Note: Descriptions are shown in the official language in which they were submitted.
CA 02362897 2001-09-18
DEMANDES OU BREVETS VOLUMtNEUX
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JUMBO APPLICATIONS/PATENTS I
THIS SECTION OF TiiE APPL.ICATION1PATENT CONTAINS MORE
THAN ONE VOLUME
THIS IS VOLUME ( _ DF s
WOTE: i=or additiona't voiumes~please contact'the Canadian Patent Office -
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CA 02362897 2001-09-18
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PLANT CHROMOSOME COMPOSITIONS AND METHODS
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BACKGROUND OF THE INVENTION
This application claims the priority of U.S. Provisional Application Ser. No.
60/125.219. filed March l8, 1999, U.S. Provisional Application Ser. No.
60/127.409.
filed April I. 1999. U.S. Provisional Application Ser. No. 60/134,770. filed
May l8.
1999, U.S. Provisional Application Ser. No. 60/153.584, filed September 13.
1999. U.S.
Provisional Application Ser. No. 60/154.603. filed September 17. 1999 and U.S.
Provisional Application Ser. No. --/---. - . filed December 16, 1999, each of
which
disclosures is specifically incorporated herein by reference in its entirety.
The government owns rights in the invention pursuant to U.S. Department of
Agriculture Grant No. 96-35304-3491. National Science Foundation Grant No.
9872641
and Grant No. DOEDE-FG05-920822072 from the Consortium for Plant
Biotechnology.
I. Field of the Invention
The present invention relates generally to the field of molecular biology.
More
particularly, it concerns plant chromosome compositions and methods for using
the same.
II. Description of Related Art
Two general approaches are used for introduction of new genetic information
("transformation") into cells. One approach is to introduce the new genetic
information
as part of another DNA molecule, referred to as a "vector." which can be
maintained as an
independent unit (an episome) apart from the 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 ManiatiseraL. 1982). However. only a
few
episomal vectors that function in higher eukaryotic cells have been developed.
The
available higher eukarvotic episomal vectors are based on naturally occurring
viruses and
most Function only in mammalian cells (Willard. 1997). In higher plant systems
the only
known double-stranded DNA viruses that replicate through a double-stranded
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intermediate upon which an episomal vector could be based is the gemini virus.
although
the gemini virus is limited to an approximately 800 by insert. Although an
episomal
plant vector based on the Caulit7ower Mosaic Virus has been developed. its
capacity to
carry new genetic information also is limited (Brisson et al., 1984).
The other general method of genetic transformation involves integration of
introduced DNA sequences into the recipient cell's chromosomes, permitting the
new
information to be replicated and partitioned to the cell's progeny as a part
of the natural
chromosomes. The most common form of integrative transformation is called
"transfection" and is frequently used in mammalian cell culture systems.
Transfecoon
involves introduction of relatively large quantities of deproteinized DNA into
cells. The
introduced DNA usually is broken and joined together in various combinations
before it
is integrated at random sites into the cell's chromosome (see, for example
Wigler et al., 1977). Common problems with this procedure are the
rearrangement of
introduced DNA sequences and unpredictable levels of expression due to the
location of
the transgene in the genome or so called "position effect variation'' (Shingo
et al., 1986).
Further. unlike episomal DNA. integrated DNA cannot normally be precisely
removed.
A more refined form of integrative transformation can be achieved by
exploiting naturally
occurring viruses that integrate into the host's chromosomes as part of their
life cycle.
such as retroviruses (see Cepko et crl.. 1984). In mouse, homologous
integration has
recently become common. although it is significantly more difficult to use in
plants (Lam
et al. 1996).
The most common genetic transformation method used in higher plants is hosed
on the transfer of bacterial DNA into plant chromosomes that occurs during
infemion by
the phytopathogenic soil bacterium AKrnhcrctPrrrrjrr (see Nester et ul.,
1984). By
substituting genes of interest for the naturally transferred bacterial
sequences (called
T-DNA). investigators have been able to introduce new DNA into plant cells.
However.
even this more "refined" integrative transformation system is limited in three
major ways.
First. DNA sequences introduced into plant cells using the Agrohcrcterium T-
DNA system
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are frequently rearranged (see Jones et «L, 1987). Second. the expression of
the
introduced DNA sequences varies between individual transformants (see
Jones et «L, 1980. This variability is presumably caused by rearranged
sequences and
the influence of surroundin~~ sequences in the plant chromosome (i.e.,
position effects). as
well as methylation of the transgene. A third drawback of the Agrobcrcter«rm T-
DNA
system is the reliance on a "gene addition" mechanism: the new Genetic
information is
added to the cenome (i.e., all the genetic information a cell possesses) but
does not
replace information already present in the genome.
One attractive alternative to commonly used methods of transformation is the
use
of an artificial chromosome. Artificial chromosomes are man-made linear or
circular
DNA molecules constructed from cis-acting DNA sequence elements that are
responsible
for the proper replication and partitioning of natural chromosomes (see
Murray et «L, 1983). Desired elements include: ( 1 ) Autonomous Replication
Sequences
lp (ARS) (these have properties of replication origins. which are the sites
for initiation of
DNA replication). (2) Centromeres (site of kinetochore assembly and
responsible for
proper distribution of replicated chromosomes at mitosis or meiosis), and (3)
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).
At present. the essential chromosomal element, for construction of artificial
chromosomes have been precisely characterized only from lower eukaryotic
species.
ARSs have been isolated from unicellular fun~~i. including .S«cch«ronTVCe.s
cerevisi«e
2s (breweis yeast) and Sclri:n.c«cclrarornvce.~~ ponrh~ tree Stinchcomb et
«!., 1979 and
Hsiao et «L, 1979). An ARS behaves like a replication origin allowing DNA
molecules
that contain the ARS to be replicated as an episome after introduction into
the cell nuclei
of these funCi. Plasmids containing these sequences replicate. but in the
absence of a
centromere they are partitioned randomly into daughter cells.
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Artificial chromosomes have been constructed in yeast using the three cloned
essential chromosomal elements. Murray et al., 1983. disclose a cloning system
based on
the in vitro construction of linear DNA molecules that can be transformed into
yeast.
where they are maintained as artificial chromosomes. These yeast artificial
chromosomes
(YACs) contain cloned genes, origins of replication. centromeres and telomeres
and are
segregated in daughter cells with high fidelity when the YAC is at least 100
kB in length.
Smaller CEN containing vectors may be stably segregated, however, when in
circular
form.
None of the essential components identified in unicellular organisms, however.
function in higher eukaryotic systems. For example. a yeast CEN sequence will
not
confer stable inheritance upon vectors transformed into higher eukaryotes.
While such
DNA fragments can be readily introduced. they do not stably exist as episomes
in the host
cell. This has seriously hampered efforts to produce artificial chromosomes in
higher
t 5 organisms.
In one case, a plant artificial chromosome was discussed (Richards et cal..
U.S.
Patent No. 5.270.201 ). However, this vector was based on plant telomeres, as
a
functional plant centromere was not disclosed. While tclomeres are important
in
maintaining the stability of chromosomal termini. they do not encode the
information
needed to ensure stable inheritance of an artificial chromosome. It is well
documented
that centromere function is crucial for stable chromosome! inheritance in
almost all
eukaryotic organisms (reviewed in Nicklas 1988). For example. broken
chromosomes
that lack a centromere (ucentric chromosomes) are rapidly lost from cell
lines, while
fragments that have a centromere are faithfully segregated. The centromere
accomplishes
this by attaching. via centromere binding proteins. to the spindle fibers
during mitosis and
meiosis, thus ensuring proper gene segregation during cell divisions.
In contrast to the detailed studies done in S. cerrai.cicre and S. pumhe.
little is
known about the molecular structure of functional centromeric DNA of higher
_>_
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eukaryotes. Ultrastructural studies indicate that higher eukaryotic
kinetochores. which
are specialized complexes of proteins that form on the chromosome during late
prophase,
are large structures (mammalian kinetochore plates are approximately 0.3 Eun
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.
While the above studies have been useful in elucidating the structure and
function
of centromeres, they have failed to provide a cloned centromere from a higher
eukaryotic
organism. The extensive literature indicating both the necessity of
centromeres for stable
inheritance of chromosomes. and the non-functionality of yeast centromeres in
higher
organisms, demonstrate that_clonin~ of a functional centromere from a higher
eukaryote
is a necessary first step in the production of artificial chromosomes suitable
for use in
IS higher plants and animals. The production of artificial chromosomes with
centromeres
which function in higher eukaryotes would overcome many of the problems
associated
with the prior art and represent a si_nificant breakthrough in biotechnology
research.
SUMMARY OF THE INVENTION
In one aspect of the invention. a method is provided for the identification of
plant
centromeres. In one embodiment of the invention. the method may comprise
tetrad
analysis. Briefly, tetrad analysis measures the recombination frequency
between genetic
makers and a centromere by analyzing all four products of individual meiosis.
A
particular advantage arises from the yrrcrrtet (grt Il mutation in
Arcrbidupsis, which causes
the four products of pollen mother cell meiosis in Ar«bidop.ci.c to remain
attached. The
c/u«rter mutation may also find use in accordance with the invention in
species other than
Arahidnpsis. For example. several naturally occurring plant species are also
known to
release pollen clusters. including water lilies. cattails. heath IEriccrce«e
«nd f_pacriclcc«e).
evening primrose (On«,~raceam. sundew (Urn.cer«ce«e). orchids (Orchid«ce«e),
and
-6-
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acaclaS (Mirnr~sucecre) (Preuss 1994: Smyth 1994). None of these species
however, has
been developed into an experimental systems thus severely limiting their use
for genetic
analysis. However, it is contemplated by the inventors th~u a c/unrtet
mutation could be
introduced into a host plant to enable the use of tetrad analysis in
potentially any species.
When used to pollinate a flower. one tetrad can result in the formation of
four seeds. and
the plants from these seeds can be analyzed genetically. With unordered
tetrads,
however, such as those produced by Arabiclopsis, genetic mapping using tetrad
analysis
requires that two markers be scored simultaneously.
In another aspect, the invention provides a recombinant DNA construct
comprising a plant centromere. The recombinant DNA construct rnay additionally
comprise any other desired sequences, for example. a telomere, including a
plant ielomere
such as an Arcrbiclupsis tlraliancr telomere, or alternatively. a yeast or any
other type of
telomere. One may also desire to include an autonomous replicating sequence
(ARS),
IS such as a plant ARS, including an Arabidop.ci.c thaliarra ARS. Still
further, one may wish
to include a structural gene on the construct, or multiple genes (for example,
two, three,
four. five, six, seven, eight, nine, ten, fifteen. twenty, twenty-five. fifty,
one hundred. two
hundred. five hundred, one thousand! up to and including the maximum number of
structural genes (roughly 5000) which can physically be placed on the
recombinant DNA
construct. Examples of structural genes one may wish to use include a
selectable or
screenable marker gene. an antibiotic resistance gene, a herbicide resistance
gene. a
nitrogen fixation gene, a plant pathogen defence gene, a plant stre,s-induced
gene, a toxin
gene, a receptor gene, a li~and gene, a hormone gene. an enzyme gene, an
interleukin
gene. a clotting factor gene. a cytokine gene. an antibody gene, a Growth
factor Gene and a
seed storage gene. In one embodiment of the invention, the construct is
capable of
expressing the structural gene, for example. in a prokaryote or eukaryote,
including a
lower eukaryote, or a higher eukaryote such as a plant.
In yet another aspect. the invention provides a recombinant DNA construct
comprising a plant centromere and which is a plasmid. The plasmid may contain
any
_7_
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desired sequences, sUCh as an origin of replication. including an origin of
replication
functions in bacteria, such as E. coli and Agrobacterirrrrr, or in plants or
yeast, for
example, such as S. cerevisicrc~. The plasmid may also comprises a selection
marker,
which may function in bacteria, including E. cnli anti A,yrohncterirrrrr, as
well as a
S selection marker that functions in plants or yeast. such as S. cerevisiae.
In still yet another aspect, the invention provides a recombinant DNA
construct
comprising a plant centromere and which is capable of being maintained as a
chromosome. wherein the chromosome is transmitted in dividing cells. The plant
centromere may be from any plant.
In still yet another aspect. the invention provides a plant centromere which
is
further defined as an Arcrhidnpsi.s thalicrncr centromere. In yet another
embodiment of the
invention. the plant centromere is an Arabiclopsis tlraliana chromosome 1
centromere,
IS and may still further be defined as flanked by the genetic markers T22C23-
T7 and
T3P8-SP6, or still further as flanked by the genetic markers T22C23-T7 and
TSD18,
T22C23-T7 and T3L4, TSD18 and T3P8-SP6, TSD18 and T3L4, and T3L4 and
T3P8-SP6. In vet another embodiment of the invention. the plant centromere
comprises
an Arcrbidupsis tlruliana chromosome 2 centromere. The chromosome 2 centromere
may
comprise, for example, from about 100 to about 61 1.000. about 500 to about
611,000,
about 1.000 to about 611.000, about 10.000 to about 61 1.000. about 20.000 to
about
61 1.000. about 40.000 to about 61 1.000, about 80.000 to about 61 1.000.
about ISO.OOU to
about 6I I ,000, or about 300,000 to about 61 1.000 contiguous nucleotides of
the nucleic
acid sequence of SEQ ID N0:209, including comprising the nucleic acid sequence
of
SEQ ID N0:209. The centromere may also be defined as comprising from about 100
to
about 50,959. about 500 to about S0,9S9, about 1.000 to about 50.959, about
5,000 to
about 50.959. about 10.000 to about 50.959, 20.000 to about 50.959. about
30.000 to
about 50.959. or about 40.000 to about S0.9S9 contiguous nucleotides of the
nucleic acid
sequence of SEQ ID N0:210. and may comprise the nucleic acid sequence of SEQ
ID
N0:210: The centromere may comprise sequences from both SEQ ID NOS:209 and
210.
_8_
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including the aforementioned fragments. or the entirety of SEQ ID NOS:209 and
210. In
particular embodimemnts, the inventors contemplate a 3' fru~ment of SEQ ID
\'0:209
can be fused to a ~' fra~tnent of SEQ 1D \0:210. optionally including one or
more 180
by repeat sequence disposed therebetween.
In still yet another aspect, the invention provides an Arcrhicloh.ci.c
tlrrrliancr
chromosome 3 centromere. In one embodiment of the invention. the centromere
may be
further defined as flanked by the genetic markers T9G9-SP6 and TSM 14-SP6, and
still
further defined as flanked by a pair of genetic markers selected from the
group consisting
of T9G9-SP6 and TI4H20. T9G9-SP6 and T7K14, T9G9-SP6 and T21P20, T14H20 and
T7K 14, T i 4H20 and T2 I P20, T 14H20 and TSM 14-SP6. T7K 14 and TSM 1=1-SP6.
T7K 14 and T2 I P20. and T21 P20 and TSM 14-SP6.
In still yet another aspect, the invention provides an Araliidnp.ci.c
tlrcrliana
chromosome 4 centromere. In certain embodiments of the invention. the
centromere may
comprise from about 100 to about 1,082.000. about 500 to about 1,082.000.
about 1.000
to about 1.082.000. about 5,000 to about 1.082.000, about 10,000 to about
1.082.000,
about 50,000 to about l ,082.000, about 100.000 to about 1,082,000, about
200.000 to
about 1.082.000. about 400.000 to about 1.082,000, or about 800,000 to about
1.082,000
contiguous nucleotides of the nucleic acid sequence of SEQ ID N0:2 l I ,
including
comprising the nucleic acid sequence of SEQ ID N0:21 I. The centromere may
also be
defined as comprisin« from about 100 to about 163.317. about 500 to about
163.317.
about 1.000 to about 163,317, about 5.000 to about 163.317, about 10.000 to
about
163,317, about 30.000 to about 163.317, about 50.000 to about 163.317. shout
80.000 to
about 163.317, or about 120.000 to about 163.317 contiguous nucleotides of the
nucleic
acid sequence of SEQ ID N0:212. and may be defined as comprising the nucleic
acid
sequence of SEQ 1D N0:212. The centromere may comprise sequences from both SEQ
ID NOS:21 I and ?I?. including the aforementioned fragments. or the entirety
of SEQ ID
NOS:21 1 and 212. In particular embodimemnts. the inventors contemplate a 3'
fragment
_y_
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of SEQ ID N0:211 can be fused to a ~' fragment of SEQ ID N0:212, optionally
including one or more 180 by repeat sequence disposed therebetween.
In yet another embodiment. there is provided a Arcrbidvpsi.s tlraliurrcr
chromosome
l, 3 or 5 centromere selected from the nucleic acid sequence given by SEQ ID
N0:184.
SEQ ID N0:185. SEQ ID NO: I 86. SEQ ID N0:187, SEQ ID NO: I88. SEQ ID NO: !
89.
SEQ ID N0:190. SEQ ID N0:191. SEQ ID N0:192. SEQ ID N0:193. SEQ ID NO: i94.
SEQ ID N0:195. SEQ ID N0:196, SEQ ID N0:197. SEQ ID N0:198, SEQ ID N0:199.
SEQ ID N0:200. SEQ ID N0:201, SEQ ID N0:202, SEQ ID N0:203. SEQ ID N0:204.
SEQ ID N0:205, SEQ ID N0:206. SEQ ID N0:207, SEQ ID N0:208. or traements
thereof. In one embodiment. the construct comprises at least 100 base pairs.
up to an
including the full length. of one of the preceding sequences. In addition. the
construct
may include t or more 180 base pair repeats.
IS In still yet another aspect. the invention provides an Arabiclapsis
rlurlicura
chromosome 5 centromere. The centromere may be further defined as flanked by
the
genetic markers F13K20-T7 and CUE1, and still farther defined as flanked by a
pair of
genetic markers selected from the Qroup consisting of F13K20-T7 and T18M4.
FI3K20-T7 and T18F2. F13K20-T7 and T24I20, T18M4 and T18F2. T18M4 and
T24I20. T I 8M4 and CUE 1. T 18F2 and T24I20, T 18F2 and CUE 1. and T24I20 and
CUE 1.
In still yet another aspect. the invention provides a recombinant DNA
construct
comprising a plant centromere. and further defined as comprising n copies of a
repeated
nucleotide sequence. wherein n is at least 2. Potentially any number of repeat
copies
capable of physically heine placed on the recombinant construct could be
included on the
construct. including about ~. 10. I5. 20. 30. 50. 75. 100. 150, 200. 300. 400.
500. 750.
1.000. 1.500. 2.000. 3.000. 5.000. 7.500. i 0.000. 20.000. 30.000. 40.000.
50.000. 60.000.
70.000. 80.000. 90.000 and about 100.000. including all ranges in-beUveen such
copy
nutobers. In one embodiment the repeated nucleotide sequence may be isolatable
from
_ 10-
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the nucleic acid sequence given by SEQ ID NO:18:~. SEQ ID N0:185. SEQ ID NO:1
S6.
SEQ ID N0:187. SEQ ID N0:188. SEQ ID N0:189. SEQ ID N0:190. SEQ ID N0:191.
SEQ 1D N0:192. SEQ ID N0:193. SEQ ID N0:194. SEQ ID N0:195. SEQ ID N0:196.
SEQ ID NO:197. SEQ ID N'0:198. SEQ ID N0:199. SEQ ID N0:200. SEQ ID N0:201.
SEQ ID N0:202. SEQ ID N0:203. SEQ ID N0:204. SEQ ID N'0:205, SEQ ID N0:206,
SEQ ID N0:207. SEQ ID N0:208. SEQ ID N0:209. SEQ ID N0:210. SEQ ID N0:21 1
or SEQ ID N0:212. Examples of such sequences that could be used are liven in
FIGS.
23A-23D. The length of the repeat used may vary. but will preferably range
from about
20 by to about 250 bp, from about 50 by to about 225 bp, from about 75 by to
about 210
bp. from about 100 by to about 205 bp, from about 125 by to about 200 bp. from
about
150 by to about 195 bp. from about 160 by to about 190 and from about 170 by
to about
185 by including about 180 bp.
In conjunction with SEQ ID NOS:209. 210. 211 and 212, the repeats may be
included as part of centromeric structures. The number of repeats may vary and
include
1, 2, 3, 4, 5. 6. 7. 8. 9, 10, 1 I . 12. 13, 14, 15. 16, 17. I 8, 19. 20, 21.
22. 23, 24. 25, 30. 35.
40. 45. 50. 60. 70. 80. 90. 100. 125, 150. 175, 200, 300. 400. 500 or more.
In still yet another aspect, the invention provides a minichromosome vector
comprising a plant centromere and a telomere sequence. Any additional desired
sequences may be added to the minichromosomc. such as an autonomous
replicating
sequence. a second telomere sequence and a structural gene. One or more of the
foregoing sequences may be added . up to the maximum number of such sequences
that
can physically be placed on the minichromosome. The minichromosome may
comprise
any of the centromere compositions disclosed herein. In one embodiment of the
invention. the minichromosome rnay comprise a nucleic acid sequence selected
from the
group consisting of SEQ ID NO: I . SEQ ID N0:2. SEQ ID N0:3. SEQ ID N0:4. SEQ
ID
NO:S. SEQ ID N0:6. SEQ ID N0:7. SEQ ID N0:8. SEQ ID N0:9. SEQ ID NO:10. SEQ
ID NO:I1. SEQ ID NO:I?. SEQ ID NO:I 3. SEQ ID N0:14. SEQ ID NO:I>. SEQ ID
N0:16. SEQ ID NO:17. SEQ ID N0:18, SEQ ID NO: l9. SEQ ID N0:20. and SEQ (D
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N0:21. The minichromosome also may contain "negative" selectable markers which
confer susceptibility to an antibiotic, herbicide or other agent, thereby
allowing for
selection against plants. plant cells or cells of anv other or~T;znism of
interest containing a
minichromosome. The minichromosome also may include genes which control the
copy
number of the minichromosome within a cell. One or more structural genes also
may be
included in the minichromosome. Specifically contemplated as being useful will
be as
many stnrctural genes as may be inserted into the minichromosome while still
maintaining a functional vector. This may include one. two. three. four, five.
six, seven.
eight, nine or more structural genes.
t0
In still yet another aspect. the invention provides a recombinant DNA
construct
comprising a plant centromere. The cell may be of any type. including a
prokaryotic cell
or eukaryotic cell. Where the cell is a eukaryotic cell. the cell may be, for
example. a
yeast cell or a higher eukaryotic cell, such as plant cell. The plant cell may
be from a
1 ~ dicotyledonous plant, such as tobacco. tomato, potato. soybean. canola.
sunflower, alfalfa.
cotton and Arcrbiclopsis, or may be a monocotyledonous plant cell, such as
wheat. maize,
rye. rice, turfgrass, oat. barley, sorghum. millet, and sugarcane. In one
embodiment of the
invention, the plant centromere is an Arahiclnp.si.s rlrcrlicrna centromere.
and the cell may
be an Arcrbidopsi.s tlrcrliurrcr cell. The recombinant DNA construct rnay
comprise
20 additional sequences. such as a telomere. an autonomous replicmin~ sequence
(ARS}, a
structural gene. or a selectable or screenable marker gene. including as many
of such
sequences as may physically be placed on said recombinant DNA construct. In
one
embodiment of the invention, the cell is further defined as capable of
expressing said
structural gene. In another embodiment of the invention. a plant is provided
comprising
?~ the aforementioned cells.
In still yet another aspect, the invention provides a method of preparing a
transgenic plant cell comprising contacting a starting plant cell with a
recombinant DNA
construct comprising a plant centromere, whereby said startinU plant cell is
transformed
30 with said recombinant DNA construct. The recombinant DN,-1 construct may
comprise
CA 02362897 2001-09-18
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any desired sequences. such as many structural genes as can physically be
placed on said
recombinant DNA construct. In particular embodiments. the centromere is an
Arcrhidopsis thaliaun centromere. and the plant cell may be an Arahiclop.ri.s
thcrlimrcr cell.
In still yet another aspect. the invention provides a transgenic plant
comprising a
minichromosome vector, wherein the vector comprises a plant centromere and a
telomere
sequence. The minichromosome vector may further comprise an autonomous
replicating
sequence, second telomere sequence. or a structural gene, such as an
antibiotic resistance
gene, a herbicide resistance gene, a nitrogen fixation gene, a plant pathogen
defense gene.
a plant stress-induced gene, a toxin gene. a receptor gene. a 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, and a growth factor gene. As many of such
sequence,
may be included as can physically be placed on the minichromosome. The
minichromosome vector may further comprise a nucleic acid sequence selected
from the
group CO11SISLIrI~ of SEQ ID NO: I, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ
ID
NO:~. SEQ ID N0:6. SEQ ID N0:7. SEQ ID N0:8, SEQ ID N0:9. SEQ ID NO:10. SEQ
ID NO:I I. SEQ ID N0:12. SEQ LD N0:13, SEQ ID NO:I~i. SEQ ID NO:15, SEQ ID
N0:16, SEQ ID N0:17, SEQ ID NO: ! 8. SEQ ID N0:19. SEQ ID N0:20, and SEQ ID
N0:21. The transgenic plant may be any type of plant, such as a dicotyledonous
plant.
for example, tobacco. tomato. potato. pea, carrot, cauliflower. broccoli,
soybean, canola.
sunflower. alfalfa, cotton and Anuhiclop.ris, or may be a monocotyledonous
plant. such as
wheat, maize. rye, rice. turf~~raaa. oat. barley. sorghum, millet. and
sugarcane.
In still yet another aspect, the invention provides a method of producing a
minichromosome vector comprising: (a) obtaining a first vector and a second
vector.
wherein said first vector or said second vector comprises a selectable or
screenable
marker. an origin of replication. a telomere. and a plant centromerc. and
wherein said first
vector and said second vector comprises a site for site-specific
recombination: and (b)
contactin~~ said first vector with said second vector to allow site-specific
recombination to
occur between said site for cite-specific recombination on said first vector
and said site
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for site-specific recombination on said second vector to create a
minichromosome vector
comprising said selectable or screenable marker. said origin of replication.
said telomere
and said plant centromere. The contacting may be done irr vireo or ire aivn,
ineludin~~
wherein the contacting is carried out in a prokaryotic cell such as an
A~rohucterirrnr or E.
cnli cell. or in a lower eukarvotic cell, such as a yeast cell. The contacting
may still
further be carried out in a higher eukaryotic cell. such as a plant cell,
including an
Arnbiclnpsis thalinncr cell. The contacting may be done in the presence of
potentially any
recombinase, including Cre. Flp. Gin. Pin. Sre, pinD, Int-B 13. and R. The
first vector or
second vector may comprise border sequences for A~roJ~crcter-iurn-mediated
IO transformation. In one embodiment of the invention, the plant centromere is
an
Arabiclupsis thalicrncr centromere. The telomere may be a plant telomere. Any
plant
selectable or screenable marker could be used. including GFP. GUS. BAR. PAT,
HPT or
NPTII.
I S In still yet another aspect. a method is provided of screening a candidate
centromere sequence for plant centromere activity, said method comprising the
steps of:
(a) obtaining an isolated nucleic acid sequence comprising a candidate
centromere
sequence: (b) integratively transforming plant cells with said isolated
nucleic acid: and
(c) screening for centromere activity of said candidate centromere sequence.
In the
20 method. the screening may comprise observing a phenotypic effect present in
the
integratively transformed plant cells or plants comprising the plant cells.
wherein the
phenotypic effect is absent in a control plant cell not integratively
transformed with said
isolated nucleic acid sequence. or a plant comprising said control plant cell.
Types of
phenotypic effects that could be screened for include reduced viability.
reduced efficiency
25 of said transforming. genetic instability in the inte~ratively transformed
nucleic acid.
aberrant plant sectors. increased ploidy, aneuploidy, and increased
integrative
transformation in distal or centromeric chromosome regions. The isolated
nucleic acid
sequence may comprise a bacterial artificial chromosome. which may be further
defines!
as a binary bacterial artificial chromosome. The inte~ratively transforming
may comprise
30 use of any type of transformation. such as A,yrnhncuerirrrn-mediated
transformation. In
-I=l-
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one embodiment of the invention, the control plant cell has been integratively
transformed with a nucleic acid sequence other than a candidate centromere
sequence.
In still yet another aspect. the invention provides a recombinant DNA
construct
comprising an Arabiclupsis polyubiquitin 1 1 promoter. wherein the promoter
comprises
from about 2~ to about 2.000 contiguous nucleotides of the nucleic acid
sequence of SEQ
ID N0:180. In further embodiments of the invention. the promoter may comprise
from
about 75 to about 2.000, from about 125 to about 2.000. from about 200 to
about 2.000.
from about 400 to about 2,000, from about 800 to about 2.000, from about 1,000
to about
2,000, or from about 1,500 to about 200 contiguous nucleotides of the nucleic
acid
sequence of SEQ ID N0:180, or may comprise the nucleic acid sequence of SEQ ID
N0:180. The promoter containing construct may comprise any additional desired
sequences, for example. that of an enhancer. a telomere sequence. a plant
centromere
sequence. an ARS, or a structural gene, including an antibiotic resistance
gene. a
1 ~ herbicide resistance gene. a nitrogen fixation gene, a plant pathogen
defense gene, a plant
stress-induced gene, 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. and a growth factor gene. In one embodiment of the
invention,
the promoter may be operably linked to the 5' end of the structural gene.
In still yet another aspect. the invention provides a recombinant DNA
construct
comprising an Arabidnp.sis 40S ribosomal protein S 16 promoter. wherein said
promoter
comprises from about 25 to about 2.000 contiguous nucleotides of the nucleic
acid
sequence of SEQ ID N0:182. In particular embodiments of the invention. the
promoter
2~ may comprise from about 7~ to about 2.000. from about 12~ to about 2.000.
from about
200 to about 2.000, from about 400 to about 2,000. from about 800 to about
2,000, from
about 1,000 to about 2.000 or from about I 500 to about 2.000 contiguous
nucleotides of
the nucleic acid sequence of SEQ ID I\O: 182. or may comprise the nucleic acid
sequence
of SEQ ID N0:182. The promoter containing construct may comprise any
additional
desired sequences, for example. that c>t an enhances. a telomere sequence, a
plant
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centromere sequence, an ARS. or a structural gene. including an antibiotic
resistance
gene, a herbicide resistance nene. a nitrogen fixation gene. a plant pathogen
defense gene.
a plant stress-induced nene. a toxin gene. a receptor gene. a lioand gene. a
seed storage
gene. a hormone gene. an enzyme nene. an interleukin gene, a clottinn factor
gene, a
cytokine gene. an antibody gene, and a growth factor gene. In one embodiment
of the
invention. the promoter may be operably linked to the 5' end of the structural
gene.
In still yet another aspect, the invention provides a recombinant DNA
construct
comprising an Arabidopsis polyubiquitin 11 3' regulatory sequence including
the
terminator sequence. wherein the 3' regulatory sequence comprises from about
25 to
about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID
N0:181. In
one embodiment of the invention, the 3' regulatory sequence may be further
defined as
comprising from about 75 to about 2001, from about 125 to about 2001, from
about 200
to about 2001. from about 400 to about 2001, from about 800 to about 2001, or
from
IS about 1,000 to about 2001 continuous nucleotides of the nucleic acid
sequence of SEQ ID
N0:181. and may comprise the nucleic acid sequence of SEQ ID N0:181. The
recombinant sequence may further comprise any other sequence, for example. an
enhancer, a telomere sequence, a plant centromere sequence. an ARS, and a
structural
gene, including an antibiotic resistance gene, a herbicide resistance gene, a
nitrogen
fixation gene, a plant pathonen defense gene, a plant strews-induced gene. a
toxin nene, 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 nene, an antibody gene.
and a growth
factor nene. In one embodiment of the invention. the terminator may be
operably linked
to the 3' end of the structural nene.
In still yet another aspect, the invention provides a recombinant DNA
construct
comprising an Arahiclnp.ci.c 40S ribosomal protein S 16 3~ regulatory sequence
including
the terminator sequence. wherein the 3' regulatory sequence comprises from
about 2~ to
about 2.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID
N0:183. In
particular embodiments of the invention. the 3~ regulatory sequence may
comprise from
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about 7~ to about 2.000. from about 1 ?~ to about 2,000, from about 200 to
about ?.000,
from about 400 to about 2.000, from about 800 to about 2,000, or from about
1.000 to
about ?.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:
183. and
may comprise the nucleic acid sequence of SEQ ID N0:183. The recombinant
sequence
may further comprise any other sequence. for example, an enhancer, a telomere
sequence.
a plant centromere sequence, an ARS, and a structurat gene, includin;~ an
antibiotic
resistance gene. a herbicide resistance gene. a nitrogen fixation gene, a
plant pathogen
defense gene, a plant stress-induced gene. a toxin gene, a receptor Gene, a
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, and a growth factor gene. In one
embodiment of
the invention, the terminator may be operably linked to the 3' end of the
structural gene.
In still yet another aspect. the invention provides methods for expressing
foreign
Qenes in plants. plant cells or cells of any other organism of interest. The
foreign genes
may be from any organism, including plants, animals and bacteria. It is
further
contemplated that minichromosomes could be used to simultaneously transfer
multiple
foreign genes to a plant comprising entire biochemical or regulatory pathways.
In yet
another embodiment of the invention. it is contemplated that the
minichromosomes can
be used as DNA cloning vectors. Such a vector could be used in plant and
animal
sequencing= projects. The current invention may be of particular use in the
cloning of
sequences which are "unclonable'~ in yeast and bacteria, but which may be
easier to clone
in a plant based system.
In still yet another aspect of the invention, it is contemplated that the
minichromosomes disclosed herein may be used to clone functional segments of
DNA
such as origins of DNA replication. telomeres. telomere associated genes.
nuclear matrix
attachment regions (MARS). scaffold attachment regions (SARs). boundary
elements.
enhancers. silencers, promoters. recombinational hot-spots and centromeres.
This
embodiment may be carried out by cloning DNA into a defective minichromosome
which
is deficient for one or more type of functional elements. Sequences which
complement
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such deficient elements would cause the minichromosome to be stably inherited.
A
selectable or screenable marker on the minichromosome could then be used to
select for
viable minichromosome containing cells which contain cloned functional
elements of the
type that were non-functional in the detective minichromosome.
In still yet another aspect of the invention, the sequences disclosed herein
may be
used for the isolation of centromeric sequences from plants other than Ar-
abiclop.sis. Such
techniques may employ, for example, hybridization or sequence-based analysis.
In one
embodiment of the invention, the centromere may be isolated from
agriculturally
important species such as, for example. vegetable crops, including artichokes,
kohlrabi,
arugula, leeks, asparagus, lettuce (e.g., head, leaf, romaine), bok choy,
malanga, broccoli.
melons (e.g., muskmelon. watermelon. crenshaw, honeydew. cantaloupe), brussels
sprouts, cabbage. cardoni, carrots, napa, cauliflower. okra, onions. celery.
parsley. chick
peas, parsnips. chicory, Chinese cabbage. peppers. collards. potatoes,
cucumber plants
IS (marrows, cucumbers), pumpkins, cucurbits, radishes, dry bulb onions,
rutabaga.
eggplant, salsify, escarole. shallots, endive, garlic, spinach, green onions,
squash, greens,
beet (sugar beet and fodder beet), sweet potatoes, Swiss chard. horseradish,
tomatoes.
kale. turnips, and spices. Alterantively, centromeres could be isolated from
fruit and vine
crops such as apples, apricots, cherries, nectarines. peaches. pears, plums,
prunes. quince
almonds, chestnuts. filberts, pecans. pistachios. walnuts, citrus,
blueberries,
boysenberries, cranberries. currants, loganberries. raspberries. strawberries,
blackberries,
gapes, avocados. bananas. kiwi, persimmons. pomegranate, pineapple, tropical
fruits.
pomes, melon, mango, papaya, and lychee.
In still yet another aspect of the invention. centromeres could be isolated in
accordance with the invention from field crop plants. such as evening
primrose, meadow
foam. corn (field. sweet. popcorn), hops. jojoha. peanuts. rice. safflower.
small Grains
(barley. oats, rye. wheat. etc.). sor~~hurn. tobacco. kapok. leguminous plants
(beans.
lentils. peas. soybeans). oil plants (rape. mustard. poppy. olives.
sunflowers, coconut.
castor oil plants. cocoa beans, groundnuts). fibre plants (cotton. tlax. hemp.
jute).
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lauraceae (cinnamon, camphor), or plants such as coffee, su'Tarcane. tea. and
natural
rubber plants. Still other examples of plants from which centromeres could be
isolated
include bedding plants such as flowers. cactus. succulents and ornamental
plants. as well
as trees such as forest (broad-!caved trees and evergreens, such as conifers).
Iruit.
ornamental. and nut-bearing trees, as well as shrubs and other nursery stock.
In still yet another aspect of the invention. the minichromosome vectors
described
herein may be used to perform efficient gene replacement studies. At present.
gene
replacement has been detected on only a few occasions in plant systems and has
only
been detected at low frequency in mammalian tissue culture systems (see
Thomas et al., 1986: Smithies et al., 1985). The reason for this is the high
frequency of
illegitimate nonhomologous recombination eventa relative to the frequency of
homologous recombination events (the latter are responsible for ~=ene
replacement).
Artificial chromosomes may participate in homologous recombination
preferentially.
Since the artificial chromosomes remain intact upon delivery, no
recombinogenic broken
ends will be generated to serve as substrates for the extremely efficient
illegitimate
recombination machinery. Thus, the artificial chromosome vectors disclosed by
the
present invention will be maintained in the nucleus through meiosis and
available to
participate in homology-dependent meiotic recombination. In addition. because
in
principle, artificial chromosomes of any length could be constructed using the
teaching of
the present invention, the vectors could be used to introduce extremely long
stretches of
DNA from the same or any other organism into cells. Specifically contemplated
inserts
include those from about several base pairs to one hundred megabase pairs,
including
about I kb. 25 kB. 50 kB, 100 kB, 125 kB, 150 kB. 200 kB. 300 kB. 400 kB. 500
kB. 600
kB, 700 kB. 800 kB. 900 kB. 1 MB. I .25 Mb. 1.5 Mb. 2 Mb, 3 Mb. 5 Mb, 10 Mb.
25 Mb.
50 Mb and 100 Mb.
In still yet another aspect. the present invention provides methods for the
construction of minichromosome vectors for the Uenetic: transformation of
plant cells.
uses of the vectors. and organisms transformed by them. Standard reference
works
CA 02362897 2001-09-18
WO 00/55325 PCT/USUO/07392
setting forth the General principles of recombinant DNA technology include
Lewin, 1985.
Other works describe methods and products of genetic engineering. See, e.
r,~..
~~laniatis ct crl.. 1982: Watson et al.. 1983: Setlow et «L. 1979: and Dillon
et «l.. 1985.
In still yet another aspect. the invention provides a method of preparing a
trans~enic cell. In one embodiment of the invention. the method comprises the
steps of:
a.) obtaining a nucleic acid molecule comprising Arabidnp.ci.c tlraliartcr
centromere DNA
having the following characteristics: l.) mapping to a location on an
Arnhidnpsi.c tlraliona
chromosome defined by a pair of genetic markers selected from the group
consisting of:
mi342 and T27K 12. mi310 and 84133, atpox and ATA. mi233 and mi 167, and F
13K20-
t7 and CUE1, and 2.) sorts DNA to the spindle poles in meiosis 1 in a pattern
indicating
the disjunction of homologous chromosomes, bl preparing a recombinant
construct
comprising said nucleic acid molecule: and c) transforming a recipient cell
with said
recombinant construct.
IS
The cell may be. for example, a lower eukaryotic cell including a yeast cell,
or
may be a higher eukaryotic cell. Where the cell is a higher eukaryotic cell,
the cell may
be an animal or plant cell. In one embodiment of the invention, the cell is
not an
Arnbidop.ci.c th«linn« cell. In another embodiment of the invention, the Ar-
crhidupsi.c
tlmli«rrcr centromere is defined by the marker pair mi342 and T27K 12. which
may be
further defined by the genetic marker pair T22C23-t7 and T3P8-sp6: and / or is
defined
by the marker pair mi310 and 84133, which may be further defined by the
genetic marker
pair FSJ I S-sp6 and T I SD9: and 1 or is defined by the marker pair atpox and
ATA. which
may be further defined by the genetic marker pair T9G9-sp6 and TSM 14-sp6: and
/ or is
defined by the marker pair mi233 and mi 167. which may be further defined by
the nenetic
marker pair T24H24.30k3 and F13H14-t7: and / or is defined by the genetic
marker pair
F13K20-t7 and CUEi. which may be further defined by a genetic marker pair
selected
from the croup consisting of F I 3K20-T7 and T I 8M=1. F 13K20-T7 and T 18F2.
F I 3 K20-T7 and T2=1120. T I 8M4 and T I 8F?. T I 8M=l and T24I20. T 18M4 and
CUE 1.
T 18F2 and 1'24120. T l SF2 and CUE 1. and T24120 and CUE 1.
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WO 00155325 PCT/US00/07392
In one embodiment of the invention, the transforming may comprise use of a
method selected from the group consisting of: A,s,~rnbcrcterirrrrr-mediated
transformation.
protoplast transformation. electroporation, or particle bombardment. The
recombinant
construct may comprise desired elements, including a telomere, such as an
Arcrhidnp.ci.c
thaliann or yeast telomere. The recombinant construct may also comprise an
autonomous
replicating sequence (ARS), for example, an Arnfiidop.si.c thaliana ARS. The
recombinant construct may also comprise a prokaryotic or eukaryotic selectable
or
screenable marker gene. Also desired to include with a recombinant construct
may be
one or more structural genes. Exemplary structural genes include a gene
selected from
the group consisting of an antibiotic resistance gene, a herbicide resistance
gene, a
nitrogen fixation gene, a plant pathogen defense gene, a plant stress-induced
gene. a toxin
gene. a seed storage gene, a hormone gene, an enzyme gene, an interleukin
gene. a
clotting factor gene, a cytokine gene. an antibody gene, and a growth factor
~eoe. The
method may further comprise the step of re~eneratin~ a transgenic plant from
said cell.
In still yet another aspect. the invention provides a method of identifying a
nucleic
acid molecule capable of conferring centromere activity comprising the steps
of: a)
obtaining a nucleic acid molecule comprising Arcrbiclop.ci.c thnliarra
centromere DNA.
wherein the Arnhicloh.ci.c rlrccliarra centromere is defined by a pair of
genetic markers
selected from the group consisting of mi342 and T27K12, mi310 and x4133, atpox
and
ATA. mi233 and mi167. and F13K?0-t7 and T17M11-sp6: b) preparing a recombinant
construct that comprises the nucleic acid molecule: and c) determining the
ability of the
recombinant construct to demonstrate a stable inheritance pattern. In the
method. the
ability to demonstrate a stable inheritance pattern may be determined by
preparin~z a
recombinant cell that comprises the recombinant construct. In another
embodiment of the
invention. the Arubiclop.cis rlrcrlicrrra centromere is defined by the marker
pair mi34? anti
T27K1?. which may be further defined by the ~yenetic marker pair T22C23-t7 and
T3P8-sp6: and / or is defined by the cnarker pair mi310 and 84133, which may
he turther
defined by the genetic marker pair F~J 1 ~-sp6 and T15D9: and / or is defined
by the
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WO 00/55325 PCT/US00/07392
marker pair atpox and ATA, which may be further defined by the genetic marker
pair
T9G9-sp6 and TAM 14-sp6: and / or is defined by the marker pair mi233 and mi
167,
which may be further defined by the genetic marker pair T?4H24.30k3 and F13H
I=t-t7:
and / or is defined by the genetic marker pair F13K20-t7 and CUE1, which may
be
i further defined by a genetic marker pair selected from the croup consisting
of F13K20-T7
and T 18M4. F I 3K20-T7 and T t 8F2. F I 3 K20-T7 and T24I20. T 18M4 and T
18F2.
T 18M4 and T24I?0, T l 8M4 and CUE 1, T I 8F2 and T?aI20, Tl 8F2 and CUE I .
and
T24I20 and CUE 1.
l0 In one embodiment of the invention. the recombinant construct is not
chromosomally integrated. Said obtaining may comprise obtaining a BAC or YAC
clone
comprising said Arahidopsis rlraliana centromere DNA. The DNA may be obtained
by a
method that includes the use of pulsed-field gel electrophoresis. and may be
obtained by a
method that includes positional cloninb. In another embodiment of the
invention. the
15 positional cloning may comprise identifying a contiguous set of clones
comprising said
Arnhiclnpsis thaliancr centromere DNA, wherein said set of clones is t7anked
by a pair of
genetic markers selected from the group consistiny~ of mi342 and T27K12. mi310
and
~4 E 33. atpox and ATA, mi233 and mi 167. and F I 3K20-t7 and T 17M I 1-sp6.
20 The contiguous set of clones may span the Ar«hidnpsi.c thalinua centromere.
The
recombinant construct may comprise a selectable or screenable marker and said
step of
determining may comprise determining a phenotype conferred by the selectable
or
screenable marker. The determining may comprise. for example. determining the
ability
of the recombinant construct to demonstrate a stable inheritance pattern in
mitosis and /
?~ or meiosis. In still another embodiment. the invention provides a
trans~~enic cell prepared
by a method provided by the invention. Also provided by the invention are a
transgenic
plant, plant parts and tissue cultures comprisin~~ the transaenic cell. In
another
embodiment of the invention. the Arahicl~~psus rlr«iicrn« centromere is
defined by the
marker pair mi34? and T27K 12. which may be further defined by the genetic
marker pair
30 T??C23-t7 and T3P8-sp6: and / or is defined by the marker pair mi310 and g~
133. which
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may be further defined by the genetic marker pair F~J 15-sp6 and TI SD9: and /
or is
defined by the marker pair atpox and ATA. which may be further defined by the
genetic
marker pair T9G9-sp6 and TSM 1=l-sp6: and / or is defined by the marker pair
mi233 and
mi 167, which may be further defined by the genetic marker pair T2~H2=1.30k3
and
F ! 3H 14-t7: and / or is defined by the genetic marker pair F ( 3K20-t7 and
CUE 1. which
may be further defined by a genetic marker pair selected from the group
consisting of
F13K20-T7 and T18M4. F13K20-T7 and T18F2. F13K20-T7 and T24I20, T18M4 and
T 18F2: T 18M4 and T24I20. T 18Md and CUE 1. T 18F2 and T24I20. T 18F2 and CUE
1.
and T24I20 and CUE I .
In still yet another aspect of the invention. a centromere used in accordance
with
the invention is not from Arahidopsi.s, for example, from Arahidopci.s
tlraliumr.
Similarly. a plant or plant cell comprising a centromere composition in
accordance with
the invention. may also be from a plant other than Arahidopsis.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to
further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to one or more of these drawings in combination with
the
detailed description of specific embodiments presented herein. The file of
this patent
contains at least one drawing executed in color. Copies of this patent with
color
drawin~(s) will be provided by the Patent and Trademark Office upon request
and
payment of the necessary fee.
FIG. I. Centromere mappin~ with unordered tetrads: A cross of Uvo parents
(AABB x aabb). in which "A" is on the centromere of one chromosome. and "B" is
linked to the centromere of a second chromosome. At meiosis. the A and B
chromosomes assort independently. resultin~l in equivalent numbers of parental
ditype
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WO 00/55325 PCT/US00/07392
(PD) and nonparental ditype (NPD) tetrads (recombinant progeny are shown in
gray).
Tetratype tetrads (TT) result only from a crossover between ''B" and the
centromere.
FIG. 2. Low resolution mad location of Ar«hidnp.ci.c centromeres. Trisomic
mapping was used to determine the map position of centromeres on four of the
five
Ar«bidnp.ci.c chromosomes (Kootnneef, 1983: Sears et crl.. 1970). For
chromosome 4.
useful trisomic strains were not obtained. With the methods of Koornneef and
Sears et
crl. 1983. (which rely on low-resolution deletion mapping) the centromere on
chromosome 1 was found to lie between the two visible markers. ttl and ctrl.
that are
separated by 5 cM. Centromere positions on the other chromosomes are mapped to
a
lower resolution.
FIG. 3. Physical maps of the aenetically-defined .4r-«hidon.ci.c centromeres.
Each
centromeric region is drawn to scale: physical sizes are derived from DNA
sequencing
l5 (chromosomes 11 and IV) or from estimates based on BAC fingerprinting
(Marry et «!.. 1999: Mozo et crl.. 1999) (chromosomes I, III. and V).
Indicated for each
chromosome are positions of markers (above), the number of tetratype / total
tetrads at
those markers (below), the boundaries of the centromere (thick black bars).
and the name
of conti~s derived from fingerprint analysis (Marry et «L. 1999: Mozo et crl..
1999). For
each contig, more than two genetic markers, developed from the database of BAC-
end
sequences (http://www.tigr.org/tdb/at/abe/bac end_search.hunl) were scored.
PCR
primers corresponding to these sequences were used to identify size or
restriction site
polymorphisms in the Columbia and Landsberg ecotypes (Bell and Ecker. 199=1:
Konieczny and Ausubel. 1993): primer sequences arc available
(http://genome-www.Stanford.edu/Arabidopsis/aboutcaps.html). Tetratype tetrads
resulting from treatments that stimulate crossing over (boxes); positions of
markers in
centimorsans (cMl) shared with the recombinant inbred (RI) map (ovals)
(http://nasc.nott.ac.uk/new_ri_map.html: Somerville and Somcrville. 1999): and
sequences bordering gaps in the physical map that correspond to 180 by repeats
(open
CA 02362897 2001-09-18
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circles) (Round et crl., 1997). ~S rDNA (black circles) or 160 by repeats Gray
circles) are
indicated (Copenhaver et crl., 1999).
FIG. 4. Exemplary list of seed stock used for' tetrad analysis in
Arcrbicl«p.si.s
th«licrrr«. The individual strains are identified by the strain number (column
B). The
tetrad member number (column A) indicates the tetrad source (i.e., T1
indicates seeds
from tetrad number 1, and the numbers - l . -2, -3. or -4 indicate individual
members of the
tetrad). The strains listed have been deposited with the Arabi~lopsis
Biological Resources
Center (ABRC) at Ohio State University under the name of Daphne Preuss.
FIG. 5. Marker information for centromere mapping. DNA polymorphisms used
to localize the centromeres are indicated by chromosome (Column 1 ). The name
of each
marker is shown in Column ?_ the name of the markers used by Copenhaver et «L.
1999
to position centromeres is Given in Column 3 and marker type is indicated in
Column =t.
1~ CAPS (Co-dominant Amplified Polvmorphic Sites) are markers that can be
amplified
with PCR and detected by digesting with the appropriate restriction enzyme
(also
indicated in Column 3). SSLPs (Simple Sequence Length Polymorphisms) detect
polymorphisms by amplifying different length PCR products. Column > notes if
the
marker is available on public web sites (e.,~..
http://genome-www.stanford.edu/Arabidopsis). For those markers that are not
available
on public web sites the sequences of the forward and reverse primers used to
amplity the
marker are listed in columns 6 and 7. respectively.
FIG. 6. Scoring PCR-based markers for tetrad analysis. The genotype of the
progeny from one pollen tetrad (T? i was determined for two genetic markers
(S0392 and
nga76). Analysis of the four progeny plants (T?-1 through T2-4) using PCR and
gel
electrophoresis allows the genotype of the plant to be determined. and the
genotype of the
pollen parent to be interred.
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FIG.7A-7N. Exemplary Minichromosorne vectors: The vectors shown in
FIG. 7A, FIG. 7B, FIG. 7E, FIG. 7F, FIG. 7I and FIG. 7J have an E. call origin
of
replication which can be high copy number. low copy number or sirt~~le copy.
In
FIGS. 7A-7N. the vectors include a multiple cloning site which can contain
recognition
sequences for conventional restriction endonucleases with 4-8 by specificity
as well as
recognition sequences for very rare cutting enzymes such as. for example. I-
Ppo 1. I-Cue
I. PI-Tli, PI-Psp I. Not I. and PI Sce I. In FIG. 7A-7N. the centromere is
flanked by Lox
sites which can act as targets for the site specific recombinase Cre. FIG.
7r1. Shows an
E. coli plant circular shuttle vector with a plant ARS. FIG. 7B. Shows a plant
circular
vector without a plant ARS. The vector relies on a plant origin of replication
function
found in other plant DNA sequences such as selectable or screenable markers.
FIG. 7C.
Shows a yeast-plant circular shuttle vector with a plant ARS. The yeast ARS is
included
twice. once on either side of multiple cionin~ site to ensure that large
inserts are stable.
FIG. 7D. Shows a yeast-plant circular shuttle vector without a plant ARS. The
vector
relies on a plant origin of replication function found in other plant DNA
sequences such
as selectable markers. The yeast ARS is included twice, once on either side of
the
multiple cloning site to ensure that lame inserts arc stable. FIG.7E. Shows an
E.
cnli-Agrobncterirrm-plant circular shuttle vector with a plant ARS. Vir
functions for
T-DNA transfer would be provided in trans by a using the appropriate
Agrnbncterir.r» r
strain. FIG. 7F. Shows an E. cull-A,~rnhrrcrerirr»r-plant circular shuttle
vector without a
plant ARS. The vector relies on a plant orisin of replication function found
in other plant
DNA sequences such as selectable markers Vir functions for T-DNA transfer
would be
provided in traps by a using the appropriate Ayrohcrcterirr» r strain. FIG.
7G. Shows a
linear plant vector with a plant ARS. The linear vector could be assembled in
vitro and
?~ then transferred into the plant by, for example. mechanical means such as
micro projectile
bombardment. electroporation. or PEG-mediated transformation. FIG. 7H. Shows a
linear plant vector without a plant ARS. The linear vector could be assemhled
i» vitro
and then transferred into the plant by, for example. mechanical means such as
micro
projectile bombardment. electroporation. or PEG-mediated transformation.
FIC'~s. 7I-7N.
The fi~lures are identical to FIGS. 7A-7F. respectively. with the exception
that they do not
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contain plant telomeres. These vectors will remain circular once delivered
into the plant
cell and therefore do not require telomeres to stabilize their ends.
FIG. 8. Sequence features at CEN2 (A) and CEN4 (B). Central bars depict
annotated genomic sequence of indicated BAC clones; black, genetically-defined
centromeres: white. regions flanking the centromeres. Sequences corresponding
to genes
and repetitive features, filled boxes (above and below the bars,
respectively). are defined
as in F1G. 12A-T; predicted nonmobile genes, red: genes carried by mobile
elements.
black: nonmobile pseudogenes, pink; pseudogenes carried by mobile elements,
gray:
retroelements, yellow; transposons, green: previously defined centromeric
repeats, dark
blue: 180 by repeats, pale blue. Chromosome-specific centromere features
include a
large mitochondria) DNA insertion (orange: CENZ). and a novel array of tandem
repeats
(purple: CEN=l). Gaps in the physical maps (//). unannotated regions (hatched
boxes), and
expressed ~~enes (filled circles) are shown.
FIG. 9. Method for converting_a BAC clone for any other bacterial clone) into
a
minichromosome. A portion of the conversion vector will integrate into the BAC
clone
(or other bacterial clone of interest) either through non-homologous
recombination
(transposable element mediated) or by the action of a site specific
recombinase system.
such as Cre-Lox or FLP-FRT.
FIG. 10. Method for analysis of diccntrie chromosomes in Ar-c~hidnt~.ci.c.
BiBAC
vectors containing centromere fragments 0100 kb) are integ=rated into the
Arcrbiclopsi.s
genome using A,~rnl~acterirrm-mediated transformation procedures and studied
for
adverse affects due to formation of dicentric chromosomes. l ) BiBACs
containing
centromere fragments are identified using standard protocols. 2) Plant
transformation.
3) Analysis of defects in growth and development of plants containing
dicentric
chromosomes.
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FIG. 11A-G. Method for converting a BAC clone (or any other bacterial clone)
into a minichromosome. The necessary selectable markers and origins of
replication for
propagation of genetic material in E. cull. A,~~rnhcrcterirrm and
Arcrhidnp.si.s as well as the
necessary genetic loci for Agrnhcrcterir«rr mediated transformation into
Ar«hiclnpsi.s are
cloned into a conversion vector. Using Cre/IoxP recombination, the conversion
vectors
are recombined into BACs containing centromere fragments to form
minichromosomes.
FIG. 12A-T. Properties of centromeric re~ions on chromosomes II and IV. ('top)
Drawing of genetically-defined centromeres (gray shading. CEN2, left: CEN=1.
right).
adjacent pericentromeric DNA, and a distal segment of each chromosome, scaled
in Mb
as determined by DNA sequencing (gaps in the grey shading correspond to gaps
in the
physical maps). Positions in cM on the RI map
(http://nasc.nott.ac.uk/new_ri_map.html)
and physical distances in Mb. beginning at the northern telomere and at the
centromeric
gap. are shown. (B~ttom) The density of each feature (FIGS. 12A-12T) is
plotted relative
IS to the position on the chromosome in Mb. (FIG. 12A, 12K) cM positions for
markers on
the RI map (solid squares) and a curve representing the genomic average of I
cM/221 kb
(dashed line). A single crossover within CEN4 in the RI mapping population
(http://nasc.nott.ac.uk/new_ri_map.html: Somerville and Somerville. 1999) may
reflect a
difference between male meiotic recombination monitored here and recombination
in
female meiosis. (FIGS. 12B-12E and FIGS. 12L-120) The % of DNA occupied by
repetitive elements was calculated for a 100 kb window with a sliding interval
of 10 kb.
(FIGS. 12B, 12L) 180 by repeats: (FIGS. 1?C. 12M) sequences with similarity to
retroelements. including del. Tal. Tall. copiu. Athila. LINE. Ty3. TSCL. 106B
(Athila-like). Tat 1. LTRs and Cinful: (FIGS. 12D, 12N) sequences with
similarity t~
transposons. including Tag 1. En/Spm. Ac/Ds, Tam I MuDR. Limpet. MITES and
Mariner: (FIGS. 12E, 120) previously described centromeric repeats including
163A.
164A. 164B. ''78A. I I B7RE. mi 167. pAT?7. 160-. 180- and s00-by repeats. and
telomeric sequences (Nlurata et al.. 1997: I-Ieslop-Harrison et crl.. 1999:
Brandes et «L. 1997: Franz et crl.. 1998: Wright et crl.. 1996: Konieczny et
«L, 1991:
Pelissier or «l.. 1996: Voytas and Ausubel. 1988: Chve or crl.. 1997: Tsay et
«!., 1993:
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Richards et «L. 1991: Simoens et «!., 1988: Thompson et crl.. 1996: Pelissier
et «L, 1996
Franz et crl.. 1998: Pelissier et «f., 199; Voytas and Ausubel. 1988:
Thompson et crl., 1996). (FIGS. 12F, 12P) ~l~ adenosine + thymidine was
calculated for ,t
50 kb window wish a sliding interval of 25 kb (FIGS. 12G-12J, 12Q-12T). The
number
of predicted genes or pseudogenes was plotted over a window of 100 kb with a
sliding
interval of 10 kb. (FIGS. 12G, 12I, 12Q, 12S)' predicted genes (FIGS. 12G,
12Q) and
pseudogenes (FIGS. 12I, 12S) typically not found on mobile DNA elements:
(FIGS. 12H,
12J, 12R, 12T) predicted genes (FIGS. 12H, 12R) and pseudogenes (FIGs. 12.1,
I2T)
often carried on mobile DNA. including reverse transcriptase, transposase. and
retroviral
polyproteins. Dashed lines indicate regions in which sequencing or annotation
is in
progress, annotation was obtained from GenBank records
(http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html). from the AGAD database
(http://www.ti~r.or~/tdb/at/a~ad/.), and by BLAST comparisons to the database
of
repetitive Ar«bidop.ci.c sequences
(http:l/nucleus.cshl.org/protarablAtRepBase.htm):
though updates to annotation records may change individual entries, the
overall structure
of the region will not be significantly altered.
FIG. 13. Methods for converting a BAC clone containins centromere DNA into a
minichromosome for introduction into plant cells. The specific elements
described are
provided for exemplary purposes and are not limiting. A) diagram of the BAC
clone.
noting the position of the centromere DNA (red). a site-specific recombination
site (for
example, lox P). and the F origin of replication. B) Conversion vector
containing
selectable and color markers (for example. 3~S-Bar. nptll. LAT~2-GUS.
Scarecrow-
GFP), telomeres. a site-specific recombination site (for example. lox P),
antibiotic
?5 resistance markers (for example. amp or spc/str). A,yrnf~«crerium T-DNA
borders (Aaro
Left and Right) and origin of replication (RiA4~. C) The product of site
specific
recombination with the Cre recombinase at the lox P sites yields a circular
product with
centromeric DNA and markers flanked by telomeres. D) Minichromosome
immediately
utter transformation into plants: subsequently. the left and right borders
will likely be
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removed by the plant cell and additional telomeric sequence added by the plant
telomerase.
FIG. 14A-B. Conservation of centromere DNA. BAC clones (bars) used to
S sequence CErlr? (FIG. 14A) and CE'N4 (FIG. 14B) are indicated: arrows denote
the
boundaries of the genetically-defined centromeres. PCR primer pairs yielding
products
from only Columbia (fitled circles) or from both Landsberg and Columbia (open
circles):
BACs encoding DNA with homology to the mitochondrial genome (gray bars); 180
by
repeats (gray boxes); unsequenced DNA (dashed lines); and gaps in the physical
map
(double slashes) are shown.
FIG. l~A-B. Primers used to analyze conservation of centromere sequences in
the A. tlraliarrn Columbia and Landsbera ecotypes. FIG. 15A: Primers used for
amplification of chromosome 2 sequences. FIG. 1~B: Primers used for
amplification of
chromosome 4 sequences.
FIG. 16. Sequences common to CEN2 and CFN4. Genetically-defined
centromeres (bold lines), sequenced (thin lines), and unannotated (dashed
lines) BAC
clones are displayed as in FIG. 14A, B: Repeats AtCCS 1 (A. tlraliaocr
centromere
conserved sequence) and AtCCS2 (closed and open circles. respectively), AtCCS3
(triangles), and AtCCS4-7 (4-7, respectively) are indicated (GenBank Accession
numbers
AF204874 to AF?04880). and were identified using BLAST ?.0
(http://blast.wustl.edu).
FIG. 17. Sequenced BAC clones from centromere ?. The sequenced BAC clones
2s are indicated by the horizontal lines near the top of the figure (see for
example T14A4).
The red box denotes the boundaries of centromere 2. and for the BAC clones
that
comprise the centromere. GenBank Accession numbers are ~acn in the lower right
panel.
The contiguous sequences within the red box are given by SFQ ID N0:209 and SEQ
ID
N0:210. Horizontal lines below the sequenced clones indicate additional BAC
clones:
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sequenced end points of these BACs are indicated with a closed circle. Clones
with one
or more endpoints that are undetermined are indicated by red text.
FIG. 18. Sequenced BAC clones from centromere =I. The sequenced BAC clones
from centromere 4 are indicated by the horizontal lines near the top of the
figure (see for
example T24M8). The red box denotes the boundaries of centrornere 4. and for
the BAC
clones that comprise the centromere. GenBank Accession numbers are given in
the lower
right panel. The contiguous sequences within the red box are given by SEQ ID
N0:211
and SEQ ID N0:212. Horizontal lines below the sequenced clones indicate
additional
BAC clones: sequenced end points of these BACs are indicated with a closed
circle.
Clones with one or more endpoints that are undetermined are indicated by red
text.
FIG. 19. Sequence tiling path of centromeres I. 3. and 5. The boundaries of
these centromeres was determined as described in Copenhaver et al ( 1999).
Contig
numbers refer to the fingerprint contigs assembled by Marra c r crl. ( 1999).
Some of these
clones have been sequenced and accession numbers are provided (see attached
list). In
other cases. sequencing will be finished by the Arahiclop.sis ~enome project.
FIG. 20. Position of DNA from centromere 2 carried in BiBAC vectors. Clones
were placed on the physical map by fingerprint and PCR analysis and comparison
with
the sequenced BAC clones.
FIG. 21. Exemplary methods for adding selectable or screenable markers to
BiBAC clones. The desired marker is flanked by transposon borders. anti
incubated with
the BiBAC in the presence of transposase. Subsequently. the BiBAC is
introduced into
plants. Often these BiBACs may integrate into natural chromosome, creating a
dicentric
chromosome which rnay have altered stability and may cause chromosome
breakage.
resulting in novel chromosome fragments.
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FIG. 22. Assa~of chromosome stability. The stability of natural chromosomes,
constructed minichromosome. or dicentric chromosomes can be assessed by
monitoring
the assortment of color markers through cell division. The markers are linked
to the
centromere in modified BAC or BiBAC vectors and introduced into plants.
Regulation of
the marker gene by an appropriate promoter determines which tissues will be
assayed.
For example, root-specific promoters, such as SCARECROW make it possible to
monitor
assortment in files of root cells: post-meiotic pollen-specific promoters such
as LAT52
allow monitoring of assortment through meiosis, and general promoters such as
the 35S
Cauliflower mosaic virus promoter make it possible to monitor assortment in
many other
plant tissues. Qualitative assays assess the general pattern of stability and
measure the
size of sectors corresponding to marker loss, while quantitative assays
require knowledge
of cell lineage and alhw the number of chromosome loss events to be calculated
during
mitosis and meiosis.
FIG. 23A-D. SecLuence ali«nments for 180 be repeats from centromeres 1-4. The
left hand column indicates the BAC source of the repeat copy and an
arbitrarily assigned
number given to the sequence. For example, the designation f12g6-I indicates a
repeat
copy from BAC number f12g6 and arbitrarily given a repeat number of 1. The
nucleic
acid sequences of the BACs containing the repeat copies. designated f12g6.
f~al3.
t25f1~, t12j2, t14c8, t6c20. f2li?. and f6h8 are given by SEQ ID N0:184. SEQ
1D
N0:191. SEQ ID N0:189. SEQ ID \0:20. SEQ ID N0:206. SEQ ID N0:186. SEQ ID
N0:208 and SEQ ID N0:207. respectively. FIG. 23A. Alignment of 180 by repeats
from centromere 1. FIG. 23B. Alignment of 180 by repeats from centromere 2.
FIG.
