U.S. patent application number 12/400175 was filed with the patent office on 2009-07-16 for nucleotide seqeunces mediating male fertility and method of using same.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to Marc C. Albertsen, Tim Fox, Gary Huffman, Mary Trimnell.
Application Number | 20090183275 12/400175 |
Document ID | / |
Family ID | 37591902 |
Filed Date | 2009-07-16 |
United States Patent
Application |
20090183275 |
Kind Code |
A1 |
Albertsen; Marc C. ; et
al. |
July 16, 2009 |
NUCLEOTIDE SEQEUNCES MEDIATING MALE FERTILITY AND METHOD OF USING
SAME
Abstract
Nucleotide sequences mediating male fertility in plants are
described, with DNA molecule and amino acid sequences set forth.
Promoter sequences and their essential regions are also identified.
The nucleotide sequences are useful in mediating male fertility in
plants.
Inventors: |
Albertsen; Marc C.; (Grimes,
IA) ; Fox; Tim; (Des Moines, IA) ; Huffman;
Gary; (Des Moines, IA) ; Trimnell; Mary; (West
Des Moines, IA) |
Correspondence
Address: |
PATRICIA A. SWEENEY
1835 PLEASANT ST.
WEST DES MOINES
IA
50265
US
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
Johnston
IA
|
Family ID: |
37591902 |
Appl. No.: |
12/400175 |
Filed: |
March 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11166609 |
Jun 24, 2005 |
7517975 |
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12400175 |
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10412000 |
Apr 11, 2003 |
7151205 |
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11166609 |
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09670153 |
Sep 26, 2000 |
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10412000 |
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Current U.S.
Class: |
800/278 ;
435/320.1; 435/419; 536/23.6; 800/298 |
Current CPC
Class: |
C12N 15/8231 20130101;
C07K 14/415 20130101; C12N 15/8289 20130101; C12N 15/8287
20130101 |
Class at
Publication: |
800/278 ;
536/23.6; 435/419; 800/298; 435/320.1 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C07H 21/04 20060101 C07H021/04; C12N 5/10 20060101
C12N005/10; A01H 5/00 20060101 A01H005/00; C12N 15/63 20060101
C12N015/63 |
Claims
1. An isolated polynucleotide comprising a sequence selected from
the group consisting of: (a) a sequence encoding a polypeptide of
SEQ ID NO: 22; (b) a polynucleotide comprising SEQ ID NO: 3; (c) a
polynucleotide having at least 90% identity to a sequence selected
from the group consisting of SEQ ID NO: 3, and 7; and (d) a
polynucleotide having at least 95% identity to a sequence selected
from the group consisting of SEQ ID NO: 3, and 7.
2. A plant cell comprising the polynucleotide of claim 1.
3. A plant comprising the polynucleotide of claim 1.
4. An expression vector comprising the polynucleotide of claim
1.
5. A method of controlling male fertility of a plant wherein the
method comprising modifying expression of a polynucleotide in the
plant comprising the polynucleotide of claim 1.
6. The method of claim 5 wherein the expression of the
polynucleotide is repressed.
7. The method of claim 5 wherein expression of the polynucleotide
is repressed by mutation of the nucleotide sequence.
8. The method of claim 5 further comprising delivering into the
plant a second polynucleotide which represses expression of the
polynucleotide.
9. The method of claim 5 further comprising delivering into the
plant a second polynucleotide oriented in the antisense direction
relative to the polynucleotide thereby repressing expression of the
polynucleotide.
10. The polynucleotide of claim 1, wherein said polynucleotide has
at least 90% identity to SEQ ID NO: 3.
11. The polynucleotide of claim 1, wherein said polynucleotide has
at least 95% identity to SEQ ID NO: 3.
12. The polynucleotide of claim 1, wherein said polynucleotide has
at least 90% identity to SEQ ID NO: 7.
13. The polynucleotide of claim 1, wherein said polynucleotide has
at least 95% identity to SEQ ID NO: 7.
Description
[0001] This application is a continuation of previously filed and
co-pending application U.S. Ser. No. 11/166,609 filed Jun. 24,
2005, which is a continuation-in-part of previously filed and
co-pending application U.S. Ser. No. 10/412,000 filed Apr. 11,
2003, which is a continuation of previously filed application U.S.
Ser. No. 09/670,153, filed Sep. 26, 2000, now abandoned, both of
which are incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Development of hybrid plant breeding has made possible
considerable advances in quality and quantity of crops produced.
Increased yield and combination of desirable characteristics, such
as resistance to disease and insects, heat and drought tolerance,
along with variations in plant composition are all possible because
of hybridization procedures. These procedures frequently rely
heavily on providing for a male parent contributing pollen to a
female parent to produce the resulting hybrid.
[0003] Field crops are bred through techniques that take advantage
of the plant's method of pollination. A plant is self-pollinating
if pollen from one flower is transferred to the same or another
flower of the same plant. A plant is cross-pollinated if the pollen
comes from a flower on a different plant.
[0004] In Brassica, the plant is normally self sterile and can only
be cross-pollinated. In self-pollinating species, such as soybeans
and cotton, the male and female plants are anatomically juxtaposed.
During natural pollination, the male reproductive organs of a given
flower pollinate the female reproductive organs of the same
flower.
[0005] Maize plants (Zea mays L.) present a unique situation in
that they can be bred by both self-pollination and
cross-pollination techniques. Maize has male flowers, located on
the tassel, and female flowers, located on the ear, on the same
plant. It can self or cross pollinate. Natural pollination occurs
in maize when wind blows pollen from the tassels to the silks that
protrude from the tops of the incipient ears.
[0006] A reliable method of controlling fertility in plants would
offer the opportunity for improved plant breeding. This is
especially true for development of maize hybrids, which relies upon
some sort of male sterility system and where a female sterility
system would reduce production costs.
[0007] The development of maize hybrids requires the development of
homozygous inbred lines, the crossing of these lines, and the
evaluation of the crosses. Pedigree breeding and recurrent
selection are two of the breeding methods used to develop inbred
lines from populations. Breeding programs combine desirable traits
from two or more inbred lines or various broad-based sources into
breeding pools from which new inbred lines are developed by selfing
and selection of desired phenotypes. A hybrid maize variety is the
cross of two such inbred lines, each of which may have one or more
desirable characteristics lacked by the other or which complement
the other. The new inbreds are crossed with other inbred lines and
the hybrids from these crosses are evaluated to determine which
have commercial potential. The hybrid progeny of the first
generation is designated F.sub.1. In the development of hybrids
only the F.sub.1 hybrid plants are sought. The F.sub.1 hybrid is
more vigorous than its inbred parents. This hybrid vigor, or
heterosis, can be manifested in many ways, including increased
vegetative growth and increased yield.
[0008] Hybrid maize seed can be produced by a male sterility system
incorporating manual detasseling. To produce hybrid seed, the male
tassel is removed from the growing female inbred parent, which can
be planted in various alternating row patterns with the male inbred
parent. Consequently, providing that there is sufficient isolation
from sources of foreign maize pollen, the ears of the female inbred
will be fertilized only with pollen from the male inbred. The
resulting seed is therefore hybrid (F.sub.1) and will form hybrid
plants.
[0009] Environmental variation in plant development can result in
plants tasseling after manual detasseling of the female parent is
completed. Or, a detasseler might not completely remove the tassel
of a female inbred plant. In any event, the result is that the
female plant will successfully shed pollen and some female plants
will be self-pollinated. This will result in seed of the female
inbred being harvested along with the hybrid seed which is normally
produced. Female inbred seed is not as productive as F.sub.1 seed.
In addition, the presence of female inbred seed can represent a
germplasm security risk for the company producing the hybrid.
[0010] Alternatively, the female inbred can be mechanically
detasseled by machine. Mechanical detasseling is approximately as
reliable as hand detasseling, but is faster and less costly.
However, most detasseling machines produce more damage to the
plants than hand detasseling. Thus, no form of detasseling is
presently entirely satisfactory, and a need continues to exist for
alternatives which further reduce production costs and to eliminate
self-pollination of the female parent in the production of hybrid
seed.
[0011] A reliable system of genetic male sterility would provide
advantages. The laborious detasseling process can be avoided in
some genotypes by using cytoplasmic male-sterile (CMS) inbreds. In
the absence of a fertility restorer gene, plants of a CMS inbred
are male sterile as a result of factors resulting from the
cytoplasmic, as opposed to the nuclear, genome. Thus, this
characteristic is inherited exclusively through the female parent
in maize plants, since only the female provides cytoplasm to the
fertilized seed. CMS plants are fertilized with pollen from another
inbred that is not male-sterile. Pollen from the second inbred may
or may not contribute genes that make the hybrid plants
male-fertile. Usually seed from detasseled normal maize and CMS
produced seed of the same hybrid must be blended to insure that
adequate pollen loads are available for fertilization when the
hybrid plants are grown and to insure cytoplasmic diversity.
[0012] There can be other drawbacks to CMS. One is an historically
observed association of a specific variant of CMS with
susceptibility to certain crop diseases. This problem has
discouraged widespread use of that CMS variant in producing hybrid
maize and has had a negative impact on the use of CMS in maize in
general.
[0013] One type of genetic sterility is disclosed in U.S. Pat. Nos.
4,654,465 and 4,727,219 to Brar, et al. However, this form of
genetic male sterility requires maintenance of multiple mutant
genes at separate locations within the genome and requires a
complex marker system to track the genes and make use of the system
convenient. Patterson also described a genic system of chromosomal
translocations which can be effective, but which are complicated.
(See, U.S. Pat. Nos. 3,861,709 and 3,710,511.)
[0014] Many other attempts have been made to improve on these
drawbacks. For example, Fabijanski, et al., developed several
methods of causing male sterility in plants (see EPO 89/3010153.8
publication no. 329,308 and PCT application PCT/CA90/00037
published as WO 90/08828). One method includes delivering into the
plant a gene encoding a cytotoxic substance associated with a male
tissue specific promoter. Another involves an antisense system in
which a gene critical to fertility is identified and an antisense
to the gene inserted in the plant. Mariani, et al. also shows
several cytotoxic antisense systems. See EP 89/401, 194. Still
other systems use "repressor" genes which inhibit the expression of
another gene critical to male sterility. PCT/GB90/00102, published
as WO 90/08829.
[0015] A still further improvement of this system is one described
at U.S. Pat. No. 5,478,369 (incorporated herein by reference) in
which a method of imparting controllable male sterility is achieved
by silencing a gene native to the plant that is critical for male
fertility and replacing the native DNA with the gene critical to
male fertility linked to an inducible promoter controlling
expression of the gene. The plant is thus constitutively sterile,
becoming fertile only when the promoter is induced and its attached
male fertility gene is expressed.
[0016] As noted, an essential aspect of much of the work underway
with male sterility systems is the identification of genes
impacting male fertility.
[0017] Such a gene can be used in a variety of systems to control
male fertility including those described herein. Previously, a male
fertility gene has been identified in Arabidopsis thaliana and used
to produce a male sterile plant. Aarts, et al., "Transposon Tagging
of a Male Sterility Gene in Arabidopsis", Nature, 363:715-717 (Jun.
24, 1993). U.S. Pat. No. 5,478,369 discloses therein one such gene
impacting male fertility. In the present invention the inventors
provide novel DNA molecules and the amino acid sequence encoded
that are critical to male fertility in plants. These can be used in
any of the systems where control of fertility is useful, including
those described above.
[0018] Thus, one object of the invention is to provide a nucleic
acid sequence, the expression of which is critical to male
fertility in plants.
[0019] Another object of the invention is to provide a DNA molecule
encoding an amino acid sequence, the expression of which is
critical to male fertility in plants.
[0020] Yet another object of the invention is to provide a promoter
of such nucleotide sequence and its essential sequences.
[0021] A further object of the invention is to provide a method of
using such DNA molecules to mediate male fertility in plants.
[0022] Further objects of the invention will become apparent in the
description and claims that follow.
SUMMARY OF THE INVENTION
[0023] This invention relates to nucleic acid sequences, and,
specifically, DNA molecules and the amino acid encoded by the DNA
molecules, which are critical to male fertility. A promoter of the
DNA is identified, as well as its essential sequences. It also
relates to use of such DNA molecules to mediate fertility in
plants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a locus map of the male fertility gene Ms26.
[0025] FIG. 2A is a Southern blot of the ms26-m2::Mu8 family
hybridized with a Mu8 probe;
[0026] FIG. 2B is a Southern blot of the ms26-m2::Mu8 family
hybridized with a PstI fragment isolated from the ms26 clone.
[0027] FIG. 3. is a Northern Blot analysis gel hybridized with a
PstI fragment isolated from the Ms26 gene.
[0028] FIG. 4A-4D is the sequence of Ms26 (The cDNA is SEQ ID NO:
1, the protein is SEQ ID NOS: 2 and 34)
[0029] FIG. 5A-5D is a comparison of the genomic Ms26 sequence
(Residues 1051-3326 of SEQ ID NO: 7) with the cDNA of Ms26 (SEQ ID
NO: 1).
[0030] FIG. 6A is a Northern analysis gel showing expression in
various plant tissues
[0031] FIG. 6B is a gel showing expression stages of
microsporogenesis
[0032] FIG. 7 is the full length promoter of Ms26 (SEQ ID NO:
5)
[0033] FIG. 8 is a bar graph showing luciferase activity after
deletions of select regions of the Ms26 promoter.
[0034] FIG. 9 shows essential regions of the Ms26 promoter (SEQ ID
NO: 6).
[0035] FIG. 10 is a bar graph showing luciferase activity after
substitution by restriction site linker scanning of select small
(9-10 bp) regions of the Ms26 essential promoter fragment.
[0036] FIGS. 11A and 11B is a comparison of the nucleotide sequence
(SEQ ID NO: 3) from the Ms26 orthologue from a sorghum panicle and
Ms26 maize cDNA (Residues 201-750 of SEQ ID NO: 1), and the sorghum
protein sequence (SEQ ID NO: 4) and Ms26 maize protein (Residues
87-244 of SEQ ID NO; 2).
[0037] FIG. 12 is a representation of the mapping of the male
sterility gene ms26.
[0038] FIG. 14 shows a sequence comparison of the region of
excision of the ms26-ref allele (SEQ ID NO: 8) with wild-type Ms26
(SEQ ID NO: 9).
[0039] FIG. 14A shows a translated protein sequence alignment
between regions of the CYP704B1, a P450 gene (SEQ ID NO: 12) and
Ms26 (SEQ ID NO: 13); FIG. 14B shows the phylogenetic tree analysis
of select P450 genes.
[0040] FIG. 15 demonstrates the heme binding domain frame shift,
showing the translated sequence alignment of regions of the Ms26
cDNA (SEQ ID NOS: 14 and 28-29), the genomic regions of exon 5 in
fertile plants (SEQ ID NOS: 15 and 30-31) and sterile plants (SEQ
ID NOS: 16 and 32-33).
[0041] FIG. 16 shows alignment of the Ms26 promoter of corn
(Residues 650-1089 of SEQ ID NO: 5), sorghum (SEQ ID NO: 19) and
rice (SEQ ID NO: 20).
[0042] FIG. 17 shows alignment of the maize Ms26 protein (SEQ ID
NO: 21); rice Ms26 protein (SEQ ID NO: 18) and sorghum Ms26 protein
(SEQ ID NO: 22) along with a consensus sequence (SEQ ID NO:
35).
DISCLOSURE OF THE INVENTION
[0043] All references referred to are incorporated herein by
reference.
[0044] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Unless
mentioned otherwise, the techniques employed or contemplated
therein are standard methodologies well known to one of ordinary
skill in the art. The materials, methods and examples are
illustrative only and not limiting.
[0045] Genetic male sterility results from a mutation, suppression,
or other impact to one of the genes critical to a specific step in
microsporogenesis, the term applied to the entire process of pollen
formulation. These genes can be collectively referred to as male
fertility genes (or, alternatively, male sterility genes). There
are many steps in the overall pathway where gene function impacts
fertility. This seems aptly supported by the frequency of genetic
male sterility in maize. New alleles of male sterility mutants are
uncovered in materials that range from elite inbreds to unadapted
populations. To date, published genetic male sterility research has
been mostly descriptive. Some efforts have been made to establish
the mechanism of sterility in maize, but few have been
satisfactory. This should not be surprising given the number of
genes that have been identified as being responsible for male
sterility. One mechanism is unlikely to apply to all mutations.
[0046] At U.S. Pat. No. 5,478,369 there is described a method by
which a male sterility gene was tagged and cloned on maize
chromosome 9. Previously, there has been described a male sterility
gene on chromosome 9, ms2, which has never been cloned and
sequenced. It is not allelic to the gene referred to in the '369
patent. See Albertsen, M. and Phillips, R. L., "Developmental
Cytology of 13 Genetic Male Sterile Loci in Maize" Canadian Journal
of Genetics & Cytology 23:195-208 (January 1981). The only
fertility gene cloned before that had been the Arabadopsis gene
described at Aarts, et al., supra.
[0047] Thus the invention includes using the sequences shown herein
it impacts male fertility in a plant, that is, to control male
fertility by manipulation of the genome using the genes of the
invention. By way of example, without limitation, any of the
methods described supra can be used with the sequence of the
invention such as introducing a mutant sequence into a plant to
cause sterility, causing mutation to the native sequence,
introducing an antisense of the sequence into the plant, linking it
with other sequences to control its expression, or any one of a
myriad of processes available to one skilled in the art to impact
male fertility in a plant.
[0048] The Ms26 gene described herein is located on maize
chromosome 1 and its dominant allele is critical to male fertility.
