U.S. patent application number 11/432758 was filed with the patent office on 2006-09-21 for nucleotide sequences 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 | 20060212971 11/432758 |
Document ID | / |
Family ID | 24689201 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060212971 |
Kind Code |
A1 |
Albertsen; Marc C. ; et
al. |
September 21, 2006 |
Nucleotide sequences 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: |
PAT SWEENEY;ATTN: PHI
1835 PLEASANT ST.
WEST DES MOINES
IA
50265
US
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
Johnston
IA
|
Family ID: |
24689201 |
Appl. No.: |
11/432758 |
Filed: |
May 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10412000 |
Apr 11, 2003 |
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11432758 |
May 11, 2006 |
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09670153 |
Sep 26, 2000 |
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10412000 |
Apr 11, 2003 |
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Current U.S.
Class: |
800/287 ;
435/412; 435/468; 536/23.6; 800/320.1 |
Current CPC
Class: |
C12N 15/8231 20130101;
C12N 15/8222 20130101; C12N 15/8217 20130101; C12N 15/8289
20130101; C07K 14/415 20130101 |
Class at
Publication: |
800/287 ;
800/320.1; 435/412; 435/468; 536/023.6 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07H 21/04 20060101 C07H021/04; C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04; A01H 1/00 20060101
A01H001/00 |
Claims
1-17. (canceled)
18. A method of impacting fertility of a plant comprising
inactivating in a plant a first nucleotide sequence, operably
linking a second nucleotide sequence to an inducible promoter and
introducing said second sequence into the plant such that the plant
is constitutively male sterile and fertility is induced by inducing
the promoter, wherein the first and second nucleotide sequence
comprises a sequence selected from the group consisting of: (a) a
sequence encoding the amino acid sequence of any of SEQ ID NO: 2,
or 4; (b) the nucleotide sequences of any of SEQ. ID NO: 1, 3, or
7; (c) a sequence which hybridizes to any of said sequences of (a)
or (b) under conditions of a wash in 0.1 SSC, 1% (w/v) SDS at
65.degree. C.; (d) a sequence having at least 90% identity to any
of said sequences of (a) or (b); and (e) a fragment of any of the
foregoing sequences which fragment is a sequence critical to male
fertility in a plant.
19-24. (canceled)
25. A method of reproducing a plant produced according to the
method of claim 18, further comprising planting seed of the plant
to provide growing male sterile plants; inducing conversion of the
growing plants to male fertile form under conditions which induce
the promoter to express the second DNA molecule; and
open-pollinating the growing plants in isolation to produce seed;
and harvesting the seed.
26. A controllably male sterile plant produced according to the
method of claim 18.
27-58. (canceled)
Description
[0001] This application is a continuation 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 and application
U.S. Ser. No. 09/670,153, filed Sep. 26, 2000, now abandoned.
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 (F1) 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 F1 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.
Further objects of the invention will become apparent in the
description and claims that follow.
SUMMARY OF THE INVENTION
[0022] 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
[0023] FIG. 1. is a locus map of the male sterility gene
SBMu200.
[0024] FIG. 2. is a gel of a Southern Blot analysis of ECORI
digested DNA from a Mu family segregating for male sterility and
hybridized with a Mu8 probe.
[0025] FIG. 3. is a Northern Blot analysis gel hybridized with a
PstI fragment isolated from the SBMu2003.1 clone.
[0026] FIG. 4A-4D is the sequence of SBMu200 (The cDNA is SEQ ID
NO: 1, the protein is SEQ ID NO: 2)
[0027] FIG. 5A-5C is the genomic SBMu200 sequence (also referred to
as SEQ ID NO: 7).
[0028] FIG. 6A-6D is a comparison of the genomic SBMu200 sequence
(SEQ ID NO: 7) with the cDNA of SBMu200 (SEQ ID NO: 1).
[0029] FIG. 7. is a Northern analysis gel showing developmental
gene expression in microsporogenesis of the gene SBMu200.
[0030] FIG. 8 is the full length promoter of SBMu200 (SEQ ID No.
5)
[0031] FIG. 9. is a bar graph showing luciferase activity after
deletions of select regions of the SbMu200 promoter.
[0032] FIG. 10 shows an essential region of the SBMu200 promoter
(SEQ ID NO. 6).
[0033] FIG. 11 is a bar graph showing luciferase activity after
substitution by restriction site linker scanning of select small
(9-10 bp) regions of the SBMu200 essential promoter fragment.
[0034] FIG. 12A and B is a comparison of SBMu200 sorghum tassel
nucleotide sequence (SEQ ID NO. 3) and SBMu200 maize cDNA 8.1 (SEQ
ID NO: 1), and sorghum tassel protein sequence (SEQ ID NO: 4) and
SBMu200 maize protein (SEQ ID NO; 2).
