Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR TRANSFORMING PLASTIDS
FIELD OF THE INVENTION
This invention relates to the application of genetic engineering techniques to
plants. More specifically, the invention relates to methods for the
transformation of
plant cell plastids.
BACKGROUND
The plastids of higher plants are an attractive target for genetic
engineering.
Plant plastids (chloroplasts, amyloplasts, elaioplasts, chromoplasts, etc.)
are the major
biosynthetic centers that in addition to photosynthesis are responsible for
production
of industrially important compounds such as amino acids, complex
carbohydrates,
fatty acids, and pigments. Plastids are derived from a common precursor known
as a
2 0 proplastid and thus the plastids present in a given plant species all have
the same
genetic content. Plant cells contain 500-10,000 copies of a small 120-i60
kilobase
circular genome, each molecule of which has a large (approximately 25kb)
inverted
repeat. Thus, it is possible to engineer plant cells to contain up to 20,000
copies of a
particular gene of interest which potentially can result in very high levels
of foreign
2 5 gene expression.
Current plastid transformation methods are inefficient, as such there is need
for constructs and methods which improve plastid transformation.
SUMMARY OF THE INVENTION
By this invention, methods which allow for the improved transformation of a
foreign DNA into plant cell plastids are provided. Such methods generally
involve
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utilizing a plant tissue source which contains cells with an altered plastid
morphology
in the transformation methods. The alteration in the plant plastid morphology
includes, inter alia, plastid size and number. By utilizing tissue derived
from such
plants in plastid transformation methods, efficiency of transformation of a
foreign
DNA into the plant cell plastid may be increased.
As exemplified herein, constructs useful for genetic engineering of plant
cells
to provide for a method of increasing plastid transformation efficiency are
provided.
The constructs include nucleic acid sequences coding for protein sequences
involved
in controlling division of plant cell organelles. The expression of such
nucleic acid
sequences in a plant cell provides for an altered number andlor size of the
chloropiasts
within the host cell.
DNA sequences, also referred to herein as polynucleotides, for use in
transformation contain an expression construct comprising a promoter region
which is
functional in a plastid, and a DNA sequence encoding a gene involved in
controlling
the division of plant cell organelles.
Methods for the use of transformed plants with altered plastid morphology are
described. Such methods include plant breeding or transformation methods to
provide
plant cells having both the nuclear and plastid constructs.
The present invention also provides methods for increasing the efficiency of
2 0 chloroplast transformation. The method generally comprises transforming
the plastids
of a plant tissue which has been modified to have an altered number and/or
size of
plastids contained within the plant cell.
The present invention also provides a mechanism for enhancing the efficiency
of chloroplast transformation in plant species.
2 5 The present invention also provides methods for improving the
selectability of
plant comprising, transforming a plant cell source having an altered plastid
morphology with a construct comprising a promoter functional in a plant cell
plastid
operably associated with a nucleic acid sequence encoding a selectable marker.
Selectable markers of interest in the present invention include herbicide
tolerance
3 0 genes such as glyphosate tolerance genes, and antibiotic resistance genes.
Glyphosate
tolerance genes include the CP4 gene from Agrobacterium.
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Another aspect of the present invention provides methods for preparing a
plant cell source with increased plastid transformation efficiency comprising,
transforming a plant cell with a construct comprising a promoter functional in
plant
cell operably associated with a nucleic acid sequence encoding aFtsZ protein.
Also considered part of this invention are the plants and plant cells obtained
using the methods described herein.
DESCRIPTION OF THE FIGURES
10 Figure 1 provides an amino acid sequence alignment of the Arabidopsis FtsZl
(SEQ ID N0:2), the Brassica FtsZI (SEQ ID NO:b), the tobacco FtsZl (SEQ ID
N0:9), the Soybean FtsZl (SEQ ID N0:72) and the corn FtsZl (SEQ ID N0:73)
protein sequences.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the subject invention, methods are provided which allow
for the improved transformation of a foreign DNA into plant cell plastids.
Such
2 0 methods generally involve utilizing a plant cell source which contains an
altered plant
plastid morphology. By utilizing tissue derived from such plants in plastid
transformation methods, efficiency of transformation of a foreign DNA into the
plant
cell plastid can be increased.
In one embodiment of the instant invention, plant tissue containing altered
25 plant plastid morphology is used for plastid transformation methods. Such
alterations
in plant plastid morphology include, but are not limited to. alterations in
the plastid
size, shape and number in respect to a wild-type plastid morphology from the
target
plant cell. In general, a wild-type plastid morphology consists of small,
round
organelles contained within the plant cell, depending on the species.
Furthermore, a
3 0 plant cell typically contains between about 50 and about i00 plastids.
The plant tissue source used in plastid transformation methods of the present
invention contains an increase in the size of the plastids contained in the
plant cells.
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Such increases in the size of the plastids provides for a larger surface area
for the
foreign DNA to penetrate the plastid membrane during transformation.
The large plastids preferably contain approximately the same number of
plastid genomes as would be contained in corresponding number of wild-type
plastids. For example, in a wild-type plant cell containing 100 plastids per
cell and
100 copies of the plastid genome in each plastid (a total of 10,000 copies of
the
plastid genome per cell), the corresponding mutant tissue source would
preferably
contain about the same number of plastid genomes, only contained in one, or
several
large plastid(s).
Alternatively, a plant tissue source with an increased number of plastids,
with
a corresponding reduced size, can also find use in the plastid transformation
methods
of the present invention.
As is understood in the art, additional methods for obtaining plants with
alterations in the plastid size and number are known. The skilled artisan will
recognize that a number of methods are available for providing for an
alteration in
plastid cell division. Such methods are described, for example, by Strepp, et
al.
( 1998) Proc. Natl. Acad. Sci. USA, 95:4368-4.373.
Cell division, also referred to as cytokinesis, has been the focus of studies
in
many organisms such as bacterial, fungal, and animal cells. Division of
bacterial cells
2 0 occurs through the formation of an FtsZ ring (also referred to as a Z
ring) at the site of
division (Lutkenhaus, et al. ( 1997) Ann. Rev. Biochem. 66:93-116). The
positioning
and formation of the Z ring acts to further drive septation (cytokinesis). The
ring is
composed of a tubulin-like FtsZ protein which has GTPase activity. Mutations
in the
ftsZ gene in E. toll leads to the production of a temperature-sensitive
filaments with
2 5 regularly spaced nucleoids at certain temperatures (Lutkenhaus ( 1992) In
Prokaryotic
Structure and Function: A New Perspective, ed. S Mohan, C Dow, pp 123-152.
Cambridge: Cambridge Univ. Press}. Such mutations in bacteria leads to the
inability
to divide correctly.
The plant cell plastid as well as the mitochondria are derived from
prokaryotic
3 0 ancestors, and thus, the division apparatus of these organelles resembles
that of
bacteria. Recently, identification of ftsZ related sequences in Arabidopsis
and
Physcomitrella patens have been reported (Osteryoung, et al: ( 1995) Nature,
376:473-
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474; and Strepp, et al. ( 1998), supra). The protein encoded by the
Arabidopsis ftsZ
gene was found to be imported into the chloroplast amd was therefore
specuiated to be
a component of the plastid division machinery {Osteryoung, et al. ( 1995},
supra).
More recently, the involvement of FtsZ in plastid division was directly
demonstrated.
The disruption of the ftsZ gene in a lower plant, Physcomitrella patens,
impeded
plastid division, thereby giving rise to mutant cell lines with one or a few
large
plastids {Strepp, et al. (1998), supra).
The use of plants with an altered number andlor size of plastids containing
one
or few large plastids could therefore be used as targets for plastid
transformation of
any plant species. Such plants containing an altered size and/or number can be
obtained using various methods, including mutagenesis, antisense suppression,
or co-
suppression. Methods for the mutagenesis of plant genomes are well known in
the art,
and include chemical, such as ethylmethane sulfonate {EMS) and
nitrosoguanidine
(NTG), as well as physical mutagenesis methods such as fast neutron
bombardment.
Other means for obtaining a plant source with an alteration in the size and/or
number of plastids contained in the cell are also contemplated. For example,
tissue
for use in the transformation methods of the present invention can be obtained
from
plants grown in culture conditions which provide for such altered plastid
content. For
example, tissue obtained from plants grown in vitro under cuiture conditions
in which
inhibitors of bacterial cell division, such as S,5'-bis-(8-anilino-1-
naphtaienesulfonate)
(Yu, et al. ( 1998} J. Biol Chem. 273:10216-10222), are present, can be
utilized as a
cell source for the plastid transformation methods of the present invention.
In a preferred embodiment, such plants containing cells with an alteration in
the size and/or number of plastids are generated by anti-sense expression of
the FtsZ
2 5 gene. Once plastid transformation is achieved and homoplasmic plants are
identified,
the anti-sense transgene can be eliminated by out-crossing and the wild-type
condition
of 50 to 100 plastids per cell restored. Similarly, plants regenerated from
plastid
transformed tissue containing an altered number and/or size of plastids from
mutations can also be reverted to the wild-type plastid conditions using such
out-
3 0 crossing methods.
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In the case of the use of culture conditions for obtaining plant cells with an
altered number and/or size of plastids, wild-type plastids can be obtained by
releasing
the tissue from such culture conditions.
In another embodiment of the present invention, novel nucleic acid sequences
are provided which encode proteins related to proteins involved in bacterial
cell and
plastid division.
In particular, novel nucleic acid sequences fromArabidopsis, soybean, corn,
Brassica are provided which encode FtsZ related proteins. Such nucleic acid
sequences find use in the preparation of DNA constructs. Such constructs find
use in
the production of plants with an altered number and/or size of chloroplasts.
The skilled artisan will recognize that other DNA sequences useful for the
production of plants with an altered number and/or size of chloroplasts are
available
in the art. The sequences include but are not limited to, ftsA, ftsL, ftsI,
ftsQ, ftsN,
ftsW, ftsK {Lutkenhaus, et al. (1997) supra), and the arc genes (Pyke, et al.
{1992)
Plant Physiol. 99:1005-1008; Pyke et al. (1994) Plant Physiol. 104:201-207;
and
Pyke ( 1997) Am. J. Botany 84:1017-1027).
In order to obtain additional ftsZ sequences, a genomic or other appropriate
library prepared from the candidate plant source of interest can be probed
with
conserved sequences from one or more plant andlor bacterial ftsZ sequences) to
2 0 identify homoiogously related sequences. Positive clones can be analyzed
by
restriction enzyme digestion and/or sequencing. When a genomic library is
used, one
or more sequences can be identified providing both the coding region, as well
as the
transcriptional regulatory elements of the ftsZ gene from such plant source.
Probes
can also be considerably shorter than the entire sequence. Oligonucleotides
can be
2 5 used, for example, but should be at least about 10, preferably at least
about 15, more
preferably at least 20 nucleotides in length. When shorter length regions are
used for
comparison, a higher degree of sequence identity is required than for longer
sequences. Shorter probes are often particularly useful for polymerase chain
reactions
(PCR), especially when highly conserved sequences can be identified. (See,
Gould, et
3 0 al., PNAS USA ( 1989) 86:1934-1938.)
When longer nucleic acid fragments (,100 bp) are employed as probes,
especially when using complete or large cDNA sequences, one can still screen
with
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moderately high stringencies (for example using 50% formamide at 37oC with
minimal washing) in order to obtain signal from the target sample with 20-50%
deviation, i.e., homologous sequences. (For additional information regarding
screening techniques see Beltz, et al., Meth. Enzymology ( 19$3) 100:266-285).
Another aspect of the present invention relates to isolated FtsZ
polynucleotides. The polynucleotide sequences of the present invention include
isolated polynucleotides that encode the polypeptides of the invention having
a
deduced amino acid sequence selected from the group of sequences set forth in
the
Sequence Listing and to other polynucleotide sequences closely related to such
sequences and variants thereof.
The invention provides a polynucleotide sequence identical over its entire
length to each coding sequence as set forth in the Sequence Listing. The
invention
also provides the coding sequence for the mature polypeptide or a fragment
thereof, as
well as the coding sequence for the mature poiypeptide or a fragment thereof
in a
reading frame with other coding sequences, such as those encoding a leader or
secretory sequence, a pre-, pro-, or prepro- protein sequence. The
polynucleotide can
also include non-coding sequences, including for example, but not limited to,
non-
coding S' and 3' sequences, such as the transcribed, untranslated sequences,
termination signals, ribosome binding sites, sequences that stabilize mRNA,
introns,
2 0 polyadenylation signals, and additional coding sequence that encodes
additional
amino acids. Fox example, a marker sequence can be included to facilitate the
purification of the fused polypeptide. Polynucleotides of the present
invention also
include polynucleotides comprising a structural gene and the naturally
associated
sequences that control gene expression.
2 5 The invention also includes polynucleotides of the formula:
X-(R 1 )n-(R2)-(R3)n-Y
wherein, at the 5' end, X is hydrogen, and at the 3' end, Y is hydrogen or a
metal, R~
and R3 are any nucleic acid residue, n is an integer between 1 and 3000,
preferably
between 1 and 1000 and R~ is a nucleic acid sequence of the invention,
particularly a
3 0 nucleic acid sequence selected from the group set forth in the Sequence
Listing and
preferably SEQ ID NOs: i,3,5,7,8,and 10-31. In the formula, R~ is oriented so
that its
5' end residue is at the left, bound to R,, and its 3' end residue is at the
right, bound to
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R~. Any stretch of nucleic acid residues denoted by either R group, where R is
greater
than l, may be either a heteropolymer or a homopolymer, preferably
aheteropolymer.
The invention also relates to variants of the polynucleotides described herein
that encode for variants of the polypeptides of the invention. Variants that
are
fragments of the poiynucleotides of the invention can be used to synthesize
full-length
polynucleotides of the invention. Preferred embodiments are polynucieotides
encoding polypeptide variants wherein 5 to 10, 1 to 5, l to 3, 2, 1 or no
amino acid
residues of a polypeptide sequence of the invention are substituted, added or
deleted,
in any combination. Particularly preferred are substitutions, additions, and
deletions
that are silent such that they do not alter the properties or activities of
the
poiynucleotide or polypeptide.
Further preferred embodiments of the invention that are at least 50%, 60%, or
70% identical over their entire length to a polynucleotide encoding a
polypeptide of
the invention, and polynucleotides that are complementary to such
polynucleotides.
More preferable are polynucleotides that comprise a region that is at least
80%
identical over its entire length to a palynucleotide encoding a polypeptide of
the
invention and polynucleotides that are complementary thereto. In this regard,
polynucleotides at least 90% identical over their entire length are
particularly
preferred, those at least 95% identical are especially preferred. Further,
those with at
2 0 least 97% identity are highly preferred and thaw with at least 98% and 99%
identity
are particularly highly preferred, with those at least 99% being the most
highly
preferred.
Preferred embodiments are polynucleotides that encode polypeptides that
retain substantially the same biological function or activity as the mature
polypeptides
2 5 encoded by the polynucleotides set forth in the Sequence Listing.
The invention further relates to polynucleotides that hybridize to the above-
described sequences. In particular, the invention relates to polynucleotides
that
hybridize under stringent conditions to the above-described polynucleotides.
As used
herein, the terms "stringent conditions" and "stringent hybridization
conditions" mean
3 0 that hybridization will generally occur if there is at Least 95% and
preferably at least
97% identity between the sequences. An example of stringent hybridization
conditions is overnight incubation at 42°C in a solution comprising 50%
formamide,
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5x SSC ( 150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20
microgramslmilliiiter
denatured, sheared salmon sperm DNA, followed by washing the hybridization
support in O.lx SSC at approximately 65°C. Other hybridization and wash
conditions
are well known and are exemplified in Sambrook, et al., Molecular Cloning: A
Laboratory Manual, Second Edition, cold Spring Harbor, NY ( 1989),
particularly
Chapter 11.
The invention also provides a polynucleotide consisting essentially of a
polynucleotide sequence obtainable by screening an appropriate library
containing the
complete gene for a polynucleotide sequence set for in the Sequence Listing
under
stringent hybridization conditions with a probe having the sequence of said
polynucleotide sequence or a fragment thereof; and isolating said
polynucleotide
sequence. Fragments useful for obtaining such a polynucleotide include, for
example,
probes and primers as described herein.
25 As discussed herein regarding polynucleotide assays of the invention, for
example, polynucleotides of the invention can be used as a hybridization probe
for
RNA, cDNA, or genomic DNA to isolate full length cDNAs or genomic clones
encoding a polypeptide and to isolate cDNA or genomic clones of other genes
that
have a high sequence similarity to a polynucleotide set forth in the Sequence
Listing.
2 0 Such probes will generally comprise at least 15 bases. Preferably such
probes will
have at least 30 bases and can have at least 50 bases. Particularly preferred
probes
will have between 30 bases and 50 bases, inclusive.
The coding region of each gene that comprises or is comprised by a
polynucleotide sequence set forth in the Sequence Listing may be isolated by
2 5 screening using a DNA sequence provided in the Sequence Listing to
synthesize an
oligonucleotide probe. A labeled oligonucleotide having a sequence
complementary
to that of a gene of the invention is then used to screen a library of cDNA,
genomic
DNA or mRNA to identify members of the library which hybridize to the probe.
For
example, synthetic oligonucleotides are prepared which correspond to the FtsZ
EST
3 0 sequences. The oiigonucleotides are used as primers in polymerase chain
reaction
(PCR) techniques to obtain 5' and 3' terminal sequence of FtsZ genes.
Alternatively,
where oligonucleotides of low degeneracy can be prepared from particularFtsZ
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peptides, such probes may be used directly to screen gene libraries forFtsZ
gene
sequences. In particular, screening of cDNA libraries in phage vectors is
useful in
such methods due to lower levels of background hybridization.
Typically, a FtsZ sequence obtainable from the use of nucleic acid probes will
show 60-70% sequence identity between the target FtsZ sequence and the
encoding
sequence used as a probe. However, lengthy sequences with as little as 50-60%
sequence identity may also be obtained. The nucleic acid probes may be a
lengthy
fragment of the nucleic acid sequence, or may also be a shorter,
oligonucleotide probe.