The locus map is represented at FIG. 1. It can be used in the
systems described above, and other systems impacting male
fertility.
[0049] The maize family cosegregating for sterility was named
ms*-SBMu200 and was found to have an approximately 5.5 Kb EcoRI
fragment that hybridized with a Mu8 probe (2A). A genomic clone
from the family was isolated which contained a Mu8 transposon. A
probe made from DNA bordering the transposon was found to hybridize
to the same .about.5.5 Kb EcoR1 fragment (2B). This probe was used
to isolate cDNA clones from a tassel cDNA library. The cDNA is 1906
bp, and the Mu insertion occurred in exon 1 of the gene. This probe
was also used to map the mutation in an RFLP mapping population.
The mutant mapped to the short arm of chromosome 1, near Ms26.
Allelism crosses between ms26-ref and ms*-SBMu200 showed that these
were allelic, indicating that the mutations occurred in the same
gene. The ms*-SBMu200 allele was renamed ms26-m2::Mu8. Two
additional alleles for the Ms26 gene were cloned, one containing a
Mutator element in the second exon, named ms26-m3::Mu*, and one
containing an unknown transposon in the fifth exon from the
ms26-ref allele. SEQ ID NO: 7 (discussed further below) represents
the genomic nucleotide sequence. Expression patterns, as determined
by Northern analysis, show tassel specificity with peak expression
at about the quartet to quartet release stages of
microsporogenesis.
[0050] Further, it will be evident to one skilled in the art that
variations, mutations, derivations including fragments smaller than
the entire sequence set forth may be used which retain the male
sterility controlling properties of the gene. One of ordinary skill
in the art can readily assess the variant or fragment by
introduction into plants homozygous for a stable male sterile
allele of Ms26, followed by observation of the plant's male tissue
development.
[0051] The sequences of the invention may be isolated from any
plant, including, but not limited to corn (Zea mays), canola
(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa),
rice (Oryza sativa), rye (Secale cereals), sorghum (Sorghum
bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat
(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana
tabacum), millet (Panicum spp.), potato (Solanum tuberosum),
peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet
potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee
(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),
citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia
sinensis), banana (Musa spp.), avocado (Persea americana), fig
(Ficus casica), guava (Psidium guajava), mango (Mangifera indica),
olive (Olea europaea), oats (Avena sativa), barley (Hordeum
vulgare), vegetables, ornamentals, and conifers. Preferably, plants
include corn, soybean, sunflower, safflower, canola, wheat, barley,
rye, alfalfa, rice, cotton and sorghum.
[0052] Sequences from other plants may be isolated according to
well-known techniques based on their sequence homology to the
homologous coding region of the coding sequences set forth herein.
In these techniques, all or part of the known coding sequence is
used as a probe which selectively hybridizes to other sequences
present in a population of cloned genomic DNA fragments (i.e.
genomic libraries) from a chosen organism. Methods are readily
available in the art for the hybridization of nucleic acid
sequences. An extensive guide to the hybridization of nucleic acids
is found in Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes, Part I,
Chapter 2 "Overview of principles of hybridization and the strategy
of nucleic acid probe assays", Elsevier, N.Y. (1993); and Current
Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds.,
Greene Publishing and Wiley-Interscience, New York (1995).
[0053] Thus the invention also includes those nucleotide sequences
which selectively hybridize to the Ms26 nucleotide sequences under
stringent conditions. In referring to a sequence that "selectively
hybridizes" with Ms26, the term includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to the specified nucleic acid target sequence
to a detectably greater degree (e.g., at least 2-fold over
background) than its hybridization to non-target nucleic acid.
[0054] The terms "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are target-sequence-dependent and
will differ depending on the structure of the polynucleotide. By
controlling the stringency of the hybridization and/or washing
conditions, target sequences can be identified which are 100%
complementary to a probe (homologous probing). Alternatively,
stringency conditions can be adjusted to allow some mismatching in
sequences so that lower degrees of similarity are detected
(heterologous probing). Generally, probes of this type are in a
range of about 1000 nucleotides in length to about 250 nucleotides
in length.
[0055] An extensive guide to the hybridization of nucleic acids is
found in Tijssen, Laboratory Techniques Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays", Elsevier, N.Y. (1993); and Current
Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds.,
Greene Publishing and Wiley-Interscience, New York (1995). See also
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd
ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0056] In general, sequences that correspond to the nucleotide
sequences of the present invention and hybridize to the nucleotide
sequence disclosed herein will be at least 50% homologous, 70%
homologous, and even 85% homologous or more with the disclosed
sequence. That is, the sequence similarity between probe and target
may range, sharing at least about 50%, about 70%, and even about
85% sequence similarity.
[0057] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. Generally, stringent wash
temperature conditions are selected to be about 5.degree. C. to
about 2.degree. C. lower than the melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The melting
point, or denaturation, of DNA occurs over a narrow temperature
range and represents the disruption of the double helix into its
complementary single strands. The process is described by the
temperature of the midpoint of transition, Tm, which is also called
the melting temperature. Formulas are available in the art for the
determination of melting temperatures.
[0058] Preferred hybridization conditions for the nucleotide
sequence of the invention include hybridization at 42.degree. C. in
50% (w/v) formamide, 6.times.SSC, 0.5% (w/v) SDS, 100 (g/ml salmon
sperm DNA. Exemplary low stringency washing conditions include
hybridization at 42.degree. C. in a solution of 2.times.SSC, 0.5%
(w/v) SDS for 30 minutes and repeating. Exemplary moderate
stringency conditions include a wash in 2.times.SSC, 0.5% (w/v) SDS
at 50.degree. C. for 30 minutes and repeating. Exemplary high
stringency conditions include a wash in 0.1.times.SSC, 0.1% (w/v)
SDS, at 65.degree. C. for 30 minutes to one hour and repeating.
Sequences that correspond to the promoter of the present invention
may be obtained using all the above conditions. For purposes of
defining the invention, the high stringency conditions are
used.
[0059] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", and (d) "percentage of sequence identity."
[0060] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0061] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, or 100 nucleotides in length, or
longer. Those of skill in the art understand that to avoid a high
similarity to a reference sequence due to inclusion of gaps in the
polynucleotide sequence a gap penalty is typically introduced and
is subtracted from the number of matches.
[0062] Methods of aligning sequences for comparison are well-known
in the art. Thus, the determination of percent sequence identity
between any two sequences can be accomplished using a mathematical
algorithm. Non-limiting examples of such mathematical algorithms
are the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the
local alignment algorithm of Smith et al. (1981) Adv. Appl. Math.
2: 482; the global alignment algorithm of Needleman and Wunsch
(1970) J. Mol. Biol. 48: 443-453; the
search-for-local-alignment-method of Pearson and Lipman (1988)
Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin and
Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:
5873-5877.
[0063] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys Inc., 9685
Scranton Road, San Diego, Calif., USA). Alignments using these
programs can be performed using the default parameters. The CLUSTAL
program is well described by Higgins et al. (1988) Gene 73: 237-244
(1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al.
(1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS
8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331.
The ALIGN program is based on the algorithm of Myers and Miller
(1988) supra. A PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4 can be used with the ALIGN program
when comparing amino acid sequences. The BLAST programs of Altschul
et al (1990) J. Mol. Biol. 215: 403 are based on the algorithm of
Karlin and Altschul (1990) supra. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to a nucleotide sequence
encoding a protein of the invention. BLAST protein searches can be
performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to a protein or polypeptide
of the invention. To obtain gapped alignments for comparison
purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25: 3389.
Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an
iterated search that detects distant relationships between
molecules. See Altschul et al. (1997) supra. When utilizing BLAST,
Gapped BLAST, PSI-BLAST, the default parameters of the respective
programs (e.g., BLASTN for nucleotide sequences, BLASTX for
proteins) can be used. See http://www.ncbi.nlm.nih.gov. Alignment
may also be performed manually by inspection.
[0064] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using GAP Weight of 50 and Length Weight of 3
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using GAP Weight of 8 and Length Weight
of 2; and the BLOSUM62 scoring matrix or any equivalent program
thereof. By "equivalent program" is intended any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version
10.
[0065] GAP uses the algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48: 443-453, to find the alignment of two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. GAP considers all possible alignments and gap
positions and creates the alignment with the largest number of
matched bases and the fewest gaps. It allows for the provision of a
gap creation penalty and a gap extension penalty in units of
matched bases. GAP must make a profit of gap creation penalty
number of matches for each gap it inserts. If a gap extension
penalty greater than zero is chosen, GAP must, in addition, make a
profit for each gap inserted of the length of the gap times the gap
extension penalty. Default gap creation penalty values and gap
extension penalty values in Version 10 of the GCG Wisconsin
Genetics Software Package for protein sequences are 8 and 2,
respectively. For nucleotide sequences the default gap creation
penalty is 50 while the default gap extension penalty is 3. The gap
creation and gap extension penalties can be expressed as an integer
selected from the group of integers consisting of from 0 to 200.
Thus, for example, the gap creation and gap extension penalties can
be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65 or greater.
[0066] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the GCG Wisconsin Genetics Software Package is
BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci.
USA 89:10915).
[0067] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0068] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0069] The use of the term "polynucleotide" is not intended to
limit the present invention to polynucleotides comprising DNA.
Those of ordinary skill in the art will recognize that
polynucleotides can comprise ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides
and ribonucleotides include both naturally occurring molecules and
synthetic analogues. The polynucleotides of the invention also
encompass all forms of sequences including, but not limited to,
single-stranded forms, double-stranded forms, hairpins,
stem-and-loop structures, and the like.
[0070] Identity to the sequence of the present invention would mean
a polynucleotide sequence having at least 65% sequence identity,
more preferably at least 70% sequence identity, more preferably at
least 75% sequence identity, more preferably at least 80% identity,
more preferably at least 85% sequence identity, more preferably at
least 90% sequence identity and most preferably at least 95%
sequence identity.
[0071] Promoter regions can be readily identified by one skilled in
the art. The putative start codon containing the ATG motif is
identified and upstream from the start codon is the presumptive
promoter. By "promoter" is intended a regulatory region of DNA
usually comprising a TATA box capable of directing RNA polymerase
II to initiate RNA synthesis at the appropriate transcription
initiation site for a particular coding sequence. A promoter can
additionally comprise other recognition sequences generally
positioned upstream or 5' to the TATA box, referred to as upstream
promoter elements, which influence the transcription initiation
rate. It is recognized that having identified the nucleotide
sequences for the promoter region disclosed herein, it is within
the state of the art to isolate and identify further regulatory
elements in the region upstream of the TATA box from the particular
promoter region identified herein. Thus the promoter region
disclosed herein is generally further defined by comprising
upstream regulatory elements such as those responsible for tissue
and temporal expression of the coding sequence, enhancers and the
like. In the same manner, the promoter elements which enable
expression in the desired tissue such as male tissue can be
identified, isolated, and used with other core promoters to confirm
male tissue-preferred expression. By core promoter is meant the
minimal sequence required to initiate transcription, such as the
sequence called the TATA box which is common to promoters in genes
encoding proteins. Thus the upstream promoter of Ms26 can
optionally be used in conjunction with its own or core promoters
from other sources. the promoter may be native or non-native to the
cell in which it is found.
[0072] The isolated promoter sequence of the present invention can
be modified to provide for a range of expression levels of the
heterologous nucleotide sequence. Less than the entire promoter
region can be utilized and the ability to drive anther-preferred
expression retained. However, it is recognized that expression
levels of mRNA can be decreased with deletions of portions of the
promoter sequence. Thus, the promoter can be modified to be a weak
or strong promoter. Generally, by "weak promoter" is intended a
promoter that drives expression of a coding sequence at a low
level. By "low level" is intended levels of about 1/10,000
transcripts to about 1/100,000 transcripts to about 1/500,000
transcripts. Conversely, a strong promoter drives expression of a
coding sequence at a high level, or at about 1/10 transcripts to
about 1/100 transcripts to about 1/1,000 transcripts. Generally, at
least about 30 nucleotides of an isolated promoter sequence will be
used to drive expression of a nucleotide sequence. It is recognized
that to increase transcription levels, enhancers can be utilized in
combination with the promoter regions of the invention. Enhancers
are nucleotide sequences that act to increase the expression of a
promoter region. Enhancers are known in the art and include the
SV40 enhancer region, the 35S enhancer element, and the like.
[0073] The promoter of the present invention can be isolated from
the 5' region of its native coding region of 5' untranslation
region (5'UTR). Likewise the terminator can be isolated from the 3'
region flanking its respective stop codon. The term "isolated"
refers to material such as a nucleic acid or protein which is
substantially or essentially free from components which normally
accompany or interact with the material as found in it naturally
occurring environment or if the material is in its natural
environment, the material has been altered by deliberate human
intervention to a composition and/or placed at a locus in a cell
other than the locus native to the material. Methods for isolation
of promoter regions are well known in the art.
[0074] "Functional variants" of the regulatory sequences are also
encompassed by the compositions of the present invention.
Functional variants include, for example, the native regulatory
sequences of the invention having one or more nucleotide
substitutions, deletions or insertions. Functional variants of the
invention may be created by site-directed mutagenesis, induced
mutation, or may occur as allelic variants (polymorphisms).
[0075] As used herein, a "functional fragment" is a regulatory
sequence variant formed by one or more deletions from a larger
regulatory element. For example, the 5' portion of a promoter up to
the TATA box near the transcription start site can be deleted
without abolishing promoter activity, as described by
Opsahl-Sorteberg, H-G. et al., "Identification of a 49-bp fragment
of the HvLTP2 promoter directing aleruone cell specific expression"
Gene 341:49-58 (2004). Such variants should retain promoter
activity, particularly the ability to drive expression in male
tissues. Activity can be measured by Northern blot analysis,
reporter activity measurements when using transcriptional fusions,
and the like. See, for example, Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2nd ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.), herein incorporated by
reference.
[0076] Functional fragments can be obtained by use of restriction
enzymes to cleave the naturally occurring regulatory element
nucleotide sequences disclosed herein; by synthesizing a nucleotide
sequence from the naturally occurring DNA sequence; or can be
obtained through the use of PCR technology See particularly, Mullis
et al. (1987) Methods Enzymol. 155:335-350, and Erlich, ed. (1989)
PCR Technology (Stockton Press, New York).
[0077] Sequences which hybridize to the regulatory sequences of the
present invention are within the scope of the invention. Sequences
that correspond to the promoter sequences of the present invention
and hybridize to the promoter sequences disclosed herein will be at
least 50% homologous, 70% homologous, and even 85% homologous or
more with the disclosed sequence.
[0078] Smaller fragments may yet contain the regulatory properties
of the promoter so identified and deletion analysis is one method
of identifying essential regions. Deletion analysis can occur from
both the 5' and 3' ends of the regulatory region. Fragments can be
obtained by site-directed mutagenesis, mutagenesis using the
polymerase chain reaction and the like. (See, Directed Mutagenesis:
A Practical Approach IRL Press (1991)). The 3' deletions can
delineate the essential region and identify the 3' end so that this
region may then be operably linked to a core promoter of choice.
Once the essential region is identified, transcription of an
exogenous gene may be controlled by the essential region plus a
core promoter. By core promoter is meant the sequence called the
TATA box which is common to promoters in all genes encoding
proteins. Thus the upstream promoter of Ms26 can optionally be used
in conjunction with its own or core promoters from other sources.
The promoter may be native or non-native to the cell in which it is
found.
[0079] The core promoter can be any one of known core promoters
such as the Cauliflower Mosaic Virus 35S or 19S promoter (U.S. Pat.
No. 5,352,605), ubiquitin promoter (U.S. Pat. No. 5,510,474) the
IN2 core promoter (U.S. Pat. No. 5,364,780) or a Figwort Mosaic
Virus promoter (Gruber, et al. "Vectors for Plant Transformation"
Methods in Plant Molecular Biology and Biotechnology) et al. eds,
CRC Press pp. 89-119 (1993)).
[0080] The regulatory region of Ms26 has been identified as
including the 1005 bp region upstream of the putative TATA box. See
FIG. 7. Further, using the procedures outlined above, it has been
determined that an essential region of the promoter includes the
-180 bp upstream of the TATA box and specifically, the -176 to -44
region is particularly essential.
[0081] Promoter sequences from other plants may be isolated
according to well-known techniques based on their sequence homology
to the promoter sequence set forth herein. In these techniques, all
or part of the known promoter sequence is used as a probe which
selectively hybridizes to other sequences present in a population
of cloned genomic DNA fragments (i.e. genomic libraries) from a
chosen organism. Methods are readily available in the art for the
hybridization of nucleic acid sequences.
[0082] The entire promoter sequence or portions thereof can be used
as a probe capable of specifically hybridizing to corresponding
promoter sequences. To achieve specific hybridization under a
variety of conditions, such probes include sequences that are
unique and are preferably at least about 10 nucleotides in length,
and most preferably at least about nucleotides in length. Such
probes can be used to amplify corresponding promoter sequences from
a chosen organism by the well-known process of polymerase chain
reaction (PCR). This technique can be used to isolate additional
promoter sequences from a desired organism or as a diagnostic assay
to determine the presence of the promoter sequence in an organism.
Examples include hybridization screening of plated DNA libraries
(either plaques or colonies; see e.g. Innis et al., eds., (1990)
PCR Protocols, A Guide to Methods and Applications, Academic
Press).