23C. Alignment of 180 by repeata from centromere 3. FIG. 23D. Alignment of 180
by
repeats from centromere 4
DETAILED DESCRIPTION OF THE INVENTION
The inventors have overcome the deficiencies in the prior art by providing.
for the
first time. the nucleic acid sequence of a plant chromosome. The significance
of this
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achievement relative to the prior art is exemplified by the general lack tit
detailed
information in the art regarding the centromeres of multicellular organisms in
general.
To date. the most extensive and reliable characterization of centromere
sequences has
come from studies of lower eukaryotes such as S. cerevi.sine and S. ponrhe.
where the
ability to analyze centromere functions has provided a clear picture of the
desired DNA
sequences. The S. cereoisioe centromere consists of three essential regions,
CDEI,
CDEII, and CDEIII, totaling only 125 bp, or approximately 0.006 to 0.06% of
each yeast
chromosome (Carbon et ul.. 1990: Bloom 1993). S. pomhe centromeres are between
40
and 100 kB in length and consist of repetitive elements that comprise 1 to 3%
of each
chromosome (Baum et nl.. 1994). Subsequent studies, using tetrad analysis to
follow the
segregation of artificial chromosomes. demonstrated that less than 1/5 of the
naturally
occurring S. ponrbe centromere is sufficient for centromere function (Baum cu
crl., 1994).
In contrast, the centromeres of mammals and other higher eukaryotes are poorly
defined. Although DNA fragments that hybridize to centromeric regions in
higher
eukaryotes have been identified, little is known regarding the functionality
of these
sequences (see Tyler-Smith er crl.. 1993). In many cases centromere repeats
correlate with
centromere location, with probes to the repeats mapping both cytologically and
genetically to centromere regions. Many of these sequences are tandemly-
repeated
satellite elements and dispersed repeated sequences in arrays ranging from 300
kB to
5000 kB in length (Willard 1990). To date. only one of these repeats. a 171 by
element
known as the alphoid satellite. has been shown by in situ hybridization to be
present at
each human centromere (Tyler-Smith er crl.. 1993). Whether repeats themselves
represent
functional centromeres remains controversial, as other genomic DNA is required
to
confer inheritance upon a region of DNA ( Willard. 1997). Alternatively, the
positions of
some higher eukaryotic centromeres have been estimated by analyzing the
segregation of
chromosome fragments. This approach is imprecise. however. because a limited
set of
fragments can be obtained. and hecause normal centromere function is
intlueneed by
surrounding chromosomal sequences (for example. see Koomneef. 1983: FIG. ?).
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A more precise method for mapping centromeres that can be used in intact
chromosomes is tetrad analysis (Mortimer et crl.. 1981 ). which provides a
functional
definition of a centromere ih its native chromosomal context. At present. the
only
centromeres that have been mapped in this manner are from lower eukaryotes.
including
S the yeasts Sacchcrru»rvces cerevisiue, Sclri=osncclurro»ryces pombc:, and
Kluyvero»rvce.s
Icrcti.s (Carbon et al.. 1990; Hegemann et al., 1993). In these systems.
accurate mapping
of the centromeres made it possible to clone ceniromeric DNA. using a
chromosome
walking strategy (Clarke et crl., 1980). Subsequently. artificial chromosome
assays were
used to define more precisely the centromere sequences (Hegemann et al.. 1993;
Baum et crl.. 1994).
Attempts to develop a reliable centromeric assay in mammals have yielded
ambiguous results. For example. Hadlaczky et crl.. ( 1991 ) identified a 1=l
kB human
fragment that can. at low frequency, result in cle »ovo centromere formation
in a mouse
cell line. In situ hybridization studies, however. have shown that this
fragment is absent
from naturally occurring centromeres, calling into question the reliability of
this approach
for testing centromere function (Tyler-Smith et nl.. 1993). Similarly,
transfection of
alphoid satellites into cell lines results in the formation of new
chromosomes. yet these
chromosomes also contain host sequences that could contribute centromere
activity
(Haaf et crl.. 1992: Willard. 1997). Further. the novel chromosomes can have
alphoid
DNA spread throughout their length yet have only a single centromeric
constriction.
indicating that a block of alphoid DNA alone may be insufficient for
centromere function
(Tyler-Smith et crl., 1993).
Although plant centromeres can be visualized easily in condensed chromosomes.
they have not been characterized as extensively as centromeres froth yeast or
mammals.
Genetic characterization has relied on seyre~Tation analysis of chromosome
traaments, and
in particular on analysis of trisomic strains that carry a Genetically marked.
telocentric
fragment (for example. see Koornneef 1983: FIG. ? t. In addition. repetitive
elements
have heen identified that are either ~~eneticallv f Richards et crl.. 1991 )
or physically
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(Alfenito c~r crl.. 1993: Maluszynska et crl.. 19911 linked to a centromere.
In no case.
however. has the functional significance of these sequences been tested.
Cytology in Arabidop.si,s tlrcrliaraa has served to correlate centromere
structure
with repeat sequences. A fluorescent dye. DAPI. allows visualization of
centromeric
chromatin domains in metaphase chromosomes. A t7uorescence ur srrrr
hybrottzatton
(FISH) probe based on 180 by pAL ( repeat sequences colocalized with the DAPI
signature near the centromeres of all five Arabidnpsis chromosomes
(Maluszynska et nl.. 1991; Martinez-Zapater et ul., 1986). Although a
functional role for
pALI has been proposed, more recent studies have failed to detect this
sequence near the
centromeres in species closely related to Arcrbiclupsis thcrliorrn
(Maluszynska er crl.. 1993).
These results are particularly troubling because one of the species tested. A.
prnuilu, is
thought to be an amphidiploid. derived from a cross between A. tlrulrcnrcr and
another
close relative (Maluszynska et ul., 1991: Price et al.. 1990. Another
repetitive sequence,
pAtTl2. has been genetically mapped to within 5 cM of the centromere on
chromosome 1
and to the central region of chromosome ~ (Richards et crl.. 1991 ), although
its presence
on other chromosomes has not been established. Like pALI. a role for pAtTl2 in
centromere function remains to be demonstrated.
Due to the tact that kinetochores constitute a necessary link between
centromeric
DNA and the spindle apparatus. the proteins that are associated with these
structures
recently have been the focus of intense investigation (Bloom 1993: Earnshaw
1991 ).
Human autoantibodies that bind specifically in the vicinity of the centromere
have
facilitated the clonin~~ of centromere-associated proteins (CENPs, Rattner
1991 ). and at
least one of these proteins belongs to the kinesin superfamily of microtubule-
haled
motors (Yen 1991 ). Yeast centromere-binding proteins also have been
identified. both
through Genetic and biochemical studies (Bloom 1993: Lechner et crl., 1991 1.
The centromeres of Arcrbiclop.si.s rlrcrlicrrur have been mapped using
trisomic
strains, where the ae~7regation of chromosome fragments (Koornneef 191;3 i or
whole
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chromosomes (Sears et al.. 1970) way used to localize tour of the centromeres
to within
5, 12. 17 and 38 cM. respectively (F1G. 2). These positions have not been
refined by
more recent studies hecause the method is limited the difficulty of obtaining
viable
trisomic strains (Koornneef 19831. These factors introduce significant error
into the
calculated position of the centromere. and in Arabidopsis, where I cM
corresponds
roughly to 200 kB (Koornneef 1987: Hwang et al.. 1991 ), this method did not
map any of
the centromeres with sufficient precision to make chromosome walking
strategies
practical. Mapping of the Arabidopsi.s genome was also discussed by (Hauge m
nl.,
1991).
f0
I. Tetrad Analysis
With tetrad analysis. the recombination frequency between genetic markers and
a
centromere can be measured directly (FIG. 1 ). This method requires analysis
of all four
products of individual meiosis. and it has not been applied previously to
multicellular
eukaryotes because their meiotic products typically are dissociated.
Identification of the
c/uurtet mutation makes tetrad analysis possible for the first time in a
higher eukaryotic
system (Preuss et nl., !99=l). The grrnrtet (yrt I ) mutation causes the four
products of
pollen mother cell meiosis in Arnbidup.sis to remain attached. When used to
pollinate a
flower, one tetrad can result in the formation of four seeds, and the plants
from these
seeds can be analyzed genetically.
With unordered tetrads. such as those produced by S. cereaisicre or
Arcrbidnpsi.s.
genetic mapping usinU tetrad analysis requires that two markers be scored
simultaneously
(Whitehouse 1950). Tetrads fall into different classes depending on whether
the markers
are in a parental (nonrecombinant) or nonparental (recombinant) configuration
(FIG. I)
A tetrad with only nonrecombinant members is referred to as a parental ditype
(PD): one
with only recombinant members as a nonparental ditype (NPD): and a tetrad with
two
recombinant and two nonrecombinant members as a tetratype (TT) (Perkins 193).
If two
genetic loci are on different chromosomes. and thus assort independently. the
frequency
of tetratype (crossover products) versus parental or nonparental assortment
ditype
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(noncrossover products) depends on the frequency of crossover between each of
the two
loci and their respective centromeres.
Tetratype tetrads arise only when a crossover has occurred between a marker in
question and its centromere. Thus, to identity genes that are closely linked
to the
centromere, markers are examined in a pair-wise fashion until the TT frequency
approaches zero. The genetic distance (in centimorgans, cM) between the
markers and
their respective centromeres is defined by the function [( 1/2)TTJ/100
(Mortimer et «l.. 1981 ). Because positional information obtained by tetrad
analysis is a
representation of physical distance between two points. as one approaches the
centromere
the chance of a recombination event declines.
Tetrad analysis has been used to genetically track centromeres in yeasts and
other
fungi in which products of a single meioses can be collected. The budding
yeast
l~ S«cch«romvce.r cerevi.si«e lacks mitotic condensation and thus cytogenetics
(Hegemann et «L, 1993), yet due to tetrad analysis. has served as the vehicle
of discovery
for ceniromere function. Meiosis is followed by the generation of four spores
held within
an ascus and these can be directly assayed for gene segre~=anon.
The recessive grtl mutation makes it possible to perform tetrad analysis in
Arcrhiclnpsi.s by causing the four products of meiosis to remain attached
(Preuss et «l.. 1994; and Smythe 1994: both incorporated herein by reference).
As
previously shown, within each tetrad. genetic loci segre~~ate in a 2:? ratio
(FIG. 6).
Individual tetrads can be manipulated onto f)owers with a fine brush (at a
rate of 20
~> tetrads per hour). and in 30% of such crosses. tour viable seeds can be
obtained
( Preuss et «l.. 1994 ).
Mapping centromeres with high precision requires a dense ~~enetic map. and
although the current Ar«hidnp.si.s map contains many visible markers, it would
be
_laborious to cross each into the gril background. Alternatively. hundreds of
DNA
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polymorphisms can be introduced simultaneously by crossing two different
strains. both
containing the drtl nuUation_ A dense RFLP map (Chant et «l., 1988) and PCR-
based
maps (Koniecznv et «L. 1993: Bell et crl.. 1990 have been generated in
Ar«hidnp.ci.c from
crosses of the Landsber~ and Columbia strains (An«hidnp.ci.c map and genetic
marker data
is available from the Internet at http://genome-www.stanford.edu/Arabidopsis
and
http://cbil.humgen.upenn.edu/at~c/sslp_info/sslp.html). These strains differ
by I% at the
DNA sequence level and have colinear genetic maps (Chano et crl., 1988:
Koornneef.
I 987).
Centromere mapping with tetrad analysis requires simultaneous analysis of two
markers. one of which must be centromere-linked (FIG. l ). To identify these
centromere-linked markers, markers distributed across all ~ chromosomes were
scored
and compared in a pairwise fashion.
Initially, ~ienetic markers that can be scored by PCR analysis were tested
(Konieczny et crl.. 1993; Bell et crl., 1994). Such markers are now
sufficiently dense to
map any locus an as additional PCR-detectable polymorphisms are identified
they are
incorporated into the analyses. In addition. as described in FIG. ~. new CAPS
and SSLP
markers useful for mapping the centromere can be readily identified.
A collection of Arcrbidop.cis tetrad sets was prepared by the inventors for
use in
tetrad analysis. To date. progeny plants from > l .000 isolated tetr<~d seed
sets have been
Uerminated and leaf tissue collected and stored from each of the tetrad
progeny plants.
The leaf tissue from individual plants was used to make DNA for PCR based
marker
analysis. The plants also were allowed to self-fertilize and the seed they
produced was
collected. From each of these individual seed sets. seedlings can be
germinated and their
tissues utilized for makin~~ genomic Di\A. Tissue pooled from multiple
seedlings is
useful for making Southern genornic DNA blots for the analysis of restriction
fragment
length polvmorphisms (RFLPs). An exemplary liar of the aced stock of
informative
individuals used for tetrad analysis is liven in FIG. -1.
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II. l~Iappin~ Strate~v
Previous DNA fingerprint and hybridization analysis of two bacterial
artificial
chromosome (BAC) libraries had led to the assembly of physical mops covering
nearly all
single-copy portions of the Arafiiclap.si.s ~enome (Marry et nl.. 1999).
However. the
presence of repetitive DNA near the Arabidapsis centromeres. including 180 by
repeats.
retroelements. and middle repetitive sequences complicated efforts to anchor
centromeric
BAC contigs to particular chromosomes (Murata et crl., 1997: Heslop-Harrison
et crl.,
1999: Brandes et crl., 1997; Franz et al.. 1998: Wright et crl.. 1996;
Koniecznyet crl.. 1991:
Pelissier et ul., 1995; Voytas and Ausubel. 1988; Chye et ul., 1997: Tsay et
crl., 1993:
Richards et al., 1991: Sirnoens et crl.. 1988: Thompson et crl., 1996:
Pelissier et al.. 1996).
The inventors used genetic mapping to unambiguously assign these unanchored
contias to specific centromeres. scoring polymorphic markers in =18 plants
with
crossovers informative for the entire genorne (Copenhaver et al.. 1998). In
this manner.
several centromeric contigs were connected to the physical maps of the
chromosome arms
(see EXAMPLE 6), and a large set of DNA markers defining centromere boundaries
were
generated. DNA sequence analysis confirmed the structure of the conti~s for
chromosomes II and IV (Lin et al.. 1999).
CEN2 and CEN=1 were selected in particular for analysis. Both reside on
structurally similar chromosomes with a 3.~ Mb rDNA arrays on their distal
tips, with
regions measuring 3 and 2 Mb. respectively, between the rDNA and centromeres,
and 16
and 13 Mb regions on their long arms (Copenhaver and Pikaard. 1996).
The virtually complete and annotated sequence of chromosomes tl and IV was
used to conduct an analysis of centromeres at the nucleotid; level
(http://www.ncbi.nlm.nih.~~ov/Entrez/nucleotide.html). The sequence
composition was
analyzed within the genetically-defined centromere boundaries and compared to
the
adjacent pericentromeric rc~Tion~ ( FIGs. l 2A-T). Analysis of the two
centrornerec
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facilitated comparisons of sequence patterns and identification of conserved
sequence
elements.
The centromere sequences were found to harbour 180 by repeat sequences. These
sequences were found to reside in the gaps of each centromeric contig (FIG. 3.
FIGS. 12B.
12L), with few repeats and no long arrays elsewhere in the genome. BAC clones
near
these gaps have end sequences corresponding to repetitive elements that likely
constitute
the bulk of the DNA between the contigs, including I80 by repeats. SS rDNA or
160 by
repeats (FIG. 3). Fluorescent irr situ hybridization has shown these
repetitive sequences
are abundant components of Ar«hi~lnp.si.s centromeres (Murals et «!.. 1997:
Heslop-Harrison et «l.. 1999: Brandes et «!., 1997). Genetic mapping and
pulsed-field
gel electrophoresis indicate that many 180 by repeats reside in long arrays
measuring
between 0.4 and I .4 Mb in the centromeric regions (Round et «L. 1997);
sequence
analysis revealed additional interspersed copies near the gaps. The inventors
specifically
1S contemplate the use of such 180 by repeats for the construction of
minichromosomes.
The annotated sequence of chromosomes II and IV identified regions with
homology to
middle repetitive DNA. both within the functional centromeres and in the
adjacent
regions (FIGS. 12B-12E and 12L-120).
In a 4.3 Mb sequenced re~Tion that includes CENZ and a 2.8 Mb sequenced region
that includes CEN4. retrotransposon homology was found to account for > 10% of
the
DNA sequence. with a maximum of 62CO and 709c, respectively (FIGS. 12C. l2M).
Sequences with similarity to transposons or middle repetitive elements were
found to
occupy a similar zone. but were less common (29% and 1 I % maximum density for
chromosomes II and 1V respectively (FIGS. 12D-12E and FIG. 12N-120). Finally.
unlike
in the case of Drn.cnplril« and Ne rrro.cpnr« centromeres l Sun et «L. I 997:
Cambareri et crl., 1998) low complexity DNA. including microsatellites,
homopolvmer
tracts. and AT rich isochores. were not found to be enriched in the
centromeres of
Arzrhidnp.sis. Near CEN2. simple repeat sequence densities were comparable to
those on
the distal chromosome arms. oceupyin~~ l.ir~ of the sequence within the
centromere.
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3.2% in the flanking regions. and ranging from 20 to 319 by in length (71 by
on average).
Except for an insertion of mitochondria) DNA at CEN2 the DNA in and around the
centromeres did not contain any large regions that deviated significantly From
the
eenomic average of - 64% A + T (FIGs. 12F. I2P) (Bevan et ul., 1999).
Unlike the 180 by repeats, all other repetitive elements near CEN2 and CEN4
were less abundant within the genetically-defined centromeres than in the
flanking
regions. The high concentration of repetitive elements outside of the
functional
centromere domain suggest they may be insufficient for centromere activity.
Thus,
identifying segments of the Arahidopsi.c genome that are enriched in these
repetitive
sequences does not pinpoint the regions that provide centromere function: a
similar
situation may occur in the genomes of other higher eukaryotes.
The repetitive DNA flanking the centromeres may play an important role,
forming
1 ~ an altered chromatin conformation that serves to nucleate or stabilize
centromere
structure. Alternatively, other mechanisms could result in the accumulation of
repetitive
elements near centromeres. Though evolutionary models predict repetitive DNA
accumulates in regions of . low recombination (Charlesworth et crl., 1986;.
Charlesworth er crl., 1994). many Arcrhidopsi.s repetitive elements are more
abundant in
the recombinationally active pericentromeric regions than in the centromeres
themselves.
Instead. retroelements and other transposons may preferentially insert into
regions
flanking the centromeres or be eliminated from the rest of the aenome at a
higher rate.
III. Centromere Compositions
Certain aspects of the present invention concern isolated nucleic acid
segments
and recombinant vectors comprisin= a plant centromere. In one embodment of the
invention. the plant eentromere is an Arcrhidohsi.c thalicuru centromere. In a
further
embodiment of the invention. nucleic acid sequences comprising an .-1.
tlrnlicrna
chromosome ? centrotnere are provided. The sequence of the Arcrhidop.cis
rhalicrrrcr
chromosome 2 centromere is exemplified by the nucleic acid sequences of SEQ ID
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N0:209 and SEQ ID N0:210. As shown in FIG. 17, the nucleic acid sequences of
SEQ
ID N0:209 and SEQ ID N0:210 flank a series of 180 by repeats in centromere 2
of A.
~lrcrlicuur. As such, the chromosome 2 centromere may further be defined as
comprising n
number of repeats linked to a nucleic acid sequence included in SEQ ID N0:209
or SEQ
ID N0:210. or sequences isolated from both of those sequences. In particular
embodiments of the invention, the number of repeats (n), is about 2. 4. 8, 1~.
25, 40. 70,
100, 200. 400. 600. 800, 1,000. I .500. 2.000. 4.000, 6.000. 8000. 10,000.
30,000. 50,000
or about 100,000. The actual repeat sequence used may vary. Representative
samples of
repeat sequences that could be used are given in FIGS. 23A-23D and included in
the
nucleic acid sequences given by SEQ )D NOs 184-208. The length of the repeat
used
may also vary. and may include repeats of. for example. about 10 bp. 20 bp. 40
bp, 60 bp.
80 hp, 100 bp. I20 bp. 140 bp, I50 bp. 160 bp. 170 bp, 180 bp. l90 bp. or
about 200 by
or larger or a repeat sequence, for example, as listed in F1G. 23A-F1G.23D and
included
in the nucleic acid sequences given by SEQ ID NOs 184-208
IS
Isolated segments of the nucleic acid sequences of SEQ ID N0:209 and SEQ ID
N0:210 are also contemplated to be of use with the invention, either with or
without
beinv linked to a series of repeats. Particularly, contiguous nucleic acid
segments of
about 100. 200. 400. 800. 1,500. 3,000. x.000, 7.500, 10,000, 15.000. 25.000.
40.000.
75.000. 100.000. 125.000, 150,000. 250.000. 350.000. 40.000. 600.000. 700.00
and
about 800.000 by of the nucleic acid sequences of SEQ ID N0:209 or SEQ ID
N0:210
specifically form part of the instant invention. In particular embouiments of
the
invention. such nucleic acid sequences may be linked to n number of repeated
sequences.
for example. where n is 2. 4. 8, 15, 2~. 40. 70. 100, 200. 400. 600, 800. i
.000. 1.500.
2.000. 4.000. 6.000. 8000, 10,000. 50.000 or about 100.000. The repeat
sequence may
comprise. for example. about 10 bp. '0 bp. 40 bp, 60 bp. 80 bp, 100 bp. I 20
bp. 140 hp.
1 ~0 bp. l60 bp. 170 hp. ! 80 bp. I 90 hp. or about 200 by or a larger segment
of
contiguous nucleotides of. for example. a repeat listed in FIG. 23A-FIG.?3D
and included
in the nucleic acid sequences given by SEQ ID NOs 184-208.
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In another embodiment of the invention. nucleic acid sequences comprising r»r
A.
tlr«licr»cr chromosome 4 centromere are provided. The sequence of the
Ar«hidnp.ci.c
tlr«li«»« chromosome 4 centromere is exemplified by the nucleic acid sequences
of SEQ
ID N0:21 I and SEQ ID N0:212. As shown in FIG. 18, the nucleic acid sequences
oC
SEQ ID N0:211 and SEQ ID N0:212 in Ar«hi~lnpsi.s flank a series of repeated
sequences. As such, the chromosome 4 centromere may further be defined as
comprising
n number of repeats linked to a nucleic acid sequence included in SEQ ID N0:21
1 or
SEQ ID N0:212, or sequences from both SEQ ID N0:21 I and SEQ ID N0:212. In
particular embodiments of the invention, the number of repeats (n), is about
?. 4. 8. I5,
25. 40. 70. I 00, 200, 400. 600, 800, 1,000, 1,500, 2,0(10, 4,000, 6,000,
8000. I 0.000.
50,000. or about 100,000. The actual repeat sequence used may vary.
Representative
samples of repeat sequences that could be used are given in FIGS. 23A-23D.
wherein
these sequences are included in the nucleic acid sequences given by SEQ ID NOs
184-
208. The length of the repeat used may also vary, and may include repeats of,
for
example. about 10 bp. 20 bp, 40 bp. 60 bp, 80 bp, 100 bp, 120 bp, 140 bp. 150
bp. 160
bp, l70 bp, 180 bp, I 90 bp, or about 200 by or larger.
Isolated segments of the nucleic acid sequences of SEQ ID N0:21 1 and SEQ ID
N0:21? are also contemplated to be of use with the invention. either with or
without
being linked to a series of repeated sequences. Particularly. contiguous
nucleic acid
segments of about 100. 200. 400. 800. 1.,00. 3,OOC1, 5.000, 7.500. 10.000.
15.000.
25.000. 40.000, 75,000. 100.000. 125.000. 150.000, 250.000, 350,000, 450.000.
600.000.
700.00 by of the nucleic acid sequences of SEQ ID N0:21 l or SEQ (D N0:212
specifically form part of the instant invention. In particular embodiments of
the
invention. such nucleic acid sequences may be linked to n number of repeated
sequences.
for example. where n is 2. 4. 8. L ~. 25. 40. 70. ! 00, 200. 400. 600. 800.
1.000. l .500.
2.000. -4.000. 6.000. 8000. 10,000. 50,000 or shout 100.000. The repeat
sequence may
comprise. for example. about 10 bp. 20 bp. 40 bp. 60 bp. 80 bp, 100 bp. l 20
bp. l40 bp.
150 bp. 160 bp, 170 bp. l80 bp. 190 bp. or about 200 by or a larger segment of
contiguous nucleotides of the sequence of SEQ ID N0:184-208.
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Also provided by the invention are regulatory regions from the Arcrhiclopsi.c
polyubiquitin I 1 gene. including promoter anti terminator sequences thereof.
The nucleic:
acid sequences of these regulatory regions are exemplified by the nucleic acid
sequences
of SEQ ID Ivr0:180 and SEQ ID N0:181. Also included with such sequences are
contiguous stretch of from about 10. 15. 20, 25. 30. =t0. 50. 75, 100. 125, I
50. 200. 300.
500, 750, 1,000, 1.500. and about 2,000 nucleotides of the nucleic acid
sequence of SEQ
ID N0:180 and SEQ ID N0:181. In particular embodiments of the invention. it
may be
desirable to operably link the Arnbidoh.ci.c polyubiquitin 1 1 promoter
sequences to the 5'
end of a coding sequence. It may also be desirable to operably link the
Arcrhiclopsis
polyubiquitin I 1 terminator sequence to the 3~ end of a coding sequence.
Still further provided by the invention are regulatory regions from the
Arubidnhsi.c
40S ribosomal protein S 16 gene. including promoter and terminator sequences
thereof.
The nucleic acid sequences of these regulatory regions are exemplified by the
nucleic acid
sequences of SEQ lD N0:182 and SEQ ID N0:183. Also included with such
sequences
are continuous stretch of from about l0. I5. ?0. ?5. 30. ~10, 50. 75. 100.
1'_'S, 150. 200.
300. 500. 750. 1,000. 1.500, and about ?.000 nucleotides of the nucleic acid
sequence of
SEQ 1D N0:182 and SEQ ID N0:183. In particular embodiments of the invention,
it
may be desirable to operably link the Arcrhiclmj~.ci.s =lOS ribosomal protein
S 16 gene
.cequences to the 5' end of a coding sequence. It may also be desirable to
operably (ink
the Arcrbidopsi.c 40S ribosomal protein S 16 gene sequence to the 3' end of a
coding
sequence.
Still further provided by the invention are «ene sequences and related
regulatory
elements and sequences with other functions from centromere regions. In
particular. the
invention includes the centromere sequences <_iven by SEQ ID NO:1. SEQ ID
NO:?. SEQ
ID N0:3. SEQ ID N0:4. SEQ ID NO:~. SEQ ID N0:6. SEQ ID N0:7. SEQ ID N0:8.
SEQ ID N0:9. SEQ ID NO:10. SEQ ID NO: I I . Sf-:Q ID NO: l?. SEQ ID N0:13. SEQ
ID
NO: l~l. SEQ 1D NO:15. SEQ ID NO: f f,. SEQ 1D X0:17. SEQ ID N0:18. SEQ ID
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N0:19_ SEQ ID N0:20. and SEQ ID N0:21, as well as lengths of about 5, 10. 15.
20. 25.
30, 40, 50. 60. 70, 80, 90, 100. 1 I 0. 12~. I 50. ! 7~. 200. 250. 300, 350,
400. 500, 550.
590. 1,000. and about 1.500 contiguous nucleotides of these sequences, up to
and
including the full length of the sequences.
Centromere-containing nucleic acid sequences may be provided with other
sequences for the creation and use of recombinant minichromosomes. Such
nucleic acid
sequences specifically within the scope of the invention include the nucleic
acid
sequences listed in the sequence listing provided herewith.
The present invention concerns nucleic acid segments, isolatable from A.
tlzcrliana
cells, that are enriched relative to total ~enomic DMA or other nucleic acids
and are
capable of conferring centromere activity to a recombinant molecule when
incorporated
into the host cell. As used herein, the term "nucleic acid segment" refers to
a nucleic acid
molecule that has been purified from total genomic nucleic acids of a
particular species.
Therefore. a nucleic acid segment conferring centromere function refers to a
nucleic acid
segment that contains centromere sequences yet is isolated away from, or
purified free
from, total genomic nucleic acids of A. thaliana. Included within the term
"nucleic acid
segment". are nucleic acid segments and smaller fragments of such segments,
and also
recombinant vectors, including, for example, BACs. ~'ACs, plasmids, cosmids,
phage,
viruses. and the like.
Similarly, a nucleic acid segment compriwng an isolated or purified
centromeric
sequence refers to a nucleic acid segment including centromere sequences and.
in certain
aspects. regulatory sequences, isolated substantially away from other
naturally occurring
sequences. or other nucleic acid sequences. In this respect. the term "gene"
is used for
simplicity to refer to a functional nucleic acid see ment. protein,
polypeptide or peptide
encoding unit. As will be understood by those in the art. this functional term
includes
both genomic sequence,, cDNA sequences and smaller engineered gene se~~ments
that
may express. or may be adapted to express. proteins. polypeptides or peptides.
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"Isolated substantially away from other sequences" means that the sequences of
interest, in thin case centromere sequences. are included within the genomic
nucleic acid
clones provided herein. Of course. this refers to the nucleic acid segment as
originally
isolated. and does not exclude genes or coding regions later added to the
segment by the
hand of man.
In particular embodiments, the invention concerns isolated nucleic acid
segments
and recombinant vectors incorporating nucleic acid sequences that encode a
centromere
functional sequence that includes a contiguous sequence from the centromeres
of the
current invention. In certain other embodiments, the invention concerns
isolated nucleic
acid segments and recombinant vectors that include within their sequence a
contiguous
nucleic acid sequence from an A. tlratiana centromere. Again. nucleic acid
segments that
exhibit centromere function activity will be most preferred.
The nucleic acid segments of the present invention. regardless of the length
of the
sequence itself, may be combined with other nucleic acid sequences, such as
promoters.
polyadenylation signals, additional restriction enzyme sites. multiple cloning
sites, other
coding segments, and the like, such that their overall length may vary
considerably. It is
therefore contemplated that a nucleic acid fragment of almost any length may
be
employed, with the total length preferably being limited by the ease of
preparation and
use in the intended recombinant DNA protocol.
(i) Prinrer.c card Probes
In addition to their use in the construction of recombinant constructs,
includin~~
minichromosomes. the nucleic acid sequences disclosed herein may find a
variety of
other uses. For example, the centromere sequences described herein may find
use as
probes or primers in nucleic acid hybridization embodiments. As such. it is
contemplated
that nucleic acid segments that comprise a sequence region that consists of at
least a 14
nucleotide long contiguous sequence that has the same sequence as. or is
complementary
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to, a l4 nucleotide long contiguous DNA segment of a centromere sequence of
the
current invention. for example, of the sequences given by SEQ ID NOS:1-2l?.
and
particularly. SEQ 1D NOS: l-? 1 and SEQ ID NOS:180-212, will find particular
utility.
Loner contiguous identical or complementary sequences. e.y., those of about
20. 30. 40.
50, 100. 200. 500, 1.000. 2.000, 5.000 bp, etc., including all intermediate
lengths and up
to and including the full-length sequence of the sequences given in SEQ ID
NOS:I-212.
also will be of use in certain embodiments.
As described in detail herein. the ability of such nucleic acid probes to
specifically
hybridize to centromeric sequences will enable them to be of use in detectin'
the presence
of similar, partially complementary sequences from other plants or animals.
However.
other uses are envisioned. including the use of the centromeres for the
preparation of
mutant species primers. or primers for use in preparing other genetic
constructions.
Nucleic acid fragments having sequence regions consisting of contiguous
nucleotide stretches of 8. 9, 10. I I, 12, 13, 14, 15, 16, 1?, 18, 19. 20. 21.
22, 23, 24, 25. 26,
?7: 28, 29, 30, 31. 32, 33, 34. 35. 36. 37, 38. 39, 40, 41. 42, 43, 44. 45,
46, 47. 48. 49. 50.
55, 60. 65. 70, 75. 80, 85. 90. 9>. 100 or even of IOl-200 nucleotides or so.
identical or
complementary to a centromere sequence of the current invention, including the
sequences given in SEQ ID NOS:I-212. are particularly contemplated as
hybridization
probes for use in, e.,~., Southern and Northern blotting. Smaller fragments
will generally
find use in hybridization embodiments. wherein the length of the contiguous
complementary region may be varied. such as between about 10-14 and about 100
or 200
nucleotides, but larber contiguous complementarily stretches also may be used.
according
to the length compiernentary sequences one wishes to detect.
Of course. fragments rnay also be obtained by other techniques such as. e.,~..
by
mechanical shearing or by restriction enzyme di~~estion. Small nucleic acid
segments or
fragments may be readily prepared by. for example. directly synthesizing the
fragment by
chemical meam. as is commonly practiced using an automated oligonucleotide
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synthesizer. Also. fragments may be obtained by application of nucleic acid
reproduction
technology, such as the PCR~~" technology of U. S. Patents 4.683.195 and
4.683,202
(each incorporated herein by reference). by introducin~_ selected sequences
into
recombinant vectors for recombinant production. and by other recombinant DNA
techniques generally known to those of skill in the art of molecular biology.
Accordingly, the centromere sequences of the current invention may be used for
their ability to selectively form duplex molecules with complementary
stretches of DNA
fragments. Depending on the application envisioned. one will desire to employ
varying
conditions of hybridization to achieve varying degrees of selectivity of
probe.towards
target sequence. For applications requiring high selectivity. one will
typically desire to
employ relatively stringent conditions to form the hybrids, e.g., one will
select relatively
low salt and/or high temperature conditions. such as provided by about 0.02 M
to about
0.15 M NaCI at temperatures of about 50°C to about 70°C. Such
selective conditions
IS tolerate little, if any, mismatch between the probe and the template or
target strand, and
would be particularly suitable for isolating centromeric DNA segments. Nucleic
acid
sequences hybridizing under these conditions and the conditions below to the
nucleic acid
sequences provided by the invention, including those given by SEQ ID NOS:I-
212. form
a part of the invention. Detection of nucleic acid segments via hybridization
is
well-known to those of skill in the art. and the teachings of U. S. Patents
4,965.188 and
5.176.995 (each specifically incorporated herein by reference in its entirety)
are
exemplary of the methods of hybridization analyses. Teachings such as those
found in
the texts of Maloy et nl.. 1991: Seeal. 1976: Prokop. 1991: and Kuby, 1994,
are
particularly relevant.
ZS
Of course. for some applications. for example. where one desires to prepare
mutants employing a mutant primer strand hybridized to an underlying template
or where
one seeks to isolate centromere function-conferring sequences from related
species,
functional equivalents. or the like. less stringent hybridization conditions
will typically he
needed in order to allow formation of the heteroduplet. In these
circumstances, one may
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desire to employ conditions such as about 0.15 M to about 0.9 M salt. at
temperatures
ranging from about 20°C to about 55°C. Cross-hybridizing species
can thereby be readily
identified ;ts positively hybridizing signals with respect to control
hybridizations. 1n any
case. it is generally appreciated that conditions can be rendered more
stringent by the
addition of increasing amounts of formamide. which serves to destabilize the
hybrid
duplex in the same manner as increased temperature or decreased salt. Thus.
hybridization conditions can be readily manipulated. and thus will generally
be a method
of choice depending on the desired results.
In certain embodiments, it will be advantageous to employ nucleic acid
sequences
of the present invention in combination with an appropriate means, such as a
label, for
determining hybridization. A wide variety of appropriate indicator means are
known in
the art, including fluorescent. radioactive. enzymatic or other iigands, such
as
avidin/biotin, which are capable of giving a detectable signal. In preferred
embodiments.
I S one will likely desire to employ a fluorescent label or an enzyme ta'r.
such as urease.
alkaline phosphatase or peroxidase, instead of radioactive or other
environmentally
undesirable reagents. In the case of enzyme tags. colorimetric indicator
substrates are
known that can be employed to provide a means visible to the human eye or
spectrophotometrically. to identify specific hybridization with complementary
nucleic
acid-containing samples.
In general. it is envisioned that the hybridization probes described herein
will be
useful both as reagents in solution hybridization as well as in embodiments
employing a
solid phase. In embodiments involving a solid phase. the test DNA (or RNA) is
adsorbed
?5 or otherwise affixed to a selected matrix or surface. This fixed. single-
stranded nucleic
acid is then subjected to specific hybridization with selected probes under
desired
conditions. The selected conditions will depend on the particular
circumstances based on
the particular criteria required (depending. for example. on the G+C content.
type of
target nucleic acid. source of nucleic acid. size of hybridization probe,
orc.l. Following
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CA 02362897 2001-09-18
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washing of the hybridized surface so as to remove nonspecifically bound probe
molecules, specific hybridization is detected. or even quantitated, by means
of the label.
(ii) Lame Nucleic Acicl Segnrertts
Using the markers t7ankin~ each centromere (see FIG. 3) it may be possible to
purify a contiguous DNA fragment that contains both t7anking markers and the
centromere encoded between those markers. In order to carry this out. very
large DNA
fragments up to the size of an entire chromosome are prepared by embedding
Aruhiclopsis
tissues in agarose using, for example. the method described by Copenhaver et
al.. ( 1995).
These large pieces of DNA can be digested in the agarose with any restriction
enzyme.
Those restriction enzymes which will be particularly useful for isolating
intact
centromeres include enzymes which yield very lame DNA fragments. Such
restriction
enzymes include those with specificities greater than six base pairs such as.
for example.
Asc I. Bae I. BbvC I. Fse I. Not I. Pac I. Pme I. PpuM I, Rsr I1, SanD I. Sap
I. SexA I, Sfi
1~ I, Sgf 1. SgrA 1, Sbf I, Srf 1. Sse8387 I. Sse8647 I. Swa. UbaD I, and UbaE
1. or any other
enzyme that cuts at a low frequency within the Arabiclopsis genome, and
specifically
within the centromeric region. Alternatively, a partial digest with a more
frequent cutting
restriction enzyme could be used.
Alternatively. lame DNA fragments spanning some or all of a centromere could
be produced using RecA-Assisted Restriction Endonuclease (RARE) cleav~t~~e
(Ferrin.
1991 ). In order to carry this out. very large DNA fragments up to the size of
an entire
chromosome are prepared by embedding Aruhidop.si.s tissues in agarose using.
for
example. the method described by Copenhaver et nl.. ( 19951. Single stranded
DNA
oligomers with sequences homologous to sites flanking the region of DNA to be
purified
are made to form triple stranded complexes with the agarose embedded DNA using
the
recombinase enzyme RecA. The DNA is then treated with a site specific
methylase such
as, for example. Alu 1 methvlase. BamH I methylase, dam methylase. EcoR I
methylase.
Hae III methylase. Hha I methvlase. Hpa II methylase. or ivlsp methviase. The
methyiasc
will modify all the sites specified by its recognition sequence except those
within the
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WO 00/~~325 PCT/US00/07392
triplex region protected by the RecA/DNA oli~omer complex. The RecA/DNA
oli~lomet
complex are then removed from the a~arose embedded DNA and the DNA is then
cleaved with the restriction enzyme corresponding to the methylase used. for
example. if
EcoRI methylase was used then EcoRl restriction endonuclease would be used to
perform
the cleavage. Only those sites protected from modification will be subject to
cleavage by
the restriction endonuclease. Thus by choosing targets flanking the
centromeric regions
that contain the recognition sequence of a site specific methylase/restriction
endonuclease
pair RARE can be used to cleave the entire region from the rest of the
chromosome. It is
important to note that this method can be used to isolate a DNA fragment of
unknown
composition by using sequence information flanking it. Thus, this method may
be used
to isolate the DNA contained within any gaps in the physical map for the
centromeres.
'The DNA isolated by this method can then be sequenced.
The large DNA fragments produced by digestion with restriction enzymes or by
IS RARE cleavage are then separated by size using pulsed-field gel
electrophoresis (PFGE)
(Schwartz et al., 1982). Specifically. Contour-clamped Homogeneous Electric
Field
(CHEF) electrophoresis (a variety of PFGE) can be used to separate DNA
molecules as
large as 10 Mb (Chu et crl., 1985). Large DNA fragments resolved on CHEF gels
can
then be analyzed using standard Southern hybridization techniques to identify
and
measure the size of those fragments which contain both centromere flanking
markers and
therefor, the centromere. After determining the size of the centromere
containing
fragment by comparison with known size standards, the region from the ~~cl
that contains
the centromere fragment can be cut out of a duplicate ael. This centromeric
DNA can
then be analyzed. sequenced. and used in a variety of applications, as
described below.
including the construction of minichromosomes. As indicated in detail below.
minichromosornes can be constructed by attaching telomeres and selectable
markers to
the centromere fragment cut from the a~am;e gel using standard techniques
which allow
DNA ligation within the gel slice. Plant cells can then be transformed with
this hybrid
DNA molecule using the techniques described herein below.
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IV. ##Recombinant Constructs Comprising Centromere Seguences ##
In light of the instant disclosure it will be possible for those of ordinary
skill in the
art to construct the recombinant DNA constructs described herein. Useful
construction
methods are well-known to those of skill in the art (see. for example,
Maniatis et al., 198?). As constructed. the minichromosome will preferably
include an
autonomous replication sequence (ARS) functional in plants, a centromere
functional in
plants, and a telomere functional in plants.
The basic elements in addition to a plant centromere that may be used in
constructing a minichromosome vector are known to those of skill in the art.
For
example, one type of telomere sequence that could be used is an Arabiclopsis
telomere,
which consists of head to tail arrays of the monomer repeat CCCTAAA totaling a
few
(for example 3-4) kb in length. The telomeres of ,4rabidopsis, like those of
other
organisms. vary in length and do not appear to have a strict length
requirement. An
1~ example of a cloned telomere can be found in GenBank accession number
M20I58
(Richards and Ausubel, 1988). Yeast telomere sequences have also been
described (see.
e.,y., Louis. 1994: Genbank accession number S70807). Additionally. a method
for
isolating a higher eukaryotic telomere from Al'(fI7lCIlJ(JSlS IIrcrIrC111C!
WaS described by
Richards and Ausubel ( 1988).
It is commonly believed that higher eukaryotes do not posses a specific
sequence
that is used as a replication origin. but instead replicate their DNA from
random sites
distributed along the chromosome. In Arclhiclnp.ci.c, it is thought that the
cell will form
origins of replications about once every 70 kb (Van't Hot, 1978). Thus.
because higher
2~ eukaryotes have origins of replication at potentially random positions on
each
chromosome. it is not possible to describe a specific origin sequence. but it
may generally
be assumed that a segment of plant DNA of a sufficient size will be recognized
by the cell
and origins will be generated on the construct. For example, any piece of
Arcrhidnp,ci.c
~enomic DNA larger than 70 kb would be expected to contain an ARS. By
including
such a segment of DNA on a recombinant vectc>r. ARS function may be provided
to the
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vector. Additionally, many S. cerevi.sicre autonomous replicating sequences
have been
sequenced and could be used to fulfill the ARS function. One example is the
Saccharonrvce.s cerevi.sicre autonomously replicating sequence ARS 131 A
(GenBank
number L25319). Many origins of replications have been also been sequenced and
cloned from E. cnli and could be used with the invention. for example. the Col
E I origin
of replication (Ohmori and Tomizawa. 1979: GenBank number V00270). One
Agrobucterirrrrr origin that could be used is RiA4. The localization of
origins of
replication in the plasmids of Agrobcrcterirr»r rlri:.o,qeues strain A4 was
described by
Jouanin et al. ( 1985).
(i) Corrsideratiorts in the Preparation oJReconrbinaru Cortstrrtcts
In addition to the basic elements, positive or negative selectable plant
markers
(e.X.. antibiotic or herbicide resistance genes). and a cloning site for
insertion of foreign
DNA may be included. In addition. a visible marker. such as green fluorescent
protein.
also may be desirable. In order to propagate the vectors in E. coli, it is
necessary to
convert the linear molecule into a circle by addition of a stuffer fragment
between the
telomeres. Inclusion of an E. coli plasmid replication origin and selectable
marker also
may be preferred. It also may be desirable to include A~robcrcterium sequences
to
improve replication and transfer to plant cells. The inventors have described
a number of
exemplary minichromosome constructs in FIGS. 7A-7EI. although it will be
apparent to
those in skill art that many changes may be made in the order and types of
elements
present in these constructs and still obtain a functional minichromosome
within the scope
of the instant invention.
Artificial plant chromosomes which replicate in yeast also may be constructed
to
take advantage of the large insert capacity and stability of repetitive DNA
inserts afforded
by this system (see Burke et nl.. 1987). In this case. yeast ARS and CEN
sequences may
be added to the vector. The artificial chromosome is maintained in yeast as a
circular
molecule using a atuffer fragment to separate the telorneres.
CA 02362897 2001-09-18
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A fragment of DNA. from any source whatsoever, may be purified and inserted
into a minichromosome at any appropriate restriction endonuclease cleavage
site. The
DNA se~~ment usually will include various regulatory signals for the
expression of
proteins encoded by the fragment. Alternatively. regulatory signals resident
in the
S minichromosome may be utilized.
The techniques and procedures required to accomplish insertion are well-known
in the art (see Maniatis et crl., 1982). Typically. this is accomplished by
incubating a
circular plasmid or a linear DNA fragment in the presence of a restriction
endonuclease
such that the restriction endonuclease cleaves the DNA molecule. Endonucleases
preferentially break the internal phosphodiester bonds of polynucleotide
chains. They
may be relatively unspecific. cutting polynucleotide bonds regardless of the
surroundinyT
nucleotide sequence. However. the endonucleases which cleave only a specific
nucleotide sequence are tailed restriction enzymes. Restriction endonucleases
generally
internally cleave DNA molecules at specific recognition sites, making breaks
within
"recognition" sequences that in many, but not all. cases exhibit two-fold
symmetry around
a given point. Such enzymes typically create double-stranded breaks.
Many of these enzymes make a staggered cleavage. yielding DNA fragments with
protruding single-stranded ~' or 3' termini. Such ends are said to be "sticky"
or
"cohesive" because they will hydrogen bond to complementary 3' or 5~ ends. As
a result.
the end of any DNA fragment produced by an enzyme. such as EcoRI, can anneal
with
any other fragment produced by that enzyme. This properly allows splicing of
foreign
genes into plasmids, for example. Some restriction endonucleases that may be
particularly useful with the current invention include HinclIIl. Pstl, EcoRl,
and BamHl.
Some endonucleases create fragments that have blunt ends. that is. that lack
any
protruding sin~_le strands. An alternative way to create blunt ends rs to use
a restnctron
enzyme that leaves overhangs, but to fill in the overhangs with a polymerase,
such .m
klenovy. thereby resulting in blunt ends. When DNA has heen cleaved with
restriction
CA 02362897 2001-09-18
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enzymes that cut across both strands at the same position, blunt end ligation
can be used
to join the fragments directly together. The advantage of this technique is
that any pair of
ends may be joined together. irrespective of sequence.
Those nucleases that preferentially break oft terminal nucleotides are
referred to
as exonucleases. For example. small deletions can be produced in any DNA
molecule by
treatment with an exonuclease which starts from each 3' end of the DNA and
chews away
single strands in a 3' to 5' direction, creating a population of DNA molecules
with
single-stranded fragments at each end, some containing terminal nucleotides.
Similarly,
exonucleases that digest DNA from the 5' end or enzymes that remove
nucleotides from
both strands have often been used. Some exonucleases which may be particularly
useful
in the present invention include Bal3l, S1. and E.r~~III. These nucleolytic
reactions can be
controlled by varying the time of incubation. the temperature. and the enzyme
concentration needed to make deletions. Phosphatases and kinases also may be
used to
control which fragments have ends which can be joined. Examples of useful
phosphatases include shrimp alkaline phosphatase and calf intestinal alkaline
phosphatase. An example of a useful kinase is T4 polynucleotide kinase.
Once the source DNA sequences and vector sequences have been cleaved and
modified to generate appropriate ends they are incubated together with enzymes
capable
of mediating the ligation of the two DNA molecules. Particularly useful
enzymes for this
purpose include T4 lipase. E. cnli lipase, or other similar enzymes. The
action of these
enzymes results in the sealing of the linear DNA to produce a larger DNA
molecule
containing the desired fragment (see. for example. U.S. Patent Nos. 4.237.224:
4.264.731; 4,273,875: 4.322,499 and 4,336,336. which are specifically
incorporated
herein by reference).
It is to be understood that the termini of the linearized plasmid and the
termini of
the DNA fragment being inserted must be complementary or blunt in order for
the
ligation reaction to be successful. Suitable complementarily can be achieved
by choosing
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appropriate restriction endonucleases (i.e., if the fragment is produced by
the same
restriction endonuclease or one that generates the same overhand as that used
to linearize
the Plasmid, then the termini of both molecules mill be complementary). As
discussed
previously. in one embodiment of the invention. at least two classes of the
vectors used in
the present invention are adapted to receive the foreign oligonucleotide
fragments in only
one orientation. After joining the DNA segment to the vector. the resulting
hybrid DNA
can then be selected from among the large population of clones or libraries.
A method useful for the molecular cloning of DNA sequences includes in vitro
joining of DNA segments, fragmented from a source of high molecular weight
genomic
DNA, to vector DNA molecules capable of independent replication. The cloning
vector
may include plasmid DNA (see Cohen er «l., 1973). phaoe DNA (see
Thomas et «L, 1974), SV40 DNA (see Nussbaum er «L. 1976), yeast DNA. E. cull
DNA
and most Significantly, plant DNA.
IS
A variety of processes are known which may be utilized to effect
transformation;
i.e., the inserting of a heterologous DNP. sequences into a host cell, whereby
the host
becomes capable of efficient expression of the inserted sequences.
(ii) Re~ulntorv Elenrent.s
In one embodiment of the invention, constructs may include a plant promoter,
for
example, the CaMV 35S promoter (Odell et «L. 1985), or others such as CaMV 19S
(Lawton et «l., 1987), no.r (Ebert et «L. 1987). Adh (Walker er «!.. 1987),
sucrose
synthase (Yang & Russell. 1990), a-tubulin. actin (Wang et «l.. 1992), cob
(Sullivan er
«L, 1989). PEPCase (Hudspeth & Grula. 1989) or those associated with the R
gene
complex (Chandler er «L, 1989). Tissue specific promoters such as root cell
promoters
(Conkling et crl., 1990) and tissue specrtrc enhancers tt-romm et «L. ty~su)
are also
contemplated to be useful. as are inducible promoters such as ABA- and turaor-
inducible
promoters. In particular embodiments of the invention. a Lat52 promoter may be
used
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WO 00/55325 PCT/US00/07392
(Twell et al.. 1991). A particularly useful tissue specific promoter is the
SCARECROW
(Scr) root-specific promoter (DiLaurenzio et al., i 996).
As the DNA sequence between the transcription initiation site and the start of
the
coding sequence, i.e., the untranslated leader sequence, can intluence gene
expression.
Therefore. one may also wish to employ a particular leader sequence.
It is envisioned that a functional gene could be introduced under the control
of
novel promoters or enhancers, etc., or perhaps even homologous or tissue
specific (for
example. root-, collar/sheath-, whorl-, stalk-. earshank-. kernel- or leaf-
specific)
promoters or control elements. In particular embodiments of the invention, the
functional
gene may be in an antisense orientation relative to the promoter.
(ii) Terntittcttors
IS It may also be desirable to link a functional gene to a 3' end DNA sequence
that
acts as a signal to terminate transcription and allow for the poly-adenylation
of the mRNA
produced by coding sequences. Such a terminator may be the native terminator
of the
functional gene or. alternatively, may be a heterologous 3' end. Examples of
terminators
that could be used with the invention are those from the nopaline synthase
gene of
Agrvbacterittm tcmtefctciet~s (nos 3' end) (Bevan et ol., 1983). the
terminator for the T7
transcript from the octopine synthase gene of Ayrnbacteri~uu tutnc/acimes. and
the 3' end
of the protease inhibitor 1 or I1 genes from potato or tomato.
(iii) Murder Gertes
It may be desirable to use one or more marker genes in accordance with the
invention. Such markers may be adapted for use in prokaryotic, lower
eukaryotic or
higher eukaryotic systems. or may be capable of use in any combination of the
foregoin~~
classes of organisms. By employing a selectable or screenable marker protein.
one can
provide or enhance the ability to identify transformants. "Marker ~~ene;" are
~~enes that
impart a distinct phenotype to cells expressing the marker protein and thus
allow such
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transformed cells to be distinguished from cells that do not have the marker.
Such genes
may encode either a selectable or screenable marker, depending on whether the
marker
confers a trait which one can "select" for by chemical means, i.e., through
the use of a
selective agent (e.g., a herbicide. antibiotic, or the like), or whether it is
simply a trait that
one can identify through observation or testing, i.e., by "screening"'
(e.,~~., the green
fluorescent protein). Of course. many examples of suitable marker proteins are
known to
the art and can be employed in the practice of the invention.
Included within the terms selectable or screenable markers also are genes
which
encode a "secretable marker" whose secretion can be detected as a means of
identifying or
selecting for transformed cells. Examples include markers which are secretable
antigens
that can be identified by antibody interaction. or even secretable enzymes
which can be
detected by their catalytic activity. Secretable proteins fall into a number
of classes.
including small, diffusible proteins detectable, e.R., by ELISA: small active
enzymes
detectable in extracellular solution (e.g.. a-amylase, (3-lactamase,
phosphinothricin
acetyltransferase): and proteins that are inserted or trapped in the cell wall
(e.g., proteins
that include a leader sequence such as that found in the expression unit of
extensin or
tobacco PR-S).
With regard to selectable secretabie markers, the use of a gene that encodes a
protein that becomes sequestered in the cell wall. and which protein includes
a unique
epitope is considered to be particularly advantageous. Such a secreted antigen
marker
would ideally employ an epitope sequence that would provide low background in
plant
tissue. a promoter-leader sequence that would impart efficient expression and
targeting
across the plasma membrane. and would produce protein that is bound in the
cell wall and
yet accessible to antibodies. A normally secreted wall protein modified to
include a
unique epitope would satisfy all such requirements.
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1. Selectable Markers
Many selectable marker ~=enes may be used in accordance with invention
including. hut not limited to. rrc~u (Potrvkus cr «l.. 1985). which provides
kanamvcin
resistance and can be selected for using kanamycin. G=118, paromomycin. e~c.;
bcrr. which
confers bialaphos or phosphinothricin resistance: a mutant EPSP synthase
protein
(Hinchee et crl.. 1988) conferring glyphosate resistance; a nitrilase such as
burr from
Kleh.siellcr n:.aerure which confers resistance Eo bromoxynil (Stalker et nl..
1988); a
mutant acetolactate synthase (ALS) which confers resistance to imidazolinone,
sulfonylurea or other ALS inhibiting chemicals (European Patent Application
154.204,
1985); a methotrexate resistant DNFR (Thillet et «l.. 1988), a dalapon
dehalogenase that
confers resistance to the herbicide dalapon: or a mutated anthranilate
synthase that
confers resistance to 5-methyl tryptophan. Where a mutant EPSP synthase is
employed,
additional benefit may be realized through the incorporation of a suitable
chloroplast
transit peptide. CTP (U.S. Patent No. 5.188.6=l2> or OTP (U.S. Patent No.
x.633.448) and
IS use of a modified maize EPSPS (PCT Application WO 97/04103).
An illustrative embodiment of selectable marker capable of being used in
systems
to select transformants are those that encode the enzyme phosphinothricin
acetyltransferase, such as the hcrr gene from Srreprnmyce.r Irvgrn.ccopicrrs
or the put gene
from Streptnmvces viriclnclrrnnro,~mr~.s. The enzyme phosphinothricin acetyl
transferase
(PAT) inactivates the active ingredient in the herbicide bialaphos,
phosphinothricin
(PPT). PPT inhibits glutamine synthetase. (Murakami er «L. 1986: Twell et
crl.. 1989)
causing rapid accumulation of ammonia and cell death. The use of bar as a
selectable
marker gene and for the production of herbicide-resistant rice plants from
protopiasts was
described by Rathore c crl., ( (993).
A number of S. cerevisicre marker genes arc also known and could be used with
the invention, such as. for example. the HIS4 gene lDonahue et «l.. 1982:
GenBank
number J01331 ). An example of an E. cr~li marker gene which has been cloned
and
sequenced and could be used in accordance with the invention is the Ap gene,
which
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CA 02362897 2001-09-18
WO 00/~~32~ PCT/US00/07392
confers resistance to beta-lactam antibiotics such as ampacillin (nucleotides
4618 to X478
of GenBank accession number U66885).
2. Screenable Markers
Screenable markers that may be employed include a ~i-glucuronidase (GUS) or
triclA gene which encodes an enzyme for which various chromogenic substrates
are
known: an R-locus gene, which encodes a product that regulates the production
of
anthocyanin pigments (red color) in plant tissues lDellaporta et al., 1988); a
(3-lactamase
gene (Sutcliffe, 1978), which encodes an enzyme for which various chromogenic
substrates are known (e.g., PADAC, a chromogenic cephalosporin}; a .rylE gene
(Zukowsky et al., 1983) which encodes a catechol dioxygenase that can convert
chromogenic catechols: an a-amylase gene (Ikuta et al., 1990); a tyrosinase
Gene (Katz et
crl.. 1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone which in tum condenses to form the easily-detectable compound
melanin; a
~3-galactosidase gene, which encodes an enzyme for which there are chromogenic
substrates: a luciferase (lrex) gene (Ow et ul.. 1986). which allows for
bioluminescence
detection: an aequorin gene (Prasher et crl., 1985) which may be employed in
calcium
sensitive bioluminescence detection; or a gene encoding for green fluorescent
protein
(Sheen et al.. 1995: Haseloff et al., 1997: Reichel et al., 1996: Tian et ul..
1997: WO
97141228).
Genes from the maize R gene complex can also be used as screenable markers.
The R gene complex in maize encodes a protein that acts to regulate the
production of
anthocyanin pigments in most seed and plant tissue. Maize strains can have
one. or as
2~ many as tour. R alleles which combine to regulate pigmentation in a
developmental and
tissue specific manner. Thus, an R gene introduced into such cells will cause
the
expression of a red pigment and, if stably incorporated. can be visually
scored as a red
sector. If a maize line carries dominant alleles for genes encoding= for the
enzymatic
intermediates in the anthocvanin biosynthetic pathway (C2. A l . A?. Bo I and
Bz2), but
carries a recessive allele at the R locus. transformation of any cell from
th,rt line with R
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will result in red pigment formation. Exemplary lines include Wisconsin 22
which
contains the rg-Stadler allele and TR 112. a K55 derivative which is r-g, b,
Pl.
Alternatively. any genotype of maize can be utilized if the C l and R alleles
are introduced
together.
Another screenable marker contemplated for use in the present invention is
firefly
luciferase, encoded by the lrrx gene. The presence of the lox gene in
transformed cells
may be detected using, for example. X-ray film, scintillation counting,
fluorescent
spectrophotometry, low-light video cameras, photon counting cameras or
multiwell
luminometry. It also is envisioned that this system may be developed for
populational
screening for bioluminescence, such as on tissue culture plates, or even for
whole plant
screening. The gene which encodes green fluorescent protein (GFP) is
contemplated as a
particularly useful reporter gene (Sheen et al., 1995; Haseloff et al., 1997;
Reichel et crl..
1996; Tian et crl., 1997; WO 97/41228). Expression of green fluorescent
protein may be
IS visualized in a cell or plant as fluorescence following illumination by
particular
wavelengths of light.
3. Negative Selectable Markers
Introduction of genes encoding traits that can be selected against may be
useful for
eliminating minichromosomes from a cell or for selecting against cells which
comprise a
particular minichromosome. An example of a negative selectable marker which
has been
investigated is the enzyme cytosine deaminase (Stouggard. 1993). In the
presence of this
enzyme the compound 5-f7uorocytosine is converted to 5-fluorouracil which is
toxic to
plant and animal cells. Therefore. cells comprising a minichromosome with this
gene
could be directly selected against. Other genes that encode proteins that
render the plant
sensitive to a certain compound will also be useful in this context. For
example. T-DNA
gene 2 from AS~rnlprcterirrm ternrefaciens encodes a protein that catalyzes
the conversion
of u-naphthalene acetamide (NAMI to a-naphthalene acetic acid (NAA> renders
plant
cells sensitive to high concentrations of NAM (Depicker er nl.. 1988).
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V. Isolation of Centromeres From Plants
The inventors have provided, for the first time, the nucleic acid sequence of
a
plant centromere. This will allow one of skill in the art to obtain centromere
sequences
from potentially any species. The inventors specifically provide herein below
a number
of methods which may be employed to isolate such centromeres.
(i) Utilization of Conserved Segerence.c
Numerous of the centromere sequences identified by the inventors were also
shown by the inventors to be highly conserved (see e.g., Example SB. Table 3,
and Table
4). The novel finding of the inventors that a number of genes reside within
the
Arabidopsis centromere can therefore be used to find syntenic genes in other
organisms
(i.e., evolutionarily conserved relationships in gene order from species to
species). For
example, the sequence of each Arahidopsis gene can be used to search Through
sequence
databases from other plants. An exemplary list of such sequences that could be
used is a
sequence given by SEQ ID NO: I , SEQ 1D N0:2. SEQ ID N0:3, SEQ ID N0:4. SEQ ID
NO:S, SEQ ID N0:6, SEQ ID N0:7. SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10. SEQ
ID NO:11, SEQ ID N0:12, SEQ ID N0:13. SEQ ID N0:14. SEQ ID NO:15, SEQ ID
NO: l6, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, and SEQ ID
N0:21. Also useful would be the genes listed in Tables 3 and 4. Finding
identical or
similar genes would identify candidates that may reside within or near
centromeric
regions. Mapping these genes using linked markers would identify potential
centromeric
regions.
Where hybridization is used to obtain centromere sequences, it may be
desirable
2~ to use less stringent hybridization conditions to allow formation of a
heteroduplex. In
these circumstances, one may desire to employ conditions such as about 0.15 M
to about
0.9 M salt. at temperatures ranging from about 20°C to about
55°C. Cross-hybridizing
species can thereby be readily identified as positively hybridizing signals
with respect to
control hybridizations. In any case, it is generally appreciated that
conditions can be
rendered more stringent by the addition of increasing amounts of formamide.
which
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serves to destabilize the hybrid duplex in the same manner as increased
temperature or
decreased salt. Thus, hybridization conditions can be readily manipulated. and
thus will
generally be a method of choice depending on the desired results.
(ii) ldertti/icatiorr of Cerrtromere-A.c.sncicrted Clrcrructeristics
The second method takes advantage of the unique DNA properties that the
inventors have discovered at the Arabidopsi.r centromere and adjacent
pericentromere
regions. The centromeres are composed of long arrays of 180 by repeats flanked
by
regions that are 10-70% retroelements, up to 15% pseudogenes and up to 29%
!0 transposons see FIGS. 12A-T). This is unique to the centromere since
retroelements,
transposons and pseudogenes are very rare outside the centromere and
pericentromere
region. Furthermore, gene density decreases from an average of a gene every
4.5 kb on
the chromosomal arm down to one in 150 kb at the centromere. This unique
centromere
composition could be exploited in a number of ways to find centromere regions
in other
species, for example:
1 ) Markers specific for retroelements, transposons, repeat DNA elements and
pseudogenes can be devised to genetically map regions which are dense with
similar
elements.
2) The second method involves in situ hybridization. and preferably.
fluorescent
in situ hybridization (FISH). Fluorescently labeled DNA probes consisting of
retroelements. transposons and/or repetitive DNA native to a particular
species can be
combined with microscopy to identify parts of a chromosome with a similar
percentage of
2~ DNA elements as that found at the Arabiclopsis centromere.
3) Utilizing sequence databases. regions of genomes that have increased
numbers
of repetitive DNA, pseudogenes, retroelements and transposons, similar to the
composition of Arubidopsis identified by the inventors. can be used to
identify regions of
an organisms' chromosome that are centromeric.
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(iii) Utili:.atio» n/~Ce»trnmere-Assacintecl Protei»s
The third method involves immunoprecipitatin~ known centromere proteins or
kinetochore proteins and analyzing bound DNA. Antibodies specific to
centromere
proteins can be incubated with proteins extracted from cells. Extracts can be
native or
previously treated to cross-link DNA to proteins. The antibodies and bound
proteins can
be purified away from the protein extracts and the DNA isolated. The DNA can
then be
used as a probe for F1SH (as talked about above) or to probe libraries to find
neighboring
centromere sequences.
1. Centromere-Associated Protein Specific Antibodies
By identifying. for the first time, centromere-associated genes, the inventors
have
enabled the production of antibodies to the proteins encoded by such
centromere-
associated genes. The antibodies may be either monoclonal or polyclonal which
bind to
centromere-associated proteins of the current invention. The centromere-
associated
protein targets of the antibodies, include proteins which bind to the
centromere region.
Further, it is specifically contemplated that these centromere-associated
protein specific
antibodies would allow for the further isolation and characterization of the
centromere-associated proteins. For example, proteins may be isotated which
are
encoded by the centromeres. Recombinant production of such proteins provides a
source
of antigen for production of antibodies.
Alternatively. the centromere may be used as a ligand to isolate, using
affinity
methods, centromere binding proteins. Once isolated. these protein can be used
as
antigens for the production polyclonal and monoclonal antibodies. A variation
on this
technique has been demonstrated by Rattner ( 1991 ), by cloning of centromere-
associated
proteins through the use of antibodies which bind in the vicinity of the
centromere.
Means for preparing and characterizing antibodies are well known in the art
(see.
c. g., Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory. 1988;
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incorporated herein by reference). The methods for generating monoclonal
antibodies
(mAbs) generally begin along the same lines as those for preparing polyclonal
antibodies.
Briefly, a polyclonal antibody is prepared by immunizing an animal with an
immunogenic
composition in accordance with the present invention and collecting antisera
from that
immunized animal. A wide range of animal species can be used for the
production of
antisera. Typically the animal used for production of antisera is a rabbit, a
mouse, a rat, a
hamster, a guinea pig or a goat. A rabbit is a preterred choice for production
of
polyclonal antibodies because of the ease of handling, maintenance and
relatively large
blood volume.