When longer nucleic acid fragments are employed as probes (greater than about
I00
bp), one may screen at lower stringencies in order to obtain sequences from
the target
sample which have 20-50% deviation (i.e., 50-80% sequence homology) from the
sequences used as probe. Oligonucieotide probes can be considerably shorter
than the
entire nucleic acid sequence encoding an FtsZ enzyme, but should be at least
about I0,
preferably at least about I5, and more preferably at least about 20
nucleotides. A
higher degree of sequence identity is desired when shorter regions are used as
opposed
to longer regions. It may thus be desirable to identify regions of highly
conserved
amino acid sequence to design oligonucleotide probes for detecting and
recovering
other related FtsZ genes. Shorter probes are often particularly useful for
polymerase
chain reactions (PCR), especially when highly conserved sequences can be
identified.
2 0 (See, Gould, et al., PNAS USA ( 1989) 86:1934-1938.).
Another aspect of the present invention relates to FtsZ polypeptides. Such
polypeptides include isolated polypeptides set forth in the Sequence Listing,
as well as
polypeptides and fragments thereof, particularly those polypeptides which
exhibit
FtsZ activity and also those polypeptides which have at least 50%, 60% or 70%
2 5 identity, preferably at least 80% identity, more preferably at least 90%
identity, and
most preferably at least 95% identity to a polypeptide sequence selected from
the
group of sequences set forth in the Sequence Listing, and also include
portions of such
polypeptides, wherein such portion of the polypeptide preferably includes at
least 30
amino acids and more preferably includes at least 50 amino acids.
3 0 "Identity", as is well understood in the art, is a relationship between
two or
more polypeptide sequences or two or more polynucleotide sequences, as
determined
by comparing the sequences. In the art, "identity" also means the degree of
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relatedness between polypeptide or polynucleotide sequences, as determined by
the
match between strings of such sequences. "Identity" can be readily calculated
by
known methods including, but not limited to, those described in Computational
Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York ( 1988);
Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic
Press,
New York, 1993; Computer Analysis of Sequence Data, Part l, Griffin, A.M. and
Griffin, H.G., eds., Humana Press, New Jersey ( 1994); Sequence Analysis in
Molecular Biology, von Heinje, G., Academic Press ( 1987); Sequence Analysis
Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York {1991);
and
Carillo, H., and Lipman, D., SIAM J Applied Math, 48:1073 ( 1988}. Methods to
determine identity are designed to give the largest match between the
sequences
tested. Moreover, methods to determine identity are codified in publicly
available
programs. Computer programs which can be used to determine identity between
two
sequences include, but are not limited to, GCG {Devereux, J., et al., Nucleic
Acids
Research I2{ 1 ):387 ( 1984); suite of five BLAST programs, three designed for
nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed
for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in
Biotechnology, 12: 76-80 ( 1994); Birren, et al., Genome Analysis, 1: 543-559
( 1997)).
The BLAST X program is publicly available from NCBI and other sources {BLAST
2 0 Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, MD 20894; Altschul,
S., et
al., J. Mal. Biol., 215:403-410 ( 1990)). The well known Smith Watenman
algorithm
can also be used to determine identity.
Parameters for polypeptide sequence comparison typically include the
following:
2 5 Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 ( 1970}
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.
Acad. Sci USA 89:10915-10919 (1992)
Gap Penalty: 12
Gap Length Penalty: 4
3 0 A program which can be used with these parameters is publicly available as
the "gap" program from Genetics Computer Group, Madison Wisconsin. The above
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parameters along with no penalty for end gap are the default parameters for
peptide
comparisons.
Parameters for polynucleotide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 4$:443-453 ( 1970)
Comparison matrix: matches = +I0; mismatches = 0
Gap Penalty: 50
Gap Length Penalty: 3
A program which can be used with these parameters is publicly available as
the ''gap" program from Genetics Computer Group, Madison Wisconsin. The above
parameters are the default parameters for nucleic acid comparisons.
The invention also includes polypeptides of the formula:
X-(R~ )n-(R2)-(R3)n-Y
wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y
is
hydrogen or a metal, Rl and R3 are any amino acid residue, n is an integer
between 1
and 1000, and RZ is an amino acid sequence of the invention, particularly an
amino
acid sequence selected from the group set forth in the Sequence Listing and
preferably
SEQ ID NOs: 2,4,6, and 9. In the formula, R~ is oriented so that its amino
terminal
residue is at the left, bound to R~, and its carboxy terminal residue is at
the right,
bound to R3. Any stretch of amino acid residues denoted by either R group,
where R
2 0 is greater than I, may be either a heteropoiymer or a homopolymer,
preferably a
heteropolymer.
Polypeptides of the present invention include isolated polypeptides encoded by
a polynucleotide comprising a sequence selected from the group of a sequence
contained in the Sequence Listing set forth herein .
The polypeptides of the present invention can be mature protein or can be part
of a fusion protein.
Fragments and variants of the polypeptides are also considered to be a part of
the invention. A fragment is a variant polypeptide which has an amino acid
sequence
that is entirely the same as part but not all of the amino acid sequence of
the
3 0 previously described polypeptides. 'The fragments can be "free-standing"
or
comprised within a larger polypeptide of which the fragment forms a part or a
region,
most preferably as a single continuous region. Preferred fragments are
biologically
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active fragments which are those fragments that mediate activities of the
polypeptides
of the invention, including those with similar activity or improved activity
or with a
decreased activity. Also included are those fragments that antigenic or
immunogenic
in an animal, particularly a human.
Variants of the polypeptide also include polypeptides that vary from the
sequences set forth in the Sequence Listing by conservative amino acid
substitutions,
substitution of a residue by another with like characteristics. In general,
such
substitutions are among Ala, Val, Leu and lle; between Ser and Thr; between
Asp and
Glu; between Asn and Gln; between Lys and Arg; or between Phe and Tyr.
Particularly preferred are variants in which 5 to 10; i to 5; 1 to 3 or one
amino acids)
are substituted, deleted, or added, in any combination.
Variants that are fragments of the polypeptides of the invention can be used
to
produce the corresponding full length polypeptide by peptide synthesis.
Therefore,
these variants can be used as intermediates for producing the full-length
poiypeptides
of the invention.
The polynucleotides and polypeptides of the invention can be used, for
example, in the transformation of host cells, such as plant host cells, as
further
discussed herein.
The invention also provides polynucleotides that encode a polypeptide that is
a
2 0 mature protein plus additional amino or carboxyl-terminal amino acids, or
amino
acids within the mature polypeptide {for example, when the mature form of the
protein has more than one polypeptide chain}. Such sequences can, for example,
play
a role in the processing of a protein from a precursor to a mature form, allow
protein
transport, shorten or lengthen protein half life, or facilitate manipulation
of the protein
2 S in assays or production. It is contemplated that cellular enzymes can be
used to
remove any additional amino acids from the mature protein.
A precursor protein, having the mature form of the polypeptide fused to one or
more prosequences may be an inactive form of the polypeptide. The inactive
precursors generally are activated when the prosequences are removed. Some or
all of
3 0 the prosequences may be removed prior to activation. Such precursor
protein are
generally called proproteins.
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Homologous sequences are found when there is an identity of sequence and
can be determined upon comparison of sequence information, nucleic acid or
amino
acid, or through hybridization reactions between a known FtsZ and a candidate
source.
Conservative changes, such as Glu/Asp, VallIle, Ser/Thr, Arg/Lys and Gin/Asn
can
also be considered in determining sequence homology. Typically, a lengthy
nucleic
acid sequence can show as little as 50-60% sequence identity, and more
preferably at
least about 70% sequence identity, between the target sequence and the given
FtsZ
sequence of interest excluding any deletions which can be present, and still
be
considered related. Amino acid sequences are considered homologous by as
little as
25% sequence identity between the two complete mature proteins. (See
generally,
Doolittle, R.F., OF URFS and ORFS (University Science Books, CA, 1986.)
In addition, not only can sequences provided herein be used to identify
homologous FtsZ sequences, but the resulting sequences obtained therefrom can
also
provide a further method to obtain FtsZ sequences from other plant and/or
bacterial
sources. In particular, PCR can be a useful technique to obtain related FtsZ
sequences
from sequence data provided herein. One skilled in the art will be able to
design
oligonucleotide probes based upon sequence comparisons or regions of typically
highly conserved sequence.
Once the nucleic acid sequence is obtained, the transcription, or
transcription
2 0 and translation (expression), of the FtsZ sequence in a host cell is
desired to produce a
ready source of the enzyme and/or modify the number and/or size of the
plastids
found therein. Other useful applications can be found when the host cell is a
plant
host cell, in vitro and in vivo.
Nucleic acids (genomic DNA, plasmid DNA, cDNA, synthetic DNA, mRNA,
etc.) encoding FtsZ or amino acid sequences of the purified enzymes, which
permit
design of nucleic acid probes facilitating the isolation of DNA coding
sequences
therefor, are known in the art and are available for use in the methods of the
present
invention. It is generally recognized to an artisan skilled in the field to
which the
present invention pertains that the nucleic acid sequences provided herein and
the
3 0 amino acid sequences derived therefrom can be used to isolate other
potential FtsZ
genes from GenBank using DNA and peptide search techniques generally known in
the art.
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In addition to the sequences described in the present invention, DNA coding
sequences useful in the present invention can be derived from algae, fungi,
bacteria,
plants, etc: Homology searches in existing databases using signature sequences
corresponding to conserved nucleotide and amino acid sequences of FtsZ can be
employed to isolate equivalent, related genes from other sources such as
plants and
microorganisms. Searches in EST databases can also be employed. Furthermore,
the
use of DNA sequences encoding enzymes functionally enzymatically equivalent to
those disclosed herein, wherein such DNA sequences are degenerate equivalents
of
the nucleic acid sequences disclosed herein in accordance with the degeneracy
of the
genetic code, is also encompassed by the present invention. Demonstration of
the
functionality of coding sequences identified by any of these methods can be
carried
out by complementation of mutants of appropriate organisms, such as E. coli:
The
sequences of the DNA coding regions can be optimized by gene resynthesis,
based on
cadon usage, for maximum expression in particular hosts.
The nucleic acid sequences which encode FtsZ can be used in various
constructs, for example, as probes to obtain further sequences. Alternatively,
these
sequences can be used in conjunction with appropriate regulatory sequences to
increase levels of the respective FtsZ sequence of interest in a host cell for
recovery or
study of 'the enzyme in vitro or in vivo or to decrease levels of the
respective FtsZ
2 0 sequence of interest for some applications when the host cell is a plant
entity,
including plant cells, plant parts (including but not limited to seeds,
cuttings or
tissues) and plants.
Thus, depending upon the intended use, the constructs can contain the nucleic
acid sequence which encodes the entire FtsZ protein, or a portion thereof. For
2 5 example, where antisense inhibition of a given FtsZ protein is desired,
the entire FtsZ
sequence is not required. Furthermore, where FtsZ constructs are intended for
use as
probes, it can be advantageous to prepare constructs containing only a
particular
portion of a FtsZ encoding sequence, for example a sequence which is
discovered to
encode a highly conserved FtsZ region.
3 0 As discussed above, nucleic acid sequence encoding a plant or other FtsZ
proteins of this invention can include genomic, cDNA or rnRNA sequence. By
"encoding" is meant that the sequence corresponds to a particular amino acid
sequence
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either in a sense or anti-sense orientation. By "extrachromosomal" is meant
that the
sequence is outside of the plant genome of which it is naturally associated.
By
"recombinant" is meant that the sequence contains a genetically engineered
modification through manipulation via mutagenesis, restriction enzymes, and
the like.
A cDNA sequence may or may not contain pre-processing sequences, such as
transit peptide sequences or targeting sequences to facilitate delivery of the
FtsZ
protein to a given organelle or membrane location. The use of any such
precursor
FtsZ DNA sequence is preferred for uses in plant cell expression. A genomic
FtsZ
sequence can contain the transcription and translation initiation regions,
introns,
and/or transcript termination regions of the plant FtsZ, which sequences can
be used
in a variety of DNA constructs, with or without the FtsZ structural gene.
Thus,
nucleic acid sequences corresponding to the FtsZ sequences of this invention
can also
provide signal sequences useful to direct grotein delivery into a particular
organellar
or membrane location, 5' upstream non-coding regulatory regions (promoters)
having
useful tissue and timing profiles, 3' downstream non-coding regulatory regions
useful
as transcriptionai and translational regulatory regions, and may lend insight
into other
features of the gene.
Once the desired plant or other FtsZ nucleic acid sequence is obtained, it can
be manipulated in a variety of ways. Where the sequence involves non-coding
2 0 flanking regions, the flanking regions can be subjected to resection,
mutagenesis, etc.
Thus, transitions, transversions, deletions, and insertions can be performed
on the
naturally occurring sequence. in addition, all or part of the sequence can be
synthesized. In the structural gene, one or more codons can be modified to
provide
for a rnadified amino acid sequence, or one or more codon mutations can be
2 5 introduced to provide for a convenient restriction site or other purpose
involved with
construction or expression. The structural gene can be further modified by
employing
synthetic adapters, linkers to introduce one or more convenient restriction
sites, or the
like.
For the most part, the constructs will involve regulatory regions functianal
in
3 0 plants which provide for altered size and number of plastids in a plant
cell. The open
reading frame, coding for the FtsZ protein, FtsZ-related protein or functional
fragment
thereof will be joined at its 5' end to a transcription initiation regulatory
region such as
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the wild-type sequence naturally found 5' upstream to theFtsZ or FtsZ-related
structural gene, or to a heterologous regulatory region from a gene naturally
expressed
in plant tissues. Examples of useful plant regulatory gene regions include
those from
T-DNA genes, such as nopaline or octopine synthase, plant virus genes, such as
CaMV 35S, or from native plant genes.
The DNA sequence encoding a plant or other FtsZ protein of this invention
can be employed in conjunction with all or part of the gene sequences normally
associated with the FtsZ. In its component parts, a DNA sequence encoding FtsZ
is
combined in a DNA construct having, in the 5' to 3' direction of
transcription, a
transcription initiation control region capable of promoting transcription and
translation in a host cell, the DNA sequence encoding plant FtsZ and a
transcription
and translation termination region.
Potential host cells include both prokaryotic and eukaryotic cells. A host
cell
can be unicellular or found in a multicellar differentiated or
undifferentiated organism
depending upon the intended use. Cells of this invention can be distinguished
by
having a FtsZ sequence foreign to the wild-type cell present therein, for
example, by
having a recombinant nucleic acid construct encoding a FtsZ protein therein
not native
to the host species.
Depending upon the host, the regulatory regions will vary, including regions
2 0 from viral, plasmid or chromosomal genes, or the like. For expression in
prokaryotic
or eukaryotic microorganisms, particularly unicellular hosts, a wide variety
of
constitutive or regulatable promoters can be employed. Expression in a
microorganism can provide a ready source of the plant enzyme. Among
transcriptional initiation regions which have been described are regions from
bacterial
2 5 and yeast hosts, such as E. colt, B. subtilis, Sacchromyces cerevisiae,
including genes
such as beta-galactosidase, T7 polymerase, tryptophan E and the like.
In a preferred embodiment, the constructs will involve regulatory regions
functional in plants which provide for modified production of plant FtsZ, and.
possibly, modification of the plant cell plastid. The open reading frame
coding for the
3 0 plant FtsZ or functional fragment thereof will be joined at its 5' end to
a transcription
initiation regulatory region. In embodiments wherein the expression of the
FtsZ
protein is desired in a plant host, the use of all or part of the complete
plant FtsZ gene
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is desired; namely all or part of the 5' upstream non-coding regions
(promoter)
together with the structural gene sequence and 3' downstream non-coding
regionscan
be employed.
If a different promoter is desired, such as a promoter native to the plant
host of
interest or a modified promoter, i.e., having transcription initiation regions
derived
from one gene source and translation initiation regions derived from a
different gene
source, numerous transcription initiation regions are available which provide
for a
wide variety of constitutive or regulatable, e.g., inducible, transcription of
the
structural gene functions. The transcription/translation initiation regions
corresponding to such structural genes axe found immediately 5' upstream to
the
respective start codons. Among transcriptional initiation regions used for
plants are
such regions associated with the T-DNA structural genes such as for nopaline
and
mannopine synthases, the 19S and 35S promoters from CaMV, and the 5' upstream
regions from other plant genes such as napin, ACP, SSU, PG, zero, phaseolin E,
and
the like. Enhanced promoters, such as double 35S, are also available for
expression of
FtsZ sequences. Fox such applications when 5' upstream non-coding regions are
obtained from other genes regulated during seed maturation, those
preferentially
expressed in plant embryo tissue, such as ACP and napin-derived transcription
initiation control regions, are desired. Such "seed-specific promoters" can be
obtained
and used in accordance with the teachings of issued U.S. Patent Numbers
5,608,152
and 5,530,194, which references are hereby incorporated by reference.
Transcription
initiation regions which are preferentially expressed in seed tissue, i.e.,
which are
undetectable in other plant parts, are considered desirable for TAG
modifications in
order to minimize any disruptive or adverse effects of the gene product.
2 5 Regulatory transcript termination regions can be provided in DNA
constructs
of this invention as well. Transcript termination regions can be provided by
the DNA
sequence encoding the plant FtsZ or a convenient transcription termination
region
derived from a different gene source, for example, the transcript termination
region
which is naturally associated with the transcript initiation region. Where the
transcript
3 0 termination region is from a different gene source, it will contain at
least about 0.25
kb, preferably about 1-3 kb of sequence 3' to the structural gene from which
the
termination region is derived.
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Plant expression or transcription constructs having a plant FtsZ as the DNA
sequence of interest for increased or decreased expression thereof can be
employed
with a wide variety of plant life, particularly, plant life involved in the
production of
vegetable oils for edible and industrial uses. Mast especially preferred are
temperate
oilseed crops. Plants of interest include, but are not limited to, rapeseed
{Canola and
High Erucic Acid varieties), sunflower, safflower, cotton, soybean, peanut,
coconut
and oil palms, and corn. Depending on the method for introducing the
recombinant
constructs into the host cell, other DNA sequences can be required.
Importantly, this
invention is applicable to dicotyledenous and monocotyledenous species alike
and
will be readily applicable to new andlor improved transformation and
regulation
techniques.
The method of transformation is not critical to the instant invention; various
methods of plant transformation are currently available. As newer methods are
available to transform crops, they can be directly applied hereunder. For
example,
25 many plant species naturally susceptible to Agrobacterium infection can be
successfully transformed via tripartite or binary vector methods
ofAgrobacterium-
mediated transformation. In addition, techniques of microinjection, DNA
particle
bombardment, and electroporation have been developed which allow for the
transformation of various monocot and dicot plant species.