DISCLOSURE OF THE INVENTION
[0035] All references referred to are incorporated herein by
reference.
[0036] 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.
[0037] 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.
[0038] At U.S. Pat. No. 5,478,369 there is described a method by
which a male sterility gene was tagged on maize chromosome 9.
Previously, the only described male sterility gene on chromosome 9
was MS2, which has never been cloned and sequenced. 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 (Jan. 1981). The only fertility gene cloned before that
had been the Arabadopsis gene described at Aarts, et al.,
supra.
[0039] The SBMu200 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.
[0040] The maize family cosegregating for sterility was named
SBMu200 and was found to have an approximately 5.5 Kb EcoRI
fragment that hybridized with a Mu8 probe. 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 EcoRI fragment. This probe was used to isolate
cDNA clones from a tassel cDNA library. The cDNA for SBMu200 is
1906 bp, and the Mu insertion occurred in exon 1 of the gene. FIG.
9 (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.
[0041] 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.
[0042] The invention also includes those nucleotide sequences which
selectively hybridize to the SBMu200 nucleotide sequences under
stringent conditions. In referring to a sequence that "selectively
hybridizes" with SBMu200, 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.
[0043] 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.
[0044] An extensive guide to the hybridization of nucleic acids is
found in Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes, Part I,
Chapter 2 "Overview of principles of hybridization and the strategy
of nucleic acid probe assays", Elsevier, New York (1993); and
Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,
Eds., Greene Publishing and Wiley-Interscience, New York (1995).
See also Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual (2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0045] 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.
[0046] 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.
[0047] 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 2.times.SSC, 0.5% (w/v)
SDS, at 65.degree. C. for 30 minutes 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.
[0048] Methods of aligning sequences for comparison are well-known
in the art. Gene comparisons can be determined by conducting BLAST
( Basic Local Alignment Search Tool; Altschul, S. F., et al.,
(1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for
identity to sequences contained in the BLAST "GENEMBL" database. A
sequence can be analyzed for identity to all publicly available DNA
sequences contained in the GENEMBL database using the BLASTN
algorithm under the default parameters. 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.
[0049] 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 fo 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.
[0050] 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.
[0051] Sequences which hybridize to the 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.
[0052] 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. 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)).
[0053] The regulatory region of SBMU200 has been identified as
including the 1005 bp region upstream of the putative TATA box. See
FIG. 8. 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.
[0054] 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.
[0055] 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 20 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).
[0056] Further, the promoter of the present invention can be linked
with nucleotide sequences other than the SBMu200 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 SBMu200 promoter include complementary
nucleotidic units such as antisense molecules (callase antisense
RNA, bamase 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.
[0057] The invention includes vectors with the SBMu200 gene. A
vector is prepared comprising the SBMu200gene, 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.
[0058] 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).
[0059] 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: picomavirus
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.
[0060] 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).
[0061] 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.
[0062] 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).
[0063] 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.
[0064] 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).
[0065] 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).
[0066] 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.
[0067] 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/Technolo 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).
[0068] 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.
[0069] 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 SBMu200
[0070] 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 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.
[0071] 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 F2 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. 2. This EcoRI band
segregated in the fertile plants suggesting a heterozygous wild
type condition for the allele.
EXAMPLE 2
Library Construction and Screening
[0072] The process of cDNA 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
Laboratorv Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor Lab Press, Plainview, N.Y. (1989). Libraries were created as
follows.
[0073] 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. 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.
EXAMPLE 3
Expression Analysis and cDNA Isolation
[0074] 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
SBMu200-8.1, was isolated from the library.
EXAMPLE 4
Sequence Analysis
[0075] The SBMu200-3.1 genomic clone and the cDNA clone,
SBMu200-8.1, 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 FIGS. 4 and 5 and the comparison is
at FIG. 6. 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 SBMu200-8.1 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. A comparison
of the predicted protein from SBMu200-8.1 to the consensus amino
acid sequence of these domains showed 1) 92% identity to the
dioxygen binding domain, 2) 85% identity to the tridecapeptide
domain (steroid binding), and 3) 100% identity to the C-terminal
heme attachment domain. Further expression studies were done using
the SBMu200-8.1 cDNA probe against a northern containing mRNA at
discrete stages of microsporogenesis. Signal is detected from
meiosis II/quartet to late-uninucleate, with maximal signal being
observed from early-uninucleate through late-uninucleate as shown
at FIG. 7.
EXAMPLE 5
Identification of Promoter and its Essential Regions
[0076] 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).
[0077] 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.
[0078] 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
.beta.-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).
[0079] 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.
[0080] 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).
[0081] Upstream of the likely translational start codon of SBMu200,
1088 bp of DNA was present in the genomic clone SBMu200-3.1.
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.