As is well known in the art, a given composition may vary in its
immunogenicity.
It is often necessary therefore to boost the host immune system. as may be
achieved by
coupling a peptide or polypeptide immunogen to a carrier. Exemplary and
preferred
carriers are keyhole limpet hemocvanin (KLH) and bovine serum albumin (BSA).
Other
IS albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin
also can be
used as carriers. Means for conju~atin~ a polypeptide to a carrier protein are
well known
in the art and include glutaraldehyde. nr-maleimidobencoyl-N-
hydroxysuccinimide ester,
carbodimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immuno~en
composition can be enhanced by the use of non-specific stimulators of the
immune
response. known as adjuvants. Exemplary and preferred adjuvants include
complete
Freund's adjuvant (a non-specific stimulator of the immune response containing
killed
Mvcobacteriunr trrbercrrlosi.s). incomplete Freund's adjuvants and aluminum
hydroxide
adjuvant.
The amount of immuno«en composition used in the production of polyclonal
antihodies varies upon the nature of the immunogen as well as the animal used
for
immunization. A variety of routes can be used to administer the immuno~en
(subcutaneous. intramuscular. mtradermal, intravenous and intraperitoneal ).
The
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production of polyclonal antibodies may be monitored by sampling blood of the
immunized animal at various points following immunization. A second, booster.
injection also may be given. The process of boostin' and titering is repeated
until a
suitable titer is achieved. When a desired level of immunogenicity is
obtained, the
immunized animal can be bled and the serum isolated and stored, and/or the
animal can
be used to generate mAbs.
Monoclonal antibodies may be readily prepared through use of well-known
techniques, such as those exemplified in U. S. Patent 4,196.265, incorporated
herein by
reference. Typically, this technique involves immunizing a suitable animal
with a
selected immunogen composition, e.y., a purified or partially purified
minichromosome
-associated protein, polypeptide or peptide. The immunizing composition is
administered
in a manner effective to stimulate antibody producing cells. Rodents such as
mice and
rats are preferred animals, however. the use of rabbit, sheep, or frog cells
also is possible.
IS The use of rats may provide certain advantages (coding 1986), but mice are
preferred,
with the BALB/c mouse being most preferred as this is most routinely used and
generally
gives a higher percentage of stable fusions.
Following immunization. somatic cells with the potential for producing
antibodies, specifically B lymphocytes (B cells), are selected for use in the
mAb
generating protocol. These cells may be obtained from biopsied spleens,
tonsils or lymph
nodes, or from a peripheral blood sample. Spleen cells and peripheral blood
cells are
preferred. the former because they are a rich source of antibody-producing
cells that are in
the dividing plasmablast stage, and the latter because peripheral blood is
easily accessible.
2i Often, a panel of animals will hove been immunized and the spleen of animal
with the
highest antibody titer will be removed and the spleen lymphocytes obtained by
homogenizing the spleen with a aynn~_e. Typically. a spleen from an immunized
mouse
contains approximately ~ X 10' to ? x l Oh lymphocytes.
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The antibody-producing B lymphocytes from the immunized animal are then
fused with cells of an immortal myeloma cell. generally one of the same
species as the
animal that was immunized. Myeloma cell linen suited for use in hybridoma-
producing
fusion procedures preferably are non-antibody-producing. have high fusion
efficiency,
and enzyme deficiencies that render them incapable of growing in certain
selective media
which support the growth of only the desired fused cells {hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of
skill
in the art (coding I986; Campbell 1984). For example. where the immunized
animal is a
mouse, one may use P3-X63/AgB, X63-Ag8.653. NS1/l.Ag 4 1, Sp210-Agl4, FO,
NSO/U. MPC-I 1, MPC11-X45-GTG 1.7 and S 194/~XXO Bul; for rats, one may use
R2 I O.RCY3, Y3-Ag 1.2.3, IR983F and 4B210: and U-266, GM 1500-GRG2,
LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell
fusions.
IS One preferred marine myeloma cell is the NS-1 myeloma cell line (also
termed
P3-NS-1-Ag4-I), which is readily available from the NIGMS Human Genetic Mutant
Cell Repository by requesting cell line repository number GM3573. Another
mouse
myeloma cell line that may be used is the 8-azaguanine-resistant mouse marine
myeloma
SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node
cells
and mveloma cells usually comprise mixing somatic cells with myeloma cells in
a 2:1
ratio, though the ratio may vary from about 20:1 to about 1:1. respectively,
in the
presence of an agent or agents (chemical or electrical) that promote the
fusion of cell
membranes. Fusion methods using Sendai virus have been described
(Kohler c>r «l.. 197: 1976). and those using polyethylene glycol (PEG). such
as 37% (v/v)
PEG, fGefter et «l.. 1977). The use of electrically induced fusion methods
also is
appropriate (coding 1986).
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Fusion procedures usually produce viable hybrids at low frequencies. about 1 x
10-~ to l x 10-x. However, this does not pose a problem, as the viable, fused
hybrids are
differentiated from the parental. unfused cells (particularly the unfused
myeloma cells
that would normally continue to divide indefinitely) by culturing in a
selective medium.
The selective medium is generally one that contains an agent that blocks the
de uovo
synthesis of nucleotides in the tissue culture media. Exemplary and preferred
agents are
aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block
cle novo
synthesis of both purines and pyrimidines, whereas azaserine blocks only
purine
synthesis. Where aminopterin or methotrexate is used, the media. is
supplemented with
hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where
azaserine
is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating
nucleotide salvage pathways are able to survive in HAT medium. The myeloma
cells are
defective in key enzymes of the salvage pathway, e.g., hypoxanthine
phosphoribosyl
transferase (HPRT), and they cannot survive. The B-cells can operate this
pathway, but
they have a limited life span in culture and generally die within about two
weeks.
Therefore, the only cells that can survive in the selective media are those
hybrids formed
from mveloma and B-cells.
This culturing provides a population of hybridomas from which specific
hybridoma, arc selected. Typically. selection of hybridomas is performed by
culturing
the cells by single-clone dilution in microtiter plates, followed by testing
the individual
clonal supernatants (after about two to three weeks) for the desired
reactivity. The assay
should be sensitive, simple and rapid. such as radioimmunoassays. enzyme
immunoassays, cytotoxicity assays. plaque assays. dot immunobindina assays.
and the
like.
The selected hyhridomas would then be serially diluted and cloned into
individual
antibody-producing cell lines. which clones can then be propagated
indefinitely to
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provide mAbs. The cell lines may be exploited for mAb production in two basic
ways. A
sample of the hybridoma can be injected (often into the peritoneal cavity)
into a
histocompatiblc animal of the type that was used to provide the somatic and
myeloma
cells for the original fusion. The injected animal develops tumors secreting
the specific
monoclonal antibody produced by the fused cell hybrid. The body fluids of the
animal,
such as serum or ascites tluid, can then be tapped to provide mAbs in high
concentration.
The individual cell lines also could be cultured in vitro, where the mAbs are
naturally
secreted into the culture medium from which they can be readily obtained in
high
concentrations. mAbs produced by either means may be further purified, if
desired. using
filtration. centrifugation and various chromatographic methods such as HPLC or
affinity
chromatography.
2. ELISAs and Immunoprecipitation
ELISAs may be used in conjunction with the invention, for example. in
IS identifying expression of a centromere-associated protein in a candidate
centromere
sequence. Such an assay could thereby facilitate the isolation of centromeres
from
species other than Arcrbiclopsi.r. By identifying conserved, eentromere-
associated coding
sequences, the inventors have provided the essential tools for such a screen.
In an ELISA assay, proteins or peptides comprising minichromosome-encoded
protein antigen sequences are immobilized onto a selected surface, preferably
a surface
exhibiting a protein affinity such as the wells of a polystyrene microtiter
plate. After
washing to remove incompletely adsorbed material. it is desirable to bind or
coat the
assay plate wells with a nonspecific protein that is known to be anti~enically
neutral with
regard to the test antisera such as bovine serum albumin (BSA), casein or
solutions of
milk powder. This allows for blocking of nonspecific adsorption sites on the
immobilizin~~ surface and thus reduces the background caused by nonspecific
binding of
antisera onto the surface.
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After binding of antigenic material to the well, coating with a non-reactive
material to reduce background, and washing to remove unbound material. the
immobilizing surface is contacted with the antisera or clinical or biological
extract to be
tested in a manner conducive to immune complex (antigen/antibody) formation.
Such
conditions preferably include diluting the antisera with diluents such as BSA,
bovine
gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween°. These
added
agents also tend to assist in the reduction of nonspecific background. The
layered
antisera is then allowed to incubate for from about 2 to about 4 hours, at
temperatures
preferably on the order of about 25° to about 27°C. Following
incubation, the
antisera-contacted surface is washed so as to remove non-immunocomplexed
material. A
preferred washing procedure includes washing with a solution such as
PBS/Tween°, or
borate buffer.
Following formation of specific immunocomplexes between the test sample and
the bound antigen, and subsequent washing. the occurrence and even amount of
immunocomplex formation may be determined by subjecting same to a second
antibody
having specificity for the first. To provide a detecting means, the second
antibody will
preferably have an associated enzyme that will ~~enerate color or light
development upon
incubating with an appropriate chromogenic suhstrate. Thus, for example. one
will desire
to contact and incubate the antisera-bound surface with a urease or peroxidase-
conjugated
anti-human IgG for a period of time and under conditions which favor the
development of
immunocomplex formation (e.g., incubation for 2 hours at room temperature in a
PBS-containing solution).
After incubation with the second enzyme-tagged antibody, and subsequent to
washing to remove unbound material. the amount of label is quantified by
incubation
with a chromogenic substrate such as urea and bromoeresol purple or
~.~'-azino- _di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and HBO,. in
the case of
peroxidase as the enzyme label. Quantitation i~ then achieved by measuring the
degree of
color generation. e.,r,~.. using a visible spectra spectrophotometer.
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3. Western Blots
Centromere- associated antibodies may find use in immunoblot or western blot
analysis, for example. for the identification of proteins immobilized onto a
solid support
matrix. such as nitrocellulose, nylon or combinations thereof. In conjunction
with
immunoprecipitation. followed by gel electrophoresis. these may be used as a
single step
reagent for use in detecting antigens against which secondary reagents used in
the
detection of the antigen cause an adverse background. This is especially
useful when the
antigens studied are immunoglobulins (precluding the use of immunoglobulins
binding
bacterial cell wall components), the antigens studied cross-react with the
detecting agent.
or they migrate at the same relative molecular weight as a cross-reacting
signal.
lmmunologically-based detection methods for use in conjunction with Western
blotting include enzymatically-, radiolabel-. or fluorescently-tagged
secondary antibodies
against the protein moiety are considered to be of particular use in this
regard.
(iv) Genetic Mappirt,~ Bcr.sed Approaches
The genetic mapping techniques outlined here for the identification of
centromeres in Arnbidopsis may find use in other species. In one aspect, this
may
comprise actual use of the mapping data provided herein. based on synteny
between
Arcrbidnp.ci.c chromosomes and those of other specie;. Further. new mapping
data may be
obtained using the techniques described herein. For example. in any plant that
makes
tetrads. the detailed methodology described herein for tetrad analysis could
be used for
the isolation of centromeres. Briefly, tetrad analysis measures the
recombination
2~ frequency between genetic makers and a centromere by analyzing all four
products of
individual meiosis. A particular advantage arises from the c/uartet (c/rt I )
mutation in
Arnhidnp.ci.r. which causes the four products of pollen mother cell meiosis in
Ar-u(ridopsi.s
to remain attached.
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Several naturally occurring plant speciea in addition to Arabiclnp.cis are
known to
release pollen clusters, including water lilies, cattails, heath (Ericaceae
arrcl Ehacriclceuc ).
evening primrose (Orrcr,t~rcrcecre). sundews (Dru.ceracenc~). orchids
(Orchiclcrceae ). and
acacias (Nlirrroscrcene) (Preuss 1994. Smyth 199=l). However, none of these
species has
been developed into an experimental system. limiting their use for Genetic
analysis.
However. it is contemplated by the inventors that the cloning and introduction
of the
drrortet mutation. or an antisense copy of a non-mutated Quartet gene, could
allow the use
of tetrad analysis in potentially any species.
Southern ~enomic DNA blots in combination with RFLP analysis may be used to
map centromeres with a high degree of resolution. The stored seedling tissue
provides
the necessary amount of DNA for analysis of the restriction fragments.
Southern blots are
hybridized to probes labeled by radioactive or non-radioactive methods.
IS It may, in many cases. be desired to identify new polymorphic DNA markers
which are closely linked to the target region. In some cases this can be
readily done. For
example, in many plant genomes, a polymotphic Scur3A site can be found for
about every
8 to 20 kB surveyed. Subtractive methods are available for identifying such
polymorphisms (Rosenber~ et al., 1994). and these subtractions may be
performed using
DNA from selected. centromeric YAC or BAC clones. Screens for RFLP markers
potentially linked to centromeres also can be performed using DNA fragments
from a
centromere-linked YAC clone to probe blots of genomic DNA from a tar~~et
organism
that has been digested with a panel of restriction enzymes.
To be certain that an entire centromeric region has been cloned, clones or a
seriea
of clones, are identified that hybridize to markers on either side of each
centromere.
These efforts can be complicated by the presence of repetitive DNA in the
centromere. as
well as by the potential instability of centromere clones. Thus,
identification of lar~~e
clones with unique sequences that will serve as useful probes simplifies a
chromoaomc
walkinG strate~l-.
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Blot hybridization allows comparison of the structure of the clones with that
of
senomic DNA. and thug determines whether the clones have suffered deletions or
rearrangements. The ccntromeric clones identified are useful for hybridization
experiments that can be used to determine whether they share common sequences.
whether they localize irr situ to the cytologically defined centromeric
region, and whether
they contain repetitive sequences thought to map near Ar«hiclopsi.s
centromeres
(Richards et ctl.. 1991: Maluszynska et crl., 1991 ).
Exemplary methods for conducting PFGE and YAC genome analysis described
(Eeker, 1990). A large insert YAC library for genome mapping in Arahiclop.sis
tlrcrli«ircr
was described in Creusot ( 1995). The analysis of clones carrying repeated DNA
sequences in two YAC libraries of Ar«biclop.si.s th«licrrr« DNA was discussed
by Schmidt
et crl., ( 1994}. The construction and characterization of a yeast artificial
chromosome
I S library of Arabidopsi.s was described by Grill and SomerviIle ( 1991 ).
A particularly useful type of clone is the bacterial artificial chromosome
(BAC).
as data has suggested that YAC clones may sometimes not span centromeres
(Willard,
1997). The construction and characterization of a bacterial artificial
chromosome library
from, for example. Ar«bidnpsi.s rlr«licrrr« has been described (Choi et crl.,
1995). The
complementation of plant mutants with lame ~~enomic DNA fragments can be
achieved
using transformation-competent minichromosome vectors, thereby speeding
positions!
cloning. (Liu et «l.. 1999). The construction and characterization of the IGF
,9rcrhidopsi.s
BAC library was described by Mozo et «L, ( 1998.). A complete BAC-based
physical
map of the Ar«hiclop.sis tlrcrlicrn« genome has been described (Mozo et «L.
1998).
VI. Site Specific Integration and Excision of Nucleic Acid Segments
It is specifically contemplated by the inventors that one could employ
techniques
for the site-specific integration or excision of nucleic acid segments for the
construction
of minichromosomes (see. e.g.. Example 8B_ below). Such techniques also could
be used
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for the site-specific integration or excision of trans'~enes which are
introduced into a
plant, including minichromosome vectors.
Site-specific integration or excision of nucleic acid molecules can be
achieved by
Wmeans of homologous recombination (see, for example. U.S. Patent No.
5,527.695.
specifically incorporated herein by reference in its entirety). Homologous
recombination
is a reaction between any pair of DNA sequence, having a similar sequence of
nucleotides, where the two sequences interact (recombine) to form a new
recombinant
DNA species. The frequency of homologous recombination increases as the length
of the
shared nucleotide DNA sequences increases, and is higher with linearized
plasmid
molecules than with circularized plasmid molecules. Homologous recombination
can
occur between two DNA sequences that are less than identical, but the
recombination
frequency declines as the divergence between the two sequences increases.
Introduced DNA sequences can be targeted via homologous recombination by
linking a DNA molecule of interest to sequences sharing homology with
endogenous
sequences of the host cell. Once the DNA enters the cell. the two homologous
sequences
can interact to insert the introduced DNA at the site where the homologous
genomic
DNA sequences were located. Therefore, the choice of homologous sequences
contained
on the introduced DNA will determine the site where the introduced DNA is
integrated
via homologous recombination. For example. if the DNA sequence of interest is
linked
to DNA sequences sharing homology to a single copy Gene of a host plant cell,
the DNA
sequence of interest will be inserted via homologous recombination at only
that single
specific site. However. if the DNA sequence of interest is linked to DNA
sequences
2~ sharing homology to a muiticopy gene of the host eukaryotic cell, then the
DNA sequence
of interest can be inserted via homologous recombination at each of the
specific sites
where a copy of the Gene is located.
DNA can be inserted into a host chromosome or vector by a homologous
recombination reaction involving either a single reciprocal recombination
(resulting in the
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insertion of the entire length of the introduced DNA) or through a double
reciprocal
recombination (resulting in the insertion of only the DNA located between the
two
recomhination events 1. For example. if one wishes to inert ,i f~orei~n gene
into the
Qenomic site where a selected gene is located. the introduced DNA should
contain
sequences homologous to the selected gene. A single homolo~ot.ts recombination
event
would then result in the entire introduced DNA sequence bein« inserted into
the selected
gene. Alternatively, a double recombination event can be achieved by flanking
each end
of the DNA sequence of interest (the sequence intended to be inserted into the
genome)
with DNA sequences homologous to the selected gene. A homologous recombination
l0 event involving each of the homologous flanking regions will result in the
insertion of the
foreign DNA. Thus only those DNA sequences located between the two regions
sharing
genomic homology become integrated into the ~enome.
Although introduced sequences can be targeted for insertion into a specific
site via
15 homologous recombination, in higher eukaryotes homologous recombination is
a
relatively rare event compared to random insertion events. In plant cells.
foreign DNA
molecules find homologous sequences in the cell's genome and recombine at a
frequency
of approximately 0.~-4.2X 10'~'. Thus any transformed cell that contains an
introduced
DNA sequence integrated via homologous recombination will also likely contain
20 numerous copies of randomly integrated introduced DNA sequences. Therefore.
it may
be desirable to use more precise mechanisms for site-specific recombination. A
preferred
manner for carrying out site-specific recombination comprises use of a site-
specific
recombinase system. In general. a site specific recombinase system consists of
three
elements: two pairs of DNA sequence (first and second site-specific
recombination
sequences) and a specific enzyme (the site-specific recombinase). The site-
specific
recombinase will catalyze a recombination reaction only between two site-
specific
recombination sequences.
A number of different site specific recombinase system; could be employed in
30 accordance with the instant invention. including, but not limited to. the
Crc/lox system of
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bacteriophage P I (Hoess et crl.. 1982: U.S. Patent No. 5,658.772,
specifically
incorporated herein by reference in its entirety). the FLP/FRT system of yeast
(colic and
Lindquist. 1989). the Gin recombinase of phage Mu (Maeser and Kahmann. 1991 ).
the
Pin recombinase of E. coli (Enomoto et crl.. 1983). the recombinase encoded by
the sre
gene (ORF469) and which is capable of mediating integration of the R=1 phage
genome.
(Matsuura et al., 1996), the site-specific recombinase encoded by pinD of
Slri~ella
clvsenterine (Tominaga, 1997). the site-specific recombinase encoded in the
major
'pathogenicity island' of Salmonella hphi (Zhang et al., 1997) the Int-B 13
site-specific
recombinase of the bacteriophage P4 inte~rase family (Ravatn et al., 1998). as
well as the
and the R/RS system of the pSR 1 plasmid (Araki et ccl.. 1992). The
bacteriophage PI
Cre/lox and the yeast FLP/FRT systems constitute two particularly useful
systems for site
specific recombination. In these systems. a recombinase (Cre or FLP) will
interact
specifically with its respective site-specific recombination sequence (lox or
FRT.
respectively) to invert or excise the intervening sequences. The sequence for
each of
these two systems is relatively short (34 by for lox and 47 by for FRT) and
therefore.
convenient for use with transformation vectors.
The FLP/FRT recombinase system has been demonstrated to function efficiently
in plant cells, but could also be used in. for example, a bacterial cell or in
aitrr~. The
performance of the FLP/FRT system indicates that FRT site structure. and
amount of the
FLP protein present affect excision activity. In general. short incomplete FRT
sites lead
to higher accumulation of excision products than the complete full-length FRT
sites. The
systems can catalyze both infra- and intermolecular reactions, indicating
their utility for
DNA excision as well as integration reactions. The recombination reaction is
reversible
and this reversibility can compromise the efficiency of the reaction in each
direction.
Altering the structure of the site-specific recombination sequences is one
approach to
remedying this situation. The cite-specific recombination sequence can he
mutated in a
manner that the product of the recombination reaction is no loner recognized
as a
substrate for the reverse reaction. thereby stabilizing the integration or
excision event.
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In the Cre-lox system. discovered in bacteriophage P1, recombination between
loxP sites occurs in the presence of the Cre recombinase (see, e.g.. U.S.
Patent No.
x.658.77?. specifically incorporated herein by reference in its entirety).
This system has
been utilized to excise a gene located between two lox sites which had been
introduced
into a yeast genome (Saner. 1987). Cre was expressed from an inducible yeast
GALI
promoter and this Cre gene was located on an autonomously replicating yeast
vector.
Since the lox site is an asymmetrical nucleotide sequence, lox sites on the
same
DNA molecule can have the same or opposite orientation with respect to each
other.
Recombination between lox sites in the same orientation results in a deletion
of the DNA
Segment located between the two lox sites and a connection between the
resulting ends of
the original DNA molecule. The deleted DNA segment forms a circular molecule
of
DNA. The original DNA molecule and the resultin~~ circular molecule each
contain a
single lox site. Recombination between lox sites in opposite orientations on
the same
IS DNA molecule result in an inversion of the nucleotide sequence of the DNA
segment
located between the two lox sites. In addition, reciprocal exchange of DNA
segments
proximate to lox sites located on two different DNA molecules can occur. All
of these
recombination events are catalyzed by the product of the Cre coding region.
VII. Transformed Host Cells and Transeenic Plants
Methods and compositions for transformin~~ a bacterium, a yeast cell. a plant
cell.
or an entire plant with one or more minichromosomes are further aspects of
this
disclosure. A transgenic bacterium. yeast cell. plant cell or plant derived
from such a
transformation process or the progeny and seedy from such a transgenic plant
also are
further embodiments of the invention.
Means for transforming bacteria and yeast cells are well known in the art.
Typically, means of transformation are similar to those well known means used
to
transform other bacteria or yeast such as E grill or Scrc~ hcrrumvce.s
cerevisiae. Methods
for DNA transformation of plant cells include A,s;rnhcrcterirrrn-mediated
plant
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transformation. protoplast transformation (,as used herein "protoplast
transformation"
includes PEG-mediated transformation. electroporation and protoplast fusion
transformation). gene transfer into pollen. injection into reproductive
organs. injection
into immature embryos and particle bombardment. Each of these methods has
distinct
advantages and disadvantages. Thus. one particular method of introducing genes
into a
particular plant strain may not necessarily be the most effective for another
plant strain.
but it is well known in the art which methods are useful for a particular
plant strain.
There are many methods for introducing transforming DNA segments into cells.
but not all are suitable for delivering DNA to plant cells. Suitable methods
are believed
to include virtually any method by which DNA can be introduced into a cell.
such as by
A~=robcrcteriruo infection. direct delivery of DNA such as, for example. by
PEG-mediated
transformation of protoplasts (Omirulleh et «L. 1993), by
desiccationlinhibition-mediated
DNA uptake. by electroporation, by agitation with silicon carbide fibers, by
acceleration
of DNA coated particles, etc. In certain embodiments. acceleration methods are
preferred
and include, for example, microprojectile bombardment and the like.
Technology for introduction of DNA into cells is well-known to those of skill
in
the art. Four general methods for deliverinVC a gene into cells have been
described: ( 1 )
chemical methods (Graham et «l., 1973; Zatloukal et «l.. 1992); (2) physical
methods
such as microinjection (Capecchi, 1980). elcctroporation l Wong et «l.. 1982:
Fromm et «L. 1985; U. S. Patent No. 5.384.253) and the gene gun (Johnston et
crl.. 199=1:
Fynan et crl.. 1993); (3) viral vectors (Clapp 1993: Lu et «L. 1993: Eglitis
et «L, 1988x:
1988b): and (4) receptor-mediated mechanisms (Curiel et «L, 1991; 1992;
Wagner et crl.. 1992).
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(i) Elecmnpnration
The application of brief, high-voltage electric pulses to a variety of animal
and
plant cells leach to the formation of nanometer-sized pores in the plasma
membrane.
DNA is taken directly into the cell cytoplasm either through these pores or as
a
consequence of the redistribution of membrane components that accompanies
closure of
the pores. Electroporation can be extremely efficient and can be used both for
transient
expression of cloned genes and for establishment of cell lines that carry
integrated copies
of the gene of interest. Electroporation, in contrast to calcium phosphate-
mediated
transfection and protoplast fusion, frequently gives rise to cell lines that
carry one, or at
most a few. integrated copies of the foreign DNA.
The introduction of DNA by means of electroporation, is welt-known to those of
skill in the art. In this method, certain cell wall-de~radin~ enzymes. such as
pectin-de~radin~ enzymes, are employed to render the target recipient cells
more
1~ susceptible to transformation by electroporation than untreated cells.
Alternatively.
recipient cells are made more susceptible to transformation, by mechanical
woundins. To
effect transformation by electroporation one may employ either friable tissues
such as a
suspension culture of cells, or embryogenic callus. or alternatively. one may
transform
immature embryos or other organized tissues directly. One would partially
degrade the
~0 cell walls of the chosen cells by exposing them to pectin-degrading enzymes
(pectolyases)
or mechanically wounding in a controlled manner. Such cells would then he
recipient to
DNA transfer by electroporation, which may be carried out at this stake. and
transformed
cells then identified by a suitable selection or screening protocol dependent
on the nature
of the newly incorporated DNA.
(ii) Micr«prnjectile Brnnhordnzent
A further advant~~eous method for delivering transformin« DNA segments to
plant cells is microprojectile bombardment. In this method. particles may he
coated with
nucleic acids and delivered into cells by a propelling force. Exemplary
particles include
s0 those comprised ~f tunssten. fold. platinum. and the like.
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An advanta~Te of microprojectile bombardment, in addition to it being an
effective
means of reproducibly stably transforming monocots. is that neither the
isolation of
protoplasts (Cristou er ul.. 1988) nor the susceptibility to Agrnhcrcrer-iunr
infection is
required. An illustrative embodiment of a method for delivering DNA into maize
cells by
acceleration is a Biolistics Particle Deliven~ System, which can be used to
propel particles
coated with DNA or cells through a screen. such as a stainless steel or Nytex
screen, onto
a filter surface covered with plant cells cultured in suspension. 'The screen
disperses the
particles so that they are not delivered to the recipient cells in large
aggregates. It is
believed that a screen intervening between the projectile apparatus and the
cells to be
bombarded reduces the size of projectiles aggregate and may contribute to a
higher
frequency of transformation by reducing damage inflicted on the recipient
cells by
projectiles that are too lame.
For the bombardment, cells in suspension are preferably concentrated on
filters or
solid culture medium. Alternatively, immature embryos or other target cells
may be
arranged on solid culture medium. The cells to be bombarded are positioned at
an
appropriate distance below the macroprojectile stopping plate. If desired, one
or more
screens also are positioned between the acceleration device and the cells to
be
bombarded. Through the use of techniques set forth herein one may obtain up to
I .000 or
more foci of cells transiently expressing a marker gene. The number of cells
in a focus
which express the exo~~enous gene product 48 hours post-bombardment often
range from
1 to 10 and average I to 3.
In bombardment transformation. one may optinuze the prebombardment culturing=
conditions and the hombardment parameters to yield the maximum numbers of
stable
transformants. Both the physical and biological parameters for bombardment are
important in this technology. Physical factors are those that involve
manipulating the
DNA/microprojectile precipitate or those that affect the flight and velocity
of either the
macro- or microprojectiles. Biological factors include all steps involved in
manipulation
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of cells before and immediately after bombardment. the osmotic adjustment of
target cells
to help alleviate the trauma associated with bombardment, and also the nature
of the
transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is
believed
that pre-bombardment manipulations are especially important for successful
transformation of immature embryos.
Accordingly, it is contemplated that one may wish to adjust various of the
bombardment parameters in small scale studies to fully optimize the
conditions. One
may particularly wish to adjust physical parameters such as gap distance,
flight distance,
tissue distance, and helium pressure. One also may minimize the trauma
reduction
factors (TRFs) by modifying conditions which influence the physiological state
of the
recipient cells and which may therefore influence transformation and
imegration
efficiencies. For example. the osmotic state. tissue hydration and the
subculture stake or
cell cycle of the recipient cells may be adjusted for optimum transformation.
The
I S execution of other routine adjustments will be known to those of skill in
the art in Ii~Tht of
the present disclosure.
)iii) Agrohacteritrnr-Mediated Trcrnsjer
AKrohacteriunt-mediated transfer is a widely applicable system for introducing
genes into plant cells because the DNA can be introduced into whole plant
tissues.
thereby bypassing the need for regeneration of an intact plant from a
protoplast. The use
of Agrnhcrcteriernr-mediated plant inte~~rating vectors to introduce DNA into
plant cells is
well known in the art. See. for example. the methods described (Fraley et al.,
1985;
Rogers et ul., 1987). Advances in Ayrr~bacrerirrm-mediated transfer now allow
introduction of large segments of DNA (Hamilton. 1997: Hamilton et al.. 1996).
Using conventional transformation vectors. chromosomal integration is required
for stable inheritance of the foreign DNA. However, the vector described
herein may be
used for transformation with or without integration. as the centromere
function required
for stable inheritance is encoded within the minichrornosome. In particular
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embodiments.. transformation events in which the minichromosome is not
chromosomally integrated may be preferred. in that problems with site-specific
variations
in expression and insertional mutagenesis may be avoided.
The integration of the Ti-DNA is a relatively precise process resulting in few
rearrangements. The region of DNA to be transferred is defined by the border
sequences.
and intervening DNA is usually inserted into the plant genome as described
(Spielmann et «!., 1986: Joraensen et crl., 1987}. Modern Agrohcrctericrnr
transformation
vectors are capable of replication in E. cnli as well as A~~robcrcterium,
allowing for
convenient manipulations as described (Klee et «l., 1985}. Moreover. recent
technological advances in vectors for Agrnh«ctcrirrnr-mediated gene transfer
have
improved the arrangement of genes and restriction sites in the vectors to
facilitate
construction of vectors capable of expressing various polypeptide coding
genes. The
vectors described (Ropers et «L, 1987). have convenient multi-linker regions
t7anked by a
t 5 promoter and a polyadenvlation site for direct expression of inserted
polypeptide coding
genes and are suitable for present purposes. In addition, A,~rohcrcrerieorr
containing both
armed and disarmed Ti genes can be used for the transformations. In those
plant strains
where A,yrnhcrcterirrm-mediated transformation is efficient, it is the method
of choice
because of the facile and defined nature of the gene transfer.
Agrohcrcteri«m-mediated transformation of leaf disks and other tissues such as
cotyledons and hypocotvis appears to be limited to plants that Ayroh«crerirrnr
naturally
infects. Agruh«cterirrnr-mediated transformation is most efficient in
dicotyledonous
plants. Few monocots appear to be natural hosts for Ayrohcrcterirrnr. although
transgenic
plants have been produced in asparagus and more signiticantly in maize using
A,~rnhacterirrrn vectors as described (Bytebier et «!., 1987: U.S. Patent No.
S.s91.616,
specifically incorporated herein by reference). Therefore. commercially
important cereal
Brains such as rice. corn. and wheat must usually be transformed using
alternative
methods. However. as mentioned above. the transformation of asparagus using
fl,y~nbncterirurt also can be achieved (see, for example. Bytebier et «L,
1987).
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A~rnbcrcterirrnr-mediated transfer may be made more efficient through the use
of a mutant
that is defective in integration of the A,yrnbacterirrm T-DNA but competent
for delivery
of the DNA into the cell (Mysore et ul.. ?OOOa). Additionally. even in
Arcrhidnp.ci.s
ecotypes and mutants that are recalcitrant to Agrobcrcter-irrrrr root
transformation, germ-
line transformation may be carried out (Mysore er nl.. ?OOOb)
A transgenic plant formed using Agrobacterium transformation methods typically
contains a single gene on one chromosome. Such Iransgenic plants can be
referred to as
being= hemizYgous for the added gene. A more accurate name for such a plant is
an
l0 independent segregant, because each transformed plant represents a unique T-
DNA
integration event.
More preferred is a transaenic plant that is homozygous for the added foreign
DNA: i.e.. a transgenic plant that contains two copies of a transgene. one
gene at the same
I S locus on each chromosome of a chromosome pair. A homozygous transoenic
plant can
be obtained by sexually mating (selfing) an independent segregant trans~~enic
plant that
contains a single added transgene, germinating some of the seed produced and
analyzing
the resulting plants produced for enhanced activity relative to a control
(native.
non-transgenic) or an independent segregant transaenic plant.
Even more preferred is a plant in which the minichrornosome has not been
chromosomallv integrated. Such a plant may be termed ?n + x. where ?n is the
diploid
number of chromosomes and where x is the number of minichromosomes. Initially.
transformants may be 2n+l. i.e. having 1 additional minichromosome. In this
case. it
may be desirable to self the plant or to cross the plant with another 2n + 1
plant to yield a
plant which is 2n + ?. The 2n + 2 plant is preferred in that it is expected to
pass the
minichromosome through meiosis to all its offspring.
It is to be understood that two different transgenic plants also can he mated
to
produce offsprin~_~ that cont.rin two independently segre«atin~~ added.
exogenous
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minichromosomes. Selfing of appropriate progeny can produce plants that are
homozygous for both added. exogenous minichromosomes that encode a polvpeptide
of
interest. Back-crossins to a parental plant and out-crossing with a non-
tr~tns~enic plant
also are contemplated.
(iv) Otlrer Trcrn.c/~rntati~nr A~fetltods
'Transformation of plant protoplasts can be achieved using methods based on
calcium phosphate precipitation, polyethylene glycol treatment,
electroporation, and
combinations of these treatments (see. e.k.. Potrykus et nl.. 1985: Lorz et
nl., 1985;
Fromm et crl., 1986; Uchimiya et al., 1986: Callis et ul., 1987: Marcotte er
ul., 1988).
Application of these systems to different plant strains for the purpose of
making
transgenic plants depends upon the ability to regenerate that particular plant
strain from
protoplasts. Illustrative methods for the regeneration of cereals from
protoplasts are
described (Fujimura et ul., 198: Toriyama et al.. 1986; Yamada et ul., 1986;
Abdullah et al.. 1986).
To transform plant strains that cannot be successfully regenerated from
protoplasts, other ways to introduce DNA into intact cells or tissues can be
utilized. For
example, regeneration of cereals from immature embryos or explants can be
effected as
described (Vasil 1988). In addition. "particle gun" or high-velocity
microprojectile
technology can be utilized (Vasil 199? >.
Using that latter technology. DNA is carried through the cell wall and into
the
cytoplasm on the surface of small metal particles its described (Klein et nl..
1987:
Klein et ul.. 1988: McCabe et al.. 1988). The metal particles penetrate
through several
layers of cells and thus allow the transformation of cells within tissue
explants.
Protoplast fusion. for example. could be used to integrate a minichromosomc
constructed in a host cell. such as a vesst cell, and then fuse those cells to
plant
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protoplasts. The chromosomes lacking plant centromeres (such as yeast
chromosomes in
this example) would be eliminated by the plant cell while the minichromosome
would be
stable maintained. Numerous examples ~f protocols for protoplast fusion that
could be
used with the invention have been described (see. e.~y.. Negrutiu ee ul..
199?. and
Peterson).
Liposome fusion could be used to introduce a recombinant construct comprising
a
centromere, such as a minichromosome. by. for example, packaging the
recombinant
construct into small droplets of lipids (liposomes) and then fusing these
liposomes to
plant protoplasts thus delivering the AC into the plant cell (see Lurqui and
Rollo, 1993).
VIII. Exogenous Genes for Expression in Plants
One particularly important advance of the present invention is that it
provides
methods and compositions for expression of exogenous genes in plant cells. One
advance
of the constructs of the current invention is that they enable the
introduction of multiple
genes. potentially representing an entire biochemical pathway. Significantly,
the current
invention allows for the transformation of plant cells with a minichromosome
comprising
a number of structural genes. Another advantage is that more than one
minichromosome
could be introduced. allowing combinations of genes to be moved and shuffled.
Moreover. the ability to eliminate a minichromosome from a plant would provide
additional flexibility. making it possible to alter the set of genes contained
within a plant.
Further. by using site-specific recombinases, it should be possible to add
genes to an
existing minichromosome once it is in a plant.
Added genes often will be genes that direct the expression of a particular
protein
or polvpeptide product. but they also may be non-expressible DNA segments.
e.g..
transposons such as Ds that do not direct their own transposition. As used
herein. an
'expressible gene" is any gene that is capable of being transcribed into RNA
(e.b~..
mRNA. antisense RNA. etc.) or translated into a protein. expressed as a trait
of interest.
c>r the like. erc.. and is not limited to selectahle. screenable or non-
selectable marker
CA 02362897 2001-09-18
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genes. 'The inventors also contemplate that. where both an expressible gene
that is not
necessarily a marker gene is employed in combination with a marker gene. one
may
employ the separate genes on either the same or different DNA se~rnents for
transformation. In the latter case, the different vectors are delivered
concurrently to
recipient cells to maximize cotransformation.
The choice of the particular DNA segments to be delivered to the recipient
cells
often will depend on the purpose of the transformation. One of the major
purposes of
transformation of crop plants is to add some commercially desirable,
agronomically
important traits to the plant. Such traits include. but are not limited to,
herbicide
resistance or tolerance: insect resistance or tolerance; disease resistance or
tolerance
(viral. bacterial. funr'al, nematode): stress tolerance and/or resistance. as
exemplified by
resistance or tolerance to drought. heat. chilling. freezing. excessive
moisture, salt stress;
oxidative stress: increased yields: food content and makeup: physical
appearance: male
l~ sterility; drydown; standability; prolificacy; starch quantity and quality:
oil quantity and
quality: protein quality and quantity: amino acid composition: and the like.
One may
desire to incorporate one or more genes conferring any such desirable trait or
traits, such
as, for example. a gene or genes encoding herbicide resistance.
In certain embodiments, the present invention contemplates the transformation
of
a recipient cell with minichromosomes comprising more than one exogenous gene.
As -
used herein. an "exogenous gene." is a gene not normally found in the host
'~enome in an
identical context. By this, it is meant that the gene may be isolated from ~
different
species than that of the host genome. or alternatively. isolated from the host
Uenome but
?s operably linked to one or more regulatory regions which differ from those
found in the
unaltered. native ~=ene. Two or more exogenous genes also can he supplied in a
single
transformation event using either distinct transgene-encoding vectors. or
using a single
vector incorporating two or more gene coding sequences. For example. plasmids
bearing
the bcn- and « rr~A expression units in either convergent. divergent, or
colinear orientation.
are considered to be particularly useful. Further preferred combinations are
those of an
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insect resistance acne. such as a Bt gene. along with a protease inhibitor
gene such as
Mull. or the use of bar in combination with either of the above genes. Of
course. any two
or more trans~enes of any description. such as those conferring herbicide.
insect. disease
(viral. bacterial. fungal. nematode) or drought resistance. male sterility.
drydown.
S standability. prolificacy. starch properties. oil quantity and quality, or
those increasin_T
yield or nutritional quality may be employed as desired.
(i) Her-bicidc~ R~~sistance
The genes encoding phosphinothricin acetyltransferase (bar and pert).
~lyphosate
tolerant EPSP synthase genes. the glyphosate degradative enzyme gene ,~n.r
encoding
glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that
inactivates
dalapon). herbicide resistant (c~.g., sulfonylurea and imidazolinone)
acetolactate svnthase.
and hxn genes (encodin_ a nitrilase enzyme that degrades bromoxynil) are food
examples
of herbicide resistant genes for use in transformation. The bnr and hat ~~enes
code for an
enzyme, phosphinothricin acetyltransferase (PAT). which inactivates the
herbicide
phosphinothricin and prevents this compound from inhibiting ~lutamine
synthetase
enzymes. The enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP
Synthasel, is
normally inhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).
However.
genes are known that encode glyphosate-resistant EPSP synthase enzymes. These
'zenes
are particularly contemplated for use in plant transformation. The deh gene
encodes the
enzyme dalapon dehalo~enase and confers resistance to the herbicide dalapon.
The b.mr
gene codes for a specific nitrilase enzyme that converts bromoxynil to a non-
herbicidal
degradation product.
2> (ii) Irr.cect Re.ci.ctrurce
Potential insect resistance genes that can be introduced include l3crcillrr.,
flrrlr'rrr~~~IC'Ir.Cl.1 Cr'ySI:lI toxin genes or Bt genes (Watrud et ol..
1985). Bt y==ones m,m provide
resistance to lepidopteran or coleopteran pests such as European Corn Borer I
ECB ).
Preferred Bt toxin genes for use in such embodiments include the Cwlf1(h) and
Crwl.=plc l
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genes. Endotoxin genes from other species of B. tlrurirr,~ien.ci.s which
affect insect growth
or development also may be employed in this regard.
It is contemplated that preferred Bt genes for use in the transformation
protocols
S disclosed herein will be those in which the coding sequence has been
modified to eftect
increased expression in plants. and more particularly. in monocot plants.
Means for
preparing synthetic genes are well known in the art and are disclosed in. for
example.
U.S. Patent No. S.S00.365 and U.S. Patent Number No. 5.689.OS2> 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 CrvIA(h) gene
(Perlak et ul., 1991 ), and the synthetic Cr,~IA(c) gene termed 1800b (PCT
Application
WO 9S/06128). Some examples of other Bt toxin genes known to those of skill in
the art
are given in Table 1 below.
I S Table 1: Bacillus thurin;~iensis 8-Endotoxin Genes'
New Nomenclature Old l~omenclature GenBank Accession
Cr IAa CrvIA(a) M112S0
Cr I Ab C IA( b ) M 13898
C i Ac CrvIAlc ) M 1 1068
Cr IAd CrvIAtd) M732S0
C 1 Ae CrvIAle ) M6S2S2
Cr IBa CrvB X06711
Cry I Bb ETS L32020
Crv 1 Bc PEG ~ 246442
C IBd CrvEl U70726
C iCa CrvlC X07S18
Crv 1 Cb CrvlCi b ) M97880
C 1 Da C ID X54160
Cr I Db PrtB Z22S I 1
Cr 1Ea Cr IE XS398S
C 1 Eb Cr ~IEt b ) M732S3
Crv l Fa Cr IF M63897
Cr 1Fb ~ PrtD 22251?
Cr IGa PrtA 222510
Cr lGb Cr H? U7072S
Crv 1 Ha PrtC Z2?S 13
CrvlHb U3S780
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WO 00/55325 PCT/US00107392
New Nomenclature Dld Nomenclature GenBank Accession
__
Crv 1 la Cr V ._ X62821
Crv 11b CryV 007642
lJa ET4 L32019
Crv
_ ETI f_.i31527
Cr 11b
1 K 028801
Crv
_ C IIA M31738
C 2Aa
Crv2Ab C IIB M23724
Crv2Ac Cr IIC X57252
Cry3A C IIIA M22472
Cr 3Ba Cr IIIB X 17123
Cr 3Bb Cr II1B2 M89794
Cr 3C Cr II1D X59797
Cry4A Cr IVA Y00423
C 4B Cr IVB X07423
Cr 5Aa CrvVA(a) L07025
CrvSAb C VAIb) L07026
Crv6A CryVIA L07022
Crv_ 6B Cr VIB L0702=t
Cr 7Aa Cr 111C M64478
Cr 7Ab CrylIlCb 004367
Cr 8A Cr IIIE 004364
CrvBB C 11IG 004365
Cr 8C Cr II1F 004366
Cr 9A CrvlG X58120
Cr 9B Cr IX X75019
Crv9C CrvlH 237527
Cry I OA CrvIVC M 12662
CrvllA Cr IVD M31737
Crv 1 I B Jes80 X 86902
Crv 12A CryVB L07027
C l3A C VC L07023
Crv l4A CrvVD ~~ I 3955
Cr ~15A 34kDa M76442
Crv 16A cbm71 X94146
C 17A cbm71 X99478
CrvlBA C BP1 X990=i9
Crv 19A Je~65 Y089?0
Cvt I Aa C tA X03 I 82
Cvt I Ab CvtM X98793
Cvt?A CvtB Z I 41 ~#7
Cvt? B CvtB U 5?04 3
_gc~_
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''Adapted from:
http://epunix.biols.susx.ac.uk/Home/Isieil_Crickmore/Bt/index.html
Protease inhibitors also may provide insect resistance (Johnson m crl.. 1989).
and
will thus have utility in plant uansformation. The use of a protease inhibitor
II gene,
pirrll. from tomato or potato is envisioned to be particularly useful. Even
more
advantageous is the use of a hinll gene in combination with a Bt toxin gene,
the
combined effect of which has been discovered to produce synergistic
insecticidal activity.
Other genes which encode inhibitors of the insect's digestive system, or those
that encode
enzymes or co-factors that facilitate the production of inhibitors. also may
be useful. This
group may be exemplified by oryzacystatin and amylase inhibitors such as those
from
wheat and barley.
Also. genes encoding lectins may confer additional or alternative insecticide
properties. Lectins (originally termed phytohemagglutinins) are multivalent
IS carbohydrate-binding proteins which have the ability to ag~;lutin~ue red
blood cells from a
range of species. Lectins have been identified recently as insecticidal agents
with activity
against weevils. ECB and rootworm (Murdock el crl.. 1990: Czapla & Lang.
1990).
Lectin genes contemplated to he useful include, for example. barley and wheat
germ
a~~(utinin (WGA) and rice lectins (Gatehouse et crl., 1984). with WGA being
preferred.
Genes controlling the production of large or small polypeptides active against
insects when introduced into the insect pests. such as. e. y.. lyric 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
2~ specific insect pests. also may result in inseeticidal activity. or perhaps
cause cessation of
metamorphosis (Hammock et crl.. 1990).
Transoenic plants expressing genes which encode enzymes that affect the
integrity
of the insect cuticle form yet another aspect of the invention. Such genes
include those
encoding, e. ~., chitinasc. proteases. lipases and also genes for the
production of
nikkomycin_ a compound that inhibits chitin synthesis. the introduction of any
of which is
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contemplated to produce insect resistant plants. Genes that code for
activities that affect
insect molting, such as those affecting the production of ecdysteroid UDP-
glucosvl
transferase. also fall within the scope of the useful trans«enes of the
present invention.
Genes that code for enzymes that facilitate the production of compounds that
reduce the nutritional quality of the host plant to insect pests also are
encompassed by the
present invention. It may be possible. for instance, to confer insecticidal
activity on a
plant by altering its sterol composition. Sterols are obtained by insects from
their diet and
are used for hormone synthesis and membrane stability. Therefore alterations
in plant
sterol composition by expression of novel genes. e.g., those that directly
promote the
production of undesirable sterols or those that convert desirable sterols into
undesirable
forms, could have a negative effect on insect growth and/or development and
hence
endow the plant with insecticidal activity. Lipoxyaenases are naturally
occurring plant
enzymes that have been shown to exhibit anti-nutritional effects on insects
and to reduce
IS the nutritional quality of their diet. Therefore, further embodiments of
the invention
concern transgenic plants with enhanced lipoxygenase activity which may be
resistant to
insect feeding.
Trips«crr»r d«cwlnicle.s is a species of grass that is resistant to certain
insects.
including corn root worm. It is anticipated that genes encoding proteins that
are toxic to
insects or are involved in the biosynthesis of compounds toxic to insects will
be isolated
from Tripscrcr»» and that these novel genes will be useful in conferring
resistance to
insects. It is known that the basis of insect resistance in Trip.r«crr»r is
genetic. because
said resistance has been transferred to Ze« »r«v.c via sexual crosses (Branson
and Guns.
?5 197?). It is further anticipated that other cereal. monocot or dicot plant
species may have
genes encoding proteins that are toxic to insects which would be useful for
producing
insect resistant plants.
Further genes encoding proteins characterized as having potential insecticidal
activity also rnay be used as transgenea in accordance herewith. Such ;~enea
include. for
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example. the cowpea trypsin inhibitor (CpTI: Hilder et crl., 1987) which may
be used as a
rootworm deterrent; genes encoding avermectin (Avenrrectin trot! Ah«nrectin..
Campbell.
W.C.. Ed.. 1989; lkeda c:t «L. 19871 which may prove particularly useful as a
corn
rootworm deterrent; ribosome inactivating protein genes: and even genes that
regulate
plant structures. Transgenic plants including anti-insect antibody genes and
genes that
code for enzymes that can convert a non-toxic insecticide (pro-insecticide)
applied to the
outside of the plant into an insecticide inside the plant also are
contemplated.
(iii) Environment or Stress ReSI.SIClIIG'e
Improvement of a plants ability to tolerate various environmental stresses
such as.
but not limited to. drought, excess moisture. chilling. freezing, high
temperature. salt, and
oxidative stress. also can be effected through expression of novel genes. It
is proposed
that benefits may be realized in terms of increased resistance to freezing
temperatures
through the introduction of an "antifreeze" protein such as that of the Winter
Flounder
I S (Cutler et «l.. 1989) or synthetic gene derivatives thereof. Improved
chilling tolerance
also may be conferred through increased expression of ~Ivcerol-3-phosphate
acetyltransferase in chloroplasts (Wolter et «L, 1992). Resistance to
oxidative stress
(often exacerbated by conditions such as chilling temperatures in combination
with hi~~h
light intensities) can be . conferred by expression of superoxide dismutase
(Gupta et «l.. 1993), and may be improved by ~~lutathione reductase (Bowler et
«l., 1992).
Such strategies may allow for tolerance to freezing in newly emerged fields as
well as
extending later maturity higher yieldin~l varieties to earlier relative
maturity zones.
It is contemplated that the expression of novel genes that favorably effect
plant
water content, total water potential, osmotic potential. and tumor will
enhance the ability
of the plant to tolerate drought. As used herein, the terms "drought
resistance" and
"drought tolerance" are used to refer to a plants increased resistance or
tolerance to stress
induced by a reduction in water availability. as compared to normal
circumstances. and
the ability of the plant to function and survive in lower-water environments.
In this
aspect of the invention it is proposed. for cxarnple. that the expression of
genes encodin~T
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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 dehydro~enase (Lee and Saicr. 1982) and trehalose-6-
phosphate
synthase (Kaasen et al., 1992). Through the subsequent action of native
phosphatases in
the cell or by the introduction and coexpression of a specific phosphatase.
these
introduced genes will result in the accumulation of either mannitol or
trehalose,
respectively. both of which have been well documented as protective compounds
able to
mitigate the effects of stress. Mannitol accumulation in transgenic tobacco
has been
verified and preliminary results indicate that plants expressing high levels
of this
metabolite are able to tolerate an applied osmotic stress (Tarczynski et al..
1992, 1993).
Similarly, the efficacy of other metabolites in protecting either enzyme
function
(c.~~.. alanopine or propionic acid) or membrane integrity (e.~~., alanopine)
has been
documented (Loomis et al.. 1989), and therefore expression of genes encoding
for the
biosynthesis of these compounds might confer drought resistance in a manner
similar to
or complimentary to mannitol. Other examples of naturally occurring
metabolites that are
osmotically active and/or provide some direct protective effect during drought
and/or
desiccation include fructose, erythritol (Coxson et al., 1992), sorbitol,
dulcitol
(Karsten et crl.. 1992). glueosylglycerol (Reed et crl.. 198=x: ErdMann et
crl.. 1992),
sucrose. stachyose lKoster and Leopold, 1988: Blackman er al., 1992),
raffinose
(Bernal-Lugo and Leopold, 1992), proline (Rensburg et ul.. 1993). '~lycine
betaine,
ononitol and pinitol (Vernon and Bohnert. 1992). Continued canopy Qrowth and
increased reproductive fitness durin~~ times of stress will be augmented by
introduction
and expression of genes such as those controlling the osmotically active
compounds
2> discussed above and other such compounds. Currently preferred genes which
promote
the synthesis of an osmotically active polyol compound are genes which encode
the
enzymes rnannitol-I-phosphate dehydrooenase, trehalose-6-phosphate synthase
and
myoinositol 0-methyltransferase.
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It is contemplated that the expression of specific proteins also may increase
drought tolerance. Three classes of Late Embryogenic Proteins have been
assigned based
on structural similarities (see Dure er crl.. 1989). All three classes of LEAs
have been
demonstrated in maturing (i.e. desiccating) seeds. Within these 3 types of LEA
proteins.
the Type-II (dehydrin-type) have Generally been implicated in drought and/or
desiccation
tolerance in vegetative plant parts (i.e. Mundy and Chua, 1988: Piatkowski er
crl.. 1990:
Yamaguchi-Shinozaki etal.. 1992). Recently, expression of a Type-III LEA (HVA-
1) in
tobacco was found to influence plant height, maturity and drought tolerance
(Fitzpatrick,
1993). In rice. expression of the HVA-1 gene influenced tolerance to water
deficit and
salinity (Xu et al., 1996). Expression of stntctural genes from all three LEA
groups may
therefore confer drought tolerance. Other types of proteins induced during
water stress
include thiol proteases. aldolases and transmembrane transporters (Guerrero er
crl.. 1990).
which may confer various protective and/or repair-type functions during
drou~~ht stress. It
also is contemplated that genes that effect lipid biosynthesis and hence
membrane
composition might also be useful in conferring drought resistance on the
plant.
Many of these genes for improving drought resistance have complementary modes
of action. Thus. it is envisaged that combinations of these genes might have
additive
and/or synergistic effects in improving drought resistance in plants. Many of
these genes
also improve freezing tolerance (or resistance): the physical stresses
incurred during
freezing and drought are similar in nature and may be mitigated in similar
fashion.
Benefit may be conferred via constitutive expression of these genes. but the
preferred
means of expressing these novel genes may be through the use of a tumor-
induced
promoter (such as the promoters for the turgor-induced genes described in
Guerrero er crl.. 1990 and Shagan et crl.. 1993 which are incorporated herein
by reference).
Spatial and temporal expression patterns of these Genes may enable plants to
better
withstand stress.
It is proposed that expression of genes that are involved with specific
morphological traits that allow for increased water extractions from drying
soil would he
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of benefit. For example. introduction and expression of genes that alter root
characteristics may enhance water uptake. It also is contemplated that
expression of
genes that enhance reproductive fitness during times of stress would be of
significant
value. For example. expression of genes that improve the synchrony of pollen
shed and
receptiveness of the female flower parts, i.c~.. silks. would be of benefit.
In addition it is
proposed that expression of genes that minimize kernel abortion during times
of stress
would increase the amount of Grain to be harvested and hence be of value.
Given the overall role of water in determining yield, it is contemplated that
enabling plants to utilize water more efficiently. through the introduction
and expression
of novel genes. will improve overall performance even when soil water
availability is not
limiting. By introducing genes that improve the ability of plants to maximize
water usage
across a full range of stresses relating to water availability, yield
stability or consistency
of yield performance may be realized.
(ia) Di.sea.ce Resistance
It is proposed that increased resistance to diseases may be realized through
introduction of genes into plants, for example. into monocotyledonous plants
such as
maize. It is possible to produce resistance to diseases caused by viruses,
bacteria, fund
and nematodes. It also is contemplated that control of mycotoxin producing
organisms
may be realized through expression of introduced genes.
Resistance to viruses may be produced through expression of novel genes. For
example, it has been demonstrated that expression of a viral coat protein in a
transgenic
2i plant can impart resistance to infection of the plant by that virus and
perhaps other closely
related viruses (Cuozzo et ctl., 1988. Hemenway et al.. (988. Abel et al.,
1986). It is
contemplated that expression of antisense ~lenes targeted at essential viral
functions may
also impart resistance to viruses. For example. an antisense gene targeted at
the ~~ene
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
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the use of antisense genes also may increase resistance to viruses. Further,
it is proposed
that it may be possible to achieve resistance to viruses through other
approaches.
including. but not limited to the use of satellite viruses.
It is proposed that increased resistance to diseases caused by bacteria and
fund
may be realized through introduction of novel genes. It is contemplated that
genes
encoding so-called "peptide antibiotics." pathogenesis related (PR) proteins,
toxin
resistance, and proteins affecting host-pathogen interactions such as
morphological
characteristics will be useful. Peptide antibiotics are polypeptide sequences
which are
inhibitory to growth of bacteria and other microorganisms. For example, the
classes of
peptides referred to as cecropins and magainins inhibit growth of many species
of
bacteria and fungi. It is proposed that expression of PR proteins in
monocotyledonous
plants such as maize may be useful in conterrin~ 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 J3-I, 3-glucanases, chitinases. and osmotin and other
proteins that are
believed to function in plant resistance to disease or~unisms. Other genes
have been
identified that have antifungal properties, e.,~.. UDA (stinging nettle
lectin) and hevein
(Broakaert et ctl.. 1989; Barkai-Golan et ul.. 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 inactiv~ttin~T the phytotoxin. It also is
contemplated
that expression of novel genes that alter the interactions between the host
plant and
pathogen may be useful in reducing the ability of the disease organism to
invade the
2~ tissues of the host plant, e.y.. an increase in the waxiness of the leaf
cuticle or other
morphological characteristics.
(a) Plnut A,~rmtnmic Cltat-ctctcistic.c
Two of the factors determinin~7 where crop pUants can be grown are the average
daily temperature during the ~rowin~ season and the len~lth of time between
frosts.
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Within the areas where it is possible to grow a particular crop, there are
varying
limitations on the maximal time it is allowed to brow to maturity and be
harvested. For
example. a variety to be gown in a particular area is selected for its ability
to mature and
dry down to harvestable moisture content within the required period of time
with
maximum possible yield. Therefore, crops of varying maturities is developed
for
different growing locations. Apart from the need to dry down sufficiently to
permit
harvest, it is desirable to have maximal drying take place in the field to
minimize the
amount of energy required for additional drying post-harvest. Also, the more
readily a
product such as grain can dry down, the more time there is available for
growth and
kernel fill. It is considered that genes that influence maturity and/or dry
down can be
identified and introduced into plant lines using transformation techniques to
create new
varieties adapted to different growing locations or the same 'rowing location,
but having
improved yield to moisture ratio at harvest. Expression of genes that are
involved in
regulation of plant development may be especially useful.
It is contemplated that genes may be introduced into plants that would improve
standability and other plant growth characteristics. Expression of novel genes
in plants
which confer stronger stalks, improved root systems, or prevent or reduce ear
droppage
would be of great value to the farmer. It is proposed that introduction and
expression of
genes that increase the total amount of photoassimilate available by, for
example,
increasing light distribution and/or interception would he advantageous. In
addition, the
expression of genes that increase the efficiency of photosynthesis and/or the
leaf canopy
would further increase wins in productivity. 1t is contemplated that
expression of a
phytochrome gene in crop plants may be advantageous. Expression of such a gene
may
reduce apical dominance, confer semidwarfism on a plant. and increase shade
tolerance
(U.S. Patent No. 5.268.526). Such approaches would allow for increased plant
populations in the field.
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(vi) rVrrtrient Utili:.crtinrr
The ability to utilize available nutrients may be a limiting factor in growth
of crop
plants. It is proposed that it would be possible to alter nutrient uptake,
tolerate pH
extremes. mobilization through the plant. storage pools. and availability for
metabolic
S activities by the introduction of novel genes. These modifications would
allow a plant
such as maize to more efficiently utilize available nutrients. It is
contemplated that an
increase in the activity of, for example. an enzyme that is normally present
in the plant
and involved in nutrient utilization would increase the availability of a
nutrient. An
example of such an enzyme would be phytase. It is further contemplated that
enhanced
nitrogen utilization by a plant is desirable. Expression of a glutamate
dehydrogenase
Gene in plants. e.,Q., E~. cnli gdlrA genes. may lead to increased fixation of
nitrogen in
organic compounds. Furthermore. expression of gdlrA in plants may lead to
enhanced
resistance to the herbicide glufosinate by incorporation of excess ammonia
into
Glutamate, thereby detoxifying the ammonia. It also is contemplated that
expression of a
novel gene may make a nutrient source available that was previously not
accessible. c.~.,
an enzyme that releases a component of nutrient value from a more complex
molecule.
perhaps a macromolecule.
(vii) Male Sterilim
Male sterility is useful in the production of hybrid seed. 1t is proposed that
male
sterility may be produced through expression of novel genes. For example. it
has been
shown that expression of ~=enes that encode proteins that interfere with
development of
the male inflorescence and/or sametophyte result in male sterility. Chimeric
ribonuclease
genes that express in the anthem of transgenic tobacco and oilseed rape have
been
demonstrated to lead to male sterility (Mariani et al.. 1990).
A number of mutations were discovered in maize that confer cytoplasmic male
sterility. One mutation in particular. referred to as T cytoplasm. also
correlates with
sensitivity to Southern corn leaf blight. A DNA sequence. designated TURF-13
(Levings, 1990), was identified that correlates with T cytoplasm. it is
proposed that it
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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 gain production. it is proposed that
genes encoding
restoration of male fertility also rnav be introduced.
(viii) Inrpr-ovecl Nutritional Corrterrt
Genes may be introduced into plants to improve the nutrient quality or content
of
a particular crop. Introduction of genes that alter the nutrient composition
of a crop may
greatly enhance the feed or food value. For example. the protein of many
grains is
suboptimal for feed and food purposes. especially when fed to pigs, poultry.
and humans.
The protein is deficient in several amino acids that are essential in the diet
of these
species, requiring the addition of supplements to the grain. Limiting
essential amino
acids may include lysine, methionine. tryptophan. threonine, valine, arginine,
and
histidine. Some amino acids become limiting only after corn is supplemented
with other
inputs for feed formulations. The levels of these essential amino acids in
seeds and grain
may be elevated by mechanisms which include, but are not limited to. the
introduction of
genes to increase the biosynthesis of the amino acids. decrease the
degradation of the
amino acids, increase the storage of the amino acids in proteins, or increase
transport of
the amino acids to the seeds or grain.
The protein composition ~f a crop may be altered to improve the balance of
amino
acids in a variety of ways including elevating expression of native proteins,
decreasing
expression of those with poor composition. changing the composition of native
proteins.
or introducing genes encoding entirely new proteins possessing superior
composition.
The introduction of genes that alter the oil content of a crop plant may also
be of
value. Increases in oii content may result in increases in metabolizable-
energy-content
and density of the seeds for use in feed and food. The introduced genes may
encode
enzymes that remove or reduce rate-limitations or regulated steps in fatty
acid or lipid
biosynthesis. Such. genes may include. but are not limited to, those that
encode acetyl-
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CoA carboxylase. ACP-acvltransferase, ~i-ketoacyl-ACP synthase. plus other
well known
fatty acid biosynthetic activities. Other possibilities are genes that encode
proteins that do
not possess enzymatic activity such as acyl carrier protein. Genes may be
introduced that
alter the balance of fatty acids present in the oil providing a more healthful
or nutritive
feedstuff. The introduced DNA also may encode sequences that block expression
of
enzymes involved in fatty acid biosynthesis, altering the proportions of tatty
acids present
tn crops.
Genes may be introduced that enhance the nutritive value of the starch
component
of crops, for example by increasing the degree of branching. resulting in
improved
utilization of the starch in livestock by delaying its metabolism.
Additionally. other
major constituents of a crop may be altered. including genes that affect a
variety of other
nutritive, processing, or other quality aspects. For example. pigmentation may
be
increased or decreased.
Feed or food crops may also possesses insufficient quantities of vitamins.
requiring supplementation to provide adequate nutritive value. Introduction of
genes that
enhance vitamin biosynthesis may be envisioned including, for example.
vitamins A. E,
B,,. choline, and the like. Mineral content may also be sub-optimal. Thus
genes that
affect the accumulation or availability of compounds containing phosphorus.
sulfur,
calcium. manganese, zinc. and iron among others would be valuable.
Numerous other examples of improvements of crops may be used with the
invention. The improvements may not necessarily involve grain. but may, for
example.
?5 improve the value of a crop for silage. Introduction of DNA to accomplish
this might
include sequences that alter lignin production such as those that result in
the "brown
midrih" phenotype associated with superior feed value for cattle.
In addition to direct improvements in feed or food value. genes also may be
introduced which improve the proceaainy of crops and improve the value of the
products
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resulting from the processing. One use of crops if via wetmillin~. Thus novel
genes that
increase the efficiency and reduce the cost of such processing, for example by
decreasing
steeping time. may also find use. Improving the value of wetmillino products
may
include altering the quantity or quality of starch, oil. corn gluten meal. or
the components
of gluten feed. Elevation of starch may be achieved through the identification
and
elimination of rate limiting steps in starch biosynthesis or by decreasing
levels of the
other components of crops resulting in proportional increases in starch.
Oil is another product of wetmilling, the value of which may be improved by
introduction and expression of genes. Oil properties may be altered to improve
its
performance in the production and use of cooking oil, shortenings, lubricants
or other oil-
derived products or improvement of its health attributes when used in the food-
related
applications. Novel fatty acids also may be synthesized which upon extraction
can serve
as starting materials for chemical syntheses. The changes in oil properties
may be
achieved by altering the type, level, or lipid arrangement of the fatty acids
present in the
oil. This in turn may be accomplished by the addition of genes that encode
enzymes that
catalyze the synthesis of novel fatty acids and the lipids possessing them or
by increasing
levels of native fatty acids while possibly reducing levels of precursors.