2 0 In developing the DNA construct, the various components of the construct
or
fragments thereof will normally be inserted into a convenient cloning vector
which is
capable of replication in a bacterial host, e.g., E. coli. Numerous vectors
exist that
have been described in the literature. After each cloning, the plasmid can be
isolated
and subjected to further manipulation, such as restriction, insertion of new
fragments,
2 5 iigation, deletion, insertion, resection, etc., so as to tailor the
components of the
desired sequence. Once the construct has been completed, it can then be
transferred to
an appropriate vector for further manipulation in accordance with the manner
of
transformation of the host cell.
Normally, included with the DNA construct will be a structural gene having
3 0 the necessary regulatory regions for expression in a host and providing
for selection of
transforrnant cells. The gene can provide for resistance to a cytotoxic agent,
e.g.
antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to
an
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auxotrophic host, viral immunity or the like. Depending upon the number of
different
host species in which the expression construct or corriponents thereof are
introduced,
one or more markers can be employed, where different conditioxis for selection
are
used for the different hosts. A number of markers have been developed for use
for
selection of transformed plant cells, such as those which provide resistance
to various
antibiotics, herbicides, or the like. The particular marker employed is not
essential to
this invention, one or another marker being preferred depending on the
particular host
and the manner of construction.
As mentioned above, the manner in which the DNA construct is introduced
into the plant host is not critical to this invention. Any method which
provides for
efficient transformation can be employed. Various methods for plant cell
transformation include the use of Ti- or Ri-plasmids, microinjection,
eiectroporation,
DNA particle bombardment, liposome fusion, or the like. In many instances, it
will
be desirable to have the construct bordered on one or both sides by T-DNA,
particularly having the left and right borders, more particularly the right
border. This
is particularly useful when the construct uses A. tumefaciens or A. rhizogenes
as a
mode for transformation, although the T-DNA borders can find use with other
modes
of transformation.
Once a transgenic plant is obtained which contains cells with altered numbers
2 0 and/or sizes of chloroplasts, tissue containing such cells can then be
used in plastid
transformation experiments. For example, utilizing tissue containing cells
with larger
plastids provides for a larger target in plastid transformation methods, thus
allowing
for an increased probability of introduction of the foreign DNA into the plant
cell
plastid.
2 5 The DNA sequences, or polynucleotides, for use in plastid transformation
of
this invention will contain a plastid expression construct generally
comprising a
promoter functional in a plant cell plastid. and a DNA sequence of interest to
be
expressed in the transformed plastid cells.
Constructs and methods for use in transforming the plastids of higher plants
3 0 are described in Zoubenko et al. (Nuc Acid Res ( 1994) 22{ 19):3819-3824),
Svab et al.
(Proc. Natl. Acad. Sci.(1990) 87:8526-8530 and Proc. Natl: Acad. Sci.(1993)
90:913-
917) and Staub et al. (EMBO J. (i993) 12:601-606). Constructs and methods for
use
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in transforming plastids of higher plants to express DNA sequences under the
control
of a nuclearly encoded, plastid targeted T7 polymerase are described in U.S.
Patent
Number 5,576,198. The complete DNA sequences of the plastid genome of tobacco
are reported by Shinozaki et al. (EMBO J. ( 1986) 5:2043-2049).
Stable transformation of tobacco plastid genomes by particle bombardment is
reported (Svab et.al. ( 1990), supra) and Svab et al. ( 1993), supra). The
methods
described therein can be employed to obtain plants homoplasrnic for plastid
expression constructs using the methods described herein. Briefly, such
methods
involve DNA bombardment of a target host explant, preferably from a tissue
which is
rich in metabolically active plastid organelles, such as green plant tissues
including
leaves, and cotyledons. The bombarded tissue is then cultured for --2 days on
a cell
division promoting media. The plant tissue is then transferred to a selective
media
containing an inhibitory amount of the particular selective agent, as well as
the
particular hormones and other substances necessary to obtain regeneration for
that
particular plant species. For example, in the above publications and the
examples
provided herein, the selective marker is the bacterial aadA gene and the
selective
agent is spectinomycin. The aadA gene product allows for continued growth and
greening of cells whose chloroplasts comprise the marker gene product. Cells
which
do not contain the marker gene product are bleached. The bombarded explants
will
2 0 form green shoots in approximately 3-8 weeks. Leaves from these shoots are
then
subcultured on the same selective media to ensure production and selection of
homoplasmic shoots. As an alternative to a second round of shoot formation,
the
initial selected shoots can be grown to mature plants and segregation relied
upon to
provide transformed plants homopiastic for the inserted gene construct.
2 5 The transformed plants so selected can then be analyzed to determine
whether
the entire plastid content of the plant has been transformed (homoplastic
transforrnants). Typically, following two rounds of shoot formation and
spectinomycin selection, approximately 50% of the transgenic plantiets
analyzed are
homoplastic as determined by Southern blot analysis of plastid DNA. These
plantlets
3 0 are selected for further cultivation, both for analysis of the transgenic
plastid
phenotype (where the nuclear viral polymerase expression construct is also
present in
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the plastid transformant), or for use in methods to transform the viral
polymerase
construct into the nucleus of the transplastomic plants.
The methods of the present invention provide for a more efficient approach to
obtaining homoplasmic plants. Wild-type plant cells typically contain 50 to
i00
plastids per cell. However, once a transplastomic plant is obtained, the DNA
sequence contained in the plant cell nucleus can be crossed away from the
transplastornic cells. The DNA sequence transformed into the nucleus encoding
for
the alteration can be crossed away from the plant containing the transformed
plastids.
Once the DNA sequence has been crossed out, the plastids in the host plant
cell can
divide and revert back to normal (i.e. wild-type) plastid size and numbers. By
applying the selective agent for which the plastid expression constructs
provides
resistance, cells containing a pure population of the plastids containing the
foreign
DNA can be obtained.
The vectors for use in plastid transformation preferably include means for
providing a stable transfer of the plastid expression construct and selectable
marker
construct into the plastid genome. This is most conveniently provided by
regions of
homology to the target plastid genome. The regions of homology flank the
construct
to be transferred and provide for transfer to the plastid genome by homologous
recombination, via a double crossover into the genome. The complete DNA
sequence
2 0 of the plastid genome of tobacco has been reported (Shinozaki et al., EMBO
J. ( 1986)
5:2043-2049). Complete DNA sequences of the plastid genomes from liverwort
(Ohyama et al., Nature ( 1986) 322:572-574) and rice (Hiratsuka et al.. Mol.
Gen.
Genet. (1989) 217:185-194), have also been reported.
Where the regions of homology are present in the inverted repeat regions of
the plastid genome {known as IRA and IRB), two copies of the transgene are
expected
per transformed plastid. Where the regions of homology are present outside the
inverted repeat regions of the plastid genome, one copy of the transgene is
expected
per transformed plastid. The regions of homology within the plastid genome are
approximately lkb in size. Smaller regions of homology can also be used, and
as little
3 0 as 100 by can provide for homologous recombination into the plastid
genome.
However, the frequency of recombination and thus the frequency of obtaining
plants
having transformed plastids decreases with decreasing size of the homology
regions.
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Examples of constructs comprising such regions of homology for tobacco plastid
transformation are described in Svab et.al. (1990 supra) and Svab and Maliga
(1993
supra). Regions useful for recombination into tobacco and Brassica plastid
genomes
are also described in the following examples. Similar homologous recombination
and
selection constructs can be prepared using plastid DNA from the target plant
species.
Other means of transfer to the plastid gename are also considered herein, such
as by methods involving the use of transposable elements. For example, the
constructs to be transferred into the plastid genome can be flanked by the
inverted
repeat regions from a transposable marker which functions in plant plastids. A
DNA
construct which provides for transient expression of the transposase required
to
transfer the target DNA into the plastids is also introduced into the
chloroplasts. In
this manner, a variety of phenotypes can be obtained in plants transformed
with the
same expression construct depending on positional effects which can result
from
insertion of the expression constructs into various locations an the plastid
genome.
1 S Appropriate transposons for use in such plastic transformation methods
include
bacterial TnlO, bacteriophage Mu and various other known bacterial
transposons.
The DNA sequence of interest in the plastid promoter expression constructs
can be an encoding sequence which is oriented for expression of a particular
structural
gene, such that the protein encoded by the structural gene sequence is
produced in the
2 0 transformed plastid. In addition, the DNA sequence of interest can include
a number
of individual structural gene encoding regions such that an operon for
expression of a
number of genes from a single plastid promoter region is produced. Thus, it is
possible to introduce and express multiple genes from an engineered or
synthetic
operon or from a pre-existing prokaryotic gene cluster. Such a method would
allow
2 5 large scale and inexpensive production of valuable proteins and fine
chemicals in a
particular desired plant tissue or a particular stage of development,
depending upon
the promoter used to drive nuclear expression of the specific viral
polymerase. Such
an approach is not practical by standard nuclear transformation methods since
each
gene must be engineered into a monocistron including an encoded transit
peptide for
3 0 plastid uptake and appropriate promoter and terminator signals. As a
result, gene
expression levels would be expected to vary widely between cistrons, and
generation
of a number of transgenic plant lines would be required. UItirnately crosses
would be
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required to introduce all of these cistrons into one plant to get expression
to the target
biochemical pathway.
Alternatively, the DNA sequence of interest in the plastid construct can be a
fragment of an endogenous plastid gene oriented such that an RNA complementary
to
the endogenous gene mRNA is produced in the transformed plastid. Such
antisense
constructs can be used to decrease the expression of the target plastid gene.
In order to provide a means of selecting the desired plant cells following
plastid transformation, the polynucieotides for plastid transformation will
also contain
a construct which provides for expression of a marker gene. Expression of the
marker
gene product allows for selection of plant cells comprising plastid organelles
which
are expressing the marker protein. in the examples provided herein, a
bacterial aadA
gene is expressed under the regulatory control of chloroplast 5' promoter and
3'
transcription termination regions. The use of such an expression construct for
plastid
transformation of plant cells has been described by Svab and Maiiga { 1993,
supra).
Expression of the aadA gene confers resistance to spectinomycin and
streptomycin,
and thus allows for the identification of plant cells expressing this marker
gene.
Selection for the aadA marker gene is based on identification of plant cells
which are
not bleached by the presence of streptomycin, or more preferably
spectinomycin, in
the plant growth medium. Other genes which encode a product involved in
2 0 chioroplast metabolism can also be used as selectable markers. For
example, genes
which provide resistance to plant herbicides such as giyphosate, bromoxynil or
imidazolinone can find particular use. Such genes have been reported by
Stalker et al.
(J. Biol. Chem. (1985) 260:4724-4728; glyphosate resistant EPSP), Stalker et
al. (J.
Biol. Chem. ( 1985) 263:6310-6314; bromoxynil resistant nitrilase gene), and
2 5 Sathasivan et al. (Nucl. Acids Res. { 1990) 18:2188; AHAS imidazolinone
resistance
gene).
The present invention also provides methods for obtaining a plastid
transformed plant on medium containing glyphosate. At the initial event of
transformation only a few plastids out of the many present in a plant cell are
3 0 transformed and therefore are able to express glyphosate resistant marker
gene
product. The rest of the untransformed plastids within the cell remains
vulnerable to
the effect of giyphosate. Therefore, although the cell contains transformed
plastids, it
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is unable to divide and sort out the transformed plastid resulting in lack of
recovery of
transformed callus tissue which would give rise to the transformed
regenerants. Thus,
any method that reduces plastid number to one or few within the cell has the
potential
to survive the effect of glyphosate and be useful as selectable marker for
plastid
transformation.
The following examples are provided by way of illustration and not by way of
limitation.
EXAMPLES
Example 1: Identification of Plant fts2 Sequences
In order to obtain a plant tissue source with an altered number and/or size of
plastids using antisense and/or sense expression of the bacterial FtsZ plant
homologues, public as well as proprietary sequence databases are queried for
homologous sequences in soybean, rice, Arabadsopsis, corn and Brassica. Two
types
of plant FtsZ proteins have been previously identified in GenBank, type IFtsZ
proteins exemplified by accession gi11079731 (SEQ ID N0:32), appear to be
imported
2 0 into the plastid, while type II FtsZ proteins, exemplified by accession
gi13608494
(SEQ ID N0:33) and gi1683524 (SEQ ID N0:34), appear to remain in the
cytoplasm.
Homologs of both the type I FtsZ sequence as well as homologues of type IIFtsZ
genes are described below. The sequences used to search against the databases
are:
type I FtsZ homologue search was (SEQ ID N0:32), and for type IIFtsZ searches,
2 5 (SEQ ID N0:33} is used.
Searches performed in proprietary databases containing sequences obtained
from Arabidopsis identified DNA sequences which are related to the FtsZl
sequence.
The sequence of SEQ ID NO:1 is identified as AtFtsZ 1. The deduced amino acid
sequence encoded by SEQ ID NO: l is provided in SEQ ID N0:2. In addition, one
3 0 sequence (SEQ ID N0:3) was identified as related to the FtsZ2 sequence.
The
deduced amino acid sequence encoded by SEQ ID N0:3 is provided in SEQ ID N0:4.
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Sequences were also identified in databases containing sequences obtained
from Brassica. One sequence was identified as related to theArabidopsis FtsZl
sequence. Based on sequence alignments between the two sequences,
approximately
170 amino acids were predicted to be missing from the BrasSica sequence at the
N-
terminus. To obtain a full length coding sequence for the Brassica FtsZl
(BnFtsZl)
gene; RACE PCR using DNA obtained from Brassica leaves was performed using the
primers SC258 (SEQ ID N0:35) and SC259 (SEQ ID N0:36). One reaction product
was found to contain the most 5' sequence (SEQ ID NO:70) and was used to
produce
a full length sequence referred to as BnFtsZl (SEQ ID N0:5). The deduced amino
acid sequence encoded by BnFtsZl is provided in SEQ ID N0:6)
A FtsZI homoiog was also identified in tobacco with PCR using primers
designed to the conserved amino acid domains of the Arabidopsis FtsZl
sequence.
The PCR primers used are identified as SC252 (SEQ ID N0:37), SC253 (SEQ ID
N0:38), SC254 (SEQ ID N0:39) and SC255 {SEQ ID N0:40). The reaction products
were cloned into TOPO TA (Invitrogen), and a single clone, referred to as
xanthil-26-
contig (SEQ ID N0:7), contained the most sequence. Additional primers were
designed for use in RACE PCR to obtain full length coding sequence for the
tobacco
FtsZl homolog. For amplification of the 5' region, primers SC291 (SEQ ID
N0:41)
and SC292 (SEQ ID N0:42) were used, and for amplification of the 3' sequence,
primers SC293 (SEQ ID N0:43) and SC294 (SEQ ID N0:44) were used. The PCR
products were cloned in TOPO TA and sequenced. Clone xanftsZl-5'-15 (SEQ ID
N0:7 I ) was chosen to be the best for the 5' tobacco FtsZ I sequence since it
contained
the greatest amount of 5' sequence and overlap with xanthil-26-contig. This
sequence
was combined with the xanthil-26-contig to produce xanFtsZ1 (SEQ ID N0:8). The
2 5 deduced amino acid sequence is provided in SEQ ID N0:9.
FtsZ homolog sequences were identified in databases containing DNA
sequences obtained from corn by BLAST searches using the Arabidopsis FtsZ1 and
FtsZ2 amino acid sequences. Ten sequence were identified as related to these
FtsZ
sequences, provided in SEQ ID NOs:lO-19. The clones, when aligned, revealed
six
3 0 contigs, and the best representative clone for each were chosen for
further analysis.
Sequence analysis of SEQ ID NO:10 revealed a high homology to AtFtsZl, and was
estimated to be missing 158 amino acids at the N-terminal end when compared to
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Arabidopsis FtsZl. Clone SEQ ID N0:13 was found to overlap perfectly with SEQ
ID NO:10 for 153 nt at the 5'end and in addition had '167 nt additional nt at
the 5' end
that had amino acid-homology with theArabidopsis FtsZl. However, this clone
was
also not predicted to encode the full-length FtsZ, and was still missing 1 I3
amino
acids at the N-terminal end when compared to Arabidopsis FtsZl. Interestingly,
for
clone SEQ ID N0:13, its homology with SEQ ID NO:10, ends at position 167nt and
diverges. This could either be indicative of the presence of intronic sequence
or a new
class of FtsZ protein. Primer SC321 {SEQ ID N0:45) was designed to pull out
the
missing maize FtsZ 1 sequence by RACE PCR.
Sequence analysis of SEQ ID N0:18 revealed its high homology to FtsZ2, and
was also predicted to not to be full-length and 'missing about 286 amino acids
at the
N-terminal end when compared to Arabidopsis FtsZ2. Primer SC322 {SEQ ID
N0:46) was designed to pull out the missing maize FtsZ2 sequence by RACE PCR.
Although SEQ ID N0;14 and SEQ ID N0:15 were identified with the highest BLAST
scores with FtsZ2.
Soybean FtsZ homolog sequences were identified in databases by BLAST
searches with Arabidopsis FtsZl and FtsZ2 amino acid sequences. Twelve
sequences
were obtained, and are provided in SEQ ID NOs:20-31. Sequence analysis of SEQ
ID
N0:20, SEQ ID N0:24 and SEQ ID N0:25 revealed high homology to FtsZl and
2 0 none to be full-length when compared to Arabidopsis FtsZ 1. SEQ ID N0:25
had the
longest sequence at the N-terminal end and is predicted to be missing 64 amino
acids
at the N-terminal when compared to Arabidopsis FtsZl sequence. Sequences of
SEQ
ID N0:20; SEQ iD N0:24 and SEQ ID NO:25 were used to correct the overlapping
region. RACE PCR primers can now be designed to amplify the ends for obtaining
a
2 S full length DNA sequence.
A sequence alignment between the Arabidopsis, Brassica, tobacco, soybean,
and corn FtsZl protein sequences is provided in figure 1.
Example 2: Preparation of Plant Expression Constructs
2A. Nuclear Expression Constructs
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Constructs are prepared for transformation into a plant cell nucleus for
alteration of the plastid size and/or number in the transformed plant cell.
Constructs
can be prepared to alter the plastids constitutively, or in a tissue specific
manner, for
example, in leaf tissue, or seed tissue.