[0082] A reasonable TATA box was observed by inspection, about
83-77 bp upstream of the translational start codon. The genomic
clone SBMu200-3.1 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 SBMu200 MRNA a 5'-nontranslated leader of about 48-58 nt.
The total SBMu200 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 FIG. 8, which is 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.
[0083] 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 SBMu200 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 SBMu200 core
promoter.
[0084] 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.
[0085] The foregoing procedures were used to identify essential
regions of the SBMu200 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. 9. 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.
[0086] 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 cosnidered a minimal
promoter, which is further represented at FIG. 10. 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 -44bp 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".
[0087] 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. 10, and the
expression levels of the mutants relative to the wild type sequence
are shown in FIG. 11. 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 LS 10, 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 TAATA box, particularly the -52 to -71 region.
EXAMPLE 6
SBMu200 Sorghum Tassel RT-PCR and SBMu200 Maize cDNA Comparison
[0088] As noted above, SBMu200 is a male fertility gene. When it is
mutated, male sterility will result. A homologue of SBMu200 was
identified in sorghum. The sorghum-SBMu200 cDNA was isolated by
using the maize SBMu200 gene primers in a polymerase chain reaction
with sorghum tassel cDNA as the template. The resultant cDNA
fragment was sequences by methods described supra and then compared
to the SBMu200 cDNA from maize. Nucleotide sequence comparisons are
set forth in FIG. 10 and show 90% identity.
[0089] As is evident from the above, the SBMu200 gene is critical
to male fertility in plants.
[0090] Thus it can be seen that the invention achieves at least all
of its objectives.
Sequence CWU 1
1
7 1 1906 DNA Zea mays CDS (1..1638, 1642..1767) 1 gaa ttc ggc acg
agg gaa gct cac ctc acg ccg gcg acg cca tcg cca 48 Glu Phe Gly Thr
Arg Glu Ala His Leu Thr Pro Ala Thr Pro Ser Pro 1 5 10 15 ttc ttc
cca cta gca ggg cct cac aag tac atc gcg ctc ctt ctg gtt 96 Phe Phe
Pro Leu Ala Gly Pro His Lys Tyr Ile Ala Leu Leu Leu Val 20 25 30
gtc ctc tca tgg atc ctg gtc cag agg tgg agc ctg agg aag cag aaa 144
Val Leu Ser Trp Ile Leu Val Gln Arg Trp Ser Leu Arg Lys Gln Lys 35
40 45 ggc ccg aga tca tgg cca gtc atc ggc gca acg gtg gag cag ctg
agg 192 Gly Pro Arg Ser Trp Pro Val Ile Gly Ala Thr Val Glu Gln Leu
Arg 50 55 60 aac tac cac cgg atg cac gac tgg ctt gtc ggg tac ctg
tca cgg cac 240 Asn Tyr His Arg Met His Asp Trp Leu Val Gly Tyr Leu
Ser Arg His 65 70 75 80 agg aca gtg acc gtc gac atg ccg ttc act tcc
tac acc tac atc gct 288 Arg Thr Val Thr Val Asp Met Pro Phe Thr Ser
