Note: Descriptions are shown in the official language in which they were submitted.
CA 02392722 2002-08-15
WO 01/60997 PCT/USO1/04527
MALE TISSUE-PREFERRED REGULATORY REGION AND
METHOD OF USING SAME
FIELD OF THE INVENTION
The present invention is related to isolated DNA sequences which act as
regulatory
regions in eukaryotic cells. More specifically, the present invention is
related to isolated
DNA sequences from maize which act as male tissue-preferred regulatory regions
and play
a role in the expression of genes in male tissues. The present invention is
also directed to a
method for conferring on a gene, which may or may not be normally expressed in
male
tissues, the ability to be expressed in a male tissue-preferred manner.
BACKGROUND OF THE INVENTION
Tissue- and temporal-specific gene expression and regulation are found, inter
alia,
during sexual reproduction in eukaryotes. In plant gametogenesis, important
cytological
and biochemical changes occur during pollen development when the asymmetric
mitotic
division of the haploid microspore results in the formation of two cells; each
with different
developmental fates. The vegetative cell supports pollen growth while the
generative cell
undergoes mitosis and develops into sperm cells. Messenger RNAs specific to
both
pathways within pollen have been identified in plants such as maize, tomato,
tobacco, rice
and pansy; and messages specific to pollen or to one or more other cell types
within anther
such as tapetum, epidermis and stomium have also been identified.
Pollen gene expression during differentiation involves an estimated 24,000
genes
(Willing, et al., "An Analysis of the Quantity and Diversity of mRNA's From
Pollen and
Shoots of Zea mays"; Theor. Appl. Genet.; Vol. 75; pp. 751-753; (1988)),
however only
10% of clones from a cDNA library are male-specific (Stinson, et al., "Genes
Expressed in
the Male Gametophyte and Their Isolation"; Plant Physiol.; Vol. 83; pp. 442-
447; (1987))
and the percentage of genes expressed in pollen that are pollen-specific is
between 5% and
80% (Willing, et al., "An Analysis of the Quantity and Diversity of mRNA's
From Pollen
and Shoots of Zea mays"; Theor. Appl. Genet.; Vol. 75; pp. 751-753; (1988)).
This
complex process of microsporogenesis involves the sequential production of
many gene
products.
To date male-specific genes have been cloned from plants: two of these, the
maize
Ms45 gene (U.S. Pat. No. 5,478,369) and the Arabidopsis Ms2 gene (Mark, G.M.,
et al.,
Nature; Vol. 363; pp. 715-717; (1993)), have been shown to be required for
pollen
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-2-
development. Other examples of male-specific promoters in plants include ZM13,
PG,
SGB6, and 5126.
The Zm13 promoter is disclosed in U.S. Pat. No. 5,086,169. It consists of 1315
base pairs and is from a pollen specific gene described by Hanson, et al.,
Plant Cell; Vol. 1;
pp. 173-179; (1989). This gene hybridizes to mRNA found only in pollen.
Another pollen-specific promoter has been isolated and characterized upstream
of
the pollen-specific polygalacturonase gene (PG) U.S. Pat. No 5,412,085. This
promoter
region encompasses 2687 base pairs and is expressed predominantly in pollen
and
emergent tassel, but not in pre-emergent tassel. U.S. Pat. No. 5,545,546, also
from Allen
and Lonsdale, describes another pollen-specific promoter from the maize
polygalacturonase gene. It is only expressed in pollen and in emergent tassel.
U.S. Pat. No. 5,470,359 describes a regulatory region from the SGB6 gene of
maize
which confers tapetum specificity. The tissue of expression, the tapetum, is a
layer of cells
that surrounds the microsporogenous cells in the anther and provides nutrients
thereto.
The regulatory region of 5126 is described in U.S. Pat. No. 5,837,851. This
promoter preferentially expresses in the anther region of the plant.
Nine anther-specific cDNA and genomic clones from tobacco are described in
U.S.
Pat. No. 5,477,002. The cDNA clones were anther-specific by Northern analysis,
yet
differed in developmental profiles. Clone Ant32 is tapetal-specific.
European Pat. No. 0 420 819 Al describes the method of producing male sterile
plants with the wunl gene.
PCT WO 90/08825 describes anther-specific cDNAs TA13, TA26 and TA29 and
their use in a male sterility system.
PCT WO 90/08825 explains male-sterility genes pMS 10, pMS 14 and pMS 18 and
their use with the GUS reporter gene.
U.S. Pat. No. 5,589,610 details a promoter corresponding to anther-specific
cDNA
and anther-preferred cDNA AC444.
The use of a plant promoter and an exogenous gene to effect a change in the
genetic
make-up of plants is known in the art (U.S. Pat. Nos. 5,432,068, 5,412,085,
5,545,546,
5,470,359 and 5,478,369) These patents discuss plant expression cassettes with
a tissue-
specific promoter linked to a gene to effect male sterility, fertility or
otherwise express a
gene in a specific tissue. However, these patents do not teach the use of this
male tissue-
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-3-
preferred regulatory region or the use of this male tissue-preferred
regulatory region with
an exogenous gene as a method of controlling male sterility.
The present invention is directed to a male tissue specific regulatory region
and
methods of using the same. Expression of an exogenous gene in a male tissue-
preferred
manner can mediate male fertility and is useful in many systems such as in
hybrid seed
production.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for expressing
exogenous genes in a male tissue-preferred manner using an expression vector
that confers
male tissue-preferred expression to an exogenous gene. This process may be
used to
restore (as in a male sterility system) or to otherwise impact fertility, as
in hybrid seed
production. It is a further object of this invention to provide a DNA
regulatory region that
confers male tissue-preferred gene expression. It is also an object of this
invention to
provide a male tissue-preferred regulatory region or those with sequence
identity thereto
preferably of about 70%, 75%, or 80%, more preferably of about 85%, or 90%,
and most
preferably of about 95% or 99%.
It is an object of this invention to provide an isolated nucleic acid sequence
encoding the Ms45 male tissue-preferred regulatory regions.
It is an object of this invention to provide an isolated nucleic acid sequence
encoding an Ms45 male tissue-preferred regulatory region from Zea mays
comprising a
nucleic acid sequence shown in SEQ ID NO: 1 or those with sequence identity
thereto and
fragments thereof that retain male tissue preferred expression. It is also an
object of this
invention to provide an isolated nucleic acid sequence encoding a Ms45 male
tissue-
preferred regulatory region from Zea mays comprising a nucleic acid sequence
shown in
SEQ ID NO: 2 or those with sequence identity thereto and fragments thereof
that retain
male tissue preferred expression..
An object of the invention is to provide important or essential regulatory
regions of
the MS45 promoter which may be used in the control of male tissue preferred
expression
of a gene.
It is an object of this invention to provide a recombinant expression vector
comprising the isolated nucleic acid sequence shown in SEQ ID NO: 1, or those
with
sequence identity thereto, and fragments thereof operably linked to a
nucleotide sequence
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-4-
encoding an exogenous gene such that said nucleotide sequence is expressed in
a male
tissue-preferred manner in such a way that it promotes the expression of the
exogenous
gene.
It is an object of this invention to provide an exogenous gene, wherein said
exogenous gene is Ms45.
It is an object of this invention to provide a method of producing a
transformed
plant that expresses an exogenous nucleotide sequence in a male tissue-
preferred manner
comprising the steps of introducing into a plant said exogenous nucleotide
sequence
operably linked to a male tissue-preferred regulatory region comprising a
nucleotide
sequence which is shown at SEQ ID NO: 1 or those with sequence identity
thereto and
fragments thereof The method wherein said introduction step may be performed
by
microprojectile bombardment, may utilize Agrobacterium or a transfer vector
comprising a
Ti plasmid. Also, there may be more than one copy of said exogenous nucleotide
sequence
operably linked to a male tissue-preferred regulatory region.
It is an object of this invention to provide a method wherein said regulatory
region
expresses in a male tissue-preferred manner in tissues selected from the group
consisting of
pollen, tapetum, anther, tassel, pollen mother cells and microspores.
It is an object of this invention to provide a transformed plant expressing an
exogenous nucleotide sequence in a male tissue-preferred manner having an
exogenous
nucleotide sequence operably linked to a male tissue-preferred regulatory
region shown at
SEQ ID NO: 1 or SEQ ID NO: 2, those with sequence identity thereto and
fragments
thereof. Said plant is a monocot or a dicot. Any plant capable of being
transformed may be
used, including, for example, maize, sunflower, soybean, wheat, canola, rice
and sorghum.
This invention also provides the transformed tissue of the transformed plant.
By way of
example, the tissue may be pollen, ears, ovules, anthers, tassels, stamens
pistils and plant
cells. The transformed plant may contain more than one copy of said exogenous
nucleotide sequence operably linked to a male tissue-preferred regulatory
region.
It is an object of this invention to provide a method of mediating fertility
in a plant
wherein the male tissue-preferred regulatory region expresses said exogenous
nucleotide
sequence such that fertility is impacted. This exogenous nucleotide sequence
can be any
sequence impacting male fertility and can be, by way of example, a
complementary
nucleotidic unit encoding such antisense molecules as callase antisense RNA,
bamase
antisense RNA and chalcone synthase antisense RNA, Ms45 antisense RNA, or
ribozymes
CA 02392722 2003-03-03
75529-61(S)
and external guide sequences, or aptamers 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
5 regulatory system. Also, this exogenous nucleotide sequence
is a male sterility gene or the MS45 structural gene and
this plant is a monocot or a dicot.
It is an object of this invention to provide a
method of producing hybrid seed, comprising planting in
cross pollinating juxtaposition, a male fertile plant and a
male infertile plant produced according to the method above,
allowing said cross pollination to occur and harvesting the
resulting seed. The plants can be maize plants.
In another embodiment of the invention, it is
possible for the gene impacting male fertility to be
hemizygous dominant, that is where a single allele causes
sterility or heterozygous dominant, where there are two
alleles, with one allele causing sterility. This can be
useful in certain situations where, for example, the avidin
or streptavidin gene is the gene impacting male fertility.