Alternatively,
DNA sequences may be introduced which slow or block steps in fatty acid
biosynthesis
resulting in the increase in precursor fatty acid intermediates. Genes that
might be added
include desaturases. epoxidases, hydratases. dehydratases. and other enzymes
that
catalyze reactions involving fatty acid intermediates. Representative examples
of
catalytic steps that might be blocked include the desaturations from stearic
to oleic acid
and oleic to linolenic acid resulting in the respective accumulations of
stearic and oleic
acids. Another example is the blockage of elongation steps resulting in the
accumulation
of C~ to C,, saturated fatty acids.
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(i.r) Prncl«ctio» nr As.s«»rlatro» Ot'Clrt'Jricals or Biologicals
It may further be considered that a trans~enic plant prepared in accordance
with
the invention may be used for the production or manufacturing of useful
hiolo~ical
compounds that were either not produced at all. or not produced at the same
level. in the
corn plant previously. Alternatively, plants produced in accordance with the
invention
may be made to metabolize certain compounds. such as hazardous wastes, thereby
allowing bioremediation of these compounds.
The novel plants producing these compounds are made possible by the
introduction and expression of one or potentially many genes with the
constructs
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 tlavonoids or vitamina. enzymes that could produce
pharmaceuticals.
and for introducing enzymes that could produce compounds of interest to the
manufacturing industry such as specialty chemicals and plastics. The compounds
may be
produced by the plant, extracted upon harvest and/or processing, and used for
any
presently recognized useful purpose such as pharmaceuticals, fragrances, and
industrial
enzymes to name a few.
(x) Nn»-Prntei»-E.yres.si»y Sec/«e»cw.c
DNA may be introduced into plants for the purpose of expressing RNA
transcripts
that function to affect plant phenotype yet are not translated into protein.
Two examples
are antisense RNA and RNA with rihozyme activity. Both may serve possihfe
functions
in reducing or eliminating expression of native or introduced plant genes.
However. as
detailed below. DNA need not be expressed to effect the phenotype of a plant.
CA 02362897 2001-09-18
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I. Antisense RNA
Genes may be constructed or isolated. which when transcribed, produce
antisense
RNA that is complementary to all or parts) of a targeted messenger RNA(s). The
antisense RNA reduces production of the polypeptide product of the messenger
RNA.
The polypeptide product may be any protein encoded by the plant genome. The
aforementioned genes will be referred to as antisense genes. An antisense gene
may thus
be introduced into a plant by transformation methods to produce a novel
transgenic plant
with reduced expression of a selected protein of interest. For example, the
protein may be
an enzyme that catalyzes a reaction in the plant. Reduction of the enzyme
activity may
reduce or eliminate products of the reaction which include any enzymatically
synthesized
compound in the plant such as fatty acids, amino acids, carbohydrates, nucleic
acids and
the like. Alternatively, the protein may be a storage protein, such as a zein,
or a structural
protein. the decreased expression of which may lead to changes in seed amino
acid
composition or plant morphological changes respectively. The possibilities
cited above
I S are provided only by way of example and do not represent the full range of
applications.
2. Ribozymes
Genes also may be constructed or isolated. which when transcribed. produce RNA
enzymes (ribozymes) which can act as endoribonucleases and catalyze the
cleavage of
RNA molecules with selected sequences. The cleavage of selected messenger RNAs
can
result in the reduced production of their encoded polypeptide products. These
Genes may
be used to prepare novel transgenic plants which possess them. The transgenic
plants
may possess reduced levels of polypeptidcs including, hut not limited to. the
polypeptides
cited above.
Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-
specific
fashion. Ribozymes have specific catalytic domains that possess endonuclease
activity
(Kim and Cech, 1987: Gerlach et crl.. 1987: Forster and Symons, 1987). For
example, a
large number of ribozymes accelerate phosphoester transfer reactions with a
high decree
of specificity. often cleaving only one of several phosphoesters in an
oligonucleotide
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substrate (Cech et «t., 1981; Michel and Westhof~, 1990: Reinhold-Hurek and
Shub.
1992). This specificity has been attributed to the requirement that the
substrate bind vicr
specific base-pairing interactions to the internal wide sequence ("IGS") of
the ribozyme
prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific
cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et «L,
1981 ). For
example, U. S. Patent 5.354.855 reports that certain ribozymes can act as
endonucleases
with a sequence specificity greater than that of known ribonucleases and
approaching that
of the DNA restriction enzymes.
Several different ribozyme motifs have been described with RNA cleavage
activity (Syrnons, 1992). Examples include sequences from the Group I self
splicing
introns including Tobacco Ringspot Virus (Prody et crl.. 1986), Avocado
Sunblotch
Viroid (Palukaitis et «l., 1979: Symons, 1981 ). and Lucerne Transient Streak
Virus
(Forster and Symons, 1987j. Sequences from these and refuted viruses are
referred to as
hammerhead ribozyme based on a predicted folded secondary structure.
Other suitable ribozymes include sequences from RNase P with RNA cleavage
activity (Yuan et «!.. 1992. Yuan and Altman. 1994. L1. S. Patents 5,168.053
and
5.624,824). hairpin ribozyme structures (Berzal-Herranz et crl.. 1992:
Chowrira et «l.. 1993) and Hepatitis Delta virus based ribozymes (U. S. Patent
5.625,047). The general design and optimization of ribozyme directed RNA
cleavage
activity has been discussed in detail (Haseloff and Geriach, 1988. Symons.
1992.
2~ Chowrira et «1.. 1994: Thompson et «l., 1995).
The other variable on ribozyme design is the selection of a cleava~~e site on
a
given target RNA. Ribozymes are targeted to a given sequence by virtue of
annealing to
a site by complimentary base pair interactions. Two stretches of homolo<,y are
required
for this targetinU. These stretches of homolo~~ous sequences t7ank the
catalytic ribozyme
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structure defined above. Each stretch of homologous sequence can vary in
length from 7
to 15 nucleotides. The only requirement for defining the homologous sequences
is that.
on the target RVA. they are separated by a specific sequence which is the
cleavage site.
For hammerhead ribozyme, the cleavage site is a dinucleotide sequence on the
target
RNA is a uracil (U) followed by either an adenine. cytosine or uracil (A.C or
U)
(Perriman et «l., 199?: Thompson et al., 1995). The frequency of this
dinucleotide
occurring in any given RNA is statistically 3 out of 16. Therefore. for a
given target
messenger RNA of 1.000 bases. 187 dinucleotide cleavage sites are
statistically possible.
Designing and testing ribozymes for efficient cleavage of a target RNA is a
process well known to those skilled in the art. Examples of scientific methods
for
designing and testing ribozymes are described by Chowrira et «l., ( 1994) and
Lieber and
Strauss ( I995 i. each incorporated by reference. The identification of
operative and
preferred sequences for use in down regulating a Qiven gene is simply a matter
of
preparing and testing a given sequence, and is a routinely practiced
"screening" method
known to those of skill in the art.
3. Induction of Gene Silencing
It also is possible that genes may be introduced to produce novel transgenic
plants
which have reduced expression of a native gene product by the mechanism of
co-suppression. It has been demonstrated in tobacco. tomato. and petunia
(Goring et ciL, 1991: Smith et «L. 1990: Napoli et «l.. 1990: van der Krol et
«!., 1990) that
expression of the sense transcript of a native gene will reduce or eliminate
expression of
the native gene in a manner similar to that observed for antisense Genes. The
introduced
gene may encode all or part of the targeted native protein but its translation
may not be
required for reduction of levels of that native protein.
=1. Non-RICA-Expressing Sequences
DNA elements includin~7 those of transposable elements such as Ds. Ac. or Mu.
may be inserted into a gene to cause mutations. These DNA elements may be
inserted in
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order to inactivate (or activate) a gene and therehy "tag" a particular trait.
In this instance
the transposable element does not cause instability of the tagged mutation.
because the
utility of the element does not depend on its ability to move in the genome.
Once a
desired trait is tagged, the introduced DNA sequence may be used to clone the
corresponding gene, c~.y., using the introduced DNA sequence as a PCR primer
together
with PCR gene cloning techniques (Shapiro, 1983: Dellaporta et al., 1988).
Once
identified. the entire genes) for the particular trait, including control or
regulatory regions
where desired, may be isolated, cloned and manipulated as desired. The utility
of DNA
elements introduced into an organism for purposes of gene tagging is
independent of the
DNA sequence and does not depend on any biological activity of the DNA
sequence. i.e.,
transcription into RNA or translation into protein. The sole function of the
DNA element
is to disrupt the DNA sequence of a gene.
It is contemplated that unexpressed DNA sequences. including novel synthetic
sequences, could be introduced into cells as proprietary "labels" of those
cells and plants
and seeds thereof. It would not be necessary for a label DNA element to
disrupt the
function of a gene endogenous to the host organism, as the sole function of
this DNA
would be to identify the origin of the organism. For example, one could
introduce a
unique DNA sequence into a plant and this DNA element would identify all
cells, plants.
and progeny of these cells as having arisen from that labeled source. It is
proposed that
inclusion of label DNAs would enable one to distinguish proprietary ~ermplasm
or
aertnplasm derived from such, from unlabelled ~ermplasm.
Another possible element which may be introduced is a matrix attachment region
2~ element (MAR). such as the chicken Ivsozyme A element (Stief, 1989). which
can be
positioned around an expressible gene of interest to effect an increase in
overall
expression of the bene and diminish position dependent effects upon
incorporation into
the plant genome (Stief et uL. 19R9: Phi-Van et al.. 1990).
s. Other
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Other examples of non-protein expressing sequences specifically envisioned for
use with the invention include tRNA sequences. for example, to alter colon
usage. and
rRNA variants, for example. which may confer resistance to various agents such
as
antibiotics.
IX. Biological Functional Eguivalents
Modification and changes may be made in the centromeric DNA segments of the
current invention and still obtain a functional molecule with desirable
characteristics.
The following is a discussion based upon changing the nucleic acids of a
centromere to
create an equivalent, or even an improved. second-generation molecule.
In particular embodiments of the invention, mutated centromeric sequences are
contemplated to be useful for increasing the utility of the centromere. It is
specifically
contemplated that the function of the centromeres of the current invention may
be based
l5 upon the secondary structure of the DNA sequences of the centromere and /
or the
proteins which interact with the centromere. By changing the DNA sequence of
the
centromere. one may alter the affinity of one or more centromere-associated
proteins) for
the centromere and / or the secondary structure of the centromeric sequences.
thereby
changing the activity of the centromere. Alternatively. changes may be made in
the
centromeres of the invention which do not effect the activity of the
centromere. Changes
in the centromeric sequences which reduce the size of the DNA segment needed
to confer
centromere activity are contemplated to be particularly useful in the current
invention. as
would changes which increased the fidelity with which the centromere was
transmitted
during mitosis and meiosis.
X. Plants
The term "plant." as used herein. refers to any type of plant. The inventors
have
provided below an exemplary description of some planta that may be used with
the
invention. However, the list is not in any way limiting. as other types of
plants will be
known to those of skill in the art and could be used with the invention.
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A common class of plants exploited in agriculture are vegetable crops.
including
artichokes. kohlrabi, anrgula. leeks, asparagus. lettuce (e. y.. head. leaf,
romaine). bok
choy, malanga. broccoli. melons (e.,~~., muskmelon. watermelon, crenshaw,
honeydew,
cantaloupe). Brussels sprouts, cabbage, cardoni, carrots, napa. cauliflower.
okra. onions.
celery, parsley. chick peas, parsnips. chicory. Chinese cabbage, peppers,
collards,
potatoes. cucumber plants (marrows, cucumbers). pumpkins. cucurbits. radishes,
dry bulb
onions, rutabaga, eggplant, salsify, escarole. shallots. endive. garlic,
spinach, green
onions, squash. greens, beet (sugar beet and fodder beet), sweet potatoes.
Swiss chard.
horseradish, tomatoes. kale, turnips, and spices.
Other types of plants frequently finding commercial use include fruit and vine
crops such as apples. apricots. cherries, nectarines. peaches, pear. plums.
prunes, quince
almonds, chestnuts, filberts, pecans, pistachios. walnuts, citrus.
blueberries.
1 ~ boysenberries, cranberries. currants. loganberries. raspberries.
strawberries. blackberries,
grapes, avocados, bananas, kiwi, persimmons, pomegranate. pineapple. tropical
fruits,
pomes. melon. mango, papaya. and lychee.
Many of the most widely grown plants are field crop plants such as evening
primrose, meadow foam: corn (field. sweet. popcorn). hops. jojoba. peanuts,
rice,
safflower. small grains (barley. oats, rye. wheat. etc. ). sorghum, tobacco,
kapok,
leguminous plants (beans. lentils, peas, soybeans). oil plants (rape, mustard.
poppy,
olives. sunflowers, coconut. castor oil plants. cocoa beans. groundnuts).
fibre plants
(cotton, flax. hemp, jute), lauraceae (cinnamon, camphor). or plants such as
coffee.
sugarcane, tea. and natural rubber plants.
Still other examples of plants include bedding plants such as flowers. cactus.
succulents and ornamental plants, as well a; trees such as forest ( broad-
leaned trees and
evergreens, such as conifers). fruit. ornamental, and nut-bearing trees. as
well as shrubs
and other nursery stock.
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XI. Definitions
Ac used herein. the terms ''autonomous replicating sequence" or ''ARS" or
''origin
of replication " refer to an origin of DNA replication recognized by proteins
that initiate
DNA replication.
As used herein, the terms "binary BAC" or "binary bacterial artificial
chromosome" refer to a bacterial vector that contains the T-DNA border
sequences
necessary for Al;rahacterir.nn mediated transformation (see, for example.
Hamilton et ul..
1996: Hamilton. 1997: and Liu et al., 1999.
As used herein, the term "candidate centromere sequence" refers to a nucleic
acid
sequence which one wishes to assay for potential centromere function.
As used herein, a "centromere" is any DNA sequence that confers an ability to
segregate to daughter cells through cell division. In one context, this
sequence may
produce a segregation efficiency to daughter cells ranging from about 1 % to
about 100%.
including to about ~°~o, 10%, 20%, 30%. 40%, 50%. 60%, 70%, 80%, 90% or
about 95%
of daughter cells. Variations in such a segregation efficiency may find
important
applications within the scope of the invention: for example. mini-chromosomes
carrying
centromeres that confer 100% stability could be maintained in all daughter
cells without
selection. while those that confer I °lc stability could be temporarily
introduced into a
transgenic organism. but be eliminated when desired. In particular embodiments
of the
invention. the centromere may confer stable segregation of a nucleic acid
sequence.
2~ including a recombinant construct comprising the centromere, through
mitotic or meiotic
divisions. including through both meiotic and meitotic divisions. A plant
centromere is
not necessarily derived from plants. but has the ability to promote DNA
seare~ation in
plant cells.
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As used herein. the term "centromere-associated protein" refers to a protein
encoded by a sequence of the centromere or a protein which is encoded by host
DNA and
binds with relatively high affinity to the centromere.
As used herein, "eukaryote" refers to living organisms whose cells contain
nuclei.
A eukaryote may be distinguished from a "prokaryote" which is an organism
which lacks
nuclei. Prokaryotes and eukaryotes differ fundamentally in the way their
emetic
information is organized, as well as their patterns of RNA and protein
synthesis.
As used herein, the term "expression" refers to the process by which a
structural
gene produces an RNA molecule. typically termed messenger RNA (mRNAj. The
mRNA is typically, but not always, translated into polypeptide(s).
As used herein, the term "~enome" refers to all of the genes and DNA sequences
that comprise the genetic information within a eiven cell of an organism.
Usually, this is
taken to mean the information contained within the nucleus, but also includes
the
organelles.
As used herein, the term "higher eukaryote" means a multicellular eukaryote,
typically characterized by its greater complex physiological mechanisms and
relatively
large size. Generally, complex organisms such as plants and animals are
included in this
category. Preferred higher eukaryotes to be transformed by the present
invention include.
for example. monocot and dicot angiosperm species, gymnosperm species, fern
species,
plant tissue culture cells of these species. animal cells and algal cells. It
will of course be
understood that prokaryotes and eukarvotes alike may be transformed by the
methods of
this invention.
As used herein, the term "host refers to any organism that is the recipient of
a
replicable plasmid, or expression vector comprising a plant chromosome.
Ideally. host
strains used for cloning experiments should he free of anv restriction enzyme
activity that
might degrade the foreign DNA used. Preferred examples of host cells for
cloning, useful
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in the present invention. are bacteria such as E.cchericlricr coli.
Bcrcillrr.o .srrhtili,s,
Pscrulcmrrnrra.s, Streptcnrrvces. Salrrronella. and yeast cells such as S.
c'L'I'( 1'lSlclE.'. Host cells
which can be targeted for expression of .~ minichromosome may be plant cells
of any
source and specifically include Arnhiclnp.vi.c. maize. rice, sugarcane.
sorghum. barley.
soybeans. tobacco. wheat. tomato, potato. citrus. or any other agronomieally
or
scientifically important species.
As used herein. the term "hybridization" refers to the pairing of
complementary
RNA and DNA strands to produce an RNA-DNA hybrid. or alternatively. the
pairing of
two DNA single strands from genetically different or the same sources to
produce a
double stranded DNA molecule.
As used herein. the term "linker" refers to a DNA molecule. generally up to i0
or
60 nucleotides long and synthesized chemically. or cloned from other vectors.
In a
preferred embodiment. this fragment contains one. or preferably more than one.
restriction enzyme site for a blunt-cutting enzyme and a staggered-cutting
enzyme. such
as BamHl. One end of the linker fragment is adapted to be ligatable to one end
of the
linear molecule and the other end is adapted to be ligatable to the other end
of the linear
molecule.
As used herein. a "library" is a pool of random DNA fragments which are
cloned.
In principle. any gene can be isolated by screening the librarw with a
specific
hybridization probe (see. for example. Youna er nl., 1977). Each library may
contain the
DNA of a given organism inserted as discrete restriction enzyme-generated
fragments or
as randomly sheered fragments into many thousands of plasmid vectors. For
purposes of
the present invention, E. coli. yeast, and Salrrronellcr plasmids are
particularly useful
when the ~lenome inserts come from other orsanisms.
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As used herein. the term "lower eukaryote" refers to a eukaryote characterized
by
a comparatively simple physiology and composition. and most often
unicellularity.
Examples of lower eukaryotes include flagellates. ciliates, and yeast.
As used herein. a "minichromosome'' is a recombinant DNA construct including a
centromere and capable of transmission to daughter cells. The stability of
this construct
through cell division could range between from about 1 % to about 100%.
including about
5%, l0%. 209. 30%' 40%, 50%, 60%, 70%, 80%, 90% and about 95%. The
minichromosome construct may be a circular or linear molecule. It may include
elements
such as one or more telomeres, ARS sequences. and genes. The number of such
sequences included is only limited by the physical size limitations of the
construct itself.
It could contain DNA derived from a natural centromere, although it may he
preferable to
limit the amount of DNA to the minimal amount required to obtain a segre=anon
efficiency in the range of 1-100%. The minichromosome may be inherited through
mitosis or meiosis. or through both meiosis and mitosis. As used herein. the
term
minichromosome specifically encompasses and includes the terms "plant
artificial
chromosome~~ or "PLAC." and all teachings relevant to a PLAC or plant
artificial
chromosome specifically apply to constructs within the meaning of the term
minichromosome.
As used herein, by ''minichromosome-encoded protein" it is meant a polypeptide
which is encoded by a sequence of a minichromosome of the current invention.
This
includes sequences such as selectable markers. telomeres. c~c.. as well as
those proteins
encoded by any other selected functional genes on the minichromosome.
A "180 hale pair repeat" is defined as any one of the specific repeats
disclosed in
SEQ ID NOS: l84-212. or a "consensus'' sequence derived therefrom. Thus. a
liven "180
base pair repeat" may include more or less than l80 base pairs. and may
retlect a
sequence not represented by any of the specific sequences provided herein.
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As used herein. the term "plant" includes plant cells, plant protoplasts.
plant calli.
and the like, as well as whole plants regenerated therefrom.
As used herein. the term "plasmid" or "cloning vector" refers to a closed
covalently circular extrachromosomal DNA or Linear DNA which is able to
autonomously
replicate in a host cell and which is normally nonessential to the survival of
the cell. A
wide variety of plasmids and other vectors are known and commonly used in the
art (see,
for example. Cohen er nl., U.S. Patent No. 4.468.464. which discloses examples
of DNA
plasmids, and which is specifically incorporated herein by reference).
As used herein, a "probe" is any biochemical reagent (usually tagged in some
way
for ease of identification), used to identify or isolate a gene. a gene
product, a DNA
segment or a protein.
As used herein, the term "recombination" refers to any genetic exchange that
involves breaking and rejoininD of DNA strands.
As used herein the term ''regulatory sequence" refers to any DNA sequence that
influences the efficiency of transcription or translation of any gene. The
term includes,
but is not limited to, sequences comprising promoters, enhancers and
terminators.
As used herein. a "selectable marker" is a gene whose presence results in a
clear
phenotype, and most often a growth advantage for cells that contain the
marker. Thin
erowth advantage may he present under standard conditions. altered conditions
such as
elevated temperature. or in the presence of certain chemicals such as
herbicides or
antibiotics. Use of selectable markers is described, for example. in Broach et
al. ( 1979).
Examples of selectable markersthethymidine kinasegene, the
include cellular
adenine-phosphoribosyltramferaseandthe dihydrylfolatereductase
gene gene.
hygromycin phosphotransferasebargene and neomycinphosphotransferase
genes. the
eenes, among others. Preferred selectable markers in the present invention
include gene;
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whose expression confer antibiotic or herbicide resistance to the host cell.
sufficient to
enable the maintenance of a vector within the host cell, and which facilitate
the
manipulation of the plasmid into new host cells. Of particular interest in the
present
invention are proteins conferring cellular resistance to ampicillin,
chloramphenicol.
tetracycline. G-418. bialaphos, and glyphosate for example.
As used herein, a "screenable marker" is a gene whose presence results in an
identifiable phenotype. This phenotype may be observable under standard
conditions.
altered conditions such as elevated temperature, or in the presence of certain
chemicals
used to detect the phenotype.
As used herein, the term "site-specific recombination" refers to any genetic
exchange that involves breaking and rejoining of DNA strands at a specific DNA
sequence.
I$
As used herein, a "structural gene" is a sequence which codes for a
potypeptide or
RNA and includes 5' and 3' ends. The structural gene may be from the host into
which
the structural gene is transformed or from another species. A structural gene
will
preferably, but not necessarily, includC one or more regulatory sequences
which modulate
the expression of the structural gene. such as a promoter. terminator or
enhancer. A
structural gene will preferably. but not necessarily. confer some useful
phenotype upon an
organism comprising the structural gene. for example, herbicide resistance. In
one
embodiment of the invention. a structural gene may encode an RNA sequence
which is
not translated into a protein. for example a tRNA or rRNA gene.
As used herein, the term 'oelomere" 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.
As used herein. the terms "transformation" or "transfection" refer to the
acquisition in cells of new DNA sequences through the chromosomal or extra-
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chromosomal addition of DNA. This is the process by which naked DNA. DNA
coated
with protein, or whole minichromosomes are introduced into a cell, resulting
in a
potentially heritable change.
XII. Examples
The following examples are included to demonstrate preferred embodiments of
the invention. It should be appreciated by those of skilled the art that the
techniques
disclosed in the examples which follow represent techniques discovered by the
inventors
to function well in the practice of the invention, and thus can be considered
to constitute
preferred modes for its practice. However, those of skill in the art should,
in light of the
present disclosure, appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar result
without
departing from the concept. spirit and scope of the invention. More
specifically, it will be
apparent that certain agents which are both chemically and physiologically
related may be
IS substituted for the agents described herein while the same or similar
results would be
achieved. All such similar substitutes and modifications apparent to those
skilled in the
art are deemed to be within the spirit, scope and concept of the invention as
defined by
the appended claims.
EXAMPLE I
Generation of an Arabidopsis tl:aliaua Mapping Population
To generate a pollen donor plant, two parental lines carrying grtl were
crossed to
one another. The grtl-I allele was in the Landsbero ecotype background and the
c/rtl-2
allele was in the Columbia ecotype background. The Landsber~~ ecotype was
readily
2~ discernible from the Columbia ecotype because it carries a recessive
mutation, erecta,
which causes the stems to thicken, infloresences to be more compact, and the
leaves to be
more rounded and small than wildtype. To utilize this as a marker of a donor
plant. clrtl-2
pollen was crossed onto a c/rtl-l female stigma. The F, progeny were
heterozygous at all
molecular markers yet the progeny retain the grrnrtet phenotype of a tetrad of
fused pollen
gains. In addition, progeny display the ERECTA phenotype of the Columbia
plant. This
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visible marker serves as an indication that the crossing was successful in
generating
plants segregating ecotype specific markers. Further testing was done to the
donor plants
by performing PCR analysis to insure that progeny were heterozygous at
molecular loci.
Due to the fact that the pollen Grains cannot be directly assayed for marker
segregation and because of the desire to create a long-term resource available
for multiple
marker assays, it was necessary to cross individual tetrads generated by the
donor plant.
This created sets of progeny plants which yielded both large quantities of
tissue and seed.
These crosses were accomplished efficiently by generating a recipient plant
homozygous
for male sterility (msl ). The recessive mutant m.sJ was chosen to guard
against the
possibility of the recipient plant self-fertilizing and the progeny being
mistaken for tetrad
plants. Due to the fact that the homozygous plant does not self, a stock seed
generated by
a heterozygous nrale sreriliw I plant needs to be maintained from which
sterile recipient
plants can be selected.
EXAMPLE 2
Tetrad Poltinations
Tetrad pollinations were carried out as follows. A mature flower was removed
from the donor plant and tapped upon a glass microscope slide to release
mature tetrad
pollen grains. This slide was then placed under a 20-40x Zeiss dissecting
microscope.
To isolate individual tetrad pollen grains. a small wooden dowel was used to
which an
eyebrow hair with rubber cement was mounted. Using the light microscope. a
tetrad
pollen unit was chosen and touched to the eyebrow hair. The tetrad
preferentially adhered
to the eyebrow hair and was thus lifted from the microscope slide and
transported the
recipient plant stigmatic surface. The transfer was carried out without the
use of the
microscope. and the eyebrow hair with adhering tetrad was then placed against
the
recipient stigmatic surface and the hair was manually dragged across the
stigma surface.
The tetrad then preferentially adhered to the stigma of the recipient and the
cross
pollination was completed.
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Initially. S7 tetrad seed sets consisting of 3-4 seeds each, were collected.
Plants
were crown from these tetrad seed sets. and tissue was collected. DNA was
extracted
from a small portion of the stored tissue for PCR based segregation analysis.
Additionally the segregation of the visible erect« phenotype was scored. When
the plants
S set seed. the seed was collected as n source for the larger amounts of DNA
required to
analyze RFLP segregation by Southern blotting.
EXAMPLE 3
Preparation and Analysis of Centromere-Spanning Contigs
IO Previously. DNA fingerprint and hybridization analysis of two bacterial
artificial
chromosome (BAC) libraries led to the assembly of physical maps covering
nearly all
single-copy portions of the Ar«hidopsi.s genome (Marra et «l., 1999). However,
the
presence of repetitive DNA near the Ar«hidnpsis centromeres, including 180 by
repeats.
retroelements. and middle repetitive sequences complicated efforts to anchor
centromeric
15 BAC contigs to particular chromosomes (Murata et «l., 1997; Heslop-Harrison
et «l..
1999: Brandes et crl., 1997; Franz et «l.. 1998: Wright et «l., 1996;
Koniecznyet crl., 1991;
Pelissier et crl., 1995; Voytas and Ausubel. 1988; Chye et «L. 1997; Tsay et
«l., 1993;
Richards et «L. 1991; Simoens et crl.. 1988: Thompson et «l.. 1996; Pelissier
et «l.. 1996).
The inventors used genetic mapping to unambiguously assign these unanchored
contigs to
20 specific centromeres, scoring polymorphic markers in 48 plants with
crossovers
informative for the entire genomc (Copenhaver et «L, 1998). In this manner.
several
centromeric contigs were connected to the physical maps of the chromosome arms
(see
EXAMPLE 6 and Table 4), and a lame set of DNA markers defining centromere
boundaries were generated. DNA sequence analysis confirmed the structure of
the
25 conti~s for chromosomes I1 and IV 1 Lin et «l.. 1999).
CEN2 and CEN4 were selected in particular for analysis. Both reside on
structurally similar chromosomes with a 3.S Mb rDNA arrays on their distal
tips, with
regions measuring 3 and ? Mb, respectively. between the rDNA and centromeres.
and l6
30 and 13 Mb regions on their lon'1 arms (Copenhaver and Pikaard, 1996).
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The virtually complete and annotated sequence of chromosomes II and IV was
used to conduct an analysis of centromeres at the nucleotide level
(http://www.ncbi.nlm.nih.gov/Entrezlnucleotide.html). The sequence composition
was
analyzed within the genetically-defined centromere boundaries and compared to
the
adjacent pericentromeric regions (FIGS. 12A-T). Analysis of the two
centromeres
facilitated comparisons of sequence patterns and identification of conserved
sequence
elements.
The centromere sequences were found to harbour 180 by repeat sequences. These
sequences were found to reside in the gaps of each centromeric contig (FIG. 3,
FIGS. 12B,
12L), with few repeats and no long arrays elsewhere in the genome. BAC clones
near
these gaps have end sequences corresponding to repetitive elements that likely
constitute
the bulk of the DNA between the contigs, including 180 by repeats, 5S rDNa or
I60-by
IS repeats (FIG. 3). Fluorescent iu Biter hybridization has shown these
repetitive sequences
are abundant components of Arobidopsis centromeres (Murata et al., 1997:
Heslop-Harrison et al., 1999: Brandes et al., 1997). Genetic mapping and
pulsed-field
gel electrophoresis indicate that many 180 by repeats reside in long arrays
measuring
between 0.4 and 1.4 Mb in the centromeric regions (Round et al., 1997):
sequence
analysis revealed additional interspersed copies near the gaps. The inventors
specifically
contemplate the use of such 180 by repeats for the construction of
minichromosomes.
The annotated sequence of chromosomes II and IV identified regions with
homology to
middle repetitive DNA, both within the functional centromeres and in the
adjacent
regions (FIGS. 12B-12E and 12L-120).
In a 4.3 Mb sequenced region that includes CEN2 and a 2.8 Mb sequenced region
that includes CEN4, retrotransposon homology was found to account for > 10% of
the
DNA sequence. with a maximum of 6290 and 70%. respectively (FIGS. 12C, 12M).
Sequences with similarity to transposons or middle repetitive elements were
found to
occupy a similar zone. but were lesa common (29% and l l °lo maximum
density for
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chromosomes II and IV respectively (FIGS. 12D-12E and FIG. 1?N-120). Finally.
unlike
in the case of Drosophilcr and Nerrro.sporcr centromeres (Sun et crl., 1997;
Cambareri et crl.. 1998) low complexity DNA. including microsatellites,
homopolymer
tracts, and AT rich isochores, were not found to be enriched in the
centromeres of
Arnbidopsis. Near CEN2, simple repeat sequence densities were comparable to
those on
the distal chromosome arms, occupying 1.5% of the sequence within the
centromere,
3.2°lo in the flanking regions, and ranging from 20 to 319 by in length
(71 by on average).
Except for an insertion of mitochondria) DNA at CEN2 the DNA in and around the
centromeres did not contain any large regions that deviated significantly from
the
~enomic average of - 64% A + T (FIGS. 12F, 12P) (Bevan et al., 1999).
Unlike the 180 by repeats, all other repetitive elements near CE'N? and CEN=l
were less abundant within the genetically-defined centromeres than in the
flanking
regions. The high concentration of repetitive elements outside of the
functional
centromere domain suggest they may be insufficient for centromere activity.
Thus,
identifyinJ segments of the Arubidopsi.s genome that are enriched in these
repetitive
sequences does not pinpoint the regions that provide centromere function; a
similar
situation may occur in the genomes of other higher eukaryotes.
The repetitive DNA flanking the centromeres may play an important role.
Forming
an altered chromatin conformation that serves to nucleate or stabilize
centromere
structure. Alternatively. other mechanisms could result in the accumulation of
repetitive
elements near centromeres. Though evolutionary models predict repetitive DNA
accumulates in regions of low recombination (Charlesworth et crl., 1986:.
Charlesworth et ul.. 1994). many Arabidop.si.s repetitive elements are more
abundant in
the recombinationally active pericentromeric regions than in the centromeres
themselves.
Instead, retroelements and other transposons may preferentially insert into
regions
tlankin~ the centromeres or be eliminated from the rest of the Qenome at a
higher rate.
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EXAMPLE 4
Genetic Niapping of Centromeres
To map centromeres, F, plants which were heterozygous for hundreds of
polymorphic DNA markers were generated by crossing c/rrartet mutants from the
Landsber~ and Columbia ecotypes (Chang et crl. 1988: Ecker, 1994: Konieczy and
Ausubel. 1993). In tetrads from these plants, genetic markers segregate in a
?:2 ratio
(FIG. 6; Preuss et al. 1994). The segregation of markers was then determined
in plants
which were generated by crossing pollen tetrads from the F, plants onto a
Landsberg
homozygote. The genotype of the pollen grains within a tetrad was inferred
from the
genotype of the progeny. Initially, seeds were generated from greater than l00
successful
tetrad pollinations, and tissue and seeds were collected from 57 of these.
This provided
sufficient material for PCR, as well as seeds necessary for producing the
large quantities
of tissue required for Southern hybridization and RFLP mapping. In order to
obtain a
more precise localization of the centromeres the original tetrad population
was increased
from 57 tetrads to over > 1,000 tetrads.
PCR analysis was performed to determine marker segregation. To account for the
contribution of the Landsberg background from the female parent, one Landsberg
complement from each of the four tetrad plants was subtracted. As shown in
FIG. 5,
markers from sites spanning the entire genome were used for pair-wise
comparisons of all
other markers. Tetratypes indicate a crossover between one or both markers and
their
centromeres where as ditypes indicate the absence of crossovers (or presence
of a double
crossover).
Thus. at every genetic locus, the resulting diploid progeny was either L/C or
C/C.
The map Venerated with these plants is based solely on male meioses. unlike
the existing
map, which represents an average of recombination's in both males and females.
Therefore. several well-established genetic distances were recalculated and
thus will
determine whether recombination frequencies are significantly altered.
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The large quantities of genetic data generated by the analysis must be
compared
pair-wise to perform tetrad analysis. All of the data was managed in a
Microsoft Excel
spread-sheet format, assigning Landsberg alleles a value of "1'' and Columbia
alleles a
value of "0". Within a tetrad, the segregation of markers on one chromosome
was
compared to centromere-linked reference loci on a different chromosome (see
Table 2
below). Multiplying the values of each locus by an appropriate reference, and
adding the
results for each tetrad easily distinguished PD, NPD, and TT tetrads with
values of 2. 0,
and 1, respectively.
Monitoring the position of crossovers in this population identified
chromosomal
regions that could be separated by recombination from centromeres (tetratype),
as well as
regions that always cosegregated with centromeres (ditype) (Copenhaver et ul..
I 998;
Copenhaver et cr1..1999). Tetratype frequencies decrease to zero at the
centromere:
consequently, centromere boundaries were defined as the positions that
exhibited small
but detectable numbers of tetratype patterns. By scoring the segregation of
centromere
linked markers in approximately 400 tetrads, centromeres 1-5 were localized to
regions
on the physical map corresponding to contigs of 550, 1445, 1600, 1790 and 1770
kb,
respectively (FIG. 3). Additionally, for each centromeric interval, a number
of useful
recombinants were identified. The results of the analysis indicated that
centromeres
reside within large domains that restrict recombination machinery activity and
that the
transition between these domains and the surrounding recombination-proficient
DNA is
markedly abrupt.
Table 2: Scoring protocol for tetratypes.
Individual- _
members Locus Reference ReferenceLocus Reference
of a 1 Locus Locus Locus 3 Locus
tetrad 2
A 1 x 1= 1 0 x I= 0 x I= 0
0
B 1 x 1= 1 0 x 1= 1 x 1= 1
0
C 0 x 0= 0 1 x 0= 0 x 0= 0
0
D 0 x 0= 0 1 x 0= 1 x 0= 0
0
2 0
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PD NPD TT
Analysis of polymorphisms corresponding to 180 by repeats (RCEN markers.
Round et al, 1997) confirmed that these repeats map within the genetically-
defined
centromeres. Polymorphisms associated with the 180 by repeats were analyzed by
pulsed
field gel electrophoresis as described previously (Round et al., 1997).
Segregation of
these polymorphisms in tetrads with informative crossovers confirmed complete
linkage
of a 180 by repeat array at each centromere. In genetic units, the centromere
intervals
averaged 0.44 cM, (% recombination = 1/2 tetratype frequency), reflecting
recombination
rates at least 10-30 fold below the genomic average of 221 kb/cM (Somerville
and
Somerville, 1999; http://nasc.nott.ac.uk/new_ri_map.html).
The low recombination frequencies typically observed near higher eukaryotic
centromeres may be due to DNA modifications or unusual chromatin states (Choo,
1998:
Puechberty, 1999; Mahtani and Willard. 1998; Charlesworth et crl.. 1986;
Charlesworth et al., 1994). To modify these states. and thus improve
centromere
mapping resolution by raising recombination frequencies. FI Landsberg/Columbia
plants
were treated with one of a series of compounds known to cause DNA damage,
modify
chromatin structure, or alter DNA modifications. Fl Landsberg qrtl / Columbia
gutl
plants were grown under 24 hour light in 1" square pots and treated with
methanesulfonic
acid ethyl ester (0.05%), 5-aza-2'-deoxycytidine (25 or 100 mg/1), Zeocin ( 1
uglml),
methanesulfonic acid methyl ester (75 ppm). cis-diamminedichloro-platinum (20
ug/ml),
mitomycin C (lOmg/1), n-nitroso-n-ethylurea ( 100 uM), n-butyric acid (20 uM).
trichostatin A ( 10 uM), or 3-methoxybenzamide (? mM ). Plants were watered
and
flower-bearing stems were immersed in these solutions. Alternatively, plants
were
exposed to 350 nm UV (7 or 10 seconds), or heat shock (38 or 42°C for 2
hours). Pollen
tetrads from these plants were used to pollinate Landsherg stigmas 3-5 days
after each
treatment: the FI plants were subsequently subjected to additional treatments
(up to 5
times per plant, every 3-5 days).
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Tetrads from treated plants were crossed to Landsber~ stigmas, and progeny
from
8-107 tetrads subjected to each treatment were recovered and analyzed,
yielding >600
additional tetrads. These tetrads exhibited higher recombination in regions
immediately
flanking the centromeres (1.6 vs. 3.4% recombination in untreated and treated
plants.
respectively). although the sample size was insufficien«o determine if any
individual
treatment had a profound affect. The map locations of centromeres were refined
on
chromosomes 2 to 5 (FIG. 1), yielding intervals spanned by condos of 880. I
I50. 1260.
and 1070 kb. respectively, with all tetrads consistently localizing centromere
functions to
the same region (Copenhaver et al., 1999).
Efforts to increase recombination yielded a lar~~e number of tetrads with
crossovers near the centromeres; these crossovers clustered within a narrow
region at the
centromere boundaries. Five crossovers occurred over a 70 kb region near CEN2,
and 7
over a 200 kb region near CENI , yet no crossovers were detected in the
adjacent
l5 centromeric intervals of 880 and 550 kb respectively (FIG. 3). Thus, the
centromeres
were found within large domains that restrict recombination machinery
activity; the
transition between these domains and surrounding. recombination-proficient DNA
is
remarkably abrupt (FIG. 12A and K). Although analysis of more tetrads would
yield
additional recombination events, the observed distribution of crossovers
indicate that
centromere positions would not be sicnificantly refined.
EXAMPLE 5
Sequence Analysis of Arabidopsis Centromeres
A. Abundance of genes in the centromeric regions
Expressed genes are located within 1 kb of essential centromere sequences in
S.
cerevisiae, and multiple copies of tRNA genes reside within an 80 kb fragment
necessary
for centromere function in S. pnrnbe (Kuhn et al., 1991 ). In contrast. genes
are thought to
be relatively rare in the centromeres of higher eukaryotea. though there are
notable
exceptions. The Drn.coplrila light. coucertincr, respouclc-r, and rolled loci
atl map to the
centromeric region of chromosome ?, and translocations that remove light from
its native
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heterochromatic context inhibit gene expression. 1n contrast. many Dunsophilcr
and
human genes that normally reside in euchromatin become inactive when they are
inserted
near a ceniromere. Thus. genes that reside near centromeres likely have
special control
elements that allow expression (Karpen. 199:f: Lohe and I-lilliker, 1990. The
sequences
of Arcrhiclop.sis CEN2 and CEN.~, provided herein, provide a powerful resource
for
understanding how gene density and expression correlate with centromere
position and
associated chromatin.
Annotation of chromosome II and IV (http://
www.ncbi.nlm.nih.~ov/Entrez/nucleotide.html) identified many genes within and
adjacent to CEN2 and CEN4 (FIG. 8, FIGS. 12A-12T). The density of predicted
genes on
Arnhiclopsis chromosome arms averages 2~ per 100 kb. and in the repeat-rich
regions
flanking CEN2 and CEN4 this decreases to 9 and 7 genes per 100 kb,
respectively
(Bevan et al., 1999). Many predicted genes also reside within the
recombination-deficient, genetically-defined centromeres. Within CEN2, there
were
predicted genes per 100 kb: while CEN4 was strikingly different, with 12 Genes
per 100
kb.
There was strong evidence that several of the predicted centromeric Genes are
transcribed. The phosphoenolpyruvate gene (CUE! ) defines one CENT border:
mutations
in this gene cause defects in light-regulated gene expression (Li et crl.,
1995). Within the
sequenced portions of CE_N2 and CEN4, 17% (?7/160) of the predicted genes
shared
>959o identity with cloned cDNAs (SSTs). with three-fold more matches in CEN4
than in
CEN2 (http://www.tigr.org/tdb/ac/agad/). Twenty-four of these genes have
multiple
exons, and four correspond to single-copy genes with known functions. A list
of the
predicted genes identified is given in Table 3. below. A list of additional
genes encoded
within the boundaries of CEN~i are listed in Table :~. The identification of
these genes is
significant in that the genes may themselves contain unique regulatory
elements or may
reside in genomic locations tlanking unique control or regulatory elements
involved in
centromere function or gene expression. In particular, the current inventors
contemplate
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use of these genes, or DNA sequences 0 to 5 kb upstream or downstream of these
sequences, for insertion into a gene of choice in a minichromosome. It is
expected that
such elements could potentially viefd beneficial regulatory controls of the
expression of
these genes, even when in the unique environment of a centromere.
To investigate whether the remaining 23 senes were uniquely encoded at the
centromere, a search was made in the database of annotated ~enomic
Arcrbiclopsi.c
sequences. With the exception of two genes, no homologs with >95% identity
were
found elsewhere in the 80% of the genome that has been sequenced. The number
of
independent eDNA clones that correspond to a single-copy gene provides an
estimate of
the level of gene expression. On chromosome II, predicted genes with high
quality
matches to the cDNA database (> 95% identity) match an average of four
independent
cDNA clones (range I-78). Within CEN2 and CEN4. I I/27 genes exceed this
average
(Table 3). Finally, genes encoded at CEN2 and CEN4 are not members of a single
gene
family, nor do they correspond to genes predicted to play a role in centromere
functions,
but instead have diverse roles.
Many genes in the Arabiciopsi.r centromeric regions are nonfunctional due to
early
stop codons or disrupted open reading frames, but few pseudogenes were found
on the
chromosome arms. Though a large fraction of these pseudogenes have homology to
mobile elements, many correspond to genes that are typically not mobile (FIGS.
12I-J and
FIGS. 12S-T). Within the genetically-defined centromeres there were 1.0
(CEA'?/ and 0.7
(CEN4) of these nonmobile pseudogenes per 100 kb: the repeat-rich regions
bordering the
centromeres have I.5 and 0.9 per 100 kb respectively. The distributions of
pseudogenes
and transposable elements are overlapping, indicting that DNA insertions in
these regions
contributed to gene disruptions.
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Table 3: Predicted genes within CEN2 and CEN4 that correspond to the cDNA
database.
Putative function GenBank protein # of EST
accession matchesx
CEN2
Unknown AAC69124 1
SH3 domain protein AAD15528 5
Unknown AAD I 5529 I
unknownt AAD37022 1
RNA helicase~ AAC26676 2
40S ribosomal protein S 16 AAD22696 9
CEN4
Unknown AAD36948 1
Unknown AAD36947 4
leu cyl tRNA synthetase AAD36946 4
aspartic protease AAD29758 6
Peroxisomal membrane protein AAD297~9 S
(PPM2) ~
5'-adenylylsulfate reductase AAD29775 14
$
symbiosis-related protein AAD29776 3
ATP synthase gamma chain I (APCl)AAD489 3
~
protein kinase and EF hand AAD034~3 3
ABC transporter AAD03441 1
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Transcriptional regulator AAD03444 14
Unknown AAD03446 12
human PCF 1 1 p homolo~~ AAD03447 6
NSF protein AAD17345
l.3-beta-glucan synthase AAD48971 2
pyridine nucleotide-disulphide AAD48975 4
oxidoreductase
Polyubiquitin (UBQIl) ,~~' AAD48980 72
wound induced protein AAD48981 6
short chain dehydrogenase/reductaseAAD489_59 7
SL I St AAD48939
WD40-repeat protein AAD48948
* Independent cDNAs with >95% identity. t related gene present in non-
centromeric
DNA, - potentially associated with a mobile DNA element. ~ characterized gene
(B.
Tugal. 1999; J.F. Gutierrez-Marcos, 1996; N. Inohara, 1991: J. Callis. 1995).
Table 4: List of additional genes encoded within the boundaries of CEN4.
Putative Function GenBank Nucleotide
accession Position
3'(2').5'-Bisphosphate NucleotidascAC012392 71298 -73681
Transcriptional re~ultor AC012392 8061 I -81844
Equilibrative nucleoside transporterAC01239? 88570 -90739
1
Equilibrative nucleoside transporterAC012392 94940 -96878
I
Equilibrative nucleoside transporterAC012392 98929 -101019
I
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Equilibrative nucleoside transporterAC012392 1 13069 -I
1 15262
unknown AC012392 122486 -
f 24729
4-coumarate--CoA liaase AC012392 126-505 -128601
ethylene responsive protein AC012392 130044 -
I 31421
Oxygen-evolving enhancer proteinAC012392 134147 -135224
precursor
Kinesin AC012392 l 37630 -141536
receptor-like protein kinase AC012392 141847 -144363
LpxD-like protein AC012392 14492 I -146953
hypersensitivity induced proteinAC012392 147 I 58
-147838
ubiquitin AC012392 149057 -149677
unknown AC012392 I 50254 -15
I 072
ubiquitin-like protein AC012392 E53514 -154470
ubiquitin-like protein ACU12392 155734 -156513
ubiquitin-like protein AC012392 156993 -157382
unknown AC012392 159635 -165559
unknown AC012392 166279 -166920
unknown ACO 12392 I 67724 -1702
l 2
ubiquitin-like protein AC012392 176819 -178066
polyubiquitin ( UBQIO) ACO 12392 I 80613 -
J 82007
phosphatidylinositol-3.=1.5-triphosphatcACO(2477 893Ei4 -91?91
hindin~
protein
Mitochondrial ATPase AC012477 9430? -9=1677
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RING-H2 finer protein AC012477 9?2 -96142
unknown AC012477 104717 -105196
Mitochondrial ATPase AC012477 10i7~8 -10659
ferredoxin--NADP+ reductase AC012477 1074 1 -109095
unknown AC012477 109868 -1
10620
U3 snoRNP-associated protein AC012477 11 1841 -I
14133
UV-damaged DNA binding factor AC012477 1 14900 -121275
Glucan endo-1.3-Beta-GlucosidaseAC012477 122194 -122895
precursor
D123 -like protein AC012477 125886 -126887
Adrenodoxin Precursor AC012477 127660 -129246
N7 like-protein AC012477 129718 -131012
N7 like-protein AC012477 131868 -133963
N7 like-protein AC012477 1342 I ~
-136569
N7 like-protein AC012477 13966 -140864
characterized gene (3. Callis.
1995).
B. Conservation of centromeric UNA
To investigate the conservation of CEN? and CEN4 sequences. PCR primer pairs
were designed that correspond to unique regions in the Columbia sequence and
used to
survey the centromeric regions of Landsbcrg and Columbia at -20 kb intervals
(FIGS.
14A, Bj. The primers used for the analysis are listed in FIGS. 15A. B.
Amplification
products of the appropriate lensth were ohtained in both ecotypes for most
primer pairs
18570). indicating that the amplified re~iona were highly similar. In the
remaining cases.
primer pairs amplified Columbia. but not Landsber~ DNA. even at very low
strinQencics.
In these regions. additional primers were desi«ned t~ determine the extent of
nonhomology. In addition to a large insertion of mitochondrial DNA in CEU_.
two other
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non-conserved regions were identified (FIGS. 14A. B). Because this DNA is
absent from
Landsberg centromeres. it is unlikely to be required for centromere function:
consequently. the relevant portion of the centromeric sequence is reduced to
577 kb
(CEN2) and 1250 kb (CENT). The high degree of sequence conservation between
Landsber~ and Columbia centromeres indicated that the inhibition of
recombination
frequencies was not due to lame regions of nonhomoto~y. but instead was a
property of
the centromeres themselves.
C. Sequence similarity between CEN2 and CEN4
In order to discern centromere function. a search was conducted for novel
sequence motifs shared between CEN2 and CEN4, excluding from the comparison
retroelements. transposons. characterized ~:entromeric repeats, and coding
sequences
resembling mobile genes. After masking simple repetitive sequences. including
homopolymer tracts and microsatellites. contigs of unique sequence measuring d
17 kb
t5 and 851 kb for CENZ and CEN4, respectively, were compared with BLAST
(http://blast.wustl.edu).
The comparison showed that the complex DNA within the centromere regions
was not homologous over the entire sequence length. However. 16 DNA segments
in
CEN2 matched 11 regions in CEN=I with >60'70 identity (F1G. 16). The sequences
were
~roupcd into families of related sequences. and were desi«nated AtCCSI-7
(Arcrhi~lopsi.c
thalicrrrcr centromere conserved sequences 1-7). These sequences were not
previously
known to be repeated in the Arnhidopsi.c ~enome. The sequences comprised a
total of 17
kb (4%) of CEN? DNA. had an average length of 1017 bp. and had an A + T
content of
?5 6590. Based on similarity. the matching sequences were sorted into groups.
including
two families containin_ S sequences each (AtCCS 1 and AtCCS2: SEQ ID NOS:I-
la). 3
sequences from a small family encoding a putative open reading frame (AtCCS3:
SEQ ID
NOS:21-?2)), and 4 sequences found once within the centromeres (AtCCS~-AtCCS7:
SEQ ID NOS:15-ZO). one of which (AtCCS6: SEQ ID N0:17) corresponds to
predicted
CEN? and CEN4 proteins with similarity throe<Thout their exons and introns
(FIG. 16).
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Searches of the Aruhiclnp.ci.s genomic sequence database demonstrated that
AtCCS I - AtCCSS were moderately repeated sequences that appear in centromeric
and
pericentromeric regions. The remaining seqmnces were present only in the
genetically-defined centromeres. Similar comparisons of all 16 S. cerevisiae
centromeres
defined a consensus consisting of a conserved 8 by CDEI motif, an AT-rich 85
by CDEII
element. and a 26 by CDEII region with 7 highly conserved nucleotides
(Fleig et crl., 1990. In contrast, surveys of the three S. pombe centromeres
revealed
conservation of overall centromere structure. but no universally conserved
motifs (Clark.
1998).
EXAMPLE 6
Mapping Results: Arabidopsis Chromosomes 1-5
The centromere on chromosome 1 was mapped between mi342 (56.7 cM) and
T27K12 (59.1 cM). A more refined position places the centromere between the
marker
T22C23-t7 (-58.5 cM) and T3P8-sp6 (-59.1 cM). Contained within this interval
are the
previously described markers EKR1V and RCEN 1.
The centrornere on chromosome ? was mapped between mi310 ( 18.6 cM ) and
x4133 (23.8 cM). A more refined position places the centromere between the
markers
FSJ15-sp6 (-19.1 cM) and T15D9 019.3 cM). The following sequenced
(http://www.ncbi.nlm.nih.aov/Entrez/nucleotide.html ) BAC (bacterial
artificial
chromosome) clones are known to span the region between the markers FSJ 1 i-
sp6 and
T 1 SD9: T l 3E 1 1. F27C2 l . F9A ! 6, TSM2. T 17H 1. T I 8C6. TSE7. T 1212.
F27 B2?.
T6C20, T f 4C8. F7B f 9. and T 1 SD9.
There is a gap in BAC coverage between T12J2 and F27B22. RARE cleavage.
pulse field vela or DN.A sequence tiling will be used to isolate DNA in the
'~.rp for
sequencing.
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The centromere on chromosome 3 was mapped between atpox (48.6 cM > and
ATA (53.S cM). A more refined position places the centromere between the
marker
T9G9-sp6 (-53.1 cM) and TAM I=1-sp6 (-X3.3 cM). Contained within this interval
i; the
previously described marker: RCEN3.
The centromere on chromosome =l was mapped between mi233 ( 18.8 cM > and
mi167 (21.5 cM). A more refined position places the centromere between the
markers
T24H24.30k3 (-20.3 cM) and F13H14-t7 (-21.0 eM). The following sequenced
(http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html) BAC (bacterial artificial
chromosome) clones are known to span the region between the markers FSJI~-sp6
and
T6A I 3-sp6: T27D20, T 19B 17. T26N6. F4H6. T 19J 18. T4B21, T 1 J 1, T32N4. C
l 7L7.
C6L9, F6H8, F21I2. F14G16.and F28D6.
There is a gap in BAC coverage between F2I12 and F14G16. RARE cleavage.
pulse field gels or DNA sequence tiling will be used to isolate DNA in the gap
for
sequencing.
The centromere on chromosome ~ was mapped between nga76 (71.6 cM) and
PhyC (74.3 cM). A more refined position places the centromere between the
markers
FI3K20-t7 (-69.4 cM) and CUEI (-69.~ cM). Contained within thin interval are
the
publicly available markers: urn~79D. mi?91 b. CMS 1.
A table listing the BAC clones known to reside within the centromeres on
chromosomes I-V given as well as Genbank Accession numbers for the sequences
of
2~ these clones, is liven below. in Table ~ and Table 6.
Genetic positions (i.e. cM values correspond to the Lister and Dean
Recombinant
Inbred Genetic map, available on-line at
http://nasc.nott.ac.uk/new_ri_map.html Markers
are available at http://~enome-wwvv.Stanford.edulArabidopsis/aboutcaps.html.
1 3?_
CA 02362897 2001-09-18
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Table ~: BAC clones residing within A. thaliarra centromeres and associated
Genbank accession numbers
CENTROMERE 1 GENBANK ACCESSION #
F24P1 823044'
F13J4 AL08696T and AL086966*
F7G10 AL083686' and AL083685*
F28L22 AC007505
F17A20 823767*
F13G14 AL086828* and AL086827'
F13018 AL087175* and AL087174'
F24A15 AQ011599* and B98125* and B98124*
F25C4 823065*and 823064'
F3A6 none
T32E20 AC020646
F2007 B22665 and B22664*
F16K23 897718' and 825748' and B23714*
F8L2 AL084364'and AL084363'
F6C2 AL083089' and AL083088'
F1H9 AL080601"and AL080600'
F27022 AQ01 1488* and B25518*
F15P3 B97045* and 822971' and B22970*
F2406 B23041*
F20P22 AQ251396* and AQ251287'
F2C1 AL081001' and AL081000'
F 15F 11 B23547*
-l33-
CA 02362897 2001-09-18
W O I111/sS32s PCT/US00/07392
F1 F24 AL080554* and AL080553*
F6J1 AL083277*
F26H20 none
F16J24 none
F19M18 AQ011034*
F20K7 AG~251392* and AQ251282*
F 1266 AC007781 t
F23F21 none
F28G17 none
F28G 13 none
F27A14 AQ251243* and AG~251137'
F28G9 B23346* and 823345'
F21 F1 B95997* and B22704*
F16K24 B97719* and B25749*
F20C15 AQ251381' and AG~251272*
F9G18 AL084752* and AL084751* and B26534*
F10G23 AL085268* and AL085267*
F22016 AQ250131' and AQ249777* and B96460' and
B96459* and B12588* and B08235*
~F23P24 AQ011594* and B98116* and 8981 15'
!F24A9 AQ010513* and B96134* and B96133*
F26B21 825313*
I F28019 B25706*
j F 19J21 AQ011011 '
F28E 13 B25592* and 825591
' F24G 19 828443*
F15H9 822577' and 822576'
- I 3:~-
CA 02362897 2001-09-18
WO 00/~532~ PCT/US00/07392
F28A11 825540'
F26N17 825374'
F15J24 AQ011049* and 897568'
F25J4 B23109* and 823108'
F28P16 AQ011538* and 825713'
F12E11 AL086267* and AL086266*
F28G8 823344' and 823343'
F22L3 B22875* and B22874*
F25C2 B23063*
F22B13 829456'and B28433*
F13114 AL086945* and AL086944*
F11 L16 AL085969' and AL085968*
F25B1 B23057* and 823056'
F26H18 AQ010880* and AQ010879*
F20P4 822672'
F11 K13 AL085923* and AL085922*
F 1965 AQ251104'
F15F7 822200'and 822199'
F16C4 898549' and 898548' and 823399'
GAP
F 19M 16 AQ011032'
F22M21 896432' and 896431 *
F27K16 none
F21K24 897937'
F13P3 AL087187' and AL087186*
F15P18 none
F28G 19 825637'
F5E5 I AL082645* and AL082644*
CA 02362897 2001-09-18
WO 00/5325 PCT/US00/07392
F5K9 AL082841' and AL082840*
F5E 12 AL082657' and AL082656*
F21 N 15 861476'
F5L13 AL082880* and AL082879*
F17L20 B23905* and B23904*
F14K1 AL087586* and AL087585*
F16J4 B98573*
F15M18 none
F14116 AL087535' and AL087534*
F21K13 none
F16E23 none
F14~5 AL087748' and AL087747*
F20G9 822553' and B22552*
F27119 AQ01 1427' and B25464*
F1118 AL080658'
F16C8 898552' and B22985*
F2001 822655'
F13H12 AL086902' and AL086901*
F13B12 none
F27D7 none
I F21 B 16 824625'
F8F1 AL084170' and AL084169*
F9A 12 none
F22111 824855' and B24854*
~F16N17 825774' and B23737*
F 17H 11 823833'
'F15A12 none
F20M21 none
F19E19 824191'
F25015 825275' and 825274'
F27J13 AQ011435' and 825468*
~ ~(,_
CA 02362897 2001-09-18
WO 00/5532, PCT/US00/07392
F15J7 822603*and B22602*
F13J1 AL086961* and AL086960*
F9D18 AC007183t
F9M8 AL084923*
F519 AL082775*
F3L22 AL081822* and AL081821
F5P23 AL083021*
F10023 AL085527' and AL085526'
F20J1 AQ010790* and 822625'
F7K22 AL083828' and AL083827*
F6J23 AL083299* and AL083298*
T4121 AC022456t
F116 AL080639' and AL080638*
F28B8 AQ010984"
F20B1 B22488* and B22487*
F26F14 None
F18C13 B28362*
F20K13 AQ011116* and 824519"
F10K7 AL085379*
F5A 13 AC008046
F12B23 AL086177'
F9121 AL084816" and AL084815"
F17120 B23850* and 823849'
CENTROMERE 2
T13E11 AC006217
F27C21 AC006527
F9A 16 AC007662
T5M2 AC007730
T17H1 AC007143
T18C6 AC007729
T5E7 AC006225
T12J2 AC004483
GAP
T14C8 AC006219
-137-
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
F7B 19 AC006586
T15D9 AC007120 _
CENTROMERE 3
F6H5 AL083229' and AL083228*
F6G 13 AL083215* and AL083214*
F21 G23 B97922* and B24664*
F3J24 B19129* and B12732*
F2517 AQ010570* and 823104'
F14012 B22064* and B22004*
F1010 AL080869* and AL080868*
F11N16 AL086039* and AL086038*
F19M19 none
F301 AL081890* and AL081889*
F1 D9 B21602* and 821631 ' and AQ248831 * and
AL080449' and AL080450*
F8F8 none
F23A15 none
F201 AL081375* and AL081374*
F711 AL083741 * and AL083740*
F25D24 B25156* and B25155*
F10L19 AL085429' and AL085428*
F28J14 825860*and 825859*
F17D19 823796* and B23795*
F2701 1 B25508*
F27P23 AO011498* and 825537'
F11N11 B28323* and 828322'
F 16117 B97693*
_~3g_
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
GAP _
F1L15 AL080750* and AL080749*
F2A9 AL080941 ' and AL080940*
F2D1 AL081028* and AL081027*
F2D22 AL081046' and AL081045*
F208 AL081387* and AL081386*
F2014 AL081393' and AL081392*
F3G24 none
F9A7 AL084546* and AL084545*
F10N9 AL085473* and AL085472
*
T1415 AL088212* and AL088211'B19832* and B19707*
and
T1J6 AL088233* and AL088232*B19834* and 819709'
and
T2G 13 AL088663' and AL088662*
T6D10 AL090573* and AL090572*B27383* and B27382*
and
and 819977" and B19790*
T7K14 AL091315' and B27422*
and 827421 * and B20115*
and B19895'
T8012 B21405* and B21348"
T9J24 AL092268* and AL092267*820132" and 81991
and 1 '
T9K2 AL092269' and B20133*
and B19912*
;9_
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
T10F10 AL092618* and AL092617' and B20076* and
B19918*
T15N4 AL095818' and AL095817* and B20044* and
819856'
T16C1 AL095981 * and AL095980'
T16F22 AL096108*
T16M9 AL096289' and AL096288* and B20053* and
819865'
T18P7 860875' and 860874'
T21124 B62398* and 820320" and B20288*
T22E7 861351 * and 820426' and B20394*
T2419 B67385* and B67384* and 820450' and B20419*
T2405 B67422* and 820454'
T25C15 AQ225286* and B67937* and 820460
T25 F 15 AC009529t
F23H6 AC01 1621
T26J6 B76816* and 876815'
T28G 19 AC009328t
GAP
F6K8 AL083310* and AL083309'
F25M24 825253'
F25F9 823085'
F28F20 825620"
~F16C22 897681 * and 823646'
-!.. -.