A plasmid containing the napin cassette derived from pCGN3223 (described
in USPN 5,639,790, the entirety of which is incorporated herein by reference)
was
modified to make it more useful for cloning large DNA fragments containing
multiple
restriction sites, and to allow the cloning of multiple napin fusion genes
into plant
binary transformation vectors. An adapter comprised of the self annealed
oligonucleotide of sequence
CGCGATTTAAATGGCGCGCCCTGCAGGCGGCCGCCTGCAGGGCGCGCCAT
TTAAAT (SEQ ID N0:47} was Iigated into the cloning vector pBC SK+ (Stratagene)
after digestion with the restriction endonuclease BssHII to construct vector
pCGN7765. Plamids pCGN3223 and pCGN7765 were digested with NotI and
Iigated together. The resultant vector, pCGN7770, contains the pCGN7765
backbone
with the napin seed specific expression cassette from pCGN3223:
The cloning cassette, pCGN7787, essentially the same regulatory elements as
pCGN7770, with the exception of the napin regulatory regions of pCGN7770 have
been replaced with the double CAMV 35S promoter and the tml polyadenylation
and
2 0 transcriptional termination region.
A binary vector for plant transformation, pCGN5139, was constructed from
pCGN 1558 (McBride and Summerfelt, ( 1990) Plant Molecular Biology, 14:269-
276).
The polylinker of pCGN 1558 was replaced as a HindIIT/Asp718 fragment with a
polylinker containing unique restriction endonuclease sites, AscI, PacI, XbaI,
SwaI,
2 5 BamHI,and NotI. The Asp718 and HindIII restriction endonuclease sites are
retained
in pCGN5139.
A series of turbo binary vectors are constructed to allow for the rapid
cloning
of DNA sequences into binary vectors containing transcriptional initiation
regions
(promoters) and transcriptional termination regions.
3 0 The plasmid pCGN8618 was constructed by ligating oligonucleotides 5'-
TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID N0:48) and 5'-
TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID N0:49) into
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SaII/XhoI-digested pCGN7770. A fragment containing the napin promoter,
polylinker
and napin 3' region was excised from pCGN8618 bydigestion with Asp718I; the
fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment
then
ligated into pCGN5139 that had been digested with Asp718I and HindIII and
blunt-
s ended by filling in the 5' overhangs with Klenow fragment. A plasmid
cantaining the
insert oriented so that the napin promoter was closest to the blunted Asp718I
site of
pCGN5139 and the napin 3' was closest to the blunted HindIII site was
subjected to
sequence analysis to confirm both the insert orientation and the integrity of
cloning
junctions. The resulting plasmid was designated pCGN8622.
The plasmid pCGN8619 was constructed by Iigating oligonucleotides 5'-
TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC -3' (SEQ ID NO:50) and S'-
TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID N0:51) into
SaII/Xhol-digested pCGN7770. A fragment containing the napin promoter,
polylinker
and napin 3' region was removed frorri pCGN8619 by digestion with Asp718I; the
fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment
then
Iigated into pCGN5139 that had been digested with Asp718I and HindIII and
blunt-
ended by filling in the 5' overhangs with HIenow fragment. A plasmid
containing the
insert oriented so that the napin promoter was closest to the blunted Asp718I
site of
pCGN5139 and the napin 3' was closest to the blunted HindIII site was
subjected to
2 0 sequence analysis to confirm both the insert orientation and the integrity
of cloning
junctions. The resulting plasmid was designated pCGN8623.
The plasmid pCGN8620 was constructed by ligating oligonucleotides 5'-
TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGGAGCT -3' (SEQ ID N0:52}
and 5'-CCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID N0:53) into
2 5 SaII/SacI-digested pCGN7787. A fragment containing the d35S promoter,
polylinker
and tml 3' region was removed from pCGN8620 by complete digestion with Asp718I
and partial digestion with NotI. The fragment was blunt-ended by filling in
the 5'
overhangs with Klenow fragment then ligated into pCGN5139 that had been
digested
with Asp718i and HindIII and blunt-ended by filling in the S' overhangs with
Klenow
3 0 fragment. A plasmid containing the insert oriented so that the d35S
promoter was
closest to the blunted Asp718I site of pCGN5139 and the tml 3' was closest to
the
blunted HindIII site was subjected to sequence analysis to confirm both the
insert
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orientation and the integrity of cloning junctions. The resulting plasmid was
designated pCGN8624.
The plasmid pCGN8621 was constructed by ligating oligonucleotides 5'-
TCGACCTGCAGGAAGCTTGCGGCCGCGGATCCAGCT -3' (SEQ ID N0:54)
and 5'-GGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID N0:55) into
SaII/SacI-digested pCGN7787. A fragment containing the d35S promoter,
polylinker
and tml 3' region was removed from pCGN862I by complete digestion with Asg718I
and partial digestion with NotI. The fragment was blunt-ended by filling in
the 5'
overhangs with Klenow fragment then ligated into pCGN5139 that had been
digested
with Asp718I and HindIII and blunt-ended by filling in the 5' overhangs
withKlenow
fragment. A plasmid containing the insert oriented so that the d35S promoter
was
closest to the blunted Asp718I site of pCGN5139 and the tml 3' was closest to
the
blunted HindIII site was subjected to sequence analysis to confirm both the
insert
orientation and the integrity of cloning junctions. The resulting plasmid was
designated pCGN8625.
The plasmid construct pCGN8640 is a modification of pCGN8624 described
above. A 938bp PstI fragment isolated from transposon Tn7 which encodes
bacterial
spectinomycin and streptomycin resistance (Fling et al. ( 1985}, Nucleic Acids
Research 13(I9):7095-7106), a determinant for E. coli and Agrobacterium
selection,
2 0 was blunt ended with Pfu polymerase. The blunt ended fragment was ligated
into
pCGN8624 that had been digested with SpeI and blunt ended with Pfu polymerase.
The region containing the PstI fragment was sequenced to confirm both the
insert
orientation and the integrity of cloning junctions.
The spectinomycin resistance marker was introduced into pCGN8622 and
2 5 pCGN8623 as follows. A 7.7 Kbp AvrII-SnaBI fragment from pCGN8640 was
ligated to a 10.9 Kbp AvrII-SnaBI fragment from pCGN8623 or pCGN8622,
described above. The resulting plasmids were pCGN8641 and pCGN8643,
respectively.
The Arabidopsis FtsZl nucleotide sequence was used to construct the sense
3 0 expression vector pCGN649S for use in transformation of Arabidopsis.
Brassica and
tobacco. For this construct, the Arabidopsis ftsZl sequence was PCR amplified.
To
monitor protein expression of FtsZ1 in transformed lines, a c-myc tag
(EQKLISEEDL
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(SEQ ID N0:56)), was translationally fused to FtsZl at the C-terminal end. The
PCR
amplification was done by first round of amplification with primers SC247 (SEQ
iD
N0:57) and SC260 (SEQ ID N0:58} followed by amplification with SC247 (SEQ ID
NO:S9) and SC261 (SEQ ID N0:60) using the product of the first amplification
as the
5 template DNA, using standard amplification parameters. The final
amplification
product, FtsZl/c-myc fusion was cloned in the nuclear transfarmation vector
pCGN8624 to create pCGN6495, which was used to nuclear transform Arabidopsis,
canola and tobacco using standard protocols.
The turbo vector pCGN8624 was used for the antisense constructs such that
10 the antisense sequence is driven from d3SS promoter. For Arabidopsis the
coding
sequence (from ATG to TAG) was amplified with primers SC248 (SEQ ID N0:61)
and SC2S0 (SEQ ID N0:62) using AtFtsZl as template. For Brassical primers
SC276 (SEQ ID N0:63) and SC268 (SEQ ID N0:64) Were used with PCR fragment
SC3-1-1 (SEQ ID N0:70} as template DNA to generate aHindIIIlPstI fragment and
15 cloned in pBSKS (Stratagene) to generate pCGN6528. Primer SC276 was
designed to
be located 140 bases downstream from ATG due to the presence of nonhomologous
stretch of sequence compared to Arabidopsis FtsZl contained in the first 140
bases
sequence fragment. The 3' half of the coding sequence was PCR amplified using
primers SC269 (SEQ ID N0:65) and SC270 (SEQ iD N0:66) to produce a PstI/NotI
2 0 fragment, and subsequently cloned in pCGN6528 to generate pCGN6529. The
HindIIIlPstI fragment containing BnFtsZl sequence (from 140b downstream of ATG
to TAG} was cloned in turbo vector pCGN8624 to generate final transformation
vectors pCGN6S30 and pCGN6611. The HindIIIINotI fragment containing BnFtsZl
sequence was also cloned into pCGN8643 vector for seed-specific antisense
FtsZl
2 5 expression. For tobacco, primers SC30S and SC306 were designed to PCR
amplify
FtsZ 1 sequence to produce a SseIINotI fragment using 5' RACE PCR library DNA
made from leaf RNA> and cloned into TOPO TA2.1 to produce pCGN6565. The
SseIlNotI fragment from pCGN6565 was cloned in the turbo vector pCGN8624 to
generate final transformation vector pCGN6566.
2A. Plastid Expression Constructs
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Constructs and methods for use in transforming the plastids of higher plants
are described in Zoubenko et al. (Nuc Acid Res {I994) 22(19):3819-3824), Svab
et al.
(Proc. Natl. Acid. Sci. ( 1990) 87:8526-8530 and Proc. Natl. Acid. Sci. (
1993) 90:913-
917) and Staub et al. (EMBO J. (1993) 12:601-606): Constucts and methods for
use
in transforming plastids of higher plants to express DNA sequences under the
control
of a nuclearly encoded, plastid targeted T7 polymerise are described in U.S.
Patent
Number 5,576,198. The complete DNA sequences of the plastid genome of tobacco
are reported by Shinozaki et al. (EMBO J. ( 1986) 5:2043-2049).
A plastid expression construct, pMON49218, was constructed to express the
synthetic CP4 sequence with the 14 amino acid GFP fusion from the promoter
region
of the l6SrDNA operon having the nuclear-encoded RNA polymerise region
(PrrnPEP+NEP), and the terminator region from the plastid rps 16 gene. The DNA
sequence of the Prrn/NEP/G l OL:: I4aaGFP fusion SEQ ID N0:67.
Example 3: Plant Transformation And Analysis
Constructs for the expression of sense or antisense sequences are transformed
into tobacco cells using the methods described by Ursin et. al. (1991) Plant
Cell
2 0 3:583-591.
Transgenic tobacco plants containing the nuclear FtsZ constructs were
analyzed for alterations in plastid morphology. including size and number of
plastids
present in the plant cell.
Fifty-eight initial transformants {TI generation) obtained from transformation
with FtsZl expression construct pCGN6495 were screened for the large plastid
phenotype and divided into three categories. Thirty-four (34) lines contained
less than
5 large plastids, 8 lines contained between 5-20 plastids and 16 lines more
than 20
(wild-type# and more than wild-type#) plastids. One line, Nt6495-61, contained
a
single large plastid.
3 0 The screening method involved examining isolated mesophyll protoplasts at
100X magnification under light microscope. The large plastid containing
transgenic
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plants appear to be phenotypically indistinguishable from wild-type under
culture and
greenhouse conditions.
Estimation of plastid DNA copy number from several large plastid Iines
revealed no difference when compared to wild-type. Southern analysis was used
to
estimate transgene copy number in the large plastid lines and several lines
with single
integration events were identified. Western analysis of the large plastid
lines with c-
rnyc antibody confirmed expression of the introduced transgene (tagged by c-
myc). T2
seeds were collected from selected plants from each of the three categories.
Example 4: Plastid Transformation and Analysis
Leaf material from three transgenic lines, Nt6495-30 (with <5plastids/cell},
Nt6495-16 (with 5-20 plastids/cell) and Nt&495-69 {with 5-20 plastids/cell);
were
obtained for evaluation of plastid transformation efficiency and direct
glyphosate
selection. Plastid transformation vector pMON49218 which contains aadA gene
for
spectinomycin selection and GFP as a marker was used to bombard 15 leaf
explants of
each of the three transgenic lines. For each series of bombardment of the
transgenic
line 15 wild type control leaves were used. The order of bombardment for the
2 0 transgenic line and the wild type leaves were randomized to eliminate any
bias.
Transformation frequency of one event Nt6495-30 was approximately double
that of the wild type control producing 7 versus 3 transformants respectively.
Nt6495-
16 and Nt6495-69 had approximately the same transformation frequency (3
transformants) as the control. Thus, our preliminary analysis reveals that
plastid
transformation efficiency can have been enhanced by reducing the plastid
number
from wild type to less than 5 plastids per cell. Interestingly, alI of the
plastid
transformant regenerants from Nt6495 Lines were very much slower in growth and
size compared to those from wild type. It appears that the presence of the
selectable
antibiotic spectinomycin dihydrochloride at a concentration of 500mg/ml can
have
3 0 affected the regenerability of cells in the Nt6495 lines. Thus, it is
possible that there
could be more plastid transformed cells in the transgenic Nt6495 lines which
were
susceptible to the antibiotic and could not regenerate. To check if this was
the case,
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kill curves with lower concentrations of spectinomycin dihydrochloride (S0,
100, 200,
300, 400 and 500mg/mI) can be used with each of the Nt6495-30, Nt6495-16 and
Nt6495-69 lines to establish the concentration at which the regeneration of
shoots are
as good as in wild type. This concentration of spectinomycin dihydrochloride
will
then be used to repeat transformation frequency tests with the three Nt6495
lines.
To analyze for direct glyphosate selection, kill curves with varying levels of
glyphosate will be established with the Nt6495 lines to find the best
selection level.
Plastid transformation vector pMON49218 will be used to bombard the Nt6495
lines
and tested for direct selection using the optimized glyphosate level.
In Arabidopsis, FtsZl nuclear expression construct pCGN6495 was used to
transform Columbia ecotype. T1 seeds were collected and about i00 kanamycin
resistant seedlings were analyzed for alteration in plastid size and number
following
the same protocols as outlined for the tobacco section of this report. The
transgenic
plants were divided into three groups based on plastid number-I) 20
independent
lines containing few large plastid ( 1-5),{II) 23 lines lines containing 5-20
plastids and
(III) 50 lines containing wild-type plastid number were obtained. Selected T2
plants
from each category were analyzed for number of transgene integration loci and
sent to
the growth chamber for T3 seed collection to identify homozygous plants. Such
2 0 plants can be used in plastid transformations as described by Sikdar, et
al. { 1998)
Plant Cell Reports, 18:20-24.
Transformed plants selected for aadA marker gene expression or glyphosate
resistance are analyzed to determine whether the entire plastid content of the
plant has
been transformed (homoplasmic transformants). Typically, following two rounds
of
2 5 shoot formation and spectinomycin selection, approximately 50°l0 of
the transgenic
plantlets which are analyzed are homoplasmic, as determined by Southern blot
analysis of plastid DNA. Homoplasmic plantlets are selected for further
cultivation.
Southern blot analysis is used to confirm the integration of the chimeric
expression cassettes in the plastid genome. Preparation, electrophoresis, and
transfer
3 0 of DNA to filters is as described (Svab et al., ( 1993 supra)). Total
plant cellular DNA
can be prepared as described by Dellaporta et al. ( 1983) Plant Mol. Biol.
Rep. 1:19-
21).
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To visually observe the expression of marker genes such as GFP from the
chloroplasts of transformed plants, various tissues are visualized utilizing a
dissecting
microscope. Protoplasts and chloroplasts are isolated as described in Sidorov,
et al.
( 1994) ~'heor. Appl. Genet. 88:525-529.
The above results demonstrate that the sequences of the present invention
provide an efficient means for the production of plastid transformed plants.
Furthermore, such methods fmd use in plastid transformation methods involving
the
selection of transplastomic plants on herbicides, for example glyphosate.
All publications and patent applications mentioned in this specification are
indicative of the level of skill of those skilled in the art to which this
invention
pertains. Ail publications and patent applications are herein incorporated by
reference
to the same extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
obvious
that certain changes and modifications rnay be practiced within the scope of
the
appended claim.
