U.S. patent application number 14/660110 was filed with the patent office on 2016-01-14 for sugarcane centromere sequences and minichromosomes.
The applicant listed for this patent is CHROMATIN INC.. Invention is credited to Pierluigi Barone, Shawn R. Carlson, Gregory P. Copenhaver, Song Luo, Jennifer M. Mach, Daphne Preuss.
Application Number | 20160010098 14/660110 |
Document ID | / |
Family ID | 43499430 |
Filed Date | 2016-01-14 |
United States Patent
Application |
20160010098 |
Kind Code |
A1 |
Preuss; Daphne ; et
al. |
January 14, 2016 |
SUGARCANE CENTROMERE SEQUENCES AND MINICHROMOSOMES
Abstract
The invention is generally related to Sugarcane mini-chromosomes
and recombinant chromosomes containing Sugarcane centromere
sequences. In addition, the invention provides for methods of
generating Sugarcane plants transformed with these Sugarcane
mini-chromosomes. Sugarcane mini-chromosomes with novel
compositions and structures are used to transform Sugarcane cells
which are in turn used to generate Sugarcane plants. Methods for
generating Sugarcane plants include methods for delivering the
Sugarcane mini-chromosomes into Sugarcane cell to transform the
cell, methods for selecting the transformed cell, and methods for
isolating Sugarcane plants transformed with the Sugarcane
mini-chromosome or recombinant chromosome.
Inventors: |
Preuss; Daphne; (Chicago,
IL) ; Copenhaver; Gregory P.; (Chapel Hill, NC)
; Mach; Jennifer M.; (Chicago, IL) ; Barone;
Pierluigi; (Charleston, IL) ; Carlson; Shawn R.;
(Bondville, IL) ; Luo; Song; (Chicago,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHROMATIN INC. |
Chicago |
IL |
US |
|
|
Family ID: |
43499430 |
Appl. No.: |
14/660110 |
Filed: |
March 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13386366 |
Jan 20, 2012 |
8981183 |
|
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PCT/US10/43052 |
Jul 23, 2010 |
|
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14660110 |
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61228019 |
Jul 23, 2009 |
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Current U.S.
Class: |
800/298 ;
435/320.1; 435/419; 536/23.6; 536/24.2 |
Current CPC
Class: |
C12N 2800/20 20130101;
C12Q 2600/16 20130101; C12Q 2600/156 20130101; C12N 15/8257
20130101; C12N 15/82 20130101; C12Q 1/6895 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A polynucleotide comprising a nucleotide sequence selected from
the group consisting of: (a) the nucleotide sequence of SEQ ID NO:
204; and (b) a nucleotide sequence that is at least 98% identical
to the nucleotide sequence of SEQ ID NO: 204.
2. The polynucleotide of claim 1, wherein the polynucleotide
comprises the nucleotide sequence of SEQ ID NO:204.
3. A nucleic acid comprising an array comprising at least two
copies of the polynucleotide of claim 1.
4-5. (canceled)
6. A nucleic acid comprising an array comprising from 2 to 1000
copies of the polynucleotide of claim 1.
7. (canceled)
8. The nucleic acid of claim 1, wherein the array is from 1 to 200
kb in length.
9. (canceled)
10. A sugarcane centromere comprising the polynucleotide of claim
1.
11. (canceled)
12. A sugarcane artificial chromosome comprising the polynucleotide
of claim 1.
13. (canceled)
14. A sugarcane artificial chromosome comprising the sugarcane
centromere of claim 10.
15. The sugarcane artificial chromosome of claim 12, wherein the
sugarcane artificial chromosome comprises an exogenous nucleic
acid.
16. (canceled)
17. The sugarcane artificial chromosome of claim 15, wherein at
least one exogenous nucleic acid is operably linked to a
heterologous regulatory sequence functional in a sugarcane plant
cell.
18-19. (canceled)
20. A vector or cell comprising the polynucleotide of claim 1.
21-22. (canceled)
23. A vector or cell comprising the sugarcane artificial chromosome
of claim 12.
24-27. (canceled)
28. A cell comprising the vector of claim 20.
29-33. (canceled)
34. A sugarcane plant tissue or sugarcane plant comprising the
plant cell of claim 28.
35-36. (canceled)
37. A sugarcane seed obtained from the sugarcane plant of claim
28.
38. A sugarcane plant progeny comprising a sugarcane artificial
chromosome, wherein the plant progeny is the result of breeding a
plant of claim 34 comprising the sugarcane artificial
chromosome.
39. A method of using a sugarcane plant of claim 34, wherein the
sugarcane plant comprises a sugarcane artificial chromosome
comprising an exogenous nucleic acid encoding a recombinant
protein, the method comprising growing the plant to produce the
recombinant protein.
40. The method of claim 39, further comprising the step of
harvesting or processing the sugarcane plant.
Description
RELATED APPLICATION INFORMATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/228,019, filed Jul. 23, 2009, the disclosure of
which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention generally relates to Sugarcane
mini-chromosomes and recombinant chromosomes containing Sugarcane
centromere sequences as well as Sugarcane cells and plants
comprising the same.
BACKGROUND OF THE INVENTION
[0003] Two general approaches are used for introduction of new
heritable 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., "Molecular Cloning: a
Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring
Harbor, N. Y. 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
bp 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., Nature,
310:511, 1981).
[0004] 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 can be 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.,
Cell, 11:223, 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 integration site
in the host genome or so called "position effect variegation"
(Shingo et al., Mol. Cell. Biol., 6:1787, 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 Chepko et al., Cell, 37:1053, 1984).
[0005] One 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., Ann. Rev. Plant
Phys., 35:387-413, 1984). By substituting genes of interest for a
portion of 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., Mol Gen.
Genet., 207:478, 1987). Second, the expression of the introduced
DNA sequences varies between individual transformants (see Jones et
al., Embo J., 4:2411-2418, 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 the transgene. Finally, insertion of extra
elements into the genome can disrupt the genes, promoters or other
genetic elements necessary for normal plant growth and
function.
[0006] Another widely used technique to genetically transform
plants involves the use of microprojectile bombardment to integrate
DNA sequences into the genome. In this process, a nucleic acid
containing the desired genetic elements to be introduced into the
plant's native chromosome is deposited on or in small metallic
particles, e.g., tungsten, platinum, or preferably gold, which are
then delivered at a high velocity into the plant tissue or plant
cells. However, similar problems arise as with
Agrobacterium-mediated gene transfer, and as noted above expression
of the inserted DNA can be unpredictable and insertion of extra
elements into the genome can disrupt and adversely impact plant
processes.
[0007] One attractive alternative to the commonly used methods of
transformation is the use of an artificial chromosome. Artificial
chromosomes are episomal nucleic acid molecules that exist
autonomously from the native chromosomes of the host genome. They
can be linear or circular DNA molecules that are comprised of
cis-acting nucleic acid sequence elements that provide replication
and partitioning activities (see Murray et al., Nature,
305:189-193, 1983). Desired elements include: (1) origin of
replication, which are the sites for initiation of DNA replication,
(2) centromeres (site of kinetochore assembly and responsible for
proper distribution of replicated chromosomes into daughter cells
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).
An additional desired element is a chromatin organizing sequence.
It is well documented that centromere function is crucial for
stable chromosomal inheritance in almost all eukaryotic organisms
(reviewed in Nicklas 1988). 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.
[0008] Artificial chromosomes have been engineered using one of two
approaches. The first approach identifies and assembles the desired
chromosomal elements into an artificial construct. This approach
has been described as "bottom-up" and involves the use of a
heterologous system (i.e. bacteria or fungal) to perform the
various cloning steps necessary to assemble the artificial
chromosome. Artificial chromosomes of this type will be referred to
in this application as "mini-chromosomes". The second approach
derives the artificial chromosome from existing chromosomes through
chromosome fragmentation and, optionally, subsequent addition of
desired elements including transgenes. For example, an existing
chromosome can be induced to undergo breakage events that result in
chromosomal fragments. Minimal fragments that possess the elements
for replication and segregation during cell division (e.g.
centromere, origins of replication and/or telomeres) can be
identified. These derived artificial chromosomes can then be used
as targets for further manipulation including the addition of one
or more transgenes. This approach has been described as "top-down"
and does not require the use of a heterologous system (e.g.
bacterial or fungal) since it doesn't require in vitro-based
cloning steps. Artificial chromosomes of this type will be referred
to in this application as "recombinant chromosomes".
[0009] The essential chromosomal elements for construction of
artificial chromosomes have been precisely characterized in lower
eukaryotic species, and more recently in mouse and human.
Autonomous replication sequences (ARSs) have been isolated from
unicellular fungi, including Saccharomyces cerevisiae (brewer's
yeast) and Schizosaccharomyces pombe (see Stinchcomb et al., 1979
and Hsiao et al., 1979). An ARS behaves like an origin of
replication allowing DNA molecules that contain the ARS to be
replicated in concert with the rest of the genome after
introduction into the cell nuclei of these fungi. DNA molecules
containing these sequences replicate, but in the absence of a
centromere they are not partitioned into daughter cells in a
controlled fashion that ensures efficient chromosome
inheritance.
[0010] Artificial chromosomes have been constructed in yeast using
the three cloned essential chromosomal elements (see Murray et al.,
Nature, 305:189-193, 1983). None of the essential components
identified in unicellular organisms, however, function in higher
eukaryotic systems. For example, a yeast centromere sequence will
not confer stable inheritance upon vectors transformed into higher
eukaryotes.
[0011] In contrast to the detailed studies done in yeast, less is
known about the molecular structure of functional centromeric DNA
of higher eukaryotes. Ultrastructural studies indicate that higher
eukaryotic kinetochores, which are specialized complexes of
proteins that form on the centromere during late prophase, are
large structures (mammalian kinetochore plates are approximately
0.3 .mu.m 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.
[0012] While the above studies have been useful in elucidating the
structure and function of centromeres, it was not known whether
information derived from lower eukaryotic or mammalian higher
eukaryotic organisms would be applicable to Sugarcane. There exists
a need for cloned centromeres from Sugarcane, which would represent
a first step in the production of Sugarcane artificial chromosomes,
or in the identification of Sugarcane recombinant chromosomes.
There further exists a need for Sugarcane cells, plants, seeds and
progeny containing functional, stable, and autonomous artificial or
recombinant chromosomes capable of carrying a large number of
different genes and genetic elements.
SUMMARY OF THE INVENTION
[0013] In one aspect, the present invention addresses Sugarcane
mini-chromosomes comprising a Sugarcane centromere having one or
more repeated nucleotide sequences, described in further detail
herein. In some embodiments, such mini-chromosomes comprise a
centromere comprising one or more selected repeated nucleotide
sequences derived from Sugarcane, including those isolated from
Sugarcane genomic DNA and synthetic arrays of repeat sequences. In
other embodiments, the invention addresses Sugarcane recombinant
chromosomes.
[0014] In another aspect, the invention provides modified or
"adchromosomal" Sugarcane plants, containing functional, stable,
autonomous mini-chromosomes or recombinant chromosomes.
[0015] The invention provides for isolated Sugarcane
mini-chromosomes comprising a centromere, wherein the centromere
comprises at least two copies of a repeated nucleotide sequence(s),
and wherein the centromere confers the ability to segregate to
daughter cells. The repeated nucleotide sequence(s) may be short
Sugarcane satellite sequences such as those sequences set out as
SEQ ID NOS: 1-201, the consensus Sugarcane satellite sequence set
out as SEQ ID NO: 202, or the block of Sugarcane satellite sequence
set out as SEQ ID NO:204. The repeated nucleotide sequences may be
longer sequences such as the Sugarcane retrotransposon sequence
CRS, set out as SEQ ID NO: 203.
[0016] The invention also provides cells comprising a
polynucleotide, nucleic acid, vector, Sugarcane centromere,
Sugarcane artificial mini-chromosome and/or Sugarcane recombinant
chromosome of the invention. In embodiments, the cell is an
isolated cell. In other representative embodiments, the cell is a
Sugarcane cell.
[0017] Accordingly, as one aspect, the invention provides a
polynucleotide comprising a nucleotide sequence selected from the
group consisting of (a) the nucleotide sequence of any of SEQ ID
NOS: 1-204; (b) a nucleotide sequence that is at least about 80%,
85%, 90%, 95%, 97%, 98% or 99%/identical to the nucleotide sequence
of any of SEQ ID NOS: 1-204, optionally wherein the nucleotide
sequence is functional as a sugar cane plant centromere (e.g.,
confers the ability to segregate to a daughter cell); and/or (c) a
nucleotide sequence that hybridizes to the nucleotide sequence of
any of SEQ ID NOS: 1-204 under stringent conditions comprising
hybridization at 65.degree. C. and washing three times for 15
minutes with 0.25.times.SSC, 0.1% SDS at 65.degree. C., optionally
wherein the nucleotide sequence is functional as a sugar cane plant
centromere.
[0018] As another aspect, the invention provides a nucleic acid
comprising an array comprising at least about two, at least about
ten, at least about 100, from about 2 to about 1000, or from about
5 to about 250 copies of a polynucleotide of the invention. In
representative embodiments, the array is from about 1 to about 200
kb in length, optionally from about 15 to about 28 kb in length. In
exemplary embodiments, the nucleic acid is functional as a sugar
cane plant centromere.
[0019] The invention further encompasses a Sugarcane centromere
comprising a polynucleotide or nucleic acid of the invention.
[0020] Also provided by the invention is a Sugarcane artificial
chromosome comprising a polynucleotide, nucleic acid or centromere
of the invention. In embodiments of the invention, the Sugarcane
artificial chromosome further comprises an exogenous nucleic acid
(e.g., at least three exogenous nucleic acids), at least one of
which may optionally be linked to a heterologous regulatory
sequence functional in Sugarcane plant cells.
[0021] In further representative embodiments, the Sugarcane
artificial chromosomes of the invention exhibit a mitotic
segregation efficiency in Sugarcane plant cells of at least about
60%, 70%, 80%, 85%, 90%, 95% or more.
[0022] Also encompassed by the present invention is a vector
comprising a polynucleotide, nucleic acid, Sugarcane centromere, or
Sugarcane artificial chromosome of the invention.
[0023] As still another aspect, the invention provides a cell
comprising a polynucleotide, nucleic acid, Sugarcane centromere,
Sugarcane artificial chromosome, or vector of the invention. In
representative embodiments, the cell is a Sugarcane plant cell.
[0024] In particular embodiments, the invention provides a
Sugarcane plant cell comprising a Sugarcane artificial chromosome,
wherein the Sugarcane artificial chromosome is not integrated into
the genome of the Sugarcane plant cell. The Sugarcane plant cell
can optionally comprise a Sugarcane artificial chromosome that
comprises an exogenous nucleic acid, wherein the Sugarcane plant
cell exhibits an altered phenotype associated with the expression
of the exogenous nucleic acid. The altered phenotype can be any
phenotypic change of interest that can be detected and, optionally,
measured. In an exemplary embodiment, the altered phenotype
comprises altered expression (e.g., increased or decreased
expression) of a native gene. In other embodiments, the altered
phenotype comprises altered expression of an exogenous gene.
[0025] As yet a further aspect, the invention provides a Sugarcane
plant tissue, a Sugarcane plant, and/or a Sugarcane plant part
comprising a Sugarcane plant cell of the invention.
[0026] The invention also provides a Sugarcane seed obtained from a
Sugarcane plant of the invention.
[0027] The invention also contemplates a Sugarcane plant progeny
comprising a Sugarcane artificial chromosome, wherein the plant
progeny is the result of breeding a Sugarcane plant of the
invention that comprises the sugarcane artificial chromosome.
[0028] As still another aspect, the invention provides a method of
using a Sugarcane plant of the invention, wherein the Sugarcane
plant comprises a Sugarcane artificial chromosome comprising an
exogenous nucleic acid encoding a recombinant protein, the method
comprising growing the plant to produce the recombinant protein.
The method can optionally further comprise the step of harvesting
and/or processing the Sugarcane plant.
[0029] In exemplary embodiments, the invention provides for a
Sugarcane plant cell comprising a Sugarcane mini-chromosome
comprising a Sugarcane centromere that comprises at least two
repeat nucleotide sequences that have a sequence that hybridizes
under conditions comprising hybridization at 65.degree. C. and
washing three times for 15 minutes with 0.25.times.SSC, 0.1% SDS at
65.degree. C. to a nucleotide sequence selected from the group
consisting of SEQ ID NOS: 1-204, and wherein the centromere confers
the ability to segregate to daughter Sugarcane cells.
Alternatively, the hybridization conditions may comprise
hybridization at 65.degree. C. and washing three times for 15
minutes with 0.25.times.SSC, 0.1% SDS at 65.degree. C.
[0030] In another exemplary embodiment, the invention provides for
a Sugarcane plant cell comprising a Sugarcane mini-chromosome
comprising a Sugarcane centromere, wherein the centromere comprises
at least two copies of a repeated nucleotide sequence(s) that has a
sequence that is at least 80% identical to a nucleotide sequence
selected from the group consisting of SEQ ID NOS: 1-204, and
wherein the centromere confers the ability to segregate to daughter
Sugarcane cells. The invention also provides for a Sugarcane plant
cell comprising a Sugarcane mini-chromosome wherein the repeated
nucleotide sequence comprises a sequence that is at least 85%
identical, or 90% identical, or 95% identical or 98% identical to a
nucleotide sequence selected from the group consisting of SEQ ID
NOS: 1-204.
[0031] In another embodiment, the invention provides for a
Sugarcane plant cell comprising a Sugarcane Applied Mini-chromosome
comprising at least two copies of a repeated nucleotide sequence
that is at least 80% identical to the nucleotide sequence of any
one of SEQ ID NOS: 1-204 or hybridizes to the nucleotide sequence
of any one of SEQ ID NOS: 1-204 under stringent conditions
comprising hybridization at 65.degree. C. and washing three times
for 15 minutes with 0.25.times.SSC, 0.1% SDS at 65.degree. C., and
a Transgene Expression Cassette.
[0032] In a further embodiment, the invention provides for a
Sugarcane plant cell comprising a Sugarcane mini-chromosome
comprising a Sugarcane centromere, wherein the centromere comprises
(a) at least two copies of a Sugarcane satellite nucleotide
sequence (e.g., SEQ ID NO:204), and (b) at least two copies of a
Sugarcane CRS nucleotide sequence (SEQ ID NO: 203), and wherein the
centromere confers the ability to segregate to daughter Sugarcane
cells. In another embodiment, the invention provides for a
Sugarcane plant cell comprising a Sugarcane mini-chromosome
comprising a Sugarcane centromere, wherein the centromere comprises
(a) at least one array of Sugarcane satellite nucleotide sequences,
and (b) at least one array of a Sugarcane CRS nucleotide sequence
(SEQ ID NO: 203), and wherein the centromere confers the ability to
segregate to daughter Sugarcane cells. The Sugarcane satellite
nucleotide sequence may be one of the sequences set out as SEQ ID
NOS: 1-202 or SEQ ID NO: 204, or a sequence that hybridizes under
conditions comprising hybridization at 65.degree. C. and washing
three times for 15 minutes with 0.25.times.SSC, 0.1% SDS at
65.degree. C. to a nucleotide sequence selected from the group
consisting of SEQ ID NOS: 1-202 and SEQ ID NO: 204, or a sequence
that is at least 80% identical to a nucleotide sequence selected
from the group consisting of SEQ ID NOS: 1-202 and SEQ ID NO:
204.
[0033] In addition, the invention provides for a Sugarcane plant
cell comprising a Sugarcane Applied Mini-chromosome comprising a
Sugarcane centromere, wherein the Sugarcane centromere comprises
(a) at least 5 copies of a repeated nucleotide sequence within 1 kb
of nucleotide sequence, wherein the repeated nucleotide sequence is
at least 80% identical to the nucleotide sequence of any one of SEQ
ID NOS: 1-202 or SEQ ID NO: 204 or hybridizes to the nucleotide
sequence of any one of SEQ ID NOS: 1-202 or SEQ ID NO: 204 under
stringent conditions comprising hybridization at 65.degree. C. and
washing three times for 15 minutes with 0.25.times.SSC, 0.1% SDS at
65.degree. C., and (b) at least 2 copies of a repeated nucleotide
sequence that is at least 80% identical over its length to the
nucleotide sequence of any one of SEQ ID NO: 203 or hybridizes to
the nucleotide sequence of any one of SEQ ID NO: 203 under
stringent conditions comprising hybridization at 65.degree. C. and
washing three times for 15 minutes with 0.25.times.SSC, 0.1% SDS at
65.degree. C.
[0034] In another embodiment, the invention provides a Sugarcane
plant cell comprising (a) a polynucleotide sequence that is
transcribed as a first RNA, (b) a polynucleotide sequence that is
transcribed as a second RNA, and (c) a polynucleotide sequence that
is transcribed as a third RNA, wherein transcription of the
polynucleotide sequences results in increased biomass of a
Sugarcane plant.
[0035] In an additional embodiment, the invention provides for a
Sugarcane plant cell comprising a transgene expression cassette not
integrated into the plant cell genome, wherein the Transgene
Expression Cassette comprises (a) a polynucleotide sequence that is
transcribed as a first RNA, (b) a polynucleotide sequence that is
transcribed as a second RNA, and (c) a polynucleotide sequence that
is transcribed as a third RNA, wherein transcription of the
polynucleotide sequences results in increased biomass of a
Sugarcane plant.
[0036] The invention provides for a Sugarcane plant cell comprising
a recombinant chromosome comprising at least two copies of a
repeated nucleotide sequence(s), and wherein the centromere confers
the ability to segregate to daughter cells. The repeated nucleotide
sequence(s) may be short Sugarcane satellite sequences such as
those sequences set out as SEQ ID NOS: 1-201, the consensus
Sugarcane satellite sequence set out as SEQ ID NO: 202, or the
block of Sugarcane satellite sequence set out as SEQ ID NO: 204.
The repeated nucleotide sequences may be longer sequences such as
the Sugarcane retrotransposon sequence CRS, set out as SEQ ID NO:
203.
[0037] In exemplary embodiments, the invention provides for a
Sugarcane plant cell comprising a recombinant chromosome comprising
a Sugarcane centromere that comprises at least two repeat
nucleotide sequences that have a sequence that hybridizes under
conditions comprising hybridization at 65.degree. C. and washing
three times for 15 minutes with 0.25.times.SSC, 0.1% SDS at
65.degree. C. to a nucleotide sequence selected from the group
consisting of SEQ ID NOS: 1-204, and wherein the centromere confers
the ability to segregate to daughter Sugarcane cells.
Alternatively, the hybridization conditions may comprise
hybridization at 65.degree. C. and washing three times for 15
minutes with 0.25.times.SSC, 0.1% SDS at 65.degree. C.
[0038] In another exemplary embodiment, the invention provides for
a Sugarcane plant cell comprising a recombinant chromosome
comprising at least two copies of a repeated nucleotide sequence(s)
that has a sequence that is at least 80% identical to a nucleotide
sequence selected from the group consisting of SEQ ID NOS: 1-204,
and a transgene expression cassette comprising at least three
exogenous nucleic acids. The invention also provides for Sugarcane
recombinant chromosomes wherein the repeated nucleotide sequence
comprise a sequence that is at least 85% identical, or 90%
identical, or 95% identical or 98% identical to a nucleotide
sequence selected from the group consisting of SEQ ID NOS:
1-204.
[0039] In a further embodiment, the invention provides for a
Sugarcane plant cell comprising a Sugarcane recombinant chromosome
comprising a Sugarcane centromere, wherein the centromere comprises
(a) at least two copies of a Sugarcane satellite nucleotide
sequence, and (b) at least two copies of a Sugarcane CRS nucleotide
sequence (SEQ ID NO: 203), and wherein the centromere confers the
ability to segregate to daughter Sugarcane cells. In another
embodiment, the invention provides for a Sugarcane recombinant
chromosome comprising a Sugarcane centromere, wherein the
centromere comprises (a) at least one array of Sugarcane satellite
nucleotide sequences, and (b) at least one array of a Sugarcane CRS
nucleotide sequence (SEQ ID NO: 203), and wherein the centromere
confers the ability to segregate to daughter Sugarcane cells. The
Sugarcane satellite nucleotide sequence may be one of the sequences
set out as SEQ ID NOS: 1-202 or SEQ ID NO: 204, or a sequence that
hybridizes under conditions comprising hybridization at 65.degree.
C. and washing three times for 15 minutes with 0.25.times.SSC, 0.1%
SDS at 65.degree. C. to a nucleotide sequence selected from the
group consisting of SEQ ID NOS: 1-202 and SEQ ID NO: 204, or a
sequence that is at least 80% identical to a nucleotide sequence
selected from the group consisting of SEQ ID NOS: 1-202 and SEQ ID
NO: 204.
[0040] Alternatively, the invention provides for Sugarcane plant
cells comprising a recombinant chromosome that has not been
maintained in a cell of a heterologous organism.
[0041] In another embodiment, the invention provides for a
Sugarcane plant cell comprising (a) at least two copies of a
repeated nucleotide sequence that is at least 80% identical to the
nucleotide sequence of any one of SEQ ID NOS: 1-204 or hybridizes
to the nucleotide sequence of any one of SEQ ID NOS: 1-204 under
stringent conditions comprising hybridization at 65.degree. C. and
washing three times for 15 minutes with 0.25.times.SSC, 0.1% SDS at
65.degree. C., and (b) a Transgene Expression Cassette comprising
at least three exogenous nucleic acids, wherein the nucleotide
sequence and the Transgene Expression Cassette are not integrated
into the genome of the Sugarcane plant cell.
[0042] The invention also provides for a Sugarcane plant cell
comprising a Sugarcane mini-chromosome comprising a Sugarcane
centromere, wherein the centromere comprises at least two synthetic
repeat sequences or a synthetic array of repeated nucleotide
sequence, wherein the array comprises at least two copies of a
repeated nucleotide sequence, and wherein the centromere confers
the ability to segregate to daughter Sugarcane cells. These
artificially synthesized repeated nucleotide sequences may be based
on sequence information from natural Sugarcane centromere
sequences, combinations or fragments of natural Sugarcane
centromere sequences including a combination of repeats of
different lengths, a combination of different sequences, a
combination of both different repeat lengths and different
sequences, a combination of different artificially synthesized
sequences or a combination of natural Sugarcane centromere
sequence(s) and artificially synthesized Sugarcane sequence(s). The
polynucleotides comprising synthetic arrays of Sugarcane repeat
sequences and synthetic arrays of Sugarcane repeat sequences may be
generated using any technique known in the art including PCR from
Sugarcane genomic DNA (or a clone thereof) or by custom
oligonucleotide synthesis.
[0043] The invention provides for any of the Sugarcane
mini-chromosomes or recombinant chromosomes described herein having
a centromere comprising an array of repeated nucleotide sequence
that ranges from about 1 kb to about 200 kb in length, 1 kb to
about 100 kb in length, about 1 kb to about 10 kb in length, about
2 kb to about 12 kb in length, about 5 kb to about 25 kb in length,
about 10 kb to about 50 kb in length, about 25 kb to 100 kb in
length.
[0044] The invention further contemplates any of the Sugarcane
mini-chromosomes or recombinant chromosomes of the invention having
centromeres comprising at least 300 bp, 400 bp, 500 bp, 600 bp, 700
bp, 750 bp, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb,
5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5
kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb,
14 kb, 14.5 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 25 kb, 30
kb, 35 kb, 40 kb, 45 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb,
110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190
kb, 200 kb, 225 kb, 250 kb, 275 kb, 300 kb, 325 kb, 350 kb or 375
kb.
[0045] In another embodiment, any of the Sugarcane mini-chromosomes
or recombinant chromosomes of the invention comprise centromeres
having n copies of a repeated nucleotide sequence, wherein n is
less than 2000, less than 1500, less than 1000, less than 500, less
than 400, less than 300, less than 250, less than 200, less than
100, less than 90, less than 80, less than 70, less than 60, less
than 50, less than 40, less than 30, less than 25, less than 20,
less than 15, less than 10, less than 9, less than 8, less than 7,
less than 6 or less than 5. In exemplary embodiments, the
centromeres of the Sugarcane mini-chromosomes of the invention
comprise n copies of a repeated nucleotide sequence, wherein n is
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
550, 600, 650, 700, 750, 800, 850, 900 or 1000. In additional
exemplary embodiments, the centromeres of the Sugarcane
mini-chromosomes or recombinant chromosomes of the invention
comprise n copies of a repeated nucleotide sequence where n ranges
from 2 to 10, 2 to 20, 2 to 50, 2 to 100, 2 to 250, 2 to 500, 2 to
1000, 5 to 15, 5 to 25, 5 to 50, 5 to 100, 5 to 250, 5 to 500, 5 to
1000, 15 to 25, 15 to 50, 15 to 100, 15 to 250, 15 to 500, 15 to
1000, 25 to 50, 25 to 100, 25 to 250, 25 to 500, 25 to 1000, 50 to
100, 50 to 250, 50 to 500, 50 to 1000, 100 to 250, 100 to 500, 100
to 1000, 250 to 500, 250 to 1000, or 500 to 1000.
[0046] In an embodiment of the invention, any of the Sugarcane
mini-chromosomes or recombinant chromosomes of the invention
comprise a centromere having at least 5 consecutive repeated
nucleotide sequences (e.g., SEQ ID NO: 204) in "head to tail
orientation." In an embodiment of the invention, any of the
Sugarcane mini-chromosomes or recombinant chromosomes of the
invention comprise a centromere having at least 5 consecutive
repeated nucleotide sequences in "tandem," in which one repeat
sequence is immediately adjacent to another repeat sequence in any
orientation, e.g. head to tail, tail to tail, or head to head. The
invention also provides for any of the Sugarcane mini-chromosomes
or recombinant chromosomes of the invention comprising a centromere
having at least 5 repeated nucleotide sequences that are
consecutive. The term "consecutive" refers to the same or similar
repeated nucleotide sequences (e.g., at least 70% identical) that
follow one after another without being interrupted by other
significant sequence elements. Consecutive repeated nucleotide
sequences may be in any orientation, e.g. head to tail, tail to
tail, or head to head, and need not be directly adjacent to each
other (e.g., may be 1-50 bp apart).
[0047] The invention further provides for any of the Sugarcane
mini-chromosomes or recombinant chromosomes of the invention
comprising a centromere having at least 5 of the consecutive
repeated nucleotide sequences (e.g., SEQ ID NO: 204) separated by
less than n number of nucleotides, wherein n ranges from 1 to 10,
or 1 to 20, or 1 to 30, or 1 to 40, or 1 to 50 or wherein n is less
than 10 bp or n is less than 20 bp or n is less than 30 bp or n is
less that 40 bp or n is less than 50 bp.
[0048] The invention also provide for any of the Sugarcane
mini-chromosomes or recombinant chromosomes of the invention
comprising a centromere having at least two arrays of consecutive
repeated nucleotide sequences (e.g., SEQ ID NO: 204), wherein the
array comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,
30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,
500, 600, 700, 800, 900, 1000 or 2000 repeated nucleotide
sequences. The repeats within an array may be in tandem in any
orientation, e.g. head to tail, tail to tail, or head to head, or
consecutive in any orientation, e.g. head to tail, tail to tail, or
head to head. The arrays may be separated by less than n number of
nucleotides, wherein n ranges from 1 to 10, or 1 to 20, or 1 to 30,
or 1 to 40, or 1 to 50, or 1 to 60, or 1 to 70, or 1 to 80, or 1 to
90, or 1 to 100, or wherein n is less than 10 bp or n is less than
20 hp or n is less than 30 bp or n is less that 40 bp or n is less
than 50 bp. The two arrays may comprise the same repeated
nucleotide sequence or two different repeated nucleotide sequences
(i.e. the first array can be comprised of repeat type 1 and the
second array can be comprised of repeat type 2--here "type 1" and
"type 2" are arbitrary designations).
[0049] In one embodiment, the Sugarcane mini-chromosomes or
recombinant chromosomes of the invention are 1000 kb or less in
length, 900 kb or less in length, 800 kb or less in length or 700
kb or less in length. In exemplary embodiments, the Sugarcane
mini-chromosome is 600 kb or less in length, 500 kb or less in
length, 250 kb or less in length, 100 kb or less in length, 50 kb
or less in length, 10 kb or less in length, 5 kb or less in length,
or 1 kb or less in length. For example, the Sugarcane
mini-chromosomes of the invention are 50 to 250 kb in length, 50 to
100 kb in length, 50 to 75 kb in length, 50 to 100 kb in length, 60
kb to 85 kb in length, 70 to 90 kb in length, 75 to 100 kb in
length, 100 to 250 kb in length, 250 to 500 kb in length, 500 to
1000 kb in length. In an exemplary embodiment, the Sugarcane
mini-chromosome is 28 kb in length, 42 kb in length, 82 kb in
length, 87 kb in length, 88 kb in length, 97 kb in length, 130 kb
in length, 150 kb in length, 200 kb in length or ranges from 28-200
kb in length. The mini-chromosome of the invention preferably has a
segregation efficiency during mitotic division of at least 60%, at
least 80%, at least 90% or at least 95% and/or a transmission
efficiency during meiotic division of, e.g., at least 60%, at least
80%, at least 85%, at least 90% or at least 95%.
[0050] The Sugarcane mini-chromosome or recombinant chromosomes of
the invention preferably has a segregation efficiency during
mitotic division of at least 60%, at least 80%, at least 90% or at
least 95% and/or a transmission efficiency during meiotic division
of, e.g., at least 60%, at least 80/%, at least 85%, at least 90%
or at least 95%.
[0051] In another embodiment, the Sugarcane mini-chromosomes or
recombinant chromosomes of the invention comprise a site for
site-specific recombination.
[0052] The invention also provides for a Sugarcane mini-chromosome,
wherein the mini-chromosome is derived from a donor clone or a
centromere clone and has substitutions, deletions, insertions,
duplications or arrangements of one or more nucleotides in the
mini-chromosome compared to the nucleotide sequence of the donor
clone or centromere clone. In one embodiment, the Sugarcane
mini-chromosome is obtained by passage of the Sugarcane
mini-chromosome through one or more hosts. In another embodiment,
the mini-chromosome is obtained by passage of the mini-chromosome
through two or more different hosts. The host may be selected from
the group consisting of viruses, bacteria, yeasts. In another
embodiment, the Sugarcane mini-chromosome is obtained from a donor
clone by in vitro methods that introduce sequence variation during
template-based replication of the donor clone, or its complementary
sequence. In one embodiment this variation may be introduced by a
DNA-dependent DNA polymerase. In a further embodiment a Sugarcane
minichromosome derived by an in vitro method may be further
modified by passage of the mini-chromosome through one or more
hosts.
[0053] The invention also provides for a Sugarcane mini-chromosome
or recombinant chromosome, wherein the mini-chromosome comprises at
least one exogenous nucleic acid. In further exemplary embodiments,
the Sugarcane mini-chromosome or recombinant chromosome comprises
at least two or more, at least three or more, at least four or
more, at least five or more, at least ten or more, at least 20 or
more, at least 30 or more, at least 40 or more, at least 50 or more
exogenous nucleic acids.
[0054] In one embodiment, at least one exogenous nucleic acid of
any of the Sugarcane mini-chromosomes or recombinant chromosomes of
the invention is operably linked to a heterologous regulatory
sequence functional in plant cells, including but not limited to a
plant regulatory sequence. The invention also provides for
exogenous nucleic acids linked to a non-plant regulatory sequence,
such as an arthropod, viral, bacterial, vertebrate or yeast
regulatory sequence. The invention also provides for exogenous
nucleic acids linked to a regulatory sequence from Sugarcane.
[0055] The invention also provides for a mini-chromosome or
recombinant chromosome comprising a gene or group of genes that act
to improve the total recoverable sugar from Sugarcane. Such genes
may act to increase the sugar concentration of the stem juice,
increase the amount of juice, increase the stem strength to improve
yield, or increase total biomass of the plant. Such genes may be
derived from bacterial sequences such as a sucrose isomerase or
from animal, plant fungal, or protist sequences. Such genes from
plants may include genes involved in sugar metabolism or transport
or genes of unknown function or genes not known to be associated
with sugar metabolism or transport but that have been shown to
quantitatively increase total recoverable sugar. Such genes may
also include genes that affect plant height, stem diameter, water
metabolism or total biomass. Such genes may also include those that
regulate the equilibrium between starch and sugar. Several genes
have been shown to improve sugar accumulation. For example,
expression of a bacterial sucrose isomerase can increase Sugarcane
sugar content by as much as two-fold (Birch, R. G., and Wu, L.
(2007).
[0056] Doubled sugar content in Sugarcane plants modified to
produce a sucrose isomer. Plant Biotechnology Journal 5: 109-117).
The lignin-deficient "brown midrib" mutations improve sorghum sugar
content via their effects on lignin; this phenotype is caused by
mutations in cinnamyl alcohol dehydrogenase (CAD), and 14 CAD-like
genes are present in the sorghum genome (Saballos, A., Ejeta, G.,
Sanche, E., Kang, C., and Vermerris, W. (2008). A Genome-Wide
Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum
(Sorghum bicolor (L.) Moench) Identifies SbCAD2 as the Brown
midrib6 Gene. Genetics).
[0057] In another embodiment, the Sugarcane mini-chromosome or
recombinant chromosome comprises an exogenous nucleic acid that
comprises a QTL that confers a desirable trait. QTLs that affect
total recoverable sugars have been mapped in Sugarcane (Murray, S.
C., Sharma, A., Rooney, W. L., Klein, P., Mullet, J. E., Mitchell,
S. E., Kresovitch, S. (2008) Genetic Improvement of Sorghum as a
Biofuel Feedstock: I. QTL for Stem Sugar and Grain Nonstructural
Carbohydrates. Crop Sci. 48:2165-2179).
[0058] In another embodiment, the Sugarcane mini-chromosome or
recombinant chromosome comprises an exogenous nucleic acid that
confers herbicide resistance, insect resistance, disease
resistance, or stress resistance on the Sugarcane plant. The
invention provides for Sugarcane mini-chromosomes or recombinant
chromosomes comprising an exogenous nucleic acid that confers
resistance to phosphinothricin or glyphosate herbicide. Nonlimiting
examples include an exogenous nucleic acid that encodes a
phosphinothricin acetyltransferase, glyphosate acetyltransferase,
acetohydroxyadic synthase or a mutant enoylpyruvylshikimate
phosphate (EPSP) synthase. Nonlimiting examples of exogenous
nucleic acids that confer insect resistance include a Bacillus
thuringiensis toxin gene or Bacillus cereus toxin gene. In related
embodiments, the Sugarcane mini-chromosome or recombinant
chromosome comprises an exogenous nucleic acid conferring herbicide
resistance, an exogenous nucleic acid conferring insect resistance,
and optionally at least one additional exogenous nucleic acid.
[0059] The invention further provides for Sugarcane
mini-chromosomes or recombinant chromosomes comprising additional
copies of genes already found in the Sugarcane genome. The
invention also provides for the additional copies of Sugarcane
genes carried on the Sugarcane mini-chromosome or recombinant
chromosomes to be operably linked to either their native regulatory
sequences or to heterologous regulatory sequences.
[0060] The invention further provides for Sugarcane
mini-chromosomes or recombinant chromosomes comprising an exogenous
nucleic acid that confers resistance to drought, heat, chilling,
freezing, excessive moisture, ultraviolet light, ionizing
radiation, toxins, pollution, mechanical stress or salt stress. The
invention also provides for a Sugarcane mini-chromosome that
comprises an exogenous nucleic acid that confers resistance to a
virus, bacteria, fungi or nematode.
[0061] The invention provides for Sugarcane mini-chromosomes or
recombinant chromosomes comprising an exogenous nucleic acid
selected from the group consisting of a nitrogen fixation gene, a
plant stress-induced gene, a nutrient utilization gene, a gene that
affects plant pigmentation, a gone that encodes an antisense or
ribozyme molecule, a gene encoding a secretable antigen, a toxin
gene, a receptor gene, a ligand gene, a seed storage gene, a
hormone gene, an enzyme gene, an interleukin gene, a clotting
factor gene, a cytokine gene, an antibody gene, a growth factor
gene, a transcription factor gene, a transcriptional repressor
gene, a DNA-binding protein gene, a recombination gene, a DNA
replication gene, a programmed cell death gene, a kinase gene, a
phosphatase gene, a G protein gene, a cyclin gene, a cell cycle
control gene, a gene involved in transcription, a gene involved in
translation, a gene involved in RNA processing, a gene involved in
RNAi, an organellar gene, a intracellular trafficking gene, an
integral membrane protein gene, a transporter gene, a membrane
channel protein gene, a cell wall gene, a gene involved in protein
processing, a gene involved in protein modification, a gene
involved in protein degradation, a gene involved in metabolism, a
gene involved in biosynthesis, a gene involved in assimilation of
nitrogen or other elements or nutrients, a gene involved in
controlling carbon flux, a gene involved in respiration, a gene
involved in photosynthesis, a gene involved in light sensing, a
gene involved in organogenesis, a gene involved in embryogenesis, a
gene involved in differentiation, a gene involved in meiotic drive,
a gene involved in self incompatibility, a gene involved in
development, a gene involved in nutrient, metabolite or mineral
transport, a gene involved in nutrient, metabolite or mineral
storage, a calcium-binding protein gene, or a lipid-binding protein
gene.
[0062] The invention also provides for a Sugarcane mini-chromosome
or recombinant chromosome comprising an exogenous enzyme gene
selected from the group consisting of a gene that encodes an enzyme
involved in metabolizing biochemical wastes for use in
bioremediation, a gene that encodes an enzyme for modifying
pathways that produce secondary plant metabolites, a gene that
encodes an enzyme that produces a pharmaceutical, a gene that
encodes an enzyme that improves changes in the nutritional content
of a plant, a gene that encodes an enzyme involved in vitamin
synthesis, a gene that encodes an enzyme involved in carbohydrate,
polysaccharide or starch synthesis, a gene that encodes an enzyme
involved in mineral accumulation or availability, a gene that
encodes a phytase, a gene that encodes an enzyme involved in fatty
acid, fat or oil synthesis, a gene that encodes an enzyme involved
in synthesis of chemicals or plastics, a gene that encodes an
enzyme involved in synthesis of a fuel, a gene that encodes an
enzyme involved in synthesis of a fragrance, a gene that encodes an
enzyme involved in synthesis of a flavor, a gene that encodes an
enzyme involved in synthesis of a pigment or dye, a gene that
encodes an enzyme involved in synthesis of a hydrocarbon, a gene
that encodes an enzyme involved in synthesis of a structural or
fibrous compound, a gene that encodes an enzyme involved in
synthesis of a food additive, a gene that encodes an enzyme
involved in synthesis of a chemical insecticide, a gene that
encodes an enzyme involved in synthesis of an insect repellent, or
a gene controlling carbon flux in a plant.
[0063] In another embodiment of the invention, any of the Sugarcane
mini-chromosomes or recombinant chromosomes of the invention
comprise a telomere.
[0064] The invention also provides embodiments wherein any of the
Sugarcane mini-chromosomes or recombinant chromosomes of the
invention are linear or circular.
[0065] In one embodiment, the invention provides for Sugarcane
plants or plant cells comprising any of the Sugarcane
mini-chromosomes or recombinant chromosomes of the invention. The
invention also provides for Sugarcane plant tissue and Sugarcane
seed obtained from the Sugarcane plants of the invention.
[0066] In another embodiment, the invention provides for Sugarcane
plants comprising any of the Sugarcane mini-chromosomes or
recombinant chromosomes of the invention, which may be referred to
herein as "adchromosomal" Sugarcane plants. In addition, the
invention provides for Sugarcane plant cells, tissues and seeds
obtained from these modified plants.
[0067] In one embodiment, the invention provides for a Sugarcane
plant cell comprising any of the Sugarcane mini-chromosomes or
recombinant chromosomes of the invention that (i) is not integrated
into the Sugarcane plant cell genome and (ii) confers an altered
phenotype on the Sugarcane plant cell associated with the
expression of at least one structural gene within the Sugarcane
mini-chromosome. The altered phenotype comprises increased
expression of a native gene, decreased expression of a native gene,
or expression of an exogenous gene. In a further embodiment, these
Sugarcane plant cells also comprise one or more integrated
exogenous structural gene(s).
[0068] Another embodiment of the invention is a part of any of the
Sugarcane plants of the invention. Exemplary Sugarcane plant parts
of the invention include a pod, root, sett root, shoot root, root
primordial, shoot, primary shoot, secondary shoot, tassle, panicle,
arrow, midrib, blade, ligule, auricle, dewlap, blade joint, sheath,
node, internode, bud furrow, leaf scar, cutting, tuber, stem,
stalk, fruit, berry, nut, flower, leaf, bark, wood, epidermis,
vascular tissue, organ, protoplast, crown, callus culture, petiole,
petal, sepal, stamen, stigma, style, bud, meristem, cambium,
cortex, pith, sheath, silk, ovule or embryo. Other exemplary
Sugarcane plant parts are a meiocyte or gamete or ovule or pollen
or endosperm of any of the plants described herein. Other exemplary
plant parts are a seed, seed-piece, embryo, protoplast, cell
culture, any group of plant cells organized into a structural and
functional unit, ratoon, or propagule of any of the Sugarcane
plants of the invention.
[0069] An embodiment of the invention is a progeny of any of the
Sugarcane plants of the invention. These progeny of the invention
may be the result of self-breeding, cross-breeding, apomyxis or
clonal propagation. In exemplary embodiments, the invention also
provides for progeny that comprise a Sugarcane mini-chromosome or
recombinant chromosome that is descended from a parental Sugarcane
mini-chromosome or recombinant chromosome that contained a
centromere less than about 1000 kilobases in length, less than
about 750 kilobases in length, less than about 600 kilobases in
length, less than about 500 kilobases in length, less than about
400 kilobases in length, less than about 300 kilobases in length,
less than about 250 kilobases in length, less than about 200
kilobases in length, less than about 150 kilobases in length, less
than about 100 kilobases in length, less than about 90 kilobases in
length, less than about 85 kilobases in length, less than about 80
kilobases in length, less than about 75 kilobases in length, less
than about 70 kilobases in length, less than about 65 kilobases in
length, less than about 60 kilobases in length, less than about 55
kilobases in length, less than about 50 kilobases in length, less
than about 45 kilobases in length, less than about 40 kilobases in
length, less than about 35 kilobases in length, less than about 30
kb in length, less than about 25 kilobases in length, less than
about 20 kb in length, less than about 15 kilobases in length, less
than about 12 kilobases in length, less than about 10 kb in length,
less than about 7 kb in length, less than about 5 kb in length, or
less than about 2 kb in length.
[0070] In another aspect, the invention provides for methods of
making a Sugarcane mini-chromosome for use in any of the Sugarcane
plants of the invention. In representative embodiments, these
methods comprise identifying a centromere nucleotide sequence in a
Sugarcane genomic DNA library using a multiplicity of diverse
probes, and constructing a Sugarcane mini-chromosome comprising the
centromere nucleotide sequence. These methods may further comprise
determining hybridization scores for hybridization of the
multiplicity of diverse probes to genomic clones within the
Sugarcane genomic nucleic acid library, determining a
classification for genomic clones within the Sugarcane genomic
nucleic acid library according to the hybridization scores for at
least two of the diverse probes, and selecting one or more genomic
clones within one or more classifications for constructing the
Sugarcane mini-chromosome.
[0071] The invention also contemplates methods of using any of the
Sugarcane plants of the invention to produce a recombinant protein,
by growing a Sugarcane plant comprising a Sugarcane mini-chromosome
or recombinant chromosome that comprises an exogenous nucleic acid
encoding the desired recombinant protein. Optionally the Sugarcane
plant is harvested and the desired protein product is isolated from
the plant. Exemplary protein products include industrial enzymes
such as those useful for biofuel production.
[0072] The invention also contemplates methods of using any of the
Sugarcane plants of the invention to produce a chemical product, by
growing a Sugarcane plant comprising a Sugarcane mini-chromosome or
recombinant chromosome that comprises an exogenous nucleic acid
encoding an enzyme involved in the synthesis of the chemical
product. Optionally the Sugarcane plant is harvested and the
desired chemical product is isolated from the plant. Exemplary
chemical products include sugars, lipids and carbohydrates useful
in the production of biofuels.
[0073] Another aspect of the invention provides for methods of
using any of the Sugarcane plants of the invention comprising a
Sugarcane mini-chromosomes or recombinant chromosome for a food
product, a pharmaceutical product or chemical product, according to
which a suitable exogenous nucleic acid is expressed in Sugarcane
plants or plant cells and the plant or plant cells are grown. The
plant may secrete the product into its growth environment or the
product may be contained within the plant, in which case the plant
is harvested and desirable products are extracted.
[0074] Thus, the invention contemplates methods of using any of the
Sugarcane plants of the invention comprising a Sugarcane
mini-chromosome or recombinant chromosome to produce a modified
food product, for example, by growing a plant that expresses an
exogenous nucleic acid that alters the nutritional content of the
plant, and harvesting or processing the Sugarcane plant.
[0075] The invention also provides for methods of constructing a
synthetic array of repeated nucleotide sequence having Sugarcane
centromere function comprising the steps of: (a) PCR amplifying a
Sugarcane satellite sequence, (b) cloning the PCR amplified
satellite sequence into a cloning vector, (c) sequencing the cloned
satellite DNA, (d) using a restriction enzyme with an asymmetric
recognition sequence to excise the cloned satellite sequence from
the cloning vector, (e) ligating the satellite sequence to one
another forming a synthetic tandem array, and (f) ligating the
synthetic array into a Sugarcane mini-chromosome backbone vector.
The invention also provides for an isolated Sugarcane
mini-chromosome comprising a synthetic array of repeated nucleotide
sequence constructed according to the method of the invention, and
Sugarcane plant cells and plants comprising these
mini-chromosomes.
[0076] In another embodiment, the invention provides for methods of
contacting a Sugarcane cell with a Sugarcane mini-chromosome
comprising the steps of (a) delivering the mini-chromosome to
immature differentiated leaves of the apical region of the stem of
a Sugarcane plant, wherein the mini-chromosome comprises a
selectable marker gene, and (b) selecting the Sugarcane cells
expressing the marker gene, wherein expression of the marker gene
indicates transformation with the mini-chromosome. The leaves used
in this method are immature but are fully differentiated, such as
the inner immature leaves of the Sugarcane stem. In an exemplary
embodiment, the mini-chromosome may be delivered by bombarding the
immature leaves with micro-particles comprising the Sugarcane
mini-chromosome.
[0077] The invention also provides for methods of regenerating a
Sugarcane plant transformed with a Sugarcane mini-chromosome
comprising the steps of (a) obtaining a callus comprising a
Sugarcane cell that is transformed by any of the methods of the
invention, and (b) growing the callus in media that may comprise
1%-3% polyvinylpyrrolidone to form a plantlet, wherein the cells of
the plantlet are transformed with the Sugarcane mini-chromosome. In
a further embodiment, the methods of culturing the callus comprise
growing the cells in liquid media for a time period and
subsequently culturing the cells in a solid culture media. In an
exemplary embodiment, the Sugarcane mini-chromosome comprises a
growth regulating gene such as a gene in the auxin biosynthesis or
perception pathways. Such genes may include iaaM (Trp
mono-oxygenase), iaaH (Indolo-3-acetamide hydrolase), and ipt (AMP
iso-pentenyl transferase). When these three genes are expressed on
a mini-chromosome(s), IaaMconverts Trp into indole-3-acetamide,
which IaaH converts into auxin. Ipt converts AMP into a cytokinin.
The expression of all three genes allows a cultured cell to grow in
the absence of exogenously supplied hormones.
[0078] A further embodiment of the invention is a sugar cane
artificial chromosome comprising at least two repeats of a nucleic
acid with at least 98% identity to SEQ ID NO: 204. A still further
embodiment of the invention is plant cell comprising a sugar cane
artificial chromosome comprising at least two repeats of a nucleic
acid with at least 98% identity to SEQ ID NO: 204. The plant cell
can be a sugar cane plant cell. A further embodiment is a sugar
cane artificial chromosome comprising at least two repeats of a
nucleic acid with at least 98% identity to SEQ ID NO: 204 wherein
the orientation of the repeats is selected from the group
consisting of head to tail, tail to tail, and head to head. In
other embodiments, the percent identity of the nucleic acid to SEQ
ID NO: 204 is at least 95%. In still other embodiments, the percent
identity of the nucleic acid to SEQ ID NO: 204 is at least 99%.