[0083] Further, a promoter of the present invention can be linked
with nucleotide sequences other than the Ms26 gene to express other
heterologous nucleotide sequences. The nucleotide sequence for the
promoter of the invention, as well as fragments and variants
thereof, can be provided in expression cassettes along with
heterologous nucleotide sequences for expression in the plant of
interest, more particularly in the male tissue of the plant. Such
an expression cassette is provided with a plurality of restriction
sites for insertion of the nucleotide sequence to be under the
transcriptional regulation of the promoter. These expression
cassettes are useful in the genetic manipulation of any plant to
achieve a desired phenotypic response. Examples of other nucleotide
sequences which can be used as the exogenous gene of the expression
vector with the Ms26 promoter include complementary nucleotidic
units such as antisense molecules (callase antisense RNA, barnase
antisense RNA and chalcone synthase antisense RNA, Ms45 antisense
RNA), ribozymes and external guide sequences, an aptamer or single
stranded nucleotides. The exogenous nucleotide sequence can also
encode auxins, rol B, cytotoxins, diptheria toxin, DAM methylase,
avidin, or may be selected from a prokaryotic regulatory system. By
way of example, Mariani, et al., Nature; Vol. 347; pp. 737; (1990),
have shown that expression in the tapetum of either Aspergillus
oryzae RNase-T1 or an RNase of Bacillus amyloliquefaciens,
designated "barnase," induced destruction of the tapetal cells,
resulting in male infertility. Quaas, et al., Eur. J. Biochem. Vol.
173: pp. 617 (1988), describe the chemical synthesis of the
RNase-T1, while the nucleotide sequence of the barnase gene is
disclosed in Hartley, J. Molec. Biol.; Vol. 202: pp. 913 (1988).
The rolB gene of Agrobacterium rhizogenes codes for an enzyme that
interferes with auxin metabolism by catalyzing the release of free
indoles from indoxyl-.beta.-glucosides. Estruch, et al., EMBO J.
Vol. 11: pp. 3125 (1991) and Spena, et al., Theor. Appl. Genet.;
Vol. 84: pp. 520 (1992), have shown that the anther-specific
expression of the rolB gene in tobacco resulted in plants having
shriveled anthers in which pollen production was severely decreased
and the rolB gene is an example of a gene that is useful for the
control of pollen production. Slightom, et al., J. Biol. Chem. Vol.
261: pp. 108 (1985), disclose the nucleotide sequence of the rolB
gene. DNA molecules encoding the diphtheria toxin gene can be
obtained from the American Type Culture Collection (Rockville,
Md.), ATCC No. 39359 or ATCC No. 67011 and see Fabijanski, et al.,
E.P. Appl. No. 90902754.2, "Molecular Methods of Hybrid Seed
Production" for examples and methods of use. The DAM methylase gene
is used to cause sterility in the methods discussed at U.S. Pat.
No. 5,689,049 and PCT/US95/15229 Cigan, A. M. and Albertsen, M. C.,
"Reversible Nuclear Genetic System for Male Sterility in Transgenic
Plants". Also see discussion of use of the avidin gene to cause
sterility at U.S. Pat. No. 5,962,769 "Induction of Male Sterility
in Plants by Expression of High Levels of Avidin" by Albertsen et
al.
[0084] The invention includes vectors with the Ms26 gene. A vector
is prepared comprising Ms26, a promoter that will drive expression
of the gene in the plant and a terminator region. As noted, the
promoter in the construct may be the native promoter or a
substituted promoter which will provide expression in the plant.
Selection of the promoter will depend upon the use intended of the
gene. The promoter in the construct may be an inducible promoter,
so that expression of the sense or antisense molecule in the
construct can be controlled by exposure to the inducer.
[0085] Other components of the vector may be included, also
depending upon intended use of the gene. Examples include
selectable markers, targeting or regulatory sequences, stabilizing
or leader sequences, etc. General descriptions and examples of
plant expression vectors and reporter genes can be found in Gruber,
et al., "Vectors for Plant Transformation" in Method in Plant
Molecular Biology and Biotechnology, Glick et al eds; CRC Press pp.
89-119 (1993). The selection of an appropriate expression vector
will depend upon the host and the method of introducing the
expression vector into the host. The expression cassette will also
include at the 3' terminus of the heterologous nucleotide sequence
of interest, a transcriptional and translational termination region
functional in plants. The termination region can be native with the
promoter nucleotide sequence of the present invention, can be
native with the DNA sequence of interest, or can be derived from
another source. Convenient termination regions are available from
the Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also, Guerineau et al.
Mol. Gen. Genet. 262:141-144 (1991); Proudfoot, Cell 64:671-674
(1991); Sanfacon et al. Genes Dev. 5:141-149 (1991); Mogen et al.
Plant Cell 2:1261-1272 (1990); Munroe et al. Gene 91:151-158
(1990); Ballas et al. Nucleic Acids Res. 17:7891-7903 (1989); Joshi
et al. Nucleic Acid Res. 15:9627-9639 (1987).
[0086] The expression cassettes can additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region), Elroy-Stein et al. Proc. Nat. Acad. Sci. USA
86:6126-6130 (1989); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus), Allison et al.; MDMV leader (Maize Dwarf
Mosaic Virus), Virology 154:9-20 (1986); human immunoglobulin
heavy-chain binding protein (BiP), Macejak et al. Nature 353:90-94
(1991); untranslated leader from the coat protein mRNA of alfalfa
mosaic virus (AMV RNA 4), Jobling et al. Nature 325:622-625 (1987);
Tobacco mosaic virus leader (TMV), Gallie et al. (1989) Molecular
Biology of RNA, pages 237-256; and maize chlorotic mottle virus
leader (MCMV) Lommel et al. Virology 81:382-385 (1991). See also
Della-Cioppa et al. Plant Physiology 84:965-968 (1987). The
cassette can also contain sequences that enhance translation and/or
mRNA stability such as introns.
[0087] In those instances where it is desirable to have the
expressed product of the heterologous nucleotide sequence directed
to a particular organelle, particularly the plastid, amyloplast, or
to the endoplasmic reticulum, or secreted at the cell's surface or
extracellularly, the expression cassette can further comprise a
coding sequence for a transit peptide. Such transit peptides are
well known in the art and include, but are not limited to, the
transit peptide for the acyl carrier protein, the small subunit of
RUBISCO, plant EPSP synthase, and the like. One skilled in the art
will readily appreciate the many options available in expressing a
product to a particular organelle. For example, the barley alpha
amylase sequence is often used to direct expression to the
endoplasmic reticulum (Rogers, J. Biol. Chem. 260:3731-3738
(1985)). Use of transit peptides is well known (e.g., see U.S. Pat.
Nos. 5,717,084; 5,728,925).
[0088] In preparing the expression cassette, the various DNA
fragments can be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers can be
employed to join the DNA fragments or other manipulations can be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction
digests, annealing, and resubstitutions, such as transitions and
transversions, can be involved.
[0089] As noted herein, the present invention provides vectors
capable of expressing genes of interest under the control of the
promoter. In general, the vectors should be functional in plant
cells. At times, it may be preferable to have vectors that are
functional in E. coli (e.g., production of protein for raising
antibodies, DNA sequence analysis, construction of inserts,
obtaining quantities of nucleic acids). Vectors and procedures for
cloning and expression in E. coli are discussed in Sambrook et al.
(supra).
[0090] The transformation vector comprising the promoter sequence
of the present invention operably linked to a heterologous
nucleotide sequence in an expression cassette, can also contain at
least one additional nucleotide sequence for a gene to be
cotransformed into the organism. Alternatively, the additional
sequence(s) can be provided on another transformation vector.
[0091] Reporter genes can be included in the transformation
vectors. Examples of suitable reporter genes known in the art can
be found in, for example, Jefferson et al. (1991) in Plant
Molecular Biology Manual, ed. Gelvin et al. (Kluwer Academic
Publishers), pp. 1-33; DeWet et al. Mol. Cell. Biol. 7:725-737
(1987); Goff et al. EMBO J. 9:2517-2522 (1990); Kain et al.
BioTechniques 19:650-655 (1995); and Chiu et al. Current Biology
6:325-330 (1996).
[0092] Selectable marker genes for selection of transformed cells
or tissues can be included in the transformation vectors. These can
include genes that confer antibiotic resistance or resistance to
herbicides. Examples of suitable selectable marker genes include,
but are not limited to, genes encoding resistance to
chloramphenicol, Herrera Estrella et al. EMBO J. 2:987-992 (1983);
methotrexate, Herrera Estrella et al. Nature 303:209-213 (1983);
Meijer et al. Plant Mol. Biol. 16:807-820 (1991); hygromycin,
Waldron et al. Plant Mol. Biol. 5:103-108 (1985); Zhijian et al.
Plant Science 108:219-227 (1995); streptomycin, Jones et al. Mol.
Gen. Genet. 210:86-91 (1987); spectinomycin, Bretagne-Sagnard et
al. Transgenic Res. 5:131-137 (1996); bleomycin, Hille et al. Plant
Mol. Biol. 7:171-176 (1990); sulfonamide, Guerineau et al. Plant
Mol. Biol. 15:127-136 (1990); bromoxynil, Stalker et al. Science
242:419-423 (1988); glyphosate, Shaw et al. Science 233:478-481
(1986); phosphinothricin, DeBlock et al. EMBO J. 6:2513-2518
(1987).
[0093] The method of transformation/transfection is not critical to
the instant invention; various methods of transformation or
transfection are currently available. As newer methods are
available to transform crops or other host cells they may be
directly applied. Accordingly, a wide variety of methods have been
developed to insert a DNA sequence into the genome of a host cell
to obtain the transcription or transcript and translation of the
sequence to effect phenotypic changes in the organism. Thus, any
method which provides for efficient transformation/transfection may
be employed.
[0094] Methods for introducing expression vectors into plant tissue
available to one skilled in the art are varied and will depend on
the plant selected. Procedures for transforming a wide variety of
plant species are well known and described throughout the
literature. See, for example, Miki et al, "Procedures for
Introducing Foreign DNA into Plants" in Methods in Plant Molecular
Biotechnology, supra; Klein et al, Bio/Technology 10:268 (1992);
and Weising et al., Ann. Rev. Genet. 22: 421-477 (1988). For
example, the DNA construct may be introduced into the genomic DNA
of the plant cell using techniques such as microprojectile-mediated
delivery, Klein et al., Nature 327: 70-73 (1987); electroporation,
Fromm et al., Proc. Natl. Acad. Sci. 82: 5824 (1985); polyethylene
glycol (PEG) precipitation, Paszkowski et al., EMBO J. 3: 2717-2722
(1984); direct gene transfer WO 85/01856 and EP No. 0 275 069; in
vitro protoplast transformation U.S. Pat. No. 4,684,611; and
microinjection of plant cell protoplasts or embryogenic callus.
Crossway, Mol. Gen. Genetics 202:179-185 (1985). Co-cultivation of
plant tissue with Agrobacterium tumefaciens is another option,
where the DNA constructs are placed into a binary vector system.
See e.g., U.S. Pat. No. 5,591,616; Ishida et al., "High Efficiency
Transformation of Maize (Zea mays L.) mediated by Agrobacterium
tumefaciens" Nature Biotechnology 14:745-750 (1996). The virulence
functions of the Agrobacterium tumefaciens host will direct the
insertion of the construct into the plant cell DNA when the cell is
infected by the bacteria. See, for example Horsch et al., Science
233: 496-498 (1984), and Fraley et al., Proc. Natl. Acad. Sci. 80:
4803 (1983).
[0095] Standard methods for transformation of canola are described
at Moloney et al. "High Efficiency Transformation of Brassica napus
using Agrobacterium Vectors" Plant Cell Reports 8:238-242 (1989).
Corn transformation is described by Fromm et al, Bio/Technology
8:833 (1990) and Gordon-Kamm et al, supra. Agrobacterium is
primarily used in dicots, but certain monocots such as maize can be
transformed by Agrobacterium. See supra and U.S. Pat. No.
5,550,318. Rice transformation is described by Hiei et al.,
"Efficient Transformation of Rice (Oryza sativs L.) Mediated by
Agrobacterium and Sequence Analysis of the Boundaries of the T-DNA"
The Plant Journal 6(2): 271-282 (1994, Christou et al, Trends in
Biotechnology 10:239 (1992) and Lee et al, Proc. Nat'l Acad. Sci.
USA 88:6389 (1991). Wheat can be transformed by techniques similar
to those used for transforming corn or rice. Sorghum transformation
is described at Casas et al, supra and sorghum by Wan et al, Plant
Physicol. 104:37 (1994). Soybean transformation is described in a
number of publications, including U.S. Pat. No. 5,015,580.
[0096] Further detailed description is provided below by way of
instruction and illustration and is not intended to limit the scope
of the invention.
EXAMPLE 1
Identification and Cosegregation of ms26-m2::Mu8
[0097] Families of plants from a Mutator (Mu) population were
identified that segregated for plants that were mostly male
sterile, with none or only a few extruded abnormal anthers, none of
which had pollen present. Male sterility is expected to result from
those instances where a Mu element has randomly integrated into a
gene responsible for some step in microsporogenesis, disrupting its
expression. Plants from a segregating F.sub.2 family in which the
male sterile mutation was designated ms26*-SBMu200, were grown and
classified for male fertility/sterility based on the above
criteria. Leaf samples were taken and DNA subsequently isolated on
approximately 20 plants per phenotypic classification, that is male
fertility vs. male sterility.
[0098] Southern analysis was performed to confirm association of Mu
with sterility. Southern analysis is a well known technique to
those skilled in the art. This common procedure involves isolating
the plant DNA, cutting with restriction endonucleases, fractioning
the cut DNA by molecular weight on an agarose gel, and transferring
to nylon membranes to fix the separated DNA. These membranes are
subsequently hybridized with a probe fragment that was
radioactively labeled with P.sup.32P-dCTP, and washed in an SDS
solution. Southern, E., "Detection of Specific Sequences Among DNA
Fragments by Gel Electrophoresis," J. Mol. Biol. 98:503-317 (1975).
Plants from a segregating F.sub.2 ms26*-SBMu200 family were grown
and classified for male fertility/sterility. Leaf samples and
subsequent DNA isolation was conducted on approximately 20 plants
per phenotypic classification. DNA (.about.7 ug) from 5 fertile and
12 sterile plants was digested with EcoRI and electrophoresed
through a 0.75% agarose gel. The digested DNA was transferred to
nylon membrane via Southern transfer. The membrane was hybridized
with an internal fragment from the Mu8 transposon. Autoradiography
of the membrane revealed cosegregation of a 5.5 Kb EcoRI fragment
with the sterility phenotype as shown in FIG. 1. This EcoRI band
segregated in the fertile plants suggesting a heterozygous wild
type condition for the allele
EXAMPLE 2
Library Construction, Screening, and Mapping
[0099] The process of genomic library screenings is commonly known
among those skilled in the art and is described at Sambrook, J.,
Fritsch, E. F., Maniatis T., et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor Lab Press, Plainview, N.Y. (1989). Libraries were created as
follows.
[0100] DNA from a sterile plant was digested with EcoRI and run on
a preparative gel. DNA with a molecular weight between 5.0 and 6.0
Kb was excised from the gel, electroeluted and ethanol
precipitated. This DNA was ligated into the Lambda Zap vector
(Stratagene.TM.) using the manufacturer's protocol. The ligated DNA
was packaged into phage particles using Gigapack Gold
(Stratagene.TM.). Approximately 500,000 PFU were plated and lifted
onto nitrocellulose membranes. Membranes were hybridized with the
Mu8 probe. A pure clone was obtained after 3 rounds of screening.
The insert was excised from the phage as a plasmid and designated
SBMu200-3.1. A PstI border fragment from this clone was isolated
and used to reprobe the orginal EcoRI cosegregation blot as shown
in FIG. 2B. The 5.5 kb EcoRI fragment is homozygous in all the
sterile plants, which confirms that the correct Mu fragment was
isolated. Three of the fertile plants are heterozygous for the 5.5
kb EcoRI band and a 4.3 Kb EcoRI band. Two of the fertile plants
are homozygous for the 4.3 kb EcoRI band, presumably the wild type
allele.
[0101] The PstI probe was used to map the ms*-SBMu200 mutation in
an RFLP mapping population. The mutant mapped to the short arm of
chromosome 1, near the male sterile locus, Ms26 (Loukides et al.,
(1995) Amer. J. Bot 82, 1017-1023). To test whether ms*-SBMu200 was
an allele of ms26-ref, ms*-SBMu200 and ms26-ref were crossed with
each other using a known heterozygote as the pollen donor. The
testcross progeny segregated male-sterile and wild-type plants in a
1:1 ratio, indicating allelism between ms*-SBMu200 and ms26-ref.
The ms*-SBMu200 allele was designated ms26-m2::Mu8. The map
location is shown in FIG. 12.
EXAMPLE 3
Identification and Cloning of Additional ms26 Alleles
[0102] Three additional Mu insertion mutations in Ms26 were
identified by using a polymerase chain reaction (PCR) primer for Mu
and a gene specific primer for Ms26. Sequence analyses of the PCR
products showed that all three Mu insertions occurred in the second
exon (FIG. 1). The F.sub.2 seeds from one of these families were
grown and examined for male fertility/sterility. Southern blot
analyses of this family confirmed the cosegregation of the Mu
insertion in Ms26 with the male-sterile phenotype.
[0103] The ms26 allele described in Loukides et al., (1995) Amer.
J. Bot 82, 1017-1023 and designated ms26-ref was also investigated.
To analyze the mutation in ms26-ref, Ms26 genomic sequences were
cloned from ms26-ref sterile and fertile plants. Ms26 was cloned as
a .about.4.2 kb EcoRI fragment and ms26-ref cloned as a .about.6 kb
HindIII fragment and an overlapping .about.2.3 kb EcoRI fragment
from the sterile plant. Sequence analysis revealed the presence of
a new segment (1,430 bp) in the last exon of the ms26-ref allele
shown in FIG. 1. An 8 bp host site duplication (GCCGGAGC) was found
that flanks the inserted element and the element also contains a 15
bp terminal inverted repeat (TIR) (TAGGGGTGAAAACGG; SEQ ID NO: 23).