In such an instance, it is desirable to provide for the
hybrid seed to segregate for fertility, in order to provide
pollen for pollination in the farmer's field.
These and other objects are achieved, in
accordance with one embodiment of the present invention by
the provision of an isolated DNA molecule wherein the DNA
molecule comprises a nucleotide sequence shown at SEQ ID NO:
1, SEQ ID NO: 2, those with sequence identity thereto and
fragments thereof that retain the male tissue preferred
expression of a gene.
CA 02392722 2008-04-29
75529-61
5a
In one aspect, there is described an isolated
nucleic acid other than SEQ ID NO: 1 or 2, comprising a
region defined by: (a) base 1155 to base 1311 of SEQ ID
NO: 1 or 2; or (b) base 1169 to base 1198 of SEQ ID NO: 1
or 2; or (c) base 1239 to base 1278 of SEQ ID NO: 1 or 2; or
(d) SEQ ID NO: 4; or (e) SEQ ID NO: 5; or (f) a sequence
which hybridizes under conditions of high stringency to any
of (a) to (e), wherein conditions of high stringency are
represented by a wash stringency of 50% formamide with 5X
Denhardt's solution, 0.5% SDS and 1X SSPE at 42 C; and
wherein the region confers to operably-linked nucleotide
sequences a preference for expression in plant male tissue.
In another aspect, there is described a plant cell
comprising a nucleic acid other than SEQ ID NO: 1 or 2, the
nucleic acid comprising a region defined by: (a) base 1155
to base 1311 of SEQ ID NO: 1 or 2; or (b) base 1169 to
base 1198 of SEQ ID NO: 1 or 2; or (c) base 1239 to
base 1278 of SEQ ID NO: 1 or 2; or (d) SEQ ID NO: 4; or (e)
SEQ ID NO: 5; or (f) a sequence which hybridizes under
conditions of high stringency to any of (a) to (e); wherein
conditions of high stringency are represented by a wash
stringency of 50% formamide with 5X Denhardt's solution,
0.5% SDS and 1X SSPE at 42 C; wherein the region confers to
operably-linked nucleotide sequences a preference for
expression in plant male tissue; and wherein the region is
in operable linkage with an exogenous promoter.
In another aspect, there is described a method of
mediating male fertility in a plant comprising introducing
into cells of the plant a nucleic acid other than SEQ ID
NO: 1 or 2, the nucleic acid comprising a region defined by:
(a) base 1155 to base 1311 of SEQ ID NO: 1 or 2; or (b)
base 1169 to base 1198 of SEQ ID NO: 1 or 2; or (c)
base 1239 to base 1278 of SEQ ID NO: 1 or 2; or (d) SEQ ID
CA 02392722 2008-04-29
75529-61
5b
NO: 4; or (e) SEQ ID NO: 5; or (f) a sequence which
hybridizes under conditions of high stringency to any of (a)
to (e); wherein conditions of high stringency are represented
by a wash stringency of 50% formamide with 5X Denhardt's
solution, 0.5% SDS and 1X SSPE at 42 C; wherein the region
confers to operably-linked nucleotide sequences a preference
for expression in plant male tissue; and wherein the region
is in operable linkage with an exogenous promoter selected
from CaMV35S, SGB6, and 5126; the nucleic acid further
comprising an exogenous gene operably linked to the
promoter; and wherein the exogenous gene and promoter
control male fertility of the plant.
In another aspect, there is described a method of
producing hybrid seeds comprising: (a) producing a first
parent plant comprising a nucleic acid operably linked to an
exogenous gene impacting male fertility of the plant such
that the plant is male sterile; (b) producing a second
parent plant which is male fertile; and (c) cross-
fertilizing the first parent plant and the second parent
plant to produce hybrid seeds; wherein the nucleic acid is
other than SEQ ID NO: 1 or 2 and comprises a region defined
by: (a) base 1155 to base 1311 of SEQ ID NO: 1 or 2; or (b)
base 1169 to base 1198 of SEQ ID NO: 1 or 2; or (c)
base 1239 to base 1278 of SEQ ID NO: 1 or 2; or (d) SEQ ID
NO: 4; or (e) SEQ ID NO: 5; or (f) a sequence which
hybridizes under conditions of high stringency to any of (a)
to (e); wherein conditions of high stringency are represented
by a wash stringency of 50% formamide with 5X Denhardt's
solution, 0.5% SDS and 1X SSPE at 42 C; wherein the region
confers to operably-linked nucleotide sequences a preference
for expression in plant male tissue; and wherein the region
is in operable linkage with an exogenous promoter.
CA 02392722 2008-08-06
75529-61
5c
In accordance with a further embodiment of the
present invention, there has been provided an expression
vector comprising an exogenous gene, wherein the expression
of the exogenous gene is under the control of a male tissue-
preferred regulatory region, and where the product of the
exogenous gene impacts male fertility.
In accordance with a further embodiment of the
present invention, there has been provided a method of using
such an expression vector to produce a male-sterile plant,
comprising the step of introducing an expression vector into
plant cells, wherein the exogenous gene of the expression
vector may be a complementary nucleotidic unit 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
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-6-
encode auxins, rol B, cytotoxins, diptheria toxin, DAM methylase, avidin,
streptavidin, or
may be selected from a prokaryotic regulatory system.
In accordance with a further embodiment of the present invention, there has
been
provided a method of using a male tissue-preferred regulatory region to
produce a male-
fertile hybrid plant comprising the steps of:
a) producing a first parent male-sterile plant comprising an expression vector
that
comprises a male tissue-preferred regulatory region and a first exogenous
gene,
wherein the male tissue-preferred regulatory region controls the expression of
the first exogenous gene, and wherein the product of the first exogenous gene
disrupts male fertility.
b) producing a second parent plant comprising an expression vector that
comprises
a second exogenous gene, wherein the regulatory region controls the expression
of the second exogenous gene so that it can be expressed in male tissues;
c) cross-fertilizing the first parent with the second parent to produce a
hybrid
plant, wherein the male tissues of the hybrid plant express the second
exogenous gene, and wherein the product of the second exogenous gene
prevents the disruption of male tissue function by the product of the first
exogenous gene, thereby producing a male-fertile hybrid plant.
In accordance with a further embodiment of the present invention, there has
been
provided a method of using a male tissue-preferred regulatory region to
produce a male-
fertile hybrid plant comprising the steps of:
a) producing a first parent male-sterile plant wherein a first gene involved
in
expression of male fertility is disrupted;
b) producing a second parent plant comprising an expression vector that
comprises
a male tissue-preferred regulatory region and an exogenous gene wherein the
male tissue-preferred regulatory region controls the expression of the
exogenous gene so that it can be expressed in male tissues and could
functionally complement the function of the gene disrupted in a);
CA 02392722 2002-08-15
WO 01/60997 PCT/USO1/04527
-7-
c) cross-fertilizing the first parent with the second parent to produce a
hybrid
plant, wherein the male tissues of the hybrid plant express the exogenous
gene,
and wherein the product of the exogenous gene prevents the disruption of the
tassel function, thereby producing a male-fertile hybrid plant.
In still another embodiment of the invention, a method or producing a hybrid
plant
producing seeds with one or more grain or seed traits of interest is provided,
such as
improved oil, starch or protein composition, including the steps of:
u) producing a first male-sterile plant comprising an expression vector that
comprises a male tissue-preferred regulatory region and a first exogenous
gene,
wherein the male tissue-preferred regulatory region controls the expression of
the first exogenous gene, and wherein the product of the first exogenous gene
disrupts male fertility;
b) producing a second plant which does not contain the first exogenous gene;
c) cross-fertilizing the first plant with the second plant to produce a hybrid
first
parent plant;
cl) producing a second parent plant comprising an expression vector that
comprises
a second exogenous gene expressing a grain or seed trait of interest;
e) cross-fertilizing the first parent plant with the second parent plant
thereby
producing a hybrid plant which produces grain having the grain or seed trait
of
interest.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a diagram of Ac 4.1 Ms45 genomic clone and restriction sites.
Figure 2 is a plasmid map of PHP6045.
Figure 3 is an autoradiogram of the primer extension products indicating the
start of
transcription of Ms45. Lanes labeled G, A, T, C, correspond to sequencing
reactions with
dideoxynucleotides ddGTP, ddATP, ddTTP, and ddCTP, respectively. Lanes 1-4
correspond to primer extension reactions with mRNA from (1) tassels, (2)
leaves, (3)
anthers, and (4) leaves.
CA 02392722 2003-03-03
75529-61(S)
-$-
Figure 4 shows an anther mRNA Northern analysis gel hybridized with the male
fertility gene Ms45.
Figure 5 is a bar graph illustrating the stage specificity of the Ms45 Male
Tissue-
Preferred Regulatory Region.
Figure 6 illustrates tissue specificity illustrated by lack of activity in non-
male
1o tissue. Figure 7 shows the results of a mutational analysis of TATA box.
Figure 8 identifies mutations introduced by linker scanning mutagenesis into
the
region upstream of the MS45 promoter.
Figure 9 is a graph representing the effect of 5' deletions (the deletion
point shown
on the x-axis) on activity of the MS45 promoter with BgIII site number.3
(CAATCCATTAA to ATGATCTATTAAA) (the y axis showing luciferase activity
normalized to GUS as a percent of the wild type full length activity). Figure
10 is a.
graph representing the effect of linker scanning mutational analysis of the
MS45 promoter,
with the linker scanning mutant of the promoter (the mutation point referred
to in Figure 8
represented on the x-axis)fused to the luciferase reporter (the y axis showing
luciferase
activity normalized to GUS as a percent of the wild type full length
activity).
DISCLOSURE OF THE INVENTION
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
herein are
standard methodologies well known to one of ordinary skill in the art. The
materials,
methods and examples are illustrative only and not limiting.
In the description that follows, a number of terms are used extensively. The
following
definitions are provided to facilitate understanding of the invention.
