Note: Descriptions are shown in the official language in which they were submitted.
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MAIZE ALTERNATIVE OXIDASE GENES AND USES THEREOF
TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More
specifically, it relates to nucleic acids and methods for modulating their
expression in
plants.
BACKGROUND OF THE INVENTION
There are several obstacles facing the development of crop varieties. These
1C~ challenges include developing varieties with greater cold tolerance;
developing varieties
with greater pathogen resistance; achieving male sterility for hybrid crops
through new
methods; overcoming limitations of selectable markers for transformation; and
engineering
protein targeting to the mitochondria.
A major determinant of hybrid seed corn performance in northern climates is
15 seedling vigor in cold soil or in the cold weather conditions of spring.
Cold soils and
weather also affect the performance of other crops such as wheat, soybeans,
rice, barley,
oats, sunflower, and rye. Improving seedling vigor in cold conditions will
increase the
seedling stand count, extend the effective growing season for a. given cold
climatic zone,
and increase yield in general. Moreover, it will create new opportunities for
the use of
2C~ higher yielding and genetically modified varieties in those regions.
Cold conditions inhibit normal respiration in plants. Cellular respiration is
vital to
aerobic life and occurs in three principal steps: a) carbohydrates, fatty
acids, and some
amino acids are oxidized to 2-carbon chemical subunits and presented in the
form of
acetyl-CoA; b) these acetyl groups enter the citric acid cycle, yielding
carbon dioxide and
25 hydrogen protons and electrons; and c) these high energy electrons cascade
down the
respiratory chain producing ATP and finally reducing oxygen 1:o form water.
The terminal
oxidase of the normal cyanide-sensitive respiration pathway is cytochrome
oxidase. This
pathway is also known simply as the cytochrome pathway. Cy~tochrome is cold-
labile, and
performs poorly in cold conditions. When normal respiration is inhibited, as
in cold
3a conditions, movement of carbohydrates through the citric acid cycle is
slowed, thereby
slowing cellular respiration and causing an accumulation of various
intermediates, such as
citric acid, which can be stressful or toxic to the cells.
Further, an inhibition of normal cellular respiration can result in an
accumulation of
free radicals, reactive oxygen species such as hydrogen peroxide, superoxide,
or hydroxyl
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radicals. These radicals can cause cellular damage by their high chemical
reactivity. They
can also dramatically change gene expression, including activation of defense
systems,
which may be stressful or toxic to the plant.
An alternative respiration pathway has been noted in bacteria, plants and some
fungi. This pathway involves an enzyme known as alternative oxidase. Increased
expression of alternative oxidase can cause elevated temperatures of plant
organs. This
increase is most dramatic in thermogenic plants that evolve heat during
flowering to
volatilized odoriferous compounds that attract insect pollinators. These
thermogenic
plants include Sauromautum guttatum, Symplocarpus foetidus, and Arum
maculaturn. For
these plants there is a massive increase in alternative oxidase mRNA and
protein which is
regulated by salicylic acid. Pathogen infected plant tissue has also been
observed to have
elevated alternative oxidase levels and increased organ temperature.
An increase in alternative oxidase protein levels is correlated with an
elevated level
of activity of the alternative pathway (Vanlerberghe, 1992a). Cold causes
elevated
expression of alternative oxidase and activation of the alternative oxidase
pathway in
plants. Species and varieties exhibiting better cold tolerance have been
observed to have
higher alternative oxidase pathway expression. For example, winter wheat has
higher
levels of expression than spring wheat. (McCaig et al., 1977) In cold-gown
tobacco, as
much as 45% of respiration is through the alternative pathway. (Vanlerberghe,
1992a)
Cold-grown maize seedlings also show increased alternative oxidase pathway
activity
(Stewart et al., 1990a). Two rice alternative oxidase genes are induced in
expression at the
level of mRNA in cold conditions. (Ito et al., 1997) The alternative oxidase
pathway is
adaptive in cold conditions because it allows the citric acid cycle and
respiration to
proceed, it alleviates chemical species stress, and it evolves heat.
Increasing the resistance of crop species to pathogen attack is another area
of
concern in the development of new crop varieties. Every year, large portions
of crops are
lost due to susceptibility to various pathogens. Increased resistance to such
pathogens
would decrease these losses.
There is increasing information relating alternative oxidase to plant
responses to
pathogen attack. This involvement centers around reactive chemical species,
such as
reactive oxygen species (ROS) like hydrogen peroxide, superoxide, and
hydroxide
radicals. Various conditions which produce ROS, such as inhibition of
cytochrome
respiration, saturation of cytochrome respiration, or normal plant responses
to pathogens,
result in activation of alternative oxidase expression. The regulation of
alternative oxidase
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expression is redox sensitive. There are reports that plant alternative
oxidase genes are
transcriptionally activated, and the proteins themselves are post-
transcriptionally activated,
by ROS stress.
There is additional data implicating alternative oxidase in plant responses to
S pathogens. It has been known for some time that salicylic acid is a chemical
inducer of
alternative oxidase expression in therrnogenic flowers. It has also been known
that
salicylic acid is an inducer of pathogenesis-related protein expression,
inducible resistance
to pathogens, and systemic acquired resistance to pathogens. It has more
recently been
demonstrated that salicylic acid treatment of tobacco leaves causes an
increase in
alternative oxidase expression and flux through the alternate respiratory
pathway.
Importantly, SHAM (salicylhydroxamic acid), which blocks alternative oxidase
activity,
also blocks induced and systemic acquired pathogen resistance (as to tobacco
mosaic virus
in tobacco). It is less clear what role it may play in resistance to bacteria
and fungi
(Chivasa, et al., 1997). Salicylate does not appear to be the determinant of
non-induced
steady-state levels of alternative oxidase, but rather it determines the
induced expression
levels (Lennon, et al., 1997). Other factors, such as ROS, may also play a
role. In
summary, alternative oxidase expression is contributing to pathogen resistance
by some
unknown mechanism.
Another challenge in the development of new crop varieties is the special
requirements involved in developing hybrid seed. The production of hybrid seed
for crop
plants involves the crossing of two parent varieties to yield a more
desirable, usually
higher-yielding, hybrid progeny. The production of this hybrid seed is costly
in terms of
both time and money. Hybrid seed corn production often involves manual or
mechanical
emasculation of the parent serving as the female and the donation of pollen to
it from the
other parent. Male sterile lines are desired because they would greatly
simplify hybrid
seed production by eliminating the need for the physical emasculation. Various
male
sterile parents have been proposed and a few implemented, with varied success.
The need
for more diverse and more effective approaches for producing male sterility is
clear.
There is evidence that mitochondria are involved in male fertility. In fact,
some
male sterility is cytoplasmically inherited by virtue of genetic abnormalities
in the
mitochondria. The Texas CMS (Cytoplasmic male sterile) is one famous example.
Some
male sterile plants involve abnormally low levels of alternative oxidase
expression and/or
activity (Connett and Hanson, 1990; Musgrave et al., I 986). The manipulation
of the
alternative oxidase pathway has been used to generate male sterility in
tobacco
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(International Patent Application WO 9b/31113). A partial sequence for an
alternative
oxidase, corresponding to ZmAOX2 in this application, has been published
(Polidoros,
A.N.. GenBank Direct Submission 30-DEC-1997, Accession AF0405b6, bases 1 to
447).
Genetic engineering of crop plants has been limited in both the variety and
volume
of genetic engineering events possible by limitations of selectable markers
during
transformation. There are relatively few selectable markers employed by plant
transformation protocols. This causes several problems. One such problem is
that some of
the markers are not cleanly selectable. Another is that with so few markers,
stacking of
multiple genetic engineering traits becomes problematic because the plant to
be
transformed with a new gene may already possess a transgene construct with the
same
selectable marker.
There is indication that alternative oxidase could be used as a selectable
marker.
For example, it has been shown that tobacco cells treated with inhibitors for
the
cytochrome respiration pathway, such as potassium cyanide, are able to survive
by virtue
of respiration from the alternative oxidase pathway, but their growth is slow.
Such
cyanide-treated tobacco cells grew faster when transformed with the
alternative oxidase
gene under the direction of the 35S promoter which causes a high (higher than
normal)
expression of the alternative oxidase gene (Vanlerberghe et al., 1997a),
presumably
because of elevated respiration carried on by the alternative pathway.
Protein targeting to the mitochondria is another area in which further
advances are
needed in the art. The alternative oxidase genes are nuclear-encoded, but the
protein is
localized to the mitochondria. The import of the protein into the mitochondria
is
dependent upon transit peptides located at the N-termini of the primary
peptide transcripts.
These transit peptides are subsequently cleaved off upon entry into the
mitochondria.
Direct N-terminal sequencing of the Sauromatum guttatum mature AOX peptide
indicates
the start site of the mature peptide (Rhoads and IVIcIntosh, 1993). This
splice-site region is
highly conserved in other plant alternative oxidases (Whelan, et al., 1995).
The mature
peptide usually starts with "XST", and the transit peptide has an arginine
residue (R) at the
minus 2 amino acid position, which is necessary for import (mutagenesis of
this arginine
inhibits import).
What is needed in the art are the alternative oxidase sequences needed to
provide a
means for using the alternative oxidase pathway to improve seedling vigor in
cold
conditions. Further, what is needed in the art are means to increase
resistance to pathogen
attack, means to generate male sterility, means to improve plant
transformation efficiency,
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new selectable markers, and means to engineer protein targeting to the
mitochondria. The
present invention provides these and other advantages.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids
and
proteins relating to Zea mays Alternative Oxidase 1, Zea mays Alternative
Oxidase 2, and
Zea mays Alternative Oxidase 3, hereby referred to as ZmAOX 1, ZmAOX2 and
ZmAOX3, respectively. It is an object of the present invention to provide
transgenic
plants comprising the nucleic acids of the present invention, and methods for
modulating,
in a transgenic plant, the expression of the nucleic acids of the present
invention.
Therefore, in one aspect the present invention relates to an isolated nucleic
acid
comprising a member selected from the group consisting of (a) a polynucleotide
having a
specified sequence identity to a polynucleotide encoding a polypeptide of the
present
invention; (b) a polynucleotide which is complementary to the polynucleotide
of (a); and,
1 S {c) a polynucleotide comprising a specified number of contiguous
nucleotides from a
polynucleotide of (a) or (b). The isolated nucleic acid can be DNA.
In other aspects the present invention relates to: 1 ) recombinant expression
cassettes, comprising a nucleic acid of the present invention operably linked
to a promoter,
2) a host cell into which has been introduced the recombinant expression
cassette, and 3) a
transgenic plant comprising the recombinant expression cassette. The host cell
and plant
are optionally a maize cell or maize plant, respectively.
Definitions
Units, prefixes, and symbols may be denoted in their SI accepted form. Unless
otherwise indicated, nucleic acids are written left to right in 5' to 3'
orientation; amino acid
sequences are written left to right in amino to carboxy orientation,
respectively. Numeric
ranges recited within the specification are inclusive of the numbers defining
the range and
include each integer within the defined range. Amino acids may be referred to
herein by
either their commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted single-letter codes.
Unless
otherwise provided for, software, electrical, and electronics terms as used
herein are as
defined in The New IEEE Standard Dictionary of Electrical and Electronics
Terms (5'"
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edition, 1993). The terms defined below are more fully defined by reference to
the
specification as a whole.
By "amplified" is meant the construction of multiple copies of a nucleic acid
sequence or multiple copies complementary to the nucleic acid sequence using
at least one
of the nucleic acid sequences as a template. Amplification systems include the
polymerase
chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid
sequence
based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase
systems,
transcription-based amplification system (TAS), and strand displacement
amplification
(SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and
Applications, D. H.
Persing et al., Ed., American Society for Microbiology, Washington, D.C.
(1993). The
product of amplification is termed an amplicon.
As used herein, "antisense orientation" includes reference to a duplex
polynucleotide sequence that is operably linked to a promoter in an
orientation where the
antisense strand is transcribed. The antisense strand is sufficiently
complementary to an
endogenous transcription product such that translation of the endogenous
transcription
product is often inhibited.
By "encoding" or "encoded", with respect to a specified nucleic acid, is meant
comprising the information for translation into the specified protein. A
nucleic acid
encoding a protein may comprise non-translated sequences (e.g., introns)
within translated
regions of the nucleic acid, or may lack such intervening non-translated
sequences (e.g., as
in cDNA). The information by which a protein is encoded is specified by the
use of
codons. Typically, the amino acid sequence is encoded by the nucleic acid
using the
"universal" genetic code. However, variants of the universal code, such as are
present in
some plant, animal, and fungal mitochondria, the bacterium Mycoplasma
capricolum, or
the ciliate Macronucleus, may be used when the nucleic acid is expressed
therein.
When the nucleic acid is prepared or altered synthetically, advantage can be
taken
of known codon preferences of the intended host where the nucleic acid is to
be expressed.
For example, although nucleic acid sequences of the present invention may be
expressed in
both monocotyledonous and dicotyledonous plant species, sequences can be
modified to
account for the specific eodon preferences and GC content preferences
ofmonocotyledons
or dicotyledons as these preferences have been shown to differ (Murray et al.
Nucl. Acids
Res. 17: 477-498 ( 1989)). Thus, the maize preferred codon for a particular
amino acid
may be derived from known gene sequences from maize. Maize codon usage for 28
genes
from maize plants is listed in Table 4 of Murray et al., supra.
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As used herein "full-length sequence" in reference to a specified
polynucleotide or
its encoded protein means having the entire amino acid sequence of, a native
(non-
synthetic), endogenous, biologically active form of the specified protein.
Methods to
determine whether a sequence is full-length are well known in the art
including such
exemplary techniques as northern or western blots, primer extension, S 1
protection, and
ribonuclease protection. See, e.g., Plant Molecular Biology: A Laboratory
Manual, Clark,
Ed., Springer-Verlag, Berlin (1997). Comparison to known full-length
homologous
(orthologous and/or paralogous) sequences can also be used to identify full-
length
sequences of the present invention. Additionally, consensus sequences
typically present at
the 5' and 3' untranslated regions of mRNA aid in the identification of a
polynucleotide as
full-length. For example, the consensus sequence ANNNNAUGG, where the
underlined
codon represents the N-terminal methionine, aids in determining whether the
polynucleotide has a complete S' end; consensus sequences at the 3' end, such
as
polyadenylation sequences, aid in determining whether it has a complete 3'
end.
The term "gene activity" refers to one or more steps involved in gene
expression,
including transcription, translation, and the functioning of the protein
encoded by the gene.
As used herein, "heterologous" in reference to a nucleic acid is a nucleic
acid that
originates from a foreign species, or, if from the same species, is
substantially modified
from its native fornn in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a heterologous
structural gene is
from a species different from that from which the structural gene was derived,
or, if from
the same species, one or both are substantially modified from their original
form. A
heterologous protein may originate from a foreign species or, if from the same
species, is
substantially modified from its original form by deliberate human
intervention.
By "host cell" is meant a cell which contains a vector and supports the
replication
and/or expression of the vector. Host cells may be prokaryotic cells such as
E. coli, or
eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.
Preferably, host
cells are monocotyledonous or dicotyledonous plant cells. A particularly
preferred
monocotyledonous host cell is a maize host cell.
The term "introduced" in the context of inserting a nucleic acid into a cell,
means
"transfection" or "transformation" or "transduction" arid includes reference
to the
incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where
the nucleic acid
may be incorporated into the genome of the cell (e.g., chromosome, plasrnid,
plastid or
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mitochondria) DNA), converted into an autonomous replicon, or transiently
expressed
(e.g., transfected mRNA).
The term "isolated" refers to material, such as a nucleic acid or a protein,
which is:
{1) substantially or essentially free from components which normally accompany
or
S interact with it as found in its natural environment. The isolated material
optionally
comprises material not found with the material in its natural environment; or
(2) if the
material is in its natural environment, the material has been synthetically
altered or
synthetically produced by deliberate human intervention and/or placed at a
different
location within the cell. The synthetic alteration or creation of the material
can be
performed on the material within or apart from its natural state. For example,
a naturally-
occurnng nucleic acid becomes an isolated nucleic acid if it is altered or
produced by non-
natural, synthetic methods, or if it is transcribed from DNA which has been
altered or
produced by non-natural, synthetic methods. See, e.g., Compounds and Methods
for Site
Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Patent No. 5,565,350; In
Vivo
Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al.,
PCT/LJS93/03868.
The isolated nucleic acid may also be produced by the synthetic re-arrangement
("shuffling") of a part or parts of one or more allelic forms of the gene of
interest.
Likewise, a naturally-occurring nucleic acid (e.g., a promoter) becomes
isolated if it is
introduced to a different locus of the genome. Nucleic acids which are
"isolated," as
defined herein, are also referred to as "heterologous" nucleic acids.
Unless otherwise stated, the term "ZmAOXl, ZmAOX2, or ZmAOX3 nucleic
acid" represents a nucleic acid of the present invention and means a nucleic
acid
comprising a polynucleotide of the present invention (a "ZmAOXl, ZmAOX2, or
ZmAOX3 polynucleotide") encoding a ZmAOXI, ZmAOX2, or ZmAOX3 polypeptide.
A "ZmAOXl, ZmAOX2, or ZmAOX3 gene" is a gene of the present invention and
refers
to a full-length ZmAOXI, ZmAOX2, or ZmAOX3 polynucleotide.
As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or
ribonucleotide polymer, or chimeras thereof, in either single- or double-
stranded form, and
unless otherwise limited, encompasses known analogues having the essential
nature of
natural nucleotides in that they hybridize to single-stranded nucleic acids in
a manner
similar to naturally occurnng nucleotides (e.g., peptide nucleic acids).
By "nucleic acid library" is meant a collection of isolated DNA or RNA
molecules
which comprise and substantially represent the entire transcribed fraction of
a genome of a
specified organism or of a tissue from that organism. Construction of
exemplary nucleic
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acid libraries, such as genomic and cDNA libraries, is taught in standard
molecular biology
references such as Berger and Kimmel, Guide to Molecular Cloning Technigues,
Methods
in Enzymology, Vol. 152, Academic Press, Inc., San Diego, CA (Berger);
Sambrook et al.,
Molecular Cloning - A Laboratory Manual, 2nd ed., Vol. 1-3 ( 1989); and
Current
Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols,
a joint
venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.
(1994).
As used herein "operably linked" includes reference to a functional linkage
between a promoter and a second sequence, wherein the promoter sequence
initiates and
mediates transcription of the DNA sequence corresponding to the second
sequence.
Generally, operably linked means that the nucleic acid sequences being linked
are
contiguous and, where necessary to join two protein coding regions, contiguous
and in the
same reading frame.
As used herein, the term "plant" includes reference to whole plants, plant
parts or
organs (e.g., leaves, stems, roots, etc.), plant cells, seeds and progeny of
same. Plant cell,
as used herein, further includes, without limitation, cells obtained from or
found in: seeds,
suspension cultures, embryos, meristematic regions, callus tissue, leaves,
roots, shoots,
gametophytes, sporophytes, pollen, and microspores. Plant cells can also be
understood to
include modified cells, such as protoplasts, obtained from the aforementioned
tissues. The
class of plants which can be used in the methods of the invention is generally
as broad as
the class of higher plants amenable to transformation techniques, including
both
monocotyledonous and dicotyledonous plants. A particularly preferred plant is
Zea mays.
As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide,
ribopolynucleotide, or chimeras or analogs thereof that have the essential
nature of a
natural deoxy- or ribo- nucleotide in that they hybridize, under stringent
hybridization
conditions, to substantially the same nucleotide sequence as naturally
occurring
nucleotides and/or allow translation into the same amino acids) as the
naturally occurring
nucleotide(s). A polynucleotide can be full-length or a subsequence of a
native or
heterologous structural or regulatory gene. Unless otherwise indicated, the
term includes
reference to the specified sequence as well as the complementary sequence
thereof. Thus,
DNAs or RNAs with backbones modified for stability or for other reasons are
"polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs
comprising
unusual bases, such as inosine, or modified bases, such as tritylated bases,
to name just two
examples, are polynucleotides as the term is used herein. It will be
appreciated that a great
variety of modifications have been made to DNA and RNA that serve many useful
purposes
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known to those of skill in the art. The term polynucleotide as it is employed
herein embraces
such chemically, enzymatically or metabolically modified forms of
polynucleotides, as well
as the chemical forms of DNA and RNA characteristic of viruses and cells,
including among
other things, simple and complex cells.
The terms "polypeptide", "peptide" and "protein" are used interchangeably
herein
to refer to a polymer of amino acid residues. The terms apply to amino acid
polymers in
which one or more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to naturally
occurring amino acid
polymers. The essential nature of such analogues of naturally occurnng amino
acids is
that, when incorporated into a protein, that protein is specifically reactive
to antibodies
elicited to the same protein but consisting entirely of naturally occurring
amino acids. The
terms "polypeptide", "peptide" and "protein" are also inclusive of
modifications including,
but not limited to, glycosylation, lipid attachment, sulfation, gamma-
carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation. Further, this
invention
contemplates the use of both the methionine-containing and the methionine-less
amino
terminal variants of the protein of the invention.