The transposon sequence is shown in SEQ ID NO: 10. The ms26-ref
genomic sequence in its entirety is shown in SEQ ID NO: 11. A
variant of the ms26-ref allele was also found. Sequence analysis of
this allele, designated ms26'-0406, was found to have lost the 1430
bp segment found in the last exon of the ms26-ref allele but left
an 8 bp footprint at the site of insertion. Plants homozygous for
the ms26'-0406 allele were male sterile. A comparison of the
excision allele, ms26'-0406 (SEQ ID NO: 8) with the region in the
wild-type Ms26 gene (SEQ ID NO: 9) is shown in FIG. 13.
EXAMPLE 4
Expression Analysis and cDNA Isolation
[0104] Northern analysis can be used to detect expression of genes
characteristic of anther development at various states of
microsporogenesis. Northern analysis is also a commonly used
technique known to those skilled in the art and is similar to
Southern analysis except that mRNA rather than DNA is isolated and
placed on the gel. The RNA is then hybridzed with the labeled
probe. Potter, E., et al., "Thyrotrotropin Releasing Hormone Exerts
Rapid Nuclear Effects to Increase Production of the Primary
Prolactin in RNA Transcript," Proc. Nat. Acad. Sci. USA
78:6662-6666 (1981), Lechelt, et al., "Isolation & Molecular
Analysis of the Plows," Mol. Gen. Genet. 219:225-234 (1989). The
PstI fragment from the SBMu200-3.1 clone was used to probe a
Northern blot containing kernel, immature ear, seedling and tassel
RNA. A signal was seen only in tassel RNA at approximately the
quartet stage of microsporogenesis, as reflected in FIG. 3. The
transcript is about 2.3 kb in length. The same probe was also used
to screen a cDNA library constructed from mRNA isolated from
meiotic to late uninucleate staged anthers. One clone, designated
Ms26-8.1, was isolated from the library.
EXAMPLE 5
Sequence and Expression Analysis
[0105] The SBMu200-3.1 genomic clone and the Ms26-8.1 cDNA clone
were sequenced by Loftstrand Labs Limited. Sanger, F., Nicklen, S.,
Coulson A. R. (1977) "DNA sequencing with chain terminating
inhibitors" Proc. Natl. Acad. Sci. USA 74:5463-5467. The sequences
are set forth in FIG. 4 and the comparison is at FIG. 5. The
cDNA/genomic comparison reveals five introns are present in the
genomic clone. The Mu8 insertion occurs in exon 1. Testing for
codon preference and non-randomness in the third position of each
codon was consistent with the major ORF in the cDNA being the
likely protein-coding ORF. There is a putative Met start codon at
position 1089 in the genomic clone. The cDNA homology with respect
to the genomic clone begins at nucleotide 1094. Thus Ms26-8.1 does
not represent a full length clone and lacks 5 bases up to the
putative Met start codon. A database search revealed significant
homology to P450 enzymes found in yeast, plants and mammals. P450
enzymes have been widely studied and three characteristic protein
domains have been elucidated. The Ms26 protein contains several
structural motifs characteristic of eukaryotic P450's, including
the heme-binding domain FxxGxRxCxG (domain D; SEQ ID NO: 24),
domain A A/GGXD/ETT/S (dioxygen-binding), domain B
(steroid-binding), and domain C. The highly conserved heme-binding
motif was found in MS26 as FQAGPRICLG (SEQ ID NO: 25), 51 amino
acids away from C-terminus. The dioxygen binding domain AGRDTT (SEQ
ID NO: 26) was located between amino acids 320-325. The
steroid-binding domain was found as LVYLHACVTETLR (SEQ ID NO: 27),
amino acids 397-409. The most significant homologous sequence
detected in Genebank database is a deduced protein sequence from
rice (GeneBank accession number 19071651). The second highest
homologous sequence is a putative Arabidopsis P450 gene (CYP704B1)
whose function is also unknown. FIG. 14A shows a sequence alignment
between CYP704B1 (SEQ ID NO: 12) and Ms26 (SEQ ID NO: 13).
Phylogenetic tree analysis of some P450 genes revealed that Ms26 is
most closely related to P450s involved in fatty acid
omega-hydroxylation found in Arabidopsis thaliana and Vicia sativa
(FIG. 14B). The translational frame shift caused in the ms26'-0406
excision mutation is believed to destroy the activity of the heme
binding domain, thus resulting in sterility. See the comparison at
FIG. 15 (Ms26 cDNA at SEQ ID NO: 14; fertile exon 5 region at SEQ
ID NO: 15 and sterile exon 5 region is SEQ ID NO: 16).
[0106] Further expression studies were done using the Ms26 cDNA
probe against a northern containing mRNA at discrete stages of
microsporogenesis. FIG. 6A shows a Northern blot with RNA samples
from different tissues including root (1), leaf (2), husk (3), cob
(4), ear spikelet (5), silk (6), immature embryo (7) mature embryo
(8), and tassel from, fertile plant (9), ms26-m2::Mu8 sterile plant
(10), ms26-ref sterile plant (11) and fertile plant (12). A
hybridization signal using Ms26 cDNA was detected only in tassel
tissues. FIG. 6B shows a Northern blot containing mRNA at discrete
stages of microsporogenesis. Hybridization signals using Ms26 cDNA
were detected from meiosis II/quartet stage (4) to late-uninucleate
stage (10), with the maximal signal being observed from
early-uninucleate through late-uninucleate stage (10).
EXAMPLE 6
Identification of Promoter and its Essential Regions
[0107] A putative TATA box can be identified by primer extension
analysis as described in by Current Protocols in Molecular Biology,
Ausubel, F. M. et al. eds; John Wiley and Sons, New York pp.
4.8.1-4.8.5 (1987).
[0108] Regulatory regions of anther genes, such as promoters, may
be identified in genomic subclones using functional analysis,
usually verified by the observation of reporter gene expression in
anther tissue and a lower level or absence of reporter gene
expression in non-anther tissue. The possibility of the regulatory
regions residing "upstream" or 5' ward of the translational start
site can be tested by subcloning a DNA fragment that contains the
upstream region into expression vectors for transient expression
experiments. It is expected that smaller subgenomic fragments may
contain the regions essential for male-tissue preferred expression.
For example, the essential regions of the CaMV 19S and 35S
promoters have been identified in relatively small fragments
derived from larger genomic pieces as described in U.S. Pat. No.
5,352,605.
[0109] The selection of an appropriate expression vector with which
to test for functional expression will depend upon the host and the
method of introducing the expression vector into the host and such
methods are well known to one skilled in the art. For eukaryotes,
the regions in the vector include regions that control initiation
of transcription and control processing. These regions are operably
linked to a reporter gene such as UidA, encoding--glucuronidase
(GUS), or luciferase. General descriptions and examples of plant
expression vectors and reporter genes can be found in Gruber, et
al., "Vectors for Plant Transformation" in Methods in Plant
Molecular Biology and Biotechnology; Glick, et al. eds; CRC Press;
pp. 89-119; (1993). GUS expression vectors and GUS gene cassettes
are commercially available from Clonetech, Palo Alto, Calif., while
luciferase expression vectors and luciferase gene cassettes are
available from Promega Corporation, Madison, Wis. Ti plasmids and
other Agrobacterium vectors are described in Ishida, Y., et al.,
Nature Biotechnology; Vol. 14; pp. 745-750; (1996) and in U.S. Pat.
No. 5,591,616 "Method for Transforming Monocotyledons" (1994).
[0110] Expression vectors containing putative regulatory regions
located in genomic fragments can be introduced into intact tissues
such as staged anthers, embryos or into callus. Methods of DNA
delivery include microprojectile bombardment, DNA injection,
electroporation and Agrobacterium-mediated gene transfer (see
Gruber, et al., "Vectors for Plant Transformation," in Methods in
Plant Molecular Biology and Biotechnology, Glick, et al. eds.; CRC
Press; (1993); U.S. Pat. No. 5,591,616; and Ishida, Y., et al.,
Nature Biotechnology; Vol. 14; pp. 745-750; (1996)). General
methods of culturing plant tissues are found in Gruber, et al.,
supra and Glick, supra.
[0111] For the transient assay system, staged, isolated anthers are
immediately placed onto tassel culture medium (Pareddy, D. R. and
J. F. Petelino, Crop Sci. J.; Vol. 29; pp. 1564-1566; (1989))
solidified with 0.5% Phytagel (Sigma, St. Louis) or other
solidifying media. The expression vector DNA is introduced within 5
hours preferably by microprojectile-mediated delivery with 1.2
.mu.m particles at 1000-1100 Psi. After DNA delivery, the anthers
are incubated at 26.degree. C. upon the same tassel culture medium
for 17 hours and analyzed by preparing a whole tissue homogenate
and assaying for GUS or for lucifierase activity (see Gruber, et
al., supra).
[0112] Upstream of the likely translational start codon of Ms26,
1088 bp of DNA was present in the genomic clone ms26-m2::Mu8.
Translational fusions via an engineered NcoI site were generated
with reporter genes encoding luciferase and .beta.-glucuronidase to
test whether this fragment of DNA had promoter activity in
transient expression assays of bombarded plant tissues. Activity
was demonstrated in anthers and not in coleoptiles, roots and
calli, suggesting anther-preferred or anther-specific promoter
activity.
[0113] A reasonable TATA box was observed by inspection, about
83-77 bp upstream of the translational start codon. The genomic
clone ms26-m2::Mu8 thus includes about 1005 bp upstream of the
possible TATA box. For typical plant genes, the start of
transcription is 26-36 bp downstream of the TATA box, which would
give the Ms26 mRNA a 5'-nontranslated leader of about 48-58 nt. The
total ms26-m2::Mu8 subgenomic fragment of 1088 bp, including
nontranslated leader, start of transcription, TATA box and
sequences upstream of the TATA box, was thus shown to be sufficient
for promoter activity. See SEQ ID NO: 5. The putative TATA box
(TATATCA) is underlined. Thus, the present invention encompasses a
DNA molecule having a nucleotide sequence of SEQ ID NO: 5 (or those
with sequence identity) and having the function of a male
tissue-preferred regulatory region.
[0114] Deletion analysis can occur from both the 5' and 3' ends of
the regulatory region: fragments can be obtained by site-directed
mutagenesis, mutagenesis using the polymerase chain reaction, and
the like (Directed Mutagenesis: A Practical Approach; IRL Press;
(1991)). The 3' end of the male tissue-preferred regulatory region
can be delineated by proximity to the putative TATA box or by 3'
deletions if necessary. The essential region may then be operably
linked to a core promoter of choice. Once the essential region is
identified, transcription of an exogenous gene may be controlled by
the male tissue-preferred region of Ms26 plus a core promoter. The
core promoter can be any one of known core promoters such as a
Cauliflower Mosaic Virus 35S or 19S promoter (U.S. Pat. No.
5,352,605), Ubiquitin (U.S. Pat. No. 5,510,474), the IN2 core
promoter (U.S. Pat. No. 5,364,780), or a Figwort Mosaic Virus
promoter (Gruber, et al., "Vectors for Plant Transformation" in
Methods in Plant Molecular Biology and Biotechnology; Glick, et al.
eds.; CRC Press; pp. 89-119; (1993)). Preferably, the promoter is
the core promoter of a male tissue-preferred gene or the CaMV 35S
core promoter. More preferably, the promoter is a promoter of a
male tissue-preferred gene and in particular, the Ms26 core
promoter.
[0115] Further mutational analysis, for example by linker scanning,
a method well known to the art, can identify small segments
containing sequences required for anther-preferred expression.
These mutations may introduce modifications of functionality such
as in the levels of expression, in the timing of expression, or in
the tissue of expression. Mutations may also be silent and have no
observable effect.
[0116] The foregoing procedures were used to identify essential
regions of the Ms26 promoter. After linking the promoter with the
luciferase marker gene deletion analysis was performed on the
regions of the promoter upstream of the putative TATA box, as
represented in FIG. 8. The x-axis of the bar graph indicates the
number of base pairs immediately upstream of the putative TATA box
retained in a series of deletion derivatives starting from the 5'
end of the promoter. The y-axis shows the normalized luciferase
activity as a percent of full-length promoter activity.
[0117] As is evident from the graph, approximately 176 bp
immediately upstream of the TATA box was sufficient, when coupled
to the core promoter (putative TATA box through start of
transcription), plus 5' nontranslated leader, for transient
expression in anthers. By contrast, luciferase activity was minimal
upon further deletion from the 5' end to 91 bp upstream of the
putative TATA box. This 176 bp upstream of the putative TATA box
through the nontranslated leader can be considered a minimal
promoter, which is further represented at FIG. 9. The TATA box is
underlined. Deletion within the full-length promoter from -176
through -92 relative to the TATA box reduced activity to about 1%
of wild type. Deletion of -39 through -8 did not greatly reduce
activity. Therefore the -176 to -44 bp region contains an essential
region and thus would constitute an upstream enhancer element
conferring anther expression on the promoter, which we refer to as
an "anther box".
[0118] Linker scanning analysis was conducted across the anther box
in 9-10 bp increments. The locations of the linker scanning
substitutions in this region are shown in FIG. 9, and the
expression levels of the mutants relative to the wild type sequence
are shown in FIG. 10. The most drastic effect on transient
expression in anthers was observed for mutants LS12 and LS13, in
the region 52-71 bp upstream of the putative TATA box. A major
effect on transient expression in anthers was also observed for
mutants LS06, LS07, LS08 and LS10, within the region 82-131 bp
upstream of the putative TATA box. Sequences within the anther box
required for wild type levels of transient expression in anthers
are thus demonstrated in the -52 to -131 region relative to the
putative TATA box, particularly the -52 to -71 region.
EXAMPLE 7
Ms26 Sorghum, Rice and Maize Comparison
[0119] As noted above, Ms26 is a male fertility gene in maize. When
it is mutated, and made homozygous recessive, male sterility will
result. An orthologue of Ms26 was identified in sorghum. The
sorghum orthologue of the Ms26 cDNA was isolated by using the maize
Ms26 gene primers in a polymerase chain reaction with sorghum
tassel cDNA as the template. The resultant cDNA fragment was
sequenced by methods described supra and then compared to the Ms26
cDNA from maize. Nucleotide sequence comparisons are set forth in
FIG. 11 and show 90% identity. An orthologue from rice was also
identified and the predicted coding sequence is SEQ ID NO: 17 and
protein is SEQ ID NO: 18. It has one intron less than the maize and
sorghum Ms26, and the coding sequences are highly conserved.
[0120] Identification of the sorghum and rice promoters was
accomplished. FIG. 16 shows an alignment of the Ms26 promoter of
corn (SEQ ID NO: 5), sorghum (SEQ ID NO: 19) and rice (SEQ ID NO:
20). The last three bases of the corn promoter shown in the figure
is the ATG start of translation.
[0121] Alignment as reflected in FIG. 17 of the maize Ms26 protein
(SEQ ID NO: 2), rice Ms26 protein (SEQ ID NO: 18) and sorghum Ms26
protein (SEQ ID NO: 4), and a consensus sequence (SEQ ID NO: 35).
The comparison of protein sequences shows the protein is highly
conserved among the orthologues, with the rice protein sharing 92%
similarity and 86% identity when compared to the maize orthologue.
The predicted tissue specificity in rice and sorghum is further
reflected in a comparison of the Ms26 protein in the sorghum and
rice EST database derived from panicle (flower) libraries. Sorghum
sequences producing significant alignments (GenBank accession
numbers B1075441.1; B1075273.1; B1246000.1; B1246162.1; BG948686.1;
B1099541.1 and BG948366.1, among others) all were sequences from
immature panicle of sorghum, and sequences showing significant
alignment in rice (GenBank accession numbers C73892.1; CR290740.1,
among others) were also from rice immature panicle.
[0122] As is evident from the above, nucleotide sequences which map
to the short arm of chromosome 1 of the Zea mays genome, at the
same site as the Ms26 gene, ms26-m2::Mu8 and its alleles, are genes
critical to male fertility in plants, that is, are necessary for
fertility of a plant, or, when mutated from the sequence found in a
fertile plant, cause sterility in the plant.
[0123] Thus it can be seen that the invention achieves at least all
of its objectives.