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SEQUENCE LISTING
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CA 02352464 2001-05-24
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Met Ala Ile Ile Pro Leu Ala Gln Leu Asn Glu Leu Thr I1e Ser Ser
1 5 10 15
Ser Ser Ser Ser Phe Leu Thr Lys Ser Ile Ser Ser His Ser Leu His
20 25 30
Ser Ser Cys Ile Cys Ala Ser Ser Arg Ile Ser Gln Phe Arg Gly Gly
35 40 45
Phe Ser Lys Arg Arg Ser Asp Ser Thr Arg Ser Lys Ser Met Arg Leu
50 55 60
Arg Cys Ser Phe Ser Pro Met Glu Ser Ala Arg Ile Lys Val Ile Gly
65 70 75 80
Val Gly Gly Gly Gly Asn Asn A1a Val Asn Arg Met Ile Ser Ser Gly
g5 90 95
Leu Gln Ser Val Asp Phe Tyr Ala Ile Asn Thr Asp Ser Gln Ala Leu
100 105 110
Leu Gln Phe Ser Ala Glu Asn Pro Leu Gln Ile Gly Glu Leu Leu Thr
115 120 125
Arg G1y Leu Gly Thr Gly Gly Asn Pro Leu Leu Gly Glu Gln Ala Ala
130 235 140
Glu Glu Ser Lys Asp Ala Ile Ala Asn Ala Leu Lys Gly Ser Asp Leu
145 150 155 160
Val Phe I1e Thr Ala Gly Met Gly Gly Gly Thr Gly Ser Gly Ala Ala
165 170 175
Pro Val Val Ala Gln Ile Ser Lys Asp Ala Gly Tyr Leu Thr Val G1y
180 185 190
Va1 Val Thr Tyr Pro Phe Ser Phe Glu Gly Arg Lys Arg Ser Leu Gln
195 200 205
Ala Leu Glu Ala Ile Glu Lys Leu Gln Lys Asn Val Asp Thr Leu Ile
210 215 220
Val Ile Pro Asn Asp Arg Leu Leu Asp Ile AIa Asp Glu Gln Thr Pro
225 230 235 240
Leu Gln Asp Ala Phe Leu Leu Ala Asp Asp Val Leu Arg Gln Gly Val
245 250 255
Gln Gly Ile Ser Asp Ile Ile Thr Ile Pro Gly Leu Val Asn Val Asp
260 265 270
Phe Ala Asp Val Lys Ala Val Met Lys Asp Ser Gly Thr Ala Met Leu
275 280 285
G1y Val Gly Val Ser Ser Ser Lys Asn Arg Ala Glu Glu Ala Ala Glu
290 295 300
Gln Ala Thr Leu Ala Pro Leu Ile Gly Ser Ser Ile Gln Ser Ala Thr
305 310 315 320
Gly Val Va1 Tyr Asn I1e Thr Gly Gly Lys Asp Ile Thr Leu Gln Glu
325 330 335
Val Asn Arg Val Ser Gln Val Val Thr Ser Leu Ala Asp Pro Ser Ala
340 345 350
Asn Ile Ile Phe Gly Ala Val Va1 Asp Asp Arg Tyr Thr Gly Glu Ile
355 360 365
His Val Thr Ile Ile Ala Thr Gly Phe Ser Gln Ser Phe Gln Lys Thr
370 375 380
Leu Leu Thr Asp Pro Arg Ala Ala Lys Leu Leu Asp Lys Met Gly Ser
385 390 395 400
Ser Gly Gln Gln G1u Asn Lys Gly Met Ser Leu Pro His Gin Lys Gln
405 410 415
Ser Pro Ser Thr Ile 5er Thr Lys Ser Ser Ser Pro Arg Arg Leu Phe
420 425 430
Phe
- 2 -
CA 02352464 2001-05-24
WO 00132799 PCT/US99/2$103
<210>
3
<211>
1611
<212>
DNA
<213>
Arabidopsis
sp
<400>
3
tgttgttgccgctcagaaatctgaatcttctccaatcagaaactctccacggcattacca60
aagccaagctcaagatcctttcttgaaccttcacccggaaatatctatgcttagaggtga120
agggactagtacaatagtcaatccaagaaaggaaacgtcttctggacctgttgtcgagga180
ttttgaagagccatctgctccgagtaactacaatgaggcgaggattaaggttattggtgt240
gggaggtggtggatcaaatgctgtgaatcgtatgatagagagtgaaatgtcaggtgtgga300
gttctggattgtcaacactgatatccaggctatgagaatgtctcctgttttgcctgataa360
taggttacaaattggtaaggagttgactaggggtttaggtgctggaggaaatccagaaat420
cggtatgaatgctgctagagagagcaaagaagttattgaagaagctctttatggctcaga480
tatggtctttgtcacagctggaatgggcggtggaactggcactggtgcagcccctgtaat540
tgcaggaattgccaaggcgatgggtatattgacagttggtattgccacaacgcctttctc600
gtttgagggtcgaagaagaactgttcaggctcaagaagggcttgcatctctcagagacaa660
tgttgacactctcatcgtcattccaaatgacaagttgcttacagctgtctctcagtctac720
tccggtaacagaagcatttaatctagctgatgatatactccgtcagggggttcgtgggat780
atctgatatcattacgattcctggtttggtgaatgtggattttgctgatgtgagagctat840
aatggcaaatgcggggtcttcattgatgggaataggaactgcgacaggaaagagtcgggc900
aagagatgctgcgctaaatgcaatccaatcccctttgttagatattgggattgagagagc960
cactggaattgtttggaacattactggcggaagtgacttgacattgtttgaggtaaatgc1020
tgctgcggaagtaatatatgatcttgtcgatccaactgccaatcttatattcggtgctgt1080
tgtagatccagccctcagcggtcaagtaagcataaccctgatagctacgggtttcaaacg1140
acaagaagagggagaaggacgaacagttcagatggtacaagcagatgctgcgtcagttgg1200
agctacaagaagaccctcttcttcctttagagaaagcggttcagtggagatcccagagtt1260
cttgaagaagaaaggcagctctcgttatccccgagtctaaagcccaatctaatcactacc1320
ctgcacactgcagcaataacaaacgtgtgtgtactggtagtctggtactgccttctggga1380
tacagcaagatgtgttgatgtatgatcaagaatctgtgtgggtgtgtatatgttctgtca1440
ctgcctctggtcgtgttcttgaataggttgttttagaaatcggagtttctctctatgtca1500
cttccaaaacaaaaaaggagaagaagaatcacacttctcgaaccataaacatacttataa1560
gattatgagagttttagcagaaattattgtcaaaaaaaaaaaaaaaaaaaa 1611
<210>
4
<211>
397
<212>
PRT
<213>
Arabidopsis
sp
<400>
4
Met Leu Asn Pro
Arg Arg Lys
Gly G1u
Glu
Gly
Thr
Ser
Thr
Ile
Val
1 5 10 15
Thr Ser Glu Pro
Ser Ser Ala
Gly Pro
Pro
Val
Val
G1u
Asp
Phe
Glu
20 25 30
Ser Asn Lys Val Gly Val
Tyr Ile Gly Gly
Asn Gly
Glu
Ala
Arg
Ile
35 40 45
Gly Ser Ile Glu Glu Met
Asn Ser Ser Gly
Ala Val
Val
Asn
Arg
Met
50 55 60
Glu Phe Ile Gln Met Arg
Trp Ala Met Ser
Ile Pro
Val
Asn
Thr
Asp
65 70 75 80
Val Leu Ile Gly Glu Leu
Pro Lys Thr Arg
Asp Gly
Asn
Arg
Leu
Gln
85 90 95
Leu Gly a Gly Ile Gly Asn Ala
Al Gly Asn Met Ala Arg
Pro G1u Glu
- 3 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
200 105 110
Ser Lys Glu Val Ile Glu Glu Ala Leu Tyr Gly Ser 'Asp Met Val Phe
115 120 125
Val Thr Ala Gly Met Gly Gly Gly Thr Gly Thr Gly Ala Ala Pro Val
130 135 140
Ile Ala Gly Ile Ala Lys Ala Met Gly Ile Leu Thr Val Gly Ile A1a
145 150 155 160
Thr Thr Pro Phe Ser Phe Glu Gly Arg Arg Arg Thr Val Gln Ala Gln
165 170 175
Glu Gly Leu A1a Ser Leu Arg Asp Asn Val Asp Thr Leu Ile Val Ile
180 185 190
Pro Asn Asp Lys Leu Leu Thr Ala Val Ser Gln Ser Thr Pro Val Thr
195 200 205
Glu Ala Phe Asn Leu Ala Asp Asp Ile Leu Arg Gln Gly Val Arg Gly
210 215 220
Ile Ser Asp Ile ITe Thr Ile Pro Gly Leu Val Asn Val Asp Phe Ala
225 230 235 240
Asp Val Arg Ala Ile Met Ala Asn Ala Gly Ser Ser Leu Met Gly Ile
245 250 255
Gly Thr Aia Thr Gly Lys Ser Arg Ala Arg Asp Ala Ala Leu Asn A1a
260 265 270
Ile Gln Ser Pro Leu Leu Asp Ile Gly Ile Glu Arg Ala Thr Gly Ile
275 280 285
Val Trp Asn Ile Thr Gly Gly Ser Asp Leu fihr Leu Phe Glu Val Asn
290 295 300
Ala Ala Ala Glu Val Ile Tyr Asp Leu Val Asp Pro Thr Ala Asn Leu
305 310 315 320
Ile Phe Gly Ala Val Val Asp Pro Ala Leu Ser Gly Gln Val Ser Ile
325 330 335
Thr Leu Ile Ala Thr Gly Phe Lys Arg Gln Glu Glu Gly Glu Gly Arg
340 345 350
Thr Val Gln Met Val Gln Ala Asp Ala Ala Ser Val Gly Ala Thr Arg
355 360 365
Arg Pro Ser Ser Ser Phe Arg Glu Ser Gly Ser Val Glu Ile Pro Glu
370 375 380
Phe Leu Lys Lys Lys Gly Ser Ser Arg Tyr Pro Arg Val
385 390 395
<210> 5
<211> 1450
<222> DNA
<213> Brassica sp
<400>
atggcgattagtccgttggcacagcttaacgagctaccagtctcttcctcgtttcttgcg 60
acatcccactcgctgcacagtaccagaatcagtggcggcttctcaaaacaaaggtttaag 120
caaacacggttgagatgctccttctctccgatggagtctgcgaggattaaggtggttggt 180
gtcggcggtggtggtaacaatgccgtcaatcgcatgatttccagcggcttacagagtgtt 240
gatttctatgcgataaacacggactctcaagctctcttgcagtcttctgcgcagaaccct 300
cttcaaattggagagctcctaactcgtggccttgggactggtgggaacccgcttctagga 360
gaacaagctgctgaggaatctaaagacgcgattgctaatgctcttaaaggatctgacctt 420
gytttcattactgctggtatgggtggtggcactggctccggtgctgctcctgttgttgct 480
cagatctcgaaagacgctggttatttgaccgttggtgttgttacctatcccttcagcttc 540
gaaggtcgtaaaagatctttgcaggcacttgaagccattgaaaagctgcagaagaacgtg 600
gataccctcatcgtgataccaaatgatcgtctcctagatattgctgatgaacagacgcct 660
- 4 -
CA 02352464 2001-05-24
WO 00/32799 PCT/(1599/28103
cttcaagacgcttttcttctcgcggatgatgttttgcggcaaggagttcaaggaatctct720
gatattattactatacctggactggtcaatgtagattttgcggatgtgaagtcggttatg780
aaagattccggaactgcgatgctcggggtgggtgtttcttcaagcaagaaccgagcagaa840
gaagcagctgagcaagccactttggctccattgatcggatcatccattcaatcagctact900
ggtgtcgtctacaacatcaccggtggaaaagacattactttgcaggaagtgaaccgagta960
tctcaggtggtgacaagtttggcagacccatcggccaacatcatatttggagctgttgtg1020
gatgatcgatacactggagagattcatgtaacgataatagccacggggttctcacagtct1080
ttccagaagacacttctcagtgatccaagagcagctaaactactcgacaaaacgggatca1140
tcaggtcaacaacaagagaacaaaggcagtcaccagaggcagtctcctgcaactatcaac1200
accaaatcatcttctccccgtagattg.ttcttctagtatcttttgttttttaagcatatt1260
cctttatcaaaaatgtaacgatcttcaggctcaaatatcaattacttttctccagattat1320
ctcaaaagaagtaatttgttaaaccaaaaaaaaaaaaaaagggcggccgctctagaggat1380
ccaagcttacgtacgcgtgcatgcgacgtcatagctcttctatagtgtcacctaaattca1440
attcactggc
1450
<210> 6
<211> 411
<212> PRT
<213> Brassica sp
<220>
<221> VARIANT
<222> (1)...(411
<223> Xaa = Any Amino Acid
<400> 6
Met Ala Ile Ser Pro Leu Ala Gln Leu Asn Glu Leu Pro Val Ser Ser
1 5 10 15
Ser Phe Leu Ala Thr Ser His Ser Leu His Ser Thr Arg Ile Ser Gly
20 25 30
Gly Phe Ser Lys G1n Arg Phe Lys Gln Thr Arg Leu Arg Cys Ser Phe
35 40 45
Ser Pro Met Glu Ser Ala Arg Ile Lys Val Val Gly Val Gly Gly Gly
50 55 60
Gly Asn Asn Ala Val Asn Arg Met Ile Ser Ser Gly Leu Gln Ser Val
65 70 75 80
Asp Phe Tyr Ala I1e Asn Thr Asp Ser Gln Ala Leu Leu Gln Ser Ser
85 90 95
Ala Gln Asn Pro Leu Gln Ile Gly Glu Leu Leu Thr Arg Gly Leu Gly
100 105 110
Thr Gly Gly Asn Pro Leu Leu G1y Glu Gln Ala Ala Glu Glu Ser Lys
115 120 125
Asp Ala Ile Ala Asn Ala Leu Lys Gly Ser Asp Leu Xaa Phe Ile Thr
130 135 140
Ala Gly Met G1y Gly Gly Thr Gly Ser Gly Ala Ala Pro Val Val Ala
145 150 155 160
Gln Ile Ser Lys Asp Ala Gly Tyr Leu Thr Val Gly Val Val Thr Tyr
165 170 175
Pro Phe Ser Phe Glu Gly Arg Lys Arg Ser Leu Gln Ala Leu Glu Ala
180 185 190
Ile Glu Lys Leu Gln Lys Asn Val Asp Thr Leu Ile Val Ile Pro Asn
195 200 205
Asp Arg Leu Leu Asp Ile Ala Asp Glu Gln Thr Pro Leu Gln Asp Ala
210 215 220
Phe Leu Leu Ala Asp Asp Val Leu Arg Gln Gly Val Gln Gly Ile Ser
- 5 -
CA 02352464 2001-05-24
WO 00/32799 PCTIUS99I2$i03
225 230 235 240
Asp Ile Thr 21e Pro Gly Leu Val Asp Phe Asp Val
Ile Asn Val Ala
245 250 255
Lys Ser Met Lys Asp Ser Gly Thr Leu Gly Gly Val
Val Ala Met Val
260 265 270
Ser Ser Lys Asn Arg Ala Glu Glu Glu Gln Thr Leu
Ser Ala Ala Ala
275 280 285
Ala Pro Ile Gly Ser Ser Ile Gln Thr Gly Val Tyr
Leu Ser Ala Val
290 295 300
Asn Ile Gly Gly Lys Asp I1e Thr Glu Val Arg Val
Thr Leu Gln Asn
305 310 315 320
Ser Gln Val Thr Ser Leu Ala Asp Ala Asn Ile Phe
Val Pro Ser I7.e
325 330 335
Gly Ala Val Asp Asp Arg Tyr Thr Ile His Thr Ile
Val Gly Glu Val
340 345 350
Ile Ala Gly Phe Ser Gln Ser Phe Thr Leu Ser Asp
Thr Gln Lys Leu
355 360 365
Pro Arg Ala Lys Leu Leu Asp Lys Ser Ser Gln Gln
Ala Thr Gly Gly
370 375 380
Gln Glu Lys Gly Ser His Gln Arg Pro Ala Ile Asn
Asn Gln Ser Thr
385 390 395 400
Thr Lys Ser Ser Pro Arg Arg Leu
Ser Phe Phe
405 410
<210> _
7
<211>
2295
<212>
DNA
<213> iana sp
Nicot
<400>
7
tgccgttaaccggatgattt caagcggttt acagggtgttgacttctatgctataaacac 60
ggatgctcaagcactgctac agtctgctgc tgaaaacccgcttcaaattggagaacttct 120
gactcgtgggcttggtactg gtggtaatcc tcttttaggggaacaggcagtggaggagtc 180
gaaggaagccattgcaaatt ctctaaaagg ttcagatatggtgttcataacagcaggaat 240
gggtggaggtacaggatctg gtgctgctcc tgttgtggctcaaatagcaaaagaagcagg 300
ctatttgactgttggtgttg tcacataccc attcagctttgaaggacgtaaaagatccgt 360
gcaggctctggaagcaattg aaaaacttca gaaaaatgtagatacccttatagtaattcc 420
aatgaccgtctgctagatat tgctgatgag cagacaccacttcaagatgcttttcttctt 480
gctgatgatgtattacgcca aggtgtccaa ggaatttccgatataattactatacctggg 540
cttgtaaatgtggattttgc cgatgtaaag gtagtgatgaaagattctggaactgctatg 600
cttggagttggggtttcatc aagcaagaac cgtgctgaagaagcagccgaacaagcaact 660
cttgcccctcttaattggat cgtccattca atcgccactggggtagtatccaccattcca 720
ggaggaaaagaccataactt tgcagaaagt gaatagggtgtctcaggttgttacagtctg 780
gctgatccctcccgctaaca tcatatttgg tgctgttgtggatgagcgctacaatggcga 840
aatacacgtgaccataattg caactggttt tacccagtcttttcagaagactcttctctc 900
tgacccacgaggtgcaaagc ttgttgataa aggcccagtaatccaagaaagcatggcatc 960
acctgttaccctgaggtcat caacctcacc ttcgacaacatcacgaacacctactcggag 1020
gctgttcttttagctccttt atatagtttg ttacggcttcatttttctcttttcttactt 1080
ttttcttttttactttcttt gtatttacat,gttttgctgattggtgtttgcatttggctg 1140
tagacatagtgatgattctt atcaagtgca tcacattcatactcgaaaaaaaaaaaaaaa 1200
aaaaaaagtactctgcgttg ttacccactg ttaagggcgaattctgcagatatcccatca 1260
cactggcggccgctcgagca tgcatctaga gggcc 2295
<210>
8
<211>
1255
- 6 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/2$103
<212> DNA
<213> Nicotiana sp
<400> 8
atggccacca tctcaaaccc agcagagatagcagcttcttctccttcctttgctttttac60
cactcttcct ttattcctaa acaatgctgcttcaccaaagctcgccggaaaagcttatgt120
aaacctcaac gtttcagcat ttcaagttcatttactccttttgattctgctaagattaag180
gttatcggcg tcggtggcgg tggtaacaatgccgttaaccggatgatttcaagcggttta240
cagggtgttg acttctatgc tataaacacggatgctcaagcactgctgcagtctgctgct300
gaaaacccgc ttcaaattgg agaacttctgactcgtgggcttggtactggtggtaatcct360
cttttagggg aacaggcagc ggaggagtcgaaggaagccattgcaaattctctaaaaggt420
tcagatatgg tgttcataac agcaggaatgggtggaggtacaggatctggtgctgctcct480
gttgtggctc aaatagcaaa agaagcaggctatttgactgttggtgttgtcacataccca540
ttcagctttg aaggacgtaa aagatccgtgcaggctctggaagcaattgaaaaacttcag600
aaaaatgtag atacccttat agtaattcccaatgaccgtctgctagatattgctgatgag660