1. Definitions
Sequence identity or similarity, as known in the art, are relationships
between two
polypeptide sequences or two polynucleotide sequences, as detennined by
comparing the
sequences. In the art, identity also means the degree of sequence relatedness
between two
polypeptide or two polynucleotide sequences as determined by the match between
two
strings of such sequences. Both identity and similarity can be readily
calculated
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-9-
(Computational Molecular Biology; Lesk, A.M. ed.; Oxford University Press, New
York;
(1988); Biocomputing: Informatics and Genome Projects; Smith, D.W. ed.;
Academic
Press, New York; (1993); Computer Analysis of Sequence Data (Part I); Griffin,
A.M. and
H.G. Griffin eds.; Humana Press, New Jersey; (1994); von Heinje, G., Sequence
Analysis
in Molecular Biology; Academic Press; (1987); and Sequence Analysis Primer;
Gribskov,
M. and J. Devereux eds.; M Stockton Press, New York; (1991)). While there
exist a
number of methods to measure identity and similarity between two
polynucleotide or two
polypeptide sequences, both terms are well known to skilled artisans (von
Heinje, G.,
Sequence Analysis in Molecular Biologv; Academic Press; (1987); Sequence
Analysis
Primer; Gribskov, M. and J. Devereux eds.; M Stockton Press, New York; (1991);
and
Carillo, H., and D. Lipman, SIAM, J. Applied Math.; Vol. 48; pp: 1073;
(1988)). Methods
commonly employed to determine identityo or similarity between two sequences
include,
but are not limited to those disclosed in Carillo, H., and D. Lipman, SIAM J.
Applied
Math.; Vol. 48; pp. 1073; (1988). Preferred methods to determine identity are
designed to
give the largest match between the two sequences tested. Methods to determine
identity
and similarity are codified in computer programs. Preferred computer program
methods to
determine identity and similarity between two sequences include, but are not
limited to,
GCG program package (Devereux, J., et al., Nucleic Acids Research; Vol. 12(1);
pp. 387;
(1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F., et al., J. Molec. Biol.;
Vol. 215;
pp. 403; (1990)).
Male tissue consists of tissues made of collections of cells that are directly
involved
or supportive of the reproduction of the male gametes such as pollen, tapetum,
anther,
tassel, pollen mother cells and microspores. The tapetum is the tissue in the
anther in
closest contact with the pollen mother cells and microspores and is likely
involved with the
nutrition of the developing pollen grains. The pollen mother cells undergo two
meiotic
divisions to produce a tetrad of haploid microspores. Microspores undergo two
mitoses
to mature into a pollen grain. Pollen or pollen grains are mature male
gametophytes that
can have the ability to fertilize plants that are compatible. The anther is
that portion of the
stamen in which pollen is produced, the remainder of the stamen consisting of
the filament,
from which the anther depends. The stamen is the male organ of the flower.
The male tissue-preferred regulatorv region is a nucleotide sequence that
directs a
higher level of transcription of an associated gene in male tissues than in
some or all other
tissues of a plant. For example, the Ms45 gene, described herein, is detected
in anthers
CA 02392722 2002-08-15
WO 01/60997 PCT/USO1/04527
-10-
during quartet, quartet release and early uninucleate stages of development.
For details
regarding stages of anther development see Chang, M. T. and M.G. Neuffer,
"Maize
Microsporogenesis"; Genome; Vol. 32; pp. 232-244; (1989). The male tissue-
preferred
regulatory region of the Ms45 gene directs the expression of an operably
linked gene in male
tissues. The preferred tissues of expression are male, not for example, root
or coleoptile
tissue. Predominant expression is in male tissues such as, but not limited to,
pollen, tapetum,
anther, tassel, pollen mother cells and microspores. This male tissue-
preferred expression
refers to higher levels of expression in male tissues, but not necessarily to
the exclusion of
other tissues.
To mediate is to influence in a positive or negative way or to influence the
outcome, such as with fertility or any other trait.
Male fertility is impacted when non-normal fertility is experienced; this can
be as
reduced fertility or increased fertility or fertility that is different in
terms of timing or other
characteristics.
Isolated means altered "by the hand of humans" from its natural state; i.e.,
that, if it
occurs in nature, it has been changed or removed from its original
environment, or both. For
example, a naturally occurring polynucleotide or a polypeptide naturally
present in a living
organism in its natural state is not "isolated," but the same polynucleotide
or polypeptide
separated from the coexisting materials of its natural state is "isolated", as
the term is
employed herein. For example, with respect to polynucleotides, the term
isolated means that
it is separated from the chromosome and cell in which it naturally occurs. As
part of or
following isolation, such polynucleotides can be joined to other
polynucleotides, such as
DNAs, for mutagenesis, to form fusion proteins, and for propagation or
expression in a host,
for instance. The isolated polynucleotides, alone or joined to other
polynucleotides such as
vectors, can be introduced into host cells, in culture or in whole organisms.
Introduced into
host cells in culture or in whole organisms, such DNAs still would be
isolated, as the term is
used herein, because they would not be in their naturally occurring form or
environment.
Similarly, the polynucleotides and polypeptides may occur in a composition,
such as media
formulations, solutions for introduction of polynucleotides or polypeptides,
for example, into
cells, compositions or solutions for chemical or enzymatic reactions, for
instance, which are
not naturally occurring compositions, and, therein remain isolated
polynucleotides or
polypeptides within the meaning of that term as it is employed herein.
CA 02392722 2002-08-15
WO 01/60997 PCT/USOl/04527
- 1 I -
An exo2enous gene refers in the present description to a DNA sequence that is
introduced or reintroduced into an organism. For example, any gene, even the
MS45
structural gene, is considered to be an exogenous gene, if the gene is
introduced or
reintroduced into the organism.
2. Isolation of a Male Tissue-Preferred Regulatory Region
Although anther-specific promoters and genes active in male tissues are known
in
the art, (McCormick, et al., "Anther-Specific Genes: Molecular
Characterization and
Promoter Analysis in Transgenic Plants," in Plant Reproduction: From Floral
Induction to
Pollination; Lord, et al. eds.; pp. 128-135; (1989); Scott, et al.,
International Application
Publication No. WO 92/11379 (1992); van der Meer, et al., The Plant Cell; Vol.
4; pp. 253;
(1992)), there are no generally accepted principles or structural criteria for
recognizing DNA
sequences that confer male tissue expression to gene expression in maize.
Consequently, it is
not possible to isolate a male tissue-preferred regulatory region directly
from a maize
genomic library by screening for a consensus sequence that confers male tissue-
preferred
expression.
For example, hybridization of such sequences may be carried out under
conditions of
reduced stringency, medium stringency or even highly stringent conditions
(e.g., conditions
represented by a wash stringency of 35-40% Formamide with 5X Denhardt's
solution, 0.5%
SDS and 1 x SSPE at 37 C; conditions represented by a wash stringency of 40-
45%
Formamide with 5X Denhardt's solution, 0.5% SDS and 1X SSPE at 42 C; and
conditions
represented by a wash stringency of 50% Formamide with 5X Denhardt's solution,
0.5%
SDS and 1 X SSPE at 42 C, respectively). Medium stringency in a standard
hybridization of
nucleic acids would be useful in identifying the male tissue-preferred
regulatory regions
disclosed herein as well as other genes (see e.g. Sambrook, J., et al.,
Molecular Cloning: a
Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.;
(1982)). In general, sequences which code for a male tissue-preferred
regulatory region will
have sequence identity thereto of preferably 70%, 75%, or 80%, more preferably
of 85%,
or 90%, and most preferably of 95% or 99%.
Methods are readily available in the art for the hybridization of nucleic acid
sequences. Hybridization screening of plated DNA libraries (see e.g. Sambrook,
J., et al.,
Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory Press,
Cold Spring
Harbor, N.Y.; (1982)) or amplifying coding sequences using the polymerase
chain reaction
CA 02392722 2002-08-15
WO 01/60997 PCT/USO1/04527
-i2-
(see e.g. Innis, et al., PCR Protocols, a Guide to Methods and Applications;
Academic Press;
(1990)) are well known techniques for isolating genomic DNA..
Regulatory regions may be identified in the genomic subclones using functional
analysis, usually verified by the observation of reporter gene expression in
anther tissue
and the reduction or absence of reporter gene expression in non-anther tissue.
This general
approach is illustrated in Example 3, below. The possibility of the regulatory
regions
residing "upstream" or 5' ward of the transcriptional start site can be tested
by subcloning
a DNA fragment that contains the upstream region and subcloning small
fragments into
expression vectors for transient expression experiments. It is expected that
smaller
fragments nlay contain 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.
In general, sequences which code for a male tissue-preferred regulatory region
will
have sequence identity thereto of preferably 70%, 75%, or 80%, more preferably
of 85%,
or 90%, and most preferably of 95% or 99%.
Deletion analysis can occur from both the 5' and 3' ends of the regulatory
region:
fragments can be obtained by linker-scanning mutagenesis, mutagenesis using
the
polymerase chain reaction, and the like (Directed Mutagenesis: A Practical
Approach; IRL
Press; (1991)). The 3' deletions can delineate the male tissue-preferred
regulatory region and
identify the 3' end so that this 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 Ms45 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 Biotechnoloyy; 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 Ms45 core promoter.
CA 02392722 2003-03-03
75529-61(S)
- 13-
Further mutational analysis can 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.
3. Insertion of the region into an expression vector
1o 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 the
(3-
glucuronidase (GUS) gene 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 Biolo-gy and Biotechnolog y;
Glick, et al.
eds; CRC Press; pp. 89-119; (1993). Gus expression vectors and Gus gene
cassettes are
commercially available from Clonetech, Palo Alto, CA, while luciferase
expression
vectors and luciferase gene cassettes are available from Promega Corporation,
Madison,
WI. Ti plasmids and other Agrobacterium vectors are described in Ishida, Y.,
et al., Nature
Biotechnolo~y; Vol. 14; pp. 745-750; (1996) and in U.S. Pat. No. 5,591,616
Method for
Transforming Monocotyledons, filed May 3d, 1994.
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 Method for Transforming
Monocotyledons, filed May 3d, 1994, and Ishida, Y., et al., Nature
Biotechnoloev; Vol. 14;
pp. 745-750; (1996)). General methods of culturing plant tissues are found in
Gruber, et
al., "Vectors for Plant Transformation," in Methods in Plant Molecular Biology
and
Biotechnoloey; Glick, et al. eds.; CRC Press; (1993).