F24M20 825096'
F27B5 823236' and 823235' -
- l=lU-
CA 02362897 2001-09-18
WO 00/~~325 PCT/US00/07392
F21 A 14 AC016828t
T4P3 AC009992
T14A11 AC012327
T26P13 AC009261
T18B3 AC011624t
F12P5 AL086610* and AL086609'
F22N7 AQ251226*
F21 N 12 B24707*
F7N6 none
F12E16 none
F21J13 AQ251199* and AQ011170*
F25 M 18 825251
F9B18 AL084600* and AL084599'
F20J23 AQ011113* and 824515'
F1 G6 AL080561 * and AL080560' and AQ251107
*
F704 AL083940* and AL083939'
F1 D4 AL080441 * and AL080440' and B22163*
F19P10 AQ251376* and AQ251268'
F4P10 AL082481 *and AL082480'
F9123 AL084818* and AL084817'
IF3118 AL081711 * and AL081710*
F13K14 AL087018* and AL087017'
F13K8 AL087008* and AL087007'
F13J3 AL086965* and AL086964'
F20F5 822533'
F1 K22 AL080723* and AL080722*
F3H 19 AL081679* and AL081678'
F23M13 B98039*
-1~1-
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
F23N10 B98054* and B98053*
F8M14 AL084410* and AL084409'
F7C16 AL083567* and AL083566*
F26D5 none
F10J2 AL085340*
F16L6 B23418*
F26P16 B25396* and B25395*
GAP
F28D17 none
F27E12 AQ251248* and AG~251142* and AQ011376*
and
AQ011375*
F4M19 AL082399* and AL082398*
T27B3 AL137079
F26B15 AL138645
T14K23 AL132909
T32A11 AL138653
F3021 AL081924* and AL081923*
F3114 AL081705' and AL081704*
F20C5 AQ251382* and AQ251273'
F14B7 AL087267* and AL087266*
F14K13 AL087604* and AL087603*
F21L14 897938'and B24690*
F23012 898080* and 898079*
F14G1 AL087450* and AL087449*
F19117 AQ225333*
F7C3 AL083548* and AL083547'
!F4111 AL082258* and AL082257*
F7J17 AL083789* and AL083788*
_ j:~?_
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
F18L6 B22332* and B22331*
F16N 18 B25775*
F28J6 823358'
F7C6 AL083554* and AL083553*
F28C1 B23304* and B23303*
F18117 B24063*
F10P16 AL085555* and AL085554*
F24G 17 none
F4K4 AL082320* and AL082319*
F26B15 B25309* and B25308*
F12P9 AL086614*and AL086613*
F8C3 AL084070* and AL084069*
F25D21 825153' and B25152*
F27C7 AQ010648' and AQ010647* and B23240*
F23G13 none
F15B16 AL087857'and AL087856*
CENTROMERE 4
T27D20 AF076274
T19B17 AF069441
T26N6 AF076243
F4H6 AF074021
T19J18 AF149414
T4B21 AF118223
T1J1 AF128393
T32N4 AF162444
C17L7 none
C6L9 none
T1J24 AF147263
F6H8 AF178045
F2112 AF 147261
GAP
F14G 16 AF 147260
F28D6 AF147262
- I 4s-
CA 02362897 2001-09-18
WO 00/~s32s PCT/US00/07392
CENTROMERE 5
F3F24 AC018632 _
F13K20 AL087030* and AL087029*
F6L19 none
F23C8 AC018928
F18F14 810562'
F22D5 AQ251214'
F12P18 none
F6C14 none
GAP
F28N5 823377*
F2C13 none
F12P1 AL086602*
F9K2 AL084855'
F23F23 AL086757
F13D7 AL086757' and AL086~56*
F4C11 AL082053" and AL082052*
F28G24 none
F7C4 AL083550' and AL083549*
F4B15 AL082023* and AL082022*
F19111 AQ010999*
F3M22 AL081848* and AL081847*
F1 M22 AL080803' and AL080802'
F21 A22 B24614* and B24613*
F8P23 AL084535* and AL084534'
F17M7 B22216' and B22215*
F21B21 B24632*
F17G22 B23828* and B23827*
F11 P4 AL086088" and AL086087*
IIF14J11 AL087566' and AL087565'
F7J19 AL083792* and AL083791'
F20G20 j none
-~4=1-
CA 02362897 2001-09-18
WO 00/~~325 PCT/US00/07392
F27H14 AQ251251* and AQ251145*
F25E10 none
F24123 B25815* and B25066*
T3D5 AL089085* and AL089084*
T17G5 AL096632* and AL096631
F20C16 B24433*
F27M22 none
F27K1 B23257*
F21 N24 B61479* and B24716*
F11 F13 AL085745' and AL085744*
F5015 AL082980* and AL082979*
F8G 15 AL084218* and AL084217*
F9A17 B12265* and B10646'
F25E19 none
F24C5 AQ010525* and AQ010524*
F27L2 AQ010708* and B96166*
F10A6 AL085056* and AL085055*
F23B23 AQ011184*
F1 E3 AL0804828' and AL080481' and B22171'
and
B22170*
GAP
F20J17 AQ011108' and 824510'
F21022 B24736* and B24735*
F26021 n one
F25M11 B25245* and B25244*
F18F8 B26318* and B22290*
F17M12 823910'
F22M20 896430'
iF9K6 A L084860'
-IdS-
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
F13J20 AL086992"and AL086991'
F12E24 AL086282* and AL086281
F26K6 AQ010623' and AQ010622*
F12L5 AL086477* and AL086476*
F11 B6 AL085606* and AL085605*
F21 M 19 824701
F3N7 AL081864* and AL081863*
F10J11 none
F11 F9 AL085739* and AL085738*
F3G22 AL081647' and AL081646*
F15E15 823535'
F10K18 AL08539T and AL085396'
F5B20 AL082559* and AL082558*
F1 F13 AL080535'
F26M 13 none
F18D9 826307' and 822283"
F28D1 823312'
F13C19 AL086736* and AL086735*
F2811 none
F26D1 823180'
F16J19 B97706'and 825740"
F2D20 AL081042'
F22N6 898712" and 898711
F27K3 AQ010703'
F 19124 AQ011005'
F19J19 none
F24E18 AQ011661* and AQ011660* and 825052"
~F27K6 AQ010706* and AQ010705* and 896164" and
823259'
- I 46-
CA 02362897 2001-09-18
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F25L7 AQ010583*
F28M5 823516' and 823371
F18L3 none
F14C23 AL087326* and AL087325*
F11 C6 AL085640* and AL085639*
F6024 AL083442* and AL083441
F1 M8 AL080782' and AL080781
F16J23 897710*and B23709*
F1809 898639' and B98638* and 898691 * and
B22349*
F26L23 AQ011321 * and AQOi 1320*
F3B13 AL081491 * and AL081490*
F22D12 B24795*
F1G16 none
F 1 OM21 AL085461 '
F2A14 AL080946* and AL080945'
F13M20 AL087096* and AL087095*
F19J6 none
F9015 AL085006" and AL085005'
F5A6 AL082510* and AL082509'
F17D12 B97751 * and B23790*
F11C12 AL085648* and AL085647*
F26P20 B25400' and B25399*
F13118 AL086953* and AL086952*
F2122 B12725" and B08590*
F21811 824621 ' and 824620 "
'~F28A24 AQ011507* and 825554'
-I-t7-
CA 02362897 2001-09-18
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F13014 AL087167* and AL087166*
F14A22 AL087257* and AL087256'
F21 G 14 897912*
F18M12 B09450* and B09052*
F3D18 AL081552*
F28K14 B25874* and B25873*
F28L21 B25895* and 825894'
F1 D3 AL080439* and AL080438*
F16019 897731 *
F15115 AQ251156* and A0251026*
F27G 1 AQ010677* and B23247* and B23246*
F22C19 B97947*
F1 E16 AG~251175*
F 18F 18 AQ251089
F12P2 AL086604* and AL086603*
F15018 823621 * and 823620'
F13D8 AL086759* and AL086758'
F23J22 AQ011543* and A0011257'
F3K18 none
F17022 A0251082*
F25A22 B25136*
~,F15G12 A0251153* and AQ251023*
'F23A7 895912' and 895911 *
IF26L22 A0011319* and AQ01 1318' and 862693'
F11820 AL085623' and AL085622*
T28K13 861711'
T19L12 861940' and 861939'
-14S-
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
F25A15 AQ251405* and AQ251342*
F22 H 10 AQ251219*
F3N13 AL081870* and AL081869"
F27F24 AQ251249* and AQ251143*
F27J 18 AQ011439*
F20K22 AQ011121 * and B24528"
F2J19 AL081240* and AL081239* and B26437*
F9F4 AL084708* and AL084707* and B30281
F8P17 AL084523* and AL084522*
F7E 14 AL083629* and AL083628*
F26J23 AQ011270
F19N2 None
F27G5 AQ010682* and AQ010681 *
" = partial (BAC
end) sequence
t = full sequence
in more than one
part
Table 6: Fully sequenced BAC clones containing A. thaliana centromere
sequences*
Clone Genbank Date Of' AvailabilityComment
Accession
No.
CENTROMERE 1
F28L22 AC007505 Feb 7. 2000:
May 6.
1999
T32E20 .AC020646 10 Feb. 2000:
Jan 8.
2000
F12G6 AC007781 Jun 1 1. 1999 3 unordererd
pieces
- I 49-
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
F9D18 AC007183 ~~Iar 30. 19996 unordererd
pieces
T4121 AC0224~6 Feb ?8. 2000:
Feb 3,
?000
FSA 13 AC008046 Feb 8, 2000:
Jul l4.
1999
CENTROMERE
T 13E I 1 AC006217 Dec 17, 1999:
Dec 24,
1998
F27C21 AC006527 Dec 17. 1999:
Feb 5,
1999
F9A 16 AC007662 Dec 17. I 999:
May
27. 1999
TSM2 AC007730 Dec 17, 1999:
Jun 5,
1999
T 17H 1 AC007143 Dec 17. 1999;
Mar 17,
1999
T I 8C6 AC007729 Dec 17. 1999:
Jun ~,
1999
TSE7 AC006225 Dec 17. 1999;
Jun 5.
1999
T12J2 AC004483 Dec 17. 1999;
Jul 17,
1999
GAP
T6C20 AC005898 Mar 20, 1999; 10 unordererd
Dec 7, pieces
1998
T14C8 AC006219 Dec 17, 1999:
Feb 9,
1999
F7B 19 AC006~86 Dec 17. 1999:
Feb 19.
1999
T 15D9 AC007120 Dec 17. 1999:
Mar 19,
1999
entire chromosomeAE002093 Dec 17. 1999:
Il Dec 16,
1999
CENTROMERE s
T25F1~ AC009~29 Dec 3. 1999: ? unordererd
Auk l6. pieces
1999
F23H6 AC0 f I 62 Nov 24. 1999:
I Oct 8.
1999
- I ~ 0-
CA 02362897 2001-09-18
WO 00/~~325 PCT/US00/07392
T28G 19 AC009328 Oct 26. I 999: 16 unordererd
Auk 16, pieces
1999
T I 8B3 ACO l I 624 Nov 18, I 999: 14 unordererd
Oct 8. pieces
1999
T26P13 AC009261 Nov 3. 1999:
Aug 10,
1999
T 14A 1 1 AC012327 Nov 20, 1999;
Oct 23,
1999
T4P3 AC009992 Oct 2 I , 1999;
Sep 9.
1999
F21A14 AC016828 Jan 13. 2000: 6 unordererd
Dec 3, pieces
1999
T27B3 AL137079 Jan 21, 2000
F26B 15 AL 138645 Feb 2. 2000
T 14K23 AL 132909 Nov 12. 1999
T32A11 AL138653 Feb 2 2000
CHROMOSOME 4
T27D20 AF076274 Au~ 3. 1998
T 19B 17 AF069441 Jun 3, 1999
T26N6 AF076243 Ma 1 1, I 999
F4H6 AF074021 Ma 11.1999
T 19J 18 AF I 49414 Au~ I 3. 1999
T4B21 AF118223 Aua 10. 1999:
Jan 7,
1999
T 1 J l AF I 28393 Nov 12. 1999
T32N4 AF 162444 Au~ 13. 1999
C17L7 AC012392 Oct 27. 1999
C6L9 AC012477 Nov 6. 1999
TIJ24 AF147263 Au~ 13. 1999
F6H8 AF178045 Au. 19. 1999
'F21I2 AF147261 Mav I1. 1999
GAP
F14G16 AFI47?60 Au~ 13. 1999
IF28D6 AF14726? Au~ 13. 1999
entire chromosomehttp://websvr.mips.Dec 17. 1999
IV biochem.
mp~.de/p
roj/thal/chr4_anno
uncement/
,CENTROMERE
s
!F3F24 ACO I 863? Dec 1 >. 1999
F23C8 AC0189?8 Dec ?4. 1999
-151-
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'' The sequences for clones from centromeres I . 3 and ~ are given in SEQ ID
NOS:184-
208. Sequences for contigs including the centromere ? and 4 clones are given
by SEQ ID
NOS:209-212.
' BAC clone number desi~Tnations are given. The centromere number origin of
the clone
i is as indicated.
~ Where a second date is given. the second date indicates the date for the
revised
sequence.
I O EXAMPLE 7
Constructing BAC Vectors for Testing Centromere Function
A BAC clone may be retrofitting with one or more plant telomeres and
selectable
markers together with the DNA elements necessary for Agrohcrcteriunr
transformation
(FIG. 9). This method will provide a means to deliver any BAC clone into plant
cells and
15 to test it for centromere function.
The method works in the following way. The conversion vector contains a
retrofitting cassette. The retrofitting cassette is flanked by Tn 10. Tn~.
Tn7. Mu or other
transposable elements and contains an origin of replication and a selectable
marker for
20 A~yrnhrrcrerium, a plant telomere array followed by T-DNA right and left
borders
followed by a second plant telomere array and a plant selectable marker (FIG.
9). The
conversion vector is transformed into an E. c.wli strain carrying the tar~~et
BAC. The
transposable elements flanking the retrofitting cassette then mediate
transposition of the
cassette randomly into the BAC clone. The retrofitted BAC clone can now be
2~ translormed into an appropriate strain of A,yrnhcrcrerirnn and then into
plant cells where it
can be tested for hish fidelity meiotic and mitotic transmission which would
indicate that
the clone contained a complete functional plant centromerc.
EXAMPLE 8
30 Construction of Plant Nlinichromosomes
Minichromosomes are constructed by combining the previously isolated essential
chromosomal elements. Exemplary rninichromosome vectors include those designed
to
be "shuttle vectors": i.e.. they can be maintained in a convenient host wuch
aE. coh.
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CA 02362897 2001-09-18
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A~r~hcrcrcnirnrr or yeast) as well as plant cells.
A. General Techniques for Minichromosome Construction
A minichromosome can be maintained in E. c«li or other bacterial cells as a
circular molecule by placing a removable stuffer fragment between the
telomeric
sequence blocks. The stuffer fragment is a dispensable DNA sequence. bordered
by
unique restriction sites, which can be removed by restriction digestion of the
circular
DNAs to create linear molecules with telomeric ends. The linear minichromosome
can
then be isolated by. for example, gel electrophoresis. In addition to the
stuffer fragment
and the plant telomeres, the minichromosome contains a replication origin and
selectable
marker that can function in plants to allow the circular molecules to be
maintained in
bacterial cells. The minichromosomes also include a plant selectable marker. a
plant
centromere, and a plant ARS to allow replication and maintenance of the DNA
molecules
in plant cells. Finally, the minichromosome includes several unique
restriction sites
where additional DNA sequence inserts can be cloned. The most expeditious
method of
physically constructing such a minichromosome. i.e.. ligating the various
essential
elements together for example, will be apparent to those of ordinary skill in
this an.
A number of minichromosome vectors have been designed by the current
inventors and are disclosed herein for the purpose of illustration (FIGS. 7A-
7H). These
vectors are not limiting however. as it will be apparent to those of skill in
the art that
many changes and alterations may be made and still obtain a functional vector.
B. Modified Technique for Minichromosome Construction
A two step method was developed for construction of minichromosomes. which
allows adding essential elements to BAC clones containing centromeric DNA.
These
procedures can take place in viva. eliminating problems of chromosome breakage
that
often happen in the test tube. The details and advantages of the techniques
are as follows:
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1.) One plasmid can be created that contains markers. origins and border
sequences for Ayrol»rcterimo transfer, markers for selection and screening in
plants. plant telomeres. and a loxP site or other site useful for site-
specific
recombination in viva or i~r aitrm. The second plasmid can be an existing BAC
clone, isolated from the available ~enomic libraries (FIG. 11A).
2.) The two plasmids are mixed. either within a single E. cnli cell. or in a
test
tube, and the site-specific recombinase cre is introduced. This will cause the
two
plasmids to fuse at the IoxP sites (FIG. I 1B).
3.) If deemed necessary. useful restriction sites (AseI/PacI or Not 1) are
included to remove excess material. (for example other selectable markers or
replication origins)
4.) Variations include vectors with or without a KanR gene (FIGS. 1 IB. 1 1C),
with or without a LAT~2 GUS «ene. with a LAT52 GFP gene, and with a GUS
Gene under the control of other plant promoters. (FIGS. 1 I C, 1 1 D and I 1
E).
C. l~~Iethod for Preparation of Stable Non-Integrated Minichromosomes
A technique has been developed to ensure that minichromosomes do no integrate
into the host genome (FIG. 1 1F). In particular. minichromosomes must be
maintained as
distinct elements separate from the host chromosomes. To ensure that the
introduced
minichromosome does not integrate. the inventors envision a variety that would
encode a
lethal plant gene (such as diptheria toxin or any other gene product that,
when expressed.
causes lethality in plants). This gene could be located between the right
Ayrobcrctericm
border and the telomere. Minichromosomes that enter a plant nucleus and
integrate into a
host chromosome would result in lethality. However. if the minichromosome
remains
separate. and further, if the ends of thin construct are degraded up to the
telomeres. then
the lethal gene would be removed and the cells would survive.
_ I ;.t_
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EXAMPLE 9
In Vivo Screen of Centromere Activity by the Analysis of Dicentric Chromosomes
A method was designed for the screening of centromere activity (FIG. 10). In
the
method. plants are first transformed with binary BAC clones that contain DNA
from the
genetically-defined centromeric regions. By allowing the DNA to integrate into
the host
chromosomes. it is expected that this integration will result in a chromosome
with two
centromeres. This is an unstable situation which often leads to chromosome
breakage, as
single chromosomes harboring two or more functional centromeres will often
times break
at junctions between the two centromeres when pulled towards opposite poles
during
mitotic and meiotic events. This can lead to severe growth defects and
inviable progeny
when genes important or essentially for cellular and developmental processes
are
disrupted by the breakage event. Therefore, regions having centromere function
could be
identified by looking for clones that exhibit. upon introduction into a host
plant. any of
the following predicted properties: reduced efficiencies of transformation:
causation of
genetic instability when integrated into natural chromosomes such that the
transformed
plants show aberrant sectors and increased lethality: a difficulty to
maintain, particularly
when the transformed plants are gown under conditions that do not select for
maintenance of the transgenes: a tendency to integrate into the genome at the
distal tips of
chromosomes or at the centromeric regions. In contrast, clones comprising
non-centromeric DNA will be expected to integrate in a more random pattern.
Confirmation of a resulting distribution and pattern of integration can be
determined by
sequencing the ends of the inserted DNA.
The screen is performed by identifying clones of greater than l00 kb that
encode
2~ centromere DNA in a BiBAC library (binary bacterial artificial chromosomes)
(Hamilton.
1997). This is done by screening filters comprisin« a BiBAC ~enomic library
for clones
that encode DNA from the centromeres lFIG. 10. step I ). The BiBAC vector is
used
because it can contain large inserts of ,~trahirln/~ci.s ~lenomic material and
also encodes the
binary sequences needed for ~l,y-nbacterirrrrr-mediated transformation. The
centromere
sequence containing BiBAC vectors are then directly integrated into
chromosomes by
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Ayrnhcrcterirrnr-mediated transformation (FIG. 10. step ?l. As a control.
BiBAC
constructs containing non-centromeric DNA also are used for transformation.
BiBACs
harborin~~ sequences with centromere function will result in forming dicentric
chromosomes. Progeny from transformed plants will be analyzed for nonviability
and
gross morphological differences that can be attributed to chromosomal breaks
due to the
formation of dicentric chromosomes (FIG. 10. step ~ ). Non-centromere
seduences are
expected to show little phenotypic differences from wildtype plants
EXAMPLE 10
Refined Centromere Mapping with Treatment for Increased Recombination
In order to achieve a more refined map position for the centromeres in
Arcrhidopsi.c thctlictrrn. various chemical and environmental treatments were
used to
stimulate recombination. The treatments were used on pollen donors in crosses
performed to create the tetrad sets of plants (see EXAMPLE 2). Pollen donor
plants were
IS planted individually in 1 inch square pots and grown under 24 hr light in a
growth room
until f7owerin~. Flowering plants were then dipped in one of the following
solutions and
watered with 50 ml of the same solution.
-156-
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
..
s
G~ 4
y G r ~ O
; G =a.
y-. r,!
r ~ ~ =
C c'I _ m, ~~ - ~ C
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J j L .
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G . ~ J .r _
-
T
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n',:J U r ,U
v
;J -J = ~~ - _J v
.Ur_~ :J J ~ ~ ~, U C
U r U 'v ..
'' G ~ ~ :r ~ ~ Q
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.C' J ~ ., = ~ r-
'~~ ~ _ ~ D ' C 4 y ~
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r ~ . 0
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1 ~ _ V O
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Z C G = . l C = G ~~ G ~ J O
Q O L G = G O = 1 .G ~ C ~
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.
-157-
CA 02362897 2001-09-18
WO 00155325 PCT/L1S00/07392
J _
O
O J
f J
C
~iC ~'r
c
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O J _r
Q,- V J
I~J r ~
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OT
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OT ~V '~
J.i.Q D (J
-158-
CA 02362897 2001-09-18
WO 00/~~325 PCT/US00/07392
Following treatment. plants were then returned to the growth room and grown
under standard conditions for 2-~ days. Pollen was then collected from newly
opened
flowers and used to pollinate receptive stigmas as described in Example ?.
Then the
pollen donor plants were again treated as described above and used in another
round of
pollination. Pollen donor plants were typically subjected to 5-10 rounds of
treatment and
pollen collection.
Treatments were also performed using non-chemical agents. As above, the
treatments were used to achieve more refined map positions for the centromeres
in
Arabidop.sis by stimulating recombination in additional pollen donor plants.
The
treatments were as follows:
Table 8: Non-Chemical Treatment Agents.
TREATMENT TREATMENT PARAMETERS
heat shock: about 35 C to about 48 C, and preferably.
about 42 C
UV exposure (350 about 1 second to about 50 seconds. and
nm): preferably. about 7
seconds
Gamma radiation: about 0. I kRads to about 20 kRads, and
preferably, about 10
kRads
Magnetic field about 1 to 20 Tesla for 1 h to continuous
cold stress about -10 to I SC for I rein to continuous
l5 Heat shock treatments were performed by placing the pot containing the
pollen
donor plants in shallow dishes filled with water (to prevent desiccation). and
placing the
plant-containing dishes in incubators of the appropriate temperature. UV
exposure was
performed by placing the pollen donor plants in a BioRad CIV chamber and
illuminatin~~
the plants at the appropriate wave length for varying amounts of time. Both
the UV and
heat shock plants were subjected to several rounds of treatment and pollen
collection.
Plants exposed to a gamma radiation source (Cobalt-60) were treated only once
and then
discarded to prevent the accumulation of deleterious chromosomal
rearrangements.
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CA 02362897 2001-09-18
WO 00/5325 PCT/US00/07392
Following treatment, plants were then returned to the growth room and grown
under standard conditions for 2-5 days. Pollen was then collected from newly
opened
flowers and used to pollinate receptive stigmas as described in Example ?.
Then the
pollen donor plants were again treated as described above and used in another
round of
pollination. Pollen donor plants were typically subjected to i-10 rounds of
treatment and
pollen collection. The results are shown at Table 9 below.
Table 9: Results of Recombination After Treatments
Treatment Tetrads Obs Exp (O-E) '/E=X''
n-butyric acid 43 11 2.5 28.9**
UV exposure 350 nm 57 12 3.2 24.2**
Methanesulfonic acid ethyl10 5" 0.6 32.2**
ester
5-aza-2'-deoxycytidine 68 16 3.9 37.5*'
heat shock 23 7 I .3 25.0**
3-methoxybenzamide 44 8 ?.5 12.1 **
Zeocin !06 14 6.0 10.6**
Untreated 384 22 N/A N/A
** indicates significant by X~ (df=I )
EXAMPLE 11
Facilitation of Genetic Introgression
It is also contemplated by the inventors that one could employ techniques or
treatments which stimulate recombination to facilitate iotrooressi~n.
lntroaression
describes a breeding technique whereby one or more desired trait, is
transferred into
one strain (A) from another (B), the trait is then isolated in the genetic
background of
the desired strain (A> by a series of backcrosses to the ame strain fA). The
number of
backcrosses required to isolate the desired trait in the desired genetic
hack~round is
dependent on the frequency of recombination in each backcross.
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Backcrossing transfers a specific desirable trait from one source to an inbred
or
other plant that lacks that trait. This can be accomplished, for example. by
first crossing a
superior inbred (A) (recurrent parent) to a donor inbred (non-recurrent
parent), which
carries the appropriate genes) for the trait in question. for example. a
construct prepared
in accordance with the current invention. The progeny of this cross first are
selected in
the resultant progeny for the desired trait to be transferred from the non-
recurrent parent.
then the selected progeny are mated back to the superior recurrent parent (A).
After five
or more backcross generations with selection for the desired trait, the
progeny are
hemizygous for loci controlling the characteristic being transferred. but are
like the
superior parent for most or almost all other genes. The last backcross
veneration would
be selfed to give progeny which are pure breeding for the genes) being
transferred, i.e.
one or more transformation events.
Therefore. through a series a breeding manipulations. a selected transgene may
be
moved from one line into an entirely different line without the need for
further
recombinant manipulation. Transgenes are valuable in that they typically
behave
genetically as any other gene and can be manipulated by breeding techniques in
a manner
identical to any other corn gene. Therefore. one may produce inbred plants
which are true
breeding for one or more transgenes. By crossing different inbred plants. one
may
produce a large number of different hybrids with different combinations of
transgenes. !n
this way, plants may be produced which have the desirable agronomic properties
frequently associated with hybrids Whybrid vigor"). as well as the desirable
characteristics imparted by one or more transgene(s).
Breeding also can be used to transler an entire minichromosome from one plant
to
another plant. For example, by crossine a first plant having a minichromosome
to a
second plant lacking the minichronu>some. progeny of any generation of thin
cross may
be obtained having the minichromosome. or any additional number of desired
minichromosomes. Through a series of hackcrosses. a plant may be obtained that
has the
genetic backUround of the second plant hm hat the minichromosome from the
first plant.
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s; >: ,~c :< r W. ;e x :e x :~: :E: :j:
All of the compositions and methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present disclosure.
While the
compositions and methods of thin invention have been described in terms of
preferred
embodiments, it will be apparent to those of skill in the art that variations
may be applied
to the compositions and methods and in the steps or in the sequence of steps
of the
method described herein without departing from the concept, spirit and scope
of the
invention. More specifically, it will be apparent that certain agents which
are both
chemically and physiologically related may be substituted for the agents
described herein
while the same or similar results would he achieved. All such similar
substitutes and
modifications apparent to those skilled in the art are deemed to be within the
spirit. scope
and concept of the invention as defined by the appended claims.
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CA 02362897 2001-09-18
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-176-
CA 02362897 2001-09-18
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SEQUENCE LIST.' .'3
<110> PREUSS, DAPHNE
COPENHAVER, GREGORY
KEITH, KEVIN
<120> CHROMOSOME COMPOSITIONS AND METHODS
<130> ARCD:309PZ6
<140> UNKNOWN
<141> 1999-12-10
<160> 179
<170> PatentIn Ver. 2.0
<210> 1
<211> 1038
<212> DNA
<213> Arabidopsis thaliana
<400> 1
tgactatgtg atatggttca aattacctat aactactctc tcaaataaga gatcaattgc 60
agttttttag gatcgaattc acggagttct tttgttcaaa cagtgagtta aatgtcgaga 120
ttaagctagc aggatatgat tgaaaataaa agagaacaaa gtaagaaaac agcagattga 180
ttttgttgta aacgatttaa taaagagcta ggaacagggt attctcacga aactattggt 240
tagtagatct aatgaaagct aggttgtgat caaactaCtc ttaaactcaa actctaatta 300
tggaacaaca ggtaggcgtg ccgcgaaact ccctatatct atagctaata ataaccggag 360
aagccgagaa actatcaacc taaatatgca ttcttaacga gttcaattgt tcatcttact 420
agataggccg attcttatta cacacctata aaccagactc atcaaataat agatccaatt 480
acagatacct atgatgggca tatctagtgt ctggattcaa gatctagtta attactctag 540
atctagcatt aagcatagat gaagaactct acagataacc tagcagaggg ggcaatctac 600
taaaccatat gaatccctaa tgaaaaaccc tattcctaac aagcagatta ctcagacata 660
ttggatggag caaacaacat aattgacctt agcttttgct ccaaaatgtc tccttatctc 720
cattgttgtc ccattgcata aaatacctga aaagacacca aaaagactcg agagataaca 780
taacgactca aaatcctata cctaaaacat ggatacaatc agtaaaaatc gggttatatc 840
aactccccga gacttagctt ttgcttcccc tcaaacaaaa cacaaaagca aaacccgtgg 900
aagaggtttt gaaaacaaag gaactcccaa cattctctag cctattgcca tgatcatcca 960
aactaagtcc atatgcctaa caagtctaat caaatcctaa ccaacatgta cttctctgat 1020
tgatttttcc agttcttt 1032
<210> 2
<211> 601
<212> DNA
<213> Arabidopsis thaliana
<400> 2
tgatatggtt caaattacct ataactactc tctcaaataa gagatcaatt gcagtttttt 60
aggatcgaat tcacggagtt cttttgttca aacagtgagt taaatgtcga gattaagcta 120
gcaggatatg attgaaaata aaagagaaca aagtaagaaa acagcagatt gattttgttg 180
taaacgattr aataaagagc taggaacagg gtattctcac gaaactattg gttagtagat 240
ctaatgaaag ctaggttgtg atcaaactat tcttaaactc aaactctaat tatggaacaa 300
caggtaggcg tgccgcgaaa ctccctatat ctatagctaa taataaccgg agaagccgag 360
aaactatcaa cctaaatatg cattcttaac gagttcaatt gttcatctta ctagataggc 420
cgattcttac tacacaccta taaaccagac tcatcaaata atagatccaa ttacagatac 480
ctatgatggg catatctagt gtctggattc aagatctagt taattactct aga~c~agca 54C
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
ttaagcatag atgaagaact ctacagataa cctagcagar_,ggggcaatct actaaaccat 600
a 601
<210> 3
<211> 885
<212> DNA
<213> Arabidopsis thaliana
<400> 3
tttttttgtc cacacaatga gttgaatgtc aagattaagc tagtagagat tgattgtaat 60
aagaagtaaa caaagtaaaa agacaacgga ttgattggtt gtaaacgata aaataaagag 120
gtaggaacaa ggtattctca ggagactatt ggttagtaga tctaatgaaa gctaggttgt 180
tatcgaacca ttattaaaca caaattttaa ttatggaata accggtggtg ttctgcaaaa 240
cttttgtgcc tatagctaag aataaccgca gaagccgaga gatctttaac ctaaacatgc 300
attctaaacg agttcaattg ttcaccttag tatataggcc gattcttatt acacacctat 360
aaaccagact catcaaataa tagatccaac tacatatacc tatggtgggt atatctagtg 420
tctggattca agatctagtt aattactcta gatctagcat taagattaat tctacacata 480
atttagcaag ggggtgatct actaaaccat atgaatccct aatgaaaaac tcaattccta 540
acaagaaact actcagacag attgattgaa acaaacaaca taaatgaata agaaagcata 600
aacacaacaa ataaaattag ggaatgaaag gatctcttca ctgtaatgag aactgaatga 660
atctctgaag aacaacggat gattagctta tgtctctctg aaaataggga ttaaaaactt 720
gataaaagga acttaggtct aaacaatgac ctttaaaact atatataaac cctataaaac 780
gtccagggac taataatgca aatagggaag tcttttgggg caaatttcca cttttgtaaa 840
cttgaaagcg tattggactt ttctgggccg aaactggtgt cgatc 8g5
<210> 4
<211> 1072
<212> DNA
<213> Arabidopsis thaliana
<400> 4
tatcttgata tggttcaaat taccctaaga actactctct caaataagag atccattgcg 60
gtatttaagg atcgaattcc acaaagttct tttcttcaaa caataagttc aatgtcaaga 120
ttaagctaga agggtatgat cgaaataata agaaaacaaa ggaagaaaac agtagattgt 180
ttcgttgtaa acgattaaat aaaaagctag gaacagggta ttctcatgaa actattggtt 240
agtagatcta atgaaagcta ggttgttatc gaaccattct taaactcaaa ctctaattat 300
ggaataactg gtggtgttcc gcaaaactcc ctataattat agctaagaat aaccggagaa 360
ttcgagagat tattaaccta aatatgcatt cttaacgagt tcgattgttc accttagtag 420
ataggccaat ttttattaca cacctataaa ccaggctcat caaataatag atccaactac 480
agatacctat ggtggacata tctattgtct ggattcaaga tctagttaat tactctagat 540
ctagcattaa gcataatcaa agatgaagaa ttctacagat aacctagcaa aggggaaaaa 600
ttactaaacc atatgaatcc ctagtgagaa accctattcc taacaagcag attactcaga 660
catattgatt gaagcgaaca acataattgt gtatgaaagg tccaaaatcg tccttagctt 720
ccttttcctt acctcttgct cgaaatgtct cctcatctcc attgttgtcc cgttgcacag 780
aatacctgaa aagacactac aaagactcga gaaataacat aaagactcaa aatccattac 840
caaacacata gataaaatcg gtgaaaatag gatatatcaa ctccccaaga cttagctttt 900
gcttgccctc aagcaaaaca caaaagtcga acccgtggaa gagattttga aaacaaagga 96C
actcccaaca tcctctagac tattgccatg atcatccaaa ctaaatccac atgcctagca 1020
agtctaatca aatcctaacc aacatgtact tctctaccca agctttgtaa tt 1072
<210> 5
<211> 591
<212> DNA
<213> Arabidopsis thaliana
<400> 5
tgatatggtt caaattaccc caagaactac tctctcaaat aagagatcca ttgcggtatt 50
taaggatcga attccacaaa gttcttttct tcaaacaata agttcaatgt caagattaag 120
ctagaagggt atgatcgaaa taataagaaa acaaaggaag aaaacagtag attgtttcgt 180
tgtaaacgat taaataaaaa gctaggaaca gggtattctc atgaaactat tggttaatag 240
atctaatgaa agctaggttg ttatcgaacc: attcttaaac tcaaactcta attatggaat 300
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
aactggtggt gttccgcaaa actccctata attatagcta agaataaccg gagaattcga 360
gagattatta acctaaatat gcattcttaa cgagttcgat tgttcacctt agtagatagg 420
ccaattttta ttacacacct ataaaccagg ctcatcaaat aatagatcca actacagata 480
cctatggtgg acatatctat tgtctggatt caagatctag ttaattactc tagatctagc 540
attaagcata atcaaagatg aagaattcta cagataacct agcaaagggg a 591
<210> 6
<211> 650
<212> DNA
<213> Arabidopsis thaliana
<400> 6
taagaactat tatctcaaat atttaaggat cgaatttcac aaagtttttt tgttcacaca 60
atgaattaaa tatcgagatt aagctagtag ggaatgattg aaaataaagg agaacaaagt 120
aaaaagacag cagattaatt ggttgtaaac gattaaataa agagttagga acatggtatt 180
ctcaggaaac tattggttag tagatctaat gaaagctagg ttgttatcga accattctta 240
aactcaaact ctaattatgc aataaccggt ggtgttccgc caaactccct atgcttatag 300
ctaagaataa ccggagaagc cgagagatct ttaacctaaa catgcattct aaacgagttc 360
aattgttcac cttactagat agaccgattc ttattacaca cctataaacc aggttaatca 420
aataataaat ccaattacag atacctatgg tgggcatatc tattgtctgg cttcaagatc 480
tagttaattt ctgtagatct accattaagt ataatcaaag atgaagaatt ctacagataa 540
cctagaaaag gaggcaatat actaaaccat atgaatcccc aatgagaaac cctattccta 600
acaagcaaac tactcagaca tattgaatga aacaaacaac ataattgagt 650
<210> 7
<211> 856
<212> DNA
<213> Arabidopsis thaliana
<400> 7
taaatatgca tttttaatga gttcgtttgt tcaccttagt agataggccg attcttatta 60
cacacctaaa aaccagactc atcaaataat agatccaact acatatactt atggtgggca 120
tatctattgt ctggattcaa gatctagtta gttactctag atctagcatt aagcataatt 180
aaagatgaag aattctacag ataacctagc aaagggggca atctactaaa ctatatgaat 240
ccctaatgag aaaccctatt cctaacaagc agactact_ca gacatattga ttgaaggaaa 300
caacataatt gagtatgaaa acataaacac ggcaaataga tttaagggaa agaagggatc 360
tcttcactgt attaggaact gaatcaatct ctgaaaacac tcgatgaata gcttatgtct 420
ctcagtaaca gggtttgcaa aaagcttgat aaaaaacttg ataatgaaaa cttaggtcta 480
aacaatgtat atacaccctc taaaaacgtc tagggactaa taatgtaaat agaaaagttt 540
tctagggcaa atttcctctt ctgtaaactt gaaagcgtct aggactttgc tgggccgaaa 600
ctggtgtcga tcgacactag gagtgtgtcg atcgacactc ctcttgattc gtgaaaccaa 660
agtcgtcctt accttacttt ttcttagctt ttgctccaaa atgtctcctt atctccattg 720
ttgtcccact gcatagaata cctgaaaaga caccaaaaag actcgagaaa taacataaag 780
actcaagatc ctatacctaa aacatagata aaatcagtta aaataaggat atatcaatca 840
ccacaatcta catatt 856
<210> 8
<211> 736
<212> DNA
<213> Arabidopsis thaliana
<400> 8
aactatgatt ttagagtaac cgatggcgtt ccgcgaaact c:cctatgctt atagctaaga 60
ataaccggag aagccgagag atctttaact taaacatgca ;:tattatcaa atttgattag 120
ttcacctagt atctaaacca gagcccttat atgagcctac ctgttctttc ttaaatgcct 180
aggctcatct atgatagatc aaatagcaaa tacctatggt gggcatacct attatctaat 240
atcaagttct agttagctac tctagaacta ccaataagaa caattaagat gaagaatcat 300
atagataacc tagcaagggg caatctacta aatcatctaa <xtctctaatg agaaacccta 360
aacctaacaa gtggattact aagacatgat caaagaaaca caaatcatat tctgaataag 420
aaataaatga tgaaaataac aagagaaaag agtaagaaag atccaaaagg gagttttcac 480
aggtttttgc tctccaaagt acaaaagaga tccaggaaat ~acctcccaa agcttacggg 540
CA 02362897 2001-09-18
WO 00155325 PCT/US00/07392
tctaaaacaa tgacctaaaa actatatata tgtcttaaaa acatgatggg ccttaattaa 600
acataggaga aagttctggg ccgaatttgg aaatctccaa aacatcaata agttgcgtct 650
cgaaattgca gtcaggtatt agtgttgctc gacactaggg gtggtgtcgt tcgacaccca 720
cgtgcatttt cgtctc 736
<210> 9
<211> 679
<222> DNA '
<213> Arabidopsis thaliana
<400> 9
tatcgcaccg ttctcgaact caaactatga ttttagagta acctgtggtg ttccgcgaaa 60
ctctctatac ttagagctaa gataaccgaa gaagccgaga aatcttatac taaaccatgc 120
atttttatca agaatgatta gttcacctag tatctaaact agagcccttc tatgagccta 180
tctgttcttt cttaaatgcc taggcccatc tatgatggat caaatagcaa gtacctatgg 240
tggacatacc tattatctaa tatcaagttc tagtcagcta ctctataact agcattaaga 300
acaatcaaga taaagaactc tacagataac ctagcaaggg ggcaatctac taaatcatct 360
aaatccctaa tgagaaaccc taaacctaac aagtgaatta ctcagacatg atcaaagaaa 420
cacaaatcat agtctgaata aggaatcaat aatagcaaga aaaagagtaa agaagatctt 480
ctccaaaggg agtctccaca gggttttgct cccgaagtac aaaaaaatag agaatatcct 540
ttccaagctt agatctaaac aatgacccta aaaacctaat tatatgtcta aaacacgtga 600
tgggccttca ttaaacatag gagaaagttc tgggccgaat ttggaaatct tcaaaacatc 660
aataagttgt gtctcgaaa 679
<210> 10
<211> 1198
<212> DNA
<213> Arabidopsis thaliana
<400> 10
aagacatatc aatgtgctat gtgatatagg tcaaattaat cataagacca ctctctcaaa 60
taaagaggtc aattgcagcg cttagggatc gaataacaaa gttctcggat cacgcaatag 120
actaacggca aatcgaatta tgctaagtaa aatagtaaat aaaataaaga gaaacaaaag 180
tatgcaatag caatcgattg gatggttgtg aaacaagata agaaaagcgt caggcttagg 240
ctattctcag gaaatagatg atagtagatc tagaaatagc taggttatta tcgcaccgtt 300
ctcgaactca aactatgatt ttagagtaac ctgtggtgtt ccgcgaaact ctctatactt 360
agagctaaga taaccgaaga agccgagaaa tcttatacta aaccatgcat ttttatcaag 420
aatgattagt tcacctagta tctaaactag agccctccta tgagcctatc tgttctttct 480
taaatgccta ggcccatcta tgatggatca aatagcaagt acctatggtg gacataccta 540
ttatctaata tcaagttcta gttagctact ctataactag cattaagaac aatcaagata 600
aagaactcta cagataacct agcaaggggg caatccacta aatcatctaa atccctaatg 660
agaaacccta aacctaacaa gtgaattact cagacatgat caaagaaaca caaatcatag 720
tctgaataag gaatcaataa tagcaagaaa aagagtaaag aagatcttct ccaaagggag 780
tctccacagg gttttgctcc cgaagtacaa aaaaatagag aatatccttt ccaagcttag 840
atctaaacaa tgaccctaaa aacctaatta tatgtctaaa acacgtgatg ggccttcatC 9C0
aaacatagga gaaagttctg ggccgaattt ggaaatcttc aaaacatcaa taagttgtgt 960
ctcgaaattg ccgtcaagta atggtttcgc tcgacaccta agttcatttt cgtctccgga 1020
ttgtttctgc agcttaaatt ctctgttttc ctccagaatg ctccattatc tccaaatgaa 1080
tccaaacatg taaagacctg aaaaggacta gaaaagactc tagaaataac aattagactc 1190
taaaacctat atctaaaaca tacttaaatt aggaaaaaca gggatatatc acttatat 1198
<210> 11
<211> 696
<212> DNA
<213> Arabidopsis thaliana
<400> 11
tccaactatg attttagagt aaccgatggc gttccgcgaa actscctatg cttatagcta 60
agaataaccg gagaagccga gagatcttta acttaaacnt gcattattat caaatttgat 120
tagttcacct agtatccaaa ccagagccct tatatgagcc tacctgttct ttcttaaatg 180
cctaggctca Cctatgatag atcaaatagc aaatacctat ygtgggcata cctattatct 240
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
aatatcaagt tctagttagc tactctagaa ctaccaataa gaacaattaa gatgaagaat 300
catatagata acctagcaag gggcaatcta ctaaatcatc taaatctcta atgagaaacc 360
ctaaacctaa caagtggatt actaagacat gatcaaagaa acacaaatca tattctgaat 420
aagaaataaa tgatgaaaat aacaagagaa aagagtaaga aagatccaaa agggagtttt 480
cacaggtttt tgctctccaa agtacaaaag agatccaggg aatagcctcc caaagcttac 540
gggtctaaaa caatgaccta aaaactatat atatgtctta aaaacatgat gggccttaa~ 600
taaacatagg agaaagttct gggccgaatt tggaaatctt caaaacatca ataagttgcg 660
tctcgaaatt gcagtcaggt attagtgttg ctcgac 696
<210> 12
<211> 670
<212> DNA
<213> Arabidopsis thaliana
<400> 12
agttgatata gctcaaattg ccttaagctt actccctcaa ttaagagatc gtcgttagca 60
cttaagggtc gaattccatt gagctctcga tgttcacaca atagacttat gatattgttt 120
aataagctaa atgaagtaat tgaatattaa aggcaaataa gcaagtaaat gagttgtaga 180
ttcaagtgat taaagcgtca ggtctaagga attatctcgg gagatagata aattgtagat 240
ctagataata tcaggattgt tatcgcaccg ttctcatact caaactataa ttctagagta 300
atcagtggcg ttccgcaaaa ctctctatac ttacaactaa gataaccgga gaagccgaga 360
aatcctatgc taaagcatgc attgttaata agcttgaata gttcacctag tatctaaacc 420
agagcccttc tatgaaccta cctgtttttt cttaaatgcc taggctcatc tatgatggtt 480
caaataacaa atacctatgg cgggaatacc tattatctaa tatcaagctc taggtgatca 540
atctaaaact agcattaaga ataatcaaga tgaagaacta taagaataat cctaagggct 600
tttcgatcta ctaatccatc taaatcccta ttgagactcc gtagacccaa caaggtgatt 660
actcaaacat 670
<210> 13
<211> 687
<212> DNA
<213> Arabidopsis thaliana
<400> 13
tcgatccccg gcaacggcgc caaatttgat atagctcaaa tcgccttaag cttactccct 60
caaataagag ttgtcgttag cacttaagtg tcgaattcca ctgagctctc gatgttcaca 120
caatagaatt atgatgttat taaataatct agacaaagta attgaatgta aaagacaa~t 180
aaccaagtaa acgaagtgta gattcaagtg attaaagcgt cgggtctaag gaattgtctc 240
gggagataga taaattgtag atctagctaa tataaggatt gttatggcac cgttctcaaa 300
ctcaaactaa gattctagag taaccggtga tgttccacaa aactctcttt acttagagct 360
aagataaccg gagaaaccga gaaatcttat actaaagcat gcattgttat caagcttgac 920
tagttcacct agtatctaaa ccagagccct tctatgagcc tacatgttct ttcttaaatg 480
cctaaactca tctatgatag ttcaaacaac aagtacctat ggtgggcata cctattatct 540
aatatcacgt tctaggtgat caatctaaaa ctagcaataa gaataatcaa gatgaagaac 500
tataagaata atcttaaggg gttttcgatc tactaatcca tctaaatccc tattgagact 660
ccctaaaccc aacaaggtga ttactca 687
<210> 19
<211> 802
<212> DNA
<213> Arabidopsis thaliana
<400> 14
tctcaatctg aaggtacctg aaaaacaaga gaccaataga caaagaaata cgtgaatacg 60
tggtagaaaa gttgaattta gacttaaaag gacacctagt ccaatggtga actaaaagag 120
aacttcgcca acggggccaa atttgatata gctcaaattg ccttaagctt actccgtcaa 180
ttaagagatc atcgttagca cttaaggatc gaattccatt gagctctcga tgttcacaca 240
atagacttat gatgttgttt aataagctaa atgaagtaat tgaatattaa aggcaaacaa 300
gcaagtaaat gagttgtaca ttcaagtgat taaagagtca ggtctaagga attgtctcc~ 360
gagatagata aattgtagat ctagataata tcaggattgc catcgcaccg ttcccaaact 420
caaactataa ttctagagta acagtggcgt tccgcgaaac tctctatgct tacaactaag 480
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
ataaccggag aagccgagaa atccgatgct aaagcatgca ttgttaataa gcttgattag 540
ttcacctagt atctaaacca gagcccttct atgagcctac ctgttctttc ttaaatgtct 600
aggctcatct atgatggttc aaataacaaa tacctatggc gggaatacct attatctaat 660
ataaagttct aggtgatcaa tctaaaacta gctataagaa taatcaagat gaagaactct 720
aagaataatc ctaaggggtt ttcgatctac taatccatct aaatccctat tgagactccc 780
tagacccaac aaggtgat=a ct gp2
<210> 15
<211> 821
<212> DNA
<213> Arabidopsis t~:aliana
<400> 15
acaaagtctt aatagtacct gttttaaata taatagagaa gattttataa aaacgatgga 60
aacaagtctg gtattgatgt tttccgttct catcaacaac ttcacctatt tcagctcgtt 120
gcattcgttc aaggaattga gtcctgaaac agtagcaaaa aaagaaggaa ataaatagcc 180
aagaataaaa ttatttataa tacactaaac agttaagaga taatgaaaat ataaacgttc 290
ttacgtgatg cgatccatgt taatctctgg gtaactttaa ttgaatgtaa ttcttgaagc 300
accattatgt gtgattagac attgacgacc taaaatattt atgtttttat tatatgcatt 360
agctataaaa aaaacatatg ar_gagaagag agttaaattt accttcatga tcagcaacgt 420
tccaaaatcg aagcacgcac gttataggat tcataccgaa ggtccactga taaccaaggt 480
gtagaccaag ttccctccac aaacgcacaa acaaatcaca agtatatcca cttgcaacac 540
atgtaagagt ttttccccca at.aagaatat cttaattatt tctctctaac atctaagttc 600
tataaattaa gccacctata aagataacaa tgcttactat gtttctacat taaaatatat 660
aaccaaaaat atgtcgaact atatagtcgg aaatactaac caagtatttt taagctcaaa 720
ctcgagaaat tgagaaagtc gaggttttcg gaatattgag gaacataatc tggtcttgtt 780
atattcccaa ctcgcacg~c aacaccataa gcatctacaa a 821
<210> 16
<211> 672
<2I2> DNA
<213> Arabidopsis thaliana
<400> 16
cctcccaagt gagcttgttt aaagtcatta gcttgactcc tttaatcatc aagaagctcc 60
tggagatgaa ccgatggcac ctctggaagg atttggtctg ctaagtattt cttgagcctt 120
tgaccattta ctgtaaaatc tccactctta ccagctagag tgactgctcc ataaggacgg 180
acctcagtga tgtagaaggg gccagaccat ctggatttaa gttttcctgg aaagagtttc 240
aagcgagagt tgaatagcag cacctgatca ccaaccttga aatccttaat gatgatcttc 300
ttgtcatgga aaagcttggt tctctccttg tagattttag aactctcata agcttctaga 360
cggatctcat cgaggttac~ tagttggatc aatcgcttct cctcagcagt ttttatgtca 420
aagttcaaaa gttttaccgc caacattgct ttgtactcga gctcaacggg tagatgacat 480
gattttccat agagaagat-_ gaaaggagtt gtacctatgg gggtcttgaa ggctgtcctg 540
taagcccata atgcatcatc tagctttaca gaccagtctt tccttgtaat cccaacagtc 600
ttttctagaa ttgtttttG~ ct~cctatcg gagatctcaa cctgcccgct tgtctgtgga 650
tctatgacca gc 672
<210> 17
<211> 2954
<212> DNA
<213> Arabidopsis t:~a_iana
<400> 17
taacatgaaa atattatc~c atgtatctta taatacaaac tttctgcaat cttcttaaga 60
acatggctaa atagcaaaca tcgctatcaa ttggtgaatt taaaaaacaa agagtcactg 120
attacataag aacatccgcg gttggatggc agctttcgca cttgtaagac tcaagtagta 180
cgttcttctg gagaaaaggc attgaacgtt tcgatagcgt actctttagc tgcccctcgc 240
atcctctctt cgtctagtt.- agcacaatag tccatagcgt taactgaatg tagctcaagt 300
aagatcacac aagcaattcc aa~:aaggctt gaaaatcaca cgtgaaatat atatgtcaca 360
cagtcaaata tgaaatac=~ at<:ganacaa gataatgaaa gacaaccata cctaacgacg 420
aacaattaaa ctgtaagt-_g ggtctactca ttgtcataag atcgagcctc tacgttagca 480
CA 02362897 2001-09-18
WO 00/~s325 PCT/US00/07392
ttcttccgtt tacaccatgc ttttgaataa ggtacaagag catgatttta tgtgttctat 540
gtacttcgcc aactgctcgt ctttcaacac ttcactcttg cagtctaata caatgatttt 600
ctatttcatc aaatcgatga caagcccaac ccaatgctgc ctctcaacct gcattgggca 660
gtagatgaca tcaacctcat caaaccattt tggtcgcacc atggatggga ctcttgtaag 720
cgtggtgaag taaaaaactc tcttacctga agctgcacaa aatttgttgt attggcgtcg 780
tagctctgac aagaaacttg atggtagaaa atcaaaatgc agcggcttct ttttatcccg 840
ccgcatctga atgaacctca ctaaaagatc tggtccttgt ggaagtcact ttttttatta 900
gcacacacct ttaatactta gttttctact aaaaaagcag tgagcacaca gttttatttg 960
aaacaacaca gattgcatgt ataacttact acttcatcta gaagtgtttg cgcctcgtaa 1020
atagttagaa attcagtatt ctccattaca acgccatcag cctgaaaata agatctacat 1080
ggtaccatat accagcaagt tagttgcgaa agcaaataag ttcttctgaa aatatagggg 1140
ctataaatac ttacactctc cgttctattt gtttaagaaa acatttcact ctctctgcac 1200
ttggaacagc gaatcggtca tatggaccaa tggtggtgga tttggaggta tcttctcccc 1260
ttttcctggc ctaacaacat gaactttagt cgaccctcta acaggtgttt gtcgctgctg 1320
taccggaatg gtaggccgac tctctcttct gttgtacatg acaggactgt ccctaatccg 1380
gtcgctcgac cgcaacacta tgacttttgt ggggaatgtc ttcttcttac cttttggagg 1440
taacctgctg atgtcagcaa caatttcttg cgttaggcca acaaaacgtt tggggcaata 1500
acatcacaat catcttccaa gttcttttct gcatcaatat cctgcataca tatacccatg 1560
tacatagttt agcatatata catcatttac ctatagttta acatctttgt catggcttac 1620
cgggttggtt gcatgcaatt cttctttgtt tctaatctgt aatattcaat tagagaaagt 1680
aagtgggttt cattcaaaaa atataagtgc tagtggatta aatatacaga cggatatagt 1740
aataccttac tgcttgacac ctgctccttc tctttagctc ttttaggtgg ccgggtagat 1800
tgggcgtcca tatcagaaat ggattttttt ctccgtcctt tttgagaagc agcgtcacct 1860
cagacctagt agtacatcac atttctatgt catatttact cattaaaaca aatgagtaac 1920
agtctttacc gtatgctaac ctcctcaaga gatactcttg ctgccatctg acacacttaa 1980
tccatcagtt atgccacaaa cacccgctac actgtcactt cctccaggta gccaaatatc 2040
aagttcatcc ggctgggaac caacatgcgt atgccttggc tggtcagagc ttctatgttc 2100
ctatttcatg aattgcaatt gtaacataag tatattatgt cacttaaaat ggaatggaac 2160
ataaatgttc agttttctaa gacatataca acaaatcaag gtaacttaac ctttgtagag 2220
catgactctg ctacacattt gttgcattcc cctttttgag catgttgtat attttgccca 2280
cttaccagtt ccctaacaga ttcagcaatc attgccacca attctttctt atgcttatct 2340
agttgagcat caaagtatgc cttcaagtca actgtgttcg cctgttgaat cttgagaaaa 2400
cgtttttcaa attcatcagc tgcctctttg atcaatgcac tactactatg ttgcctttcc 2460
ccagcaatcg ggacatcatg ctgcaaatct tctctagcct aaaaactgtc atcatttttt 2520
tgtggttgtc catcatcaac aggaacatca acacccatgc cgtcttcaca tacaccggtt 2580
gcaccctctt gtctctcttc tgcagcacca ccatggacat cagcctccat ctcatcttca 2640
ccatcaatct tagacgaatt cacaccatca tctgcaagaa ctcatcatca gatggttgta 2700
caatatcaca agcgtccatg ccaccactcc aactgttatg ctcgtatggg tgctcctcta 2760
ttattaaaga tagcaggtgt gatacttctt catcctcctc ctcatcagat aaagataagt 2820
cgatatcttt tgcaagtagt ttttcattca gcagtgatgt gacccagtcc tacaatataa 2880
agttgaattg gatagttaag gcatgcacat acaattttaa taccaaaatc aagtagctga 2940
catacaatat aaag 2954
<210> 18
<211> 1129
<212> DNA
<213> Arabidopsis thaliana
<400> 18
catgcgggaa cccttgtatg gctcttgtat cttggggaag ttccttggtt gttctttgtt 60
ttgcgcttct aattgtagaa agaaacgagg ttctgcccat ggatatttaa gaaatgcaac 120
aacatcttta accatctcta catgttcagg attaagattg ttcccgcaca tgtcaggcaa 180
agaattccat caactattag aagcaaggct aggcgaaacc gggttagcgg gtctttgtat 240
ttcttcccca tagaaagctt tcctacaatc cattttgaag ttgaccttct ctctttacca 300
aatagtgact tgtagaagct tgtaggattc tgaaccgctt tcacattccc tctagttctt 360
gcatccttct tgttgaagtc agctttgctg aagttaagac cagtcaccag ctcaaaatca 420
tcaatgcaaa agtgaattgg tgtcccaaca aacaaccacc gcatctcata cttcttattt 480
gtcaccaact gtctagatag aatgtaatgc aagaacatta cagaaaatcg atgggttccc 540
atttgcaaga tacttcgaaa tttggaacca tccaaattct gcaaaatgct cttcatctag 6C0
tgcagccttt ataatctcaa tccacctaag tgtaaagtaa ttgtttatcc tctttcgacc 660
ttcgggttca gaacaaagcc gaaagaaacg ctttgtcaac tcatcaccc:c cc<3tttaaca 720
CA 02362897 2001-09-18
WO UU/sS32~ PCT/US00/07392
gataccctga aattcaaagg taccattatc acttcttttc gcttacgaga tacccaaaac 780
agatgtatac aaatcatgta atctaaactg gcactaaatg ttcaataact caccatatag 840
gtggttaaac cacacatgca atatgtagcc atctttccat tagtttccta gtcgcaactc 900
aaattcgacg attatatagt ccccgccaca aattgcacac ccggaagttg cttactcgac 960
ttcaccgtca ccagccctgt cgccgttatg cgaaaccccc aaatcgaaaa accggatacc 1020
cttgcatcgc cactctaacg gtgtcgatct atatcactct tcgaaacttt cccaagttgc 1080
ccgttttatg tatccacgct ttattttggg tattgtaaat tctctgcaa 1129
<210> 19
<211> 713
<212> DNA
<213> Arabidopsis thaliana
<400> 19
ctaggttatg aacccacgct acgcatgggt ttatttgtat aacttttaat aattttttgt 60
gtgattagat tttttattga aagttttgaa agatatgttt ttatatgtct acttgttgtt 120
aatattataa actctttgaa ttttaaaatt accatgacaa atagttacaa tttaaataca 180
taactaaaaa taactttaat acaactttta cgtttacaac atttatcaaa tgaacatttt 240
tttggttcat gactctctct ttatctttag atgtttagga taacgacgtt ttttacgtat 300
gaaataaatt gttgtatgga aaataatttt gacgactttc tcatcttggt acccgcaaat 360
ttactatcgt ttttcgttta tcttcgctcc attagttagt atagctcttc taaaaataag 420
atattatcta gaagtacatt cataacctca tctacacttt cttttaacaa ctcatacttt 480
tctgactgtc ttttcaatat ggatatgaat ttctgtcttc acaatttttt acagagagtt 540
atcactcttc ctatcttttt gcaagcactc gttttatttg tttcattttt tcttacagtg 600
tctctttcca tcctcttatg atttgtctaa gcaatatgtg tcaaactttc taaaattttc 660
acatgcttca tattttttct tcgaatatct cttccttttc ttgttagtat aag 713
<210> 20
<211> 1023
<212> DNA
<213> Nrabidopsis thaliana
<400> 20
ttatctttca aaacaaaaaa aaacttcctt ttgaaagata tggagcctat ccacaagctc 60
actgatttga atgtaaactt gaccgaatgg aaggtttatg tgaagatcct atctatctgg 120
aatcatcctc caaagaatca tggtgaagtt acaaccatga ttttggttga ttaaaaggtt 180
tggatccttt tattttttgg ttcatttatc aaattttctt tgttacgatt gaaatagagg 240
cttttttctt ctttaaaaga gagttttttc ttttcttCtt acctgggtac tcgaatagat 300
gcaaccatac cttaaaaaaa ctataaatat cctttctgac ccattctcaa gccggacatc 360
tgctttcatc tttctgattt tcgagtcatt tatccaacaa acagagttaa ttattctttg 420
tttcatgtcc aaatcaagtt tatttgggga acaagtgttt agcctgttct agtaattaag 480
aaaagtaatt tctttgattt catttttccc atagacatta agtatccttc gtcttaggaa 540
gttgagtatg tcactggtaa gtgtatagag aatttaagtt gttatcattt aaatctgctt 600
atatttgtta gtatgtgatg catttaattt ttatataatc tatcaacatc ttctgttcca 660
cttagacgct atgggtgtgg tgtcaaatat ctctgctatt aagaattttc catttgttgg 720
tcatcagggt gagactgact ataagtacat gtatgtctct ttcgacattg tggacactat 780
gtaagttgtt gtatttatag gtgtataaag aatcctgctg tcttatgaat atgaaactta 840
gaagaaataa gtattgcaga ttgaatcata ttgtctaggg acagaaaatc aagtgctttc 900
ttgtggaaaa atgttgtgaa ctgtttgtta taaagtggac taaacgtatt gttcagtttc 960
atatagcaat aaaccaattc ttgcaattgt ccgcttctag agaaacacag aggttgaggg 1020
t to
1023
<210> 21
<211> 1955
<2i2> DNA
<213> Arabidopsis thaliana
<400> ~l
taacagaaat aagtataact ataactatac ttttatgtaa tcgttgcaaa ctttgatagg 60
agtttg~ttg gagtttcttt acgtcatatt tgtaaacttt gttaggagtt taacaaaaag 120
taagatagag aaaaatccaa cgggaacaca ataattaaat aggttcaaaa cataagcaaa 180
CA 02362897 2001-09-18
WO 00/55325 PCT/US110/07392
gtttaacaaa aaaaacaatt tgcaatgaca aagtttggag aaacttaaaa gaagttcaaa 240
acataattta aagaagaata agagctgcca ggtcaaactc ctaaatattt tcaatcactt 300
ccatcctttg gttctctgga acttcataat aatcttcgaa accgcggctg taaaagagaa 360
aaataaaaat ttttattgat taattgatgt tatggttaac taatacaatt aaaaaatgtt 420
aaaagtctta catgtaactt cgaatatcta tctcagggaa agtggttgga ttgatgtaaa 480
ttcttgaaca tccaaattca ctcatcaaaa cattctcacc tgttacaaaa tttttattta 540
aagttatata atacaaataa aaatcataag taaataggat tacaatagta ttgaaaataa 600
ctaaccctca atctcagcaa ttctccaaaa cctcacatgc ctctgaactg atacatttcg 660
agaattgtgt aacaaagatt tcacagcatc ttccaacagc atagcatttc attttctctc 720
ccctaaccaa tatgataaaa cattacacta agcaatacat ttttaagctc aattttcaga 780
cggaagacac attactaaat atacttataa acactaaaac ttacatatta cccaaaagat 840
cgaaagagac atatctcgat tcatagtcgg tttcaccctg gcgacaaaca aatgagaact 900
tcttaatagc cgagatattt gacactacac ccaacattgc atctaagtat agaataatat 960
attaatagag tagttcaaaa aaataataac ataaactact aacaaaaatt cattctttaa 1020
atgagaagaa catagattga ctaaaaactt acccgtgata taatcccaat cctcaagaca 1080
tggatactta aggtttatgg gaaaaatgaa atcaaagaaa tctcttcacc agttccggaa 1140
gaggcaaaac acttgtttcc caaatgaact tgatgtggaa acaaaacgaa gaatacctaa 1200
ccctttcttg aggaacaacg acccgaaagt cagagatgtg aatccatgtg tcaggaattg 1260
catctacacg attattctga aaaaattccc agataactca aagattaaat aatttaaaat 1320
gatctgtttc aataataaca aagtaaactt ggattaaaag aacgaaacaa tcaaaaatac 1380
aaaccttgtc atcatgaaga atcatggttg taatttcacc atgactgttt ggaggatgat 1440
tccagattga caggatcttc acgttaatct tccaattagt cgagttatcc ctcaaatcag 1500
tgagtttgtg aacagacatc atgtttatcc aaggaagttt ttttttttgt ttgaaagata 1560
agagttaaac ctaattgttt taggcttgag gtttttgatt ttatataatt acgaaaagat 1620
acaatgatgg acagttgatg tgtaaaaagg aaacgaattg tccacaacca ataggatttc 1680
ttataggtta gaacaataat gttgatattt atttcttagc ctgttactaa aaaattgtaa 1740
ttatttaaag gagaatgtat gaaaaaatag gaataggttt gtttttcaag attctcatga 1800
ttaatcatat ttaataggat tgattattta gtgtacaaaa tctttcctaa ttctgatt~t 1860
gttggttttt tttgtttaaa atgaccaaaa gtgtttatag ttctccgtct gattaacatt 1920
gtaatataaa agaatatgtt tcttaatcat aaata 1955
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 22
cgccaaagac tacgaaatga tc 22
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Desc=iption of Artificial Sequence: Synthetic
Primer
<400> 23
ataatagata aagagcccca cac 2;
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
c)
CA 02362897 2001-09-18
WO 00/55326 PCT/US00/07392
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 24
gggtctggtt agccgtgaag 20
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 25
gttttactta gtccaatggt ag
22
<220> 26
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 26
aaatggccaa cgatcagaag aatag 25
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 27
gaagtccggc atgttatcac ccaag 25
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 28
caagtcgcaa acggaaaatg 20
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthet_c
CA 02362897 2001-09-18
WO 00/5532 PCT/US00/07392
Primer
<400> 29
aaactacgcc taaccactat tctc 24
<210> 3v
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 30
gaagtacagc ggctcaaaaa gaag 24
<210> 3I
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 31
ttgctgccat gtaataccta agtg 24
<210> 32
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 32
gttgacttgt atttgatttc tttttc 26
<210> 33
<211> 22
<212> DNA
<213> i-artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 33
cgagtgattt ccttttgcta cc 22
<210> 34
<211> 22
<212> DNa
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<400> 34
aagataaagc agcgaatgtg 22
tc
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Sequence:Synthetic
Artificial
Primer
<400> 35
cgaaagccgt aactagataa 24
taag
<2I0> 36
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Sequence:Synthetic
Artificial
Primer
<400> 36
taccagcata caggagaacg 20
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Sequence:Synthetic
Artificial
Primer
<400> 37
cctgattgca gttttattta 22
cc
<210> 38
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Sequence:Synthetic
Artificial
Primer
<400> 38
tccataccta agttccacaa c 21
<210> 39
<211> 19
<212> DNA
<213> Artificial Seauence
<220>
<223> Description o~ Artificial Sea_uence: Synthetic
Prime.
1?