[0079] Another embodiment of the invention is a sugar cane
artificial chromosome comprising at least 15 kbp of a nucleic acid
with at least 98% identity to SEQ ID NO: 204. A further embodiment
of the invention is a plant cell comprising at least 15 kbp of a
nucleic acid with at least 98% identity to SEQ ID NO: 204. In other
embodiments, the percent identity of the nucleic acid to SEQ ID NO:
204 is at least 95%. In still other embodiments, the percent
identity of the nucleic acid to SEQ ID NO: 204 is at least 99%. The
plant cell can be a sugar cane plant cell.
[0080] Another embodiment of the invention is a sugar cane
artificial chromosome comprising at least 28 kbp of a nucleic acid
with at least 98% identity to SEQ ID NO: 204. A further embodiment
of the invention is a plant cell comprising a sugar cane artificial
chromosome comprising at least 28 kbp of a nucleic acid with at
least 98% identity to SEQ ID NO: 204. In other embodiments, the
percent identity of the nucleic acid to SEQ ID NO: 204 is at least
95%. In still other embodiments, the percent identity of the
nucleic acid to SEQ ID NO: 204 is at least 99%. The plant cell can
be a sugar cane plant cell.
[0081] Another embodiment of the invention is a sugar cane
artificial chromosome comprising a centromere wherein the
centromere comprises at least two repeats of a nucleic acid with at
least 98% identity to SEQ ID NO: 204. A further embodiment of the
invention is a plant cell comprising a sugar cane artificial
chromosome comprising a centromere wherein the centromere comprises
at least two repeats of a nucleic acid with at least 98% identity
to SEQ ID NO: 204. The plant cell can be a sugar cane plant cell. A
further embodiment is a sugar cane artificial chromosome comprising
a centromere wherein the centromere comprises at least two repeats
of a nucleic acid with at least 98% identity to SEQ ID NO: 204
wherein the orientation of the repeats is selected from the group
consisting of head to tail, tail to tail and head to head. In other
embodiments, the percent identity of the nucleic acid to SEQ ID NO:
204 is at least 95%. In still other embodiments, the percent
identity of the nucleic acid to SEQ ID NO: 204 is at least 99%.
[0082] Another embodiment of the invention is a sugar cane
artificial chromosome comprising a centromere wherein the
centromere comprises at least 15 kbp of a nucleic acid with at
least 98% identity to SEQ ID NO: 204. A further embodiment of the
invention is a plant cell comprising a centromere wherein the
centromere comprises at least 15 kbp of a nucleic acid with at
least 98% identity to SEQ ID NO: 204. The plant cell can be a sugar
cane plant cell. In other embodiments, the percent identity of the
nucleic acid to SEQ ID NO: 204 is at least 95%. In still other
embodiments, the percent identity of the nucleic acid to SEQ ID NO:
204 is at least 99%.
[0083] Another embodiment of the invention is an isolated nucleic
acid comprising at least 98% identity to SEQ ID NO: 204. A further
embodiment of the invention is a plant cell comprising an isolated
nucleic acid comprising at least 98% identity to SEQ ID NO: 204.
The plant cell can be a sugar cane plant cell. In other
embodiments, the percent identity of the nucleic acid to SEQ ID NO:
204 is at least 95%. In still other embodiments, the percent
identity of the nucleic acid to SEQ ID NO: 204 is at least 99%.
[0084] Another embodiment of the invention is an isolated nucleic
acid comprising at least two repeats of a nucleic acid with at
least 98% identity to SEQ ID NO: 204. A further embodiment of the
invention is a plant cell comprising an isolated nucleic acid
comprising at least two repeats of a nucleic acid with at least 98%
identity to SEQ ID NO: 204. The plant cell can be a sugar cane
plant cell. A further embodiment is an isolated nucleic acid
comprising at least two repeals of a nucleic acid with at least 98%
identity to SEQ ID NO: 204 wherein the orientation of the repeats
is selected from the group consisting of head to tail, tail to tail
and head to head. In other embodiments, the percent identity of the
nucleic acid to SEQ ID NO: 204 is at least 95%. In still other
embodiments, the percent identity of the nucleic acid to SEQ ID NO:
204 is at least 99%.
[0085] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising at
least two repeats of a nucleic acid with at least 98% identity to
SEQ ID NO: 204; transforming a sugar cane plant cell with the
nucleic acid; and obtaining a sugar cane plant cell wherein the
nucleic acid is autonomously replicating during sugar cane plant
cell division. In other embodiments, the percent identity of the
nucleic acid to SEQ ID NO: 204 is at least 95%. In still other
embodiments, the percent identity of the nucleic acid to SEQ ID NO:
204 is at least 99%.
[0086] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising at
least 15 kbp of a nucleic acid with at least 98% identity to SEQ ID
NO: 204; transforming a sugar cane plant cell with the nucleic
acid; and obtaining a sugar cane plant cell wherein the nucleic
acid is autonomously replicating during sugar cane plant cell
division. In other embodiments, the percent identity of the nucleic
acid to SEQ ID NO: 204 is at least 95%. In still other embodiments,
the percent identity of the nucleic acid to SEQ ID NO: 204 is at
least 99%.
[0087] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising at
least 28 kbp of a nucleic acid with at least 98% identity to SEQ ID
NO: 204; transforming a sugar cane plant cell with the nucleic
acid; and obtaining a sugar cane plant cell wherein the nucleic
acid is autonomously replicating during sugar cane plant cell
division. In other embodiments, the percent identity of the nucleic
acid to SEQ ID NO: 204 is at least 95%. In still other embodiments,
the percent identity of the nucleic acid to SEQ ID NO: 204 is at
least 99%.
[0088] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising a
centromere comprising at least two repeats of a nucleic acid with
at least 98% identity to SEQ ID NO: 204; transforming a sugar cane
plant cell with the nucleic acid; and obtaining a sugar cane plant
cell wherein the nucleic acid is autonomously replicating during
sugar cane plant cell division. In other embodiments, the percent
identity of the nucleic acid to SEQ ID NO: 204 is at least 95%. In
still other embodiments, the percent identity of the nucleic acid
to SEQ ID NO: 204 is at least 99%.
[0089] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising a
centromere comprising at least 15 kbp of a nucleic acid with at
least 98% identity to SEQ ID NO: 204; transforming a sugar cane
plant cell with the nucleic acid; and obtaining a sugar cane plant
cell wherein the nucleic acid is autonomously replicating during
sugar cane plant cell division. In other embodiments, the percent
identity of the nucleic acid to SEQ ID NO: 204 is at least 95%. In
still other embodiments, the percent identity of the nucleic acid
to SEQ ID NO: 204 is at least 99%/o.
[0090] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising a
centromere comprising at least 28 kbp of a nucleic acid with at
least 98% identity to SEQ ID NO: 204; transforming a sugar cane
plant cell with the nucleic acid; and obtaining a sugar cane plant
cell wherein the nucleic acid is autonomously replicating during
sugar cane plant cell division. In other embodiments, the percent
identity of the nucleic acid to SEQ ID NO: 204 is at least 95%. In
still other embodiments, the percent identity of the nucleic acid
to SEQ ID NO: 204 is at least 99%.
[0091] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising at
least two repeats of a nucleic acid with at least 98% identity to
SEQ ID NO: 204; transforming a sugar cane plant cell with the
nucleic acid; and obtaining a sugar cane plant cell wherein the
nucleic acid segregates to daughter sugar cane cells during sugar
cane cell division. In other embodiments, the percent identity of
the nucleic acid to SEQ ID NO: 204 is at least 95%. In still other
embodiments, the percent identity of the nucleic acid to SEQ ID NO:
204 is at least 99%.
[0092] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising at
least 15 kbp of a nucleic acid with at least 98% identity to SEQ ID
NO: 204; transforming a sugar cane plant cell with the nucleic
acid; and obtaining a sugar cane plant cell wherein the nucleic
acid segregates to daughter sugar cane cells during sugar cane cell
division. In other embodiments, the percent identity of the nucleic
acid to SEQ ID NO: 204 is at least 95%. In still other embodiments,
the percent identity of the nucleic acid to SEQ ID NO: 204 is at
least 99%.
[0093] Another embodiment is a method of stably incorporating an
autonomously replicating nucleic acid in a sugar cane plant cell
comprising the steps of obtaining a nucleic acid comprising at
least 28 kbp of a nucleic acid with at least 98% identity to SEQ ID
NO: 204; transforming a sugar cane plant cell with the nucleic
acid; and obtaining a sugar cane plant cell wherein the nucleic
acid segregates to daughter sugar cane cells during sugar cane cell
division. In other embodiments, the percent identity of the nucleic
acid to SEQ ID NO: 204 is at least 95%. In still other embodiments,
the percent identity of the nucleic acid to SEQ ID NO: 204 is at
least 99%.
SEQUENCES OF THE INVENTION
[0094] The following list indicates the identity of the SEQ ID NOs
in the sequence listing:
[0095] SEQ ID NOS: 1-201--Sugarcane satellite sequences
[0096] SEQ ID NO: 202--Consensus Sugarcane satellite sequence
[0097] SEQ ID NO: 203--Sugarcane CRS sequence
[0098] SEQ ID NOS: 204--Block of Sugarcane satellite repeat
sequence
[0099] SEQ ID NOS: 205-221--Primer sequences
[0100] SEQ ID NOS: 222-241--Promoter sequences
[0101] SEQ ID NOS: 242-251--Primers
DETAILED DESCRIPTION OF THE INVENTION
[0102] While this invention is susceptible of embodiment in many
different forms, and will be described herein in detail, specific
embodiments thereof with the understanding that the present
disclosure is to be considered as an exemplification of the
principles of the invention and is not intended to limit the
invention to the specific embodiments illustrated.
[0103] The invention provides novel, functional, stable, autonomous
Sugarcane mini-chromosomes and recombinant chromosomes comprising
centromeres comprising Sugarcane repeat sequences including
synthetic sequences. Optionally, the Sugarcane mini-chromosome or
recombinant chromosome is "isolated." The invention also provides
for "adchromosomal" Sugarcane plants described in further detail
herein.
[0104] One aspect of the invention is related to Sugarcane plants
containing functional, stable, autonomous Sugarcane
mini-chromosomes or recombinant chromosomes, optionally carrying
one or more exogenous nucleic acids or carrying extra copies of a
nucleic acid that already exists in the Sugarcane genome. Such
plants carrying Sugarcane mini-chromosomes or recombinant
chromosomes are contrasted to transgenic plants whose genome has
been altered by integrating exogenous nucleic acid transgenes into
the native Sugarcane chromosomes. In representative embodiments,
expression of the exogenous nucleic acid, either constitutively or
in response to a signal (which may be induced by challenge or a
stimulus), or tissue specific expression, or time specific
expression, results in an altered phenotype of the plant.
[0105] The invention provides for Sugarcane mini-chromosomes or
recombinant chromosomes comprising at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
250, 500, 1000 or more exogenous nucleic acids.
[0106] The invention contemplates that Sugarcane plants may be used
to carry the autonomous Sugarcane mini-chromosomes or recombinant
chromosomes as described herein. A related aspect of the invention
is Sugarcane plant parts or plant tissues, including a pod, root,
sett root, shoot root, root primordial, shoot, primary shoot,
secondary shoot, tassle, panicle, arrow, midrib, blade, ligule,
auricle, dewlap, blade joint, sheath, node, internode, bud furrow,
leaf scar, cutting, tuber, stem, stalk, fruit, berry, nut, flower,
leaf, bark, wood, epidermis, vascular tissue, organ, protoplast,
crown, callus culture, petiole, petal, sepal, stamen, stigma,
style, bud, meristem, cambium, cortex, pith, sheath, silk, ovule or
embryo. Other exemplary Sugarcane plant parts are a meiocyte or
gamete or ovule or pollen or endosperm of any of the plants of the
invention. Other exemplary plant parts are a seed, seed-piece,
embryo, protoplast, cell culture, any group of plant cells
organized into a structural and functional unit, ratoon or
propagule of any of the Sugarcane plants of the invention.
[0107] In one embodiment, the exogenous nucleic acid is primarily
expressed in a specific location or tissue of a Sugarcane plant,
for example, stem, epidermis, vascular tissue, meristem, cambium,
cortex, pith, leaf, sheath, flower, root or seed. Tissue-specific
expression can be accomplished with, for example, localized
presence of the Sugarcane mini-chromosome or recombinant
chromosome, selective maintenance of the Sugarcane mini-chromosome
or recombinant chromosomes, or with promoters that drive
tissue-specific expression.
[0108] Another related aspect of the invention is Sugarcane
meiocytes, pollen, ovules, endosperm, seed, somatic embryos,
apomyctic embryos, embryos derived from fertilization, vegetative
propagules and progeny of the originally adchromosomal plant and of
its filial generations that retain the functional, stable,
autonomous Sugarcane mini-chromosome or recombinant chromosome.
Such progeny include clonally propagated Sugarcane plants, embryos
and plant parts as well as filial progeny from self- and
cross-breeding, and from apomnyxis.
[0109] In representative embodiments, the Sugarcane mini-chromosome
or recombinant chromosome is transmitted to subsequent generations
of viable daughter cells during mitotic cell division with a
transmission efficiency of at least 60%, 70%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99%.
[0110] In embodiments of the invention, during meiotic division,
the Sugarcane mini-chromosome or recombinant chromosome is
transmitted to viable gametes with a transmission efficiency of at
least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% when more
than one copy of the Sugarcane mini-chromosome recombinant
chromosome is present in the gamete mother cells of the plant. The
Sugarcane mini-chromosome or recombinant chromosome can optionally
be transmitted to viable gametes during meiotic cell division with
a transmission frequency of at least 1%, 10%, 20%, 30%, 40%, 45%,
46%, 47%, 48%, or 49% when one copy of the mini-chromosome or
recombinant chromosome is present in the gamete mother cells of the
Sugarcane plant. According to embodiments of the invention, for
production of seeds via sexual reproduction or by apomyxis the
Sugarcane mini-chromosome or recombinant chromosome is transferred
into at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
of viable embryos when cells of the plant contain more than one
copy of the Sugarcane mini-chromosome or recombinant chromosome.
For production of seeds via sexual reproduction or by apomyxis from
Sugarcane plants with one mini-chromosome or recombinant chromosome
per cell, the Sugarcane mini-chromosome or recombinant chromosome
is optionally transferred into at least 1%, 10%, 20%, 30%, 40%,
45%, 46%, 47%, 48%, or 49% of viable embryos.
[0111] In representative embodiments of the invention, a Sugarcane
mini-chromosome or recombinant chromosome that comprises an
exogenous selectable trait or exogenous selectable marker can be
employed to increase the frequency in subsequent generations of
adchromosomal cells, tissues, gametes, embryos, endosperm, seeds,
plants or progeny that comprise the Sugarcane minichromosome or
recombinant chromosome. In particular embodiments, the frequency of
transmission of Sugarcane mini-chromosomes or recombinant
chromosome into viable cells, tissues, gametes, embryos, endosperm,
seeds, plants or progeny can be at least 95%, 96%, 97%, 98%, 99% or
99.5% after mitosis or meiosis by applying at least one selection
that favors the survival of adchromosomal cells, tissues, gametes,
embryos, endosperm, seeds, plants or progeny over such cells,
tissues, gametes, embryos, endosperm, seeds, plants or progeny
lacking the mini-chromosome or recombinant chromosome.
[0112] Transmission efficiency may be measured as the percentage of
Sugarcane progeny cells or Sugarcane plants that carry the
Sugarcane mini-chromosome or recombinant chromosome as measured by
one of several assays taught herein including detection of reporter
gene fluorescence, PCR detection of a sequence that is carried by
the mini-chromosome or recombinant chromosome, RT-PCR detection of
a gene transcript for a gene carried on the Sugarcane
mini-chromosome or recombinant chromosome, Western analysis of a
protein produced by a gene carried on the Sugarcane mini-chromosome
or recombinant chromosome, Southern analysis of the DNA (either in
total or a portion thereof) carried by the Sugarcane
mini-chromosome or recombinant chromosome, fluorescence in situ
hybridization (FISH) or in situ localization by repressor binding,
to name a few. Any assay used to detect the presence of the
Sugarcane mini-chromosome (or a portion of the mini-chromosome) or
recombinant chromosome may be used to measure the efficiency that a
parental cell or plant transmits the mini-chromosome or recombinant
chromosome to its progeny. Efficient transmission as measured by
some benchmark percentage should indicate the degree to which the
Sugarcane mini-chromosome or recombinant chromosome is stable
through the mitotic and meiotic cycles.
[0113] Sugarcane plants of the invention may also contain
chromosomally integrated exogenous nucleic acid in addition to the
autonomous Sugarcane mini-chromosomes or recombinant chromosome.
The modified Sugarcane plants or plant parts, including plant
tissues of the invention may include Sugarcane plants that have
chromosomal integration of some portion of the mini-chromosome
(e.g. exogenous nucleic acid or centromere sequences) or
recombinant chromosome in some or all cells of the plant. In one
aspect of the invention, the autonomous Sugarcane mini-chromosome
or recombinant chromosome can be isolated from integrated exogenous
nucleic acid by crossing the modified Sugarcane plant containing
the integrated exogenous nucleic acid with Sugarcane plants
producing some gametes lacking the integrated exogenous nucleic
acid and subsequently isolating offspring of the cross, or
subsequent crosses, that are modified but lack the integrated
exogenous nucleic acid. This independent segregation of the
Sugarcane mini-chromosome or recombinant chromosome is one measure
of the autonomous nature of the mini-chromosome.
[0114] Another aspect of the invention relates to methods for
producing and, optionally, isolating such modified Sugarcane plants
containing functional, stable, autonomous Sugarcane
mini-chromosomes.
[0115] In one embodiment, the invention contemplates improved
methods for isolating native Sugarcane centromere sequences. In
another embodiment, the invention contemplates methods for
generating variants of native or artificial Sugarcane centromere
sequences by passage through other host cells such as bacterial or
fungal hosts.
[0116] In a further embodiment, the invention contemplates methods
for delivering the Sugarcane mini-chromosome into Sugarcane plant
cells or tissues to transform the cells or tissues, optionally
detecting mini-chromosome presence or assessing mini-chromosome
performance, and optionally generating a Sugarcane plant from such
cells or tissues.
[0117] Exemplary assays for assessing Sugarcane mini-chromosome or
recombinant chromosome performance include lineage-based
inheritance assays, use of chromosome loss agents to demonstrate
autonomy, exonuclease digestion, global mitotic mini-chromosome
inheritance assays (sectoring assays) with or without the use of
agents inducing chromosomal loss, assays measuring expression
levels of genes (including marker genes) carried by the Sugarcane
mini-chromosome over time and space in a Sugarcane plant, physical
assays for separation of autonomous Sugarcane mini-chromosomes or
recombinant chromosomes from endogenous nuclear chromosomes of
Sugarcane plants, molecular assays demonstrating conserved
Sugarcane mini-chromosome structure or recombinant chromosomes,
such as PCR, Southern blots, Sugarcane mini-chromosome rescue,
cloning and characterization of Sugarcane mini-chromosome sequences
present in the Sugarcane plant, cytological assays detecting
Sugarcane mini-chromosome or recombinant chromosome presence in the
Sugarcane cell's genome (e.g. FISH) and meiotic Sugarcane
mini-chromosome or recombinant chromosome inheritance assays, which
measure the levels of mini-chromosome or recombinant chromosome
inheritance into a subsequent generation of Sugarcane plants via
meiosis and gametes, embryos, endosperm or seeds.
[0118] Another aspect of the invention relates to methods for using
Sugarcane plants containing a Sugarcane mini-chromosome or
recombinant chromosome for producing food products, pharmaceutical
products, biofuels and chemical products by appropriate expression
of exogenous nucleic acid(s) contained within the
mini-chromosome(s) or recombinant chromosome(s).
[0119] Yet another aspect of the invention provides novel
autonomous Sugarcane mini-chromosomes with novel compositions and
structures which are used to transform plant cells which are in
turn used to generate a plant (or multiple plants). Exemplary
Sugarcane mini-chromosomes of the invention are contemplated to be
of a size 2000 kb or less in length. Other exemplary sizes of
Sugarcane mini-chromosomes include less than or equal to, e.g.,
1500 kb, 1000 kb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 450 kb,
400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 150 kb, 100 kb, 80 kb, 60
kb, 40 kb, 35 kb in length. In an exemplary embodiment, the
mini-chromosome is about 28 kb in length, 42 kb in length, 82 kb in
length, 87 kb in length, 88 kb in length, 97 kb in length, 130 kb
in length, 150 kb in length, 200 kb in length or ranges from 28 kb
to 200 kb in length.
[0120] In a related aspect, novel Sugarcane centromere compositions
as characterized by sequence content, size or other parameters are
provided. Optionally, the minimal size of Sugarcane centromeric
sequence is utilized in mini-chromosome construction. Exemplary
sizes include a Sugarcane centromeric nucleic acid segment derived
from a portion of Sugarcane genomic DNA or synthesized based on a
Sugarcane satellite repeat sequence, that is less than or equal to
1000 kb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 400 kb, 300 kb,
200 kb, 190 kb, 150 kb, 100 kb, 95 kb, 90 kb, 85 kb, 80 kb, 75 kb,
70 kb, 65 kb, 60 kb, 55 kb, 50 kb, 45 kb, 40 kb, 35 kb, 30 kb, 28
kb, 25 kb, 20 kb, 17 kb, 15 kb, 12 kb, 10 kb, 7, kb, 6.4 kb, 5 kb,
or 2 kb in length. Exemplary inserts may range in size from 80 kb
to 100 kb, 7 kb to 190 kb, 7 kb to 12 kb, 5 kb to 10 kb, 3 kb to 10
kb, 3 kb to 7 kb, 5 kb to 7 kb, 10 to 30 kb, 15 to 30 kb, and 15 to
28 kb. Another related aspect is the novel structure of the
Sugarcane mini-chromosome, particularly structures lacking
bacterial sequences (e.g., sequences required for bacterial
propagation), referred to as backbone-free Sugarcane
mini-chromosomes.
[0121] In other exemplary embodiments, the invention contemplates
Sugarcane mini-chromosomes or other vectors comprising centromeric
nucleotide sequence that when hybridized to 1, 2, 3, 4, 5, 6, 7, 8
or more of the probes described in the examples herein, under
hybridization conditions described herein, e.g. low, medium or high
stringency, provides relative hybridization scores. Exemplary
stringent hybridization conditions comprise hybridization at
65.degree. C. and washing three times for 15 minutes with
0.25.times.SSC, 0.1% SDS at 65.degree. C. Additional exemplary
stringent hybridization conditions comprise hybridization in 0.02 M
to 0.15 M NaCl at temperatures of about 50.degree. C. to 70.degree.
C. or 0.5.times.SSC 0.25% SDS at 65.degree. for 15 minutes,
followed by a wash at 65.degree. C. for a half hour or
hybridization at 65.degree. C. for 14 hours followed by 3 washings
with 0.5.times.SSC, 1% SDS at 65.degree. C. Probe hybridization can
be scored visually to determine a binary (positive versus negative)
value, or the probes can be assigned a score based on the relative
strength of their hybridization on a 10 point scale. For example,
relative hybridization scores of 5 may be used to select clones
that hybridize well to the probe. Alternatively, a hybridization
signal greater than background for one or more of these probes can
be used to select clones. Modified or adchromosomal Sugarcane
plants or plant parts containing such Sugarcane mini-chromosomes
are contemplated.
[0122] The advantages of the present invention include: provision
of an autonomous, independent genetic linkage group for
accelerating Sugarcane breeding; lack of disruption of host
Sugarcane genome; multiple gene "stacking" of large and potentially
unlimited numbers of genes; uniform genetic composition of
exogenous DNA sequences in plant cells and plants containing
autonomous Sugarcane mini-chromosomes; defined genetic context for
predictable gene expression; and higher frequency occurrence and
recovery of Sugarcane plant cells and plants containing stably
maintained exogenous DNA due to elimination of an inefficient
integration step. In addition, Sugarcane mini-chromosomes that
increase total recoverable sugars or enhance the utility of
modified Sugarcane plants for use in biofuel production are
specifically envisioned.
I. Composition of Mini-Chromosomes and Mini-Chromosome
Construction
[0123] The Sugarcane mini-chromosome vector of the present
invention may contain a variety of elements, including (1)
sequences that function as Sugarcane centromeres, (2) one or more
exogenous nucleic acids, including, for example, plant-expressed
genes, or genes for non-coding RNAs, (3) sequences that function as
an origin of replication, which may be included in the region that
functions as a plant centromere, (4) optionally, a bacterial
plasmid backbone for propagation of the plasmid in bacteria, (5)
optionally, sequences that function as plant telomeres, (6)
optionally, additional "stuffer DNA" sequences that serve to
physically separate the various components on the Sugarcane
mini-chromosome from each other, (7) optionally "buffer" sequences
such as MARs (Matrix Attachment Regions) or SARs (Scaffold
Attachment Regions), (8) optionally marker sequences of any origin,
including but not limited to plant and bacterial origin, (9)
optionally, sequences that serve as recombination sites, and (10)
optionally, "chromatin packaging sequences" such as cohesion and
condensing binding sites.
[0124] The Sugarcane mini-chromosomes of the present invention may
be constructed to include various components which are novel, which
include, but are not limited to, the Sugarcane centromere
comprising novel repeating centromeric sequences, as described in
further detail below.
Novel Centromere Compositions
[0125] The centromere in the mini-chromosome of the present
invention may comprise novel repeating Sugarcane centromeric
sequences.
[0126] Vectors comprising one, two, three, four, five, six, seven,
eight, nine, ten, 15 or 20 or more of the elements contained in any
of the exemplary vectors described in the examples below are also
contemplated.
[0127] The invention specifically contemplates the alternative use
of fragments or variants (mutants) of any of the nucleic acids
described herein that retain the desired activity, including
nucleic acids that function as Sugarcane centromeres, nucleic acids
that function as promoters or other regulatory control sequences,
or exogenous nucleic acids. Variants may have one or more
additions, substitutions or deletions of nucleotides within the
original nucleotide sequence or consensus sequence. Variants
include nucleic acid sequences that are at least 50%, 55%, 60, 65,
70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, or 100% identical to the original nucleic acid
sequence. Variants also include nucleic acid sequences that
hybridize under low, medium, high or very high stringency
conditions to the original nucleic acid sequence. Similarly, the
invention also contemplates the alternative use of fragments or
variants of any of the polypeptides described herein.
[0128] The comparison of sequences and determination of percent
identity between two nucleotide sequences can be accomplished using
a mathematical algorithm. For example, the percent identity between
two nucleotide sequences can be determined using the Needleman and
Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm which has been
incorporated into the GAP program in the GCG software package
(available at www.gcg.com), using either a Blossum 62 matrix or a
PAM250 matrix. The parameters can be set so as to maximize the
percent identity.
[0129] As used herein, the term "hybridizes under low stringency,
medium stringency, and high stringency conditions" describes
conditions for hybridization and washing. Guidance for performing
hybridization reactions can be found in Current Protocols in
Molecular Biology (1989) John Wiley & Sons, N.Y., 6.3.1-6.3.6,
which is incorporated by reference. Aqueous and non-aqueous methods
are described in that reference and either can be used. Specific
hybridization conditions referred to herein are as follows: 1) low
stringency hybridization conditions in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
two washes in 0.5.times.SSC, 0.1% SDS, at least at 50.degree. C.;
2) medium stringency hybridization conditions in 6.times.SSC at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 55.degree. C.; 3) high stringency
hybridization conditions are hybridization at 65.degree. C. for
12-18 hours and washing three times for 15-90 minutes with
0.25.times.SSC, 0.1% SDS at 65.degree. C. Additional exemplary
stringent hybridization conditions comprise 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 65.degree. C. Other exemplary highly selective or
stringent hybridization conditions comprise 0.02 M to 0.15 M NaCl
at temperatures of about 50.degree. C. to 70.degree. C. or
0.5.times.SSC 0.25% SDS at 65.degree. for 12-15 hours, followed
three washes at 65.degree. C. for 15-90 minutes each.
Sugarcane Mini-Chromosome Sequence Content and Structure
[0130] Sugarcane-expressed genes from non-plant sources may be
modified to accommodate Sugarcane codon usage, to insert preferred
motifs near the translation initiation ATG codon, to remove
sequences recognized in plants as 5' or 3' splice sites, or to
better reflect plant GC/AT content. Plant genes typically have a GC
content of more than 35%, and coding sequences which are rich in A
and T nucleotides can be problematic. For example, ATTTA motifs may
destabilize mRNA; plant polyadenylation signals such as AATAAA at
inappropriate positions within the message may cause premature
truncation of transcription; and monocotyledons such as Sugarcane
may recognize AT-rich sequences as splice sites.
[0131] Each exogenous nucleic acid or Sugarcane-expressed gene may
include a promoter, a coding region and a terminator sequence,
which may be separated from each other by restriction endonuclease
sites or recombination sites or both. Genes may also include
introns, which may be present in any number and at any position
within the transcribed portion of the gene, including the 5'
untranslated sequence, the coding region and the 3' untranslated
sequence. Introns may be natural plant introns derived from any
plant, or artificial introns based on the splice site consensus
that has been defined for plant species. Some intron sequences have
been shown to enhance expression in plants. Optionally the
exogenous nucleic acid may include a plant transcriptional
terminator, non-translated leader sequences derived from viruses
that enhance expression, a minimal promoter, or a signal sequence
controlling the targeting of gene products to plant compartments or
organelles.
[0132] The coding regions of the genes can encode any protein,
including but not limited to visible marker genes (for example,
fluorescent protein genes, other genes conferring a visible
phenotype to the plant) or other screenable or selectable marker
genes (for example, conferring resistance to antibiotics,
herbicides or other toxic compounds or encoding a protein that
confers a growth advantage to the cell expressing the protein) or
genes which confer some commercial or agronomic value to the
modified or adchromosomal Sugarcane plant. Multiple genes can be
placed on the same Sugarcane mini-chromosome vector. The genes may
be separated from each other by restriction endonuclease sites,
homing endonuclease sites, recombination sites or any combinations
thereof. Alternatively, the cloning process can be executed in a
manner that destroys the intervening restriction sites. Any number
of genes can be present.
[0133] The Sugarcane mini-chromosome vector may also contain a
bacterial plasmid backbone for propagation of the plasmid in
bacteria such as E. coli, A. tumefaciens, or A. rhizogenes. The
plasmid backbone may be that of a low-copy vector or in other
embodiments it may be desirable to use a mid to high level copy
backbone. In one embodiment of the invention, this backbone
contains the replicon of the F' plasmid of E. coli. However, other
plasmid replicons, such as the bacteriophage P1 replicon, or other
low-copy plasmid systems such as the RK2 replication origin, may
also be used. The backbone may include one or several
antibiotic-resistance genes conferring resistance to a specific
antibiotic to the bacterial cell in which the plasmid is present.
Bacterial antibiotic-resistance genes include but are not limited
to kanamycin-, ampicillin-, chloramphenicol-, streptomycin-,
spectinomycin-, tetracycline- and gentamycin-resistance genes.
[0134] The Sugarcane mini-chromosome vector may also contain plant
telomeres. An exemplary telomere sequence is TTTAGGG or its
complement. Telomeres are 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 (Richards et al., Cell, 1988 Apr. 8; 53(1):127-36;
Ausubel et al., Current Protocols in Molecular Biology, Wiley &
Sons, 1997).
[0135] Additionally, the Sugarcane mini-chromosome vector may
contain "stuffer DNA" sequences that serve to separate the various
components on the mini-chromosome (centromere, genes, telomeres)
from each other. The stuffer DNA may be of any origin, prokaryotic
or eukaryotic, and from any genome or species, plant, animal,
microbe or organelle, or may be of synthetic origin. The stuffer
DNA can range from 100 bp to 10 Mb in length and can be repetitive
in sequence, with unit repeats from 10 to 1,000,000 bp. Examples of
repetitive sequences that can be used as stuffer DNAs include but
are not limited to: rDNA, satellite repeats, retroelements,
transposons, pseudogenes, transcribed genes, microsatellites, tDNA
genes, short sequence repeats and combinations thereof.
Alternatively, the stuffer DNA can consist of unique,
non-repetitive DNA of any origin or sequence. The stuffer sequences
may also include DNA with the ability to form boundary domains,
such as but not limited to scaffold attachment regions (SARs) or
matrix attachment regions (MARs). The stuffer DNA may be entirely
synthetic, composed of random sequence. In this case, the stuffer
DNA may have any base composition, or any A/T or G/C content. For
example, the G/C content of the stuffer DNA could resemble that of
the plant (.about.30-40%), or could be much lower (0-30%) or much
higher (40-100%). Alternatively, the stuffer sequences could be
synthesized to contain an excess of any given nucleotide such as A,
C, G or T. Different synthetic stuffers of different compositions
may also be combined with each other. For example a fragment with
low G/C content may be flanked or abutted by a fragment of medium
or high G/C content, or vice versa.
[0136] In one embodiment of the invention, the Sugarcane
mini-chromosome has a circular structure without telomeres. In
another embodiment, the Sugarcane mini-chromosome has a circular
structure with telomeres. In a third embodiment, the Sugarcane
mini-chromosome has a linear structure with telomeres, for example,
as would result if a "linear" structure were to be cut with a
unique endonuclease, exposing the telomeres at the ends of a DNA
molecule that contains all of the sequence contained in the
original, closed construct with the exception of an
antibiotic-resistance gene. In a fourth embodiment of the
invention, the telomeres could be placed in such a manner that the
bacterial replicon, backbone sequences, antibiotic-resistance genes
and any other sequences of bacterial origin and present for the
purposes of propagation of the Sugarcane mini-chromosome in
bacteria, can be removed from the plant-expressed genes, the
centromere, telomeres, and other sequences by cutting the structure
with, for example, a unique endonuclease. This results in a
Sugarcane mini-chromosome from which much of, or even all,
bacterial sequences have been removed. In this embodiment,
bacterial sequence present between or among the plant-expressed
genes or other Sugarcane mini-chromosome sequences would be excised
prior to removal of the remaining bacterial sequences by cutting
the Sugarcane mini-chromosome with an endonuclease and re-ligating
the structure such that the antibiotic-resistance gene has been
lost. The unique endonuclease site may be the recognition sequence
of any of a number of endonucleases including but not limited to
restriction endonucleases, meganucleases, or homing endonuclease.
Alternatively, the endonucleases and their sites can be replaced
with any specific DNA cutting mechanism and its specific
recognition site such as rare-cutting endonuclease or recombinase
and its specific recognition site, as long as that site is present
in the Sugarcane mini-chromosomes only at the indicated
positions.
[0137] Various structural configurations are possible by which
Sugarcane mini-chromosome elements can be oriented with respect to
each other. A Sugarcane centromere can be placed on a Sugarcane
mini-chromosome either between genes or outside a cluster of genes
next to one telomere or next to the other telomere. Stuffer DNAs
can be combined with these configurations to place the stuffer
sequences inside the telomeres, around the centromere between genes
or any combination thereof. Thus, a large number of alternative
Sugarcane mini-chromosome structures are possible, depending on the
relative placement of centromere DNA, genes, stuffer DNAs,
bacterial sequences, telomeres, and other sequences. The sequence
content of each of these variants is the same, but their structure
may be different depending on how the sequences are placed. These
variations in architecture are possible both for linear and for
circular Sugarcane mini-chromosomes.
Exemplary Centromere Components
[0138] Sugarcane centromere components may be isolated or derived
from a native plant genome, for example, modified through
recombinant techniques or through the cell-based techniques
described below. Alternatively, wholly artificial centromere
components may be constructed using as a general guide the sequence
of native Sugarcane centromeres such as native satellite repeat
sequences. Combinations of centromere components derived from
natural sources and/or combinations of naturally derived and
artificial components are also contemplated.
[0139] In one embodiment, the Sugarcane centromere contains n
copies of a repeated nucleotide sequence obtained by the methods
disclosed herein; wherein n is at least 2. In another embodiment,
the Sugarcane centromere contains n copies of interdigitated
repeats. An interdigitated repeat is a DNA sequence that consists
of two distinct repetitive elements that combine to create a unique
permutation. 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. Moreover, the copies, while largely
identical, can vary from each other. Such repeat variation is
commonly observed in naturally occurring centromeres. The length of
the repeat may vary, and can range from about 20 bp to about 360
bp, from about 20 bp to about 250 bp, from about 50 bp to about 225
bp, from 20 bp to 137 bp, from about 75 bp to about 210 bp, from
about 100 bp to about 205 bp, from about 125 bp to about 200 bp,
from about 150 bp to about 195 bp, from about 160 bp to about 190
and from about 170 bp to about 185 bp including about 180 bp.
Exemplary repeats include without limitation a 92 bp repeat, a 97
bp repeat or a 100 bp repeat. Larger repeats including those up to
3,465 bp or 3,500 bp or 3,600 bp or 3,700 bp are also anticipated
by the current invention.
[0140] The invention contemplates that two or more of these
Sugarcane repeated nucleotide sequences, or similar Sugarcane
repeated nucleotide sequences, may be oriented head to tail within
the centromere. The term "head to tail" refers to multiple
consecutive copies of the same or similar repeated nucleotide
sequence (e.g., at least 70% identical) that are in the same 5'-3'
orientation. The invention also contemplates that two or more of
these repeated nucleotide sequences may be consecutive within the
Sugarcane centromere. The term "consecutive" refers to the same or
similar repeated nucleotide sequences (e.g., at least 70%
identical) that follow one after another without being interrupted
by other significant sequence elements. Such consecutive repeated
nucleotide sequences may be in any orientation, e.g. head to tail,
tail to tail, or head to head, and may be separated by n number of
nucleotides, wherein n ranges from 1 to 10, or 1 to 20, or 1 to 30,
or 1 to 40, or 1 to 50. Exemplary repeated nucleotide sequences
derived from Sugarcane, and identified by the methods described
herein, are set out as SEQ ID NOS: 1-202, SEQ ID NO: 204 and CRS
(SEQ ID NO: 203).
[0141] Modification of Sugarcane Centromeres Isolated from Native
Plant Genome
[0142] Modification and changes may be made in the Sugarcane
centromeric DNA segments specifically described herein and still
obtain a functional molecule with desirable characteristics. Such
modified Sugarcane centromeres are also encompassed by the present
invention. The following is a discussion based upon changing the
nucleic acids of a Sugarcane centromere to create an equivalent, or
even an improved, second generation molecule.
[0143] In particular embodiments of the invention, mutated
Sugarcane centromeric sequences are contemplated to be useful for
increasing the utility of the Sugarcane centromere. Without being
bound by any theory of the invention, it is specifically
contemplated that the function of the Sugarcane centromeres of the
current invention may be based in part or in whole upon the
secondary structure of the DNA sequences of the Sugarcane
centromere, modification of the DNA with methyl groups or other
adducts, and/or the proteins which interact with the Sugarcane
centromere. By changing the DNA sequence of the Sugarcane
centromere, one may alter the affinity of one or more
centromere-associated protein(s) for the Sugarcane centromere
and/or the secondary structure or modification of the Sugarcane
centromeric sequences, thereby changing the activity of the
Sugarcane centromere. Alternatively, changes may be made in the
Sugarcane centromeres of the invention which do not affect the
activity of the Sugarcane centromere. Changes in the Sugarcane
centromeric sequences which reduce the size of the DNA segment
needed to confer Sugarcane centromere activity are contemplated to
be particularly useful in the current invention, as would changes
which increased the fidelity with which the Sugarcane centromere
was transmitted during mitosis or meiosis.
[0144] Modification of Sugarcane Centromeres by Passage Through
Bacteria Sugarcane or Other Hosts or Processes
[0145] In the methods of the present invention, the resulting
Sugarcane mini-chromosome DNA sequence may also be a derivative of
the parental clone or Sugarcane centromere clone having
substitutions, deletions, insertions, duplications and/or
rearrangements of one or more nucleotides in the nucleic acid
sequence. Such nucleotide mutations may occur individually or
consecutively in stretches of about 1, 2, 3, 4, 5, 6, 7, 8, 9 10,
15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175,
200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000,
4000, 8000, 10000, 50000, 100000, and about 200000, including all
ranges in-between.
[0146] Variations of Sugarcane mini-chromosomes may arise through
passage of Sugarcane mini-chromosomes through various hosts
including virus, bacteria, yeast, or another prokaryotic or
eukaryotic organism and may occur through passage of multiple hosts
or an individual host. Variations may also occur by replicating the
Sugarcane mini-chromosome in vitro.
[0147] Derivatives may be identified through sequence analysis, or
variations in Sugarcane mini-chromosome molecular weight through
electrophoresis such as, but not limited to, CHEF gel analysis,
column or gradient separation, or any other methods used in the
field to determine and/or analyze DNA molecular weight or sequence
content. Alternately, derivatives may be identified by the altered
activity of a derivative in conferring Sugarcane centromere
function to a Sugarcane mini-chromosome.
[0148] Production or Synthesis of Synthetic Sugarcane Centromere
Repeat Sequences
[0149] These artificially synthesized repeated nucleotide sequences
of the invention may be derived from natural Sugarcane centromere
sequences, combinations or fragments of natural Sugarcane
centromere sequences including a combination of repeats of
different lengths, a combination of different sequences, a
combination of both different repeat lengths and different
sequences, a combination of different artificially synthesized
sequences or a combination of natural Sugarcane centromere
sequence(s) and artificially synthesized sequence(s). The synthetic
nucleotide sequences and arrays of these synthetic repeat sequences
may be generated using any technique known in the art including PCR
from genomic DNA, e.g. the methods described in Example 1, or by
custom polynucleotide synthesis.
[0150] Polynucleotide synthesis is the non-biological, chemical
synthesis of defined sequences of nucleic acids using automated
synthesizers. Oligonucleotides may be chemically synthesized,
purified and then these oligonucleotides are connected by specific
annealing and standard ligation or polymerase reactions. Exemplary
ligation methods include ligation of phosphorylated overlapping
oligonucleotides (Gupta et al. Proc. Natl Acad. Sci. USA, 60,
1338-1344, Fuhrmann et al. Plant J. 1999 August; 19(3):353-61), the
FokI method (Mandecki et al. Gene, 68, 101-107) and a modified form
of ligase chain reaction for gene synthesis. In addition, PCR
assembly approaches may be used which generally employ
oligonucleotides of 40-50 nt long that overlap each other. These
oligonucleotides are designed to cover most of the sequence of both
strands, and the full-length molecule is generated progressively by
overlap extension PCR (Stemmer et al. Gene, 164, 49-53).,
thermodynamically balanced inside-out PCR (Gao et al. Nucleic Acids
Res. 2003 Nov. 15; 31(22):e143) or combined approaches (Young et
al. Nucleic Acids Res. 2004 Apr. 15; 32(7):e59).
Exemplary Exogenous Nucleic Acids Including Plant-Expressed
Genes
[0151] Of particular interest in the present invention are
exogenous nucleic acids which when introduced into Sugarcane plants
will alter the phenotype of the plant, a plant organ, plant tissue,
or a portion of the plant. Exemplary exogenous nucleic acids encode
polypeptides. Other exemplary exogenous nucleic acids alter
expression of exogenous or endogenous genes, either increasing or
decreasing expression, optionally in response to a specific signal
or stimulus.
[0152] As used herein, the term "trait" can refer either to the
altered phenotype of interest or the nucleic acid which causes the
altered phenotype of interest.
[0153] One of the major purposes of transformation of Sugarcane is
to add some commercially desirable, agronomically important traits
to the plant. Such traits include, but are not limited to, enhanced
production of total recoverable sugars; utility for production of
biofuels; herbicide resistance or tolerance, insect (pest)
resistance or tolerance; disease resistance or tolerance (viral,
bacterial, fungal, nematode or other pathogens); stress tolerance
and/or resistance, as exemplified by resistance or tolerance to
drought, heat, chilling, freezing, excessive moisture, salt stress,
mechanical stress, extreme acidity, alkalinity, toxins, UV light,
ionizing radiation or oxidative stress; increased yields, increased
biomass, whether in quantity or quality; enhanced or altered
nutrient acquisition and enhanced or altered metabolic efficiency;
enhanced or altered nutritional content and makeup of plant tissues
used for food, feed, fiber or processing; physical appearance; male
sterility; drydown; standability; prolificacy; starch quantity and
quality; oil quantity and quality; protein quality and quantity;
amino acid composition; modified chemical production; altered
pharmaceutical or nutraceutical properties; altered bioremediation
properties; increased biomass; altered growth rate; altered
fitness; altered biodegradability; altered CO.sub.2 fixation;
presence of bioindicator activity; altered digestibility by humans
or animals; altered allergenicity; altered mating characteristics;
altered pollen dispersal; improved environmental impact; altered
nitrogen fixation capability; 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, food
additives, carbohydrates, RNAs, lipids, fuels, dyes, pigments,
vitamins, scents, flavors, vaccines, antibodies, hormones, and the
like; and alterations in plant architecture or development,
including changes in developmental timing, photosynthesis, signal
transduction, cell growth, reproduction, or differentiation.
Additionally one could create a library of an entire genome (or a
portion thereof) from any organism or organelle including mammals,
plants, microbes, fungi, or bacteria, represented on Sugarcane
mini-chromosomes.
[0154] In one embodiment, the Sugarcane plant comprising a
Sugarcane mini-chromosome or recombinant chromosome may exhibit
increased or decreased expression or accumulation of a product of
the plant, which may be a natural product of the plant or a new or
altered product of the plant. Exemplary products include an enzyme,
an RNA molecule, a nutritional protein, a structural protein, an
amino acid, a lipid, a fatty acid, a polysaccharide, a sugar, an
alcohol, an alkaloid, a carotenoid, a propanoid, a phenylpropanoid,
or terpenoid, a steroid, a flavonoid, a phenolic compound, an
anthocyanin, a pigment, a vitamin or a plant hormone. In another
embodiment, the Sugarcane plant comprising a Sugarcane
mini-chromosome or recombinant chromosome has enhanced or
diminished requirements for light, water, nitrogen, or trace
elements. In another embodiment the Sugarcane plant comprising a
Sugarcane mini-chromosome or recombinant chromosome has an enhanced
ability to capture or fix nitrogen from its environment. In yet
another embodiment, the Sugarcane plant comprising a Sugarcane
mini-chromosome or recombinant chromosome is enriched for an
essential amino acid as a proportion of a protein fraction of the
plant. The protein fraction may be, for example, total seed
protein, soluble protein, insoluble protein, water-extractable
protein, and lipid-associated protein. The Sugarcane
mini-chromosome or recombinant chromosome may include genes that
cause the overexpression, underexpression, antisense modulation,
sense suppression, inducible expression, inducible repression, or
inducible modulation of another gene.
[0155] A brief summary of exemplary improved properties and
polypeptides of interest for either increased or decreased
expression is provided below.
[0156] (1) Herbicide Resistance
[0157] An herbicide resistance (or tolerance) trait is a
characteristic of a Sugarcane plant comprising a Sugarcane
mini-chromosome or recombinant chromosome that is resistant to
dosages of an herbicide that is typically lethal to a wild type
plant. Exemplary herbicides for which resistance is useful in a
plant include glyphosate herbicides, phosphinothricin herbicides,
oxynil herbicides, imidazolinone herbicides, dinitroaniline
herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos
herbicides, sulfonamide herbicides and glufosinate herbicides.
Other herbicides would be useful as would combinations of herbicide
genes on the same Sugarcane mini-chromosome or recombinant
chromosome.
[0158] The genes encoding phosphinothricin acetyltransferase (bar),
glyphosate tolerant EPSP synthase genes, glyphosate
acetyltransferase, the glyphosate degradative enzyme gene gox
encoding glyphosate oxidoreductase, deh (encoding a dehalogenase
enzyme that inactivates dalapon), herbicide resistant (e.g.,
sulfonylurca and imidazolinone) 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 gene codes for an enzyme, phosphinothricin
acetyltransferase (PAT), which inactivates the herbicide
phosphinothricin and prevents this compound from inhibiting
glutamine synthetase enzymes. The enzyme 5 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. The glyphosate
acetyl transferase gene inactivates the herbicide glyphosate and
prevents this compound from inhibiting EPSP synthase.
[0159] Polypeptides that may produce plants having tolerance to
plant herbicides include polypeptides involved in the shikimate
pathway, which are of interest for providing glyphosate tolerant
plants. Such polypeptides include polypeptides involved in
biosynthesis of chorismate, phenylalanine, tyrosine and
tryptophan.
[0160] (ii) Insect Resistance
[0161] Potential insect resistance (or tolerance) genes that can be
introduced include Bacillus thuringiensis toxin genes or Bt genes
(Watrud et al., In: Engineered Organisms and the Environment,
1985). Bt genes may provide resistance to lepidopteran or
coleopteran pests such as European Corn Borer (ECB). Exemplary Bt
toxin genes for use in such embodiments include the CryIA(b) and
CryIA(c) genes. Endotoxin genes from other species of B.
thuringiensis which affect insect growth or development also may be
employed in this regard.
[0162] It is contemplated that in some embodiments Bt genes for use
in the Sugarcane mini-chromosomes or recombinant chromosomes
disclosed herein will be those in which the coding sequence has
been modified to effect increased expression in plants, and for
example, in monocot plants including Sugarcane. Means for preparing
synthetic genes are well known in the art and are disclosed in, for
example, U.S. Pat. No. 5,500,365 and U.S. Pat. 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 CryIA(b) gene (Perlak et al., Proc.
Natl. Acad. Sci. USA, 88:3324-3328, 1991), and the synthetic
CryIA(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.
TABLE-US-00001 TABLE 1 Bacillus thuringiensis Endotoxin Genes.sup.a
New Nomenclature Old Nomenclature GenBank Accession Cry1Aa CryIA(a)
M11250 Cry1Ab CryIA(b) M13898 Cry1Ac CryIA(c) M11068 Cry1Ad
CryIA(d) M73250 Cry1Ae CryIA(e) M65252 Cry1Ba CryIB X06711 Cry1Bb
ET5 L32020 Cry1Bc PEG5 Z46442 Cry1Bd CryE1 U70726 Cry1Ca CryIC
X07518 Cry1Cb CryIC(b) M97880 Cry1Da CryID X54160 Cry1Db PrtB
Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b) M73253 Cry1Fa CryIF
M63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1Gb CryH2 U70725
Cry1Ha PrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1Ib CryV
U07642 Cry1Ja ET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2Aa
CryIIA M31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252 Cry3A
CryIIIA M22472 Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2 M89794 Cry3C
CryIIID X59797 Cry4A CryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa
CryVA(a) L07025 Cry5Ab CryVA(b) L07026 Cry6A CryVIA L07022 Cry6B
CryVIB L07024 Cry7Aa CryIIIC M64478 Cry7Ab CryIIICb U04367 Cry8A
CryIIIE U04364 Cry8B CryIIIG U04365 Cry8C CryIIIF U04366 Cry9A
CryIG X58120 Cry9B CryIX X75019 Cry9C CryIH Z37527 Cry10A CryIVC
M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902 Cry12A CryVB L07027
Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34 kDa M76442 Cry16A
cbm7l X94146 Cry17A cbm71 X99478 Cry18A CryBP1 X99049 Cry19A Jeg65
Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt2A CytB Z14147
Cyt2B CytB U52043 .sup.aAdapted from:
http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html
[0163] Protease inhibitors also may provide insect resistance
(Johnson et al., Proc Natl Acad Sci USA. 1989 December; 86(24):
9871-9875), and will thus have utility in Sugarcane transformation.
The use of a pinII gene in combination with a Bt toxin gene, the
combined effect of which has been discovered to produce synergistic
insecticidal activity is envisioned to be particularly useful.
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.
[0164] Amylase inhibitors are found in various plant species and
are used to ward off insect predation via inhibition of the
digestive amylases of attacking insects. Several amylase inhibitor
genes have been isolated from plants and some have been introduced
as exogenous nucleic acids, conferring an insect resistant
phenotype that is potentially useful ("Plants, Genes, and Crop
Biotechnology" by Maarten J. Chrispeels and David E. Sadava (2003)
Jones and Bartlett Press).