Tyr Thr Tyr Ile Ala 85 90 95 gac ccg gtg aat gtc gag cat gtc ctc
aag act aac ttc acc aat tac 336 Asp Pro Val Asn Val Glu His Val Leu
Lys Thr Asn Phe Thr Asn Tyr 100 105 110 ccc aag gga atc gtg tac aga
tcc tac atg gac gtg ctc ctc ggt gac 384 Pro Lys Gly Ile Val Tyr Arg
Ser Tyr Met Asp Val Leu Leu Gly Asp 115 120 125 ggc atc ttc aac gcc
gac ggc gag ctg tgg agg aag cag agg aag acg 432 Gly Ile Phe Asn Ala
Asp Gly Glu Leu Trp Arg Lys Gln Arg Lys Thr 130 135 140 gcg agt ttc
gag ttc gcc tcc aag aac ctg agg gat ttc agc gcc att 480 Ala Ser Phe
Glu Phe Ala Ser Lys Asn Leu Arg Asp Phe Ser Ala Ile 145 150 155 160
gtg ttc aga gag tac tcc ctg aag ctg tcg ggt ata ctg agc cag gca 528
Val Phe Arg Glu Tyr Ser Leu Lys Leu Ser Gly Ile Leu Ser Gln Ala 165
170 175 tcc aag gca ggc aaa gtt gtg gac atg cag gaa ctt tac atg agg
atg 576 Ser Lys Ala Gly Lys Val Val Asp Met Gln Glu Leu Tyr Met Arg
Met 180 185 190 acg ctg gac tcc atc tgc aag gtt ggg ttc ggg gtc gag
atc ggc acg 624 Thr Leu Asp Ser Ile Cys Lys Val Gly Phe Gly Val Glu
Ile Gly Thr 195 200 205 ctg tcg cca gat ctc ccc gag aac agc ttc gcg
cag gcg ttc gat gcc 672 Leu Ser Pro Asp Leu Pro Glu Asn Ser Phe Ala
Gln Ala Phe Asp Ala 210 215 220 gcc aac atc atc atc acg ctg cgg ttc
atc gac ccg ctg tgg cgc atc 720 Ala Asn Ile Ile Ile Thr Leu Arg Phe
Ile Asp Pro Leu Trp Arg Ile 225 230 235 240 aag agg ttc ttc cac gtc
ggg tca gag gcc ctc cta gcg cag agc atc 768 Lys Arg Phe Phe His Val
Gly Ser Glu Ala Leu Leu Ala Gln Ser Ile 245 250 255 aag ctc gtg gac
gag ttc acc tac agc gtg atc cgc cgg agg aag gcc 816 Lys Leu Val Asp
Glu Phe Thr Tyr Ser Val Ile Arg Arg Arg Lys Ala 260 265 270 gag atc
gtc gag gtc cgg gcc agc ggc aaa cag gag aag atg aag cac 864 Glu Ile
Val Glu Val Arg Ala Ser Gly Lys Gln Glu Lys Met Lys His 275 280 285
gac atc ctg tca cgg ttc atc gag ctg ggc gag gcc ggc gac gac ggc 912
Asp Ile Leu Ser Arg Phe Ile Glu Leu Gly Glu Ala Gly Asp Asp Gly 290
295 300 ggc ggc ttc ggg gac gat aag agc ctc cgg gac gtg gtg ctc aac
ttc 960 Gly Gly Phe Gly Asp Asp Lys Ser Leu Arg Asp Val Val Leu Asn
Phe 305 310 315 320 gtg atc gcc ggg cgg gac acg acg gcg acg acg ctg
tcg tgg ttc acg 1008 Val Ile Ala Gly Arg Asp Thr Thr Ala Thr Thr
Leu Ser Trp Phe Thr 325 330 335 cac atg gcc atg tcc cac ccg gac gtg
gcc gag aag ctg cgc cgc gag 1056 His Met Ala Met Ser His Pro Asp
Val Ala Glu Lys Leu Arg Arg Glu 340 345 350 ctg tgc gcg ttc gag gcg
gag cgc gcg cgc gag gag ggc gtc acg ctc 1104 Leu Cys Ala Phe Glu
Ala Glu Arg Ala Arg Glu Glu Gly Val Thr Leu 355 360 365 gtg ctc tgc
ggc ggc gct gac gcc gac gac aag gcg ttc gcc gcc cgc 1152 Val Leu
Cys Gly Gly Ala Asp Ala Asp Asp Lys Ala Phe Ala Ala Arg 370 375 380
gtg gcg cag ttc gcg ggc ctc ctc acc tac gac agc ctc ggc aag ctg
1200 Val Ala Gln Phe Ala Gly Leu Leu Thr Tyr Asp Ser Leu Gly Lys
Leu 385 390 395 400 gtc tac ctc cac gcc tgc gtc acc gag acg ctc cgc
ctg tac ccc gcc 1248 Val Tyr Leu His Ala Cys Val Thr Glu Thr Leu
Arg Leu Tyr Pro Ala 405 410 415 gtc cct cag gac ccc aag ggg atc ctg
gag gac gac gtg ctg ccg gac 1296 Val Pro Gln Asp Pro Lys Gly Ile
Leu Glu Asp Asp Val Leu Pro Asp 420 425 430 ggg acg aag gtg agg gcc