Sequence CWU 1
1
3511906DNAZea maysCDS(1)..(1638) 1gaa ttc ggc acg agg gaa gct cac
ctc acg ccg gcg acg cca tcg cca 48Glu Phe Gly Thr Arg Glu Ala His
Leu Thr Pro Ala Thr Pro Ser Pro 1 5 10 15ttc ttc cca cta gca ggg
cct cac aag tac atc gcg ctc ctt ctg gtt 96Phe Phe Pro Leu Ala Gly
Pro His Lys Tyr Ile Ala Leu Leu Leu Val20 25 30gtc ctc tca tgg atc
ctg gtc cag agg tgg agc ctg agg aag cag aaa 144Val Leu Ser Trp Ile
Leu Val Gln Arg Trp Ser Leu Arg Lys Gln Lys35 40 45ggc ccg aga tca
tgg cca gtc atc ggc gca acg gtg gag cag ctg agg 192Gly Pro Arg Ser
Trp Pro Val Ile Gly Ala Thr Val Glu Gln Leu Arg50 55 60aac tac cac
cgg atg cac gac tgg ctt gtc ggg tac ctg tca cgg cac 240Asn Tyr His
Arg Met His Asp Trp Leu Val Gly Tyr Leu Ser Arg His65 70 75 80agg
aca gtg acc gtc gac atg ccg ttc act tcc tac acc tac atc gct 288Arg
Thr Val Thr Val Asp Met Pro Phe Thr Ser Tyr Thr Tyr Ile Ala85 90
95gac ccg gtg aat gtc gag cat gtc ctc aag act aac ttc acc aat tac
336Asp Pro Val Asn Val Glu His Val Leu Lys Thr Asn Phe Thr Asn
Tyr100 105 110ccc aag gga atc gtg tac aga tcc tac atg gac gtg ctc
ctc ggt gac 384Pro Lys Gly Ile Val Tyr Arg Ser Tyr Met Asp Val Leu
Leu Gly Asp115 120 125ggc atc ttc aac gcc gac ggc gag ctg tgg agg
aag cag agg aag acg 432Gly Ile Phe Asn Ala Asp Gly Glu Leu Trp Arg
Lys Gln Arg Lys Thr130 135 140gcg agt ttc gag ttc gcc tcc aag aac
ctg agg gat ttc agc gcc att 480Ala Ser Phe Glu Phe Ala Ser Lys Asn
Leu Arg Asp Phe Ser Ala Ile145 150 155 160gtg ttc aga gag tac tcc
ctg aag ctg tcg ggt ata ctg agc cag gca 528Val Phe Arg Glu Tyr Ser
Leu Lys Leu Ser Gly Ile Leu Ser Gln Ala165 170 175tcc aag gca ggc
aaa gtt gtg gac atg cag gaa ctt tac atg agg atg 576Ser Lys Ala Gly
Lys Val Val Asp Met Gln Glu Leu Tyr Met Arg Met180 185 190acg ctg
gac tcc atc tgc aag gtt ggg ttc ggg gtc gag atc ggc acg 624Thr Leu
Asp Ser Ile Cys Lys Val Gly Phe Gly Val Glu Ile Gly Thr195 200
205ctg tcg cca gat ctc ccc gag aac agc ttc gcg cag gcg ttc gat gcc
672Leu Ser Pro Asp Leu Pro Glu Asn Ser Phe Ala Gln Ala Phe Asp
Ala210 215 220gcc aac atc atc atc acg ctg cgg ttc atc gac ccg ctg
tgg cgc atc 720Ala Asn Ile Ile Ile Thr Leu Arg Phe Ile Asp Pro Leu
Trp Arg Ile225 230 235 240aag agg ttc ttc cac gtc ggg tca gag gcc
ctc cta gcg cag agc atc 768Lys Arg Phe Phe His Val Gly Ser Glu Ala
Leu Leu Ala Gln Ser Ile245 250 255aag ctc gtg gac gag ttc acc tac
agc gtg atc cgc cgg agg aag gcc 816Lys Leu Val Asp Glu Phe Thr Tyr
Ser Val Ile Arg Arg Arg Lys Ala260 265 270gag atc gtc gag gtc cgg
gcc agc ggc aaa cag gag aag atg aag cac 864Glu Ile Val Glu Val Arg
Ala Ser Gly Lys Gln Glu Lys Met Lys His275 280 285gac atc ctg tca
cgg ttc atc gag ctg ggc gag gcc ggc gac gac ggc 912Asp Ile Leu Ser
Arg Phe Ile Glu Leu Gly Glu Ala Gly Asp Asp Gly290 295 300ggc ggc
ttc ggg gac gat aag agc ctc cgg gac gtg gtg ctc aac ttc 960Gly Gly
Phe Gly Asp Asp Lys Ser Leu Arg Asp Val Val Leu Asn Phe305 310 315
320gtg atc gcc ggg cgg gac acg acg gcg acg acg ctg tcg tgg ttc acg
1008Val Ile Ala Gly Arg Asp Thr Thr Ala Thr Thr Leu Ser Trp Phe
Thr325 330 335cac atg gcc atg tcc cac ccg gac gtg gcc gag aag ctg
cgc cgc gag 1056His Met Ala Met Ser His Pro Asp Val Ala Glu Lys Leu
Arg Arg Glu340 345 350ctg tgc gcg ttc gag gcg gag cgc gcg cgc gag
gag ggc gtc acg ctc 1104Leu Cys Ala Phe Glu Ala Glu Arg Ala Arg Glu
Glu Gly Val Thr Leu355 360 365gtg ctc tgc ggc ggc gct gac gcc gac
gac aag gcg ttc gcc gcc cgc 1152Val Leu Cys Gly Gly Ala Asp Ala Asp
Asp Lys Ala Phe Ala Ala Arg370 375 380gtg gcg cag ttc gcg ggc ctc
ctc acc tac gac agc ctc ggc aag ctg 1200Val Ala Gln Phe Ala Gly Leu
Leu Thr Tyr Asp Ser Leu Gly Lys Leu385 390 395 400gtc tac ctc cac
gcc tgc gtc acc gag acg ctc cgc ctg tac ccc gcc 1248Val Tyr Leu His
Ala Cys Val Thr Glu Thr Leu Arg Leu Tyr Pro Ala405 410 415gtc cct
cag gac ccc aag ggg atc ctg gag gac gac gtg ctg ccg gac 1296Val Pro
Gln Asp Pro Lys Gly Ile Leu Glu Asp Asp Val Leu Pro Asp420 425
430ggg acg aag gtg agg gcc ggc ggg atg gtg acg tac gtg ccc tac tcg
1344Gly Thr Lys Val Arg Ala Gly Gly Met Val Thr Tyr Val Pro Tyr
Ser435 440 445atg ggg cgg atg gag tac aac tgg ggc ccc gac gcg gcg
agc ttc cgg 1392Met Gly Arg Met Glu Tyr Asn Trp Gly Pro Asp Ala Ala
Ser Phe Arg450 455 460ccg gag cgg tgg atc aac gag gat ggc gcg ttc
cgc aac gcg tcg ccg 1440Pro Glu Arg Trp Ile Asn Glu Asp Gly Ala Phe
Arg Asn Ala Ser Pro465 470 475 480ttc aag ttc acg gcg ttc cag gcg
ggg ccg agg atc tgc ctg ggc aag 1488Phe Lys Phe Thr Ala Phe Gln Ala
Gly Pro Arg Ile Cys Leu Gly Lys485 490 495gac tcg gcg tac ctg cag
atg aag atg gcg ctg gcc atc ctc ttc cgc 1536Asp Ser Ala Tyr Leu Gln
Met Lys Met Ala Leu Ala Ile Leu Phe Arg500 505 510ttc tac agc ttc
cgg ctg ctg gag ggg cac ccg gtg cag tac cgc atg 1584Phe Tyr Ser Phe
Arg Leu Leu Glu Gly His Pro Val Gln Tyr Arg Met515 520 525atg acc
atc ctc tcc atg gcg cac ggc ctc aag gtc cgc gtc tct agg 1632Met Thr
Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Arg530 535
540gcc gtc tgatgtcatg gcgatttgga tatggatatc gtcccgctta atccacgaca
1688Ala Val545aataacgctc gtgttacaaa tttgcatgca tgcatgtaag
ggaaagcgat gggtttcatt 1748ggtggcttgg cttaagcctt aaaaactccg
tcgggtcttg cgaaccacca catcactagt 1808gttttgtact ctactcctca
gtggaagtgt agtgacagca tacaagttca tcatatatat 1868tatcctcttt
cttaaaaaaa aaaaaaaaaa aactcgag 19062546PRTZea mays 2Glu Phe Gly Thr
Arg Glu Ala His Leu Thr Pro Ala Thr Pro Ser Pro1 5 10 15Phe Phe Pro
Leu Ala Gly Pro His Lys Tyr Ile Ala Leu Leu Leu Val20 25 30Val Leu
Ser Trp Ile Leu Val Gln Arg Trp Ser Leu Arg Lys Gln Lys35 40 45Gly
Pro Arg Ser Trp Pro Val Ile Gly Ala Thr Val Glu Gln Leu Arg50 55
60Asn Tyr His Arg Met His Asp Trp Leu Val Gly Tyr Leu Ser Arg His65
70 75 80Arg Thr Val Thr Val Asp Met Pro Phe Thr Ser Tyr Thr Tyr Ile
Ala85 90 95Asp Pro Val Asn Val Glu His Val Leu Lys Thr Asn Phe Thr
Asn Tyr100 105 110Pro Lys Gly Ile Val Tyr Arg Ser Tyr Met Asp Val
Leu Leu Gly Asp115 120 125Gly Ile Phe Asn Ala Asp Gly Glu Leu Trp
Arg Lys Gln Arg Lys Thr130 135 140Ala Ser Phe Glu Phe Ala Ser Lys
Asn Leu Arg Asp Phe Ser Ala Ile145 150 155 160Val Phe Arg Glu Tyr
Ser Leu Lys Leu Ser Gly Ile Leu Ser Gln Ala165 170 175Ser Lys Ala
Gly Lys Val Val Asp Met Gln Glu Leu Tyr Met Arg Met180 185 190Thr
Leu Asp Ser Ile Cys Lys Val Gly Phe Gly Val Glu Ile Gly Thr195 200
205Leu Ser Pro Asp Leu Pro Glu Asn Ser Phe Ala Gln Ala Phe Asp
Ala210 215 220Ala Asn Ile Ile Ile Thr Leu Arg Phe Ile Asp Pro Leu
Trp Arg Ile225 230 235 240Lys Arg Phe Phe His Val Gly Ser Glu Ala
Leu Leu Ala Gln Ser Ile245 250 255Lys Leu Val Asp Glu Phe Thr Tyr
Ser Val Ile Arg Arg Arg Lys Ala260 265 270Glu Ile Val Glu Val Arg
Ala Ser Gly Lys Gln Glu Lys Met Lys His275 280 285Asp Ile Leu Ser
Arg Phe Ile Glu Leu Gly Glu Ala Gly Asp Asp Gly290 295 300Gly Gly
Phe Gly Asp Asp Lys Ser Leu Arg Asp Val Val Leu Asn Phe305 310 315
320Val Ile Ala Gly Arg Asp Thr Thr Ala Thr Thr Leu Ser Trp Phe
Thr325 330 335His Met Ala Met Ser His Pro Asp Val Ala Glu Lys Leu
Arg Arg Glu340 345 350Leu Cys Ala Phe Glu Ala Glu Arg Ala Arg Glu
Glu Gly Val Thr Leu355 360 365Val Leu Cys Gly Gly Ala Asp Ala Asp
Asp Lys Ala Phe Ala Ala Arg370 375 380Val Ala Gln Phe Ala Gly Leu
Leu Thr Tyr Asp Ser Leu Gly Lys Leu385 390 395 400Val Tyr Leu His
Ala Cys Val Thr Glu Thr Leu Arg Leu Tyr Pro Ala405 410 415Val Pro
Gln Asp Pro Lys Gly Ile Leu Glu Asp Asp Val Leu Pro Asp420 425
430Gly Thr Lys Val Arg Ala Gly Gly Met Val Thr Tyr Val Pro Tyr
Ser435 440 445Met Gly Arg Met Glu Tyr Asn Trp Gly Pro Asp Ala Ala
Ser Phe Arg450 455 460Pro Glu Arg Trp Ile Asn Glu Asp Gly Ala Phe
Arg Asn Ala Ser Pro465 470 475 480Phe Lys Phe Thr Ala Phe Gln Ala
Gly Pro Arg Ile Cys Leu Gly Lys485 490 495Asp Ser Ala Tyr Leu Gln
Met Lys Met Ala Leu Ala Ile Leu Phe Arg500 505 510Phe Tyr Ser Phe
Arg Leu Leu Glu Gly His Pro Val Gln Tyr Arg Met515 520 525Met Thr
Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Arg530 535
540Ala Val5453494DNASorghum sp.modified_base(351)a, c, t, g,
unknown or other 3ggaattcggc ttatgccgtt cacttcctac acctacatcg
ctgacccggt gaatgtcgag 60catgtcctca agactaactt caccaattac cccaaggggg
acgtgtacag atcctacatg 120gatgtgctcc tcggtgacgg catattcaac
gctgacggcg agctgtggag gaagcagagg 180aagacggcga gtttcgagtt
cgcctccaag aacctgaggg atttcagtgc caatgttttc 240agagagtact
ccctgaagct gtcgggcata ctgagtcagg catccaaggc aggcaaagtt
300gttgacatgc aggaacttta catgaggatg acactggact cgatctgcaa
ngttgggttc 360ggggtcnana tcggcacgct gtcnccggat ctccccgaga
acagcttcnc ccaagcgttc 420gatgccgcta acatcatcgt cacnctgcgg
ttcatccacc cnctgtggcg catccagaag 480ttcttccccn gtca
4944158PRTSorghum sp.MOD_RES(113)Any amino acid 4Met Pro Phe Thr
Ser Tyr Thr Tyr Ile Ala Asp Pro Val Asn Val Glu1 5 10 15His Val Leu
Lys Thr Asn Phe Thr Asn Tyr Pro Lys Gly Asp Val Tyr20 25 30Arg Ser
Tyr Met Asp Val Leu Leu Gly Asp Gly Ile Phe Asn Ala Asp35 40 45Gly
Glu Leu Trp Arg Lys Gln Arg Lys Thr Ala Ser Phe Glu Phe Ala50 55
60Ser Lys Asn Leu Arg Asp Phe Ser Ala Asn Val Phe Arg Glu Tyr Ser65
70 75 80Leu Lys Leu Ser Gly Ile Leu Ser Gln Ala Ser Lys Ala Gly Lys
Val85 90 95Val Asp Met Gln Glu Leu Tyr Met Arg Met Thr Leu Asp Ser
Ile Cys100 105 110Xaa Val Gly Phe Gly Val Xaa Ile Gly Thr Leu Ser
Pro Asp Leu Pro115 120 125Glu Asn Ser Phe Xaa Gln Ala Phe Asp Ala
Ala Asn Ile Ile Val Thr130 135 140Leu Arg Phe Ile His Pro Leu Trp
Arg Ile Gln Lys Phe Phe145 150 15551092DNAZea mays 5gaattccaag
cgaggccctt gtagcagaga gtgttgctga tgcagtcggc ggaaatgagt 60gcgtgctgag
agcaacgctg aggggttcca gggatggcaa tggctatggc aatcggctag
120aggtggagga caaggtggtg aggattggga gggcaaccta tggcaagttg
gtgaagaggc 180acgcaatgag agatctattc agacttacac tggatgccgc
caacaaattc aacctttaga 240ttttgatact gtcactccta ctttattcct
tggttgggca acttccaata ggctcatgtt 300aatcaatgat tagtgattat
tcagcaaata ttcttgtttg tttgacattt ataatatgtg 360gggtgagacg
gattaaatat catccatgag agctttatct tcatgctctc ttgattttgg
420tttcagatca ttctttcagt gttcacaaga attttctcag tttggtccat
gtaatttttg 480aagtgaggtt ccttaaattt cattatgctt cctttctttt
ctagactagc aactgcatga 540cttttcactt tgggttcaca aattgactca
caagaaaaca aattcacttt tgggttcaca 600aattcctctt caggatgtac
ttttcacttg aactgtcatg tataggaaca aggaatggct 660cagtttttaa
ggaacaatgt acagatttca tttcagaact ctttctggtt ggttgagttt
720cagacttttt gtaccaagct gatggatcac aatacttgtt tccaaagtct
gataacagaa 780actggcaact cctaattgat aataaaaaga ataaaataca
gtatcagata tctcattttc 840ttggttggca gatcacaaaa aggaacacaa
aggctaagcc tcctacttgt tcgggagtta 900ggtcagggac accatatgaa
tgaaagaaat cttaatttgg ggtcacacca agattgtctc 960tctcgaggtt
ggggggtccc taaggttggt agtagcaata cccaatatat cacctaacaa
1020acccaatcca tgctacatac atacatagca tccatcactt gtagactgga
cccttcatca 1080agagcaccat gg 10926267DNAZea mays 6ccccatctca
ttttcttggt tggcagatca caaaaaggaa cacaaaggct aagcctccta 60cttgttcggg
agttaggtca gggacaccat atgaatgaaa gaaatcttaa tttggggtca
120caccaagatt gtctctctcg aggttggggg gtccctaagg ttggtagtag
caatacccaa 180tatatcacct aacaaaccca atccatgcta catacataca
tagcatccat cacttgtaga 240ctggaccctt catcaagagc accatgg
26773897DNAZea mays 7gaattccaag cgaggccctt gtagcagaga gtgttgctga
tgcagtcggc ggaaatgagt 60gcgtgctgag agcaacgctg aggggttcca gggatggcaa
tggctatggc aatcggctag 120aggtggagga caaggtggtg aggattggga