[0165] Genes encoding lectins may confer additional or alternative
insecticide properties. Lectins 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 with activity against weevils, ECB
and rootworm (Murdock et al., Phytochemistry, 29:85-89, 1990,
Czapla & Lang, J. Econ. Entomol., 83:2480-2485, 1990). Lectin
genes contemplated to be useful include, for example, barley and
wheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., J.
Sci. Food. Agric., 35:373-380, 1984).
[0166] 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., Nature, 344:458-461, 1990).
[0167] Genes that 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 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 exogenous nucleic acids of the
present invention.
[0168] Genes that code for enzymes that facilitate the production
of compounds that reduce the nutritional quality of the host
Sugarcane plant to insect pests also are encompassed by the present
invention. It may be possible, for instance, to confer insecticidal
activity on a Sugarcane 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 modified plants with enhanced lipoxygenase
activity which may be resistant to insect feeding.
[0169] Tripsacum dactyloides is a species of grass that is
resistant to certain insects, including 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 conferring 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, Proceedings North Central Branch Entomological
Society of America, 27:91-95, 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 Sugarcane plants.
[0170] Further genes encoding proteins characterized as having
potential insecticidal activity also may be used as exogenous
nucleic acids in accordance herewith. Such genes include, for
example, the cowpea trypsin inhibitor (CpTI; Hilder et al., Nature,
330:160-163, 1987) which may be used as a rootworm deterrent; genes
encoding avermectin (Avermectin and Abamectin., Campbell, W. C.,
Ed., 1989; Ikeda et al., J. Bacteriol., 169:5615-5621, 1987) which
may prove particularly useful as a corn rootworm deterrent;
ribosome inactivating protein genes; and even genes that regulate
plant structures. Sugarcane plants comprising a Sugarcane
mini-chromosome or recombinant chromosome comprising 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.
[0171] Polypeptides that may improve Sugarcane tolerance to the
effects of plant pests or pathogens include proteases, polypeptides
involved in anthocyanin biosynthesis, polypeptides involved in cell
wall metabolism, including cellulases, glucosidases, pectin
methylesterase, pectinase, polygalacturonase, chitinase,
chitosanase, and cellulose synthase, and polypeptides involved in
biosynthesis of terpenoids or indole for production of bioactive
metabolites to provide defense against herbivorous insects. It is
also anticipated that combinations of different insect resistance
genes on the same Sugarcane mini-chromosome or recombinant
chromosome will be particularly useful.
[0172] Vegetative Insecticidal Proteins (VIP) are another class of
proteins originally found to be produced in the vegetative growth
phase of the bacterium, Bacillus cereus, but do have a spectrum of
insect lethality similar to the insecticidal genes found in strains
of Bacillus thuringiensis. Both the vip1a and vip3A genes have been
isolated and have demonstrated insect toxicity. It is anticipated
that such genes may be used in modified plants to confer insect
resistance ("Plants, Genes, and Crop Biotechnology" by Maarten J.
Chrispeels and David E. Sadava (2003) Jones and Bartlett
Press).
[0173] (ii) Environment or Stress Resistance
[0174] Improvement of a Sugarcane plant's 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., J. Plant Physiol., 135:351-354, 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., The
EMBO J., 4685-4692, 1992). Resistance to oxidative stress (often
exacerbated by conditions such as chilling 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., Ann Rev. Plant
Physiol., 43:83-116, 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.
[0175] It is contemplated that the expression of novel genes that
favorably affect Sugarcane plant water content, total water
potential, osmotic potential, or 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
Sugarcane plant's 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 at al., J.
Bacteriology, 174:889-898, 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 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., Science, 259:508-510, 1993, Tarczynski et al
Proc. Natl. Acad. Sci. USA, 89:1-5, 1993).
[0176] 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., J. Expt. Zoology, 252:9-15, 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., Biotropica, 24:121-133, 1992), sorbitol, dulcitol
(Karsten et al., Botanica Marina, 35:11-19, 1992), glucosylglycerol
(Reed et al., J. Gen. Microbiology, 130:1-4, 1984; Erdmann et al.,
J. Gen. Microbiology, 138:363-368, 1992), sucrose, stachyose
(Koster and Leopold, Plant Physiol., 88:829-832, 1988; Blackman et
al., Plant Physiol., 100:225-230, 1992), raflinose (Bernal Lugo and
Leopold, Plant Physiol., 98:1207-1210, 1992), proline (Rensburg at
al., J. Plant Physiol., 141:188-194, 1993), glycine betaine,
ononitol and pinitol (Vernon and Bohnert, The EMBO J.,
11:2077-2085, 1992). Continued growth and increased reproductive
fitness during times of stress may be augmented by introduction and
expression of genes such as those controlling the osmotically
active compounds discussed above and other such compounds. Genes
which promote the synthesis of an osmotically active polyol
compound include genes which encode the enzymes mannitol 1
phosphate dehydrogenase, trehalose 6 phosphate synthase and
myoinositol 0 methyltransferase.
[0177] It is contemplated that the expression of specific proteins
also may increase drought tolerance in Sugarcane. Three classes of
Late Embryogenic Abundant (LEA) Proteins have been assigned based
on structural similarities (see Dure et al., Plant Molecular
Biology, 12:475-486, 1989). All three classes of LEAs have been
demonstrated in maturing (e.g. 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 (e.g. Mundy and Chua, The EMBO J.,
7:2279-2286, 1988; Piatkowski et al., Plant Physiol., 94:1682-1688,
1990; Yamaguchi Shinozaki et al., Plant Cell Physiol., 33:217-224,
1992). Expression of a Type III LEA (HVA 1) in tobacco was found to
influence plant height, maturity and drought tolerance
(Fitzpatrick, Gen. Engineering News, 22:7, 1993). In rice,
expression of the HVA 1 gene influenced tolerance to water deficit
and salinity (Xu et al., Plant Physiol., 110:249-257, 1996).
Expression of structural genes from any of the three LEA groups may
therefore confer drought tolerance. Other types of proteins induced
during water stress include thiol proteases, aldolases or
transmembrane transporters (Guerrero et al., Plant Molecular
Biology, 15:11-26, 1990), which may confer various protective
and/or repair type functions during drought stress. It also is
contemplated that genes that effect lipid biosynthesis and hence
membrane composition might also be useful in conferring drought
resistance in Sugarcane.
[0178] 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 Sugarcane. 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. Benefits may be conferred
via constitutive expression of these genes; alternatively, one
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., Plant Molecular
Biology, 15:11-26, 1990 and Shagan et al., Plant Physiol.,
101:1397-1398, 1993 which are incorporated herein by reference).
Spatial and temporal expression patterns of these genes may enable
plants to better withstand stress.
[0179] 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, e.g., 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.
[0180] Given the overall role of water in determining yield, it is
contemplated that enabling Sugarcane 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 Sugarcane plants to maximize water usage across a full
range of stresses relating to water availability, yield stability
or consistency of yield performance may be realized.
[0181] Polypeptides that may improve stress tolerance in Sugarcane
under a variety of stress conditions include polypeptides involved
in gene regulation, such as serine/threonine-protein kinases, MAP
kinases, MAP kinase kinases, and MAP kinase kinase kinases;
polypeptides that act as receptors for signal transduction and
regulation, such as receptor protein kinases; intracellular
signaling proteins, such as protein phosphatases, GTP binding
proteins, and phospholipid signaling proteins; polypeptides
involved in arginine biosynthesis; polypeptides involved in ATP
metabolism, including for example ATPase, adenylate transporters,
and polypeptides involved in ATP synthesis and transport,
polypeptides involved in glycine betaine, jasmonic acid, flavonoid
or steroid biosynthesis; and hemoglobin. Enhanced or reduced
activity of such polypeptides in Sugarcane plants comprising a
Sugarcane mini-chromosome or recombinant chromosome will provide
changes in the ability of the plants to respond to a variety of
environmental stresses, such as chemical stress, drought stress and
pest stress.
[0182] Other polypeptides that may improve Sugarcane tolerance to
cold or freezing temperatures include polypeptides involved in
biosynthesis of trehalose or raffinose, polypeptides encoded by
cold induced genes, fatty acyl desaturases and other polypeptides
involved in glycerolipid or membrane lipid biosynthesis, which find
use in modification of membrane fatty acid composition, alternative
oxidase, calcium-dependent protein kinases, LEA proteins or
uncoupling protein.
[0183] Other polypeptides that may improve Sugarcane tolerance to
heat include polypeptides involved in biosynthesis of trehalose,
polypeptides involved in glycerolipid biosynthesis or membrane
lipid metabolism (for altering membrane fatty acid composition),
heat shock proteins or mitochondrial NDK.
[0184] Other polypeptides that may improve Sugarcane tolerance to
extreme osmotic conditions include polypeptides involved in proline
biosynthesis.
[0185] Other polypeptides that may improve Sugarcane tolerance to
drought conditions include aquaporins, polypeptides involved in
biosynthesis of trehalose or wax, LEA proteins or invertase.
[0186] (iv) Disease Resistance
[0187] It is proposed that increased resistance (or tolerance) to
diseases may be realized through introduction of genes into
Sugarcane. It is possible to produce resistance to diseases caused
by viruses, viroids, bacteria, fungi and nematodes. It also is
contemplated that control of mycotoxin producing organisms may be
realized through expression of introduced genes. Resistance can be
affected through suppression of endogenous factors that encourage
disease-causing interactions, expression of exogenous factors that
are toxic to or otherwise provide protection from pathogens, or
expression of factors that enhance Sugarcane's own defense
responses.
[0188] Resistance to viruses may be produced through expression of
novel genes in Sugarcane. For example, it has been demonstrated
that expression of a viral coat protein in a modified plant can
impart resistance to infection of the plant by that virus and
perhaps other closely related viruses (Cuozzo et al.,
Bio/Technology, 6:549-553, 1988, Hemenway at al., The EMBO J.,
7:1273-1280, 1988, Abel et al., Science, 232:738-743, 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.
[0189] It is proposed that increased resistance to diseases caused
by bacteria and fungi Sugarcane may be realized through
introduction of novel genes. It is contemplated that genes encoding
so called "peptide antibiotics," pathogenesis related (PR)
proteins, toxin resistance, or 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 Sugarcane 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 beta 1,3 glucanases,
chitinases, and osmotin and other 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), or 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
Sugarcane host 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.
[0190] Polypeptides useful for imparting improved disease responses
to Sugarcane include polypeptides encoded by cercosporin induced
genes, antifungal proteins and proteins encoded by R-genes or SAR
genes.
[0191] Agronomically important diseases in Sugarcane include but
are not limited to: pineapple disease of Sugarcane, pokkah boeng
disease of Sugarcane, Sugarcane eye spot disease, Sugarcane leaf
scald disease, Sugarcane mosaic virus disease, Sugarcane ratoon
stunting disease, Sugarcane red rot Disease, Sugarcane rust
Disease, Sugarcane smut disease, Metarhizium anisopliae, Ustilago
scitaminea, Colletotrichum falcatum, Fusarium moniliformae,
Cephalosporium sacchari, Certocystis paradoxa, Cercospora,
Helminthosporium and Leptosphaeria, Puccinia. graminicolum,
Puccinia aphanidermatum and Puccinia catenulatum, Xanthomonas
albilineans, Leifsonia xyli, Sugarcane mosaic virus (SCMV)
(Potyvirdae), Sugarcane bacilliform virus (SCBV)
(Pararetroviridae), Sugarcane yellow leaf syndrome (YLS), and
Sugarcane yellow loaf virus (ScYLV).
[0192] (v) Plant Agronomic Characteristics
[0193] Temperature also influences where Sugarcane can be grown.
Within the areas where it is possible to grow Sugarcane, 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 within the
required period of time with maximum possible yield. It is
considered that genes that influence maturity can be identified and
introduced into Sugarcane lines to create new varieties adapted to
different growing locations or the same growing location, but
having improved yield at harvest. Expression of genes that are
involved in regulation of plant development may be especially
useful.
[0194] It is contemplated that genes may be introduced into
Sugarcane that would improve standability and other plant growth
characteristics. Expression of novel genes in Sugarcane which
confer stronger stalks, improved root systems, or prevent or reduce
ear droppage or shattering 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
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 phylochrone gene in Sugarcane may be advantageous.
Expression of such a gene may reduce apical dominance, confer
semidwarfism on a plant, or increase shade tolerance (U.S. Pat. No.
5,268,526). Such approaches would allow for increased Sugarcane
populations in the field.
[0195] (vi) Nutrient Utilization
[0196] The ability to utilize available nutrients may be a limiting
factor in growth of Sugarcane. 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 Sugarcane plant 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 or decrease the availability of an
antinutritive factor. An example of such an enzyme would be
phytase. It is further contemplated that enhanced nitrogen
utilization by Sugarcane 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 Sugarcane 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.
[0197] Polypeptides useful for improving nitrogen flow, sensing,
uptake, storage and/or transport include those involved in
aspartate, glutamine or glutamate biosynthesis, polypeptides
involved in aspartate, glutamine or glutamate transport,
polypeptides associated with the TOR (Target of Rapamycin) pathway,
nitrate transporters, nitrate reductases, amino transferases,
ammonium transporters, chlorate transporters or polypeptides
involved in tetrapyrrole biosynthesis.
[0198] Polypeptides useful for increasing the rate of
photosynthesis include phytochrome, ribulose bisphosphate
carboxylase-oxygenase, Rubisco activase, photosystem I and II
proteins, electron carriers, ATP synthase, NADH dehydrogenase or
cytochrome oxidase.
[0199] Polypeptides useful for increasing phosphorus uptake,
transport or utilization include phosphatases or phosphate
transporters.
[0200] (vii) Male Sterility
[0201] Male sterility is useful in the production of hybrid
varieties of Sugarcane. 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, RNAs, or
peptides 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 at
al., Nature, 347:737-741, 1990).
[0202] 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, Science,
250:942-947, 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.
[0203] (viii) Altered Nutritional Content
[0204] Genes may be introduced into Sugarcane to improve or alter
the nutrient quality or content. Introduction of genes that alter
the nutrient composition may greatly enhance the feed, food or
forage value. Limiting essential amino acids may include lysine,
methionine, tryptophan, threonine, valine, arginine, and histidine.
The levels of these essential amino acids 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 particular tissues.
[0205] Polypeptides useful for providing increased protein quantity
and/or quality include polypeptides involved in the metabolism of
amino acids in Sugarcane, particularly polypeptides involved in
biosynthesis of methionine/cysteine and lysine, amino acid
transporters, amino acid efflux carriers, seed storage proteins,
proteases, or polypeptides involved in phytic acid metabolism.
[0206] The protein composition of Sugarcane 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.
[0207] The introduction of genes that alter the oil content of
Sugarcane may also be of value. Increases in oil content may result
in increases in metabolizable-energy-content. 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 acetyl-CoA
carboxylase, ACP-acyltransferase, alpha-ketoacyl-ACP synthase, or
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 Sugarcane.
[0208] Genes may be introduced that enhance the nutritive value of
Sugarcane, or of foods derived from Sugarcane by increasing the
level of naturally occurring phytosterols, or by encoding for
proteins to enable the synthesis of phytosterols in Sugarcane. The
phytosterols from these Sugarcane can be processed directly into
foods, or extracted and used to manufacture food products.
[0209] Genes may be introduced that enhance the nutritive value or
energy value of the starch component of Sugarcane, for example by
altering the degree of branching of starch molecules, resulting in
improved utilization of the starch in biofuel or feedstock
applications. Additionally, other major constituents of Sugarcane
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.
[0210] Carbohydrate metabolism may be altered, for example by
increased sucrose production and/or transport. Polypeptides useful
for affecting carbohydrate metabolism include polypeptides involved
in sucrose or starch metabolism, carbon assimilation or
carbohydrate transport, including, for example sucrose transporters
or glucose/hexose transporters, enzymes involved in
glycolysis/gluconeogenesis, the pentose phosphate cycle, or
raffinose biosynthesis, or polypeptides involved in glucose
signaling, such as SNF1 complex proteins.
[0211] Sugarcane 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, B12, choline, or 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, or iron among others would be valuable.
[0212] Numerous other examples of improvements of Sugarcane may be
used with the invention. 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. Other genes may encode for enzymes
that alter the structure of extracellular carbohydrates, or that
facilitate the degradation of the carbohydrates so that it can be
efficiently fermented into ethanol or other useful
carbohydrates.
[0213] It may be desirable to modify the nutritional content of
Sugarcane by reducing undesirable components such as fats,
starches, etc. This may be done, for example, by the use of
exogenous nucleic acids that encode enzymes which increase plant
use or metabolism of such components so that they are present at
lower quantities. Alternatively, it may be done by use of exogenous
nucleic acids that reduce expression levels or activity of native
Sugarcane enzymes that synthesize such components.
[0214] Likewise the elimination of certain undesirable traits may
improve the food or feed value of Sugarcane. Many undesirable
traits must currently be eliminated by special post-harvest
processing steps and the degree to which these can be engineered
into Sugarcane prior to harvest and processing would provide
significant value. Examples of such traits are the elimination of
anti-nutritionals such as phytates and phenolic compounds which are
commonly found in many crop species. Also, the reduction of fats,
carbohydrates and certain phytohormones may be valuable for the
food and feed industries as they may allow a more efficient
mechanism to meet specific dietary requirements.
[0215] In addition to direct improvements in feed or food value,
genes also may be introduced which improve the processing of
Sugarcane and improve the value of the products resulting from the
processing. Novel genes that increase the efficiency and reduce the
cost of such processing, for example by decreasing the time
required at a particular step, may also find use. Improving the
value of products derived from processed Sugarcane may include
altering the quantity or quality of sugar, starch, oil, fiber,
gluten, or other components. Elevation of sugar or starch may be
achieved through the identification and elimination of rate
limiting steps in starch and sugar biosynthesis by expressing
increased amounts of enzymes involved in biosynthesis or by
decreasing levels of the other components resulting in proportional
increases in sugar or starch. In addition, Sugarcane can be
modified by introducing or expressing a gene or genes that produce
novel products, such as secondary plant metabolites or
pharmaceutical products, that could be purified during the
processing step. Using Sugarcane mini-chromosomes or recombinant
chromosomes to both introduce genes for new products and optionally
for improving processing steps could provide a cost effective
option to produce these novel products.
[0216] Oil is another product of processing, 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 (e.g. fatty acid elongases,
desaturases) and the lipids possessing them or by increasing levels
of native fatty acids while possibly reducing levels of precursors
or breakdown products. 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, or 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 or 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 C8 to C12 saturated fatty acids.
[0217] Polypeptides useful for providing increased oil quantity
and/or quality include polypeptides involved in fatty acid and
glycerolipid biosynthesis, beta-oxidation enzymes, enzymes involved
in biosynthesis of nutritional compounds, such as carotenoids and
tocopherols.
[0218] Polypeptides involved in production of galactomannans or
arabinogalactans are of interest for providing plants having
increased and/or modified reserve polysaccharides for use in food,
pharmaceutical, cosmetic, paper and paint industries.
[0219] Polypeptides involved in modification of
flavonoid/isoflavonoid metabolism in plants include
cinnamate-4-hydroxylase, chalcone synthase or flavones synthase.
Enhanced or reduced activity of such polypeptides in Sugarcane
plants comprising a Sugarcane mini-chromosome will provide changes
in the quantity and/or speed of flavonoid metabolism in plants and
may improve disease resistance by enhancing synthesis of protective
secondary metabolites or improving signaling pathways governing
disease resistance.
[0220] Polypeptides involved in lignin biosynthesis are of interest
for increasing Sugarcane's resistance to lodging and for increasing
the usefulness of plant materials as biofuels.
[0221] (ix) Production or Assimilation of Chemicals or
Biologicals
[0222] It may further be considered that Sugarcane plants
comprising a Sugarcane mini-chromosome or recombinant chromosome
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 Sugarcane 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.
[0223] The novel Sugarcane plants producing these compounds are
made possible by the introduction and expression of one or
potentially many genes with the constructs 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 falconoid 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 Sugarcane 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.
[0224] (x) Other Characteristics
[0225] Cell cycle modification: Polypeptides encoding cell cycle
enzymes and regulators of the cell cycle pathway are useful for
manipulating growth rate in Sugarcane to provide early vigor and
accelerated maturation. Improvements in quality traits, such as
seed oil content, may also be obtained by expression of cell cycle
enzymes and cell cycle regulators. Polypeptides of interest for
modification of the cell cycle pathway include cycling and
EIF5.alpha. pathway proteins, polypeptides involved in polyamine
metabolism, polypeptides which act as regulators of the cell cycle
pathway, including cyclin-dependent kinases (CDKs), CDK-activating
kinases, cell cycle-dependent phosphatases, CDK-inhibitors, Rb and
Rb-binding proteins, or transcription factors that activate genes
involved in cell proliferation and division, such as the E2F family
of transcription factors, proteins involved in degradation of
cyclins, such as cullins, and plant homologs of tumor suppressor
polypeptides.
[0226] Plant growth regulators: Polypeptides involved in production
of substances that regulate the growth of various plant tissues are
of interest in the present invention and may be used to provide
Sugarcane plants comprising a Sugarcane mini-chromosome having
altered morphologies and improved plant growth and development
profiles leading to improvements in yield and stress response. Of
particular interest are polypeptides involved in the biosynthesis,
or degradation of plant growth hormones, such as gibberellins,
brassinosteroids, cytokinins, auxins, ethylene or abscisic acid,
and other proteins involved in the activity, uptake and/or
transport of such polypeptides, including for example, cytokinin
oxidase, cytokinin/purine permeases, F-box proteins, G-proteins or
phytosulfokines.
[0227] Transcription factors in plants: Transcription factors play
a key role in plant growth and development by controlling the
expression of one or more genes in temporal, spatial and
physiological specific patterns. Enhanced or reduced activity of
such polypeptides in Sugarcane plants comprising a Sugarcane
mini-chromosome will provide significant changes in gene
transcription patterns and provide a variety of beneficial effects
in plant growth, development and response to environmental
conditions. Transcription factors of interest include, but are not
limited to myb transcription factors, including helix-turn-helix
proteins, homeodomain transcription factors, leucine zipper
transcription factors, MADS transcription factors, transcription
factors having AP2 domains, zinc finger transcription factors,
CCAAT binding transcription factors, ethylene responsive
transcription factors, transcription initiation factors or UV
damaged DNA binding proteins.
[0228] Homologous recombination: Increasing the rate of homologous
recombination in Sugarcane is useful for accelerating the
introgression of transgenes into breeding varieties by
backcrossing, and to enhance the conventional breeding process by
allowing rare recombinants between closely linked genes in phase
repulsion to be identified more easily. Polypeptides useful for
expression in plants to provide increased homologous recombination
include polypeptides involved in mitosis and/or meiosis, DNA
replication, nucleic acid metabolism, DNA repair pathways or
homologous recombination pathways including for example,
recombinases, nucleases, proteins binding to DNA double-strand
breaks, single-strand DNA binding proteins, strand-exchange
proteins, resolvases, ligases, helicases and polypeptide members of
the RAD52 epistasis group.
Enhanced Biofuel Conversion
[0229] Biofuels may be produced from the conversion of Sugarcane
biomass into liquid or gaseous fuels by converting the biomass into
sugars, or by direct extraction of sugars, that can be fermented or
chemically converted to form a biofuel. Biofuels can also be
generated by extracting oils from the biomass. Exemplary biofuels
are ethanol, propanol, butanol, methanol, methane,
2,5-dimethylfurqan, dimethyl ether, biodiesel (short chain acid
alkyl esters), biogasoline, paraffins (alkanes), other hydrocarbons
or co-products of hydrogen.
[0230] The invention provides for Sugarcane mini-chromosomes or
recombinant chromosomes expressing at least one gene that enhances
or increases sugar production or extractability, enhances or
increases biomass, enhances the conversion of biomass to sugars or
enhances sugar fermentation to biofuels. It may further be
considered that a modified Sugarcane plant prepared in accordance
with the invention may be used as biomass for the production of
biofuels or the plant may facilitate conversion of biomass to
sugars or facilitate fermentation of sugars to biofuels.
[0231] Enzymes that may be useful for biofuel production include
those that break down glucans. In some embodiments, the enzymes are
selected from the group consisting of: endo-.beta.(1,4)-glucanase,
cellobiohydrolase, .beta.-glucosidase, .alpha./.beta.-glucosidase,
mixed-linked glucanase, endo-.beta.(1,3)-glucanase,
exo-.beta.(1,3)-glucanase and .beta.-(1,6)-glucanase. In other
embodiments the enzymes break down xyloglucans, xylans, mannans or
lignins.
[0232] The enzyme genes may be controlled by inducible promoters
that may be inactive until a desired time, such as at harvest or
when the plant is added to the biofuels process (e.g. inactive at
physiological conditions, then activated by heat or pH), or
sequestered by subcellular localization. The enzymes may also be
controlled by a tissue-specific promoter which may be active only
in specific tissues (e.g. seeds or leaves).
Non-Protein-Expressing Exogenous Nucleic Acids
[0233] Sugarcane plants with decreased expression of a gene of
interest can also be achieved, for example, by expression of
antisense nucleic acids, dsRNA or RNAi, catalytic RNA such as
ribozymes, sense expression constructs that exhibit cosuppression
effects, aptamers or zinc finger proteins.
[0234] Antisense RNA reduces production of the polypeptide product
of the target messenger RNA, for example by blocking translation
through formation of RNA:RNA duplexes or by inducing degradation of
the target mRNA. Antisense approaches are a way of preventing or
reducing gene function by targeting the genetic material as
disclosed in U.S. Pat. Nos. 4,801,540; 5,107,065; 5,759,829;
5,910,444; 6,184,439; and 6,198,026, all of which are incorporated
herein by reference. In one approach, an antisense gene sequence is
introduced that is transcribed into antisense RNA that is
complementary to the target mRNA. For example, part or all of the
normal gene sequences are placed under a promoter in inverted
orientation so that the complementary strand is transcribed into a
non-protein expressing antisense RNA. The promoter used for the
antisense gene may influence the level, timing, tissue,
specificity, or inducibility of the antisense inhibition.
[0235] Autonomous Sugarcane mini-chromosomes or recombinant
chromosomes may comprise exogenous DNA flanked by recombination
sites, for example lox-P sites, that can be recognized by a
recombinase, e.g. Cre, and removed from the Sugarcane
mini-chromosome or recombinant chromosome. In cases where there is
a homologous recombination site or sites in the host genomic DNA,
the exogenous DNA excised from the Sugarcane mini-chromosome or
recombinant chromosome may be integrated into the genome at one of
the specific recombination sites and the DNA flanked by the
recombination sites will become integrated into the host DNA. The
use of a Sugarcane mini-chromosome or recombinant chromosome as a
platform for DNA excision or for launching such DNA integration
into the host genome may include in vivo induction of the
expression of a recombinase encoded in the genomic DNA of a
transgenic host, or in a Sugarcane mini-chromosome or recombinant
chromosome.
[0236] RNAi gene suppression in plants by transcription of a dsRNA
is described in U.S. Pat. No. 6,506,559, U.S. patent application
Publication No. 2002/0168707, WO 98/53083, WO 99/53050 and WO
99/61631, all of which are incorporated herein by reference. The
double-stranded RNA or RNAi constructs can trigger the
sequence-specific degradation of the target messenger RNA.
Suppression of a gene by RNAi can be achieved using a recombinant
DNA construct having a promoter operably linked to a DNA element
comprising a sense and anti-sense element of a segment of genomic
DNA of the gene, e.g., a segment of at least about 23 nucleotides,
optionally about 50 to 200 nucleotides where the sense and
anti-sense DNA components can be directly linked or joined by an
intron or artificial DNA segment that can form a loop when the
transcribed RNA hybridizes to form a hairpin structure.
[0237] Catalytic RNA molecules or ribozymes can also be used to
inhibit expression of the target gene or genes or facilitate
molecular reactions. Ribozymes are targeted to a given sequence by
hybridization of sequences within the ribozyme to the target mRNA.
Two stretches of homology are required for this targeting, and
these stretches of homologous sequences flank the catalytic
ribozyme structure. It is possible to design ribozymes that
specifically pair with virtually any target mRNA and cleave the
target mRNA at a specific location, thereby inactivating it. A
number of classes of ribozymes have been identified. One class of
ribozymes is derived from a number of small circular RNAs that are
capable of self-cleavage and replication in plants. The RNAs
replicate either alone (viroid RNAs) or with a helper virus
(satellite RNAs). Examples include Tobacco Ringspot Virus (Prody et
al. Science, 231:1577-1580, 1986), Avocado Sunblotch Viroid
(Palukaitis et al., Virology, 99:145-151, 1979; Symons, Nucl. Acids
Res., 9:6527-6537, 1981), and Lucerne Transient Streak Virus
(Forster and Symons, Cell, 49:211-220, 1987), and the satellite
RNAs from velvet tobacco mottle virus, Solanum nodiflorum mottle
virus and subterranean clover mottle virus. The design and use of
target RNA-specific ribozymes is described in Haseloff, et al.,
Nature 334:585-591 (1988). Several different ribozyme motifs have
been described with RNA cleavage activity (Symons, Annu. Rev.
Biochem., 61:641-671, 1992). Other suitable ribozymes include
sequences from RNase P with RNA cleavage activity (Yuan et al.,
Proc. Natl. Acad. Set. USA, 89:8006-8010, 1992; Yuan and Allman,
Science, 263:1269-1273, 1994; U.S. Pat. Nos. 5,168,053 and
5,624,824), hairpin ribozyme structures (Berzal-Herranz et al.,
Genes and Devel., 6:129-134, 1992; Chowrira et al., J. Biol. Chem.,
269:25856-25864, 1994) and Hepatitis Delta virus based ribozymes
(U.S. Pat. No. 5,625,047). The general design and optimization of
ribozyme directed RNA cleavage activity has been discussed in
detail (Haseloff and Gerlach, 1988, Nature. 1988 Aug. 18;
334(6183):585-91, Chowrira et al., J. Biol. Chem., 269:25856-25864,
1994).
[0238] Another method of reducing protein expression utilizes the
phenomenon of cosuppression or gene silencing (for example, U.S.
Pat. Nos. 6,063,947; 5,686,649; or 5,283,184; cach of which is
incorporated herein by reference). Cosuppression of an endogenous
gene using a full-length cDNA sequence as well as a partial cDNA
sequence are known (for example, Napoli et al., Plant Cell
2:279-289 [1990]; van der Krol et al., Plant Cell 2:291-299 [1990];
Smith et al., Mol. Gen. Genetics 224:477-481 [1990]). The
phenomenon of cosuppression has also been used to inhibit plant
target genes in a tissue-specific manner.
[0239] In some embodiments, nucleic acids from one species of plant
are expressed in another species of plant to effect cosuppression
of a homologous gene. The introduced sequence generally will be
substantially identical to the endogenous sequence intended to be
repressed, for example, about 65%, 80%, 85%, 90%, 95% or even 98%
or greater identical. Higher identity may result in a more
effective repression of expression of the endogenous sequence. A
higher identity in a shorter than full length sequence compensates
for a longer, less identical sequence. Furthermore, the introduced
sequence need not have the same intron or exon pattern, and
identity of non-coding segments will be equally effective.
Generally, where inhibition of expression is desired, some
transcription of the introduced sequence occurs. The effect may
occur where the introduced sequence contains no coding sequence per
se, but only intron or untranslated sequences homologous to
sequences present in the primary transcript of the endogenous
sequence.
[0240] Yet another method of reducing protein activity is by
expressing nucleic acid ligands, so-called aptamers, which
specifically bind to the protein. Aptamers may be obtained by the
SELEX (Systematic Evolution of Ligands by Exponential Enrichment)
method. See U.S. Pat. No. 5,270,163, incorporated herein by
reference. In the SELEX method, a candidate mixture of single
stranded nucleic acids having regions of randomized sequence is
contacted with the protein and those nucleic acids having an
increased affinity to the target are selected and amplified. After
several iterations a nucleic acid with optimal affinity to the
polypeptide is obtained and is used for expression in modified
plants.
[0241] A zinc finger protein that binds a polypeptide-encoding
sequence or its regulatory region is also used to alter expression
of the nucleotide sequence. Transcription of the nucleotide
sequence may be reduced or increased. Zinc finger proteins are, for
example, described in Beerli et al. (1998) PNAS 95:14628-14633., or
in WO 95/19431, WO 98/54311, or WO 96/06166, all incorporated
herein by reference.
[0242] 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.
[0243] It is contemplated that unexpressed DNA sequences, including
novel synthetic sequences, could be introduced into Sugarcane 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 Sugarcane 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.
Exemplary Plant Promoters, Regulatory Sequences and Targeting
Sequences
[0244] Exemplary classes of plant promoters are described
below.
[0245] Constitutive Expression promoters: Exemplary constitutive
expression promoters include the ubiquitin promoter (e.g.,
sunflower-Binet et al. Plant Science 79: 87-94 (1991);
maize--Christensen et al. Plant Molec. Biol. 12: 619-632 (1989);
and Arabidopsis--Callis et al., J. Biol. Chem. 265: 12486-12493
(1990) and Norris et al., Plant Mol. Biol. 21: 895-906 (1993)); the
CaMV 35S promoter (U.S. Pat. Nos. 5,858,742 and 5,322,938); or the
actin promoter (e.g., rice--U.S. Pat. No. 5,641,876; McElroy et al.
Plant Cell 2: 163-171 (1990), McElroy et al. Mol. Gen. Genet. 231:
150-160 (1991), and Chibbar et al. Plant Cell Rep. 12: 506-509
(1993)). Exemplary promoters for use in Sugarcane include the maize
polyubiquitin 1 (Mubi-1) and the Sugarcane polyubiquitin 9 (SCubi9)
promoters (Wang M L, Goldstein C, Su W, Moore P H, Albert H H.
Production of biologically active GM-CSF in Sugarcane: a secure
biofactory. Transgenic Res. 2005, 14:167-78); and the Sugarcane
polyubiquitin 4 (ubi4) promoter (Wei H, Wang M L, Moore P H, Albert
H H. Comparative expression analysis of two Sugarcane polyubiquitin
promoters and flanking sequences in transgenic plants. J Plant
Physiol. 2003, 160:1241-51).
[0246] Inducible Expression promoters: Exemplary inducible
expression promoters include the chemically regulatable tobacco
PR-1 promoter (e.g., tobacco--U.S. Pat. No. 5,614,395;
Arabidopsis-Lebel et al., Plant J. 16: 223-233 (1998); maize--U.S.
Pat. No. 6,429,362). Various chemical regulators may be employed to
induce expression, including the benzothiadiazole, isonicotinic
acid, and salicylic acid compounds disclosed in U.S. Pat. Nos.
5,523,311 and 5,614,395. Other promoters inducible by certain
alcohols or ketones, such as ethanol, include, for example, the
alcA gene promoter from Aspergillus nidulans (Caddick et al. (1998)
Nat. Biotechnol 16:177-180). A glucocorticoid-mediated induction
system is described in Aoyama and Chua (1997) The Plant Journal 11:
605-612 wherein gene expression is induced by application of a
glucocorticoid, for example a dexamethasone. Another class of
useful promoters are water-deficit-inducible promoters, e.g.
promoters which are derived from the 5' regulatory region of genes
identified as a heat shock protein 17.5 gene (HSP 17.5), an HVA22
gene (HVA22), and a cinnamic acid 4-hydroxylase (CA4H) gene of Zea
mays. Another water-deficit-inducible promoter is derived from the
rab-17 promoter as disclosed by Vilardell et al., Plant Molecular
Biology, 17(5):985-993, 1990. See also U.S. Pat. No. 6,084,089
which discloses cold inducible promoters, U.S. Pat. No. 6,294,714
which discloses light inducible promoters, U.S. Pat. No. 6,140,078
which discloses salt inducible promoters, U.S. Pat. No. 6,252,138
which discloses pathogen inducible promoters, and U.S. Pat. No.
6,175,060 which discloses phosphorus deficiency inducible
promoters.
[0247] As another example, numerous wound-inducible promoters have
been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588
(1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier
& Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al.
Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3:
191-201 (1993)). Logemann et al., describe 5' upstream sequences of
the potato wunl gene. Xu et al. show that a wound-inducible
promoter from the dicotyledon potato (pin2) is active in the
monocotyledon rice. Rohrmeier & Lehle describe maize Wipl cDNA
which is wound induced and which can be used to isolate the cognate
promoter. Firek et al. and Warner et al. have described a
wound-induced gene from the monocotyledon Asparagus officinalis,
which is expressed at local wound and pathogen invasion sites.
[0248] Tissue-Specific Promoters: Exemplary promoters that express
genes only in certain Sugarcane tissues are useful according to the
present invention. For example root specific expression may be
attained using the promoter of the maize metallothionein-like (MTL)
gene described by de Framond (FEBS 290: 103-106 (1991)) and also in
U.S. Pat. No. 5,466,785, incorporated herein by reference. U.S.
Pat. No. 5,837,848 discloses a root specific promoter. Another
exemplary promoter confers pith-preferred expression (see Int'l.
Pub. No. WO 93/07278, herein incorporated by reference, which
describes the maize trpA gene and promoter that is preferentially
expressed in pith cells). Leaf-specific expression may be attained,
for example, by using the promoter for a maize gene encoding
phosphoenol carboxylase (PEPC) (sce Hudspeth & Grula, Plant
Molee Biol 12: 579-589 (1989)). Pollen-specific expression may be
conferred by the promoter for the maize calcium-dependent protein
kinase (CDPK) gene which is expressed in pollen cells (WO
93/07278). U.S. Pat. Appl. Pub. No. 20040016025 describes
tissue-specific promoters. Pollen-specific expression may be
conferred by the tomato LAT52 pollen-specific promoter (Bate et.
al., Plant Mol Riol. 1998 July; 37(5):859-69).
[0249] See also U.S. Pat. No. 6,437,217 which discloses a
root-specific maize RS81 promoter, U.S. Pat. No. 6,426,446 which
discloses a root specific maize RS324 promoter, U.S. Pat. No.
6,232,526 which discloses a constitutive maize A3 promoter, U.S.
Pat. No. 6,177,611 which discloses constitutive maize promoters,
U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter
that are aleurone and seed coat-specific promoters, U.S. Pat. No.
6,429,357 which discloses a constitutive rice actin 2 promoter and
intron, U.S. patent application Pub. No. 20040216189 which
discloses an inducible constitutive leaf specific maize chloroplast
aldolase promoter.
[0250] Optionally a plant transcriptional terminator can be used in
place of the plant-expressed gene native transcriptional
terminator. Exemplary transcriptional terminators are those that
are known to function in plants and include the CaMV 35S
terminator, the tm1 terminator, the nopaline synthase terminator
and the pea rbcS E9 terminator. These can be used in both
monocotyledons and dicotyledons.
[0251] Various intron sequences have been shown to enhance
expression, particularly in monocotyledonous cells. For example,
the introns of the maize Adh1 gene have been found to significantly
enhance expression. Intron 1 was found to be particularly effective
and enhanced expression in fusion constructs with the
chloramphenicol acetyltransferase gene (Callis et al., Genes
Develop. 1: 1183-1200 (1987)). The intron from the maize bronze1
gene also enhances expression. Intron sequences have been routinely
incorporated into plant transformation vectors, typically within
the non-translated leader. U.S. Patent Application Publication
2002/0192813 discloses 5', 3' and intron elements useful in the
design of effective plant expression vectors.
[0252] A number of non-translated leader sequences derived from
viruses are also known to enhance expression, and these are
particularly effective in dicotyledonous cells. Specifically,
leader sequences from Tobacco Mosaic Virus (TMV, the
"omega-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa
Mosaic Virus (AMV) have been shown to be effective in enhancing
expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711
(1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)). Other
leader sequences known in the art include, but are not limited to:
picornavirus leaders, for example, EMCV leader
(Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O.,
Fuerst, T. R., and Moss, B. PNAS USA 86:6126-6130 (1989));
potyvirus leaders, for example, TEV leader (Tobacco Etch Virus)
(Allison et al., 1986); MDMV leader (Maize Dwarf Mosaic Virus);
Virology 154:9-20); human immunoglobulin heavy-chain binding
protein (BiP) leader, (Macejak, D. G., and Sarnow, P., Nature 353:
90-94 (1991); untranslated leader from the coat protein mRNA of
alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L.,
Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV),
(Gallie et al., Molecular Biology of RNA, pages 237-256 (1989); or
Maize Chlorotic Mottle Virus leader (MCMV) (Lommel et al., Virology
81:382-385 (1991). See also, Della-Cioppa et al., Plant Physiology
84:965-968 (1987).
[0253] A minimal promoter may also be incorporated. Such a promoter
has low background activity in plants when there is no
transactivator present or when enhancer or response element binding
sites are absent. One exemplary minimal promoter is the Bzl minimal
promoter, which is obtained from the bronze1 gene of maize. Roth et
al., Plant Cell 3: 317 (1991). A minimal promoter may also be
created by use of a synthetic TATA element. The TATA element allows
recognition of the promoter by RNA polymerase factors and confers a
basal level of gene expression in the absence of activation (see
generally, Mukumoto (1993) Plant Mol Biol 23: 995-1003; Green
(2000) Trends Biochem Sci 25: 59-63).
[0254] Sequences controlling the targeting of gene products also
may be included. For example, the targeting of gene products to the
chloroplast is controlled by a signal sequence found at the amino
terminal end of various proteins which is cleaved during
chloroplast import to yield the mature protein (e.g. Comai et al.
J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can
be fused to heterologous gene products to effect the import of
heterologous products into the chloroplast (van den Broeck, et al.
Nature 313: 358-363 (1985)). DNA encoding for appropriate signal
sequences can be isolated from the 5' end of the cDNAs encoding the
RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2
protein or many other proteins which are known to be chloroplast
localized. Other gene products are localized to other organelles
such as the mitochondrion and the peroxisome (e.g. Unger et al.
Plant Molec. Biol. 13: 411-418 (1989)). Examples of sequences that
target to such organelles are the nuclear-encoded ATPases or
specific aspartate amino transferase isoforms for mitochondria.
Targeting cellular protein bodies has been described by Rogers et
al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)). In addition,
amino terminal and carboxy-terminal sequences are responsible for
targeting to the ER, the apoplast, and extracellular secretion from
aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)).
Additionally, amino terminal sequences in conjunction with carboxy
terminal sequences are responsible for vacuolar targeting of gene
products (Shinshi et al. Plant Molec. Biol. 14: 357-368
(1990)).
[0255] Another possible element which may be introduced is a matrix
attachment region element (MAR), such as the chicken lysozyme A
element, which 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 (Stiefet al., Nature, 341:343, 1989; Phi-Van et al.,
Mol. Cell. Biol., 10:2302-2307.1990).
Use of Non-Plant Promoter Regions Isolated from Drosophila
melanogaster and Saccharomyces cerevisiae to Express Genes in
Plants
[0256] The promoter in the Sugarcane mini-chromosome or recombinant
chromosome of the present invention can be derived from plant or
non-plant species. In one embodiment, the nucleotide sequence of
the promoter is derived from non-plant species for the expression
of genes in plant cells. In one embodiment, the non-plant promoters
are constitutive or inducible promoters derived from insect, e.g.,
Drosophila melanogaster or yeast, e.g., Saccharomyces cerevisiae.
Table 2 lists nonlimiting examples of promoters from Drosophila
melanogaster and Saccharomyces cerevisiae that can be used to
derive the examples of non-plant promoters in the present
invention. Promoters derived from any animal, protist, or fungi are
also contemplated. SEQ ID NOS: 222-241, or fragments, mutants,
hybrid or tandem promoters thereof, are examples of promoter
sequences derived from Drosophila melanogaster or Saccharomyces
cerevisiae. These non-plant promoters can be operably linked to
nucleic acid sequences encoding polypeptides or
non-protein-expressing sequences including, but not limited to,
antisense RNA and ribozymes, to form nucleic acid constructs,
vectors, and host cells (prokaryotic or eukaryotic), comprising the
promoters.
TABLE-US-00002 TABLE 2 Drosophila melanogaster Promoters
(Information obtained from the Flybase Web Site at
http://flybase.bio.indiana.edu/ which is a database of the
Drosophila Genome) SEQ Standard promoter Chromo- ID NO: SSym
Flybase ID gen name Gene Product some 222 Pgd FBgn0004654
Phosphogluconate 6-phosphogluconate X dehydrogenase dehydrogenase
223 Grim FBgn0015946 grim grim-P138 3 224 Uro FBgn0003961 Urate
oxidase Uro-P1 2 225 Sna FBgn0003448 Snail sna-P1 2 226 Rh3
FBgn0003249 Rhodopsin 3 Rh3 3 227 Lsp-1 .gamma. FBgn0002564 Larval
serum protein 1 Lsp1.gamma.-P1 3 Saccharomyces cerevisiae Promoters
(Information obtained from the Saccharomyces Genome Database Web
site at http://www.yeastgenome.org/SearchContents.shtml Seq
Systematic Standard promoter Chromo- No. SSymbol Name gen name Gene
Product some 228 Tef-2 YBR118W TEF2 (Translation Translation
elongation 2 elongation factor fact EF-1 alpha promoter) 229 Leu-1
YGL009C LEU1 (LEUcine isopropylmalate 7 biosynthesis) isomerase 230
Met16 YPR167C METhionine 3'phosphoadenylyl- 16 requiring sulfate
reductase 231 Leu-2 YCL018W LEU2 (leucine beta-IPM (isopropylmalate
3 biosynthesis) dehydrogenase) 232 His-4 YCL030C HIS4 (HIStidine
histidinol 3 requiring) dehydrogenase 233 Met-2 YNL277W MET2
(methionine L-homoserine-O- 14 requiring) acetyltransferase 234
Ste-3 YKL178C STE3 (alias DAF2 a-factor receptor 11 Sterile) 235
Arg-1 YOL058W ARG1(alias ARG10 arginosuccinate 15 ARGinine
requiring) synthetase 236 Pgk-1 YCR012W PGK1 (phosphoglycerate
phosphoglycerate kinase 3 kinase) 237 GPD-1 YDL022W GPD1 (alias
glycerol-3-phosphate 4 DAR1/HOR1/OSG1/O dehydrogenase R5:
glycerol-3-phospha dehydrogenase activity 238 ADH1 YOL086C ADH1
(alias ADC1) alcohol dehydrogenase 15 239 GPD-2 YOL059W GPD2 (alias
GPD3: glycerol-3-phosphate 15 glycerol-3-phosphate dehydrogenase
dehydrogenase activity 240 Arg-4 YHR018C ARGinine requiring
argininosuccinate lyase 8 241 Yat-1 YAR035W YAT-1(carnitine
carnitine 1 acetyltransferase) acetyltransferase indicates data
missing or illegible when filed
[0257] In the Sugarcane mini-chromosomes or recombinant chromosome
of the present invention, the promoter may be a mutant of the
promoters having a substitution, deletion, and/or insertion of one
or more nucleotides in the nucleic acid sequence of SEQ ID NOS:
222-241, hybrid or tandem promoters.
[0258] The techniques used to isolate or clone a nucleic acid
sequence comprising a promoter of interest are known in the art and
include isolation from genomic DNA. The cloning procedures may
involve excision or amplification, for example by polymerase chain
reaction, and isolation of a desired nucleic acid fragment
comprising the nucleic acid sequence encoding the promoter,
insertion of the fragment into a vector molecule, and incorporation
of the recombinant vector into the plant cell.
DEFINITIONS
[0259] The term "adchromosomal" Sugarcane plant or plant part as
used herein means a Sugarcane plant or plant part that contains
functional, stable and autonomous Sugarcane mini-chromosomes.
Adchromosomal Sugarcane plants or plant parts may be chimeric or
not chimeric (chimeric meaning that Sugarcane mini-chromosomes are
only in certain portions of the plant, and are not uniformly
distributed throughout the plant). An adchromosomal Sugarcane plant
cell contains at least one functional, stable and autonomous
Sugarcane mini-chromosome.
[0260] The term "autonomous" as used herein means that when
delivered to plant cells, at least some Sugarcane mini-chromosomes
are transmitted through mitotic division to daughter cells and are
episomal in the daughter plant cells, i.e. are not chromosomally
integrated in the daughter plant cells. Daughter plant cells that
contain autonomous mini-chromosomes can be selected for further
replication using, for example, selectable or screenable markers.
During the introduction into a cell of a mini-chromosome, or during
subsequent stages of the cell cycle, there may be chromosomal
integration of some portion or all of the DNA derived from a
mini-chromosome in some cells. The mini-chromosome is still
characterized as autonomous despite the occurrence of such events
if a plant may be regenerated that contains episomal descendants of
the mini-chromosome, optionally distributed throughout its parts,
or if gametes or progeny can be derived from the plant that contain
episomal descendants of the mini-chromosome distributed through its
parts.
[0261] 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 transmission
efficiency to daughter cells ranging from about 1% to about 100%,
including to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
or about 95% of daughter cells. Variations in transmission
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 transmission to
daughter cells of a nucleic acid sequence, including a recombinant
construct comprising the centromere, through mitotic or meiotic
divisions, including through both meiotic and meiotic divisions. A
plant centromere is not necessarily derived from plants, but has
the ability to promote DNA transmission to daughter plant
cells.
[0262] 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.
[0263] The term "co-delivery" as used herein refers to the delivery
of two nucleic acid segments to a cell. In co-delivery of plant
growth inducing genes and mini-chromosomes, the two nucleic acid
segments are delivered simultaneously using the same delivery
method. Alternatively, the nucleic acid segment containing the
growth inducing gene, optionally as part of an episomal vector,
such as a viral vector or a plasmid vector, may be delivered to the
plant cells before or after delivery of the mini-chromosome, and
the mini-chromosome may carry an exogenous nucleic acid that
induces expression of the earlier-delivered growth inducing gene.
In this embodiment, the two nucleic acid segments may be delivered
separately at different times provided the encoded growth inducing
factors are functional during the appropriate time period.
[0264] The term "coding sequence" is defined herein as a nucleic
acid sequence that is transcribed into mRNA which is translated
into a polypeptide when placed under the control of promoter
sequences. The boundaries of the coding sequence are generally
determined by the ATG start codon located at the start of the open
reading frame, near the 5' end of the mRNA, and TAG, TGA or TAA
stop codons at the end of the coding sequence, near the 3' end of
the mRNA, and in some cases, a transcription terminator sequence
located just downstream of the open reading frame at the 3' end of
the mRNA. A coding sequence can include, but is not limited to,
genomic DNA, cDNA, semisynthetic, synthetic, or recombinant nucleic
acid sequences.
[0265] 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 the construction of mini-chromosomes.
[0266] The term "exogenous" when used in reference to a nucleic
acid, for example, is intended to refer to any nucleic acid that
has been introduced into a recipient cell, regardless of whether
the same or similar nucleic acid is already present in such a cell.
Thus, as an example, "exogenous DNA" can include an additional copy
of DNA that is already present in the plant cell, DNA from another
plant, DNA from a different organism, or a DNA generated
externally, such as a DNA sequence containing an antisense message
of a gene, or a DNA sequence encoding a synthetic or modified
version of a gene. An "exogenous gene" can be a gene not normally
found in the host genome in an identical context, or an extra copy
of a host gene. 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.
[0267] The term "functional" as used herein to describe a
mini-chromosome means that when an exogenous nucleic acid is
present within the mini-chromosome the exogenous nucleic acid can
function in a detectable manner when the mini-chromosome is within
a plant cell; exemplary functions of the exogenous nucleic acid
include transcription of the exogenous nucleic acid, expression of
the exogenous nucleic acid, regulatory control of expression of
other exogenous nucleic acids, recognition by a restriction enzyme
or other endonuclease, ribozyme or recombinase; providing a
substrate for DNA methylation. DNA glycolation or other DNA
chemical modification; binding to proteins such as histones,
helix-loop-helix proteins, zinc binding proteins, leucine zipper
proteins, MADS box proteins, topoisomerases, helicases,
transposases, TATA box binding proteins, viral protein, reverse
transcriptases, or cohesins; providing an integration site for
homologous recombination; providing an integration site for a
transposon, T-DNA or retrovirus; providing a substrate for RNAi
synthesis; priming of DNA replication; aptamer binding; or
kinetochore binding. If multiple exogenous nucleic acids are
present within the mini-chromosome, the function of one or more of
the exogenous nucleic acids can be detected under suitable
conditions permitting function thereof. An "isolated
polynucleotide" or "isolated nucleic acid" (and similar terms) can
refer to a nucleotide sequence (e.g., DNA or RNA) that is not
immediately contiguous with nucleotide sequences with which it is
immediately contiguous (one on the 5' end and one on the 3' end) in
the naturally occurring genome of the organism from which it is
derived. Thus, in one embodiment, an isolated nucleic acid includes
some or all of the 5' non-coding (e.g., promoter) sequences that
are immediately contiguous to a coding sequence. The term
"isolated" can refer to a polynucleotide or nucleic acid that is
substantially free of cellular material, viral material, and/or
culture medium (when produced by recombinant DNA techniques), or
chemical precursors or other chemicals (when chemically
synthesized). "Isolated" does not necessarily mean that the
preparation is technically pure (homogeneous), but it is
sufficiently pure to provide the polynucleotide or nucleic acid in
a form in which it can be used for the intended purpose. In certain
embodiments, the isolated polynucleotide or nucleic acid is at
least about 50% pure, e.g., at least about 60%, 70%, 80%, 90%, 95%,
96%, 97%, 98%, or 99% or more pure.
