatgcacga agccaacctt
cggtcgggaa ggcgtgcaga gaagcattgc 240 acatgaaagg tgaaagggta
ttaaccaaag ttgatagctt cgatgacagc aaaggagctt 300 cagatacaag
ccaaaggaaa aggcgacgaa ggcctaaatc cgagcagccg aagatgggga 360
aaatacgcta ctgccctaac aacatttgta aacagtgagg ggtacaattg taattatgta
420 ctaagtcggt tcgtctcccc tataaataga tgaacagtaa cccgcataaa
ttacattttg 480 ccaggtgcta cagctttgta tagctcaggc tccaaaacac
attcgtgcta tcttgcacta 540 agaagtcaat ggtatgattg taaacttgtt
ttctataaga gaaatgaaat tctaaggcac 600 atgagatgag ttctcatatc
ttcgtcatgt ttttatgtat tctagtcgat tacatccaac 660 cttcgtcctt
gagtagttat cccaaagact taacacttca aggatgaagg cttctacttt 720
ttaacattgt gttgtcttgt tttttatttc atttagcaat taaaagcaag tgactaacac
780 atggttaaac ccaagatccg aaaagaggct aaaattgagc aagaatgaac
aaaagttggt 840 aagaggaaca taaaccaacc tttcttagca acattcttcc
aaaaaaagaa gatcaaaaca 900 tgtacccttg tattttgtga aaactggatc
tccaaaattg cctacaatgg aaggtggcta 960 cgagaaacgg ttataatcga
ggaggtagag agaattttat gctacaacct tcacaggcgg 1020 tttccctaag
aaacatccac tctaaatgtc tttgcacata cggttcactt aaaaaaccgc 1080
aaatgcaaat tgttcatttt cactggaggt tttttaagcg aaccgctaga ggaaatctca
1140 tttgcaccgg cgatccttaa gacatatcat gagcgaggtt gccttggaag
ccggaagagt 1200 tggtcaatga cctataaaaa gcagaggaca caggagtgcc
ctattcaagc attgcctaaa 1260 aatagcaaag gccaaacgac catttcgtgt
acatagcaaa cggtgctcct ctctctcaag 1320 aaaggatatc ttcgggaaca
tccatccatc cccaatcccc aaaggcgagg agagaactag 1380 caaaggggaa
atggctgctt ccacaaacaa cactctcagg gtgctgttca ccctaatggt 1440
tgtatgcgcc gcagtatgca cagcgaaaag gactgtagca aaggcaggag acttggcgcc
1500 agcccctgct ccgttaggag caggaggcgc caccgcagcc ccagaaggtg
cggctagagc 1560 cagcaggacg ttcgacatat cgaagttcgg cgcgaccagc
gacggcaaga cggactcgac 1620 acaggttgca ttgcattgca ttgcattgca
ttgcatgttg gcacggtggt gtgactgatg 1680 cactggttca atgatctatc
aggcagtcca ggacacgtgg acgtcagcgt gcggagcgat 1740 gggagacgca
acgatgctca tccccaaggg cgactacctg gtgggccctc tctactt 1797 95 828 DNA
Zea mays 95 ccgtgacttt ccacgggtac acatatgggc cctaccatgg ctctcttatc
aactgggcct 60 cgaagcctag ttagttgatg gcttgcataa ttgcattgca
taattgcgct tctccctacc 120 atgtgcctgt ttgtttcggc ttctgacagc
ttctggccac caaaagctgc tgcggactgc 180 caaacgctct gcttttcagt
cagcttctat aaaattcgtt ggggcaaaaa ccatccaaaa 240 tcaatataaa
cacataatcg gttgagtcgt tgtaatagtt ggaatccgtc actttctaga 300
tattgaaccc tatgaacaac tttatcttcc tccacacgta atcgtaatga tactcagatt
360 ctttccacag ccaaattccc ccacagccaa attttcagaa aagctggtca
gaaaaaagct 420 gaaccaaaca ggcccatggt ctcctctgct ccgtccggct
gagcgattct tccggtggga 480 gagctgcagc tgttgcatgg cgatggatgg
cagcgaggtg gtccaagaat ttctccccgg 540 catgtcctct cctccagacc
tccaccgatg cagcaggctc ctggtagagc taactaaatc 600 ggggacccct
tctcaagttt tcatcactat atatgcagca gatacctaga agagcacgac 660
cgagctagga gaagcgcgaa cgccgtgcat gcgcagacgt tgaggtcgag ggacacggta
720 tctctgagct tcatcggaga gcgacccgcc accgccacgc ttggccgcaa
gccgagaaga 780 gtgccgggcc gggagaccgg acgattattg atccgtagca gattcgct