cagacaccac ttcaagatgc ttttcttcttgctgatgatgtattacgccaaggtgtccaa720
ggaatttccg atataattac tatacctgggcttgtaaatgtggattttgccgatgtaaag780
gtagtgatga aagattctgg aactgctatgcttggagttggggtttcatcaagcaagaac840
cgtgctgaag aagcagccga acaagcaactcttgcccctcttattggatcgtccattcaa900
tcagccactg gggtagtatc caccattccaggaggaaaagacataactttgcagaaagtg960
aatagggtgt ctcaggttgt tacagtctggctgatccctcccgctaacatcatatttggt1020
gctgttgtgg atgagcgcta caatggcgaaatacacgtgaccataattgcaactggtttt1080
acccagtctt ttcagaagac tcttctctctgacccacgaggtgcaaagcttgttgataaa1140
ggcccagtaa tccaagaaag catggcatcacctgttaccctgaggtcatcaacctcacct1200
tcgacaacat cacgaacacc tactcggaggctgttcttttagctcctttatatag 1255
<210> 9
<211> 413
<212> PRT
<213> Nicotiana sp
<400> 9
Met Ala Thr Ile Ser Asn Glu Ile Ala Ser Pro Ser
Pro Ala Ala Ser
1 5 10 15
Phe Ala Phe Tyr His Ser Ile Pro Gln Cys Phe Thr
Ser Phe Lys Cys
20 25 30
Lys Ala Arg Arg Lys Ser Lys Pro Arg Phe Ile Sex
Leu Cys Gln Ser
35 40 45
Ser Ser Phe Thr Pro Phe Lys Val Gly Val
Asp Ser Ala Lys Ile Ile
50 55 60
Gly Gly Gly Gly Asn Asn Ile Ser G1y Leu
Ala Va1 Asn Arg Met Ser
65 70 75 80
Gln Gly Val Asp Phe Tyr Ala Gln Leu Leu
Ala Ile Asn Thr Asp Ala
85 90 95
Gln Ser Ala Ala Glu Asn Gln Ile Glu Leu Thr Arg
Pro Leu Gly Leu
100 105 110
Gly Leu Gly Thr Gly Gly Leu Leu Glu Gln Ala Glu
Asn Pro Gly Ala
115 120 125
Glu Sex Lys Glu Ala Ile Ser Leu Gly Sex Met Val
Ala Asn Lys Asp
130 135 140
Phe Tle Thr Ala Gly Met Gly Thr Ser Gly Ala Pro
Gly Gly Gly Ala
145 150 155 160
Val Va1 Ala Gln Ile Ala Ala Gly Leu Thr Gly Val
Lys Glu Tyr Val
165 170 175
Val Thr Tyr Pro Phe Ser Gly Arg Arg Ser Gln Ala
Phe Glu Lys Val
_ 7 _
CA 02352464 2001-05-24
WO 00/32799 ~ PCT/US99/28103
180 185 190
Leu Glu Ile Glu Lys Leu Gln Lys Asp Thr Ile Val
Ala Asn Val Leu
195 200 205
Ile Pro Asp Arg Leu Leu Asp Ile Glu Gln Pro Leu
Asn Ala Asp Thr
210 215 220
Gln Asp Phe Leu Leu Ala Asp Asp Arg Gln Val Gln
Ala Val Leu Gly
225 230 235 240
Gly Ile Asp Ile Ile Thr Ile Pro Val Asn Asp Phe
Ser Gly Leu Val
245 250 255
A1a Asp Lys Val Val Met Lys Asp Thr Ala Leu Gly
Val Ser Gly Met
260 265 270
Val Gly Ser Ser Sex Lys Asn Arg Glu Ala Glu Gln
Val Ala G1u Ala
275 280 285
Ala Thr Ala Pro Leu Ile Gly Ser Gln Ser Thr Gly
Leu Ser Ile Ala
290 295 300
Val Val Thr Ile Pro Gly Gly Lys Thr Leu Lys Val
Sex Asp Ile Gln
305 310 315 320
Asn Arg Ser Gln Val Va1 Thr Val Ile Pro Ala Asn
Val Trp Leu Pro
325 330 335
Ile Ile Gly Ala Val Val Asp Glu Asn Gly Ile His
Phe Arg Tyr Glu
340 345 350
Val Thr Ile A1a Thr Gly Phe Thr Phe Gln Thr Leu
Ile Gln Ser Lys
355 360 365
Leu Ser Pro Arg Gly Ala Lys Leu Lys Gly Val Ile
Asp Val Asp Pro
370 375 380
Gln Glu Met Ala Ser Pro Val Thr Ser Ser Ser Pro
Ser Leu Arg Thr
385 390 395 400
Ser Thr Ser Arg Thr Pro Thr Arg Phe Phe
Thr Arg Leu
405 410
<210> 10
<211> 12?8
<212> DNA
<213> Zea
mays
<220>
<221> misc_feature
<222> (1)...(1278)
<223> n ,T,C or G
= A
<400> 10
gatcttgtcttcataacagc tgggatggga gggggtactggatctggtgctgctccagtt60
gttgcccagatatcaaagga agctggttat cttactgttggtgttgtcacctatccattc120
agtttcgagggccgtaagcg ctctgtacag gcattggaagcactagagaagctggaaaag180
agtgtagacacacttattgt gattccaaat gataagttattagatgttgcggatgaaaac240
atgcccttgcaagatgcatt tctccttgca gatgatgtccttcgtcagggtgttcaagga300
atatcagacatcatcacaat accgggactt gtcaatgttgattttgctgatgtaaaagct360
gtcatgaaaaactctggaac tgccatgctc ggtgttggtgtttcttccagcaaaaatcgg420
gcccaagaagctgctgaaca ggcaacactt gctcctttgattggatcatccatcgaggca480
gctactggcgttgtgtataa tattactggt gggaaggacatcactttgcaagaagtgaac540
aaggtgtcccagattgtgac aagcctagct gacccatctgcgaacataattttcggtgct600
gtcgttgatgaccgttacac tggtgagata catgtgacaatcattgcgacaggatttcca660
cagtccttccagaaatccct tttggcggat ccaaagggagcacgtatagtggaatccaaa720
gagaaagcagcaaccctcgc ccataaagca gcagcagctgcagttcaaccggtccctgct780
tctgcttggtctcgaagact cttctcctga gaagctcatttggtgaaccgtgactcgtag840
_ g _
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
tgcattagatttgcatttagcgtgttgagggcagtccctaaggtgatcttcggatatctg 900
gagatttatagcttgggctagtgttcggtagtggtagaataagtttcagtgtatgtatcg 960
ttgctttgctttatgtttttgaggatcaggcggtgaggctgagagaagtgctcagcaact 1020
caacattgaactgttgtagaagatctttgattgcttttattgctgcaacatgccaacaac 1080
cctctgttggattcamcmnaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1140
aaaaaaaaaaaaaaaaaaaaaaaanncaaaaaaaaaaaaaaaaaaaaagggcggccgccg 1200
actagtgagctcgtcgacccgggaattaattccggaccggtacctgcaggcgtaccagct 1260
ttccctatagtgagtcgt 1278
<210>
11
<211>
283
<212>
DNA
<213>
Zea mays
<400>
11
gctccagttgttgcccagatatcaaaggaagctggttatcttactgttggtgttgtcacc 60
tatccattcagtttcgagggccgtaagcgctctgtacaggcattggaagcactagagaag 120
ctggaaaagagtgtagacacacttattgtgattccaaatgataagttattagatgttgcg 180
gatgaaaacatgcccttgcaagatgcatttctccttgcagatgatgtccttcgtcagggt 240
gttcaaggaatatcagacatcatcacaataccgggacttgtca 283
<210>
12
<211>
287
<212>
DNA
<213>
Zea mays
<220>
<221> feature
misc
_
<222>
(1).
.(287)
<223>
n = A,T,C
or G
<400>
12
gggccgtaagcgctctgtacaggcattggaagcactagagaagctggaaaagagtgtaga 60
cacacttattgtgattccanatnatnngttattagatgttgcggatgaaaacatgccctt 120
gcaagatgcatttctccttgcagatgatgtccttcgtcagggtgttcaaggaatatcaga 180
catcatcacaataccgggacttgtcaatgttgattttgctgatgtaaaagctgtcatgaa 240
aaactctggaactgccatgctcggtgttggtgtttcttccagcaaaa 287
<210>
13
<211>
1122
<212>
DNA
<213>
Zea mays
<400>
13
gctataaacaccgattcccaagcccttattaattcacaagcgcaatatcctctgcaaatt 60
ggagagcagttgacccgcggcttaggtgccggtggaaatccgaatttgggagagcaggct 120
gctgaggaatcaagagaaaccatagccactgccctgagggattcagatcttgtcttcata 180
acagctgggatgggagggggtactggatctggtgctgctccagttgttgcccagatatca 240
aaggaagctggttatcttactgttggtgttgtcacctatccattcagtttcgagggccgt 300
aagcgctctgtacaggcaaagtatctgagccccccttcactcctgaattttaattcaaac 360
tgtcatatctcgttctgcgactttcttttgctcgatggaagcattagtttgtagtcataa 420
caatgacatccagccacatttattgctgatgatgtatacaatggtaggtcaaagaaatgt 480
agcatcatgccatcacctgtagttcatctcatcattttgttcctacttttctgcgtggtt 540
gatgcccaaaacaatatacaactatgtggttgtactgttgcattgccttgtggagggatg 600
tttatgttgtgaaatatttcaaaacacatgtcattatgaatattccctcctgtggttgtg 660
_ g _
CA 02352464 2001-05-24
WO 00132799 PCT/US99/28103
gggacttgtttcaaatgctatgaattaagaacaaggcaacataaagtgttaaatgttaac720
cgtctttcgtccatgaaacattattcccttgaggataatgggccttggacaaaggctgat780
gagagtataattaccaagcttaaatcttcgtaataaaatttcaatagatattgtaagata840
acataaaataaagggtataaaaaggggtaaataaatcatagacgaattatattatattta900
cttaatatattgaatcattgaatacaataatacctctgccttggcaaaggttggattccg960
aaaaatgtgattgcaagttaccagaatgcgtgaacagtaaaggaatactgttcactattt1020
ataggcacaggacacagcctgtggaggaattcaattatacccgtcataagagtttacaca1080
ttgacttagacctttatggactaaaagatcattgctatcttt 1122
<210>
14
<211>
291
<212>
DNA
<213> mays
Zea
<220>
<221> feature
misc
<222> _
(1). .(291)
<223> A,T,C or
n = G
<400>
14
aaaatagtgtggacaccctaatcgtcatcccaaatgataagttgctgtctgctgtttctc60
caaatacacctgtaactgaagcatttaatctggctgatgatattcttcgtcaaggcattc120
gtggcatatctgatataattacggttcctgggnaggttaatgttgattttgctgacgtac180
gtgctatcatgcaaaatgcagggtcatccttgatgggtatagggactgctacaggaaagt240
caagagcaagggatgctgctcttaacgccatccagtcgccgctgcttgata 291
<210>
15
<211>
415
<212>
DNA
<213> mat's
Zea
<220>
<221> feature
misc
<222> _
(1). .(415)
<223> A,T,C or
n = G
<400>
15
gagcaagggatgctgctcttaacgccatccagtcgccgctgcttgatattggaattgaaa60
gagccacaggcattgtgtggaatatcactgggggaactgacctgactttgtttgaggtga120
atgctgcggccgaaattatctacgaccttgtcgatccaaacgctaatctgatatttggcg180
ccgtcatagacccgtcactgagtgggcaggtgagcataaccttgatagctactggcttca240
.
aacggcaggatgaaccagaaggccgcgtgtcgaagggtgggcaacaaggtgagaatggcc300
gacgcccatccccagcanagggcaacaacacggtggaaattccaaaattcccgccaacaa360
aaagggcccttccnncttcccacnattttgactggtcctgtctgcacctgtatga 415
<210>
16
<211>
744
<212>
DNA
<213> mat's
Zea
<220>
<221> feature
misc
<222> _
(1). .(744)
<223> A,T,C or
n = G
- 10 -
CA 02352464 2001-05-24
WO OOI32799 PCT/US99/28103
<400>
16
aattcccgggtcgacccacgcgtcccgcggacgcgtgggtggaatatcactggagggaac 60
gatctaaccttgacagaggtgaatgctgcagctgaagtaatctatgatcttgttgaccct 120
ggtgcaaatctgatttttggctctgttatagatccgtcatacactggtcaagtgagcata 180
actctaattgcaactggtttcaaacgccaggaggaaagtgagagccggtcttcacaggct 240
ggaggagacaagcaaccgcggtcgctcggctggttttctcccacttcccaggaggaaggt 300
catgcattgcaaatcccanagttcctacagaggaaagggcgtccagggtttcacgagtct 360
gaacacactttggatcaatgtttttcttgtcatagtttggtacgatgcaggtttggtttc 420
tgggtctcttaggtagcaaggtagaacagatgttcctgaacccgcacatactaatctgtg 480
tgcaaacttcngccgctgagtaccattggcttgggctgctttgcttctcangaacctgca 540
gtgaggtctcaatttgctagttagtatgattaaaagtnaagcgctgagaccaaattatac 600
gttccgtgtgaatgattacttgctcnctgccattttcttttcaaaaaaaaaaaaaaaaaa 660
aaaaaggcggcgctntanaggatccaagcttacttcccctgcatncgacncanagctntt 720
ntatagngtnacctaaattcaatc 744
<210>
17
<211>
230
<212>
DNA
<213>
Zea mays
<220>
<221> feature
misc -
<222>
(lj..
.(230)
<223>
n = A,T,C
or G
<400>
17
ggctgctgaggaatcaagagaaaccatagccactgccctgagggattcagatcttgtttt 60
cataacagctgggatgnnagggggtgctgctccaattgttgcccagatatcaaaggaagc 120
tggttatcttactgttggtgttgtcacctatccattcaatttcgagggccgtaagcgctc 180
tttacaggcaagtatctgagccccccttcactcctgaattagaattcaaa 230
<210>
18
<211>
318
<212>
DNA
<213>
Zea mays
<220>
<221> feature
misc
<222> _
(1j. .(318)
<223>
n = A,T,C
or G
<400>
18
caggcattgtgtggaatatcactgggggaactgacctaactttgtttgaggtgaatgctg 60
cggccgaaattatctacgaccttgtcgatccaaatgctaatctgatatttggtgccgtca 220
tagacccgtcactgagtgggcaggtgagcataacctgatagctactggcttcaaacggca 180
ggatgaaccagaaggccgcgtgtcgaagggtgggcaacaaagtgagaatggccgacgccc 240
gtcccccgcagagggcagcagcacggtggagttccagagtcctgcgacgtagagganctt 300
ctcgcttcccagagttga 318
<210>
19
<211>
471
<212>
DNA
<213>
Zea mays
<220>
- 11 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
<221> misc feature
<222> {1)...(472)
<223> n = A,T,C or G
<400> 19
cgacgcccaaggtgacgaatgctgtcagccacgctgtgctacacgggggaaacaatgcaa60
anacattacctgcctcactcntgcttgctcctgtaaatataatgatngtcgctgctacat120
natatttactcctgctgctgcttgaggccattattctgtacgtaaatgaagccactacta180
ctctcacacagcatgcgccggccgacgacgtacgtacgtgtattatatacgctctacccc240
gtgagcttttgttcgagtgatacgtgatccatccatgcatggatgcttatgtatgtatat300
gtgttagtcgtctcagggaaccgggcancanaagggggtgttgtattanatttacgtctt360
ctggtgattaaataanaaaggggtatgttggatgtgtgcaaaaaaaaaaaaaaaanaaaa420
aaaaaaaaaaaaaaaaaaagggcggccgccgactagtgagctcgtcgaccc 471
<210>
20
<211>
1085
<212>
DNA
<213>
Glycine
sp
<220>
<221> feature
misc
_
<222>
(1).
.{1085)
<223>
n =
A,T,C
or G
<400>
20
cggctcgaggaaccctattaaaattggagaagttctgactcgtggattaggtacgggcgg60
gaatccacttttgggggaacaagctgcagaggaatcaagagatgctattgctgatgctct120
taaaggatcagatttggtgtttataacggctgggatgggtgggggaaccgggtctggtgc180
tgccccagttgtagcccaaatatcaaaagaggcaggttacttgactgtaggtgttgttac240
ctatcccttcagttttgaaggacgtaagagatccttgcaggcctttgaagccatcgaaag300
gctgcagaaaaatgttgacacmmttatagtgawtccmaatgmccgtctgcttgacawagy360
tratragcaratgcctcttcaaggatgctttccgytttgcagatgacgttytmsggcaag420
gagtmcagggaatatcagacattatamctgtacctggacttkkcaaatgtggattttgca480
agatgtaaaagctgtgatgaaagactctgggactgcaatgcttggagtaggtgtttccty540
cggtaaaaaaccgagcagaagaagcagccgaacaggctactttggctcctttaattggat600
cctctattcagtcaagctactggggtagtgtataatattactggagggaaaggacataac660
cctgcaggaagtgracagggtttytmaggtkgkgacyarkttggctgatccttctgctaa720
.
tattatatttggggctgtcgttgatgatcgctacacgggggagattcacgtgactatcat780
tgcaactggcttctcacagtcttttcagaagaagttgctaacagatccaagggcttgcaa840
agctgcttgacaaggtggctgagggccaagaaagcaaggcagtccctcctcccctcaagt900
cctcaaacaaggttgaatctagaccatccccgcgaaagctctttttttagttgcatggtt960
.
ctttttaccctttttcatttttccaattattattattatattatatnggccgatcaaaaa1020
aaaaaaaaaaggcggccgccgactagtgagctcgtcgacccgggaattaattccggaccg1080
gtacc
1085
<210>
21
<211>
797
<212>
DNA
<213>
G3.ycine
sp
<400>
21
ccagctggcgaaaggggatgtgctgcaaggcgattaagttgggtacgcagggttttccca60
gtcacgacgttgtaaaacgacggcagtgaattgaatttaggtgacactatagaagagcta120
tgacgtcgcatgcacgcgtacgtaagctcggaattcggctcgagaggctactttggctcc180
tttaattggatcctctattcagtcagctactggggtagtgtataatattactggaggaaa240
- 12 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
ggacataaccctgcaggaagtgaacagggtttctcaggttgtgactagtttggctgatcc 300
ttctgctaatattatatttggggctgtcgttgatgatcgctacactggggagattcacgt 360
gactatcattgcaactggcttctcacagtcttttcagaagaagttgctaacagatccaag 420
ggctgcaaagctgcttgacaaggtggctgagggccaagaaagcaaggtagtccctcctcc 480
cctcaagtcctcaaacaaggttgaatctagaccatccccgcgaaagctcttttttttagt 540
tgcatggttctttttaccctttttcatttttccaattattattattatattatattggcc 600
gatcaaaaaaaaaattattatattatattgtaggacacaatgatcttgatgcttaattaa 660
gtgagatatcattctcttgatgttaaaaaaaaaaaaaaagggcggccgccgactagtgag 720
ctcgtcgacccgggaattaattccggaccggtacctgcaggcgtaccagctttccctata 780
gtgagtcgtattagagg 797
<210>
22
<211>
714
<212>
DNA
<213>
Glycine
sp
<220>
<221> feature
misc
_ .(714)
<222>
(1).