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<400> 39
aggggcgagt aaatcaatc 1'3
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<900> 40
gaagtgcgga tctgtttgaa g 21
<210> 41
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 41 _
ataaaaagcc ggagatggtt g 2'-
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Arti_'icial Sequence: Synthetic
Primer
<400> 42
attcatgagt gcaaagggta gag 23
<210> 43
<211> 23
<212> DNA
<213> Artificial Sea_uence
<220>
<223> Description of Art_~icial Sequence: Synthetic
Primer
<400> 43
ctcagccaaa gaatcaagta gag 23
<210> 44
<211> 21
<212> DNA
<213> Artificial Sec_uer.c-
<220>
<223> Description. o~ Art-=icial Sequence: Synthetic
Primer
is
CA 02362897 2001-09-18
WO UO/55325 PCT/US00/07392
<400> 44
aagcttcatt ctgtggtttt g 21
<210> 45
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 45
agaatcctta gccgtcctg 19
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 46
gccttggatg atcagtggtg 20
<210> 47
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 97
agcccttgga tcatattctt tagc 24
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 48
ggctactggt caaatcattc 20
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Prime.
l =l
CA 02362897 2001-09-18
W O 00/55325 PCT/US00/07392
<400> 49
gaatctttgc aaacgagtgg
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 50
gcggctgatg atctccacct c 21
<210> 51
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 51
ttaccccgca ggaaaaagta tg 22
<210> 52
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 52
acttcatcac ttgcgggact g 21
<210> 53
<212> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 53
ggcccaagaa gcccacaaca c 21
<210> 54
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> _Description of Artificial Seauenc~: Synthetic:
Primer
l~
CA 02362897 2001-09-18
WO OO/s~32s PCT/US00/07392
<400> 54
accgcaagtg tggctgttc 19
<210> 55
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 55
ctattctaga agattgttag gagttac 27
<210> 56
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 56
atgcctattt agccttttta tag 23
<210> 57
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 57
cgtctgtatg gattcgtagc 20
<210> 58
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 58
tgagaggtgc aaaatcataa cag 23
<210> 5°
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
CA 02362897 2001-09-18
WO 00/55325 PCT/IJS00/07392
<400> 59
accgcgtcgt tggagc 16
<210> 60
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 60
ggccgcgtaa gaggagac 18
<210> 61
<211> 26
<212> DNA
<2I3> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 61
aaactgatat tgtagatgtg tattcg 26
<210> 62
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 62
cgttcgaagc gtttgttc 18
<210> 63
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtiLicial Sequence: Synthetic
Primer
<400> 63
attacagttt tgcctagaag atgg 24
<210> 64
<211> 26
<212> DNR
<213> Rrtiricial Sequence
<220>
<223> Description o' Artvficial Sequence: Synthetic
Primer
li
CA 02362897 2001-09-18
WO 00/5532 PCT/US00/07392
<400> 64
aagttgattt tctactgttt atttag 26
<210> 65
<211> 20
<212> DNv
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 65
catcgtcata tggcttgttc 20
<210> 66
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 66
taacgttccc acatgagc 18
<210> 67
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 67
aactctgtac ctgctgga 18
<220> 68
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 68
catctccatg aaggtgaata g 21
<210> 69
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description or Artificial Sequence: Synthetic
Primer
I
CA 02362897 2001-09-18
WO 00/5532 PCT/US00/07392
<400> 6°
aagttatgca aaacgttatg acg 23
<210> 70
<211> 26
<212> DNa
<213> P.rtificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 70
gagcccttct atgagcctac ctgttc 26
<210> 71
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 71
agagatcccc tgttactaaa gcctattctg 30
<210> 72
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 72
atatttcgtc gatcgtgttt g 21
<210> 73
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 73
gtgcc;.cagg gacttcac 18
<210> 74
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Descrio_tion of Artificial Sequence: Synthetic
?Timer
<400> 74
19
CA 02362897 2001-09-18
WO 00/55325 PCT/USUO/07392
ggtaacagcc ttcactcgcc 20
<210> 75
<211> 22
<212> DNR
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 75
aaagacttgt atttgggatt tg 22
<210> 76
<211> 25
<212> DNA
<213> Artificia2 Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 76
tctttccctt aatctatctg ttgtg 25
<210> 77
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 77
aaacgattgt tttcctgcag tg 22
<210> 78
<211> 24
<212> DNA
<213> Artificial Seauence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 78
tctctgtgct ttctctttcc tgac 24
<210> 79
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 79
gcaatgctac cgctctgata g 2i
7U
CA 02362897 2001-09-18
VVO 00/55325 PCT/US00/07392
<210> 80
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Sequence:Synthetic
Artificial
Primer
<400> 80
ttgtttttct aggttttgtt 25
gtaag
<210> 81
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Sequence:Synthetic
Artificial
Primer
<400> 81
atgctgcgat gtttgtaagg 20
<210> 82
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Sequence:Synthetic
Artificial
Primer
<400> 82
agtcgatgtc taggctcttc 20
<210> 83
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Sequence:Synthetic
Artificial
Primer
<400> 83
cttccatttc ttgatccagt tc 22
<210> 84
<211> 25
<212> DNA
<213> Artificial Sea_uence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 84
actaaggccc gtgttgacgt ccctc
?I
CA 02362897 2001-09-18
WO 00/~~325 PCT/US00/07392
<210> 85
<21I> 2i
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 85
aaccgcttcc cattcgtctt c 21
<210> 86
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 86
ggcgaccttg gacctgtata cg 22
<210> 87
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 87
aaccgccatt ttcatttcta tc 22
<210> 88
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 88
taggggacat atcaaaccaa c 21
<210> 89
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 89
gtctaaaacc atcttcacca tact 24
<210> 90
77
CA 02362897 2001-09-18
WO 00/~s325 PCT/US00/07392
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> _Description of Artificial Sequence: Synthetic
Primer
<400> 90
atgcctaact attcgctgac 20
<210> 91
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 91
ttctgtagtt ctttgtgagt gc 22
<210> 92
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 92
ggcattaatt gggaaggtc lg
<210> 93
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 93
tataacatca aaagcggtca tcag 24
<210> 94
<211> 20
<2i2> DNA
<213> Artificial Sequence
<220>
<223=~ Description of Artificial Sequence: Synthetic
?rimes
<400 > 94
gcattaaaga caaaaagccc 20
<210> 95
<21i> 21
CA 02362897 2001-09-18
WO 00/5532 PCT/US00/07392
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: SynChetic
Primer
<400> 95
cgttgacccc gagaagatta c 21
<210> 96
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 96
ttcgggaatc atggtctaca ag 22
<210> 97
<211> 24
<212> DNA
<213> Artificial Seauence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 97
tgtcacatac acggtttctc ttag 24
<210> 98
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 9s
caagcttcat ggggactag 19
<210> 99
<211> 23
<212> DNA
<213> Artificial Seauence
<220>
<223> Description or Artificial Sequence: Synthetic
Primer
<400> 99
taatacggga caatctacaa cac 23
<210> 100
<211> 22
<212> DNA
CA 02362897 2001-09-18
WO 00/~~325 PCTlUS00/07392
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 100
ctaattgtaa cggagaagag ag 22
<210> 101
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 101
aagcatgtta cgtgggattg 20
<210> 102
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 102
caaatgatgt ctggtctatc ttc 23
<210> 103
<211> 25
<212> DNA
<213> Artificial Seauence
<220>
<223> Description of Art=ficial Sequence: Synthetic
Primer
<400> 103
aatttaaaag gaatcagaga actac 25
<210> 104
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
Primer
<400> 104
atgatcaaag ggggacgagg 20
<210> 105
<2-~1> 24
<212> DNA
<213> Artificial Sequence
CA 02362897 2001-09-18
WO 00/~s32s PCT/US00/07392
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 1G~
aaggaaacac caccaaacga aaac 24
<210> 106
<212> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 106
gagacagagg atttggaac 19
<210> 107
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 107
gaaaccctct cctcaaac 18
<210> 108
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 108
cccctcccgc cctaaaccta c 21
<210> 109
<21i= 25
<212=~ DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 109
ttccg~=taca tggccttcta ccttg 25
<210= 110
<211=~ 24
<212> DNA
<213> Ar~i'icial Sequence
?O
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 110
cgtattcccc tgaaaagtga cctg 24
<210> 111
<221> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 111
acatccggcc ttcccattg 19
<210> 112
<21I> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 1I2
attcttttgc tttatgggac ttc 23
<210> 113
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 113
aaacatgctg cagcttgatt ag 22
<210> 114
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 114
aggacgatga tacgcttgtg gag 23
<210> 115
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
77
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 115
atcatgggga cgctgctttt c 21
<210> 116
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 116
ttggttttaa ggctttggtg tagg 24
<210> 117
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 117
atgcgcagaa gagacgatga tag 23
<210> 118
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 118
gtttaaattt ttatgtcatg tctgtttc 28
<210> 119
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 119
ctttgggcga tgtaggagta g 21
<210> 120
<211> 21
<212> DNA
<213> Artificial Seauence
<220>
<223> Description or Artificial SequencF~: Synthetic
y
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
Primer
<400> 120
cgcgacctta gccttgttgt g 21
<210> 121
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 121
tgtgggcagg gtaatggatg 20
<210> 122
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 122
atatccggct ccgaacttgt gg 22
<210> 123
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 123
ccgcgagatg gatgtgatga c 21
<210> 124
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 124
tgagggggct gacatttctt c 21
<210> 125
<2I1> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of .4rtificial Sequence: Synt~e~ic
Primer
7 c)
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<400> 125
ttcccccgag gcgactgac 1'~
<210> 126
<221> 31
<212> DNa
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:Synthetic
Primer
<400> 126
tcggttgggg atagaaaatg
g 21
<210> 127
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:Synthetic
Primer
<400> 127
gtggcacgat cgtatgagtt 23
agc
<210> 128
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:Synt:~etic
Primer
<400> 128
ctctcatcga ccctcactct 24
caag
<210> 129
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence:Synthetic
Primer
<400> 129
agtcccaaca aaaccaaaaa 27
cataaac
<210> 130
<211> 21
< 212 > DDIr',
<213> Artificial Sequence
<220>
Descrio_tion of ArtificialSequence:Synthe~ic
<223> _
?timer
CA 02362897 2001-09-18
WO 00/~~325 PCT/US00/07392
<400> 130
ggcctccatg ctaccaacaa c 21
<210> 131
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 131
cacaaaatgc cacccctact acc 23
<210> 132
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 132
tggcagcaga gttatttgac gag 23
<210> 133
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 133
atgcgcgact gaaggacacc 20
<210> 134
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 134
ggcctgccca taaacctg 1g
<210> 135
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ~.rti=vcia- Sequence: Synthetic
Primer
<400> 135
31
CA 02362897 2001-09-18
WO 00/5532 PCT/US00/07392
ccgctgtgga acctgaaag 19
<210> 136
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 136
aaacgccgcc aaaatcagaa c 21
<210> 137
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 137
acaaccttag ccccatccat tc 22
<210> 138
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence. Synthetic
Primer
<400> 138
ctgcgagcga ggtcaatg 18
<210> 139
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 139
gcagccgtgt ggatggag 18
<210> 140
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 140
aatcaattgg tttctacttt ttag 24
37
CA 02362897 2001-09-18
WO 00/~532~ PCT/US00/07392
<210> 141
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 141
aactccgact gaaggtatag c 21
<210> 192
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 142
tttgcaccgc ctatgttacc 20
<210> 143
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 143
gaggacgttt tgcagagtg 19
<210> 144
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 144
tcgactagat ttattatttc tctcag 26
<210> 145
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial sequence: Synthetic
Primer
<400> 145
tttggcttga ctctgtgaac 20
CA 02362897 2001-09-18
WO 00/i532s PCT/LJS00/07392
<210> 146
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 146
gcgaattcct tgccactaag 20
<210> 147
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 147
aagaagaaga ggaggaagaa gatgtc 26
<210> 148
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 148
agtggacgcc ttcttcaatg tg 22
<210> 149
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sea_uence: Synthetic
Primer
<400> 149
tggtccgtcg tagggcaac 19
<210> 150
<211> 21
<212> DNA
<213> Artificial Sequence
<220=
<22?% Description of Artificial Sequence: Synthetic
Primer
<40C> 150
cttcacgctg ccttcactct c 21
<2i0> 151
3=l
CA 02362897 2001-09-18
WO 00/55325 PCT/USO(I/07392
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 151
gatacgctcg ttcccactcg 20
<210> 152
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 152
caaaaccaaa tccgcgaaga ac 22
<210> 153
<211> 24
<212> DNR
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<900> 153
agtggccagc cttcttaaca tacc 24
<210> 154
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> I54
tttgtgcaat ttattagggt ag 22
<210> 155
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Prime.
<400> 155
atttgca_qaa gctgaagttg gtc ?3
<21G> 156
<211> 24
37
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 156
ccgtggtcga gagttgagtt agtc 24
<210> 157
<211> 24
<212> DNA
<213> Artificial Sequence
<220> "
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 157
acccggagta gtttttcagt gttc 24
<210> 158
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 158
agcttcgata acaaactcac c 21
<210> 159
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 159
agaagataaa tcaactaaac aaaatg 26
<210> 160
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 160
aacgcttatc ctctttctct tttac 25
<210> 161
<211> 22
<212> DNA
~6
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 161
acggttgccc atcttatcag tg 22
<210> 162
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 162
tctcgttctg atggctcctg tg 22
<210> 163
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 163
gtgtaaccgg tgatactctc gcc 23
<210> 164
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 164
cgacgaagca gtggaggaac 20
<210> 165
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Syr.theCic
Primer
<400> 165
gcgagaaaac gtgaagagat ag 22.
<210> 166
<211> 22
<212> DNA
<2i3> Artificial Sequence
37
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 166
agctactacc cgaatgtgaa tc 22
<210> 167
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 167
ttggtgtgtt aagaagagtg g 21
<210> 168
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sea_uence: Synthetic
Primer
<400> 168
taggacgcaa atcagagaag 20
<210> 169
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 169
ctaatcatgt gtctttaggc tatc 24
<210> 170
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 170
ttgggctggc gtggaatc 18
<210> 171
<211> 18
<212> DNA
<213> Artificial Sequence
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
<220>
<223> Description oz Artificial Sequence: Synthetic
Primer
<400> 1?1
18
agggcagaaa gcgtcagg
<210> 172
<211> 20
<212> DNA
<213> Artificial Seauence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 172
gctgcgaagg ttgaatgaag 20
<210> 173
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 173
tcgccgggaa aaacagtaac 20
<210> 174
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 174
caccgacgtt atctgggaaa g 21
<210> 175
<2i1> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description oz ._tificial Sequence: Synthetic
Primer
<400> 175
aaaagttagg tagtaggaaa gaaagaag 26
<210> 176
<211> 21
<212> DNA
<213> Artificial Seauence
<220>
39
CA 02362897 2001-09-18
WO 00/5325 PCT/US00/07392
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 176
gagcgtgctt ttggagtttt g 21
<210> 177
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 177
aaccctagat cgcccttttt tc 22
<210> 178
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 178
gactcatatg tggcgttttc 20
<210> 179
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Primer
<400> 179
aggattcact ggcggttg 18
1
CA 02362897 2001-09-18
WO 00/ss32i PCT/US00/0'7392
SEQ ID NO_ 180
Arnbiclup.sis lhnlia~tn
UBQ I I upstream regulatory sequences
cataacacaat~~ctttgtccaaagaaagaca~a~aagggct«tttttgtttgtttgtlaaa~~tgtga~_~cat~_~'
cat~,~,
tgggtcaagtitagatcattca~aggagataaccatgaagaguctacacttcgtccattaacaaaatcatca~
attatt
ccttcctaattacta~tcataccctaaatattcacttacattaatttclggtattctctutg~tagt~etcgctagatt
tnttgg~tcaaaattaaacttcatctactttatttcagtaagatcatgtttcattgtgagaatgcca~ttcaataacca
ttaacacaatctcttgaccattttttttttggtttaattgatacttaaacaaaactc~aattggttcaaagctcaaatt
~=
elacatga«ataagccaaactcaaaactgaaatacugcclaaaacacacagattagcaaaaatcatcatc~=~taatct
ccccaataaccaaatcaaaactagttttttcaacaaaaaagaaaattgtaatcgctatggctccgtagatcgtaaaaca
a
gatccacaggttcctatcagtaagcgaaacacgtacatttgtttatgtctaatatagcgtcgagttaggatcattgccc
c
aaaaaaaagagctaccttcgacgtggcatcagacaatttctacaatttagaagaaaacaaaaagaaaaaaccctcagcg
g
acccttgtcaaatcaaccacacagcagacacagaacgaattcagtcacgctagctgttgtgtaaagtgtcatacggcac
t
tcctatcttttgggattgcttc«catcgggtttataacaatcaaaactcgtaaccaaacattaaacgatttcaagtaaa
cagagaaccaaaagaagaaaaaactaagatagaagaaaacgttgttgaagatatgagaaagagggagaagagaatctgt
a
tttatatagagggtatttgtggcggcataacttaaagccctagcgtgtacgaggcggcatataacttgaaaccctaacg
t
gtacgaggcggcataacttcaaacccaagcgtttgatcgag«accatatagcccattaagagcctatatgtat«tggg
ctgaataaaatatta«gggccagtagattgtggtagtagaaagagaagagctctaactacaaagactactagtct«gt
tccltctacgatgattggeacagaaaccaaaccattggttcaa
caaatgtggtcccacgtgtooatctcatg~ctcta~g
gaaaccac«ggatgacgctgcaaggagttgatcitlatgctgcggttccgtt«gtgttgtgcatataatc«agagaa
aggtactgtcttctcatgtactcttgttttataggtttttcctclcttttttaagaaaagigtgtataattgtgttaca
a
atg«~cagatggagaegaaattgagtttgaatcaaacaaatcagagtttagtgaa~ga~aaggctctg«catatcaga
actt~osaatcttQCtgatagactcagcatcttgcagtctcctctacatctcattgtattgtcccct~tttttgctt~
ca
GCv.
catgtttttgaaa~~t~cttaaaacattgtttcaaattcgcactgcactttactggcacacttataaggtgatattg«
la
tcacacactgtcaagaagttaaaaccattagatgttgacaggactgaacattagatagagactctgtac«ctcacactc
atcaaactaagcaeatcatataaaga~acaacgat~_acaaaagtgaatcgaaagagataattgcaataaa~aataagt
tc
oatttgttg«tgaaagaaaagagagatgaaagaaacggaaacatagtcgatcaattatttatcagaggctgtacatggc
cccaaaacalaaaccaccaaagtactagatgaaacgatacatgaacttggttcagtaaccalaagagagagagacaagg
t
SEQ ID NO: 181
Arabidnp.si.s thalicma
UBQI 1 downstream regulatory sequences
cataacacaatgct«gtccaaagaaagacagagaa~ggcttttttttgttt~ttt~ttaaagt~tgaggcatg,'citg
g
tgggtcaa~«tai=atcattcagaggagataaccatgaagagttctacac«c~tccattaacaaaatcatca~atta«
ccucctaattacta~tcatacc:ctaaatattcacitaca«aat«etggtauctcttttggta~ts~tc~ctagatt
tt«t«g~tcaaaattaaacttcatctactttatttcagtaagatcat~tttcattgl~a~aat~ccasttcaataacca
ttaacacaatctctt~accattttttttttggtttaattgatacttaaacaaaactc~aatt~_gucaaagctcaaatt
~,
~tacatgattataaeccaaactcaaaactgaaatactigcctaaaacacacagaltagcaaaaatcatcatc~'gtaat
ct
ccccaataaccaaatcaaaactagttttttcaacaaaaaagaaaattgtaatcgctatg;ctccgtagatcgtaaaaca
a
gatccacaggttcctatcagtaagcgaaacacgtacatttgtttatgtctaatatagcgtcgagttaggatcatteccc
c
aaaaaaaa~a~,ctaccttcgacgtggcatcagacaatttctacaattta~aagaaaacaaaaagaaaaaac~ctcagc
g~~
acccttgtcaaatcaaccacacagcagacacagaacgaattcagtcacgctagctg«gtgtaaagtgtcatacggcact
tcctatcttttggga«gcttcttcatcgggtttataacaatcaaaactcgtaaccaaacattaaacgatttcaagtaaa
cagagaaccaaaagaagaaaaaactaagatagaagaaaacgttgttgaa~_atatgagaaaga~=ggagaa~'agaatc
t''ta
tttatataga~g~ta«t~t~gcggcataacttaaagccctagc~tgtacga«gcg~catataacttgaaaccctaacgt
YIaC~~ig~C~~~CataacttCaaaCCCaa~CgtttgatCgagttaccatata~CCCattaagagcctatat~tatttt
gg~
ctgaataaaatattattgggccagtagattgtggtagtagaaagagaa''agctctaactacaaagactactagtcttt
gt
tccttctac~'at~attgggacagaaaccaaaccattggttcaacaaatgt~~tcccacgtgt~gatctcatggctcta
~g
gaaaccactt~s~at~ac~ctgcaag~~agttgatctttatgct~'c~g«cc~titt~t~ttgtocatataatctta~a
gaa
a~~tactgtc«ctcat~tactcugttttatag~tttttcctctctttutaaoaaaa~_t~t~talaatt~t_ttacaa
atg«
gca~at~';=a~_a~~aaattga~tttgaatcaaacaaatca~agttta~tgaae~a~aa~yctct~_ttcatatca~a
aCll~'~~~alC:lt~_Cioata~_aCICa~CalCllgCa~ICICCICIaCaIClC:allolalt~lCCCCt~lttll?
Ctl~Ca
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
catgttt«~aaa~=t~cttaaaaeattgt«caaattcgcact~cactttactg~cacacttataa~gtgatattgtlta
IC:ICaCaCIoIC aa~;aa~llaa:laCl:allag:llgllgaCag YaC
l~aaCall:l~alagilg:lClClglaClll:lCilCaC lC
:llCaaalaaa~=l;aCalCaIalaaaga'_:1C8:1CgillgaCa:laagt~'aatc~=aaagagataattgcaata~
_a~'aal:l:1';llC
aralltgll~'lit,';laa~'aaaa~;ai=agalgaaagaaacggaaacataglC!_atc:aallalllalca!?:1~
'~'Cl'=lacal~'~'C
CCC:IaaaCalaaaC(::lCCa;l;l~taCl:lgal''aaaCgatacatgaactt
_'_IIC:I''laaCCal:l:lg:li':1'':1'_aL'a(:a:l''qt
SEQ ID NO: I 82
Arabidopsi.s rlraliana
S16 upstream re~_ulatory sequences
cgttttttttttgY~=aaatacgaaatcgaaaggctaaaaacaactcaattcaat~gccaggattaaagecgagtaccc
aa
atttttaatacca~gtaaaatgatccgcgtgttttctaagtgaaaclgcgaaagcgaacgactaaaaacaa«caattga
atggccaagattagaagllatgacccattattttaaaatctgggtcaaaagoatccgc~tgttttgtaagtgaaacccc
g
aaatcgaacagctaaaaacaattcaattaaatgaccaagattagaag«atgacccattattttaaaatttgggtcaaaa
~gatccgcgcgtttctaaaaaggtttttttcatgacataaaalttttaaccttatggcaaatttattattttagcgaaa
t
tcatgcatact«attaaacgtaattatcaaataatagagtagattataattgaatagtataaacalattttcattttat
ttttgttttlgttlaactaagctcacagcgacacaattccgaalatlagcaattg
gagaaagaataaaagacacagagca
ccatattattcttttaYCCCaaaaatttccacaastacaaatccYYcaaaaacacatacataeaccaattttt~~itta
c
tgacgaaattttcagatca~aaaaccggatttgattcaagtg~~tccctaaaccgtcggttttaaacaatattttcaac
g
atYaYctttttcaaYtcaaactaYcoaaacaYCtatcaoacclasacaaaaca«aeaaacacaatcat:.ratYattal
ct
tgatcgtcttta~'atg~actattaggcatagatclaagcattcttggt~aatctaagctttttctcaagatctgtclc
at
CgCagagatC:la:tl~lCIlgICa2atICQalg lYo~_Ctl .1
~:IlllgaaCflaItaCaClIgIlCatglgICICgCCa
g ,._,. g~ ga
tagCItCIIC f ICl gIl ~LlaaaBaICIIIfIgIICICaIgaClIf
gleClICaCagCIlCa~aaCaaaalCCaEa~=alC
catttccctatgtaac~ctctcttatcatctctgtgtacgattctgtacactcglcg
gttaatccgcaacagtcacacgt
caccgaatcta~ctcatc
~_ctggatalttgtgtcaccggaaccgtt~a~ataatta«acatccggtgtt~aaattaaca
ttgttggalcactaa~=lacactatcagacatggtltcttgatttaaaaccctgltgagaaaat«cgatcaacaatctg
a
ttcllac«caac:uaaoaaactalaaaeaaaccaagaggagaagttttatcagta'_cagaagatgagaagataacgca
a
taagcltgtgatatatcttclttgttttatctctg«tctgaaacagagaaatggtttttaaaaag~gattctlaaaaga
aallggaatacatt«t«It««aacg«cgltttgccactctclgtc«ctaaattactttcalctcc:gagaaaaat
ttcacaaaacaaaacca«~caaaaacagagtattaaaaatgctttttatcntggcttttcttgaaaacaaatagaaaa
agttaccptttctatttacttcacggaaaaaacattagaagtlcalaaaaaticactcgaatctclcgttacttragaa
g
agagaaaaaaaaaa~tattgtt«ggaaatatatgaggaaga~aaaat~a~agaagaagtagtgcag~~c~aagYaYaag
c
aatgtttggtgttttataagagagacgataglagtaactagtgacglggaatattgat«taattttttagtittat«t
atttattgaacaltttalcaaaac~cc~cgtattagaga~'galgc
ccta~'ntaat~tctt~~tataaaagcetatattt
tecatt«tcatcmactctctYce«a~Ytttcattita~c~~caYac~aYaYaYcaaaa'_caa~Ya"aaaccctaacc
_ _ . ._ _ ~ ~_
SEQ ID N0 183
Arabidnp.si.s rhaliarra
S 16 downstream re~ulatnry sequences
aacaaagctggg«««ttltttcaatttcgattcatcteaaggt««~gagtttttgaltatg«aett~=t~_agactc
ttagaatttctgttttat«atttatt~_~ataactgtgattctccaa~aacttal~tcttalatgautt~tcttcattt
c
gIlgCIlCltettlelI~CCl~l~al~lall~CaIClgCala~_llg~all~itaa~IlayvllCllog~lllataata
a~o
CIYIYaCIIYaIYIIaCIIa~CCaI~YCt~atcaaatate_'IC
I;IaCa;la!'l_YYLCt_'LI:ICIc:IYIIaIall'~~IICi'c
~laaalltl~~IlaCB~ItI~ItICC~3~lI~'=IaICIC~taagIC~IaaC~'lllYlgl:lCltallCaCI''aaaa
Caa~t
tplatlYIIYIIaYICIIIIYIIaCCICICCIB'_laCYallllllllYlfIYIYaaCaa~alCaYaaaCIYYYaCIYI
<_'a
attt_taccYataamaaaYaaccaaataacttttYatccaaaaatcYa_~taattaataaccYaaaaYatctttctact
aa
~ttttttcctacltc~acetYatttneactle«~caaaatYttatYctrttt~'t~c~ttccaccactmc~taYl'_aa
aectc«_Ytalcc~taaaYcaaaaa~'tcaacccltltYa!'tt«t_ovate_'caat«ctYcacatttttratt~ra_
ecc
IIYCCdIYYaY:IC:tCCal<_IYIICCCaa~CllCaYC'_~_~Caa_'aC'_tCC
~_l:lY:l:lCClllC'ICa'?ICaCCIICI~_aa'_t'.a
auccYYaacaa~r~rYaatacYcacYttcatesataat<_atatc_'aea'_a_'_'cc;laat~'atca_'terma_'
mcatacaa~_
caalccYaYaam«m~~ttt~'cveltYta!rtettYtC~IaaYacatat~_calctlcaaaY«l<_t«tttY~~ali'a
~ttY~ta
CA 02362897 2001-09-18
WO 00/5532 PCT/US00/07392
taltctttataccca~=tatcat''attcatataggtaaagaaacaoag«acac:«aaatgcgtatacacaaa~~atat
ggl
~tttgt~ctgattugctctuacttgttg«ttcaaacttat~'aaaaga~atalalcara~=a~=I~=tat~aaccalaa
cc
~lal::ll~'ICCCaa:lllC:l:laCaa~lCClClalCgaglgt~Itl~:ICS'CCC:IaC'l:tlll~=:Illa:lt
~'aglli'aflaC~C~:l
I~=glglcllgflagcaatagcaaatat~=atcggcragccccaaaacc:7lC1'_:hall'=a;l:Il:l~la:IC~;
~=a''CI~IICaC
_coal'r'rCalC~,aatt~_IIICLICa''C''~aIIICCICICCI1CCICCIC''''II1'L:I~:IIC':1':aC:
l~aa''Cl~'Ca:la:l~=:I'~C
(LgCCCICCa:IICCIC4'aaatttglCCa.lglCC~l~aIaICIIIaICa:lg _raai (ItCI~=allC;
g:IIC:ICCIIg~IIaCIC
actccgctctctgtttt~=ggcgatgltggtagactcgaatcc;cga~=cttcaa~'agaatatc''tcacaacattgt
tcaacat
_Icgactttcgaggagaataaaacactacttgctgaaaaccalcaagtgatrccl~_tgc«gcaaagittatgaaec:a
ga
agtcagtt~t~acaagaagaaac~ctacatt~acttgagtgtcactttla~aeatlgattclaacaagatcatcatagg
g
aactcagta_~cactcaaggctctyatagatcttatc~~gea~tl~"ate'acttawa~'clactcatgaa_ctclttg
cgc
t_ttatatacctctgttgtgatgaaatggagaactegaaaaa~~caatctc;;«gggg«gactcctt«gcagtcagaa
atatcaaagcaagaagaaattcgtttgactc~ttagttatcttg~=cattgatltctitacacgaacgc~ttatccagg
aa
ctggctaatgggttatctatgatct~ttaa~tatctt~altcttgaggaacaaaa~tt~catg~tgacttgtgaeaacg
t
SEQ ID N0:184
Arabidnpsis thaliana
contains 3 contigs, I-4960, 4961-13005, 13006-403-19
BAC F12G6
tacgctcactatacg~cgaattcttcatctttaattat~cctaat~ctaoatcta~a~taattaacta~atcatgaatc
c
agacaatagatatgcccgccataggtatctgtaglcgoatctatlattt~at~'a~_ICt~=gl«ata~gt~tgtaata
a_a
atcggcctatgtaclaagatgaacaatteaacttgttaagaat~c~tat«a~gtt~ata~_«tatcg~c«ctcca~tt
atta«agctatagatatagggag«tcgcggcacgcccaccggtta«ccataa«ag:l~tu~aotttaagaatggtt
c=ataacaacctagctttcatlagatctactaaccaatagt«cctgaaaataccat~_Itcctagctttttattaaatc
k
tttacaacccaatcaatct~_ctgttttcttattttgttitctaactattttacteatacr«aata~~cttaatctc~a
ca
tttaactcactgtttgaacaaaagaactccgcgaattcgatcctaaaatottgcaatt~=atctcttatttgagagagt
ag
ttctaggtaatttgaaccatatcaaatttggcgccgtlgcc
gaagltcttgggaatcaUattgagtttagtttagagat
lllglClagIIIIIaCItClalttlgllaClC:ICgCgItlCttgt111C'llClttICC~'l~=Itlc
a~glYtalgCCla~~'
caaacaaggagcacaaaavagaaggagcttatcaacttglclg«ctgc~amac
~zgaLaacugaacgc:ltcaaccaaa:l
aactaaaaaattagcaatcgctgacagagtcatcagagct~at~magaa~~_a~_l«t~=a~_a~at
~a~gat~ggcaagcct
acaatga~~cto~ttaga~attggatgatcatg~~actcatcctaccc,=a~=~=ac~ccaat~=aggatca~gc~tgc
ctcaat
gcgcaattagctactagagacgcagcagggctcggtgtcgagcgataccaact~~=gt~=tcgalcgacaccatcggga
gca
scacgcagatgcaatc~gtgtcgaacgacacaataga~~t~tc~atc«aca<'c:aacaccacattcctgcagctattc
ctc
cagctgcggaaccacgtcgtactcttg~saatttcaacaagccag:lcctgltctatgctaatcgatcagcgattgtac
cc
tcaccattltagaggaacgactatgagctgaagcatggctac«tgctttagltg~'ccagcatcctttccate~~c«at
c
tcatgagcagccaatggatcatatcgagcgatttgaggalctlgttliga~mueaa~_~_c
taalggag«tcagagtttg
aaagctctagggaagcutcaa~ataQcc«c«gaacaatttctac~_at~_at~mtaa~«t~'a"oa~lt~=agaaacaa
y
tgtctacc«cacacaa~_gtccagtlgaagctttcaaggctgc«~==gttcg«lcaa~=~_a~=taccaac~_agattg
tcca
catcateatttcagt~_ag~tacaacttctag~taccttcttca~a~=~a~lt~=att~~_a~ataccagatggctcta
~atgc
agcga~caatggtaacttcaatacacettacccagct~_algctaca;=ccttsataga~aacettgcttgcagcaaca
gta
ccaagaatgctgattttgagaggaagaaeattgctggagctg«tca~gaactct~atagca~aggtgaalgctaagmg
gattcagttcataacttgctcacagggaagaagcatgtccattttgca~cl~aagaa~a~
actattgagcctgagcctga
otcagtggaaggagtctt«acatagatggtcagggatacagga:tat«g~~ca~'ccmaaggcaacltcagtggcaaca
gatttgcage~aaccag~ettcctcalactacactccaaagcct~'ctttcca~aa~am«tcllcaaagcagctttcag
aggacclatggaaattcaacttaccaagcccccccccccctcagca~'agacaa~'aat~'e;l;ttcaatgc«~'agt
agattc
ttgagagccaaacaaagc«cttgtggagtllaatYgCaa~lll~;llL'Cl~'lC
l;le:l~:l~alCllaal''g~aagBICgac
aalClgagClc:lCac:ll~'aagilil~Cla~atgta(:a~gla~ClCaaaClYC:aCa:llllafl:aa~_agacaa
gaagoftIICI
tCCl~olaalCC'teal'L'Cl:taCCaaagaaagagCtgtaal_c:c:«Ctt~_afC;l~~a_';1;1_x=a~_aiea
l~=I~Iggga=oaal
tggacactgaagacgagugga~~ctlgtagttgcagagat=glatC:gacca;leaccc:letatgtcgatcgacacca
tat
gggacgcttttctcagagacgacccaclacgacg gtgtcgatteatarcc ema~lalc
~_atc~acactggatctggac
I~CaCCIIoIYavYtvCa2tvta1C1:1[a~8:lllc:llC:llClIlaaClal ~(:llaaf
~'W;tYaWa~a~taattaacta~
:llcal~aafcca~:IC:lala~:Ilel~Clc:~cC:lla~~lalcl~tagllL';=ai11:111;1t1(~'al~=a~=
lcl~_glllalag~l
~l~l:lal88~ilalt
~'uCCIBIIIaCaaa~al~'aaCaall~aaCICltfaa~'aal~y_l;lltla~'~=lI~=alal'IIICIC~=
CIICICC~_Il:tlfall:tgc:lalai'alala~'~_~a~IIII~=C~~CaC~eC C':1C~;1;=tt;ltlc
tataatla,3aglll~a~Il
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
taagaatggttcgataacaacctagctttcaltagatctattaaccaatagtttcclga'_aatacctt,~ttccta~c
gtt
ttattaaatcgtttacaacccaatcaatclgci~ttctcttatttt~_ttttcttacta«teactcatacc«ycta~ct
~
taalCIlgaCat«aaCICallg«lgaalaaaaoaaOlCCglgaattcgatcctaaaatael~taatt'atctcttatt
tgagagagtagttctag~~taatttgaaccatatcactcctaatW
alclctaaclr«at:unla~'uaat~=cattggat
tet~acacatt«"accat~aaaacacaaacaaaactg«tactgattctaa~'eaattttlt~'lt,'~_ttla~~'ttc
tttt
~agaaaatgggtataagtgttttctaaacaclcctaatccaactctaactcttatcalt;l_'tc:aaat~~catt~~a
tt~
tgacacatt«gaccatagaaacagta~caaagctatuactgcW
ctaagcaaattttt"tt~'~'t«ta<ecctcttttg
~~lacaaaatgggtataattatt'=gctaaacactcttaatccatclctaactcttalaa«a~_tc
aaat~_t:attagatttl
sacctattttaaccataaaatcactaacaaaacagtttcctgcttctaagcaat~_tttt''ltg ~Ilnalcctct«
tgl
sagaaaatgggtataagcg«g~ctaaatcactcttaatccatctctaactcnataatta~
tcaaatgcattagattgt
eacacattttgaccatagaaacactaaaaaatattaactgcttctaagctaatlltlgttggtittaacctctttt~;g
ga
gaaaat~=ggtataagtg«atctaaacactcctaatccatctctaactcnataalta~=ICaaaa~ctitg_;Ittgtg
ac
acattttgaccatagaaacactaacaaagctatttagtgcttctaagcaatt««g«gg«tlagcctc«ttgggac
aaaataggtataagtattgtctaaacactcctaatccatctttaactcttacaattaueaaatyatlgga«gtgaca
cattttgaccataaaaacaclaacaaaactgtatactgcttctaagttatttt«gttg gtt«agcctc«ttgggaga
aaataggtataagtgatgtctaaacactcctaatccatcictaactcttataatta~tcaaa~'gct«~~attgtgaca
c
attttgagcataaaaacactaacaaagctatttactacttctaagttattttttctt~gttttaecctttttt~=~_ga
gaa
attgggtataagtgttgtccaaacattcctaatctatctttatctcttataattagtcaaatocatt~~attgtgacac
a
ttttgaccatagaaacactaacaaaact~gttagtgcttttaagcaattttttottg~ttttactct~ttttaggagaa
a
atg~gtataagtgtttctaaacactcctaatccatctctaactcttataattagttaaa~oct«g~attgl~acacatt
ttgaccatagaaacactaacaaagctatttactgcttctaagttattttttgtt~~lltt;,=c~tctlll~~a~;lga
aaat
~ggtataagt~_tt~~tccaaacactcctaatcaatctctaaclcttataatlaatcaaat''eallggattgtgacac
atll
tgaccataaaaacactaacaaagctgtctactgtttcaaaacaattt«gtiggttttagcctct«tca<_a~aaaaaa~
glataagtgttglctaaacacmctaatccatctctaactcuataattagtcaaat~_ca«~~~attu~_acacatt«g
accataaaaacactaacaaaactgtttacl~_cttctaa~caattitttttt~at«ta~=cctc««~e~la~_aaaatg
~g
tataagtgtt~tctaa~~cactcctaatccatctctaactcttataattagtcgaalLC;alI~~vttCty;ICaCatt
ttoa
ccataaaacactaaaaaaactgtttactgctcctaagcaatttttgtggtttagclt«ttga=maccaaacagctggc
ctacagccaccaagaaggattactaccacgcccccggaattgacccctcclgycaaacc
c:eaaataaccaattctaggtt
ttttctcaacccgggaagatattcttgaaaaatttcccctctgcggcacagaaa~~aaaaalcactlctccccccctca
tt
_gtgcccgcac~ccatcgcattgttgcagccttcgtgtg~gtgcaaacaattc««taccy~ttclccctaggagga~'
accacaaacgggctatatggtgctttggttattcgacggtcatctttag~cttccatc«tot~=c~'ccacaaaalcga
ca
at~atcttctt«tc~tcttctttcca~aaaa~catacaattgttcgagcaaacatcaatc"tatgalalgaaagtccta
aattgcccatcaacttttcggtttggtaatgtgaactggtagcctggttlccttct~=gtaaaaaatctgtaaacattt
cg
caaacagaatccaccaact«tcactcatattataatccgtcttgttctgcatea~_tce'agal'_claaegataattg
cea
ctggccttcacgacaaccatcgtacaatggatlatttgcaccttctaacat~tc~ta~=aa«tl«I~_aat~=~«acgg
a
ccggttcttccatactctgataactactatcct~atgataattatcctgat~ataattatcamaaccccac~_ttaaaa
t
aaaatgcgtcgllcaecatatccacatatg_atttlctacaacattacccac«cttcageatta,'aactaaaatacgt
c
atalccgtgtaactagttcctatatccatattcaattc«ctccat~=cttataccaaacataat;latc;a~gcalaaa
tcc
tlgactaaacaaatgactactaaclcttctacccgagatgatgtgtttctcattc«gcalac
amacaag'_acaatgaa
atttacctc~actactct~lactata~~ct~actattascgaat~'tcatgaactcmeeaetcc
~'y_acatattcg~ct
sacaaatttcccgttacttcatcaaatctcttgtacatccaatatcgatagaaacy~ccac
cacy'ccataaltgaaott
lcccsccattataocataaactaaacttatattttttctttcaaaatcggttttttttccutt~'atl~_t~~'««~at
t
tegtggtgaatgagaatgaagact~cc~lg~attatatagaggatttcttat~=actattalac~amaaacatt~taga
c
oClCagaaallaggt gIaCC IaCIC;IgCaaCgCgaaaaaC gtl«CCaaCCatattataC _'aC aattat
~C~actatgtl
acgacaaaggcaaaatagtcgtattatcgtcgccgaatgtacgactattntctgtagtcg<eatat~ta;=tcgtcaaa
cta
cgactactuacgactacatattgttctcagtaatgttgtcgtccgata~tc~=taatt~~ac~>actat~caccgacta
aa
lallglagtCgc;l3;t;lalaC~:ICLalglaaCg1ClaalIgtaClgll~tC~laaa<_IC~~lC2lCCa~_CaaIC
~la:lgll;l
Y.
ICgCaaaaa;l~gaCgaCtilC;lltgllaglCgCCItICgla'=lC'~tC~IItICIi'tItIClf,_la'=li'CiC
aaa~C;l''C
algglcl~lalagaCagIgaCCllggalCCaaccaaata~c«ct'~aacttctcaaat~C
_'a;UaCa;ll.':1~C18oaa'_ct
ccitllca~tt~ttgcatatcncctt~~aettttatccaac~=tc;_~ct~=~cotaatatlt~acyg;lace
ml~acta
tittctggcclagaacagrtcclacggcataatct~ac~tatcacacataatctt~!aac~~ata~lctc;tatla~'~
a~ct
cgaacaacag~:t~cagataccaaagctttcttgacggtgtgaaa~yattt«a~=gcat~_tecae:ltc~';rutl~a
acta;~g
tctccttgacaatagtctagttaac~~cct~'gctatctt~_~=a~aa~tccttaataaatcttct~ta,'aatc:ca~
'catgac
C;Iag~aa;lCIlIliTal~IClllgacaa«t«~'~=tgi'Cl~~aalac;llCafCaCttC ~altlllC
l'CIIYICa:ICCICII
IaCCCIICICt~'alatCll~llaCCCaaCattal'L'ccatccuCaCaI~aa;ll~'aCal:lllll;l:C;l:lll:
l:li'Fl:lCaa~a1
lad'(CICIIC~CaCCI~_~;ll:aalaC('I;IC'ecaa~aflcaactaacal~a~~a1';Ia:l~a1' _'1";l
C:ll;l~':lCCa:laaaalC'
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
tc;catgaagac«cc:ICCaICICCtc~'attaaatctaaaaatataea~gtcatacacctrtgaaal~«
yca~'~';=acatt
'_cataa;lccaaata:laa«ctctaataa~_caauaetmcataa~~acatotgaaa~'t~~IUtc
tc«";Itcatta~=~~_t
~,aa«~'~'taI«
~~aaa~'aaaccaclalatcc;alcaaaaaagcaata~tat~'a~t~a«a~'etaa~'c~_«cta~cattt~a
tcaatgaat~~~taaa~'aaaaatgatctttccta~atgca~calttagctitctataatcaatalacattctat~
cccaat
tata~=«cta''ta;~a«amaattcatct«ttcatttttaacaacaytcattccgccclttttae~'~~;m«ctat~m:
lct~_
ga~aa~cctaa~_tactatta~a~ata~_~~gtaaatgacaccagcatcaa~ca;_tttcaa«ta«mlc a
etacttat«
caa~tta~~~~=tttaacc;a~~'t~ata~~ylaaat~acacaagcatcaagt~~ttcaatama"aala_'~a«c~tt«
caa
g~=tggatcctateau~cataaacta~_t~aaattcccttaatgtcaaataat~'aataac:caatl~'ctctcc:lat
acttt
etaaottca~ataatagcagzttcacttcatcatcatttaactcaacattaat~atcac~~_~ataaela~a,~ltt~g
acc
aaggaatot~ttpoagct~gatttccctcaag~~aatccacctgcgaaaatacca~~cca«cccactaa~cccaccaa~
ta
gag~acclgc~caacl~_atgtgcccacgtacgcaaatgg~lacttgagcct~ataaycr~~tcacct~=atoaag~~'
a~a
taagcga~taacaacctaggaactaacgggga~atattc~cggagcggacctoatatctc~gaactgacct~ccaaata
t
cca~gagaatctcc~cagatcctggacg~agctaccacgtgt~tagacctatataa~~aa~caag_'ac~'ac«g~aag
ac
acattcaatgca~ctttacattcacacttacacttttacacttgtaatctcattccacattgtaaaa~ W
ttactatcat
caatacaaaa~tctcttcettctta~caatataactctcaaagttcatetgaa~atctagccctcc«tctcataatclc
agttacaaatctctact~tg~atttcagaacccacatttggcactgtctgtggegacggaaatagalca«acaacacca
aaactaacacatcaattgt~accaacgatcat~gatcccaatttatcc~ attcaacaa~atcl ~gc
~tacca~ttcaagt
cttaaacgataac~tctcg~acg~~cacctcccaac~~atctctctttc~gaatcca~~tt~at~ctamcat~acatig
atgcegaeaacatctcccaaaactc~aattcttct=aatctgaccaaatcctccccccctt~caaacaa~~=pacg~ge
aa
caaaacgtcacgatacagcctcctaacggagataaccgggctaacccetctaatca~ctccctcc~~a~acct~ccaac
;a
act~~c~~'a"ctcy=cgcaateatcctgaacctaatcaacataagcaaaaaccaagaaa«~'c~catc~caacctcgc
ao
aaat~~taag~aaattcctcccc~c~aaact~ggattcctgccca~atccccatagatct~aca~'atacc~tctcccc
oa
ca~atcccactccaa~;=actc~tcta~acttcglctc~atapa~~tctcacctcctctcaacc~~eag~'tc~'ccu«
Ia
caggcacccgtgcacactctc~=tc~aaaacccaacaacagg~'aat~a~acac~_taeate~=cctaccncccaa~z~
ctacc
gctcccacaga~aacctcotac~tgac~ccctcgaat~~ttt~ctaetctc~aa~=~saactctatt~'atacaetact
ce~
tactcatcgtaaacaggacgtctgaagcc~acctct~gggtctcacccaaattcaeaac~aatccct~_c~'atcltac
atc
caaaaattcaaa_caatcaaatcaaagatt~cgaacctaaacga~gaggtagccataacc~'cccltc~_c;lac~~'C
II~I~
~tIIICCICIa~~'f IC! ~I ~aL~'aaClC:lc:Cg«ag~cacccgg(liClllagpal~'CCl l
tcacilaag_eICIlCaclll
eccaaapcc~aag~~~_agcacaccgttttggcccaaagattcaaa~aatctaaa~CaCa88aCOCtICCCII~caact
aa
aatacccttcaaaaa'=~aaaaccaaacacaag~~cagcataccctattc~caata~aa~a~~ca~'tc~a~gac~t~a
gtc
cagaactt~acctc~aaaaatatt~caa~taccacaaaaagagag=atatttcacctaa~aatatc~_recc~=«acaa
aa
ttaalceca~~cegg~=lcaaaactaaaaaa~gttcaaaccccaaagctaaaacccctcctccc~at~a~ca~gaggag
sa
~caaactcccaaamaaaeaaacgggaccgaacactgeaapoaggtgactctc:ctceacct~'ctag~'aaa~aac~ca
ta~
accta~ccttt~=cap'aarlc_'aacl~gaaggca~gacaaaaa~gtca~tcactacacctccacccgcc;m
gvaaaa~aaa
aacac~a~'~'«mca«acs'cctccc~aaattct~=cagaaacgcaacc~a~'cacclctcmc~aaac~'calc~~:at
lcat
catgggag~atctca~ctcl~'cata~actcaatcaattcgatcaaaactcatca~a~oaa~=~'ccaa«calacacaa
aa~_
ecailaa_ccc~at~at!_~=cacct~atcacca~atcacatttt~~~aaagtgaaaccacl~acclc~ataaacctca
c~at
~aI~CICIC~IIBICC~;I;IIC~aC~I;IYCCaaCI;lC~aaCICICCC~Ialaitl~BlC~aCaCo°.de
gree._aa~ClCa~'lt~aC~l
aCICIICIaI~:ICIC:ICCICC:I:I'.:lC~ICa;IC~CCICCICCC,'CCiICCa~~~a;lYlC:l;l;~ttaCC~
~;ICCI~'=al~~TaaCC;l
aC IClaalaaaaaala''C:IC C IC:C:IC ~tCailC Y~C:ICCtccaatccacaa~~';IICC ~,'allC
C' ~it:tt;IC:I;ICICBCI.'lC C'
CCCdCC''' ~ L;la~'IC:aIC~'aaCaal(:''IIC.(: ~al:Il'WatlCaaC~CalCaaBC:
aala'<I'?ll:l=;l''L:IC: ~a;l'L'CCI:IC;I;I
fICI~~cattaa~clagclcaa~aaal~=gvoalcC~_cgaaatacacgcccatagcgaemca;ICtC;~lC:ilCCa~
CCa~'l
IC~aalC~lll~aaClaaCCil~~aICCCCa~a~~agaaaacacctc~~Ca~aC~CCfI~_~Ca~CICII~;CIICIaa
aaCC
ICIIItao~a~'ailaal~~~fal;la~l~Il~tctaaatactcctaattcatctctaaclCttataa«a~W
a;la~'=CIII
~~'attot~aCaCallIlaaCCala~aaaeaataacaaagctatttact~IIICIaa~CaaIIfIII~IIC~=Illla_
'CCI
CIItIc°c°'.-' - -a.°-aaaaL°-
°°tataa~l~Il~tclaaacactcctaatccatctttaactcttacaatta_=ICaaal~_Call
~=
~iltlu9~aCaIalIlI~atcata~aaacactaacatatatattQaCl~CltCIal~'Caattlttl~ltl'=~tltla
~CCCC
l[llovvilvaaaall'_otataa~l,_It~=tctaataactcctaatccatctttaactcttataatta~tcaaat~C
afli'
all of ~aCaC;lll Il~al'C alaaaaC llaCaaaac:llaC aICICaI~aaIClCitIl;i~'aClC:l~
~:IaI;IIC;Io ~=atCC ~ ~T
aaa~aC~~ll~alalC~'_°C'CIIC~~:I~CIaaCaC~laC~llClll~~;lCCalC;laaata;lCL'~t
C'aatcttC'CaccaW
caa~aacayac aalaaclmaC catcatatc~~ac~ataattatgatcaaagtcaa~a~c«cc«clta~tagcta«a
cattccaaaacac~at«ctaaaca~ataatcaaaaata~tatta~aaca~utaattta~~taattcaaatlaaactct
ca ila~ail'~:laactt:lcW
111~1c:1cll8llll~aaaQlla~Cl.'lCaaalCa;lC~al;~l~~lltl_'Cll:lc'~lallal~
Ill;ll(: a9llC'lCC aaCaIC':Ila ~;IIC YCIC CC
aCC'aa~lC;lC(:aaaaall;lC;l;i;laalla;lllaaaala;lalC:l~a~IC
aaalatataCa:Il l ~;tl:lll ~;Ial;l:ll ~;lC;laa;lC l:lal ~aaCaClaa~'al ~C al
Y:ll;Ila:l:l:llil;Il~:l:lC':lC;laCaaill
lCalaBaaalClC:ICC~~;ICl:l:l;t:IC:;l~;llllillC~:IIIIC;IICaIC~L'illilalC~'aa;l;l'
?;1:1.''':Il~',~l;l'';I:l,!ala~=l<_a
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
esaatagtg~=gtacca~amg«aalct~=ac~aca~~tgtcaacagaaaa«caa«ttgaa__~_tat~a««
~;ttggcct
~acaggaccc~=agttctcalc:l:ICCaaaccttgttatc:~ltgcccattctcttttattaataaaW
ct«tta~;t,~at
ctgacatcitgtatccaacagta,'c«
caatagtcalaccctacggatcaaacatactaatcaaaactctaaaacatgca
aaCCtaaaattalc;a«acaaaacaytcaatactlacat~=ttcgtcgac~'agaagCaaaCCCal:laCClII!_aag
algga
aCCa''Cv~aLiClaa~aal!'ICCtCaCaaCiClC:ICaC°aataCaCCiIICCIi'la:ll:l~_aC~'~
'lllC:laC:l''l'=la:lga~;~_
al~aaaCgallllalCIIIIC~CI~:la:llttClla~'tlalillllgfatlCgfllIali'a~ali'c:laatgata
ccgalal
atatatataaga~=a"agaaatac:llCglaY:I~'~Cal~'llagal~Ca:lcatttaaaaaa~'ta:uaata~_tl:
:llg:ll~_ll(C
aagtcatttcgtaatcll~_aala«clglaalatallltoaataaclcaattattaataaaattaaaaatttggcgagt
c
llacallllaga~Taaalaatalaaafatlftlleg«a«altaaltagagltgtlalalaaafacCaaliglc:ltaaC
l
=Cgtg gataaaltagtaacaalcacat
galcctataatgtttgagacatlt:1.ll:l.ICaaaaIIICCalaa:lalllaalaa
ttatggagagaaataagtaataaalattitlcataltttttataatggaagtga«acaaa«aggaaacaatatatatg
caaatatatigctaacaaaa~aatctataatltaa«ctattatggcacaacatacagaatggata«ctaaltlagttt
tctactctatta«ggggaaattggaacacaaattaagggatgcttgattaagagataagagttgaaataatttattttt
icaacttagaag~=ttcctctatgaag~t~attttacgacatatttctttggacaaatcitttaataaactrctagggg
tt
gggttttggtgttcttttgtttaataaggtta~atatctaactctttatitgcgtgtccgatgggatacaatatttgcc
g
ccccatacctctttg«taacccc~c~c~attucaccctaattgttagactcacaaaaggcagcagttgactcagaggt
ltacaagggcttgatggggtclgacaeaaatgttatgaaagctaatgacgtaagtlcaacacatgctcgaccaactacl
t
ccaccaacaactcgaagaaecctccaactt''tcccgaaagcicatgctcgaccaaacaactcaaggcaagcactcaga
tg
tcagatgattggttagaactcaaggagagatctaaatggcaagacaaagccatacaagagttaacacatattgtgaaag
a
gctaaaggatcaaatcaaggagctcaatggaaaagccaaccaagtaccactcaacatcaaggacgcccctgacgatgga
g
acctgtcaagaaatcclaatga~lalgat~alcactctactaaagaaggagaggagacctcatttcctctetacaaccc
a
ccgtaagcctaatga~tat~a~'aagtcaa~cttaaagacatacaacaagctcactt~_~ga~caaatcccat~_tcta
tctt
tgtacatatci«atttlcc«~'tt~=««tsalgcat«~~ttagt~=ttttcag
gagataaglalaaaga_ct~_agtga
agtg_attct~~gctctga~a=tacaacaclalactcoaccacaaagcaatgaaggatactc«accat~ttctagctta
tc
CaCgCCaggagalCa(:l _ -
CgaCt~C~~IgC'lgaCCgcagcagaagaatggagalCga~laIaClCaIggCgoLgCtHHCCa
ccatagaggcactgag
'~IyegIaCCCacatc'Caggagctgagacagaaca~~~gCg~L'CICgtCl:ll~_gCClgggagC:lal
cacat~'cagCCallgacgaccaac«
r~'IICgIICIlcgaglgaggtaagcgtctcacttcaccattL'tattatatcatc
tcttgtgatltg«c«cat«t~«tclgtga«ggatttglcctgagtactctcttccaa~lttattcacacagtgga
ctgigigalltaaglllgo~e~a~~=~clcaggaagtgtgtgttgcactgtgtatattttttagtctgcattcatttaa
g~
catagaaaaaccaaaacaatttoaaaaatttea~aaaatgatttcataaaaaa~a~=tgttaatgtagtt~cattacat
tt
aggatcgagtcgagagtgttgcatttaggattottgcataagcataggueat~atgatgagatagccttgtaatcattt
t
gg«caccggataaactcaglgccmcgtt~ctagttgtctgtt~cctagtcaatgaatttgaaataaaact~aaccat~
cctagattgctctactctacca~actgmatgatttgataccactccctalcaat«gaacrt~zaatca~tatc«
laatt
atcatgtct~_catcaaat«~aaclcal~~ataccctaaaatacttggattttcttactcatttt~atcactc«gttaa
tccaagtaoctcactctrcctatta~=agca~«aacccgaacctaaacctaoac«tctmaaaccrtatatcactt~~t
sagtgtttgteag~tcttamc~altaa~ctt~=yta~=aaagtgtta~gttcgtaarcaca~a~ata~t~tctcat~ta
~
ttclagltcscatl«tc~oacta<_ata~sacta~~to~~=lecttatacttigggttgggatgtgtttaaaaaaaaaa
aa
aagglggallC:lflgafaa~=aaaa~~la:laa~'aClClagYlaaagl:lBgCl~:la:l~Ca!'a:l:l:l:l~'l
Cl:l~I:laaggllll
gggalll glaa:l~:l:laa~l:l:l~:l~tl(:l l'' llagC Iaal gaagaa'=~ gC aaila_=CCCIC
~gllll:la:latllla:laaCa~=
aaaccttagttg«ac:Ig:l:l:mca:laccm~'agaaagcttctcct~gagitaa~a~aaaa~aaaagaal~attaga
aa
aagggc«aaatgattcateactgcaaagg_tagagttaagttcttaatttgggactagagataggatlacca«agagc
ttcattggttatactct~g~ta~alg~ aatcitatctctgtatgcatagcttg
g~acttaccttta~~cattctawaat~
cttaattatttttt~_agagattcc~l~ttact~aa~cctattctataagg~=accatctu~tctctt~~accittatc
tta
ggcaaatgag«ca«gat~atgratt~cttaa«cact«ccagaactaatgaatQ«aaagggattg Tlagatltgaa
aacatgt~taggtc~a~calac~actc~_~att~
att~=ataacaa~_ectgactaac~ttttl~agta~aattt~=atcata
tcgcagcltagaactacraac«~y_acattga«tcatctgatttatctagtgctttggctctaagtccccgctttcaaa
cctcacctcca~ctt~ttc«tatt~ttt~c«~=ag~~caa~caaagactaagttt~~~g~a~ttgataagt~t~cat«
tacacatttlsa~'catcca«tgtcatc:am«a~=catcatatcatcactgttttataccacttcmatcatttotcam
allll;calgtttaggata~l111~_CaI~Cal~lt~C~lalllgfoLLQItIICFI~'~'lgalll~=~a~Cl~llgg
C~':IgCI
aattg~aa~aaact~~cca~=atcatatcaalacattl~accacacaglC~=a~ta~at~~cttcac~ucatcaacaaa
cca
cttgaccccctg~=tc~a~ta,'aa~=~~cltcatcacttcacaccaccactt~accccaa~~tc~~'=talcatcalc
tccacc:
acct~atcataactc~atcacaccactc~=accccca~atc~aetacctccatcaccttcactccatcawcactc~att
a
cactiaccgaotcccaccatcatcaracactc~=aca~atcactctaccaccaagcc~agtactarcatctcc:atcac
tcg:l
ctlcatactroact=yca~=cttca~=a~_tctmclattcc~=caclcaarca~amctc~age~caag;._aa~aaaa«
aa~_a
CICC:I~_CIIaIC:ICIC~aCCallCaCtC'~'~c:aaWY°~=lC~a~lalC~TIlCllaalCC'~lC::l
Ca:ll:lll,'C'=lC!~llll
2aYl:lllawotllCi'olalalllL'Oi'lal:la°Ia~=c'al~lalIlCaCaCIIICi':l:l~:la:l
a~llllaalW C~=CI~:IC:I
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
ctgtgttctt~=acattttt_taatccaga«tcuuaatrtamagtattca~tattc~c«ttcattitc~_tttact
aa~_ttgttcatcctattatcatcugcttttattct~=«gclatcat=tt«rattctatc~=tttat~_ctttatgcaa
tg
aI~'tctaag«Igtgactag~'tltclga?gal~=g°ll:ly~_lagttta~'aattctea~=tat~_clil
~'~=l~'~'ll~'a~llll:ll
t««ag:IlClCllClaCatta'llllgllcllaal~ccl:111~'c:Illll;_alcaaCl~'~=aafllgil~!CCt'
:1'_:IC:IIIIW "c
aCCCaaaail'_tgttCgillYaaal'_ICl'=aaCCaCl:lallCal:'=:1'=aIIC'_lnL'CICt'_IaOC;ICI
B:IIC;IIaCaC:ll'':IC
alCailLlCltallllaClCCaaaa~aClaaaCaa~CIllIlCtI~CIICICaaa'=IItI~aI~~'l~La~CC:l:la
~lC(:~l
aI~=a~!lclllg~'clllgtalcllcalacaa''gaaacac;laCll:lg~'CIIIga:l~'alca~~a:llgtl~=l
lcl:Igllclaitla
ClCaalCafaCaCalgaCaICIa;lCalalII~aCICC;IIaaCallaaaCaaga(ICalIllaaIIC:.ICaalaC(l
lgal
ggt~=tagccgaattccatatgattct«ggettt~talctacaaacaaagaaacactactta~'gcttltlagatccgg
ta
sc~_'tt_='cta~ttcttalactcaatcatacacatecaatcta~tcatattt~a«ccaaaacactaaccaa'~cttc
ttctt
octtctcgaa~ctttgatggtgtagct~aacttc~tatgagictlloaatttgtatcttctaacaaagaaacactaatt
a
egcttttaagatcatgttgcggttctagttctlalaclcaatcatacacacgatatrtagtcalaltlaactccaaaac
a
claaagaggcttcttcttggttctcaaaocttt~ctc~totl~_ccatagttcttatltgtcttttgctttgtatcttc
ta
acatggaaacactacagaggcttttaaoatccg~tttcagttct~~ttcttatacttaatcatacal:atgacatctag
tc
atattt~actccaaaacactatcaaccttc«ctt~cttctccca!'ctttsate~tQta~'ccstaatct~tat~aatc
tt
tegctttgtatcttctaacaaggaaacacta~gcttttaaatcag~ttacagttctaa~tcttatactcaatcatacac
a
tgacatctagtcatat«gactcgaaaacactaaccaagg«clccctgcttctcaaagcttlgatggtgtagccaaagt
ccgtataagtctttlactttgcatcttctaacaaggaaacactacttaggctttlacgattcagttgcggttctagttc
t
tatactcaatcatacacatgacatttcgtcatata«tgactccaaaatactaaccatgc«cttcttggttclcaaaac
ttt~atggtgtaeccgaagtcgtatga~tctll~~c«tctalctictaagaaggaaacacttcttaggcttttaagatc
cggttgcggttctaagtcttatacttaatcatacacal~acatcaa~tcatcttt~actccaaaacaaaaataaa~ctt
c
ltcttgcttctaaaagctttgatggtgtagtaaaas«t~tac_'a~tcttt~tcttcatatcltctaacaa~gaaacac
t
acttat~cttttaa_'~'tccaett~cc~otctasttcttatacttaatcatac~catmaatcta_'tcatattt~act
cca
aaagactaatcaagcttcttctt~cttcta~aagtttt~atggtgtasct~aaotccgtatgagtctltaaa«totatc
ttttaacaaggaaacactacttaggcttttaagatccatttgcagttctagttcttatactcaatcatagacatgatat
a
tagtcatatttgattccaaaacartaaaaa~~=c«attc«gg«ctcgaaocntgctcgt~_tt~
ccatagttctlatt
tgtctttcgctttgtatcttctaacatggaaacactacalaggct«taagatcc~
atttcagttccaottcttatactc
aatcatacacatsccatctaetcatatttsaatccaaaatactatcaa~tttcttctt~cttctccaa~cttt'_ate~
t~
tagccgaa«gcgtatgaatctttggctttctatcttctaacaa~gaaacacta~gctaatatg~tcta~tt~cggttct
aettcttalactcaatcatacacataacatctastcatottloactcoaaaacactaaccaa~cttctttctacttctc
a
aa~CIII~CIE_~t~L:IECCOaa~ICC~IaI'!a~IC;lII~~CIII_l?CatCllCla:lC:l:l~~aaaCal:laC
llilY~CIllla
c~'attca_ttac~~_ttctaettcttatactcaatcatacacateacauta~tcatatttoactccaaaacacaaacc
aa
scttcttttt~cttctaaaaccttt'_ata~tclaclcsaa~lccatacea~tcatt~~'ct«ctatcttctaaaaa~a
aa
acattacatgtgcttttaagatccg~tt~c~altcta~ttcltatalacaatcatacacat~'acatcta~=tcatatt
t~a
cttcaacaatctaacaaa~ettcttcttecuctcata~'cttt~at:.'~t'ztacceaa<_tec~tal~a_'tcttt~
sctttc
tatcttctaacaa~oaaacaclacttaeectt«aasattc~~ttacoottctaa~tcttatacttaatcatucacat~a
catcaagtcatttllgactccaaaccacaaaccaa~cttcllctt~cttctaaaatctttaatgo~tactcaaaVtccg
t
acaa_tcttt'~~ctttotatcttctaal:aa9~aaacactac;eaa~_'c««aam'I
cat'tt~_c;aatlaa2lc:atatti2
actccaaaacactaacata~cttcttctt~_e«clc~a;lecttl~attet~ta~l:l~'aa'_lca>=tat~a'_tcl
tt~aatt
t~tatcttctaacaa~taaaaactacttae~_c««aa~atcl
a'_«~t~~tlcla~Itcttatactaaatlalaaaeat
~atatcta~tcatattt~acctcaaaacactaaaaae~cttettcu_~«ctc~aa'_c«t~rtc~l'_tl~'ccata~_
tt
chat!'c~tccttcaattt~tatcttctaacat~caaacactacata~'_c«ttaa'_atcc~~tttct~ttcta~ttc
tt
atactcaatcatacacatgtatcta~tcatattt!_actccaaaacaatatcaa~tttcttctt~cltctccaa~cttl
,=a
l~oc~Iil~cceaaatccetat~aatatu~~cttt~tatcttctaacaa~saaacama~~c«ttaa~atcat~tta'~a
ettctaa~tcttaactcaatcatacacatoacama~lrattt'_l~actaoaaaaaactaaccaa~c«
ettc:ctactt
ctcaaagctttggtggtgtagacgaastccgtat~agtatttggctltgcatcttctaacaa~~aaacal
acttatgct
llI:lCQallC~olloC~~IICIaHIICIIaIaCllaalCalaCaCalpaCallla~lLalalll~a;l(c
C:l:la_~I;tCla:l
CCaa~cllcllCll~~IfIIcaaa~cllleal_e__'tela~Ic:aaB~tccatat~a~tClll~iCIIICI:IICII(
:laaCaa
'~_oaaaCallaCll VoYClllla:l!'aICC l9ll_~C
alll'l:l_,'Ilc:IIaI:ICIYata~a_':t_2_~:1_;,';t:l'?C:l:l_~~la:lCaW
la$OOOttt~a~atctcca~~aaaCaill:BaaCIC_!':l~CllY~aICII:ICllaICICL'al_~;Ila:Il:Yla
tC~:l~'a:lil~Cili1
caaaewa[~il~_~:llCCl(:lC(:ICai'vYClaa_~a;I:ICICIIIaalYlil:t;tCl_'?:Iallllall:ll
C'1a21a~(:_~l~alleCa
aCoBlgalgaal~aa~~!'ClaCllala~a~a;llaaa~:1'L':Ita:IC:IaC~:I:II:ICI(''laaaYCl:1;1
1Ci1lall:~aaYaIlCat
IallalClaaa:l:lC:lCaa_~aaaaa~UFlt~aaaal~fa:ll:l_>~_':I:ICallCla:l:IC
~:IIIC'll:;ll:ll;tC~l_~:ItILI:CC:IIYali1
C~la~aC_~:I~il:l_~CCCill~aaClCllaa_YaIYC~_Cl_i'Call':lY~l(.'C(:Cll'_Y~_'l~_'1':
l~;l=_~:IIIC~':IC:ICI_~aalC:l:l
CCaCI:ICaII~ICiICCiIC~
ila:IC~_~:l_Ya_YaY!'I:ICIICaCL'll'_;I;IC;:lCall',_';IC_YICC'~I;I:IaIY_1'Yva_~_v
aaa~lli'
aCICI~'IaCCCaIICICatl~:ll~C~'lll:(;:la~':1CIICaa_'l'_',:1W:I;lICIIiC:_".Y;Il~ll
_'a_'ll:l:llli'1;7CIC:11_' '
CA 02362897 2001-09-18
WO 00/5325 PCT/US00/07392
:l~=~=t~=~_~aaac~_Itcac~g~aga~cactacccaeaccaaatctcca~~'attaaaaa~caattccc~ac~ctt
atealca~_
cl~_ca«ctt~~tactt~~~=tcetc~~c~_~cltccaaattartel'=I~catccttgt_aact«rt~caactctmaa
caaa~
tccaca~=cattaccat~catoc~a~=ttttatca~~~a~,a~,ll~tcaaatccaaa=~t~cac_l~=~'~acaaa:c
y'tacac
cacacaaaac~=~'a~_agcatcraacactty=~taaaaa~~cat~all~=f~l~caa:lclc~~clleacata~'c«a
ac~tcaC
:lyclcttaat~=Italcmcaactaaacatc~_aa~=aagalctccaa~a~'ctcgatt~'ctaacttc~
~«tg;u:~alcy=a~
t,=a~_~'at~ataagca~'tactcatatccaaectagl~eaaa~taacttccacaaa«atc~ccaaaaal~~amc
ay'aatc~'
a~tatclc~=gtcacacacaata''aa~'ta~~caaaccat~taaac~ataaatttctc~=aaa~aa~=a~=laa~_~'
macylaa
ca~calct~_tc~ttttcltacaa~gaataaaatgagccat«t~~~'agaa~c~alccaccaccac:aaa4'ala~_a,
_lcgm
cc~c~ltga~tac~a~~~a~lcccaaaac~aagtccalactaacatco~tccatg~ctQc~l~°~aala~'~
=~avav~:la~'
ata~ao~CCC~c'.all~!~a~~?=fIIIfCCIII~~'C:ta~fl~=oca~atcc~,~~aac~ttccacaaaacoIIC~
aCC~t(:at~'~_
C'=Ca~loaa~~CCaaaaalaa~al QII QI QaCYa~L'Cl~CaaI~ICCI ~ICaC°CCCIaC~I
~~CCIICatI:ll ~'1 aaltC
acaaataatct~aa~tc~~a~~ct~caatct~~aacacaaa~cc~caaacctttgaa~a~~aatcc~ttttataol~ta
t
attcatc~tttaactcattctc~ac~tcpcttaatatcc~a~a~aagaa~~tgtct~taeagtataastccga~aagaa
c
caagattg~c~ctgtc~ttaacttgt~ctt~ataa~~tgtaaac~cttcatccgcttctttc~tccat~Igaccttgcc
al
ccttaatgcaactc~t~att~gtgctacaatocagctgaagtgat~aacaaaccg~caata~aaaga~~ctaattcgle
~~
a:l=CI~~C~IaC:CIC=YllaCIQIII~I~!'R:ICCo~CCaa~aCCEaaCI~Clll:aal8lll~IllCalC~:tCI
laf:lalCC(:
ttatccgatatgacata~cccaaaaaaa~aacctgtgagacaccaaactc~lafllaleEC~azCl~caaacaacttcl
c
citccgtaaattgctagaacagcttgealgtggttclcgtgttcctcatgtgacga~cteaagat~a~aalatramaaa
atacacaaccacgaatttcccgat_aaagtactt~attcatgacgc~cateaacgtacttggg~cgtlo~l~a~eccaa
a
'_~rgcat~acaaeccattcaaaaaetccttctcgtgtttigaazgcggttttccactcatctccc~s~tcoaatcc~;
aattl
~alg~«ccc~ctttttaoatcaatcttt~aaaa~aga~at~eclt~ccsatu~~lccaatae~tcalcaagtc~l~gl
at~gegaagcgafa~cecaccgtaatt«~tlgafa~ctcgactatctacacacatac~ccac~ttcc~tc«tct«gg
:lath:l~aaL~~L'C:1~'vlaCaaCYC:lai'°aCl~a~~clttctttaatat~aCCltll~'Cla~aa~
tlCllc:laCLlYlcuvC
YCaaCal ~ICaIQIICII~C ~~aCICaI~'CaaIa~L'l~l~~ ~'lC~~lII~~LTIiI~C ~ll''CiICC
~='~ YCaCa:IC'alC I;II YI Y:1
l~'llgaatatcll~a'~l~uloo[B~CIC(:lY~?IaBCICII~L'C!'YaaaCaC~ICl~Ca88tICllY:lalIaC
~ ItatCY:la
IatYYtYIYaYctaC_'_eteQatataYlceClelaafl'_ate~taacactcctaatLCaaatccttcac«Icca~_C
lcfl
tctcaaacaCaaaaat~.iaacacaacat~'_'=a~tgl~=IaCaIg~CCI~IC~Taa'=«arat"~~~'Cal~a~a~l
t~~~CII~L'C
YIaCC o~IooanolvaC ovaIIC~'_a~
aaa~'c~~I'=l'=ICI'l~agat~'~aa~caocacaatcllat~a=l IICC(:a~:lla
aattcatatgtattcgca~ccccatcat~caIaBICIIIC~atcatactcccaa~gtc~lcccaa~at~av~'l
~avl~:lC
allcala~~l~la:lcatcgaaataa~t~cgg[CCll~l:l~aall~CCCIaI~ ~t'a~!'8ila~.'aaa(:la~
oaCIC~'aCEt oaaa
czc~=aac~aaacgccatcctgcaaceaaglcaugcatat;~t~ttggatgllcticaaat~~laayc~ca~cttlc~e
aca~cttcctc~_~caattacattac~.:~xaacttccc~_aafcaafaacaaaagt_ca~acte~_~=cct«~att~'
tgcaagl
a~aacgaaaaat~tta~tgcgaaeccaat~ttttfcac~al~ct~tg~a~t~a~=acyaaaaatc~t~ctacc:lacat
at
_~accttcatcacccttggtottatgc~tttcaataaac~cttcctct~Iftg~tttgoacacQct~ttt~olll,'ea
~:l~
YCIQlIIoIC( _
_~tgaccaagttccccacaa~catagcaacEtaa~~Cattc~u~CvuvtCv;tlColC~Ialll=~~~oCl~
l~'t'aICIIC:IICIIII~Ca~l~3C8C~a~Io~'l~llaa~~i'Cl:lClll~IlCl~lalClll:lC~:IIY;ICC
O:Ia:ICCa~CI~,
aalll~alfl:lelclCa~atgglcgleaaCgl~~:la:lgala~:l~utg«
lga~agcaaaacl°ll~(:lCa:laa~aaCal
'=l:~C'ICIQIYa''cclccgccacagt~=~'aa~gatcgaalaet~alaac~'C'~Illl~~a~ll~a~lIC~Ca_I
aCICCI;II
~aagl~l~a~accaa«gtacctcact~=ICalagat"tcattacfl~taacaa~3aa~_ataaaattcitclc
C;_Iaafc;~l
clacaYttcta~aCCCIt~ICICa~atlttYCaYYCYattatacalc:YtYcYatcYlaaft'ltYaYYaaYaaaaYlY
tc1
Cl~a~tttcncttcaat«tlcccaaga~C~aata~~t~CIllacICoIaC'~t~CCCtaaac~lCll~aatt~Ct~C'C
a
ccaa~aagctgcat~lCC:aCgeaatc'=li'laoCCaCCaaC~aIaCIC~ICI(:ICa~CaU~l;lCaCaCfl~:l:l
:IleC:lal~
tftcctctaca~ccacaa~ccaatcaa~ta~IQCaICICCCC~~ataca~CCll~:laaIIC'lvv_aalv(C~aClll
c?aaC
Cff~IIICCCaaC~lafptcttgaaacatcl~Ilcclgglgicla~'cc~gca~aaaatc~_l~L'IlY°fl
l~~:IIIY!_c:laa
~actaau~atta~geaQctggtcac~:1''YltYOfl~IvCII
lYC~2ICYalCl~aaC::l~C~lClI~IIYIC~I~~a''CC:1
C~~C:~aCI~CI~CI~ga=°C~'=lla~Cl~CaIC:I:1''al''lCl~allaaC''lal~:t<;laa~'CI
C:aC;~aafl~aa'.'.CCf~C
aaC~CaUlClgagao~C~3~a8~:lYfall.'~C~B:lallC:~lIll:Cilall~al~~ll~~~lCC!'YaaaCll~;
lC'=CCaaC~Il
llCYll('IIIYYaYYCalI!'tlYaIlCa:latllCaa!'CCaaYa:taaactaeaat(:IYataccaaet~ala~a'
,d~_'_c~~~:1
a_2Ca88_Yla:lc:aa2C:f_2~~T2lll2a~alCICC:l~2aaaCaaCaaaCIC~a_QCIIC~aICfIaCIIatCIC
''_al'_alaal_'
:llC~:l~aa2l~Caalaaa~~=Y2l~a~talCl:ICICCICa~'o~Ilaa~aa~CIClItaal~laaalt~_a~Iltl
aIfalCla
aaYCYl~allacaacgalgalgeal~:l:l'gclacttlla~~'eaataaa~a~
ataacaacgaatacictaaay'taatcat
:llC!':lat'alll:l~'lallalCCIaaIaBCCtaa~aaaaa'_~a~aaaaCCtaalt_~aaca«cfaaaC~aIIC'
lcalalaC.T
l_YaaYCCCfIt_Yalal
YIaYaCYataaYCC(::llY:IaCIClfaa_~'aIYCYCIYCaICalaI;lCICaIICaIaCaCa1 ~_aC:al
(:I:IYlC:llalllY:lClCl:a:ICa:llCl:laC:l:l:l~C'ilClllll~ClIC1(:ala'_CIIIY:I:I_Y~
l_Yla_~'CC~aa_'LCC~_lal
Y:I~ICII~~~'CIllClalCflelaaCaaYi'aaac
aClaclll~'«Clttcell~C~'~~IlClaaG'IC(Iafacllaalc:ll
:lCaCalYaCalCaa_'lCallIllYaCICCaaaCCaCaaaCCa:l~_CIICtICtll'.CIICI:Iaaa~Clllaal~
__'l_'l;l~_lC
CA 02362897 2001-09-18
WO 00/»325 PCT/US00/07392
aaa~accgtacgagtctttgacttt~tatcttotaaeaa~caaacactactaaggcttttaag~tccagu~~caatcla
~_
tcatattt~attataaaacactaaeeaa~cltcttctt~cttctcgaa~ctttgatggtgta~ctgaa~'tca"tat~'
a~~t
cttt~aa«~~tatcttctaacaa~tcaacactacua~'gcttttaaoatcca~«gcggltctagttcttattctaaat
cataaacatgatatctagtcalattttaclccaaaac:lctaaaaa~'~cttcttctt~~_ttctt~aagcttt~_cty
't~=tt
gccatagttc«attt~=tcgtte~attt;_tatc«ctaacatggaaacattacalaggtatltaagatcc~_~ttly~~
_«
ctagttcttatacyaattataeacatactatcta~icataut_actccaalacaclaacaag«tcttctl~=c«c'tc
caagctttgat~=~_c~_ta~=ccgaaatca~tatgaatat«g~'ctttgtatcttctaacaaggaaacactag"mt«a
a~a
tcatgtigceattctaagtcttatactcaatcatacacaagacatctt~_Icatatttgactagaaaactaaccaagct
tc
ttcct~cttctcaaaactttgatggtgta~ccgaagtccgtatgaggcttt~gctttgcatcttctaacaag~
aaacact
acttat~cttttacgattcagtt~cggltctagttcttatactcaatcatacacatgacatctagtcatattt~actc~
a
aaacactaaccaagcttcttcgt~cttctcaaagctitgatg gtgtagcagaagtccgtalgagtcattg
~ct«~catc
ttctaacaagaaaaaactattaggctlttacgattcggttgcgcttctagttcttatactcaatcatacacatgacatt
t
agtcatatttgactccaaaacactaaccaagcttcttcttggttctcaacgctttgatggtgtagttgaagtccatatg
a
otc«tggctttctatcttctaagaaggaaacattagtlcggcttttaagatccggttgcgattctagttcttatactca
atcatacacatgacatctagtcatattcgattccaacaatataacaaagcatcttcttgcttctcaaagctttgatggt
~
tagcaaaaatctgtatgagtcutgaatttgtalctgctaacaaggatacactacttaggcttataagatcc~g«tce
ttatag«c«atactcaattatacacatgccatcatgtcatat«gactccaaaacacaatcaagtttcttctt
gcttc:
tccaagc«tgatggtgtatccaaaattcgtatgaatc«tggctttgtatc«ctaacaagsaaacactaggc«ttaa
gatccggttgcggttctaagtcttatattcaatcatccacatgacattitgtcatalttgactcgaaaacatttaccaa
g
tttcttc:ctgcttctcaaagctttgatggtgtagcaggaclccgtatgagtctttggctttgcatcttctaacaa~ga
aa
cactactta~gcttttacaattcgacct~actttgatcctag~attagaggatccttaggttctgctlccatatcaagc
a
caacgaaatacgtaggcacttccactccattaatca«actggtagatcttttagcaggccgaaaggttttcttgaaaae
ctatcagcaaagatcaaagtaaggtcgcaaggcctgtactgagtgaattccagcttccttgccacaaaaagt
~'~~c;lttaa
ectlactgaa"ctcClaagICaC:ICaa:lCaa(:IgClgaaa;CCaaltgCC(:aitlggaaCalgglaga~_l~_a:
laY:llCaC~'
alc:llc:CagCtllt(:ll~ilalaatcctcttgaCa~tilgctctagaaogallagCalCaCallCg(C:7talgaa
tCCtItC
ca~_aatt~aatnctcacatctttgtgcgg~tcc~_ggattaggtt~a~_aacttctatcagaggcatcaccgc«ccat
ct
Iatcaaoctscttctctacaa~aeactt~tatctttctatccacect«cctsaatttccatsseasa~caest~:lae~
t
aacggtgctggc«gaatcg«laaccatcctctcaatgatggctgctttctcgtccaaggtggacttagtttgaaagcg
aoatccagccgaatggtcgagtccaacaactgaaatttcaatctgagtagaaacctccccgtcclgaacatcact~tcc
t
~agtgaugagttg'~agacatettgagtaggcagctctceatcatgYCagatatlgatagcgtgag'c~
gt_gcgtattct
ttc~'QottctYtat~'aaC«CCCI>;'L'aaatcCt«aacltt~aeaQaaea~otcQac~ca~ut~tccitcca~~ta
tet
~acc«agaggtaaotgcctcaag«tgatgttaagatcgttgaaagtgcaatccaccttgttgtgga«tctgcvatcl
ttttggcgagatccattactcctgctccttgtccctg~_agaatctgctgtagcatgttcttcaaatctgaatct~rga
g«
g«~aaatcg~'ttcttgttgctgctgtccaaacccatgttgtggaagttgctgctgatagttgccctgalactactgcl
t
tegaacatacccctyaccttggttgtatggaacaaa~gggltgg~ttgattatgctgctgtgaagggta~=ac«~~tca
t
etgggtttgcaacattgttacttctotaagacagattgggatgg«ctgcttgaagttg«gaaaccl«ttt~ laacct
ccttggt«tgcacataactgagttcttctg:lctatagagtc;tccccatcctgaacttegaac~~tctcatcatccec
ea~
aaaat~aac~t_'l«ct'~ttecacta=~astaacttetcca~ctt~tcattcata~ctttcatatccct~ta_'tett
tct
calca~aatca~a~tt~~teca~at~cttctatc~taatcitcattsta~ttaccatcc~act~t~ccaaauctctacc
aectc~catccttcttcaac~tcc«
~u~zaeaaa~ttcccatte~aa~ca~tatcaa~_aascatcct~'atctt~'~~:tae
_acarctca~=Iaga'_agtgctga'=aagtgaa,'ctttt«eaacccgtoatvaoovCatt[ootClv:p;IvCCIIt
f,':la<'C
~CICCCaIgcttcaaa~=aaggtttca~cattctgCtgIgIgaatCCggatatct:la«Ct''ga~lgl~'C'C:I~ll
l:f~~aa~r
IIgg3~aaaaalltagCCaggaaggCltlCll~Ca~lCglCCCag~llglgaClgalCCCl~~oola~CotCIICICCC
a
Cagatgggcttt~tClICaaglEaaaaCggYaaCaggCgaagCllgaaaCCalC«caCIaaCW Ca«aatt«ti'lla
~gCIElitga~~CCIIICaaaCIC~ICCa~glgalC~aglgg~llallCalf~oCaoglCgt~aaattt~llCCCCI~=
aaCi
alagCaalaa~aW~ClCtlgalC:lC;aa:lalt~t'IlgttttYaacc'~~'CggagggaraatgCCattaC~~c;t'
;~llal~<_ll
ol_2t_vv_vaaotCoCCaaCYCCiIaI_~tl_~ll~~'_2Ca_vllCll~ctcatctacaac:el2a2CCalCQtatt
a~_fal~l~ICI
YIICICIIaY:Ira,'Ci'a~C~:ll~'CuY.lCgal~li~ICglig;lad'a_ga~ottCtaatglCCtl'=ggaCC~
l~llt'=cat:l
caactcatatca~cac:a~cuaaaeacaccaatca~acact<_~tcca~c~aa~tt~!'teaatat~a~'tacctaa:l
acaca
caaacacat~a:laaa>?acaca'_a'_ma~taacuelaacaaaceaactt~atcttaac~tttccaaaaatcte:;ta
:uu
a~caaa~aaacac:ctaatl~~caac_'c'c~ccaaattsataata~ett«tca~atcaattaatttlaaa~rartatc
at~r
tc~«eta~taet~aa~~t~tcaalccaaateealaloateca~acaaat~a~atateatcasaa~tt~claa~wa;wry
.