[0268] An "isolated" cell refers to a cell that is at least
partially separated from other components with which it is normally
associated in its natural state. For example, an isolated cell can
be a cell in culture medium.
[0269] As used herein, a "library" is a pool of cloned DNA
fragments that represents some or all DNA sequences collected,
prepared or purified from a specific source. Each library may
contain the DNA of a given organism inserted as discrete
restriction enzyme generated fragments or as randomly sheared
fragments into many thousands of plasmid vectors. For purposes of
the present invention, E. coli, yeast, and Salmonella plasmids are
particularly useful for propagating the genome inserts from other
organisms. In principle, any gene or sequence present in the
starting DNA preparation can be isolated by screening the library
with a specific hybridization probe (see, for example, Young et
al., In: Eukaryotic Genetic Systems ICN-UCLA Symposia on Molecular
and Cellular Biology, VII, 315-331, 1977).
[0270] As used herein, the term "linker" refers to a DNA molecule,
generally up to 50 or 60 nucleotides long and composed of two or
more complementary oligonucleotides that have been synthesized
chemically, or excised or amplified from existing plasmids or
vectors. In a representative embodiment, this fragment contains
one, or more than one, restriction enzyme site for a blunt cutting
enzyme and/or a staggered cutting enzyme, such as BamHI. One end of
the linker is designed to be ligatable to one end of a linear DNA
molecule and the other end is designed to be ligatable to the other
end of the linear molecule, or both ends may be designed to be
ligatable to both ends of the linear DNA molecule.
[0271] As used herein, a "mini-chromosome" is a recombinant DNA
construct including a centromere that is capable of transmission to
daughter cells. A 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, origin of
replication sequences, stuffer sequences, buffer sequences,
chromatin packaging sequences, linkers 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 from the natural centromere to the minimal amount
required to obtain a transmission 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 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.
[0272] The term "non-protein expressing sequence" or "non-protein
coding sequence" is defined herein as a nucleic acid sequence that
is not eventually translated into protein. The nucleic acid may or
may not be transcribed into RNA. Exemplary sequences include
ribozymes or antisense RNA.
[0273] As used herein, "nucleic acid," "nucleotide sequence," and
"polynucleotide" are used interchangeably and encompass both RNA
and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g.,
chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The
term polynucleotide, nucleotide sequence, or nucleic acid refers to
a chain of nucleotides without regard to length of the chain. The
nucleic acid can be double-stranded or single-stranded. Where
single-stranded, the nucleic acid can be a sense strand or an
antisense strand. The nucleic acid can be synthesized using
oligonucleotide analogs or derivatives (e.g., inosine or
phosphorothioate nucleotides). Such oligonucleotides can be used,
for example, to prepare nucleic acids that have altered
base-pairing abilities or increased resistance to nucleases. The
present invention further provides a nucleic acid that is the
complement (which can be either a full complement or a partial
complement) of a nucleic acid, nucleotide sequence, or
polynucleotide of the invention.
[0274] The term "operably linked" is defined herein as a
configuration in which a control sequence, e.g., a promoter
sequence, directs transcription or translation of another sequence,
for example a coding sequence. For example, a promoter sequence
could be appropriately placed at a position relative to a coding
sequence such that the control sequence directs the production of a
polypeptide encoded by the coding sequence.
[0275] "Phenotype" or "phenotypic trait(s)", as used herein, refers
to an observable property or set of properties resulting from the
expression of a gene. The set of properties may be observed
visually or after biological or biochemical testing, and may be
constantly present or may only manifest upon challenge with the
appropriate stimulus or activation with the appropriate signal.
[0276] The term "recombinant chromosome" refers to an engineered or
artificial chromosome that has been constructed by fragmenting a
natural chromosome and identifying fragmentation products that are
capable of segregation through mitotic and/or meiotic cell
divisions. Recombinant chromosomes are distinct from
mini-chromosomes in that they are not constructed in vitro from
constituent parts and have not been passaged through an
heterologous cell such as a bacteria or fungus (as is commonly used
in standard cloning techniques). Recombinant chromosomes may the
used as targets for addition of transgene expression cassettes.
[0277] The term "Sugarcane" refers to any species or hybrid of the
genus Saccharum including but not limited to: S. acinaciforme, S.
aegyptiacum, S. alopecuroides (Silver Plume Grass), S.
alopecuroideum, S. alopecuroidum (Silver Plumegrass), S.
alopecurus, S. angustifolium, S. antillarum, S. arenicola, S.
argenteum, S. arundinaceum (Hardy Sugar Cane (Usa)), S.
arundinaceum var. trichophyllum, S. asper, S. asperum, S.
atrorubens, S. aureum, S. balansae, S. baldwini, S. baldwinii
(Narrow Plumegrass), S. barberi (Cultivated Sugarcane), S.
barbicoatatum, S. beccarii, S. bengalense (Munj Sweetcane), S.
benghalense, S. bicorne, S. bflorum, S. boga, S. brachypogon, S.
bructealum, S. brasiliamum, S. brevibarbe (Short-Beard Plume
Grass), S. brevibarbe var. brevibarbe (Shortbeard Plumegrass), S.
brevibarbe var. contortum (Shortbeard Plumegrass), S. brevifolium,
S. brunneum, S. caducum, S. canaliculatum, S. capense, S. casi, S.
caudatum, S. cayennense, S. cayennene var. genuinum, S. cayennense
var. laxiusculum, S. chinense, S. ciliare, S. coarclatum
(Compressed Plumegrass), S. confertum, S. conjugatum, S. contortum,
S. contortum var. contortum, S. contractum, S. cotuliferum, S.
cylindricum, S. cylindricum var. contractum, S. cylindricum var.
longifolium, S. deciduum, S. densum, S. diandrum, S. dissitiflorum,
S. distichophyllum, S. dublum, S. ecklonli, S. edule, S. elegans,
S. elephantinum, S. erlantholdes, S. europaeum, S. exaltatum, S.
fasciculalum, S. fasllgiatum, S. fatuum, S. fllifolium, S.
filiforme, S. floridulum, S. formosanum, S. fragile, S. fidulvum,
S. fuscum, S. giganteum (Sugarcane Plume Grass), S. glabrum, S.
glaga, S. glaucum, S. glaza, S. grandiflorum, S. griffithii, S.
hildebrandtii, S. hirsulum, S. holcoides, S. holcoides var.
warmingianum, S. hookeri, S. hybrid, S. hybridum, S. indum, S.
infirmum, S. insulare, S. irritans, S. jaculalorium, S. jamaicense,
S. japonicum, S. juncifolium, S. kajkalense, S. kanashiroi, S.
klagha, S. koenigii, S. laguroides, S. longifolium, S.
longisetosum, S. longiretosum var. hookeri, S. longisetum, S. lota,
S. luzonicum, S. macilentum, S. macrantherum, S. maximum, S.
mexicanum, S. modhara, S. monandrum, S. moonja, S. munja, S.
munroanum, S. muticum, S. narenga (Narenga Sugarcane), S.
negrosense, S. obscurum, S. occidentale, S. officinale, S.
oficinalis, S. officinarum (Cultivated Sugarcane), S. officinarum
`Choribon`, S. oficinarum `Otaheite`, S. officinarum `Pele's Smoke`
(Black Magic Repellent Plant), S. officinarum L. `Laukona`, S.
officinarum L. `Violaceum`, S. officinarum var. brevipedicellatum,
S. officinarum var. oficinarum, S. officinarum var. violaceum
(Burgundy-Leaved Sugarcane), S. pallidum, S. paniceum, S.
panicosum, S. pappiferum, S. parviflorum, S. pedicellare, S.
perrieri, S. polydactylum, S. polystachyon, S. polystachyum, S.
purphyrocomum, S. procerum, S. propinquum, S. punctatum, S. rara,
S. rarum, S. ravennae (Hardy Pampas Plume Grass), S. repens, S.
reptans, S. ridleyi, S. robustum (Wild New-Guinean Cane), S.
roseum, S. rubicundum, S. rufum, S. sagittatum, S. sanguineum, S.
sape, S. sara, S. scindicus, S. semidecumbens, S. sibiricum, S.
sikkimense, S. sinense (Cultivated Sugarcane), S. sisca, S.
sorghum, S. speciosissimum, S. sphacelatum, S. spicatum, S.
spontaneum (Wild Sugar Cane), S. spontaneum var. insulare, S.
spontanum, S. stenophyllum, S. stewartii, S. strictum, S.
tenerijfae, S. ternatum, S. thunbergii, S. tinctorium, S.
tridentatum, S. trinii, S. tristachyum, S. velutinum, S.
versicolor, S. viguieri, S. villosum, S. violaceum, S. wardii, S.
warminglanum, S. williamsii.
[0278] As used herein, the term "Sugarcane Basic Mini-Chromosome"
is defined as a recombinant DNA construct that when present within
a Sugarcane cell is capable of mitotic and/or meiotic transmission
to Sugarcane daughter cells under appropriate conditions and
comprises a Sugarcane Assembled Centromere and, optionally, one or
more of the following: [0279] (a) one or more telomeres; [0280] (b)
one or more sequences for regulating, maintaining, or imparting
topological or chromatin structure, molecular integrity, or
stability of gene expression or inheritance in Sugarcane; [0281]
(c) the required vector DNA that allows for propagation of the
mini-chromosome in Sugarcane and DNA that facilitates the selective
removal of unwanted portions of the mini-chromosome prior to or
after Sugarcane transformation; or [0282] (d) a Transgene
Expression Cassette, wherein the Transgene Expression Cassette
serves only to regulate, maintain, or impart function or stability
to a mini-chromosome in Sugarcane.
[0283] A "Sugarcane Basic Mini-Chromosome" does not include a
Sugarcane Transgene Expression Cassette that imparts one or more
functions other than those expressly set forth in subsection (d),
above.
[0284] As used herein, a "Sugarcane Assembled Centromere" means a
polynucleotide sequence having the properties of a Centromere that
is assembled from one or more fragments of native Centromere(s)
and/or other polynucleotide sequence, which are (i) isolated from a
plant cell, and/or based on plant Centromere sequence motifs, (ii)
inserted into a vector (e.g. a plasmid vector) that is propagated
and maintained in a cell of a heterologous organism, and (iii)
delivered back into a Sugarcane plant cell as part of a Sugarcane
Basic or Sugarcane Applied Mini-Chromosome. A Sugarcane Assembled
Centromere may possibly be modified by an endogenous in vivo
process after it is delivered into a Sugarcane plant cell such that
its sequence now differs from that contained in the parental
Sugarcane Basic or Sugarcane Applied Mini-Chromosome as propagated
in a cell of a heterologous organism. For the avoidance of doubt a
Sugarcane Assembled Centromere does not include derivatives or
deletions of native Sugarcane Centromeres that are constructed
within the Sugarcane plant cell, and are never maintained in their
entirety in a cell of a heterologous organism.
[0285] As used herein, a "Sugarcane Applied Mini-Chromosome" means
a genetic construct formed by integrating one or more Transgene
Expression Cassettes into a Sugarcane Basic Mini-Chromosome,
wherein said Transgene Expression Cassettes impart one or more
functions other than to regulate, maintain, or impart function or
stability to a mini-chromosome.
[0286] The term "plant part" as used herein includes a pod, root,
sett root, shoot root, root primordial, shoot, primary shoot,
secondary shoot, tassle, panicle, arrow, midrib, blade, ligule,
auricle, dewlap, blade joint, sheath, node, internode, bud furrow,
leaf scar, cutting, tuber, stem, stalk, fruit, berry, nut, flower,
leaf, bark, wood, epidermis, vascular tissue, organ, protoplast,
crown, callus culture, petiole, petal, sepal, stamen, stigma,
style, bud, meristem, cambium, cortex, pith, sheath, silk, ovule or
embryo. Other exemplary Sugarcane plant parts are a meiocyte or
gamete or ovule or pollen or endosperm of any of the plants of the
invention. Other exemplary plant parts are a seed, seed-piece,
embryo, protoplast, cell culture, any group of plant cells
organized into a structural and functional unit, ratoon or
propagule.
[0287] The term "promoter" is defined herein as a DNA sequence that
allows the binding of RNA polymerase (including but not limited to
RNA polymerase I, RNA polymerase II and RNA polymerase III from
eukaryotes) and directs the polymerase to a downstream
transcriptional start site of a nucleic acid sequence encoding a
polypeptide to initiate transcription. RNA polymerase effectively
catalyzes the assembly of messenger RNA complementary to the
appropriate DNA strand of the coding region.
[0288] A "promoter operably linked to a heterologous gene" is a
promoter that is operably linked to a gene that is different from
the gene to which the promoter is normally operably linked in its
native state. Similarly, an "exogenous nucleic acid operably linked
to a heterologous regulatory sequence" is a nucleic acid that is
operably linked to a regulatory control sequence to which it is not
normally linked in its native state.
[0289] The term "hybrid promoter" is defined herein as parts of two
or more promoters that are fused together to generate a sequence
that is a fusion of the two or more promoters, which is operably
linked to a coding sequence and mediates the transcription of the
coding sequence into mRNA.
[0290] The term "tandem promoter" is defined herein as two or more
promoter sequences each of which is operably linked to a coding
sequence and mediates the transcription of the coding sequence into
mRNA.
[0291] The term "constitutive active promoter" is defined herein as
a promoter that allows permanent stable expression of the gene of
interest.
[0292] The term "Inducible promoter" is defined herein as a
promoter induced by the presence or absence of a biotic or an
abiotic factor.
[0293] The term "polypeptide" does not refer to a specific length
of the encoded product and, therefore, encompasses peptides,
oligopeptides, and proteins. The term "exogenous polypeptide" is
defined as a polypeptide which is not native to the plant cell, a
native polypeptide in which modifications have been made to alter
the native sequence, or a native polypeptide whose expression is
quantitatively altered as a result of a manipulation of the plant
cell by recombinant DNA techniques.
[0294] As used herein, the term "pseudogene" refers to a
non-functional copy of a protein-coding gene; pseudogenes found in
the genomes of eukaryotic organisms are often inactivated by
mutations and are thus presumed to be non-essential to that
organism; pseudogenes of reverse transcriptase and other open
reading frames found in retroelements are abundant in the
centromeric regions of Arabidopsis and other organisms and are
often present in complex clusters of related sequences.
[0295] 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.
[0296] As used herein the term "repeated nucleotide sequence"
refers to any nucleic acid sequence of at least 25 bp present in a
genome or a recombinant molecule, other than a telomere repeat,
that occurs at least two or more times and that are optionally at
least 80% identical either in head to tail or head to head
orientation either with or without intervening sequence between
repeat units.
[0297] As used herein, the term "retroelement" or "retrotransposon"
refers to a genetic element related to retroviruses that disperse
through an RNA stage; the abundant retroelements present in plant
genomes contain long terminal repeats (LTR retrotransposons) and
encode a polyprotein gene that is processed into several proteins
including a reverse transcriptase. Specific retroelements (complete
or partial sequences) can be found in and around plant centromeres
and can be present as dispersed copies or complex repeat clusters.
Individual copies of retroelements may be truncated or contain
mutations; intact retroelements are rarely encountered.
[0298] As used herein the term "satellite DNA" refers to short DNA
sequences (typically <1000 bp) present in a genome as multiple
repeats, mostly arranged in a tandemly repeated fashion, as opposed
to a dispersed fashion. Repetitive arrays of specific satellite
repeats are abundant in the centromeres of many higher eukaryotic
organisms.
[0299] 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. The use of a screenable marker allows for
the use of lower, sub-killing antibiotic concentrations and the use
of a visible marker gene to identify clusters of transformed cells,
and then manipulation of these cells to homogeneity. Illustrative
screenable markers of the present include genes that encode
fluorescent proteins that are detectable by a visual microscope
such as the fluorescent reporter genes DsRed, ZsGren, ZsYellow,
AmCyan, Green Fluorescent Protein (GFP) and modifications of these
reporter genes to excite or emit at altered wavelengths. An
additional screenable marker gene is lac.
[0300] Alternative methods of screening for modified plant cells
may involve use of relatively low, sub-killing concentrations of a
selection agent (e.g. sub-killing antibiotic concentrations), and
also involve use of a screenable marker (e.g., a visible marker
gene) to identify clusters of modified cells carrying the
screenable marker, after which these screenable cells are
manipulated to homogeneity. 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. This
growth advantage may be present under standard conditions, altered
conditions such as elevated temperature, specialized media
compositions, or in the presence of certain chemicals such as
herbicides or antibiotics. Use of selectable markers is described,
for example, in Broach et al. Gene, 8:121-133, 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,
neomycin phosphotransferase genes and phosphomannose isomerase
gene, among others. Nonlimiting examples of selectable markers in
the present invention include genes whose expression confer
antibiotic or herbicide resistance to the host cell, or proteins
allowing utilization of a carbon source not normally utilized by
plant cells. Expression of one of these markers should be
sufficient to enable the survival of those cells that comprise a
vector within the host cell, and facilitate the manipulation of the
vector into new host cells. Of particular interest in the present
invention are proteins conferring cellular resistance to kanamycin,
G 418, paramomycin, hygromycin, bialaphos, and glyphosate for
example, or proteins allowing utilization of a carbon source, such
as mannose, not normally utilized by plant cells.
[0301] The term "stable" as used herein means that the
mini-chromosome can be transmitted to daughter cells over at least
8 mitotic generations. Some embodiments of mini-chromosomes may be
transmitted as functional, autonomous units for less than 8 mitotic
generations, e.g. 1, 2, 3, 4, 5, 6, or 7. According to
representative embodiments, the mini-chromosome can be transmitted
over at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 generations, for example,
through the regeneration or differentiation of an entire plant, and
may even be are transmitted through meiotic division to gametes.
Other representative mini-chromosomes can be further maintained in
the zygote derived from such a gamete or in an embryo or endosperm
derived from one or more such gametes. A "functional and stable"
mini-chromosome is one in which functional mini-chromosomes can be
detected after transmission of the mini-chromosomes over at least 8
mitotic generations, or after inheritance through a meiotic
division. During mitotic division, as occurs occasionally with
native chromosomes, there may be some non-transmission of
mini-chromosomes; the mini-chromosome may still be characterized as
stable despite the occurrence of such events if an adchromosomal
plant that contains descendants of the mini-chromosome distributed
throughout its parts may be regenerated from cells, cuttings,
propagules, or cell cultures containing the mini-chromosome, or if
an adchromosomal plant can be identified in progeny of the plant
containing the mini-chromosome.
[0302] 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 may
optionally, 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 may
optionally, 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.
[0303] As used herein, the term "telomere" or "telomere DNA" 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. Telomeres can
include naturally occurring telomere sequences or synthetic
sequences. Telomeres from one species may confer telomere activity
in another species. An exemplary telomere DNA is a heptanucleotide
telomere repeat TITAGGG (and its complement) found in the majority
of plants.
[0304] "Transformed," "transgenic," "modified," and "recombinant"
refer to a host organism such as a plant into which an exogenous or
heterologous nucleic acid molecule has been introduced, and
includes meiocytes, seeds, zygotes, embryos, endosperm, or progeny
of such plant that retain the exogenous or heterologous nucleic
acid molecule but which have not themselves been subjected to the
transformation process.
[0305] When the phrase "transmission efficiency" of a certain
percent is used, transmission percent efficiency is calculated by
measuring mini-chromosome presence through one or more mitotic or
meiotic generations. It is directly measured as the ratio
(expressed as a percentage) of the daughter cells or plants
demonstrating presence of the mini-chromosome to parental cells or
plants demonstrating presence of the mini-chromosome. Presence of
the mini-chromosome in parental and daughter cells can be
demonstrated with assays that detect the presence of an exogenous
nucleic acid carried on the mini-chromosome. Exemplary assays can
be the detection of a screenable marker (e.g. presence of a
fluorescent protein or any gene whose expression results in an
observable phenotype), a selectable marker, or PCR amplification of
any exogenous nucleic acid carried on the mini-chromosome.
Constructing Mini-Chromosomes by Site-Specific Recombination
[0306] Sugarcane mini-chromosomes may be constructed using
site-specific recombination sequences (for example those recognized
by the bacteriophage P1 Cre recombinase, or the bacteriophage
lambda integrase, or similar recombination enzymes). According to
this embodiment, a compatible recombination site, or a pair of such
sites, is present on both the Sugarcane centromere containing DNA
clones and the donor DNA clones. Incubation of the donor clone 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 Sugarcane mini-chromosomes contain
Sugarcane centromere sequences as well as mini-chromosome vector
sequences. The DNA molecules formed in such recombination reactions
are introduced into E. coli, other bacteria, yeast or Sugarcane
cells by common methods in the field including, but not limited to,
heat shock, chemical transformation, electroporation, particle
bombardment, whiskers, or other transformation methods followed by
selection for marker genes including chemical, enzymatic, color, or
other marker, allowing for the selection of transformants harboring
mini-chromosomes.
II. Methods of Detecting and Characterizing Mini-Chromosomes in
Plant Cells or of Scoring Mini-Chromosome Performance in Plant
Cells:
Identification of Candidate Centromere Fragments by Probing BAC
Libraries
[0307] Sugarcane centromere clones are identified from a large
Sugarcane genomic insert library such as a Bacterial Artificial
Chromosome library. Probes are labeled using nick-translation in
the presence of radioactively labeled dCTP, dATP, dGTP or dTTP as
in, for example, the commercially available Rediprime kit
(Amersham) as per the manufacturer's instructions. Other labeling
methods familiar to those skilled in the art could be substituted.
The libraries are screened and deconvoluted. Sugarcane genomic
clones are screened by probing with small centromere-specific
clones. Other embodiments of this procedure would involve
hybridizing a library with other centromere sequences. Of the BAC
clones identified using this procedure, a representative set are
identified as having high hybridization signals to some probes, and
optionally low hybridization signals to other probes. These are
selected, the bacterial clones grown up in cultures and DNA
prepared by methods familiar to those skilled in the art such as
alkaline lysis. The DNA composition of purified clones is surveyed
using for example fingerprinting by digesting with restriction
enzymes such as, but not limited to, HinfI or HindIII. In a
representative embodiment the restriction enzyme cuts within the
tandem centromere satellite repeat (see below). A variety of clones
showing different fingerprints are selected for conversion into
mini-chromosomes and inheritance testing. It can also be
informative to use multiple restriction enzymes for fingerprinting
or other enzymes which can cleave DNA.
Fingerprinting Analysis of BACs and Mini-Chromosomes
[0308] Sugarcane centromere function may be associated with large
tandem arrays of satellite repeats. To assess the composition and
architecture of the centromere BACs, the candidate BACs are
digested with a restriction enzyme, such as HindII, which cuts with
known frequency within the consensus sequence of the unit repeat of
the tandemly repeated centromere satellite. Digestion products are
then separated by agarose gel electrophoresis. Large insert clones
containing a large array of tandem repeats will produce a strong
band of the unit repeat size, as well as less intense bands at
2.times. and 3.times. the unit repeat size, and further multiples
of the repeat size. These methods are well-known and there are many
possible variations known to those skilled in the art.
Determining Sequence Composition of Mini-Chromosomes by Shotgun
Cloning/Sequencing, Sequence Analysis
[0309] To determine the sequence composition of the Sugarcane
mini-chromosome, the centromeric region of the Sugarcane
mini-chromosome is sequenced. To generate DNA suitable for
sequencing. Sugarcane mini-chromosomes are fragmented, for example
by using a random shearing method (such as sonication,
nebulization, etc). Other fragmentation techniques may also be used
such as enzymatic digestion. These fragments are then cloned into a
vector (e.g., a plasmid) and sequenced. The resulting DNA sequence
is trimmed of poor-quality sequence and of sequence corresponding
to the vector. The sequence is then compared to the known DNA
sequences using an algorithm such as BLAST to search a sequence
database such as GenBank.
[0310] To determine the consensus of the Sugarcane satellite repeat
in the Sugarcane mini-chromosome, the sequences containing the
satellite repeat are aligned using a DNA sequence alignment program
such as ContigExpress from Vector NTI. The sequences may also be
aligned to previously determined repeats for that species. The
sequences are trimmed to unit repeat length using the consensus as
a template. Sequences trimmed from the ends of the alignment are
realigned with the consensus and further trimmed until all
sequences are at or below the consensus length. The sequences are
then aligned with each other. The consensus is determined by the
frequency of a specific nucleotide at each position; if the most
frequent base is three times more frequent than the next most
frequent base, it was considered the consensus.
[0311] Methods for determining consensus sequence are well known in
the art, see, e.g., U.S. Pat. App. Pub. No. 20030124561; Hall &
Preuss (2002). These methods, including DNA sequencing, assembly,
and analysis, are well-known and there are many possible variations
known to those skilled in the art. Other alignment parameters may
also be useful such as using more or less stringent definitions of
consensus.
Non-Selective Mini-Chromosome Mitotic Inheritance Assays
[0312] The following list of assays and potential outcomes
illustrates how various assays can be used to distinguish
autonomous events from integrated events.
Assay #1: Transient Assay
[0313] Sugarcane mini-chromosomes are tested for their ability to
become established as chromosomes and their ability to be inherited
in mitotic cell divisions. In this assay, Sugarcane
mini-chromosomes are delivered to Sugarcane plant cells, for
example suspension cells in liquid culture. The cells used can be
at various stages of growth. Optionally, a population in which some
cells are undergoing division can be used. The Sugarcane
mini-chromosome is then assessed over the course of several cell
divisions, by tracking the presence of a screenable marker, e.g. a
visible marker gene such as a fluorescent protein. Sugarcane
mini-chromosomes that are established and inherited well may show
an initial delivery into many single cells; after several cell
divisions, these single cells divide to form clusters of
mini-chromosome-containing cells. Other exemplary embodiments of
this method include delivering Sugarcane mini-chromosomes to other
mitotic cell types, including roots and shoot meristems.
Assay #2: Non-Lineage Based Inheritance Assays on Modified
Transformed Cells and Plants
[0314] Sugarcane mini-chromosome inheritance is assessed on
modified cell lines and plants by following the presence of the
mini-chromosome over the course of multiple cell divisions. An
initial population of Sugarcane mini-chromosome containing cells is
assayed for the presence of the Sugarcane mini-chromosome, by the
presence of a marker gene, including but not limited to a
fluorescent protein, a colored protein, a protein assayable by
histochemical assay, and a gene affecting cell morphology. In the
use of a DNA-specific dye, all nuclei are stained with a dye
including but not limited to DAPI, Hoechst 33258, OliGreen, Giemsa
YOYO, or TOTO, allowing a determination of the number of cells that
do not contain the mini-chromosome. After the initial determination
of the percent of cells carrying the Sugarcane mini-chromosome, the
remaining cells are allowed to divide over the course of several
cell divisions. The number of cell divisions, n, is determined by a
method including but not limited to monitoring the change in total
weight of cells, and monitoring the change in volume of the cells
or by directly counting cells in an aliquot of the culture. After a
number of cell divisions, the population of cells is again assayed
for the presence of the Sugarcane mini-chromosome. The loss rate
per generation is calculated by the equation:
Loss rate per generation=1-(F/I).sup.1/n
[0315] The population of Sugarcane mini-chromosome-containing cells
may include suspension cells, callus, roots, leaves, meristems,
flowers, or any other tissue of modified plants, or any other cell
type containing a mini-chromosome.
[0316] These methods are well-known and there are many possible
variations known to those skilled in the art; they have been used
before with human cells and yeast cells.
Assay #3: Lineage Based Inheritance Assays on Modified Cells and
Plants
[0317] Sugarcane mini-chromosome inheritance is assessed on cell
lines and plants comprising Sugarcane mini-chromosomes by following
the presence of the Sugarcane mini-chromosome over the course of
multiple cell divisions. In cell types that allow for tracking of
cell lineage, including but not limited to root or leaf cell files,
trichomes, and leaf stomata guard cells, Sugarcane mini-chromosome
loss per generation does not need to be determined statistically
over a population, it can be discerned directly through successive
cell divisions. In other manifestations of this method, cell
lineage can be discerned from cell position, or methods including
but not limited to the use of histological lineage tracing dyes,
and the induction of genetic mosaics in dividing cells.
[0318] In one simple example, the two guard cells of the stomata
are daughters of a single precursor cell. To assay Sugarcane
mini-chromosome inheritance in this cell type, the epidermis of the
leaf of a Sugarcane plant containing a Sugarcane mini-chromosome is
examined for the presence of the Sugarcane mini-chromosome by the
presence of a marker gene, including but not limited to a
fluorescent protein, a colored protein, a protein assayable by
histochemical assay, and a gene affecting cell morphology. The
number of loss events in which one guard cell contains the
Sugarcane mini-chromosome (L) and the number of cell divisions in
which both guard cells contain the Sugarcane mini-chromosome (B)
are counted. The loss rate per cell division is determined as
L/(L+B). Other lineage-based cell types are assayed in similar
fashion. These methods are well-known and there are many possible
variations known to those skilled in the art; they have been used
before with yeast cells (though, instead of observing the marker in
stomates, a color marker was observed in yeast colonies).
[0319] Linear Sugarcane mini-chromosome inheritance may also be
assessed by examining leaf or root files or clustered cells in
callus over time. Changes in the percent of cells carrying the
Sugarcane mini-chromosome will indicate the mitotic
inheritance.
Assay #4: Inheritance Assays on Modified Cells and Plants in the
Presence of Chromosome Loss Agents
[0320] Any of the above three assays can be done in the presence of
chromosome loss agents (including but not limited to colchicine,
colcemid, caffeine, etopocide, nocodazole, oryzalin, trifluran). It
is likely that an autonomous Sugarcane mini-chromosome will prove
more susceptible to loss induced by chromosome loss agents;
therefore, autonomous mini-chromosomes should show a lower rate of
inheritance in the presence of chromosome loss agents. These
methods have been used to study chromosome loss in fruit flies and
yeast; there are many possible variations known to those skilled in
the art.
III. Transformation of Plant Cells and Plant Regeneration
[0321] Various methods may be used to deliver DNA into plant cells.
These include biological methods, such as Agrobacterium, E. coli,
and viruses, physical methods such as biolistic particle
bombardment, nanocopoea device, the Stein beam gun, silicon carbide
whiskers and microinjection, electrical methods such as
electroporation, and chemical methods such as the use of
poly-ethylene glycol and other compounds known to stimulate DNA
uptake into cells. Examples of these techniques are described by
Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al.,
Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology
4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987).
Transformation using silicon carbide whiskers, e.g. in maize, is
described in Brisibe, J. Exp. Bot. 51(343):187-196 (2000) and
Dunwell, Methods Mol. Biol. 111:375-82 (1999) and U.S. Pat. No.
5,464,765.
Agrobacterium-Mediated Delivery
[0322] Agrobacterium-mediated transformation is one method for
introducing a desired genetic element into a plant. Several
Agrobacterium species mediate the transfer of a specific DNA known
as "T-DNA" that can be genetically engineered to carry a desired
piece of DNA into many plant species. Plasmids used for delivery
contain the T-DNA flanking the nucleic acid to be inserted into the
plant. The major events marking the process of T-DNA mediated
pathogenesis are induction of virulence genes, processing and
transfer of T-DNA.
[0323] There are three common methods to transform plant cells with
Agrobacterium. The first method is co-cultivation of Agrobacterium
with cultured isolated protoplasts. This method requires an
established culture system that allows culturing protoplasts and
plant regeneration from cultured protoplasts. The second method is
transformation of cells or tissues with Agrobacterium. This method
requires (a) that the plant cells or tissues can be modified by
Agrobacterium and (b) that the modified cells or tissues can be
induced to regenerate into whole plants. The third method is
transformation of seeds, immature or mature embryos, apices or
meristems with Agrobacterium. This method requires exposure of the
meristematic cells of these tissues to Agrobacterium and
micropropagation of the shoots or plant organs arising from those
meristematic cells.
[0324] Those of skill in the art are familiar with procedures for
growth and suitable culture conditions for Agrobacterium as well as
subsequent inoculation procedures. Liquid, solid, or semi-solid
culture media can be used. The density of the Agrobacterium culture
used for inoculation and the ratio of Agrobacterium cells to
explant can vary from one system to the next, as can media, growth
procedures, timing and lighting conditions.
[0325] Transformation of dicotyledons using Agrobacterium has long
been known in the art, and transformation of monocotyledons using
Agrobacterium has also been described. See, WO 94/00977 and U.S.
Pat. No. 5,591,616, both of which are incorporated herein by
reference. See also, Negrotto et al., Plant Cell Reports 19:
798-803 (2000), incorporated herein by reference.
[0326] A number of wild-type and disarmed strains of Agrobacterium
tumefaciens and Agrobacterium rhizogenes harboring Ti or Ri
plasmids can be used for gene transfer into plants. In embodiments
of the invention, the Agrobacterium hosts contain disarmed Ti and
Ri plasmids that do not contain the oncogenes that cause
tumorigenesis or rhizogenesis. Exemplary strains include
Agrobacterium lumefaciens strain C58, a nopaline-type strain that
is used to mediate the transfer of DNA into a plant cell,
octopine-type strains such as LBA4404 or succinamopine-type
strains, e.g., EHA101 or EHA105. The use of these strains for plant
transformation has been reported and the methods are familiar to
those of skill in the art.
[0327] U.S. Application No. 20040244075 published Dec. 2, 2004
describes improved methods of Agrobacterium-mediated
transformation. The efficiency of transformation by Agrobacterium
may be enhanced by using a number of methods known in the art. For
example, the inclusion of a natural wound response molecule such as
acetosyringone (AS) to the Agrobacterium culture has been shown to
enhance transformation efficiency with Agrobacterium tumefaciens
(Shahla et al., (1987) Plant Molec. Biol. 8:291-298).
Alternatively, transformation efficiency may be enhanced by
wounding the target tissue to be modified or transformed. Wounding
of plant tissue may be achieved, for example, by punching,
maceration, bombardment with microprojectiles, etc. (See e.g.,
Bidney et al., (1992) Plant Molec. Biol. 18:301-313).
[0328] In addition, another recent method described by Broothaerts,
et. al. (Nature 433: 629-633, 2005) expands the bacterial genera
that can be used to transfer genes into plants. This work involved
the transfer of a disarmed Ti plasmid without T-DNA and another
vector with T-DNA containing the marker enzyme beta-glucuronidase,
into three different bacteria. Gene transfer was successful and
this method significantly expands the tools available for gene
delivery into plants.
Microprojectile Bombardment Delivery
[0329] Another widely used technique to genetically transform
plants involves the use of microprojectile bombardment. In this
process, a nucleic acid containing the desired genetic elements to
be introduced into the plant is deposited on or in small dense
particles, e.g., tungsten, platinum, or 0.5 to 1.0 micron gold
particles, which are then delivered at a high velocity into the
plant tissue or plant cells using a specialized biolistics device.
Many such devices have been designed and constructed; one in
particular, the PDS1000/He sold by BioRad, is the instrument most
commonly used for biolistics of plant cells. The advantage of this
method is that no specialized sequences need to be present on the
nucleic acid molecule to be delivered into plant cells; delivery of
any nucleic acid sequence is theoretically possible.
[0330] For the bombardment, cells in suspension are concentrated on
filters, petri dishes or solid culture medium. Alternatively,
immature embryos, seedling explants, or any plant tissue or target
cells may be arranged on filters, petri dishes or solid culture
medium. The cells to be bombarded are positioned at an appropriate
distance below the microprojectile stopping plate.
[0331] Various biolistics protocols have been described that differ
in the type of particle or the manner in which DNA is coated onto
the particle. Any technique for coating microprojectiles that
allows for delivery of transforming DNA to the target cells may be
used. For example, particles may be prepared by functionalizing the
surface of a gold particle by providing free amine groups. DNA,
having a strong negative charge, will then bind to the
functionalized particles.
[0332] Parameters such as the concentration of DNA used to coat
microprojectiles may influence the recovery of transformants
containing a single copy of the transgene. For example, a lower
concentration of DNA may not necessarily change the efficiency of
the transformation but may instead increase the proportion of
single copy insertion events. In this regard, ranges of
approximately 1 ng to approximately 10 .mu.g (10,000 ng),
approximately 5 ng to 8 .mu.g or approximately 20 ng, 50 ng, 100
ng, 200 ng, 500 ng, 1 .mu.g, 2 .mu.g, 5 .mu.g, or 7 g of
transforming DNA may be used per each 1.0-2.0 mg of starting gold
particles (in the 0.5 to 1.0 micron range).
[0333] Other physical and biological parameters may be varied, such
as manipulation of the DNA/microprojectile precipitate, factors
that affect the flight and velocity of the projectiles,
manipulation of the cells before and immediately after bombardment
(including osmotic state, tissue hydration and the subculture stage
or cell cycle of the recipient cells), the orientation of an
immature embryo or other target tissue relative to the particle
trajectory, and also the nature of the transforming DNA, such as
linearized DNA or intact supercoiled plasmids. One may also want to
use agents to protect the DNA during delivery. One may particularly
wish to adjust physical parameters such as DNA concentration, gap
distance, flight distance, tissue distance, and helium
pressure.
[0334] The particles delivered via biolistics can be "dry" or
"wet." In the "dry" method, the mini-chromosome DNA-coated
particles such as gold are applied onto a macrocarrier (such as a
metal plate, or a carrier sheet made of a fragile material such as
mylar) and dried. The gas discharge then accelerates the
macrocarrier into a stopping screen, which halts the macrocarrier
but allows the particles to pass through; the particles then
continue their trajectory until they impact the tissue being
bombarded. For the "wet" method, the droplet containing the
mini-chromosome DNA-coated particles is applied to the bottom part
of a filter holder, which is attached to a base which is itself
attached to a rupture disk holder used to hold the rupture disk to
the helium egress tube for bombardment. The gas discharge directly
displaces the DNA/gold droplet from the filter holder and
accelerates the particles and their DNA cargo into the tissue being
bombarded. The wet biolistics method has been described in detail
elsewhere but has not previously been applied in the context of
plants (Mialhe et al., Mol Mar Biol Biotechnol. 4(4):275-83, 1995).
The concentrations of the various components for coating particles
and the physical parameters for delivery can be optimized using
procedures known in the art. A variety of Sugarcane cells/tissues
are suitable for transformation, including immature embryos,
scutellar tissue, suspension cell cultures, immature inflorescence,
shoot meristem, epithelial peels, nodal explants, callus tissue,
hypocotyl tissue, cotyledons, roots, leaves, meristem cells, and
gametic cells such as microspores, pollen, sperm and egg cells. It
is contemplated that any cell from which a fertile plant may be
regenerated is useful as a recipient cell. Callus may be initiated
from tissue sources including, but not limited to, immature
embryos, seedling apical meristems, microspore-derived embryos,
roots, hypocotyls, cotyledons and the like. Those cells which are
capable of proliferating as callus also are recipient cells for
genetic transformation.
[0335] Any suitable plant culture medium can be used. Examples of
suitable media would include but are not limited to MS-based media
(Murashige and Skoog, Physiol. Plant, 15:473-497, 1962) or N6-based
media (Chu et al., Scientia Sinica 18:659, 1975) supplemented with
additional plant growth regulators including but not limited to
auxins such as picloram (4-amino-3,5,6-trichloropicolinic acid),
2,4-D (2,4-dichlorophenoxyacetic acid), naphalene-acetic acid (NAA)
and dicamba (3,6-dichloroanisic acid), cytokinins such as BAP
(6-benzylaminopurine) and kinetin, and gibberellins. Other media
additives can include but are not limited to amino acids,
macroelements, iron, microelements, vitamins and organics,
carbohydrates, undefined media components such as casein
hydrolysates, an appropriate gelling agent such as a form of agar,
a low melting point agarose or Gelrite if desired. Those of skill
in the art are familiar with the variety of tissue culture media,
which when supplemented appropriately, support plant tissue growth
and development and are suitable for plant transformation and
regeneration. These tissue culture media can either be purchased as
a commercial preparation, or custom prepared and modified. Examples
of such media would include but are not limited to Murashige and
Skoog (Murashige and Skoog, Physiol. Plant, 15:473-497, 1962), N6
(Chu et al., Scientia Sinica 18:659, 1975), Linsmaier and Skoog
(Linsmaier and Skoog, Physio. Plant., 18:100, 1965), Uchimiya and
Murashige (Uchimiya and Murashige, Plant Physiol. 15:473, 1962),
Gamborg's B5 media (Gamborg et al., Exp. Cell Res., 50:151, 1968),
D medium (Duncan et al., Planta, 165:322-332, 1985), Mc-Cown's
Woody plant media (McCown and Lloyd, HortScience 6:453, 1981),
Nitsch and Nitsch (Nitsch and Nitsch, Science 163:85-87, 1969), and
Schenk and Hildebrandt (Schenk and Hildebrandt, Can. J. Bot.
50:199-204, 1972) or derivations of these media supplemented
accordingly. Those of skill in the art are aware that media and
media supplements such as nutrients and growth regulators for use
in transformation and regeneration and other culture conditions
such as light intensity during incubation, pH, and incubation
temperatures can be varied.
[0336] Those of skill in the art are aware of the numerous
modifications in selective regimes, media, and growth conditions
that can be varied depending on the plant system and the selective
agent. Typical selective agents include but are not limited to
antibiotics such as geneticin (G418), kanamycin, paromomycin or
other chemicals such as glyphosate or other herbicides.
Consequently, such media and culture conditions disclosed in the
present invention can be modified or substituted with nutritionally
equivalent components, or similar processes for selection and
recovery of transgenic events, and still fall within the scope of
the present invention.
Sugarcane Mini-Chromosome Delivery without Selection
[0337] The Sugarcane mini-chromosome is delivered to Sugarcane
plant cells or tissues, e.g., plant cells in suspension to obtain
stably modified callus clones for inheritance assay. Suspension
cells are maintained in a growth media, for example Murashige and
Skoog (MS) liquid medium containing an auxin such as
2,4-dichlorophenoxyacetic acid (2,4-D). Cells are bombarded using a
particle bombardment process, such as the helium-driven PDS-1000/He
system, and propagated in the same liquid medium to permit the
growth of modified and non-modified cells. Portions of each
bombardment are monitored for formation of fluorescent clusters,
which are isolated by micromanipulation and cultured on solid
medium. Clones modified with the mini-chromosome are expanded and
homogenous clones are used in inheritance assays, or assays
measuring mini-chromosome structure or autonomy.
Sugarcane Mini-Chromosome Transformation with Selectable Marker
Gene
[0338] Isolation of Sugarcane mini-chromosome-modified cells in
bombarded calluses or explants can be facilitated by the use of a
selectable marker gene. The bombarded tissues are transferred to a
medium containing an appropriate selective agent for a particular
selectable marker gene. Such a transfer usually occurs between 0
and about 7 days after bombardment. The transfer could also take
place any number of days after bombardment. The amount of selective
agent and timing of incorporation of such an agent in selection
medium can be optimized by using procedures known in the art.
Selection inhibits the growth of non-modified cells, thus providing
an advantage to the growth of modified cells, which can be further
monitored by tracking the presence of a fluorescent marker gene or
by the appearance of modified explants (modified cells or explants
may be green under light in selection medium, while surrounding
non-modified cells are weakly pigmented). In plants that develop
through shoot organogenesis, the modified cells can form shoots
directly, or alternatively, can be isolated and expanded for
regeneration of multiple shoots transgenic for the Sugarcane
mini-chromosome. In plants that develop through embryogenesis,
additional culturing steps may be necessary to induce the modified
cells to form an embryo and to regenerate in the appropriate media.
Sugarcane is generally regenerated through embryogenesis but can
also be regenerated by shoot organogenesis.
[0339] Useful selectable marker genes are well known in the art and
include, for example, herbicide and antibiotic resistance genes
including but not limited to neomycin phosphotransferase II
(conferring resistance to kanamycin, paramomycin and G418),
hygromycin phosphotransferase (conferring resistance to
hygromycin), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS,
conferring resistance to glyphosate), phosphinothricin
acetyltransferase (conferring resistance to
phosphinothricin/bialophos), MerA (conferring resistance to
mercuric ions). Selectable marker genes may be transformed using
standard methods in the art.
[0340] The first step in the production of Sugarcane plants
containing novel genes involves delivery of DNA into a suitable
plant tissue (described in the previous section) and selection of
the tissue under conditions that allow preferential growth of any
cells containing the novel genes. Selection is typically achieved
with a selectable marker gene present in the delivered DNA, which
may be a gene conferring resistance to an antibiotic, herbicide or
other killing agent, or a gene allowing utilization of a carbon
source not normally metabolized by plant cells. For selection to be
effective, the plant cells or tissue need to be grown on selective
medium containing the appropriate concentration of antibiotic or
killing agent, and the cells need to be plated at a defined and
constant density. The concentration of selective agent and cell
density are generally chosen to cause complete growth inhibition of
wild type plant tissue that does not express the selectable marker
gene; but allowing cells containing the introduced DNA to grow and
expand into adchromosomal clones. This critical concentration of
selective agent typically is the lowest concentration at which
there is complete growth inhibition of wild type cells, at the cell
density used in the experiments. However, in some cases,
sub-killing concentrations of the selective agent may be equally or
more effective for the isolation of plant cells containing
mini-chromosome DNA, especially in cases where the identification
of such cells is assisted by a visible marker gene (e.g.,
fluorescent protein gene) present on the Sugarcane mini-chromosome.
Such sub-killing concentrations of the selective agent may be
administered during part or all of the selection timing.
[0341] In some species (e.g., tobacco or tomato), a homogenous
clone of modified cells can also arise spontaneously when bombarded
cells are placed under the appropriate selection. An exemplary
selective agent is the neomycin phosphotransferase II (nptII)
marker gene, which is commonly used in plant biotechnology and
confers resistance to the antibiotics kanamycin, 418 (geneticin)
and paramomycin. In other species, or in certain plant tissues or
when using particular selectable markers, homogeneous clones may
not arise spontaneously under selection; in this case the clusters
of modified cells can be manipulated to homogeneity using the
visible marker genes present on the mini-chromosomes as an
indication of which cells contain mini-chromosome DNA.
Regeneration of Modified Plants from Explants to Mature, Rooted
Plants
[0342] For Sugarcane, regeneration of a whole plant typically
occurs via an embryogenic step that is not necessary for plant
species where shoot organogenesis is more efficient. The explant
tissue is cultured on an appropriate media for embryogenesis, and
the embryo is cultured until shoots form. The regenerated shoots
are cultured in a rooting medium to obtain intact whole plants with
a fully developed root system. These plants are potted in soil and
grown to maturity in a greenhouse.
[0343] Generally, regeneration and tissue culture of Sugarcane
plant parts and whole plants is challenging as Sugarcane produces
phenolic compounds while in culture. The present invention provides
for methods of culturing Sugarcane cells and tissues in media
containing polyvinylpyrrolidone (PVP), as described in Example 4.
The PVP acts as a sink for the phenolic compounds produced by
Sugarcane and enhances callus growth during selection as well as
facilitating callus and plantlet regeneration. Furthermore,
generation of Sugarcane callus can be facilitated by delivering to
the plant cells and/or tissues mini-chromosomes of the invention
that contain auxin genes. The presence of the auxin genes will
facilitate callus induction of the transformed tissue. The
invention also provides for tissue culture methods which cycle
between the liquid culture media and solid culture media in order
to promote the frequency and the morphogenic competence of the
regenerable Sugarcane callus.
[0344] For plants that develop through shoot organogenesis,
regeneration of a whole plant involves culturing of regenerable
explant tissues taken from sterile organogenic callus tissue,
seedlings or mature plants on a shoot regeneration medium for shoot
organogenesis, and rooting of the regenerated shoots in a rooting
medium to obtain intact whole plants with a fully developed root
system. These plants are potted in soil and grown to maturity in a
greenhouse.
[0345] Explants are obtained from any tissues of a plant suitable
for regeneration. Exemplary tissues include hypocotyls, internodes,
roots, cotyledons, petioles, cotyledonary petioles, leaves and
peduncles, prepared from sterile seedlings or mature plants.
[0346] Explants are wounded (for example with a scalpel or razor
blade) and cultured on a shoot regeneration medium (SRM) containing
Murashige and Skoog (MS) medium as well as a cytokinin, e.g.,
6-benzylaminopurine (BA), and an auxin, e.g.,
.alpha.-naphthaleneacetic acid (NAA), and an anti-ethylone agent,
e.g., silver nitrate (AgNO.sub.3). For example, 2 mg/L of BA, 0.05
mg/L of NAA, and 2 mg/L of AgNO.sub.3 can be added to MS medium for
shoot organogenesis. The most efficient shoot regeneration is
obtained from longitudinal sections of internode explants.
[0347] Shoots regenerated via organogenesis are rooted in a MS
medium. Plants are potted and grown in a greenhouse to sexual
maturity for seed harvest.
[0348] To regenerate a whole Sugarcane plant with a Sugarcane
mini-chromosome, explants are pre-incubated for 1 to 7 days (or
longer) on the shoot regeneration medium prior to bombardment with
mini-chromosome (see below). Following bombardment, explants are
incubated on the same shoot regeneration medium for a recovery
period up to 7 days (or longer), followed by selection for
transformed shoots or clusters on the same medium but with a
selective agent appropriate for a particular selectable marker gene
(described herein).
Method of Co-Delivering Growth Inducing Genes to Facilitate
Isolation of Modified Plant Cell Clones
[0349] Another method used in the generation of Sugarcane cell
clones containing Sugarcane mini-chromosomes involves the
co-delivery of DNA containing genes that are capable of activating
growth of plant cells, or that promote the formation of a specific
organ, embryo or plant structure that is capable of self-sustaining
growth. In one embodiment, the recipient Sugarcane cell receives
simultaneously the Sugarcane mini-chromosome and a separate DNA
molecule encoding one or more growth promoting,
organogenesis-promoting, embryogenesis-promoting or
regeneration-promoting genes. Following DNA delivery, expression of
the plant growth regulator genes stimulates the plant cells to
divide, or to initiate differentiation into a specific organ,
embryo, or other cell types or tissues capable of regeneration.
Multiple plant growth regulator genes can be combined on the same
molecule, or co-bombarded on separate molecules. Use of these genes
can also be combined with application of plant growth regulator
molecules into the medium used to culture the Sugarcane cells, or
of precursors to such molecules that are converted to functional
plant growth regulators by the plant cell's biosynthetic machinery,
or by the genes delivered into the Sugarcane cell.
[0350] The co-bombardment strategy of Sugarcane mini-chromosomes
with separate DNA molecules encoding plant growth regulators
transiently supplies the plant growth regulator genes for several
generations of Sugarcane cells following DNA delivery. During this
time, the Sugarcane mini-chromosome may be stabilized by virtue of
its Sugarcane centromere, but the DNA molecules encoding plant
growth regulator genes, or organogenesis-promoting,
embryogenesis-promoting or regeneration-promoting genes will tend
to be lost. The transient expression of these genes, prior to their
loss, may give the cells containing Sugarcane mini-chromosome DNA a
sufficient growth advantage, or sufficient tendency to develop into
plant organs, embryos or a regenerable cell cluster, to outgrow the
non-modified cells in their vicinity, or to form a readily
identifiable structure that is not formed by non-modified Sugarcane
cells. Loss of the DNA molecule encoding these genes will prevent
phenotypes from manifesting themselves that may be caused by these
genes if present through the remainder of Sugarcane plant
regeneration. In rare cases, the DNA molecules encoding plant
growth regulator genes will integrate into the Sugarcane genome or
into the Sugarcane mini-chromosome.