gggcaaccta tggcaagttg gtgaagaggc 180acgcaatgag agatctattc
agacttacac tggatgccgc caacaaattc aacctttaga 240ttttgatact
gtcactccta ctttattcct tggttgggca acttccaata ggctcatgtt
300aatcaatgat tagtgattat tcagcaaata ttcttgtttg tttgacattt
ataatatgtg 360gggtgagacg gattaaatat catccatgag agctttatct
tcatgctctc ttgattttgg 420tttcagatca ttctttcagt gttcacaaga
attttctcag tttggtccat gtaatttttg 480aagtgaggtt ccttaaattt
cattatgctt cctttctttt ctagactagc aactgcatga 540cttttcactt
tgggttcaca aattgactca caagaaaaca aattcacttt tgggttcaca
600aattcctctt caggatgtac ttttcacttg aactgtcatg tataggaaca
aggaatggct 660cagtttttaa ggaacaatgt acagatttca tttcagaact
ctttctggtt ggttgagttt 720cagacttttt gtaccaagct gatggatcac
aatacttgtt tccaaagtct gataacagaa 780actggcaact cctaattgat
aataaaaaga ataaaataca gtatcagata tctcattttc 840ttggttggca
gatcacaaaa aggaacacaa aggctaagcc tcctacttgt tcgggagtta
900ggtcagggac accatatgaa tgaaagaaat cttaatttgg ggtcacacca
agattgtctc 960tctcgaggtt ggggggtccc taaggttggt agtagcaata
cccaatatat cacctaacaa 1020acccaatcca tgctacatac atacatagca
tccatcactt gtagactgga cccttcatca 1080agagcaccat ggaggaagct
cacatcacgc cggcgacgcc atcgccattc ttcccactag 1140cagggcctca
caagtacatc gcgctcctcc tggttgtcct ctcatggatc ctggtccaga
1200ggtggagcct gaggaagcag aaaggcccga gatcatggcc agtcatcggt
gcaacggtgg 1260agcagctgag gaactaccac cggatgcacg actggcttgt
cgggtacctg tcacggcaca 1320ggacagtgac cgtcgacatg ccgttcactt
cctacaccta catcgctgac ccggtgaatg 1380tcgagcatgt cctcaagact
aacttcacca attaccccaa ggtaaatgac ctgaactcac 1440tgatgttcag
tcttcggaaa tcagagctga aagctgaatc gaatgtgcct gaacaccgtg
1500tagggaatcg tgtacagatc ctacatggac gtgctcctcg gtgacggcat
cttcaacgcc 1560gacggcgagc tgtggaggaa gcagaggaag acggcgagtt
tcgagttcgc ctccaagaac 1620ctgagggatt tcagcgccat tgtgttcaga
gagtactccc tgaagctgtc gggtatactg 1680agccaggcat ccaaggcagg
caaagttgtg gacatgcagg tgagatcact gctcccttgc 1740cattgccaac
atgagcattt caacctgaga cacgagagct accttgccga ttcaggaact
1800ttacatgagg atgacgctgg actccatctg caaggttggg ttcggggtcg
agatcggcac 1860gctgtcgccg gatctccccg agaacagctt cgcgcaggcg
ttcgatgccg ccaacatcat 1920cgtcacgctg cggttcatcg acccgctgtg
gcgcatcaag aggttcttcc acgtcgggtc 1980agaggccctc ctagcgcaga
gcatcaagct cgtggacgag ttcacctaca gcgtgatccg 2040ccggaggaag
gccgagatcg tcgaggcccg ggccagcggc aaacaggaga aggtacgtgc
2100acatgactgt ttcgattctt cagttcatcg tcttggccgg gatggacctg
atcctgattg 2160attatatatc cgtgtgactt gtgaggacaa attaaaatgg
gcagatgaag cacgacatcc 2220tgtcacggtt catcgagcta ggcgaggccg
gcgacgacgg cggcggcttc ggggacgaca 2280agagcctccg ggacgtggtg
ctcaacttcg tgatcgccgg gcgggacacg acggcgacga 2340cgctgtcgtg
gttcacgcac atggccatgt cccacccgga cgtggccgag aagctgcgcc
2400gcgagctgtg cgcgttcgag gcggagcgcg cgcgcgagga gggcgtcgcg
ctcgtgccct 2460gcggcggcgc tgacgccgac gacaaggcgt tcgccgcccg
cgtggcgcag ttcgcgggcc 2520tcctcaccta cgacagcctc ggcaagctgg
tctacctcca cgcctgcgtc accgagacgc 2580tccgcctgta ccccgccgtc
cctcaggtga gcgcgcccga cacgcgacct ccggtccaga 2640gcacagcatg
cagtgagtgg acctgaatgc aatgcacatg cacttgcgcg cgcgcaggac
2700cccaagggga tcctggagga cgacgtgctg ccggacggga cgaaggtgag
ggccggcggg 2760atggtgacgt acgtgcccta ctcgatgggg cggatggagt
acaactgggg ccccgacgcg 2820gcgagcttcc ggccggagcg gtggatcaac
gaggatggcg cgttccgcaa cgcgtcgccg 2880ttcaagttca cggcgttcca
ggcggggccg aggatctgcc tgggcaagga ctcggcgtac 2940ctgcagatga
agatggcgct ggccatcctc ttgcgcttct acagcttccg gctgctggag
3000gggcacccgg tgcagtaccg catgatgacc atcctctcca tggcgcacgg
cctcaaggtc 3060cgcgtctcta gggccgtctg atgtcatggc gatttgggat
atcatcccgc ttaatcctta 3120aaaatttgca tgcatgcatg taagggaaag
cgatgggttt cattggtggc ttggcttaag 3180ccttaaaaac tccgtcgggt
cttgcgaacc accacatcac tagtgttttg tactctactc 3240ctcagtggaa
gtgtagtgac agcatacaag ttcatcatat atattatcct ctttcttcgc
3300cggatgcttc ccgggacctt ttggagacca ttactgacag gcgtgtgaaa
aaaaggcttc 3360ttctgcggcg aagttttggg ttcagagtct tggcgtcttt
gcagcagaaa aaaggtttgg 3420aaggatctga accctgaacc gaaaatggct
tcggaaatat gctcgcatcg gggcggggcc 3480gtcactcggg atgacgacaa
gcccacaagc agtgagagcg aagcgatctt tggagtttgg 3540agacactctc
ggacccctcg gcgctccgcg agctcatctt cgcctcctct gtcgtgtccg
3600tggcggcacc gcgcccgccc gcctcgtgtt cgaccaaatc ccgcgccccg
accggttcgt 3660gtacaacacc ctcatccgcg gcgccgcgcg cagtgacacg
ccccgggacg ccgtatacat 3720ctataaatca tggtattgta ctttattttc
aaacggcctt aacacaacca tatttttatg 3780gtaaacacgt tcaaaattga
cacaaattta aaacaggcac aaaccgtagc taaacataag 3840agaatgagag
acaacccaaa ggttagagat gaaataagct gagtaaacga cgaattc 38978360DNAZea
mays 8caggacccca aggggatcct ggaggacgac gtgctgccgg acgggacgaa
ggtgagggcc 60ggcgggatgg tgacgtacgt gccctactcg atggggcgga tggagtacaa
ctggggcccc 120gacgcggcga gcttccggcc ggaggcccgg agcggtggat
caacgaggat ggcgcgttcc 180gcaacgcgtc gccgttcaag ttcacggcgt
tccaggcggg gccgaggatc tgcctgggca 240aggactcggc gtacctgcag
atgaagatgg cgctggccat cctcttgcgc ttctacagct 300tccggctgct
ggaggggcac ccggtgcagt accgcatgat gaccatcctc tccatggcgc
3609352DNAZea mays 9caggacccca aggggatcct ggaggacgac gtgctgccgg
acgggacgaa ggtgagggcc 60ggcgggatgg tgacgtacgt gccctactcg atggggcgga
tggagtacaa ctggggcccc 120gacgcggcga gcttccggcc ggagcggtgg
atcaacgagg atggcgcgtt ccgcaacgcg 180tcgccgttca agttcacggc
gttccaggcg gggccgagga tctgcctggg caaggactcg 240gcgtacctgc
agatgaagat ggcgctggcc atcctcttcc gcttctacag cttccggctg
300ctggaggggc acccggtgca gtaccgcatg atgaccatcc tctccatggc gc
352101440DNAZea mays 10cggagctagg ggtgaaaacg ggtagggtac ccgaaacggg
taccggatac ggatactgat 60tcgggaccat ttttcggata cggatacggg tattttttag
attcgggacg gatacgggta 120atacccggat agtatggctt cggattcggg
tcggatacgg agcgagtact acccggtaaa 180tacccggata ctcgggtcgg
ataccgggta cccggaattc gggtacccgt tttttctttt 240tctgcaaaat
aatatagtta taaaatcata acttttacat atgaaatcgg atgaagataa
300agtttatatg aaaattgtag agctcgaaga gatctataac tttgtagtac
atcacatttt 360tgtttaaaca tatctttagg ccaaaatcat taaaataatg
tctaaattta tatcaaaata 420atagacttta tcattttcat gtggggactt
aagattatat ccatgtggga acttaggatt 480atctttttat aaactattta
ttaatattgg taacttattt gcaattttcg gtcgacgcta 540caatattttt
atgaatttaa ttgtattttg atgattttct acaacaagaa attaataata
600caccaaatag cctaaaaaat tcatggattt ttacggggac acaacatata
tccacatata 660gttctcaaaa acatttggac tataaaatcc acaagatgtt
ggtgtttctt ccattctact 720cccacttatt gcgtgagtta catgtgaaat
cattttatgt atcgaagttt caacataatt 780aatatttcac ttatcatttt
catgtggcga cttgaggttt tatttgaata gaatgtttat 840ttgttttggt
aagctttttg cattttggat caaactagtg tatttatgaa ttttaattat
900actttgatga ttttatgtag aaagaaatta ataatgtata aatagcctca
gaaatctatg 960aaattatacg aaggtacaac atatggccac atatagtcat
aacaaataat gggaccataa 1020aatccacagg atgtcaacgt ttcttctatt
ttatttccac ttattgcgtg agttacacgt 1080gaaatcactc taagtatcca
agtttcaaca taatcaatac ttcactttac catttttacg 1140tgggaacttg
agattatctt ctattaaatg cttattagta ttaatttact tgcaatttcg
1200tggtcgaaca agaatatttt ttgataacca attaatgcat tatccgacaa
gtatccgata 1260tccgatcaaa taatatccgt atccgtcact tatccgctcg
gataaatatc cggtccctgt 1320atccgtatcc gtcccgtttc taactatccg
tatccgatcc cgaatccgtt ttaaatacat 1380tagggtagga tacaggatga
gctaatatcc gtccgtatcc gcccgttttc acccctagcc 1440114182DNAZea mays
11aactgcatga cttttcactt tgggttcaca aattgactca caagaaaaca aattcacttt
60tgggttcaca aattcctctt caggatgtac ttttcacttg aaactgtcat gtataggaac
120aaggaatggc tcagttttta aggaacaatg tacagatttc atttcagaac
tctttctggt 180tggttgagtt tcagactttt tgtaccaagc tgatggatca
caatacttgt ttccaaagtc 240tgataacaga aactggcaac tcctaattga
taataaaaag aataaaatac agtatcagat 300atctcatttt cttggttggc
agatcacaaa aaggaacaca aaggctaagc ctcctacttg 360ttcgggagtt
aggtcaggga caccatatga atgaaagaaa tcttaatttg gggtcacacc
420aagattgtct ctctcgaggt tggggggtcc ctaaggttgg tagtagcaat
acccaatata 480tcacctaaca aacccaatcc atgctacata catacatagc
atccatcact tgtagactgg 540acccttcatc aagagcacca tggaggaagc
tcacatcacg ccggcgacgc catcgccatt 600cttcccacta gcagggcctc
acaagtacat cgcgctcctc ctggttgtcc tctcatggat 660cctggtccag
aggtggagcc tgaggaagca gaaaggcccg agatcatggc cagtcatcgg
720tgcaacggtg gagcagctga ggaactacca ccggatgcac gactggcttg
tcgggtacct 780gtcgcggcac aggacagtga ccgtcgacat gccgttcact
tcctacacct acatcgctga 840cccggtgaat gtcgagcatg tcctcaagac
taacttcacc aattacccca aggtaaatga 900cctgaactca ctgatgttca
gtcttcggaa atcagagctg aaagctgaat cgaatgtgcc 960tgaacaccgt
gtagggaatc gtgtacagat cctacatgga cgtgctcctc ggtgacggca
1020tcttcaacgc cgacggcgag ctgtggagga agcagaggaa gacggcgagt
ttcgagttcg 1080cctccaagaa cctgagggat ttcagcgcca ttgtgttcag
agagtactcc ctgaagctgt 1140cgggtatact gagccaggca tccaaggcag
gcaaagttgt ggacatgcag gtgagatcac 1200tgctcccttg ccattgccaa
catgagcatt tcaacctgag acacgagagc taccttgccg 1260attcaggaac
tttacatgag gatgacgctg gactccatct gcaaggttgg gttcggggtc
1320gagatcggca cgctgtcgcc ggatctcccc gagaacagct tcgcgcaggc
gttcgatgcc 1380gccaacatca tcgtcacgct gcggttcatc gacccgctgt
ggcgcatcaa gaggttcttc 1440cacgtcgggt cagaggccct cctagcgcag
agcatcaagc tcgtggacga gttcacctac 1500agcgtgatcc gccggaggaa
ggccgagatc gtcgaggtcc gggccagcgg caaacaggag 1560aaggtacgtg
tacatgactg tttcgattct tcagttcatc gtcttggccg ggatggacct
1620gatcctgatt gattatatat ccgtgtgact tgtgaggaca aattaaaatg
ggcagatgaa 1680gcacgacatc ctgtcacggt tcatcgagct aggcgaggcc
ggcgacgacg gcggcggctt 1740cggggacgac aagagcctcc gggacgtggt
gctcaacttc gtgatcgccg ggcgggacac 1800gacggcgacg acgctgtcgt
ggttcacgca catggccatg tcccacccgg acgtggccga 1860gaagctgcgc
cgcgagctgt gcgcgttcga ggcggagcgc gcgcgcgagg agggcgtcgc
1920gctcgtgccc tgcggcggcg ctgacgccga cgacaaggcg ttcgccgccc
gcgtggcgca 1980gttcgcgggc ctcctcacct acgacagcct cggcaagctg
gtctacctcc acgcctgcgt 2040caccgagacg ctccgcctgt accccgccgt
ccctcaggtg agcgcgcccg acacgacctc 2100cggtccgcga tgcaacgcat
atgtggctgt ccgcagagca cagcatgcag tgagtggacc 2160tgaatgcact
atgcaatgca cttgcgcgcg cgcaggaccc caaggggatc ctggaggacg
2220acgtgctgcc ggacgggacg aaggtgaggg ccggcgggat ggtgacgtac
gtgccctact 2280cgatggggcg gatggagtac aactggggcc ccgacgcggc
gagcttccgg ccggagctag 2340gggtgaaaac gggtagggta cccgaaacgg
gtaccggata cggatactga ttcgggacca 2400tttttcggat acggatacgg
gtatttttta gattcgggac ggatacgggt aatacccgga 2460tagtatggct
tcggattcgg gtcggatacg gagcgagtac tacccggtaa atacccggat
2520actcgggtcg gataccgggt acccggaatt cgggtacccg ttttttcttt
ttctgcaaaa 2580taatatagtt ataaaatcat aacttttaca tatgaaatcg
gatgaagata aagtttatat 2640gaaaattgta gagctcgaag agatctataa
ctttgtagta catcacattt ttgtttaaac 2700atatctttag gccaaaatca
ttaaaataat gtctaaattt atatcaaaat aatagacttt 2760atcattttca
tgtggggact taagattata tccatgtggg aacttaggat tatcttttta
2820taaactattt attaatattg gtaacttatt tgcaattttc ggtcgacgct
acaatatttt 2880tatgaattta attgtatttt gatgattttc tacaacaaga
aattaataat acaccaaata 2940gcctaaaaaa ttcatggatt tttacgggga
cacaacatat atccacatat agttctcaaa 3000aacatttgga ctataaaatc
cacaagatgt tggtgtttct tccattctac tcccacttat 3060tgcgtgagtt
acatgtgaaa tcattttatg tatcgaagtt tcaacataat taatatttca
3120cttatcattt tcatgtggcg acttgaggtt ttatttgaat agaatgttta
tttgttttgg 3180taagcttttt gcattttgga tcaaactagt gtatttatga
attttaatta tactttgatg 3240attttatgta gaaagaaatt aataatgtat
aaatagcctc agaaatctat gaaattatac 3300gaaggtacaa catatggcca
catatagtca taacaaataa tgggaccata aaatccacag 3360gatgtcaacg
tttcttctat tttatttcca cttattgcgt gagttacacg tgaaatcact
3420ctaagtatcc aagtttcaac ataatcaata cttcacttta ccatttttac