ca~_'ca~aat«g~lt~Cltttcaaaca~~tcctaatgttaat~=cagaaaaca~taaaaagtcata~aa:u~=act,'
atat:I
CIC~aIC;'C:1~?wIC~:ICI'=Cl;:l~;lad'~'~aCCCI'lCatCi'a';tacaaca'=gat~aca~a~'a~t:l
:l;l:lal!_laYaaal'''r
:ICCaYtIIaC(Cl:llC;IC:ICa~ICIC;ICt~_(taL'la~aCl~~tHl~atC~aCta~a:lt~°ll~a
:l:laC:lY;1;1:1:1;1I21C'l:l
aaallCaala'~C:1:1~1~:I;Ilk;la,'Ca~~laa'=taaaca'l:lga:tatcaaat~~al~'a~a:l;lllCCl
;l:l,"_;It'r'r;r!_:1:1:11C'
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
~aatagtetggtctccct~atcaattcagtt~gttalc
aaalclcaattaagctatccctagacaacaagttcaacaaca
~attaattcattccc~t~=atagaaatcctcaagcata~'cta"acact~aCCtaattcccaltagcgatctctaactt
agc
aggcaataagactgacig«ilaagccaataacc~ctctaaac_~'ccaagcccltg~_~_ataga~'acgattatcact
ggaat
tagtagatcraal:gttccattaaatgccttttgagc~
tt~,~~,~,~atc~_gatlccaottgatcagaaaggaataagcagta
a:IlClaCaC«aaCCa:iC''gallCgaaCal,~_~~«Cl:lCiIaCIaCICtalCCCIICCICaaaaalCClaatCaC
laClCl
~~acaacat~=cgtlaagacaataacatagaaacgattgaa~,attccatacitaagatattaaagg~~aatgaaacaa
agat,=
aacaagcaaatgaaagcaaaatctaaataac«agaa~aaaaatcgaaatacaaagtt~_cacgaatgaaaa~_c~=gcl
ttt
gaataata~_tgcaaaaltcgtgtctcaaaagtgcalgtctcacggcgaaaccctlaaacacttatttatactaagata
ag
aaa~cgggcttcca~cccatttaacgacccttggacgacgtgatcaagagt~tcggctcaaagtgggcttcctgatggg
c
ttcgaacg~=gctltgaatgigccttctcttctc«tccgtt~=clcagg«giclgg«gaglgcagattgatcagagggl
Cgagglggt~olCoala~gtllgltgaCa~=aalgglCl~~gc:lgCIICIIggCCICLgIgaCCgagtatglglglCa
gCI
~tca~~at~~actagttcttctcctctacgalcgagtagaactggtg_actggcaaaglaggactgc«ggtcgcclctg
v CD Cff
cgatcgaglagaactglaaaatggactgcctcctctcclclgcgatcgagtataaglgtctggatggaclactgagclg
a
clgcttccalclctgctactggtgatcgaglalgttggtggaaatgtgact«cig«ctglttttcctctttctgagca
caaatagctccaaagcacclgaaacaactccagaatgcagaatgtatgcaagactagtcclatactcgactaaagacgc
a
caatgtacaaaaatgatgatgaatgctatgaaagatgggt~aaactatactcaaaatggtgataaaaggatgtgtaaaa
c
atgcaaattatagacalatcacagagataalatcccatctaccctgagtataaccaatgaagctctaatggtaalccca
t
clccaatcccaatalaagaacttaactctaccctttgca«catgaatcttttaatccctllltctaatcattctlttct
lttctattaactctaggagaagctttctcaacactttgatacatctagcggattt~satttctttaacaactaaggttc
c
tgtttaaaaatttlaaaarcgagggcll«glc«tcttca«agctaal:aagaactctttcttttcttlacaaalccca
aaacclltactagaatttttctgcttcttlagcltacttcacctaoagtctltlaccttttc«atcaatgaatccacct
ttttlttctlltagacacattccaacccaaagtataagcgcccacctaglcctatctagtccggalaacgcgaactaga
a
clacafgagacactalctctgtcattacgaacctaacact«claccaa~cttaatcgyaataagacctcacaaacactc
acaagtgatatagggcltgaaagaaagtttaggtltga'=ttcggglllaactgctctaataaggagagtcagctactt
gg
attaacaagagtggttaaaatgagtaaaaaaatccaagta«ttaggglatccalaagttcaaatlgatag
ggaatggta
lcagatcatgacagtgtggtagaatagagcaatctaggcalggtgcagtlttacttaaatttcattgactatgcaacag
a
caactagcaacgagggeaetgagtttalccggtgaacaaaaatgc«acaaggctatctcatcattatcccclatgcata
tgcagcaatcctaaafgaaacaclclagaclcgatcclaaa«aaatgcaactatatgaacactctgtttttgtgaaatc
attttctcacatttttcaaaaacaatttatttttttatgcctta~=atgaat~cagactcaagagtatatacaatgcaa
ca
cacac«c«cclgagccctcccccaaacttaaatcacatgtgctgcacatcctctagtaatccaagttg~~ccagagta
atcaagcaag gatatcatgtactgcaaaactc
~ucgcct~atatacmctcaaatglcaataggttagtctcaaagaaa
acaatgaaaccaaaattgttatcaacagacaalgaaatggactatattggtcgagtacaatctagtactcgagtatact
g
aacttttCCCga~ttctlctttaacgatttgaatc~a~t~gaaca~aaatC~gBggICgaglalalClgllgagglCga
g
ttcagccagtgaagtcaagt~=~=tcgaetaaactatca~tc~acg~tcae~=aatttgcagggacggaaccatgaatc
gacg
~tcgagtagatggtgcacitggttacggttgt~a~at~~lcga~tcat~tg~itgtaalcgagtagttcttaaggcttt
a
acggtgaacgagatggggaactcgactgcggtgec~casa~~l~~tc~aetacggcga~ttgtgctcaagtgic«lgct
CIICgC gglC g:IglgllglggCgagaaCaaaglgaC ggt ~'aCagCtlC ItCll ggt:CttC~=aglgtag
~'~ ICgI glgaaa
~~tagagtgaagtggtcga~talggcgac~aaamga~t~aaat~gl~'tcaaaacgagtaattctttggacgagtgaga
g
afg~Cgalllgtgglgaaggilg8~aaattg~agl~~(C~=~_l~_'a~a~alg:l:lg:lg:lgill~'ggl(:ga~l
agBVlllac:Illl
~aatagagtga~ggCgaa«ca~aag~l~l~ll~~aaaletaltctatLatICtctclal:alCll:lIC(lC:lggaag
ililil
acgcPt~;tgglccatgccctg«gllttgtctttaagcttag«ccct~_«gactgggaaaacctgaaatcctctgtaag
tctctgttagtcgaglatatgg«gaatataaaggatclgaatcatcaaattcactcgactaaagatcctcatcagcacg
tgaccaatgagatcgagtgacaaatgaaacctt~tttatactc~at«cacagagaaactcgatctccacagcatglggt
cgagtgaaacactgagatt~=gatccattgcacttaaatatt~gcagctamctgaaaacacta~=taaaacactagtaa
a
ggcatgggacttcctcccaagtaagcltgtlltaagtcattagc«gactccc«attctlltgatcatcaagaag«cc
lggagatgaaccgacgtcacctctggaaggattteatctgctaa~ta«tc«~aotc«tgaccatttactgigaaatc
tctactcetaccaoctasa~teaccsctccataa~~ac~aac«caleataca~aa~o~acc~~actatctasacttaa
~ctttcctggaaaga~tttcaagcgagagataaaaaaaacaccttatcacc:aacctg~=aaatccgaa~tgatgatct
tct
I_olCalooaaaa~CIIa2llClCICCIl~la2alllt:tYa>_,c:lilt:ll:l:l_'CllClaaaCai'alc;ICa
lCaa_~9IC:iCIi'
agttggalCaaCCgCIICICCICagCgi'lllllal~lCaill~ll~ilY~it~llll:IICYC
CC:ec'alagCIlIYIaCICga~
ttcaac:aggta_gtgacalgatt«CC:I«lgaeilagallgilailgg:l.llgtaclaalgv~C~llCllgaa:l'!
Ct~'ICCaaI
aagccgaCa~~l~C~llitlCgagCllllc il~aCIaYlClllCellYla;ItCiIC;I;IC
ilglClllICCaaaall~CltItalC
tccclattggaaatclcaacttgcccgcgtglctglgg:il~=:Il;fil~,u;l~'tyv~y_aCCllglgCllIaClc
calgClICII
Ca'.'.aaggtlll(;aaa:laC(:ll~Il!!al!'ilatll~C:lllt~(;;i(;W
IIC:IC'll;IlC:;1(:I;IC'Cll~'=''aa(;ICC'.aaaICIC.''_gga
a'~allal~~tCII~ aaCa~CIllil~C _Ca:ICIIII_'Call'_ll_~ _'IaY_~:IC I
_'YC':I:IIa<_CtIC ~aCCC iICIII_~al:ll:l
IaalClaCaaC~al (:a~lel:il:lCfl~lla~~~lill~'e:l~at'_L' ~'il;lt_'aal'C
c:;ll:i:laatl~:lIlCC~I :llaCill~tlil:l
CA 02362897 2001-09-18
WO 00/5s32s PCT/US00/07392
atclcaactttcaagattYggttctgaYgcatcteatttcttctact~at~ugccc«tctct~acatgaalcacatt
ttaaaacaaactcctga~cgtccttaaacattgtta~'ccaccaaaaacct~m«~ea~_aatrlttaacacl~~tctt~
'aal
_~tc_cgaagtgaccaccatat~'cg~'a~~ccat~=gt:lat~_ea''caggataccttctac«catcttccga_aca
catctcct
ata_atcttatctttacagagaytgtaaagataa'rYclcatcueagtattaatggtgtat~_tccctgaaaaacttct
icc
tttcalaactggtgaag«g~~~_ag~~ctctactct~=cat~maaataatt~'acs~teatca"cataccat~="a~at
cttccc
tcaaca~~cgtUa~~ttggtg~aactctttccc~'ct~'tagctcccacc~aaaaallclacaaecatta~_tt~_ctc
ttct~=~=
(:aflgaglC''IClalCtgaafagglICllCaaIlClCaICCIIi'aCa~al_'YICa~CI~CaCCaIIIICaaIgCI
IICII
YtcIlclgt«ccatgtcaaacttcta
caacagaagtatccatctcaata_'tctlg~_Itla«falccltcllagaataca
tatgcctccactacaagilaaacaggc~tttaccgactacaacacta~_tc~tttcata~tcgtaaacagaaataatal
:gac
taatagaCgilCtaittcagagtaEtcgtatatatatagtc~Ctaa~_caacata~Itcot:ltottagtCgl:la;Ig
ltaCgaC1
aaClIaCgaClaC glaaCaaICg gCalgaaalaglC gIC fall' SIC ~laaCallt ogC
uaCIaCg:llaC gallaalla8
cctgtttatgggggattattcgtgtagtcgtaaacta~tcgtactmcatt~
acgaclacttlgcgactagtttacgatc
aa«gtattaaaclctgggcactattctggttgtcgtatcttaglcgctaaataatatacgactaatgtacgacgccttt
ac~actacctaacgactctcttatattgicagacgatagty«aatatl~~t~actat«tacYactatgatacgacta
tacgac~actattggacgacaatttgacgacgacattttgt~'tccaaataa~aatttggtcatatcttgatttcttt~
tt
ggttggtcatcgattctgagaattcggtitgtttgttgttctaaalgctataaagtataacaaaaacacatttttatig
a
aaatcctatcaaatacataaaacacaacttacacttaaggttcaaagtactaaacacacacacaaacattatt
~ttcaaa
gttcaagtgaaaagggactgaaatctaaacctaltgatcattatgtggagtagctggtagaggcictgtggttglcgaa
g
altgagaggccatgaagtcaaggaatlgcggatctgt«gtc ggagatacttctccactaac
gacaagtcaaggaaltcc
aaagallcgggcagtctaagcltcacgcgcaacaatctcagaalcacgcttctcattgtagYclacctgctcctcaatc
t
tgcgttgcgautcttcagccattcttctaaatctaggaa~~_aa~at~_ate~augtccag_gagatlgcagttgYcac
aY
cctaatgt~tctttgagactgccaagiccataagYatt~_uetctg~aatcccttty=gt~_~actgaaaagaaataaa
gaa
=agattagtgaatgaataaccuaaaactcaaagtcaa~a~=~ttta~'att~=caacattttacct~gagaaagatgYt
IYtc
aactcclcattt=tcaYcaeaaYcttccYCCtctaYctYtYacaatclatctclcaeattct~ctcataa~ttt~a~ca
a
tcatctccgccttccgatcaacatacgtaccatccgac«agtalgagtctctacaaacacclcaccaagagtgactgit
cttcccaatttctcctcctgcactgaaaccaacacagctcaaatactcaaaaacttacalgtgaggtaactgaaagagg
t
aacaeaaaaacttactaattcgtcttggatttcctgaaaa~ac«tg~ccca~~aeaagtY;algtgaggaccgagaccg
t
tacggtcagagagacaagct«ggaata~gtccgactcr~_tt«tgt~ct~~ttc~=~t~tcccaetaagcgcacataac
a
ectgcaccctaetlctccttgcggtgctaaccatgtctttcaagcgcc~utacaaacctcattgaagtaatact~caca
gtctctgttattaagggatcccaactgtgagttttctacaaaaaatt~tagc«~a~tta~t~aaatttaaaagattaag
clggaaag«aagaaagtaagagaaatgcalgacagcttaccgcaaaclcaagaaataacctctctcttttattcactgg
tacacatgaccagttatagtasg~agcatitgtgtaaaaatcc~'a"tgatct«claaccaact«gatcccct~tcgcg
aglaaacctccaaacacatgaaacaatgaaa~ataaaaacatgttacatta~tgaaactactcaacataaagaatagca
a
gtcaccacaacagaacacaagagagcaagccaccacaacagaacacaa~_tat=ctagacatgaccactatgttcacag
aa
=atatagcaagcgaccacaacagaacacaaglaagcaagccaccacaacagaacaaaccgccacaacagaacacaagag
a
Ytaa~ccacracaacagaacacaagatagcaa~~ccaccacaacaeaacacaa~ata~_caa~ccaccacaaca~aaca
aac
cgccacaacagaacacaagagagtaagccaccacaacagaacacaa~_ctcatcaa~=acatsaccacaatgttcacac
aao
atagcaagcaaccacaatctaacacaagctcatcaagacagta~tattacaeaa~ctaatcaagacacaatccaatgaa
a
caatctaatacaa~=cagatcaagacacaatatatgttcacaacaaattaacaaa~c~'Yac:,caatgaYeaalccav
c;acaa
tctaacacag~ata~caapccaccacaaca~aacacaa~'a~~a~taa"ccac:cacaaca~aacacaagctcatcaa~
acat
YilYclttatsttcacacaata~caatc~atcaaaatctaacacaa~ctcatcaa_'acaacaa~~ctaatcaaYacac
aatc
caccacaatctaacacaaQcaeatcaaeacaacaaYrtaatcaaeacacaacctaaeac;aaectaattatt~ctYcaa
cc
taagacaagcauctcaagacaacaagctagaggatctcaacetaacacaa~ctaatcatt~ttgcaacctaacacaa~c
t
tatacagaalctaaaacaagctaataagttgaggaagagcacataccattcagtcttcgecttgs~=aataggasacag
ta
cg~tgYtccacatttccctcccgggaaccataagtatgtc~'ga~aga~=ccctc~cc~a~'tcttctt~~tagclccg
acaat
Yattgaggagctccctcttgaaagttgttgccctgagaagatggatggctgtgmcctgaaagttg''galttggagact
c
C~~aggCICIIgglIlagaggalCllClggCgglgYallaaaCl~'C~'CCI(:llCaVaIC~CaoCg~CgvCaoalCa
aCaC
a_Cf'raolnotoYaYCIClg~ilafagllgat~_C~~'C~~Ci"_al;l~ll~'CY°_I;IYI~~~~a~ct
aac~=tt~a~~CICCa~°,C°
YCIIga'_~C~IaI','.C~ICagaggagClIgaQ~CgYaI~CYll:a~a==;1~'CIl'';1';~C:1CC~'CCgCC~
~:I~l=:la~'tC~l~
llgctlag~_ca~_C~=acgclgglcllccaacr~~Iglg~~BICCICC~'~I:IIICI~L~'la;lC~ll~~gwiCvl
CII(:(:lCl
llC~l~a3CCICCaC~aCC~~CgataQoCal~Yllguaa~'augaa~2l:l~:l~~:IIIIY;I:I~aa~a;la~a~aa
gill~L'allC~
acgaa~aaaga~aa~atgattcaaagaagaag:lay';liclaat~aa~'a~;lagag~'C~'aa~a:lg:lagll~ca
~aagaagatc
'aa~aa~_aa~'aacttgaa~aagaagatcgaattagatc~;Ia"aa~~tlmnl=aa,'aa~'aagttgaagaa~ata~
atc~aa
oaantlaaCCIIIYYaIYaaalYaYaa_YY~_aal('Y;IIC l.'a:lll;l'~_'_cWLIC'lll:ll:ICaC
LIaYICYC:IaI_TIaYIC_Yl
alit(a~lCaCfaaIIIICII~~otIlY_onCCoCOaaal;la.'aa:l.'CCC;IaC;l;ll;t;l:l_~;IC'aafl
_YICL'CCYaBtaYIC~
tlYaalaolCYIIaCIltllallYYCllli'_'YCIICYaaatlaClaa'~CCCaaC;l:l;Illt;llYYt_'ICIC_
'c;ItYllilvl' '
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
cgtaatatcgtCgtlagCttt:lll'_';gIIIgggcttcggaattactaagccc:l:lC:Oa~l:ia:la:1'_CCIg
IC~Caaglla~,
lCgl:tllala!?lC~_C:ICat(attlt''nllllaggllC'~aaalfaaalagCCCa:ltalCalIa~aC'=ICI''
lC''lCg~ll:l
~ICi'IaCIICa~IC
~=IlaallllaltgggCll~~g~=CllgggalglCgaCaYCCCaaCaaallaallalalll:l~'aagiat
tcal~'alaa(tataaaalllctalaalalllallcaaataatlaaacalgatat~attcttaaacaaaaaatnc:iag
la
:lafa:la8a:t~l(:lafatacatataagc:iCal:altlagaaClcllCaICaICIIC~:IIIICaIC:IIC'_ICI
'_a:l!'aC'':lta
aallaC:IlCaa:lallCaICCICIggttc:lgCatclCCtaCalCglaalCga~l:ilg:lll"=~IIIa:l~'CIII
CIaCC~'ga
ttgtgta~'lace:igalCllcaall,_lagtcagcaacacagcttcatcattttctg«t,=caaaa~':i~'lC~_
calcatl~~ll
llCalallC:lCCagaaat<'tIICCCCg:lggattaacttttaaaacatttagcc:laat'!C:IIIItY~ItICIII
~ talalg
!'atacggaatataacaaactlgatctgcgtgcgatgctgaatacacaaattatacattaataaalatacaaaattatt
ta
ataa«tatagagaacctlatgtattcltaattacatacctagaataaatggctcofalltatt~latttccta~atgat
a~aacgtclacaacaccaccatggcttc~ccgagt~=cctctaccaacctttgg~tcataccacttacarttaaaaagt
of
gat«tcaaattgacgatccccicatactcgagttctatgatatcagttaaggtaccgta"aaatcc~=cttcatca~at
g
aatccgcgtaa««clcc«taacacataccccatagttacatgtttttcgtccagctccalgatttttcgtatgaaac
aaalalcctcgtolgaaatacattggccaagttttaaccttgttcaaaggcccatgcacaatctcttgaacrcaaatag
g
aaatagagaa~tag~~ttccaaaatgtlacctacataataaataaacaatatcaatatcaacataatttaaatgatcac
t
tttaaaaattactaaaatgatgtagactlacatattct«aactcattgataatattcttcagctctt«cgcgtaatgt
tctttttcggacaaaettggatatttcaaagaaagaaaatcctcgaacatactatatttacaacc:gaagataaataaa
tc
aaagttatcataatgaaaattttaaattaagaagtgatttaaaatacctctcaagtggttgaaaata~
Icaraatttctl
aaaacatatgcatgt~c~=ctctggtagtctttctctgtaagccattctacgtgcalttcaccacttggacggtcgacg
tg
cataaatatgttagotactccagggacatgatatacaggaacttctccttcatcgaatcttg«aatctgaltcatgtaa
ttgatucaattctgaa~tatagattgcgaatcgtaaaataaatagttatatttacctt~attttgttt~tatatgatc~
'
~_c;aaagtagtgagtcaccaaacagct~ct
SEQ ID NO: I85
Arnbidopsis rlraliunn
BAC F9D18
saaticactca~lata~~cctt~cYaccteacttt~atccttQCtsataa~lc«caaaaaaacccttt~_crl~ctaca
agatclaccaotaal~=attaatggagtggaagtacctacagatttcgttgtgctlgaaatggaagcagaacctaagga
tc
ctctaatccta~~~a~accttttcttagcctccgtgggagcgatgatagat~tcaaagacl~gagaataagtcttaacc
t
lgggaaecacatgaagctgcagtttgacatcaatgaaacttcgcaaageacagctgtagaagaaaagatcagggclc:a
ac
ctcaavcttc~sgattcaatcaacagaccaagcacagcctctacacctgacttgcgagatctcaaaaagaaatctgatg
ag
caagaagaaal:catagagaagclagctcagacagttgaggaactlaagagtaaacto~=atcat~~tgcaagagaaae
cma
atcaaaalYCa~oatt~:lcactatccc~aeaaaaaa~attacitcaa~at'_etct~a~__=aeataeattatcaacc
a,_aa,_
a~aaa'~a~s~eeta«IC~a~eaaa~aaeaattca~tatlct~ctactcatcW
caaea~_a~~at,_ct'_aatat~at~at
oa'~atca~a,_ac_actat~ca~~atcttctctatcacccattttcttcttaat~a'_t~toa~oaalcaa~cta~a~
acl«
aaacaa~ctcac«~_'=a,_~aattcccaa~actetttct~laaataaaacttllatlttctlstta«ttt_'ac«~tt
t
tt~~ttgt~Tl«~toattCtca~gaacagagaaacagcgto~agglagaglaaaaalllaaaaattlIlaCIClaOaga
g
caacagg~gatr~aetat~tcagaaattcaagagtttgaaaaacttctgtt~cactaaga~~ccat_'aggtc~a~taa
~l
t~~tcga~tattagtgatgattttaaaaaacaaaatlttgaaattatacttataclcgaccaaca~aagctacagagac
t
tecatggagtttaacaagtttacaga~=gatgac:Igaagattctagtcaacag'agaacaglgcttcaggacaaaca~
'arac
agc=lggcc~amacctctcacctttgttcccccacgcgtttttagagataacaaaatctc«ccctccttccccaccac
tc~atcmac:cctatcctttcccacc~acatcatctctcttcctctccaaac~clcaacctc~aacaclcactctcact
c
acgtctctcacal
gaccaaac«cactcaccatctctcttcctcaccaaamclcgaccacmcatlacactr'_accaca
cc~ctcacte_~'ateCCacacctcc_~~'cttclccatctctcac~~cltc~tclcccateccac
c~accaWaCaca~eC,_
C1~_CC~_ClI_'C~CC':lCl_'(:C:(lC:iIII;IIICfItC:ll:ll~aCC~~ItIC~CCaII:ICICaICaIIC
It~aCCtIL'lC'?Il'
C:ICIC~aCa:lClWi'CCIIICaCtIC;~'l:lg~a8g(:lllaCCnICll:aaC'TICIIC:ICCafClIl:aCIC~
aII~~C:IaC''aC
,_CtCCICICC:IaIC:lal~l:alal:IC1'all:YCC~'_aCIIIC~_CCaICICCCtL'ICaC,'l'l_~I,~IIC
aCIC,_aC('ICaCC~I(
CCa~CIICa(:CalIICICaCC_~CIC~I_~tCICCICIICactcaacCLTclacac~aCCaafICICCCIICIC'~CC
aIIC~I
C~tICallI~CC:i~:lecilCICICICaIICtcamaacacIC~aCC~CIC:IICICICCaCC,_<_'a~IIC_'aa!.
TaOICICa;l
ICaICIaI~'CttaCIC~aCCICaa,_CC_:IIICaCIC,_,~~ILaa~Clla_'_Cll:_aCCnC(CIfCaaCCC'_C
CaCIaltlCl
C;_"_aL'aanIIIaCCI'_aCCIC°CC~'CaC;lal:C,_caaacaclCYacCaCa~ICCIICIal',_aa
Caaaal'aCCYaaYC(:aC'
aatltCaCICIaCIC,_aW
''_lattaml:eaCI~C,_IaCtt~aCC~<_'llla~t~lfl,_C2tIlIalila_':ICI:IaCal:ilf _ _
CA 02362897 2001-09-18
WO 00/5532, PCT/US00/07392
eatat«~gcl«oa~llaca«cu«tcaggaaa«aatatgaotaactaca~t~r~cgaatcctccat ~gat~c~~~~at
tacaac~tc~at~aa,,ct~aatctt~~tcaact;gaccagagagagagcgacag~~cttat~=a~a~'cucagagct~
_a~ac
ccaacgctta~'ta~~clcgaycaat~agaaga~aeclgaga~tgcta~ra~gaaaga«agcaat~'acca~~cagata
t~'a~'a
tgatc~ac;=aa~a[att~=ac;_tcga~_tat~~agcctga~=tcat~~gcaca~'aoaeacgaa~=ct~'tt~,aar
ag~_cctgat~aa
gt[aca~=t~r~_a~=~ra~'mcam;y~act««~~a~'ctca:ltgacttct~r~'g~'aac~_;y'gtacccct~'tl
atca'arttlg,~c
ccagct~~~~l~,ctact~~=ag~_act'tacaacacttattc~a~aaglgtcatct~'_ag:lc~'ct~at~~tctta
cecatac~«~=
c«acaagaa;=~aaacaata~_agtttctctctactctgcaagtggagalgtafcaa~'~'acttaca~;cagateagc
tggag
a~tgaa~e~tt~=e~=;=tlcttgactttttcagtgaacgagcagcgttaccagctatctafcaa~=a~cit~~aaa~_
gatt~«
tggtttccccaat~~aaag~~=aactaaacccaagttcga~a~'ggaagagttgaaaaatltglggttaaccattggga
acg
atatagc~ctcaactct~c~a~~tctaa~a~caaccagattcgaagccct~tagcgaat~ttct~lactctag~~aatc
t
acaggcatcgt~tctaacaca~=ac:a[~~=agatgat(gattctgcac[caagggcattctccgcagaacaaagggga
agaa
gglcctgaaggoc~acctcaateatgcacca~'cgg«atgctlctgttgatccacct~_t~tg~=atacagoaa~tggg
c°c
acaccaacgagaagaagagggtgcgaggagccctttgtgtaggtggtg«gtgacaccgattctgattgcatglggtgta
ccgctcatgtctccaggg«tgatcc gag gatgatggatttagalcatttgcgtc
gttgtgagtttctggagcacgacat
ggttggcgatttctalcgctacaaattcgagcactccttgacccgaacagccaacattctgc«ccctgcaccgaggcca
taaccatacttcagggcgaaaaca«gacttcaatcctgcgcgtgattacctctactttgagagcgctccaccgactgat
eacaacg~=ccctacaaaagaaectaca~ag~atgagatt~ctgaQaca~at~aggatagaaag~a~gagtacgalacg
ae
catgtatcatttca~lgagaatgtacctccagcgcgggagagcaagagctt~a~cgaagctcacagaaacaacagtaag
t
lgcagaggtggtgcaagaaacaagataggctactcatcaagtgc[tcaaggccatcaagtttctgaca~~acaagctaa
gc
tgctcctcttctaccacagctattcegca~ogagagcctcctcaggacat~ccctc~aagagatatgac~=cgccagag
cc
aactcgccact~~cct~a~'ctaa~tcaccacaggcctgagcctagtgaccgagtagtcccaccagtccctgtgtg~ca
tt
catcattcaagcctcgg~'agcaegggaoaaagaagaaggctgcactcgctcggtctggcagtag~ragtacacgactt
ctc
CagICCC ola~Cfl;IC gCaaCC aC ~~lgatggccgcilgcataagaaCagBgglC
gagl:IlC:lll::l~':t~CgClg(:I~~CC
CgaCga:lggagC:lgit~~lC~:IgIaCC(:CCilgggggaagClaagaCdCaaCa~~ggagallC[IC"aIggCClg
ggagCaal
C:BCaagCa'C:Iall~aCuaCCaaC[CCOCICCI[CIICCaCI~ag~taagcacctatCtCCaccattgtaatatacc
alc
tcctgtttttattitotttlt~_tgal~_to«ttgtcctgagtactctcttccaaatttgstcacacagtggact~t~t
sa
titaa~ttt~~~=~~a~,'~ctca~~aa~l~~t~tgtlgcaltgtatataatcttgaQtctgcattcatctgaa~cata
gaaa
aacccaaaaaaattgaaaaatttcaeaaratgatltcacaaaaalagagtgttcatglagltgcattgca«taggalcg
agtctagagtgtttc~tttag~alt~tl~catatgcataggggataatgatgagatagtcttgtaagcattttggttca
c
cagataagctcagtgccctcgttgttagttgtttgatacglagtcaataaaatlgaaglaaaactgcaccatgcctaca
t
tgctctac[cgaccacacggltaggatctgalacca«ccctatcaa«tgaacltgaatctø'~altta~;aattatcat
gt
cctg gca«~_aattt
caactcatg~_ataccctaaaatacttggattttcttactcattttaaccactcttgltaatccaa
gtagctaactctcc«attaga~ca~=tlaacccgaarccaaacctaaartttctttcaagccctatatcacttgtgagt
g
tttgt~~a~gtcttatttcca«~a~'ctt~~taoaaagtgttagQttc~taacgacagagatagtgtctca[gtagttc
ta
oltt~CglltIIC~~aCI~'=ala!_~aCfauClgggCgCIIaIaIC:algv~[[irLrvatvlvlll:lil:laL:l:
l:laa~~lgeal
lcallgttgataa~~aaag~r~aaa~aatlclaggggaattaagctaaagaagttagaaaaaacaaatctagtaaaggQ
II
llggaalgllaaa~tlaea~raal~:IwlICtty[laa3gacaaaCICIlagaaaaCaaaalatal;7Cailtaa~'al
aftaC[
alaaatacatatalall:talttll;it:«aa«taltalcaaacalatatacaa~~acat«at«clacaaaaa~ala'_
l
aIICafaa~lacaaala~alltaatt~'alcacaclaalg«caaatatttitatglaa~aaa«tcatcclaatataa«t
ctttt~attataatct~ae'_agataaactaaaanatttlcttgctttttcplttttsacttcaaaatatatatalaat
ac
aacca«tgtaacaaa~_cataaacamaaaaacaaaattaa~_tcaaaatcatasaaatgactalta~caa~aaaatgtt
a
atCgtt~tatCaga~l~lt~a~>aaa~CItCtccta~'agttaa~agaaaa~aaaa~aalgalat~Jaaaaa~
aglll~a:laY
aIICaI~3~l~caaa~~g[a~~a~Itang(tCtIMallgggaClggagllggg:tllaCC:lll:l~~:I~CI[C:lll
~llala~
tctgsgtagatgggatcttatctctgtatgcataacttgg~t'aCII:IOClllaac;Iac;la:la~c;llaafCalI
CflYl~a~
aICCCCI~I(aCllaa~Cll:lllClEli'avvvaCCglC:IIIBIC(CII~aCCfICaCCIIa~'CCaaalga~_ttc
att~al
~aIQCaII~Clf~aflC;lC~[W
a~aaCtaatgaat~_uaaag~~aflg~lagatll~aaa~Catgt~~l:l~glC~aafa
IaagagaC~~lalt~all~=alaaCaa~~C:lt~~'ClaaC~tllllga~taaaattcaatcatatc~catctfa~'aa
ctaCCa
aCllggacatt~allllalllC'CLC:t;IClI~atlllll~~Cl~a~tccCCacctlc:laacctCICCIICaaC[;l
lolall
l;llalllgCll~awvCa;1_Ca:Ia.;ICla:l~lll~~ggagaatt~_alal~IClaIa~tCtgC:1t~111ICa't
'=ICCaIIC
alCalC
~IIII~a:IICta~Iltt;~f:IlL:1110:IlC:ICIalIllatalCalllClCaflgltall~C:7IaCIII=Cal=
at
lage'ala~llll~c;al:lC:WUI~C;ItIIei~a~
Il~llllCa~_gtaatttyla~Cl~llt_C~a~~CaallIeL'aa~aaa
cgagcctgaaecagaae:matactcgaccaccaggtcgc~=tgcatccagcaccatacltgacmrct~_~'I
a~_tgactt
t~~a~ccattc«cccatctactcg;lc~cccaygtc~aolaaccteatctca~,~ccacty'at~_ayrceam~_~=cc
~cc
CI~aIC'~a~lallaClIC!_'C'C:IC:ICC;ICCI~BI:C:IC:ICIC~'n[CvllC3CICl:ICC;:ICCII;ICI
C~=aCWCCI~!_ll:''a';
lalC~lCaC(C::IC(:;ICC::1;lC:;lc:C;lll':ICIC:sac;aacacaclC~alC;lCafcllcata~.:lcl
actcaaatctclal(C~~al
IaCCCC:IICCII:I~~:l;4IC'ft'Iy_IC
J;ltlc,n:1[tIClfI~llIlalC~lIaCC!?ClaaalC~_IICICatf~_ell~IllC
li
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
itcgittltccaatfactcgatcaacttaclcgaccacacccagtgtcttgcaaca'=actgt_la~,tc~,a~~tac~
~ttot
ctcata~;acts;ttt~ttacatg:tcca~tatactc~~accacacccagtylcigyaacagact~=t~_cagtc~~ag
tatatct
glltCaIalCalgtill~llaClcY:ICClgoactcgaccacatcclaCIICIiT~?lalCa~'CCf~ll'=I'~~(C~
tlgl~aCl
Il:igtaattctgttatlalttclgtlltcl~~cal~_Illgctta~'~=aftgttagaaaccccaaaact~tlalIL,
CIIg''Cl
l;~aclf:lft~aCllClgatcaCalCtClt
'ti,lll'=Cafe;ICaCCIatII~"_atI~:ICaCCl:laaalaCfaCaaCi':ICaI
_att~~,tgtttta~~ataatt~'aClaaaaaCClaltafCatuaacat~,~aa~_c;lc'IaCIIIiICItfIC~~oa
ICIILII
~ag~~tttta~ttttatattcaatcataC,~aata:lcat~=tt~'tcatatttcacict~aaac~_cta:fecaaaat
ttttclt_
cttctttaagtattatagtatatttgctcctaaacactaaacctaaaccctacacctfaaalcccaaaccctaaafcta
a
ttccttaaccclaaaacctaaacectaaaccclaaaccctaaaccctaac~ctaaaccttaaaccctaacfctaaacca
t
aaaccgtacacccaaaaccttaaaacataaacactaaacgcaaaalcttaaccttaaaccttaaaccctaaatcctact
t
taacttcctggttctttttgcgtttcta~=«citagactgaat~ataaacaaaacatcaa~_tcatattt~actacaaa
aa
cacaaaccaagctlcttcltgattclcaaagcItIQatggtgaagccgaagttcttat'_aottttug~tfttgaatca
t
alaacaaggaagcactaclttact«tcagtatctcgttga~g«ctagttttata«caa«atacacatgacalclag
tcatatttcactccgaaacgctaarcaagattcttcltgctttltaaagtatcafactataltlgatcctailacacta
aa
cctaaactctacaccctalatccca~aacctaaaatcgaacccctaaaccgtaactcat~aaccctaaatcttaaaccc
g
aaaccgtaaaccctaaactcttaaccctaaaccclcaaacctaaaccctaaacclfagafcccaaactttaaaatctaa
a
tcctactttaggc«ccggaatcgagttgcggttctagttcitatgctcaatgatacacaaagcatctagtcatatttga
caacaaatccgctaacgaagtttcttcttlattctcaaaggtttgatggtgaagccaaaattcttatgagttttcagtg
l
tttgaatcgtttaacaaggaagcactactttcactctaaaacgataaccaacattcttcttgcttcttaaa~ttttata
g
latatttgctcctaaactclaaaacttaaactctacaccataaatcccaaaacctaaaatctaacccctaaaccctcaa
c
cctaaaccctaaaccttaaataccaaatcctaaaccctaaaccctaaaccataaaccctaaaccctaaactcttaaccc
t
aaacccttaaacctaaat~ataa~t~t~talt«
~~cats=tltt~a~catccattt~tcatcact«agcatcatatcatc
actettttataccatttctcatcattt~tcatcactuacatglttaggatagatct~cat~_cat~tt~,catattigt
gt
t~atttcaa~tgatttggagct~,tt~ac~agctatct~gaagagcaavctgatcat~tcaaaccacteeaccccgagg
tc
~agtagaagacatcaccacttcacctcaccactc~tccacgagglcgagtgtcctcatctcratcacct~laccatcac
tc
~atcacatcactcgaccccgaggtc~a=_Igfcttcacctccattatcaaaccaccactcgatctcaccactctgcctt
ga
agtcgagtatcaccatcaccaccactc~act~c~tactc~atoaaaa~cuca~=a~=cctlcttcaficc~~cactcaa
cca
gacactc~agcacgaggaa~=aaaagaa~actccagctactcactc~accactt~~~tcgactaca=ttc«aatccglc
c
caatacttcgtcgttttataagtagcatgtac«cacattttcgaaaacaagtttttatcfa~ttttattcc~cagacct
tgt_ttctagacctt«gtaatctggatttrtctttatcfafmagtattcagtattca~=cttttgttctt~atttc~tt
tactattgttcattctgttatcatcctgct~ttacact~«~ttatcat~ttttcaacttg«caac~tttatgctttct
gttatgatgtctga~tagt~_aatag~ttfct~a~~at~,_~tfa~a~ta~f~Ia
~aattclca~tatgctaggtgattgae
tattga«gafagatcccftctagatfaott~ttcttaat~cctatt~cutc;
~atcaactggaattt~_a~rcccagaca
Ettctgcgcccaaaa~gtgttc~atgaaalgtctgaaCCactaattcta~a~a«c~t~=accat~_tacraa~gtattg
gt
IoCagggagC~llllggCllIaaCIf~tf~allC~lael~tTCCl~ltaggllaoClCIC'=ICaat~~l'~alloaol
tlg<._o
actaggttaactgg:lgaglClCI~tlgc_~'t~_~'CaCItaglllltggllaal~ra:lCtlgIt~Tlcla~lnutt
aafflatlga
gCalgtcaatcacctctcgggaattctttatct~allyaaflc:lll~_llfallCiaClYlfoIttaCl~CafClI~t
,ttal
CIgIC~CIIaaIIIClaca~IlCIIICII~IIaI'IC
~!aC(:aCCLa~tI~ICI~=~=Ca:ICa~:Ill~~l~rCaalCoaolalClp
l~llflaftffCl~ICtaffaCIC~aCC I~IC;tCIC ~acctcacrta~l~'elCl~
l:aaaYa"CI~'IYI1~~'lC~aglgll
Ilacl~rlllcl~Cllgaalllc~gflllclgc:ll~ltCaclla~!aact~ctagaacararc:ICaaCCl~rIl:ll
l~Clt~~
CIIgacllaglgacI~CI~amacatctl~allYltay:llcacaaccat«~.t~allVacaaccuca;f:lClalBaC~
a
Calgalaglgcttta~'gtttaattgalllaaaa:ICCtatt:llcaCtaaacaclaaar~tcaaaccala''aCCC(:
a:laCll
taaaCCtaaalCClaCttacotfta~l~caccaaac:atcnnnnnnnnnnnnnnnnnnnnacCI~aIftla~~'ttaaf
ag~
tatecccaccttaggiatctattactg~aacaatcita~at'~Igtgctagc~'tc~attacltagat,,aacltatca
aacc
l«allallalgCtl~CfIaoIlCla~~=~alttaC:l:l~H"a~lf~
~YalCaalac:lalfaa'=Cala~ll~a~llll~C~~a
acacfcatcggttactttatatta~=I~atll~l~=ttct:l_'aatg~~Itcaataataatcctaatttact~=ctag
atctact
attct«atatttcctgagaatttccctaaaccceacamtaatc:atctgaatcaaaaac;caactttaa«~ctttctt~
ICfIifaCflttalaalallllll~flla~CIIIatIl~aaaclallaalClafwfulu,tttl~'ay:luCtltolno
aat
ttgacccfaaagtactacaafcgalcfcttat«~'aya~=a~tg~tctta~=~anaa«t~~acct;u;ucattaagett
ta
a~ggltta~~gttaaga~fata~r~~Itta,»~tltac~=~'utca~atua~gaml~r~=a«ca_~~~'~«a~~~ltta
~U
~gttgoatt«aggttttgggalua~~~f'=tai'ayttta"attaa~tcuta"aamaa;natactat~~atactttaa~
a~gcaa~aa~aatctt~etta~c~tttc~;=f_=t~;aaatat~actacat;_tcat,~tgta~gattyamafaaaact
agaac
ctcaacga~atcccaaaaa~taaa~taet~~cne~«~_«atac~_altcaaoaccaaaaaamcalaa~aacatcagctt
caccatcaaa~cttl~a~aatcaaoaacaagctt~=~nta_ma~=atltafn~_tcaaatayacta~~atg:uttot~t
atce
ttgagcataaaaacta~aaccya:ueca~'a«c~=a~=aa~ttaaa~_tai'~~acta~=«=nita~,~atclat~~«t
c~'~=ottt
auyytlla~oatctat~~~[lla~~~~111:1~2c'Ilfaf~_'_tll~'°o°_illa'raa11I
;1~'"'_Ill;l'_'=l~ll:l;;~_~Ill:l~''r
I~
CA 02362897 2001-09-18
WO 00/5532 PCT/US00/07392
a'~,tag~~atttlagguug''«at[ta~~=,=tgtagagitta;gtttaatgtttag~=atcaaatatacfat,=ata
c« Bata
IaaglCaaaflaal44laC''accactctctct~,alaacacta~'attttaacCattttaa~'ccett~tttucatYC
attt
ag4ttaglltcaagtatctttlcat~caltccta~-tctcttlt44tafctttt4ac:atutgcatt'_c
att~,cataccat
:l4llgCa«lagalla«Ifacag~Ig:1;114:lagIaCIIIII~gaCICaaClf~~aC~tltCla~'CCC:Ia~'=a~
IggilCl
;t'_Cf4gaglIlCIg tgatcaaagga;li';'aC;il,'_=aa'=lal:l4lC'_g ~~lalllaC IC,'i'CI
~'aa;=alc:;ta~l'~_'a~'4aC~;C
~lgaggaaCaag~agl;llaCIC~aCl:alai'4a4fC~~Cl;1';aa~'1L';a'=l~a~'_,'aaalaaa''~'aga
aY:lY;I:l~Cla4l4
gaCC:lagCll;CIag'.'.CClgaa'=l4
gaglgal;I=acaaaga,;ga'?CI.4lCC'aC[1114[aCICYi'44I;IYaC4L:~aglg
~Ig[I~YCg4Cg84lltCllaIfIlafCIIIIICCCaaIfaY~~lalC4fagtatt~=catataaata_,ac4tcttat
g«t
Illg4CCaaaalalC«t_ClltglCIaCIggaIa(laggg«4llagallIClaIIgCa[[ltC(W'[Ivtlgl~il''a
gaalllgaall(gaIIfCIICIgCaaaCIIgtttatcuattaa~_~atclal4alll~ctaa~Ilfala:It~;ltflC
l~_g~
t«tatgattt«ctgagtlttgttclatgat~«ctlcattgttctlgagta~'tc«cttcaaa~ttc«~~aggat~~g~
[faggttta~~gtgga«cctctg«ctllagcta~ctaa~«ta[aaat~catg~«ccc«cl~'t« ta"~a~ttctta
algClalfIlCIItctaatcaattgaaaattgattttagactttctccccctaataagtgttt~at~aaatgttigaac
c:
aaclaafltcagagatttgtaga«ctagcccaagacctlggatgtttagac[gc«~«~aatltaa[a~~attegtg[I
tcatcaatgaaaattgagatlaggaagtggtaaagttalgagaatcattatcacgaaagt~
ea«~attt~gttctgaat
ctattgtctagggataacttgattgagtttg«ag«ag4taalggattgaggagtccactagaaccaa«accccattt
ttaagagclctgttlcgttlcattg(gcaaatctcgigtctttaglactctgttcgttttgtttagttctlgtg4aaaa
t
tggtglttttagtattctactcpacctatttaagfaatatgctc_gccgagtaccttacttagt~_ctto~tttacttt
cc
tgttcttgcacttgacagttccttgattttaglt[catttatgl««atcacttglgctct«acc«clgcttaglal
agtlt4atttccgaattlgattlgcatattaacclattgtita~gattagtaeaaacacaccaaacc;tattcacactt
gg
c«gacttagtattcictgaccacatcctgattgtlagcaattccacccaa«a~att~a4acctlaaalgctacaatga
cafaagatgcalttagggaattgacacacaaaacc«glatcaclctcaaataaaga'=gtca~'ttgca~_cacttagc
~al
c~_aattcacaaagttclcggalcacgcaa«Igactaatggctattgaatttagctaa~,taaaata~laat~=aaaat
aaau
eaaacaaaaagtaagcaatatcaatcaattgtgagttgtgaaaacaagataataaaagcgtcaggtta'ggtannnnnn
n
nnnnnnnnnnnnn~'atgtttggacactaaea4taaacclaaactctaca4cctaaatcccaaaacctaaaatc4aacc
cc
taaa4cclaac44cggaa4cctaaaagclaaacccaaaaccgcaaac4ctaaacccttaaacctaaacc4taaaccata
g
atc4taaactftaaaa4claaalcc[actttaggcttccggaatctggttgcggitctac«4«atg4t4aatgataca
caaagcatctagtcatattt4actacaaatcagctaa4caagccta«cttgattctcaaa~cut~~ataalgaa~=ccg
a
at[ctlatgagtlttittgttttgaalcgtttaacaaggaagcactactttacttttcgggat~fggttaa~~ttcta~
t
ttfatattlaatcatacacatgacaacacgtggtcatattt4a4ac4gaaacg4taacaaagattttcttgcttcttaa
a
~tatta[aatatatttgctcclaaacactaaacctaaaacctccactctaaatc4tgaaccctaaaatctaatccctaa
a
tcctaa4gctaaat4ttaaaccctaaaccctaactctaaaccctaaaccgtacaccctaaacc«aaaac4taaacc4ga
aacgcaaaalgtlaa44ttaaaccttaaaccttgaatcctalt«aacttcc~~~ttat««gt~tttcta~'«tttat
actgaatgalaat4aaaatatcaagtcatatttgactacaaaaacacaaacgaa~cttcttcal~,attctcaaa~ctt
tg
at~~gtgaagccgaaguc«aa4agtttttggttttgaatcgtataacaaggaa~cactac«tactc«cgc~atctc~~
tt~a~~ttctaettttatattctatcatacacat~acat4utclc:«atttcactct~aaat~
ctaaf4aasafcttt4t
t~cttcttaaa~fatcatattQ[at«eatcclaaa4aclcaac4taaact4ta4accclaaalcccaaaa4ctaaaat4
caacccctaaaccctaaccct~~aaccctaaaacctaaacc4aaaacc~~caaacmaaacccttaaa4claaaccclaa
ctcaatcaccattgacgagagctaacctaaca~~ca«ac~_aatcaa4aa~«aaa~c:caaaac~zmc44tycaaccaa
t
accttggtacagggccac~aatctctgeaatta~t~~«caoacatttcatc~aacacct«t~_g~c~,ca~aaatgtct
g
ggclcaaatt4cagttgatcaaaaagcaataggcatlaagaacaactaatccataa,=ggatctat4aatcaatactca
at
4a44ta=catactaa~aattctacactactc[aac4catcctcaaacctatt4actactca<_acatcataaca~aaa~
ca
taaacgttgaacaagttgaaaa4atgataa4aacagl~taacagcaggatgataacagaat~aacaaca~taaacgaaa
t
taagaacaaaaactgaatactgaata«gaatagataaagagaaatccggattacaaaaggtctcgaacacaaggtctgc
~~aataaaacta~aaaaaaaactt~ttt~ceaaaat~t~aaetacatectacttata~ccaaatatccwaaaccctaat
aClCaaaaCgaCgaaglatlggga4ggatlaagaactgtactcgac4Caagl~gl4ga~tgg~al,~'tC~';l~I~aa
IagCl
'_oaCICIICIIIICIICaIf~'I~CICLa'~lL'ICI_'_~lI'_a~,t_~C~YiIBt~'aa~a;l_~FiCl4l_~;l
;l~'4llV4Cal(:u_,~l:ll
~Ia~IC~aalIglggfgalgglgafaCICgaCflC:lag~lag;lglg~t~TagillC~a~lil~l=~141~~4;1:11
Y:la~~l~a
a~aCaCICi'aCCaC~'_nntC~a~[~~[lIYatCYa~l°al'~i'ICa~~I!,'al~"_a'_aI'_:1'_~
alaClC=;IO4lc:~l~~IC~'
a~_tg,'[gaggtgaaglgglgatttcurtactc~,ac4tgg~'g,;tc~~agt
g'=t4tctt~,agatcttyaa~ctc ctagtc~'a
cctc~~g~lc~aglgctttgacat~_atcag~[ccgctfrttccagatagct4~tcaaca~ctc4uaatc:lc4tyaaa
tca
acacaaatat'_caacatsca['~taaatctalcctaaaaat~taaaeteat'_acaaat~at~a~aa;Ue~tataaaa
ca~t
'_atcatat_at=ctaaast_~at~a4aaat~=at~ciraaaacatecaaaataca4acttatea~'ta:uetm:laat
t4t;l
t4aa ~coagtaa~~~tctg~[~~'factatcttc~ ~atcc4aaaa~atlagaala~tact4aac
aamat~'cwtt"atctg
atcaacatfaaccacac~atccc:cattalcccttct'_a<~aaac«aataa'_a«ct'~a~cctn:u~_a'_caa~'e
~c~act4
t'_l~eaataatctt~t'_tta~calcacc« cct«aaccat«~attct~_,ac«
4[~ttt<_'tuaaa'_eauma~a~,~a
li
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
~~cagc=aa~aaattccaatttlt~'c~~~'raacat~ttra~=ccctgaataagaagtca~_ace~'att~'<~acal
caatt~a~~c
ttgaatattactgaa,'ccacgtctattca,'accts;taca~=gccttcttagcctcttitaaga~_ttuctaa~'~r
aaaaca
t;aaagaacccaeagctatctccttctgccac~wa~mcaaaata~'aa_agataaaatcccgat~=egtc,':laagaa
agaa
aa~'laCll~aaaCIIllctIIIII~';laa~a~'~'c~'~':l~;l~=II:lIlga~~aatgaccatacacgel'-
C~f~~tC:l'_a~ICLCI
:1~_~:1~'~=~lCaaal'a1;1''Ca'_aC'_CC1'll~'~'c'a:l,~la~Caa''CCa;lC;l:lCCatta:IC:I
aI~HCICI:IIC;Ia';CllCl'IIa
_';lall~'gallalClli'CIA'uti'a11;1~~aCC::la~~l~'tacagcacacccctacacg~'tao;llCCaCa
a,'L'ICaCI'.,'ICICtC
:Ilacaa~~Cttgcag~'W
ctccaa~_ccu~'la;lggaaatattagacgg'catgagagagtaat~'ctrt,'I~=aca~'aa~_caat
tt~yttaaagtctccaacaacaa~ccal~ga~~aa«acaaagag_a;aggaa~rca~acaacc«gtaatatcatcccaa
a
~agaccttctctca;~'cacaotaltcctmcataaacaaaaagtaacagcaaac_actaagcaatatca~'gaa«ttaa
l
cgagcagaccattaactgatca~=tcctgctgaaaaccaaaacagaaartoacggatcccaaactatcca~atccgtcc
ca
ectctgaacagcaataattagaatcratcctccaacct~~ta~agtagcagccaaaaca~aactagcalt~tcctaa~c
~
acgtgagttaccaaaaaataacctaccag~_agattattc~'aagcaalccagc«cttactatccgttg~'cga~=acg
_act
a«aaggcctctaacatttcaaaaacagatcgccataa~Ttaacccaaaagatgataatog~aacaactamaatg~aaao
agaggactggcg;~~galcclagagtcatcacatctcaactta~acaaaaagaactctctcttgaaatga~cgct~ccg
~
«olgccacatttttcaa«gc«c«cttctcclta«ggacatcgcaccccaacca~~a~_sa~_gag~aaa:le~'aggat
caacta~ctgatccctt~t~~accgaatetcuc~acacacagactagttggacctccatc~~a~acctttgaaatagaa
ggcgccaccttag~gatctcaeaaagagggctaagaccagcttgaacaacagtaa~a~aa~aaeaaettactgtlatg~
~
tgccttcacgtctgatttgggaatccatttctgt«aggcaccgctgutaacatcccggataaeagctcttt~=,'atcc
at
ctgatgacttgataggaccaega~~caa~ct~t«gatctciltt«gaacgggacctccttcttttgaac«~=ata«I
atattgaatcatacatataacatctagtcatatttcactctgaaacgctaaccaaaattctttttgcttcttaaa~tat
t
atagtata«tgctctaaaacactgaacctaaactctactccataaatcccaaaaactaaaatttaacccctaaacccta
aacctaaaccctaaaccclaa;ltccaaaaccttaaactcttaaccctaaacccttaaaactaaacactaaaccmaaac
c
atagaccccaaactttaaaacctaaatcctact:ttagcttccgaaattcggttgogtttctattttttatactaaalg
at
atacaaa~=catttaetcatalttgactacaaatccgctaaccaagcttcttoatt~'tcaaaccttttat~~_t~'aa
~'cc~_;1
atttcttatgaetttttt~tttt~aatc~tacaataaggaacctatactttacctttcgg~attt~~tt~=a~~'«cta
~_t
«latattcaatcatacacat~acat~ta~tcatatttcactccoaaact~_taatagtgcacaaactactnnnnnnnnn
n
nnnnnnnnnncttctgtggtatgctgatcacgtcaattacctggttagtgotgaagagcecccaaatc«tyagctatg
agaagaagaagttcttcaaggacattaaccatttttactga~acoaaccttalclctacacacW
~Caaaaat:l;lgalc
tacaa~agat~tQtctc;lgaagac~aaatc:gaaggCatCCtactoCaCIgCCatL~;~ICI~=CClal~'~~iL~CC
aC:ItIYC
aacgttcaagacagtgtcaaaaatccttcaagctggtttttggtggccgacaatgtttaaggatgcicaa_'aat«atc
t
cgaaatgtgartcatgtcagaeaa~_agggaacataagcagaaggaatgagatgcctca«aatcc~atccta~'aa~_t
tgag
atc«tgacgtttgo~gaattgattttatg~~lctatttccttctlcttacgggaacaagtacatactgglcecagtaga
ctatetatcaaaatgg~t~eaa~ccataactaeccccaccaatgatgctaga~ltgtettaaa~ct~ucaa~acaatta
tcttccctagatttgga~tcccga~a~«gtaatcagt~ac~gaggoaaacattttatcaacaaggtutt~a~~aacc«
ttaaagaagcatggagtaaaggacaaggtagccactccttatcalccacagacga=rgg~'ca_gtggaaalctcagac
a~_
ggagatacaagcaattctagagaaaaca;tgggaa«acaaggagagaltgglcttctaaactc~'at~~ac~'cactal
gg~
cttaca~aacagcmtcaaaacccctalt~~cac~actcctttcaacctcctctatg~aaaatcct
~=ICa«t~cct~tt
~aactcgagtataaa~_cvat~t~'"~_ca;_ttaaactcct~=aacittgacattaaaaccgtc~'a~Ua~aa~~c,T
~=tl~atcta
act~aac~atctcaac~a~attcgcata~aa~ettat~'aga~ttccaaaatctacaa~~ayc~aaccaa~'tctttcc
at~
acaagaaeata~tctcaa~=a~attttaa«~~tt~'a't~atcaa_t~tt~~ct~ttcaacletc~cctaa"~'ctt«t
ccaay'
aagmc;tagtcta~ataetcto;=tcctttctctguact~=ca~tct~=accuat~~_t~'clatcactcta~cl~~;v
:taga;l
lggagacttcacagtcaat~~ctaaco~ctcaasaaatacat~ata~atca~ttlattcca~_aa_~agmct~'«cc:l
t
t~~aggagcctataaacgcttaat~aotat~a~~agtcaasttagagacctaaaacaapctcactt~~~'~=a~~~agl
clca
IoCCtatCIllgtaC:IIaICIIIaaItllCCII~II~tIIIIYaI~CaICII~IIa~t~IfIICa~~a~ataaolal~
aa
~agctaagvvaaalagaltclggClll!_:lg~_gaacaaagalacactcgaccac°gaottatcaa~=~_a
rarlc~acattgt
tct~~tttccccacccca~gaaatcactc~accacacccaacagg~accgaat~ac~atet~atc~_a~tat~~_e~_a
otta
t~=caatcaaaacttclccatgleagtaaatcactcgac:ci'c~'glgl:lgi'cCgcapcagaa~aa~=a4'a'_~=
tC~_a~=talcat
ca~a~Cf~IQCt~gccgteaCYaa~~a~Ca~a~'~Il~aataccccca~gc~~aa~Ct~a~aCOCaaca~=~Cra~a«c
ttc
nat~~cct~'_<_a'_caatcaca_'_'camtatl~am'accaaat«~'CIICIICIICCaC:Ii';t!'_'(aa~CaC
CICacICCac
catt~taatatarcalctcct~«tuatltl~lllClal_~'atclIlll~lcCl_Ya~l;lclcllllcCaaa«t~at:
l
CaCa~l~'~_aCl~t~'I~allfaaolll~ouu~ra~oaClc
a~~aa~l~l~l!'ItIC:lll~lalal:lalCll~'a~lCl;=Calt
catclgag~cata~aaaaa:lccaaaaaa:ltl,'aaaaalllragaaa;ll~alllcaC;laaaal:l~a~I~llcal
~la~=tl~'
CaIl~Callla~~'OIC~a~I~Ia~'a;_falllC~lllaY~~allCIl~CaIaIL'cata~woalaat~al~a,'ala
~CCt!''