[0351] Alternatively the genes promoting plant cell growth may be
genes promoting shoot formation or embryogenesis or giving rise to
any identifiable organ, tissue or structure that can be regenerated
into a Sugarcane plant. In this case, it may be possible to obtain
embryos or shoots harboring Sugarcane mini-chromosomes directly
after DNA delivery, without the need to induce shoot formation with
growth activators supplied into the medium, or lowering the growth
activator treatment necessary to regenerate Sugarcane plants. The
advantages of this method are more rapid regeneration, higher
transformation efficiency, lower background growth of non-modified
tissue, and lower rates of morphologic abnormalities in the
regenerated Sugarcane plants (due to shorter and less intense
treatments of the tissue with chemical plant growth activators
added to the growth medium).
Determination of Min-Chromosome Structure and Autonomy in Sugarcane
Adchromosomal Plants and Tissues
[0352] The structure and autonomy of the Sugarcane mini-chromosome
in modified Sugarcane plants and tissues can be determined by
methods including but not limited to: conventional and pulsed-field
Southern blot hybridization to genomic DNA from modified tissue
subjected or not subjected to restriction endonuclease digestion,
dot blot hybridization of genomic DNA from modified tissue
hybridized with different mini-chromosome specific sequences,
mini-chromosome rescue, exonuclease activity, PCR on DNA from
modified tissues with probes specific to the mini-chromosome, or
Fluorescence In Situ Hybridization to nuclei of modified cells.
Table 3 below summarizes these methods.
TABLE-US-00003 TABLE 3 Examples of methods to determine
mini-chromosome structure and autonomy Assay Assay details
Potential outcome Interpretation Southern Restriction digest of
Native sizes and patter Autonomous or blot genomic DNA* of bands
integrated via CEN compared to purified fragment mini-C Altered
sizes or pattern Integrated or rearrange of bands CHEF gel
Restriction digest of Native sizes and patter Autonomous or
Southern genomic DNA compar of bands integrated via CEN blot to
purified mini-C fragment Altered sizes or pattern Integrated or
rearrange of bands Native genomic DNA Mini-C band migrating
Autonomous circles or (no digest) ahead of genomic DNA linears
present in plant Mini-C band Integrated co-migrating with genomic
DNA >1 mini-C bands Various possibilities observed Exonuclease
Exonuclease digestion Signal strength close to Autonomous circles
assay genomic DNA followe that w/o exonuclease present by detection
of circular No signal or signal Integrated mini-chromosome by
strength lower that w/o PCR, dot blot, or exonuclease restriction
digest optional), electrophoresis and southern blot (useful fo
circular mini- chromosomes) Mini- Transformation of plan Colonies
isolated only Autonomous circles chromosome genomic DNA into E.
from mini-C plants wit present, native mini-C rescue coli followed
by mini-Cs, not from structure selection for antibiotic controls;
mini-C resistance genes on structure matches that mini-C of the
parental mini-C Colonies isolated only Autonomous circles from
mini-C plants wit present, rearranged mini-Cs, not from mini-C
structure OR controls; mini-C mini-Cs integrated via structure
different from centromere fragment parental mini-C Colonies
observed both Various possibilities in mini-C-modified plants and
in controls PCR PCR amplification of All mini-c parts detecte
Complete mini-C various parts of the mi by PCR sequences present in
chromosome plant Subset of mini-c parts Partial mini-C sequenc
detected by PCR present in plant FISH Detection of mini- Mini-C
sequences autonomous chromosome sequences detected, free of genom
in mitotic or meiotic Mini-C sequences integrated nuclei by
fluorescence detected, associated wi in situ hybridization genome
Mini-C sequences Both autonomous and detected, both free and
integrated mini-C associated with genom sequences present No mini-C
sequences Mini-C DNA not detected visibl by FISH *Genomic DNA
refers to total DNA extracted from plants containing a
mini-chromosome indicates data missing or illegible when filed
[0353] Furthermore, Sugarcane mini-chromosome structure can be
examined by characterizing mini-chromosomes `rescued` from
Sugarcane adchromosomal cells. Circular Sugarcane mini-chromosomes
that contain bacterial sequences for their selection and
propagation in bacteria can be rescued from a Sugarcane
adchromosomal plant or plant cell and re-introduced into bacteria.
If no loss of these sequences has occurred during replication of
the Sugarcane mini-chromosome in plant cells, the mini-chromosome
is able to replicate in bacteria and confer antibiotic resistance.
Total genomic DNA is isolated from the Sugarcane adchromosomal
plant cells by any method for DNA isolation known to those skilled
in the art, including but not limited to a standard
cetyltrimethylammonium bromide (CTAB) based method (Current
Protocols in Molecular Biology (1994) John Wiley & Sons, N.Y.,
2.3). The purified genomic DNA is introduced into bacteria (e.g.,
E. coli) using methods familiar to one skilled in the art (for
example heat shock or electroporation). The transformed bacteria
are plated on solid medium containing antibiotics to select
bacterial clones modified with Sugarcane mini-chromosome DNA.
Modified bacterial clones are grown up, the plasmid DNA purified
(by alkaline lysis for example), and DNA analyzed by restriction
enzyme digestion and gel electrophoresis or by sequencing. Because
plant-methylated DNA containing methylcytosine residues will be
degraded by wild-type strains of E. coli, bacterial strains (e.g.
DH10B) deficient in the genes encoding methylation restriction
nucleases (e.g. the mcr and mrr gene loci in E. coli) are suitable
for this type of analysis. Sugarcane mini-chromosome rescue can be
performed on any plant tissue or clone of plant cells comprising a
mini-chromosome.
Sugarcane Mini-Chromosome Autonomy Demonstration by In Situ
Hybridization (ISH)
[0354] To assess whether the Sugarcane mini-chromosome is
autonomous from the native Sugarcane chromosomes, or has integrated
into the plant genome, In Situ Hybridization is carried out
(Fluorescent In Situ Hybridization or FISH is particularly well
suited to this purpose). In this assay, mitotic or meiotic tissue,
such as root tips or meiocytes from the anther, possibly treated
with metaphase arrest agents such as colchicines or nitrous oxide
is obtained, and standard FISH methods are used to label both the
Sugarcane centromere and sequences specific to the Sugarcane
mini-chromosome. For example, a Sugarcane centromere is labeled
using a probe from a sequence that labels all Sugarcane
centromeres, attached to one fluorescent tag (Molecular Probes
Alexafluor 568, for example), and sequences specific to the
Sugarcane mini-chromosome are labeled with another fluorescent tag
(Alexafluor 488, for example). All Sugarcane centromere sequences
are detected with the first tag; only Sugarcane mini-chromosomes
are detected with both the first and second tag. Chromosomes are
stained with a DNA-specific dye including but not limited to DAPI,
Hoechst 33258, OliGreen, Giemsa YOYO, and TOTO. An autonomous
Sugarcane mini-chromosome is visualized as a body that shows
hybridization signal with both Sugarcane centromere probes and
Sugarcane mini-chromosome specific probes and is separate from the
native Sugarcane chromosomes.
Determination of Genes Expression Levels
[0355] The expression level of any gene present on the Sugarcane
mini-chromosome can be determined by methods including but not
limited to one of the following. The mRNA level of the gene can be
determined by Northern Blot hybridization, Reverse
Transcriptase-Polymerase Chain Reaction, binding levels of a
specific RNA-binding protein, in situ hybridization, or dot blot
hybridization.
[0356] The protein level of the gene product can be determined by
Western blot hybridization, Enzyme-Linked Immunosorbant Assay
(ELISA), fluorescent quantitation of a fluorescent gene product,
enzymatic quantitation of an enzymatic gene product,
immunohistochemical quantitation, or spectroscopic quantitation of
a gene product that absorbs a specific wavelength of light.
[0357] Use of Exonuclease to Isolate Circular Sugarcane
Mini-Chromosome DNA from Sugarcane Genomic DNA
[0358] Exonucleases may be used to obtain pure Sugarcane
mini-chromosome DNA, suitable for isolation of Sugarcane
mini-chromosomes from E. coli or from Sugarcane cells. The method
assumes a circular structure of the Sugarcane mini-chromosome. A
DNA preparation containing Sugarcane mini-chromosome DNA and
Sugarcane genomic DNA from the source organism is treated with
exonuclease, for example lambda exonuclease combined with E. coli
exonuclease I, or the ATP-dependent exonuclease (Qiagen Inc).
Because the exonuclease is only active on DNA ends, it will
specifically degrade the linear Sugarcane genomic DNA fragments,
but will not affect the circular Sugarcane mini-chromosome DNA. The
result is Sugarcane mini-chromosome DNA in pure form. The resultant
Sugarcane mini-chromosome DNA can be detected by a number of
methods for DNA detection known to those skilled in the art,
including but not limited to PCR, dot blot followed by
hybridization analysis, and southern blot followed by hybridization
analysis. Exonuclease treatment followed by detection of resultant
circular Sugarcane mini-chromosomes may be used as a method to
determine Sugarcane mini-chromosome autonomy.
Structural Analysis of Sugarcane Mini-Chromosomes by BAC-End
Sequencing
[0359] BAC-end sequencing procedures, known to those skilled in the
art, can be applied to characterize Sugarcane mini-chromosome
clones for a variety of purposes, such as structural
characterization, determination of sequence content, and
determination of the precise sequence at a unique site on the
chromosome (for example the specific sequence signature found at
the junction between a centromere fragment and the vector
sequences). In particular, this method is useful to prove the
relationship between a parental Sugarcane mini-chromosome and the
mini-chromosomes descended from it and isolated from plant cells by
mini-chromosome rescue, described above. This method also fosters
identification of specific Sugarcane mini-chromosomes if more than
one unique Sugarcane mini-chromosome is present in a plant cell
simultaneously.
Methods for Scoring Meiotic Sugarcane Mini-Chromosome
Inheritance
[0360] A variety of methods can be used to assess the efficiency of
meiotic Sugarcane mini-chromosome transmission. In one embodiment
of the method, gene expression of genes encoded by the Sugarcane
mini-chromosome (marker genes or non-marker genes) can be scored by
any method for detection of gene expression known to those skilled
in the art, including but not limited to visible methods (e.g.
fluorescence of fluorescent protein markers, scoring of visible
phenotypes of the plant), scoring resistance of the Sugarcane
plants or plant tissues to antibiotics, herbicides or other
selective agents, by measuring enzyme activity of proteins encoded
by the mini-chromosome, or measuring non-visible plant phenotypes,
or directly measuring the RNA and protein products of gene
expression using microarray, northern blots, in situ hybridization,
dot blot hybridization, RT-PCR, western blots, immunoprecipitation,
Enzyme-Linked Immunosorbant Assay (ELISA), immunofluorescence and
radio-immunoassays (RIA). Gene expression can be scored in the
post-meiotic stages of microspore, pollen, pollen tube or female
gametophyte, or the post-zygotic stages such as embryo, seed, or
progeny seedlings and plants. In another embodiment of the method,
the Sugarcane mini-chromosome can be directly detected or
visualized in post-meiotic, zygotic, embryonal or other cells by a
number of methods for DNA detection known to those skilled in the
art, including but not limited to fluorescence in situ
hybridization, in situ PCR, PCR, southern blot, or by Sugarcane
mini-chromosome rescue described above.
FISH Analysis of Sugarcane Mini-Chromosome Copy Number in
Meiocytes, Roots or Other Tissues of Modified Sugarcane Plants
[0361] The copy number of the Sugarcane mini-chromosome can be
assessed in any cell or plant tissue by In Situ Hybridization
(Fluorescent In Situ Hybridization or FISH is particularly well
suited to this purpose). In an exemplary assay, standard FISH
methods are used to label the Sugarcane centromere, using a probe
which labels all Sugarcane chromosomes with one fluorescent tag
(Molecular Probes Alexafluor 568, for example), and to label
sequences specific to the Sugarcane mini-chromosome with another
fluorescent tag (Alexafluor 488, for example). All Sugarcane
centromere sequences are detected with the first tag; only
Sugarcane mini-chromosomes are detected with both the first and
second tag. Nuclei are stained with a DNA-specific dye including
but not limited to DAPI, Hoechst 33258, OliGreen, Giemsa YOYO, and
TOTO. Sugarcane mini-chromosome copy number is determined by
counting the number of fluorescent foci per cell that label with
both tags.
Induction of Callus and Roots from Modified Sugarcane Plant Tissues
for Inheritance Assays
[0362] Sugarcane mini-chromosome inheritance is assessed using
callus and roots induced from transformed Sugarcane plants. To
induce roots and callus, tissues such as leaf pieces are prepared
from adchromosomal Sugarcane plants and cultured on a Murashige and
Skoog (MS) or Chus's N6 (N6) medium that may contain a cytokinin,
e.g., 6-benzylaminopurine (BA), and an auxin, e.g.,
.alpha.-naphthaleneacetic acid (NAA). Any tissue of a modified
Sugarcane plant can be used for callus and root induction, and the
medium recipe for tissue culture can be optimized using procedures
known in the art.
Clonal Propagation of Modified Sugarcane Plants
[0363] To produce multiple clones of plants from a
mini-chromosome-transformed Sugarcane plant, any tissue of the
plant can be tissue-cultured for shoot organogenesis using
regeneration procedures described herein for regeneration of plants
from explants. Alternatively, multiple auxiliary buds can induced
from a mini-chromosome-modified Sugarcane plant by excising the
shoot tip, which can be rooted and subsequently be grown into a
whole plant; each auxiliary bud can be rooted and produce a whole
plant. Additionally, multiple shoots that result from one plant can
be subdivided in culture to produce multiple individual plants.
Scoring of Antibiotic- or Herbicide Resistance in Seedlings and
Plants (Progeny of Self- and Out-Crossed Transformants
[0364] Progeny seeds harvested from Sugarcane plants comprising a
mini-chromosome can be scored for antibiotic- or herbicide
resistance by seed germination under sterile conditions on a growth
media (for example Murashigc and Skoog (MS) medium) containing an
appropriate selective agent for a particular selectable marker
gene. Only seeds containing the Sugarcane mini-chromosome can
germinate on the medium and further grow and develop into whole
plants. Alternatively, Sugarcane seeds can be germinated in soil,
and the germinating seedlings can then be sprayed with a selective
agent appropriate for a selectable marker gene. Sugarcane seedlings
that do not contain a Sugarcane mini-chromosome do not survive;
only seedlings containing a mini-chromosome can survive and develop
into mature plants.
Genetic Methods for Analyzing Sugarcane Mini-Chromosome
Performance:
[0365] Though Sugarcane is typically propagated vegitatively, it is
possible to use sexual propagation techniques as well. In addition
to direct transformation of a Sugarcane plant with a Sugarcane
mini-chromosome, Sugarcane plants containing a Sugarcane
mini-chromosome can be prepared by crossing a first Sugarcane plant
containing the functional, stable, autonomous Sugarcane
mini-chromosome with a second Sugarcane plant lacking the Sugarcane
mini-chromosome.
[0366] Fertile Sugarcane plants comprising Sugarcane
mini-chromosomes can be crossed to other Sugarcane plant lines to
study mini-chromosome performance and inheritance. In the first
embodiment of this method, pollen from an adchromosomal Sugarcane
plant can be used to fertilize the stigma of a non-adchromosomal
Sugarcane plant. Sugarcane mini-chromosome presence is scored in
the progeny of this cross using the methods outlined in the
preceding section. In the second embodiment, the reciprocal cross
is performed by using pollen from a non-adchromosomal Sugarcane
plant to fertilize the flowers of an adchromosomal Sugarcane plant.
The rate of Sugarcane mini-chromosome inheritance in both crosses
can be used to establish the frequencies of meiotic inheritance in
male and female meiosis. In a third embodiment of this method,
pollen for an adchromosomal plant is used to fertilize another or
the same adchromosomal plant (e.g. self or sibling pollination). In
the fourth embodiment of this method, the progeny of one of the
crosses just described are back-crossed to a non-adchromosomal
Sugarcane parental line, and the progeny of this second cross are
scored for the presence of genetic markers in the plant's natural
chromosomes as well as the Sugarcane mini-chromosome. Scoring of a
sufficient marker set against a sufficiently large set of progeny
allows the determination of linkage or co-segregation of the
Sugarcane mini-chromosome to specific chromosomes or chromosomal
loci in the plant's genome. Genetic crosses performed for testing
genetic linkage can be done with a variety of combinations of
parental lines; such variations of the methods described are known
to those skilled in the art.
[0367] 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.
Example 1
Sugarcane Centromere Discovery from Genomic DNA
Identification of Sugarcane Satellite Repeat Sequences
[0368] Centromere satellite repeats were amplified from Sugarcane
(Saccharum officinarum.times.Saccharum spontaneum or Saccharum
officinarum) genomic DNA using standard PCR methods. Briefly, PCR
reaction was carried under the following conditions: 1 cycle at
95.degree. C. for 3 minutes, 10 cycles of 94.degree. C. for 15
seconds, 55.degree. C. for 15 seconds, and 72.degree. C. for 30
seconds, and 25 cycles at 94.degree. C. for 15 seconds, 52.degree.
C. for 15 seconds and 72.degree. C. for 30 seconds, followed by 1
cycle at 72.degree. C. for 5 minutes. The sequences of primers used
for amplifying satellite repeats were: forward:
5'-gtcacccagcagttccatcgggtgc-3' (SEQ ID NO: 205 and reverse:
5'-actgctgggtgacgtggctcaagt-3' (SEQ ID NO: 206). After PCR,
amplified satellite repeats were cloned into a standard cloning
vector (pCR2; Invitrogen Corp.; Carlsbad, Calif.; USA). Colonies
with insertions were cultured, DNA was extracted and sequenced.
This PCR analysis identified 201 satellite sequences, set out at
SEQ ID NOS: 1-201.
[0369] To determine the consensus of the identified satellite
repeat sequences, these sequences were aligned using a DNA sequence
alignment program (CONTIGEXPRESS.RTM. from VECTOR NTI.RTM.
(Invitrogen)). The sequences were trimmed to unit repeat length
using the consensus as a template. Sequences trimmed from the ends
of the alignment were realigned with the consensus and further
trimmed until all sequences were at or below the consensus length.
The consensus was determined by the frequency of a specific
nucleotide at each position; if the most frequent base is three
times more frequent than the next most frequent base, it was
considered the consensus.
[0370] The Sugarcane centromere specific retrotransposon sequence
CRS (Centromere Retrotransposon in Sugarcane--see Nagaki &
Murata, Chromosome Research, 2005, 13:195-203) was PCR amplified
and sequenced using primers located in different region of the CRS
sequence. The PCR reaction was carried out as described above using
the following primer sequences:
TABLE-US-00004 (SEQ ID NO: 207) CRSF 5'-gggaagtaca gggacgaaga gc-3'
(SEQ ID NO: 208) CRSF1 5'-actaacaatg cacgggaagg-3' (SEQ ID NO: 209)
CRSF2 5'-gtaggccatg gcagtttgat-3' (SEQ ID NO: 210) CRSF3
5'-aacacaccac ccaatccaat-3' (SEQ ID NO: 211) CRSF4 5'-ccaaacaagc
gtgttatgat tgt-3' (SEQ ID NO: 212) CRSF5 5'-aggttatgtg cgtcagtctc
ttag-3' (SEQ ID NO: 213) CRSF6 5'-ggcaaacctg ttgcatactt tag-3' (SEQ
ID NO: 214) CRSF7 5'-accatgtcat aaaactgatg atg-3' (SEQ ID NO: 215)
CRSR 5'-tgcaaccaaa ccaaatcacc ag-3' (SEQ ID NO: 216) CRSR1
5'-caagcgaaca atctcacgaa-3' (SEQ ID NO: 217) CRSR2 5'-aaatcatcat
cgtgcgcata-3' (SEQ ID NO: 218) CRSR3 5'-aacacaccac ccaatccaat-3'
(SEQ ID NO: 219) CRSR4 5'-gaacgctcct tgatgacac-3' (SEQ ID NO: 220)
CRSRS 5'-gtacccacta cgcaaatcaa cc-3' (SEQ ID NO: 221) CRSR6
5'-caacttcagt ttgaccatca gtt-3'
[0371] The sequence for CRS is set out as SEQ ID NO: 203. The
primer pairs CRSF 5'-gggaagtacagggacgaagagc-3' (SEQ ID NO: 207) and
CRSR 5'-tgcaaccaaaccaaatcaccag-3' (SEQ ID NO: 215) can be used to
amplify CRS from Sugarcane genomic DNA.
BAC Library Construction
[0372] A Bacterial Artificial Chromosome (BAC) library was
constructed from Sugarcane genomic DNA. The Sugarcane genomic DNA
was isolated from cultivar R570 (P1 504632), a hybrid between S.
officinarum and S. spontaneum, and digested with the restriction
enzymes Mbo I. These enzymes were chosen because they are
methylation insensitive and therefore can be used to enrich BAC
libraries for centromere DNA sequences.
Probe Identification and Selection
[0373] Three groups of Sugarcane repetitive genomic DNA, including
specific centromere-localized sequences, were initially compiled as
candidate probes for hybridization with the BAC libraries. Four
probes were picked to interrogate the BAC libraries. These probes
represent different groups of commonly found repetitive sequences
in the Sugarcane genome. The four probes were: SCEN, SCRM and High
Me/Low Methylation (HiMe and LoMe). The SCEN and SCRM probes were
each pooled PCR products. Probes were prepared and labeled with
standard molecular methods. The HiMe and LoMe probes were pooled
genomic DNA cut with a methylation-sensitive enzyme (BfuC1); large
DNA fragments were isolated for the "HighMe" probe and small DNA
fragments were isolated for the "LowMe" probe. Positives reported
are for the HighMe probe.
Library Interrogation and Data Analysis
[0374] The BAC clones from the libraries were spotted onto filters
for further analysis. The filters were hybridized with each of the
probes to identify specific BAC clones that contain DNA from the
group of sequences represented by the probes. Hybridization
conditions were: hybridization at 65.degree. C. for 12-15 hours and
washing three times for 15-90 minutes with 0.25.times.SSC, 0.1% SDS
at 65.degree. C. Other exemplary stringent hybridization conditions
could be used, such as hybridization at 65.degree. C. 0.5.times.SSC
0.25% SDS for 15 minutes, followed by a wash at 65.degree. C. for a
half hour.
[0375] A total of 18,453 BAC clones from the library was
interrogated with each of the 4 probes (SCEN, SCRM, HiMe, and
LoME), and the hybridization intensities of the BAC clones with
each probe were examined to quantify hybridization intensity for
each clone. Scores of 1 to 10 (based on the hybridization
intensities, with 10 being the strongest hybridization) were
assigned and entered into a spreadsheet for classification. The
spreadsheet contained a total of 3 tables, 1 for each probe used in
the interrogation (values from HiMe and LoMe) probes were entered
in a single table for comparison. Each table contained the
hybridization scores of each BAC clone from the Mbo I library, to
one of the 4 probes. Data analysis found BACs that contained
different groups of repetitive sequences.
Classification and Selection of BAC Clones for Mini-Chromosome
Construction
[0376] BAC clones containing centromeric/heterochromatic DNA were
identified by their visual hybridization scores to different
probes. The goal was to select BAC clones that contained a diverse
set of various repetitive sequences. Seven classes of centromeric
BAC clones were eventually chosen to cover the broadest possible
range of centromeric/heterochromatic sequences for Sugarcane
mini-chromosome (MC) construction. 658 unique clones that hybridize
with one or more of the probes were isolated from one filter, which
comprised 18,432 clones. They fell into classes as set out in Table
4 below.
TABLE-US-00005 TABLE 4 Classification of Centromere containing BACs
Probe Hybridization Range Class SCEN SCRM HiMe # clones identified
I + 472 II + 360 III + 398 IV + + 219 V + + 343 VI + + 166 VII + +
+ 156 * Probes with significant hybridization signals as determined
by visual scoring are indicated with a "+"
Example 2
Construction of Sugarcane MCs Containing Genomic DNA
[0377] A subset of BAC clones identified in Example 1 were grown,
and DNA was extracted for MC construction using a NUCLEOBOND.RTM.
purification kit from Clontech Laboratories, Inc. (Mountain View,
Calif.; USA). To determine the molecular weight of centromere
fragments in the BAC libraries, a frozen sample of bacteria
harboring a BAC clone was grown in selective liquid media, and the
BAC DNA harvested using a standard alkaline lysis method. The
recovered BAC DNA was restriction digested and resolved on an
agarose gel. Centromere fragment size was determined by comparing
to a molecular weight standard.
Donor DNA Containing Gene Stacks for MC Construction
[0378] Several donor DNA plasmids containing gene stacks for
testing of MCs in plant tissues were built, varying depending on
the specific fluorescent protein marker used for detection of
transgenic events. One set of MCs was built that contained the gene
stack from donor plasmid CHROM5798, which genetic elements are set
out in Table 5. Another set of MCs was built that contained the
gene stack from donor plasmid CHROM5434, which genetic elements are
the same as in donor plasmid CHROM5798 except that the nuclear
localized GFP gene was replaced with an AmCyan (Clontech)
fluorescent protein gene. Another set of MCs was built that
contained the gene stack from donor plasmid CHROM5436, which
genetic elements are the same as in donor plasmid CHROM5798, except
that the nuclear localized GFP gene was replaced with a ZsGreen
(Clontech) fluorescent protein gene.
TABLE-US-00006 TABLE 5 Donor Components of CHROM5798 Genetic Size
Location Element (bp) (bp) Details YAT1 yeast 2000 6271-8270 PCR
amplified YAT1 promoter promoter from chromosome I of Saccharomyces
cerevisiae for expression of NptII in Sugarcane Arabidopsis 360
5898-6257 PCR amplified Arabidopsis UBQ10 thaliana intron from
UBQ10 gene Intron (At4g05320) for stabilization of NptII gene
transcript and increase protein expression level NPTII 795
5076-5870 Neomycin phosphotransferase II plant selectable marker
Rps16A 489 4524-5012 Amplified from Arabidopsis terminator thaliana
40S ribosomal protein S16 (At2g09990) for termination of NptII gene
Bacterial 817 3525-4341 Bacterial kanamycin selectable kanamycin
marker Terminator 6 332 3049-3380 Terminator 6 AcGFP (nuc) 831
2084-2914 Nuclear localized green fluores- cent protein. UBQ10 2038
10-2047 PCR amplified Arabidopsis Promoter thaliana promoter from
UBQ10 gene (At4g05320) for stabilization of DsRedI gene transcript
and increase protein expression level LoxP 34 8290-8319
Recombination site for Cre and mediated recombination (Arenski
10802-10831 et al 1983, Abremski et al 1984)
Preparation of Donor DNA for Retrofitting
[0379] Cre recombinase-mediated exchange was used to construct
Sugarcane MCs by combining the Sugarcane centromere fragments
cloned in pBeloBAC11 with the donor plasmid CHROM5798 (Table 5).
The recipient BAC vector carrying the Sugarcane centromere fragment
contained a loxP recombination site; the donor plasmid contained
two such sites, flanking the sequences to be inserted into the
recipient BAC.
[0380] Sugarcane MCs were constructed using a two-stop method.
First, the donor plasmid was linearized to allow free contact
between the two loxP sites; in this step the backbone of the donor
plasmid is eliminated. In the second step, the donor molecules were
combined with Sugarcane centromere BACs and were treated with Cre
recombinase, generating circular Sugarcane MCs with all the
components of the donor and recipient DNA. Sugarcane MCs were
delivered into E. coli and selected on medium containing kanamycin
and chloramphenicol. Only vectors that successfully ere recombined
and contained both selectable markers survived in the medium. To
determine the molecular weight of the Sugarcane centromere
fragments in the Sugarcane MCs, three bacterial colonies from each
transformation event were independently grown in selective liquid
media and the Sugarcane MC DNA was harvested using a standard
alkaline lysis method. The recovered Sugarcane MC was restriction
digested and resolved on an agarose gel. Sugarcane centromere
fragment size was determined by comparison to molecular weight
standards. When variation in Sugarcane centromere size was noted,
the Sugarcane MC with the largest Sugarcane centromere insert was
used for further experimentation. All 84 MCs subjected to further
testing had the features described in Table 6.
TABLE-US-00007 TABLE 6 MC constructs tested in sugarcane callus
Original Strength of CEN Donor MC Satellite Strength of Strength of
fragment plasmid CHROM# BAC name signal CRS signal FISH signal size
(KB) CHROM# 5800 3G17 L L n/a 90 5798 5801 5B12 H N good 115 5798
5802 18E23 H M good 80 5798 5803 19H6 M H good 102 5798 5804 24L19
L M good 35 5798 5805 21L3 M M good 120 5798 5806 4H1 H N good 90
5798 5807 3O5 M H good 100 5798 5808 1K10 L H good 150 5798 5809
17H17 H H good 110 5798 5810 21A4 H H good 150 5798 5811 18F12 H H
good 110 5798 5812 20H10 H H n/a 150 5798 5813 21B1 H H good 130
5798 5814 24J1 H H good 65 5798 5815 19J18 H H good 100 5798 5816
17B4 H L good 70 5798 5817 18P24 H L good 120 5798 5818 20A3 H L
n/a 160 5798 5819 24F15 H L n/a 120 5798 5820 17C9 H M good 125
5798 5821 19J8 H N n/a 120 5798 5822 18C14 H N good 130 5798 5823
17P2 H N good 110 5798 5824 17M9 H N good 150 5798 5825 24J17 H N
n/a 170 5798 5826 24C6 H N good 75 5798 5827 3I9 L H good 120 5798
5828 22H16 L H n/a 135 5798 5829 6C15 L H good 70 5798 5830 20C8 L
L good 90 5798 5831 19K22 L M n/a 105 5798 5832 17E7 L M good 105
5798 5833 24M21 L M n/a 130 5798 5834 18J2 M H good 105 5798 5835
17N22 M H n/a 145 5798 5836 7E2 M H good 230 5798 5837 1L6 M H n/a
90 5798 5838 1P13 M H good 90 5798 5839 4H14 M L good 70 5798 5840
17E9 M L n/a 110 5798 5841 3P16 M L good 100 5798 5842 22I13 M L
n/a 130 5798 5843 23F24 M L n/a 90 5798 5844 1K6 M L good 80 5798
5845 1P14 M L good 135 5798 5846 19H7 M M n/a 150 5798 5847 18F16 M
M n/a 160 5798 5848 22D19 M M n/a 100 5798 5849 6C9 M M n/a 120
5798 5850 23I19 M N good 65 5798 5851 3F1 M N n/a 75 5798 5852 2A7
M N n/a 75 5798 5853 20A22 N N none CEN 100 5798 5854 19A22 N N n/a
115 5798 5855 17A22 N N n/a 120 5798 5856 21A22 N N none CEN 80
5798 5857 18A22 N N n/a 120 5798 5858 1A4 L H n/a 65 5798 5859 1M10
N N n/a 110 5798 5860 7D11 M M n/a 80 5798 5861 7J24 M L n/a 100
5798 5862 1P1 H L n/a 125 5798 5863 21B11 H N good 150 5798 5864
8O2 M L good 70 5798 5865 8I7 M H good 135 5798 5866 17H17 H H good
110 5434 5867 21A4 H H good 140 5434 5868 19J18 H H good 95 5434
5869 18P24 H L good 120 5434 5870 17C9 H M good 120 5434 5871 18E23
H M good 85 5434 5874 4H14 M L good 85 5434 5876 17P2 H N good 100
5434 5878 6C15 L H good 80 5434 5881 17C9 H M good 120 5436 5882
17P2 H N good 100 5436 5883 4H14 M L good 85 5436 5884 18E23 H M
good 85 5436 5885 6C15 L H good 80 5436 5886 18P24 H L good 120
5436 5887 19J18 H H good 95 5436 5888 21A4 H H good 140 5436 5889
17H17 H H good 110 5436 H indicates high hybridization signal in
the original filter hybridization, M indicates medium signal, and L
indicates low signal. N indicates no signal was observed. In column
labeled "Strength of FISH signal," "good" indicates strong
hybridization to centromeres observed in root tip spread, n/a
indicates "not determined," and "none CEN" means no hybridization
was observed to the centromeric region of any chromosomes.
Example 3
MC Delivery into Sugarcane Cells and Regeneration
[0381] The Sugarcane MCs from Example 2 were tested in several
Sugarcane cells, including Saccharum officinarum and a hybrid
between S. officinarum and S. spontaneum, and the procedure was
optimized for antibiotic selection, cell pre-treatments, and
bombardment conditions. MCs were tested both in leaf-roll tissue
directly, or callus tissue that was initiated from leaf-rolls. The
presence of MCs was determined both by direct molecular assays or
indirect measurement of fluorescent cells. Preliminary results
identified several MCs that successfully generated fluorescent cell
clusters in Saccharum cells.
Sugarcane Transformation, Selection and Regeneration.
[0382] Prior to delivery of the two-gene stack containing MCs from
Example 2, Sugarcane callus was initiated from leaf roll tissue.
Sugarcane tops were collected from greenhouse-grown plants for
preparing explants. The Sugarcane tops (minimally 3-6 months old)
were cut below the highest visible node. The older leaves (approx
3-4) were removed until the internode was visible and cut about 2''
below this internode, and disinfected by submerging in 20% bleach
for 20 min (5-10 tops in 3 L bleach solution). Subsequently, the
cane tops were rinsed with sterile distilled water 3-4 times to
remove excess bleach. In a tissue culture hood, more external
leaves were removed from the cane tops, and the top portion was cut
off leaving about 10 to 12 cm above the internode and 1 cm below
the internode. Thin stem sections (approx 1 to 1.5 mm thickness)
were sliced from the lower edge of the internodes. During this
process, tools were frequently dipped in antioxidant mixture
(PhytoTechnology Laboratories; Shawnee Mission, Kans.; USA) to
avoid browning at cut sites. Those sections with orange centers
were avoided, and only those sections with green centers were used
for callus induction. Approximately 9 pieces of thin stem sections
were placed per plate on MS3 Medium (Murashige and Skoog, 1962.
Physiologia Plantarum, 15:472-497), supplemented with 500 mg/L
casein hydrolysate, 20 g/L sucrose and 3 mg/L 2,4-D, pH to 5.8 and
solidified with 2.5 g/L GELRITE@(Sigma-Aldrich; Saint Louis, Mo.;
USA) or 6 g/L Phytoblend (Caisson Laboratories; North Logan, Utah;
USA). The callus was sub-cultured once after a 15-day interval onto
the same medium or MS1 (MS; 4.3 g/l MS salts and vitamins
supplemented with 20 g/l sucrose, 0.5 g/l casein, 1 mg/l 2,4-D, pH
to 5.8) medium. Callus was generally sufficiently established for
bombardment after 2-3 months. Prior to bombardment with MCs, the
white, nodular and embryogenic calli were subcultured for 3-4 days
on MS3 Medium, and then transferred onto Sugarcane Osmotic Medium
(SCOM) prior to bombardment (4-5 hours at 28.degree. C.). Sugarcane
Osmotic Medium consists of MS3 medium supplemented with 500 mg/L
casein hydrolysate, 20 g/L sucrose and 3 mg/L 2,4-D. pH to 5.8 and
solidify with 2.5 g/L GELRITE.RTM. or 6 g/L Phytoblend with the
addition of 36.4 g/L sorbitol, and 36.4 g/L mannitol.
[0383] Precipitation of MC DNA onto gold particles for the purpose
of delivery using the biolistic method was performed as follows:
1.8 mg of sterile, washed gold (0.6 .mu.M diameter was preferred)
was combined with desired amount of MC DNA (in 1.times.TE). Careful
handling of DNA was critical; wide bore tips were used for all
pipetting, and solutions were preferentially dispensed into the
bottom of the tube to assist with the gentle mixing process. The
volume was brought to 250 .mu.l with cold (4.degree. C.) sterile
water and 250 .mu.l of cold (4.degree. C.) 2.5M CaCl.sub.2 was
added immediately, followed by addition of 50 .mu.l of filter
sterilized 0.1M Spermidine (free base, filter sterilized). The
mixture was gently finger vortexed 1-2.times. to ensure even mixing
of all solutions, and DNA was allowed to precipitate onto the gold
particles on ice for 1.5 hours; with finger vortexing 1-2.times.
after 45 min. The gold/DNA mixture was pelleted (5 min, 800 rpm,
RT) and washed once with 100% ethanol, and 36 .mu.l 100% ethanol
was added to the gold/DNA pellet, and mixed gently. Typically 6
.mu.l of gold/DNA/ethanol was used per macrocarrier (i.e., one
bombardment shot). The absolute number of molecules delivered per
shot was varied by precipitating a varying amount of DNA onto the
gold particles.
[0384] Bombardment conditions using the BioRad PDS-1000/He
biolistic transformation system Bio-Rad Laboratories; Hercules,
Calif.) were as follows. A rupture disk rating of 900-1800 psi;
1100 or 1300 psi was preferred, with one shot per plate. The
preferred gap distance (distance from rupture disk to macrocarrier)
was 6 mm. The target shelf for tissue was L2-L4; L2 (3rd shelf from
the bottom) was preferred. Vacuum pressure of 27.5-28 in Hg; 27.5
in Hg was preferred. These bombardment conditions were tested with
R570 callus, other conditions (pressure, rupture disk rating, gap
distance, target shelf, duration of osmotic and rest treatments,
etc.) can of course be modified for other genotypes and/or tissues.
Following bombardment, callus was allowed to recover at 28.degree.
C. in the dark for an additional 12-18 hours on SCOM. Tissues were
transferred onto MS3 medium for recovery for 3-4 days, and the
number of calli present on each plate used for bombardment was
counted. At the end of the recovery period, calli were checked for
transient expression of fluorescent marker genes under the
microscope, and the transient transformation efficiency was
calculated.
[0385] Transient expression of MC encoded green fluorescent protein
(GFP) gene AcGFPnuc was demonstrated in Sugarcane (cv. L97-128)
callus induced from immature leaf tissue. Approximately
2.5.times.10.sup.9 DNA molecules for 9 different MC DNAs were
delivered into the callus tissues 4 hours after osmotic treatment
per plate, and the bombarded tissues were examined for GPF
expression 4 days later. The results are summarized in Table 7.
Calli expressing GFP were observed in 6 out of the 9 MCs delivered
in this experiment, with a frequency ranging from 1.7% for MC
CHROM5889 to 20% for MC CHROM5886. However, no GFP expressing calli
were observed for 3 MC constructs. These results demonstrated that
MC DNA had been successfully delivered into sugarcane callus cells
and the MC encoded fluorescent protein gone AcGFPnuc was expressed
and biologically functional.
TABLE-US-00008 TABLE 7 Transient expression of AcGFPbuc in
Sugarcane MC containing transgenic callus # of calli % GFP BAC # of
plates # of calli expressing expressing MC # Cen observed observed
GFP callus 5881 17C9 7 140 5 3.6 5882 17P2 9 92 4 4.3 5883 4H14 12
136 0 0.0 5884 18E24 12 140 6 4.3 5885 6C15 10 120 3 2.5 5886 18P24
4 60 12 20.0 5887 19J18 5 82 0 0.0 5998 21A4 5 94 0 0.0 5889 17H17
6 120 2 1.7
[0386] For selection of transgenic events, bombarded calli were
transferred onto sub-lethal selection medium (ChromMS3G30), and
culture at 28.degree. C., in the dark, for 2 weeks. ChromMS3G30
medium consists of MS3 medium supplemented with 30 mg/l G418
sulfate (Geneticin (Sigma)) after autoclaving. The calli were
broken up into small pieces and transferred onto lethal selection
medium (second round of selection) MS3G50, and culture at
28.degree. C., in the dark, for 4 weeks. MS3G50 Medium consists of
MS3 medium supplemented with 50 mg/l G418 sulfate after
autoclaving. Tissue growth was visually assessed to identify
resistant callus. Resistant calli were subcultured for another
round of selection on ChromMS3G50 for an additional 4 weeks.
[0387] For plant regeneration, surviving calli (putative resistant
calli) were transferred onto RSCG25 medium to initiate regeneration
and were cultured at 26.degree. C., low light (16 hour day length,
26.degree. C.) for 3-4 weeks. RSCG25 Medium consists of MS3 medium
supplemented with 500 mg/L casein hydrolysate, 20 g/L sucrose and
0.5 mg/L kinetin, pH to 5.8 and solidified with 2.5 g/L GELRITE@,
and further supplemented with 25 mg/L G418 sulfate after
autoclaving. Developing plantlets were transferred to RtSC medium
in sundae cups (Solo Cup Company; Lake Forest, Ill.; USA) for
plantlet growth and root development and cultured at 26.degree. C.,
16 hour day length. RtSC medium consists of MS medium supplemented
with 25 g/L sucrose, pH to 5.8 and solidify with 2.5 g/L
GELRITE.RTM., further supplemented with 20 mg/L G418 sulfate after
autoclaving. Finally, plantlets were transferred into pre-moistened
soil-less mix (LCI, BFG Supply Company; Joliet, Ill.; USA) under a
humidome in an 18-well flat in a growth chamber (28.degree. C., 16
hour day length). The dome was cracked open slightly to slowly
reduce humidity 3-4 days after transplanting. The dome was removed
completely 2 days later and plantlets were transferred to a
greenhouse (28.degree. C., 16 hour day length). Plants were watered
from trays beneath the pots when the soil began to dry.
Identification of MC Containing Transgenic Events
[0388] More than 1200 putative transgenic Sugarcane events were
generated from mini-chromosome transformation. A total of 920
(.about.76%) of putative transgenic calli events were analyzed
using diagnostic PCR for the several DNA fragments carried on the
gene stack. The presence or absence of the nptII, AcGFPnuc or
ZsGreen (depending on the mini-chromosome used in transformation),
and the ubiquitin (UBQ10) promoter was determined and compared to
amplification of the endogenous genomic internal control ADH. The
events thus screened cover a collection of 51 MCs with an average
size ranging between 65 and 187 kb. Transgenic events were derived
from the bombardment of six different sugarcane genotypes (R570,
L97-128, Q117, NCo310, Pindar and Q63). Of the 920 events, 33% were
derived from the callus bombarded with the DNA concentration of
1.times.10.sup.9 molecules (90-200 ng of DNA) per shot, 8.7% from
2.5.times.10.sup.9 molecules (200-500 ng of DNA), 37.6% from
5.times.10.sup.9 molecules (450 ng-1 .mu.g of DNA) and 6.2% from
1.times.10.degree. molecules (800 ng-1.2 .mu.g of DNA) per shot.
The putative transgenic events were obtained after selecting the
bombarded calli on different levels of G418 concentration ranging
from 22.5 mg to 50 mg/l (100% potency) for a period of 4-5 months
with a minimum of 4 rounds of selection. For PCR screening, total
genomic DNA was isolated from approximately 40-60 mg of callus
tissue by the DNA preparation method of Krysan et al. (Krysan P I
I, Young J C, Tax F, Sussman M R (1996) Identification of
transferred DNA insertions within Arabidopsis genes involved in
signal transduction and ion transport. Proc Natl Acad Sci USA 93:
8145-8150). The concentration of DNA was measured, normalized
uniformly and used for either single-step or multiplex PCR
analysis. The optimized PCR conditions, primers and reagents that
were used for detecting the 4 PCR fragments in all the calli events
generated is as follows.
[0389] Single-step PCR conditions used an initial denaturation at
95.degree. C. for 2 minutes, followed by 35 cycles each consisting
of 0.3 minutes denaturation at 94.degree. C., 0.3 minutes annealing
at 52.degree. C., and 1.2 minutes extension at 72.degree. C.,
followed by a final extension for 4 minutes at 72.degree. C., after
which the samples were kept at 4.degree. C. indefinitely. The PCR
reaction was performed in a total volume of 25 .mu.l, consisting of
19.2 .mu.l water, 2.5 .mu.l 10.times.NEB Buffer, 0.4 .mu.l dNTP mix
(40 Mm), 0.2 .mu.l F-Primer (20 .mu.M), 0.2 .mu.l R-primer (20
.mu.M), 0.50.2 .mu.l NEB Taq Polymerase and 2 .mu.l DNA (100-200
ng). Multiplex PCR conditions used an initial denaturation at
95.degree. C. for 1 minutes, followed by 40 cycles each consisting
of 0.1 minutes denaturation at 95.degree. C., 0.1 minutes annealing
at 55.degree. C. (and increasing 0.1.degree. C. with each
subsequent cycle), and 1.5 minutes extension at 72.degree. C.,
followed by a final extension for 5 minutes at 72.degree. C., after
which the samples were kept at 4.degree. C. indefinitely. The PCR
reaction was performed in a total volume of 25 .mu.l, consisting of
15 .mu.l water, 5 .mu.l 5.times. Multiplex Master Mix (New England
Biolabs; Ipswich, Mass.; USA), 2 .mu.l F-Primer (20 .mu.M), 2 .mu.l
R-primer (20 .mu.M), and 1 .mu.l DNA (100-200 ng). The expected PCR
product sizes were 470 bp (ADH), 662 bp (AcGFPnuc), 886 bp (UBQ10),
1000 bp (nptII), and 924 hp (ZsCreen). The PCR reaction was carried
out as described above using the following primer sequences:
TABLE-US-00009 (SEQ ID NO: 242) ADH1 CHSL-285 5'-aagtcggcag
agagcaacat-3' (SEQ ID NO: 243) ADH1 CHSL-286 5'-cagatgcaaa
cccaacacac-3' (SEQ ID NO: 244) AcGFPnuc CHSL-132 5'-cgattttctg
ggtttgatcgtt ag-3' (SEQ ID NO: 245) AcGFPnuc CHSL-199 5'-cattgtgggc
gttgtagttg-3' (SEQ ID NO: 246) UBQ10 CHSL-468 5'-gttgtggtty
gtgctttcct-3' (SEQ ID NO: 247) UBQ10 CHSL-469 5'-ccactttgac
gccgtttatt-3' (SEQ ID NO: 248) NPTII CHSL-132 5'-cgattttctg
ggtttgatcgttag-3' (SEQ ID NO: 249) NPTII CHND7 5'-gaactcgtca
agaaggcgata-3' (SEQ ID NO: 250) ZsGreen CHSL-132 5'-cgattttctg
ggtttgatcgttag-3' (SEQ ID NO: 251) ZsGreen CHSL-201 5'-tcagggcaat
gcagatcc-3'
[0390] Among the 920 events analyzed, 358 events (38.9%) showed the
presence of all three PCR amplicons (nptII, AcGFPnuc or ZsGreen,
and UBQ10) (summarized in Table 8). Based on this analysis the
escape frequency is around 61%. The observed escape rate appears
the highest in genotype R570, in which most of the transformations
were performed.
TABLE-US-00010 TABLE 8 Sugarcane MC containing transgenic callus
Events Positive events MC # analyzed for all amplicons 5800 24 12
5801 9 0 5802 42 18 5803 5 1 5804 10 2 5805 3 1 5806 4 1 5809 11 3
5810 7 5 5812 4 0 5814 88 62 5816 29 14 5817 42 12 5819 75 43 5820
53 42 5821 4 1 5822 18 1 5823 4 0 5824 12 0 5825 7 0 5827 6 0 5830
5 0 5834 20 3 5835 4 1 5837 12 1 5839 7 0 5840 3 0 5842 2 0 5844 7
0 5846 14 0 5850 14 11 5851 14 0 5852 5 0 5854 26 3 5856 11 3 5857
6 1 5858 25 1 5859 14 0 5860 26 4 5862 11 0 5863 11 0 5864 50 8
5873 2 2 5874 14 8 5881 47 44 5882 19 13 5883 12 4 5884 35 16 5885
23 8 5888 2 2 5889 22 7 Total 920 358
[0391] MC transgenic events were obtained in all genotypes tested,
and significant genotype dependence was noted for both
transformation efficiency and escape rate for G418 selection. Table
9 summarizes transformation results for several genotypes.
TABLE-US-00011 TABLE 9 Sugarcane MC transgenic events in multiple
genotypes No. of putative No. of PCR Escape frequency Genotype
events produced positive events (%) R570 554 94 83 Q117 151 95 37
L97-128 182 142 22 NCo310 6 6 0 Pindar 23 18 21.7 Q63 4 3 25 Total
920 358
Evaluation of Autonomous MCs
[0392] To evaluate whether the candidate Sugarcane MCs were
maintained autonomously, fluorescence in situ hybridization (FISH)
was performed on mitotic metaphase chromosome spreads from callus
tissue. FISH was performed essentially as described in Kato et al.
Proc. Natl. Acad. Sci. U.S.A. 101: 13554-13559, 2004, using probes
labeled with ALEXA FLUOR.RTM. 488 ("Alexa488") and ALEXA
FLUOR.RTM.568 ("Alexa568;" Invitrogen). Alexa488 labeled CHROM5798
DNA was used as a MC-specific probe. Alexa568 labeled pBeloBAC11
DNA was used as a second MC-specific probe. Alexa568 labeled PCR
amplified centromere sequences from BAC 18E23 were used as a
centromere-specific probe. The latter probe was also expected to
stain centromere regions on the endogenous chromosomes.
[0393] For FISH evaluation, freshly growing callus tissue was
collected following a recent transfer to fresh media. Depending on
genotype, different morphologies were apparent. Generally, tissue
was nodular and firm, and met forceps with resistance. Using
forceps or scalpel, a very small cluster of nodules was excised and
transferred to a 1.7 mL microfuge tube. A few microliters of
dH.sub.2O were added to the tube to keep the tissue moist. The tube
was covered with cap containing a small puncture to allow exposure
to nitrous oxide in next step. Callus tissue was placed in the
pressure chamber under 160 psi for 4.5 hours. Tissue was fixed in
90% acetic acid, and spread onto poly-lysine coated glass slides by
squashing thin cross sections. Following hybridization, slides were
counter-stained with DAPI (0.04 mg/ml) and .gtoreq.15 metaphase
cells were evaluated per callus using a Zeiss Axio-Imager (Carl
Zeiss MicroImaging, Inc.; Thornwood, N.Y.; USA) equipped with
rhodamine, FITC, and DAPI filter sets (excitation BP 550/24,
emission BP 605/70; excitation BP 470/40, emission: BP525/50; and
excitation G 365, emission BP 445/50, respectively). Gray-scale
images were captured in each panel, merged and adjusted with
pseudo-color using Zeiss AxioVision (Version 4.5; Carl Zeiss
MicroImaging, Inc.) software; fluorescent signals from
doubly-labeled MCs were detected in both the red and green
channels.
[0394] Extra-chromosomal signals were considered to indicate
autonomous Sugarcane MCs if the images showed co-localization of
the Alexa488 (green) and Alexa568 (red) signals within 1 nuclear
diameter of the endogenous metaphase Sugarcane chromosomes, and the
signals were clearly distinct from the DAPI-stained host
chromosomes. Typical Autonomous MC signals in FISH hybridization
show overlapping distinct Alexa488 (green) and Alexa568 (red)
signals that in computer-generated merged images, the overlap shows
a yellow signal. Integrated constructs result in two distinct FISH
signals, each on a replicated metaphase chromatid, and usually
these FISH signals do not overlap with the centromere region.
Autonomous MCs were found to co-exist in the presence of integrated
constructs, indicating the ability of a specific MC to produce a
purely autonomous event in transgenic lines obtained in parallel
with the event characterized here, or obtained from future
transformation experiments under different transformation
conditions. Table 10 summarizes the preliminary FISH evaluation of
selected transgenic lines that were confirmed to contain MCs
identified by PCR, and demonstrated that both autonomous only
(category A) and autonomous and integrated events (category A+I)
were obtained for a significant number of Sugarcane MCs.
