Note: Descriptions are shown in the official language in which they were submitted.
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SPECIFICATION
TITLE OF THE INVENTION
"METHODS FOR GENERATING OR INCREASING
REVENUES FROM CROPS"
This application is a continuation-in-part of U.S. Patent Application Serial
No.
09/553,231, filed March 13, 2002, which is a continuation of U.S. Patent
Application
Serial No. 09/090,051, filed June 3, 1998, now U.S. Patent No. 6,156,953 which
claims the priority of U.S. Provisional Patent Application Serial No.
60/048,451, filed
June 3, 1997; and U.S. Provisional Patent Application Serial No. 60/073,741,
filed
February 5, 1998, both of the disclosures of which are specifically
incorporated herein
by reference in their entirety. This application is also a continuation-in-
part of U.S.
Patent Application Serial No. 09/531,120, filed March 17, 2000, which claims
the
priority of U.S. Provisional Application Ser. No. 60/125,219, filed March 18,
1999;
U.S. Provisional Application Ser. No. 60/127,409, filed April 1, 1999; U.S.
Provisional Application Ser. No. 60/134,770, filed May 18, 1999; U.S.
Provisional
Application Ser. No. 60/153,584, filed September 13, 1999, U.S. Provisional
Application Ser. No. 60/154,603, filed September 17, 1999 and U.S. Provisional
Application Ser. No. 60/172,493, filed December 16, 1999, each of which
disclosures
is specifically incorporated herein by reference in its entirety.
The government owns rights in the present invention pursuant to U.S.
Department of Agriculture Grant No. 96-35304-3491 and Grant No. DE-FC05-
920R22072 from the Consortium for Plant Biotechnology Research, National
Science
Foundation Grant No. 9872641, and Department of Energy Small Business
Innovation
Research Grants DE-FG02-OlER83163, DE-FG02-OlER83165, and DE-FG02-
O 1 ER83166.
BACKGROUND OF THE INVENTION
The present invention relates generally to methods of doing business. More
specifically, the present invention relates to methods of generating and/or
increasing
the revenue derived from crops.
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To improve agricultural traits or produce certain products, it has been known
to
add a gene to an organism such as a plant. Although genes, for a vast array of
products, have been proposed or identified, the technologies in the industry,
heretofore, have severely curtailed delivery of several genes into a plant.
Two general approaches are used for introduction of new genetic information
("transformation") into cells. One approach is to introduce the new genetic
information as part of another DNA molecule, referred to as an "episomal
vector," or
"mini chromosome", which can be maintained as an independent unit (an episome)
apart from the host chromosomal DNA molecule(s). Episomal vectors contain all
the
necessary DNA sequence elements required for DNA replication and maintenance
of
the vector within the cell. Many episomal vectors are available for use in
bacterial
cells (for example, see Maniatis et al., 1982). However, only a few episomal
vectors
that function in higher eukaryotic cells have been developed. Higher
eukaryotic
episomal vectors were primarily based on naturally occurring viruses. In
higher plant
systems gemini viruses are double-stranded DNA viruses that replicate through
a
double-stranded intermediate upon which an episomal vector could be based,
although
the gemini virus is limited to an approximately 800 by insert. Although an
episomal
plant vector based on the Cauliflower Mosaic Virus has been developed, its
capacity to
carry new genetic information also is limited (Brisson et al., 1984).
The other general method of genetic transformation involves integration of
introduced DNA sequences into the recipient cell's chromosomes, permitting the
new
information to be replicated and partitioned to the cell's progeny as a part
of the natural
chromosomes. The introduced DNA usually is broken and joined together in
various
combinations before it is integrated at random sites into the cell's
chromosome (see,
for example Wigler et al., 1977). Common problems with this procedure are the
rearrangement of introduced DNA sequences and unpredictable levels of
expression
due to the location of the transgene in the genome or so called "position
effect
variation" (Shingo et al., 1986). Further, unlike episomal DNA, integrated DNA
cannot normally be precisely removed. A more refined form of integrative
transformation can be achieved by exploiting naturally occurring viruses that
integrate
into the host's chromosomes as part of their life cycle, such as retroviruses
(see
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Cepko et al., 1984). In mouse, homologous integration has recently become
common,
although it is significantly more difficult to use in plants (Lam et al.
1996).
The most common genetic transformation method used in higher plants is
based on the transfer of bacterial DNA into plant chromosomes that occurs
during
infection by the phytopathogenic soil bacterium Agrobacterium (see
Nester et al., 1984). By substituting genes of interest for the naturally
transferred
bacterial sequences (called T-DNA), investigators have been able to introduce
new
DNA into plant cells. However, even this more "refined" integrative
transformation
system is limited in three major ways. First, DNA sequences introduced into
plant
cells using the Agrobacterium T-DNA system are frequently rearranged (see
Jones et al., 1987). Second, the expression of the introduced DNA sequences
varies
between individual transformants (see Jones et al., 1985). This variability is
presumably caused by rearranged sequences and the influence of surrounding
sequences in the plant chromosome (i.e., position effects), as well as
methylation of
1 S the transgene. A third drawback of the Agrobacterium T DNA system is the
reliance
on a "gene addition" mechanism: the new genetic information is added to the
genome
(i.e., all the genetic information a cell possesses) but does not replace
information
already present in the genome.
One attractive alternative to commonly used methods of transformation is the
use of an artificial chromosome. Artificial chromosomes are man-made linear or
circular DNA molecules constructed from cis-acting DNA sequence elements that
provide replication and partitioning of the constructed chromosomes (see
Murray et al., 1983). Desired elements include: (1) Autonomous Replication
Sequences (ARS) (these have properties of replication origins, which are the
sites for
initiation of DNA replication), (2) Centromeres (site of kinetochore assembly
and
responsible for proper distribution of replicated chromosomes at mitosis or
meiosis),
and (3) if the chromosome is linear, telomeres (specialized DNA structures at
the ends
of linear chromosomes that function to stabilize the ends and facilitate the
complete
replication of the extreme termini of the DNA molecule).
The essential chromosomal elements for construction of artificial chromosomes
have been precisely characterized in lower eukaryotic species, and more
recently in
mouse and human. ARSs have been isolated from unicellular fungi, including
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Saccharomyces cerevisiae (brewer's yeast) and Schizosaccharomyces pombe (see
Stinchcomb et al., 1979 and Hsiao et al., 1979). An ARS behaves like a
replication
origin allowing DNA molecules that contain the ARS to be replicated as an
episome
after introduction into the cell nuclei of these fungi. DNA molecules
containing these
sequences replicate, but in the absence of a centromere they are partitioned
randomly
into daughter cells.
Artificial chromosomes have been constructed in yeast using the three cloned
essential chromosomal elements. Murray et al., 1983, disclose a cloning system
based
on the in vitro construction of linear DNA molecules that can be transformed
into
yeast, where they are maintained as artificial chromosomes. These yeast
artificial
chromosomes (YACs) contain cloned genes, origins of replication, centromeres
and
telomeres and are segregated in daughter cells with high fidelity when the YAC
is at
least 100 kB in length. Smaller CEN-containing vectors may be stably
segregated,
however, when in circular form.
None of the essential components identified in unicellular organisms, however,
function in higher eukaryotic systems. For example, a yeast CEN sequence will
not
confer stable inheritance upon vectors transformed into higher eukaryotes.
While such
DNA fragments can be readily introduced, they do not stably exist as episomes
in the
host cell. This has seriously hampered efforts to produce artificial
chromosomes in
higher organisms.
In one case, a plant artificial chromosome was discussed (Richards et al.,
U.S.
Patent No. 5,270,201). However, this vector was based on plant telomeres, as a
functional plant centromere was not disclosed. While telomeres are important
in
maintaining the stability of chromosomal termini, they do not encode the
information
needed to ensure stable inheritance of an artificial chromosome. It is well
documented
that centromere function is crucial for stable chromosomal inheritance in
almost all
eukaryotic organisms (reviewed in Nicklas 1988). For example, broken
chromosomes
that lack a centromere (acentric chromosomes) are rapidly lost from cell
lines, while
fragments that have a centromere are faithfully segregated. The centromere
accomplishes this by attaching, via centromere binding proteins, to the
spindle fibers
during mitosis and meiosis, thus ensuring proper gene segregation during cell
divisions.
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In contrast to the detailed studies done in S. cerevisiae and S. pombe, less
is
known about the molecular structure of functional centromere DNA of higher
eukaryotes. Ultrastructural studies indicate that higher eukaryotic
kinetochores, which
are specialized complexes of proteins that form on the chromosome during late
prophase, are large structures (mammalian kinetochore plates are approximately
0.3
pro in diameter) which possess multiple microtubule attachment sites (reviewed
in
Rieder, 1982). It is therefore possible that the centromeric DNA regions of
these
organisms will be correspondingly large, although the minimal amount of DNA
necessary for centromere function may be much smaller.
The above studies have been useful in elucidating the structure and function
of
centromeres. The extensive literature indicating both the necessity of
centromeres for
stable inheritance of chromosomes, and the non-functionality of yeast
centromeres in
higher organisms, demonstrate that cloning of a functional centromere from a
higher
eukaryote is a necessary Frost step in the production of artificial
chromosomes suitable
for use in higher plants and animals. The production of artificial chromosomes
with
centromeres which function in higher eukaryotes would overcome many of the
problems associated with the prior art and represent a significant
breakthrough in
biotechnology research.
SUMMARY OF THE INVENTION
The present invention provides methods for improving crops as well as
reducing the time necessary to produce new crops. Pursuant to the present
invention,
methods are provided that allow one to increase revenues associated with
crops,
develop new crops, develop new avenues for generating revenues from crops, and
provide new services to a third party.
By allowing for the isolation and identification of plant centromere DNA
sequences from the total genomic DNA of an organism or fractions thereof it is
possible to construct chromosomes having functional centromeres and carrying
large
number of genes. As noted above, genes for producing a vast set of products
have
been identified, but technologies used within the industry severely limit the
delivery of
these genes to plant cells. One or at most a few genes are typically inserted
into
random locations in the host chromosomes, which can irreversibly disrupt host
gene
functions while causing variable and uncontrolled expression of the introduced
genes.
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The present invention makes it possible to overcome the technical limitations
associated with gene delivery in crop species, thereby allowing for the
ability to
shorten the time required for crop development.
To this end, in an embodiment, the present invention provides a method for
providing a service. The method comprises the steps of: identifying a sequence
associated with a centromere of a crop; using a gene or a number of genes and
the
sequence to create a minichromosome; and introducing the minichromosome into a
cell of the commercial plant to create a transgenic plant having a desirable
characteristic not present in the crop .
In an embodiment, the sequence is isolated after it is identified.
In an embodiment, the commercial plant is chosen from the group consisting of
vegetable crop, fruit and vine crops, and field crop plants.
In an embodiment, the desirable characteristic is chosen from the group
consisting of insect resistances, disease resistances, herbicide resistances,
environmental resistances, agronomic characteristics, nutrient utilization,
nutrient
content, production of bioproducts, production of pharmaceutical products,
production
of chemical products, chemical assimilation, and reduction of length to
maturity.
In an embodiment, the gene is selected from the group consisting of a
selectable marker gene, a screenable marker gene, an antibiotic resistance
gene, a
ligand gene, an enzyme gene, a herbicide resistance gene, a nitrogen fixation
gene, a
plant pathogen defense gene, a plant stress-induced gene, a toxin gene, a
receptor gene,
an interleukin gene, a clotting factor gene, a cytokine gene, a growth factor
gene, a
gene encoding an enzyme, a gene encoding an antibody, a gene encoding an
antigen
for a vaccine, a transcription factor, a cytoskeletal protein, a DNA-binding
protein, a
protease, an endonuclease, a lipid, a seed storage gene, and a biosynthetic
gene for
producing pharmaceutically active proteins, small molecules with medicinal
properties, chemicals with industrial utility, nutraceuticals, carbohydrates,
RNAs,
lipids, fuels, dyes, pigments, vitamins, scents, flavors, vaccines,
antibodies, and
hormones.
In an embodiment, the new commercial plant has at least two desirable
characteristics not present in the commercial plant.
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In an embodiment, the minichromosome includes at least two genes each
producing a different desirable characteristic.
In an embodiment, the method includes the step of producing seeds that
generate the new commercial plant.
In an embodiment, the method includes the step of extracting the desired
product from the new commercial plant.
In a further embodiment, the present invention provides a method of increasing
revenues from a species of plant. The method comprises the steps o~
identifying a
sequence associated with a centromere of the species of plant; using a gene or
a
number of genes and the sequence to create a minichromosome; and introducing
the
minichromosome into the species of plant to create a plant having a desirable
characteristic not naturally present in the species plant or to increase or
decrease the
level of a naturally occurring characteristic.
In yet another embodiment of the present invention, a method for increasing
the value of a specific crop is provided. The method comprises the steps of:
identifying a sequence associated with a centromere of the crop; using a gene
or a
number of genes and the sequence to create a minichromosome; and introducing
the
minichromosome into the crop to create a transgenic plant having a
characteristic that
causes the modified crop to be more valuable.
Still further, in an embodiment, a method for increasing crop yield is
provided.
The method comprises the steps of: identifying a sequence associated with a
centromere of the crop; using a gene or a number of genes and the sequence to
create a
minichromosome; and introducing the minichromosome into the crop to create a
transgenic plant having a desirable characteristic not naturally present in
the crop or to
increase or decrease the level of a naturally occurring characteristic.
Moreover, in an embodiment, the present invention provides a method for
producing a new seed. The method includes the steps of: identifying a sequence
associated with a centromere of the commercial plant; using a gene or a number
of
genes and the sequence to create a minichromosome; introducing the
minichromosome
into the commercial plant to create a transgenic plant having a desirable
characteristic
not present in the commercial plant or to increase or decrease the level of a
naturally
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occurnng characteristic; allowing the transgenic plant to mature; and
collecting seeds
from the mature plant.
Further, the present invention provides a method for generating revenue based
on agricultural products. The method comprises the steps of: developing a new
crop
based on a minichromosome comprising a sequence of a centromere; and receiving
a
fee for the new crop.
In an embodiment, the fee is received as a royalty payment.
In an embodiment, the fee is received through sales of seed generated by the
new crop.
In an embodiment, the fee is based on sales of a product made from the new
crop.
In an embodiment, the fee is based on licensing of the new crop or product.
In an embodiment, the fee is based on the receipt of products or services.
In addition, in an embodiment, the present invention provides a method for
performing a service comprising the steps of: developing a modified plant
having a
desired characteristic that does not naturally occur in a non-modified plant
by inserting
into a cell of the non-modified plant a minichromosome comprising a sequence
of a
centromere and a gene expressing the desired characteristic.
In a still further embodiment, the present invention provides a method for
performing a service for a third party including the step o~ isolating from a
total
genomic DNA of a plant a nucleotide sequence relating to the plant's
centromere and
using the nucleotide sequence to make a minichromosome for the third party.
Yet another embodiment of the present invention provides methods for
establishing strategic relationships with a third party. The method can
comprise the
steps of initiating a research and development program with a third party to
develop a
crop that includes a mini chromosome comprising a gene and a centromere
sequence.
If desired, the method can include the step of working with the third party to
develop the crop.
In an embodiment, the method includes the step of licensing to the third party
at least a portion of the technology necessary to develop the crop.
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In order to obtain a centromere DNA sequence from a selected organism, in an
embodiment, the method that is used comprises the steps of preparing a sample
of
genomic DNA from a selected organism, obtaining a plurality of nucleic acid
segments
from the genomic DNA and screening the nucleic acid segments to identify one
or
more centromere nucleic acid sequences. In an embodiment, the method of
obtaining
the plurality of nucleic acid segments comprises contacting said genomic DNA
with a
restriction endonuclease and selecting nucleic acid segments containing
repetitive
DNA to obtain said plurality of nucleic acid segments. In another embodiment,
the
method of obtaining the plurality of nucleic acid segments comprises
contacting said
genomic DNA with a methylation sensitive restriction endonuclease and
selecting
nucleic acid segments exhibiting resistance to cleavage with said methylation
sensitive
restriction endonuclease to obtain said plurality of nucleic acid segments. In
yet
another embodiment, the method of obtaining the plurity of nucleic acid
segments
comprises contacting said genomic DNA with a restriction endonuclease or
physically
shearing said genomic DNA and selecting nucleic acid segments that anneal
rapidly
after denaturation to obtain said plurality of nucleic acid segments.
In another aspect, the invention provides a method for identifying a
centromere
nucleic acid sequence from a dataset of the genomic sequences of an organism.
The
method comprises the steps of (1) providing a first dataset consisting of the
genomic
sequences, or a representative fraction of genomic sequence, of the organism;
(2)
identifying and eliminating known non-centromeric repeat sequences from the
first
dataset by using the BLAST sequence comparison algorithm to create a second
dataset; (3) comparing each sequence in the second dataset to itself by using
the
BLAST sequence comparison algorithm, obtaining a BLAST score for each pair of
sequence compared, and collecting high score pairs to create a third dataset;
(4)
examining the BLAST score of each high score pair in the third dataset and,
eliminating the pairs having a score greater than 10-2° to create a
fourth dataset; (5)
eliminating the high score pairs in the fourth dataset having less than 80 by
or more
than 250 by to create a fifth dataset; (6) examining the nucleotide position
of each high
score pair in the fifth dataset and eliminating pairs having 100% identity as
well as
identical nucleotide positions to create a sixth dataset; (7) examining the
nucleotide
position of each high score pair in the sixth dataset and eliminating pairs
having
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opposite orientation of the nucleotides to create a seventh dataset; (8)
examining the
nucleotide position of both sequences for each high score pair in the seventh
dataset
and eliminating sequences that are overlapping to create an eighth dataset;
and (9)
examining the nucleotide position of each sequence in the eighth dataset and
eliminating sequences not having at least one neighboring sequence within 250
by to
create a ninth dataset; and (10) comparing each sequence in the ninth dataset
to all
other sequences in the ninth dataset by using the BLAST sequence comparison
algorithm and selecting the most common sequence as a centromere sequence of
the
organism. In one embodiment, the known non-centromeric repeat sequence in the
second step is a ribosomal DNA.
A number of centromeres can be used pursuant to the present invention. In an
aspect of the present invention, a Brassica oleracea centromere comprising
Brassica
oleracea centromere DNA is used. In one embodiment, the Brassica oleracea
centromere is defined as comprising n copies of a repeated nucleotide
sequence,
wherein n is at least 2. Potentially any number of repeat copies capable of
physically
being placed on the recombinant construct could be included on the construct,
including about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750,
1,000,
1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000, 40,000, 50,000,
60,000,
70,000, 80,000, 90,000 and about 100,000, including all ranges in-between such
copy
numbers. In one embodiment, the repeated nucleotide sequence is isolated from
Brassica oleracea given by SEQ ID NO:l, 2, 3, or 4.
In yet another aspect, a Glycine max centromere comprising glycine max
centromere DNA is used. In an embodiment, the Glycine max centromere is
defined as
comprising n copies of a repeated nucleotide sequence, wherein n is at least
2.
Potentially any number of repeat copies capable of physically being placed on
the
recombinant construct could be included on the construct, including about 5,
10, 15,
20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000,
5,000,
7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000
and
about 100,000, including all ranges in-between such copy numbers. In one
embodiment, the repeated nucleotide sequence is isolated from Glycine max
given by
SEQ ID NO:S, 6, 7, or 8.
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In yet another aspect, a Lycopersicon esculentum centromere comprising
Lycopersicon esculentum centromere DNA is used. In an embodiment, the
Lycopersicon esculentum centromere is defined as comprising n copies of a
repeated
nucleotide sequence, wherein n is at least 2. Potentially any number of repeat
copies
capable of physically being placed on the recombinant construct could be
included on
the construct, including about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300,
400, 500,
750, 1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000, 40,000,
50,000,
60,000, 70,000, 80,000, 90,000 and about 100,000, including all ranges in-
between
such copy numbers. In one embodiment, the repeated nucleotide sequence is
isolated
from Lycopersicon esculentum given by SEQ ID N0:9 or 10.
In yet another aspect, a Zea mays centromere comprising Zea mays centromere
DNA is used. In an embodiment, the centromere is defined as comprising n
copies of
a repeated nucleotide sequence, wherein n is at least 2. Potentially any
number of
repeat copies capable of physically being placed on the recombinant construct
could be
included on the construct, including about S, 10, 15, 20, 30, 50, 75, 100,
150, 200, 300,
400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000,
30,000,
40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about 100,000, including
all
ranges in-between such copy numbers. In one embodiment, the repeated
nucleotide
sequence is isolated from Zea mays given by SEQ ID NO:11, 12 or 13. ,
In yet another aspect, recombinant DNA construct comprising a plant
centromere sequence is used. The recombinant DNA construct may additionally
comprise any other desired sequences, for example, a telomere. Still further,
one may
wish to include a structural gene on the construct, or multiple genes.
Examples of
structural genes one may wish to use include a selectable or screenable marker
gene,
an antibiotic resistance gene, a ligand gene, an enzyme gene, a herbicide
resistance
gene, a nitrogen fixation gene, a plant pathogen defense gene, a plant stress-
induced
gene, a toxin gene, a receptor gene, a gene encoding an enzyme, a gene
encoding an
antibody, a gene encoding an antigen for a vaccine, a transcription factor, a
cytoskeletal protein, a DNA-binding protein, a protease, an endonuclease, a
lipid, a
seed storage gene, an interleukin gene, a clotting factor gene, a cytokine
gene, a
growth factor gene and a biosynthetic gene for producing pharmaceutically
active
proteins, small molecules with medicinal properties, chemicals with industrial
utility,
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nutraceuticals, carbohydrates, RNAs, lipids, fuels, dyes, pigments, vitamins,
scents,
flavors, vaccines, antibodies, and hormones.
In still yet another aspect, a recombinant DNA construct comprising a plant
centromere sequence and which is capable of being maintained as a chromosome,
wherein the chromosome is transmitted in dividing cells is used. The plant
centromere
may be from any plant or may be from any other source of DNA or may be
partially or
entirely synthetic in origin.
In yet another aspect, a recombinant DNA construct comprising a plant
centromere sequence and which is a plasmid is used. The plasmid may contain
any
desired sequences, such as an origin of replication. The plasmid may also
comprise a
selection marker.
In still yet another aspect, a mini chromosome comprising a plant centromere
sequence and may also contain a telomere sequence is used. Any additional
desired
sequences may be added to the mini chromosome, such as an autonomous
replicating
sequence and a structural gene such as those described above. The mini
chromosome
may comprise any of the centromere compositions disclosed herein.
The mini chromosome also may contain "negative" selectable markers which
confer susceptibility to an antibiotic, herbicide or other agent, thereby
allowing for
selection against plants, plant cells or cells of any other organism of
interest containing
a mini chromosome. The mini chromosome also may include genes or other
sequences which control the copy number of the mini chromosome within a cell.
One
or more structural genes also may be included in the mini chromosome.
Specifically
contemplated as being useful will be as many structural genes as may be
inserted into
the mini chromosome.
In still yet another aspect, a cell transformed with a recombinant DNA
construct comprising a plant centromere sequence is used. The cell may be of
any
type, including a prokaryotic cell or eukaryotic cell. Where the cell is a
eukaryotic
cell, the cell may be, for example, a yeast cell or a higher eukaryotic cell,
such as plant
cell. The plant cell may be from a dicotyledonous plant, such as tobacco,
tomato,
potato, soybean, canola, sunflower, alfalfa, cotton and Arabidopsis, or may be
a
monocotyledonous plant cell, such as wheat, maize, rye, rice, turfgrass, oat,
barley,
sorghum, millet, and sugarcane. In one embodiment of the invention, the plant
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centromere is a centromere chosen from the group consisting of Brassica
oleracea,
Glycine max, Lycopersicon esculentum, and Zea mays and the cell may be a cell
chosen from one of the above species or any other species. The recombinant DNA
construct may comprise additional sequences, such as a telomere, an autonomous
replicating sequence (ARS), a structural gene or genes, or a selectable or
screenable
marker gene or genes, including as many of such sequences as may physically be
placed on said recombinant DNA construct. In one embodiment of the invention,
the
cell is further defined as capable of expressing said structural gene. In
another
embodiment of the invention, a plant is provided comprising the aforementioned
cells.