As used herein "promoter" includes reference to a region of DNA upstream from
the start of transcription and involved in recognition and binding of RNA
polymerase and
other proteins to initiate transcription. A "plant promoter" is a promoter
capable of
initiating transcription in plant cells whether or not its origin is a plant
cell. Exemplary
plant promoters include, but are not limited to, those that are obtained from
plants, plant
viruses, and bacteria which comprise genes expressed in plant cells such
Agrobacterium or
Rhizobium. Examples of promoters under developmental control include promoters
that
preferentially initiate transcription in certain tissues, such as leaves,
roots, or seeds. Such
promoters are referred to as "tissue preferred". Promoters which initiate
transcription only
in certain tissue are referred to as "tissue specific". A "cell type" specific
promoter
primarily drives expression in certain cell types in one or more organs, for
example,
vascular cells in roots or leaves. An "inducible" or "repressible" promoter is
a promoter
which is under environmental control. Examples of environmental conditions
that may
effect transcription by inducible promoters include anaerobic conditions or
the presence of
light. Tissue specific, tissue preferred, cell type specific, and inducible
promoters
represent the class of "non-constitutive" promoters. A "constitutive" promoter
is a
promoter which is active under most environmental conditions.
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The term "ZmAOXI, ZmAOX2, or ZmAOX3 polypeptide" means a polypeptide
of the present invention and refers to one or more amino acid sequences, in
glycosylated or
non-glycosylated form. The term is also inclusive of fragments, variants,
homologs,
alleles or precursors (e.g., preproproteins or proproteins) thereof. A
"ZmAOXI,
ZmAOX2, or ZmAOX3 protein" is a protein of the present invention and comprises
a
ZmAOXI, ZmAOX2, or ZmAOX3 polypeptide.
As used herein "recombinant" includes reference to a cell or vector, that has
been
modified by the introduction of a heterologous nucleic acid or that the cell
is derived from
a cell so modified. Thus, for example, recombinant cells express genes that
are not found
in identical form within the native (non-recombinant) form of the cell or
express native
genes that are otherwise abnormally expressed, under-expressed or not
expressed at all as a
result of deliberate human intervention. The term "recombinant" as used herein
does not
encompass the alteration of the cell or vector by naturally occurring events
(e.g.,
spontaneous mutation, natural transformation/transductionltramsposition) such
as those
1 S occurring without deliberate human intervention.
As used herein, a "recombinant expression cassette" is a nucleic acid
construct,
generated recombinantly or synthetically, with a series of specified nucleic
acid elements
which permit transcription of a particular nucleic acid in a host cell. The
recombinant
expression cassette can be incorporated into a plasmid, chromosome,
mitochondria) DNA,
plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant
expression
cassette portion of an expression vector includes, among other sequences, a
nucleic acid to
be transcribed, and a promoter.
The term "residue" or "amino acid residue" or "amino acid" are used
interchangeably herein to refer to an amino acid that is incorporated into a
protein,
polypeptide, or peptide (collectively "protein"). The amino acid may be a
naturally
occurring amino acid and, unless otherwise limited, may encompass non-natural
analogs of
natural amino acids that can function in a similar manner as naturally
occurring amino
acids.
The term "selectively hybridizes" includes reference to hybridization, under
stringent hybridization conditions, of a nucleic acid sequence to a 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 sequences and to the substantial
exclusion of
non-target nucleic acids. Selectively hybridizing sequences typically have
about at least
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80% sequence identity, preferably 90% sequence identity, and most preferably
100%
sequence identity (i.e., complementary) with each other.
The term "stringent conditions" or "stringent hybridization conditions"
includes
reference to conditions under which a probe will selectively 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 sequence-dependent and will be different
in
different circumstances. By controlling the stringency of the hybridization
and/or washing
conditions, target sequences can be identified which are 100% complementary to
the probe
(homologous probing). Alternatively, stringency conditions can be adjusted to
allow some
1 fl mismatching in sequences so that lower degrees of similarity are detected
(heterologous
probing). Generally, a probe is less than about 1000 nucleotides in length,
optionally less
than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration
is less
than about 1.5 M Na ion, typically about 0.01 to L0-M Na ion concentration (or
other
salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for
short probes (e.g., 1. U
to 50 nucleotides) and at least about 60°C for long probes (e.g.,
greater than 50
nucleotides). Stringent conditions may also be achieved with the addition of
destabilizing
agents such as formamide. Exemplary low stringency conditions include
hybridization
with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium
dodecyl
sulphate) at 37°C, and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCI/0.3
M trisodium
citrate) at 50 to 55°C. Exemplary moderate stringency conditions
include hybridization in
40 to 45% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.5X to IX
SSC at 55 to
60°C. Exemplary high stringency conditions include hybridization in 50%
formamide, I
M NaCI, 1% SDS at 37°C, and a wash in O.1X SSC at 60 to
65°C.
Specificity is typically the function of post-hybridization washes, the
critical
factors being the ionic strength and temperature of the final wash solution.
For DNA-
DNA hybrids, the Tm can be approximated from the equation of Meinkoth and
Wahl, Anal.
Biochem., 138:267-284 (1984): Tm = 81.5 °C + 16.6 (log M) + 0.41 (%GC) -
0.61 (%
form) - 500/L; where M is the molarity of monovalent cations, %GC is the
percentage of
guanosine and cytosine nucleotides in the DNA, % form is the percentage of
formamide in
the hybridization solution, and L is the length of the hybrid in base pairs.
The Tm is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary
target sequence hybridizes to a perfectly matched probe. Tm is reduced by
about 1°C for
each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be
adjusted
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to hybridize to sequences of the desired identity. For example, if sequences
with >90%
identity are sought, the Tm can be decreased 10°C. Generally, stringent
conditions are
selected to be about 5°C lower than the thermal melting point (Tm) for
the specific
sequence and its complement at a defined ionic strength and pH. However,
severely
stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4
°C lower than
the thermal melting point (Tm); moderately stringent conditions can utilize a
hybridization
and/or wash at 6, 7, 8, 9, or 10 °C lower than the thermal melting
point (Tm); low
stringency conditions can utilize a hybridization and/or wash at 11, 12, 13,
14, 1 S, or 20 °C
lower than the thermal melting point (Tm). Using the equation, hybridization
and wash
compositions, and desired Tm, those of ordinary skill will understand that
variations in the
stringency of hybridization and/or wash solutions are inherently described. If
the desired
degree of mismatching results in a Tn, of less than 45 °C (aqueous
solution] or 32 °C
(formamide solution) it is preferred to increase the SSC concentration so that
a higher
temperature can be used. 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).
As used herein, "transgenic plant" includes reference to a plant which
comprises
within its genome a heterologous polynucleotide. Generally, the heterologous
polynucleotide is stably integrated within the genome such that the
polynucleotide is
passed on to successive generations. The heterologous polynucleotide may be
integrated
into the genome alone or as part of a recombinant expression cassette.
"Transgenic" is used
herein to include any cell, cell line, callus, tissue, plant part or plant,
the genotype of which
has been altered by the presence of heterologous nucleic acid including those
transgenics
initially so altered as well as those created by sexual crosses or asexual
propagation from
the initial transgenic. The term "transgenic" as used herein does not
encompass the
alteration of the genome (chromosomal or extra-chromosomal) by conventional
plant
breeding methods or by naturally occurring events such as random cross-
fertilization, non-
recombinant viral infection, non-recombinant bacterial transformation, non-
recombinant
transposition, or spontaneous mutation.
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As used herein, "vector" includes reference to a nucleic acid used in
introduction of
a polynucleotide of the present invention into a host cell. Vectors are often
replicons.
Expression vectors permit transcription of a nucleic acid inserted therein.
The following terms are used to describe the sequence relationships between a
polyr~ucleotide/polypeptide of the present invention with a reference
polynucleotide/polypeptide: (a) "reference sequence", (b) "comparison window",
(c)
"sequence identity", and (d) "percentage of sequence identity".
(a) As used herein, "reference sequence" is a defined sequence used as a basis
for
sequence comparison with a polynucleotide/polypeptide of the present
invention. A
reference sequence may be a subset or the entirety of a specified sequence;
for example, as
a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene
sequence.
(b) As used herein, "comparison window" includes reference to a contiguous and
specified segment of a polynucleotide/polypeptide sequence, wherein the
polynucleotide/polypeptide sequence may be compared to a reference sequence
and
wherein the portion of the polynucleotide/polypeptide sequence in the
comparison window
may comprise additions or deletions (i.e., gaps) compared to the reference
sequence
(which does not comprise additions or deletions) for optimal alignment of the
two
sequences. Generally, the comparison window is at least 20 contiguous
nucleotides/amino
acids residues in length, and optionally can be 30, 40, 50, 100, or longer.
'those of skill in
the art understand that to avoid a high similarity to a reference sequence due
to inclusion
of gaps in the polynucleotide/polypeptide sequence, a gap penalty is typically
introduced
and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well-known in the art.
Optimal alignment of sequences for comparison may be conducted by the local
homology
algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the
homology
alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by
the
search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:
2444
(1988); by computerized implementations of these algorithms, including, but
not limited
to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View,
California;
GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin,
USA;
the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244
(1988);
Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids
Research
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16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences
8: 155-65
(1992), and Pearson, et al., Methods in Molecular-Biology 24: 307-331 (1994).
The BLAST family of programs which can be used for database similarity
searches
includes: BLASTN for nucleotide query sequences against nucleotide database
sequences;
BLAS TX for nucleotide query sequences against protein database sequences;
BLASTP for
protein query sequences against protein database sequences; TBLASTN for
protein query
sequences against nucleotide database sequences; and TBLASTX for nucleotide
query
sequences against nucleotide database sequences. See, Current Protocols in
Molecular
Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-
Interscience, New
York (1995).
Unless otherwise stated, sequence identity/similarity values provided herein
refer
to the value obtained using the BLAST 2.0 suite of programs using default
parameters.
Altschul et al., J. Mol. Biol., 215:403-410 (1990); Altschul et al., Nucleic
Acids Res:
25:?389-3402 (1997).
Software for performing BLAST analyses is publicly available, e.g., through
the
National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
This
algorithm involves first identifying high scoring sequence pairs (HSPs) by
identifying
short words of length W in the query sequence, which either match or satisfy
some
positive-valued threshold score T when aligned with a word of the same length
in a
database sequence. T is referred to as the neighborhood word score threshold.
These
initial neighborhood word hits act as seeds for initiating searches to find
longer HSPs
containing them. The word hits are then extended in both directions along each
sequence
for as far as the cumulative alignment score can be increased. Cumulative
scores are
calculated using, for nucleotide sequences, the parameters M (reward score for
a pair of
matching residues; always > 0) and N (penalty score for mismatching residues;
always <
0). For amino acid sequences, a scoring matrix is used to calculate the
cumulative score.
Extension of the word hits in each direction are halted when: the cumulative
alignment
score falls off by the quantity X from its maximum achieved value; the
cumulative score
goes to zero or below, due to the accumulation of one or more negative-scoring
residue
alignments; or the end of either sequence is reached. The BLAST algorithm
parameters
W, T, and X determine the sensitivity and speed of the alignment. The BLASTN
program
(for nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of
10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino
acid
sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an
expectation
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(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989)
Proc.
Natl. Acad. Sci. USA 89:1091 S).
In addition to calculating percent sequence identity, the BLAST algorithm also
performs a statistical analysis of the similarity between two sequences (see,
e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5877 (1993)). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(N)), which
provides
an indication of the probability by which a match between two nucleotide or
amino acid
sequences would occur by chance.
BLAST searches assume that proteins can be modeled as random sequences.
However, many real proteins comprise regions of nonrandom sequences which may
be
homopolymeric tracts, short-period repeats, or regions enriched in one or more
amino
acids. Such low-complexity regions may be aligned between unrelated proteins
even
though other regions of the protein are entirely dissimilar. A number of low-
complexity
filter programs can be employed to reduce such low-complexity alignments. For
example,
the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU
(Claverie
and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be
employed
alone or in combination.
GAP can also be used to compare a polynucleotide or polypeptide of the present
invention with a reference sequence. GAP uses the algorithm of Needleman and
Wunsch
(J. ~~lol. Biol. 48: 443-453, 1970) to find the alignment of two complete
sequences that
maximizes the number of matches and minimizes the number of gaps. GAP
considers all
possible alignments and gap positions and creates the alignment with the
largest number of
matched bases and the fewest gaps. It allows for the provision of a gap
creation penalty
and a gap extension penalty in units of matched bases. GAP must make a profit
of gap
creation penalty number of matches for each gap it inserts. If a gap extension
penalty
greater than zero is chosen, GAP must, in addition, make a profit for each gap
inserted of
the length of the gap times the gap extension penalty. Default gap creation
penalty values
and gap extension penalty values in Version 10 of the Wisconsin Genetics
Software
Package for protein sequences are 8 and 2, respectively. For nucleotide
sequences the
default gap creation penalty is SO while the default gap extension penalty is
3. The gap
creation and gap extension penalties can be expressed as an integer selected
from the
group of integers consisting of from 0 to 100. Thus, for example, the gap
creation and gap
extension penalties can each independently be: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 30, 40,
50, 60 or greater.
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GAP presents one member of the family of best alignments. There may be many
members of this family, but no other member has a better quality. GAP displays
four
figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The
Quality is the
metric maximized in order to align the sequences. Ratio is the quality divided
by the
number of bases in the shorter segment. Percent Identity is the percent of the
symbols that
actually match. Percent Similarity is the percent of the symbols that are
similar. Symbols
that are across from gaps are ignored. A similarity is scored when the scoring
matrix value
for a pair of symbols is greater than or equal to 0.50, the similarity
threshold. The scoring
matrix used in Version 10 of the Wisconsin Genetics Software Package is
BLOSUM62
(see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. U'SA 89:10915).
(c) As used herein, "sequence identity" or "identity" in the context of two
nucleic
acid or polypeptide sequences includes reference to the residues in the two
sequences
which are the same when aligned for maximum correspondence over a specified
comparison window. When percentage of sequence identity is used in reference
to
proteins it is recognized that residue positions which are not identical often
differ by
conservative amino acid substitutions, where amino acid residues are
substituted for other
amino acid residues with similar chemical properties (e.g. charge or
hydrophobicity) and
therefore do not change the functional properties of the molecule. Where
sequences differ
in conservative substitutions, the percent sequence identity may he adjusted
upwards to
correct for the conservative nature of the substitution. Sequences which
differ by such
conservative substitutions are said to have "sequence similarity" or
"similarity". Means
for making this adjustment are well-known to those of skill in the art.
Typically this
involves scoring a conservative substitution as a partial rather than a full
mismatch,
thereby increasing the percentage sequence identity. Thus, for example, where
an identical
amino acid is given a score of 1 and a non-conservative substitution is given
a score of
zero, a conservative substitution is given a score between zero and 1. The
scoring of
conservative substitutions is calculated, e.g., according to the algorithm of
Meyers and
Miller, ComputerApplic. Biol. Sci., 4: 11-17 (1988) e.g., as implemented in
the program
PC/GENE (Intelligenetics, Mountain View, California, USA).
(d) As used herein, "percentage of sequence identity" means the value
determined
by comparing two optimally aligned sequences over a comparison window, wherein
the
portion of the polynucleotide sequence in the comparison window may comprise
additions
or deletions (i.e., gaps) as compared to the reference sequence (which does
not comprise
additions or deletions) for optimal alignment of the two sequences. The
percentage is
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calculated by determining the number of positions at which the identical
nucleic acid base
or amino acid residue occurs in both sequences to yield the number of matched
positions,
dividing the number of matched positions by the total number of positions in
the window
of comparison and multiplying the result by 100 to yield the percentage of
sequence
identity.
DETAILED DESCRIPTION OF THE INVENTION
Overview
The present invention provides, among other things, compositions and methods
for
modulating (i.e., increasing or decreasing) the level of polynucleotides and
polypeptides of
the present invention in plants. In particular, the polynucleotides and
polypeptides of the
present invention can be expressed temporally or spatially, e.g., at
developmental stages, in
tissues, and/or in quantities, which are uncharacteristic of non-recombinantly
engineered
plants. Thus, the present invention provides utility in such exemplary
applications as
enhancing cold tolerance in plants, enhancing disease resistance in plants,
achieving male
sterility in plants, developing selectable genetic markers for transgenic
plant production,
and engineering protein targeting to the mitochondria.
Cold Tolerance
The present invention can be used to enhance cold tolerance in plants. The
coding
regions for any of the three maize alternative oxidase genes will be expressed
in a
transgenic plant, such as maize, under the direction of tissue-preferred and
developmental
promoters that cause high level expression in seedlings. Two such promoters
are those for
the GA-regulated alpha-amylase genes and that of a cysteine protease, which
cause high-
level expression in the seed (scutellum and aleurone). A third promoter, that
for the beta-
glucosidase (Glul ) gene, causes high-level expression in the seedling
mesocotyl,
coleoptile, and young leaves. Those of skill in the art will recognize that
other promoters
are possible. Upon germination, these plants have elevated levels of
alternative oxidase
and enhanced cold tolerance, which will be manifested as better, more complete
stands
when germinated in cold soil or cold weather conditions.
Alternatively, a modified coding region of any of the alternative oxidase
genes to
produce an alternative oxidase with higher intrinsic activity can be used in
the same
process described above. This is accomplished using a directed protein
engineering
strategy involving the alteration of one or both of the cysteine residues at
amino acid
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positions 120 and 170 in ZmAOX 1 and 102 and 152 in ZxnAOX2 with a non-
sulfhydryl-
containing amino acid such as serine. These cysteine residues are responsible
for
dimerization of alternative oxidase. The dimeric form is less active than the
monomeric
form, and also less susceptible to pyruvate stimulation. Upon reintroduction
of the
modified alternative oxidase gene, there will be enhanced alternative oxidase
activity and
enhanced cold tolerance. Since the enzyme will be intrinsically more active,
other
seedling preferred/specific promoters can be used that need not have such high
levels of
expression.
Another option for modifying either of the alternative oxidase genes to
produce an
i 0 alternative oxidase with higher intrinsic activity is using a directed
protein engineering
strategy involving sequence shuffling of the maize alternative oxidases along
with coding
regions or synthetic oligonucleotides for other plant and non-plant
alternative oxidases.
Increased activity may be first assessed by complementing microbes, such as E.
toll or
yeast, that are mutated in their own alternative oxidase, with the modified
alternative
oxidases. Those complemented with more active alternative oxidases will be
expected to .
grow more actively in vitro. The recovered clones are then reiraoduced into
the plant.
Once again, since the enzyme will be more active. other seedling
preferred/specific
promoters pan be us-~d which need not have such a high level of expression.
Enhancing Disease Resistance
The present invention can also be used to enhance disease resistance in
plants. Any
of the three maize alternative oxidase sequences can be used to enhance
disease resistance
in plants to various pathogens. An exemplary plant is maize. Exemplary
pathogens
include, but are not limited to, viral pathogens.
In preferred embodiments, this invention enhances inducible resistance to
pathogens, but it can be used in constitutive resistance mechanisms as well.
Inducible
resistance mechanisms involve enhancing the level and timing of alternative
oxidase
expression following pathogen attack in order to increase expression and thus
resistance.
The present invention can be used in any number of ways to accomplish this
goal.
A. Native ZmAOX
The coding regions for any of the maize alternative oxidase genes can be
expressed
in a transgenic plant under the direction of pathogen-inducible promoters that
cause
elevated expression (where possible, highly elevated expression) following
pathogen
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attack. Maize is a particularly preferred transgenic plant. Exemplary
inducible promoters
include inducible maize promoters as disclosed in U.S. Patent Application No.
09/257;583
entitled "Inducible Maize Promoters", filed Feb. 25, 1999. Those of skill will
recognize
other promoters are possible, including promoters having some tissue or
developmental
preference to their expression that would focus their inducibility to tissues
or stages
particularly susceptible to the target pathogen. Upon pathogen attack, these
transgenic
plants have elevated levels of alternative oxidase and enhanced disease
resistance.
B. Site-Specific Engineered ZmAOX
Alternatively, the coding region of any of the alternative oxidase genes can
be
modified to produce an alternative oxidase with higher intrinsic activity.