ggc ggg atg gtg acg tac gtg ccc tac tcg 1344 Gly Thr Lys Val Arg
Ala Gly Gly Met Val Thr Tyr Val Pro Tyr Ser 435 440 445 atg ggg cgg
atg gag tac aac tgg ggc ccc gac gcg gcg agc ttc cgg 1392 Met Gly
Arg Met Glu Tyr Asn Trp Gly Pro Asp Ala Ala Ser Phe Arg 450 455 460
ccg gag cgg tgg atc aac gag gat ggc gcg ttc cgc aac gcg tcg ccg
1440 Pro Glu Arg Trp Ile Asn Glu Asp Gly Ala Phe Arg Asn Ala Ser
Pro 465 470 475 480 ttc aag ttc acg gcg ttc cag gcg ggg ccg agg atc
tgc ctg ggc aag 1488 Phe Lys Phe Thr Ala Phe Gln Ala Gly Pro Arg
Ile Cys Leu Gly Lys 485 490 495 gac tcg gcg tac ctg cag atg aag atg
gcg ctg gcc atc ctc ttc cgc 1536 Asp Ser Ala Tyr Leu Gln Met Lys
Met Ala Leu Ala Ile Leu Phe Arg 500 505 510 ttc tac agc ttc cgg ctg
ctg gag ggg cac ccg gtg cag tac cgc atg 1584 Phe Tyr Ser Phe Arg
Leu Leu Glu Gly His Pro Val Gln Tyr Arg Met 515 520 525 atg acc atc
ctc tcc atg gcg cac ggc ctc aag gtc cgc gtc tct agg 1632 Met Thr
Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Arg 530 535 540
gcc gtc tga tgt cat ggc gat ttg gat atg gat atc gtc ccg ctt aat
1680 Ala Val Cys His Gly Asp Leu Asp Met Asp Ile Val Pro Leu Asn
545 550 555 cca cga caa ata acg ctc gtg tta caa att tgc atg cat gca
tgt aag 1728 Pro Arg Gln Ile Thr Leu Val Leu Gln Ile Cys Met His
Ala Cys Lys 560 565 570 575 gga aag cga tgg gtt tca ttg gtg gct tgg
ctt aag cct taaaaactcc 1777 Gly Lys Arg Trp Val Ser Leu Val Ala Trp
Leu Lys Pro 580 585 gtcgggtctt gcgaaccacc acatcactag tgttttgtac
tctactcctc agtggaagtg 1837 tagtgacagc atacaagttc atcatatata
ttatcctctt tcttaaaaaa aaaaaaaaaa 1897 aaactcgag 1906 2 588 PRT Zea
mays 2 Glu Phe Gly Thr Arg Glu Ala His Leu Thr Pro Ala Thr Pro Ser
Pro 1 5 10 15 Phe Phe Pro Leu Ala Gly Pro His Lys Tyr Ile Ala Leu
Leu Leu Val 20 25 30 Val Leu Ser Trp Ile Leu Val Gln Arg Trp Ser
Leu Arg Lys Gln Lys 35 40 45 Gly Pro Arg Ser Trp Pro Val Ile Gly
Ala Thr Val Glu Gln Leu Arg 50 55 60 Asn Tyr His Arg Met His Asp
Trp Leu Val Gly Tyr Leu Ser Arg His 65 70 75 80 Arg Thr Val Thr Val
Asp Met Pro Phe Thr Ser Tyr Thr Tyr Ile Ala 85 90 95 Asp Pro Val
Asn Val Glu His Val Leu Lys Thr Asn Phe Thr Asn Tyr 100 105 110 Pro
Lys Gly Ile Val Tyr Arg Ser Tyr Met Asp Val Leu Leu Gly Asp 115 120
125 Gly Ile Phe Asn Ala Asp Gly Glu Leu Trp Arg Lys Gln Arg Lys Thr
130 135 140 Ala Ser Phe Glu Phe Ala Ser Lys Asn Leu Arg Asp Phe Ser
Ala Ile 145 150 155 160 Val Phe Arg Glu Tyr Ser Leu Lys Leu Ser Gly
Ile Leu Ser Gln Ala 165 170 175 Ser Lys Ala Gly Lys Val Val Asp Met
Gln Glu Leu Tyr Met Arg Met 180 185 190 Thr Leu Asp Ser Ile Cys Lys
Val Gly Phe Gly Val Glu Ile Gly Thr 195 200 205 Leu Ser Pro Asp Leu
Pro Glu Asn Ser Phe Ala Gln Ala Phe Asp Ala 210 215 220 Ala Asn Ile
Ile Ile Thr Leu Arg Phe Ile Asp Pro Leu Trp Arg Ile 225 230 235 240
Lys Arg Phe Phe His Val Gly Ser Glu Ala Leu Leu Ala Gln Ser Ile 245
250 255 Lys Leu Val Asp Glu Phe Thr Tyr Ser Val Ile Arg Arg Arg Lys
Ala 260 265 270 Glu Ile Val Glu Val Arg Ala Ser Gly Lys Gln Glu Lys
Met Lys His 275 280 285 Asp Ile Leu Ser Arg Phe Ile Glu Leu Gly Glu