1n still yet another aspect, a method for preparing a transgenic plant cell is
used. The method comprises the steps of contacting a starting plant cell with
a
recombinant DNA construct comprising a plant centromere sequence of the
present
invention, whereby the starting plant cell is transformed with the recombinant
DNA
construct.
In still yet another aspect, a transgenic crop comprising a mini chromosome,
wherein the mini chromosome comprises a plant centromere sequence of the
present
invention is used. The mini chromosome may further comprise a telomere
sequence,
an autonomous replicating sequence or a structural gene, such as a selectable
or
screenable marker gene, an antibiotic resistance gene, a ligand gene, an
enzyme gene,
a herbicide resistance gene, a nitrogen fixation gene, a plant pathogen
defense gene, a
plant stress-induced gene, a toxin gene, a receptor gene, a gene encoding an
enzyme, a
gene encoding an antibody, a gene encoding an antigen for a vaccine, a
transcription
factor, a cytoskeletal protein, a DNA-binding protein, a protease, an
endonuclease, a
lipid, a seed storage gene, an interleukin gene, a clotting factor gene, a
cytokine gene,
a growth factor gene and a biosynthetic gene for producing pharmaceutically
active
proteins, small molecules with medicinal properties, chemicals with industrial
utility,
nutraceuticals, carbohydrates, RNAs, lipids, fuels, dyes, pigments, vitamins,
scents,
flavors, vaccines, antibodies, and hormones. The transgenic crop may be any
type of
crop, such as a dicotyledonous plant, for example, tobacco, tomato, potato,
pea, carrot,
cauliflower, broccoli, soybean, canola, sunflower, alfalfa, cotton and
Arabidopsis, or
may be a monocotyledonous plant, such as wheat, maize, rye, rice, turfgrass,
oat,
barley, sorghum, millet, and sugarcane.
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In still yet another aspect, the method includes preparing a transgenic crop
tissue. The method comprises the steps of contacting a starting crop tissue
with a
recombinant DNA construct comprising a plant centromere sequence of the
present
invention, whereby the starting crop tissue is transformed with the
recombinant DNA
construct.
In still yet another aspect, the method includes preparing a transgenic crop
seed. The method comprises the steps of contacting a starting crop, crop
tissue, or
crop cell, with a recombinant DNA construct comprising a plant centromere
sequence
of the present invention, whereby the starting crop, crop tissue, or crop cell
is
transformed with the recombinant DNA construct. These transformed crops, crop
tissues, or crop cells are allowed to develop into mature crops, using
standard
agricultural techniques. Transgenic seed is then collected from these crops.
In still yet another aspect, the method includes preparing an extract of a
transgenic crop, crop tissue, crop seed, or crop cell. The method comprises
the steps
of contacting a starting crop, crop tissue, or crop cell with a recombinant
DNA
construct comprising a plant centromere sequence of the present invention,
whereby
the starting crop cell is transformed with the recombinant DNA construct. The
resulting transgenic crop, crop tissue, crop seed, or crop cell is then
extracted and
processed to yield the desirable product. One preferred desirable product is a
food
product. Another preferred desirable product is a pharmaceutical product. Yet
another
preferred desirable product is a chemical product.
Additional features and advantages of the present invention are described in,
and will be apparent from, the following Detailed Description of the Invention
and the
figures.
BRIEF DESCRIPTION OF THE FIGURES
The following drawings form part of the present specification and are included
to further demonstrate certain aspects of the present invention. The invention
may be
better understood by reference to one or more of these drawings in combination
with
the detailed description of specific embodiments presented herein.
FIG. lA-F Consensus sequences ofrepeats from Brassica oleracea. FIG. 1A is
the consensus sequence of ChrBol. This consensus was assembled from 33
sequences
collected by the inventors. The length of this repeat is 180 ~ 0.86 base pairs
and A and
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T compose 60% of the consensus. FIG. 1B is the consensus sequence of ChrBo2.
This consensus was assembled from 7 sequences collected by the inventors. The
length of this repeat is 180 ~ 0.45 base pairs and A and T compose 63% of the
consensus. FIG. 1C is a comparison of the consensus sequences of ChrBol and
ChrBo2. The two repeats (ChrBol and ChrBo2) were alligned to each other using
the
ClustalX program (ClustalX is a free multiple sequence alignment program for
Windows. Those sites with significant differences between the two sequences
(Chi-
squared, P<0.05) are highlighted. FIG. 1D is a revised consensus sequence of
ChrBol. This consensus was assembled from 33 DNA sequences collected by the
inventors and 18 sequences from Genbank, identified by the assession numbers:
M30962 M30963 M31436 M31435
M31438 M31434 M31439 M31437
X68786 X12736 X07519 X16589
X15291 X68783 X68784 X61_ 583
AJ228348222947
FIG. 1E is a revised consensus sequence of ChrBo2. This consensus was
assembled from 7 DNA sequences collected by the inventors and 5 sequences from
Genbank, identified by the accession numbers AJ228347, M30962, X12736, X61583,
and X68785. FIG. 1F is a comparison of the revised consensus sequences of
ChrBol
and ChrBo2, aligned as for FIG. 1C.
FIG. 2A-F Consensus sequences of repeats from Glycine max. FIG. 2A is a
consensus sequence of ChrGml. This consensus was assembled from 32 sequences
collected by the inventors. The length of this repeat is 92 ~ 0.79 base pairs
and A and
T compose 63% of the consensus. FIG. 2B is a consensus sequence of ChrGm2.
This
consensus was assembled from 21 sequences collected by the inventors. The
length of
this repeat is 91 ~ 048 base pairs and A and T compose 62% of the consensus.
FIG.
2C is a comparison of the consensus sequences of ChrGml and ChrGm2. The two
repeats (ChrGml and ChrGm2) were aligned to each other using the ClustalX
program
(ClustalX is a free multiple sequence alignment program for Windows. Those
sites
with significant differences between the two sequences (Chi-squared, P<0.05)
are
highlighted. FIG. 2D is a revised consensus sequence of ChrGml. This consensus
was assembled from 32 DNA sequences collected by the inventors and 1 sequence
from Genbank, identified by the accession number 226334. FIG. 2E is a revised
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consensus sequence of ChrGm2. This consensus was assembled from 21 DNA
sequences collected by the inventors and 13 sequences from Genbank, identified
by
the accession numbers AF297983, AF297984, and AF297985. FIG. 2F is a
comparison of the revised consensus sequences of ChrGml and ChrGm2, aligned as
for FIG. 2C.
FIG. 3A-B Consensus sequences of repeats from Lycopersicon esculentum.
FIG. 3A is a consensus sequence of ChrLel. This consensus was assembled from
42
sequences collected by the inventors. The length of this repeat is 181 ~ 0.61
base pairs
and A and T compose SO% of the consensus. FIG. 3B is a revised consensus
sequence of ChrLel. This consensus was assembled from 32 sequences collected
by
the inventors and 2 Genbank sequences identified by the accession numbers
X87233
and AY007367.
FIG. 4A-C Consensus sequences of repeats from Zea mays. FIG. 4A is a
consensus sequence of ChrZml. This consensus was assembled from 38 sequences
1 S collected by the inventors. The length of this repeat is 180 ~ 1.15 base
pairs and A and
T compose 56% of the consensus. FIG. 4B is a revised consensus sequence of
ChrZml. This consensus was assembled from 38 sequences collected by the
inventors
and 26 sequences from Genbank, identified by the accession numbers:
M32521 M32522 M32523 M32524 M32525 M32526
M32527 M32528 M32529 M32530 M32531 M32532
M32533 M32534 M325375 M32536 M32537 M32538
M35408 AF030934 AF030935 AF030936 AF030937 AF030938
AF030939 AF030940
FIG. 4C is a consensus sequence of ChrZm2. This consensus was assembled
from 6 sequences collected from Genbank identified by the accession numbers:
AF078918 AF078919AF078920
AF0789121 AF078922AF078923
The length of this repeat is 158 ~ 1.6 base pairs and A and T compose 53% of
the consensus.
FIG. 5 Mini chromosome containing centromere sequences as well as mini
chromosome vector sequences
FIG. 6 Mini chromosome construct formed by mini chromosome vector
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tailing method.
FIG. 7A-7N. Exemplary Mini chromosome vectors: The vectors shown in
FIG. 7A, FIG. 7B, FIG. 7E, FIG. 7F, FIG. 7I and FIG. 7J have an E. coli origin
of
replication which can be high copy number, low copy number or single copy. 1n
FIGS.7A-7N, the vectors include a multiple cloning site which can contain
recognition sequences for conventional restriction endonucleases with 4-8 by
specificity as well as recognition sequences for very rare cutting enzymes
such as, for
example, I-Ppo I, I-Cue I, PI-Tli, PI-Psp I, Not I, and PI Sce I. In FIG. 7A-
7N, the
centromere is flanked by Lox sites which can act as targets for the site
specific
recombinase Cre. FIG. 7A. Shows an E. coli plant circular shuttle vector with
a plant
ARS. FIG. 7B. Shows a plant circular vector without a plant ARS. The vector
relies
on a plant origin of replication function found in other DNA sequences such as
selectable or screenable markers. FIG. 7C. Shows a yeast-plant circular
shuttle
vector with a plant ARS. The yeast ARS is included twice, once on either side
of
multiple cloning site to ensure that large inserts are stable. FIG.7D. Shows a
yeast-plant circular shuttle vector without a plant ARS. The vector relies on
a plant
origin of replication function found in other plant DNA sequences such as
selectable
markers. The yeast ARS is included twice, once on either side of the multiple
cloning
site to ensure that large inserts are stable. FIG.7E. Shows an E.
coli-Agrobacterium-plant circular shuttle vector with a plant ARS. Vir
functions for
T-DNA transfer would be provided in trans by a using the appropriate
Agrobacterium
strain. FIG. 7F. Shows an E. coli-Agrobacterium-plant circular shuttle vector
without
a plant ARS. The vector relies on a plant origin of replication function found
in other
plant DNA sequences such as selectable markers. Vir functions for T-DNA
transfer
would be provided in trans by a using the appropriate Agrobacterium strain.
FIG. 7G.
Shows a linear plant vector with a plant ARS. The linear vector could be
assembled
in vitro and then transferred into the plant by, for example, mechanical means
such as
micro projectile bombardment, electroporation, or PEG-mediated transformation.
FIG. 7H. Shows a linear plant vector without a plant ARS. The linear vector
could be
assembled in vitro and then transferred into the plant by, for example,
mechanical
means such as micro projectile bombardment, electroporation, or PEG-mediated
transformation. FIGS. 7I-7N. The figures are identical to FIGS. 7A-7F,
respectively,
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with the exception that they do not contain plant telomeres. These vectors
will remain
circular once delivered into the plant cell and therefore do not require
telomeres to
stabilize their ends.
FIG. 8. Sequence features at Arabidopsis CEN2 (A) and CEN4 (B). Central
S bars depict annotated genomic sequence of indicated BAC clones; black,
genetically-defined centromeres; white, regions flanking the centromeres.
Sequences
corresponding to genes and repetitive features, filled boxes (above and below
the bars,
respectively), are defined as in FIG. 11A-T; predicted nonmobile genes, red;
genes
carried by mobile elements, black; nonmobile pseudogenes, pink; pseudogenes
carried
by mobile elements, gray; retroelements, yellow; transposons, green;
previously
defined centromeric repeats, dark blue; 180 by repeats, pale blue.
Chromosome-specific centromere features include a large mitochondria) DNA
insertion (orange; CEN2), and a novel array of tandem repeats (purple; CEN4).
Gaps
in the physical maps (//), unannotated regions (hatched boxes), and expressed
genes
(filled circles) are shown.
FIG. 9. Method for converting a BAC clone (or any other bacterial clone) into
a mini chromosome. A portion of the conversion vector will integrate into the
BAC
clone (or other bacterial clone of interest) either through non-homologous
recombination (transposable element mediated) or by the action of a site
specific
recombinase system, such as Cre-Lox or FLP-FRT.
FIG. l0A-G. Method for converting a BAC clone (or any other bacterial
clone) into a mini chromosome. The necessary selectable markers and origins of
replication for propagation of genetic material in E. coli, Agrobacterium and
Arabidopsis as well as the necessary genetic loci for Agrobacterium mediated
transformation into Arabidopsis are cloned into a conversion vector. Using
Cre/loxP
recombination, the conversion vectors are recombined into BACs containing
centromere fragments to form mini chromosomes.
FIG. 11A-T. Properties of centromeric regions on chromosomes II and IV of
Arabidopsis. (Top) Drawing of genetically-defined centromeres (gray shading,
CEN2,
left; CEN4, right), adjacent pericentromeric DNA, and a distal segment of each
chromosome, scaled in Mb as determined by DNA sequencing (gaps in the grey
shading correspond to gaps in the physical maps). Positions in cM on the RI
map
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(http://nasc.nott.ac.uk/new ri map.html) and physical distances in Mb,
beginning at
the northern telomere and at the centromeric gap, are shown. (Bottom) The
density of
each feature (FIGS. 11A-11T) is plotted relative to the position on the
chromosome in
Mb. (FIG. 11A, 11K) cM positions for markers on the RI map (solid squares) and
a
curve representing the genomic average of 1 cM/221 kb (dashed line). A single
crossover within CEN4 in the RI mapping population
(http://nasc.nott.ac.uk/new ri map.html; Somerville and Somerville, 1999) may
reflect
a difference between male meiotic recombination monitored here and
recombination in
female meiosis. (FIGs. 11B-11E and FIGS. 11L-110) The % of DNA occupied by
repetitive elements was calculated for a 100 kb window with a sliding interval
of 10
kb. (FIGs. 11B, 11L) 180 by repeats; (FIGs. 12C, 12M) sequences with
similarity to
retroelements, including del, Tal, Tall, copia, Athila, LINE, Ty3, TSCL, 106B
(Athila-like), Tatl, LTRs and Cinful; (FIGS. 11D, 11N) sequences with
similarity to
transposons, including Tagl, En/Spm, Ac/Ds, Taml MuDR, Limpet, MITES and
Mariner; (FIGS. 11E, 110) previously described centromeric repeats including
163A,
164A, 164B, 278A, 11B7RE, mi167, pAT27, 160-, 180- and 500-by repeats, and
telomeric sequences. (FIGS. 11F, 11P) % adenosine + thymidine was calculated
for a
SO kb window with a sliding interval of 25 kb (FIGS. 11G-11J, 11Q-11T). The
number of predicted genes or pseudogenes was plotted over a window of 100 kb
with a
sliding interval of 10 kb. (FIGs. 11 G, 11I, 11 Q, 11 S) predicted genes
(FIGS. 11 G,
11Q) and pseudogenes (FIGs. 11I, 11S) typically not found on mobile DNA
elements;
(FIGs. 11H, 11J, 11R, 11T) predicted genes (FIGs. 11H, 11R) and pseudogenes
(FIGs. 11J, 11T) often carried on mobile DNA, including reverse transcriptase,
transposase, and retroviral polyproteins. Dashed lines indicate regions in
which
sequencing or annotation is in progress, annotation was obtained from GenBank
records (http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html), from the AGAD
database (http://www.tigr.org/tdb/at/agad/.), and by BLAST comparisons to the
database of repetitive Arabidopsis sequences
(http://nucleus.cshl.org/protarab/AtRepBase.htm); though updates to annotation
records may change individual entries, the overall structure of the region
will not be
significantly altered.
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FIG. 12. Methods for converting a BAC clone containing centromere DNA
into a mini chromosome for introduction into plant cells. The specific
elements
described are provided for exemplary purposes and are not limiting. A) diagram
of the
BAC clone, noting the position of the centromere DNA, a site-specific
recombination
site (for example, lox P), and the F origin of replication. B) Conversion
vector
containing selectable and color markers (for example, 35S-Bar, nptII, LAT52-
GUS,
Scarecrow-GFP), telomeres, a site-specific recombination site (for example,
lox P),
antibiotic resistance markers (for example, amp or spc/str), Agrobacterium T-
DNA
borders (Agro Left and Right) and origin of replication (RiA4). C) The product
of site
specific recombination with the Cre recombinase at the lox P sites yields a
circular
product with centromeric DNA and markers flanked by telomeres. D) Mini
chromosome immediately after transformation into plants; subsequently, the
left and
right borders will likely be removed by the plant cell and additional
telomeric
sequence added by the plant telomerase.
1 S FIG. 13A-B. Conservation of Arabidopsis centromere DNA. BAC clones
(bars) used to sequence CEN2 (FIG. 13A) and CEN4 (FIG. 13B) are indicated;
arrows denote the boundaries of the genetically-defined centromeres. PCR
primer
pairs yielding products from only Columbia (filled circles) or from both
Landsberg
and Columbia (open circles); BACs encoding DNA with homology to the
mitochondrial genome (gray bars); 180 by repeats (gray boxes); unsequenced DNA
(dashed lines); and gaps in the physical map (double slashes) are shown.
FIG. 14A-B. Primers used to analyze conservation of centromere sequences in
the A. thaliana Columbia and Landsberg ecotypes. FIG. 14A: Primers used for
amplification of chromosome 2 sequences. FIG. 14B: Primers used for
amplification
of chromosome 4 sequences.
FIG. 15. Sequences common to Arabidopsis CEN2 and CEN4.
Genetically-defined centromeres (bold lines), sequenced (thin lines), and
unannotated
(dashed lines) BAC clones are displayed as in FIG. 14A, B. Repeats AtCCSI (A.
thaliana centromere conserved sequence) and AtCCS2 (closed and open circles,
respectively), AtCCS3 (triangles), and AtCCS4-7 (4-7, respectively) are
indicated
(GenBank Accession numbers AF204874 to AF204880), and were identified using
BLAST 2.0 (http://blast.wustl.edu).
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DETAILED DESCRIPTION OF THE INVENTION
The present invention provides numerous methods for doing business or
providing a service. These methods include increasing revenues generated by
crops,
developing new crops, and improving existing crops. As noted above, although
it has
been known to add a gene to improve the agricultural traits of a plant, this
technology
has been severely limited. One, or at most a few genes, are typically inserted
into
random locations in the host chromosome. This technology thereby can
irreversibly
disrupt host gene functions, can cause variable and uncontrolled expression of
the
introduced genes, and is increasingly time consuming and expensive as the
number of
genes introduced increases.
The inventors have identified the centromeres of a number of crops allowing
them to overcome technical limitations associated with gene delivery in crops.
This
technology allows the inventors to develop methods for improving crops and
developing new crops. Through these methods, the inventors are able to
increase
revenues associated with a crop, generate additional revenues from crops,
provide a
multitude of services associated with crops as well as produce new products
from
crops. This technology also affords the ability to perform a number of
services for
third parties (entities that request a service from another party) based on
crops.
For example, in an embodiment, the present invention allows the inventors to
provide a service to a third party. The service includes identifying a
sequence
associated with the centromere of a commercial plant. A gene or set of genes
to be
added to the commercial plant is then chosen. The gene or set of genes and the
sequence is then utilized to create a mini chromosome. This mini chromosome is
then
introduced into a cell of the commercial plant to create a transgenic plant
having
desirable characteristics not present in the commercial plant. A variety of
different
genes can be utilized.
This method can be used to increase the revenues generated by commercial
plants. In this regard, the desirable characteristic can be chosen so that the
resultant
plant is more valuable. As used herein, the term "more valuable" can have a
variety of
meanings. In its most basic form, it means more revenue is generated by the
crop.
However, the term can also mean that the costs associated with the crop are
reduced.
In this regard, the gene or set of genes can add characteristics that make the
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commercial plant more hearty (including but not limited to characteristics
such as
standility, draught tolerance, improved stature, cold tolerance, and pest
resistance). On
the other hand, the gene or set of genes can cause the plant to mature
quicker. A
variety of characteristics can be added that can increase the value of the
commercial
plant.
In an embodiment, a method of generating revenues based on agricultural
products is provided. The method includes the step of developing a new crop
based on
a mini chromosome including a sequence of a centromere and receiving a fee for
the
new crop. The term "crop" is defined herein in the definitions section. As
used
herein, the term "new" as modifying crop means a crop, that includes a gene or
set of
genes or expresses a trait, that is in nature not typically part of the
genetic sequence of
or expressed by the crop. Additionally, the term "new" as modifying crop can
also
mean a crop that includes a gene or set of genes naturally found in the crop
but
modified so that their copy number is changed. Additionally, the term "new" as
modifying crop can also mean a crop that is modified to increase or decrease a
trait
that in nature is typically found at undesirable levels. Thus, developing a
"new
soybean" means that a soybean is provided that includes a trait or a gene in
its
sequence that is not typically found in soybeans in nature or a soybean that
has been
modified to increase or decrease an existing trait or gene product.
A variety of mechanisms can be utilized as a method of receiving a fee for the
new crop. Such methods can include a fee that is received as a royalty
payment. The
royalty payment may take a variety of forms. The royalty can be charged either
as a
one-time payment, fixed fee paid on a regular basis, or a portion of the
sales, or any
combination thereof. For example, the technology for creating a new crop or
the new
crop itself may be licensed to a third party. The third party may sell seeds
to a farmer
or other commercial entity for a set price and the fee is set as a portion of
the sales
price. For example, a fee of 1 % of the sales of the seeds can be charged as
the fee. If
desired, the fee can be based on sales of a product made from the new crop,
e.g., foods,
pharmaceuticals, or chemicals. If desired, the fee can comprise, in whole or
in part, a
cross-license of technology from a third party. A variety of mechanisms can be
used
to meet the step receiving a fee. Essentially, all that is required is that a
valve is
received.
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The present invention affords a variety of different business methods. In
part,
these methods are due to the ability to introduce a number and variety of
different
genes into a crop. These genes can be introduced into the plants in a single
step.
Because the genes can be introduced independent of the host genome, this
reduces the
damaging effects that occur in transformation technologies. This independence
allows
the additional advantage that the chromosome can be eliminated or modified
when
desired. Further, because the DNA sequences surrounding the genes are defined,
copy
number and expression levels of the genes can be controlled precisely. This
technology allows one to shorten both the cost and the time required for crop
development.
Pursuant to the present invention, strategic relationships can be forged with
third parties, e.g., agricultural companies that can benefit from the use of
the
technology in crops. These relationships can be used to initiate and drive a
research
and development program using crop centromeres to introduce engineered
1 S chromosomes into crops. Such programs can be based on the ability to use
existing
genes, or genes to be developed, to produce a unique variety of seeds. It
should be
noted that these programs can be used to not only generate improved crops, but
also to
produce pharmaceuticals, chemicals, and compounds based, at least in part, on
crops.
In addition to the methods set forth above, it is also envisioned that methods
of
generating revenue can include, without limitation: selling centromeres,
possibly for
specific fields of use or exclusive use; developing chromosomes per customer
specifications; designing plants carrying custom chromosomes to enhance crop
values;
and producing high value products synthesized by genes corned on engineered
chromosomes.
The methods of the present invention are premised, in part, on the fact that
the
inventors have overcome the deficiencies in the prior art by providing the
nucleic acid
sequences of plant centromeres. The significance of this achievement relative
to the
prior art is exemplified by the general lack of detailed information in the
art regarding
the centromeres of multicellular organisms in general. To date, the most
extensive and
reliable characterization of centromere sequences has come from studies of
lower
eukaryotes such as S. cerevisiae and S. pombe, where the ability to analyze
centromere
functions has provided a clear picture of the desired DNA sequences. The S.