This is
accomplished via a directed protein engineering strategy involving alteration
of, for
example, one or both of the cysteine residues at amino acid positions 120 and
170 in
ZmAOXI and 102 and 152 in ZmAOX2, with a non-sulfhydryl-containing amino acid
such as serine. These cysteine residues aid in the dimerization of alternative
oxidase. The
:iimeric foirn is less active than the monomeric form, and also less
susceptible to pyruvate
'tirrmlation. Upon reintroduction.of the modified alternative oxidase gene in
the manner
described above, there is enhanced alternative oxidase activity and enhanced
disease
resistance. As the enzyme is more active, other tissue preferred/specific
promoters can be
used which need not have as high a level of expression.
C Seguence-Shuffled ZmAOX
Alternatively, the coding region of either of the alternative oxidase genes
can be
modified to produce an alternative oxidase with higher intrinsic activity.
This is
accomplished via a directed protein engineering strategy involving sequence
shuffling of
these two maize alternative oxidases along with coding regions or synthetic
oligonucleotides for other plant and non-plant alternative oxidases. Increased
activity can
be first assessed by complementing microbes, such as E. coli or yeast, that
are mutated in
their own alternative oxidase, with the modified maize alternative oxidases.
Those
complemented with more active alternative oxidases will grow more actively in
vitro. The
recovered clones are then reintroduced into the plant as discussed previously.
The enzyme
will be more active, therefore, other tissue preferredlspecific promoters can
be used which
need not have as high a level of expression.
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Using coding regions for the native, site-specific engineered, or sequence
shuffled
versions of the maize alternative oxidases, a non-inducible resistance is
achieved by
driving their expression with a promoter that gives appropriate levels of
alternative oxidase
expression in the desired tissue and/or developmental stages.
Male Sterility
The present invention provides methods for the creation of transgenic male
sterile
plants for the purpose of creating hybrid seed. The coding region for one of
the maize
alternative oxidase genes described herein is used.
A. Mutugenesis
Any of the maize alternative oxidase genes can be mutagenized so as to be
nonfunctional. One method which can be employed to achieve this is transposon
insertional mutagenesis. Those of skill in the art will recognize that there
are several other
procedures,whieh can also be employed. Where one or more of the maize
alternative
oxidase genes are normally expressed in the tassel, mutagenesis may result in
male
sterility. This can be useful in creating hybrids; the male parent will donate
a functional
copy of the gene so that the resulting hybrid plant is fertile.
Ft. Antisense
Any of these maize alternative oxidase genes can be expressed in an antisense
configuration under the direction of a tassel-specific promoter. Male
sterility results when
the alternative oxidase expression in the tassel is sufficiently reduced. This
strategy
requires a restorer gene in the hybrid plant to counteract the antisense
expression
suppression.
Selectable Genetic Markers for Transgenic Plant Production
Alternative oxidase can be used as a selectable marker. For example, it has
been
shown that tobacco cells treated with inhibitors for the cytochrome
respiration pathway,
such as potassium cyanide, are able to survive by virtue of respiration from
the alternative
oxidase pathway, but growth is at a reduced rate. Such cyanide-treated tobacco
cells grew
faster when transformed with the alternative oxidase gene under the direction
of the 35S
promoter which causes a high (higher than normal) expression of the
alternative oxidase
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gene (Vanlerberghe et al., 1997a), because of elevated respiration earned on
by the
alternative pathway.
Alternative oxidase genes, in particular the maize genes presented herein, can
be
used as selectable markers. In this invention, one or more of the ZmAOX genes
can be
cotransformed with the target transformation gene. The ZmAOX gene would be
under the
direction of a highly active promoter. The more rapidly growing cells, tissue,
or callus
would be subcultured, as they represent the successfully transformed tissue.
Ideally, the
promoter would be preferentially active during early developmental or
culturing stages,
and have less impact later in development, or even in succeeding generations.
There are several known inhibitors of alternative oxidase, among which are
SHAM
(salicylhydroxamic acid) and Disulfiram. It is possible to create a variant of
the maize
alternative oxidase genes that is resistant to these inhibitors. This variant
can be created by
either: a) directed protein engineering to modify specific sites or structure
involved in the
susceptibility to these inhibitors; b) sequence shuffling to create such
resistance versions,
1 ~ followed by selection for gain of resistance in E. coli or yeast mutants
lacking alternative
oxidase function; or c) more conventional mutagenesis, as by EMS, etc.,
followed by
selection for gain of resistance in E. coli or yeast mutants lacking
alternative oxidase
function. The resistant variant, under the direction of a fairly strong
constitutive promoter,
although the native ZmAOX promoter may suffice and be advantageous for later
in
development, is then reintroduced into the plant during cotransformation with
the
transforming gene of interest. The selection medium would contain the
inhibitor (as
SHAM or disulfiram), and cells which grow are those transformed with the gene
of interest
and linked to the alternative oxidase selectable marker construct. This
selection is even
more effective with the addition of SHAM or disulfuran to inhibit the
alternative pathway
plus an inhibitor of the cytochrome pathway, such as cyanide or antimycin A.
In these
conditions, respiration only occurs via the inhibitor-resistant alternative
oxidase, and only
those cells transformed with it survive.
In addition to its role as a potential selectable marker, the alternative
oxidase
transformation strategies outlined above are additionally useful for enhancing
transformation frequency. The alternative oxidase pathway is more active
following
stresses. Particle bombardment, a commonly used transformation technique, is
undoubtedly stressful on the tissue. The alternative pathway is more active in
bombarded
tissues. This increased activity is adaptive in that it contributes to
reduction of reactive
oxygen species that accumulate following stresses such as bombardment and
cause
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damage to the cells and decrease transformation efficiency. Increasing the
alternative
oxidase pathway following transformation, as by one of the methods above, or
by
transiently elevated expression of the gene's coding region by including it in
DNA or RNA
form on the transforming particles, thus enhances cell survival rates, and
hence increases
transformation frequency.
Engineering Protein Tareetin~ to the Mitochondria
The present invention provides transit peptides for the alternative oxidase
genes.
The transit peptides direct these and other proteins to the mitochondria. As
the transit
peptides are a chief determinant for direction of proteins to the
mitochondria, the transit
peptide coding region for any of the genes of the present invention can be
used to
genetically engineer direction of other proteins to the mitochondria. This is
achieved by
fusing the transit peptide coding region to the N-terminal end of the protein
destined to the
mitochondria. Upon translation, the chimeric protein is directed to the
mitochondria, and
upon entry, the transit peptide is proteolytically removed by proteases
present in the
mitochondria, and the liberated protein then resides and functions in the
mitochondria.
Depending upon the protein involved, such engineering of protein targeting to
the
mitochondria is useful in various areas including, but not limited to,
herbicide resistance,
transformation selectable markers, metabolite production, male sterility (and
restoration of
fertility), cell cycle control, and apoptosis. Regarding use in selectable
markers, it should
be noted that many antimicrobial/antiprokaryote compounds/drugs are known.
Many of
these affect prokaryotic translation. The plant mitochondria and chloroplasts
are relatively
prokaryotic-like in their translational apparatus, as they were apparently
incorporated by a
primitive eukaryotic cell via endosymbiosis. The mitochondrial transit peptide
from the
alternative oxidase genes can be used to direct genes encoding resistance to
such drugs to
the mitochondria. As such, they can be used for selectable markers in plant
transformation.
It should be noted that although the transit peptide may alone be able to
direct
proteins to the mitochondria, no single transit peptide will direct all
proteins to the
mitochondria. Other factors, such as the structure of the mature peptide
region, will render
the transit peptides more or less able to direct the protein to the
mitochondria. Moreover, a
comparison of even related AOX proteins, as these alternative oxidases from
various plant
species are, indicates that there are divergent transit peptide sequences both
within and
between species.
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The present invention also provides isolated nucleic acids comprising
polynucleotides of sufficient length and complementarity to a gene of the
present invention
to use as probes or amplification primers in the detection, quantitation, or
isolation of gene
transcripts. For example, isolated nucleic acids of the present invention can
be used as
probes in detecting deficiencies in the level of mRNA in screenings for
desired transgenic
plants, for detecting mutations in the gene (e.g., substitutions, deletions,
or additions), for
monitoring upregulation of expression or changes in enzyme activity in
screening assays
of compounds, for detection of any number of allelic variants (polymorphisms),
orthologs,
or paralogs of the gene, or for site directed mutagenesis in eukaryotic cells
(see, e.g., U.S.
Patent No. 5,565,350). The isolated nucleic acids of the present invention can
also be used
for recombinant expression of their encoded polypeptides, or for use as
immunogens in the
preparation and/or screening of antibodies. The isolated nucleic. acids of the
present
invention can also be employed for use in sense or antisense suppression of
one or more
genes of the present invention in a host cell, tissue, or plant. Attachment of
chemical
agents which bind, intercalate, cleave and/or crosslink to the isolated
nucleic acids of the
present invention can also be used to modulate transcription or translation.
The present invention also provides isolated proteins comprising a polypeptide
of
the present invention (e.g., preproenzyme, proenzyme, or enzymes). The present
invention
also provides proteins comprising at least one epitope from a polypeptide of
the present
invention. The proteins of the present invention can be employed in assays for
enzyme
agonists or antagonists of enzyme function, or for use as immunogens or
antigens to obtain
antibodies specifically immunoreactive with a protein of the present
invention. Such
antibodies can be used in assays for expression levels, for identifying and/or
isolating
nucleic acids of the present invention from expression libraries, for
identification of
homologous polypeptides from other species, or for purification of
polypeptides of the
present invention.
The isolated nucleic acids and polypeptides of the present invention can be
used
over a broad range of plant types, particularly monocots such as the species
of the family
Gramineae including Hordeum, Secale, Triticum, Sorghum (e.g., S. bicolor) and
Zea (e.g.,
Z. mays). The isolated nucleic acid and proteins of the present invention can
also be used
in species from the genera: Cucurbita, Rosa, Vitis, .luglans, Fragaria, Lotus,
Medicago,
Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot,
Daucus,
Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,
Hyoscvumus,
Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Mcajorana, Ciahorium,
Helianthus,
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Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium,
Parrieum,
Pennisetum, Ranunculus, Senecio, Salpiglossis, C'ucumis, Browallicr, Glycine,
Pisum,
Phaseolars, Lolium, Oryza, and Avena.
Nucleic Acids
The present invention provides, among other things, isolated nucleic acids of
RNA,
DNA, and analogs and/or chimeras thereof, comprising a polynucleotide of the
present
invention.
A polynucleotide of the present invention is inclusive of:
(a) a polynucleotide encoding a polypeptide of SEQ ID NOS: 2, 3, 5, 6, 8
including
exemplary polynucleotides of SEQ ID NOS: l, 4, 7;
(b) a poiynucleotide which is the product of amplification from a Zea mczys
nucleic
acid library using primer pairs which selectively hybridize under stringent
conditions to
loci within a polynueleotide selected from the group consisting of SEQ ID NOS:
1, 4, 7;
1 ~ (c) a polynucleotide which selectively hybridizes to a polynucleotide of
(a) or (b);
(d) a polynucleotide having a specified sequence identity with polynucleotides
of
(a), (b), or (c);
(e) a polynucleotide encoding a protein having a specified number of
contiguous
amino acids from a prototype polypeptide, wherein the protein is specifically
recognized
by antisera elicited by presentation of the protein and wherein the protein
does not
delectably immunoreact to antisera which has been fully immunosorbed with the
protein;
(f) complementary sequences of polynucleotides of (a), (b), (c), (d), or (e);
(g) polynucleotides comprising the sequences obtained from the clones
deposited
in a bacterial host with the American Type Culture Collection (ATCC) on
January 14,
2000, and assigned Accession Number PTA-1209; and
(h) a polynucleotide comprising at least a specific number of contiguous
nucleotides from a polynucleotide of (a), (b), (c), (d), (e), (f) or (g).
A. Polynucleotides Encoding A Polypeptide of the Present Invention
As indicated in (a), above, the present invention provides isolated nucleic
acids
comprising a polynucleotide of the present invention, wherein the
polynucleotide encodes
a polypeptide of the present invention. Every nucleic acid sequence herein
that encodes a
polypeptide also, by reference to the genetic code, describes every possible
silent variation
of the nucleic acid. One of ordinary skill will recognize that each codon in a
nucleic acid
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-z~-
(except AUG, which is ordinarily the only colon for methionine; and UGG ,
which is
ordinarily the only colon for tryptophan) can be modified to yield a
functionally identical
molecule. Thus, each silent variation of a nucleic acid which encodes a
polypeptide of the
present invention is implicit in each described polypeptide sequence and is
within the
scope of the present invention. Accordingly, the present invention includes
polynucleotides of SEQ ID NOS: l, 4, or 7, and polynucleotides encoding a
polypeptide of
SEQ ID NOS: 2, 3, 5, 6, or 8.
B. Polynucleotides Amplified from a Zea mat's Nucleic Acid Library
As indicated in (b), above, the present invention provides an isolated nucleic
acid
comprising a poiynucleotide of the present invention, wherein the
polynucleotides are
amplified from a Zea mat's nucleic acid library. Zea mat's lines B73, PHREI,
A632,
BMS-P2#10, W23, and Mol7 are known and publicly available. Other publicly
known
~znd availabi.e maize lines can be obtained from the Maize Genetics
Cooperation (Urbana,
iL). The nucleic acid library may be a cDNA library, a genomic library, or a
library
generally ~~onstructed from nuclear transcripts at any stage of intron
prace.ssing. cDNA
liuraiies can be normalized to increase the representation of relatively rare
cDNAs. In
optional embodiments, the cDNA libraxy is constructed using a full-length cDNA
synthesis method. Examples of such methods include Oligo-Capping (Maruyama, K.
and
Sugano, S. Gene 138: 171-174, 1994), Biotinylated CAP Trapper (Carninci, P.,
Kvan, C.,
et al. Genomics 37: 327-336, 1996), and CAP Retention Procedure (Edery, E.,
Chu, L.L.,
et al. Molecular and Cellular Biology 15: 3363-3371, 1995). cDNA synthesis is
often
catalyzed at 50-55°C to prevent formation of RNA secondary structure.
Examples of
reverse transcriptases that are relatively stable at these temperatures are
Superscript II
Reverse Transcriptase (Life Technologies, Inc.), AMV Reverse Transcriptase
(Boehringer
Mannheim] and RetroAmp Reverse Transcriptase (Epicentre). Rapidly growing
tissues, or
rapidly dividing cells are preferably used as mRNA sources. A preferred tissue
source
from which one can isolate alternative oxidase mRNA is cold-stressed maize
seedlings
(e.g., seedlings at V3 stage treated for up to 24 hours at 10°C).
Another preferred tissue
source is pathogen-infected maize leaves (e.g., leaves of V6 plants 48 hours
after
inoculation with Cochliobolus heterostrophus conidia). Another preferred
tissue source is
maize tassels at the early stages of pollen shed.
The present invention also provides subsequences of the polynucleotides of the
present invention. A variety of subsequences can be obtained using primers
which
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-n-
selectively hybridize under stringent conditions to at least two sites within
a
polynucleotide of the present invention, or to two sites within the nucleic
acid which flank
and comprise a polynucleotide of the present invention, or to a site within a
polynucleotide
of the present invention and a site within the nucleic acid which comprises
it. Primers are
chosen to selectively hybridize, under stringent hybridization conditions, to
a
polynucleotide of the present invention. Generally, the primers are
complementary to a
subsequence of the target nucleic acid which they amplify but may have a
sequence
identity ranging from about 85% to 99% relative to the polynucleotide sequence
which
they are designed to anneal to. As those skilled in the art will appreciate,
the sites to which
the primer pairs will selectively hybridize are chosen such that a single
contiguous nucleic
acid can be formed under the desired amplification conditions.
In optional embodiments, the primers will be constructed so that they
selectively
hybridize under stringent conditions to a sequence (or its complement) within
the target
nucleic acid which comprises the codon encoding the carboxy or amino terminal
amino
acid residue (i.e., the 3' terminal coding region and 5' terminal coding
region,
respectively) of the polynucleotides of the present invention. Optionally
within these
embodiments, the primers will be constructed to selectively hybridize entirely
within the
coding region of the target polynucleotide of the present invention such that
the product of
amplification of a cDNA target will consist of the coding region of that cDNA.
The
primer length in nucleotides is selected from the group of integers consisting
of from at
least 15 to 50. Thus, the primers can be at least 15, i 8, 20, 25, 30, 40, or
50 nucleotides in
length. Those of skill will recognize that a lengthened primer sequence can be
employed to
increase specificity of binding (i.e., annealing) to a target sequence. A non-
annealing
sequence at the 5'end of a primer (a "tail") can be added, for example, to
introduce a
cloning site at the terminal ends of the amplicon.
The amplification products can be translated using expression systems well
known
to those of skill in the art and as discussed, infra. The resulting
translation products can be
confirmed as polypeptides of the present invention by, for example, assaying
for the
appropriate catalytic activity (e.g., specific activity and/or substrate
specificity), or
verifying the presence of one or more linear epitopes which are specific to a
polypeptide of
the present invention. Methods for protein synthesis from PCR derived
templates are
known in the art and available commercially. See, e.g., Amersham Life
Sciences, Inc,
Catalog '97, p.354.
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_ZS_
Methods for obtaining S' and/or 3' ends of a vector insert are well known in
the art.
See, e.g., RACE (Rapid Amplification of Complementary Ends) as described in
Frohman,
M. A., in PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D.
H.
Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Ine., San Diego),
pp. 28-38
(1990)); see also, U.S. Pat. No. 5,470,722, and Current Protocols in Molecular
Biology,
Unit 15.6, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience,
New York
(1995); Frohman and Martin, Technigues 1:165 (1989).
C. Polynucleotides Which Selectively Hybridize to a Polynucleotide of (A) or
(B)
1 t) As indicated in (c), above, the present invention provides isolated
nucleic acids
comprising polynucleotides of the present invention, wherein the
polynucleotides
selectively hybridize, under selective hybridization conditions, to a
polynucleotide of
sections (A) or (B) as discussed above. Thus, the polynucleotides of this
embodiment can
he used for isolating, detecting, and/or quantifying nucleic acids comprising
the
polynacleotides of (A) or (B). For example, polynucleotides of the present
invention can
be used to identify, isolate, or amplify partial or full-lengtli clones in a
deposited library.
In some embodiments, the polynucleotides are genomic or cDNA sequences
isolated or
otherwise complementary to a cDNA from a dicot or monocot nucleic acid
library.
Exemplary species of monocots and dicots include, but are not limited to:
maize, cxnola,
soybean, cotton, wheat, sorghum, sunflower, alfalfa, oats, sugar cane, millet,
barley, and
rice. Optionally, the cDNA library comprises at least 30% to 95% full-length
sequences
(for example, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% full-length
sequences). The cDNA libraries can be normalized to increase the
representation of rare
sequences. Low stringency hybridization conditions are typically, but not
exclusively,
employed with sequences having a reduced sequence identity relative to
complementary
sequences. Moderate and high stringency conditions can optionally be employed
for
sequences of greater identity. Low stringency conditions allow selective
hybridization of
sequences having about 70% to 80% sequence identity and can be employed to
identify
orthologous or paralogous sequences.
D. Polynucleotides Having a Specific Seguence Identity with the
Polynucleotides of (A),
(B) or (C)
As indicated in (d), above, the present invention provides isolated nucleic
acids
comprising polynucleotides of the present invention, wherein the
polynucleotides have a
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specified identity at the nucleotide level to a polynucleotide as disclosed
above in sections
(A), (B), or (C), above. Identity can be calculated using, for example, the
BLAST or GAP
algorithms under default conditions. The percentage of identity to a reference
sequence is
at least 60% and, rounded upwards to the nearest integer, can be expressed as
an integer
selected from the group of integers consisting of from 60 to 99. Thus, for
example, the
percentage of identity to a reference sequence can be at least 70%, 75%, 80%,
85%, 90%,
or 95%.
Optionally, the polynucleotides of this embodiment will encode a polypeptide
that
will share an epitope with a polypeptide encoded by the polynucleotides of
sections (A),
(B), or (C). Thus, these polynucleotides encode a first polypeptide which
elicits
production of antisera comprising antibodies which are specifically reactive
to a second
polypeptide encoded by a polynucleotide of (A), (B j, or (C). However, the
first
polypeptide does not bind to antisera raised against itself when the antisera
has been fully
immunosorbed with the first polypeptide. Hence, the polynucleotides of this
embodiment
1 S can be used to generate antibodies for use in, for example, the screening
of expression
libraries for nucleic acids comprising polynucleotides of (A), (B), or (C), or
for
purification of, or in immunoassays for, polypeptides encoded by the
polynucleotides of
(A), (B), or {C). The polynu~leotides of this embodiment embrace nucleic acid
sequences
which.can be employed for selective hybridization to a polynucleotide encoding
a
polypeptide of the present invention.
Screening polypeptides for specific binding to antisera can be conveniently
achieved using peptide display libraries. This method involves the screening
of large
collections of peptides for individual members having the desired function or
structure.