TABLE-US-00012 TABLE 10 Preliminary FISH evaluation of Sugarcane MC
transgenic events Autonomous Integrated CHROM# Event ID# copy copy
Category 5802 RC5802-85-12-5 + - A 5802 RC5802-85-14-2 + - A 5802
RC5802-85-14-2 + - A 5802 RC5802-85-16-1 + - A 5814 BC5814-109-6-1
+ - A 5814 BC5814-109-9-1 + - A 5819 AC5819-109-29-1 + - A 5819
AC5819-109-30-2 + - A 5820 BC5820-105-13-1 + - A 5820
BC5820-105-13-1 + - A 5820 RC5820-74-6-3 + - A 5824 RC5824-77-29-1
R + - A 5837 RC5837-88-13-1 + - A 5840 RC5840-99-10-1 + - A 5860
RC5860-75-13-1 + - A 5802 RC5802-85-14-5 + + A + I 5809
RC5809-79-4-1 + + A + I 5810 BC5810-112-23-1 + + A + I 5810
BC5810-112-23-2 + + A + I 5814 AC5814-109-12-2 + + A + I 5814
AC5814-109-14-1 + + A + I 5814 AC5814-109-14-8 + + A + I 5814
AC5814-109-15-10 + + A + I 5814 AC5814-109-15-5 + + A + I 5814
RC5814-101-16-1 + + A + I 5816 BC5816-109-21-5 + + A + 1 5816
BC5816-109-21-6 + + A + I 5817 PC5817-104-1-1 + + A + I 5817
PC5817-104-2-1 + + A + I 5817 PC5817-104-5-1 + + A + I 5819
AC5819-109-26-1 + + A + I 5819 AC5819-109-27-1 + + A + I 5819
AC5819-109-29-1 + + A + I 5819 AC5819-109-29-4 + + A + I 5819
BC5819-105-3-023 + + A + I 5820 BC5820-105-11-1 + + A + I 5820
BC5820-105-15-1 + + A + I 5820 BC5820-105-15-1 + + A + I 5820
BC5820-105-15-1 + + A + I 5834 RC5834-80-2-1 + + A + I 5850
RC5850-74-16-2 + + A + I 5850 RC5850-74-16-2 + + A + I 5850
RC5850-74-16-2 + + A + I 5850 RC5850-74-18-8 + + A + I Events with
high quality FISH signals as determined by visual scoring are
indicated with a "+", a clear absence of signal is indicated by
"-".
Example 4
Construction of Sugarcane MCs Containing Synthetic Arrays of Repeat
Sequence
[0395] A synthetic array of the satellite repeat sequences was
generated using PCR and directional cloning. A block of 4 Sugarcane
satellite repeats were PCR amplified and sequenced. The sequence is
set out as SEQ ID NO: 204. This sequence was used as the basis for
building the synthetic array. Several arrays were constructed
ranging in size between 8 kb and 25 kb. Two Sugarcane MCs
containing specific synthetic arrays of 18 kb (CHROM5613) and 25 Kb
(CHROM5616) were used in further testing.
[0396] The Sugarcane MCs also contained either 5 or 8 stacked
exogenous genes. The 5- or 8-gene stacks were based on the 2-gene
stack construct from donor plasmid CHROM5738. The genetic elements
within the donor plasmid CHROM5738 are the same as in donor plasmid
CHROM5798 except that the nuclear localized GFP gene was replaced
with a nuclear localized DsRed2 fluorescent protein gene
(DsRed2+NLS), described in Table 11.
TABLE-US-00013 TABLE 11 Donor Components of CHROM5738 Size Genetic
(base Location Element pair) (bp) Details YAT1 yeast 2000 7110-9109
PCR amplified YAT1 Promoter promoter from chromo- some I of
Saccharomyces cerevisiae for expression of NptII in Sugarcane
Arabidopsis 360 9123-9482 PCR amplified Arabidopsis UBQ10 Intron
thaliana intron from UBQ10 gene (At4g05320) for stabilization of
NptII gene transcript and increase protein expression level nptII
795 9510-10304 Neomycin phosphotransfer- ase II plant selectable
marker Rps16A 489 10368-10856 Amplified from Arabidopsis terminator
thaliana 40S ribosomal protein S16 (At2g09990) for termination of
NptII gene Bacterial 817 11039-11855 Bacterial kanamycin Kanamycin
selectable marker Terminator 6 332 12000-12331 Terminator 6 DsRed2
+ NLS 780 12466-13245 Nuclear localized red fluorescent protein
from Discosoma sp. (Matz, M et. al Nat Biotechnol 1999 December;
17(12): 1227). UBQ10 2038 13282-15319 PCR amplified Arabidopsis
Promoter thaliana promoter from UBQ10 gene (At4g05320) for
stabilization of DsRedI gene transcript and increase protein
expression level LoxP 34 7057-7090 Recombination site for Cre and
mediated recombination 15335-15368 (Arenski et. al 1983, Abremski
et. al 1984)
[0397] The 5-gene stack included the marker genes Anthocyanin,
ZsGreen, and ZsYellow in addition to NptII, DsRed in CHROM5738. The
8-gene stack included those genes in the 5-gene stack plus three
additional genes from the Agrobacterium tumefaciens tumor-inducing
(Ti) pathway. These were the iaaM (Trp mono-oxygenase), iaaH
(Indole-3-acetamide hydrolase), and ipt (AMP iso-pentenyl
transferase) genes.
[0398] In order to investigate whether the Sugarcane MCs could
accommodate a large number of genes, Sugarcane MCs containing a
gene stack, a synthetic array of repeat nucleotide sequence and
about 20 kb of A. thaliana DNA was constructed. The total size of
these Sugarcane MCs ranged between 82 kb and 87 kb. In addition,
Sugarcane MCs with a gene stack with 2 genes in addition to a 28 kb
synthetic Sugarcane centromere repeat array and an approximately 50
kb insertion of A. thaliana DNA was constructed using the methods
described above. The Sugarcane MC demonstrated that the Sugarcane
MCs of the invention can accommodate a large payload of genes, as
50 kb of the A. thaliana DNA includes a wide variety of genes. The
total size of these MCs were 82 and 87 kb.
Sugarcane Transformation and Regeneration with Synthetic MCs.
[0399] To enhance the efficiency with which Sugarcane cells
transformed with Sugarcane MCs can be regenerated into Sugarcane
plants, Sugarcane MCs containing the auxin gone pathway were
delivered into fully differentiated leaf rolls rather than
undifferentiated tissue, e.g., embryos. In addition, the growth
conditions were altered in order to facilitate generation of
transformed Sugarcane callus. Functional Testing of Sugarcane MCs
Using Transient Assays
[0400] Sugarcane (variety R570; Saccharum
oficinarum.times.Saccharum spontaneum) was grown in the greenhouse
for 6 months without floral initiation due to the growth time as
well as the day-length settings on greenhouse supplemental
lighting. Stalks from several (clonal) plants were used to generate
leaf rolls that were purely leaf tissue and did not include any
developing meristematic tissue.
[0401] Sugarcane MCs with a synthetic Sugarcane centromere were
delivered to leaf rolls. One Sugarcane MC contained an 8-gene stack
(denoted herein as the "8-gene MC"), and one Sugarcane MC contained
a 5-gene stack (denoted herein as the "5-gene MC"). These Sugarcane
MCs are described in detail in Example 3. In addition, a control
plasmid (lacking a centromere) containing eight genes was also
delivered, in which the 8-gene stack is identical to that delivered
on the 8-gene Sugarcane MC.
[0402] The 8-gene Sugarcane MC included A. tumefaciens tumor
inducing (Ti) pathway genes (iaaM, iaaH, and ipt). The inclusion of
these genes minimized the time the transformed cells were in tissue
culture. IaaM converts Trp into indole-3-acetamide, which IaaH
converts into auxin. Isopentenyl transferase (Ipt) converts
3',5'-adenosine monophosphate (AMP) into a cytokinin. The presence
of the hormone auxin was used in cell culture to stimulate plant
cells to form callus. Media with auxin promotes callus growth from
plant cells whereas plant cells cultured on media lacking auxin
either germinate (for embryogenic material) or are unable to grow
(non-embryogenic or meristematic tissue such as leaf tissue). Thus,
the 8-gene MC induced callus formation without supplementing the
media with auxin.
[0403] A biolistic delivery method using dry gold particles was
carried out to deliver MCs to the Sugarcane leaf rolls. For this
method, Sugarcane MC DNA (in 1.times.TE) was precipitated onto 2.1
mg of sterilized and washed 0.6 pt gold particles. The
DNA-containing gold particles were re-suspended in cold sterile
water and 2.5 M CaCl.sub.2. Filter-sterilized 0.1 M spermidine
(free base) was added to the mixture. Subsequently, the mixture was
allowed to precipitate on ice for 1.5 hours, with gentle finger
vortexing (3.times.) after 45 minutes. The precipitated DNA was
then washed with 100% ethanol, resuspended in 100% ethanol which
was allowed to fully evaporate prior to bombardment.
[0404] The apical region of the Sugarcane stem was collected (20-30
cm long), after removing the outermost mature leaves, the remaining
leaves were sterilized by submersion in a solution of 50 ml bleach
in 1 liter of water for 10 minutes. The remaining mature leaves
were aseptically removed and the young inner immature leaves were
sliced into sections/discs approximately 2-3 mm thick. The leaf
rolls were placed in SCOM at 28.degree. C. for 4-5 hours before
bombardment.
[0405] The 3 constructs wore each initially tested by delivery into
16 leaf rolls. For delivery, the leaf rolls were bombarded with the
MC DNA using the BioRad PDS-1000/He with a rupture disk rating of
900-1800 psi (1350 psi was preferred with one shot per plate). The
gap distance (distance from rupture disk to macrocarrier) was 6 mm.
Target shelf for tissue was L2-L4; L2 or L3 was preferred. The
vacuum pressure of 25-29 in Hg; 27.5 in Hg was preferred. The
bombarded leaf rolls were stored at 28.degree. C. (dark) for an
additional 16-18 hours on SCOM.
[0406] Subsequently, the bombarded leaf rolls were transferred to
MSO (4.3 g/l Murashige and Skoog (MS) salts and vitamins,
supplemented with 20 g/l sucrose, 0.5 g/1 casein, with NO 2,4-D, pH
to 5.8 and solidified with 2 g/L GELRITE.RTM.) and stored at
28.degree. C. in the dark for 2 weeks. The leaf rolls were visually
assessed for callus production 2 and 4 weeks after bombardment.
Control leaf rolls not subjected to bombardment, and the leaf rolls
that were bombarded with the 5-gene stack MC showed no sign of
growth or callus formation. Two of the 16 bombarded leaf rolls that
were bombarded with the 8-gene stack MC produced callus (12.5% of
the total explants).
[0407] Callus from the tissue was phenotypically evaluated for
DsRed expression using a fluorescent dissecting microscope. DsRed
was observed in the tissue. The resulting calluses were transferred
to Regeneration Sugarcane Medium (RSCM; 43 g/l MS salts and
vitamins supplemented with 20 g/l sucrose, 0.5 g/l casein, 0.5 mg/l
kinetin. pH to 5.8 and solidify with 2 g/L GELRITE.RTM.) in low
light (16 hour day length, 26.degree. C.) to initiate regeneration.
This media did not contain auxin. After 2 additional weeks of
culture, callus had grown, but had also started to differentiate
into root (primarily) and shoot material. Such differentiation is
not expected in the presence of auxin, suggesting either silencing
or loss of the 3 genes from the A. tumefaciens Ti pathway. After 2
additional weeks on media, PCR evaluation of this material was done
for presence of the DsRed gene. PCR results were negative, which
further suggested loss of the entire MC and verified that the genes
were not silenced. Because of the loss of the entire MC, these
tissues were not regenerated into plants.
[0408] The advantages of including the genes of the Ti pathway on a
MC are that the non-meristematic tissues were transformed and the
need for callus initiation prior to DNA delivery was eliminated. In
addition, the time in culture was reduced and as a result
somaclonal variation, endogenous chromosome number changes and the
like were also reduced. Furthermore, the inclusion of the Ti
pathway genes eliminated the need for selectable marker genes.
There was an observed 12.5% transformation efficiency of Sugarcane
in the initial experiments.
[0409] In a separate experiment, 5-gene (NptII, DsRed, Anthocyanin,
ZsGreen, and ZsYellow) MCs were delivered into Sugarcane callus
generated from Sugarcane variety R570; (Saccharum
officinarum.times.Saccharum sponlaneum) These MCs were delivered to
the Sugarcane callus cells using the wet biolistic method as
described above. In brief, the droplet containing the MC DNA-coated
particles was delivered to cells using a RioRad PDS-1000/He with a
rupture disk rating of 400-1800 psi (650 psi was preferred with one
shot per plate) adapted for a filter holder, which was attached to
a base which was itself attached to a rupture disk holder used to
hold the rupture disk to the helium egress tube for bombardment.
The gas discharge directly displaced the DNA/gold droplet from the
filter holder and accelerated the particles and their DNA cargo
into the tissue being bombarded.
[0410] Following delivery, callus from the tissue was
phenotypically evaluated for DsRed expression using a fluorescent
dissecting microscope. DsRed was observed in the tissue. The
resulting calluses were transferred to Selection Sugarcane Medium
MS3-50; (4.3 g/l MS salts and vitamins supplemented with 20 g/l
sucrose, 0.5 g/l casein, 3.0 mg/l 2,4-D, 0.5 g/l
polyvinylpyrrolidone (PVP). pH to 5.8 with 2 g/l GELRITE.RTM.;
further supplemented with 50 mg/l G418 after autoclaving) for
initial selection for 2 weeks. All calluses were subsequently
transferred to additional selection on Selection Sugarcane Medium
MS3-75 (4.3 g/l MS salts and vitamins supplemented with 20 g/l
sucrose, 0.5 g/l casein, 3.0 mg/l 2,4-D, 0.5 g/l
polyvinylpyrrolidone (PVP) pH to 5.8 with 2 g/l GELRITE.RTM.;
further supplemented with 75 mg/l G418 after autoclaving) for 4
additional weeks. Tissue was then visually assessed for Sugarcane
callus tissue that was able to grow on this selection. Those
identified events were transferred to Regeneration Sugarcane Medium
RSCM-25 (4.3 g/l MS salts and vitamins supplemented with 20 g/l
sucrose, 0.5 g/l casein, 0.5 mg/l kinetin. pH to 5.8 and solidify
with 2 g/L GELRITE.RTM.; further supplemented with 25 mg/l G418
sulfate after autoclaving) in low light (16 hour day length,
26.degree. C.) to initiate regeneration. Simultaneous with
initiating regeneration, this callus tissue was evaluated by PCR
for presence of the genes on the MC. PCR confirmed that these genes
were indeed present in the majority of the events. After 2
additional weeks of culture, the callus events had started to
differentiate into plantlets (shoot material).
[0411] After an additional 4-6 weeks on regeneration, plantlets
(with and without initial root initiation) were transferred to
Rooting Medium RtSC-25 (2.15 g MS salts and vitamins supplemented
with 20 g/l sucrose, 0.5 g/l casein, 0.5 mg/l kinetin. pH to 5.8
and solidify with 2 g/L GELRITE.RTM.; further supplemented with 25
mg/l G418 sulfate after autoclaving). Rooting occurred in 2 to 6
additional weeks of culture with 16 hour day length at 26.degree.
C. in sundae cups (Solo Cup Company). Plantlets with well
established root systems were transferred into pre-moistened
soil-less mix (LC1, BFG Supply Company) under a humidome in 18-well
flats in a growth chamber (28.degree. C., 16 hour day length). The
dome was opened slightly 3-4 days after transplanting to slowly
reduce humidity. The dome was removed completely 2 days later and
the plantlets were then transferred to a greenhouse (28.degree. C.,
16 hour day length). The plants were watered from trays beneath the
pots when the soil began to dry. The plants were subsequently
transplanted and grown to maturity in 1.6 gallon pots with
Soil:Peat:Perlite (1:1:1) supplemented with OSMOCOTE.RTM.
fertilizer (The Scotts Miracle-Gro Company; Marysville, Ohio;
USA).
[0412] Sugarcane callus and tissues produced phenolic compounds
while in tissue culture, and these phenolic compounds appeared to
reduce or inhibit callus growth and plantlet regeneration. In order
to promote Sugarcane plantlet regeneration in culture, the media
described above (MSO, MS3 and variants thereof and RSCM) were
supplemented with polyvinylpyrrolidone (PVP) at a concentration
ranging from 1% to 3% w/v according to the intensity of the
exudation of the phenolic compounds. The PVP acted as a sink for
phenolic compounds and enhanced subsequent callus growth and
plantlet regeneration.
[0413] In order to promote the frequency and the morphogenetic
competence of regenerable Sugarcane callus, the cells were cycled
from a liquid culture to a solid culture. The apical region of the
Sugarcane stem (20-30 cm long) was collected, and the mature leaves
were removed. The stem was surface-sterilized by submerging the
tissue in a solution of 50 ml bleach in 1 liter of water for 10
minutes. The remaining outermost mature leaves were aseptically
removed, and the young inner immature leaves were sliced into
sections/discs approximately 2-3 mm thick.
[0414] The resulting leaf roll discs were placed on Sugarcane MS3
Medium at 28.degree. C. for 2 weeks in the dark. The resulting
regenerable Sugarcane callus (white nodular embryogenic pieces) was
then removed and placed into liquid Sugarcane MS1 medium at
28.degree. C. for 2 weeks on a rotating orbital shaker (100-150
rpm) in the dark. After the two week culture in the liquid MS1
medium, the regenerable Sugarcane callus (white nodular embryogenic
pieces) was removed and sub-cultured back onto Sugarcane MS3 Medium
(MS3) at 28.degree. C. for 2 additional weeks in the dark.
Sugarcane callus can be sub-cultured in 2-week intervals between
solid MS medium containing 3 mg/l 2,4-D and liquid MS medium
containing 1 mg/l 2,4-D to maintain embryogenic callus.
[0415] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference. Applicant intends that the sequence listing filed
herewith forms a part of the description of the specification and
is hereby incorporated by reference in its entirety.
[0416] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
2511137DNASaccharum officinarum 1cccagcagtt ccatcgggtg catcctagat
aaattttgag ccgatggtac attcgacaca 60aaacgcgcac ctatcttgcg tcaagattgg
cactatctcc aaacggactc aaacaagcat 120ccacttgagc ctcatca
1372136DNASaccharum officinarum 2cctaggagta tcgtcgggtg cgtccaaaac
aatttctgag ccgatggtac gttcggtgga 60aaccgtgcac ctatattgca ccaacactaa
cactgtctcc aaacggaccg aaagaagatt 120ccacatgacc cactcg
1363136DNASaccharum officinarum 3tcttggactt ccaccgggtg cattcaagac
gatatctgag cctatggtac gtttggcaca 60aaccgtgcac ctatattgca ccgacactga
cactgtctcc aaatggaccg aaaggagatt 120ccacatgacc cactcg
1364136DNASaccharum officinarum 4tctaggactt ccatcaggtg cgttcaagat
gatttccgag cctatggtac atttggcaca 60aaccgcacac atatcttgcg tcaagttagc
actatctcca aacggactca aacaagcttc 120cacttgagcc acgtca
1365137DNASaccharum officinarum 5cccagcagtt ccatcgggtg cctccaatac
tatttccaag cctacggtat gtttgacgca 60aaccatgcac ctatcttgcg tcaagattag
cactatctcc aaacggaccc aaccgagctt 120ccacttgagc cacgtca
1376137DNASaccharum officinarum 6cccagcagtt ccatcgggtg catccaaatt
gatttctgag cctatggtat gtatggcgca 60gaccgtgcaa ttatcttgca ccgacactaa
acctatatcc aaacggactt aaacgagatt 120ccacatggcc ccctgca
1377137DNASaccharum officinarum 7cctaggagtt ccattgggtg cgtccaaaat
gattctcgag cctccgatac gttctgcaca 60aaccgtgcac gtatcttaca gaaagattag
caccatctcc aaacggaacg aaacgagctt 120ccacttgagc ctcgtca
1378137DNASaccharum officinarum 8ccaagtggta ccatcgagtg tgttcaaaat
gatttttgag ccaatggtac gtctagcgca 60aaccatgcac ccatcttgca ctggcactaa
cactatctcc aaacagaaag aatcaagatg 120ccacatgacc catgtca
1379137DNASaccharum officinarum 9cccagcagtt ccatcgggtg cctccaatac
tatttccgag cctacggcac attcgacgca 60aatcgagcac caatcgtgca tcaagagtag
cactatctcc aaacgtaccg aatcgagctt 120ccacttgagc cacgtca
13710137DNASaccharum officinarum 10cccagcagtt ccatcgggtg cctccaatac
tatttccgag cctacggcac attcgacgta 60aaccgagcac caatcttgca tcaagagtag
cactatctcc aaacgtaccg aatcgagctt 120ccacttgagc ctcatca
13711137DNASaccharum officinarum 11cctaggagtc ccctagagtg cgtccaaaac
gatttctatg cctatgctac atttggtgca 60aaccaagcac caatcttgca tcaagagaag
cactatcccc aaacggaccg acctaagctt 120ccacttgagc cacgtca
13712128DNASaccharum officinarum 12cccagcagtt ccatcgggtg cgtccaaaac
tatttatgac ctatggtacg ttcaatgcaa 60accgtggacc tatcttctgt agcactatct
ccaaacggac caaacagatc tccaccaggc 120cctcatca 12813136DNASaccharum
officinarum 13cctaggagta ccacgggtgc ttctaaaacg atttatgaac
caatggtgcg tttgacgaaa 60actgagcacc tatcttgcac cgaccctaac actgtttcaa
aatagatcga aacaagattc 120cacatgaccc acgtaa 13614137DNASaccharum
officinarum 14cctaggagtt ccttcgggtg cctccaatac tatttctaag
cctactatac gtttgacgca 60aaccttgcac caatcgtgca tcaagagtag cactatcccc
aaacggaccg aaccgagctt 120ccacttgagc cacgtca 13715137DNASaccharum
officinarum 15cccagcagtt ccatcgggtg catctaaaat gatttccgag
cctacggtac gtccggcaca 60aactgtgcac ctatcttgaa acaagattag cacaatctcc
aaactaacgg aaatgagctt 120ccacttgagc cacgtca 13716136DNASaccharum
officinarummisc_feature(1)..(9)n is a, c, g, or t 16nnnnnnnnnc
catcgggtgc gttcaagatg atttctgggc ctannnaang ntaggcacaa 60actgcccatc
tatctcgtgt caagattagc actatctcca aacggactca aacgagcttc
120cacttgagcc ttctca 13617137DNASaccharum officinarum 17cctaggagta
ccaccgggtg cgtccaaaat gattttcgag cctgtggtac gttcggcgca 60aaccgtgcac
ctatcttgcg tcaagattag cactatctcc aaatgaacta aatcaagcat
120ccacttgagc cacgtca 13718137DNASaccharum officinarum 18cccagcagtt
ccatcgggtg cctccaatac tatttctgag cctaaggtat gtttgacgca 60aactgtgcac
caatcgtgca tcaagagtag tactatctcc aaacggaccg aaccgagctt
120ccacttgagc ctcatca 13719137DNASaccharum officinarum 19cctaggagtc
ccatcgagtg cgtccaaaat gatttctatg cctatggtat gttcggtgca 60aaccgtgcac
ctctcttgca ctgacattaa cactgtctcc aaatggacta aaatgaaatt
120ccacatgacc cacgcca 13720136DNASaccharum officinarum 20cttatgtgtt
ccattgggtg cgtccacaac tatttatgag cctatgctac cttcaaagca 60aaccgtgcac
ctatcttgtg ttaagattgg cactatctcc aaacggacca aacagggctc
120cactagaccc tcgtca 13621138DNASaccharum officinarum 21cttaggagta
ccattgggtg cttttaaaat gatttatgag actatggtac gttttgacgc 60aaattgagca
cttatcttgg accgacgcta acactgtctc aaaactgatc gaaacaagat
120tccacaagac ccacgtta 13822137DNASaccharum officinarum
22actaggagtt ccatcgggtg cctccaatac tatttacgag cctacggtac atttgacgca
60aaccgtgcac caatcgtgca tcaagagtag cactatctcc aaacggaccg aaccgagctt
120ccacttgagc cacgtca 13723137DNASaccharum officinarum 23cccagcagtt
ccatcgggtg cctctaatac tatttctgag cctattgtac gtttgatgca 60aaccgtacac
caatcgtgca tcaagagtag cactatcccc aaacggaccg aaccgagctt
120ccacttgagc cacgtca 13724137DNASaccharum officinarum 24cccagcagtt
ccatcgggtg cggtcaacac gacttccgag ccgatgatac gttcggctca 60aaccgcgcac
ctttcttgtg tcaagattac cactatctcc aaatagactc aaacaagctt
120ccacttgagc ctcatca 13725136DNASaccharum officinarum 25tctaggagta
tcgtcgggtg cctccaaaac aatttctgag ccgatggtac gttcggcgca 60aaccatgcac
ctatattgca ccgacactaa cactgtctcc aaacgaaccg aaatgagatt
120ccacttgacc cactcg 13626137DNASaccharum officinarum 26tctaggactt
ccatcgggtg cgttcaagac gatttccaag cctatggtac gtttgacaca 60aaccgcgcac
ctatcttgcg tcaaaattag cactatctcc aaacggactc aaacaagctt
120ccacttgagc cacgtca 13727137DNASaccharum officinarum 27cccagcagtt
ccatcgggtg cgtccaacat gatttccgag ccaatggtac gttcgacaca 60aaccacgcat
atttcttaca tcaagattag cactatttcc aaacagactc aaacaagctt
120ccacttgagc cacgtca 13728137DNASaccharum officinarum 28cccagcagtt
ccatcgggtg cgtccaagat gattttcggg cctacggaaa gctaggcaca 60aactgcacac
ctatattgtg tcaagattgg cactatctcc aaacggactc aaacaagctt
120ccacttgagc cttgtca 13729137DNASaccharum officinarum 29cctaggagta
ccaccaggtg catccaaaat gatttctgag cctatggtac gttcaacgcg 60aactgtggac
ctatcttgcg gcaagattat caccatctcc aaacagaacg aaacgagctt
120ccacttgagc cacgtca 13730137DNASaccharum officinarum 30cccagcagtt
ccatcgggtg cctccaatac tatttccgag cctacggcac attcgacgca 60aatcgagcac
caatcttgca tcaagagtag cactatctcc aaacgtaccg aatcgagctt
120ccacttgagc ctcgtca 13731137DNASaccharum officinarum 31cctacgagtc
ccctcgagtg cgtccaaaac gattcctatg cctatgctac atttggtgca 60aaccaagcac
caatcttgca tcaagagaag cactatcatc aaacggaccg agccaagctt
120ccacttgagc ctcatca 13732137DNASaccharum officinarum 32cctaggagtc
ccatcgagtg tgtccaaaat gatttctatg cccatggtac gttcagtgca 60aaccgtgcac
ctatcttgca tcgacgctaa cactgtctca aaatagatct aaatgagatt
120ccacatgacc tatgtca 13733116DNASaccharum officinarum 33cccagcagtt
ccatcgggtg cgtccacaac tatttatgtg cctatggtac cttcatttca 60aaccatgcac
ctatcttgtg tcaagattag cactatcatc aaacggaccc atgtca
11634137DNASaccharum officinarum 34cccagcagtt ccatcgggtg cctccaatac
tatttccgag cctaaggtac gtttgacgca 60aaccgtgcac caatcgtgca tcaaggatag
tactatctct aaacggacca aaccgagctt 120caacttgagc cacgtca
13735137DNASaccharum officinarum 35cccagcagtt ccatcgggtg catcctaaat
gatttccaag cctatggtac gttcaacgca 60aaccatccac ctatcttgcg ataagattag
cactatctcc aaacagacag aaacaagctt 120ccacttgagc ctcaaca
13736126DNASaccharum officinarum 36cctaggagaa ccgtcgagtg cgtctaaaat
tatttctatg tttggcgcaa accgtgtacc 60tatcttgcac tgacactaat atcgtctgca
aacagatctt aacgagactt gacttgaccc 120acatca 12637137DNASaccharum
officinarum 37actaggagtt ctatcgggtg tgtccaaaat gatttccgag
cctatggtac attcggcgta 60aacaatgctc ctatcttgca tcaagattag cactatctcc
aaatggaccg aaacgagctt 120ccacttgagc cacgtca 13738137DNASaccharum
officinarum 38cccagcagtt ccatcgggtg cctccaatac tatatccgag
cctacggtat gttcgacaca 60aaccatgcac caatcatgca tcaatagtgg cactatctcc
aaatggaccg taccgagctt 120ccacttgagc ctcgtca 13739137DNASaccharum
officinarum 39cctaggagtc ccatcgagtg tgtccaaaac gatttctatg
cctatggtac gttcagtgca 60aaccatgcac ctatcttgca ccgacactaa tactgtctca
aaatagatca aaacgagatt 120ccacaagacc cacgtca 13740137DNASaccharum
officinarum 40cccagcagtt ccatcgggtg cctccaatac tatttctgag
cctacggaac attcgatgca 60aaccgagcac caatcttgca tcaagagtag cactatctcc
aaacgtatcg aatcgagctt 120ccacttgagc cacgtca 13741136DNASaccharum
officinarum 41cccagcagtt ccatcgggtg cgtccaaaaa catttctgag
cttacggtac ggtcggcgca 60aaccgtgccc tatcttgagt caagataagc actatctcca
aacgaaccga aacgagcttc 120gatttgagcc ccgtcg 13642137DNASaccharum
officinarum 42ccatggagta ccatccggtg cgtccaaaac gatttctgag
gctatggtac atttggcgca 60aaccgtgcac ctaccttaaa ctaaaactta cactgtctcc
aaacagaccg aaacgatctt 120ccacttgagc cacgtca 13743137DNASaccharum
officinarum 43cccagcagtt ccatcgggtg cctccaaatc gatttctgag
cctacggtat gtttggcgca 60gaccgtgcaa ttatcttgca ccgacactaa acctatctcc
aaacggactt aaacgagatt 120ccacatggcc cccgaca 13744137DNASaccharum
officinarum 44cctaggagtt ccattggttg catccaaaat gattctcgag
cctccggtac gttctgcgca 60aaccgtgcac ctgtcttgca gaaagattag cgccatctcc
aaacggaacg aaacgagctt 120ccacttgagc ctcgtca 13745137DNASaccharum
officinarum 45cctatgagtt ccattaggtg tgtccaaaat gatttctaag
cctatggtat gtttgttgca 60aaccgtgcac ctatcttgca tcaagtttag caccatctcc
aatcggaccg taacgtgctt 120tcacttgagc cacgtca 13746137DNASaccharum
officinarummisc_feature(6)..(6)n is a, c, g, or t 46cccagnagtt
ccatcgggtg catctaaaac gatttctgag cctagggtat gttcaacgga 60aaccgtgcac
gtatcttgcg tcaagatttg cactatctca aaacatacca aaacgagcat
120ccacttgagc cacgtca 13747137DNASaccharum officinarum 47cccagcggtt
ccatcgggtg catgtaaaat gatttttgag cttatggtat gttcggcgca 60aaccatgaac
ctatcttgcg tcaagattgg cactatcaac aagtggaccg aaaggagctt
120tgacttgagc cttgtca 13748136DNASaccharum officinarum 48cccagcagtt
ccatcgggtg cgtccaaaat gatttctgag cctgtgctac gttcggcgaa 60accttgcacc
catcttgtgt catgattagc tctatgtcca aacagaccaa aacgagcatc
120cacgtgtgcc tcgtca 13649137DNASaccharum officinarum 49cctaggagta
ccatcgagtg cgtccaaaat gactttcaag cctatggtac gttcgacgca 60aaccgtgcac
ctatcttgcg tcaagattag cactatctcc aaacatacaa aaatgagcat
120ccacttgagc cacgtca 13750137DNASaccharum officinarum 50cccagcagtt
ccatcgggtg cgtccaacac gatttctgtg cctatggtac gtttagcgca 60aaccgtgcac
gtaacttgca ctaacactgc cactgtcacc aactggattg aaacgagatt
120cctcatgacc aaagtca 13751138DNASaccharum officinarum 51cataggagat
ccatcgggtg catccaaaac gattttctga gcctatgcta cgttcggcgc 60aaaccgtgca
cctatcttgc gtcaaggtta gcactatgtc taaacagacc aaaacgagca
120tccacttgag ccttatca 13852137DNASaccharum officinarum
52cctaggagta ctatcgagtg cgtccaaaat gatttccgag cctatggtac gtttgacgca
60aatcgtgcac caatctcgca tcaaggttag cactatcccc aaacagacca aaacgagctt
120ccacttgagc cacgtca 13753137DNASaccharum officinarum 53cccagcagtt
ccatcgggtg catcaaaaac aatttctgag cctatgctac atttggcgca 60aaccattcac
ctttcttgcg tcaagattag cacaatctcc aaatggattg aaacgagctt
120cctgttgagc ctcgtaa 13754137DNASaccharum officinarum 54cctaagagta
ccatcgggtg tgtccaaaat gatttatgag cctagggtac gtttggcgca 60aaccgtgcac
ctatcttgga ttgaaactac cattgtctcc aaatggattg aaatgagatt
120ccacatgacc aatgtca 13755137DNASaccharum officinarum 55catagtagat
ctatcgggtg cgtccacatg gatttctgac cctatgttac gttcggcgca 60aaccgtgcat
ctatcttgcg tcaagattag cactatctcc aaatagactt aaacaagtaa
120ccacttgagc cacgtca 13756137DNASaccharum officinarum 56cccagcagtt
ccatcgggtg cctccaaaac tattttcgag cctacggcac attcgacgta 60aaccgagcac
caatcttgca tcaagagtag cactatctcc aaacataccg aatcgagctt
120ccacttgagc ctcgtca 13757137DNASaccharum officinarum 57cctaggagtc
ccctagagtg cgtccaaaat gatttctgtg cctatgctac atttggtgca 60aaccaagcac
caatcttgca tcaagagaag cactatcccc aaacggaccg acctgagctt
120ccacttgagc cacgtca 13758137DNASaccharum officinarum 58cccagcagtt
ccatcgggtg cctccaaagc tatttatgag cctacggtac gtttgacaca 60aaccgttcac
caatcttgca ccaagagcag cactatctcg aaatagacgg aaccgagctt
120ccacttgggc ctcgtca 13759137DNASaccharum officinarum 59cctaggaacc
taccgagtgc atccaaaata attttccatg cctatggtat gttcggtgca 60aaccgtgcac
ctatcttaca ttgatactaa cactatctcc aaatggacta aaacgagatt
120ccacatgacc cacgtca 13760136DNASaccharum officinarum 60cttacgagtt
ccattgggtg cgtccaaaac tatttatgag cctatagtac gtttagtgca 60aaccatgcac
ctatcttgca ttaagattag cactatctcc aaacggatcg aacagagctc
120caccagagcc acgtca 13661137DNASaccharum officinarum 61cctagaagta
ccatcgggtg cgtctgaaac gacttatgag cctatggtac gtttgatgca 60aaccgagcac
ctatcttgca ccaacggtaa cactgtctca aaaactatcg aaaagggatt
120ctacatgacc cacgtca 13762137DNASaccharum officinarum 62cctaggagtt
ccatcggttg cctcaaatac tatttccgag cctacagtac gttcgacgca 60aaccgtgcac
caatctttca tcaagagtag cactacctcc aaacggaccg aaccgagctt
120ccacttgagc cacgtca 13763134DNASaccharum officinarum 63cccagcagtt
ccatcgggtg cgtccaaaat aatttctaag cctatggtaa attcagcaca 60aaccatgcac
ctatcttgca ccggcactaa cactgtctcc aaatgaacca aaacgatcca
120cttgacccac gtta 13464121DNASaccharum officinarum 64cctaggagtt
ccatcaggtg cgtccaaaat gattttcgag cctatggaat gttcggcaca 60aaccgtgcac
ctatcttgct caagatttgg accgaaacga tcttccactt gagcctcatc 120t
12165137DNASaccharum officinarum 65ccttggagta ccatcgggtg catccaaatt
gatttataag cctctggtac gtttggtgca 60aaccctggac ctatcttgca ctgatagtaa
cactgtctcc aaatggaccg aaatgagatt 120ccatatgacc cacgtca
13766137DNASaccharum officinarum 66cctaggagtt ccattgggtg cgtccacaat
aattttccga gcctatggtg cgttcggtgc 60aaactatgca cctatcttgt gttatgatta
gtactatcgc caatcgtacc gaaatagctt 120ccacttgagc cacgtca
13767128DNASaccharum officinarum 67cccagcagtt ccatcgggtg cgtccaaaac
tatttatgac ctatggtacg ttcaatgcaa 60accgtgcacc tatcttctac agcactatct
ccaaacggac caaacagatc tccaccagac 120cctcgtca 12868135DNASaccharum
officinarum 68cctaggagta ccgtgggtgc ttctaaaatg atttatgaac
ctatggtgcg tttgacgcaa 60accgtgcacc tatattgcac cgacactaac actgtctcca
aacggatcga aacatgattc 120cacttgaccc actcg 13569137DNASaccharum
officinarum 69tctaggactt ccatcgcgtg cgttcaagat gatttctgag
cctatggtgc gtttggcaca 60aaccacgcag ctatcttgca ttaaaattag cactatctcc
aaatggactc aaacaagctt 120tcacttgagc cacgtca 13770137DNASaccharum
officinarummisc_feature(4)..(4)n is a, c, g, or t 70cccngnagtt
ccatcgggtg cctccaatac tatttctgag cctactgtcc gtttgacgca 60aaccgtgcac
caatcgtgca tcaagagtag cactatcccc aaatggaccg aaccgagctt
120ccacttgagc cacgtca 13771137DNASaccharum officinarum 71cccagcagtt
ccatcgggtg cgttcaaaac gatatctgag cctacggtat gtttggcgca 60aaccgtgcgc
ctatcttaca ccgatactaa cactgtctct aaacagaccg aaatgagatt
120ccacatgacc cacatca 13772135DNASaccharum officinarum 72cctaggtgtt
ccatctggtg tgtccaaaat gattttcaag cctatggtac gctcggcgca 60agccgtgcac
ctatcttgct
ccaatactaa gactctccaa acaaactgaa ataagattcc 120acatgaccca ggtca
13573137DNASaccharum officinarum 73cctaggtgtt ctgtcgggtg tgtctaaagc
gattttggag cctgtggtac gttcagggca 60aacagtgcac ctatcttgcg ttaagaatag
tagtatctct aaacggaccg aaacgagcat 120ccacttgagc cacgtca
13774137DNASaccharum officinarum 74cccagcagtt ccatcgggtg cgtccaaaat
gatttctgag cccgtggaaa gttcggcgca 60aactgtgcac caatcttgcg tcaagattag
cactatctcc aaacagaccg aatcgaactt 120ccacttgagc cacgtca
13775137DNASaccharum officinarum 75cccagcagtt ccatcgggtg catccagaac
gatttccgag cctatggtat attcggcaca 60aacaatgcac ctatcttgca ccgacactaa
cactgtctcc aaacggacct aaatgagaat 120ccacacgacc cacgtca
13776138DNASaccharum officinarum 76cctaggagct tcatcaggtg cctgtaaaaa
ctattctcga gcctttggta tgttcggcgc 60aaaccatgcc cctatcctac atcaagatta
gcagtatctc caaacggacc gaaacgagtt 120tccacttgag ccacgtca
13877137DNASaccharum officinarum 77cccagcagtt ccatcgggtg cgtccaaaat
gatttctaag cctatggtac attcggcgca 60aaccgtgcaa ctatcttgtg tcaagattag
ctctatgtcc aaacagacca aaactagcat 120ccatgtgtgc ctcgtca
13778130DNASaccharum officinarum 78cctaggagta ccatcaggtg cgtccaaaat
gatttctaac cctatggtac atttggcaca 60aaccttgcac ctatcaagat tagctctatc
tccaaacaga ccaaaaggac tatccaattg 120agcctcgtca 13079137DNASaccharum
officinarum 79cctaggagta gcatcagata cgtccaaaat gatttctgag
cctatggtac gttcaatgca 60aaccatgcac ctatcttgcg tcaagattag cactgtctcc
aaacatacca aaatgagcat 120ccacttaagc cacgtca 13780137DNASaccharum
officinarum 80cctaggagta ccatcgggtg cgtccaaaat gatttctgag
cctatggtat gtttggcgca 60aattgtgcac ctatcttgca tcaagtttag caacatctcc
aatcggaccg taacgagctt 120tcacttgagc cacgtca 13781135DNASaccharum
officinarum 81cccagcagtt ccatcgggtg cgtccaaaac aatttctatg
cctatggtac gtttggtgca 60aacagtgcac ctatcttgca ctgacagtac cactgtcttc
taatgttgaa atgagattcc 120acatgaccaa tgtca 13582137DNASaccharum
officinarum 82ccttagagat ccattggttg cgtccaaaac gatttctgac
cctaagttac gttcggcgca 60aaccgtgcaa caatcttgcg tcaagattag cactatctcc
aaacagacca aaacgagcat 120ccacttgagc ctcatca 13783136DNASaccharum
officinarum 83ccaagagtac catcaggtgt gtctgaaacg attttcaagc
ttgtggtacg ttcgccgcaa 60accgtgcacc tatcttgcgt caagattagt actatctcca
aatagcctaa aataaataac 120cacttgagcc acgtca 13684137DNASaccharum
officinarum 84cccagcagtt ccatcgggtg cctccagtac tatttccaag
cgtacggtac gtttgacgca 60aaccgtgcac caatcgtgca tcaagagtag cactatctcc
aaatggactg aaccaagctt 120ccacttgagc cacgtca 13785137DNASaccharum
officinarum 85cccagcagtt ccatcgggtg cctccaatac tatttctgag
cctatgattt atccgatgca 60aaccatgcat caatcatgcg tcaagagtag cactatctcc
aaatggaccg taccgagctt 120ccacttgagc ctcgtca 13786137DNASaccharum
officinarum 86cctaggagtc ccatcgagtg tgtccaaaac gatttatatg
cctatggtat gtttagtgca 60aaccgtgcac ctatcttgca ctgacactaa cactgtctta
aagtagatca aaacgagatt 120ccacaagacc cacgtca 13787137DNASaccharum
officinarum 87cctaggagtt ccaccgggtg cctccaatac tatttccgag
cctacggcac attcgacgca 60aaccgagcac caatcttgca tcaagagtag cactatctcc
aaacataccg aatcgagatt 120ccacttgagc cacgtca 13788131DNASaccharum
officinarum 88cccagcagtt ccatcgggtg catctaaacc aatttccgag
cctatggtag gttccacgca 60acccgtgcac ctcgtcaaga ttagcactat ctccaaacgg
accaaaacaa gcttccactt 120cagcctcgtc a 13189138DNASaccharum
officinarum 89cctatgagtt ccatggggtg tgtccaaaat gattttccaa
gcctatggta tgtttggcgt 60aatccgtgca cctatcttgc atcaagttta gcaccatctc
caatcggacc gtaacgagct 120tccacttgag ccatgtca 13890137DNASaccharum
officinarum 90cccagcagtt ccatcgggtg cctccaaaac tatttccgag
cctacgacac attcgacgta 60aaccgagcac caatcttgca tcaagagtag cactatctcc
aaacgtaccg aatcgagctt 120ccacttgagc cacgtca 13791137DNASaccharum
officinarum 91cccagcagtt ccatcgggtg cgctcaaaat gatgttcgag
cctacggtac atttggcgca 60aaccatgcac ctatcttgtg tcaacattag tactatctcc
aaatagaccg aaataagctt 120tcacttgagc cacgtca 13792137DNASaccharum
officinarum 92cccagcagtt ccatcgggtg cgtccaaaat gatttctgac
cgtatgttac gttcggcgca 60aatcgtgcat ctatcttgcg tcaagattag cactatctcc
aaagagacca aaacgagcat 120ccacttgagc ctcttca 13793137DNASaccharum
officinarum 93gcaaggagta ccatcgggtg tgtctgaaat gagtttcaag
cttgtggtac gttcggcgca 60aaccgtgcaa ctatcttgca tcaagattag cactatctct
aaacagacta aaacaagtaa 120ccacttgagc cacgtca 13794137DNASaccharum
officinarum 94cccagcagtt ccatcgggtg cctccaatac tagttccgag
cctacagtac gttcaacgca 60aaccgtgcac caatcatgca tcaagagtag cactatctcc
aaatggactg taccgagctt 120ccacttgagc ctcgtca 13795136DNASaccharum
officinarum 95cctaggagtc ccatcgagtg tgtccaaaat gatttttatg
cctatggtac gttcagtgca 60aaccgtgcac ctatcttgca ccgaagctaa cactgtctca
aaatagatca aaatgagatt 120ccacatgacc cacgcc 13696137DNASaccharum
officinarum 96cctaggagtt ccattgggtg cctccaatac tatttccgag
cctacggcac attcgacgca 60aatcgagcac caatcttgca tcaagagtag cactatctcc
aaacgtaacg aatcgaactt 120ccacttgagc cacgtca 13797128DNASaccharum
officinarum 97cccagcagtt ccatcgggtg cgtccaaaac tatttatgac
ctatggtacg ttcaatgcaa 60accgtgcacc tatcttctgt agcgctatct ccaaacggac
cgaacagatc tccactagac 120cctcatca 12898136DNASaccharum officinarum
98ccaaagagta ccgcgggtgc ttctaaaacg atttattaac ctatggtacg tttgacgcaa
60accgagcacc tatcttgcac tgacgctaat actgtctcaa aatagatcaa aacaagattc
120cacatgaccc atgtca 13699137DNASaccharum officinarum 99cctaggagtt
ccattgggtg cctccaatac tatttccgag cctacggcac attcgacgca 60aaccgagcac
caatcttgca tcaagagtag cactatctcc aaacgcaccg aatcgagctt
120ccacttgagc cacgtca 137100137DNASaccharum officinarum
100cccagcagtt ccatcgggtg cgtccaagac gattttcgag tcgatggtac
aatgaacaca 60aaccaggcac ctatcttgca tcaagaatag cactatctcc aaacggactc
aaacaagctt 120ccacttgagc ctcgcca 137101128DNASaccharum officinarum
101cctagtagta tcatcaggcg tgtccaaagt gatttcaggg ccaatggcac
attctgagca 60aaccatttgc accgacacta acactatccc caaacagacc gaaatgagct
tccacttgat 120cctcatca 128102137DNASaccharum officinarum
102cctaggagta ccatcagatg cgcccaaaat gattgctaag cctatgacat
gtttcacgca 60aaccatgcac ctatcttgca ccgacgcaaa ctctgtctcc aaacagacca
aaacaagttt 120ccacatgacc cacatca 137103134DNASaccharum officinarum
103cctaggagtt ccatcgggtg cgtccaaaac gattttcgag cctatggtac