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cerevisiae centromere consists of three essential regions, CDEI, CDEII, and
CDEIII,
totaling only 125 bp, or approximately 0.006 to 0.06% of each yeast chromosome
(Carbon et al., 1990; Bloom 1993). S. pombe centromeres are between 40 and 100
kB
in length and consist of repetitive elements that comprise 1 to 3% of each
chromosome
(Baum et al., 1994). Subsequent studies, using tetrad analysis to follow the
segregation of artificial chromosomes, demonstrated that less than 1/S of the
naturally
occurring S. pombe centromere is sufficient for centromere function
(Baum et al., 1994).
In contrast, the centromeres of mammals and other higher eukaryotes are less
understood. Although DNA fragments that hybridize to centromeric regions in
higher
eukaryotes have been identified, in many cases, little is known regarding the
functionality of these sequences (see Tyler-Smith et al., 1993). Centromere
repeats
often correlate with centromere location, with probes to the repeats mapping
both
cytologically and genetically to centromere regions. Many of these sequences
are
tandemly-repeated satellite elements and dispersed repeated sequences in
arrays
ranging from 300 kB to 5000 kB in length (Willard 1990). To date, only one of
these
repeats, a 171 by element known as the alphoid satellite, has been shown by in
situ
hybridization to be present at each human centromere (Tyler-Smith et al.,
1993).
Whether repeats themselves represent functional centromeres remains
controversial, as
other genomic DNA can be required to confer efficient inheritance upon a
region of
DNA (Willard, 1997). Alternatively, the positions of some higher eukaryotic
centromeres have been estimated by analyzing the segregation of chromosome
fragments. This approach is imprecise, however, because a limited set of
fragments
can be obtained, and because normal centromere function is influenced by
surrounding
chromosomal sequences (for example, see Koornneef, 1983; FIG. 2).
A more precise method for mapping centromeres that can be used in intact
chromosomes is tetrad analysis (Mortimer et al., 1981), which provides a
functional
definition of a centromere in its native chromosomal context. Centromeres that
have
been mapped in this manner include those from the yeasts Saccharomyces
cerevisiae,
Schizosaccharomyces pombe, and Kluyveromyces lactis (Carbon et al., 1990;
Hegemann et al., 1993). In many of these systems, accurate mapping of the
centromeres made it possible to clone centromeric DNA, using a chromosome
walking
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strategy (Clarke et al., 1980). Subsequently, artificial chromosome assays
were used
to define more precisely the centromere sequences (Hegemann et al., 1993;
Baum et al., 1994).
Attempts to develop a reliable centromeric assay in mammals have yielded
ambiguous results. For example, Hadlaczky et al., (1991) identified a 14 kB
human
fragment that can, at low frequency, result in de novo centromere formation in
a mouse
cell line. In situ hybridization studies, however, have shown that this
fragment is
absent from naturally occurring centromeres, calling into question the
reliability of this
approach for testing centromere function (Tyler-Smith et al., 1993).
Similarly,
transfection of alphoid satellites into cell lines results in the formation of
new
chromosomes, yet some of these chromosomes also required host sequences that
could
contribute centromere activity (Haaf et al., 1992; Willard, 1997). Further,
the novel
chromosomes can have alphoid DNA spread throughout their length yet have only
a
single centromeric constriction, indicating that a block of alphoid DNA alone
may be
insufficient for centromere function (Tyler-Smith et al., 1993).
Although plant centromeres can be visualized easily in condensed
chromosomes, they have not been characterized as extensively as centromeres
from
yeast or mammals. Genetic characterization has relied on segregation analysis
of
chromosome fragments, and in particular on analysis of trisomic strains that
carry a
genetically marked, telocentric fragment (for example, see Koornneef 1983). In
addition, repetitive elements have been identified that are either genetically
(Richards et al., 1991) or physically (Alfenito et al., 1993; Maluszynska et
al., 1991)
linked to a centromere. In no case, however, has the functional significance
of these
sequences been tested.
Cytology in Arabidopsis thaliana has served to correlate centromere structure
with repeat sequences. A fluorescent dye, DAPI, allows visualization of
centromeric
chromatin domains in metaphase chromosomes. A fluorescence in situ
hybridization
(FISH) probe based on 180 by pALl repeat sequences colocalized with the DAPI
signature near the centromeres of all five Arabidopsis chromosomes
(Maluszynska et al., 1991; Martinez-Zapater et al., 1986). Although a
functional role
for pALI has been proposed, more recent studies have failed to detect this
sequence
near the centromeres in species closely related to Arabidopsis thaliana
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(Maluszynska et al., 1993). These results are particularly troubling because
one of the
species tested, A. pumila, is thought to be an amphidiploid, derived from a
cross
between A. thaliana and another close relative (Maluszynska et al., 1991;
Price et al., 1995). Another repetitive sequence, pAtTl2, has been genetically
mapped
to within 5 cM of the centromere on chromosome 1 and to the central region of
chromosome 5 (Richards et al., 1991), although its presence on other
chromosomes
has not been established. Like pALI, a role for pAtTl2 in centromere function
remains to be demonstrated.
Due to the fact that kinetochores constitute a necessary link between
centromeric DNA and the spindle apparatus, the proteins that are associated
with these
structures recently have been the focus of intense investigation (Bloom 1993;
Earnshaw 1991 ). Human autoantibodies that bind specifically in the vicinity
of the
centromere have facilitated the cloning of centromere-associated proteins
(CENPs,
Rattner 1991), and at least one of these proteins belongs to the kinesin
superfamily of
1 S microtubule-based motors (Yen 1991 ). Yeast centromere-binding proteins
also have
been identified, both through genetic and biochemical studies (Bloom 1993;
Lechner et al., 1991 ).
The centromeres of Arabidopsis thaliana have been mapped using trisomic
strains, where the segregation of chromosome fragments (Koornneef 1983) or
whole
chromosomes (Sears et al., 1970) was used to localize four of the centromeres
to
within 5, 12, 17 and 38 cM, respectively. These positions have not been
refined by
more recent studies because the method is limited the difficulty of obtaining
viable
trisomic strains (Koornneef 1983). These factors introduce significant error
into the
calculated position of the centromere, and in Arabidopsis, where 1 cM
corresponds
roughly to 200 kB (Koornneef 1987; Hwang et al., 1991 ), this method did not
map any
of the centromeres with sufficient precision to make chromosome walking
strategies
practical. Mapping of the Arabidopsis genome was also discussed by (Hauge et
al.,
1991 ).
I. Isolation of centromere clones
The present invention relates to methods of isolating and identifying
centromere DNA sequences from total genomic DNA of an organism without genetic
mapping of the organism. Centromere DNA can be purified from total genomic DNA
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using several methods which include: 1 ) digesting genomic DNA with
restriction
enzymes and separating the fragments on agarose gels, to reveal major classes
of
repetitive DNA; 2) digesting genomic DNA with restriction enzymes sensitive to
DNA
methylation and separating the fragments on agarose gels to reveal the heavily
methylated fraction of the genome; and 3) collecting the rapidly annealing
fraction of
denatured genomic DNA. These three methods isolate centromere DNA; therefore,
these methods are expected to independently isolate the same sequences, thus
validating the sequences' centromere origin. It is anticipated that each of
these
methods can be applied to genomic DNA from any organism, including some lower
organisms such as yeasts, as well as higher organisms such as plants and
animals.
Each of these methods is described in detail below.
1. Isolation of repetitive DNA
Centromere regions often contain many copies of the same DNA sequence
(repetitive DNA); such repeats can range in size from a few nucleotides long
to
hundreds or thousands of bases. Such repetitive DNA can be identified
following
digestion of genomic DNA with restriction endonucleases. Digestion of non-
repetitive
genomic DNA with a particular restriction enzyme produces a distribution of
size
fragments; in contrast, digestion of repeats with a restriction enzyme that
cuts within
each repeat produces a fragment of a typical size. Thus, genomic DNA that has
been
cut with a restriction enzyme can be size fractionated by agarose gel
electrophoresis to
reveal repetitive DNA elements; after staining the gel to reveal the DNA, the
repetitive
fragment can be excised and purified using conventional techniques or
commercial
kits. Such repeats can be introduced into cloning vectors and characterized as
described below. By using this method with a variety of restriction enzymes,
different
repetitive elements can be purified from genomic DNA.
2. Purification of methylated DNA
This method is disclosed in detail in co-pending U.S. Patent Application
Serial
No.09/888,220, filed June 22, 2001, the disclosure of which is incorporated
herein by
reference in its entirety and made a part hereof. Plant centromere DNA is
often
extensively modified by methylation; the presence of this methylation can be
used to
purify centromere fragments. Digestion of genomic DNA with a methylation-
sensitive
restriction endonuclease (for example Sau3A or HpaII) yields a range of
fragment
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sizes; endonuclease sites that are methylated are protected from digestion.
Heavily
methylated DNA molecules, such as centromere DNA, yield large fragments after
digestion and can therefore be separated from the lightly or non-methylated
fraction by
virtue of their size. For example agarose gel electrophoresis, acrylamide gel
electrophoresis, sucrose gradient fractionation, or other size fractionation
techniques
can be used to separate these fragments into pools of "large" (7-l2kb) and
"smaller"
fragments (3-7 kb and 0-3kb).
3. Isolation of rapidly annealing DNA.
The rapidity with which denatured single stranded DNA can reanneal with
another single stranded DNA molecule of complementary sequence upon
renaturation
is dependant upon its abundance. Therefore when genomic DNA is denatured and
allowed to renature, the repetitive fraction of the genome, including
centromere DNA,
will renature before the unique and low copy fractions of the genome. Thus by
fragmenting purified genomic DNA, denaturing it, collecting fractions at
specific time
points (such as 2, 4, 6, 8, and 10 minutes) during renaturation and treating
those
fractions to remove unannealed DNA it is possible to purify repetitive DNA
from total
genomic DNA. Several methods can be used to remove unannealed from annealed
DNA including treatment of the sample with an enzyme, such as S 1 nuclease,
that
degrades single-stranded DNA or exposure to an agent that binds single-
stranded DNA
such as hydroxylapatite. By varying the time at which fractions are collected
during
renaturation it is possible to separate DNA fragments into highly repetitive,
moderately repetitive, and non-repetitive fractions.
II. Cloning and seguencing small fragments of centromere DNA
Repetitive or methylated DNA fragments isolated using the methods described
above can be ligated (using T4 DNA ligase, for example) to a plasmid vector
and
cloned by transformation into E. coli. These clones can then propagated,
sequenced,
used to assemble mini chromosomes, or used to identify larger centromere
clones,
generate molecular markers that facilitate genetic mapping of centromeres, or
create
probes for chromosome mapping experiments such as fluorescent in situ
hybridization
(FISH).
III. Identifying centromere clones in ~enomic libraries
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A genomic library can be screened for clones carrying centromere DNA by
arraying the clones onto solid supports, such as membrane filters, and probing
with
labeled fragments of purified centromere DNA, including cloned repetitive or
methylated DNA fragments described above, or alternatively, the entire set of
rapidly
annealing genomic DNA or highly methylated genomic DNA fragments. Probes can
be used singly or in combination. Typically these probes are labeled by
incorporation
of radionucleotides, fluorescent nucleotides, or other chemical or enzymatic
ligands
that enable easy detection. The labeled probe DNA is denatured and hybridized
to the
arrayed library using standard molecular biology techniques. Hybridization is
performed at a temperature that will discourage non-specific DNA annealing
while
promoting the hybridization of the labeled probe to complementary sequences.
After
incubation, the arrayed library is washed to remove unannealed probe, and a
detection
method appropriate to the label incorporated in the probe is used. For
example, if the
probe is radiolabeled, the labeled filter is exposed to X-ray film.
To identify centromere clones, the results of several hybridization
experiments
are quantitated and compared. In some cases, centromere clones may hybridize
to only
one probe; in other cases, the clones will hybridize to multiple probes. The
hybridization intensity of each clone to each probe can be measured and stored
in a
database. A preferred method for this analysis is to use software that
digitizes the
hybridization signals, assigns each signal to its corresponding clone address,
ensures
that duplicate copies of the clones successfully hybridized, and enters the
resulting
information into a relational database (MySQL for example). Another possible
method
for this analysis is to examine the hybridization results visually, estimate
the
hybridization intensity, and tabulate the resulting information.
The results of each hybridization experiment can be classified by grouping
clones that show hybridization to each probe above a threshold value. For
example, a
computerized relational database can be queried for clones giving
hybridization signals
above a certain threshold for individual probes or for multiple probes. Based
on these
hybridization patterns, clones can be grouped into categories, and
representative
members of each category can be tested in mini chromosomes.
IV. Identifying Centromere Seguences of an Organism from Geneomic Seguence
Datasets
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It is possible to devise computational algorithms to search databases of
genomic sequences and select centromere sequences by identifying those with
the
characteristics of centromeres. For example, by selecting the most abundant
tandem
repeat of a particular size will yield centromere sequences. Other sets of
S characteristics could also be useful. The following is a computational
algorithm
designed to extract centromere sequences from genomic sequence datasets. It is
important to note that this algorithm examines primary sequence data and does
not rely
on prior annotation of the sequence. The algorithm consists of steps 1 through
10.
However, not all the steps must occur in the listed order without altering the
output.
Other rearrangements are easily recognizable by one skilled in the art. The
following
terms are used in describing the algorithm. BLAST is Basic Local Alignment
Search
Tool, a family of freely available algorithms for sequence database searches.
BLAST
aligns two sequences and yields an estimate of the probability that this
alignment is
significant, i.e. that it did not occur by chance. The two sequences compared
by
BLAST are called the 'query', usually a single sequence of interest, and the
'subject',
often part of a large database of sequences that are compared to the query.
The query
sequence (query) can also be part of a database of sequences. The outputs of
BLAST
are High Scoring Pairs (HSPs) that are alignments of subject and query
sequences.
Nucleotide position describes the position of a given nucleotide within the
sequence,
relative to the first nucleotide of the sequence. BLAST score (e value) is the
likelihood that a given sequence alignment is significant (the lower the value
the
higher the significance). The algorithm is as follows:
(1) provide a first dataset consisting of the genomic sequences, or a
representative fraction of genomic sequence, of the organism of interest;
(2) identify and eliminate known non-centromeric repeat sequences from the
first dataset by using the BLAST sequence comparison algorithm to create a
second
dataset;
(3) compare each sequence in the second dataset to itself by using the BLAST
sequence comparison algorithm, obtain a BLAST score for each pair of sequence
compared, and collect high score pairs to create a third dataset;
(4) examine the BLAST score of each high score pair in the third dataset and
eliminate the pairs having a score greater than 10-2° to create a
fourth dataset;
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(5) eliminate the high score pairs in the fourth dataset having less than 80
by or
more than 250 by to create a fifth dataset;
(6) examine the nucleotide position of each high score pair in the fifth
dataset
and eliminate pairs having 100% identity and identical nucleotide positions
(i.e. self
matches) to create a sixth dataset;
(7) examine the nucleotide position of each high score pair in the sixth
dataset
and eliminate pairs having opposite orientation of the nucleotides to create a
seventh
dataset;
(8) examine the nucleotide position of both sequences for each high score pair
in the seventh dataset and eliminate sequences that are overlapping to create
an eighth
dataset; and
(9) examine the nucleotide position of each sequence in the eighth dataset and
eliminate sequences not having at least one neighboring sequence within 250 by
to
create a ninth dataset; and
(10) compare each sequence in the ninth dataset to all other sequences in the
ninth dataset by using the BLAST sequence comparison algorithm and select the
most
common sequence as a centromere sequence of the organism.
Optimally, the dataset used in step (1) in the above algorithm would be the
whole genome dataset such as the Arabidopsis genome which was derived by
methodical sequencing of mapped clones or the rice genome dataset which was
derived by shotgun sequencing. Alternatively, the algorithm would also work
well on
representative genome datasets. By the term "representative gemone datasets",
it is
meant that the genomic sequences in the dataset is a subset of the sequences
of the
whole genome collected from the whole genome without bias, such as bias toward
coding sequences. These sequences would be representative of the genome as a
whole. For example, the use of a O.SX or even a O.1X library of Arabidposis
with
representative genome datasets would return a true positive result. On the
contrary,
the use of a subset of genomic sequences of the whole genome which are not
representative of the whole genome and biased toward certain sequences, such
as the
coding sequence, would return false positive results.
V. Centromere Compositions
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The present invention concerns nucleic acid segments, isolatable from various
plant cells, that are enriched relative to total genomic DNA, or isolated from
other
sources or chemically synthesized with a novel sequence, or other nucleic
acids that
are capable of conferring centromere activity to a recombinant molecule when
S incorporated into the host cell. As used herein, the term "nucleic acid
segment" refers
to a nucleic acid molecule that has been purified from total genomic nucleic
acids of a
particular species. Therefore, a nucleic acid segment conferring centromere
function
refers to a nucleic acid segment that contains centromere sequences yet is
isolated
away from, or purified free from, total genomic nucleic acids. Included within
the
term "nucleic acid segment", are nucleic acid segments and smaller fragments
of such
segments, and also recombinant vectors, including, for example, mini
chromosomes,
artificial chromosomes, BACs, YACs, plasmids, cosmids, phage, viruses, and the
like.
Similarly, a nucleic acid segment comprising an isolated or purified
centromeric sequence refers to a nucleic acid segment including centromere
sequences
and, in certain aspects, regulatory sequences, isolated substantially away
from other
naturally occurnng sequences, or other nucleic acid sequences. In this
respect, the
term "gene" is used for simplicity to refer to a protein, polypeptide or
peptide-encoding
unit. As will be understood by those in the art, this functional term includes
both
genomic sequences, cDNA sequences and smaller engineered gene segments that
may
express, or may be adapted to express, proteins, polypeptides or peptides.
"Isolated substantially away from other sequences" means that the sequences
of interest, in this case centromere sequences, are included within the
genomic nucleic
acid clones provided herein. Of course, this refers to the nucleic acid
segment as
originally isolated, and does not exclude all genes or coding regions.
In particular embodiments, the invention concerns isolated nucleic acid
segments and recombinant vectors incorporating nucleic acid sequences that
encode a
centromere functional sequence that includes a contiguous sequence from the
centromeres of the current invention. Again, nucleic acid segments that
exhibit
centromere function activity will be most preferred.
In still yet another aspect, the invention provides a plant centromere which
is
further defined as an Arabidopsis thaliana centromere. In yet another
embodiment of
the invention, the plant centromere comprises an Arabidopsis thaliana
chromosome 2
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centromere. The chromosome 2 centromere may comprise, for example, from about
100 to about 611,000, about S00 to about 611,000, about 1,000 to about
611,000, about
10,000 to about 611,000, about 20,000 to about 611,000, about 40,000 to about
611,000, about 80,000 to about 611,000, about 150,000 to about 611,000, or
about
300,000 to about 611,000 contiguous nucleotides of a first nucleic acid
sequence
flanking a first series of 180 by repeats in centromere 2 of A. thaliana. The
centromere may also be defined as comprising from about 100 to about 50,959,
about
S00 to about 50,959, about 1,000 to about 50,959, about 5,000 to about 50,959,
about
10,000 to about 50,959, 20,000 to about 50,959, about 30,000 to about 50,959,
or
about 40,000 to about 50,959 contiguous nucleotides of a second nucleic acid
sequence flanking a second series of 180 by repeats in centromere 2 of A.
thaliana.
The centromere may comprise sequences from both of the third and the fourth
sequences, including the aforementioned fragments, or the entirety of these
sequences.
In particular embodiments, the inventors contemplate a 3' fragment of the
first
sequence can be fused to a 5' fragment of the second sequence, optionally
including
one or more 180 by repeat sequence disposed therebetween.
In still yet another aspect, the invention provides an Arabidopsis thaliana
chromosome 4 centromere. In certain embodiments of the invention, the
centromere
may comprise from about 100 to about 1,082,000, about S00 to about 1,082,000,
about
1,000 to about 1,082,000, about 5,000 to about 1,082,000, about 10,000 to
about
1,082,000, about 50,000 to about 1,082,000, about 100,000 to about 1,082,000,
about
200,000 to about 1,082,000, about 400,000 to about 1,082,000, or about 800,000
to
about 1,082,000 contiguous nucleotides of a third nucleic acid sequence
flanking a
third series of repeated sequences, including comprising the nucleic acid
sequence of
the third sequence. The centromere may also be defined as comprising from
about 100
to about 163,317, about 500 to about 163,317, about 1,000 to about 163,317,
about
5,000 to about 163,317, about 10,000 to about 163,317, about 30,000 to about
163,317, about 50,000 to about 163,317, about 80,000 to about 163,317, or
about
120,000 to about 163,317 contiguous nucleotides of the nucleic acid sequence
of a
fourth sequence flanking a fourth series of repeated sequences, and may be
defined as
comprising the nucleic acid sequence of the fourth sequence. The centromere
may
comprise sequences from both the third and the fourth sequences, including the
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aforementioned fragments, or the entirety of the third and the fourth
sequences. In
particular embodiments, the inventors contemplate a 3' fragment of the third
sequence
can be fused to a 5' fragment of the fourth sequence, optionally including one
or more
180 by repeat sequence disposed therebetween.
In yet another embodiment, there is provided a Arabidopsis thaliana
chromosome 1, 3 or 5 centromere selected from the nucleic acid sequence given
by
one of the repeated sequences in these chromosomes, or fragments thereof. The
length
of the repeat used may vary, but will preferably range from about 20 by to
about 250
bp, from about 50 by to about 225 bp, from about 75 by to about 210 bp, from
about
100 by to about 205 bp, from about 125 by to about 200 bp, from about 150 by
to
about 195 bp, from about 160 by to about 190 and from about 170 by to about
185 by
including about 180 bp. In one embodiment, the construct comprises at least
100 base
pairs, up to an including the full length, of one of the preceding sequences.
In
addition, the construct may include 1 or more 180 base pair repeats.
In one embodiment, the centromere n copies of a repeated nucleotide sequence
obtained by the method disclosed herein, wherein n is at least 2. Potentially
any
number of repeat copies capable of physically being placed on the recombinant
construct could be included on the construct, including about 5, 10, 15, 20,
30, 50, 75,
100, 150, 200, 300, 404, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000, 7,500,
10,000,
20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about
100,000,
including all ranges in-between such copy numbers. Moreover, the copies, while
largely identical, can vary from each other. Such repeat variation is commonly
observed in naturally occurring centromeres.
In another embodiment, the centromere is a Brassica oleracea centromere
comprising Brassica oleracea centromere DNA. In one embodiment, the Brassica
oleracea centromere is defined as comprising n copies of a repeated nucleotide
sequence, wherein n is at least 2. Potentially any number of repeat copies
capable of
physically being placed on the recombinant construct could be included on the
construct, including about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400,
500, 750,
1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000, 40,000,
50,000,
60,000, 70,000, 80,000, 90,000 and about 100,000, including all ranges in-
between
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such copy numbers. In one embodiment, the repeated nucleotide sequence is
isolated
from Brassica oleracea given by SEQ ID NO:1, 2, 3, or 4.
In yet another embodiment, the centromere is a Glycine max centromere
comprising glycine max centromere DNA. In an embodiment, the Glycine max
centromere is defined as comprising n copies of a repeated nucleotide
sequence,
wherein n is at least 2. Potentially any number of repeat copies capable of
physically
being placed on the recombinant construct could be included on the construct,
including about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750,
1,000,
1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000, 40,000, 50,000,
60,000,
70,000, 80,000, 90,000 and about 100,000, including all ranges in-between such
copy
numbers. In one embodiment, the repeated nucleotide sequence is isolated from
Glycine max given by SEQ ID NO:S, 6, 7, or 8.