Ala Gly Asp Asp Gly 290 295 300 Gly Gly Phe Gly Asp Asp Lys Ser Leu
Arg Asp Val Val Leu Asn Phe 305 310 315 320 Val Ile Ala Gly Arg Asp
Thr Thr Ala Thr Thr Leu Ser Trp Phe Thr 325 330 335 His Met Ala Met
Ser His Pro Asp Val Ala Glu Lys Leu Arg Arg Glu 340 345 350 Leu Cys
Ala Phe Glu Ala Glu Arg Ala Arg Glu Glu Gly Val Thr Leu 355 360 365
Val Leu Cys Gly Gly Ala Asp Ala Asp Asp Lys Ala Phe Ala Ala Arg 370
375 380 Val Ala Gln Phe Ala Gly Leu Leu Thr Tyr Asp Ser Leu Gly Lys
Leu 385 390 395 400 Val Tyr Leu His Ala Cys Val Thr Glu Thr Leu Arg
Leu Tyr Pro Ala 405 410 415 Val Pro Gln Asp Pro Lys Gly Ile Leu Glu
Asp Asp Val Leu Pro Asp 420 425 430 Gly Thr Lys Val Arg Ala Gly Gly
Met Val Thr Tyr Val Pro Tyr Ser 435 440 445 Met Gly Arg Met Glu Tyr
Asn Trp Gly Pro Asp Ala Ala Ser Phe Arg 450 455 460 Pro Glu Arg Trp
Ile Asn Glu Asp Gly Ala Phe Arg Asn Ala Ser Pro 465 470 475 480 Phe
Lys Phe Thr Ala Phe Gln Ala Gly Pro Arg Ile Cys Leu Gly Lys 485 490
495 Asp Ser Ala Tyr Leu Gln Met Lys Met Ala Leu Ala Ile Leu Phe Arg
500 505 510 Phe Tyr Ser Phe Arg Leu Leu Glu Gly His Pro Val Gln Tyr
Arg Met 515 520 525 Met Thr Ile Leu Ser Met Ala His Gly Leu Lys Val
Arg Val Ser Arg 530 535 540 Ala Val Cys His Gly Asp Leu Asp Met Asp
Ile Val Pro Leu Asn Pro 545 550 555 560 Arg Gln Ile Thr Leu Val Leu
Gln Ile Cys Met His Ala Cys Lys Gly 565 570 575 Lys Arg Trp Val Ser
Leu Val Ala Trp Leu Lys Pro 580 585 3 494 DNA Sorghum sp.
modified_base (1)..(494) "n" bases may be a, t, c, g, other or
unknown 3 ggaattcggc ttatgccgtt cacttcctac acctacatcg ctgacccggt
gaatgtcgag 60 catgtcctca agactaactt caccaattac cccaaggggg
acgtgtacag atcctacatg 120 gatgtgctcc tcggtgacgg catattcaac
gctgacggcg agctgtggag gaagcagagg 180 aagacggcga gtttcgagtt
cgcctccaag aacctgaggg atttcagtgc caatgttttc 240 agagagtact
ccctgaagct gtcgggcata ctgagtcagg catccaaggc aggcaaagtt 300
gttgacatgc aggaacttta catgaggatg acactggact cgatctgcaa ngttgggttc
360 ggggtcnana tcggcacgct gtcnccggat ctccccgaga acagcttcnc
ccaagcgttc 420 gatgccgcta acatcatcgt cacnctgcgg ttcatccacc
cnctgtggcg catccagaag 480 ttcttccccn gtca 494 4 158 PRT Sorghum sp.
MOD_RES (1)..(158) "Xaa" may be any, other or unknown amino acid 4
Met Pro Phe Thr Ser Tyr Thr Tyr Ile Ala Asp Pro Val Asn Val Glu 1 5
10 15 His Val Leu Lys Thr Asn Phe Thr Asn Tyr Pro Lys Gly Asp Val
Tyr 20 25 30 Arg Ser Tyr Met Asp Val Leu Leu Gly Asp Gly Ile Phe
Asn Ala Asp 35 40 45 Gly Glu Leu Trp Arg Lys Gln Arg Lys Thr Ala
Ser Phe Glu Phe Ala 50 55 60 Ser Lys Asn Leu Arg Asp Phe Ser Ala
Asn Val Phe Arg Glu Tyr Ser 65 70 75 80 Leu Lys Leu Ser Gly Ile Leu
Ser Gln Ala Ser Lys Ala Gly Lys Val 85 90 95 Val Asp Met Gln Glu
Leu Tyr Met Arg Met Thr Leu Asp Ser Ile Cys 100 105 110 Xaa Val Gly
Phe Gly Val Xaa Ile Gly Thr Leu Ser Pro Asp Leu Pro 115 120 125 Glu
Asn Ser Phe Xaa Gln Ala Phe Asp Ala Ala Asn Ile Ile Val Thr 130 135
140 Leu Arg Phe Ile His Pro Leu Trp Arg Ile Gln Lys Phe Phe 145 150
155 5 1092 DNA Zea mays 5 gaattccaag cgaggccctt gtagcagaga
gtgttgctga tgcagtcggc ggaaatgagt 60 gcgtgctgag agcaacgctg
aggggttcca gggatggcaa tggctatggc aatcggctag 120 aggtggagga