Antibody screening of peptide display libraries is well known in the art. The
displayed
peptide sequences can be from 3 to 5000 or more amino acids in length,
frequently from 5-
100 amino acids long, and often from about 8 to 15 amino acids long. In
addition to direct
chemical synthetic methods for generating peptide libraries, several
recombinant DNA
methods have been described. One type involves the display of a peptide
sequence on the
surface of a bacteriophage or cell. Each bacteriophage or cell contains the
nucleotide
sequence encoding the particular displayed peptide sequence. Such methods are
described
in PCT patent publication Nos. 91/17271, 91/18980, 91/19818, and 93/08278.
Other
systems for generating libraries of peptides have aspects of both in vitro
chemical
synthesis and recombinant methods. See, PCT Patent publication Nos. 92/05258,
92/14843, and 97/20078. See also, U.S. Patent Nos. 5,658,754; and 5,643,768.
Peptide
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PCT1US00/01847
display libraries, vectors, and screening kits are commercially available from
such
suppliers as Invitrogen (Carlsbad, CA).
E. Polynucleotides Encoding a Protein Having a Subseguence J~rom a Prototype
Polypeptide and is Cross-Reactive to the Prototype Polypeptide
As indicated in (e), above, the present invention provides isolated nucleic
acids
comprising polynucleotides of the present invention, wherein the
polynucleotides encode a
protein having a subsequence of contiguous amino acids from a prototype
polypeptide of
the present invention such as are provided in (a), above. The length of
contiguous amino
acids from the prototype polypeptide is selected from the group of integers
consisting of
from at least 10 to the number of amino acids within the prototype sequence.
Thus, for
example, the polynucleotide can encode a polypeptide having a subsequence
having at
least 10, 15, 20, 25, 30, 35, 40, 45, or 50, contiguous amino acids from the
prototype
polypeptide. Further, the number of such subsequences encoded by a
polynucleotide of
i 5 the instant embodiment can be any integer selected from the group
consisting of from I to
20, such as 2, 3, 4, or 5. The subsequences can be separated by any integer of
nucleotides
from 1 to the number of nucleotides in the sequence such as at least 5. 10,
15, 25, 50, 100,
or 200 nucleotides.
The proteins encoded by polynucleotides of this embodiment, when presented as
an
immunogen, elicit the production of polyclonal antibodies which specifically
bind to a
prototype polypeptide such as but not limited to, a polypeptide encoded by the
polynucleotide of (a) or (b), above. Generally, however, a protein encoded by
a
polynucleotide of this embodiment does not bind to antisera raised against the
prototype
polypeptide when the antisera has been fully immunosorbed with the prototype
Z5 polypeptide. Methods of making and assaying for antibody binding
specificity/affinity are
well known in the art. Exemplary immunoassay formats include ELISA,
competitive
immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent
assays and
the like.
In a preferred assay method, fully immunosorbed and pooled antisera which is
elicited to the prototype polypeptide can be used in a competitive binding
assay to test the
protein. The concentration of the prototype polypeptide required to inhibit
SO% of the
binding of the antisera to the prototype polypeptide is determined. If the
amount of the
protein required to inhibit binding is less than twice the amount of the
prototype protein,
then the protein is said to specifically bind to the antisera elicited to the
immunogen.
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Accordingly, the proteins of the present invention embrace allelic variants,
conservatively
modified variants, and minor recombinant modifications to a prototype
polypeptide.
A polynucleotide of the present invention optionally encodes a protein having
a
molecular weight as the non-glycosylated protein within 20% of the molecular
weight of
the full-length non-glycosylated polypeptides of the present invention.
Molecular weight
can be readily determined by SDS-PAGE under reducing conditions. Optionally,
the
molecular weight is within 15% of a full length polypeptide of the present
invention, more
preferably within 10% or 5%, and most preferably within 3%, 2%, or I % of a
full length
polypeptide of the present invention.
Optionally, the polynucleotides of this embodiment will encode a protein
having a
specific enzymatic activity at least 50%, 60%, 80%, or 90% of a cellular
extract
comprising the native, endogenous full-length polypeptide of the present
invention.
Further, the proteins encoded by polynucleotides of this embodiment will
optionally have a
substantially similar affinity constant (Km ) and/or catalytic activity (i.e.,
the microscopic
rate constant, k~at) as the native endogenous, full-length protein. Those of
skill in the art
will recognize that ?c~at/Km value determines the specificity for competing
substrates and is
often referred to as the specificity constant. Proteins of this embodiment can
have a
kca~/Km value at least 10% of a full-length polypeptide of the present
invention as
determined using the endogenous substrate of that polypeptide: Optionally, the
k~a,~Km
value will be at least 20%, 30%, 40%, 50%, and most preferably at least 60%,
70%, 80%,
9U%, or 95% the k~at/Km value of the full-length polypeptide of the present
invention.
Determination of k~a,, Km , arid k~a,lK", can be determined by any number of
means well
known to those of skill in the art. For example, the initial rates (i.e., the
first 5% or less of
the reaction) can be determined using rapid mixing and sampling techniques
(e.g.,
continuous-flow, stopped-flow, or rapid quenching techniques), flash
photolysis, or
relaxation methods (e.g., temperature jumps) in conjunction with such
exemplary methods
of measuring as spectrophotometry, spectrofluorimetry, nuclear magnetic
resonance, or
radioactive procedures. Kinetic values are conveniently obtained using a
Lineweaver-
Burk or Eadie-Hofstee plot.
F. Polynucleotides Complementary to the Polynucleotides of (A)-(E)
As indicated in (fJ, above, the present invention provides isolated nucleic
acids
comprising polynucleotides complementary to the polynucleotides of paragraphs
A-E,
above. As those of skill in the art will recognize, complementary sequences
base-pair
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throughout the entirety of their length with the polynucleotides of sections
(A)-(E) (i.e.,
have 100% sequence identity over their entire length). Complementary bases
associate
through hydrogen bonding in double stranded nucleic acids. For example, the
following
base pairs are complementary: guanine and cytosine; adenine and thyrnine; and
adenine
and uracil.
G. Polynucleotides Which are Subsequences of the Polvnucleotides of (A)-(F)
As indicated in (g), above, the present invention provides isolated nucleic
acids
comprising polynucleotides which comprise at least 15 contiguous bases from
the
1 U polynucleotides of sections (A) through (F) as discussed above. The length
of the
polynucleotide is given as an integer selected from the group consisting of
from at least 15
to the length of the nucleic acid sequence from which the polynucleotide .is a
subsequence
of. Thus, for example, polynucleotides of the present invention are inclusive
of
polynucleotides comprising at least 15, 2.0, 25, 30, 40, 50, 60, 75, or 100
contiguous
nucleotides in length from the polynucleotides of (A)-(F). Optionally, the
number of such
subsequences encoded by a polynucleotide of the instant embodiment can be any
integer
selected from the group consisting of from l to 20, such as 2, 3, 4, or 5. The
subsequerces
can be separated by any integer of nucleotides from 1 to the number of
nucleotides in the
sequence such as at least 5, 10, 15, 25, 50, 100, or 20U nucleotides.
The subsequences of the present invention can comprise structural
characteristics
of the sequence from which it is derived. Alternatively, the subsequences can
lack certain
structural characteristics of the larger sequence from which it is derived
such as a poly (A)
tail. Optionally, a subsequence from a polynucleotide encoding a polypeptide
having at
least one linear epitope in common with a prototype polypeptide sequence as
provided in
(a), above, may encode an epitope in common with the prototype sequence.
Alternatively,
the subsequence may not encode an epitope in common with the prototype
sequence but
can be used to isolate the larger sequence by, for example, nucleic acid
hybridization with
the sequence from which it's derived. Subsequences can be used to modulate or
detect
gene expression by introducing into the subsequences compounds which bind,
intercalate,
cleave and/or crosslink to nucleic acids. Exemplary compounds include
acridine, psoralen,
phenanthroline, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.
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Construction of Nucleic Acids
The isolated nucleic acids of the present invention can be made using (a)
standard
recombinant methods, (b) synthetic techniques, or combinations thereof. In
some
embodiments, the polynucleotides of the present invention will be cloned,
amplified, or
S otherwise constructed from a monocot. In preferred embodiments the monocot
is Zea
mays.
The nucleic acids may conveniently comprise sequences in addition to a
polynucleotide of the present invention. For example, a multi-cloning site
comprising one
or more endonuclease restriction sites may be inserted into the nucleic acid
to aid in
i0 isolation of the polynucleotide. Also, translatable sequences may be
inserted to aid in the
isolation of the translated polynucleotide of the present invention. For
example, a hexa-
histidine marker sequence provides a convenient means to purify the proteins
of the
present invention. A polynucleotide of the present invention can be attached
to a vector,
adapter, or linker for cloning and/or expression of a polynucleotide of the
present
invention. Additional sequences may be added to such cloning and/or expression
sequences to optimize their function in cioning and/or expression, to aid in
isolation of the
polynucleo tide, or to improve the introduction of the polynucleotide into a
cell. Typically,
the length of a nucleic acid of the present invention less the length of its
polynucleotide of
the present invention is less than z0 kilobase pairs, often less than 15 kb,
anal frequently
20 less than 10 kb. Use of cloning vectors, expression vectors, adapters, and
linkers is well
known and extensively described in the art. For a description of various
nucleic acids see,
for example, Stratagene Cloning Systems, Catalogs 1995, s 996, 1997 (La Jolla,
CA); and,
Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, IL).
25 A. Recombinant Methods, for Constructing Nucleic Acids
The isolated nucleic acid compositions of this invention, such as RNA, eDNA,
genomic DNA, or a hybrid thereof, can be obtained from plant biological
sources using
any number of cloning methodologies known to those of skill in the art. In
some
embodiments, oligonucleotide probes which selectively hybridize, under
stringent
30 conditions, to the polynucleotides of the present invention are used to
identify the desired
sequence in a eDNA or genomic DNA library. Isolation of RNA, and construction
of
cDNA and genomic libraries is well known to those of ordinary skill in the
art. See, e.g.,
Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,
Berlin
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(1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Eds.,
Greene
Publishing and Wiley-lnterscience, New York (1995).
A number of cDNA synthesis protocols have been described which provide
substantially pure full-length cDNA libraries. Substantially pure full-length
cDNA
S libraries are constructed to comprise at least 90%, and more preferably at
least 93% or
95% full-length inserts amongst clones containing inserts. The length of
insert in such
libraries can be from 0 to 8, 9, 10, 11, 12, 13, or more kilobase pairs.
Vectors to
accommodate inserts of these sizes are known in the art and available
commercially. See,
e.g., Stratagene's lambda ZAP Express (eDNA cloning vector with 0 to 12 kb
cloning
capacity). An exemplary method of constructing a greater than 95% pure full-
length
cDNA library is described by Carninci et al., CJenomics, 37:327-336 ( 1996).
Other
methods for producing full-length libraries are known in the art. See, e.g.,
Edery et al.,
Mol. Cell Bio1.,15(6):3363-3371 (1995); and, PC'T Application WO 96/34981.
1 S Al. Normalized or Subtracted cDNA Libraries
A non-normalized cDNA library represents the mRNA population of the tissue it
was made from. Since unique clones are out-numbered by clones derived from
highly
expressed genes their isolation can be laborious. Normalization of a cDNA
library is the
process of creating a library in which each clone is more equally represented.
Construction of normalized libraries is described in Ko, Nucl. Acids. Res.,
18(19):5705-
5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A., 88:1943-1947 (1991);
U.S. Patents
5,482,685, and 5,637,685. In an exemplary method described by Soares et al.,
normalization resulted in reduction of the abundance of clones from a range of
four orders
of magnitude to a narrow range of only 1 order of magnitude. Proc. Natl. Acad.
Sci. USA,
91:9228-9232 (1994).
Subtracted cDNA libraries are another means to increase the proportion of less
abundant cDNA species. In this procedure, cDNA prepared from one pool of mRNA
is
depleted of sequences present in a second pool of mRNA by hybridization. The
cDNA:rnRNA hybrids are removed and the remaining un-hybridized cDNA pool is
enriched for sequences unique to that pool. See, Foote et al. in, Plant
Molecular Biology:
A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and
Zarbl,
Technique, 3(2):58-63 (1991); Sive and St. John, Nucl. Acids Res.,
16(22):10937 (1988);
Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene
Publishing and
Wiley-Interscience, New York (1995); and, Swaroop et al., Nucl. Acids Res.,
19)8):1954
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(1991 ). cDNA subtraction kits are commercially available. See, e.g., PCR-
Select
(Clontech, Palo Alto, CA).
To construct genomic libraries, large segments of genomic DNA are generated by
fragmentation, e.g. using restriction endonucleases, and are ligated with
vector DNA to
form concatemers that can be packaged into the appropriate vector.
Methodologies to
accomplish these ends, and sequencing methods to verify the sequence of
nucleic acids are
well known in the art. Examples of appropriate molecular biological techniques
and
instructions sufficient to direct persons of skill through many construction,
cloning, and
screening methodologies are found in Sambrook, et al., Molecular Cloning: t1
Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory Vols. I-3 (1989), Methods in
Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and
Kimmel,
Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular
Biology,
Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York
(1995); Plant
Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (
1997).
1 S Kits for constmction of genomic libraries arP also commercially available.
'The cDNA or genomic library can be screened using a probe based upon the
sequence of a polynucleotide of the present invention such as those disclosed
herein.
Probes may be used to hybridize with genomic DriA or cDNA sequences to isolate
homologous genes in the same or different plant species. 'Those of skill in
the art will
appreciate that various degrees of stringency of hybridization can be employed
in the
assay; and either the hybridization or the wash medium can be stringent.
The nucleic acids of interest can also be amplified from nucleic acid samples
using
amplification techniques. For instance, polymerase chain reaction (PCR)
technology can
be used to amplify the sequences of polynucleotides of the present invention
and related
genes directly from genomic DNA or cDNA libraries. PCR and other in vitro
amplification methods may also be useful, for example, to clone nucleic acid
sequences
that code for proteins to be expressed, to make nucleic acids to use as probes
for detecting
the presence of the desired mRNA in samples, for nucleic acid sequencing, or
for other
purposes. The T4 gene 32 protein (Boehringer Mannheim) can be used to improve
yield of
long PCR products.
PCR-based screening methods have been described. Wilfinger et al. describe a
PCR-based method in which the longest cDNA is identified in the first step so
that
incomplete clones can be eliminated from study. BioTechnigues, 22(3): 481-486
(1997).
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Such methods are particularly effective in combination with a full-length cDNA
construction methodology, above.
B. Synthetic Methods for Constructing Nucleic Acids
The isolated nucleic acids of the present invention can also be prepared by
direct
chemical synthesis by methods such as the phosphotriester method of Narang et
al., Meth.
Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al., Meth.
Enzymol.
68: 109-1 S 1 ( 1979); the diethylphosphoramidite method of Beaucage et al.,
Tetra. Lett. 22:
1859-1862 ( 1981 ); the solid phase phosphoramidite triester method described
by Beaucage
and Camthers, Tetra. Letts. 22(20): 1859-1862 ( 1981 ), e.g., using an
automated
synthesizer, e.g., as described in Needham-VanDevanter et al., Nucleic Acids
Res., 12:
6159-6168 ( 1984); and, the solid support method of U.S. Patent No. 4,458,066.
Chemical
synthesis generally produces a single stranded oligonucleotide. This may be
converted
into double stranded DNA by hybridization witl-~ a complementary sequence, or
by
1 ~ polymerization with a DNA polyrnerase using the single strand as a
template. One of skill
will recognize that while chemical synthesis of DNA is best emplomed for
sequences of
about 100 bases or less, longer sequences may be obtained by the ligation or
shorter
sequences.
Recombinant Expression Cassettes
The present invention further provides recombinant expression cassettes
comprising a nucleic acid of the present invention. A nucleic acid sequence
coding for the
desired polypeptide of the present invention, for example a cDNA or a genornic
sequence
encoding a full length polypeptide of the present invention, can be used to
construct a
2~ recombinant expression cassette which can be introduced into the desired
host cell. A
recombinant expression cassette will typically comprise a polynucleotide of
the present
invention operably linked to transcriptional initiation regulatory sequences
which will
direct the transcription of the polynucleotide in the intended host cell, such
as tissues of a
transformed plant.
For example, plant expression vectors may include (I) a cloned plant gene
under
the transcriptional control of 5' and 3' regulatory sequences and (2) a
dominant selectable
marker. Such plant expression vectors may also contain, if desired, a promoter
regulatory
region (e.g., one conferring inducible or constitutive, environmentally- or
developmentally-regulated, or cell- or tissue-specific/selective expression),
a transcription
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_3~_
initiation start site, a ribosome binding site, an RNA processing signal, a
transcription
termination site, and/or a polyadenylation signal.
A plant promoter fragment can be employed which will direct expression of a
polynucleotide of the present invention in all tissues of a regenerated plant.
Such
promoters are referred to herein as "constitutive" promoters and are active
under most
environmental conditions and states of development or cell differentiation.
Examples of
constitutive promoters include the cauliflower mosaic virus (CaMV) 35S
transcription
initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium
tumefaciens, the ubiquitin i promoter, the Smas promoter, the cinnamyl alcohol
dehydrogenase promoter (U.S. Patent No. 5,683,439), the Nos promoter, the pEmu
promoter, the rubisco promoter, the GRP 1-8 promoter, and other transcription
initiation
regions from various plant genes known to those of skill.
Alternatively, the plant promoter can direct expression of a polynucleotide of
the
present invention in a specific tissue or may be otherwise under more precise
environmental or developmental control. Such promoters are referred to here
a.s
"inducible" promoters. Environmental conditions that may effect transcription
by
inducible promoters include pathogen attack, anaerobic conditions, or the
presence of
light. ES;amples of inducible promoters are the Adhl promoter which is
inducible by
hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress,
and the
PPDIC prorr~oter which is inducible by light.
Examples of promoters under developmental control include promoters that
initiate
transcription only, or preferentially, in certain tissues, such as leaves,
roots, fruit, seeds, or
Bowers. Exemplary promoters include the anther specific promoter 5126 (U.S.
Patent Nos.
5,689,049 and 5,689,051), glob-1 promoter, and gamma-zero promoter. The
operation of a
promoter may also vary depending on its location in the genome. Thus, an
inducible
promoter may become fully or partially constitutive in certain locations.
Both heterologous and non-heterologous (i.e., endogenous) promoters can be
employed to direct expression of the nucleic acids of the present invention.
These
promoters can also be used, for example, in recombinant expression cassettes
to drive
expression of antisense nucleic acids to reduce, increase; or alter
concentration and/or
composition of the proteins of the present invention in a desired tissue.
Thus, in some
embodiments, the nucleic acid construct will comprise a promoter functional in
a plant
cell, such as in Zea mays, operably linked to a polynucleotide of the present
invention.
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Promoters useful in these embodiments include the endogenous promoters driving
expression of a polypeptide of the present invention.
In some embodiments, isolated nucleic acids which serve as promoter or
enhancer
elements can be introduced in the appropriate position (generally upstream) of
a non-
heterologous form of a polynucleotide of the present invention so as to up- or
down-
regulate expression of a polynucleotide of the present invention. For example,
endogenous
promoters can be altered in vivo by mutation, deletion, and/or substitution
(see, Kmiec,
LLS. Patent x,565,350; Zarling et al., PCT/LJS93/03868), or isolated promoters
can be
introduced into a plant cell in the proper orientation arid distance from a
gene of the
present invention so as to control the expression of the gene. Gene expression
can be
modulated under conditions suitable for plant growth so as to alter the total
concentration
and/or alter the composition of the polypeptides of the present invention in
plant cell.
Thus, the present invention provides compositions, and methods for making,
heterologous
promoters and/or enhancers operably linked to a native, endogenous (i.e., non-
1 S heterologous) .form of a polynucleotide of the present invention.
If polypeptide expression is desired, it is generally desirable to include a
rolyadenylation region at the 3'-end of a polynucleotide coding region. The
polyadexoylation region can be derived from the natural gene, frorr~ a variety
of other plant
genes, or from T-DNA. The 3' end sequence to be added can be derived from, fo:
2G example, the nopaline synthase or octopine synthase genes, or alternatively
from another
plant gene, or less preferably from any other eukaryotic gene.
An intron sequence can be added to the 5' untranslated region or the coding
sequence of the partial coding sequence to increase the amount of the mature
message that
accumulates in the cytosol. Inclusion of a spliceable intron in the
transcription unit in both
25 plant and animal expression constructs has been shown to increase gene
expression at both
the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol.
8: 4395-
4405 ( 1988); Callis et al., Genes Dev. 1: 1183-1200 ( 1987). Such intron
enhancement of
gene expression is typically greatest when placed near the 5' end of the
transcription unit.
Use of maize introns Adhl-S intron 1, 2, and 6, the Bronze-I intron are known
in the art.