In yet another embodiment, the centromere is a Lycopersicon esculentum
centromere comprising Lycopersicon esculentum centromere DNA. In an
embodiment,
the Lycopersicon esculentum centromere is defined as comprising n copies of a
repeated nucleotide sequence, wherein n is at least 2. Potentially any number
of repeat
copies capable of physically being placed on the recombinant construct could
be
included on the construct, including about 5, 10, 15, 20, 30, S0, 75, 100,
150, 200, 300,
400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000,
30,000,
40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about 100,000, including
all
ranges in-between such copy numbers. In one embodiment, the repeated
nucleotide
sequence is isolated from Lycopersicon esculentum given by SEQ ID N0:9 or 10.
In yet another embodiment, the centromere is a Zea mays centromere
comprising Zea mays centromere DNA. In an embodiment, the centromere is
defined
as comprising n copies of a repeated nucleotide sequence, wherein n is at
least 2.
Potentially any number of repeat copies capable of physically being placed on
the
recombinant construct could be included on the construct, including about 5,
10, 15,
20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000,
5,000,
7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000
and
about 100,000, including all ranges in-between such copy numbers. In one
embodiment, the repeated nucleotide sequence is isolated from Zea mays given
by
SEQ ID NO:11, 12 or 13.
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The centromere can additionally be defined as the region of the chromosome
where the sister chromatids pair during cell division. The centromere is also
the
chromosomal region where the kinetochore (the chromosomal attachment structure
for
the spindle) and the spindle (the cellular machinery that provides the motive
force for
chromosome segregation) attach to the chromosome during mitosis and meiosis.
The
centromere is also defined as the region of the primary constriction in a
condensed
chromosome. The DNA of the centromere is characteristically heavily
methylated,
repetitive, and condensed (heterochromatic).
VI. Mini chromosome construction
Mini chromosomes are constructed by combining fragments of centromere
DNA with other DNA sequences useful for propagation of the resultant
recombinant
DNA molecule in E. coli, other bacteria, yeast or plants. Recombinant plasmids
containing large fragments of centromere DNA are referred to as centromere
clones.
Centromere sequences removed from centromere clones, or centromere sequences
derived directly from genomic DNA, are referred to as centromere fragments.
Recombinant constructs containing DNA sequences necessary for the propagation,
delivery, selection, and detection of mini chromosomes will be referred to as
mini
chromosome vector sequences or mini chromosome vectors; these sequences can
include but are not limited to selectable marker genes, visible marker genes,
origins of
replication, restriction endonuclease recognition sites, homing endonuclease
recognition sites, sequences recognized by site specific recombinase enzymes,
telomere sequences, and sequences required for delivery of mini chromosomes
into
bacteria, yeast or plant cells. Recombinant constructs containing both large
centromere
fragments as well as mini chromosome vector sequences are referred to as mini
chromosomes. The process of assembling mini chromosomes from centromere
clones/fragments and mini chromosome vector sequences can be done in several
ways,
and involves techniques that are common practice among those trained in
molecular
biology:
1) Joining centromere fragments to mini chromosome vector sequences:
Centromere DNA fragments and mini chromosome vector DNA fragments are
generated and purified using conventional techniques, some of which include
restriction enzyme digestion, agarose gel electrophoresis, gel purification of
specific
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fragments, anion-exchange purification and ethanol precipitation. The
resulting
purified centromere and vector fragments are enzymatically joined in vitro,
using for
example T4 DNA ligase. The ends of the fragments can be cohesive, as the
result of
digestion with compatible restriction endonucleases or from the addition of
compatible
S oligonucleotide linkers; alternatively the ends of the fragments can be
blunt and can be
directly joined. Following ligation, the resulting mini chromosomes are
introduced into
E. coli, other bacteria, yeast, or plant cells using chemical or physical
transformation
methods. The structure of the resulting mini chromosomes can be determined by
recovering them from the host organism and assessing DNA fragment size and
composition.
2) Transfer of mini chromosome vector sequences into centromere clones by site-
specific recombination:
The mini chromosome vector sequences can be constructed to include site
specific recombination sequences (for example those recognized by the
bacteriophage
P1 Cre recombinase, or the bacteriophage lambda integrase, or similar
recombination
enzymes). A compatible recombination site, or a pair of such sites, can also
be
included in the centromere clones. Incubation of the mini chromosome vector
and the
centromere clone in the presence of the recombinase enzyme causes strand
exchange
to occur between the recombination sites in the two plasmids; the resulting
mini
chromosomes contain centromere sequences as well as mini chromosome vector
sequences (FIG. 5). Introducing the DNA molecules formed in such recombination
reactions into E. coli, other bacteria, yeast or plant cells can be followed
by selection
for marker genes present on both parental plasmids, allowing the isolation of
mini
chromosomes.
3) Mini chromosome vector tailing method for mini chromosome construction:
Centromere DNA fragments isolated from genomic DNA or from centromere
clones can be modified on their ends by treatment with restriction
endonucleases, or by
ligation with DNA molecules including, but not limited to, oligonucleotide
linkers, or
by the addition of nucleotides, to produce a desired cohesive or blunt end.
These
fragments are size-fractionated by, agarose gel electrophoresis or other
methods, and
the centromere fragments purified using conventional techniques. Mini
chromosome
vector fragments are generated and purified in a similar manner, resulting in
linear
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mini chromosome vector sequences with DNA ends compatible with those on the
centromere fragments. Compatible ends in this case are defined by ends that
can be
joined in vitro by the action of a ligase enzyme. As shown in FIG. 6, the two
fragments
are then mixed so that the mini chromosome vector molecules are present in at
least
two-fold molar excess over the centromere fragments. The fragments are joined
by the
addition of a ligase enzyme (for example bacteriophage T4 DNA ligase),
resulting in
the formation of DNA molecules in which mini chromosome vector molecules have
been joined to both ends of the same centromere fragment. Digestion of the
ligation
mixture with a rare-cutting restriction or homing endonuclease (for example
endonucleases with recognition sequences of 8 or more bases) results in linear
mini
chromosome precursors consisting of a fragment of the original mini chromosome
vector attached to each end of the centromere fragment. The ends of this
hybrid
molecule are compatible because they were created by the same restriction
enzyme.
This linear mini chromosome precursor is purified, for example, by agarose gel
electrophoresis followed by gel purification of the DNA fragments of the
expected
length. The purified DNA molecules are circularized by joining the ends, for
example
by treatment with a DNA ligase enzyme. The resulting mini chromosome molecules
can be introduced into E. coli, other bacteria, yeast or plant cells, followed
by
purification and characterization using conventional methods.
VII. Use of mini chromosomes for plant transformation
1) Delivery of mini chromosomes into plant cells:
Mini chromosomes are purified and delivered into plant cells, either
individually or as a mixture. The mini chromosomes can be either circular or
linear or
mixtures thereof. The plant cells used for mini chromosome delivery can be
either
intact seedlings, immature or mature plants, parts of seedlings or plants,
specific plant
tissues (for example leaves, stems, roots, flowers, fruits), differentiated
tissues cultured
in vitro (for example roots), or undifferentiated cells (for example callus)
cultured in
vitro. The mini chromosome DNA can be delivered into plant cells by a variety
of
methods including but not limited to the following: electroporation;
Agrobacterium-
mediated DNA delivery; virus-mediated DNA delivery; delivery mediated by salts
or
lipids that facilitate the cellular uptake of DNA; microinjection of DNA;
manipulation
into a cell of DNA-coated or DNA-containing particles, droplets, micelles,
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microspheres, or chemical complexes using a variety of techniques, including
biolistic
particle bombardment, optical tweezers, particle beams, and electrospray
apparatus;
manipulation of DNA-coated magnetic particles into the cells by magnetic
fields;
DNA delivery into cells by cell wounding using micro-needles (for example
silicon
carbide needles); sonication or other acoustic treatment of the cells to
facilitate DNA
uptake; fusion of plant cells with other cell types carrying a mini
chromosome,
including bacterial, yeast, or other plant cells; any other electrical,
chemical, physical,
or biological mechanism that results in the introduction of mini chromosome
DNA
into the plant cell
2) Isolating plant cells containing mini chromosomes:
Following mini chromosome delivery, plant cells, plant tissues, or complete
plants carrying the mini chromosome can be isolated by a variety of selection
methods.
Selection involves subjecting the plant cells, tissues or plants to chemical,
environmental, or mechanical treatments that enrich for those cells, tissue or
plants
that contain a mini chromosome. The selection methods include but are not
limited to:
fluorescence-activated cell sorting of cells, cell clumps, or cell protoplasts
based on
expression of a marker protein encoded by the mini chromosome (for example, a
fluorescent protein such as DsRed); affinity purification of cells, cell
clumps, or
protoplasts based on expression of a cell wall protein, membrane protein, or
membrane-associated protein encoded by the mini chromosome; any cell
fractionation
method capable of separating cells based on their density, size or shape to
enrich for
cells with a property that differs from that of the starting population and is
conferred
by the mini chromosome; selection of cells for resistance to an antibiotic
conferred by
the mini chromosome; selection of cells for resistance to an herbicide
conferred by the
mini chromosome; selection of cells for resistance to a toxic metal, salt,
mineral or
other substance conferred by the mini chromosome; selection of cells for
resistance to
abiotic stress (for example heat, cold, acid, base, osmotic stress) conferred
by the mini
chromosome; selection of cells capable of utilizing a carbon source or other
nutrient
source not normally utilized by plant cells, this utilization function being
conferred by
the mini chromosome. As a result of the treatment, a population of plant cells
can be
obtained that contain mini chromosomes. Individual clones or sub-populations
of these
cells can be expanded in culture for further characterization.
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Alternatively, plant cells, plant tissues, or complete plants that carry mini
chromosomes can be identified by direct screening. Such methods involve
subjecting
each cell, plant, or tissue to diagnostic tests indicative of the presence of
the mini
chromosome. These tests can include direct assays for the presence of mini
chromosome DNA, or indirect assays for properties conferred by the mini
chromosome. Direct assays for the presence of the mini chromosome DNA include
but
are not limited to: staining of cells with DNA-binding molecules to allow
detection of
an additional chromosome; in situ hybridization with labeled DNA probes
corresponding to sequences present on the mini chromosome; southern blots or
dot
blots of DNA extracted from the cells, plant or tissue and probed with labeled
DNA
sequences corresponding to sequences present on the mini chromosome;
electrophoresis of genomic DNA extracted from the cells, plant or tissue under
conditions that allow identification of the mini chromosome; amplification of
specific
sequences present on the mini chromosome from genomic DNA extracted from the
cells, plant or tissue using the polymerase chain reaction. Indirect assays
for properties
conferred by the mini chromosome include but are not limited to: detection of
the
expression of a fluorescent marker encoded by the mini chromosome by
fluorescence
microscopy, flow cytometery or fluorimetry; detection of the expression of a
protein
encoded by the mini chromosome by use of specific antibodies, or any other
reagent
capable of specifically binding to the protein; use of cell fractionation
methods capable
of detecting a specific density, size or shape of the cells or tissues, that
is conferred by
the mini chromosome; growth of cells, seedlings, plants or tissues on an
antibiotic-
containing medium to determine the presence of an antibiotic-resistance gene
encoded
by the mini chromosome; growth of cells, seedlings, plants or tissues on an
herbicide-
containing medium to determine the presence of an herbicide-resistance gene
encoded
by the mini chromosome; growth of cells, seedlings, plants or tissues on a
medium
containing a toxic metal, salt, mineral or other substance to determine the
presence of
an gene conferring resistance to this substance encoded by the mini
chromosome;
growth of cells, tissues or plants under conditions of abiotic stress (for
example heat,
cold, acid, base, osmotic stress) to determine the presence of a gene
conferring
resistance to this stress encoded by the mini chromosome; growth of cells on a
medium containing a carbon source or other nutrient source normally not
utilized by
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plant cells, to determine the presence of a utilization function conferred by
the mini
chromosome.
3) Characterization of plant cell clones containing mini chromosomes
Plant cells, tissues, or entire plants containing mini chromosomes can be
further characterized to determine whether the mini chromosome is an
autonomous
DNA molecule, or whether it is associated with one of the plant cell's
chromosomes
by integration. The methods used for this analysis include, but are not
limited to, the
following:
1) Detection of marker protein expression by microscopy, flow cytometry,
fluorimetry,
enzymatic assays, cell staining or any other technique that allows the
detection of a
marker protein having a specific enzymatic activity, or confernng a specific
color, or
fluorescence property onto the cells. For example, if a cell line has been
selected for
containing a mini chromosome by selecting for the function of a resistance
gene
encoded by the mini chromosome, and if a marker protein is also encoded by the
mini
chromosome, then expression of this marker protein in the selected cells is an
indication of the presence of the entire mini chromosome, and could indicate
autonomy of this mini chromosome from the cell's other chromosomes.
2) Use of gel electrophoresis to detect a mini chromosome in genomic DNA
isolated
from the plant cells, tissue or entire plants. For example, genomic DNA
isolated from
the cells, tissues or plants can be fractionated by gel electrophoresis,
either intact or
following digestion with restriction endonucleases or homing endonucleases,
allowing
the detection of a mini chromosome or a fragment of a mini chromosome.
3) Use of southern blots or dot blots of DNA extracted from the cells, tissue
or plants
to detect the presence of specific sequences contained on the mini chromosome.
For'
example, digestion of genomic DNA extracted from the cells, tissues or plants
can be
fractionated by agarose gel electrophoresis, blotted onto a DNA-binding
membrane,
and probed with labeled DNA sequences corresponding to sequences present on
the
mini chromosome to detect specific fragments of mini chromosome DNA, and thus
allowing the determination of the autonomous, or integrated structure of the
mini
chromosome.
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4) Cytological techniques for directly visualizing the mini chromosome in the
transformed cells, such as staining of cells with DNA-binding dyes or in situ
hybridization with labeled DNA probes corresponding to sequences present on
the
mini chromosome.
5) Genetic analysis of marker segregation by scoring marker inheritance in
progeny of
a plant containing a mini chromosome. For example, markers present on an
autonomous mini chromosome will segregate independently from markers on the
arms
of the host chromosomes in a population of F2 progeny generated from a cross
between a line carrying a mini chromosome and a second marked line that
doesn't
carry the mini chromosome. Markers include but are not limited to: visible
markers
confernng a visible characteristic to the plant; selectable markers,
conferring resistance
to an antibiotic, herbicide, or other toxic compound; enzymatic markers,
conferring an
enzymatic activity that can be assays in the plant or in extracts made from
the plant;
protein markers, allowing the specific detection of a protein expressed in the
plant;
molecular markers, such as restriction fragment length polymorphisms,
amplified
fragment length polymorphisms, short sequence repeat (microsatellite) markers,
presence of certain sequences in the DNA of the plant as detected by the
polymerase
chain reaction, single nucleotide polymorphisms or cleavable amplified
polymorphic
sites.
4) Plant regeneration from transformed cell clones:
Plant cells or tissues that harbor mini chromosomes can be used to regenerate
entire plants. This will be accomplished with standard techniques of plant
regeneration
from differentiated tissues or undifferentiated cells. Typically, transformed
tissues or
callus are subjected to a series of treatments with media containing various
mixtures of
plant hormones and growth regulators that promote the formation of a plant
embryo,
specific plant tissues or organs, or a complete plant (roots and shoot) from
the starting
cells or tissues. Following plant regeneration, the plant can be grown either
in sterile
media or in soil.
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VIII. Testing mini chromosome inheritance in plant cells
The inheritance of mini chromosomes can be measured through one or more
cell divisions. After isolating cells, tissues, or entire plants that contain
the mini
chromosome, the population of cells is allowed to grow (either with or without
S selection), and the presence of the mini chromosome is monitored as the
cells divide.
Mini chromosomes can be detected in cells by a variety of methods, including
but not
limited to: detection of fluorescence or any other visual characteristic
arising from a
marker protein gene present on the mini chromosome; resistance to an
antibiotic,
herbicide, toxic metal, salt, mineral or other substance, or abiotic stress as
outlined
above (Isolating plant cells containing mini chromosomes); staining of cells
with
DNA-binding molecules to allow detection of an additional chromosome; in situ
hybridization with labeled DNA probes corresponding to sequences present on
the
mini chromosome; southern blots or dot blots of DNA extracted from the cell
population and probed with labeled DNA sequences corresponding to sequences
present on the mini chromosome; expression of a marker enzyme encoded by a
gene
present on the mini chromosome (i.e. luciferase, alkaline phosphatase, beta-
galactosidase, etc.) that can be assayed in the cells or in an extract made
from the cells.
The percentage of cells containing the chromosome is determined at regular
intervals during this growth phase. The change in the fraction of cells
harboring the
mini chromosome, divided by the number of cell divisions, represents the
average mini
chromosome loss rate. Mini chromosomes with the lowest loss rates have the
highest
level of inheritance.
IX. Recovery of mini chromosomes from plant cells
Recovery of mini chromosomes from plant cells can be achieved by a variety
of techniques, including, but not limited to, the following:
1) Extracting the genomic DNA of transformed plant cells and introducing that
DNA
into E. coli, other bacteria or yeast and selecting for the antibiotic
resistance genes
present on the mini chromosome.
2) Isolation of chromosomes from cells, tissues or plants containing mini
chromosomes, and sorting these by flow cytometry to allow the separation of
chromosomes of different size;
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3) Isolation of individual chromosomes from a cell harboring mini chromosomes
by
micro-manipulation involving mechanical devices such as needles made of glass,
metal or other suitable substances, or other techniques such as optical
tweezers, or
micro-suction devices.
4) Combinations of the above, for example chromosome isolation by flow
cytometry
or micromanipulation followed by introduction into E. coli, other bacteria,
yeast or
plant cells.
The resulting mini chromosomes "rescued" in this fashion may differ from
their parental molecules in total size, size of the centromere, presence or
absence of
additional sequences, and overall arrangement of the sequences. These
procedures
allow the isolation of DNA molecules capable of replicating and segregating in
plant
cells without having to test mini chromosomes individually. For example, after
delivery of pools of mini chromosomes, or pools of centromere clones into
plant cells,
tissues or whole plants, and recovering them by the methods listed above,
facilitates
the selection of specific mini chromosomes or centromere clones that remain
autonomous in plant cells. Whereas plant transformation with mini chromosomes
relies on the sequences contributed by mini chromosome vectors, the recovery
methods do not necessarily require mini chromosome vector sequences; as a
result,
pools of centromere clones can be delivered into plant cells followed by
recovery of
the ones that replicated and persist.
X. Exogenous Genes for Expression in Plants
One particularly important advance of the present invention is that it
provides
methods and compositions for expression of exogenous genes in plant cells. One
advance of the constructs of the current invention is that they enable the
introduction
of multiple genes (often referred to as gene "stacking"), potentially
representing an
entire biochemical pathway, or any combination of genes encoding different
biochemical processes or pathways. Significantly, the current invention allows
for the
transformation of plant cells with a mini chromosome comprising a number of
structural genes. Another advantage is that more than one mini chromosome
could be
introduced, allowing combinations of genes to be moved and shuffled. Moreover,
the
ability to eliminate a mini chromosome from a plant would provide additional
flexibility, making it possible to alter the set of genes contained within a
plant.
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Further, by using site-specific recombinases, it should be possible to add
genes to an
existing mini chromosome once it is in a plant.
Added genes often will be genes that direct the expression of a particular
protein or polypeptide product, but they also may be non-expressible DNA
segments,
S e.g., transposons such as Ds that do not direct their own transposition. As
used herein,
an "expressible gene" is any gene that is capable of being transcribed into
RNA (e.g.,
mRNA, antisense RNA, etc.) or translated into a protein, expressed as a trait
of
interest, or the like, etc., and is not limited to selectable, screenable or
non-selectable
marker genes. The inventors also contemplate that, where both an expressible
gene
that is not necessarily a marker gene is employed in combination with a marker
gene,
one may employ the separate genes on either the same or different DNA segments
for
transformation. In the latter case, the different vectors may be delivered
concurrently
to recipient cells to maximize cotransformation or may be delivered
sequentially.
The choice of the particular DNA segments to be delivered to the recipient
cells often will depend on the purpose of the transformation. One of the major
purposes of transformation of crop plants is to add some commercially
desirable,
agronomically important traits to the plant. Such traits include, but are not
limited to,
herbicide resistance or tolerance; insect resistance or tolerance; disease
resistance or
tolerance (viral, bacterial, fungal, nematode); stress tolerance and/or
resistance, as
exemplified by resistance or tolerance to drought, heat, chilling, freezing,
excessive
moisture, salt stress; oxidative stress; increased yields; food content and
makeup;
physical appearance; male sterility; drydown; standability; prolificacy;
starch quantity
and quality; oil quantity and quality; protein quality and quantity; amino
acid
composition; the production of a pharmaceutically active protein; the
production of a
small molecule with medicinal properties; the production of a chemical
including those
with industrial utility; the production of nutraceuticals, carbohydrates,
RNAs, lipids,
fuels, dyes, pigments, vitamins, scents, flavors, vaccines, antibodies,
hormones, and
the like. Additionally one could create a library of an entire genome from any
organism or organelle including mammals, plants, microbes, fungi, bacteria,
represented on mini chromosomes. Furthermore one could incorporate a desired
genomic segment such as one that includes a quantitative trait onto a mini
chromosome. One may desire to incorporate one or more genes conferring any
such
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desirable trait or traits, such as, for example, a gene or genes encoding
herbicide
resistance.
In certain embodiments, the present invention contemplates the transformation
of a recipient cell with mini chromosomes comprising more than one exogenous
gene.
S An "exogenous gene," can be a gene not normally found in the host genome in
an
identical context, or alternatively, the mini chromosome could be used to
introduce
extra copies of host genes into a cell. The gene may be isolated from a
different
species than that of the host genome, or alternatively, isolated from the host
genome
but operably linked to one or more regulatory regions which differ from those
found in
the unaltered, native gene. Two or more exogenous genes also can be supplied
in a
single transformation event using either distinct transgene-encoding vectors,
or using a
single vector incorporating two or more gene coding sequences. For example,
plasmids bearing the bar and aroA expression units in either convergent,
divergent, or
colinear orientation, are considered to be particularly useful. Further
preferred
combinations are those of an insect resistance gene, such as a Bt gene, along
with a
protease inhibitor gene such as pinll, or the use of bar in combination with
either of
the above genes. Of course, any two or more transgenes of any description,
such as
those confernng herbicide, insect, disease (viral, bacterial, fungal,
nematode) or
drought resistance, male sterility, drydown, standability, prolificacy, starch
properties,
oil quantity and quality, modified chemical production, pharmaceutical or
nutraceutical properties, bioremediation properties, increased biomass,
altered growth
rate, altered fitness, altered salinity tolerance, altered thermal tolerance,
altered growth
form, altered composition, altered metabolism, altered biodegradability,
altered COZ
fixation, altered stress tolerance, presence of bioindicator activity, altered
digestibility
by humans or animals, altered allergenicity, altered mating characteristics,
altered
pollen dispersal, altered appearance, improved environmental impact, nitrogen
fixation
capability, or those increasing yield or nutritional quality may be employed
as desired.
(i) Herbicide Resistance
The genes encoding phosphinothricin acetyltransferase (bar and pat),
glyphosate tolerant EPSP synthase genes, the glyphosate degradative enzyme
gene gox
encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that
inactivates dalapon), herbicide resistant (e.g., sulfonylurea and
imidazolinone)
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acetolactate synthase, and bxn genes (encoding a nitrilase enzyme that
degrades
bromoxynil) are good examples of herbicide resistant genes for use in
transformation.
The bar and pat genes code for an enzyme, phosphinothricin acetyltransferase
(PAT),
which inactivates the herbicide phosphinothricin and prevents this compound
from
inhibiting glutamine synthetase enzymes. The enzyme S-enolpyruvylshikimate
3-phosphate synthase (EPSP Synthase), is normally inhibited by the herbicide
N-(phosphonomethyl)glycine (glyphosate). However, genes are known that encode
glyphosate-resistant EPSP synthase enzymes. These genes are particularly
contemplated for use in plant transformation. The deh gene encodes the enzyme
dalapon dehalogenase and confers resistance to the herbicide dalapon. The bxn
gene
codes for a specific nitrilase enzyme that converts bromoxynil to a non-
herbicidal
degradation product.