caaggtggtg aggattggga gggcaaccta tggcaagttg gtgaagaggc 180
acgcaatgag agatctattc agacttacac tggatgccgc caacaaattc aacctttaga
240 ttttgatact gtcactccta ctttattcct tggttgggca acttccaata
ggctcatgtt 300 aatcaatgat tagtgattat tcagcaaata ttcttgtttg
tttgacattt ataatatgtg 360 gggtgagacg gattaaatat catccatgag
agctttatct tcatgctctc ttgattttgg 420 tttcagatca ttctttcagt
gttcacaaga attttctcag tttggtccat gtaatttttg 480 aagtgaggtt
ccttaaattt cattatgctt cctttctttt ctagactagc aactgcatga 540
cttttcactt tgggttcaca aattgactca caagaaaaca aattcacttt tgggttcaca
600 aattcctctt caggatgtac ttttcacttg aactgtcatg tataggaaca
aggaatggct 660 cagtttttaa ggaacaatgt acagatttca tttcagaact
ctttctggtt ggttgagttt 720 cagacttttt gtaccaagct gatggatcac
aatacttgtt tccaaagtct gataacagaa 780 actggcaact cctaattgat
aataaaaaga ataaaataca gtatcagata tctcattttc 840 ttggttggca
gatcacaaaa aggaacacaa aggctaagcc tcctacttgt tcgggagtta 900
ggtcagggac accatatgaa tgaaagaaat cttaatttgg ggtcacacca agattgtctc
960 tctcgaggtt ggggggtccc taaggttggt agtagcaata cccaatatat
cacctaacaa 1020 acccaatcca tgctacatac atacatagca tccatcactt
gtagactgga cccttcatca 1080 agagcaccat gg 1092 6 267 DNA Zea mays 6
ccccatctca ttttcttggt tggcagatca caaaaaggaa cacaaaggct aagcctccta
60 cttgttcggg agttaggtca gggacaccat atgaatgaaa gaaatcttaa
tttggggtca 120 caccaagatt gtctctctcg aggttggggg gtccctaagg
ttggtagtag caatacccaa 180 tatatcacct aacaaaccca atccatgcta
catacataca tagcatccat cacttgtaga 240 ctggaccctt catcaagagc accatgg
267 7 3897 DNA Zea mays 7 gaattccaag cgaggccctt gtagcagaga
gtgttgctga tgcagtcggc ggaaatgagt 60 gcgtgctgag agcaacgctg
aggggttcca gggatggcaa tggctatggc aatcggctag 120 aggtggagga
caaggtggtg aggattggga gggcaaccta tggcaagttg gtgaagaggc 180
acgcaatgag agatctattc agacttacac tggatgccgc caacaaattc aacctttaga
240 ttttgatact gtcactccta ctttattcct tggttgggca acttccaata
ggctcatgtt 300 aatcaatgat tagtgattat tcagcaaata ttcttgtttg
tttgacattt ataatatgtg 360 gggtgagacg gattaaatat catccatgag
agctttatct tcatgctctc ttgattttgg 420 tttcagatca ttctttcagt
gttcacaaga attttctcag tttggtccat gtaatttttg 480 aagtgaggtt
ccttaaattt cattatgctt cctttctttt ctagactagc aactgcatga 540
cttttcactt tgggttcaca aattgactca caagaaaaca aattcacttt tgggttcaca
600 aattcctctt caggatgtac ttttcacttg aactgtcatg tataggaaca
aggaatggct 660 cagtttttaa ggaacaatgt acagatttca tttcagaact
ctttctggtt ggttgagttt 720 cagacttttt gtaccaagct gatggatcac
aatacttgtt tccaaagtct gataacagaa 780 actggcaact cctaattgat
aataaaaaga ataaaataca gtatcagata tctcattttc 840 ttggttggca
gatcacaaaa aggaacacaa aggctaagcc tcctacttgt tcgggagtta 900
ggtcagggac accatatgaa tgaaagaaat cttaatttgg ggtcacacca agattgtctc
960 tctcgaggtt ggggggtccc taaggttggt agtagcaata cccaatatat
cacctaacaa 1020 acccaatcca tgctacatac atacatagca tccatcactt
gtagactgga cccttcatca 1080 agagcaccat ggaggaagct cacatcacgc
cggcgacgcc atcgccattc ttcccactag 1140 cagggcctca caagtacatc
gcgctcctcc tggttgtcct ctcatggatc ctggtccaga 1200 ggtggagcct
gaggaagcag aaaggcccga gatcatggcc agtcatcggt gcaacggtgg 1260
agcagctgag gaactaccac cggatgcacg actggcttgt cgggtacctg tcacggcaca