828 96 847 DNA Zea mays 96 ccgtgacttt ccacgggtac acatatgggc
cctaccatgg ctctcttatc aactgggcct 60 cgaagcctag ttagttgatg
gcttgcataa ttgcattgca taattgcgct tctccctacc 120 atgtgcctgt
ttgtttcggc ttctgacagc ttctggccac caaaagctgc tgcggactgc 180
caaacgctct gcttttcagt cagcttctat aaaattcgtt ggggcaaaaa ccatccaaaa
240 tcaatataaa cacataatcg gttgagtcgt tgtaatagtt ggaatccgtc
actttctaga 300 tattgaaccc tatgaacaac tttatcttcc tccacacgta
atcgtaatga tactcagatt 360 ctttccacag ccaaattccc ccacagccaa
attttcagaa aagctggtca gaaaaaagct 420 gaaccaaaca ggcccatggt
ctcctctgct ccgtccggct gagcgattct tccggtggga 480 gagctgcagc
tgttgcatgg cgatggatgg cagcgaggtg gtccaagaat ttctccccgg 540
catgtcctct cctccagacc tccaccgatg cagcaggctc ctggtagagc taactaaatc
600 ggggacccct tctcaagttt tcatcactat atatgcagca gatacctaga
agagcacgac 660 cgagctagga gaagcgcgaa cgccgtgcat gcgcagacgt
tgaggtcgag ggacacggta 720 tctctgagct tcatcggaga gcgacccgcc
accgccacgc ttggccgcaa gccgagaaga 780 gtgccgggcc gggagaccgg
acgattattg atccgtagca gattcgctaa tggcggagac 840 ggcggac 847 97 727
DNA Zea mays 97 aaaaaaccca cgggttcacg ggtttgggta ctataggaac
aaacccgtac caataaaccc 60 gtcgggtata gatttatgcc cattaacaaa
cccatggata tgaaaattga tccaaacccg 120 taccctaata gggtaaaaac
ccatcgggtt tcgggtttcg agtacccatt gtcatcttta 180 acaggaagtg
agtcatgggc ctcttgtgcg tttgcgcttc tcgcttcatg gtccgtgact 240
ttccacgggt acacatatgg gccctaccat ggctctctta tcaactgggc ctcgaagcct
300 agctagttga tggcttgcat aattgcattg catggtctcc tctgctccgt
ccgactgagc 360 gattcttccg gtaggggagc tgcagtgcag ctggtgcatg
gcgatggatg gctgcgagtg 420 gtccaagaat ttctccccgg catgtcctct
cctccagacc tccaccgatg cagcaggctc 480 ctggtagagc taactaaatc
ggggacccct tctcaagttt tcatcactat atatgcagca 540 gatacctaga
agagcacgac cgagctagga gaagcgcgaa cgcgtgcatg cgcagacgtt 600
gaggtcgagg gacacggtat ctctgagctt catcggagag cgacccgcca ccgccacgct
660 tggccgcaag ccgagaagag tgccgggccg ggagaccgga cgattattga
tccgtagcag 720 attcgct 727 98 746 DNA Zea mays 98 aaaaaaccca
cgggttcacg ggtttgggta ctataggaac aaacccgtac caataaaccc 60
gtcgggtata gatttatgcc cattaacaaa cccatggata tgaaaattga tccaaacccg
120 taccctaata gggtaaaaac ccatcgggtt tcgggtttcg agtacccatt
gtcatcttta 180 acaggaagtg agtcatgggc ctcttgtgcg tttgcgcttc
tcgcttcatg gtccgtgact 240 ttccacgggt acacatatgg gccctaccat
ggctctctta tcaactgggc ctcgaagcct 300 agctagttga tggcttgcat
aattgcattg catggtctcc tctgctccgt ccgactgagc 360 gattcttccg
gtaggggagc tgcagtgcag ctggtgcatg gcgatggatg gctgcgagtg 420
gtccaagaat ttctccccgg catgtcctct cctccagacc tccaccgatg cagcaggctc
480 ctggtagagc taactaaatc ggggacccct tctcaagttt tcatcactat
atatgcagca 540 gatacctaga agagcacgac cgagctagga gaagcgcgaa
cgcgtgcatg cgcagacgtt 600 gaggtcgagg gacacggtat ctctgagctt
catcggagag cgacccgcca ccgccacgct 660 tggccgcaag ccgagaagag
tgccgggccg ggagaccgga cgattattga tccgtagcag 720 attcgctaat
ggcggatacg gcggac 746
* * * * *
References