(ii) Insect Resistance
Potential insect resistance genes that can be introduced include Bacillus
thuringiensis crystal toxin genes or Bt genes (Watrud et al., 1985). Bt genes
may
provide resistance to lepidopteran or coleopteran pests such as European Corn
Borer
(ECB). Preferred Bt toxin genes for use in such embodiments include the
CrylA(b)
and CrylA(c) genes. Endotoxin genes from other species of B. thuringiensis
which
affect insect growth or development also may be employed in this regard.
It is contemplated that preferred Bt genes for use in the transformation
protocols disclosed herein will be those in which the coding sequence has been
modified to effect increased expression in plants, and more particularly, in
monocot
plants. Means for preparing synthetic genes are well known in the art and are
disclosed in, for example, U.S. Patent No. 5,500,365 and U.S. Patent Number
No.
5,689,052, each of the disclosures of which are specifically incorporated
herein by
reference in their entirety. Examples of such modified Bt toxin genes include
a
synthetic Bt CrylA(b) gene (Perlak et al., 1991), and the synthetic CrylA(c)
gene
termed 1800b (PCT Application WO 95/06128). Some examples of other Bt toxin
genes known to those of skill in the art are given in Table 1 below.
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Table 1: Bacillus thuringiensis S-Endotoxin Genes$
New Nomenclature Old Nomenclature GenBank Accession
Cryl Aa CryIA(a) M 11250
Cryl Ab CryIA(b) M 13898
Cryl Ac CryIA(c) M 11068
Cryl Ad CryIA(d) M73250
Cryl Ae CryIA(e) M65252
Cryl Ba CryIB X06711
Cryl Bb ETS L32020
Cryl Bc PEGS 246442
Cryl Bd CryE 1 U70726
Cryl Ca CryIC X07518
Cryl Cb CryIC(b) M97880
CrylDa CrylD X54160
CrylDb PrtB 222511
Cryl Ea CryIE X53985
Cryl Eb CryIE(b) M73253
CrylFa CryIF M63897
Cryl Fb PrtD 222512
Cryl Ga PrtA 222510
Cryl Gb CryH2 U70725
Cryl Ha PrtC 222513
CrylHb U35780
CrylIa CryV X62821
Cryl Ib CryV U07642
Cryl Ja ET4 L32019
Cryl Jb ET 1 U31527
CrylK U28801
Cry2Aa CryIIA M31738
Cry2Ab CryIIB M23 724
Cry2Ac CryIIC X57252
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New Nomenclature Old Nomenclature GenBank Accession
Cry3A CryIIIA M22472
Cry3Ba CryIIIB X17123
Cry3Bb CryIIIB2 M89794
Cry3C CryIIID X59797
Cry4A CryIVA Y00423
Cry4B CryIVB X07423
CrySAa CryVA(a) L07025
CrySAb CryVA(b) L07026
Cry6A CryVIA L07022
Cry6B CryVIB L07024
Cry7Aa CryIIIC M64478
Cry7Ab CryIIICb U04367
CryBA CryIIIE U04364
CryBB CryIIIG U04365
CryBC CryIIIF U04366
Cry9A CryIG X5 8120
Cry9B CryIX X75019
Cry9C CryIH 237527
CrylOA CryIVC M12662
Cryl 1 A CryIVD M31737
Cryl 1B Jeg80 X86902
Cryl2A CryVB L07027
Cryl 3A CryVC L07023
Cryl4A CryVD U13955
CrylSA 34kDa M76442
Cryl 6A cbm71 X94146
Cryl7A cbm71 X99478
Cryl 8A CryBP 1 X99049
Cryl9A Jeg65 Y08920
Cyt 1 Aa CytA X03182
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New Nomenclature Old Nomenclature GenBank Accession
CytlAb CytM X98793
Cyt2A CytB Z 14147
Cyt2B CytB U52043
aAdapted from: http://epunix.biols.susx.ac.uk/Home/Neil CrickmoreBt/index.html
Protease inhibitors also may provide insect resistance (Johnson et al., 1989),
and will thus have utility in plant transformation. The use of a protease
inhibitor II
gene, pinll, from tomato or potato is envisioned to be particularly useful.
Even more
advantageous is the use of a pinll gene in combination with a Bt toxin gene,
the
combined effect of which has been discovered to produce synergistic
insecticidal
activity. Other genes which encode inhibitors of the insect's digestive
system, or those
that encode enzymes or co-factors that facilitate the production of
inhibitors, also may
be useful. This group may be exemplified by oryzacystatin and amylase
inhibitors
such as those from wheat and barley.
Also, genes encoding lectins may confer additional or alternative insecticide
properties. Lectins (originally termed phytohemagglutinins) are multivalent
carbohydrate-binding proteins which have the ability to agglutinate red blood
cells
from a range of species. Lectins have been identified recently as insecticidal
agents
1 S with activity against weevils, ECB and rootworm (Murdock et al., 1990;
Czapla &
Lang, 1990). Lectin genes contemplated to be useful include, for example,
barley and
wheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984), with
WGA
being preferred.
Genes controlling the production of large or small polypeptides active against
insects when introduced into the insect pests, such as, e.g., lytic peptides,
peptide
hormones and toxins and venoms, form another aspect of the invention. For
example,
it is contemplated that the expression of juvenile hormone esterase, directed
towards
specific insect pests, also may result in insecticidal activity, or perhaps
cause cessation
of metamorphosis (Hammock et al., 1990).
Transgenic plants expressing genes which encode enzymes that affect the
integrity of the insect cuticle form yet another aspect of the invention. Such
genes
include those encoding, e.g., chitinase, proteases, lipases and also genes for
the
production of nikkomycin, a compound that inhibits chitin synthesis, the
introduction
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of any of which is contemplated to produce insect resistant plants. Genes that
code for
activities that affect insect molting, such as those affecting the production
of
ecdysteroid UDP-glucosyl transferase, also fall within the scope of the useful
transgenes of the present invention.
Genes that code for enzymes that facilitate the production of compounds that
reduce the nutritional quality of the host plant to insect pests also are
encompassed by
the present invention. It may be possible, for instance, to confer
insecticidal activity
on a plant by altering its sterol composition. Sterols are obtained by insects
from their
diet and are used for hormone synthesis and membrane stability. Therefore
alterations
in plant sterol composition by expression of novel genes, e.g., those that
directly
promote the production of undesirable sterols or those that convert desirable
sterols
into undesirable forms, could have a negative effect on insect growth and/or
development and hence endow the plant with insecticidal activity.
Lipoxygenases are
naturally occurring plant enzymes that have been shown to exhibit anti-
nutritional
effects on insects and to reduce the nutritional quality of their diet.
Therefore, further
embodiments of the invention concern transgenic plants with enhanced
lipoxygenase
activity which may be resistant to insect feeding.
Tripsacum dactyloides is a species of grass that is resistant to certain
insects,
including corn root worm. It is anticipated that genes encoding proteins that
are toxic
to insects or are involved in the biosynthesis of compounds toxic to insects
will be
isolated from Tripsacum and that these novel genes will be useful in confernng
resistance to insects. It is known that the basis of insect resistance in
Tripsacum is
genetic, because said resistance has been transferred to Zea mays via sexual
crosses
(Branson and Guss, 1972). It is further anticipated that other cereal, monocot
or dicot
plant species may have genes encoding proteins that are toxic to insects which
would
be useful for producing insect resistant plants.
Further genes encoding proteins characterized as having potential insecticidal
activity also may be used as transgenes in accordance herewith. Such genes
include,
for example, the cowpea trypsin inhibitor (CpTI; Hilder et al., 1987) which
may be
used as a rootworm deterrent; genes encoding avermectin (Avermectin and
Abamectin., Campbell, W.C., Ed., 1989; Ikeda et al., 1987) which may prove
particularly useful as a corn rootworm deterrent; ribosome inactivating
protein genes;
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and even genes that regulate plant structures. Transgenic plants including
anti-insect
antibody genes and genes that code for enzymes that can convert a non-toxic
insecticide (pro-insecticide) applied to the outside of the plant into an
insecticide
inside the plant also are contemplated.
(iii) Environment or Stress Resistance
Improvement of a plants ability to tolerate various environmental stresses
such
as, but not limited to, drought, excess moisture, chilling, freezing, high
temperature,
salt, and oxidative stress, also can be effected through expression of novel
genes. It is
proposed that benefits may be realized in terms of increased resistance to
freezing
temperatures through the introduction of an "antifreeze" protein such as that
of the
Winter Flounder (Cutler et al., 1989) or synthetic gene derivatives thereof.
Improved
chilling tolerance also may be conferred , through increased expression of
glycerol-3-phosphate acetyltransferase in chloroplasts (Wolter et al., 1992).
Resistance to oxidative stress (often exacerbated by conditions such as
chilling
1 S temperatures in combination with high light intensities) can be conferred
by expression
of superoxide dismutase (Gupta et al., 1993), and may be improved by
glutathione
reductase (Bowler et al., 1992). Such strategies may allow for tolerance to
freezing in
newly emerged fields as well as extending later maturity higher yielding
varieties to
earlier relative maturity zones.
It is contemplated that the expression of novel genes that favorably effect
plant
water content, total water potential, osmotic potential, and turgor will
enhance the
ability of the plant to tolerate drought. As used herein, the terms "drought
resistance"
and "drought tolerance" are used to refer to a plants increased resistance or
tolerance to
stress induced by a reduction in water availability, as compared to normal
circumstances, and the ability of the plant to function and survive in lower-
water
environments. In this aspect of the invention it is proposed, for example,
that the
expression of genes encoding for the biosynthesis of osmotically-active
solutes, such
as polyol compounds, may impart protection against drought. Within this class
are
genes encoding for mannitol-L-phosphate dehydrogenase (Lee and Saier, 1982)
and
trehalose-6-phosphate synthase (Kaasen et al., 1992). Through the subsequent
action
of native phosphatases in the cell or by the introduction and coexpression of
a specific
phosphatase, these introduced genes will result in the accumulation of either
mannitol
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or trehalose, respectively, both of which have been well documented as
protective
compounds able to mitigate the effects of stress. Mannitol accumulation in
transgenic
tobacco has been verified and preliminary results indicate that plants
expressing high
levels of this metabolite are able to tolerate an applied osmotic stress
(Tarczynski et al., 1992, 1993).
Similarly, the efficacy of other metabolites in protecting either enzyme
function (e.g., alanopine or propionic acid) or membrane integrity (e.g.,
alanopine) has
been documented (Loomis et al., 1989), and therefore expression of genes
encoding
for the biosynthesis of these compounds might confer drought resistance in a
manner
similar to or complimentary to mannitol. Other examples of naturally occurring
metabolites that are osmotically active and/or provide some direct protective
effect
during drought and/or desiccation include fructose, erythritol (Coxson et al.,
1992),
sorbitol, dulcitol (Karsten et al., 1992), glucosylglycerol (Reed et al.,
1984;
ErdMann et al., 1992), sucrose, stachyose (Koster and Leopold, 1988;
Blackman et al., 1992), raffinose (Bernal-Lugo and Leopold, 1992), proline
(Rensburg et al., 1993), glycine betaine, ononitol and pinitol (Vernon and
Bohnert,
1992). Continued canopy growth and increased reproductive fitness during times
of
stress will be augmented by introduction and expression of genes such as those
controlling the osmotically active compounds discussed above and other such
compounds. Currently preferred genes which promote the synthesis of an
osmotically
active polyol compound are genes which encode the enzymes mannitol-1-phosphate
dehydrogenase, trehalose-6-phosphate synthase and myoinositol 0-
methyltransferase.
It is contemplated that the expression of specific proteins also may increase
drought tolerance. Three classes of Late Embryogenic Proteins have been
assigned
based on structural similarities (see Dure et al., 1989). All three classes of
LEAs have
been demonstrated in maturing (i.e. desiccating) seeds. Within these 3 types
of LEA
proteins, the Type-II (dehydrin-type) have generally been implicated in
drought and/or
desiccation tolerance in vegetative plant parts (i.e. Mundy and Chua, 1988;
Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992). Recently,
expression of a
Type-III LEA (HVA-1) in tobacco was found to influence plant height, maturity
and
drought tolerance (Fitzpatrick, 1993). In rice, expression of the HVA-1 gene
influenced tolerance to water deficit and salinity (Xu et al., 1996).
Expression of
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structural genes from all three LEA groups may therefore confer drought
tolerance.
Other types of proteins induced during water stress include thiol proteases,
aldolases
and transmembrane transporters (Guerrero et al., 1990), which may confer
various
protective and/or repair-type functions during drought stress. It also is
contemplated
S that genes that effect lipid biosynthesis and hence membrane composition
might also
be useful in confernng drought resistance on the plant.
Many of these genes for improving drought resistance have complementary
modes of action. Thus, it is envisaged that combinations of these genes might
have
additive and/or synergistic effects in improving drought resistance in plants.
Many of
these genes also improve freezing tolerance (or resistance); the physical
stresses
incurred during freezing and drought are similar in nature and may be
mitigated in
similar fashion. Benefit may be conferred via constitutive expression of these
genes,
but the preferred means of expressing these novel genes may be through the use
of a
turgor-induced promoter (such as the promoters for the turgor-induced genes
described
in Guerrero et al., 1990 and Shagan et al., 1993 which are incorporated herein
by
reference). Spatial and temporal expression patterns of these genes may enable
plants
to better withstand stress.
It is proposed that expression of genes that are involved with specific
morphological traits that allow for increased water extractions from drying
soil would
be of benefit. For example, introduction and expression of genes that alter
root
characteristics may enhance water uptake. It also is contemplated that
expression of
genes that enhance reproductive fitness during times of stress would be of
significant
value. For example, expression of genes that improve the synchrony of pollen
shed
and receptiveness of the female flower parts, i.e., silks, would be of
benefit. In
addition it is proposed that expression of genes that minimize kernel abortion
during
times of stress would increase the amount of grain to be harvested and hence
be of
value.
Given the overall role of water in determining yield, it is contemplated that
enabling plants to utilize water more efficiently, through the introduction
and
expression of novel genes, will improve overall performance even when soil
water
availability is not limiting. By introducing genes that improve the ability of
plants to
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maximize water usage across a full range of stresses relating to water
availability,
yield stability or consistency of yield performance may be realized.
(iv) Disease Resistance
It is proposed that increased resistance to diseases may be realized through
introduction of genes into plants, for example, into monocotyledonous plants
such as
maize. It is possible to produce resistance to diseases caused by viruses,
bacteria,
fungi and nematodes. It also is contemplated that control of mycotoxin
producing
organisms may be realized through expression of introduced genes.
Resistance to viruses may be produced through expression of novel genes. For
example, it has been demonstrated that expression of a viral coat protein in a
transgenic plant can impart resistance to infection of the plant by that virus
and
perhaps other closely related viruses (Cuozzo et al., 1988, Hemenway et al.,
1988,
Abel et al., 1986). It is contemplated that expression of antisense genes
targeted at
essential viral functions may also impart resistance to viruses. For example,
an
antisense gene targeted at the gene responsible for replication of viral
nucleic acid may
inhibit replication and lead to resistance to the virus. It is believed that
interference
with other viral functions through the use of antisense genes also may
increase
resistance to viruses. Further, it is proposed that it may be possible to
achieve
resistance to viruses through other approaches, including, but not limited to
the use of
satellite viruses.
It is proposed that increased resistance to diseases caused by bacteria and
fungi
may be realized through introduction of novel genes. It is contemplated that
genes
encoding so-called "peptide antibiotics," pathogenesis related (PR) proteins,
toxin
resistance, and proteins affecting host-pathogen interactions such as
morphological
characteristics will be useful. Peptide antibiotics are polypeptide sequences
which are
inhibitory to growth of bacteria and other microorganisms. For example, the
classes of
peptides referred to as cecropins and magainins inhibit growth of many species
of
bacteria and fungi. It is proposed that expression of PR proteins in
monocotyledonous
plants such as maize may be useful in conferring resistance to bacterial
disease. These
genes are induced following pathogen attack on a host plant and have been
divided
into at least five classes of proteins (Bol, Linthorst, and Cornelissen,
1990). Included
amongst the PR proteins are ~i-1, 3-glucanases, chitinases, and osmotin and
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proteins that are believed to function in plant resistance to disease
organisms. Other
genes have been identified that have antifungal properties, e.g., UDA
(stinging nettle
lectin) and hevein (Broakaert et al., 1989; Barkai-Golan et al., 1978). It is
known that
certain plant diseases are caused by the production of phytotoxins. It is
proposed that
resistance to these diseases would be achieved through expression of a novel
gene that
encodes an enzyme capable of degrading or otherwise inactivating the
phytotoxin. It
also is contemplated that expression of novel genes that alter the
interactions between
the host plant and pathogen may be useful in reducing the ability of the
disease
organism to invade the tissues of the host plant, e.g., an increase in the
waxiness of the
leaf cuticle or other morphological characteristics.
(v) Plant Agronomic Characteristics
Two of the factors determining where crop plants can be grown are the average
daily temperature during the growing season and the length of time between
frosts.
Within the areas where it is possible to grow a particular crop, there are
varying
limitations on the maximal time it is allowed to grow to maturity and be
harvested.
For example, a variety to be grown in a particular area is selected for its
ability to
mature and dry down to harvestable moisture content within the required period
of
time with maximum possible yield. Therefore, crops of varying maturities is
developed for different growing locations. Apart from the need to dry down
sufficiently to permit harvest, it is desirable to have maximal drying take
place in the
field to minimize the amount of energy required for additional drying post-
harvest.
Also, the more readily a product such as grain can dry down, the more time
there is
available for growth and kernel fill. It is considered that genes that
influence maturity
and/or dry down can be identified and introduced into plant lines using
transformation
techniques to create new varieties adapted to different growing locations or
the same
growing location, but having improved yield to moisture ratio at harvest.
Expression
of genes that are involved in regulation of plant development may be
especially useful.
It is contemplated that genes may be introduced into plants that would improve
standability and other plant growth characteristics. Expression of novel genes
in plants
which confer stronger stalks, improved root systems, or prevent or reduce ear
droppage would be of great value to the farmer. It is proposed that
introduction and
expression of genes that increase the total amount of photoassimilate
available by, for
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example, increasing light distribution and/or interception would be
advantageous. In
addition, the expression of genes that increase the efficiency of
photosynthesis and/or
the leaf canopy would further increase gains in productivity. It is
contemplated that
expression of a phytochrome gene in crop plants may be advantageous.
Expression of
such a gene may reduce apical dominance, confer semidwarfism on a plant, and
increase shade tolerance (LJ.S. Patent No. 5,268,526). Such approaches would
allow
for increased plant populations in the field.
(vi) Nutrient Utilization
The ability to utilize available nutrients may be a limiting factor in growth
of
crop plants. It is proposed that it would be possible to alter nutrient
uptake, tolerate
pH extremes, mobilization through the plant, storage pools, and availability
for
metabolic activities by the introduction of novel genes. These modifications
would
allow a plant such as maize to more efficiently utilize available nutrients.
It is.
contemplated that an increase in the activity of, for example, an enzyme that
is
normally present in the plant and involved in nutrient utilization would
increase the
availability of a nutrient. An example of such an enzyme would be phytase. It
is
further contemplated that enhanced nitrogen utilization by a plant is
desirable.
Expression of a glutamate dehydrogenase gene in plants, e.g., E. coli gdhA
genes, may
lead to increased fixation of nitrogen in organic compounds. Furthermore,
expression
of gdhA in plants may lead to enhanced resistance to the herbicide glufosinate
by
incorporation of excess ammonia into glutamate, thereby detoxifying the
ammonia. It
also is contemplated that expression of a novel gene may make a nutrient
source
available that was previously not accessible, e.g., an enzyme that releases a
component
of nutrient value from a more complex molecule, perhaps a macromolecule.
(vii) Male Sterility
Male sterility is useful in the production of hybrid seed. It is proposed that
male sterility may be produced through expression of novel genes. For example,
it has
been shown that expression of genes that encode proteins that interfere with
development of the male inflorescence and/or gametophyte result in male
sterility.
Chimeric ribonuclease genes that express in the anthers of transgenic tobacco
and
oilseed rape have been demonstrated to lead to male sterility (Mariani et al.,
1990).
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A number of mutations were discovered in maize that confer cytoplasmic male
sterility. One mutation in particular, referred to as T cytoplasm, also
correlates with
sensitivity to Southern corn leaf blight. A DNA sequence, designated TURF-13
(Levings, 1990), was identified that correlates with T cytoplasm. It is
proposed that it
would be possible through the introduction of TURF-13 via transformation, to
separate
male sterility from disease sensitivity. As it is necessary to be able to
restore male
fertility for breeding purposes and for grain production, it is proposed that
genes
encoding restoration of male fertility also may be introduced.
(viii) Improved Nutritional Content
Genes may be introduced into plants to improve the nutrient quality or content
of a particular crop. Introduction of genes that alter the nutrient
composition of a crop
may greatly enhance the feed or food value. For example, the protein of many
grains
is suboptimal for feed and food purposes, especially when fed to pigs,
poultry, and
humans. The protein is deficient in several amino acids that are essential in
the diet of
1 S these species, requiring the addition of supplements to the grain.
Limiting essential
amino acids may include lysine, methionine, tryptophan, threonine, valine,
arginine,
and histidine. Some amino acids become limiting only after corn is
supplemented with
other inputs for feed formulations. The levels of these essential amino acids
in seeds
and grain may be elevated by mechanisms which include, but are not limited to,
the
introduction of genes to increase the biosynthesis of the amino acids,
decrease the
degradation of the amino acids, increase the storage of the amino acids in
proteins, or
increase transport of the amino acids to the seeds or grain.
The protein composition of a crop may be altered to improve the balance of
amino acids in a variety of ways including elevating expression of native
proteins,
decreasing expression of those with poor composition, changing the composition
of
native proteins, or introducing genes encoding entirely new proteins
possessing
superior composition.
The introduction of genes that alter the oil content of a crop plant may also
be
of value. Increases in oil content may result in increases in metabolizable-
energy-
content and density of the seeds for use in feed and food. The introduced
genes may
encode enzymes that remove or reduce rate-limitations or regulated steps in
fatty acid
or lipid biosynthesis. Such genes may include, but are not limited to, those
that encode
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acetyl-CoA carboxylase, ACP-acyltransferase, [3-ketoacyl-ACP synthase, plus
other
well known fatty acid biosynthetic activities. Other possibilities are genes
that encode
proteins that do not possess enzymatic activity such as acyl carrier protein.
Genes may
be introduced that alter the balance of fatty acids present in the oil
providing a more
healthful or nutritive feedstuff. The introduced DNA also may encode sequences
that
block expression of enzymes involved in fatty acid biosynthesis, altering the
proportions of fatty acids present in crops.
Genes may be introduced that enhance the nutritive value of the starch
component of crops, for example by increasing the degree of branching,
resulting in
improved utilization of the starch in livestock by delaying its metabolism.
Additionally, other major constituents of a crop may be altered, including
genes that
affect a variety of other nutritive, processing, or other quality aspects. For
example,
pigmentation may be increased or decreased.
Feed or food crops may also possess sub-optimal quantities of vitamins,
antioxidants or other nutraceuticals, requiring supplementation to provide
adequate
nutritive value and ideal health value. Introduction of genes that enhance
vitamin
biosynthesis may be envisioned including, for example, vitamins A, E, B~2,
choline,
and the like. Mineral content may also be sub-optimal. Thus genes that affect
the
accumulation or availability of compounds containing phosphorus, sulfur,
calcium,
manganese, zinc, and iron among others would be valuable.