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% Phytagell(Sigma, St. Louis) or other
solidifying media.
The expression vector DNA is introduced within 5 hours preferably by
microprojectile-
*Trade-mark
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-14-
mediated delivery with 1.2 m particles at 1000 -1100 Psi. After DNA delivery,
the anthers
are incubated at 26 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., "Vectors for Plant Transformation," in Methods in Plant
Molecular Biology
and BiotechnoloGlick, et al. eds.; CRC Press; (1993)).
The above-described methods have been used to identify DNA sequences that
regulate gene expression in a male tissue-preferred manner. Such a region has
been
identified as the full length Ms45 male tissue-preferred regulatory region
(SEQ ID No: 1).
A TATA box mutation with sequence identity with the full length Ms45 male
tissue-
preferred regulatory region is identified in SEQ ID No: 2.
Thus, the present invention encompasses a DNA molecule having a nucleotide
sequence of SEQ ID No: 1(or those with sequence identity) and having the
function of a
male tissue-preferred regulatory region.
A putative TATA box can be identified by primer extension analysis as
described
in Example 2 below or in 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).
4. Use of a Male Tissue-Preferred Regulatory Region to Control Fertility
An object of the present invention is to provide a means to control fertility
using a
male tissue-preferred regulatory region. Importantly, this male tissue-
preferred regulatory
region can control the expression of an exogenous gene in anthers from quartet
through
early uninucleate stages of development. The practical significance of such
timing is that
the expression of a sterility-inducing gene during this developmental stage
will disrupt
anther maturation early enough to permit visual verification of the function
of the sterility-
inducing system in the field in that no anthers will be extruded. Thus, the
effects of the
sterility-inducing gene would be evident in the production field at a
sufficiently early stage
of development to allow either manual or mechanical detasseling of any
"fertile escapes"
that result from a partial or total breakdown of the sterility-inducing
system.
One approach to control male fertility is to manipulate gene expression in the
tapetum.
The tapetum is a layer of cells that surrounds sporogenous cells in the anther
and likely
provides nutrients, such as reducing sugars, amino acids and lipids to the
developing
microspores (Reznickova, C.R., Acad. Bulg. Sci.; Vol. 31; pp. 1067; (1978);
Nave, et al., J.
Plant Physiol.; Vol. 125; pp. 451; (1986); Sawhney, et al., J. Plant Physiol.;
Vol. 125; pp.
CA 02392722 2002-08-15
WO 01/60997 PCT/USOl/04527
-15-
467; (1986)). Ms45 is found to be highly expressed in the tapetal layer.
Tapetal cells also
produce 13(1,3)-glucanase ("callase") which promotes microspore release
(Mepham, et al.,
Protoplasma; Vol. 70; pp. 1; (1970)). Therefore, a delicate relationship
exists between the
tapetum and the sporogenous cells, and any disruption of tapetal function is
likely to result in
dysfunctional pollen grains. In fact, lesions in tapetal biogenesis are known
to result in male
sterility mutants (Kaul, "Male Sterility in Higher Plants" in Monographs on
Theoretical and
Applied Genetics; Frankel et al. eds.; Springer Verlag; Vol. 10; pp. 15-95;
(1988)). A
premature or late appearance of callase during the development of the tapetum
is also
associated with certain types of male sterility (Warmke, et al., J. Hered.;
Vol. 63; pp. 103;
(1972)). Therefore, the callase gene can be used to disrupt male tissue
function. Scott, et al.,
PCT WO 93/02197 (1993), discloses the nucleotide sequence of a tapetum-
specific callase.
Thus, a failure of the microspores to develop into mature pollen grains can be
induced using
a recombinant DNA molecule that comprises a gene capable of disrupting tapetal
function
under the control of tapetum-specific regulatory sequences.
One general approach to impact male fertility is to construct an expression
vector in
which the male tissue-preferred regulatory region is operably linked to a
nucleotide sequence
that encodes a protein capable of disrupting male tissue function, resulting
in infertility.
Proteins capable of disrupting male tissue function include proteins that
inhibit the synthesis
of macromolecules that are essential for cellular function, enzymes that
degrade
macromolecules that are essential for cellular function, proteins that alter
the biosynthesis or
metabolism of plant hormones, structural proteins, inappropriately expressed
proteins and
proteins that inhibit a specific function of male tissues.
For example, an expression vector can be constructed in which the male tissue-
preferred regulatory region is operably linked to a nucleotide sequence that
encodes an
inhibitor of protein synthesis, which could be but is not limited to a
cytotoxin. Diphtheria
toxin, for example, is a well-known inhibitor of protein synthesis in
eukaryotes. 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. DAM methylase, for example, is a
well
known enzyme from Escherichia coli which modifies the adenine residue in the
sequence 5'
GATC 3' to N6-methyl-adenine. Cigan and Albertsen describe how DAM methylase
could
be used to impact fertility in transgenic plants (PCT/US95/15229 Cigan, A.M.
and
CA 02392722 2002-08-15
WO 01/60997 PCT/USO1/04527
-16-
Albertsen, M.C., "Reversible Nuclear Genetic System for Male Sterility in
Transgenic
Plants"). Another example of a protein which disrupts fertility is avidin as
illustrated in U.S.
Pat. App. No. 08/475,582 " Induction of Male Sterility in Plants by Expression
of High
Levels of Avidin" by Howard, J. and Albertsen, M.C.
Alternatively, the disruption of tapetal function can be achieved using DNA
sequences
1o that encode enzymes capable of degrading a biologically important
macromolecule. For
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
amvloliquefaciens,
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-T 1, while the nucleotide sequence of the barnase gene is disclosed
in Hartley, J.
Molec. Biol.; Vol. 202; pp. 913; (1988).
RNase-T 1 and barnase genes may be obtained, for example, by synthesizing the
genes
with mutually priming long oligonucleotides. See, for example, Current
Protocols in
Molecular Biology; Ausubel, F.M., et al., eds.; John Wiley and Sons, New York;
pp. 8.2.8
to 8.2.13; (1987). Also, see Wosnick, et al., Gene; Vol. 60; pp. 115; (1987).
Moreover,
current techniques using the polymerase chain reaction provide the ability to
synthesize very
large genes (Adang, et al., Plant Molec. Biol.; Vol. 21; pp. 1131; (1993);
Bambot, et al., PCR
Methods and Applications; Vol. 2; pp. 266; (1993)).
In an alternative approach, pollen production is inhibited by altering the
metabolism of
plant hormones, such as auxins. For example, the rolB gene of AgroBacteriunz
rhizogenes
codes for an enzyme that interferes with auxin metabolism by catalyzing the
release of free
indoles from indoxyl-P-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 ro1B gene in tobacco resulted in plants having
shriveled anthers in
which pollen production was severely decreased. Therefore, 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.
In order to express a protein that disrupts male tissue funetion, an
expression vector is
constructed in which a DNA sequence encoding the protein is operably linked to
DNA
sequences that regulate gene transcription in a male tissue-preferred manner.
The general
requirements of an expression vector are described above in the context of a
transient
expression system. Here, however, the preferred mode is to introduce the
expression vector
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-17-
into plant embryonic tissue in such a manner that an exogenous protein will be
expressed at a
later stage of development in the male tissues of the adult plant. Mitotic
stability can be
achieved using plant viral vectors that provide epichromosomal replication.
An alternative and preferred method of obtaining mitotic stability is provided
by the
integration of expression vector sequences into the host chromosome. Such
mitotic stability
can be provided by the microprojectile delivery of an expression vector to
embryonic tissue
(Gruber, et al., "Vectors for Plant Transformation," in Methods in Plant
Molecular Biology
and Biotechnology; Glick, et al. eds.; CRC Press; (1993)).
Transformation methodology can be found for many plants, including but not
limited
to sunflower, soybean, wheat, canola, rice and sorghum (Knittel, N., et al.,
J. Plant Cell Rep.;
Springer International, Berlin, W. Germany; Vol. 14(2/3); pp. 81-86; (1994);
Chee, P.P., et
al., Plant Physiol.; American Society of Plant Physiologists, Rockville, MD;
Vol. 91(3); pp.
1212-1218; (1989); Hadi, M.Z., et al., J. Plant Cell Rep.; Springer
International, Berlin, W.
Germany; Vol. 15(7); pp. 500-505; (1996); Perl, A., et al., Molecular and
General Genetics;
Vol. 235(2-3); pp. 279-284; Zaghmout, O.M.F. and N.L. Trolinder, Nucleic Acids
Res.; IRL
Press, Oxford; Vol. 21(4); pp.1048; (1993); Chen, J.L. and W.D. Beversdorf,
TheorAppl.
Genet.; Springer International, Berlin, W. Germany; Vol. 88(2); pp.187-192;
(1994);
Sivamani, E., et al., Plant Cell Rep.; Springer International, Berlin, W.
Germany; Vol. 15(5);
pp. 322-327; (1996); Hagio, T., et al., Plant Cell Rep.; Vol. 10(5); pp. 260-
264; (1991)) and
are also known to those skilled in the art.
In order to select transformed cells, the expression vector contains a
selectable marker
gene, such as a herbicide resistance gene. For example, such genes may confer
resistance to
phosphinothricine, glyphosate, sulfonylureas, atrazine, imidazolinone or
kanamycin.
Although the expression vector can contain cDNA sequences encoding an
exogenous protein
under the control of a male tissue-preferred regulatory region, as well as the
selectable marker
gene under control of constitutive promoter, the selectable marker gene can
also be delivered
to host cells in a separate selection expression vector. Such a"co-
transformation" of
embryonic tissue with a test expression vector containing a male tissue-
preferred regulatory
region and a selection expression vector is illustrated below.
5. Induction of Sterility
In an alternative approach, male sterility can be induced by the use of an
expression
vector in which the male tissue-preferred regulatory region is operably linked
to a nucleotide
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-~s-
sequence that encodes a complementary nucleotidic unit. The binding of
complementary
nucleic acid molecules to a target molecule can be selected to be inhibitory.