30 See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds.,
Springer,
New York ( 1994). The vector comprising the sequences from a polynucleotide of
the
present invention will typically comprise a marker gene which confers a
selectable
phenotype on plant cells. Typical vectors useful for expression of genes in
higher plants
are well known in the art and include vectors derived from the tumor-inducing
(Ti)
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plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. in
Enzymol.,
1 X3:253-277 ( 1987).
A polynucleotide of the present invention can be expressed in either sense or
anti-
sense orientation as desired. It will be appreciated that control of gene
expression in either
sense or anti-sense orientation can have a direct impact on the observable
plant
characteristics. Antisense technology can be conveniently used to inhibit gene
expression
in plants. To accomplish this, a nucleic acid segment from the desired gene is
cloned and
operably linked to a promoter such that the anti-sense strand of RNA will be
transcribed.
The construct is then transformed into plants and the antisense strand of RNA
is produced.
In plant cells, it has been shown that antisense RNA inhibits gene expression
by preventing
the accumulation of mRNA which encodes the enzyme of interest, see, e.g.,
Sheehy et al.,
Proc. Nat'l. Acad. Sci. (USA) 85: 8805-8809 (i988); and Hiatt et al., U.S.
Patent No.
4,801,340.
Another method of suppression is sense suppression. Introduction of nucleic
acid
configured in the sense orientation has been shown to be an effective means by
which to
block the transcription of target genes. For an example of i:he nse of this
method to
modulate expression of endogenous gene's see, Napoli et ul., :t he Plant Cell
2: 279-289
(I990) and [J.S. °atent No. 5,034,323.
Catalytic RNA molecules of ribozymes can also be used to inhibit expression of
plant genes. It is possible to design ribozyrnes that specifically pair with
virtually any
target RNA and cleave the phosphodiester backbone at a specific location,
thereby
functionally inactivating the target RNA. In carrying out this cleavage, the
ribozyme is not
itself altered, and is thus capable of recycling and cleaving other molecules,
making it a
true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers
RNA-
cleaving activity upon them, thereby increasing the activity of the
constructs. The design
and use of target RNA-specific ribozymes is described in Haseloff et al.,
Nature 334: 585-
591 (1988).
A variety of cross-linking agents, alkylating agents and radical generating
species
as pendant groups on polynucleotides of the present invention can be used to
bind, label,
detect, and/or cleave nucleic acids. For example, Vlassov, V. V., et al.,
Nucleic Acids Res
( 1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA
fragment with
alkylating derivatives of nucleotides complementary to target sequences. A
report of
similar work by the same group is that by Knorre, D. G., et al., Biochimie
(1985) 67:785-
789. Iverson and Dervan also showed sequence-specific cleavage of single-
stranded DNA
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-ao-
mediated by incorporation of a modified nucleotide which was capable of
activating
cleavage (JAm Chem Soc (1987) 109:1241-1243). Meyer, R. B., et al., JAm Chem
Soc
(1989) I 11:8517-$519, effect covalent crosslinking to a target nucleotide
using an
alkylating agent complementary to the single-stranded target nucleotide
sequence. A
photoactivated crosslinking to single-stranded oligonucleotides mediated by
psoralen was
disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203. Use of
crosslinking in
triple-helix forming probes was also disclosed by Home, et al., JAm Chem Soc
(1990)
112:2435-2437. Use of N4, N4-ethanocytosine as an alkylating agent to
crosslink to
single-stranded oligonucleotides has also been described by Webb and
Matteucci, JAm
Chem Soc ( i 986) 10$:2764-2765; Nucleic Acids Res ( 1986) 14:7661-7674;
Feteritz et al.,
J. Am. Chem. Soc. 113:4000 ( 1991 ). Various compounds to bind, detect, label,
and/or
cleave nucleic acids are known in the art. See, for example, U.S. Patent Nos.
5,543,507;
5,672,593; 5,484,908; 5,256,648; and, 5,681941.
. Proteins
The isolated proteins of the present invention comprise a polypeptide having
at
least 10 amino acids encoded by any one of the polynucleotides of the present
invention as
discussed more fully, above, or polypeptides which are conservatively modified
variants
thereof. The proteins of the present invention or variants thereof can
comprise any number
of contiguous amino acid residues from a polypeptide of the present invention,
wherein
that number is selected from the group of integers consisting of from 10 to
the number of
residues in a full~~length polypeptide of the present invention. Optionally,
this subsequence
of contiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acids in
length, often
at least 50, 60, 70, 80, or 90 amino acids in length. Further, the number of
such
subsequences can be any integer selected from the group consisting of from 1
to 20, such
as 2, 3, 4, or 5.
The present invention further provides a protein comprising a polypeptide
having a
specified sequence identity with a polypeptide of the present invention. The
percentage of
sequence identity is an integer selected from the group consisting of from 50
to 99.
Exemplary sequence identity values include 60%, 65%, 70%, 75%, 80%, 85%, 90%,
and
95%. Sequence identity can be determined using, for example, the GAP or BLAST
algorithms.
As those of skill will appreciate, the present invention includes
catalytically active
polypeptides of the present invention (i.e., enzymes). Catalytically active
polypeptides
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have a specific activity of at least 20%, 30%, or 40%, and preferably at least
SO%, 60%, or
70%, and most preferably at least 80%, 90%, or 95% that of the native (non-
synthetic),
endogenous polypeptide. Further, the substrate specificity (k~ac/Km) is
optionally
substantially similar to the native (non-synthetic), endogenous polypeptide.
Typically, the
Km will be at least 30%, 40%, or 50%, that of the native (non-synthetic),
endogenous
polypeptide; and more preferably at least 60%, 70%, 80%, or 90%. Methods of
assaying
and quantifying measures of enzymatic activity and substrate specificity
(k~at~K",), are well
known to those of skill in the art.
Generally, the proteins of the present invention will, when presented as an
immunogen, elicit production of an antibody specifically reactive to a
polypeptide of the
present invention. Further, the proteins of the present invention will not
bind to antisera
raised against a polypeptide of the present invention which has been fully
immunosorbed
with the same polypeptide. Immunoassays for determining binding are well known
to
those of skill in the art. A preferred immunoassay is a competitive
immunoassay as
1 S discussed, supra. Thus, the proteins of the present invention can be
employed as
immunogens for constructing antibodies immunoreactive to a protein of the
present
invention for such exemplary utilities as immunoassays or protein purification
techniques.
Expression of Proteins in Host Cells
Using the nucleic acids of the present invemion, one may express a protein of
the
present invention in a recombinantly engineered cell such as bacteria, yeast,
insect,
mammalian, or preferably plant cells. The cells produce the protein in a non-
natural
condition (e.g., in quantity, composition, location, and/or time), because
they have been
genetically altered through human intervention to do so.
It is expected that those of skill in the art are knowledgeable in the
numerous
expression systems available for expression of a nucleic acid encoding a
protein of the
present invention. No attempt to describe in detail the various methods known
for the
expression of proteins in prokaryotes or eukaryotes will be made.
In brief summary, the expression of isolated nucleic acids encoding a protein
of the
present invention will typically be achieved by operably linking, for example,
the DNA or
cDNA to a promoter (which is either constitutive or regulatable), followed by
incorporation into an expression vector. The vectors can be suitable for
replication and
integration in either prokaryotes or eukaryotes. Typical expression vectors
contain
transcription and translation terminators, initiation sequences, and promoters
useful for
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-42-
regulation of the expression of the DNA encoding a protein of the present
invention. To
obtain high level expression of a cloned gene, it is desirable to construct
expression vectors
which contain, at the minimum, a strong promoter to direct transcription, a
ribosome
binding site for translational initiation, and a transcription/translation
terminator. One of
skill would recognize that modifications can be made to a protein of the
present invention
without diminishing its biological activity. Some modifications may be made to
facilitate
the cloning, expression, or incorporation of the targeting molecule into a
fusion protein.
Such modifications are well known to those of skill in the art and include,
for example, a
meii:ionirre added at the amino terminus to provide an initiation site, or
additional amino
I O acids (e.g.. poly His) placed on either terminus to create conveniently
located purification
sequences. Restriction sites or termination codons can also be introduced.
Transfection/Transformation of Cells
Tlre method of transfor-mation/transfection is not critical to the instant
invention;
I5 various motirods of transformation or transfection are currently available.
~"a newer
methods are available to transform crops ar other host cells they rnay he
dirertiv applied.
Accordingly. a wide variety of methods have been developed to insert a DIVA
sEquence
into the ~ enome of a host cell to obtain the transcription and,'or
translation of the sequem;e
~~ effect phenotypic changes in the organism. Thus, any method which providea
for
20 effectiv: transformation/transfection may be employed.
A. Plant Transformation
A DNA sequence coding for the desired polypeptide of the present invention,
for
example a cDNA or a genomic sequence encoding a full length protein, will be
used t~
25 constnrct a recombinant expression cassette which can be introduced into
the desired plant.
The preferred method of plant transformation for the present invention ~s by
particle
bombardment of immature maize embryos. See, e.g., Tomes, et al., Direct DNA
Transfer
into Intact Plant Cells Via Microprojectile Bombardment. pp.197-213 in Plant
Cell, Tissue
and Organ Culture, Fundamental Methods. eds. O. L. Gamborg and G.C. Phillips.
30 Springer-Verlag Berlin Heidelberg New York, 1995, and Songstad, D.D., B.M.
Hairston
and C.L. Armstrong 1993. Stable Transformation of Maize by Microprojectile
Bombardment of Immature Embryos. Agronomy Abstracts p. 183.
Isolated nucleic acid acids of the present invention can be introduced into
plants
according to techniques known in the art. Generally, recombinant expression
cassettes as
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WO 00/44920 _ 43 _ PCT/US00/01847
described above and suitable for transformation of plant cells are prepared.
Techniques for
transforming a wide variety of higher plant species are well known and
described in the
technical, scientific, and patent literature. See, for example, Weising et
al.. Ann. Rev.
Genet. 22: 421-477 (1988). For example, the DNA construct may be introduced
directly
into the genomic DNA of the plant cell using techniques such as
electroporation,
polyethylene glycol (PEG), poration, particle bombardment, silicon fiber
delivery, or
microinjection of plant cell protoplasts or embryogenic callus. See, e.g.,
Tomes, et al.,
Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment.
pp.197-
213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods. eds. O. L.
Gamborg
and G.C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995.
Alternatively, the
DNA constructs may be combined with suitable T-DNA flanking regions and
introduced
into a conventional Agrobacterium tumefaciens host vector. The virulence
functions of the
Agrobacterium tumefaciens host will direct the insertion of the construct and
adjacent
marker into the plant cell DNA when the cell is infected by the bacteria. See,
U.S. Patent
No.5,591,616.
. . The introduction of DNA constructs using PEG precipitation is described in
Paszkowski et al., Ernbo! ~: 2717-2722 (1984). Electroporation techniques are
described
in Fromm et al., Proc. Natl. Acad. Sci. (U,SA) 82: 5824 (1985). Ballistic
transformation
techniques are described in Klein et al., Nature 327: 70-73 ( 1987).
Agrobacterium tumefaciens-mediated transformation techniques are well
described in the
scientific literature. See, for example Horsch et al., Science 233: 496-498
(1984), and
Fraley et al.. Proc. Natl. Acad. Sci. (USA) 80: 4803 (1983). Although
Agrobacteriurn is
useful primarily in dicots, certain monocots can be transformed by
Agrobacterium. For
instance, Agrobacterium transformation of maize is described in U.S. Patent
No.
5,550,318.
Other methods of transfection or transformation include ( 1 ) Agrobacterium
rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller In:
Genetic
Engineering, vol. 6, PWJ Rigby, Ed., London, Academic Press, 1987; and
Lichtenstein, C.
P., and Draper, J,. 1n: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI
Press, 1985),
Application PCT/L1S87/02512 (WO 88/02405 published Apr. 7, 1988) describes the
use of
A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors
pARC8 or
pARCl6 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al., Plant Cell
Physiol.
25: 1353 (1984)), (3) the vortexing method (see, e.g., Kindle, Proc. Natl.
Acad. Sci., (USA)
87: 1228 (1990).
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DNA can also be introduced into plants by direct DNA transfer into pollen as
described by Zhou et al., Methods in Enzymology, 101:433 ( 1983); D. Hess,
Intern Rev.
Cytol., 107:367 ( 1987); Luo et al., Plant Mol. Biol. Reporter, 6:165
(1988). Expression of polypeptide coding genes can be obtained by injection of
the DNA
into reproductive organs of a plant as described by Pena et al., Nature,
325.:274 ( 1987).
DNA can also be injected directly into the cells of immature embryos and the
rehydration
of desiccated embryos as described by Neuhaus et al., Theor. Appl. Genet.,
75:30 ( 1987);
and Benbrook et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham,
Mass., pp.
27-54 (1986). A variety of plant viruses that can be employed as vectors are
known in the
art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic
virus, and
tobacco mosaic virus.
B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells
Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered
competent for transfection by various means. There are several well-known
methods of
introducing DNA into animal cells. These include: calcium phosphate
precipitation, fusion
of the recipient cells with bacterial protoplasts containing the DNA,
treatment of the
recipient cells with liposomes containing the DNA, DEAE dextran,
electroporation,
biolistics, and micro-injection of the DNA directly into the cells. The
transfected cells are
cultured by means well known in the art. Kuchler, R.J., Biochemical Methods in
Cell
Culture and Virology, Dowden, Hutchinson and Ross, lnc. ( 1977).
Synthesis of Proteins
The proteins of the present invention can be constructed using non-cellular
synthetic methods. Solid phase synthesis of proteins of less than about 50
amino acids in
length may be accomplished by attaching the C-terminal amino acid of the
sequence to an
insoluble support followed by sequential addition of the remaining amino acids
in the
sequence. Techniques for solid phase synthesis are described by Barany and
Mernfield,
Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis,
Biology.
Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield, et al., J.
Am. Chem. Soc.
85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd
ed., Pierce
Chem. Co., Rockford, Ill. ( 1984). Proteins of greater length may be
synthesized by
condensation of the amino and carboxy termini of shorter fragments. Methods of
forming
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-45-
peptide bonds by activation of a carboxy terminal end {e.g., by the use of the
coupling
reagent N,N'-dicycylohexylcarbodiimide) are known to those of skill.
Purification of Proteins
The proteins of the present invention may be purified by standard techniques
well
known to those of skill in the art. Recombinantly produced proteins of the
present
invention can be directly expressed or expressed as a fusion protein. The
recombinant
protein is purified by a combination of cell lysis (e.g., sonication, French
press) and
affinity chromatography. For fusion products, subsequent digestion of the
fusion protein
with an appropriate proteolytic enzyme releases the desired recombinant
protein.
The proteins of this invention, recombinant or synthetic, may be purified to
substantial purity by standard techniques well known in the art, including
detergent
solubilization, selective precipitation with such substances as ammonium
sulfate, column
chromatography, immunopurification methods, and others. See, for instance, R.
Scopes,
Protein Purification: Principles and Practice, Springer-Verlag: New York
(1982);
Deutscher, Guide to Protein Purification, Academic Press (1990). For example,
antibodies may be raised to the proteins as described herein. Purification
from E. coli can
be achieved following procedures described in U.S. Patent No. 4,511,503. The
protein
may then be isolated from cells expressing the protein and further purified by
standard
protein chemistry techniques as described herein. Detection of the expressed
protein is
achieved by methods known in the art and include, for example,
radioimmunoassays,
Western blotting techniques or immunoprecipitation.
Transgenic Plant Regeneration
Transformed plant cells which are derived by any of the above transformation
techniques can be cultured to regenerate a whole plant which possesses the
transformed
genotype. Such regeneration techniques often rely on manipulation of certain
phytohormones in a tissue culture growth medium. For transformation and
regeneration of
maize, see Gordon-Kamm et al., The Plant Cell, 2:603-618 (1990).
Plants cells transformed with a plant expression vector can be regenerated,
e.g.,
from single cells, callus tissue or leaf discs according to standard plant
tissue culture
techniques. It is well known in the art that various cells, tissues, and
organs from almost
any plant can be successfully cultured to regenerate an entire plant. Plant
regeneration
from cultured protoplasts is described in Evans et al., Protoplasts Isolation
and Culture,
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Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp.
124-176
( 1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press,
Boca Raton,
pp. 21-73 (1985).
The regeneration of plants containing the foreign gene introduced by
Agrobacterium from leaf explants can be achieved as described by Horsch et
al., Science,
227:1229-1231 (1985). In this procedure, transformants are grown in the
presence of a
selection agent and in a medium that induces the regeneration of shoots in the
plant species
being transformed as described by Fraley et al., Proc. Natl. Acad. Sci.
(U.S.A.), 80:4803
(1983). This procedure typically produces shoots within two to four weeks and
these
transformant shoots are then transferred to an appropriate root-inducing
medium
containing the selective agent and an antibiotic to prevent bacterial growth.
Transgenic
plants of the present invention may be fertile or sterile.
Regeneration can also be obtained from plant callus, explants, organs, or
parts
thereof. Such regeneration techniques are described generally in Klee et al.,
Ann. Rev. of
Plant Phys. 38: 467-486 (1987). The regeneration of plants from either single
plant
protoplasts or various explants is well known in the art. See, for example,
Methods Jor
Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,
Inc., San
Diego, Calif. (1988). This regeneration and growth process includes the steps
of selection
of transformant cells and shoots, rooting the transformant shoots and growth
of the
plantlets in soil. For maize cell culture and regeneration see generally, The
Maize
Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn
Improvement, 3~d edition, Sprague and Dudley Eds., American Society of
Agronomy,
Madison, Wisconsin (1988).
One of skill will recognize that after the recombinant expression cassette is
stably
incorporated in transgenic plants and confirmed to be operable, it can be
introduced into
other plants by sexual crossing. Any of a number of standard breeding
techniques can be
used, depending upon the species to be crossed.
In vegetatively propagated crops, mature transgenic plants can be propagated
by
the taking of cuttings or by tissue culture techniques to produce multiple
identical plants.
Selection of desirable transgenics is made and new varieties are obtained and
propagated
vegetatively for commercial use. In seed propagated crops, mature transgenic
plants can
be self crossed to produce a homozygous inbred plant. The inbred plant
produces seed
containing the newly introduced heterologous nucleic acid. These seeds can be
grown to
produce plants that would produce the selected phenotype.
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Parts obtained from the regenerated plant, such as flowers, seeds, leaves,
branches,
fruit, and the like are included in the invention, provided that these parts
comprise cells
comprising the isolated nucleic acid of the present invention. Progeny and
variants, and
mutants of the regenerated plants are also included within the scope of the
invention,
provided that these parts comprise the introduced nucleic acid sequences.
Transgenic
plants expressing the selectable marker can be screened for transmission of
the nucleic
acid of the present invention by, for example, standard immunoblot and DNA
detection
techniques. Transgenic lines are also typically evaluated on levels of
expression of the
heterologous nucleic acid. Expression at the RNA level can be determined
initially to
identify and quantitate expression-positive plants. Standard techniques for
RNA analysis
can be employed and include PCR amplification assays using oligonucleotide
primers
designed to amplify only the heterologous RNA templates and solution
hybridization
assays using heterologous nucleic acid-specific probes. The RNA-positive
plants can then
analyzed for protein expression by Western immunoblot analysis using the
specifically
reactive antibodies of the present invention. In addition, in situ
hybridization and
immunocytochemistry according to standard protocols can be done using
heterologous
nucleic acid specific polynucleotide probes and antibodies, respectively, to
localize sites of
expression within transgenic tissue. Generally, a number of transgenic lines
are usually
screened for the incorporated nucleic acid to identify and select plants with
the most.
appropriate expression profiles.
A preferred embodiment is a transgenic plant that is homozygous for the added
heterologous nucleic acid; i.e., a transgenic plant that contains two added
nucleic acid
sequences, one gene at the same locus on each chromosome of a chromosome pair.
A
homozygous transgenic plant can be obtained by sexually mating (selfing) a
heterozygous
transgenic plant that contains a single added heterologous nucleic acid,
germinating some
of the seed produced and analyzing the resulting plants produced for altered
expression of
a polynucleotide of the present invention relative to a control plant (i.e.,
native, non
transgenic). Back-crossing to a parental plant and out-crossing with a non-
transgenic
plant are also contemplated.
Modulating Poly~pentide Levels andlor Composition
The present invention further provides a method for modulating (i.e.,
increasing or
decreasing) the concentration or ratio of the polypeptides of the present
invention in a
plant or part thereof. Modulation can be effected by increasing or decreasing
the
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concentration and/or the ratio of the polypeptides of the present invention in
a plant. The
method comprises introducing into a plant cell a recombinant expression
cassette
comprising a polynucleotide of the present invention as described above to
obtain a
transformed plant cell, culturing the transformed plant cell under plant cell
growing
conditions, and inducing or repressing expression of a polynucleotide of the
present
invention in the plant for a time sufficient to modulate concentration and/or
the ratios of
the polypeptides in the plant or plant part.