Numerous other examples of improvements of crops may be used with the
invention. The improvements may not necessarily involve grain, but may, for
example, improve the value of a crop for silage. Introduction of DNA to
accomplish
this might include sequences that alter lignin production such as those that
result in the
"brown midrib" phenotype associated with superior feed value for cattle.
In addition to direct improvements in feed or food value, genes also may be
introduced which improve the processing of crops and improve the value of the
products resulting from the processing. One use of crops if via wetmilling.
Thus
novel genes that increase the efficiency and reduce the cost of such
processing, for
example by decreasing steeping time, may also find use. Improving the value of
wetmilling products may include altering the quantity or quality of starch,
oil, corn
gluten meal, or the components of gluten feed. Elevation of starch may be
achieved
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through the identification and elimination of rate limiting steps in starch
biosynthesis
or by decreasing levels of the other components of crops resulting in
proportional
increases in starch.
Oil is another product of wetmilling, the value of which may be improved by
introduction and expression of genes. Oil properties may be altered to improve
its
performance in the production and use of cooking oil, shortenings, lubricants
or other
oil-derived products or improvement of its health attributes when used in the
food-
related applications. Novel fatty acids also may be synthesized which upon
extraction
can serve as starting materials for chemical syntheses. The changes in oil
properties
may be achieved by altering the type, level, or lipid arrangement of the fatty
acids
present in the oil. This in turn may be accomplished by the addition of genes
that
encode enzymes that catalyze the synthesis of novel fatty acids and the lipids
possessing them or by increasing levels of native fatty acids while possibly
reducing
levels of precursors. Alternatively, DNA sequences may be introduced which
slow or
block steps in fatty acid biosynthesis resulting in the increase in precursor
fatty acid
intermediates. Genes that might be added include desaturases, epoxidases,
hydratases,
dehydratases, and other enzymes that catalyze reactions involving fatty acid
intermediates. Representative examples of catalytic steps that might be
blocked
include the desaturations from stearic to oleic acid and oleic to linolenic
acid resulting
in the respective accumulations of stearic and oleic acids. Another example is
the
blockage of elongation steps resulting in the accumulation of Cg to C~2
saturated fatty
acids.
(ix) Production or Assimilation of Chemicals or Biologicals
It may further be considered that a transgenic plant prepared in accordance
with the invention may be used for the production or manufacturing of useful
biological compounds that were either not produced at all, or not produced at
the same
level, in the corn plant previously. Alternatively, plants produced in
accordance with
the invention may be made to metabolize or absorb and concentrate certain
compounds, such as hazardous wastes, thereby allowing bioremediation of these
compounds.
The novel plants producing these compounds are made possible by the
introduction and expression of one or potentially many genes with the
constructs
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provided by the invention. The vast array of possibilities include but are not
limited to
any biological compound which is presently produced by any organism such as
proteins, nucleic acids, primary and intermediary metabolites, carbohydrate
polymers,
enzymes for uses in bioremediation, enzymes for modifying pathways that
produce
secondary plant metabolites such as flavonoids or vitamins, enzymes that could
produce pharmaceuticals, and for introducing enzymes that could produce
compounds
of interest to the manufacturing industry such as specialty chemicals and
plastics. The
compounds may be produced by the plant, extracted upon harvest and/or
processing,
and used for any presently recognized useful purpose such as pharmaceuticals,
fragrances, and industrial enzymes to name a few.
(x) Non-Protein-Expressing Sequences
DNA may be introduced into plants for the purpose of expressing RNA
transcripts that function to affect plant phenotype yet are not translated
into protein.
Two examples are antisense RNA and RNA with ribozyme activity. Both may serve
possible functions in reducing or eliminating expression of native or
introduced plant
genes. However, as detailed below, DNA need not be expressed to effect the
phenotype of a plant.
1. Antisense RNA
Genes may be constructed or isolated, which when transcribed, produce
antisense RNA that is complementary to all or parts) of a targeted messenger
RNA(s).
The antisense RNA reduces production of the polypeptide product of the
messenger
RNA. Genes may also be constructed to produce double-stranded RNA molecules
complementary to all or part of the targeted messenger RNA(s). Genes designed
in
this manner will be referred to as RNAi constructs; the double-stranded RNA or
RNAi
constructs can trigger the sequence-specific degradation of the target
messenger RNA.
The polypeptide product of the target messenger RNA may be any protein. The
aforementioned genes will be referred to as antisense genes and RNAi
constructs,
respectively. An antisense gene or RNAi construct may thus be introduced into
a plant
by transformation methods to produce a novel transgenic plant with reduced
expression of a selected protein of interest. For example, the protein may be
an
enzyme that catalyzes a reaction in the plant. Reduction of the enzyme
activity may
reduce or eliminate products of the reaction which include any enzymatically
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synthesized compound in the plant such as fatty acids, amino acids,
carbohydrates,
nucleic acids and the like. Alternatively, the protein may be a storage
protein, such as
a zero, or a structural protein, the decreased expression of which may lead to
changes
in seed amino acid composition or plant morphological changes respectively.
The
possibilities cited above are provided only by way of example and do not
represent the
full range of applications.
2. Ribozymes
Genes also may be constructed or isolated, which when transcribed, produce
RNA enzymes (ribozymes) which can act as endoribonucleases and catalyze the
cleavage of RNA molecules with selected sequences. The cleavage of selected
messenger RNAs can result in the reduced production of their encoded
polypeptide
products. These genes may be used to prepare novel transgenic plants which
possess
them. The transgenic plants may possess reduced levels of polypeptides
including, but
not limited to, the polypeptides cited above.
1 S Ribozymes are RNA-protein complexes that cleave nucleic acids in a
site-specific fashion. Ribozymes have specific catalytic domains that possess
endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and
Symons,
1987). For example, a large number of ribozymes accelerate phosphoester
transfer
reactions with a high degree of specificity, often cleaving only one of
several
phosphoesters in an oligonucleotide substrate (Cech et al., 1981; Michel and
Westhof,
1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to
the
requirement that the substrate bind via specific base-pairing interactions to
the internal
guide sequence ("IGS") of the ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific
cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al.,
1981).
For example, U. S. Patent 5,354,855 reports that certain ribozymes can act as
endonucleases with a sequence specificity greater than that of known
ribonucleases
and approaching that of the DNA restriction enzymes.
Several different ribozyme motifs have been described with RNA cleavage
activity (Symons, 1992). Examples include sequences from the Group I self
splicing
introns including Tobacco Ringspot Virus (Prody et al., 1986), Avocado
Sunblotch
Viroid (Palukaitis et al., 1979; Symons, 1981), and Lucerne Transient Streak
Virus
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(Forster and Symons, 1987). Sequences from these and related viruses are
referred to
as hammerhead ribozyme based on a predicted folded secondary structure.
Other suitable ribozymes include sequences from RNase P with RNA cleavage
activity (Yuan et al., 1992, Yuan and Altman, 1994, U. S. Patents 5,168,053
and
5,624,824), hairpin ribozyme structures (Berzal-Herranz et al., 1992;
Chowrira et al., 1993) and Hepatitis Delta virus based ribozymes (U. S. Patent
5,625,047). The general design and optimization of ribozyme directed RNA
cleavage
activity has been discussed in detail (Haseloff and Gerlach, 1988, Symons,
1992,
Chowrira et al., 1994; Thompson et al., 1995).
The other variable on ribozyme design is the selection of a cleavage site on a
given target RNA. Ribozymes are targeted to a given sequence by virtue of
annealing
to a site by complimentary base pair interactions. Two stretches of homology
are
required for this targeting. These stretches of homologous sequences flank the
catalytic ribozyme structure defined above. Each stretch of homologous
sequence can
vary in length from 7 to 15 nucleotides. The only requirement for defining the
homologous sequences is that, on the target RNA, they are separated by a
specific
sequence which is the cleavage site. For hammerhead ribozyme, the cleavage
site is a
dinucleotide sequence on the target RNA is a uracil (U) followed by either an
adenine,
cytosine or uracil (A,C or U) (Perriman et al., 1992; Thompson et al., 1995).
The
frequency of this dinucleotide occurnng in any given RNA is statistically 3
out of 16.
Therefore, for a given target messenger RNA of 1,000 bases, 187 dinucleotide
cleavage sites are statistically possible.
Designing and testing ribozymes for efficient cleavage of a target RNA is a
process well known to those skilled in the art. Examples of scientific methods
for
designing and testing ribozymes are described by Chowrira et al., (1994) and
Lieber
and Strauss (1995), each incorporated by reference. The identification of
operative
and preferred sequences for use in down regulating a given gene is simply a
matter of
preparing and testing a given sequence, and is a routinely practiced
"screening"
method known to those of skill in the art.
3. Induction of Gene Silencing
It also is possible that genes may be introduced to produce novel transgenic
plants which have reduced expression of a native gene product by the mechanism
of
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co-suppression. It has been demonstrated in tobacco, tomato, and petunia
(Goring et al., 1991; Smith et al., 1990; Napoli et al., 1990; van der Krol et
al., 1990)
that expression of the sense transcript of a native gene will reduce or
eliminate
expression of the native gene in a manner similar to that observed for
antisense genes.
The introduced gene may encode all or part of the targeted native protein but
its
translation may not be required for reduction of levels of that native
protein.
4. Non-RNA-Expressing Sequences
DNA elements including those of transposable elements such as Ds, Ac, or Mu,
may be inserted into a gene to cause mutations. These DNA elements may be
inserted
in order to inactivate (or activate) a gene and thereby "tag" a particular
trait. In this
instance the transposable element does not cause instability of the tagged
mutation,
because the utility of the element does not depend on its ability to move in
the genome.
Once a desired trait is tagged, the introduced DNA sequence may be used to
clone the
corresponding gene, e.g., using the introduced DNA sequence as a PCR primer
together with PCR gene cloning techniques (Shapiro, 1983; Dellaporta et al.,
1988).
Once identified, the entire genes) for the particular trait, including control
or
regulatory regions where desired, may be isolated, cloned and manipulated as
desired.
The utility of DNA elements introduced into an organism for purposes of gene
tagging
is independent of the DNA sequence and does not depend on any biological
activity of
the DNA sequence, i.e., transcription into RNA or translation into protein.
The sole
function of the DNA element is to disrupt the DNA sequence of a gene.
It is contemplated that unexpressed DNA sequences, including novel synthetic
sequences, could be introduced into cells as proprietary "labels" of those
cells and
plants and seeds thereof. It would not be necessary for a label DNA element to
disrupt
the function of a gene endogenous to the host organism, as the sole function
of this
DNA would be to identify the origin of the organism. For example, one could
introduce a unique DNA sequence into a plant and this DNA element would
identify
all cells, plants, and progeny of these cells as having arisen from that
labeled source. It
is proposed that inclusion of label DNAs would enable one to distinguish
proprietary
germplasm or germplasm derived from such, from unlabelled germplasm.
Another possible element which may be introduced is a matrix attachment
region element (MAR), such as the chicken lysozyme A element (Stief, 1989),
which
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can be positioned around an expressible gene of interest to effect an increase
in overall
expression of the gene and diminish position dependent effects upon
incorporation into
the plant genome (Stief et al., 1989; Phi-Van et al., 1990).
5. Other
Other examples of non-protein expressing sequences specifically envisioned
for use with the invention include tRNA sequences, for example, to alter codon
usage,
and rRNA variants, for example, which may confer resistance to various agents
such
as antibiotics.
XI. Biological Functional Eguivalents
Modification and changes may be made in the centromeric DNA segments of
the current invention and still obtain a functional molecule with desirable
characteristics. The following is a discussion based upon changing the nucleic
acids of
a centromere to create an equivalent, or even an improved, second-generation
molecule.
In particular embodiments of the invention, mutated centromeric sequences are
contemplated to be useful for increasing the utility of the centromere. It is
specifically
contemplated that the function of the centromeres of the current invention may
be
based upon the secondary structure of the DNA sequences of the centromere,
modification of the DNA with methyl groups or other adducts, and / or the
proteins
which interact with the centromere. By changing the DNA sequence of the
centromere, one may alter the affinity of one or more centromere-associated
proteins)
for the centromere and / or the secondary structure or modification of the
centromeric
sequences, thereby changing the activity of the centromere. Alternatively,
changes
may be made in the centromeres of the invention which do not affect the
activity of the
centromere. Changes in the centromeric sequences which reduce the size of the
DNA
segment needed to confer centromere activity are contemplated to be
particularly
useful in the current invention, as would changes which increased the fidelity
with
which the centromere was transmitted during mitosis and meiosis.
XII. Plants
The term "plant," as used herein, refers to any type of plant. The inventors
have provided below an exemplary description of some plants that may be used
with
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the invention. However, the list is not in any way limiting, as other types of
plants will
be known to those of skill in the art and could be used with the invention.
A common class of plants exploited in agriculture are vegetable crops,
including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g.,
head, leaf,
romaine), bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon,
crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni, carrots,
napa,
cauliflower, okra, onions, celery, parsley, chick peas, parsnips, chicory,
Chinese
cabbage, peppers, collards, potatoes, cucumber plants (marrows, cucumbers),
pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify,
escarole,
shallots, endive, garlic, spinach, green onions, squash, greens, beet (sugar
beet and
fodder beet), sweet potatoes, swiss chard, horseradish, tomatoes, kale,
turnips, and
spices.
Other types of plants frequently finding commercial use include fruit and vine
crops such as apples, apricots, cherries, nectarines, peaches, pears, plums,
prunes,
quince almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus,
bluebernes,
boysenberries, cranberries, currants, loganberries, raspberries, strawbernes,
blackberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate,
pineapple,
tropical fruits, pomes, melon, mango, papaya, and lychee.
Many of the most widely grown plants are field crop plants such as evening
primrose, meadow foam, corn (field, sweet, popcorn), hops, jojoba, peanuts,
rice,
safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, tobacco,
kapok,
leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard,
poppy,
olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts),
fibre plants
(cotton, flax, hemp, jute), lauraceae (cinnamon, camphor), or plants such as
coffee,
sugarcane, tea, and natural rubber plants.
Still other examples of plants include bedding plants such as flowers, cactus,
succulents and ornamental plants, as well as trees such as forest (broad-
leaved trees
and evergreens, such as conifers), fruit, ornamental, and nut-bearing trees,
as well as
shrubs and other nursery stock.
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XIII. Definitions
As used herein, the terms "autonomous replicating sequence" or "ARS" or
"origin of replication " refer to an origin of DNA replication recognized by
proteins
that initiate DNA replication.
As used herein, the terms "binary BAC" or "binary bacterial artificial
chromosome" refer to a bacterial vector that contains the T-DNA border
sequences
necessary for Agrobacterium mediated transformation (see, for example,
Hamilton et
al., 1996; Hamilton, 1997; and Liu et al., 1999.
As used herein, the term "candidate centromere sequence" refers to a nucleic
acid sequence which one wishes to assay for potential centromere function.
As used herein, a "centromere" is any DNA sequence that confers an ability to
segregate to daughter cells through cell division. In one context, this
sequence may
produce a segregation efficiency to daughter cells ranging from about 1 % to
about
100%, including to about S%, 10%, 20%, 30%, 40%, SO%, 60%, 70%, 80%, 90% or
about 95% of daughter cells. Variations in such a segregation efficiency may
find
important applications within the scope of the invention; for example, mini
chromosomes carrying centromeres that confer 100% stability could be
maintained in
all daughter cells without selection, while those that confer 1 % stability
could be
temporarily introduced into a transgenic organism, but be eliminated when
desired. In
particular embodiments of the invention, the centromere may confer stable
segregation
of a nucleic acid sequence, including a recombinant construct comprising the
centromere, through mitotic or meiotic divisions, including through both
meiotic and
meitotic divisions. A plant centromere is not necessarily derived from plants,
but has
the ability to promote DNA segregation in plant cells.
As used herein, the term "centromere-associated protein" refers to a protein
encoded by a sequence of the centromere or a protein which is encoded by host
DNA
and binds with relatively high affinity to the centromere.
As used herein, the term "circular permutations" refer to variants of a
sequence
that begin at base n within the sequence, proceed to the end of the sequence,
resume
with base number one of the sequence, and proceed to base n - 1. For this
analysis, n
may be any number less than or equal to the length of the sequence. For
example,
circular permutations of the sequence ABCD are: ABCD, BCDA, CDAB, and DABC.
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As used herein, the term "crop" includes any plant or portion of a plant grown
or harvested for commercial or beneficial purposes.
As used herein, "eukaryote" refers to living organisms whose cells contain
nuclei. A eukaryote may be distinguished from a "prokaryote" which is an
organism
which lacks nuclei. Prokaryotes and eukaryotes differ fundamentally in the way
their
genetic information is organized, as well as their patterns of RNA and protein
synthesis.
As used herein, the term "expression" refers to the process by which a
structural gene produces an RNA molecule, typically termed messenger RNA
(mRNA). The mRNA is typically, but not always, translated into polypeptide(s).
As used herein, the term "genome" refers to all of the genes and DNA
sequences that comprise the genetic information within a given cell of an
organism.
Usually, this is taken to mean the information contained within the nucleus,
but also
includes the organelles.
1 S As used herein, the term "higher eukaryote" means a multicellular
eukaryote,
typically characterized by its greater complex physiological mechanisms and
relatively
large size. Generally, complex organisms such as plants and animals are
included in
this category. Preferred higher eukaryotes to be transformed by the present
invention
include, for example, monocot and dicot angiosperm species, gymnosperm
species,
fern species, plant tissue culture cells of these species, animal cells and
algal cells. It
will of course be understood that prokaryotes and eukaryotes alike may be
transformed
by the methods of this invention.
As used herein, the term "host" refers to any organism that contains a
plasmid,
expression vector, or integrated construct comprising a plant centromere.
Preferred
examples of host cells for cloning, useful in the present invention, are
bacteria such as
Escherichia coli, Bacillus subtilis, Pseudomonas, Streptomyces, Salmonella,
and yeast
cells such as S. cerevisiae. Host cells which can be targeted for expression
of a mini
chromosome may be plant cells of any source and specifically include
Arabidopsis,
maize, rice, sugarcane, sorghum, barley, soybeans, tobacco, wheat, tomato,
potato,
citrus, or any other agronomically or scientifically important species.
As used herein, the term "hybridization" refers to the pairing of
complementary
RNA and DNA strands to produce an RNA-DNA hybrid, or alternatively, the
pairing
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of two DNA single strands from genetically different or the same sources to
produce a
double stranded DNA molecule.
As used herein, the term "linker" refers to a DNA molecule, generally up to 50
or 60 nucleotides long and synthesized chemically, or cloned from other
vectors. In a
preferred embodiment, this fragment contains one, or preferably more than one,
restriction enzyme site for a blunt-cutting enzyme and a staggered-cutting
enzyme,
such as BamHI. One end of the linker fragment is adapted to be ligatable to
one end of
the linear molecule and the other end is adapted to be ligatable to the other
end of the
linear molecule.
As used herein, a "library" is a pool of random DNA fragments which are
cloned. In principle, any gene can be isolated by screening the library with a
specific
hybridization probe (see, for example, Young et al., 1977). Each library may
contain
the DNA of a given organism inserted as discrete restriction enzyme-generated
fragments or as randomly sheered fragments into many thousands of plasmid
vectors.
For purposes of the present invention, E. coli, yeast, and Salmonella plasmids
are
particularly useful when the genome inserts come from other organisms.
As used herein, the term "lower eukaryote" refers to a eukaryote characterized
by a comparatively simple physiology and composition, and most often
unicellularity.
Examples of lower eukaryotes include flagellates, ciliates, and yeast.
As used herein, a "mini chromosome" is a recombinant DNA construct
including a centromere and capable of transmission to daughter cells. Mini
chromosome may remain separate from the host genome (as episomes) or may
integrate into host chromosomes. The stability of this construct through cell
division
could range between from about 1% to about 100%, including about 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% and about 95%. The mini chromosome
construct may be a circular or linear molecule. It may include elements such
as one or
more telomeres, ARS sequences, and genes. The number of such sequences
included
is only limited by the physical size limitations of the construct itself It
could contain
DNA derived from a natural centromere, although it may be preferable to limit
the
amount of DNA to the minimal amount required to obtain a segregation
efficiency in
the range of 1-100%. The mini chromosome could also contain a synthetic
centromere
composed of tandem arrays of repeats of any sequence, either derived from a
natural
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centromere, or of synthetic DNA. The mini chromosome could also contain DNA
derived from multiple natural centromeres. The mini chromosome may be
inherited
through mitosis or meiosis, or through both meiosis and mitosis. As used
herein, the
term mini chromosome specifically encompasses and includes the terms "plant
artificial chromosome" or "PLAC," or engineered chromosomes or
microchromosomes and all teachings relevant to a PLAC or plant artificial
chromosome specifically apply to constructs within the meaning of the term
mini
chromosome.
As used herein, by "mini chromosome-encoded protein" it is meant a
polypeptide which is encoded by a sequence of a mini chromosome of the current
invention. This includes sequences such as selectable markers, telomeres,
etc., as well
as those proteins encoded by any other selected functional genes on the mini
chromosome.
As used herein, the term "plant" includes plant cells, plant protoplasts,
plant
calli, and the like, as well as whole plants regenerated therefrom.
As used herein, the term "plasmid" or "cloning vector" refers to a closed
covalently circular extrachromosomal DNA or linear DNA which is able to
replicate in
a host cell and which is normally nonessential to the survival of the cell. A
wide
variety of plasmids and other vectors are known and commonly used in the art
(see, for
example, Cohen et al., U.S. Patent No. 4,468,464, which discloses examples of
DNA
plasmids, and which is specifically incorporated herein by reference).
As used herein, a "probe" is any biochemical reagent (usually tagged in some
way for ease of identification), used to identify or isolate a gene, a gene
product, a
DNA segment or a protein.
As used herein, the term "recombination" refers to any genetic exchange that
involves breaking and rejoining of DNA strands.
As used herein the term "regulatory sequence" refers to any DNA sequence
that influences the efficiency of transcription or translation of any gene.
The term
includes, but is not limited to, sequences comprising promoters, enhancers and
terminators.
As used herein, a "selectable marker" is a gene whose presence results in a
clear phenotype, and most often a growth advantage for cells that contain the
marker.
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This growth advantage may be present under standard conditions, altered
conditions
such as elevated temperature, or in the presence of certain chemicals such as
herbicides or antibiotics. Use of selectable markers is described, for
example, in
Broach et al. (1979). Examples of selectable markers include the thymidine
kinase
gene, the cellular adenine-phosphoribosyltransferase gene and the
dihydrylfolate
reductase gene, hygromycin phosphotransferase genes, the bar gene and neomycin
phosphotransferase genes, among others. Preferred selectable markers in the
present
invention include genes whose expression confer antibiotic or herbicide
resistance to
the host cell, sufficient to enable the maintenance of a vector within the
host cell, and
which facilitate the manipulation of the plasmid into new host cells. Of
particular
interest in the present invention are proteins confernng cellular resistance
to
ampicillin, chloramphenicol, tetracycline, G-418, bialaphos, and glyphosate
for
example.
As used herein, a "screenable marker" is a gene whose presence results in an
identifiable phenotype. This phenotype may be observable under standard
conditions,
altered conditions such as elevated temperature, or in the presence of certain
chemicals
used to detect the phenotype.
As used herein, the term "site-specific recombination" refers to any genetic
exchange that involves breaking and rejoining of DNA strands at a specific DNA
sequence.