For example, if
the target is an mRNA molecule, then binding of a complementary nucleotide
unit, in this
case an RNA, results in hybridization and in arrest of translation (Paterson,
et al., Proc. Nat'l.
Acad. Sci.; Vol. 74; pp. 4370; (1987)). Thus, a suitable antisense RNA
molecule, such as
one complementary to Ms45 (U.S. Pat. No. 5,478,369), would have a sequence
that is
complementary to that of an mRNA species encoding a protein that is necessary
for male
sterility (Fabijanski in "Antisense Gene Systems of Pollination Control For
Hybrid Seed
Production", U.S. Pat. App. No. 08/288,734).
For example, the production of callase antisense RNA would inhibit the
production of
the callase enzyme which is essential for microspore release. In addition,
male sterility can
be induced by the inhibition of flavonoid biosynthesis using an expression
vector that
produces antisense RNA for the 3' untranslated region of chalcone synthase A
gene (Van der
Meer, et al., The Plant Cell; Vol. 4; pp. 253; (1992)). The cloning and
characterization of the
chalcone synthase A gene is disclosed by Koes, et al., Gene; Vol. 81; pp. 245;
(1989), and
by Koes, et al., Plant Molec. Biol.; Vol. 12; pp. 213; (1989).
Alternatively, an expression vector can be constructed in which the male
tissue-
preferred regulatory region is operably linked to a nucleotide sequence that
encodes a
ribozyme. Ribozymes can be designed to express endonuclease activity that is
directed to a
certain target sequence in an mRNA molecule. For example, Steinecke, et al.,
EMBO J.;
Vol. 11; pp. 1525; (1992), achieved up to 100% inhibition of neomycin
phosphotransferase
gene expression by ribozymes in tobacco protoplasts. More recently, Perriman,
et al.,
Antisense Research and Development; Vol. 3; pp. 253; (1993), inhibited
chloramphenicol
acetyl transferase activity in tobacco protoplasts using a vector that
expressed a modified
hammerhead ribozyme. In the context of the present invention, appropriate
target RNA
molecules for ribozymes include mRNA species that encode proteins essential
for male
fertility, such as callase mRNA and Ms45 mRNA.
In a further alternative approach, expression vectors can be constructed in
which a
male tissue-preferred regulatory region directs the production of RNA
transcripts capable of
promoting RNase P-mediated cleavage of target mRNA molecules. According to
this
approach, an external guide sequence can be constructed for directing the
endogenous
ribozyme, RNase P, to a particular species of intracellular mRNA, which is
subsequently
cleaved by the cellular ribozyme (U.S. Pat. No. 5,168,053; Yuan, et al.,
Science; Vol. 263;
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-19-
pp. 1269; (1994)). Preferably, the external guide sequence comprises a ten to
fifteen
nucleotide sequence complementary to an mRNA species that encodes a protein
essential for
male fertility, and a 3'-RCCA nucleotide sequence, wherein R is preferably a
purine. The
external guide sequence transcripts bind to the targeted mRNA species by the
formation of
base pairs between the mRNA and the complementary external guide sequences,
thus
promoting cleavage of mRNA by RNase P at the nucleotide located at the 5'-side
of the base-
paired region.
Another alternative approach is to utilize aptamer technology, where the
complementary nucleotidic unit is a nucleotide that serves as a ligand to a
specified target
molecule (U.S. Pat. No. 5472841). This target could be a product essential for
male fertility
or a product disrupting male fertility. Using this method, an aptamer could be
selected for the
target molecule, Ms45 or avidin for example, that would bind and inhibit
expression of the
target. The nucleotide sequence encoding the aptamer would be part of
expression vectors
constructed so that a male tissue-preferred regulatory region directs the
production of the
aptamer.
Sterility can also be induced by interruption of a gene important in male
fertility such
as the Ms45 or the Ms2 gene (Mark, G.M., et al., Nature; Vol. 363; pp. 715-
717; (1993)).
Methods of gene interruption are well known in the art and include, but are
not limited to,
transposable element insertion and mutation induction.
6. Restoration of Male Fertility in the Fl Hybrid
The above-described methods can be used to produce transgenic male-sterile
maize
plants for the production of F1 hybrids in large-scale directed crosses
between inbred lines.
If the egg cells of the transgenic male-sterile plants do not all contain the
exogenous gene that
disrupts tapetal function, then a proportion of F1 hybrids will have a male-
fertile phenotype.
On the other hand, F1 hybrids will have a male-sterile phenotype if the
exogenous gene is
present in all egg cells of the transgenic male-sterile plants because
sterility induced by the
exogenous gene would be dominant. Thus, it is desirable to use a male
fertility restoration
system to provide for the production of male-fertile Fl hybrids. Such a
fertility restoration
system has particular value when the harvested product is seed or when crops
are self-
pollinating.
Also, such a fertility restoration system has particular value when the male
tissue-
preferred regulatory region is operatively linked to an inducible promoter
such as in WO
CA 02392722 2003-03-03
75529-61(S)
-20-
89110396 (Marianai, et al., Plants with Modified Stamen Cells) and the
inducible promoter is
responsive to extemal controls. This linked male tissue-preferred regulatory
region consists
of a male tissue-preferred regulatory region, an inducible promoter and an
exogenous gene.
One approach to male fertility restoration would be to cross transgenic male-
sterile
plants with transgenic male-fertile plants which contain a fertility
restoration gene under the
control of a male tissue-preferred regulatoryregion. For example, Fabijansla
in "Antisense
Gene Systems of Pollination Control For Hybrid Seed Production", U.S. Patent
No.
5,356,799, crossed male-fertile plants that expressed a barnase inhibitor,
designated
"barstar," with male-sterile plants that expressed barnase. Hartley, J. Mol.
Biol.; Vol. 202;
pp. 913; (1988), discloses the nucleotide sequence of barstar.
Another approach would be to cross male-sterile plants containing a disruption
in an
essential male fertility gene, to transgenic male fertile plants containing
the male tissue-
preferred regulatory region operably linked to a non-disrupted copy of the
fertility gene such
as Ms45 or Ms2 gene. The full sequence of the Ms45 gene is contained in U.S.
Pat. No.
5,478,369 and Ms2 in Mark, G.M., et al., Nature; Vol. 363; pp. 715-717;
(1993).
Alternatively, male fertility restoration can be achieved by expressing
complementary
nucleotidic units such as toxin ribozymes or aptamers in male-fertile plants
to neutralize the
effects of toxin in male-sterile plants. Thus, male fertility can be restored
in the Fl hybrids
by producing a male-fertile transgenic plant that synthesizes a particular
species of RNA
molecule or polypeptide to counteract the effects of the particular exogenous
gene expressed
in the male-sterile transgenic plants.
In an alternative method for restoring male fertility, transgenic male-sterile
plants
contain an expression vector having a male tissue-preferred regulatory region,
a prokaryotic
regulatory region (from a prokaryotic regulatory system), and an exogenous
gene that is
capable of disrupting tapetal function. Transgenic male-fertile plants are
produced that
express a prokaryotic peptide under the control of a male tissue-preferred
regulatory region.
In the resulting F1 hybrids from the male-sterile and male-fertile cross, the
prokaryotic
peptide binds to the prokaryotic regulatory sequence and represses the
expression of the
exogenous gene which is capable of disrupting male fertility. An advantage of
this method of
fertility restoration is that one form of transgenic male-fertile plant can be
used to provide Fl
fertility regardless of the identity of the exogenous gene that was used to
disrupt tapetal
function in the transgenic male-sterile plant.
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-21 -
For example, the LexA gene/LexA operator system can be used to regulate gene
expression pursuant to the present invention. See U.S. Pat. No. 4,833,080 and
Wang, et al.,
Mol. Cell. Biol.; Vol. 13; pp. 1805; (1993). More specifically, the expression
vector of the
male-sterile plant would contain the LexA operator sequence, while the
expression vector of
the male-fertile plant would contain the coding sequences of the LexA
repressor. In the F 1
l0 hybrid, the LexA repressor would bind to the LexA operator sequence and
inhibit
transcription of the exogenous gene that encodes a product capable of
disrupting male
fertility. These would include, but are not limited to, avidin, DAM methylase,
diptheria
toxin, RNase T, barnase, rol B and chalcone synthase A.
LexA operator DNA molecules can be obtained, for example, by synthesizing DNA
fragments that contain the well-known LexA operator sequence. See, for
example, U.S. Pat.
No. 4,833,080 and Garriga, et al., Mol. Gen. Genet.; Vol. 236; pp. 125;
(1992). The LexA
gene may be obtained by synthesizing a DNA molecule encoding the LexA
repressor. Gene
synthesis techniques are discussed above and LexA gene sequences are
described, for
example, by Garriga, et al., Mol. Gen. Genet.; Vol. 236; pp. 125; (1992).
Alternatively, DNA
molecules encoding the LexA repressor may be obtained from plasmid pRB500,
American
Type Culture Collection accession No. 67758. Those of skill in the art can
readily devise
other male fertility restoration strategies using prokaryotic regulatory
systems, such as the lac
repressor/lac operon system or the trp repressor/trp operon system.
7. Identification of Essential Parts of Regulatory Region
Identification of the essential parts of a regulatory region can be performed
by
deleting, adding and/or substituting nucleotides in a regulatory region by
methods well
known to one skilled in the art. Such variants can be obtained, for example,
by
oligonucleotide-directed mutagenesis, linker-scanning mutagenesis and
mutagenesis using
the polymerase chain reaction (Directed Mutagenesis: A Practical Approach; IRL
Press;
(1991)).