In some embodiments, the concentration and/or ratios of polypeptides of the
present invention in a plant may be modulated by altering, in vivo or in
vitro, the promoter
of a gene to up- or down-regulate gene expression. In some embodiments, the
coding
regions of native genes of the present invention can be altered via
substitution, addition,
insertion, or deletion to decrease activity of the encoded enzyme. See, e.g.,
Kmiec, U.S.
Patent 5,565,350; Zarling et al., PCT/US93/03868. And in some embodiments, an
isolated
nucleic acid (e.g., a vector) comprising a promoter sequence is transfected
into a plant cell.
Subsequently, a plant cell comprising the promoter operably linked to a
polynucleotide of
the present invention is selected for by means known to those of skill in the
art such as, but
not limited to, Southern blot, DNA sequencing, or PCR analysis using primers
specific to
the promoter and to the gene and detecting amplicons produced there&om. A
plant or
plant part altered or modified by the foregoing embodiments is grown under
plant-forming
conditions for a time sufficient to modulate the concentration and/or ratios
of polypeptides
of the present invention in the plant. Plant-forming conditions are well known
in the art
and discussed briefly, supra.
In general, concentration or the ratios of the polypeptides is increased or
decreased
by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a
native
control plant, plant part, or cell lacking the aforementioned recombinant
expression
cassette. Modulation in the present invention may occur during and/or
subsequent to
growth of the plant to the desired stage of development. Modulating nucleic
acid
expression temporally and/or in particular tissues can be controlled by
employing the
appropriate promoter operably linked to a polynucleotide of the present
invention in, for
example, sense or antisense orientation as discussed in greater detail, supra.
Induction of
expression of a polynucleotide of the present invention can also be controlled
by
exogenous administration of an effective amount of inducing compound.
Inducible
promoters and inducing compounds which activate expression from these
promoters are
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well known in the art. In preferred embodiments, the polypeptides of the
present invention
are modulated in monocots, particularly maize.
UTRs and Colon Preference
In general, translational efficiency has been found to be regulated by
specific
sequence elements in the 5' non-coding or untranslated region (S' UTR) of the
RNA.
Positive sequence motifs include translational initiation consensus sequences
(Kozak,
Nucleic Acids Res.15:8125 (1987)) and the 7-methylguanosine cap structure
(Drummond
et al., Nucleic Acids Res. 13:7375 ( 1985)). Negative elements include stable
intramolecular 5' UTR stem-loop structures (Muesing et al., Cell 48:691
(1987)) and AZJG
sequences or short open reading frames preceded by an appropriate AUG in the
5' UTR
(Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284 (19$8)). Accordingly,
the present
invention provides 5' and/or 3' untranslated regions for modulation of
translation of
heterologous coding sequences.
1 S Further, the polypeptide-encoding segments of the polynucleotides of the
present
invention can be modified to alter colon usage. Altered colon usage can be
employed to
alter translational efficiency and/or to optimize the coding sequence for
expression in a
desired host such as to optimize the colon usage in a heterologous sequence
for expression
in maize. Colon usage in the coding regions of the polynucleotides of the
present
invention can be analyzed statistically using commercially available software
packages
such as "Colon Preference" available from the University of Wisconsin Genetics
Computer Group (see Devereaux et al., Nucleic Acids Res. 12: 387-395 (1984))
or
MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present
invention
provides a colon usage frequency characteristic of the coding region of at
least one of the
polynucleotides of the present invention. The number of polynucleotides that
can be used
to determine a colon usage frequency can be any integer from 1 to the number
of
polynucleotides of the present invention as provided herein. Optionally, the
polynucleotides will be full-length sequences. An exemplary number of
sequences for
statistical analysis can be at least 1, S, 10, 20, 50, or 100.
Secruence Shuffling
The present invention provides methods for sequence shuffling using
polynucleotides of the present invention, and compositions resulting
therefrom. Sequence
shuffling is described in PCT publication No. WO 97120078. See also, Zhang, J.-
H., et al.
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-so-
Proc. Natl. Acad. Sci. USA 94:4504-4509 (1997). Generally, sequence shuffling
provides
a means for generating libraries of polynucleotides having a desired
characteristic which
can be selected or screened for. Libraries of recombinant polynucleotides are
generated
from a population of related sequence polynucleotides which comprise sequence
regions
which have substantial sequence identity and can be homologously recombined in
vitro or
in vivo. The population of sequence-recombined polynucleotides comprises a
subpopulation of polynucleotides which possess desired or advantageous
characteristics
and which can be selected by a suitable selection or screening method. The
characteristics
can be any property or attribute capable of being selected for or detected in
a screening
system, and may include properties of: an encoded protein, a transcriptional
element, a
sequence controlling transcription, RNA processing, RNA stability, chromatin
conformation, translation, or other expression property of a gene or
transgene, a replicative
element, a protein-binding element, or the like, such as any feature which
confers a
selectable or detectable property. In some embodiments, the selected
characteristic will be
a decreased Kr" and/or increased K~a~ over the wild-type protein as provided
herein. In
other embodiments, a protein or polynucleotide generated from sequence
shuffling will
have a ligand binding affinity greater than the non-shuffled wild-type
polynucleotide. The
increase in such properties can be at least 110%, 120%, 130%, 140% or at least
150% of
the wild-type value.
Generic and Consensus Seauences
Polynucleotides and polypeptides of the present invention further include
those
having: (a) a generic sequence of at least two homologous polynucleotides or
polypeptides,
respectively, of the present invention; and, {b) a consensus sequence of at
least three
homologous polynucleotides or polypeptides, respectively, of the present
invention. The
generic sequence of the present invention comprises each species of
polypeptide or
polynucleotide embraced by the generic polypeptide or polynucleotide sequence,
respectively. The individual species encompassed by a polynucleotide having an
amino
acid or nucleic acid consensus sequence can be used to generate antibodies or
produce
nucleic acid probes or primers to screen for homologs in other species,
genera, families,
orders, classes, phyla, or kingdoms. For example, a polynucleotide having a
consensus
sequence from a gene family of Zea ways can be used to generate antibody or
nucleic acid
probes or primers to other Gramineae species such as wheat, rice, or sorghum.
Alternatively, a polynucleotide having a consensus sequence generated from
orthologous
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genes can be used to identify or isolate orthologs of other taxa. Typically, a
polynucleotide having a consensus sequence will be at least 9, 10, I5, 20, 25,
30, or 40
amino acids in length, or 20, 30, 40, 50, 10U, or I SO nucleotides in length.
As those of
skill in the art are aware, a conservative amino acid substitution can be used
for amino
acids which differ amongst aligned sequence but are from the same conservative
substitution group as discussed above. Optionally, no more than 1 or 2
conservative amino
acids are substituted for each 10 amino acid length of consensus sequence.
Similar sequences used for generation of a consensus or generic sequence
include
any number and combination of allelic variants of the same gene, orthologous,
or
paralogous sequences as provided herein. Optionally, similar sequences used in
generating
a consensus or generic sequence are identified using the BLAST algorithm's
smallest sum
probability (P(N)). Various suppliers of sequence-analysis software are listed
in chapter 7
of Current Protocols in Molecular Biology, F.M. Ausubel et al., :Eds., Current
Protocols, a
joint venture between Greene Publishing Associates, Inc. and John Wiley &
Sons, Inc.
(Supplement 30). A polynucleotide sequence is considered similar to a
reference sequence
if the smallest sum probability iu a comparison of the test nucleic acid to
the reference
nucleic acid is less than about 0.1, more preferably less than about 0.01, or
0.001, and must
preferably less than about 0.0001. or 0.00001. Similar polynucleotides can be
aligned and
a consensus or generic sequence generated using multiple sequence alignment
software
available from a number of commercial suppliers such as the Genetics Computer
Group's
(Madison, WI) PILEUP software, Vector NTI's (North Bethesda, MD) ALIGNX, or
Genecode's (Ann Arbor, MI) SEQUENCHER. Conveniently, default parameters of
such
software can be used to generate consensus or generic sequences.
Computer Applications
The present invention provides machines, data structures, and processes for
modeling or analyzing the polynucleotides and polypeptides of the present
invention.
A. Machines and Data Structures
The present invention provides a machine having a memory comprising data
representing a sequence of a polynucleotide or polypeptide of the present
invention. The
machine of the present invention is typically a digital computer. The memory
of such a
machine includes, but is not limited to, ROM, or RAM, or computer readable
media such
as, but not limited to, magnetic media such as computer disks or hard drives,
or media such
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as CD-ROM. Thus, the present invention also provides a data structure
comprising a
sequence of a polynucleotide of the present invention embodied in a computer
readable
medium. As those of skill in the art will be aware, the form of memory of a
machine of
the present invention or the particular embodiment of the computer readable
medium is not
a critical element of the invention and can take a variety of forms.
B. Homology Searches
The present invention provides a process for identifying a candidate homologue
(i.e., an ortholog or paralog) of a polynucleotide or polypeptide of the
present invention. A
candidate homologue has statistically significant probability of having the
same biological
function (e.g., catalyzes the same reaction, binds to homologous
proteins/nucleic acids) as
the reference sequence to which it's compared. Accordingly, the
polynucleotides and
polypeptides of the present invention have utility in identifying homologs in
animals or
other plant species, particularly those in the family Gramineae such as, but
not limited to,
i 5 sorghum, wheat, or rice.
The process of the present invention comprises obtaining data representing a
polynucleotide or polypeptide test sequence. Test sequences are.generally at
least 25
amino acids in length or at least 50 nucleotides in length. Optionally, the
test sequence can
be at least 50, 100, 150, 200, 250, .'00, or 400 amino acids in length. A test
polynucleotide
can be at least 50, 100, 200, 300, 400, or 500 nucleotides in length. Often
the test
sequence will be a full-length sequence. Test sequences can be obtained from a
nucleic
acid of an animal or plant. Optionally, the test sequence is obtained from a
plant species
other than maize whose function is uncertain but will be compared to the test
sequence to
determine sequence similarity or sequence identity; for example, such plant
species can be
of the family Gramineae, such as wheat, rice, or sorghum. The test sequence
data are
entered into a machine, typically a computer, having a memory that contains
data
representing a reference sequence. The reference sequence can be the sequence
of a
polypeptide or a polynucleotide of the present invention and is often at least
25 amino
acids or 100 nucleotides in length. As those of skill in the art are aware,
the greater the
sequence identity/similarity between a reference sequence of known function
and a test
sequence, the greater the probability that the test sequence will have the
same or similar
function as the reference sequence.
The machine further comprises a sequence comparison means for determining the
sequence identity or similarity between the test sequence and the reference
sequence.
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Exemplary sequence comparison means are provided for in sequence analysis
software
discussed previously. Optionally, sequence comparison is established using the
BLAST or
GAP suite of programs.
The results of the comparison between the test and reference sequences can be
displayed. Generally, a smallest sum probability value (P(N)) of less than
0.1, or
alternatively, less than 0.01, 0.001, 0.0001, or 0.00001 using the BLAST 2.0
suite of
algorithms under default parameters identifies the test sequence as a
candidate homologue
(i.e., an allele, ortholog, or paralog) of the reference sequence. A nucleic
acid comprising
a polynucleotide having the sequence of the candidate homologue can be
constructed using
well known library isolation, cloning, or in vitro synthetic chemistry
techniques (e.g.,
phosphoramidite) such as those described herein. In additional embodiments, a
nucleic
acid comprising a polynucleotide having a sequence represented by the
candidate
homologue is introduced into a plant; typically, these polynucleotides are
operably linked
to a promoter. Confirmation of the function of the candidate homologue can be
established by operably linking the candidate homolog nucleic acid to, for
example, an
inducible promoter, or by expressing the antisense transcript, and analyzing
the plant for
changes in phenotype consistent with the presumed function of the candidate
homolog.
Optionally, the plant into which these nucleic acids are introduced is a
monocot such as
from the family Gramineae. Exemplary plants include maize, sorghum, wheat,
rice.,
canola, alfalfa, cotton, and soybean.
C'. Computer Modeling
The present invention provides a process of modeling/analyzing data
representative
of the sequence a polynucleotide or polypeptide of the present invention. The
process
comprises entering sequence data of a polynucleotide or polypeptide of the
present
invention into a machine, manipulating the data to model or analyze the
structure or
activity of the polynucleotide or polypeptide, and displaying the results of
the modeling or
analysis. A variety of modeling and analytic tools are well known in the art
and available
from such commercial vendors as Genetics Computer Group (Version 10, Madison,
WI).
Included amongst the modeling/analysis tools are methods to: 1) recognize
overlapping
sequences (e.g., from a sequencing project) with a polynucleotide of the
present invention
and create an alignment called a "contig"; 2) identify restriction enzyme
sites of a
polynucleotide of the present invention; 3) identify the products of a T1
ribonuclease
digestion of a polynucleotide of the present invention; 4) identify PCR
primers with
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minimal self complementarity; 5) compare two protein or nucleic acid sequences
and
identifying points of similarity or dissimilarity between them; 6) compute
pairwise
distances between sequences in an alignment, reconstruct phylogenetic trees
using distance
methods, and calculate the degree of divergence of two protein coding regions;
7) identify
patterns such as coding regions, terminators, repeats, and other consensus
patterns in
polynucleotides of the present invention; 8) identify RNA secondary structure;
9) identify
sequence motifs, isoelectric point, secondary structure, hydrophobicity, and
antigenicity in
polypeptides of the present invention; and, 10) translate polynucleotides of
the present
invention and backtranslate polypeptides of the present invention.
i0
Detection of Nucleic Acids
The present invention further provides methods for detecting a polynucleotide
of
the present invention in a nucleic acid sample suspected of containing a
polynucleotide of
the present invention, such as a plant cell lysate, particularly a lysate of
maize. In some
15 embodiments, a gene of the present invention or portion thereof can be
amplified prior to
the step of contacting the nucleic acid sample with a polynucleotide of the
present
invention. The nucleic acid sample is contacted with the polynucleotide to
form a
hybridization complex. The polynucleotide hybridizes under stringent
conditions to a gene
encoding a polypeptide of the present invention. Formation of the
hybridization complex is
20 used to detect a gene encoding a polypeptide of the present invention in
the nucleic acid
sample. Those of skill will appreciate that an isolated nucleic acid
comprising a
polynucleotide of the present invention should lack cross-hybridizing
sequences in
common with non-target genes that would yield a false positive result.
Detection of the
hybridization complex can be achieved using any number of well known methods.
For
25 example, the nucleic acid sample, or a portion thereof, may be assayed by
hybridization
formats including but not limited to, solution phase, solid phase, mixed
phase, or in situ
hybridization assays.
Detectable labels suitable for use in the present invention include any
composition
detectable by spectroscopic, radioisotopic, photochemical, biochemical,
immunochemical,
30 electrical, optical or chemical means. Useful labels in the present
invention include biotin
for staining with labeled streptavidin conjugate, magnetic beads, fluorescent
dyes,
radiolabels, enzymes, and colorimetric labels. Other labels include ligands
which bind to
antibodies labeled with fluorophores, chemiluminescent agents, and enzymes.
Labeling
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the nucleic acids of the present invention is readily achieved such as by the
use of labeled
PCR primers.
Example 1
This example describes the construction of the cDNA libraries.
Total RNA Isolation
Total RNA was isolated from maize tissues with TRIzoITM Reagent (Life
Technologies, Inc., Rockville, MD) using a modification of the guanidine
isothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi
(Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, l5fi (19$7)). In brief,
plant tissue
samples were pulverized in liquid nitrogen before the addition of the TRIzoI
Reagent, and
then were further homogenized with a mortar and pestle. Addition of chloroform
followed
by ce;~trifugation was conducted for separation of an aqueous phase and an
organic phase.
I ~ The total RNA was recovered by precipitation with isopropyl alcohol from
the aqueous
phase.
Poly(A)-~- RNA Isolation
The selection of poly(A)-~ R.NA from total RNA was performed using
PolyATtract~ system (Promega Curporation, Madison, WI). In brief, biotinylated
oligo(dT) primers were used to hybridize to the 3' poly(A) tails on mRNA. The
hybrids
were captured using streptavidin coupled to paramagnetic particles and a
magnetic
separation stand. The rnRNA was washed at high stringency conditions and
eluted by
RNase-free deionized water.
cDNA Library Construction
cDNA synthesis was performed and unidirectional cDNA libraries were
constructed using the SuperScriptTM Plasmid System (Life Technologies, Inc.,
Rockville,
MD). The first strand of cDNA was synthesized by priming an oligo(dT) primer
34 containing a Not I site. The reaction was catalyzed by SuperScriptTM
Reverse
Transcriptase II at 45°C. The second strand of cDNA was labeled with
alpha-3zP-dCTP
and a portion of the reaction was analyzed by agarose gel electrophoresis to
determine
cDNA sizes. cDNA molecules smaller than 504 base pairs and unligated adapters
were
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removed by Sephacryl-5400 chromatography. The selected cDNA molecules were
ligated
into pSPORT 1 vector in between of Not I and Sal I sites.
Example 2
This example describes cDNA sequencing and library subtraction.
Sequencing Template Preparation
Individual colonies were picked and DNA was prepared either by PCR with M13
forward primers and M 13 reverse primers, or by plasmid isolation. All the
cDNA clones
were sequenced using M13 reverse primers.
Q-bot Subtraction Procedure
cDNA libraries subjected to the subtraction procedure were plated out on 22 x
22
cm2 agar plate at density of about 3,000 colonies per plate. The plates were
incubated in a
37°C incubator for 12-24 hours. Colonies were picked into 384-well
plates by a robot
colony picker, Q-bot (GENETIX Limited). These plates were incubated overnight
at
37°C. Once sufficient colonies were picked, they were pinned onto 22 x
22 cmz nylon
membranes using Q-bot. Each membrane contained 9,216 colonies or 36,864
colonies.
These membranes were placed onto agar plate with appropriate antibiotic. The
plates were
incubated at 37°C for overnight. After colonies were recovered on the
second day, these
filters were placed on filter paper prewetted with denaturing solution for
four minutes, then
were incubated on tvp of a boiling water bath for additional four minutes. The
filters were
then placed on filter paper prewetted with neutralizing solution for four
minutes. After
excess solution was removed by placing the filters on dry filter papers for
one minute, the
colony side of the filters were place into Proteinase K solution, incubated at
37°C for 40-
50 minutes. The filters were placed on dry filter papers to dry overnight. DNA
was then
cross-linked to nylon membrane by UV light treatment.
Colony hybridization was conducted as described by Sambrook,J., Fritsch, E.F.
and
Maniatis, T., (in Molecular Cloning: A laboratory Manual, 2°d Edition).
The following
probes were used in colony hybridization:
1. First strand cDNA from the same tissue as the library was made from to
remove
the most redundant clones.
2. 48-192 most redundant cDNA clones from the same library based on previous
sequencing data.
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3. 192 most redundant eDNA clones in the entire maize sequence database.
4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAA
AAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA.
$. eDNA clones derived from rRNA.
The image of the autoradiography was scanned into computer and the signal
intensity and cold colony addresses of each colony was analyzed. Re-arraying
of cold-
colonies from 384 well plates to 96 well plates was conducted using Q-bot.
Example 3
This example describes identification of the gene from a computer homology
search. Gene identities were determined by conducting BLAST (Basic Local
Alignment
Search Tool; Altschul, S. F., et al., ( 1993) J. Mol. Biol. 21$:403-410; see
also
www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for similarity
to
1$ sequences contained in the BLAST "nr" database (comprising all non-
redundant GenBank
CDS translations, sequences derived from the 3-dimensional structure
Brookhaven Protein
Data Bank, the last major release of the SWISS-PROT protein sequence database,
h,MBL,
and DDBJ databases). The cDNA sequences were analyzed for similarity to all
publicly
available DNA sequences contained in the "nr" database using the BLASTN
algorithm.
The DNA sequences were translated in all reading frames and compared for
similarity to
all publicly available protein sequences contained in the "nr" database using
the BLASTX
algorithm (Gish, W. and States, D. J. Nature CJenetics 3:266-272 (1993))
provided by the
NCBI. In some cases, the sequencing data from two or more clones containing
overlapping segments of DNA were used to construct contiguous DNA sequences.
2$
Examine 4
This example provides an analysis of the transit peptides of the present
invention in
comparison to other plant alternative oxidase genes.
Table 1
CLUSTAL W ( 1.74) multiple sequence alignment of selected Plant Alternative
Oxidase Genes.
ZmAOXl Zea mat's Alternative Oxidase 1, Herein.
ZmA0X2 Zea mat's Alternative Oxidase 2, Herein.