<223>
n = A,T,C.or
G
<400>
22
aattcggctcgagacggctgcgagaagacgacagaagggggttaccgttatcatgcaagc 60
tgataatggggcctctgaagttcttgttccgttattataaaactgagtccttcactctct 120
ctcgaaccagctcacagaaacaatgatctcctacgccgacatgctcaagggatcacatgg 180
atgtcaacaacttcaactatcctccattgtcagagatgtaaactacagctgtggctcgtg 240
tggttatgagctgaacttgaactccagcaaccgcaacacttgttctctcattgactcaaa 300
gtccataaagagaggcatcatctccttcttctccgtggatgagagcaggttcactcagat 360
ccagcaacttcactggccttcttggatgccctttttcaactccaagcgccaaagaaccaa 420
gcttttttgccgcagctgtgggaaccaccttggctatgcttacactttgcctctcaatct 480
caatcccgggatggcatctctgatgattcagaatctatgatatcaaactaaccgctttgt 540
taccttctttctgcgaggaaccaagtcaaaagttagangatatgggcaaggtttgagact 600
gcatcttcctccactcttggtggtctaattcttgaaagggacagaaacatattcatcagt 660
tcttggttggttggaatgngaattaatgnattctaccttttgacattatgaagg 714
<210>
23
<211>
525
<222>
DNA
<213>
Glycine
sp
<400>
23
cgggctcgagattactggaggaaaggacataaccctgcaggaagtgaacagggtttctca 60
ggttgtgactagtttggctgatccttctgctaatattatatttggggctgtcgttgatga 120
tcgctacactggggagattcacgtgactatcattgcaactggcttctcacagtcttttca 180
gaagaagttgctaacagatccaagggctgcaaagctgcttgacaaggtggctgagggcca 240
agaaagcaaggtagtccctcctcccctcaagtcctcaaacaaggttgaatctagaccatc 300
cccgcgaaagctctttttttagttgcatggttctttttaccctttttcatttttccaatt 360
attattattatattatattggccgatcaaaaaaaaaattattatattatattgtaggaca 420
caatgatcttgatgcttaattaagtgagatatcattctcttgatgttctttcccctccaa 480
aaaaaaaaaaaaagggcggccgccgactagtgagctcgtcgaccc 525
<210>
24
<211>
1083
<212>
DNA
<213>
Glycine
sp
- 13 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28I03
<400>
24
cggctcgaggaaccctattaaaattggagaagttctgactcgtggattaggtacgggcgg 60
gaatccacttttgggggaacaagctgcagaggaatcaagagatgctattgctgatgctct 120
taaaggatcagatttggtgtttataacggctgggatgggtgggggaaccgggtctggtgc 180
tgccccagttgtagcccaaatatcaaaagaggcaggttacttgactgtaggtgttgttac 240
ctatcccttcagttttgaaggacgtaagagatccttgcaggcctttgaagccatcgaaag 300
gctgcagaaaaatgttgacacacttatagtgattccaaatgaccgtctgcttgacatagc 360
tgatgagcagatgcctcttcaggatgcttttccgtcttgcagatgacgttctacggcaag 420
gagtacagggaatatcagacattatamctgwcctggacttgtcaatgtggatttttgcag 480
atgtaaaagctgtgatgaaagactctgggactgcaatgcttggagtaggtgtttcctccg 540
gtaaaaaccgagcagaagaagcagccsaacaggctactttggctyctttaattggatcct 600
ctatttcagtcagctactgggggtagtgtataatattactggaggaaaggacataaccct 660
scaggaagtgaacagggkttctcaggttgtgactaagtttggctgatccttctgctaata 720
ttatatttggggctgtcgttgatgatcgctacacgggggagattcacgtgactatcattg 780
caactggcttctcacagtcttttcagaagaagttgctaacagatccaagggctgcaaagc 840
tgcttgacaaggtggctgagggccaagaaagcaaggcagtccctcctcccctcaagtcct 900
caaacaaggttgaatctagaccatccccgcgaaagctctttttttagttgcatggttctt 960
tttaccctttttcatttttccaattattattattatattatattggccgatcaaaaaaaa 1020
aaaaaaagggcggccgccgactagtgagctcgtcgacccgggaattaattccggaccggt 1080
acc 1083
<210>
25
<211>
1335
<212>
DNA
<213>
Glycine
sp
<220>
<221> feature
misc
_
<222>
(1?.
.(2335)
<223>
n = A,T,C
or G
<400>
25
cggctcgaggcccagaacaacaaaaattgctcctcaacgcctaagtcgtcgtttcggttc 60
ggtgagatgctcctacgcttacgtagataacgccaaaattaaggttgtcggcatcggcgg 120
tggcggcaacaatgccgttaatcgcatgatcggaagtggtttgcagggtgtagacttcta 180
tgcgataaataccgatgctcaggcactattaaattctgctgctgagaaccctattaaaat 240
tggagaagttctgactcgtggattaggtacaggtgggaatccacttttgggggaacaagc 300
tgcggaggaatccagagatgctattgctgatgctcttaaaggatcagatttggtatttat 360
aacggctgggatgggtgggggaaccgggtcttggtgctgccccagttgtagnccaaatat 420
caaaagaggcaggntactttgactgtaggtgttggtacctatcccttcagttttgaagga 480
cgtaagagatgcttgcaggcctttgaagccatcgaaaggctgcagaaaaatgttgcacac 540
ttatagttattccaaatgatcgtctgcttgacatancttgatgaaccagatgcctattca 600
aggatgctttycgytytkcarawkatgttytamcgsaargsgkacagggaatatcaagac 660
attwtaacaggtacctggacttgtmaatgtagattttgctgatgtaaaamctgkgataaa 720
gacttctgggactgcaatgcttggtgtaggtgtttcatccggtaaaaccgaccagaagaa 780
gcagcagaacagggctactttggctcctttaattggatcatctattcagtcagctactgg 840
ggtagtgtataatattactggaggaaaggacataaccctgcaggaagtgaacagggtttc 900
tcaggtggtgactagtttggctgatccttctgctaatattatatttggagcttgttgttg 960
atgatcgcttacactggggagattcacgtgactataattgcaactggcttctcacagtct 1020
tttcagaagaagttgctaacagatccaagggctgcaaagctgcttgacaaagtggctgag 1080
ggccaagaaagcaaggcagtccctcctccccccaagtcctcaatcaaggttgaatctaga 1140
ccatccccgcgaaagctctttttgtagttgcatggttcttttacccttttcttttttcca 1200
attattatattgtaagtcattctgtagtacaatgatcttgatgcttaatttagtgagata 1260
tcattctcttgatgttaaaaaaaaaaaaaaaaaaaaaaaaaaagggcggccgccgactag 1320
- 14 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
tgagctcgtc gaccc 1335
<210>
26
<211>
902
<212>
DNA
<213>
Glycine
sp
<220>
<221> feature
misc
_
<222>
(1).
.(902)
<223>
n = A,T,C
or G
<400>
26
aattcggctcgagtaccagggttggtgaatgtagattttgctgatgttcgggctataatg 60
gccaatgcaggttcttcactaatggggataggaactgcaactggaaaatcaagggcaaga 120
gatgctgcattaaatgccatccagtcacctttactggatattggtataragagggctact 180
kgaattgtttggaacawaactggtgggactgatctgrccttgtttgaggtaaacacggca 240
gcagaggttatttatgacctcgtggaccctactgctaatttaatatttggagcagtaata 300
gatccatcactcagtggtcaagtgagcataacattaattgcttactgrattcaaagcgyc 360
aagaggagagtgaagggaggcctctgcaggccagtcaactcactcaagcagacacaacct 420
tcggcaccaattggcggtcttcctctttcactgatggtggtttgtttgagataccagaat 480
tcctaaagaagaraggaggttcacgctatccgagggcgtaatctttttcatcctaatttc 540
ttttgatcccttgcatttcttcacccttggatatacatagcaattggtctagttcttarg 600
tccctgtcttgscctttttcggatttwrkcaaragttgkgkatacagttkgttcatgaaa 660
gtttattacttyccactgkccagacttatgggkctaaaccgganggtattksarcatgga 720
tgcttttcttggcatatttgaattagtttattagcttgtacagagatttcagtaatgctg 780
agagcttgttatagttctttggcatgttatagaaaattcattattattaaaaaaaaaaaa 840
aaaggcggccgccgactagtgagctcgtcgacccgggaattnattccggaccggtacctg 900
ca 902
<210>
27
<211>
856
<212>
DNA
<213>
Glycine
sp
<220>
<221> feature
misc
_
<222>
(1).
.(856)
<223>
n = A,T,C
or G
<400>
27
aattcggctcgagattggtgaaccgtagactttgctgatgttcgagctataatggccaat 60
gcagggtcttcacttatggggataggaactgcaactgggaaaacaagggcmarggawgct 120
gcattaaatgctatccagtcmccctttactggatatttggtataraaagggctactggaa 180
ttgtatggaacataacyggkggaagtgatttgaccttgtttgaaggtaaatgttgcasca 240
raagttatatatgmccttgtggmccccactgstaatttaatatttgggscagwaatagat 300
ccatcactccagtgggcaagtaagcatammwtaatcgcaactggattcaagcgtcaagag 360
gaaaagtgaagggagaccctatgcaggccagtcaactcacacaaggagatnccgttggta 420
tcaatcggcgatyttctactttcactgatggtagcttttgttggagatccctggaattct 480
taaagaagaaggggcgctcacgttatccaagagtttaatactcttttccccaactcctta 540
atccctccttgcatctctttmccaascaatttttagggatacaaatctcatcagtctaag 600
gtattagatcacggtttttgccccttttttcatttttaggttcgcattgtgcantamagt 660
tgttcattgaaagcgaagttactttccaaaaccgttgttttctgarttgaaggcttggtt 720
ggcatgttttwataagtttattagcttgtatttttgtncagagaataatatatcagtaat 780
ggtcagtgcttgttataaanccncnaaaaaaaaaaaaaaaaaaagggcggccgccgacta 840
- 15 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/281U3
gtgagctcgt cgaccc 856
<210>
28
<221>
1060
<212>
DNA
<213>
Glycine
sp
<220>
<221> feature
misc
_ .(1060)
<222>
(1).
<223>
n = A,T,C
or G
<400>
28
aattcggctcgaggtcacaacccctttttcatttgaagggcgaagaagggcagttcaagc60
acaagaaggaattgctgcattaagagataatgttgatacactgatagttattccaaatga120
caaactgctgactgcagtttctcaatctacccctgtaactgaagcattcaatctggctga180
tgatattcttagacaaggtgttcgtggtatatctgatattattacgataccaggattggt240
gaatgtagactttgcagatgttcgagctataatggccaatgcaggttcttcacttatggg300
gataggaactgcaactggcaaaacaagggcaagagacgctgcattaaatgctatccagtc360
acctttactagatattggtatagaaagggctaccggaattgtatggaacataactggtgg420
aaagwgatttgaccttgtttgaggtaaatgctgcagcagaagttatatatgaccttgtgg480
accccactgytaatttaatatttggagcagtaatagatccatcactcagtggtcaagtaa540
gcatcacattaattgcaactggattcaagccgtcaagaggaaaagtgaagggagacctat600
acaggccarccaatttacacaaggagatacggttggtatcaaccsgcgatcttcctcttt660
cactgatggkagctttgttgagayccctgaattcttaaagaaraaggggcgctcaygtta720
tccgagagcttaatactcttctccccaatttcttaatcccttgatttctttacaaagtaa780
tttttagggatacaaatctcatcagtctaggtattagatcccgttttgcccctttttttt840
ttcatttttaggttcgcattgggcatactgttgttcaagaaagcaaagtactttcaaaac900
cgttgtttactgagtcgaggcttgttggcaggttttaataagtttattagcttgtatttt960
ttgtacagagaatatatcagtaatggtcagggcttgttatnnnanncccnnnnannaaan1020
aaaaaaaaaggcggccgccgactagtgagctcgtcgaccc 1060
<210>
29
<211>
727
<212>
DNA
<223>
Glycine
sp
<220>
<221> feature
misc
-
<222>
(1).
..(727)
<223>
n = A,T,C
or G
<400>
29
atctcncaaaatgcatgncnctgtgtgtggcatatattcaaaatgacttggcccagggtg60
gggttttantttgctcttagaaaatgtgttgagcctgcacatanaagattggagttgttg120
attctcagtggattgttcaccaaggtattccctcactagggaatcagggtgantctcaaa180
caggaaagcnccatggcaggggntgagggancggtgtanaaaggagtggccatgttccag240
agtcggtggcaaatgctgaatacgcgtatcacaactccattggaattgatacatctaatt300
ccactgctcattaggtgacttcggcctaagttgacttgtaaacatattgttactaccctt360
agccttacgcgtagaattttcccttaaaaaaaaaaatatattcctatgtaacgttacgta420
catgcaatgcaatcacaatatagagtcctagctagggaccaaacatcatttcgatgtaga480
aattgctgtacttaacagtgagtaaatctagtgaagagaattattattgctgctaacgaa540
ggtgcttataggaaatggaaatgctagtgaatccttaaattggaggctgacaacgaagtt600
ctttagggtttttgggattaaagaaaacgaaatgtcataattatcatacccttgggatga660
ggagacaggactattactataaaaaaaaaaaaaaagggcggccgccgactagtgagctcg720
- 16 -
CA 02352464 2001-05-24
WO 00132799 PCT/US99/28103
tcgaccc 727
<210>
30
<211>
1185
<212>
DNA
<213>
Glycine
sp
<220>
<221> feature
misc
_
<222>
(1).
.(1185)
<223>
n = A,T,C
or G
<400>
30
cggctcgagctggaatgggtgggggaactggcacaggtggagctccaattattgctagta 60
ttgcaaagtcaatgggtatattgacggttggtattgtcaccacccctttctcgtttgaag 120
ggagaaagagatctattcaagcccaagaaggaattacagccttaagagataatgttgaca 180
cgcttatagttattccaaatgacaagctactaacggcagtttctcaatctacccctgtaa 240
ctgaagcattcaatctggctgatgatattcttcgacagggtgttcgtggcatatctgata 300
ttattacaataccagggttggtgaatgtagattttgctgatgttcgggctataatggcca 360
atgcaggttcttcactaatggggataggaactgcaactggaaaatcaagggcaagagatg 420
ctgcattaaatgccatccagtcaccwttmctggatattggtatagararggctactggaa 480
ttgtttggaacawaactggkgggactgatcttgaccttgtttgaggtaaacacggcarca 540
rraggttatttatgacctcgtggaccctactgctaatttaatatttggagcagtaataga 600
tccatcactcagtggkcaagtgagcataacattaattgctactggattcaagcgtcaaga 660
ggarartgaarggaggcctntgcaggccagtcaactcactcaagcagacacaaccttcgg 720
caccaattggcggtcttcctctttcactgatggtggtttgtttgagataccagaattcct 780
aaagaagagaggaggttcacgctatccgagggcgtaatctttttcatcctaatttctttg 840
atcccttgcatttcttcacccttggatatacatagcattggtctagttcttaggtccctg 900
tcttgccctttttcggattttagtcagagttgtgtatacagtttgttcatgaaagtttat 960
tacttcccactgtccagacttatgggtctaaccggaggtattgcagcatggatgcttttc 1020
ttggcatatttgaattagtttattagcttgtacagagatttcagtaatgctgagagcttg 1080
ttatagttctttggcatgttatagaaaattcattattattattcatcccnccaaaaaaaa 1140
aaaaaaaaaaaaagggcggccgccgactagtgagctcgtcgaccc 1185
<210>
31
<211>
700
<212>
DNA
<213>
Glycine
sp
<220>
<221> feature
misc -
<222>
(1)..