A series of 5' deletions of a regulatory region can be constructed using
existing
restriction sites. The resulting promoter fragments can be tested for activity
using an
expression vector as previously discussed. Further refinement and delineation
may be
obtained by making smaller changes, preferably of about 50 or 30 nucleotides,
more
preferably of about 20 or 10 nucleotides and most preferably of about 5 or I
nucleotides, to
the smallest restriction fragment that still confers proper expression upon
the reporter
CA 02392722 2004-11-25
75529-61 (S)
22
construct (Directed Mutagenesis: A Practical Approach; IRL
Press; (1991)). These can be introduced into the expression
vector using introduced or natural restriction sites. A
series of 3' deletions can also be generated as discussed
above or by PCR or by methods well known to one skilled in
the art (Directed Mutagenesis: A Practical Approach; IRL
Press; (1991)). Further refinement and delineation may be
obtained by making smaller changes, preferably of about 50
to 30 nucleotides, more preferably of about 20 or 10
nucleotides and most preferably of about 5 or 1 nucleotides,
to the smallest restriction fragment that still confers
proper expression upon the reporter construct (Directed
Mutagenesis: A Practical Approach; IRL Press; (1991)).
These 5' and 3' deletions therefore will delineate
the minimal region essential for mimicking the proper tissue
and temporal expression of the longer regulatory region. In
general, sequences which code for this minimal region of a
male tissue-preferred regulatory region will have sequence
identity thereto preferably of about 70%, 75%, or 80%, more
preferably of about 85%, or 90%, and most preferably of
about 95% or 99%.
Deletional analysis has demonstrated that the
functional sequences are located in the -38 to -195 region
upstream of the TATA box (nucleotide 1155 to 1312 of
SEQ ID NO: 1 or 2). Important regions in that area include
the -72 to -121 region (nucleotide 1229 to 1278 of
SEQ ID NO: 1 or 2) and -142 to -171 region (nucleotide 1208
to 1179 of SEQ ID NO: 1 or 2). An essential region is -72
to -111 (nucleotide 1239 to 1278 of SEQ ID NO: 1 or 2).
The following is presented by way of illustration
and is not intended to limit the scope of the invention.
CA 02392722 2004-11-25
75529-61(S)
22a
EXAMPLE 1
Genomic Cloning and sequencing of Ms45 promoter
The Ac tagging and identification of the Ms45
cDNA and Northern analysis is described in U.S. Pat.
No. 5,478,369.
A partial cDNA of Ms45 was used to screen a B73
maize genomic library. This library was made by cloning
SAU3A1 partials into a BAMHI digested genomic cloning
vector (Lambda Dash* II, Stratagene, La Jolla, CA).
Approximately 1x106 plaques were screened using an E. coli
strain suitable for genomic DNA (ER1647, New England
Biolabs, MA) as the host. Clone AC4.1 was purified to
homogeneity after three rounds of screening. Restriction
mapping of AC4.1 showed the clone to be about 13 kb in
length and contained two internal BAMHI sites (Figure 1).
One of these sites was also found in the Ms45 partial cDNA.
Two BAMHI fragments were subcloned to a cloning vector
(Bluescript* SK+, Stratagene, La Jolla, CA). The 5' end
clone was about 3.5 kb in length
*Trade-mark
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
_21_
and corresponded to sequence upstream (5) of the internal BAMHI site. The 3'
end clone
was 2.5kb and contained Ms45 sequence downstream of the internal BAMHI site.
Concurrently, a putative full length Ms45 cDNA was isolated and sequenced. By
sequence
comparison of the 5' end clone and the Ms45 cDNA the putative translational
start site was
identified (Figure 1).
Sequencing of the Ms45 promoter region was accomplished using the dideoxy
chain termination method of Sanger, F., et al., "DNA Sequencing with Chain
Terminating
Inhibitors"; Proc. Nat'l. Acad. Sci.; Vol. 74; pp. 5463-5467; (1977). Genomic
clone
pac4.1-5' (Figure 1) was sequenced using the universal oligo and others that
were sequence
specific using techniques well known in the art.
The male tissue-preferred regulatory region had an NCOI site introduced at the
start
codon and was cloned as an NCOI fragment into a promoterless Luci expression
vector.
This new reporter vector was designated as plasmid PHP6045 (figure 2) ATCC No:
97828 (Deposited Dec. 12, 1996; American Type Culture Collection, 12301
Parklawn Dr.,
Rockville, MD 20852).
EXAMPLE 2
Primer Extension Analysis
Total RNA was isolated from maize tassels containing quartet through early
uninucleate stage anthers. The total RNA was precipitated with ethanol and
MgCI2. One
milligram of total RNA was isolated and the poly A+ mRNA was purified by using
oligo-
dT cellulose. Poly A+ RNA was also isolated directly from 6 day old maize
seedling
leaves and maize anthers using protocols known to those skilled in the art.
A sequencing ladder was prepared using a single stranded Ms45 oligonucleotide
and incorporation of 35S-dATP in a standard sequencing procedure, using
protocols well
known to one skilled in the art.
Primer Extension was done according to the method below:
1. 5'-end labeling synthetic oligonucleotide primer.
Combined: 5 pmol primer NI 1916 (PHL11916) in1.0 gl
5 l (50 gCi) gamma 32P-ATP (>5000 Ci/mmole)
0.7 l lOX kinase buffer
0.7 l T4 polynucleotide kinase
incubated 37 C, 45 min
CA 02392722 2004-11-25
75529-61 (S)
-24-
Diluted with 20 l TE and heated to 65 C to inactivate enzyme.
lOX Kinase Buffer To make I ml
O.5M Tris-HCI, pH 7.6-8.0 0.5 ml of IM
5 mM spermidine 0.05 ml of 0.1 M
100 mM MgC12 0.1 ml of IM
100mM DTT 0.5 ml of O.SM
0.1 mg/ml gelatin or BSA 50 l of 2 mg/ml
0.1 ml water
U. Annealed primer and RNA
Kinased primers were annealed to mRNA from maize tassel, 6d maize seedling
leaves, maize anthers and 6d maize leaves. Mixed together on ice were 2 1
mRNA, 1 l
kinased oligo, 2 15X annealing buffer (1.25M KCI,10mM Tris, pH 7.9-8.15), and
I l
30 mM vanadyl. The total volume was brought to 10 l with 10mM Tris, pH 8.15.
This
mixture was heated to 65 C and cooled to 55 C for 4 hours period on
thermocycler
heating block.
llI. Primer extension
23 l primer extension mix (see recipe below) and 0.4 l reverse transcriptase
(SuperScript; BRL, MD) were added to each tube. This was mixed by gently
pipeting up-
and down and placed immediately in 48 C- and incubated 45 min. Primer
Extension Mix
consists of 10 mM MgC12, 5mM DTT, 0:33mM each dATP, dCTP, dGTP, dTTP and
DEPC water.
300 l ethanol was added and precipitated in -20C freezer overnight, then
pelleted
minutes in a microfuge. Pellets were dried in a Speed Vac and dissolved in 6
l of 0.1
30 NaOH/1 mM EDTA. Tube contents were mixed bypipetting and vortexing to
insure that
pellets were dissolved. These were left at room temp 2.5 hours, and 6 l
sequencing dye
(Stop solution from USB Sequencing kit) was-added, and the solution was
denatured at approxirnately 95 C. One half of the sample was loaded on 6%
denaturing polyacrylamide
sequencing gel with stacking buffer and run at 55 Watts for 2 hours. The gel
was dried in a
gel dryer, and exposed to Kodak X-AR film. After a three day exposure, a
transcription
product was observed in the maize tassel mRNA primer extension reaction which
corresponded to a deoxythymidine located 42 nucleotides upstream of the start
codon
(Figure 3). This position was designated as +1 (nucleotide 1350
of SEQ ID NO: 1 or 2). A minor start of transcription was also
identified at -3.
*Z`rade-mark
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-25-
EXAMPLE 3
Determination of Stage and Tissue Specificity of the Ms45 Male Tissue-
Preferred Regulatory Region
The full-length male tissue-preferred regulatory region (SEQ ID No: 1) was
fused
to the luciferase reporter gene from the firefly, Photinus pyralis, (DeWit,
T.R., et al., Proc.
Nat'l Acad. Sci. USA; Vol. 82; pp. 7870-7873; (1985)) with the PinII-3'
nontranslated
region from potato (An, G., et al., "Functional Analysis of the 3' Control
Region of the
Potato Wound-Inducible Proteinase Inhibitor II Gene"; Plant Cell; Vol. 1; pp.
115-122;
(1989)). Maize anthers at various stages of development were plated on tassel
culture
medium (Pareddy, et al., Theoret. Appl. Genet.; Vol. 77; pp. 521-526; (1989)),
solidified
with agar (Phytagar , Sigma, St. Louis). One of the three anthers from each
floret was
staged, and the remaining anthers were pooled by stage and plated for
microprojectile
bombardment, typically eight anthers per plate. The anthers were shot at 1100
p.s.i. with
1.8m tungsten particles onto which was precipitated DNA of the Ms45 male
tissue-
preferred regulatory region- luciferase reporter construct. All anthers on a
given plate were
at the same stage: premeiotic, meiosis I, meiosis II, quartet, microspore
release, early
uninucleate microspore, or mid-uninucleate microspore. Three repetitions were
shot of
each stage. Anthers were incubated overnight at 26 C for 18hr. A crude
extract was
prepared with the anthers from each plate and assayed for luciferase activity
and protein
content. The luciferase activity, normalized to protein concentration, is
graphed in Figure 5
as a function of stage of development. The major activity was at the quartet
and microspore
release stages of development, with minor activity in meiosis I and II, and
barely detectable
activity in the early uninucleate stage. No significant activity above
background was
detected in premeiotic or mid-uninucleate anthers.
In addition, embryogenic callus, cultured on MS medium containing 2.0 ug/ml of
2,4-D was bombarded in the same manner, except at 650 p.s.i. with particles
coated with a
luciferase reporter fused either to the Ms45 male tissue-preferred regulatory
region or to a
maize ubiquitin promoter (U.S. Pat. No. 5,510,474) and a uidA (GUS) reporter
fused to a
maize ubiquitin promoter. Luciferase was normalized to (3-glucuronidase. As
shown in
Figure 6, the Ms45 male tissue-preferred regulatory region was incapable of
driving
transient expression in embryogenic callus and shoots, even though the
ubiquitin promoter
was expressed. Similarly, maize seeds, imbibed and germinated in distilled
water for two
CA 02392722 2004-11-25
75529-61(S)
-26-
days and placed on wet filters, were subjected to microprojectile bombardment
and their
hypocotyls assayed for luciferase and P-glucuronidase. The ubiquitin
regulatory region
(promoter) was active, but the Ms45 male tissue-preferred regulatory region
was not.