OsAOXla Oryza sativa Alternative Oxidase 1a, Acc. AB004864
3$ OsAOXlb Oryza sativa Alternative Oxidase lb, Acc. AB004865
SgAOXl Sauromatum guttatum Alternative Oxidase 1, Acc.
NtAOXl Nicotiana tabacum Alternative Oxidase 1, Acc. X79768
NtAOX Nicotiana tabacum Alternative Oxidase, Acc. S71335
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CrAOX Catharanthus roseus
Alternative Oxidase,
Acc. AB009395
AtAOXla Arabidopsis thaliana
Alternative Oxidase
la, Acc. D89875
AtAOXlb Arabidopsis thaliana
Alternative Oxidase
lb, Acc. D89875
AtAOXlc Arabidopsis thaliana
Alternative Oxidase
lc, Acc. AB003175
S GmAOXl Glycine max Alternative
Oxidase 1, Acc.
X68702
GmAOX2 Glycine max Alternative
Oxidase 2, Acc.
U87906
GmAOX3 Glycine max Alternative
Oxidase 3, Acc.
U87907
IO ZmAOXl ---MSTRA---AGSALLRHLGPRVFG------------------PVFSPAVAPPRPLLAL
_ ---MSSRM---AGATLLRHLGPRLFAA----------------EPVYSGLAASARGVMPA
OSAOXlb_
ZmAOX2 --MMSSR----AGSILLRHAG-SRLFT----------------AAAISP----AAASRPL
_ ---MSSRM---AGSAILRHVGGVRLFT----------------ASATSPAAAAAAAARPF
OsAOXla
_ --MISSRL---AGTALCRQLSHVPVPQY---------------LPALRPTADTASSLLHR
SgAOXl-
IS NtAOXl _-_____________-____________________-____-__________________
NtAOX ---MMTRGATRMTRTVLGHMGPRYFSTAIFR-NDAGTGVMS--GAAVFMHGVPANPSEKA
_ ---MMSRGATRISRSLICQISPRYFSSAAVRGHEPSLGILTSGGTTTFLHGNPGNGSERT
CrAOX
_ --MMMSRR--YGAKLMETAVTH----------------------------SHLLNPRVPL
AtAOXlb
_ MITTLLRRSLLDASKQATSIN-----------------------------GILFHQLAP-
AtAOXIc
2O _ --MMITRGGAKAAKSLLVAAGPRLFSTVRTV-----------SSHEALSASHILKPGVTS
AtAOXla
_ MMMMMSRS---GANRVANTAM-------------------------------F-VAKGLSG
GmA0X1
_ --MKLTALNSTVRRALLNGRNQN-----------------GNRLGSAALMPYAAAETRLL
GmAOX2
_ --MKNVLVRSAAR-ALLGG---------------------GGRSYYRQLSTAAIVEQRHQ
GmAOX3
2S
ZtrtAOXl AGGGERGGALVWVRVRL~LST-SAAEAKEEVAASKGNSGS-TAAAKAEAVEAAKEGDGKRD
_ AAR-------IFPARM~ASTSSAGADVKEGAAEKLPEPAATAAAAAT------DPQNK--
OsAOXlb
_ LAGGNGVP-AVM-LRL~MSTSSPAAP---TEAKD--EAAKASKVGGD---------KKA
ZmA0X2
_ LAGGEAVP-GVWGLRL~MSTSSVAS----TEA-----AAKAEAKKADA--------EKE
OsAOXla
O _ CSAAAPAQRAGLWPPSWFSPPRH~ASTLSARAQDGGKEKAAGTAGKVPPG-----EDGGAEK
SgAOXl_
NtAOXl --MWVRH-FPVMGPRS~ASTVALND-KQHDKKVENGG-------AAASGG------GDGGDE
- WTWVRH-FPVMGSRS ~AMSMALND-KQHDKKAENGS------AAATGG------GDGGDE
NtAOX
_ ALTWIK--LPMMRARS ~ASTVATVDQKDKDEKREDKN-------GVADG--------ENGN-
CrAOX
_ VTENIRV-PAMGWRV~FSKMTFEKKKTTEEK-GSS---------GGKA-----DQGNKGE
AtAOXlb
3S _ -AKYFRV-PAVGGLRD~FSKMTFEKKKTSEEEEGSGD-------GVKV-N---DQGNKGE
AtAOXlc
_ AWIWTRA-PTIGGMRF~ASTITLGEKTPMKEEDANQKKTENESTGGDA-A---GGNNKGD
AtAOXIa
_ EVGGLRA-LYGGGVRS~ESTLALSEKEKIEKKVGLSS----------------AGGNKEE
GmAOXI
_ CAGGANGW-FFYWKRT~MVSPAEAKVPEKEKEKEK------------------AKAEKS
GmAOX2
_ HGGGAFG---SFHLRR~MSTLP-EVKDQHSEEKKNE------------------VNGTSN
GmAOX3
40 _ (Transit Peptide
Cleavage Site)
ZmAOXI KWSSYWGVA-PS-KLMNKDGAEWRWSCFRPWEAYKPDTTIDLNRHHEPKVLLDKIAYWT
4S _ KAWSYWGIQ-PP-KLVKEDGTEWKWLSFRPWDTYTSDTSIDVTKHHEPKGLPDKLAYWT
OsAOXlb
_ WINSYWGIE-QNNKLARDDGTEWKWTCFRPWETYTADTSIDLTRHHEPKTLMDKVAYWT
ZmAOX2
_ VVVNSYWGIE-QSKKLVREDGTEWKWSCFRPWETYTADTSIDLTKHHVPKTLLDKIAYWT
OsAOXIa
_ EAWSYWAVP-PS-KVSKEDGSEWRWTCFRPWETYQADLSIDLHKHHVPTTILDKLALRT
SgA0X1-
NtAOXl KSWSYWGVP-PS-KVTKEDGTEWKWNCFRPWETYKADLSIDLTKHHAPTTFLDKFAYWT
SO _ KSWSYWGVQ-PS-KVTKEDGTEWKWNCFRPWETYKADLSIDLTKHHAPTTFLDKFAYWT
NtAOX
_ KAWSYWGVE-AP-KLTKEDGTVWRWTCFRPWETYKPDTDIELKKHHVPVTLLDKVAFFT
CrAOX
_ QLIVSYWGVK-PM-KITKEDGTEWKWSCFRPWETYKSDLTIDLKKfiHVPSTLPDKLAYWT
AtAOXlb
_ QLIVSYWGVK-PM-KITKEDGTEWKWSCFRPWETYKADLTIDLKKHHVPSTLPDKIAYWM
AtAOXIc
_ KGIASYWGVE-PN-KITKEDGSEWKWNCFRPWETYKADITIDLKKHHVPTTFLDRIAYWT
AtAOXla
SS _ KVIVSYWGIQ-PS-KITKKDGTEWKWNCFSPWGTYKADLSIDLEKHMPPTTFLDKMAFWT
GmAOXl
_ WESSYWGIS--RPKWREDGTEWPWNCFMPWESYRSNVSIDLTKHHVPKNVLDKVAYRT
GmA0X2
_ AWTSYWGIT--RPKVRREDGTEWPWNCFMPWDSYHSDVSIDVTKHHTPKSLTDKVAFRA
GmAOX3
_ ***,. *: ..**: *
* .* ** ;* _, *:.
:* * . *:.*
f)OZmA0X1 VKLLRVPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEHSGGWIRALLEEA
_ VRSLAVPRDLFFQRRHASHALLLETVAGVPGMVGGMLLHLRSLRRFEQSGGWIRALLEEA
OsAOXlb
_ VKSLRFPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEQSGGWIRALLEEA
ZmA0X2
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OsAOXIa_ VKSLRFPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEQSGGWIRTLLEEA
SgAOXl_ VKALRWPTDIFFQRRYACRAMMLETVAAVPGMVGGVLLHLKSLRRFEHSGGWIRALLEEA
NtAOXI- VKALRYPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEQSGGWIKALLEEA
NtAOX_ VKSLRYPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEQSGGWIKTLLDEA
S CrAOX_ VKALRWPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEHSGGWIKALLEEA
AtAOXIb_ VKSLRWPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLVHCKSLRRFEQSGGWIKALLEEA
AtAOXlc- VKSLRWPTDLFFQRRYGCRAIMLETVAAVPGMVGGMLMHFKSLRRFEQSGGWIKALLEEA
AtAOXla- VKSLRWPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEQSGGWIKALLEEA
GmAOXl_ VKVLRYPTDVFFQRRYGCRAMMLETVAAVPGMVAGMLLHCKSLRRFEHSGGWFKALLEEA
IO GmAOX2_ VKLLRIPTDLFFKRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRKFQQSGGWIKALLEEA
GmAOX3- VKFLRVLSDIYFKERYGCHAMMLETTAAVPGMVGGMLLHLKSLRKFQHSGGWIKALLEEA
*; * * " *:.*:..:*::***;*,*****.*;*;* ;*.*;*;;****.;;**.**
ZmAOXI_ ENERMHLMTFMEVAKPKWYERALVLAVQGVFFNAYFLGYLISPKFAHRWGYLEEEAIHS
IS OsAOXlb_ ENERMHLMTFLEVMQPRWWERALVLAAQGVFFNAYFVGYLVSPKFAHRFVGYLEEEAVSS
ZmA0X2_ ENERMHLMTFMEVAKPRWYERALVITVQGVFFNAYFLGYLLSPKFAHLWGYLEEEAIHS
OsAOXla_ ENERMHLMTFMEVANPKWYERALVITVQGVFFNAYFLGYLLSPKFAHRWGYLEEEAIHS
SgAOXI_ ENERMHLMTFMEVAQPRWYERALVLAVQGVFFNAYFLGYLLSPKFAHRWGYLEEEAIHS
NtAOXl_ ENERMHLMTFMEVAKPNWYERALVFAVQGVFINAYFVTYLLSPKLAXRIVGYLEEEAIHS
ZO NtAOX_ ENERMHLMTFMEVAKPNWYERALVFAVQGVFFNAYFVTYLLSPKLAHRIVGYLEEEAIHS
CrAOX_ ENERMHLMTFMEVSKPRWYERALVFAVQGVFFNAYFLTYLASPKLAHRIVGYLEEEAIHS
AtAOXlb- ENERMHLMTFMEVAKPNWYERALVIAVQGIFFNAYFLGYLISPKFAHRMVGYLEEEAIHS
AtAOXIc- ENERMHLMTFMEVAKPKWYERALVISVQGVFFNAYLIGYIISPKFAHRMVGYLEEEAIHS
AtAOXla_ ENERMHLMTFMEVAKPKWYERALVITVQGVFFNAYFLGYLISPKFAHRMVGYLEEEAIHS
ZS GmAOXl_ ENERMHLMTFMEVAKPKWYERALVITVQGVFFNAYFLGYLLSPKFAHRMFGYLEEEAIHS
GmAOX2_ ENERMHLMTMVELVKPKWYERLLVLAVQGVFFNAFFVLYILSPKVAHRIVGYLEEEAIHS
GmAOX3- ENERMHLMTMVELVKPSWHERLLIFTAQGVFFNAFFVFYLLSPKAAHRFVGYLEEEAVIS
*********;,*: :* * ** *;. .**.*;*,... *. **; * _.*******:
3O ZtnAOXl_ YTEYLKDLF.AGKIENVPAPAIAIDYWQLPADATLKDVVVWRSDEAHHRDVNHFASDIHF
OsAOXlb- YTEYLKDLEAGKIENTPAPAIAIDYWRLPADATLKDWTVIRADEAHHRDLNHFASDIQQ
ZmAOX2_ YTEYLKDLEAGKIENVPAPAIAIDYWRLPANATLKDWTVVI2ADEAHHRDVNHFASDIHC
OsAOXla_ YTEFLKDLEAGKIDNVPAPAIAIDYWRLPANATLKDWTWRADEAHHRDVNHFASDIHY
SgAOXl_ YTEFLKDIDNGAIQDCPAPAIALDYWRLPQGSTLRDWTVVRADEAHHRDVNHFASDVHY
3S NtAOXl_ YTEFLKELDKGNIENVPAPAIATDYWRLPKDSTLRBWLVVRADEAHHRDVVFIFAPDIHY
NtAOX_ YTEFLKELDKGNIENVPAPAIAIDYCRLPKDSTLLDWLWRADEAHHRDVNHFASDTHY
CrAOX_ YSEFLNELDKGNIENVPAPAIAIDYWQMPPDSTLRDWMWRADEALHRDVNHYASDIHY
AtAOXIb_ YTEFLKELDNGNIENVPAPAIAIDYWRLEADATLRDWMWRADEAHHRDVNHYASDIHY
AtAOXlc_ YTEFLKELDNGNIENVPAPAIAVDYWRLEADATLRDVVMWRADEAHHRDVNHYASDIHY
O AtAOXla_ YTEFLKELDKGNIENVPAPAIAIDYWRLPADATLRDWMWRP~EAHHRDVNHFASDIHY
GmAOXl_ YTEFLKELDKGNIENVPAPAIAIDYWQLPPGSTLRDVVMWRADEAHHRDVNHFASDIHY
GmAOX2_ YTEYLKDLESGAIENVPAPAIAIDYWRLPKDARLKDVITVIRADEAHHRDVNHFASDIHF
GmAOX3_ YTQHLNAIESGKVENVPAPAIAIDYWRLPKDATLKDWTVIRADEAIiHRDVNHFASDIHH
*:..*: .. * ... ,*****;** * **. *;*.*** ***.**.* *.
aS .. .. . . . . . ..
ZmAOXl_ QGMQLKETPAPIEYH
OsAOXIb_ QGMKLKDTPAPIGYH
ZmAOX2_ QGMQLKQSPAPIGYH
OsAOXla_ QGMELKQTPAPIGYH
SO SgAOXl_ QDLELKTTPAPLGYH
NtAOXl- QGQQLKDSPAPIGYH
NtAOX_ QGQQLKDSPAPIGYH
CrAOX_ KGLELKEAAAPLDYH
AtAOXlb_ QGRELKEAPAPIGYH
SS AtAOXlc- QGHELKEAPAPIGYH
AtAOXla- QGRELKEAPAPIGYH
GmAOXl_ QGRELREAAAPIGYH
GmAOX2 QGKELREAPAPIGYH
~
GmAOX3_ QGKELKEAPAPIGYH
O .*. **. **
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The known or predicted transit peptide cleavage sites are denoted by a
vertical line
( ~ ). The first three amino acids of the mature peptides are underlined. The
penultimate
amino acid of the transit peptide, the conserved Arginine, is in boldface.
Please note that
the transit peptides are considerably divergent in size and sequence, whereas
the bulk of
the mature protein coding region is highly conserved. The alignment of the
maize
sequences to these other plant AOX genes sequences clearly reveals the likely
transit
peptide cleavage site, even though the transit peptides, with the possible
exception of those
for the rice AOX clones, are quite different.
Example 5
This example describes the profiling of ZmAOX3 mRNA in Zea mat's GS3 cell
suspension cultures following exposure to spores of the fungus Fusarium
moniliforme or
to chito-oligosaccharides.
Maize GS3 (HYII) cells were grown as suspension cultures to mid-log phase,
when
trey were treated with either 1 rrrl water (control), l ml Fusarium
moniliforrne spores to
give a final concentration of 100,000 spores/ml, or 1 ml chito-oligosaccharide
mixture to
give a final woncentration of 100 pg/ml. The chito-oligosaccharide mixture was
a partial
hydrolysate of crab shell chitin from CarboMer, Inc. {Westborough, MA). (See
Yalpani,
M. and D. Pantaleone (1994) Carbohydrate Research 256:159-175 for details of
preparation of the chito-oligosaccharides.)
Cells were harvested at 2 hours and 6 hours post-treatment and immediately
frozen
in liquid nitrogen and kept at -80°C until RNA extraction.
RNA extraction and polyA+RNA isolation were performed as described in
Example 1. Double-stranded cDNA was synthesized using the SuperScriptTM
Plasmid
System (Life Technologies, Inc., Roekville, MD). In-vitro transcription
labeling of eRNA
with biotin conjugated ribonucleotides was performed with the MEGAscript TM T7
kit
(Ambion, Inc., Austin, TX), followed by the QIAGEN, Inc. (Valencia, CA)
RNeasy~ mini
protocol for RNA cleanup. The resulting cRNA was fragmented and hybridized for
16
hours to a customized GeneChip~ array of Zea mat's oligonucleotides, then
washed and
stained with streptavidin, R-phycoerythrin conjugate, using the Affymetrix
GeneChip
Fluidics Station. The Hewlett-Packard G2500A Gene Array Scanner and Affymetr-
ix
GeneChip Analysis Suite software were used to analyze the results.
Data for ZmAOX3 showed the following changes in expression level in response
to the described treatment:
CA 02355616 2001-06-29
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_m _
2-hour exposure to Fusarium moniliforme spores: 4.8-fold increase
6-hour exposure to Fusarium moniliforme spores: 5.0-fold increase
2-hour exposure to chito-oligosaccharide mixture: 9.5-fold increase
6-hour exposure to chito-oligosaccharide mixture: 8.9-fold increase
S These data support the conclusion that ZmAOX genes are upregulated in
defense
situations and illustrate their potential utility in engineering plant cold
tolerance and
disease resistance.
References
Chivasa, S. et al. (1997) Plant Cell 9, 547-557.
Connett, M.B. and bIanson, M.R. ( 1990) Plant Physiol. 93, 1634-1640.
Ito, Y., et al. (1997) Gene 203(2), 121-129.
Lennon, A.M. et al. ( 1997) Plant Physiol. 115, 783-791.
McCaig, T.N. et al. (1977) Can. J. Bot. 55:549-555.
Musgrave, M.E. et al. (1986) Plant Sci. 33, 7-11.
Yolidoros, A.N. et al. (1997) GenBank Direct Submission. Accession AF040566.
Submitted (30-DEC-1997).
Rhoades, D.M. et al. (1993) Plant Mol. Bio. Int. J. Mol. Biol. Biochem. Genet.
Eng.
21, 615-624.
Stewart, C.R. et al. ( 1990a) Plant Physiol. 92, 755-760.
Stewart, C.R. et al. (1990b) Plant Physiol. 92, 761-766.
Vanlerberg~he, G.C. et al. (1992a) Plant Physiol. 100, 115-119.
Vanlerberghe, G.C. et al. ( 1992b) Plant Physiol. 100, 1846-1851.
Vanlerberghe, G.C. et al. (1997a) Plant Physiol. 113, 657-661.
Vanlerberghe, G.C. et al. (1997b) Ann Rev. Plant. Physiol. Plant Mol. Bio. 48,
703-734.
Whelan, J. et al. (1995) Plant Mol. Bio. 27, 769-778.
The above examples are provided to illustrate the invention but not to limit
its
scope. Other variants of the invention will be readily apparent to one of
ordinary skill in
the art and are encompassed by the appended claims. All publications, patents,
patent
applications, and computer programs cited herein are hereby incorporated by
reference.
The polynucleotides of SEQ ID NOS: l, 4, and 7 are contained in a deposit made
to the American Type Culture Collection (ATCC) on January 14, 2000, and
assigned
Accession Number PTA-1209. American Type Culture Collection is located at
10801
CA 02355616 2001-06-29
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University Blvd., Manassas, VA 20110-2209.
The ATCC deposit will be maintained under the terms of the Budapest Treaty on
the International Recognition of the Deposit of Microorganisms for the
Purposes of Patent
Procedure. The deposit is provided as a convenience to those of skill in the
art and is not
an admission that a deposit is required under 35 U.S.C. Section 112. The
deposited
sequences, as well as the polypeptides encoded by the sequences, are
incorporated herein
by reference and control in the event of any conflict, such as a sequencing
error, with the
description in this application.
CA 02355616 2001-06-29
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SEQUENCE LISTING
<110> Pioneer Hi-Bred International, Inc.