gtgggaactt 3480gagattatct tctattaaat gcttattagt attaatttac
ttgcaatttc gtggtcgaac 3540aagaatattt tttgataacc aattaatgca
ttatccgaca agtatccgat atccgatcaa 3600ataatatccg tatccgtcac
ttatccgctc ggataaatat ccggtccctg tatccgtatc 3660cgtcccgttt
ctaactatcc gtatccgatc ccgaatccgt tttaaataca ttagggtagg
3720atacaggatg agctaatatc cgtccgtatc cgcccgtttt cacccctagc
cggagcggtg 3780gatcaacgag gatggcgcgt tccgcaacgc gtcgccgttc
aagttcacgg cgttccaggc 3840ggggccgagg atctgcctgg gcaaggactc
ggcgtacctg cagatgaaga tggcgctggc 3900catccttctt gcgcttctac
agcttccggc tgctggaggg gcacccggtg cagtaccgca 3960tgatgaccat
cctctccatg gcgcacggcc tcaaggtccg cgtctctagg gccgtctgat
4020gtcatggcga tttgggatat catcccgctt aatccacgac aaataacgtt
cgtgttacaa 4080atttgcatgc atgcatgtaa gggaaagcga tgggtttcat
tggtggcttg gcttaagcct 4140taaaaactcc gtcgggttct tgcgaaccac
cacatcacta ga 418212505PRTArabidopsis thaliana 12Leu Val Ile Ala
Cys Met Val Thr Ser Trp Ile Phe Leu His Arg Trp1 5 10 15Gly Gln Arg
Asn Lys Ser Gly Pro Lys Thr Trp Pro Leu Val Gly Ala20 25 30Ala Ile
Glu Gln Leu Thr Asn Phe Asp Arg Met His Asp Trp Leu Val35 40 45Glu
Tyr Leu Tyr Asn Ser Arg Thr Val Val Val Pro Met Pro Phe Thr50 55
60Thr Tyr Thr Tyr Ile Ala Asp Pro Ile Asn Val Glu Tyr Val Leu Lys65
70 75 80Thr Asn Phe Ser Asn Tyr Pro Lys Gly Glu Thr Tyr His Ser Tyr
Met85 90 95Glu Val Leu Leu Gly Asp Gly Ile Phe Asn Ser Asp Gly Glu
Leu Trp100 105 110Arg Lys Gln Arg Lys Thr Ala Ser Phe Glu Phe Ala
Ser Lys Asn Leu115 120 125Arg Asp Phe Ser Thr Val Val Phe Lys Glu
Tyr Ser Leu Lys Leu Phe130 135 140Thr Ile Leu Ser Gln Ala Ser Phe
Lys Glu Gln Gln Val Asp Met Gln145 150 155 160Glu Leu Leu Met Arg
Met Thr Leu Asp Ser Ile Cys Lys Val Gly Phe165 170 175Gly Val Glu
Ile Gly Thr Leu Ala Pro Glu Leu Pro Glu Asn His Phe180 185 190Ala
Lys Ala Phe Asp Thr Ala Asn Ile Ile Val Thr Leu Arg Phe Ile195 200
205Asp Pro Leu Trp Lys Met Lys Lys Phe Leu Asn Ile Gly Ser Glu
Ala210 215 220Leu Leu Gly Lys Ser Ile Lys Val Val Asn Asp Phe Thr
Tyr Ser Val225 230 235 240Ile Arg Arg Arg Lys Ala Glu Leu Leu Glu
Ala Gln Val Lys His Asp245 250 255Ile Leu Ser Arg Phe Ile Glu Ile
Ser Asp Asp Pro Asp Ser Lys Glu260 265 270Thr Glu Lys Ser Leu Arg
Asp Ile Val Leu Asn Phe Val Ile Ala Gly275 280 285Arg Asp Thr Thr
Ala Thr Thr Leu Thr Trp Ala Ile Tyr Met Ile Met290 295 300Met Asn
Glu Asn Val Ala Glu Lys Leu Tyr Ser Glu Leu Gln Glu Leu305 310 315
320Glu Lys Glu Ser Ala Glu Ala Thr Asn Thr Ser Leu His Gln Tyr
Asp325 330 335Thr Glu Asp Phe Asn Ser Phe Asn Glu Lys Val Thr Glu
Phe Ala Gly340 345 350Leu Leu Asn Tyr Asp Ser Leu Gly Lys Leu His
Tyr Leu His Ala Val355 360 365Ile Thr Glu Thr Leu Arg Leu Tyr Pro
Ala Val Pro Gln Asp Pro Lys370 375 380Gly Val Leu Glu Asp Asp Met
Leu Pro Asn Gly Thr Lys Val Lys Ala385 390 395 400Gly Gly Met Val
Thr Tyr Val Pro Tyr Ser Met Gly Arg Met Glu Tyr405 410 415Asn Trp
Gly Ser Asp Ala Ala Leu Phe Lys Pro Glu Arg Trp Leu Lys420 425
430Asp Gly Val Phe Gln Asn Ala Ser Pro Phe Lys Phe Thr Ala Phe
Gln435 440 445Ala Gly Pro Arg Ile Cys Leu Gly Lys Asp Ser Ala Tyr
Leu Gln Met450 455 460Lys Met Ala Met Ala Ile Leu Cys Arg Phe Tyr
Lys Phe His Leu Val465 470 475 480Pro Asn His Pro Val Lys Tyr Arg
Met Met Thr Ile Leu Ser Met Ala485 490 495His Gly Leu Lys Val Thr
Val Ser Arg500 50513518PRTZea mays 13Ile Ala Leu Leu Leu Val Val
Leu Ser Trp Ile Leu Val Gln Arg Trp1 5 10 15Ser Leu Arg Lys Gln Lys
Gly Pro Arg Ser Trp Pro Val Ile Gly Ala20 25 30Thr Val Glu Gln Leu
Arg Asn Tyr His Arg Met His Asp Trp Leu Val35 40 45Gly Tyr Leu Ser
Arg His Arg Thr Val Thr Val Asp Met Pro Phe Thr50 55 60Ser Tyr Thr
Tyr Ile Ala Asp Pro Val Asn Val Glu His Val Leu Lys65 70 75 80Thr
Asn Phe Thr Asn Tyr Pro Lys Gly Ile Val Tyr Arg Ser Tyr Met85 90
95Asp Val Leu Leu Gly Asp Gly Ile Phe Asn Ala Asp Gly Glu Leu
Trp100 105 110Arg Lys Gln Arg Lys Thr Ala Ser Phe Glu Phe Ala Ser
Lys Asn Leu115 120 125Arg Asp Phe Ser Ala Ile Val Phe Arg Glu Tyr
Ser Leu Lys Leu Ser130 135 140Gly Ile Leu Ser Gln Ala Ser Lys Ala
Gly Lys Val Val Asp Met Gln145 150 155 160Glu Leu Tyr Met Arg Met
Thr Leu Asp Ser Ile Cys Lys Val Gly Phe165 170 175Gly Val Glu Ile
Gly Thr Leu Ser Pro Asp Leu Pro Glu Asn Ser Phe180 185 190Ala Gln
Ala Phe Asp Ala Ala Asn Ile Ile Ile Thr Leu Arg Phe Ile195 200
205Asp Pro Leu Trp Arg Ile Lys Arg Phe Phe His Val Gly Ser Glu
Ala210 215 220Leu Leu Ala Gln Ser Ile Lys Leu Val Asp Glu Phe Thr
Tyr Ser Val225 230 235 240Ile Arg Arg Arg Lys Ala Glu Ile Val Glu
Val Arg Ala Ser Gly Lys245 250 255Gln Glu Lys Met Lys His Asp Ile
Leu Ser Arg Phe Ile Glu Leu Gly260 265 270Glu Ala Gly Asp Asp Gly
Gly Gly Phe Gly Asp Asp Lys Ser Leu Arg275 280 285Asp Val Val Leu
Asn Phe Val Ile Ala Gly Arg Asp Thr Thr Ala Thr290 295 300Thr Leu
Ser Trp Phe Thr His Met Ala Met Ser His Pro Asp Val Ala305 310 315
320Glu Lys Leu Arg Arg Glu Leu Cys Ala Phe Glu Ala Glu Arg Ala
Arg325 330 335Glu Glu Gly Val Thr Leu Val Leu Cys Gly Gly Ala Asp
Ala Asp Asp340 345 350Lys Ala Phe Ala Ala Arg Val Ala Gln Phe Ala
Gly Leu Leu Thr Tyr355 360 365Asp Ser Leu Gly Lys Leu Val Tyr Leu
His Ala Cys Val Thr Glu Thr370 375 380Leu Arg Leu Tyr Pro Ala Val
Pro Gln Asp Pro Lys Gly Ile Leu Glu385 390 395 400Asp Asp Val Leu
Pro Asp Gly Thr Lys Val Arg Ala Gly Gly Met Val405 410 415Thr Tyr
Val Pro Tyr Ser Met Gly Arg Met Glu Tyr Asn Trp Gly Pro420 425
430Asp Ala Ala Ser Phe Arg Pro Glu Arg Trp Ile Asn Glu Asp Gly
Ala435 440 445Phe Arg Asn Ala Ser Pro Phe Lys Phe Thr Ala Phe Gln
Ala Gly Pro450 455 460Arg Ile Cys Leu Gly Lys Asp Ser Ala Tyr Leu
Gln Met Lys Met Ala465 470 475 480Leu Ala Ile Leu Phe Arg Phe Tyr
Ser Phe Arg Leu Leu Glu Gly His485 490 495Pro Val Gln Tyr Arg Met
Met Thr Ile Leu Ser Met Ala His Gly Leu500 505 510Lys Val Arg Val
Ser Arg51514128PRTZea mays 14Gln Asp Pro Lys Gly Ile Leu Glu Asp
Asp Val Leu Pro Asp Gly Thr1 5 10 15Lys Val Arg Ala Gly Gly Met Val
Thr Tyr Val Pro Tyr Ser Met Gly20 25 30Arg Met Glu Tyr Asn Trp Gly
Pro Asp Ala Ala Ser Phe Arg Pro Glu35 40 45Arg Trp Ile Asn Glu Asp
Gly Ala Phe Arg Asn Ala Ser Pro Phe Lys50 55 60Phe Thr Ala Phe Gln
Ala Gly Pro Arg Ile Cys Leu Gly Lys Asp Ser65 70 75 80Ala Tyr Leu
Gln Met Lys Met Ala Leu Ala Ile Leu Phe Arg Phe Tyr85 90 95Ser Phe
Arg Leu Leu Glu Gly His Pro Val Gln Tyr Arg Met Met Thr100 105
110Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Arg Ala
Val115 120 12515128PRTZea mays 15Gln Asp Pro Lys Gly Ile Leu Glu
Asp Asp Val Leu Pro Asp Gly Thr1 5 10 15Lys Val Arg Ala Gly Gly Met
Val Thr Tyr Val Pro Tyr Ser Met Gly20 25 30Arg Met Glu Tyr Asn Trp
Gly Pro Asp Ala Ala Ser Phe Arg Pro Glu35 40 45Arg Trp Ile Asn Glu
Asp Gly Ala Phe Arg Asn Ala Ser Pro Phe Lys50 55 60Phe Thr Ala Phe
Gln Ala Gly Pro Arg Ile Cys Leu Gly Lys Asp Ser65 70 75 80Ala Tyr
Leu Gln Met Lys Met Ala Leu Ala Ile Leu Phe Arg Phe Tyr85 90 95Ser
Phe Arg Leu Leu Glu Gly His Pro Val Gln Tyr Arg Met Met Thr100 105
110Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Arg Ala
Val115 120 1251687PRTZea mays 16Gln Asp Pro Lys Gly Ile Leu Glu Asp
Asp Val Leu Pro Asp Gly Thr1 5 10 15Lys Val Arg Ala Gly Gly Met Val
Thr Tyr Val Pro Tyr Ser Met Gly20 25 30Arg Met Glu Tyr Asn Trp Gly
Pro Asp Ala Ala Ser Phe Arg Pro Glu35 40 45Ala Arg Ser Gly Gly Ser
Thr Arg Met Ala Arg Ser Ala Thr Arg Arg50 55 60Arg Ser Ser Ser Arg
Arg Ser Arg Arg Gly Arg Gly Ser Ala Trp Ala65 70 75 80Arg Thr Arg
Arg Thr Cys Arg85171635DNAOryza sativa 17atgaagagcc ccatggagga
agctcatgca atgccagtga catcattctt cccagtagca 60ggaatccaca agctcatagc
tatcttcctt gttgtcctct catggatctt ggtccacaag 120tggagcctga
ggaaccagaa agggccaaga tcatggccaa tcatcggcgc gacagtggag
180caactgaaga actaccacag gatgcatgac tggcttgtcg agtacttgtc
gaaggacagg 240acggtgaccg tcgacatgcc tttcacctcc tacacctaca
ttgccgaccc ggtgaacgtc 300gagcatgtcc tgaagaccaa cttcaccaat
taccccaagg gtgaagtgta caggtcttac 360atggatgtgc tgctcggtga
tggcatattc aatgccgacg
gcgagatgtg gaggaagcaa 420aggaagacgg cgagcttcga gtttgcctcc
aagaacttga gagacttcag cactgtggtg 480ttcagggagt actccctgaa
gctatcaagc attctgagcc aagcatgcaa ggccggcaga 540gttgtagaca
tgcaggaatt gttcatgagg atgacactgg actcgatctg caaggtcggg
600tttggggttg agatcgggac gctgtcacct gatctcccgg agaacagctt
tgcccaggca 660ttcgacgctg ccaacatcat cgtcacgctg cggttcatcg
atcctctgtg gcgtctgaag 720aagttcttgc acgtcggatc agaggctctc
ctcgagcaga gcatgaagct ggttgatgac 780ttcacctaca gcgtgatccg
ccgccgcaag gctgagatct tgcaggctcg agccagcggc 840aagcaagaga
agatcaagca cgacatactg tcgcggttca tcgagctcgg ggaggccggc
900ggcgacgagg ggggcggcag cttcggggac gacaagagcc tccgcgacgt
ggtgctcaac 960ttcgtgatcg ccgggcgtga cacgacggcg acgacgctgt
cgtggttcac gtacatggcg 1020atgacgcacc cggccgtcgc cgacaagctc
cggcgcgagc tggccgcgtt cgaggatgag 1080cgcgcgcgcg aggagggcgt
cgcgctcgcc gacgccgccg gcgaggcgtc gttcgcggcg 1140cgcgtggcgc
agttcgcgtc gctgctgagc tacgacgcgg tggggaagct ggtgtacctg
1200cacgcgtgcg tgacggagac gctccgcctc tacccggcgg tgccgcagga
ccccaagggg 1260atcgtggagg acgacgtgct ccccgacggc accaaggtgc
gcgccggcgg gatggtgacg 1320tacgtgccct actccatggg gaggatggag
tacaactggg gccccgacgc ggcgagcttc 1380cggccggagc ggtggctcag
cggcgacggc ggcgcgttcc ggaacgcgtc gccgttcaag 1440ttcaccgcgt
tccaggccgg gccgcggatc tgcctcggca aggactccgc ctacctccag
1500atgaagatgg cgctcgccat cctcttccgc ttctacacct tcgacctcgt
cgaggaccac 1560cccgtcaagt accggatgat gaccatcctc tccatggctc
acggcctcaa ggtccgcgtc 1620tccacctccg tctga 163518544PRTOryza sativa
18Met Lys Ser Pro Met Glu Glu Ala His Ala Met Pro Val Thr Ser Phe1
5 10 15Phe Pro Val Ala Gly Ile His Lys Leu Ile Ala Ile Phe Leu Val
Val20 25 30Leu Ser Trp Ile Leu Val His Lys Trp Ser Leu Arg Asn Gln
Lys Gly35 40 45Pro Arg Ser Trp Pro Ile Ile Gly Ala Thr Val Glu Gln
Leu Lys Asn50 55 60Tyr His Arg Met His Asp Trp Leu Val Glu Tyr Leu
Ser Lys Asp Arg65 70 75 80Thr Val Thr Val Asp Met Pro Phe Thr Ser
Tyr Thr Tyr Ile Ala Asp85 90 95Pro Val Asn Val Glu His Val Leu Lys
Thr Asn Phe Thr Asn Tyr Pro100 105 110Lys Gly Glu Val Tyr Arg Ser
Tyr Met Asp Val Leu Leu Gly Asp Gly115 120 125Ile Phe Asn Ala Asp
Gly Glu Met Trp Arg Lys Gln Arg Lys Thr Ala130 135 140Ser Phe Glu
Phe Ala Ser Lys Asn Leu Arg Asp Phe Ser Thr Val Val145 150 155
160Phe Arg Glu Tyr Ser Leu Lys Leu Ser Ser Ile Leu Ser Gln Ala
Cys165 170 175Lys Ala Gly Arg Val Val Asp Met Gln Glu Leu Phe Met
Arg Met Thr180 185 190Leu Asp Ser Ile Cys Lys Val Gly Phe Gly Val
Glu Ile Gly Thr Leu195 200 205Ser Pro Asp Leu Pro Glu Asn Ser Phe
Ala Gln Ala Phe Asp Ala Ala210 215 220Asn Ile Ile Val Thr Leu Arg
Phe Ile Asp Pro Leu Trp Arg Leu Lys225 230 235 240Lys Phe Leu His
Val Gly Ser Glu Ala Leu Leu Glu Gln Ser Met Lys245 250 255Leu Val
Asp Asp Phe Thr Tyr Ser Val Ile Arg Arg Arg Lys Ala Glu260 265
270Ile Leu Gln Ala Arg Ala Ser Gly Lys Gln Glu Lys Ile Lys His
Asp275 280 285Ile Leu Ser Arg Phe Ile Glu Leu Gly Glu Ala Gly Gly
Asp Glu Gly290 295 300Gly Gly Ser Phe Gly Asp Asp Lys Ser Leu Arg
Asp Val Val Leu Asn305 310 315 320Phe Val Ile Ala Gly Arg Asp Thr
Thr Ala Thr Thr Leu Ser Trp Phe325 330 335Thr Tyr Met Ala Met Thr
His Pro Ala Val Ala Asp Lys Leu Arg Arg340 345 350Glu Leu Ala Ala
Phe Glu Asp Glu Arg Ala Arg Glu Glu Gly Val Ala355 360 365Leu Ala
Asp Ala Ala Gly Glu Ala Ser Phe Ala Ala Arg Val Ala Gln370 375