.(700)
<223>
n = A,T,C
or G
<400>
31
aattcggctcgagattgtcaccacccctttctcgtttgaagggagaaagagatctattca 60
agcccaagaaggaattacagccttaagagataatgttgacacgcttatagttattccaaa 120
tgacaagctactaacggcagtttctcaatctacccctgtaactgaagcattcaatctggc 180
tgatgatattcttcgacagggtggtccgtggcatatctgatattattacaataccagggt 240
tggtgaatgtagattttgctgatgttcgggctataatggccaatgcaggttcttcactaa 300
tggggataggaactgcaactggaaaatcaagggcaagagatgctgcattaaatgccatcc 360
agtcacctttactggatattggtatagagagggctactggaattgtttggaacataactg 420
gtgggactgatctgccttgtttgaggtaaacacngcagcaganggtatttatgacctcgn 480
ggccctactgctaattaatatttggagcagaatagatccatcctcatggcaagtgacata 540
cattnantgctctggattcaagcgtcaagangagaagtgaagggangcctttgcaggcca 600
- 17 -
CA 02352464 2001-05-24
WO 00/32799 PCTNS99/28103
gcactcactcagcagacacaaccttngnaccaattggcggcttcctctttcactgatggg 660
nggttggttgagatncnanaattcctaaagaaaaanagag 700
<210>
32
<211>
1425
<212>
DNA
<213>
Arabidopsis
sp
<400>
32
ccacgcgtccggaggaagtaaacaatggcgataattccgttagcacagcttaatgagcta 60
acgatttcttcatcttcttcttcgtttcttaccaaatcgatatcttctcattcgttgcat 120
agtagctgcatttgcgcaagttctagaatcagtcaattccgtggcggcttctctaaacga 180
agaagcgattcaacaaggtctaagtcgatgcgattgaggtgttccttctctccgatggaa 240
tctgcgagaattaaggtgattggtgtcggtggtggtggtaacaatgccgttaaccggatg 300
atttcaagcggtttacagagtgttgatttctatgcgataaacacggattcgcaagctctg 360
ttacagttttctgctgagaacccacttcaaattggagaacttttaactcgtgggcttggc 420
actggtggaaacccgcttcttggagaacaagctgcagaagaatcaaaagatgcaattgct 480
aatgctcttaaaggatcagaccttgttttcataactgctggtatgggtggtggaacaggg 540
tctggtgctgcacctgtggtagctcagatttcgaaggatgctggttatttgactgttggt 600
gttgttacctatccgtttagctttgaaggacgtaaaagatctttgcaggcactggaagct 660
attgaaaagctccaaaagaatgttgatacccttatcgtgattccaaatgatcgtctgcta 720
gatattgctgatgaacagacgccacttcaggacgcgtttcttcttgcagatgatgtttta 780
cgccaaggagtacaaggaatctcagatattattactatacctggactagtcaatgtggat 840
tttgcagatgtgaaggcagtcatgaaagattctggaactgcaatgctcggggtaggtgtt 900
tcttccagcaaaaaccgggcagaagaagcagctgaacaagcaactttggctccattgatc 960
ggatcatccatacaatcagctactggtgtcgtctacaacatcactggtggaaaagacata 1020
actttgcaggaagtgaaccgagtatcacaggtcgtgacaagtttggcagacccatcggcc 1080
aacatcatatttggagctgttgtggatgatcgctacaccggagagattcatgtaacgata 1140
atcgccacaggcttctctcagtcattccagaagacacttctgactgatccaagagcagct 1200
aaactccttgacaaaatgggatcatcaggtcaacaagagaacaaaggaatgtctctgcct 1260
caccagaagcagtctccatcaactatctctaccaaatcgtcttctccccgtagacttttc 1320
ttctagttttctttttttccttttcggtttcaagcatcaaaaatgtaacgatcttcaggc 1380
tcaaatatcaattacatttgattttcctccaaaaaaaaaaaaaaa 1425
<210>
33
<211>
1612
<212>
DNA
<213>
Arabidopsis
sp
<400>
33
tgttgttgccgctcagaaatctgaatcttctccaatcagaaactctccacggcattacca.60
aagccaagctcaagatcctttcttgaaccttcacccggaaatatctatgcttagaggtga 120
agggactagtacaatagtcaatccaagaaaggaaacgtcttctggacctgttgtcgagga 180
ttttgaagagccatctgctccgagtaactacaatgaggcgaggattaaggttattggtgt 240
gggaggtggtggatcaaatgctgtgaatcgtatgatagagagtgaaatgtcaggtgtgga 300
gttctggattgtcaacactgatatccaggctatgagaatgtctcctgttttgcctgataa 360
taggttacaaattggtaaggagttgactaggggtttaggtgctggaggaaatccagaaat 420
cggtatgaatgctgctagagagagcaaagaagttattgaagaagctctttatggctcaga 480
tatggtctttgtcacagctggaatgggcggtggaactggcactggtgcagcccctgtaat 540
tgcaggaattgccaaggcgatgggtatattgacagttggtattgccacaacgcctttctc 600
gtttgagggtcgaagaagaactgttcaggctcaagaagggcttgcatctctcagagacaa 660
tgttgacactctcatcgtcattccaaatgacaagttgcttacagctgtctctcagtctac 720
tccggtaacagaagcatttaatctagctgatgatatactccgtcagggggttcgtgggat 780
atctgatatcattacgattcctggtttggtgaatgtggattttgctgatgtgagagctat 840
aatggcaaatgcggggtcttcattgatgggaataggaactgcgacaggaaagagtcgggc 900
- 18 -
CA 02352464 2001-05-24
WO 00132799 PCTlUS99/28103
aagagatgctgcgctaaatgcaatccaatcccctttgttagatattgggattgagagagc 960
cactggaattgtttggaacattactggcggaagtgacttgacattgtttgaggtaaatgc 1020
tgctgcggaagtaatatatgatcttgtcgatccaactgccaatcttatattcgtgctgtt 1080
gtagatccagccctcagcggtcaagtaagcataaccctgatagctacgggtttcaaacga 1140
caagaagagggagaaggacgaacagttcagatggtacaagcagatgctgcgtcagttgga 1200
gctacaagaagaccctcttcttcctttagagaaagcggttcagtggagatcccagagttc 1260
ttgaagaagaaaggcagctctcgttatccccgagtctaaagcccaatctaatcactaccc 1320
tgcacactgcagcaataacaaacgtgtgtgtactggtagtctggtactgccttctgggat 1380
acagcaagatgtgttgatgtatgatcaagaatctgtgtgggtgtgtatatgttctgtcac 1440
tgcctctggtcgtgttcttgaataggttgttttagaaatcggagtttctctctatgtcac 1500
ttccaaaacaaaaaaggagaagaagaatcacacttctcgaaccataaacatacttataag 1560
attatgagagttttagcagaaattattgtcaaaaaaaaaaaaaaaaaaaaa 1611
<210> 34
<211> 299
<212> DNA
<213> Arabidopsis
sp
<220>
<221> miscfeature
_
<222> (1).
.(299)
<223> n
= A,T,C
or G
<400> 34
agtaattgaaaaatgacacatacctcttttnctacaatcaaagtttataactaagaagaa 60
caaatgcaatagtaataataaagatttgaggacatactgtgacaaagaccatatctgagc 120
cataaagagcttctncaataactgctttgctctctctagcagcattcataccgatttcng 180
gatttcctccagcacctaaacccctagtcaactccttaccaatttgtaacctattatcag 240
gcaaaacaggagacattctcatagcctggatatcagtgttgacaatccagaactccaca 299
<210> 35
<211> 25
<212> DNA
<213> Artificial
Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 35
cttgccgcaa aacatcatcc gcgag 25
<210> 36
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 36
cacccacccc gagcatcgca gttccgga 28
<210> 37
<211> 41
<212> DNA
- 19 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28183
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 37
ttggtgtcgg tggtggtggt aacaatgccg ttaaccggat g 42
<210> 38
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 38
ggtaacaatg ccgttaaccg gatgatttca agcggtttac a 41
<210> 39
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 39
gctgctcttg gatcagtcag aagtgtcttc tggaatgact g 41
<210> 40
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 40
tttgtcaagg agtttagctg ctcttggatc agtcagaag 39
<210> 41
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 41
cagcaccaga tcctgtacct ccacccat 28
<210> 42
<211> 30
<212> DNA
- 20 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 42
ctgaatgggt atgtgacaac accaacagtc 30
<210> 43
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 43
gctgatgatg tattacgcca aggtgtcc 28
<210> 44
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 44
ctggaactgc tatgcttgga gttggggt 28
<210> 45
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 45
tcgaaactga atggataggt gacaacacca acag 34
<210> 46
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 46
tggatcgaca aggtcgtaga taatttcggc cgcagc 36
<210> 47
<211> 56
<212> DNA
- 22 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 47
cgcgatttaa atggcgcgcc ctgcaggcggccgcctgcag ggcgcgccat ttaaat56
<210> 48
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 48
tcgaggatcc gcggccgcaa gcttcctgcagg 32
<210> 49
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 49
tcgacctgca ggaagcttgc ggccgcggatcc 32
<210> 50
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic 0ligonucleotide
<400> 50
tcgacctgca ggaagcttgc ggccgcggatcc 32
<210> 51
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 51
tcgaggatcc gcggccgcaa gcttcctgcagg 32
<210> 52
<211> 36
<212> DNA
- 22 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 52
tcgaggatcc gcggccgcaa gcttcctgca ggagct 36
<210> 53
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 53
cctgcaggaa gcttgcggcc gcggatcc 28
<210> 54
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 54
tcgacctgca ggaagcttgc ggccgcggat ccagct 36
<210> 55
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 55
ggatccgcgg ccgcaagctt cctgcagg 28
<210> 56
<211> 10
<222> PRT
<213> Artificial Sequence
<220>
<223> c-myc Tag
<400> 56
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 57
<221> 37
- 23 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 57
acgtgcggcc gcatggcgat aattccgtta gcacagc 37
<210> 58
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 58
actgataagc ttttgctcga agaaaagtct acgggga 37
<210> 59
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 59
acgtgcggcc gcatggcgat aattccgtta gcacagc 37
<210> 60
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 60
cgtcctgcag gctacaagtc ttcctcactg ataagctttt gctc 44
<210> 61
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 61
acgtcctgca ggatggcgat aattccgtta gcacagc 37
<210> 62
<211> 38
- 24 -
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic 0ligonucleotide
<400> 62
acgtgcggcc gcctagaaga aaagtctacggggagaag 38
<210> 63
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligoilucleotide
<400> 63
acgtaagctt tccttctctc cgatggagtctg 32
<210> 64
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 64
acgtctgcag cttttcaatg gcttcaagtgcct 33
<210> 65
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 65
acgtctgcag aagaacgtgg ataccctcatcgtg 34
<210> 66
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide
<400> 66
acgtgcggcc gcctagaaga acaatctacggggagaag 38
<210> 67
<211> 244
- 25 -
CA 02352464 2001-05-24
WO 00132799 PCT/US99/28103
<212> DNA
<213> Artificial Sequence
<220>
<223> Prrn/NEP/Gene101eader/14 amino acid from GFP
fusion
<400>
67
gaattcggtacccccgtcgttcaatgagaatggataagaggctcgtggga ttgacgtgag60
ggggcagggatggctatatttctgggagcgaactccgggcgaatactgaa gcgcttggat120
acaagttatccttggaaggaaagacaattccggatcctctagaaataatt ttgtttaact180
ttaagaaggagatatacccatgggtaaaggagaagaacttttcactggag ttgtcccaag240
catg 244
<210>
68
<211>
32
<212>
DNA
<213>
Artificial
Sequence
<220>
<223>
Synthetic
Oligonucleotide
<400>
68
gtcctgcaggatggccaccatctcaaacccag 32
<210>
69
<211>
37
<212>
DNA
<213>
Artificial
Sequence
<220>
<223>
Synthetic
Oligonucleotide
<400>
69
tagcggccgcctatataaaggagctaaaagaacagcc 37
<z1o>
7a
<211>
669
<212>
DNA
<213>
Arabidopsis
sp
<400>
70
atggcgattagtccgttggcacagcttaacgagctaccagtctcttcctc gtttsttgcg60
acatcccactcgctgcacagtaccagaatcagtggcgggcttctcaaaac aaaggtttaa120
gcaaacacggttgagatgctccttctctccgatggagtctgcgaggatta aggtggttgg180
tgtcggcggtggtggtaacaatgccgtcaatcgcatgatttccagcggct tacagagtgt240
tgatttctatgcgataaacacggactctcaagctctcttgcagtcttctg cgcagaaccc300
tcttcaaattggagagctcctaactcgtggccttgggactggtgggaacc cgcttctagg360
agaacaagctgctgaggaatctaaagacgcgattgctaatgctcttaaag gatctgacct420
tgytttcattactgctggtatgggtggtggcactggctccggtgctgctc ctgttgttgc480
tcagatctccaaagacgctggttatttgaccgttggtgttgttacctatc ccttcagctt540
cgaaggtcgtaaaagatctttgcaggcacttgaagccattgaaaagctgc agaakaacgt600
ggataccctcatcgtgataccaaatgatcgtctcctagatattgctgatg aacagacgcc660
tcttcaaga 669
- 26 -
CA 02352464 2001-05-24
WO U0/32799 PCT/US99/2$103
<210> 71
<211> 646
<222> DNA
<213> Nicotiana sp
<400>
71
ggccctctagatgcatgctcgagcggccgccagtgtgatggatatctgcagaattcgccc 60
ttaagcagtggtaacaacgcagagtacgcgggggtaaaccaaacagacagagagagcaga 120
aacagcaatggccaccatctcaaacccagcagagatagcagcttcttctccttcctttgc 180
tttttaccactcttcctttattcctaaacaatgctgcttcaccaaagctcgccggaaaag 240
cttatgtaaacctcaacgtttcagcatttcaagttcatttactccttttgattctgctaa 300
gattaaggttatcggcgtcggtggcggtggtaacaatgccgttaaccgtatgattggcag 360
tggcttacagggtgttgacttctatgctataaacacggatgctcaagcactgctgcagtc 420
tgctgctgaaaatccacttcaaattggagagcttctgactcgtgggcttggtactggtgg 480
caatcctcttttaggggaacaggcagcagaggagtcgaaggaagccattgcaaattctct 540
aaaaggttcagatatggtgttcataacagcaggaatgggtggaggtacaggatctggtgc 600
tgaagggcgaattccagcacactggcggccgttactagtggatccg 646
<210> 72
<211> 325
<212> PRT
<213> Glycine sp
<400> 72
Gly Ser Arg Pro Arg Thr Thr Lys Tle Ala Pro Gln Arg Leu Ser Arg
1 5 10 15
Arg Phe Gly Ser Val Arg Cys Ser Tyr Ala Tyr Val Asp Asn Ala Lys
20 25 30
Ile Lys Val Val Gly Ile Gly Gly Gly Gly Asn Asn Ala Val Asn Arg
35 40 45
Met Ile Gly Ser Gly Leu G1n Gly Val Asp Phe Tyr Ala Ile Asn Thr
50 55 60
Asp Ala Gln Ala Leu Leu Asn Ser Ala Ala Glu Asn Pro Ile Lys Ile
65 70 75 80
Gly Glu Val Leu Thr Arg Gly Leu Gly Thr Gly Gly Asn Pro Leu Leu
85 90 95
Gly Glu Gln Ala Ala Glu Glu Ser Arg Asp Ala Ile Ala Asp Ala Leu
100 105 110
Lys Gly Ser Asp Leu Val Phe Ile Thr Ala Gly Met Gly Gly Gly Thr
115 120 125
Gly Ser Gly Ala Ala Pro Va1 Va1 Ala Gln Ile Ser Lys Glu Ala Gly
130 135 240
Tyr Leu Thr Val Gly Val Val Th,r Tyr Pro Phe Ser Phe Glu Gly Arg
145 150 155 160
Lys Arg Ser Leu Gln Ala Phe Glu Ala Ile Glu Arg Leu Gln Lys Asn
165 170 175
Val Asp Thr Leu Ile Val Ile P'ro Asn Asp Arg Leu Leu Asp Ile Ala
180 185 190
Asp Glu Gln Met Pro Leu Gln Asp Ala Phe Pro Phe Ala Asp Asp Val
195 200 205
Leu Arg Gln Gly Val G1n Giy Ile Ser Asp Ile Ile Thr Val Pro Gly
210 215 220
Leu Val Asn Val Asp Phe Ala Asp Val Lys Ala Val Met Lys Asp Ser
225 230 235 240
Gly Thr Ala Met Leu Gly Val Gly Val Ser Ser Gly Lys Asn Arg Ala
- 27 -
CA 02352464 2001-05-24
WO 00132799 PCT/US99/28103
245 250 255
Glu Glu Ala Ala Glu Gln Ala Thr Leu Ala Pro Leu Ile Gly Ser Ser
260 265 270
I1e Gln Ser Ala Thr Gly Val Val Tyr Asn Ile Thr Gly Gly Lys Asp
275 280 285
Ile Thr Leu Gln Glu Val Asn Arg Val Ser G1n Val Val Thr Ser Leu
290 295 300
Ala Asp Pro Ser Ala Asn Ile Ile Phe Gly Ala Val Val Asp Asp Arg
305 310 315 320
Tyr Thr Gly Glu Ile
325
<210> 73
<211> 357
<212> PRT
<213> Zea mays
<220>
<221> VARIANT
<222> (1)..-.(357}
<223> Xaa = Any Amino Acid
<400> 73
Asp Leu Val Phe Ile Thr Ala Gly Met Gly Gly Gly Thr Gly Ser Gly
1 5 10 15
Ala Ala Pro Val Val Ala Gln Ile Ser Lys Glu Ala Gly Tyr Leu Thr
20 25 30
Val Gly Val Val Thr Tyr Pro Phe Ser Phe Glu Gly Arg Lys Arg Ser
35 40 45
Val Gln Ala Leu Glu Ala Leu Glu Lys Leu Glu Lys Ser Val Asp Thr
50 55 60
Leu Ile Va1 I1e Pro Asn Asp Lys Leu Leu Asp Val Ala Asp Glu Asn
65 70 75 80
Met Pro Leu Gln Asp Ala Phe Leu Leu Ala Asp Asp Val Leu Arg Gln
85 90 95
Gly Val Gln Gly Ile Ser Asp I1e Ile Thr Ile Pro Gly Leu Val Asn
100 105 110
Val Asp Phe Ala Asp Val Lys Ala Va1 Met Lys Asn Ser Gly Thr Ala
115 120 125
Met Leu Gly Val Gly Val Ser Ser Ser Lys Asn Arg Ala Gln Glu Ala
130 135 140
Ala Glu Gln Ala Thr Leu Ala Pro Leu Ile Gly Ser Ser Ile Glu Ala
145 150 155 160
A1a Thr Gly Val Val Tyr Asn Ile Thr Gly Gly Lys Asp Ile Thr Leu
165 170 175
Gln Glu Val Asn Lys Val Ser Gln Ile Val Thr Ser Leu Ala Asp Pro
180 185 190
Ser Ala Asn Ile Ile Phe Gly Ala Va1 Val Asp Asp Arg Tyr Thr Gly
195 200 205
Glu Ile His Val Thr Ile Ile Ala Thr Gly Phe Pro Gln Ser Phe Gln
210 215 220
Lys Ser Leu Leu Ala Asp Pro Lys Gly Ala Arg Ile Val Glu Ser Lys
225 230 235 240
Glu Lys Ala Ala Thr Leu Ala His Lys Ala Ala Ala Ala Ala Val GIn
245 250 255
_ 28 _
CA 02352464 2001-05-24
WO 00/32799 PCT/US99/28103
Pro Val Pro Ala Ser A1a Trp Ser Arg Arg Leu Phe Ser Xaa Glu Ala
260 265 270
His Leu Val Asn Arg Asp Ser Xaa Cys Ile Arg Phe Ala Phe Ser Val
275 280 285
Leu Arg Ala Val Pro Lys Val TIe Phe Gly Tyr Leu Glu Ile Tyr Ser
290 295 300
Leu Gly Xaa Cys Ser Val Val Val G1u Xaa Val Ser Val Tyr Val Ser
305 310 315 320
Leu Leu Cys Phe Met Phe Leu Arg Ile Xaa Arg Xaa Gly Xaa Glu Lys
325 330 335
Cys Ser Ala Thr Gln His Xaa Thr Val Xaa Lys Ile Phe Asp Cys Phe
340 345 350
Ile A1a Ala Thr Cys
355
- 29 -