<120> Maize Alternative Oxidase Genes and Uses
Thereof
<130> 0963-PCT
<150> US 60/117,776
<151> 1999-01-29
<160> 9
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 1520
<212> DNA
<213> Zea mays
<220>
<221> CDS
<222> (124)...{1164)
<400> 1
caggcaaaca ctgaaacact gagcaaacag caaagcacgc
60
agtaccacag
tagcagcgag
tctggagaaa tccctcgtct ctagtggccg tcgtccgacc
120
accttcacca
gttgaccgca
acgatg agcacccgc gcggcagga tccgccctc ctccgccac ctgggt 168
Met SexThrArg AlaAlaGly SerAlaLeu LeuArgHis LeuGly
1 5 10 15
ccgcgc gtcttcggc ccagttttt tctccggcg gtcgcgccg ccgagg 216
ProArg ValPheGly ProValPhe SerProAla ValAlaPro ProArg
20 25 30
ccactg ctggccttg gccggcggc ggggaacgg ggcggggcg ctcgtg 264
ProLeu LeuAlaLeu AlaGlyGly GlyGluArg GlyGlyAla LeuVal
35 40 45
tgggtg cgggtgcgg ctactgtcc acctccgcc gccgaggcg aaggag 312
TrpVal ArgValArg LeuLeuSer ThrSerAla AlaGluAla LysGlu
50 55 60
gaggtg gcggcgtcc aaggggaac tcaggaagc accgcggcg gcgaag 360
GluVal AlaAlaSer LysGlyAsn SerGlySer ThrAlaAla AlaLys
65 70 75
gcggag gcggtggag gccgetaag gagggtgac ggaaagaga gacaaa 408
AlaGlu AlaValGlu AlaAlaLys GluGlyAsp GlyLysArg AspLys
80 85 90 95
gtggtg agcagctac tggggcgtc gcgccgtcg aagctgatg aacaag 456
ValVal SerSerTyr TrpGlyVal AlaProSer LysLeuMet AsnLys
100 105 110
gacggc gccgagtgg aggtggtct tgcttcagg ccatgggag gcgtac 504
AspGly AlaGluTrp ArgTrpSer CysPheArg ProTrpGlu AlaTyr
115 120 125
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aag ccggacacc acgatcgat ctcaac agacaccac gaacccaag gtg 552
Lys ProAspThr ThrIleAsp LeuAsn ArgHisHis GluProLys Val
130 135 140
ctg ctcgacaag atcgcctat tggacc gtcaaatta ctgcgcgtg ccc 600
Leu LeuAspLys IleAlaTyr TrpThr ValLysLeu LeuArgVal Pro
145 150 155
acc gacatattc ttccagagg aggtac ggctgccgt getatgatg ctg 648
Thr AspIlePhe PheGlnArg ArgTyr GlyCysArg AlaMetMet Leu
160 165 170 175
gaa acagtggcg gcggtgccg gggatg gtgggcggc atgctgctt cac 696
Glu ThrValAla AlaValPro GlyMet ValGlyGly MetLeuLeu His
180 185 190
ctg cgctcgctc cgccgcttc gagcac agcggcggc tggatccgg gcg 744
Leu ArgSexLeu ArgArgPhe GluHis SerGlyGly TrpIleArg Ala
195 200 205
ctg ctggaggag gcggagaat gaacgc atgcacctc atgaccttc atg 792
Leu LeuGluGlu AlaGluAsn GluArg MetHisLeu MetThrPhe Met
210 215 220
gag gtggccaag cccaagtgg tacgag cgcgcgctt gtcctcgcc gtg 840
Glu ValAlaLys ProLysTrp TyrGlu ArgAlaLeu ValLeuAla Val
225 230 235
cag ggcgtcttc ttcaacgcc tacttc ctcggctac ctcatctcc ccc B88
Gln GlyValPhe PheAsnAla TyrPhe LeuGlyTyr LeuIleSer Pro
240 245 250 255
aag ttcgcgcac cgtgtcgtt gggtac ctcgaggag gaggccatc cac 936
Lys PheAlaHis ArgValVal GlyTyr LeuGluGlu GluAlaIle His
260 265 270
tca tataccgaa tacctcaag gacctc gaggccggc aagatcgag aac 984
Ser TyrThrGlu TyrLeuLys AspLeu GluAlaGly LysIleGlu Asn
275 280 285
gtc cccgcgccg gccattgcc atcgac tactggcag ctcccaget gat 1032
Val ProAlaPro AlaIleAla IleAsp TyrTrpGln LeuProAla Asp
290 295 300
gcg acgctcaag gatgtggtt gtcgtg gtgcgctcc gacgaggcg cac 1080
Ala ThrLeuLys AspValVal ValVal ValArgSer AspGluAla His
305 310 315
cac cgcgacgtc aatcacttt gcgtcg gacatacat ttccagggt atg 1128
His ArgAspVal AsnHisPhe AlaSer AspIleHis PheGlnGly Met
320 325 330 335
cag ctcaaggag acacctgca ccgatt gagtaccat tgaacaatcg 1174
Gln LeuLysGlu ThrProAla ProIle GluTyrHis
340 345
gggtcctgtg tgaga tccagttcatt ttggctagctgtaggta ctaaagatg1234
acgct gt g
cttgagaaat gaaaa cctggctgcta tgagtagcaaagatctc tgggttgat1294
aaaaa tg g
cctaaaatct gtgag tttgtaagtag agatacagacattgata gtgcactga1354
tttac tg c
atcttgtcca tacca gacttggttgg ttccacagagaaaactt atatccgat1414
gaaca tc t
tgtaacagag ttttttttc gaaaaggagca acgaaggagccgcgcgt gtcgatttt1474
t ct c
ctaaaaaaaa aaaaa aaaaaaaaaaa aaaaaaaaaaaaa 1520
aaaaa aa
_Z_
CA 02355616 2001-06-29
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<210> 2
<211> 347
<212> PRT
<213> Zea mays
<400> 2
Met Ser Thr Arg Ala Ala Gly Ser Ala Leu Leu Arg His Leu Gly Pro
1 5 10 15
Arg Val Phe Gly Pro Val Phe Ser Pro Ala Val Ala Pro Pro Arg Pro
20 25 30
Leu Leu Ala Leu Ala Gly Gly Gly Glu Arg Gly Gly Ala Leu Val Trp
35 40 45
Val Arg Val Arg Leu Leu Ser Thr Ser Ala Ala Glu Ala Lys Glu Glu
50 55 60
Val Ala Ala Ser Lys Gly Asn Ser Gly Ser Thr Ala Ala Ala Lys Ala
65 70 75 80
Glu Ala Val Glu Ala Ala Lys Glu Gly Asp Gly Lys Arg Asp Lys Val
85 90 95
Val Ser Ser Tyr Trp Gly Val Ala Pro Ser Lys Leu Met Asn Lys Asp
100 105 110
Gly Ala Glu Trp Arg Trp Ser Cys Phe Arg Pro Trp Glu Ala Tyr Lys
115 120 125
Pro Asp Thr Thr Ile Asp Leu Asn Arg His His Glu Pro Lys Val Leu
130 135 140
Leu Asp Lys Ile Ala Tyr Trp Thr Val Lys Leu Leu Arg Val Pro Thr
145 150 155 160
Asp Ile Phe Phe Gln Arg Arg Tyr Gly Cys Arg Ala Met Met Leu Glu
165 170 175
Thr Val Ala Ala Val Pro Gly Met Val Gly Gly Met Leu Leu His Leu
180 185 190
Arg Ser Leu Arg Arg Phe Glu His Ser Gly Gly Trp Ile Arg Ala Leu
195 200 205
Leu Glu Glu Ala Glu Asn Glu Arg Met His Leu Met Thr Phe Met Glu
210 215 220
Val Ala Lys Pro Lys Trp Tyr Glu Arg Ala Leu Val Leu Ala Val Gln
225 230 235 240
Gly Val Phe Phe Asn Ala Tyr Phe Leu Gly Tyr Leu Ile Ser Pro Lys
245 250 255
Phe Ala His Arg Val Val Gly Tyr Leu Glu Glu Glu Ala Ile His Ser
260 265 270
Tyr Thr Glu Tyr Leu Lys Asp Leu G1u Ala Gly Lys Ile Glu Asn Val
275 280 285
Pro Ala Pro Ala Ile Ala Ile Asp Tyr Trp Gln Leu Pro Ala Asp Ala
290 295 300
Thr Leu Lys Asp Val Val Val Val Val Arg Ser Asp Glu Ala His His
305 310 315 320
Arg Asp Val Asn His Phe Ala Ser Asp Ile His Phe Gln Gly Met Gln
325 330 335
Leu Lys Glu Thr Pro Ala Pro Ile Glu Tyr His
340 345
<210> 3
<211> 52
<212> PRT
<213> Zea mays
<400> 3
Met Ser Thr Arg Ala Ala Gly Ser Ala Leu Leu Arg His Leu Gly Pro
1 5 10 15
Arg Val Phe Gly Pro Val Phe Ser Pro Ala Val Ala Pro Pra Arg Pro
20 25 30
_;_
CA 02355616 2001-06-29
WO PCT/US00/01847
00/44920
Leu Leu Ala Gly Gly Ala LeuVal Trp
Leu Gly Glu Gly
Ala Gly Arg
35 40 45
Val Arg
Arg
Val
50
<210> 4
<211> 1457
<212> DNA
<213> Zea mat's
<220>
<221> CDS
<222> (108)...(1094)
<400> 4
cgaaaaccca aaaac gtggccca ccaacgattc acttccccga ggggg60
cgtct ag tccca
cggcgatcgg c ctcecacg cggcgaacac ggcagag atgatg agc 116
aattcgcaa tt
MetMet Ser
1
tcc cgg gga tccatcctc ctccgccac gccggctcc cgtctc ttc 164
gcc
Ser Arg Gly SerIleLeu LeuArgHis AlaGlySer ArgLeu Phe
Ala
5 10 15
acc gca gcg atctctccg gcggcggcc tcgaggcca ctgctc gcc 212
gcg
Thr Ala Ala IleSerPro AlaAlaAla SerArgPro LeuLeu Ala
Ala
20 25 30 35
ggc ggc ggt gttccggca gtcatgcta cggcttatg tccacg tcc 260
aat
Gly Gly Gly ValProAla ValMetLeu ArgLeuMet SerThr Ser
Asn
40 45 50
tcc ccc get cccacggag gcgaaggac gaggcagcc aaggcc tcc 308
gcc
Ser Pro Ala ProThrGlu AlaLysAsp GluAlaAla LysAla Ser
Ala
55 60 65
aag gtg gga gacaagaag gcggtggtg atcaacagc tactgg ggg 356
gga
Lt'sVal G1y AspLysLys AlaValVal IleAsnSer TyrTrp Gly
Gly
70 75 80
atc gag aac aacaagcta gcgcgggac gacggcacc gagtgg aag 404
caa
Ile Glu Asn AsnLysLeu AlaArgAsp AspGlyThr GluTrp Lys
Gln
85 90 95
tgg act ttt aggccatgg gagacgtac acggcggac acgtcc att 452
tgc
Trp Thr Phe ArgProTrp GluThrTyr ThrAlaAsp ThrSer Ile
Cys
100 105 110 115
gac ctc aga caccatgag cccaagacg ctgatggat aaggtc gca 500
acc
Asp Leu Arg HisHisGlu ProLysThr LeuMetAsp LysVal Ala
Thr
120 125 130
tac tgg gtc aagtcgctg cgcttcccc acggacatc ttcttc cag 548
acc
Tyr Trp Val LysSerLeu ArgPhePro ThrAspIle PhePhe Gln
Thr
135 140 145
agg cgg ggc tgccgggcg atgatgctg gaaacggtg getgcg gtg 596
tat
Arg Arg Gly CysArgAla MetMetLeu GluThrVal AlaAla Val
Tyr
150 155 160
cct ggg gtg ggcggcatg ctgctccac ctgcgctca ctccgc cgc 644
atg
Pro Gly Val GlyGlyMet LeuLeuHis LeuArgSer LeuArg Arg
Met
CA 02355616 2001-06-29
WO PCT/US00/01847
00/44920
165 170 175
ttcgag agcggc ggctggatc cgcgetttg ctggaggag gccgag 692
cag
PheGlu SerGly GlyTrpIle ArgAlaLeu LeuGluGlu AlaGlu
Gln
180 185 190 195
aacgag atgcac ctcatgacc ttcatggag gtggcgaag ccgagg 740
cgc
AsnGlu MetHis LeuMetThr PheMetGlu ValAlaLys ProArg
Arg
200 205 210
tggtac cgcgcg ctcgttatc accgtccag ggcgtcttc ttcaac 788
gag
TrpTyr ArgAla LeuValIle ThrValGln GlyValPhe PheAsn
Glu
215 220 225
gcatac ctcggc tacctcttg tccccgaag ttcgcgcac ctcgtc 836
ttc
AlaTyr LeuGly TyrLeuLeu SerProLys PheAlaHis LeuVal
Phe
230 235 240
gtcggc ctggag gaggaggcc atccactcg tacaccgag tacctc 884
tac
ValGly LeuGlu GluGluAla IleHisSer TyrThrGlu TyrLeu
Tyr
245 250 255
aaggat gaggcc ggcaagatc gagaacgtc cccgccccg gccatc 932
ctg
LysAsp GluAla GlyLysIle GluAsnVal ProAlaPro AlaIle
Leu
260 265 270 275
gccatc tactgg cgcctcccc getaacgcc acgctcaag gacgta 980
gac
AlaIle TyrTrp ArgLeuPro AlaAsnAla ThrLeuLys AspVal
Asp
280 285 290
gtcacc gtccgc gccgacgag getcaccac cgcgacgtc aaccac 1028
gtc
ValThr ValArg AlaAspGlu AlaHisHis ArgAspVal AsnHis
Val
295 300 305
tttgca gacatc cattgccag ggaatgcag ctgaagcag tcccct 1076
tcg
PheAla AspIle HisCysGln GlyMetGln LeuLysGln SerPro
Ser
310 315 320
gcgccg ggatac cactgaggatgtt tgtgctcttc c 1124
atc ttaattttg
AlaPro GlyTyr His
IIe
325
atcgctaata agcaattgtc ttaaggga a aaaggatg cttattgagt
tacgagtact1184
t gg
gctacggcga ttaggatatt accag gtttgaga gtgaaaccta
ttatatgtac1244
ttcca tt
gcatgttaca tgtacatatc gtgcg aggtgctt ttctggcgtt
tatcacttct1304
tctaa ag
tcctggagttcctttgttct catgtcagc tcgctaatat cgaatgtaca1364
t tgaattgggc
atttttgcataaaaaaaaaa aaaaa aaaaaaaaaaaaaa aaaaaaaaaa2424
aaaaa aaaaaa
aaaaaaaaaaaaaaaaaaaa aaaaaaaaa a 1457
a aa
<210> 5
<211> 329
<212> PRT
<213> Zeamay s
<400> 5
Met Met Ser Ser Arg Ala Gly Ser Ile Leu Leu Arg His Ala Gly Ser
1 5 10 15
Arg Leu Phe Thr Ala Ala Ala Ile Ser Pro Ala Ala Ala Ser Arg Pro
20 25 30
Leu Leu Ala Gly Gly Asn Gly Val Pro Ala Val Met Leu Arg Leu Met
35 40 45
Ser Thr Ser Ser Pro Ala Ala Pro Thr Glu Ala Lys Asp Glu Ala Ala
-5-
CA 02355616 2001-06-29
WO 00/44920 PCTNS00/01847
50 55 60
Lys Ala Ser Lys Val Gly Gly Asp Lys Lys Ala Val Va1 I1e Asn Ser
65 70 75 80
Tyr Trp Gly Ile Glu Gln Asn Asn Lys Leu Ala Arg Asp Asp Gly Thr
85 90 95
Glu Trp Lys Trp Thr Cys Phe Arg Pro Trp Glu Thr Tyr Thr Ala Asp
100 105 110
Thr Ser Ile Asp Leu Thr Arg His His Glu Pro Lys Thr Leu Met Asp
115 120 125
Lys Val Ala Tyr Trp Thr Val Lys Ser Leu Arg Phe Pro Thr Asp Ile
130 135 140
Phe Phe Gln Arg Arg Tyr Gly Cys Arg Ala Met Met Leu Glu Thr Val
145 150 155 160
Ala Ala Val Pro Gly Met Val Gly Gly Met Leu Leu His Leu Arg Ser
165 170 175
Leu Arg Arg Phe Glu Gln Ser Gly Gly Trp Ile Arg Ala Leu Leu Glu
180 185 190
Glu Ala Glu Asn Glu Arg Met His Leu Met Thr Phe Met. Glu Val Ala
195 200 205
Lys Pro Arg Trp Tyr Glu Arg Ala Leu Val Ile Thr Val Gln Gly Val
210 215 220
Phe Phe Asn Ala Tyr Phe Leu Gly Tyr Leu Leu Ser Pro Lys Phe Ala
225 230 235 240
His Leu Val Val Gly Tyr Leu Glu Glu Glu Ala Ile His Ser Tyr Thr
245 250 255
Glu Tyr Leu Lys Asp Leu Glu Ala Gly Lys Ile Glu Asn Val Pro Ala
260 265 270
Pro Ala Ile Ala Ile Asp Tyr Trp Arg Leu Pro Ala Asn Ala Thr Leu
275 280 285
Lys Asp Val Val Thr Val Val Arg Ala Asp Glu Ala His His Arg Asp
290 295 300
Val Asn His Phe Ala Ser Asp Ile His Cys Gln Gly Met Gln Leu Lys
305 310 315 320
Gln Ser Pro Ala Pro Ile Gly Tyr His
325
<210> 6
<211> 47
<212> PRT
<213> Zea mays
<400> 6
Met Met Ser Ser Arg Ala Gly Ser Ile Leu Leu Arg His Ala Gly Ser
1 5 10 15
Arg Leu Phe Thr Ala Ala Ala Ile Ser Pro Ala Ala Ala Ser Arg Pro
20 25 30
Leu Leu Ala Gly Gly Asn Gly Val Pro Ala Val Met Leu Arg Leu
35 40 45
<210> 7
<211> 420
<212> DNA
<213> Zea mays
<220>
<221> CDS
<222> (2)...(420)
<400> 7
a ccc acg cgt ccg ccc acg cgt ccg ccc acg cgt ccg gca gtc gtc agc 49
Pro Thr Arg Pro Pro Thr Arg Pro Pro Thr Arg Pro Ala Val Val Ser
1 5 10 15
_6_
CA 02355616 2001-06-29
WO 00/44920 PCT/US00/01847
tactgg atcgac acgcccaag ctcgtgaag gaagac ggcacggag 97
ggc
TyrTrp-GlyIleAsp ProLys LeuValLys GluAsp GlyThrGlu
Thr
20 25 30
tggaag accagc ttccggccg tgggacgcg tacacg tcggacacg 145
tgg
TrpLys ThrSer PheArgPro TrpAspAla TyrThr SerAspThr
Trp
35 40 45
tccatc ataggg aagcaccac gcgccgacg acgctg cccgacaag 193
gac
SerIle IleGly LysHisHis AlaProThr ThrLeu ProAspLys
Asp
50 55 60
gcggcg ctgatc gtcaagtcg ctgcgcgtg cccatg gacctcttc 241
tac
AlaAla LeuIle ValLysSer LeuArgVal ProMet.AspLeuPhe
Tyr
65 70 75 80
ttccag cggcac gccagccac gcgctgctg ctcgag acggtggcg 289
cgc
PheGln ArgHis AlaSerHis AlaLeuLeu LeuGlu ThrValAla
Arg
g5 90 95
gccgtg ggcatg gtgggcggc atgctcctc cacctg cgctccctc 337
ccg
AlaVal GlyMet ValGlyGly MetLeuLeu HisLeu ArgSerLeu
Pro
100 105 110
cgccgc gagcac agcggcggc tggatccgc gcgctg ctcgaggag 385
ttc
ArgArg GluHis SerGlyGly TrpTleArg AlaLeu LeuGluGlu
Phe
115 120 125
gccgag gagcgc atgcaactc atgacgttc tc 420
aac
AlaGlu GluArg MetGlnLeu MetThrPhe
Asn
130 135
<210> 8
<211> 140
<212> PRT
<213> Zeamays
<400> 8
ProThr ProPro ThrArgPro ProThrArg ProAla ValValSer
Arg
1 5 10 15
TyrTrp IleAsp ThrProLys LeuValLys G1uAsp GlyThrGlu
Gly
20 25 30
TrpLys ThrSer PheArgPro TrpAspAla TyrThr SerAspThr
Trp
35 40 45
SerIle IleGly LysHisHis AlaProThr ThrLeu ProAspLys
Asp
50 55 60
AlaAla LeuIle ValLysSer LeuArgVal ProMet AspLeuPhe
Tyr
65 70 75 80
PheGln ArgHis AlaSexHis AlaLeuLeu LeuGlu ThrValAla
Arg
85 90 95
AlaVal GlyMet Val31yGly MetLeuLeu HisLeu ArgSerLeu
Pro
100 105 110
ArgArg GluHis SerGly TrpIleArg AlaLeu LeuGluGlu
Phe Gly
115 120 125
AlaGlu Glu MetGln MetThr Ser
Asn Arg Leu Phe
130 135 140
<210> 9
<211> 36
<212> DNA
_7_
CA 02355616 2001-06-29
WO 00/44920 PCT/US00/01847
<213> Artificial Sequence
<220>
<223> Designed oligonucleotide based upon the adapter
sequence and poly T to remove clones which have
poly A tail but no cDNA
<400> 9
tcgacccacg cgtccgaaaa aaaaaaaaaa aaaaaa 36
s_