This result is paralleled by the results of RNA hybridization analysis. Maize
anthers at various stages of development were collected and treated as
follows.. One of the,
lo three anthers from each floret was fixed in (3:1 ethanol: glacial acetic
acid) in a well of a
microtiter plate, and two were frozen in liquid nitrogen in a well at the
corresponding .
position of another microtiter plate. Fixed anthers were staged; then, the con-
esponding
frozen anthers were pooled by stage and polyA+ RNA was isolated from 20
anthers (RNA
Micro-Quick*Prep kit, Pharmacia Uppsila Sweden). Identical volumes of RNA from-
anthers at each pooled stage were subjected to electrophoresis on 1.2% agarose
in MOPS
buffer + formaldehyde. RNA satnples were transfenred by blotting to a nylon
membrane,
fixed by UV cross-linking. (Stratalinker; Stratagene Inc., La Jolla), and
hybridized to a 32P-
labeled probe fragment consisting of all of the Ms45 cDNA coding region and 3'
region.
The results shown in Figure 4 confirna steady state Ms45 transcript detectable
in quartet
through early uninucleate stages, and possibly as early as, but not earlier
than, telophase II
in meiosis. Either transcript levels resulting from Ms45 male tissue-preferred
regulatory
region activity during meiosis do not accumulate sufficiently to be detected
by RNA
hybridization, or the meiotic stage male tissue-preferred regulatory region
activity observed
in transient assays does not occur in plants.
Thus the Ms45 male tissue-preferred regulatory region (SEQ ID NO: 1) was
characterized as having male tissue-preferred expression from at least quartet
stage of
anther development through quartet release, with lower-level expression
possible in the
meiotic and early uninucleate stages.
*Trade-mark
CA 02392722 2004-11-25
75529-61(S)
26a
EXAMPLE 4
TATA Box Analysis
Within the 1388bp fragment of DNA encoding the
Ms45 male tissue-preferred regulatory region, the major
start of transcription has been identified at +1
(nucleotide 1350 of SEQ ID NO: 1 or 2), a minor start of
transcription has been identified at -3 relative to the
major start of transcription, and a putative TATA box has
been identified at -33 (CATTAAA; nucleotide 1317-1323 of
SEQ ID NO: 1). It was noted that the sequence TAAAGAT
at -30 (nucleotide 1320-1326 of SEQ ID NO: 1 or 2) could
also be a candidate for the actual TATA box. This 1388bp
fragment was operably linked to a reporter gene cassette
comprising the luciferase
CA 02392722 2004-11-25
75529-61(S)
-27-
coding region from firefly (Pareddy, et al., Theoret. Appi. Genet.; Vol. 77;
pp. 521-526;
(1989)) followed by the 3'-nontranslated region from the proteinase inhibitor
II gene of
potato. (An, G., et al., "Functional Analysis of the 3' Control Region of the
Potato
Wound-Inducible Proteinase Inhibitor 11 Gene"; Plant Cell; Vol. 1; pp. 115-
122; (1989)).
One way that is well known in the art to analyze TATA boxes is through
mutation.
In another derivative, from one to six nucleotides of the putative TATA box
were changed
in a given derivative. A BGLII site was introduced at -38 (nucleotide
position 1312 of SEQ ID NO: 1) altered the putative TATA box fran
CATTAAA to TATTAAA, which is a closer match to the canonical TATA box
sequence TATATAA.
It will be appreciated by one skilled in the art that certain substitutions
within the
TATA box may affect the level of expression of the promoter without
influencing tissue
specificity. As shown in Figure 7, the change in the TATA box associated with
the BgIII
site introduced at -38 dramatically increased transient expression levels in
anthers and
further suggests that the sequence at -33 is the authentic TATA box.
Introduction of a
BGI,II site at -40, -43, -51 or -53 (at nucleotide positions 1310, 1307,
1299 and 1297 of SBQ ID NO: 1, respectively) did not increase activity of
the pranoter (data npt sbown), providing that the irncrease observed in
the -38 BGI,II site introduction was imrelated to the BGLII site ~er se.
Other modifications of the putative TATA box were introduced to further test
for
its functionality. Alteration of the putative TATA box sequence from CATTAAA
to
GATTAAA, CATGGAA or GGGCCCA all reduced the transient expression level in
anthers, further suggesting the importance of this sequence as a TATA box.
Surprisingly,
none of these mutations abolished transient activity; however, there have been
reports of
transient activity in other systems in the absence of a TATA-like sequence and
even of
TATA-less promoters (Guan, L., and J.G. Scandalios, Plant J.; Vol. 3; pp. 527-
536;
(1993); Close, P. S., "Cloning and Molecular Characterization of Two Nuclear
Genes for
Zea rrmays Mitochondrial Chaperonin 60"; (Dissertation); Iowa State
University, Ames,
Iowa; pp. 92, 128; (1993)).
While the foregoing describes preferred embodiments of the invention, it will
be
understood by those skilled in the art that variations and modifications may
be made and
still fall within the scope of the invention.
CA 02392722 2004-11-25
75529-61 (S)
-28-
Example 5
Essential Sequences
In determining the essential region of the MS45 promoter, the methods
described in
the specification supra were used. These methods are well known to one skilled
in the art.
In general, sequences are selectively mutated or deleted and the impact on
expression then
observed.
A series of 5' deletions in the Ms45 promoter were generated from -1349 to
various existing restriction endonuclease cleavage sites to -221. (See Figure
8) 5' deletions
from -1349 to -195, -145 and -95 were generated by introduction of restriction
sites by
PCR. A series of 3' deletions, from -38 to -195, -145 and -95 were also
generated. In these
derivatives, a Bg1II cloning site was included that modified the putative TATA
box from
CATTAAA to TATTAAA, resulting in a higher level of reporter gene activity.
Linker
scanning mutations were generated by site-directed mutagenesis of the 5'
deletion
derivative to -195. Increments of 10 bp per mutant were altered along the
length of the
region upstream of the TATA box from -195 through -39, excepting the 5'-most
substitution was 14 bp and the 3'-most substitution was 13 bp. All
substitutions consisted
of G and C residues and included an Apal restriction site for ease of
identifying the desired
products of site-directed mutagenesis. Promoter derivatives were then fused to
a luciferase
reporter gene with a 3' nontranslated region from the Proteinase Inhibitor II
(PinII) gene of
Solanum tuberosum (An, G., Mitra, A., Choi, H.K., Costa, M.A., An, K.,
Thomburg, R.W.
and Ryan, C.A. 1989. "Functional Analysis of the 3' Control Region of the
Potato Wound-
Inducible Proteinase Inhibitor II Gene" Plant Cell 1: 115-122.)
Promoter activity was measured by assaying luciferase gene expression
following
microprojectile bombardment of quartet- to early-uninucleate-staged anthers
from corn.
The luciferase gene was used as the marker gene. For control, a non-deleted
Ms45
promoter:GUS:PinII-3' construct was also bombarded into anthers. Anthers were
incubated for 16 hours at 27 C, and extracts were assayed for luciferase
activity and (3-
glucuronidase (GUS) activity. Relative promoter activity in this assay is
expressed as the
luciferase activity of the mutant, normalized for the reference GUS activity,
as a percent of
luciferase activity for the full-length promoter (to -1349), also normalized
for GUS
activity. The activity of the -1349 promoter fragment was thus defined as
100%.
CA 02392722 2002-08-15
WO 01/60997 PCT/US01/04527
-29-
Results are summarized below and in Fig. No. 8 which identifies the areas of
mutation, and by the graphs of Figures No. 9 and 10, representing expression
levels.
Figure 9 shows luciferase activity normalized to GUS as a percent of the wild
type full
length sequence, with the point of deletion in the sequence upstream of the
TATA box
identified. Figure 10 also shows luciferase activity plotted against the
linker scanning
mutant of the MS45 promoter fused to the luciferase reporter. The data showed
that
significant activity was retained in the 5' deletion to -195, but that most of
the promoter
activity was lost by further deletion to -145. (See 5' deletion graph of
Figure 9). This
indicates the presence of one or more important but not absolutely essential
sequences
between -145 and -195. The 5' deletion to -95 abolished activity, indicating
that one or
more essential promoter elements are likely to be present between -95 and -
145. All of the
3' deletion derivatives generated from the MS45 promoter were inactive
indicating that at
least the region from -38 to -95 contains essential sequences. Further
mutational analysis
by linker scanning clarified the location of functional sequences in the Ms45
promoter.
Notably, the region spanned by linker scanning mutations #09, 10, 11 and 12 (-
111 through
-72 relative to the start of transcription) appears to be essential, since
each of these linker
scanning mutations abolished promoter activity. (See linker scanning graph
Figure No. 10).
Mutation of the 10 bp immediately upstream of this essential region resulted
in a
significant reduction in promoter activity, although not its elimination,
suggesting that
important sequences are present from -121 through -72. A similar reduction in
activity was
observed for linker scanning mutations #04 and 05 (and less dramatically for
#03), which
collectively span the region from -171 through -142. Mutational analysis of
the Ms45
promoter has thus identified functional sequences between -171 and -142, and
also
between -121 and -72, with an essential region between -111 and -72.
While the foregoing describes preferred embodiments of the invention, it will
be
understood by those skilled in the art that variations and modifications may
be made and
still fall within the scope of the invention.