As used herein, a "structural gene" is a sequence which codes for a
polypeptide
or RNA and includes 5' and 3' ends. The structural gene may be from the host
into
which the structural gene is transformed or from another species. A structural
gene
will preferably, but not necessarily, include one or more regulatory sequences
which
modulate the expression of the structural gene, such as a promoter, terminator
or
enhancer. A structural gene will preferably, but not necessarily, confer some
useful
phenotype upon an organism comprising the structural gene, for example,
herbicide
resistance. In one embodiment of the invention, a structural gene may encode
an RNA
sequence which is not translated into a protein, for example a tRNA or rRNA
gene.
As used herein, the term "telomere" refers to a sequence capable of capping
the
ends of a chromosome, thereby preventing degradation of the chromosome end,
ensuring replication and preventing fusion to other chromosome sequences.
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Telomeres can include naturally occurring telomere sequences or synthetic
sequences.
Telomres from one species may confer telomere activity in another species.
As used herein, the terms "transformation" or "transfection" refer to the
acquisition in cells of new DNA sequences through the chromosomal or extra
s chromosomal addition of DNA. This is the process by which naked DNA, DNA
coated with protein, or whole mini chromosomes are introduced into a cell,
resulting in
a potentially heritable change.
As used herein the term "consensus" refers to a nucleic acid sequence derived
by comparing two or more related sequences. A consensus sequence defines both
the
conserved and variable sites between the sequences being compared. Any one of
the
sequences used to derive the consensus or any permutation defined by the
consensus
may be useful in construction mini chromosomes.
As used herein the term "repeated nucleotide sequence" refers to any nucleic
acid sequence of at least 25 by present in a genome or a recombinant molecule
that
1 S occurs at least two or more times and that are preferably at least 80%
identical either in
head to tail or head to head orientation either with or without intervening
sequence
between repeat units.
XIV. Examples
The following examples are included to demonstrate preferred embodiments of
the invention. It should be appreciated by those of skilled the art that the
techniques
disclosed in the examples which follow represent techniques discovered by the
inventors to function well in the practice of the invention, and thus can be
considered
to constitute preferred modes for its practice. However, those of skill in the
art should,
in light of the present disclosure, appreciate that many changes can be made
in the
specific embodiments which are disclosed and still obtain a like or similar
result
without departing from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are both
chemically and
physiologically related may be substituted for the agents described herein
while the
same or similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be within the
spirit,
scope and concept of the invention as defined by the appended claims.
EXAMPLE 1
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Isolation of Genomic DNA
Tissue from various plants are harvested for DNA extraction. For DNA
extraction, leaf tissue is cooled in liquid nitrogen, ground to a fine powder
and
transferred to an organic solvent-resistant test tube or beaker. Warm CTAB
extraction
S solution (2% (w/v) CTAB, 100 mM Tris-Cl, pH 9.5, 20 mM EDTA, pH 8.0, 1.4 M
NaCI, 1 % polyethylene gycol) is added in a ratio of 20 ml per gram of tissue
and
mixed thoroughly. For each 20 ml extraction buffer, 50 microliters of (3-
mercaptoethanol and 30 microliters of 30 mg/ml RNAse A are added and the
mixture
is incubated for 10-60 min. at 65°C with occasional mixing. The
homogenate is
extracted with an equal volume of chloroform, and is then centrifuged 5 min at
7500 x
g (8000 rpm in JA20; 10,000 rpm in a microcentrifuge, for smaller samples),
4°C. The
top (aqueous) phase is recovered and nucleic acids are precipitated by adding
1 volume
isopropanol. After mixing, the precipitate is pelleted at 15 min at 7500 x g,
4°C. The
pellet is washed with 70% ethanol, dried and resuspended in a minimal volume
of TE
(10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA, pH 8.0).
EXAMPLE 2
Brassica oleracea centromere repeat sequences
We purified repetitive sequences from Brassica oleracea (Brassica oleracea
fast plants, obtained from the Wisconsin Crucifer Cooperative). We set forth
herein
two centromere repeats, termed ChrBol and ChrBo2. We determined the consensus
of
each repeat as described in Example 6.
The consensus sequence of ChrBol is shown in FIG. 1A (SEQ ID NO:l). This
consensus was assembled from DNA sequences collected by the inventors. Twenty-
four of these sequences completely spanned the repeat, and nine others
partially
covered the repeat. The length of this repeat is 180 ~ 0.86 base pairs, and A
and T
comprise of 60% of the consensus.
The consensus sequence of ChrBo2 is shown in FIG. 1B (SEQ ID N0:2). This
consensus was assembled from DNA sequences collected by the inventors. Five of
these sequences completely spanned the repeat, and two others partially
covered the
repeat. The length of this repeat is 180 ~ 0.45 base pairs, and A and T
comprise 63%
of the consensus.
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The two repeats (ChrBol and ChrBo2) were aligned to each other using the
ClustalX program (ClustalX is a free multiple sequence alignment program for
Windows. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and
Higgins,
D.G. (1997) The ClustalX windows interface: flexible strategies for multiple
sequence
S alignment aided by quality analysis tools. Nucleic Acids Research, 24:4876-
4882.).
The two consensus sequences differ significantly from each other at several
bases.
Those sites with significant differences (chi-squared, P < 0.05) are
highlighted as
shown in FIG. 1 C.
The GenBank nt database (fttp://ftp.ncbi.nlm.nih.gov/blast/db/, March 29
version, downloaded on 04/07/2002) and the plant satellite DNA database
(http://w3lamc.umbr.cas.cz/PlantSat/, downloaded on 4/14/2002) were compared
to
the inventors' consensus sequences using the blastn program and an Expect
value
threshold score of -3. Consensus sequences were assembled using all inventors'
and
GenBank sequences that matched with an Expect (E) value of less than -45.
The revised consensus sequence of ChrBol is shown in FIG. 1D (SEQ ID
N0:3). This consensus was assembled from thirty-three DNA sequences collected
by
the inventors and eighteen GenBank sequences (Table 10). Thirty of these
sequences
completely spanned the repeat, and twenty-one others partially covered the
repeat.
The length of this repeat is 180 ~ 0.81 base pairs, and A and T comprise of
59% of
the consensus.
Table 1. GenBank sequences (accession numbers) that match inventors'
ChrBol consensus
M30962 M30963 M31436 M31435
M31438 M31434 M31439 M31437
X68786 X12736 X07519 X16589
X15291 X68783 X68784 X61583
AJ228348 222947
The revised consensus sequence of ChrBo2 is shown in FIG. 1 E (SEQ ID
N0:4). This consensus was assembled from seven DNA sequences collected by the
inventors and five GenBank sequences (Table 2). Seven of these sequences
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completely spanned the repeat, and five others partially covered the repeat.
The length
of this repeat is 180 t 0.44 base pairs, and A and T comprise of 63% of the
consensus.
Table 2. GenBank sequences (accession numbers) that match inventors'
ChrBo2 consensus
AJ228347 M30962X12736
X61583 ~ X68785
The two revised consensus sequences (ChrBol and ChrBo2) were aligned to
each other using the ClustalX program. The two consensus sequences differ
significantly (chi-squared, P < 0.05) from each other at several bases
(highlighted as
shown in FIG. 1 F).
A total of 20 GenBank entries match the Brassica oleracea centromere
sequences defined by the inventors. These are annotated as follows:
Xle7-2EB gene
1 S Xle4-7B gene
Xle6-14H gene
Satellite tandem repeat monomer
HindIII satellite repeat
Satellite DNA inverted direct repeat
Tandem repeated DNA
Highly repetitive DNA
They are not annotated as centromere repeats in GenBank. A completed list of
these sequences are shown in Table 3.
Table 3. GenBank entries match the Brassica oleracea centromere sequences
defined by the inventors
GenBank Annotation Repeat PositionNo. % Identity
of
Accession base
No.
airs
X68786 B. juncea Xle7-Complete472-651 180 97
2EB ene
X68786 B. juncea Xle7-Complete763-942 180 94
2EB ene
X68786 B. juncea Xle7-Partial 648-761 115 96
2EB ene
X12736 B. campestries Complete181-2 180 97
DNA for satellite
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GenBank Annotation Repeat PositionNo. % Identity
of
Accession base
No.
airs
tandem repeat
monomer
(consensus
se uence
X07519 Wild cabbage Complete179-1 179 97
satellite DNA
X61583 B. napus CanrepCompete 2-173 176 98
highly repetitive
DNA
X68783 B. juncea repetitivePartial 2-173 172 97
DNA sequence
came subfamil
A
X68784 B. juncea Xle4-7BComplete983- 180 95
ene 1162
X68784 B. juncea Xle4-7BPartial 815-986 172 94
gene
AJ228348 B. carinata Partial 2-173 172 96
DNA,
HindIII satellite
repeat (clone
Bcar3
M31438 B. oleracea Partial 176-1 176 94
satellite
DNA inverted
direct re eat
X16589 . B. nigra tandemPartial 177-1 177 94
repeat DNA (clone
BN1G 9, BN1G
23, BG1G 14
M31434 B. oleracea Partial 176-8 169 95
satellite
DNA inverted
direct re eat
M31437 B. oleracea Partial 175-1 175 94
satellite
DNA inverted
direct re eat
M30963 B. juncea tandemlyComplete181-2 180 93
re Bated DNA
M31435 B. oleracea Partial 174-8 169 94
satellite
DNA inverted
direct re eat
M31436 B. oleracea Partial 175-1 177 94
satellite
DNA inverted
direct re eat
X15291 B. juncea satellitePartial 1-161 161 95
DNA
M31439 B. oleracea Partial 176-1 177 90
satellite
DNA inverted
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GenBank Annotation Repeat PositionNo. % Identity
of
Accession base
No.
airs
direct re eat
222947 B. campestris Partial 181-347 170 90
satellite DNA
222947 B. campestris Partial 2-179 178 89
satellite DNA
M30962 B. campestris Complete181-2 180 87
tandemly repeated
DNA
X68785 B. juncea Xle6-Complete580-758 180 92
14H gene
X68785 B. juncea Xle6-Partial 404-568 165 90
14H gene
AJ228347 B. carinata Partial 177-2 176 90
DNA,
HindIII satellite
repeat (clone
BcarS
EXAMPLE 3
Glycine max centromere repeat sequences
We purified repetitive sequences from soybean (Glycine max, variety Williams
S 82), and set forth herein two centromere repeats, termed ChrGml and ChrGm2.
We
determined the consensus of each repeat as shown in Example 6.
The consensus sequence for ChrGml is shown in FIG. 2A (SEQ ID NO:S).
This consensus was assembled from DNA sequences collected by the inventors.
Seven of these sequences completely spanned the repeat, and twenty-five others
partially covered the repeat. It is 92 ~ 0.79 base pairs in length, and A and
T
comprise of 63% of the consensus.
The consensus sequence for ChrGm2 is shown in FIG. 2B (SEQ ID N0:6).
This consensus was assembled from DNA sequences collected by the inventors.
Ten
of these sequences completely spanned the repeat, and eleven others partially
covered
the repeat. It is 91 t 0.48 base pairs in length, and A and T comprise of 62%
of the
consensus.
The two repeats (ChrGml and ChrGm2) were aligned to each other using the
ClustalX program Those sites which differ significantly from each other (chi-
squared,
P < 0.05) are highlighted in FIG. 2C.
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The GenBank nt database (fttp://ftp.ncbi.nlm.nih.gov/blast/db/, March 29
version, downloaded on 04/07/2002) and the plant satellite DNA database
(http://w3lamc.umbr.cas.cz/PlantSat/ downloaded on 4/14/2002) were compared to
the inventors' consensus sequences using the blastn program and an Expect
value
S threshold of -3. Consensus sequences were built using all inventors' and
GenBank
sequences that matched with an Expect (E) value of less than -25.
The revised consensus sequence for ChrGml is shown in FIG. 2D (SEQ ID
N0:7). This consensus was assembled from thirty-two DNA sequences collected by
the inventors and one matching sequence from GenBank (accession number
Z26334).
Eight of these sequences completely spanned the repeat, and twenty-five others
partially covered the repeat. It is 92 t 0.74 base pairs in length, and A and
T
comprise of 56% of the consensus.
The revised consensus sequence for ChrGm2 is shown in FIG. 2E (SEQ ID
N0:8). This consensus was assembled from twenty-one DNA sequences collected by
the inventors and three matching sequences from GenBank (accession numbers
AF297983, AF297984, AF297985). Ten of these sequences completely spanned the
repeat, and fourteen others partially covered the repeat. It is 91 ~ 0.53 base
pairs in
length, and A and T comprise of 61 % of the consensus.
The two repeats (ChrGml and ChrGm2) were aligned to each other using the
ClustalX program Those sites with significant differences (chi-squared, P <
0.05) are
highlighted in FIG. 2F.
A total of 4 GenBank entries match the Glycine max centromere sequences
defined by the inventors. These are annotated as follows:
Satellite DNA
Tospovirus resistance protein C (Sw5-c), tospovirus resistance protein D (Sw5-
d), and tospovirus resistance protein E (Sw5-e) genes
They are not annotated as centromere repeats in GenBank. A complete list of
these sequences is shown in Table 4:
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Table 4: GenBank entries match the Glycine max centromere sequences defined
by the inventors
GenBank Annotation Repeat PositionNo. % Identity
of
Accession base
No.
airs
226334 G. max satelliteComplete92-1 92 95
DNA
AF297985 G. max clone Partial 259-173 87 93
TRS3 tandem
repetitive repeat
region
AF297985 G. max clone Partial 78-3 76 94
TRS3 tandem
repetitive repeat
region
AF297985 G. max clone Partial 168-83 86 90
TRS3 tandem
repetitive repeat
region
AF297984 G. max clone Partial 170-84 87 91
TRS2 tandem
repetitive repeat
re ion
AF297984 G. max clone Partial 260-175 86 88
TRS2 tandem
repetitive repeat
region
AF297984 G. max clone Partial 79-3 77 89
TRS2 tandem
repetitive repeat
re ion
AF297983 G. max clone Partial 77-3 75 94
TRS 1 tandem
repetitive repeat
re ion
EXAMPLE 4
Lycopersicon esculentum centromere repeat sequences
We purified repetitive sequences from tomato (Lycopersicon esculentum,
variety Microtom) and set forth herein one centromere repeat. We determined
the
consensus of this repeat as shown in Example 6.
The consensus sequence of ChrLel is shown in FIG. 3A (SEQ >D N0:9). This
consensus was assembled from forty-two DNA sequences collected by the
inventors.
Eighteen of these sequences completely spanned the repeat, and twenty-four
others
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partially covered the repeat. The repeat is 181 ~ 0.61 base pairs in length,
and A and
T comprise of 50% of the consensus.
The GenBank nt database (fttp://ftp.ncbi.nlm.nih.gov/blast/db/, March 29
version, downloaded on 04/07/2002) and the plant satellite DNA database
(http://w3lamc.umbr.cas.cz/PlantSat/, downloaded on 4/14/2002) were compared
to
the inventors' consensus sequences using the blastn program and an Expect
value
threshold value of -3. Consensus sequences were built using all inventors' and
GenBank sequences matched with an Expect (E) value of less than -40.
We determined the consensus of this repeat. The repeat is 181 ~ 0.61 base
pairs in length, and A and T comprise of 50% of the consensus.
The revised consensus sequence of ChrLel is shown in FIG. 3B (SEQ >D
NO:10). This consensus was assembled from forty-two sequences collected by the
inventors and two GenBank sequence (nt database at
fttp://ftp.ncbi.nlm.nih.gov/blast/db/. March 29 version, downloaded on
04/07/2002).
Eighteen of these sequences completely spanned the repeat, and twenty-six
others
partially covered the repeat. The GenBank sequences are accession numbers
X87233
and AY007367.
Neither of the 2 GenBank entries that match the Lycopersicon esculentum
centromere sequences defined by the inventors are complete repeats; they match
only
a portion of the sequence identified by the company. These are annotated as
follows:
Satellite DNA
Tandem repetitive repeat region
They are not annotated as centromere repeats in GenBank. A complete list of
these sequences is shown in Table 5.
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Table 5: GenBank entries match the LycopersicoH esculentum centromere
sequences defined by the inventors
GenBank Annotation Repeat PositionNo. % Identity
of
Accession base
No.
airs
X87233 L. esculentum Partial 163-1 161 93
satellite DNA
AY007367 L. esculentum Partial 12003- 154 93
tospovirus 12156
resistance protein
C (Sw5-c),
tospovirus
resistance protein
D (Sw5-d), and
tospovirus
resistance protein
E
Sw5-a enes
AY007367 L. esculentum Partial 12184- 161 90
tospovirus 12344
resistance protein
C (Sw5-c),
tospovirus
resistance protein
D (Sw5-d), and
tospovirus
resistance protein
E
Sw5-a genes
AY007367 L. esculentum Partial 12546- 155 90
tospovirus 12700
resistance protein
C (Sw5-c),
tospovirus
resistance protein
D (Sw5-d), and
tospovirus
resistance protein
E
Sw5-a enes
AY007367 L. esculentum Partial 12365- 157 89
tospovirus 12526
resistance protein
C (Sw5-c),
tospovirus
resistance protein
D (Sw5-d), and
tospovirus
resistance protein
E
Sw5-a enes
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EXAMPLE 5
Zea mays centromere repeat sequences
We purified repetitive sequences from corn (Zea mays, variety B73), and set
forth herein one centromere repeat, termed ChrZml. We determined the consensus
of
the repeat as shown in Example 5. The repeat is 180 ~ 1.15 base pairs in
length, and A
and T comprise of 56% of the consensus.
The consensus sequence of ChrZml is shown in FIG. 4A (SEQ ID NO:11).
This consensus was assembled from thirty-eight DNA sequences collected by the
inventors. Three of these sequences completely spanned the repeat, and thirty-
five
others partially covered the repeat.
The GenBank nt database (fttp://ftp.ncbi.nlm.nih.gov/blast/db/, March 29
version, downloaded on 04/07/2002) and the plant satellite DNA database
(http://w3lamc.umbr.cas.cz/PlanSat/, downloaded on 4/14/2002) were compared to
the
inventors' consensus sequences using the blastn program and an Expect value
threshold score of -3. Consensus sequences were built using all inventors' and
GenBank sequences matched with an Expect (E) value of -50.
The revised consensus sequence of ChrZml is shown in FIG. 4B (SEQ ID
N0:12). This consensus was assembled from thirty-eight DNA sequences collected
by
the inventors and twenty-six matching GenBank sequences (Table 6). Twenty of
these
sequences completely spanned the repeat, and forty-four others partially
covered the
repeat. The length of the repeat is 180 t 0.51 base pairs, and A and T
comprise the
consensus.
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Table 6. GenBank sequences that match the inventors' ChrZml consensus
M32521 M32522 M32523 M32524 M32525 M32526
M32527 M32528 M32529 M32530 M32531 M32532
M32533 M32534 M325375 M32536 M32537 M32538
M35408 AF030934 AF030935 AF030936 AF030937 AF030938
AF030939 AF030940
A total of 26 GenBank entries match the Zea mays centromere sequences
defined by the inventors. These are annotated as follows:
180-by knob-specific repeat region
heterochromatin repetitive DNA
They are not annotated as centromere repeats in GenBank. A complete list of
these sequences is shown in Table 7.
Table 7: GenBank entries match the Lycopersicon esculentum centromere
sequences
defined by the inventors
GenBank Annotation Repeat Position No. of
Accession base Identity
No.
airs
M32522 Maize 180-by Complete1-180 180 96
knob-specific
re eat re ion
M32521 Maize 180-by Complete1-180 180 96
knob-specific
re eat re ion
M32533 Z. mays subsp. Complete1-180 180 96
mexicana 180-by
knob-specific
re eat re ion
M32525 Maize 180-by Complete1-180 180 96
knob-specific
re eat re ion
M32524 Maize 180-by Complete1-180 180 96
knob-specific
re eat re ion
M32523 Maize 180-by Complete1-180 180 96
knob-specific
re eat region
M35408 Corn Complete1-180 180 96
heterochromatin
re etitive DNA
M32526 Maize 180-by Complete1-180 180 95
knob-specific
re eat re ion
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GenBank Annotation Repeat Position No. of
Accession base Identity
No.
airs
AF030939 Z. mays 180-by Complete1-180 180 95
knob-associated
tandem repeat
15-
T3-2
M32528 Maize 180-by Complete1-180 180 95
knob-specific
re eat re ion
M32534 Z. mays subsp. Complete1-180 180 94
mexicana 180-by
knob-specific
re eat region
M32527 Maize 180-by Partial 8-179 172 95
knob-specific
re eat re ion
M32538 T. dactyloides Complete1-179 179 94
(Tripsacum
dactyloides,
gams
grass) 180-by
knob-specific
re eat re ion
M32529 Maize 180-by Complete1-180 180 93
knob-specific
re eat re ion
AF030938 Z. mays 180-by Partial 4-180 177 93
knob-associated
tandem repeat
15-
T3-1
M32532 Maize 180-by Complete1-180 180 93
knob-specific
re eat re ion
AF030937 Z. mays 180-by Complete1-180 180 92
knob-associated
tandem repeat
1-
T7-2
AF030940 Z. mays 180-by Complete1-180 180 92
knob-associated
tandem repeat
15-
T7-1
AF030936 Z. mays 180-by Partial 10-180 172 93
knob-associated
tandem repeat
1-
T7-1
M32537 T. dactyloides Complete1-180 180 92
180-
by knob-specific
re eat re ion
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GenBank Annotation Repeat Position No.
of
Accession base Identity
No.
airs
M32530 Maize 180-by Complete 1-180 180 92
knob-specific
re eat re ion
M32531 Maize 180-by Complete 1-179, 180 91
knob-specific introduced
re eat re ion one a
AF030935 Z. mays 180-byPartial 1-175 175 90
knob-associated
tandem repeat
1-
T3-2
AF030934 Z. mays 180-byPartial 47-201 155 92
knob-associated
tandem repeat
1-
T3-1
M32536 T. dactyloidesComplete 1-180 180 94
180-
by knob-specific
re eat re ion
M32535 T. dactyloidesComplete 1-177, 177 91
180- 2%
by knob-specific gaps
re eat region
Six GenBank sequences of Zea mays centrometric repeat CentC were collected
(Table 13) and assigned the identifier ChrZm2. The consensus of the repeat was
determined as shown in Example 6. The repeat is 158 X1.6 base pairs in length.
A and
T comprises of 53% of the bases. All 6 sequences are of unit length.
The consensus sequence of ChrZm2 (SEQ ID N0:13) is shown in FIG. 4C.
Table 8. GenBank sequences of Zea mays centrometric repeat ChrZm2
AF078918 AF078919 AF078920
AF0789121AF078922 AF078923
EXAMPLE 6
Determining consensus sequences
Sequences were first aligned and edited in Vector NTI suite? (InforMax, 7600
Wisconsin Ave., Suite 1100, Bethesda, MD 20814) and exported as a fasta file.
A perl
program, consensus.pl, was written and used to determine the consensus for
each
position within the repeats based on the following rules:
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The most common base is designated as the consensus if it occurs three times
more frequently than the second most common base.
If the occurrence of the most common base is not three times more frequent
than the second most common base, but the combined frequency of the two most
common bases is three times that of the third most common base, and the
frequency of
the second most common base is greater than the frequency of the third most
common
base, then the second and first bases are together considered as a consensus
polymorphism, and designated using the IUPAC codes (M=A or C, R= A or G, W= A
or T, S=C or G, Y= C or T, k= G or T, V= A or C or G, H=A or C or T, D=A or G
or
T,B=CorGorT,N=GorTorCorA).
If the combined frequency of the two most common bases is not three times
greater than that of the third most common base, but the combined frequency of
the
three most common bases is three times that of the fourth most common base,
and the
third most common base is more common than the fourth most common base, and
the
frequency of occurrence of the fourth base is less than or equal to 22%, the
consensus
is assigned according to the ICTPAC ambiguity codes for the three most common
bases.
If the four bases occur approximately equally (23-27%), the consensus is
assigned as
N.