380Phe Ala Ser Leu Leu Ser Tyr Asp Ala Val Gly Lys Leu Val Tyr
Leu385 390 395 400His Ala Cys Val Thr Glu Thr Leu Arg Leu Tyr Pro
Ala Val Pro Gln405 410 415Asp Pro Lys Gly Ile Val Glu Asp Asp Val
Leu Pro Asp Gly Thr Lys420 425 430Val Arg Ala Gly Gly Met Val Thr
Tyr Val Pro Tyr Ser Met Gly Arg435 440 445Met Glu Tyr Asn Trp Gly
Pro Asp Ala Ala Ser Phe Arg Pro Glu Arg450 455 460Trp Leu Ser Gly
Asp Gly Gly Ala Phe Arg Asn Ala Ser Pro Phe Lys465 470 475 480Phe
Thr Ala Phe Gln Ala Gly Pro Arg Ile Cys Leu Gly Lys Asp Ser485 490
495Ala Tyr Leu Gln Met Lys Met Ala Leu Ala Ile Leu Phe Arg Phe
Tyr500 505 510Thr Phe Asp Leu Val Glu Asp His Pro Val Lys Tyr Arg
Met Met Thr515 520 525Ile Leu Ser Met Ala His Gly Leu Lys Val Arg
Val Ser Thr Ser Val530 535 54019436DNASorghum sp. 19aacgaatgta
tcattgtgcc taaattttta aagaattgtg gacaatttct ggtaggctga 60gtttcagact
ttcagtacca agctgatgga tcacattctg gatccgaagt atgataacat
120aatctggcaa ctcctaattg taataacaat gaataacctg caaatacagt
ataagagtgg 180ctcattttct tggttggcag atcacaaaaa ggaacacaaa
ggctaagcgc caacttgtcc 240gggagttagg tcatggatac catatgaatg
aaagaaatct taatttccgg tcacaccaag 300attgtctctc tcaaggttgg
taacagcaat acccaatata tcacctaaca aacccagaca 360acactacata
cataacatcc atcacttgga gactggaccc ttcatcaaga gcaccatgga
420ggaagctcac ctcatg 43620450DNAOryza sativa 20aagcctggtt
tcagttggtg acaatttaac agaattcaga tggatatggt tctgatatta 60gaaggtggca
tacctttagt cgctgcaaac gcttcagtta tctgaacaaa acaacgaact
120tggctgagca ggggaaaaaa atactgtagc attcattttg tgtttacatg
agtaacgatt 180cttttctagg tggacagatc acaaaaagaa aactaaagct
aagatccaac tcctaagggt 240gttaggttag ggacaccata tgaatgagac
aatcttaatt cttggtcaca caaagattgt 300ctcaaggttg gtagcatcag
tgcccaatat atcacctaac tatgccatcc aaaatgctac 360atagcatctc
ttgtagactg aacccttcat gaagagcccc atggaggaag ctcatgcaat
420gccagtgaca tcattcttcc cagtagcagg 45021538PRTZea mays 21Met Glu
Glu Ala His Leu Thr Pro Ala Thr Pro Ser Pro Phe Phe Pro1 5 10 15Leu
Ala Gly Pro His Lys Tyr Ile Ala Leu Leu Leu Val Val Leu Ser20 25
30Trp Ile Leu Val Gln Arg Trp Ser Leu Arg Lys Gln Lys Gly Pro Arg35
40 45Ser Trp Pro Val Ile Gly Ala Thr Val Glu Gln Leu Arg Asn Tyr
His50 55 60Arg Met His Asp Trp Leu Val Gly Tyr Leu Ser Arg His Arg
Thr Val65 70 75 80Thr Val Asp Met Pro Phe Thr Ser Tyr Thr Tyr Ile
Ala Asp Pro Val85 90 95Asn Val Glu His Val Leu Lys Thr Asn Phe Thr
Asn Tyr Pro Lys Gly100 105 110Ile Val Tyr Arg Ser Tyr Met Asp Val
Leu Leu Gly Asp Gly Ile Phe115 120 125Asn Ala Asp Gly Glu Leu Trp
Arg Lys Gln Arg Lys Thr Ala Ser Phe130 135 140Glu Phe Ala Ser Lys
Asn Leu Arg Asp Phe Ser Ala Ile Val Phe Arg145 150 155 160Glu Tyr
Ser Leu Lys Leu Ser Gly Ile Leu Ser Gln Ala Ser Lys Ala165 170
175Gly Lys Val Val Asp Met Gln Glu Leu Tyr Met Arg Met Thr Leu
Asp180 185 190Ser Ile Cys Lys Val Gly Phe Gly Val Glu Ile Gly Thr
Leu Ser Pro195 200 205Asp Leu Pro Glu Asn Ser Phe Ala Gln Ala Phe
Asp Ala Ala Asn Ile210 215 220Ile Ile Thr Leu Arg Phe Ile Asp Pro
Leu Trp Arg Ile Lys Arg Phe225 230 235 240Phe His Val Gly Ser Glu
Ala Leu Leu Ala Gln Ser Ile Lys Leu Val245 250 255Asp Glu Phe Thr
Tyr Ser Val Ile Arg Arg Arg Lys Ala Glu Ile Val260 265 270Glu Val
Arg Ala Ser Gly Lys Gln Glu Lys Met Lys His Asp Ile Leu275 280
285Ser Arg Phe Ile Glu Leu Gly Glu Ala Gly Phe Gly Asp Asp Lys
Ser290 295 300Leu Arg Asp Val Val Leu Asn Phe Val Ile Ala Gly Arg
Asp Thr Thr305 310 315 320Ala Thr Thr Leu Ser Trp Phe Thr His Met
Ala Met Ser His Pro Asp325 330 335Val Ala Glu Lys Leu Arg Arg Glu
Leu Cys Ala Phe Glu Ala Glu Arg340 345 350Ala Arg Glu Glu Gly Val
Thr Leu Val Leu Cys Gly Gly Ala Asp Ala355 360 365Asp Asp Lys Ala
Phe Ala Ala Arg Val Ala Gln Phe Ala Gly Leu Leu370 375 380Thr Tyr
Asp Ser Leu Gly Lys Leu Val Tyr Leu His Ala Cys Val Thr385 390 395
400Glu Thr Leu Arg Leu Tyr Pro Ala Val Pro Gln Asp Pro Lys Gly
Ile405 410 415Leu Glu Asp Asp Val Leu Pro Asp Gly Thr Lys Val Arg
Ala Gly Gly420 425 430Met Val Thr Tyr Val Pro Tyr Ser Met Gly Arg
Met Glu Tyr Asn Trp435 440 445Gly Pro Asp Ala Ala Ser Phe Arg Pro
Glu Arg Trp Ile Asn Glu Asp450 455 460Gly Ala Phe Arg Asn Ala Ser
Pro Phe Lys Phe Thr Ala Phe Gln Ala465 470 475 480Gly Pro Arg Ile
Cys Leu Gly Lys Asp Ser Ala Tyr Leu Gln Met Lys485 490 495Met Ala
Leu Ala Ile Leu Phe Arg Phe Tyr Ser Phe Arg Leu Leu Glu500 505
510Gly His Pro Val Gln Tyr Arg Met Met Thr Ile Leu Ser Met Ala
His515 520 525Gly Leu Lys Val Arg Val Ser Arg Ala Val530
53522532PRTSorghum sp. 22Met Pro Ala Thr Pro Leu Phe Pro Leu Ala
Gly Leu His Lys Tyr Ile1 5 10 15Ala Ile Leu Leu Val Val Leu Ser Trp
Ala Leu Val His Arg Trp Ser20 25 30Leu Arg Lys Gln Lys Gly Pro Arg
Ser Trp Pro Val Ile Gly Ala Thr35 40 45Leu Glu Gln Leu Arg Asn Tyr
His Arg Met His Asp Trp Leu Val Gly50 55 60Tyr Leu Ser Arg His Lys
Thr Val Thr Val Asp Met Pro Phe Thr Ser65 70 75 80Tyr Thr Tyr Ile
Ala Asp Pro Val Asn Val Glu His Val Leu Lys Thr85 90 95Asn Phe Thr
Asn Tyr Pro Lys Gly Asp Val Tyr Arg Ser Tyr Met Asp100 105 110Val
Leu Leu Gly Asp Gly Ile Phe Asn Ala Asp Gly Glu Leu Trp Arg115 120
125Lys Gln Arg Lys Thr Ala Ser Phe Glu Phe Ala Ser Lys Asn Leu
Arg130 135 140Asp Phe Ser Ala Asn Val Phe Arg Glu Tyr Ser Leu Lys
Leu Ser Gly145 150 155 160Ile Leu Ser Gln Ala Ser Lys Ala Gly Lys
Val Val Asp Met Gln Glu165 170 175Leu Tyr Met Arg Met Thr Leu Asp
Ser Ile Cys Lys Val Gly Phe Gly180 185 190Val Glu Ile Gly Thr Leu
Ser Pro Asp Leu Pro Glu Asn Ser Phe Ala195 200 205Gln Ala Phe Asp
Ala Ala Asn Ile Ile Val Thr Leu Arg Phe Ile Asp210 215 220Pro Leu
Trp Arg Val Lys Arg Phe Phe His Val Gly Ser Glu Ala Leu225 230 235
240Leu Ala Gln Ser Ile Lys Leu Val Asp Glu Phe Thr Tyr Ser Val
Ile245 250 255Arg Arg Arg Lys Ala Glu Ile Val Glu Ala Arg Ala Ser
Gly Lys Gln260 265 270Glu Lys Met Lys His Asp Ile Leu Ser Arg Phe
Ile Glu Leu Gly Glu275 280 285Ala Gly Asp Asp Gly Gly Phe Gly Asp
Asp Lys Ser Leu Arg Asp Val290 295 300Val Leu Asn Phe Val Ile Ala
Gly Arg Asp Thr Thr Ala Thr Thr Leu305 310 315 320Ser Trp Phe Thr
His Met Ala Met Ser His Pro Asp Val Ala Glu Lys325 330 335Leu Arg
Arg Glu Leu Cys Ala Phe Glu Ala Glu Arg Ala Arg Glu Glu340 345
350Gly Val Ala Val Pro Cys Cys Gly Pro Asp Asp Asp Lys Ala Phe
Ala355 360 365Ala Arg Val Ala Gln Phe Ala Gly Leu Leu Thr Tyr Asp
Ser Leu Gly370 375 380Lys Leu Val Tyr Leu His Ala Cys Val Thr Glu
Thr Leu Arg Leu Tyr385 390 395 400Pro Ala Val Pro Gln Asp Pro Lys
Gly Ile Leu Glu Asp Asp Val Leu405 410 415Pro Asp Gly Thr Lys Val
Arg Ala Gly Gly Met Val Thr Tyr Val Pro420 425 430Tyr Ser Met Gly
Arg Met Glu Tyr Asn Trp Gly Pro Asp Ala Ala Ser435 440 445Phe Arg
Pro Glu Arg Trp Ile Asn Glu Glu Gly Ala Phe Arg Asn Ala450 455
460Ser Pro Phe Lys Phe Thr Ala Phe Gln Ala Gly Pro Arg Ile Cys
Leu465 470 475 480Gly Lys Asp Ser Ala Tyr Leu Gln Met Lys Met Ala
Leu Ala Ile Leu485 490 495Phe Arg Phe Tyr Ser Phe Gln Leu Leu Glu
Gly His Pro Val Gln Tyr500 505 510Arg Met Met Thr Ile Leu Ser Met
Ala His Gly Leu Lys Val Arg Val515 520 525Ser Arg Ala
Val5302315DNAArtificial SequenceDescription of Artificial Sequence
Synthetic terminal inverted repeat sequence 23taggggtgaa aacgg
152410PRTZea maysMOD_RES(2)..(3)Any amino acid 24Phe Xaa Xaa Gly
Xaa Arg Xaa Cys Xaa Gly1 5 102510PRTZea mays 25Phe Gln Ala Gly Pro
Arg Ile Cys Leu Gly1 5 10266PRTZea mays 26Ala Gly Arg Asp Thr Thr1
52713PRTZea mays 27Leu Val Tyr Leu His Ala Cys Val Thr Glu Thr Leu
Arg1 5 102842PRTZea mays 28Cys His Gly Asp Leu Asp Met Asp Ile Val
Pro Leu Asn Pro Arg Gln1 5 10 15Ile Thr Leu Val Leu Gln Ile Cys Met
His Ala Cys Lys Gly Lys Arg20 25 30Trp Val Ser Leu Val Ala Trp Leu
Lys Pro35 402928PRTZea mays 29Lys Leu Arg Arg Val Leu Arg Thr Thr
Thr Ser Leu Val Phe Cys Thr1 5 10 15Leu Leu Leu Ser Gly Ser Val Val
Thr Ala Tyr Lys20 253042PRTZea mays 30Cys His Gly Asp Leu Asp Met
Asp Ile Val Pro Leu Asn Pro Arg Gln1 5 10 15Ile Thr Leu Val Leu Gln
Ile Cys Met His Ala Cys Lys Gly Lys Arg20 25 30Trp Val Ser Leu Val
Ala Trp Leu Lys Pro35 403114PRTZea mays 31Lys Leu Arg Arg Val Leu
Arg Thr Thr Thr Ser Leu Val Phe1 5 103224PRTZea mays 32Arg Trp Arg
Trp Pro Ser Ser Cys Ala Ser Thr Ala Ser Gly Cys Trp1 5 10 15Arg Gly
Thr Arg Cys Ser Thr Ala203311PRTZea mays 33Pro Ser Ser Pro Trp Arg
Thr Lys Gly Glu Phe1 5 103442PRTZea mays 34Cys His Gly Asp Leu Asp
Met Asp Ile Val Pro Leu Asn Pro Arg Gln1 5 10 15Ile Thr Leu Val Leu
Gln Ile Cys Met His Ala Cys Lys Gly Lys Arg20 25 30Trp Val Ser Leu
Val Ala Trp Leu Lys Pro35 4035548PRTArtificial SequenceDescription
of Artificial Sequence Synthetic consensus sequence 35Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Pro Xaa Thr Pro Phe1 5 10 15Phe Pro
Leu Ala Gly Ile His Lys Tyr Ile Ala Ile Leu Leu Val Val20 25 30Leu
Ser Trp Ile Leu Val His Arg Trp Ser Leu Arg Lys Gln Lys Gly35 40
45Pro Arg Ser Trp Pro Val Ile Gly Ala Thr Val Glu Gln Leu Arg Asn50
55 60Tyr His Arg Met His Asp Trp Leu Val Gly Tyr Leu Ser Arg His
Arg65 70 75 80Thr Val Thr Val Asp Met Pro Phe Thr Ser Tyr Thr Tyr
Ile Ala Asp85 90 95Pro Val Asn Val Glu His Val Leu Lys Thr Asn Phe
Thr Asn Tyr Pro100 105 110Lys Gly Asp Val Tyr Arg Ser Tyr Met Asp
Val Leu Leu Gly Asp Gly115 120 125Ile Phe Asn Ala Asp Gly Glu Leu
Trp Arg Lys Gln Arg Lys Thr Ala130 135 140Ser Phe Glu Phe Ala Ser
Lys Asn Leu Arg Asp Phe Ser Ala Ile Val145 150 155 160Phe Arg Glu
Tyr Ser Leu Lys Leu Ser Gly Ile Leu Ser Gln Ala Ser165 170 175Lys
Ala Gly Lys Val Val Asp Met Gln Glu Leu Tyr Met Arg Met Thr180 185
190Leu Asp Ser Ile Cys Lys Val Gly Phe Gly Val Glu Ile Gly Thr
Leu195 200 205Ser Pro Asp Leu Pro Glu Asn Ser Phe Ala Gln Ala Phe
Asp Ala Ala210 215 220Asn Ile Ile Val Thr Leu Arg Phe Ile Asp Pro
Leu Trp Arg Ile Lys225 230 235 240Arg Phe Phe His Val Gly Ser Glu
Ala Leu Leu Ala Gln Ser Ile Lys245 250 255Leu Val Asp Glu Phe Thr
Tyr Ser Val Ile Arg Arg Arg Lys Ala Glu260 265 270Ile Val Glu Ala
Arg Ala Ser Gly Lys Gln Glu Lys Met Lys His Asp275 280 285Ile Leu
Ser Arg Phe Ile Glu Leu Gly Glu Ala Gly Asp Asp Gly Gly290 295
300Gly Xaa Xaa Phe Gly Asp Asp Lys Ser Leu Arg Asp Val Val Leu
Asn305
310 315 320Phe Val Ile Ala Gly Arg Asp Thr Thr Ala Thr Thr Leu Ser
Trp Phe325 330 335Thr His Met Ala Met Ser His Pro Asp Val Ala Glu
Lys Leu Arg Arg340 345 350Glu Leu Cys Ala Phe Glu Ala Glu Arg Ala
Arg Glu Glu Gly Val Ala355 360 365Leu Xaa Xaa Cys Gly Xaa Xaa Xaa
Xaa Asp Asp Lys Ala Phe Ala Ala370 375 380Arg Val Ala Gln Phe Ala
Gly Leu Leu Thr Tyr Asp Ser Leu Gly Lys385 390 395 400Leu Val Tyr
Leu His Ala Cys Val Thr Glu Thr Leu Arg Leu Tyr Pro405 410 415Ala
Val Pro Gln Asp Pro Lys Gly Ile Leu Glu Asp Asp Val Leu Pro420 425
430Asp Gly Thr Lys Val Arg Ala Gly Gly Met Val Thr Tyr Val Pro
Tyr435 440 445Ser Met Gly Arg Met Glu Tyr Asn Trp Gly Pro Asp Ala
Ala Ser Phe450 455 460Arg Pro Glu Arg Trp Ile Asn Glu Asp Gly Xaa
Ala Phe Arg Asn Ala465 470 475 480Ser Pro Phe Lys Phe Thr Ala Phe
Gln Ala Gly Pro Arg Ile Cys Leu485 490 495Gly Lys Asp Ser Ala Tyr
Leu Gln Met Lys Met Ala Leu Ala Ile Leu500 505 510Phe Arg Phe Tyr
Ser Phe Xaa Leu Leu Glu Gly His Pro Val Gln Tyr515 520 525Arg Met
Met Thr Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val530 535
540Ser Arg Ala Val545
* * * * *
References