CA 02392722 2002-08-15
WO 01/60997 30 PCT/USOl/04527
Applicant's oragent's fntemational application No.
file referertce 0578R-PCT N/A
INDICATIONS RELATING TO DEPOSITED MICROORGANISM
OR O'tHER BIOLOGICAL MATERIAL
(PCT Rule 13bis)
A. The indications made below relate to the deposited microorganism or other
biological material referred to in the description
on page 23 . Iine 17-19
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional
sheet 0
Name of depositary institution
AMERICAN TYPE CULTURE COLLECTION (ATCC)
Address of depositary institution /including postal code and country)
10801 University Blvd.
Manassas, Virginia 20110-2209
United States of America
Date of deposit Accession Number
December 12, 1996 97828
C. ADDITIONAL INDICATIONS (leave blank irnor applicable) This information is
continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iftlie indications are
not for a!l desi_qnated Statest
E. SEPARATE FURNISHING OF INDICATIONS /leave blank rf not applicable)
The indications listed beiow will be submitted to the Intemational Bureau
later rspectjvthegenerai nantre ojthe rndicartonse.g.. 'Accesston
Number of Depastt')
For receiving Office use oniv ^or lnternational Bureau use only
D/This sheet was received with the international application This sheet was
received bv the Internationai Bureau on:
V
Authorized officer Authorized officer
Y1'I' ~,,?, 77'1~i`oi ~
Form P /RO/134 (Julv 1998)
CA 02392722 2002-08-15
WO 01/60997 1 PCT/USO1/04527
SEQUENCE LISTING
<110> ALBERTSEN, MARC C.
FOX, TIMOTHY W.
GARNAAT, CARL W.
HUFFMAN, GARY
KENDALL, TIMMY L.
<120> MALE TISSUE-PREFERRED REGULATORY REGION AND METHOD OF
USING SAME
<130> 578R
<140>
<141>
<150> 08/880,499
<151> 1997-06-23
<160> 24
<170> PatentIn Ver. 2.0
<210> 1
<211> 1394
<212> DNA
<213> Zea mays
<400> 1
ccatggtgtc tctatgaaaa agatgagtac aatgtgtcta tatccgtttt cttagggtcc 60
cttcttctgc cttattactg actgaatcgg ggttacaaaa aacttccacg ggtgcatgat 120
ctccatgttc cacttctccc acctcgcgtt gcacatttct tggatgtcgg tggttcccat 180
ctgaccgagg cccatcagac acctttcggg acacccatca agggcctttc ggatggccca 240
cgagacgtat cgggtcgtgg tgatccaggg gatatatgtc ccccacaatc gtcacctata 300
ttattattct ttagatatta tttaattttt ggaaaaataa caaacttata cttttgtgta 360
gggcctcagc atagattttc gcttagggcc cagaaatgcg aggaccagcc atgtctagtg 420
tccactattg gcactaccca gaacaagatt taaaaaaata accaaagtaa ctaatccact 480
cgaaagctat catgtaatgt ttaaagaaac atctattaaa accacgatcc tcttaaaaaa 540
caagcatatt tcgaaagaga caaattatgt tacagtttac aaacatctaa gagcgacaaa 600
ttatatcgaa aggtaagcta tgacgttcag atttttcttt ttcattcttg ttattttgtt 660
attgttttta tatacatttt cttctcttac aatagagtga ttttcttccg attttataaa 720
atgactataa agtcattttt atataagagc acgcatgtcg tagattctcg ttcaaaaatc 780
tttctgattt ttttaagagc tagtttggca accctgtttc tttcaaagaa ttttgatttt 840
ttcaaaaaaa attagtttat tttctcttta taaaatagaa aacacttaga aaaatagagt 900
tgccagacta gccctagaat gttttcccaa taaattacaa tcactgtgta taattatttg 960
gccagcccca taaattattt aaaccgaaac tgaaatcgag cgaaaccaaa tctgagctat 1020
ttctctagat tagtaaaaag ggagagagag aggaagaaat cagttttaag tcattgtccc 1080
tgagatgtgc ggtttggcaa cgatagccac cgtaatcata gctcataggt gcctacgtca 1140
ggttcggcag ctctcgtgtc atctcacatg gcatactaca tgcttgttca accgttcgtc 1200
ttgttccatc gtccaagcct tgcctattct gaaccaagag gatacctact cccaaacaat 1260
ccatcttact catgcaactt ccatgcaaac acgcacatat gtttcctgaa ccaatccatt 1320
aaagatcaca acagctagcg ttctcccgct agcttccctc tctcctctgc cgatcttttt 1380
cgtccaccac catg 1394
<210> 2
<211> 1394
<212> DNA
<213> Zea mays
<400> 2
ccatggtgtc tctatgaaaa agatgagtac aatgtgtcta tatccgtttt cttagggtcc 60
CA 02392722 2002-08-15
WO 01/60997 2 PCT/US01/04527
cttcttctgc cttattactg actgaatcgg ggttacaaaa aacttccacg ggtgcatgat 120
ctccatgttc cacttctccc acctcgcgtt gcacatttct tggatgtcgg tggttcccat 180
ctgaccgagg cccatcagac acctttcggg acacccatca agggcctttc ggatggccca 240
cgagacgtat cgggtcgtgg tgatccaggg gatatatgtc ccccacaatc gtcacctata 300
ttattattct ttagatatta tttaattttt ggaaaaataa caaacttata cttttgtgta 360
gggcctcagc atagattttc gcttagggcc cagaaatgcg aggaccagcc atgtctagtg 420
tccactattg gcactaccca gaacaagatt taaaaaaata accaaagtaa ctaatccact 480
cgaaagctat catgtaatgt ttaaagaaac atctattaaa accacgatcc tcttaaaaaa 540
caagcatatt tcgaaagaga caaattatgt tacagtttac aaacatctaa gagcgacaaa 600
ttatatcgaa aggtaagcta tgacgttcag atttttcttt ttcattcttg ttattttgtt 660
attgttttta tatacatttt cttctcttac aatagagtga ttttcttccg attttataaa 720
atgactataa agtcattttt atataagagc acgcatgtcg tagattctcg ttcaaaaatc 780
tttctgattt ttttaagagc tagtttggca accctgtttc tttcaaagaa ttttgatttt 840
ttcaaaaaaa attagtttat tttctcttta taaaatagaa aacacttaga aaaatagagt 900
tgccagacta gccctagaat gttttcccaa taaattacaa tcactgtgta taattatttg 960
gccagcccca taaattattt aaaccgaaac tgaaatcgag cgaaaccaaa tctgagctat 1020
ttctctagat tagtaaaaag ggagagagag aggaagaaat cagttttaag tcattgtccc 1080
tgagatgtgc ggtttggcaa cgatagccac cgtaatcata gctcataggt gcctacgtca 1140
ggttcggcag ctctcgtgtc atctcacatg gcatactaca tgcttgttca accgttcgtc 1200
ttgttccatc gtccaagcct tgcctattct gaaccaagag gatacctact cccaaacaat 1260
ccatcttact catgcaactt ccatgcaaac acgcacatat gtttcctgaa cagatctatt 1320
aaagatcaca acagctagcg ttctcccgct agcttccctc tctcctctgc cgatcttttt 1380
cgtccaccac catg 1394
<210> 3
<211> 158
<212> DNA
<213> Zea mays
<400> 3
cgtgtcatct cacatggcat actacatgct tgttcaaccg ttcgtctttg ttccatcgtc 60
caagccttgc ctattctgaa ccaagaggat acctactccc aaacaatcca tcttactcat 120
gcaacttcca tgcaaacacg cacatatgtt tcctgaac 158
<210> 4
<211> 30
<212> DNA
<213> Zea mays
<400> 4
catgcttgtt caaccgttcg tcttgttcca 30
<210> 5
<211> 50
<212> DNA
<213> Zea mays
<400> 5
ctgaaccaag aggataccta ctcccaaaca atccatctta ctcatgcaac 50
<210> 6
<211> 40
<212> DNA
<213> Zea mays
<400> 6
aggataccta ctcccaaaca atccatctta ctcatgcaac 40
<210> 7
<211> 11
<212> DNA
CA 02392722 2002-08-15
WO 01/60997 3 PCT/USO1/04527
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Illustrative
DNA
<400> 7
caatccatta a 11
<210> 8
<211> 13
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Illustrative
DNA
<400> 8
atgatctatt aaa 13
<210> 9
<211> 255
<212> DNA
<213> Zea mays
<400> 9
gcggccgcgg atcccgtgtc atctcacatg gcatactaca tgcttgttca accgttcgtc 60
ttgttccatc gtccaagcct tgcctattct gaaccaagag gatacctact cccaaacaat 120
ccatcttact catgcaactt ccatgcaaac acgcacatat gtttcctgaa cagatctatt 180
aaagatcaca acagctagcg ttctcccgct agcttccctc tctcctctgc cgatcttttt 240
cgtccaccac catgg 255
<210> 10
<211> 14
<212> DNA
<213> Zea mays
<400> 10
gcgcgggccc gcgc 14
<210> 11
<211> 10
<212> DNA
<213> Zea mays
<400> 11
gccgggcccg 10
<210> 12
<211> 10
<212> DNA
<213> Zea mays
<400> 12
gcgcgggccc 10
<210> 13
<211> 10
<212> DNA
CA 02392722 2002-08-15
WO 01/60997 4 PCT/US01/04527
<213> Zea mays
<400> 13
gcgggcccgc 10
<210> 14
<211> 10
<212> DNA
<213> Zea mays
<400> 14
cgggcccggc 10
<210> 15
<211> 10
<212> DNA
<213> Zea mays
<400> 15
cgcgggcccg 10
<210> 16
<211> 10
<212> DNA
<213> Zea mays
<400> 16
gcgcgggccc 10
<210> 17
<211> 10
<212> DNA
<213> Zea mays
<400> 17
ggccgggccc 10
<210> 18
<211> 10
<212> DNA
<213> Zea mays
<400> 18
gccggggccc 10
<210> 19
<211> 10
<212> DNA
<213> Zea mays
<400> 19
gcgggcccgc 10
<210> 20
<211> 10
<212> DNA
<213> Zea mays
<400> 20
gcgggcccgc 10
CA 02392722 2002-08-15
WO 01/60997 5 PCT/US01/04527
<210> 21
<211> 10
<212> DNA
<213> Zea mays
<400> 21
gggcccgccg 10
<210> 22
<211> 10
<212> DNA
<213> Zea mays
<400> 22
cgggcccgcg 10
<210> 23
<211> 10
<212> DNA
<213> Zea mays
<400> 23
cgggcccgcg 10
<210> 24
<211> 13
<212> DNA
<213> Zea mays
<400> 24 -
gggcccggcc gcg 13