1320 ggacagtgac cgtcgacatg ccgttcactt cctacaccta catcgctgac
ccggtgaatg 1380 tcgagcatgt cctcaagact aacttcacca attaccccaa
ggtaaatgac ctgaactcac 1440 tgatgttcag tcttcggaaa tcagagctga
aagctgaatc gaatgtgcct gaacaccgtg 1500 tagggaatcg tgtacagatc
ctacatggac gtgctcctcg gtgacggcat cttcaacgcc 1560 gacggcgagc
tgtggaggaa gcagaggaag acggcgagtt tcgagttcgc ctccaagaac 1620
ctgagggatt tcagcgccat tgtgttcaga gagtactccc tgaagctgtc gggtatactg
1680 agccaggcat ccaaggcagg caaagttgtg gacatgcagg tgagatcact
gctcccttgc 1740 cattgccaac atgagcattt caacctgaga cacgagagct
accttgccga ttcaggaact 1800 ttacatgagg atgacgctgg actccatctg
caaggttggg ttcggggtcg agatcggcac 1860 gctgtcgccg gatctccccg
agaacagctt cgcgcaggcg ttcgatgccg ccaacatcat 1920 cgtcacgctg
cggttcatcg acccgctgtg gcgcatcaag aggttcttcc acgtcgggtc 1980
agaggccctc ctagcgcaga gcatcaagct cgtggacgag ttcacctaca gcgtgatccg
2040 ccggaggaag gccgagatcg tcgaggcccg ggccagcggc aaacaggaga
aggtacgtgc 2100 acatgactgt ttcgattctt cagttcatcg tcttggccgg
gatggacctg atcctgattg 2160 attatatatc cgtgtgactt gtgaggacaa
attaaaatgg gcagatgaag cacgacatcc 2220
tgtcacggtt catcgagcta ggcgaggccg gcgacgacgg cggcggcttc ggggacgaca
2280 agagcctccg ggacgtggtg ctcaacttcg tgatcgccgg gcgggacacg
acggcgacga 2340 cgctgtcgtg gttcacgcac atggccatgt cccacccgga
cgtggccgag aagctgcgcc 2400 gcgagctgtg cgcgttcgag gcggagcgcg
cgcgcgagga gggcgtcgcg ctcgtgccct 2460 gcggcggcgc tgacgccgac
gacaaggcgt tcgccgcccg cgtggcgcag ttcgcgggcc 2520 tcctcaccta
cgacagcctc ggcaagctgg tctacctcca cgcctgcgtc accgagacgc 2580
tccgcctgta ccccgccgtc cctcaggtga gcgcgcccga cacgcgacct ccggtccaga
2640 gcacagcatg cagtgagtgg acctgaatgc aatgcacatg cacttgcgcg
cgcgcaggac 2700 cccaagggga tcctggagga cgacgtgctg ccggacggga
cgaaggtgag ggccggcggg 2760 atggtgacgt acgtgcccta ctcgatgggg
cggatggagt acaactgggg ccccgacgcg 2820 gcgagcttcc ggccggagcg
gtggatcaac gaggatggcg cgttccgcaa cgcgtcgccg 2880 ttcaagttca
cggcgttcca ggcggggccg aggatctgcc tgggcaagga ctcggcgtac 2940
ctgcagatga agatggcgct ggccatcctc ttgcgcttct acagcttccg gctgctggag
3000 gggcacccgg tgcagtaccg catgatgacc atcctctcca tggcgcacgg
cctcaaggtc 3060 cgcgtctcta gggccgtctg atgtcatggc gatttgggat
atcatcccgc ttaatcctta 3120 aaaatttgca tgcatgcatg taagggaaag
cgatgggttt cattggtggc ttggcttaag 3180 ccttaaaaac tccgtcgggt
cttgcgaacc accacatcac tagtgttttg tactctactc 3240 ctcagtggaa
gtgtagtgac agcatacaag ttcatcatat atattatcct ctttcttcgc 3300
cggatgcttc ccgggacctt ttggagacca ttactgacag gcgtgtgaaa aaaaggcttc
3360 ttctgcggcg aagttttggg ttcagagtct tggcgtcttt gcagcagaaa
aaaggtttgg 3420 aaggatctga accctgaacc gaaaatggct tcggaaatat
gctcgcatcg gggcggggcc 3480 gtcactcggg atgacgacaa gcccacaagc
agtgagagcg aagcgatctt tggagtttgg 3540 agacactctc ggacccctcg
gcgctccgcg agctcatctt cgcctcctct gtcgtgtccg 3600 tggcggcacc
gcgcccgccc gcctcgtgtt cgaccaaatc ccgcgccccg accggttcgt 3660
gtacaacacc ctcatccgcg gcgccgcgcg cagtgacacg ccccgggacg ccgtatacat
3720 ctataaatca tggtattgta ctttattttc aaacggcctt aacacaacca
tatttttatg 3780 gtaaacacgt tcaaaattga cacaaattta aaacaggcac
aaaccgtagc taaacataag 3840 agaatgagag acaacccaaa ggttagagat
gaaataagct gagtaaacga cgaattc 3897
* * * * *
References