gctcagcgca 60aatcgtgctt tcttgcatca atgttagccc tatctccaaa tggaccgaaa
cgagcttcca 120cttgagccac gtca 134104137DNASaccharum officinarum
104cccagcagtt ccatcgggtg cgtccaatat gatttctgag cctatggtac
gttcggcata 60aaccatgcac caatcttgct tccagattag cactatctct aaatggacca
aaatgagctt 120ccacttaagc ctcatca 137105137DNASaccharum officinarum
105cctaggtgta ccaccgggtg cgtccaaaac gattactgag cctatggtat
gtttgaccca 60aaccatgcac ctatcttgta ctaacactaa caccatctcc aaacggacca
aaacgagctt 120tgacacaacc caagtca 137106137DNASaccharum officinarum
106cctaggggtt ccttcgggtg cgtccaaaat aatttttaag cctatggtac
gttcagcgga 60aactgtgcat ctgtcttgtg tcaagattag cactgtcttc aaatggacca
aaacgagctt 120gcacttgagc cacgtca 137107132DNASaccharum officinarum
107cccagcagtt ccatcgggtg catcccaaaa ggatttctga tctacgtttc
gcgcaaaccg 60tgcaccgatc ttgcactgac tctaatactg tctccaaatg gattgaaaca
agattccaca 120cgaccaatgt ga 132108137DNASaccharum officinarum
108ccgaggagat ccatcgggtg cgtccaaaat gatttctgag cctgtcctac
gttcggagca 60aaccttgcac ccatcttgtg tcatgattag ctctatgtcc aaacagacca
aaacgagcat 120gcacgtgtgc ctcatca 137109137DNASaccharum officinarum
109cctaggagta ccatcgagtg cgacaaaaat gattttcgag cctatggtac
gttcgacgta 60aaccgtgcac ctatcttgtg tcaatattag cactatgtct aaatagactg
aaacaaataa 120ccacttgagc cacgtca 137110137DNASaccharum officinarum
110cccagcagtt ccatcgggtg cgtccaaaat gatttctgag cccgtggtaa
gttcggcgca 60aacagtgcac caatcttgcg tcaagattag cactatctcc aaacagaccg
aatcgaactt 120ccacttgagc cacgtca 137111136DNASaccharum officinarum
111cccagcagtt ccatcgggtg cgtccaaaat gatttccaag ctatgatacg
tttggtgcaa 60actgtatacc tatcttgcgt aaagattagt actatctcca aacggatcga
aacgagcttc 120tacttgagcc acgtca 136112137DNASaccharum officinarum
112cccagcagtt ccatcgggtg cattcaaaat gattgccgag cttatggtac
attcggcgca 60aactgtgcac ctatcttgag tcaaggttag cactatctac aaaccaactg
aaacgagccc 120ccatttgagc ctcgtga 137113137DNASaccharum officinarum
113cctaggagtt ccattcggtg tgtccaaaac aatttacgag cctatggtac
gtttggcgca 60aactatgcac ctatcttgca gtgaaactaa cactatctcc aaaaggaccg
aaaggagatt 120ctacatgacc cacatca 137114137DNASaccharum officinarum
114cctagaagta ccatatggtg tgtccaaaat gatttctgag cctatgggac
atttggcgca 60aactgtgccc ccatcttgca ctgaaactaa cacagtctcc aaacacaccg
aaacgagctt 120ccacttgagc cacgtca 137115137DNASaccharum officinarum
115cacagcagtt ccatcgggtg cgtccaaaat aacttctgag cctatggtac
gttcgacgca 60gactgtgtac ctatcttgcg tcaggattag aactatctcc aattggaccg
aaccaagctt 120ccacttgagc cacgtca 137116137DNASaccharum officinarum
116cccagcagtt ccatcgggtg cgtccaaaac aatttctgag gctatggtac
gttcgacaca 60aaacgtgcac ctttcttgtg tcaagattag cactatcttt gaacggtccg
aatcgaggtt 120cgacttcagc cacatca 137117137DNASaccharum officinarum
117cctagcagta ccatcgggtg cacccaaaat gatttctcag cctatggtac
attcggtgca 60aaccgtacac ctatcttgcg tcaagattag cactatctcc aaacaaactg
aatcaagcat 120ccacttgagc cacgtca 137118128DNASaccharum officinarum
118cccagcagtt ccatcgggtg cctccaaaaa tttctatgac ctatggtacg
ttcaatgcaa 60accgtgcacc catcttctgt agcattatct ccaagtggac caaacagatc
tccaccagac 120cctcatca 128119136DNASaccharum officinarum
119cctaggagta ccgcgggcgc ttcgaaaaca atttatgaac ctatggtaca
tttgacgcaa 60accgagcacc tatcttgcac cgacgctaac actatttcaa aatagatcga
aacaagattc 120cacatgaccc acgtaa 136120137DNASaccharum officinarum
120cctaggagtt ccatcgggtg cgtccaacac gatttctgat ctgatggtac
attcgataca 60aatcgtgcac ttttcttgca tcgagattac cactatctcc aaatggactc
aaacaagctt 120ccacttgagc cacgtca 137121137DNASaccharum officinarum
121cccagcagtt ccatcgggtg cctctaatac tatttctgag cctattgtac
gtttgatgca 60aaccgtacac caatcgtgca tcaagagtag cactatcccc aaacggaccg
aaccgagctt 120ccacttgagc cacgtca 137122137DNASaccharum officinarum
122cccagcagtt ccatcgggtg catccaaaat gatttctgat cctacggtat
gctcggcgta 60aactgtgcac ctttcttgcg tcaaggttag cactatctcc aaacagacca
gaacgagcat 120ccacttgagc cacgtca 137123137DNASaccharum officinarum
123cccagcagtt ccatcgggtg cctccaaagc tatttatgag cctacggtac
gtttgacaca 60aaccgttcac caatcttgca ccaagagcag cactatctcg aaatagacgg
aaccgagctt 120ccacttgggc ctcgtca 137124137DNASaccharum officinarum
124cctaggaacc taccgagtgc atccaaaata attttccatg cctatggtat
gttcggtgca 60aaccgtgcac ctatcttaca ttgatactaa cactatctcc aaatggacta
aaacgagatt 120ccacatgacc cacgtca 137125136DNASaccharum officinarum
125cttacgagtt ccattgggtg cgtccaaaac tatttatgag cctatagtac
gtttagtgca 60aaccatgcac ctatcttgca ttaagattag cactatctcc aaacggatcg
aacagagctc 120caccagagcc acgtca 136126137DNASaccharum officinarum
126cctagaagta ccatcgggtg cgtctgaaac gacttatgag cctatggtac
gtttgatgca 60aaccgagcac ctatcttgca ccaacggtaa cactgtctca aaaactatcg
aaaagggatt 120ctacatgacc cacgtca 137127137DNASaccharum officinarum
127cctaggagtt ccatcggttg cctcaaatac tatttccgag cctacagtac
gttcgacgca 60aaccgtgcac caatctttca tcaagagtag cactacctcc aaacggaccg
aaccgagctt 120ccacttgagc cacgtca 137128134DNASaccharum officinarum
128cccagcagtt ccatcgggtg cgtccaaaat gattttgaag catgtggtac
gtttggtgta 60aaacgtgcac taatcttgca tagccactaa cactgccttc aaatagaccg
atagattcca 120catgtcccaa gtca 134129136DNASaccharum officinarum
129cctaggagtt ccatcgggtg cgtccaaaac gatttttgag cctatggtac
gtttggcgca 60aagtgtgcac ctatcctgcg tcaagattag cactatctac aaacggatca
aatgagcttc 120cacttgagcc acgtca 136130137DNASaccharum officinarum
130cccagcagtt ccatcgggtg cttgtcaaat gatttatgag cctatggtac
gtttgacgca 60aattgagcac ctatcttgga ccgacgctaa cactgtctca aaacggatcg
aaacgagatt 120ccacaagacc cacgtca 137131137DNASaccharum officinarum
131actaggagtt ccatcgggtg cctccaatac tatttctgag cctattgtcc
gtttgatgca 60aaccgtacac caatcgtgca tctagagtag cactatcccc aaacggaccg
aaccgagctt 120ccacttgagc cacgtca 137132137DNASaccharum
officinarummisc_feature(72)..(72)n is a, c, g, or t 132cccagcagtt
ccatcgggtg cctccaatac gatttccaag actactgcac attcgacgca 60aaccgagcac
cnatcttgca tcangagtag cactatctcc aaacgtaccg aatcgagttt
120ccacttgagc ctcgtca 137133141DNASaccharum
officinarummisc_feature(18)..(19)n is a, c, g, or t 133cctaggagtc
ccctcganng cgtgcaaaat gatttctatg cctatgctat atttggtgca 60aaccaaaggc
ccatcttgca tcaagagaag cactatcccc aaacggaccg aaccgagctt
120ccacttgagc cacgggnccc a 141134137DNASaccharum officinarum
134cccagcagtt ccatcgggtg cgtccaatac gatttctgag cctacggtat
gtttggcgca 60gaccgtgcaa ttatcttgct ccgacactaa acctatctcc aaacggactt
aaacgagatt 120ccacatggcc cccgaca 137135137DNASaccharum officinarum
135ccttggagtt ccattgggtg catccaaaat gattctcgag cctccggtac
gttctgcgca 60aaccgtgcac ctatcttgca gaaagattag cgccatctcc aaacagaacg
aaacgagctt 120ccacttgagc cacgtca 137136137DNASaccharum officinarum
136cccagcagtt ccatcgggtg cgtgcaaaat aatttctaag gctatggtac
gttcggcaca 60aaccgtgcac ctatctggtg tcaagattag cactatcttt gagtggtccg
aatcgagctt 120gcacttgagc cttgtca 137137137DNASaccharum officinarum
137cccagcagtt ccatcgggtg cgttcgaaac aattgcatag ccggtggtag
gttcggcaca 60aaccgtgcag ctatcttgcg tcatgattag cactatctcc aaacgaactg
aatcaagcat 120gcacttgagc cacgtca 137138137DNASaccharum officinarum
138cccagcagtt ccatcgggtg cgtccaaaat gatttccgag cctatggtac
ttttaacata 60aactgtgcac ctatcttgcg tcaagattag cactatctct gaaaggaccg
aaacgagctt 120ccacttgagc cacgtca 137139128DNASaccharum
officinarummisc_feature(6)..(6)n is a, c, g, or t 139cccagnagtt
ccatcgggtg cgtccaaaac tgtttacgac ctatggtacg ttcaatgcaa 60accgtgcacc
tatcttctgt agcactatct ccaaacggac caaacagatc tccaccagac 120cctcatca
128140136DNASaccharum officinarum 140cctaggagta ccacaggtgc
ttctaaaaca atttatgaac ctatggtacg tttgacgcaa 60actgagcacc tatcttgcac
cgatgctaac actatttcaa aacagatcga aacaagattc 120cacatgaccc acgtaa
136141137DNASaccharum officinarum 141cctaggagtt ccatcgggtg
cctccaattc tatttctgag cctacggtac gatcgacgca 60aaccgtgcac caatcatgca
tcaaaagtag cactatctcc aaatgtaccg taccgagctt 120ccactggagc ctcgtca
137142137DNASaccharum officinarum 142cctaggagtc ccatcgagtg
tatccaaaac gatttctatg cccatggtac gttcagtgca 60aaccgtgcac ctatcttgca
ctgaagctaa cactgtctca aaatagatca aaacgagatt 120ccacatgacc cacgtca
137143137DNASaccharum officinarum 143cctaggagtt ccatcgggtg
cctccaatac tatttccaag cctacggcac attcaacgca 60aaccgagcaa caatcttgca
ccaagagtag cactatctcc aaacgtgccg tatcgagctt
120ctacttgagc cacgtca 137144137DNASaccharum officinarum
144cccagcagtt ccatcgggtg cgtcaaaaat gattgctgag cctgtggtac
gttcggcaca 60aactgtgcac ctatcttgca tcaagattcc cactatctcc aaacaaactg
aaccgagctt 120ccacttgagc cacgtca 137145137DNASaccharum
officinarummisc_feature(6)..(6)n is a, c, g, or t 145cccagnagtt
ccatcgggtg cgtccaagac gattttcgag ccgatagtac attcgacaca 60aaccacagac
ctatcttgcg tcaagtttag cactatctcc aaacaaactc aaacaagctt
120tcacttgagc cacgtca 137146126DNASaccharum officinarum
146cccagcagtt ccatcgggtg cctccaatac tatttccgag cctacggtac
atatgacgca 60aactgtgcat caagggtagc actatctcca aacggaccga accgagcttc
cacttgagcc 120ttgtca 126147137DNASaccharum officinarum
147ccaaggagtc ccatcgtgtg catccaaaac aatttctatg cctttggtat
attcggtgca 60aaccgtgcac ctgtcttgca ctgacactaa cactgtctcg aaacagacta
aaacgagatt 120ccacacgacc catgtca 137148135DNASaccharum officinarum
148cttacgagtc catcgggtgc gtcaaaaact atttgtgagc ctatggtacg
ttcagtgcaa 60accgtgcacc tatcctgtgt cacgattagc actatctcca aatgcaccga
acagagctcc 120accagaccca cgtca 135149137DNASaccharum officinarum
149cttaggagta ccatcgggtg cgtctaaact gatttatgac cctatggtac
gcttgacgca 60aatcgtgcac ctatcttgca ccgacgctaa cactatctca aaacagatcg
aaacgagatt 120ccacaagacc cacatca 137150137DNASaccharum officinarum
150actaggagtt ccaataggtg cctccaatac tatttccaag cctatggtac
gtttgacgca 60aaccgtgcac caaccatgca tcaagagtag cactatctcc aaacggaacg
aatcgagttt 120ccacttgagc cacgtca 137151137DNASaccharum officinarum
151cccagcagtt ccatcgggtg cgaccaaaat aatttctgag cctgtgttat
gtttggcgca 60aatcaagcac ctatgttgtg tcaagattag cactatctac aaacggaatg
aaacgagcat 120ccacttgagc ctcggca 137152137DNASaccharum officinarum
152cctaggagta ccatcgggtg cgtccaaaac gatttgtgag cctatggtac
gtttggcaca 60aaccgtgcaa ttatctttag tcaagaatag cattatcttc aaacggaatg
aaacgagcat 120ccacttgagc ctcgtca 137153137DNASaccharum officinarum
153cctatgagta ccatggggtg cttccaatac gatttctgag ctaatggtac
gtttggcaca 60aaccgtgcac ctatcaagcg tcaagattag cactatctcc aaatggaatg
aaatgagcat 120gcacttgagc cacgtca 137154137DNASaccharum
officinarummisc_feature(6)..(6)n is a, c, g, or t 154cccagnagtt
ccatcgggtg cgtataacaa gacttgcgag ccgatggtag gtttgacaca 60aaccacgtac
ctttcttacg tcaagattac cactatctcc aaacagactc aaacaagctt
120ccacttgagc cacgtca 137155137DNASaccharum officinarum
155cccagcagtt ccatcgggtg cgtcaaaaat gatttatgag cctgtggtac
gttcggcgca 60aactgtgcac ctatcttgcg tcaagattcc cactatctcc aaacgaactg
aatcgagctt 120ccacttgagc cacgtca 137156136DNASaccharum officinarum
156cccagcagtt ccatcgggtg catccaaaat gacatccgag cctatggtcg
ttcggagcaa 60accatgcacc tatcttggat aaggattagc actatcttca aacagactga
aatgagcttc 120cacatgaacc tcgtca 136157137DNASaccharum officinarum
157cctaggagta ccatcgggtg cgtccaaaat gatttttgag cctatggtac
gcttggcgca 60aactgggcac ctatcttgaa acgacactaa cattgtttcc aaatagaaca
aaacgagatt 120ccacatgaca cacatca 137158138DNASaccharum officinarum
158cctaggagtt ccatcggggg cgtccaaaat gaatttccga gcctatggta
tgttctgcgc 60aaaccgcgca cctatcttgc gtcaatatta gcactatctc caaacggacc
aatacgagct 120tccacttgag ccacgtca 138159137DNASaccharum officinarum
159cccagcagtt ccatcgggtg cgtgcaaaac gatttctgag catatggtac
gtttggcgca 60aaccgtgcaa caaacttgca ctaacactac cactgtctca aaatggattg
aaatgagatt 120ccacatgacc aatgtca 137160137DNASaccharum officinarum
160cctaggagat ccatcgggtg catccaaaat gatttttgaa cctttggtac
gttcgacgca 60aaccgtgcac ctatcttatg tcaagattag cactatatcc aaatagactg
aaacgagcat 120ccacttgagc ctcatca 137161137DNASaccharum officinarum
161cctaggagta ccatcgtgtg cgtccaaaac tatttctgag cctatggtac
gtttggtgca 60aaccatgcac ctatcttaga ccaatactaa cactatctcc aaatggacca
aaattatatt 120ccacctaact catgtca 137162137DNASaccharum officinarum
162cctaggtgtt ctatcgagtg tgcccaaaac tattttcatg cttatggtac
attcggcgca 60aaccgtgatc ctatcttgcg tcaggattag tgtaatcccc aaacagactg
aaacaagcat 120ccacttgagc cacgtca 137163137DNASaccharum officinarum
163cccagcagtt ccatcgggtg cgcctaaaac ggtttatgag cctatggtac
gtttgatgca 60aaccgagcac ctatcctgca atgacgctaa cactgtctca aaatagattg
aaacgagatt 120ccacatgacc cacgtca 137164137DNASaccharum officinarum
164cctaggagtt ccatcgggtg ccgccaatac tatttctgag ccaacgatac
gttcgacgca 60aaccatgcac caatcttgca tcaagtgtag cactatctcc aaatggatcg
aaccgagctt 120ccacttgagc cacgtca 137165132DNASaccharum officinarum
165cccagcagtt ccatcgggtg catcctaaaa tgatttttaa tctatgtttg
gcgcaaaccg 60tgcacctatc ttgcactgac tctaatactg tctccaaatg cattgaaaca
agattccaca 120cgaccaatgt ca 132166137DNASaccharum officinarum
166cccagcagtt ccatcgggtg cgtccaaaat gatttctgag ctaatggtac
gttcggcgca 60aagcgtgcag ctatcttgcg tcaagattac cacaatctcc aaatggagcg
aaacgagctt 120ccacttgagc cacgtca 137167137DNASaccharum officinarum
167cccagcagtt ccatcgggtg cctccaatac tatttctgag cctaaggtac
atttgacgca 60aaccatgcac caatcgtgca tcaagggtag tactatctcc aaacggacca
aaccgagctt 120ccacttgagc cacgtca 137168137DNASaccharum officinarum
168cccagcagtt ccatcgggtg cctccaaagc tatttatgag cctacggtac
gtttgacaca 60aaccgttcac caatcttgca ccaagagcag cactatctcg aaatagacgg
aaccgagctt 120ccacttgggc ctcgtca 137169137DNASaccharum officinarum
169cctaggaacc taccgagtgc atccaaaata attttccatg cctatggtat
gttcggtgca 60aaccgtgcac ctatcttaca ttgatactaa cactatctcc aaatggacta
aaacgagatt 120ccacatgacc cacgtca 137170136DNASaccharum officinarum
170cttacgagtt ccattgggtg cgtccaaaac tatttatgag cctatagtac
gtttagtgca 60aaccatgcac ctatcttgca ttaagattag cactatctcc aaacggatcg
aacagagctc 120caccagagcc acgtca 136171137DNASaccharum officinarum
171cctagaagta ccatcgggtg cgtctgaaac gacttatgag cctatggtac
gtttgatgca 60aaccgagcac ctatcttgca ccaacggtaa cactgtctca aaaactatcg
aaaagggatt 120ctacatgacc cacgtca 137172137DNASaccharum officinarum
172cctaggagtt ccatcggttg cctcaaatac tatttccgag cctacagtac
gttcgacgca 60aaccgtgcac caatctttca tcaagagtag cactacctcc aaacggaccg
aaccgagctt 120ccacttgagc cacgtca 137173137DNASaccharum officinarum
173cccagcagtt ccatcgggtg catctaaaat gatttatgag cctgtggtac
gtttgatgca 60aaccgagcac ctatctttca ccgacgataa cactacctca aaatagatcg
aagcgagatt 120ccacatgacc catgtca 137174137DNASaccharum officinarum
174cctaggagtt ccatcgggtg ccaccaatac tatttccgag cctacggtat
gttcgacgca 60aaccgtgcac caatcttgca tcaagagtag cactatctcc aaacgtaccg
aattgagctt 120ccacttgagc cacgtca 137175137DNASaccharum
officinarummisc_feature(6)..(6)n is a, c, g, or t 175cccagnagtt
ccatcgggtg cctccaatac tatttccaag tctacagtac gtttgatgcc 60aaccgtgcac
caatcttgca tcaagagtag tactatctcc aaacggaccg aaccgagctt
120ccacttgagc cacgtca 137176137DNASaccharum officinarum
176cccagcagtt ccatcgggtg catccaaaat gatttctgag cctacggtat
gtttggcaca 60aaccttgcac ctatcttatg tcaaggttag cactatctcc aaacagacca
aaacgagcat 120ctacttgagc ctcgtca 137177137DNASaccharum officinarum
177cctaggagta ccatcgggtg cgtccaaaac aatttccgag cctatggtac
gttcgacgca 60aaccatccac caatctcgca tcaagaatag cactgtctcc aaatagacca
aaacgagcat 120ccacttgagc ctcgtca 137178137DNASaccharum officinarum
178cctaggagta ccatcggttg cgtccaaaac gatttatgtg cctatggtac
gtttggcgca 60aacagtgcag ctattttgga ctgacagtac cgctatctcc aaatgcatta
aaacaagatt 120ccacatgacc aatgtca 137179137DNASaccharum officinarum
179cctaggagat ccatcgggtg cgtccgaaat gatttctaag cctatggtac
gttcggcgca 60aaccgtgcac ctatcttgca tcaaggttag cactatctcc aaacttacca
aaatgagcat 120ccacttgagc cacgtca 137180136DNASaccharum officinarum
180cccagcagtc catcgggtgc gtccaaaatg atttctgaga atatggtatg
ttcggtgcaa 60accatgcacc taccttgcgt caagattacc actatctcga aacggaccaa
aatgagcttc 120cacttgagcc acgtca 136181137DNASaccharum officinarum
181cccagcagtt ccatcgggtg cgtccaatat aatttccgag ccaatggtag
attcgacaca 60aaccgtgcac ctttcttgca tcaagattac cactatctcc aaacagactc
aaacaagctt 120ccacttgagc cacgtca 137182137DNASaccharum officinarum
182cccagcagtt ccatcgggtg cctccaatac tatttccgag cctacggtat
gttcgatgcg 60aaccgtgcac caatcatgca tcaagagtag cactatatcc aaatggaccg
taccgagctt 120ccacttgagc ctcatca 137183137DNASaccharum officinarum
183cctaggagtc ccatcgagtg tgtccaaaat gatttctatg cctatggtac
gttcggtgca 60aaccgtgcac ctatcttgca ccaacgctaa cactgtctca aaacagaacg
aaacaagatt 120tcacatgacc cacgtca 137184137DNASaccharum officinarum
184cctaggagct ccatcgggtg cctccaatac tatttctgag cctacggtac
atttgacgca 60caccgagcga aagtcatgca tcaagcgtag gactatctcc aaacgtatcg
aatcgagctt 120ccacttgagc cacgtca 137185151DNASaccharum officinarum
185cccagcagtt ccatcgggtg catccaaaat aattttggag cctcgatttc
tgagtgtatg 60gtacatttgg catgaaccat ccaactatct tgcaccgaca ctaacactgt
ctccaaatag 120actgaaacaa aattccacat ggcccacgtc a
151186131DNASaccharum officinarum 186cctcggagtt ccatcggatg
cgtccaaacc gatttccgag cctatggtag gtttggcaca 60acccgtgcac ctcgtcaaga
ttagcactat ctccaaacag accaaaacga gcttccactt 120gagccacgtc a
131187137DNASaccharum officinarum 187cccagcagtt ccatcgggtg
cgtccaaaat gatttctgag gctacagtat gttcgacgca 60aaccatgcac ctatcttaca
tcaagattag cactatctcc aaacagacga aaacgagcat 120ccacttgagc cacgtca
137188137DNASaccharum officinarum 188cccagcagtt ccatcgggtg
cgtccgaacc gatttcggag cctatggtac gttcgacgca 60gatcgtgcac ctatcttgcg
tcaagattag cactatctcc aaacggatga aattgagctt 120ccccttgagc cccgtca
137189137DNASaccharum officinarum 189cctaggagta ccatcgggtg
catccaaaat gatttcttag cctatggtat gtttggcgca 60aaccgtgcac ctatcttgca
ccgatactaa cactgtctcc aaatggactg gaacaagatt 120tcacatgacc tacatga
137190136DNASaccharum officinarum 190cgtaggtgtt ccatccgatg
cgcccaaaat gttttctaag cctatggtca ttcagtgcaa 60gccgtgcacc tatgttctat
caagtttagc actatctcca aacagaccga aacaagattt 120cacaggacct atgtca
136191137DNASaccharum officinarum 191cctaggagtt ctatcgggtg
agtccaaaat gttttctaag cctatggtat gttcggcgca 60aaccatacgc ctatcttgcg
tcaaaattag cactatctcc aaacggacta aaacaagctt 120ccacttgagc cacgtca
137192137DNASaccharum officinarum 192cccagcagtt ccatcgggtg
cgtccaaaat gatttctgag gctacagtat gttcgacgca 60aaccatgcac ctatcttaca
tcaagattag cactatctcc aaacagacga aaacgagcat 120ccacttgagc cacgtca
137193128DNASaccharum officinarum 193cccagcagtt ccatcgggtg
cgtccaaaac tatttatgac ctatggtacg ttcaatgcaa 60accgtgcacc tatcttctac
agcactatct ccaaacggac caaacagatc tccaccagac 120cctcgtca
128194135DNASaccharum officinarum 194cctaggagta ccgtgggtgc
ttctaaaatg atttatgaac ctatggtgcg tttgacgcaa 60accgtgcacc tatattgcac
cgacactaac actgtctcca aacggatcga aacatgattc 120cacttgaccc actcg
135195137DNASaccharum officinarum 195tctaggactt ccatcgcgtg
cgttcaagat gatttctgag cctatggtgc gtttggcaca 60aaccacgcag ctatcttgca
ttaaaattag cactatctcc aaatggactc aaacaagctt 120tcacttgagc cacgtca
137196131DNASaccharum officinarum 196cccagcagtt ccatcgggtg
catcccaaaa tgatttctga tctacgtttg gtgcaaaccg 60tgcatctatc ttgcactgaa
tctaaagtgt ctccagatag attgaaacga gataccaaat 120gaccaatgtc a
131197137DNASaccharum officinarum 197ctgagaagat ctatcgggtg
cgtccaaaat gttttctgag cctatggtac attcggcgca 60aatcatgcac ctatcttgca
tcgaggttag cattatctcc aagtggaatg aaacgagcat 120ccacttgagc cttgtca
137198137DNASaccharum officinarum 198cctagaagta ccatcgggtg
cgtctaaaat gatttctgag cctatggtac gtttggcgta 60aaccatgcac ctatcttgta
ccgacactaa cactgtctcc aaacggacca aaataagatt 120tgacatggcc tatgtca
137199137DNASaccharum officinarum 199ccaaggagtt ccatcgggtg
cgtccaaaat gatttctgag cctatggtgc attcggcgca 60aaccgtgcac ctatcttgca
tcaagattag cactatctcc aaacaaactg aatcgagctt 120ccacttgagc cacgtca
137200136DNASaccharum officinarum 200cccagcagtt ccatcggtgc
gtccaaaacg atttctgtgc ctatggtatg tttggcgcaa 60atagtgcacc catcttgcac
taatagtacc gctatctcca agtggattga aatgagattc 120cagatgacca atgtca
136201137DNASaccharum officinarum 201cctaggagat ccatcgggtg
tatccaaaat aatttctgag cctacggtat gtttggcgca 60aaccgtgcac atatcttgcg
tcatggttac cactatctct aaatagacca aaactagcat 120ccacttgagc cacgtca
137202137DNASaccharum officinarum 202ccyagsagtt ccatcgggtg
cgtccaaaay gatttyygag cctatggtac gttygrcgca 60aaccgtgcac ctatcttgca
tcaagabtag cactatctcc aaacggaccg aaacgagctt 120ccacttgagc cacgtca
1372033465DNASaccharum officinarum 203gggaagtacc agggacgaag
agctagcgca aggactaatg tttctgtagg gaaagctagt 60tcatggcagc agcgcacgtc
tacaactccg tctacatgta ctcctacgcc atcatctagt 120gacaagactc
gagctgcccc caccaattca gtmrcgaaga cagcgcaaaa gccggctgcg
180agtacttcat ccgtggcatc gacmggtaga acaagcaaca tacaatgtca
tcggtgcaag 240ggatatggcc atatgatgcg tgactgccca aacaagcgtg
ttatgattgt caaagatgat 300ggtgagtgtt catctgctag tgattttgat
gaagatacac ttgcattgct tgcgctgact 360atgcaggtag tgaggaacac
atagaagaac acatyaatgc aggtgacgcg gagcactatg 420agagcttgat
tgtacagcga gtgcttagtg tacaaatgga gaaggyagag caaaatcagc
480gacacacttt attccaaaca aagtgtgtca tcaaggagcg ttcgtgccgc
atgatcattg 540atggaggtag ctgcaacaac ttggcaagta gcgacatggt
acagaagctc gcctcaccac 600caaacacacc cccatcccta ctacatccaa
tggctgaaca acagtggtaa ggcaaaggta 660acagacttgt gcgaattgat
tttgccatcg gatcatacaa agatattgtt gaatgtgatg 720ttgtgcctat
gcaagcttgk aatattctgc taggtagacc atggcaattt gatagagatt
780cgatgcatca tggtagatca aatcaatatt cgtttctata ccatgatcga
aaaattgtgt 840tgcatcctat gtctcctgaa gctattatgc aatctgatgt
tactaaggct gcaaaagcaa 900agagcgagag caataaaaat gacaaatctg
ttgttgatga caaagatgag ataaaattga 960aaggacgttg tatgcttgct
acaaaatctg atattaatga gttcaatgca tccacctctg 1020ttgcttatgc
tttggtatgc aaggatgctt tgatttcatt tgaggatatg caacgttctt
1080tgccccctgt gtgctaacat tttgcaggag tattctgatg tgttccccat
tgagatacca 1140gcggggctgc caccaatacg tgggattgag caccaaattg
atcttatccc tggagcatct 1200ttgccaaacc gtgcgccata caggaccaat
ccggaagaag cgaaggaaat tcagcgacaa 1260gtgcaagaac tactagacaa
aggttatgtg cgtcagtctc ttagtccttg tgctgttcca 1320gtgattttag
tgcctaagaa agatggaaca tggcgcatgt gtgttgattg tagggctatt
1380aataatatca ccattcgata tcgaccccta ttccacgatt agatgatatg
cttgatgaat 1440tgagtggtgc tgttgtgttt tcaaaggttg atttgcgtag
tgggtaccac cagattcgta 1500tgaaattggg agatgaatgg aaaactgctt
tcaaaactaa gttcggtcta tatgagtggt 1560tagtcatgcc ttttgggtta
actaatgcac ctagtacttt catgagatta atgaacgagg 1620ttttgcgtgc
tttcattggg aaatttgttg tcgtctactt tgatgatata ctgatttata
1680gcaaatccat ggatgaacac cttgatcact tacgtgctgt tttcaatgct
ctacgtgatg 1740cacgtttgtt tggtaacctt gagaagtgca ctttctgcac
caatcgagta tcgtttcttg 1800gctatgttgt cactccacag ggaattgagg
ttgataaagc caaggtggaa gctatacaag 1860gatggcctgt acccatgacg
gtcacacaag tgcggagttt cctaggactt gctggtttct 1920atcgccgttt
ygtgaaggac ttcagcacca ttgctgcacc attgaatgag cttacaaaga
1980agggagtgcc cttttcttgg ggcaaagcac aagagcacgc cttccatgtg
ctgaaagata 2040agttaacaca tgcacctcta ctccaactcc ctgattttaa
taagactttc gagcttgaat 2100gtgatgcgag tggaattgga ttgggtggtg
ttttgttgca agaaggcaaa cctgttgcat 2160actttagtga gaaattgagc
gggcctattc ttaattattc tacatatgat aaggaattat 2220atgctctagt
gcggacatta gaaacatggc agcattattt gtggcccaaa gaatttgtta
2280tacattctga tcatgaatct ttgaagcata ttcgtagtca aggaaaactg
aatcgtagac 2340atgcaaatgg gttgaattta ttgaatcttt tccttatgtt
atcaaacaca agaaagggaa 2400agataatatc attgcagatg cattgtctag
gagatatact ttgctgaatc aacttgatta
2460cagattttgg gttaaaacaa ttaaagacga tatgctcatg atgctgattt
taaagaagtg 2520ctgctgcatt gtaaagatgg gaaaacgtgg aacaaattca
tcgtcaatga tgggtttgtg 2580tttagagcta acaagctatg cattccagct
agctccgttc gcttgttgtt gctgcaggaa 2640gcgcatggag gtggcttgat
ggggcatttt ggagcgaaga agacggagga cattcttgct 2700ggtcatttct
tttggccaaa gatgcgacga gacgtggaga gatttgttgc tcgctgcaca
2760acatgtcaaa aggctaatcc cggttgaatc cacacggttt gtatatgcct
cttcctgttc 2820ctagtgctcc ttgggaggat atttctatgg attttgtgtt
gggattgcca cgaactagga 2880aagggcgaga tagtgttttt gtggttgtgg
atcgattctc taaaatggca catttcatac 2940catgtcataa aactgatgat
gctacaaata ttgctgattt gttctttcga gaagttgttc 3000gcttgcatgg
tgtgccaaat acaattgttt ctgatcgtga tgctaaattt cttagccatt
3060tttggagaac tttatgggct aaattgggga ctaagctttt mttttccacc
acttgtcacc 3120cccaaactga tggtcaaact gagttgtgaa agaacttgtc
tacatgtagg ctttttaaag 3180aagaatatta agattgggaa gattgtttgc
ctcatttgag tttcttaaat cgtcktgcat 3240tccacgaaga tgtgtccytt
tgagtgtyta tgggttttcc acggctccat tgatttaatg 3300cmcwttgcam
stttgraaag ctasaatttt gatgctaagc aactgctaat tgatggywaa
3360catmcmtgaa aaccacyaaa gaaaacatas agcgcatgaa tgctaartac
aaatttgykg 3420gagataargg tagaargtaa ttgcttctgg acccggggaa ttggt
3465204548DNASaccharum officinarum 204cccagcagtt ccatcgggtg
cgtgcaaaac gatttctgag catatggtac gtttggcgca 60aaccgtgcaa caaacttgca
ctaacactac cactgtctca aaatggattg aaatgagatt 120ccacatgacc
aatgtcacct aggagatcca tcgggtgcat ccaaaatgat ttttgaacct
180ttggtacgtt cgacgcaaac cgtgcaccta tcttatgtca agattagcac
tatatccaaa 240tagactgaaa cgagcatcca cttgagcctc atcacctagg
agtaccatcg tgtgcgtcca 300aaactatttc tgagcctatg gtacgtttgg
tgcaaaccat gcacctatct tagaccaata 360ctaacactat ctccaaatgg
accaaaatta tattccacct aactcatgtc acctaggtgt 420tctatcgagt
gtgcccaaaa ctattttcat gcttatggta cattcggcgc aaaccgtgat
480cctatcttgc gtcaggatta gtgtaatccc caaacagact gaaacaagca
tccacttgag 540ccacgtca 54820525DNASaccharum officinarum
205gtcacccagc agttccatcg ggtgc 2520624DNASaccharum officinarum
206actgctgggt gacgtggctc aagt 2420722DNASaccharum officinarum
207gggaagtaca gggacgaaga gc 2220820DNASaccharum officinarum
208actaacaatg cacgggaagg 2020920DNASaccharum officinarum
209gtaggccatg gcagtttgat 2021020DNASaccharum officinarum
210aacacaccac ccaatccaat 2021123DNASaccharum officinarum
211ccaaacaagc gtgttatgat tgt 2321224DNASaccharum officinarum
212aggttatgtg cgtcagtctc ttag 2421323DNASaccharum officinarum
213ggcaaacctg ttgcatactt tag 2321423DNASaccharum officinarum
214accatgtcat aaaactgatg atg 2321522DNASaccharum officinarum
215tgcaaccaaa ccaaatcacc ag 2221620DNASaccharum officinarum
216caagcgaaca atctcacgaa 2021720DNASaccharum officinarum
217aaatcatcat cgtgcgcata 2021820DNASaccharum officinarum
218aacacaccac ccaatccaat 2021919DNASaccharum officinarum
219gaacgctcct tgatgacac 1922022DNASaccharum officinarum
220gtacccacta cgcaaatcaa cc 2222123DNASaccharum officinarum
221caacttcagt ttgaccatca gtt 232222139DNADrosophila melanogaster
222gttgtccgca gcggagatgc aactgatgca acccacattt cagatcaccg
acaacgtgca 60gcgcggcaac tacgccactc tgaccgacaa ggatgtggcg catttcgagc
agctcctggg 120caagaacttc gtgctcactg aggacctgga gggatacaac
atctgcttcc ttaagaggat 180tcgaggtagg ttgtgtaacc aaattcattc
acattcgtgt gccctttaat gaatttctcc 240gatgaattgc ttcaaccagg
caacagcaag ttggtgctta agcccggaag cacggcggag 300gtggccgcca
tcctgaagta ctgcaacgag cgtcgtttgg cggtggtgcc gcagggcggg
360aacacaggtc tagtgggcgg atccgtgccg atctgcgacg agattgtcct
ttctctagcg 420cgcctgaaca aggtgttatc cgtggacgag gtcaccggca
ttgctgtcgt ggaggcgggc 480tgcatcctgg agaacttcga tcagagggcc
agagaggtgg gcttgacggt gccactggac 540ctgggcgcca aggccagttg
ccacatcggg ggcaatgtgt ccacaaacgc gggcggagtg 600cgggtggtgc
gttacggcaa tctgcacggc tctgttttgg gcgtggaggc ggtgctggcc
660accggtcagg tgctggacct tatgtccaac ttcaagaagg acaacaccgg
ctaccacatg 720aagcacttgt tcataggatc cgagggcact ctgggcgtgg
tcacgaagct ttcgatgctc 780tgcccccatt cctcgcgagc ggtgaacgtg
gccttcatcg gcctgaactc cttcgacgat 840gtgctgaaga cttttgtcag
tgccaagcgt aatctgggcg agattctaag ctcctgcgag 900ctgattgacg
agcgggcctt gaacaccgcc ctcgagcagt tcaagttcct gaagtgagtt
960gcgccacctt tgtcttctct gagcgttacc aatcctgttc acaaacttat
ttcccatagc 1020tcccccattt cgggatttcc cttctacatg ctcatcgaga
cctcgggcag caacggtgac 1080cacgacgagg agaagatcaa ccagttcatt
ggggacggta tggagcgtgg cgagatccag 1140gatggcaccg taaccggtga
tcccggcaag gtgcaggaga tctggaagat ccgcgaaatg 1200gtgccgctgg
gtctgatcga gaagagcttc tgcttcaagt acgacatctc gctgcctctg
1260cgggacttct acaacattgt ggacgtgatg cgagagaggt gcggtcccct
ggccacagtt 1320gtctgcggat acggccatct gggggactct aatctgcacc
tgaacgtctc ctgcgaggag 1380tttaacggcg agatctacaa gcgggtcgaa
cccttcgtct acgagtacac ctccaagctg 1440aagggcagca ttagtgcgga
gcacggcatt ggcttcctga agaaggacta cctgcactac 1500tccaaggacc
cggtggccat tggctacatg cgcgagatga agaagctgct ggaccccaac
1560agcatcctca atccctataa ggtgcttaac tgaaggcttc tacctaatag
attctatttt 1620ttttgtttgt gtgtaatttt cataacctta taatacagaa
atggcattag aagtgaattt 1680tgttaacttg tgaagttaaa aaggaccatc
atatttggca cgaaaccaat gggcaaaact 1740tacttataaa atagtccgaa
aaaatagtat ataccagttt ttacagtacc acattatagg 1800tactcggagg
taataataga aaaaacacta tctttgcatt tactgttaca ctacgaagca
1860ctatatttag tagcagtact cattagagtc cactcacaaa attagcacca
accggcagta 1920attggtcaag gatcggcgat agcttcaaac tccgaagttc
aaagtcaaac tgccgccctg 1980cgaaagcttc gcgagtggag cttttctgca
cttatcgata gctaacattg tggcgcgact 2040atcgatcgac gagctgccgc
ttaacagtgc catatataga ttgtaacatt agaagctcaa 2100atcattgttg
gagcacaaac cacaaagaac acacgaaac 21392232191DNADrosophila
melanogaster 223aaaatatttc acctcatttt ccgcacacca tttataagca
aagttacccc caacccataa 60cttttatggt aagtaataca gaccctccaa gttcggcaaa
tcgataccca gcgaccttga 120gcttgacatt tatatatatg ccagaatata
acgaccacgt gctgtcaact gtgtcaggaa 180aagctcaccc acactttctt
tggaggagct gtgctcccta aacgaatttc attgtcaagg 240tcgcacgcac
aaaaatgaag aggaaaagct gaatgtgggt ggaaatgccg gccggcacga
300ccttgaagcc agttgggtga gaaataaaaa gcttttgccg gtaggagact
tgtggaacat 360cacccacaag tggcggactt ggccttggcg atggccttgt
tggagctccc tcagcaaaaa 420tgttacatag ggggaggaaa taagctcaat
tggctttatg ctttccgctc cctggaagtc 480cttttctgga atgttaaagt
gttaaatgac atttattgaa catttgggac agaggaggag 540ataatacaat
atacttgtct aattaaaaaa aatcgttatt atgatttatt ccatatgtaa
600gattttaatt catcatgatt gtaaataaat tatataaaac aaattcaata
aatttacatt 660attgataaaa tttatttttt catgaaatta tacccaaaaa
ttattctcaa tttttcttat 720aatcagtttt gcataagtat actttcttca
tacccctcta ccacagccac tgctttcttg 780actttgcaac tatccgggaa
cagcttatca taatggatga gctgcagcta acggaaaatg 840ggggagctgg
gatcaaacat tttccaaggt tgaaattgtc gtcagcataa tgtttgaggg
900agctggattc gcgttagctt gaaggtcaat ccatttgggt gccctttgtt
atggtcaagt 960ttaaggctgc aataggggga atcttcaagg accattacgc
aaggttttcg catcaaagat 1020ttgccgtgca agctttttga gttgaaggat
gcttaacttg aaagcgggtt agtggttcca 1080agagatttta ggtgaaggag
actccgctgt tttgaaatat attaagtatg taaagaagta 1140tactataaat
aacccaaagt gatacaatgt aagaaaagat ctcgttggtc cctggtataa
1200atttgtttgc cattaatgaa tattgaaaat aataattata ctaataatag
gtacaataag 1260caagattaaa ttgcatttaa tcaccaaaaa tcagtttcta
tgcgaaccaa aatgtcataa 1320caaacaattg ttgattcatc cgtagtgaaa
tccaagttcg aaattcgaaa tgagcatacg 1380acgaccaaac ttcccctcaa
aattgctaga ctcagctaga gcaagtacgc ccaagttaac 1440ccctgaaatt
cgaaatgaat tcgatgccgc gcttcgaaca acgaaatccc aaagagctta
1500cgttttattt gacgtagcac tcttacgtga aatgattttc cccaattccg
ctctcatttc 1560ccgagtctct caccgcttct cagccacttt cccaccccct
ttctagttcc gaagtaaagg 1620taacaaaggc agccgtgtct ttggggtggt
aaactggcgg tggtggtggc acattgtcag 1680tggtgtgggt tcctgtggtt
ggtggttcaa ttggttggtt gttggcataa acaaagcaca 1740cacacaatac
acacaaactc ccggggggtg gtggaaattg ggagggtgac attcactgcg
1800agagaggaac tcgcttccta taggaaagta caaagagagc tattttataa
atgtgactgc 1860agcaaggata tttacagtca gtccactctg aaacctcgac
gagagaacat tgaataacaa 1920gcggaagcga aaagcgcagt tgaaagttcg
tcaaaaagcg acaagtttcc tcgttcgttt 1980tcccgccaaa tgagtcagaa
aaattttcca agtgctcgat acgaaacata aagacttaca 2040agacttaaag
tgcaagcagt gaatggaata tattattcct cagcgatatt gaaatcaaac
2100attaaaaata tatgctacac taaagttata tattttttta aagattcata
cgttttgtaa 2160aatcacattt tgtattaaat taaataccgc c
21912242035DNADrosophila melanogaster 224tgggtgcgtc gcaggtttca
ctggaaaaca atttgcactt ttgtttgtgg agtcgacaac 60aaaagcattc acttgtctaa
gactctctca ttcataactc gcactttagt tcactgaacc 120gcacgcaaaa
ctttggggcg gacaacatgt tttcgaggtg ccaaaagctt cataaaacta
180ccaatccatt agattaaatt ccaggcggta catcttttgg ggatgattca
tgtggcaggg 240gttctctact cgtttacaat catatcatca tcttcaagat
catatagttt atcatatcag 300tagagtacta caatataatg cataaactaa
gccaaataac tttatgacgc gtgcttatgc 360gaaagtaaac tttattatca
aatttactta accgtgaaat caaaaccttt atataaacac 420gaatattatt
atctttgcta aataaaactc tcgcttaaca aacaatgaca cttcaattcc
480aacatagagt ttatcttaag ccaataacca aaaacggaac ttacataact
tgccaacaaa 540catatgaata tagctatttc ggatcgtggg agaccattat
gcatacaagg cacgctccta 600aaaaccgtgt taaacaaata tatgtcaaat
gtatatctta aaaaagcgcg cacatatctt 660ttgaaatatc ttcacccaga
gtatgtatga gattaaactg gattagcact aagccacagc 720ttctgtagat
agaaatttta tgcagagagt agattatttg gctgctgagc aatttgacca
780ccacaagata gcagagaaca tctgacattt tctatatcca tataataaaa
ctgacttaac 840actaagctga agtggtatgt ttaaatcctc cagctaataa
atcgagacta aacgccctat 900cttatagtga tatataatag tatctatatg
tgtattgtca tttactgttt atgagtattt 960gaaaaaacca ttctatattt
tataggttag ttaataaata ttttgatata catatgtaga 1020ttggctcaca
cgtacttatg acccactaca taataaaatt gttttgtttt ttaatagaat
1080aatggtttat aaaaagttta gactcacacg gaaatgataa actctttgca
aatacagctt 1140tcattttatt acaaattgca ctctttcaga tctgcagttg
ctatgccaac cttttattcc 1200ctttactaaa agggtatact aggcttactg
aacagtatgt aactggtaaa gtaaagcgtt 1260tccgattcta taaattatat
atctaaactt ttgatcagtc gaatccatct gaacacattc 1320tgtcacatta
gattattcca gaaactcaac ttaaacatgt gtatttttta agaccattat
1380caaggatatt aaaaatggtc tcctaaaatt taataaacaa aagtgtcaca
tcaaatttaa 1440gacgtaaatt aatatttttt ttctatggtg aaataattgt
tattttccaa tgttgtgaaa 1500taataaatgt atcttttcaa cgcacacatt
ttcaaggttt taataataat agtgactcgt 1560gcgtgaataa gagagaaatt
aagattttaa aaaagaataa aattcagaga tgtgatctgt 1620aaaaattatt
taccaatttt catttacccc cgaaagtgat gctaatggtt aaaacggcat
1680ttgcgactta tctcctacgt aatattgcaa aaataaggat ttggttagat
gagtgtgaag 1740taaacaagat gcaaagtttt ggagatagaa aacatagcct
tgagtcttgg tcatgtttac 1800ttggcaccag gccgcgatta tcagcgctac
tagtcgtaat ttgagttaga cctttaatac 1860tctaagtgag agtgatgata
tacgatttcc cagccacttg ctttctacga aatgcgctaa 1920aaaaaatccc
taactacaca aagatttgtg ttgttatcca ggtgttctga tataaaaggc
1980ggcaaggaaa ttgatggcat catcagtatc aaagtgagag tgattgcagt cacac
20352252136DNADrosophila melanogaster 225atgggacggt cctattctca
gcaaaaattg acaagaacaa caacaatgtc tatggaaaat 60cgaacttcat cccagcacct
gcagaaatcc cgagcgagtc ggggaaaaag tatttaaccc 120ccgaaagggt
tttccccaaa ataatgaagt aatgaatgaa gcggaaaaca ctggccgcca
180atctacctaa tactaatgag cgggccaacc cgaccaggaa tttttgcaag
tcaggtactt 240caacggatat atgggttcga caagtgcgga ttttcccgcg
acatcaatga ggacttggcc 300gggttatccg cggtgctcat cgggcaattc
cgcggccgag gacttcatcg tagtgatcat 360taggtagata tgtgcatgga
tgtgacatgg cgatcattgc gcggaataac acacgtaata 420accgagatat
ccgggatgac ccaccaggta ggatgtgagg acatatagaa aacccccagc
480cagtttttcc actcgtcgtg gcttgttttg cttgagtttc gctgactgcg
taattggata 540agatgggaaa ttactttaaa tccttcgctg atccacatcc
ggacattcgt cgaaggaaaa 600tccattgcag ggaaatacga aatggaaatg
cggctgggtt attggctcga catttcccat 660cttccctcac gccattggtt
gcaggatcgc ggggaattgg aattccgcgc tggaattttt 720tgtcacctct
tgggtttatc aaaacttttg ggtttgctat ggattttttc caattttacc
780accgcgcctg gttttttttt tttgacgacg cggaaaatcg gacttggcta
tgcgggcttg 840tctgtttttc cgggtacaaa gtctgcatgt cagcctccat
gcgggagtgg gagttgggaa 900agtttcccat cgatagttgg aggggtggct
tgaaagtctg gaggtgctag ctgggaaagt 960tgtgtgtgcg cgatgaggca
aggagtcaaa gatcagggga gttggaaagc gagaattgtg 1020ggaatcgtcc
aggactcagc tggatgctga ggggcagtat gatttttttt acgttatcaa
1080tcgaattgat tttaagacag cagaacttca catactaata agatgaccat
gggattagtt 1140aaaatgtgta actcgtattc gaatcgtcat tctttcacgg
accaatcgtg ggaacaggag 1200atctcttcga tccaagctca caggagactt
gacactcttc gtctattcct tgtcaagttt 1260ttaatgacat ctcctatgcc
ctgagctatg ttttcctagc tctcatcgat cgctgccaat 1320gagccactgg
agatgatcca taagtcagcg tagagtgcac cccagagttg acacttggtg
1380tctcggaatt cggctcatta tcagtgctat ttttggaaca cctctctgcg
aaggtgtcat 1440ttttgtcagt gcgtatcgct caggttcaac tccccaccaa
aaaccgaatt tagagcatcg 1500gcagatgtac ttgaagcact caatctaagt
gaggaaacca ccccatgaac gaagagtact 1560aggagtccta tttgactcgt
gcttaaaaat agaaaattac ttagggtgat ccataggtag 1620ggaggcgata
ttgtaacttg catttcggac ccggacctgc acgagttatt acgggtgggt
1680tgtgagcgta tcgggaaatt ggagagccac cagatctgtc ataacttata
cgggggatcc 1740ttattcctgg gagggtgcgc ctgcgtctgc tcttccgaga
gagaggtggg aaatggagga 1800agagagagag agagagagtg agagagcagg
tagagggaag tgagggaaat acgcaataag 1860ggtatgggaa aagtgctgtt
gttgttgcta ggtagcgacg cacacgtgcg agtgtttttc 1920tgttttgaag
aagaaccacc accaaatggc gacagcggcg tcggcagagg cgcagagttc
1980cgggtataaa agagcgtgct cgactgttga cctgtcacag ccacctcagc
tctcgttgag 2040aacgcaacca ccgctctata ctcgatcccg aactatataa
ctcgcctctc gatcgccgat 2100ctcccgattt acccatctcg atcagtaccg gaaacc
21362262015DNADrosophila melanogaster 226atttggctcc ccatcgccat
cggttgctcc aatgacacta gggaattgtg ggccgccgac 60agctgtcctt aattacatgg
aaatccacac tagattcgtg cccctcgccc cgtactcgca 120gccgaagtcc
ccacagagtc attcaccttg ccaccaccaa aaaaaaaacg aaagcaactg
180aaggaaaagt tcgattcgaa ggctgaggga tacccttaaa ggcccatttc
ccggcttcgt 240aaatcacatt tagttagcca tttagactac agcaagtctt
ttaagataca ctgcaaaata 300aataccatta cattaataga agtgtcatgt
catcggtctg tatttttgtt accacagaat 360agacttacat atatgataaa
aaaatgttca acaataagtt acatcggtag ccaattctat 420agatttaatt
ccttacgaat atagtttcgt tggaatactc aatttgtaat tgtaattaat
480tataattatt ataattttaa gaatttatat aagtaactaa aagacacggc
agacacagaa 540tgaaaacact ctatgttagg gaatgcaaaa aaacgtggcg
gaagccaaaa ggcgcaagca 600aaaatcgaaa ccaagtgaat ataacatatt
atttcaacag gcaactcatt cagcatataa 660tattaccacc catggagctt
tatgtagttg atgtacgtag tctatgatgt ggagcccacg 720ttggcggaac
tgggaatggg gattggggtt tgagagctgt ggtaaattgg ggggttgaag
780tatcaagggt ttgggttctg tagacctgcg gaatcgaggt gaataagcga
agaacacatt 840cacacacact aaaaggcaaa caaagggaaa tcaatctttg
tacatacttt tagcatatgc 900acacgtatga tctccaccca cttttccctc
ccaatgaaac aaacacacac acacatgcaa 960ggccgtacgt ttgtatatgt
gtgcggttgt cggctttgcc gggaattggg gaatatttgc 1020atgcctttgt
gtactttttc catatgattt atgacctaaa ttgttgctgc tcgcgcacat
1080ataattacac acacatcgct gtggccatgt gtgtgtgtgt cgtcttggga
cgcgcgccaa 1140agtatgctac actttttgtt ttatgagtta ataagtaggc
gtggccccag cccaattgct 1200acactctgat tatggcaccg gatacccaga
tagacgccca tccaccccac tgtaagatgg 1260gggaatttcc aaacctatat
gtatgtgcag atcagatagg atagcacaga actttttaaa 1320gtacactttt
ggggcacgca atttagaaaa tgtacctcgg tgtcggagaa attattttaa