EXAMPLE 7
Constructing BAC Vectors for Testing Centromere Function
A BAC clone may be retrofitted with one or more plant telomeres and
selectable markers together with the DNA elements necessary for Agrobacterium
transformation (FIG. 9). This method will provide a means to deliver any BAC
clone
into plant cells and to test it for centromere function.
The method ,works in the following way. The conversion vector contains a
retrofitting cassette. The retrofitting cassette is flanked by TnlO, TnS, Tn7,
Mu or
other transposable elements and contains an origin of replication and a
selectable
marker for Agrobacterium, a plant telomere array followed by T-DNA right and
left
borders followed by a second plant telomere array and a plant selectable
marker (FIG.
9). The conversion vector is transformed into an E. coli strain carrying the
target
BAC. The transposable elements flanking the retrofitting cassette then mediate
transposition of the cassette randomly into the BAC clone. The retrofitted BAC
clone
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can now be transformed into an appropriate strain of Agrobacterium and then
into
plant cells where it can be tested for high fidelity meiotic and mitotic
transmission
which would indicate that the clone contained a complete functional plant
centromere.
EXAMPLE 8
S Sequence Analysis of Arabidopsis Centromeres
A. Abundance of genes in the centromeric regions
Expressed genes are located within 1 kb of essential centromere sequences in
S.
cerevisiae, and multiple copies of tRNA genes reside within an 80 kb fragment
necessary for centromere function in S. pombe (Kuhn et al., 1991 ). In
contrast, genes
are thought to be relatively rare in the centromeres of higher eukaryotes,
though there
are notable exceptions. The Drosophila light, concertina, responder, and
rolled loci
all map to the centromeric region of chromosome 2, and translocations that
remove
light from its native heterochromatic context inhibit gene expression. In
contrast,
many Drosophila and human genes that normally reside in euchromatin become
inactive when they are inserted near a centromere. Thus, genes that reside
near
centromeres likely have special control elements that allow expression
(Karpen, 1994;
Lohe and Hilliker, 1995). The sequences of Arabidopsis CEN2 and CEN4, provided
herein, provide a powerful resource for understanding how gene density and
expression correlate with centromere position and associated chromatin.
Annotation of chromosome II and IV (http://
www.ncbi.nlm.nih.gov/Entrez/nucleotide.html) identified many genes within and
adj acent to CEN2 and CEN4 (FIG. 8, FIGs. 11 A-11 T). The density of predicted
genes
on Arabidopsis chromosome arms averages 25 per 100 kb, and in the repeat-rich
regions flanking CEN2 and CEN4 this decreases to 9 and 7 genes per 100 kb,
respectively (Bevan et al., 1999). Many predicted genes also reside within the
recombination-deficient, genetically-defined centromeres. Within CEN2, there
were 5
predicted genes per 100 kb; while CEN4 was strikingly different, with 12 genes
per
100 kb.
There was strong evidence that several of the predicted centromeric genes are
transcribed. The phosphoenolpyruvate gene (CUEI ) defines one CENS border;
mutations in this gene cause defects in light-regulated gene expression
(Li et al., 1995). Within the sequenced portions of CEN2 and CEN4, 17%
(27/160) of
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the predicted genes shared >95% identity with cloned cDNAs (ESTs), with three-
fold
more matches in CEN4 than in CEN2 (http://www.tigr.org/tdb/at/agad~. Twenty-
four
of these genes have multiple exons, and four correspond to single-copy genes
with
known functions. A list of the predicted genes identified is given in Table 9,
below. A
list of additional genes encoded within the boundaries of CEN4 are listed in
Table 10.
The identification of these genes is significant in that the genes may
themselves
contain unique regulatory elements or may reside in genomic locations flanking
unique
control or regulatory elements involved in centromere function or gene
expression. In
particular, the current inventors contemplate use of these genes, or DNA
sequences 0
to 5 kb upstream or downstream of these sequences, for insertion into a gene
of choice
in a mini chromosome. It is expected that such elements could potentially
yield
beneficial regulatory controls of the expression of these genes, even when in
the
unique environment of a centromere.
To investigate whether the remaining 23 genes were uniquely encoded at the
centromere, a search was made in the database of annotated genomic Arabidopsis
sequences. With the exception of two genes, no homologs with >95% identity
were
found elsewhere in the 80% of the genome that has been sequenced. The number
of
independent cDNA clones that correspond to a single-copy gene provides an
estimate
of the level of gene expression. On chromosome II, predicted genes with high
quality
matches to the cDNA database (> 95% identity) match an average of four
independent
cDNA clones (range 1-78). Within CEN2 and CEN4, 11/27 genes exceed this
average
(Table 9). Finally, genes encoded at CEN2 and CEN4 are not members of a single
gene family, nor do they correspond to genes predicted to play a role in
centromere
functions, but instead have diverse roles.
Many genes in the Arabidopsis centromeric regions are nonfunctional due to
early stop codons or disrupted open reading frames, but few pseudogenes were
found
on the chromosome arms. Though a large fraction of these pseudogenes have
homology to mobile elements, many correspond to genes that are typically not
mobile
(FIGS. 1 l I-J and FIGS. 11 S-T). Within the genetically-defined centromeres
there were
1.0 (CEN2) and 0.7 (CEN4) of these nonmobile pseudogenes per 100 kb; the
repeat-rich regions bordering the centromeres have 1.5 and 0.9 per 100 kb
respectively. The distributions of pseudogenes and transposable elements are
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overlapping, indicting that DNA insertions in these regions contributed to
gene
disruptions.
Table 9: Predicted genes within CEN2 and CEN4 that correspond to the cDNA
database.
Putative function GenBank protein # of EST
accession matches*
CEN2
Unknown AAC69124 1
SH3 domain protein AAD15528 5
Unknown AAD 15529 1
unknownt AAD37022 1
RNA helicase$ AAC26676 2
40S ribosomal protein S 16 AAD22696 9
CEN4
Unknown AAD36948 1
Unknown AAD36947 4
leucyl tRNA synthetase AAD36946 4
aspartic protease AAD29758 6
Peroxisomal membrane protein (PPM2)AAD29759 S
5'-adenylylsulfate reductase AAD29775 14
symbiosis-related protein AAD29776 3
ATP synthase gamma chain 1 (APCl)AAD48955 3
protein kinase and EF hand AAD03453 3
ABC transporter AAD03441 1
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Putative function GenBank protein # of EST
accession matches*
Transcriptional regulator AAD03444 14
Unknown AAD03446 12
human PCF1 1p homolog AAD03447 6
NSF protein AAD 17345 2
1,3-beta-glucan synthase AAD48971 2
pyridine nucleotide-disulphide AAD48975 4
oxidoreductase
Polyubiquitin (UBQIl) AAD48980 72
wound induced protein AAD48981 6
short chain dehydrogenase/reductaseAAD48959 7
SL1 St AAD48939 2
WD40-repeat protein AAD48948 2
* Independent cDNAs with >95% identity, t related gene present in non-
centromeric
DNA, $ potentially associated with a mobile DNA element, ~ characterized gene
(B.
Tugal, 1999; J.F. Gutierrez-Marcos, 1996; N. Inohara, 1991; J. Callis, 1995).
Table 10: List of additional genes encoded within the boundaries of CEN4.
Putative Function GenBank Nucleotide
accession Position
3'(2'),S'-Bisphosphate NucleotidaseAC012392 71298 -73681
Transcriptional regulator AC012392 80611 -81844
Equilibrative nucleoside transporterAC012392 88570 -90739
1
Equilibrative nucleoside transporterAC012392 94940 -96878
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Putative Function GenBank Nucleotide
accession Position
Equilibrative nucleoside transporterAC012392 98929 -101019
1
Equilibrative nucleoside transporterAC012392 113069 -115262
1
unknown AC012392 122486 -124729
4-coumarate--CoA ligase AC012392 126505 -128601
ethylene responsive protein AC012392 130044 -131421
Oxygen-evolving enhancer protein AC012392 134147 -135224
precursor
Kinesin AC012392 137630 -141536
receptor-like protein kinase AC012392 141847 -144363
LpxD-like protein AC012392 144921 -146953
hypersensitivity induced protein AC012392 147158 -147838
ubiquitin AC012392 149057 -149677
unknown AC012392 150254 -1
S 1072
ubiquitin-like protein AC012392 153514 -154470
ubiquitin-like protein AC012392 155734 -156513
ubiquitin-like protein AC012392 156993 -157382
unknown AC012392 159635 -165559
unknown AC012392 166279 -166920
unknown AC012392 167724 -170212
ubiquitin-like protein AC012392 176819 -178066
polyubiquitin (UBQIO) AC012392 180613 -182007
phosphatidylinositol-3,4,5-triphosphateAC012477 89384 -91291
binding
protein
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Putative Function GenBank Nucleotide
accession Position
Mitochondrial ATPase AC012477 94302 -94677
RING-H2 finger protein AC012477 95522 -96142
unknown AC012477 104747 -105196
Mitochondrial ATPase AC012477 105758 -106595
ferredoxin--NADP+ reductase AC012477 107451 -109095
unknown AC012477 109868 -110620
U3 snoRNP-associated protein AC012477 111841 -114133
UV-damaged DNA binding factor AC012477 114900 -121275
Glucan endo-1,3-Beta-Glucosidase AC012477 122194 -122895
precursor
D123 -like protein AC012477 125886 -126887
Adrenodoxin Precursor AC012477 127660 -129246
N7 like-protein AC012477 129718 -131012
N7 like-protein AC012477 131868 -133963
N7 like-protein AC012477 134215 -136569
N7 like-protein AC012477 139656 -140864
characterized gene (J. Callis,
1995).
B. Conservation of centromeric DNA
To investigate the conservation of CEN2 and CEN4 sequences, PCR primer
S pairs were designed that correspond to unique regions in the Columbia
sequence and
used to survey the centromeric regions of Landsberg and Columbia at ~20 kb
intervals
(FIGs. 13A, B). The primers used for the analysis are listed in FIGS. 14A, B.
Amplification products of the appropriate length were obtained in both
ecotypes for
most primer pairs (85%), indicating that the amplified regions were highly
similar. In
the remaining cases, primer pairs amplified Columbia, but not Landsberg DNA,
even
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at very low stringencies. In these regions, additional primers were designed
to
determine the extent of nonhomology. In addition to a large insertion of
mitochondria)
DNA in CEN2, two other non-conserved regions were identified (FIGS. 13A, B).
Because this DNA is absent from Landsberg centromeres, it is unlikely to be
required
for centromere function; consequently, the relevant portion of the centromeric
sequence is reduced to 577 kb (CEN2) and 1250 kb (CEN4). The high degree of
sequence conservation between Landsberg and Columbia centromeres indicated
that
the inhibition of recombination frequencies was not due to large regions of
nonhomology, but instead was a property of the centromeres themselves.
C. Sequence similarity between CEN2 and CEN4
In order to discern centromere function, a search was conducted for novel
sequence motifs shared between CEN2 and CEN4, excluding from the comparison
retroelements, transposons, characterized centromeric repeats, and coding
sequences
resembling mobile genes. After masking simple repetitive sequences, including
1 S homopolymer tracts and microsatellites, contigs of unique sequence
measuring 417 kb
and 851 kb for CEN2 and CEN4, respectively, were compared with BLAST
(http://blast.wustl.edu).
The comparison showed that the complex DNA within the centromere regions
was not homologous over the entire sequence length. However, 16 DNA segments
in
CEN2 matched 11 regions in CEN4 with >60% identity (FIG. 1 S). The sequences
were grouped into families of related sequences, and were designated AtCCSl-7
(Arabidopsis thaliana centromere conserved sequences 1-7). These sequences
were
not previously known to be repeated in the Arabidopsis genome. The sequences
comprised a total of 17 kb (4%) of CEN2 DNA, had an average length of 1017 bp,
and
had an A + T content of 65%. Based on similarity, the matching sequences were
sorted into groups, including two families containing 8 sequences each, 3
sequences
from a small family encoding a putative open reading frame, and 4 sequences
found
once within the centromeres, one of which corresponds to predicted CEN2 and
CEN4
proteins with similarity throughout their exons and introns (FIG. 15).
Searches of the Arabidopsis genomic sequence database demonstrated that
AtCCS 1 - AtCCSS were moderately repeated sequences that appear in centromeric
and
pericentromeric regions. The remaining sequences were present only in the
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genetically-defined centromeres. Similar comparisons of all 16 S. cerevisiae
centromeres defined a consensus consisting of a conserved 8 by CDEI motif, an
AT-rich 85 by CDEII element, and a 26 by CDEII region with 7 highly conserved
nucleotides (Fleig et al., 1995). In contrast, surveys of the three S. pombe
centromeres
revealed conservation of overall centromere structure, but no universally
conserved
motifs (Clark, 1998).
EXAMPLE 9
Construction of Plant Mini chromosomes
Mini chromosomes are constructed by combining the previously isolated
essential chromosomal elements. Exemplary mini chromosome vectors include
those
designed to be "shuttle vectors"; i.e., they can be maintained in a convenient
host (such
as E. coli, Agrobacterium or yeast) as well as plant cells.
A. General Techniques for Mini chromosome Construction
A mini chromosome can be maintained in E. coli or other bacterial cells as a
circular molecule by placing a removable stuffer fragment between the
telomeric
sequence blocks. The stuffer fragment is a dispensable DNA sequence, bordered
by
unique restriction sites, which can be removed by restriction digestion of the
circular
DNAs to create linear molecules with telomeric ends. The linear mini
chromosome
can then be isolated by, for example, gel electrophoresis. In addition to the
stuffer
fragment and the plant telomeres, the mini chromosome contains a replication
origin
and selectable marker that can function in plants to allow the circular
molecules to be
maintained in bacterial cells. The mini chromosomes also include a plant
selectable
marker, a plant centromere, and a plant ARS to allow replication and
maintenance of
the DNA molecules in plant cells. Finally, the mini chromosome includes
several
unique restriction sites where additional DNA sequence ipserts can be cloned.
The
most expeditious method of physically constructing such a mini chromosome,
i.e.,
ligating the various essential elements together for example, will be apparent
to those
of ordinary skill in this art.
A number of mini chromosome vectors have been designed by the current
inventors and are disclosed herein for the purpose of illustration (FIGs. 7A-
7H). These
vectors are not limiting however, as it will be apparent to those of skill in
the art that
many changes and alterations may be made and still obtain a functional vector.
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B. Modified Technique for Mini chromosome Construction
A two step method was developed for construction of mini chromosomes,
which allows adding essential elements to BAC clones containing centromeric
DNA.
These procedures can take place in vivo, eliminating problems of chromosome
S breakage that often happen in the test tube. The details and advantages of
the
techniques are as follows:
1.) One plasmid can be created that contains markers, origins and border
sequences for Agrobacterium transfer, markers for selection and screening in
plants, plant telomeres, and a loxP site or other site useful for site-
specific
recombination in vivo or in vitro. The second plasmid can be an existing BAC
clone, isolated from the available genomic libraries (FIG. 10A).
2.) The two plasmids are mixed, either within a single E. coli cell, or in a
test tube, and the site-specific recombinase cre is introduced. This will
cause
the two plasmids to fuse at the loxP sites (FIG. l OB).
3.) If deemed necessary, useful restriction sites (AseI/PacI or Not I) are
included to remove excess material. (for example other selectable markers or
replication origins)
4.) Variations include vectors with or without a KanR gene (FIGS. 10B,
10C), with or without a LAT52 GUS gene, with a LAT52 GFP gene, and with
a GUS gene under the control of other plant promoters. (FIGs. IOC, lOD and
10E).
C. Method for Preparation of Stable Non-Integrated Mini
chromosomes
A technique has been developed to ensure that mini chromosomes do not
integrate into the host genome (FIG. 10F). In particular, mini chromosomes
must be
maintained as distinct elements separate from the host chromosomes. In one
method
for ensuring that the introduced mini chromosome does not integrate, the
inventors
envision a variety that would encode a lethal plant gene (such as diptheria
toxin or any
other gene product that, when expressed, causes lethality in plants). This
gene could
be located between the right Agrobacterium border and the telomere. Mini
chromosomes that enter a plant nucleus and integrate into a host chromosome
would
result in lethality. However, if the mini chromosome remains separate, and
further, if
CA 02530178 2005-12-21
WO 2005/010187 PCT/US2003/020380
the ends of this construct are degraded up to the telomeres, then the lethal
gene would
be removed and the cells would survive.
It should be understood that various changes and modifications to the
presently
preferred embodiments described herein will be apparent to those skilled in
the art.
Such changes and modifications can be made without departing from the spirit
and
scope of the present invention and without diminishing its intended
advantages. It is
therefore intended that such changes and modifications be covered by the
appended
claims.
96
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Pruess, Daphne
Mach, Jennifer
Zieler, Helge
Jin, RongGuan
Copenhaver, Gregory
Keith, Kevin
(ii) TITLE OF INVENTION: METHODS FOR GENERATING OR
INCREASING REVENUES FROM CROPS
(iii) NUMBER OF SEQUENCES: 13
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bell, Boyd & Lloyd
(B) STREET: 70 West Madison
(C) CITY: Chicago
(D) STATE: Illinois
(E) COUNTRY: USA
(F) ZIP: 60602
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(B) COMPUTER: IBM PC Compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn 3.1 Version 3.1.15
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US Unknown
(B) FILING DATE: Concurrently Herewith
(C) CLASSIFICATION: Unknown
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 09/553,231
(B) FILING DATE: 2002-03-13
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 09/090,051
(B) FILING DATE: 1998-06-03
(vii) PRIOR APPLICATION DATA:
A) APPLICATION NUMBER: 60/048,451
(B) FILING DATE: 1997-06-03
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/073,741
(B) FILING DATE: 1998-02-05
CA 02530178 2005-12-21
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(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 09/531,120
(B) FILING DATE: 2000-03-17
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/125,219
(B) FILING DATE: 1999-03-18
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/127,409
(B) FILING DATE: 1999-04-O1
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/134,770
(B) FILING DATE: 1999-05-18
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/153,584
(B) FILING DATE: 1999-09-13
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/154,603
(B) FILING DATE: 1999-09-17
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/172,493
(B) FILING DATE: 1999-12-16
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Barrett, Robert M.
(B) REGISTRATION NUMBER: 30,142
(C) REFERENCE/DOCKET NUMBER: 0112283-040
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 312-807-4204
(B) TELEFAX: 312-827-1270
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180
(B) TYPE: DNA
(C) ORGANISM: Brassica oleracea
(D) SEQUENCE DESCRIPTION:
agcttgattt ggatacataa agtggtggag aatcaccagg aagttgaata aatctcatag 60
gagttggcat gaagaagtta tcccactttc aaatcaggtg attccagttt cccagtttgg 120
gaatagcaca gcttcttcgt cgttccaatc aaaccaggat gaatcwcttt gtraraagct 180
CA 02530178 2005-12-21
WO 2005/010187 PCT/US2003/020380
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180
(B) TYPE: DNA
(C) ORGANISM: Brassica oleracea
(D) SEQUENCE DESCRIPTION:
agcttgattt tgatacataa agtagtggag aatcaytwgg aagtggaata aatctcatag 60
gagttaggat gaagaagcta tcmcactttc aaatcaggtg atcccarttt tcctgtttgg 120
gaatatgaca acttmtttgt cattctaatc aaaccaggaw gaatcgcbat gtaaraagct 180
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180
(B) TYPE: DNA
(C) ORGANISM: Brassica oleracea
(D) SEQUENCE DESCRIPTION:
agcttgattt ggatacataa agtggtggag aatcaccagg aagttgaata aatctcatag 60
gagttggsat gaagaagtta tcccactttc aaatcaggtg attccagttt cccagtttgg 120
gaatagcaca gcttcttcgt cgttccaatc aaaccaggat gaatcacttt gtragaagct 180
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180
(B) TYPE: DNA
(C) ORGANISM: Brassica oleracea
(D) SEQUENCE DESCRIPTION:
agcttgattt tgatacataa agtartggag aatcayyagg aagtkgaata aatctcatag 60
gagttaggat gaagaagcta.tcccactttc aaatcaggtg atcccarttt tcctgtttgg 120
gaatakgaca rcttctttgt cattctaatc aaaccaggaw gaatcgckat gtaaraagct 180
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
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(B) TYPE: DNA
(C) ORGANISM: Glycine max
(D) SEQUENCE DESCRIPTION:
aaattcaaat ggtcataact tttmacwcgg akgtccgatt caggcgcata atatatcgag 60
acgctcgaaa ttgaacaayg gaagctctcg ag 92
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 91
(B) TYPE: DNA
(C) ORGANISM: Glycine max
(D) SEQUENCE DESCRIPTION:
CA 02530178 2005-12-21
WO 2005/010187 PCT/US2003/020380
aaattcaaac gacaataact ttttactcgg atgtcygatt gagtcccgta atatatcgag 60
acgctcgaaa ttgaatrytg aagctctgag c 91
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92
(B) TYPE: DNA
(C) ORGANISM: Glycine max
(D) SEQUENCE DESCRIPTION:
aaattcaaat ggtcataact tttmacwcgg akgtccgatt caggcgcata atatatcgag 60
acgctcgaaa ttgaacaayg gaagctctcg ag 92
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 91
(B) TYPE: DNA
(C) ORGANISM: Glycine max
(D) SEQUENCE DESCRIPTION:
aaattcaaac gacaataact ttttactcgg atgtcygatt gagtcccgta atatatcgag 60
acgctcgaaa ttgaatrytg aagctctgag c 91
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 181
(B) TYPE: DNA
(C) ORGANISM: Lycopersicon esculentum
(D) SEQUENCE DESCRIPTION:
ccatcacggg ttttctgggc crtttggaag gtcaaacgag. ccccggagcg agcatacgcc 60
tcattttgac gattttcgtg tgctattgca caccattttt tgggtgatcg ggattccgac 120
gtcaaaaatg ccaaatttgt tcgtggacgt ccgtcaagac gttgtctatg catacggttg 180
g 181
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 181
(B) TYPE: DNA
(C) ORGANISM: Lycopersicon esculentum
(D) SEQUENCE DESCRIPTION:
ccatcacggg ttttctgggc crtttggaag gtcaaacgag ccccgragcg agcatacgcc 60
tcattttgac gattttcgtg tgctattgca caccattttt tgggtgatcg ggattccgac 120
gtcaaaaatg ccaaatttgt tcgtggacgt ccgtcaagac gttgtctatg catacggttg 180
g 181
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
CA 02530178 2005-12-21
WO 2005/010187 PCT/US2003/020380
(A) LENGTH: 180
(B) TYPE: DNA
(C) ORGANISM: Zea mays
(D) SEQUENCE DESCRIPTION:
ggccacacaa cccccatttt tgtcgaaaat agccatgaac gaccattttc aataatacyr 60
aaggctaaca cctacggatt tttraccaag aaatggtctc caccagaaat ccaagaatgt 120
gatctatggc aaggaaacat atgtggggtg aggtgtayga gcctctggtc gaygatcaat 180
(2) INFORMATION FOR SEQ ID ~T0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180
(B) TYPE: DNA
(C) ORGANISM: Zea mays
(D) SEQUENCE DESCRIPTION:
ggccacacaa cccccatttt tgtcgaaaat agccatgaay gaccattttc aataataccg 60
aaggctaaca cctacggatt tttgaccaag aaatggtctc caccagaaat ccaagaatgt 120
gatctatggc aaggaaacat atgtggggtg aggtgtayga gcctctggtc gatgatcaat 180
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 158
(B) TYPE: DNA
(C) ORGANISM: Zea mays
(D) SEQUENCE DESCRIPTION:
ggttccggtg gcaaaaactc gtagctttgt atgcacccmg acacccgttt tcggaatggg 60
tgacgtgyga caacagaaat tgcgmgaaac caccccaaac atgagttttg kacctaaagt 120
agtggattgg gcatgttcgt tgygaaaaac gaagaaat 158