1380aagtcgactg aaccacctcg ttccatatgg agaagtctac gagttcaagt
ttaatggagc 1440agctgactgc actgaatttt gtagtttaat acacaaatcc
gcaaattgca tctcacttca 1500aatagcctgg tacatagtat ctactaacat
aactcatatt aaaataaagc aaccaaccag 1560agggccgaag ttctattaat
aaaactaata tttaactatt atatatacat tttatttact 1620tggtacgctt
atgataacct tcgaaagaga accaacacaa tacgctttgt catttgaaaa
1680ataaatatgc tgtaactact ttacaaggtg aaactcttgt cagaagataa
gaggctaggt 1740aagttgatta ttcaatcagt ttacttactg caacccaaaa
tggtcactgc actaaccttc 1800agatgagctg cactacaccc tcaatcgaga
atcaatgcaa acgcagtgcc agcgaaaatg 1860tcagcaaggg attaggccaa
tcccaaacgg gtaatcccgc tgcgacaatg ctaatccaat 1920tccgatgggc
cgtataaaag ccccaagctg ggctggctgt gatttcgtct tggcccgcag
1980accggagcat ggagtccggt aacgtgtcgt cgagc 20152272082DNADrosophila
melanogaster 227atcgatgacg gcatcggctt gacctctcgg agtacgtttg
attttataga acaagttttc 60tcctttctta tactataagg aaaaattata aaaattgctg
aaaatgaaac atggctagaa 120ttcgtttttt aacatttttt caatctgaga
aaaaatttcc gattagtctt aaaataacta 180aaccaattcg tatacccgtt
aatcgtagaa gaaaaatgaa attcatataa taagtagatg 240gatttgctga
cccggtgagg tatatatgta ttcctgaaca tgatcagtaa acgagtcgat
300ctggccttat ccgtatgaac gtcgagatct cgggaaatac aaaagctaga
aggttgagat 360taagtatgca gattctagaa gaagacgcag cgcaagtttg
cgactacgct gaatctactg 420ctaaaaactg ccacgcccac acttcttaag
aatttgattt attttcacaa gctgaggaac 480ggtagggtcg aggaactcga
ctacaacgtt ctgccttgtt tatttcttaa caaaaactta 540gtagccgttt
gggttggaaa ccacctgacc ttaggtctgg tagcagttat ttaatttatt
600ttttttattt tatacaactt gctcgctgtt tgttccccct agccctgaaa
cacaagctgt 660caaacggtgg aggtgataag tctaatgaat gcgataagct
ttatttcaat tcgcaatttt 720cgtgtggcat tttggcaaaa aaaaaaactc
gtcggacata catgttgcca caaacataaa 780gtgaatacat aatgttgggt
gaacgactca tacacgattg tggcaaatca aattctttta 840acacgggacg
gggaaaggcg agtgaagata ttttagcata tatttagcac atctgttaaa
900tccatttttt tactctccgt tttcggccag atatggttag aaaagaaaaa
aattagtaca 960tacccccata tataataaga aaaaaagaga gagtcagcag
aagtacgggg agcttaagtg 1020tagcaatcag aacatcacaa atagtaaata
aattaataat aataataatc atatccaaaa
1080atatttttat tcctaaccta tcgcattgtt acatcgaggg tgaaattcaa
aatagacaaa 1140aagttgggga ataaaatgtg aaaaaagtgg taaaatgttt
aatagtgtgg gcgttactgt 1200tttgtcggtg tgaggtgcgt ggccaccaaa
gtgtttttgg tataacgata gaaattggta 1260agacaaacaa tattgcgaag
aaaacccgaa gcatttttaa aaagtgcgaa cgtggcagtt 1320ttaagggttt
gtgggcgtgg caataatttt tggcaattcg ataaaaatgt acaggaccaa
1380atatatgaag aaatataaaa tatttttcaa aatgacagcc agcaaccata
catatatata 1440aataaatgtc ggagaccctt ccttctacct gtaacatact
tttccacgaa tctagtattg 1500gttgatatat aattatgctg tgtataagac
caaaatcagt gtacatttcc attggattca 1560ccaaccggat ggttccggat
ggtaatgcaa aatattcatc taagaaacga aaacacctag 1620aattaaacct
gaactgatat gacttatgca catatcagtg aggtgggcag ttcaaagcaa
1680tcacgatgct ccaagttatt atcgcagtgc agtgaaaaat tcacagtcac
cgtcgccaat 1740tgccaataaa gatcggccat tatacaacag aaccgcgttg
aagacgatcg acgaggtcgt 1800gggtcttatc ttatcaccac ctgaattgag
gcatgcctcc agaatgacga gggcatccga 1860agataatgtg gcccgctatt
ttcggccggg actggaccta tgcgacgacc tatgctgatg 1920acgggagtct
gccgctgata tggtgcaatg caaggctcca gtcgggggta taaaagaccc
1980agtttcggtg cagtcaagac aacagacttt aggtgttggt cgttgagcga
accaaagccg 2040gagcagttga ggaaccaaag aatagcagcg agaggaccaa gg
20822281999DNASaccharomyces cerevisiae 228tgtagggacc caaatccaat
tgtagtagtt accttgatta tggttggctt gtccttcgat 60agttttgcct tttccaaagc
gctagaaatg gattccatat cgtcgtctcc tttatcgact 120tccatgactt
cccaaccata tgcctcgtat cgcttcaaaa catcttcgtc gaacgagtac
180gaggttttac cgtcaatgga aatgctatta ctgtcataaa acgtaatcaa
gttacccaat 240tgcagatgtc ccgctaagga agaggtctcc gaagaaacac
cctcttgtaa gcaaccatcc 300cctacaatag caaacgtata tgagtcggaa
atgggaaagc catcctcgtt ataagtggcg 360gcaaagttgg cctgcgctat
tgccatacca acagcatttg agataccctg gcctagcgga 420ccggaagtga
tttccactcc cgctgagtgg aattctggat gacccggtgt ccttgagttt
480acttgtctaa attgtctcaa gtcctcgata gagtaatcgt atcctaatag
atggagcatt 540gagtacagaa gagcgcatga gtgaccgttc gacagaacaa
acctgtctct attgatccaa 600tgttcattgt tagggttaca gcgcagttgc
ttgaaaatta catgggcaac tggtgccaat 660cctagtggtg cacctgggtg
gccagattgt gcgctttcca cctggtcaac ggaaagtaat 720cttaaagtgg
aaaccgcaag tttatcaatg tcggagaact gtgccatttt tttgttcttt
780ttttgattag taaggtataa tcgtctacgt agaggttaca aatcgaagac
tacagtaaga 840ggggacaagc caattgaata tacgactgaa ataaatggaa
taattctgca ttattacact 900cgtttatata tccaaacagg tgatctggta
ttctcttgac aacgaatgaa gctccctata 960ttcgacactc cttattcagg
actcctccca acaaggagaa gtaggtgttc cttgagctac 1020cctttaaagc
tggggagatg agcttgccct tcctgtcatc gccattatga cgagaaaagt
1080aaaacatgta gaataaggtc cacccaaaca tgtccgagca atgacgttat
atatcgtgtt 1140ccctgttcaa agcatggcat atgtgccatt aaaggcgaat
ttttgtccct agcaaaggag 1200agacagcgag ccaccattaa gaagtgactt
gaaagcaagc gaaaatagct acacatatat 1260atcaatatat tgacctataa
acccaaaatg tgaaagaaat ttgataggtc aagatcaatg 1320taaacaatta
ctttgttatg tagagttttt ttagctacct atattccacc ataacatcaa
1380tcatgcggtt gctggtgtat ttaccaataa tgtttaatgt atatatatat
atatatatat 1440ggggccgtat acttacatat agtagatgtc aagcgtaggc
gcttcccctg ccggctgtga 1500gggcgccata accaaggtat ctatagaccg
ccaatcagca aactacctcc gtacattcat 1560gttgcaccca cacatttata
cacccagacc gcgacaaatt acccataagg ttgtttgtga 1620cggcgtcgta
caagagaacg tgggaacttt ttaggctcac caaaaaagaa agaaaaaata
1680cgagttgctg acagaagcct caagaaaaaa aaaattcttc ttcgactatg
ctggaggcag 1740agatgatcga gccggtagtt aactatatat agctaaattg
gttccatcac cttcttttct 1800ggtgtcgctc cttctagtgc tatttctggc
ttttcctatt tttttttttc catttttctt 1860tctctctttc taatatataa
attctcttgc attttctatt tttctctcta tctattctac 1920ttgtttattc
ccttcaaggt ttttttttaa ggagtacttg tttttagaat atacggtcaa
1980cgaactataa ttaactaaa 19992292001DNASaccharomyces cerevisiae
229tctgctatta ttgatgcttt gaagacctcc agacaaattt ttcacagaat
gtactcttac 60gttgtttacc gtattgcttt gtctctacat ttggaaatct tcttgggtct
atggattgct 120attttggata actctttgga cattgatttg attgttttca
tcgctatttt cgctgatgtt 180gctactttgg ctattgctta cgataatgct
ccttactctc caaagcccgt taaatggaac 240ctaccaagat tatggggtat
gtctattatt ttgggcatag ttttagctat aggttcttgg 300attaccttga
ctactatgtt cttaccaaag ggtggtatta tccaaaactt cggtgctatg
360aacggtatta tgttcttgca aatttccttg actgaaaact ggttgatttt
cattaccaga 420gctgctggtc cattctggtc ttctatccca tcctggcaat
tggctggtgc cgtcttcgct 480gtcgacatca tcgctaccat gtttacctta
ttcggttggt ggtctgaaaa ctggactgat 540attgttactg tcgtccgtgt
ctggatctgg tctatcggta tcttctgtgt tttgggtggt 600ttctactacg
aaatgtccac ttctgaagcc tttgacagat tgatgaacgg taagccaatg
660aaggaaaaga agtctaccag aagtgtcgaa gacttcatgg ctgctatgca
aagagtctct 720actcaacacg aaaaggaaac ctaatcctgt tgaagtagca
tttaatcata atttttgtca 780cattttaatc aacttgattt ttctggttta
atttttctaa ttttaatttt aattttttta 840tcaatgggaa ctgatacact
aaaaagaatt aggagccaac aagaataagc cgcttatttc 900ctactagagt
ttgcttaaaa tttcatctcg aattgtcatt ctaatatttt atccacacac
960acaccttaaa atttttagat taaatggcat caactcttag cttcacacac
acacacacac 1020cgaagctggt tgttttattt gatttgatat aattggtttc
tctggatggt actttttctt 1080tcttggttat ttcctatttt aaaatatgaa
acgcacacaa gtcataatta ttctaataga 1140gcacaattca caacacgcac
atttcaactt taatattttt ttagaaacac tttatttagt 1200ctaattctta
atttttaata tatataatgc acacacacta atttattcat taatttttta
1260ttgagtagga tttgaaaata tttggtatct ttgcaagatg tttgtataga
gggacaaaga 1320atcgtcttta ttatggtcaa ggctttacgt cataatagtt
cctgcccagc tcttctataa 1380tactttaaag atctcttctc gtttgctcca
tttggaagtc tcgcttacgt ttatgcgccc 1440atacagacac tcaagataca
cacttacatg aacgtataca aatttactaa cactacttga 1500aaatatgaac
cacagtacat catattaaga cgtagtattc gatgattgaa ggccgcctcc
1560gcgaaatacc tttactgatt ttgccggtta atcgcatcga aatttcttca
tcacaagaaa 1620gcaaacaaat cgccaggcca ttctacaagt ttccttttct
tatgaagatg taaaagctac 1680taaggcgtca ttactctaga tgactcagtt
tagtctgacc ttctatagta tactaccctg 1740gcgctatgat gatgagcggt
tcttttattg cggaaacgaa aattccggga ccggcgaaat 1800ttgcccggtt
ttgtccgtaa ccggcttcat gagtcggctt caatagtagt tgaatactta
1860tttaaacagc agaacttaac tcactcatca cgctgtttcc gctgaatttt
ctcaaaatat 1920ctaagcagtc aacaaatata aagaatattg aaattgacag
tttttgtcgc tatcgatttt 1980tattatttgc tgttttaaat c
20012302000DNASaccharomyces cerevisiae 230ccaaatcatt cttattcggt
ttccagacgg taacaatacc ctcgcccatc ccacacaaaa 60gggtatctgc tacttcggga
tcgacgaaac aaccacaaag aacttcgtcc tcctgatcat 120cgctgatcaa
aattttacca tcctcgtttc cagctacgtt cggtttggca tctttgtcgc
180gaacgtcaaa ataagctaac gttgtctggc ccaaagaaat gaatttatat
gcagatcttt 240tatcaaagtg gaaaatatcg ttgatagagt cgccaaaatg
tatcgaacga atggaatttg 300ataatgccaa gttttccgag tttattacgt
gtatattacc ggattcatcg cctattaaaa 360tgaatgggtg agtttgagag
gcgcataatt tcgtaaattt atcatttttt ttctcttcag 420aattaaacag
tgagcttaag tttacctttt tgacgacttt gccggtcata gtattggcct
480tttttaaaac attatccgat ccaacagaaa aaatattgtc acctttagaa
tcaaagcaca 540tggcacggac actaccttta tgtcttttag tcttccaaag
tgtttttacg cccaagtctt 600catcttttcc tgtttgcttt tgttgctgtt
gttcttcaat atcaacaaat ttcaaatctc 660cagtttctag gtctatatct
aatctaatcc aagggcatac acctttcttt gcatccttgc 720ctgtagttgc
agtgtcaatt ctacgtctac gatctaggtg cgattgcaac ttagcggggt
780cataacgatg gcacacaata tgtcctgtac caaagccagt tattataatg
ggcagttcag 840gatgtaaaag agactggaaa atgggagctt ttaatgatag
taattctaga atgggcaggt 900ttgttgaatc gacaacatct gttttttttt
tgctctttgc catagctgat gcgtggattg 960tttctaattt cccagctgct
tcctcttcca attgtggcga tgatgccatg atttctatgt 1020taaaattttt
ctaaccatga aatttttttt ttctagcgag aaaaaaaatc agaaaaatta
1080ctattagtga gtattggaga cattgtcaat gggagatgtt ctctttataa
tatcttcaac 1140aggttctttc aactctggaa attcatccac aatcttgtca
gcaagtgaat ctcttaattg 1200cttcaatcca tgcatcttgc ctctttgata
ttggttggat cttcttatgg cttccacgaa 1260ctctcttgtg taaatatctg
gatttctacc gtcctcaatg tattgaacaa cttccaaggg 1320aatgtccacc
ttagacaagc tggattgagg atcgttgctt ctcacgttca gcttgtacaa
1380gcgatccaca tttctttgca agttggtgat cattcccttg gtggcttctg
gagtaccagg 1440aaaatcatat atcgagacac ctaattcaac gaaggactca
ataatcgaag ccacttggtc 1500ttgagtagtg gccagttctt gctgcaattg
ttcattgtta gtgctgtttc cattcatctt 1560atcggtttat ttttctatat
atttgcctct ttctcaaaca ggagttagta gttaaaagta 1620cgaagttctt
gttctttaat gcgcgctgac aaaagaattg gataaaagag aatggtgggg
1680ggacaagaag gaaatttgtc ctagtttaac atgaatggca tcttgttacc
gggtggacat 1740cacctattga ttctaaatat ctttacggtt tatcatactg
ttctttattc cgtcgttatt 1800ctttttattt ttatcatcat ttcacgtggc
tagtaaaaga aaagccacaa catgactcag 1860caaatctcga caaagtaaaa
gctcatagag atagtattat attgatataa aaaaagtata 1920ctgtactgtt
tgtaaccttt tcaatgcttt aagatcaaaa ctaaggccag caaaggtatc
1980aacccatagc aactcataaa 20002312001DNASaccharomyces cerevisiae
231gaaaccatta aatcatattt aataaattgt tgcgacatgc aagaagttcg
cggatggtca 60tgcgtattta agaatagtca agtaacaatt tgcttattcg ttgatgatat
gatattattc 120agcaaagact taaatgcaaa taagaaaatc ataacaacac
tcaagaaaca atacgataca 180aagataataa atctgggtga aagtgataac
gaaattcagt acgacatact tggattagag 240atcaaatatc aaagaagcaa
gtacatgaaa ttaggtatgg aaaaatcctt gacagaaaaa 300ttacccaaac
taaacgtacc tttgaaccca aaaggaaaga aacttagagc tccaggtcaa
360ccaggtcatt atatagacca ggatgaacta gaaatagatg aagatgaata
caaagagaaa 420gtacatgaaa tgcaaaagtt gattggtcta gcttcatatg
ttggatataa atttagattt 480gacttactat actacatcaa cacattgctc
aaccatatac tattcccctc taggcaagtt 540ttagacatga catatgagtt
aatacaattc atgtgggaca ctagagataa acaattaata 600tggcacaaaa
acaaacctac caagccagat aataaactag tcgcaataag cgatgcttca
660tatggtaacc aaccatatta caagtcacaa attggtaaca ttttcctact
caacggaaaa 720gtgattggag gaaagtcgac aaaggcttcg ttaacatgca
cttcaactac agaagcagaa 780atacacgcgg tcagtgaagc tattccgcta
ttgaataacc tcagtcacct tgtgcaagaa 840cttaacaaga aaccaattat
taaaggctta cttactgata gtagatcaac gatcagtata 900attaagtcta
caaatgaaga gaaatttaga aacagatttt ttggcacaaa ggcaatgaga
960cttagagatg aagtatcagg taataattta tacgtatact acatcgagac
caagaagaac 1020attgctgatg tgatgacaaa acctcttccg ataaaaacat
ttaaactatt aactaacaaa 1080tggattcatt agatctatta cattatgggt
ggtatgttgg aataaaaatc aactatcatc 1140tactaactag tatttacgtt
actagtatat tatcatatac ggtgttagaa gatgacgcaa 1200atgatgagaa
atagtcatct aaattagtgg aagctgaaac gcaaggattg ataatgtaat
1260aggatcaatg aatattaaca tataaaatga tgataataat atttatagaa
ttgtgtagaa 1320ttgcagattc ccttttatgg attcctaaat cctcgaggag
aacttctagt atatctacat 1380acctaatatt attgccttat taaaaatgga
atcccaacaa ttacatcaaa atccacattc 1440tcttcaaaat caattgtcct
gtacttcctt gttcatgtgt gttcaaaaac gttatattta 1500taggataatt
atactctatt tctcaacaag taattggttg tttggccgag cggtctaagg
1560cgcctgattc aagaaatatc ttgaccgcag ttaactgtgg gaatactcag
gtatcgtaag 1620atgcaagagt tcgaatctct tagcaaccat tatttttttc
ctcaacataa cgagaacaca 1680caggggcgct atcgcacaga atcaaattcg
atgactggaa attttttgtt aatttcagag 1740gtcgcctgac gcatatacct
ttttcaactg aaaaattggg agaaaaagga aaggtgagag 1800ccgcggaacc
ggcttttcat atagaataga gaagcgttca tgactaaatg cttgcatcac
1860aatacttgaa gttgacaata ttatttaagg acctattgtt ttttccaata
ggtggttagc 1920aatcgtctta ctttctaact tttcttacct tttacatttc
agcaatatat atatatatat 1980ttcaaggata taccattcta a
20012322001DNASaccharomyces cerevisiae 232cactaccacc actacggttg
tccatgacgt atcctgcgat tttttgaatt aatgattcaa 60tagttgacat ttgctcgtca
ttggggttcg actgagctgc ggatgtcaac ttcgcaacag 120cttctgcatg
gtttccttga gaaaaatgag actcagcctc tgagattaac ttatccgtat
180ccatttcaga tctttgctat acgtttgtat cgctatatgt acgttctttt
aatgaacttt 240ctcctttctt tatcgtgtag cttgcttggg tatcttttaa
tgagttgcgg acagtgagat 300ttttcagaag ggcaattggc caagacacca
aaaacgtttg gacgagacag gcatcaaagg 360acaaggtaaa aggcgttgag
ctgtggctgg ctgtgtatgc gtttgaaata ccatggatag 420atatcaaaga
aagataggat gtttcataca aatcccaaat ttggggcgcg gacaactgaa
480atacgtgggt ccagtggaca cgaaagctgg aatgtttgct ggtgtagact
tacttgccaa 540cattggtaag aacgatggat cattcatggg gaagaagtat
tttcaaacag agtatcctca 600aagtggacta tttatccagt tgcaaaaagt
cgcatcattg atcgagaagg catcgatatc 660gcaaacctcg agaagaacga
cgatggaacc gctatcaata cccaaaaaca gatctattgt 720gaggctcact
aaccagttct ctcccatgga tgatcctaaa tcccccacac ccatgagaag
780tttccggatc accagtcggc acagcggtaa tcaacagtcg atggaccagg
aggcatcgga 840tcaccatcaa cagcaagaat ttggttacga taacagagaa
gacagaatgg aggtcgactc 900tatcctgtca tcagacagaa aggctaatca
caacaccacc agcgattgga aaccggacaa 960tggccacatg aatgacctca
atagcagcga agttacaatt gaattacgag aagcccaatt 1020gaccatcgaa
aagctacaaa ggaaacaact acactacaaa aggctactcg atgaccaaag
1080aatggtcctc gaagaagtgc aaccgacttt tgataggtat gaagccacaa
tacaagaaag 1140agagaaagag atagaccatc tcaagcaaca attggagctc
gaacgcagac agcaagccaa 1200acaaaagcag ttttttgacg ctgagaatga
acagctactt gctgtcgtaa gccaactaca 1260cgaagagatc aaagaaaacg
aagagagaaa tctttctcat aatcaaccca ctggtgccaa 1320cgaagatgtc
gaactcctga aaaaacagct ggaacaatta cgcaacatag aagaccaatt
1380tgagttacac aagacaaagt gggctaaaga acgcgaacaa ttgaaaatgc
ataacgattc 1440gctcagtaaa gaataccaaa atttgagcaa ggaactattt
ttgacaaaac cacaagattc 1500ctcatcggaa gaggtggcat ccttaacgaa
aaaacttgaa gaggctaatg aaaaaatcaa 1560acagttggaa caggctcaag
cacaaacagc cgtggaatcg ttgccaattt tcgacccccc 1620tgcaccagtc
gataccacgg caggaagaca acagtggtgt gagcattgcg atacgatggg
1680tcataataca gcagaatgcc cccatcacaa tcctgacaac cagcagttct
tctaggcagt 1740cgaactgact ctaatagtga ctccggtaaa ttagttaatt
aattgctaaa cccatgcaca 1800gtgactcacg tttttttatc agtcattcga
tatagaaggt aagaaaagga tatgactatg 1860aacagtagta tactgtgtat
ataatagata tggaacgtta tattcacctc cgatgtgtgt 1920tgtacataca
taaaaatatc atagcacaac tgcgctgtgt aatagtaata caatagttta
1980caaaattttt tttctgaata c 20012332001DNASaccharomyces cerevisiae
233acaatgagga agaacatgcc gttttacaag aattaaatag tttaacccaa
agaattaatg 60aactaggcat ggaaagtata aattcaaact ccgattcgga cagaataaac
gggtcatatt 120cacaagtgga ttttggtaac aataacgacg aggacgatat
gaacctgttc gacccagatt 180ttatggcaca agaccaattg cgtgctgaag
aaagagacta caacaaggat gatagaacac 240ccttagctaa ggtccctgcg
gcctttcaat caactggatt gggcataacc cccgatgacg 300atatcgagag
acaatacata acggaacaca gatcacgaca tgaagtgcca aagcggtctc
360ccgagaaacc ctccaacccg ctggaaatag gtaacccata cgcgaaacct
ggcacaaggt 420tgaataccac tcacacccac agcaaaactg atcgtagcat
tacccctcag aggggccagc 480cagtcccatc aggccagcag atttcctcct
acgtgcagcc agcaaacatt aatagtccta 540acaaaatgta tggtgcaaac
aactcggcaa tgggttcgcc caggaatcca aagacgagag 600cgccaccagg
tccatacaat cagggatgga ataaccgccc ctcgccttca aatatttacc
660aacgtcctca tccctcagat acacaaccac aagcatatca tctccccgga
aacccatact 720caacggggaa caggccaaac atgcaagcgc aatatcaccc
gcagcaggtg cccatgccta 780tcctgcagca gcccaatcgc ccgtaccaac
cttatgcgat gaatacgcac atgggctctc 840ctggcggata tgctggggca
gcaccaccat ttcagccagc taacgtcaac tacaatacta 900ggcctcagca
gccatggcct acacctaact caccatccgc acactaccgt ccgcccccta
960acctgaacca gcctcaaaac ggtagtgctg gttactatcg tccgccggca
ccacaattgc 1020aaaactccca agcccgtcca caaaagaagg acggattctc
acagttcatg ccatctgcaa 1080ctacgaagaa cccatatgcc cagtaactcg
accgactggt tgtaatttta caaaaagaga 1140gacaattaag aaaagaaaca
agcgccaggc ttccgtatcc cagtttttca tctcactttc 1200tgggcacgat
tgtaataata cttcatgata ataactaaac tatataagta gtgtctcatc
1260cgtaaatata catttagaca gattcttgta ttttctccgg gcaattttta
actttttttc 1320tgttagggca catgacactt gcctattatg gacagccagt
aaagatgtgc catatattgc 1380cccctttacg ctctctgcca gtattagtgg
gaaaaaaaaa actgaaaaaa aaaaaatcgc 1440agactactaa taatcacgtg
atatttcttt tcactctctt cataaagttg ctaaaaacac 1500acaatcgaat
gagcctctga gcagtataaa ttgtacttca aagcactagt catgaaaaac
1560gcttacatta gttcagtttg tcaaggttat gctattactt gtacttattt
cttgctattg 1620ttagtggctc cccacattga cgtattttca cgtgatgcgc
ctcactgcgg aaggcgccac 1680acattgcctg caaaaaattg tggatgcact
catttgatag taaactaagt catgttaatc 1740gtttggattt ggcacacacc
cacaaatata cacattacat atatatatat attcaaaata 1800cagctgcgtc
caatagatga gcttccgctt cgttgtacaa cctacctgct atcttgttca
1860cggatatttc ttgcttttaa taaacaaaag taactctaga acagtcaagt
cttcgataat 1920ttttttagtc acagggtccg tctaaagttt ctctttattt
ggaataatag aaaagaaaga 1980aaaaaacgta gtataaaagg a
20012342000DNASaccharomyces cerevisiae 234aaggatggca aatacccaat
cggaggaact cgaacacttc agtatctgtg tcttctagtg 60agtctttagc ggaagttatt
cagccatctt ccttcaaaag tgggagtagt tcattgcatt 120atctatcgtc
ttctatctca agccaacctg gttcgtacgg ttcttggttc aacaaaaggc
180caacaatttc tcagttcttt caaccaagcc cttctttaaa acacaacgag
tcgtgggaga 240ggctgcaaac aactgctgga aatatgcaaa ggacttcaag
ttcgtcttct ttgcagcaag 300caacctccag gttatcacta accactccgc
aacaatcacc gtctatcagc gaatatgatg 360agtatccttg gatgggcaca
cctggctctc ctaatgttgg agatgtgtct cacgcacccc 420cattggttaa
gaatatatca tataaatttc cactaaagaa cgttgagttg aaaagagatt
480gccaaaggat ctctcaggat gatcttttgg atgaggcttt tgaaagaata
tgtcagccct 540ctttggctga ccttaattcc acttacgaaa tttttccagg
taactcttct tatgcggata 600ttttgactac tgattctgat attgatgatg
gcttgatgaa taaacctctg gaactattgc 660cgaaatatac aatgtattta
acccatttta acaatttttt ccagttgcaa gcatgtcctg 720ctggtcaaga
atcagagagc agaataacaa attctatgaa gattgacctg ttaaaggcgg
780attacacaag aagtctatta gtatcgttac gttcaaggga cattagggat
gtcgcattga 840aaagagagtt tactggcaat aacaacaata acagcaacca
gaatatctat gatgagaatt 900ttgtcggaaa aaggaagtac gtgttgaaac
agaagaccag aaaaatcttt tcctgtggca 960agattggcaa gctaagtact
agtttggaaa actgcgttaa ttttgttgaa aatagtataa 1020agagtgcaat
gatgttatat gatgataatg gaatagatag tgagcttcgc gattcagaag
1080ctttacggat tttttcatct cttgttcatt attgtaatgc aggttaatgt
tttctccttc 1140tttacatgtt taatatattc caagttacct aagaggtgta
cgatattttt ttcttttata 1200tatatgattt tctctattca ttttttagtt
ttttttgata cataagcgaa tcgcacattg 1260cgcaacttca atttgttgat
tcgccaaagt attcttacca taaaacaacc attcgttgct 1320ttaccctttc
gtaatcattt accgtgataa ccataatcag aaacttatta tttcagccta
1380gtagaccggc caagcaggcc ttgtaatgtt tctcttgatt gcttgaatct
tttaagcagc 1440caaatctttc caaaaaaatg caattatcag aacaaaacta
tttaaggtga cttctccgta 1500tttacaccac cagaagcgtt
ctggctcccc ttttctctaa acgttaaaca ttttacaatt 1560gaaatgttac
caatcctata ttattgtacc acattgccag atttatgaac tctgggtatg
1620ggtgctaatt ttcgttagaa gcgctggtac aattttctct gtcattgtga
cactaattag 1680gaaacttctc gactatcaat gtgtaaatga aggaataatg
gcggaaactt tgaaactttg 1740tcaataattg catcattgga tgcgtttcat
ttggccgtta tcacggagag gcagagttct 1800ctccacaatt tgggcagaag
tcttttgaaa agacatatat atatatatat atgtatatga 1860gtggatgctt
aaggtaagaa taatttctga attcccaagt attcattttg tgcagtattc
1920acatattcta ttttattgct ttttaacttt agaggcaatt aaatttgtgt
aggaaaggca 1980aaatactatc aaaattttcc 20002352001DNASaccharomyces
cerevisiae 235ttgccttcaa gatctacttt cctaagaaga tcattattac
aaacacaact gcactcaaag 60atgactgctc atactaatat caaacagcac aaacactgtc
atgaggacca tcctatcaga 120agatcggact ctgccgtgtc aattgtacat
ttgaaacgtg cgcccttcaa ggttacagtg 180attggttctg gtaactgggg
gaccaccatc gccaaagtca ttgcggaaaa cacagaattg 240cattcccata
tcttcgagcc agaggtgaga atgtgggttt ttgatgaaaa gatcggcgac
300gaaaatctga cggatatcat aaatacaaga caccagaacg ttaaatatct
acccaatatt 360gacctgcccc ataatctagt ggccgatcct gatcttttac
actccatcaa gggtgctgac 420atccttgttt tcaacatccc tcatcaattt
ttaccaaaca tagtcaaaca attgcaaggc 480cacgtggccc ctcatgtaag
ggccatctcg tgtctaaaag ggttcgagtt gggctccaag 540ggtgtgcaat
tgctatcctc ctatgttact gatgagttag gaatccaatg tggcgcacta
600tctggtgcaa acttggcacc ggaagtggcc aaggagcatt ggtccgaaac
caccgtggct 660taccaactac caaaggatta tcaaggtgat ggcaaggatg
tagatcataa gattttgaaa 720ttgctgttcc acagacctta cttccacgtc
aatgtcatcg atgatgttgc tggtatatcc 780attgccggtg ccttgaagaa
cgtcgtggca cttgcatgtg gtttcgtaga aggtatggga 840tggggtaaca
atgcctccgc agccattcaa aggctgggtt taggtgaaat tatcaagttc
900ggtagaatgt ttttcccaga atccaaagtc gagacctact atcaagaatc
cgctggtgtt 960gcagatctga tcaccacctg ctcaggcggt agaaacgtca
aggttgccac atacatggcc 1020aagaccggta agtcagcctt ggaagcagaa
aaggaattgc ttaacggtca atccgcccaa 1080gggataatca catgcagaga
agttcacgag tggctacaaa catgtgagtt gacccaagaa 1140ttcccattat
tcgaggcagt ctaccagata gtctacaaca acgtccgcat ggaagaccta
1200ccggagatga ttgaagagct agacatcgat gacgaataga cactctcccc
ccccctcccc 1260ctctgatctt tcctgttgcc tctttttccc ccaaccaatt
tatcattata cacaagttct 1320acaactacta ctagtaacat tactacagtt
attataattt tctattctct ttttctttaa 1380gaatctatca ttaacgttaa
tttctatata tacataacta ccattataca cgctattatc 1440gtttacatat
cacatcaccg ttaatgaaag atacgacacc ctgtacacta acacaattaa
1500ataatcgcca taaccttttc tgttatctat agcccttaaa gctgtttctt
cgagcttttt 1560cactgcagta attctccaca tgggcccagc cactgagata
agagcgctat gttagtcact 1620actgacggct ctccagtcat ttatgtgatt
ttttagtgac tcatgtcgca tttggcccgt 1680ttttttccgc tgtcgcaacc
tatttccatt aacggtgccg tatggaagag tcatttaaag 1740gcaggagaga
gagattactc atcttcattg gatcagattg atgactgcgt acggcagata
1800gtgtaatctg agcagttgcg agacccagac tggcactgtc tcaatagtat
attaatgggc 1860atacattcgt actcccttgt tcttgcccac agttctctct
ctctttactt cttgtatctt 1920gtctccccat tgtgcagcga taaggaacat
tgttctaata tacacggata caaaagaaat 1980acacataatt gcataaaata c
20012361999DNASaccharomyces cerevisiae 236ttttgtaaga aattattcac
cgcatcttca tctggcaaac gaatgggaga ctttgaggaa 60cccaatccat ttctgaataa
cggagattta gaaatgtaaa aggtagcaaa tgtaaaaagt 120gccaggacca
tcacagcagt caatgccaac accaatttcc cttgccatga cactgttgga
180tcttttgaag gagatttgta acctggaatc tcactataat gaacacattc
accggattca 240cacttcaaag taatataagg gtcaccaaac acggtcaata
tcaaatcatt catagaaggc 300tcactgaatt tacattgcct tgtttctaaa
tcacagctga aatctcctgg cccttttatt 360gtctctgtca ggaaatccga
gatatctata gaccccttag caccacacaa cacagtgtcg 420ggaacgcatt
tgcattgaac gtcattacac ttataatggg aggtattctg ttccaagtcg
480tattcaaagg cacaatcact taagccacaa tagaagcttt ctaactgatc
tatccaaaac 540tgaaaattac attcttgatt aggtttatca caggcaaatg
taatttgtgg tattttgccg 600ttcaaaatct gtagaatttt ctcattggtc
acattacaac ctgaaaatac tttatctaca 660atcataccat tcttataaca
tgtcccctta atactaggat caggcatgaa cgcatcacag 720acaaaatctt
cttgacaaac gtcacaattg atccctcccc atccgttatc acaatgacag
780gtgtcatttt gtgctcttat gggacgatcc ttattaccgc tttcatccgg
tgatagaccg 840ccacagaggg gcagagagca atcatcacct gcaaaccctt
ctatacactc acatctacca 900gtgtacgaat tgcattcaga aaactgtttg
cattcaaaaa taggtagcat acaattaaaa 960catggcgggc atgtatcatt
gcccttatct tgtgcagtta gacgcgaatt tttcgaagaa 1020gtaccttcaa
agaatggggt cttatcttgt tttgcaagta ccactgagca ggataataat
1080agaaatgata atatactata gtagagataa cgtcgatgac ttcccatact
gtaattgctt 1140ttagttgtgt atttttagtg tgcaagtttc tgtaaatcga
ttaatttttt tttctttcct 1200ctttttatta accttaattt ttattttaga
ttcctgactt caactcaaga cgcacagata 1260ttataacatc tgcataatag
gcatttgcaa gaattactcg tgagtaagga aagagtgagg 1320aactatcgca
tacctgcatt taaagatgcc gatttgggcg cgaatccttt attttggctt
1380caccctcata ctattatcag ggccagaaaa aggaagtgtt tccctccttc
ttgaattgat 1440gttaccctca taaagcacgt ggcctcttat cgagaaagaa
attaccgtcg ctcgtgattt 1500gtttgcaaaa agaacaaaac tgaaaaaacc
cagacacgct cgacttcctg tcttcctatt 1560gattgcagct tccaatttcg
tcacacaaca aggtcctagc gacggctcac aggttttgta 1620acaagcaatc
gaaggttctg gaatggcggg aaagggttta gtaccacatg ctatgatgcc
1680cactgtgatc tccagagcaa agttcgttcg atcgtactgt tactctctct
ctttcaaaca 1740gaattgtccg aatcgtgtga caacaacagc ctgttctcac
acactctttt cttctaacca 1800agggggtggt ttagtttagt agaacctcgt
gaaacttaca tttacatata tataaacttg 1860cataaattgg tcaatgcaag
aaatacatat ttggtctttt ctaattcgta gtttttcaag 1920ttcttagatg
ctttcttttt ctctttttta cagatcatca aggaagtaat tatctacttt
1980ttacaacaaa tataaaaca 19992371999DNASaccharomyces cerevisiae
237aaacaaatgg caaaaataac gggcttcacc attgttcctg tatggtgtat
tagaacatag 60ctgaaaatac ttctgcctca aaaaagtgtt aaaaaaaaga ggcattatat
agaggtaaag 120cctacaggcg caagataaca catcaccgct ctcccccctc
tcatgaaaag tcatcgctaa 180agaggaacac tgaaggttcc cgtaggttgt
ctttggcaca aggtagtaca tggtaaaaac 240tcaggatgga ataattcaaa
ttcaccaatt tcaacgtccc ttgtttaaaa agaaaagaat 300ttttctcttt
aaggtagcac taatgcatta tcgatgatgt aaccattcac acaggttatt
360tagcttttga tccttgaacc attaattaac ccagaaatag aaattaccca
agtggggctc 420tccaacacaa tgagaggaaa ggtgactttt taagggggcc
agaccctgtt aaaaaccttt 480gatggctatg taataatagt aaattaagtg
caaacatgta agaaagattc tcggtaacga 540ccatacaaat attgggcgtg
tggcgtagtc ggtagcgcgc tcccttagca tgggagaggt 600ctccggttcg
attccggact cgtccaaatt attttttact ttccgcggtg ccgagatgca
660gacgtggcca actgtgtctg ccgtcgcaaa atgatttgaa ttttgcgtcg
cgcacgtttc 720tcacgtacat aataagtatt ttcatacagt tctagcaaga
cgaggtggtc aaaatagaag 780cgtcctatgt tttacagtac aagacagtcc
atactgaaat gacaacgtac ttgacttttc 840agtattttct ttttctcaca
gtctggttat ttttgaaagc gcacgaaata tatgtaggca 900agcattttct
gagtctgctg acctctaaaa ttaatgctat tgtgcacctt agtaacccaa
960ggcaggacag ttaccttgcg tggtgttact atggccggaa gcccgaaaga
gttatcgtta 1020ctccgattat tttgtacagc tgatgggacc ttgccgtctt
catttttttt ttttttcacc 1080tatagagccg ggcagagctg cccggcttaa
ctaagggccg gaaaaaaaac ggaaaaaaga 1140aagccaagcg tgtagacgta
gtataacagt atatctgaca cgcacgtgat gaccacgtaa 1200tcgcatcgcc
cctcacctct cacctctcac cgctgactca gcttcactaa aaaggaaaat
1260atatactctt tcccaggcaa ggtgacagcg gtccccgtct cctccacaaa
ggcctctcct 1320ggggtttgag caagtctaag tttacgtagc ataaaaattc
tcggattgcg tcaaataata 1380aaaaaagtaa ccccacttct acttctacat
cggaaaaaca ttccattcac atatcgtctt 1440tggcctatct tgttttgtcc
tcggtagatc aggtcagtac aaacgcaaca cgaaagaaca 1500aaaaaagaag
aaaacagaag gccaagacag ggtcaatgag actgttgtcc tcctactgtc
1560cctatgtctc tggccgatca cgcgccattg tccctcagaa acaaatcaaa
cacccacacc 1620ccgggcaccc aaagtcccca cccacaccac caatacgtaa
acggggcgcc ccctgcaggc 1680cctcctgcgc gcggcctccc gccttgcttc
tctccccttc cttttctttt tccagttttc 1740cctattttgt ccctttttcc
gcacaacaag tatcagaatg ggttcatcaa atctatccaa 1800cctaattcgc
acgtagactg gcttggtatt ggcagtttcg tagttatata tatactacca
1860tgagtgaaac tgttacgtta ccttaaattc tttctccctt taattttctt
ttatcttact 1920ctcctacata agacatcaag aaacaattgt atattgtaca
ccccccccct ccacaaacac 1980aaatattgat aatataaag
19992382009DNASaccharomyces cerevisiae 238ggatgagaaa cgagtgcggt
ttcgagagta gatattcaac ccacccgaag tagccttcag 60gaactggttc cgttctctct
tcctccggaa tagtctgaat gtccttaaga gaccgtggct 120cgtatactct
tctattcttg ggccgcaata gcaaaaagag ccagacaaac acgacggcgg
180taagaccgta gataatcagg gttgaaatga acgccgaagt cgaagaactg
tcagccatag 240tacgtatgtg ctataaatat ctaacctttc gctgctttga
atatgatgtg ctcaaatata 300acttaatata atagtataac aaaaaggagt
actatttgct aaatatcgta gacgtagtag 360acatagtaaa tacaataaag
gatagataac caagaaccca catcaagcga atacatacat 420atatatatac
tcgatgtata catgtttcta agcacttgcg cacatacgta tttaaagtat
480ttcagggaga ttaacgtatt aaaacaagaa gagggttgac tacatcacga
tgagggggat 540cgaagaaatg atggtaaatg aaataggaaa tcaaggagca
tgaaggcaaa agacaaatat 600aagggtcgaa cgaaaaataa agtgaaaagt
gttgatatga tgtatttggc tttgcggcgc 660cgaaaaaacg agtttacgca
attgcacaat catgctgact ctgtggcgga cccgcgctct 720tgccggcccg
gcgataacgc tgggcgtgag gctgtgcccg gcggagtttt ttgcgcctgc
780attttccaag gtttaccctg cgctaagggg cgagattgga gaagcaataa
gaatgccggt 840tggggttgcg atgatgacga ccacgacaac tggtgtcatt
atttaagttg ccgaaagaac 900ctgagtgcat ttgcaacatg agtatactag
aagaatgagc caagacttgc gagacgcgag 960tttgccggtg gtgcgaacaa
tagagcgacc atgaccttga aggtgagacg cgcataaccg 1020ctagagtact
ttgaagagga aacagcaata gggttgctac cagtataaat agacaggtac
1080atacaacact ggaaatggtt gtctgtttga gtacgctttc aattcatttg
ggtgtgcact 1140ttattatgtt acaatatgga agggaacttt acacttctcc
tatgcacata tattaattaa 1200agtccaatgc tagtagagaa ggggggtaac
acccctccgc gctcttttcc gatttttttc 1260taaaccgtgg aatatttcgg
atatcctttt gttgtttccg ggtgtacaat atggacttcc 1320tcttttctgg
caaccaaacc catacatcgg gattcctata ataccttcgt tggtctccct
1380aacatgtagg tggcggaggg gagatataca atagaacaga taccagacaa
gacataatgg 1440gctaaacaag actacaccaa ttacactgcc tcattgatgg
tggtacataa cgaactaata 1500ctgtagccct agacttgata gccatcatca
tatcgaagtt tcactaccct ttttccattt 1560gccatctatt gaagtaataa
taggcgcatg caacttcttt tctttttttt tcttttctct 1620ctcccccgtt
gttgtctcac catatccgca atgacaaaaa aatgatggaa gacactaaag
1680gaaaaaatta acgacaaaga cagcaccaac agatgtcgtt gttccagagc
tgatgagggg 1740tatctcgaag cacacgaaac tttttccttc cttcattcac
gcacactact ctctaatgag 1800caacggtata cggccttcct tccagttact
tgaatttgaa ataaaaaaaa gtttgctgtc 1860ttgctatcaa gtataaatag
acctgcaatt attaatcttt tgtttcctcg tcattgttct 1920cgttcccttt
cttccttgtt tctttttctg cacaatattt caagctatac caagcataca
1980atcaactatc tcatatacaa tgtctatcc 20092391943DNASaccharomyces
cerevisiae 239ggcagtcatc aggatcgtag gagataagca ccctgacaag
taacatgccg atgaagttgt 60ttggttcatt gggcaaaaaa atcgggattc tagaaaaccc
tgagttgaag attttttcga 120cagttttatc gtctaggatg gtatcggcac
tcattgtgaa cacgttttca atcggagtca 180tgatttcctc aaccctcttt
gcctttagat ccaaaacagc agagatgatt gtaacttcgt 240ctttagtcaa
ccgttccacc cccatggtcc tatgcaaggt gaccaaagtc tttaagccgg
300attttttgta catcgtacca tgatcttcac ccagcatata gtccaggaga
gtcgcgatcg 360gatatgcgac tgggtacatc agatacatca gtacaagaac
aaaggggcag aagaatgccc 420caacttgcag cccgtattta acacagacac
tctgcggaat aatttcaccg aagatcacaa 480ttagaatagt tgacgacact
acagcctgcc aaccaccccc aagacacctg tccaaaacaa 540taggcaatgt
ttcgttggtt ataacattag aaagcagcag tgtgactaga acccaatgct
600tccccctaga tattaggtca agcacccgct tggccagttt cttttcagaa
ttcgagcctg 660aagtgctgat taccttcagg tagacttcat cttgacccat
caaccccagc gtcaatcctg 720caaatacacc acccagcagc actaggatga
tagagataat atagtacgtg gtaacgcttg 780cctcatcacc tacgctatgg
ccggaatcgg caacatccct agaattgagt acgtgtgatc 840cggataacaa
cggcagtgaa tatatcttcg gtatcgtaaa gatgtgatat aagatgatgt
900atacccaatg aggagcgcct gatcgtgacc tagaccttag tggcaaaaac
gacatatcta 960ttatagtggg gagagtttcg tgcaaataac agacgcagca
gcaagtaact gtgacgatat 1020caactctttt tttattatgt aataagcaaa
caagcacgaa tggggaaagc ctatgtgcaa 1080tcaccaaggt cgtccctttt
ttcccatttg ctaatttaga atttaaagaa accaaaagaa 1140tgaagaaaga
aaacaaatac tagccctaac cctgacttcg tttctatgat aataccctgc
1200tttaatgaac ggtatgccct agggtatatc tcactctgta cgttacaaac
tccggttatt 1260ttatcggaac atccgagcac ccgcgccttc ctcaacccag
gcaccgcccc caggtaaccg 1320tgcgcgatga gctaatcctg agccatcacc
caccccaccc gttgatgaca gcaattcggg 1380agggcgaaaa ataaaaactg
gagcaaggaa ttaccatcac cgtcaccatc accatcatat 1440cgccttagcc
tctagccata gccatcatgc aagcgtgtat cttctaagat tcagtcatca
1500tcattaccga gtttgttttc cttcacatga tgaagaaggt ttgagtatgc
tcgaaacaat 1560aagacgacga tggctctgcc attgttatat tacgcttttg
cggcgaggtg ccgatgggtt 1620gctgagggga agagtgttta gcttacggac
ctattgccat tgttattccg attaatctat 1680tgttcagcag ctcttctcta
ccctgtcatt ctagtatttt tttttttttt ttttggtttt 1740actttttttt
cttcttgcct ttttttcttg ttactttttt tctagttttt tttccttcca
1800ctaagctttt tccttgattt atccttgggt tcttctttct actcctttag
attttttttt 1860tatatattaa tttttaagtt tatgtatttt ggtagattca
attctctttc cctttccttt 1920tccttcgctc cccttcctta tca
19432402001DNASaccharomyces cerevisiae 240tgacaacgag taccaggaaa
tcagtgcttc tgctttgaag aaggctcgta agggctgtga 60tggtttgaag aaaaaggcag
tcaagcaaaa ggaacaggag ttgaagaaac aacaaaaaga 120ggcagaaaat
gctgccaagc aattgtctgc tttgaatatc accattaagg aggacgaatc
180gctaccagct gccattaaga ctagaattta tgactcttat tccaaggtcg
gacaaagagt 240taaggtttcc ggttggatcc atagattacg ttctaacaag
aaggttattt tcgtcgtcct 300cagagacgga tctggtttca ttcaatgtgt
cttgtccggt gatttggcat tggctcaaca 360aactttggac ctgactttgg
aatccaccgt tactctgtac ggtaccatag tcaaattgcc 420tgagggtaaa
accgctccag gtggtgttga attgaatgtc gactattacg aagttgtagg
480tttggccccc ggtggtgaag actcctttac aaacaaaatc gcagagggct
cagacccttc 540tttactgttg gaccaacgtc atttggcctt gagaggagat
gccttgtctg cagtcatgaa 600agtccgtgct gctctactga aaagcgttag
acgtgtttat gatgaagaac atttgacaga 660agttacccca ccatgtatgg
tgcaaactca agtcgaaggt ggttccactt tgttcaagat 720gaactattac
ggcgaggaag cttacttgac ccaaagttcc caattatatt tagaaacctg
780tttggcctcc ctaggtgatg tttataccat ccaagaatct ttcagagctg
aaaagtccca 840cacaagaaga catttgtccg aatataccca tatcgaagct
gaattggcct tcttgacttt 900cgacgatcta ttacaacata ttgaaacttt
gatcgtcaaa tccgtgcaat acgttttgga 960agacccaatt gctggcccac
tcgtaaaaca attgaatcca aactttaagg ctccaaaggc 1020tccattcatg
agattacagt acaaggatgc cattacctgg ttgaacgaac acgacatcaa
1080gaacgaagag ggcgaagact ttaaatttgg tgacgatatt gcagaagctg
ctgaaagaaa 1140gatgaccgat accatcggcg tcccaatctt tttgacgaga
ttcccagtag aaatcaagtc 1200tttctacatg aagcgttgtt ctgacgaccc
ccgcgtcact gaatccgtcg acgttttgat 1260gccaaacgtt ggtgaaatca
ctggtggttc tatgagaatc gacgacatgg acgaactaat 1320ggcagggttt
aagcgtgagg gtattgatac cgacgcctac tactggttca ttgaccaaag
1380aaaatacggt acttgcccac atggtggtta cggtatcggt accgaacgta
ttttagcctg 1440gttgtgtgac agattcactg tcagagactg ttccttgtat
ccacgtttca gcggtagatg 1500taagccatga tctttagtta ctgaagagta
cgtgagcgct cacatatata caaatattta 1560taccgattaa tatttacgtt
cctccctctc tctaattatt cattgattta ttcaagaatt 1620agcgttataa
caataaatgg ttggcgcagg caattaattt ttctttactc ttccaaaccc
1680tctgttaacg acaatcaaat aacctgatct gccaaggctc catcatatct
ggcctagaac 1740agtttttttt tttcgattat ttgttcgttc ttgtggtggt
tactcattgg cagaatcccg 1800aaaatcatga ttagtagatg aatgactcac
tttttggata agctggcgca aattgaaaca 1860tgtgaaaaaa aaaaaaaagg
attataaaag gtcagcgaag cacagaactc tgagataaga 1920ctacctttct
ttagctaggg gagaatattc gcaattgaag agctcaaaag caggtaacta
1980tataacaaga ctaaggcaaa c 20012411999DNASaccharomyces cerevisiae
241tcctaaggac atattccgtt cgtacttgag ttattggatc tatgaaatcg
ctcgctatac 60accagtcatg attttgtccc tggtaatagg ggttttggtt ttattaatta
tattttttaa 120tgacaacgaa gcttgtgttt tcaattctgc aatatttgct
tttacttctc ttgtaggttt 180gttaataata ttaagtgatg gtaatccaaa
gctagtcagt cgtcgaaatt ttaggaccga 240gcttttagtg gatgtcatca
cacgtaaacc ggcggtagaa gggaaagaat ggaggatcat 300cacatacaac
atgaaccaat atttgtttaa tcatgggcaa tggcatactc cgtattactt
360ttacagcgat gaggattgct accgttattt tctacgcctt gttgagggag
taacccccaa 420gaagcaaaca gccacgtcaa ttggcaattc tccggtcacc
gctaagcctg aagatgccat 480cgagtcagct tctcctagtt ccagactgaa
ttatcaaaac tttttgctca aggcagcgga 540gatcgaacga caagctcagg
aaaattactg gcgaaggcgg catcccaata tcgatgcgct 600tcttaaaaag
acggaatagc ttagagacac taccatacgt aaagcgaaca taaactagag
660tatgatatat aatcagcact aactggccgg aaaacggccg aaggaagcct
cgaaaagtcg 720attcgtgttg gacccatttg ctgaacaaag tggttcattg
cctacctatt atggtagtag 780tcgtgataat cgtgtggttg gttttgtcaa
cggtgcattt gcattttcat gacaataaac 840cttgcgtttt cgttctcggg
atattacttt ccctccactt ctttcgcctc aatagctcct 900ataagcattc
tcagggcgta tgtcggtgat cgagatttcc aagcaagctt ttagtggaaa
960tcatcgcgcg caagccagcg gtaaagggaa aagaacggag gacgattaca
tacaagatga 1020acgaataaat aaattaataa taaataataa taaaaagtac
agtagcatta aatattatta 1080agtttaatga ttaaaaattg gttaattgtc
aagaaaatct aaggtattaa taaataaata 1140atactatgac aacttgcagc
gaaagcatca gccccaatga aaattaatca gaattgaatc 1200tgagcgtatt
tatttgataa cggtttacgt aactgttgga ataaaaatca actatcatct
1260actaactagt gtttacgtta ctagtatatt atcatatacg gtgttagaag
atgacgcaaa 1320tgatgagaaa tagtcatcgt tttcaacgga agctgaaata
caaggattga taatgtaata 1380ggatcaatga atatcaacat ataaaacgat
gataataata tttatagaat tgtgtagaat 1440tgcagattcc cttttatgga
ttcctaaatc ctcgagaaga acttctagta tatctacgta 1500cctaatatta
ttgccttatt aaaaatggaa tcccaacaat tatctcaaaa ttcccccaat
1560tctcatcagt aacaccccac cccgtattac ttttaccgtg atgaagattg
gcatcgttac 1620tttctaaacg taggacgtgc ggaatgacaa aaccatcagc
agtgtcacga tctctccagt 1680cacaatggca atcatgagtg catagtccaa
agtaaagggg caaggaaaag catgattgaa 1740aggactcccc atctggactc
tatatgtcat cagcggctaa aaaaaagcat atagcacaac 1800atcagcatca
gcatcagcac tagagtcatc ggcccggcgg tccgcggtca tccccgcgga
1860ctttccgtcc gcccggcggg ctgtatcagc gtcaactgga acgcgcatat
atatacaaga 1920cacacataac atagaagcac acccacgaca ataaccacac
gacaataacc acacccgccc 1980acccctcctt tccgtatac
199924220DNAArtificialPCR primer 242aagtcggcag agagcaacat
2024320DNAArtificialPCR primer 243cagatgcaaa cccaacacac
2024424DNAArtificialPCR primer 244cgattttctg ggtttgatcg ttag
2424520DNAArtificialPCR primer 245cattgtgggc gttgtagttg
2024620DNAArtificialPCR primer 246gttgtggttg gtgctttcct
2024720DNAArtificialPCR primer 247ccactttgac gccgtttatt
2024824DNAArtificialPCR primer 248cgattttctg ggtttgatcg ttag
2424921DNAArtificialPCR primer 249gaactcgtca agaaggcgat a
2125024DNAArtificialPCR primer 250cgattttctg ggtttgatcg ttag
2425118DNAArtificialPCR primer 251tcagggcaat gcagatcc 18
* * * * *
References