(aaoCaIIll~~ltlaCC:l~alaar_CICai'l~'CCCIC~~ifi'll;l~lltlllYalVc'~ta~ICaaIrlaaal
l_'aaolaa:l,i
CIi'CaCCaI~CCIa~aIl';CICIaCIC'_aWac:l'_«a~~_alCfa:II:ICC;IIICCCI:Ite:I:IIIIYa;I
CIIY;IaICIL'
atlta~aallalCatL'tLlt~'_c'all~0atlt~'aaCalal~~alaCCCIaaa;ll:lCll29;1llllCllaC;l
CatlllaaC
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
cactcttgttaatccaa~_ta~~ttgactctccttattagagcagttaacccgaacccaaacctaaactctcittcaag
ccc
tatatcacttgt~;a~_tgttlgtgaygtctta«mcattgagW
tggtagaaagtgttagg«c'_taacyaga~_agata"
lgIClCaIgIagtlClagllCgCglltllc~'~aca~=~ata~~altgglg~gCECIlalafC:llgggtl!_goal~_
Igltla
aaagaaaagggtgaattcatlgttgataa~_gaa:l~g~aaagaattclag~~ggaagtaa~
claaa;=aagtta~taaaaaaaa
aatctagtaaa~~~=ttttg~_gaatgttaaa~;aaaagaatgaggttctigttagctaaagaagaagg'gttaaaagc
ctt«~~
!«taaatattaaaaaca'_gaaccttagttgtt:laagaaalccaaatccgcttgatgtatcagagt~=«g>a~'aaag
cttc
tcctagagliaagagaaaa~_aaaceaat~=atat~'aaaaa~~agtttgaaa~aticatga~'t~~caaag~'gtaga
gttaa~~tt
cttgtattg_~actgga~tl~_ggattaccattagagcttcaltgttatactct~~g~,tagat~'a=a«tatctctgt
atgc
ataac«gggac«acctttagrattctactaaagctcaatcattctigagagatcccctg«acttaagcctattctgt
aagsoaccalctctgtctcltgaccttcacc«agccaaatgatttcattgatgatgca«gc«gattcacgttccaga
actaatgaat~ttaaa~;ggattggtagatttgaaagcatgtgtaggtc~agtataa~_a~~acggattga«galaaca
ag~
catgactaacgtttttgagtaaaattctatcatatcgcatcttagaactaccaacttggacaltgattttatttgctct
a
cctgatgctttggttctgagtccccaccttcaaacctctcc«caactatgicttcttatttgcttgagggcaagcaaaa
actaagt«ggg
ggaattgatatatctataatttgcalgttttcagtatccaticatcatcgtttteagttta~tttc~ t
atcattnnnnnnnnnnnnnnnnnnnngatg«tggtcactattgtactggag~~tggacaatgccatgac~=ctggttgt
gg
tlot~agggaagtcactagcaccaatgttgttaggttgctcttgcocatctacaatgtcagccaltgtgtctgttgctg
t
ccgttctttcagttggcgagcaatacggttgatgttatcgttgaattaggaagttclaattcccttgtgaccgtgtatg
a
catgtatcaacctgaclataacagacgaataaagtgtgttgctaagtacctgaaatacaaattctgaaaagacacaatg
t
tagtatctctlataacaaaaacgaacttgatcttaacaa«ltgaaatclcaaalatagcaaacaaacacccaattggca
acggc
gccatattgataataaatttttaatcaaltalcctaaaacacaattcatgtcattgtagtattttaggtgtcaat
ccaaatggglgloatgcaaacagatgagatgtoatcagaagtcactaaglcaagccaagcaataacagttttggegttt
c
taacagtcctaagcgaacatgcagaaaacagaagcaataacagaattaetaaaatcactcgaccacaacaggctgatgc
c
agaagta~'gatgtggtc~~agtaacaggtcgagtaacagaca~gatatgaaaca~_ctatactcoactgcaca~=tct
gtt~c
ca~acactg~'~~tgtggtcgagtataclggacgagtaacagacaggaaacaagacaatcttactcgactgcacagtct
g«
'_ccagacactgggtglggtcgagtaag«~~glcgagtgaltgtaaaaactaagaaacaggcaatgagaacgattaagc
ga
taacgataaaacagagaaataagatagacgagaaa«cclaaggat_~~ggtaalc~agtagagtggtatcctagcctat
t
cgaatggltaacaagcgcaalcaagctatcactagacaacaeg«caacaacagatcaattcactcccgtgatagaaatc
ctcaagcaaagctaacccagactaattcccattaacggaatctaactatgcaggcaataagaacatactgalaaagtca
a
taaac=ctctaaacagccaatcccttgggatagagacgcatatctcaggaacta~tggatcaagcattccatcgaacac
c
ltctogglgcgggaatgcttgggatcgaattcc;agttgatcagaaagaattaaccagtaaacgcaactagtcctaaag
gg
attcaattaattctaaaac«aaccattctacagacaacgaattecacaactactltatcccalccctaagaattctaag
tcactaclcagacaacatgctcaaagalalaaacocagataaacgataatattgcalaaglaaagagataaa~_a~~ta
gag
aaacaagatgacagatgaaatcaaatgcggaatctgaataaclt~gaaaaatctegaattacaggttgca~_aa~caaa
aa
cggcgcaacagaglaaaacagaaaataaaaclgaageaaaaattcaatgtc~actg~;atgtccc:ag~_aca~=aacc
ctaa~=
acatctat«atacaaaacgaaataaaatgata~ta~'acgg~ccaataacctaatrgctcg~_cccatagagacataat
gg
~CC~agatyvaCtlCgIgalCCgafi,~aCOICC:a~=~'C~CtICC:laaCaCIII~?1CIICICCICIICICCICCI
IgIgCIC
aoCItgICtg~llgagtgcgg:ltttgagt:l~'aclglgaa~acgl~alcgaglgtcCggtcgagtgalgctgatggt
ggtg
agt~algalaCICBaCCa~~~~gglCgagla:lcltgglaga~t~allg~'ICg:I~'l~~IlglC:l~~~'1~~~'lg
l~~gCg:lBglgal
aClCgaCCag
~o'_~ICi'a~lagI~ICaIC~a'TIC'_CCl'_ai'(:l''ai~Y/laClC~_:iCCI!?~t~=glC~a'_li~~':l
t'.',~;~aa~:!
atggctacaaagtcactcgacct=gg~~tccactal~tcggaagaatggctecaaagtcac;tcgacct'_'o'gtcga
gta
tgtcttctggttct~ytc~=«tcttccaattt~_cttecaaacagctccaaatcacctgaaaacaactca
~aaat~~caat
atgtatgcaaa~ctatcctaatcat~caaaotatgcaagaatgat~a~aaatgatataaaaca~=t~=at~'aat~ata
ceaa
actggactcaaaac~atgatgaat~gacact~aaaacatgcaaattata~acatatcaactctccraaacttagtcttt
~
cttgccctcaagcaaataagaa~acatagtt~aa~ga~ao~agtgaaggcggggactcagaaccaaagcatatgataga
~=
caaataaaattaatgtccaagttggtagttctaagalgc~atat~att~aattctactcaaaaacgttagccctgcctt
~
«atcaalcaalccgtctcttatactcgacctacacatgcttlcaaalctaccaatccctttaacattcattagtlctg~
aacgtgaatcaagcagl=calcalcaalgaaclcalll~~'Claag~lgaag~=ICa.l~agacaaagal~glccclla
caca
ata~~cltaaglaactlg~g~alaCClC:lag:1;11~2fIl~:IYClll:l~la~':latyCtaaagglaaglCl:l:
a:l~llal~Cala
la~_a~_ataa__'aICCCilICIaICC:I~:!_'lala:lC:lal~a:l_~?IIL:I:l:ll1'v[aaICCC:I:I(:
l(:CI~,ICCC:I:ICIIaa(:ICi:I
CCCttI~CaCIlal2aalCIllC:l:laClC111ItC;II:IfOatl~llllc:IlllCICII:I:ICIIIa~YaYaa~
CIIIClC:1
aCoCICICaIaC:lICta~L
2~~~lIlLT2aIIICIII:IaC:I:ICla:7Y=IICCI~IIIIIIIIIaIClll:laa:ICC:IaBY~CI
lltaaCCCIICIICIIta~ClaaCae~':IaCClCaIICIIIICIIIlaCallCC
Caaa:ICCIIIaCIa~~allllllllllC
taacmttagc«acttcccctu~aa«W ttecc«tcmcal~aacaal~~aauc;ectcttttc:««aaacarat
cccaacccat~'alataa~=cgcccacctagtectauc
agtccyaagaay_c~_:Iactagaactacat'_a~~:ICactatctcl
~tc~ttar_'aacctaacact«ctaccaaecmaalceaaataa~auemac:laacaclcacaa~_t~
atata~e~c«~'a
aa~aaa~tteas_'ttt~_~'~tale~~ltaacl~_ct~_taalaa_~~~~_a_'tca~ctam~~atcaac:aa~~a~t
~_~_« aaaat
17
CA 02362897 2001-09-18
WO 00/~~325 PCT/US00/07392
~agtaa~aaaatccaagtattttagggtatccat~'a~«caaattc~at~ccaa~_acatgataattctaaatcagatt
ca
a~tlcaaatt~ata~g~aatgatatca~atcctaacagt~rt~r~rtc~ra~tana~caalcla~~cat~gt~_ca~tt
ttactt
caat«caatgacaacgcatcaaacaactaacaac~=ag~r~c
am~_agcttatct~~rt_aactaaaat~_cttacaag~ct:,
tctcttcattalcccctatgcatatgcaacaatcctaaac«aaacaetcta~ame,!atcctaaatgcaatgcaactac
a
t~aacaWCUUttttgt~raaatcaltttctaaaaatttW
atttttute,'tttttctat~CC'.tla''alg:l:il=C:lgaC
lc::la~_all:llal:l(:aatgcaacacacacttect'':1 L'CCCti CCC:IaaC
ttaaatcacac::l~'tCC:lt:l~'I ~'1~'ilCC:la:ll
Il~'~:tat:1'':1$IQCICa~gaCaailaC:lCalCaCaaaaaC aaaal aaaaaCaa~=a'al ~
gtalatl:ICaal':;l~gag ( v:l
=c'tgcatacctcagtggaagaaggagcgg:I~II~';IC ~_ccaata~rcttCC f
~l'~all~~CICCC:I~'"CC:ILC Yac~lra:ltct
ccclgtl~=lgtctcagcttccccttggg~gtactc~accmcttcttcl~mt~zca~eca~caccgc~'~_tc~;agte
aalt
actgacatgg~=gaaglcgtgattgcatcattc~_ctatactc~=atcacatcttcattcgctccctgtag~=gtgtg~
tcea~
t«atttcctagcgtggagaagccagaacaatotcga~t~~tc«t~_att~ctctgt~_gtcgagtgtatlgtto«ccct
ca
aagccagaatccatttccctcagctcttcattcttatctcct~aaaactctaaca~~aatgtattaaaaataacaagga
aa
aataaaagatatttacgaaaatagacatg~=acucctcccaa~t~=a~~cttgtt«aagtctctagc«~=actcctcac
a
CICalaagaCtlaaggagggtgatagagaa~aatt~aaatc«ctcttcttccttt~Ca~atacaacatC;eacttagct
tgctctc«gacatgtctgtaattgagcactcaatp~ccctttctugtaaocttc«aaatttcttgtgg~tagtcttc
ttcttcacaccattctgaaacgaacttcttctttgetataacttcaalgccgagttgaggttgaatagcticcctttgc
a
tctgactcactgtatccctaacticttcaacagtgcaagclagc«ctggatge«tc«ct«gagatcagaattcctt
ttaaettctagccaatcaactatagagagcgtacta~acteagtg~ttga~cttgaaggtcgtgttggagctaaactcg
g
ttcctttactctttcagtttcaaatctttcttctgtaaccactcctt''eaccccaaaggtctgtccctctiuggtlga
tc
ttctt~gagttttgttgatgtcaaactgtaacclgatatccttcccaagatt~alttcaatc«
Iccalctttgacgtct
atcactgctccaactaaggccaagaaacgtcttccta~~ateagageatcctta~=~~ttcttcatccatctcaa~cac
aac
aaaatctgtag~cacttgtactccattaatctt~ata~~rcalatcttcta~_ta~~ccalaag~ttttcttaagg
tcctat
cagcaa~gatcaaggataggtcgtatggcttata«toaaaaatccca~cttcctt~ccacagaaa~tg~cattacgct~
=
actgaa~ctcctagatcacatagacagttgctgaaa~tca~u~~cctaata~a~~cat~r~la~aetacaa~ratcccg
gatc
ttccaacttttcttgaataatgttcttctagatgatt~cactaaactcgt~aggtaaegccactgttcctlgaacttct
t
tgatcctctctgtaatcatgtccttcacacgtttgtgtgag«cggtatta~CgCaagacaglctaclagaggcattgtg
atttcaaggtccttcaattttttttccaagagagcttcatac«c«tatca~ugcttcttaaatctccctggaaat~~
taalg~a~~ctt~tag~~tgga~~aacaaa~ac«tatcttt~_,_t«
ta~ca~atecag~ctttttaact~aa~aaa~ta
ctacttgagc«gggatcgagtactgtgatc~a~=tatgp~ctcctctaat~_ctattttagcttgaatttcattctgaa
ca
aaatcctccccttcttgaacttcactgtccctaglgatggag,'tagaggcal°lc°agtt~gca
gctctc~atcatggca
~ataltgaloECglgagCagIggCglaCICCIIa~~attCl=~aII~CIC;ICCCIggaagClg_~CCI~~=gllgllg
llaa
caeatg~f~la~C~_aggatgccttccacatatctcalccta~t~CIC;J~t~cItCYaaCIIgCIgllaagllC~aC~
L'Cll
tgtcggtc~aclttgttatttaatttacccaacttcttagcgatltccatt~ClCCI~t~"_CIIQICCII~aatgagc
ag
ctggagcatagtcattatgtctgaattctgaggc«caaclg~ct~ttgtl~'atgtggtgtgaaaccaggtgotggtta
ct
g«g«gat:ltcctccttgaaactgcatctttg~gac~aalrctt~=~=ct«g~ttofaa~~=aataaaoo~ttt~golt
gg
ttttgctg«gctgagggtagacttggtcatgaQggtlg~cgato«~gt~cttct~'tat~;aca~atttg~ollgggtc
t
gtaattgttgtaccccttgttgtagccaccttga«ct~aacatagct~taa~cacact~ratcattctccccctcttgt
a
c«ggaatggttcatcttgagagatgaggtgaaeatgl«clgclyactt~gag~attttgtctagittetcaugaga
actttcatgtctctacogtgcttgtcatcagagtcagaacl~~t~=c~=~'at~ctlctgtcataatcitca«ataa«g
cc
atcc~=act~a~caaagttttcaactagctcccaccc«rttcaacatccttctttaae:laatmcccttt~=aagc~at
_t
caa~=ta~catccttatcttag~caagacgcctctata~'a~t~t~'clga~'aa~a~raa~cugcttaaamc~~t~_a
t~ag~;~
cattttettt~ataacccttaaagc~«cccaagctmalagaaae«tcacttt~_tttct~a~_t~=aatcc~rgatatc
tc
attcc~~a~tctt~ca~ttctggagtagga~aa~aau«ycaa~'aaa~ctttttlgca~teatcccag~tcetgatt~
atccctggggta~c~tcttttcccacagat~~~clttelctcc;aa~'lgagaat~~aaacaa~c~aagcttgaaacca
tcl
tcactaactccattgattttagtaaggctaca~agcct«caaactc~=lcta~=at~atc~aotg~atcctccattggc
aa
vCCaluaaaCtt~IICCCCIgcaccatt~caat~a~aCI~CIIII~ralClCaaa~_LI~=il~llll~IBCIC~a~gl
ggaa
CaaI~CCalgacgcIgoIIglggltolga~~gaaatcaCl:I!'C;ICCaaI!!Il~lla~~lI~CICIIgC~'catct
acaat~
tla~CCatI~l~lCl~tICIgICCEItCIttCagll~oC~a=Caat:lC'"=tC2al~llalC~ll~aala~~aa~'ll
Cla
aIICCCII~_t'LraCCYlglalgaCalglalCaaCCt~aCt:lla:IC:I";IC'':Iala;l:lYl~l~ll'~Claa
~~laCClQaaSlaC
a:laflCl''a:l:la~aCaCaaIgllaglalClCII:IIaaC:la:l:l;IC''=:I:ICItYatCll:l:lC:a:l(
l(:l'?aaaICICa88lal:l
_YCa:IaC:laaCaCCCilatl~~CaaCggCYCCaaalt~ala:lla:lulllll:lalC:lallillCCl:laaaC:I
C:I:lIlC:ll~lC:1
ll~la~litlllla~gl~'lCaatCCeaaIg=clefYat~Ca:l:lCa~':Ilea,'all=alla~:lalrlCalIa:I
gIC:Ia~CCae
.C:l:lla:l(::1;IIIt~g~glllClaaCa~lCCIaa~C~:1:11;11~C:IYaa:l:lCa~=a:l~'C:I:II:Ia
C:l'aall:lC'.l:l:l:lll(::1C
IC'=:1CC:ICiIaC:t~~Clgal°CC:I~:la~la~~al~'l~''lC ":I~l:la~':1,'_rll'
Y:15.'l:l:ICa~:ICa~~al:Il~:l:l:ICa~Cl:ll
:1(:IC~aCI';CitCa~lCI~II~CC.aY:ICaCI~'vYtYl=_lCL'aC'I;llaCli'vaC:~':l~la:lC:l~a
Ca'r~aaaCaa''aC:IaIC
Il;ICIC_aCf~CaCa~ICI~II~C(::I~aCaCIY~'~l_lS';-
lya','.l:l:l'rllL'YICY:Ii'l~:lll~laaal:lClaa''a:l'?lil
CA 02362897 2001-09-18
WO 00/ss32s PCT/US00107392
g~'caat~~agaacgattaagc~~ataacgataaaaca~~il~'aaataagatagac
gagaaattcctaa~;gat';~yglaatcgag
taga;_'I~~'talcctagceta«cgaatg~ttaacaagcgcaatcaagmatccctagacaacaggucaacaacagatc
a
a«cactccc~_t~~ata~=aaatcctcaa~~caaagcta~~ccca_actaattcccattaac~=~aatctaactatgta
~gcaat
aagaae:a~'actgataaa~~tcaataaacgetetaaacagccaatccctlge~~ata~'a~=ac~'catatctca~';
~'aactagtgg
atcaa~'cattccatc~=aacaccttctg~=gt~=r~ggaalgc«~;ggatc~'aatlcca~tt~;alca~aaag:laa
taagcagt
aaacgvaarta~'tcctaailg
ooattcaatcaattctaaaacttaaecattctaca~'actac~~aattccacaactactua
tcccatccctaa~aattctaagtcactactca~acaacatgctcaaa~atataaac~'ca~ataaac~=:uaalatl~;
cata
agtaaagagataaa;agtagagaaacaagatgacagat~~aaatcaaat~c~yaatct~aataacttggaaaaatctcg
aa
ttacagg«gcagaa~_caaaaacggcgcagtagagtaaaatagaaaataaaactgaagcaaaaattcaat~tcgact~~
a
tgtcccaggacagaaccctaagacatctatttgtacaaaacgaaataaaatgata~tagacgggccaataacctaatcg
c
tcggcccatagagagataatgggccgagatgggcttcgtgatccgat~=ga~~tcca~_gccgcttccaaacactttgt
ctt
ctcctcttccctccctigigctcagc«ttctggttgagt~~cggatttgagtagact~t~aagatgtgatcgagt~tcc
o
gtcga~=tgalgctgat~gt~_gtgagtgatgatactcgaccag~~~glc~_agtaac«_~tagaglgatt~,gtc~a~
tott
atcag
gtggtgt~gcgaagtgatactcgaccagggggtcgagtagtgtcatc«agtggcctgagctgaggttactcgacc
tggggglcgagtatgtgg
gaagaatggctccaaagtcaclcgacctggggglcgaglatgtcttctggttctggctcgtt
tcttccaalttgcttgcaaacagctccaaatcacctgaaaacaactcagaaatgcaatatgtatgcaaagclatcctaa
t
catgcaaagtatgcaagcatgatgagaaatgatataaaacagtgatgaatgalacgaaactggactcaaaacgatgatg
a
atggacactgaaaatatgcaaattatagacatatcaactcccccaaacttagtctttgtttgccctcaagcaaataaga
a
gacatagttgaaggagaggagtgaaggcggggactcagaaccaaag::atatgataga~caaataaaatcaatgtccaa
gt
tggtag«ctaagatgcgatatgattgaattctactgaaaaacgttagctatgccltgttatcaatcaatctgtctctta
tactcgacclacacatgctttcaaatctaccaatctclttaacattcatta~ttctp~aacgtgaatcaa~cagtgcat
c
atcaatgaactcatttg~ctaaogtgaaggtcaagagacaaagatg~tcccttacaaaata~=~cttaagtaacagggo
al
ccctcaagaatgattgagctttagta~~aatgctaaa~;gtaagtcccaagttatgcatacata~ataa~atcccatct
acr
ca~a~tataacaatgaa~ttctaatggtaatcccaactcctgtcccaacttaactctaccctttgcactcatgaatctt
t
caaactctttttcatatcattcltttcltttclcttaactctaggagaagctttctcaacgcictgatacatctagcgg
g
lu~gatttctttaacaactaaggttcctgtttttttttatctttaaaaccaaag~ct«laaccc«cllc«tagcta
acaa~aacctcattc«Itctttaacattcccaaaacctttacta~atttttttt«ctaacttctlla~~c«
actlccc
ctaeaattcittccc«tcctcatcaacaateaattccctcttttcttttaaacacatcccaacccat~atataa~c~cc
cacctagtcctatccagtccgaagaacgcgaaclagaactacatgagacactatctctgtcgttacgaacctaacactt
t
ctaccaaactcaatcgaaataagacctcacaaactctcacaagtgatata~ggcltgaaa~aaagttcuggtttgggta
l
~ggttaactgctctaataagga~a~=tcagctacttggatcaacaagagtggttaaaatgagtaagaaaall:caagta
ttt
tagggtatccatgag«caaattcgatgccaagacatgataattctaaatca~=attcaagttcaaattgatag~~aatg
a
tatcagatcclaacagtgtggtcgagtagagcaatctaggcatg~tgcag««ac«caa«tcaatgacaacycatca
aacaactaacaacgagggcactgagcttatctggtgaactaaaat~cttacaagoctatctcttcattatcccctatgc
a
tat~~caacaatcctaaacgaaacactctagactcgatcctaaatgcaatgcaactacatga~cactctattttt~tga
aa
tcaltttctaaaat«Itcat«ttttttttgittttctatgcc«agatgaatgcagactcaagattalatacaatgca
acacacac«cct~agccctcccccaaacttaaatcacacagtccactgtgC~2lCC:aaatltggaagagaglaCl(:8
gga
caaaacacatcacaaaaacaaaataaaaacaagagtll~gtatattacaati'~=Iggilgl~a~~lgcalaCCICa_I
g~taal'
;la'_~agCg~agllg'_ICgICaalagCIgCClglgal«llICl;Ca~gl:l:all:''aC:L'aaICIC:CC:IgII
gIgICECagI:(ll
CCCllggggglaC'lC:~:1(:CICICIICIICIgCIgCiIgCCilglaCCgC~'IC';agl'_aattailgacatggg
'=aa''IC~I'=
atl2_CaICa«Ct'ctatactC~alCaC:11CI1(:aIIC~CICCCI~
laC'_'_l~l_'~°_ICc'ant~atlICClai~C9tL'~aHaa
2
CCagaaCaalglCgaglglClllgallgClCtglgglCg:lglglallgtl_uccctcaaagccagaatccatttccct
ca~ctcltcattcttatctcctgaaaacictaatagaat~tattaaaaataacaaggaaaaataaaagatatttacgaa
g
atagacatgggacttcctcccaagtgagcttgttttaagtctctagcttgaclcctcacactcataagacttaag'~ag
gg
t~ata~agaaeaatt~aaatcttctcttcttcc«agca~atacaacatc~~
aCIlagClIgCiClCllgaCalglClgl
aattgagcactcaat~=gCCCIItCCllgtaa~=cttctttaatttcttgtgggtagtcttcllCllCaCaCCaIIClg
aBa
cgaacttcttcttt'_gtataacttcaalgccgagttga~~gttgaatagcttccctttgcatctgactcactgtatcc
cta
ac«cttcaaca~~t~Caag(:lagCIlCl~gal'?gIfICIICIt~gaoalc:aYaallCClIll:lil~llc:lagCC
aalC;laC
laCa~aga~~CglaCIa~aC:lga~I~gtt~=a~!Cttgaa~'glcglvtl~'ga"c't:til;lC'tC~gItCCIIIa
CICIIIeeglll
C
aaalc:lllCIICt~taaCCaCtCCII~~acccCaaagglc;l~ICCCICI:tlgi'tlgillCIICII~gaPIIIlgl
l~aW
tcaaact~_taacctgatatccttcccaa~attgatttcaatctuc:catctuga~
gtctatactgmccaactaag~c
Caa~itaac 9IClIOCla~2al'_a~a~'_all;llla~~llClll
alCl'alCtCaa!'C:1C''aill'all;laLCI~la~l'l~Cll'~la
ctcca«aatcttgala'gcatatcttcla~=taggccataagg«tullaa~~ll:l'I:IIC;I~Cail~,?all:aag
galilg',
tC,'lal~C'Cllalatlli'aaaaatccca~lIICCtIgCCiIC21!_aaagl~~lallaC~l:(~:1(:I~ailCCt
C;ClagalCaCa
l;lYilL:lglt~'ct~'aaa~ICagII~cclaala~;l~Cal'~gla';ai'IaCaai'att;CC''~'alcllCCaa
c fIIICII~aataa
l~llc llel~~:«;=all~_cactaaactcgt~~agglain'gCl:aC'I;~IICC'II'=;I:1C'IfC
l~l~'atl:l:ICIClgtaatCat',
CA 02362897 2001-09-18
WO 00/~a32S PCT/US00/07392
tcc«c acac~rt«~tglgagmcggtattagc~~caa=acagtctactagaggca«gtgat«caag~_tcc«caaltl
Il«ICCaagagagc«catac«c«catcag«gcttc«aaatciccct~_oaaat~'~taal,~ya~_~=ctt~ta~;~y_
t~_
~a~gaacaaa~actttatcctt~~tttta~ca~at~!ca~tctttttaacl~~aa~aaa~~tactactt,!a~clt~_~
=~'atrsa
~tacm~atc~ra~'tat~r~_gctcctctaat~ctatttta~_cttgaatttcattctgaacaaaalcctccccttett
~aac
( r /' VV' VV n VV V V (1 VV O V ' VV . V 1 VV V V' V ' t
ttcact_ICC;ca;yt',u' _a~_ta:a~~cat_tc~a_tt~=ca_ctcic~atc,u~ ~ca=atatt; at,
_c_t_,l_c,y
Iggc!!t:lctcctt:y~'attct~,~atl~clttccct~gaai'et~'gccl~=~ll~'llYllaac;l,'al~r~~l
~la~Tc~;lY~al~
CCIICCaC:II;IICICaICCta~l~'CICagloallCgaaCIIgClgllaagllCg:IC~'ClllYlC~=SIC=aCII
IgIIaII
faaltlaCCCa:ICttCtla~y=alllCCatIgCICCI~?t~gCttoICCII''aalgag(:ag(a~'g:l~'Calagt
Cattat~_I
Cl~aattctga~rYC
gCa:ICIggCIgll~tt~atotggtgtgaaaccag~=tgglggllgCl~;llgltgaI:IICCICC(l~:l
aactgc:llClll~''gaCga;IllCIIggCIfI~~llgtaaggaataaa~~~'gI«gggttgglllIgCIgllgClga
gggla
gacttggtC:ll_aggg«ggcgatattggtgctlctglalgacagaltigggttgggtclgtaallgltgtaCCCC«gl
tgtagccacc:«~attctgaacala~ct~taagcacactgatcattctccccctcttatactt~gaatggttcatcttc
a
~a~atgag~tgaavatgtttctgctgcacttggaggattttgtctagtttgtcattgagagc«tcatgtctctacggl~
~
cttgtcatcagagmagaactggtgcggatgcttctgtcataatcttcattataattgccalccgaclgagcaaagtttt
caactagctcmaccc«cttcaacatccttcttlaagaaattccca«tgaagcggtgtcaagtagcatccttatctta
ggcaagac~cctct~la~agt~tgcteagaaeagaagc«gcttaaalccgtgatgagggcattttgtttgataaccc«
aaagc~ttcccaa~cttcaca~aaactttcactttgtttctgagtgaatccggatatctcatictggagtcttgca~tt
c
t~~a~ttg~a~aagaattttgccaagaaagcttttttgcagtcatcacaggtcgtgatt~atccctgggglagcgtcll
t
tcccaca~atg~
ecttt~tctccaaglgagaatggaaacaa~cgdagcttgaaaccatcttcactaactccattgatttt
a~taa~Qctaca~a~cclttcaaactc:gtctagatgatcgagtggatcctccatt~gcaa~ccat~aaactt~ltccc
ct
ecaccattgcaat~agactgcttttgatctcaaagttgtt~ttttgtactgga~gt~oaacaatgccatgacgctgg«g
tuvllvtvavvv;la~rlcactagcaccaatgIIgIldggllgclcllgcgcdlclacdalgtcagcca«gtgtctgtt
gc
I~ICIgIICIII(:;t~ll~'~~CgagCa:lt:ICggICg:llgllalCgllgaalaggaagllClBaIICCCIIg(L'
aCCglolaC
~ac: atglalCaaCC I ~:lCf:llaaCag;iCgaataaagl
glgllgCtadglaCCIgaaalaCaaa«c:l~~aaaa~acacaat
~ttaotatctcttataacaaaaac~aactteatcttaacaattcteaaatctcaaatataocaaacaaacacccaattt
_c
caac~_yc~ccaaatt~=ataataaatttttaatcaattatcctaaaacacaattcat~tcattgtagtattttagstg
lca~
atccaaatgg;_t~=lgat~caaacagatgaeatgtgatcagaagtcactaagtcaagccaagcaataaca~rutt;~~
gggu
tctaacagtcctaa'=cgaacatgcagaaaacagaagcaataacagaattactaaaatcactcgaccacaacaggctga
tg
ccagaagta~~at~=t~gtcgagtaacaggtcgagtaacagacaggatatgaaacdgctatactcgact~caca~tctg
tt
eccagacactgg=tgtgglcgaglatactggacgagtaacagacaggaaacaagacaatcttactcgactgaacaotct
g
tt~cca~'acact~~_t~t~~'lcoa~taagllgalcgagtgattgtaaaaactae~aaaca~~caataa~aay~atta
agc
~ataac~=ataaaaca~_a~aaataagata~acgagaaattcctaaegat~ggoldalcsaeta~'a~I~~tatclcag
ccta
ttcgaat~~ttaacaagr~caatcaa~ctatccctagacaacaggttcaacaaca~atcaattcactccg~tgatagaa
a
tcctcaa~caaa~_ctagcccagactaattccCattaacggaatctaactatgcaggcaataagaacagactgataaag
tc
aataaacgctctaaacg_'ccaatcccugggata_dgacgcalalctcaggaactagtggatcaagcattccatcgaac
a
CCIIC[vY,_t!'C='W~:taleCtt~~u~'atCgaatlCCagtIgalCagaaaoaadlaagCagIaaaC=CaaCla~t
cctaaa'_
ggattraatcaallctaaa:lc«aaccattctacagactacgaaltccataactactcattcccatcc:ctaagaattc
ta
aotcactactcaeacaacatmteaaa~acataaac~ca~ataaac~ataatattacataa~taaa~aeataaa~a~ta~
agaaacaagat~acagatgaaalcaaat~_rggaatctyaataacttsgaaaaatctcgaattacag~~ttgcagaagc
aaa
aac_'~caca~cat_a~taaaacaaaaaataaaacteaa~caaaaactcaat~tc~acta~al~tccc:aa~acaea:l
cc:cla
a=acatctat«atacaaaac~aaataaaatgataglacacg~gccaataacctaatcgctcggcccatagagagataal
ovoCCaaoaln~roCIIC~I~aICC:~_alg~a~y~ICCag9CCgClICCaaaCaCllt~lCllc:lCCICIICICIIC
CII~=I
clCagCltgtClg~llgBglgCagatttgagta~aCl~tgdagaCglgalCgagloICC~'~tC~_a~loat~Cl~=al
'_~tg
2lgagl~3lgalaClCgaCCag~~~aulC2a!'t:laCttggtil~agl~allgglCgaglaltolla~gl~~I~I~~C
~a3al
~ataC(C
~:ICCa_~g'=°tC~':I'=laglotcatcaagtg~CClgagClgalQIfaCIC~aCCC~~_I~LICQa~t'
~'al~gaa
agaalggClc
Ca:laglcaclCgaC(l'ru~gglCgaglalglggoaagaat~gClCCaaa~;ICaCIC'=aCC~'_'=r_~~L'IC~;
J
Yl:«gI(:llc'IggllCl~~CIC~=IllgllCCa:ItItgCIlgCaaaCdgCICCaaaICdCClgaaaaCaaCICa~a
aal~=C
aatatel;tl~Ca:lai'CfalCClaalCalecaaaf?tal~caa>_'autoaloaQaaal_~atataaaaca2t~al;
a:ll(_alaC
oaaaCtvLTaCt~;laaaCvateat~a.I(oCaCaCl~aaaacat2caaattata_acatalcaccaacccla2l~'at
a~lCl
a~totl~ovCy:t~lCvyaota~r~ICItatamattaaacactcl_i'CL_ctt«aa~aaaacaat~tatac<_Ia~Ia
lala
'?(:IIaIaC:IIIt?C::la;lQ.l'?llaC:ll:aaaaatatatctttatelCl_~3ICCll~ttaaaaatacataa
taacaaetaattt
taca~aa~caac~caaacatccaa«t«I_~caattccta~tettc~tcttrcc~ctat~ctcatcccactat~attacc
tataa~acaa~la~aamat~a~taatcldecatcccitaet_'amtl_~~lctatecac:aaataaatt~ctat_eett
taa
tcaacttaataa=taaam;l_'malc;t~_a_'atcacaaaacacctaataaac'_lattamatatat,ltataataat
acaa
~aatatyca_tcaaama~aca~at~aat_Taatcttg~~ctca~catactycaaccttaccaaaaag~'t~rcamacaa
c
atca~caaaacctccc~catacl_t~tatacatt_ttciccacac~tat~clacca~acacam_tacat~c;tcaccca
caue
ZO
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
acacgtacgtacacaactalgaacaccc~agctatagccacaaag~cttataaccg~aacatcactttattgcatcgtc
a
tycattacccaacatacactct~_agtcttgtgceatcatgtaagt~accac~_caa~atcaagac;ttactgacacaa
tcac
aaaaaaaaaatgaaggc;I«ca~cttaclctaa~catgatgetatcgg~_Iaaaccttyaataccacttagacacaata
acc
tcatgcagtactaaatata:fat:tgtttaccacaccccaaacaagtataacacaatcaaglaccaaatcacg««ttat
at
atatatatatatacalaatata~caa~=gac«a«cacaacctcatcgcatatctcatataagtataacaatclaacaag
~caagtaa~'ccctc
gaatcaaatttcaa«catca:lgtaaacttagttaagtccaaaticavtt:I«ccta~~icatgcacc
agtctattcca«ccttaatgatattaactatcaagttcttcaaatacccatca«ttaa~a«eaagttctaactta~=a
ctgaattctgatta~~ttaagtctaagtgagtaagcatgcatatgattgataagt=t~~tatlttecat~_t«tgagca
tcc
atttgtcatcattttagcatcatatcatcactgttttataccatttcacatcatctgtcatcactttgcatgt«aggat
agttttgcatgcatgttgcatat«gtgtlgttttcaggtgattttgagctgtcgat,=a~mtaactgaaagaagcggac
c
tgatcatgacaaaccactcgacctagaggtcaagtaggagcttcaagatctcaagagaccactcgacccccaggtcgag
t
agaagacaccaccactctatcacatcactc gacccccaagtc gagtttc«catctccatctcttgaccaccactc
gate
acctcactcgacccccaggtcgagtgac«catctccatcttctgaccatcactrgatcacatcacttgacccccaggtc
gagtgacticatcccattgccagacatctacacgacctcgtcactctacctggaactcgagtatcaccatcacaccact
c
gactgcatactcgatgaaaagc«cagagccttrttcattctgcactcaaccagacactcgagcacaaggaagaaaaaaa
gactccagctattcactcgacctcccactcgaccacatgggtcgagtacag«c«aatccgtcccaatacttcgtcgtt
ttgagtattagggtttcagaatat«ggctataagtagcatgtac«cacatt«cgcagacaagaag«ttttatcgag
ttitatttccgcagaccttglgttctagacttttgtaatccagatttctctttatctattcagtattcagtattcagc«
ttgttcttgatttcgttacactgttgttatcatgtcttcaacctgttcaacg«tatgctttctguatgat~~tctgagt
agtgaataggtttctgagoatgggttagagtagtgtaggattctcagtatgctaggtgattgagtattga«gatagatc
ccttatagattagttgttittaatgcctattgctttcigatcaacttgaatttgagcccagacat«ccgcgcccaaaag
gtgttcgatgaaatgtct~aaccactaattccagagattcgtgaccctgtaccaaggtattggttgcaggga~lcgttt
tg
gctttaacttgttgattcgtaalgcct~tta~ottagctctcgtcaahgtgattgagtctgggactaggttaacttgag
~~!IIICIgtlgCggtggCaClIagalftggttaalgaaCllgttgtCtaggg«aa«IaElgagCalglCaaIClCllg
taaactgagaaaacgaatctactcaattaccccatcctcgggaattctttatctgattgaattc«tgtttactctactg
ttgtttactgcatc«gttatctgtcgc«aatttctgaagttctttcttgttaclcgacc«atacmgaccacctagt
tgtctggcaacagactgtgaagtcgaglatctgtg«ICa«ttctgtctgttactcgacctgtcattcgacctcaccca
atgctctgcaaagagcacg«cacttaggactgctagaacacaccaaaacctgttattgcttg
gcttgacttagtgactt
ctgatcacatctggattgttagcatcacacccatttggattgacaacctaaaatactacaacgatatgattggtgtttt
a
ogataaatgatctgaaaaacctattatcaatttggcgtcgttgccaattgggtgtttgtttgttacatttgagatttca
g
aCtIgCttCgatCaagtICIttIlCa3tttlCafItIagIlaClgaCtglol~IlllICIIgII~ICIICICvaCCoav
a
tacatctctactatattcaaactcgatcaacaggcaaccagaacctcc«ttcaatgataacatcgaccgcctagttcgt
gaacttagag;faaggagaa:lcatagtcaacc«ataccgcagcaacgactcgaaatggctgacgaacaaattcaacag
aa
tggccctacaaacattg~tgctega~atgcaccacgggaccacc~tcaaa~gaaa~rgaattgcacetcctgctatcca
ga
acaacaacttcgagattaagagt~glcacatctcgatgattca~g~saataaattctatggtctgccaatggaggatcc
a
ctcgaccaccttgat~aattcgataggcictacaacctaacaaa~atcaatggtgtcagtgaa~acggaticaagctcc
g
tttgtttccagtctccttg~gcgacaaa~_cacacatctgg~=agaagaatctgccccatgactcaatcaccacctggg
atg
att~caagaaa~cttttttatcaaagtttttctccaatgccagaacl~caa~~actc a~aaat«a~a«tcttg
tttctca
cagaagactggt~aaagcttctgt~a~gcatg~taecgtucaa~ggttacaccaaccaat~mcrtcatcac~=gcttta
c
taaagcctctctgctcagcactatttacaaaggagtctaccacgcatcagaal~cltm~=yataccgcca~caatg~ea
a
tttccagaacaaggatgttgaagaaggctgggaattggttgagaacctcgctcaatca~~atggcaattac;aacgagg
act
gtgataggaccgtcagaggaaca~ctgaetctgatgacaaacacag~aa~~_a~=atcaaa~cgtt~aatgacaagctg
gac
aggattcttcttagccagcagaagcatgtgcac«ccttgttgatsacgagcagg«caagtccaagatggggaggataa
ccagttggaagaagtcagctacatcaacaacaatcagggtggttacaaacgatacaacaacttcaaaaccaacaacccc
a
acctctcctaccgcagcaccaacg«gctaatcct~=aagatcaagtgtatcctccacaacaacaacaaggtcagaacaa
a
cctttt~ttccctacaatcaa~=~~t«c~ttcccaa~caacaatttcag~~=gaactaccagcaaeaaccacctgggtt
tgc
acctcagcaaaaccaag~te'=tact~ctcctgatgctgaaatgaaacagatggtacaacaactgctacatggacaagc
at
ctagctccatggaaatt~ctaa~aa~ttatetgaatt~cac:cacaagct~~cct~maactacaat«accttaat~;ca
aaa
ol~gag~Cgtl~aattccaaa~tca~atac«agaaggaCaatCC
gC:ilC«CCtrtYCtCCC;laa~ttaci~a~tacttcc
aaggaagtcaa«cagaatcccaaggagtatsctaatgttcatgctatcaacata:Iggagt~~yaagtgagttatccac
tr
~agagagacccaa~tcagtcact«a~_gacagtgcataccaagatg=gga~,~atttca~'tctcaataaagatcaagc
tgae
aaacaactc~agca~ccactcgaccaytcactcgagca~ceaetcgacca~'ttactcga~ma,_ccactc~acca~IC
act
egagcagccactcgacca;_tcactcgagcagccactcgac cagteactc ~:IgC;I~CCaC'tt,'al
Ca'=ttaCtC~a~CagC
CaCIloaCa~laCIC~a~a;=CCaCICiTaCIagtCaClC~aYC;IYeC;tCICY:ICCIYIC:ICIC~':IgCI~~CC
:7ClC~aCCaC
ttcactcgaccgaccttcccagc;latatcaccaam~ctcyaa:watgtt«ctglcaaaa:maaa~_aaaaa~tcttca
t
ICCICaICCI«ICaagCCC;CCaCIIIC:IIIICCtaglCgaCala;lEaEla~_a'_ll:lY;A:lYaa;l:li'tac
a~_agclal~~llEg
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
ccgagaacatcaa~=~_aagtcgaattaaggatacctgtt~=ct~atyctctaac~=c~tatccc~,~attcccaaaag
ttcctc
aaagacttgamatg~agagaatteaaga~_~«caaaaaacaacag«ctaa~'mat~;a~t~caal~ccatcataca~_~
a
gaacgatgtcccagaaaagccaggag:tccctggttcattcactctaccat~ctecma~=o«cattgactttcaataca
t
~cctttgt~,accta~,~a~,catcagtaaatctcatgccactcgcagta~ctaa~'a~'~=tt~,~ltttcaacaa~t
ataaatac
t,caatatctcctt~lalcttg~ctgatag!'tctgtta~~actacctcat~_tgttatta~_aa~'a«t~'cctaaca
a'~att~!e
aaat~_t~=~=a~~t~c_,taca~acttcgtagtgctaaacat~~~_at~taa~aa~caaaa~'alcrml~'atatta~
~aa~~gcclt
ttttg~ctacagct«~,agcaalcatagatgttaagcaa~_gaaa~=att~atcttaam~_~_~=gaa"aattt~~aaa
tgaag
tttgatatcaatgat~=clatgaagaagccgacaatagaagaacaaac
ctlcttaeltaa~~~_aa~=t~~aacaottagct~,~,
tgaactgctagt~gaacttgaagaagcaaataattcaaaaactgttttgaccaa~_a~'lg~_taaa~~t~~~~tatct
cccta
~tgaaactttgagttctaagaagtcauagactcacacaag_cagcagtl~~gctca~a~=yc«acaa~pgcttgatggg
~
tctggcacaaaggttatgaaagctaatgaagtaagttcaacacatectc~accaactaauccactaacaactcgaagaa
gcctccaacttgtcctgagagatcatgcicgaccaaacaactcaaggcaagcacua~at~=tcaaat~attggttagaa
c
tcaaggaga~atctaaat~gcaagaaaaagccgtacaagagctaacacatattgtgaaa~a~~ctaaa~~atcaaatca
a~~
gagctcaatggaaaagcaaaccaagtactactcaacatcaaggacgcccct~_ac~at~~c~ccaclatacttgttagc
aa
agaaggatgtgaattcacgtcggaatggtctagaggagaagactatcttgataaaaaagaagcttactgtgaggacatg
a
ccactgagtacccaattgcagacatgtcaagaaaccctaatgagtatgatgatcacgctaccaaagaaggagaggagac
c
tcatttcctctctacaacccaccctaagcctaatgagtatgagaagtcaagc«agagacltaaaacaagctcacttgg_
~
agcaaaacccttgtctatctttgtacatatctttatttttccttg«gtttttgatgcattt~'~ttagtgttttcagga
g
ataagtataaagagctgagtgaagtgga«ctggctctgagagtacaacactatactcgaccacagagcaatgaaggata
ctcgaccatgttttagcttatctacggcaggagatcactcgactgt~_gtgctggccgcagca~aacaatggaggtcga
gt
atactcatggcggtgctggccgccaga~a~_ctgatgaggtc~agtacccacct~_cl~~,a~ct~atacagaacaa~g
tggt
tcgtctatggccttggagcaatcacaggca~_ccattgacgagcaactac
~_ucaucuc~att~aggtacgcgcctcac
ttcaccatt~tattataccatct~ttstoattt~tttcticattttttp«
tct~t~attt~_attt~tcct~a~tactct
cttccaagtttattcacaca~tggactgtgtgatttaagttta~rgg~,agggcte:l~g;l:l~I~l~lgltgCallg
tgtalil
Itlttgagtctgcattcatctaaggcatagaaaaacaaa:waaaattgaaaa:mtea~;l;l;t;ltgatltcacaaaa
aaaa
oagtgttcatgtagttgcatcacalttaggalcgagtctagagtg«tcat«ac'ac«g«gcatat~_cataggggatg
atgatgagatagccttgtaagcattttga«caccg ~ataaactca~~tgccclc~«~'rtag«~«t~tt~lc
gtagtca
atgaatttgaaataaaactgaaccacgccta~_att~=ctctactc~=accacacl~_tcat~atrt~ataccattccc
tatca
atttgaacctgaatctaalctttaattatcatgtctgcatcaaatttgaactcatogataccctaaaatacttggat«t
cttattcattttgatcactcttgttaatccaagtagctgactctcctta«agagcag«aacccaaacctagactttct
ttcaa~ccctatatcactt~t~a~t~tttUt«a~~tcttattccea«aa~ctt_r~ta~.aa~t~ttae~ttc~taac~
a
caga~atagtgtctcatgtagitctagttcgcatttttcggactagatag~acta~'~_t~ggc~c«atactltgggct
g~
gatgtgt«aaaagaaaaaaagggg«gaticattgat~~a~=aaaag~_taaa~,~actcta~'~=t~=aa~taa~ctaa
agaagc
agaaaaagtctagtaaag~ttttgg~atit~taaagaaaaeaaa~a~=ttc«~tta~=clatl~_~aaatg~~caaaag
ccc
ttggttttaaaatgttaaaaaca~gaacctta~=tt~_ttaaagaaatccaaatcc
"ma~at<_'taacuaa~tgttgagaaa
~cttctcctagagttaagagaaaagaaaagaateattayaaaaa~=ggcttaaaagattcatga;ltacaaa;~ggtag
agtt
aagttcttatattgg~attggasatggeattaccattagagcttcatttaacalactctgg~lagat~g~
a(cttatctc
t~tat~cala~ctt~~~acttaccuta~rca«c taetcaamtlaa«att««_'a_';matc uect_«am~~aa~cc
tattctataagggaccatctttgtctcttgacc«ttaccttagccaaatga~_l«tttt~';u~al~_catt~cuga«c
acgttccataactaatgaatgttaaagggaglgglagattl"aaaactt~_t~=ta~_~'lc'~=a~'calal
~_a~'lc~~attgatt
r~attgatgaggcatggctaacgtttttaagta~;aa«cgatc«gcagctta"aactt«aacu~~aca«tatttcat
ciggtctatctggtgttttggctctaagtccrc~;gtttcaaacclcacctcca«ctt~«cltgatt;_«tycttgagg
g
caagcaacgactaagt«ggggga~ttgataa~tgt~tattttgcatett«~a~,catccat«~tcatca«ttagcat
catatcatcactgttttataccatttcacatcatctgtcatcactttocatotttag~~ata,,tttt~cat~'catgt
t~cu
tatttgtgttgttttcaggtgatttggaactgtcgacgagctaactggaagaagcg~acctgatcatg:lc:aaaccac
tc~_
acccagaggtcga«taggagcttcaagatatcaa~agaccaclcgacccccag~lc~aglagaagacaccaccactcta
t
cacatcactc~acccccaa~tcoaet«cttcatctccatctctt~accaccactceatcac
cteacaceaccccca~~t
c~a'_teacttcatctccatcttct~accatcactc~atcacatcamc~accmca~_~me;l_t~:tctmatc:ccatt
~t
ca~atatctactc~acctc~tcactctacctteaactc_~a~tateaccatcacaue:tcm_~ac
'_catactc~ateaeaa
~CllCa~aCTCCIICIICaIICC'scactcaacca~ill;tll(:~:1~C:ICiIa~~;l;1~;1:1:1aY:l:l~';l
etCCa~C:latlCal(lQa
CCICCCaCllgaccatataggtcgag«ICaaalClliIillCIgIICCiIalaCtlL'!(C~Illl~a''falfag~~l
llCaaa '
alalllggctataa~l3~=Calg(acttcac:tilllC gCa~aC:lag;l:l~ll ~lI ll;Ilcl;I ~~l I I
I;II I tcyCa ~;lcell
ol~IlCla°aCtlllglaalCCggalllClCtItalClallCaYIallCa~lallC;!_~Cllll:lllc
ll~';IlllC_111:1
Clalt~IlClIlCt~llalC~lCCl2CI9lIaCaCl~tl2ttalC;Il9tCtl(:a;IC:CI'_ltCa:W
1'III;II'~eIIlCt21
Ial9al~ICl~a2lil~l_~aala!'_~ttlCl_';t2!':Il!_'v°-
tla,'a~_la_~'121;1.'Y:IIICIc::IL'l;ll'W l:l~_i'l~'alI_~ar_ta
Il~att~ata~alCCCltc'lg!_;111:tgltYllCIlFlal,_CCtatl~_Ctlllii'alC'a;ICIY~;t;lltt
~'a_'CCCa~alall
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
tccgc~cccaaat~'~=t~«c~'af~~aaat~'tct~'aaccactaa«cca~=agattc~'t~acc'cl~'tacc;la~
'~'t;llt~'~;lt~,
Cagg
gagCtlllli'YClllaaCll~It~allC'_l:lal"CII~II:t~~'lla~CICIC~'tcaat~'i'l'':1(l;_:I
YIC'tL'uY;7C
la~~«aactt~=a~,~gW
ct'_fl'_C~'~'l~_~_C:7Cll:7~';illlf~'=llalll~'a:t(:IELI(~lCl:l~'Y,~ltOalll:7ll'_
a'=C
at~tcaatcactt_laaact~a!'aaaat'~aaactactcaattaccccatcctc_'~_~aaumil:ltct_';Itt_'
:I:Itml
l~'utactctacl~=u~ulacl!_calcll_'u:Itch=lcnttaalucl'_cacltct«clt~llactcyauc:m:Il
:1
ctcgaccacctagttg'l:It;~caaca~'act~t_'cad=tcgagtatctgtgtttcattttetglcl,'llacty_;
leel;_tca
ttcgagclcacccagt__ctctgcalaga~'ctgl~tt~'~Ttc~'a~tgttttaclgtttct~t«~,:n«itt~_ttt
m~=r:l
I~IICaCIIa~~aCI~Cla~;laC'aCaCCaaa:lCC1'!llall~~CII~~CII~aCll2l~l~aCIICIL'alC~aC
;IICII'=all
~ttagcatcacacccaltl~~:fllgaraacctaaaatactacaucgatatgatt~'~t~'lllt;l~'~'afaallga
ctaaaaa
cctattatcaat~atctaaa~acta~cteta~~aaaaaaigcaaacaaacacacaattcgaccacaacar;lla~'all
cggf
aallCYgaa~~~lllClglglelfgggcaaaatlccfcgalcacgglC;gaCgalCCCII:ICICaI
~''ICa:l~lt''ICI(:~C
CCaCaagCgalgalIClCCagl:lllggaCagagIIICCICK:ICCBC~YYCaa~alllatl!':l;lllli',~YC:a
aYIIIICIC
ggllglggaCgagllalltgll~ggC:IgCCIIaCaCICICtCICIIgCCII(:aaa:la:l~';I:lailll~~t~"~
a~;l:l~:7~Ca1
catilalaglccacccaaggtcagClalgCagCaIIgICCCClgCCBCaIIICCgg'aCaaCl~(
l~C~~mcccat~_cgac
ag~t~tctcgaacaat~,_c~lg,~ceacagtt«gcgacg~et~tcactccacc_~ccclatcagctty_,_clacaa,
=actca
gccaaclttggclctaaggttg_teacllgagcigfaaggcactgcga~t~ae~t~tccactcat~tc~=~IILC~Coo
~C
eac~~~ocaaacttcactacaacatgcccaactltlcccalcttcgcccattcg,t~cccattcct~cctccattcct~
cc
catctttgccct«gatggl«ggc:accttttg~gcacatacggtcaacta~crat~cauc4'~_~caaea««cfca«
cgglcc«Iggccglolccgal~,cccttat~cgg~tcacta~a~gtaatctctt~t«a~tea~~t~~~_«aa~attaag
t
agatccctttltaallcgttcgccctaalgctlatftcactcagttaagtttggaattc=acrcaa~=,_tatmca~t~
tc
CaaaagacettCa:lal~~:Il~C:I:I~aICI:ICS'aattctagagattagtagaclcta~CCfalai'tIl"a~;=
laY:ll~:l(::l
aaaacaaa~ralCQacttC~fl:llgctgaaccgac=alga:tggagcatultCl'_i':IL'CI;II;IC.'C:a;lL
:1:1;1t~'IICCIQaaC
lCoIIICaCIII!_CIIIIaItlIaalll~aacllall:7lalgCICCggcccatttataatttat,'~:1;1:7CIlI
t!_'l:Iall
IICCCaaICIC_'aaafattcattlt~~eaQatactculaaali;'ca~_LECI°ail'_~aaC_'Cal_'a
:7lllilc:eLC!'aatr
actcc~clccag~ato~aaatc~ccaacclcaag~gafaaggtttctttataaatl~=~=cacl;_laattllctuatt
cct
CaCCallClalIIIIIICCIIg~Caoalalggaa«CaaCCllCgglgCICggaalCCatgW :li':Illlc'IC
Ya~Y~IC~'
aaatgalggacggtgaaattctgallgtgccgaglaggCggccllclccalCll~'cvvcllc:Ilc:l~'~'lcccaa
allcga
gCICCgIgIgCICIgaICa:IggaICfllCggalagagaCCaagaaalCllgalg
gC~lICalaf;l~'~'l~Cll~'C~_attca
acatcagaagcgtac~Clg~agaYpaf_'oaaac;lCoaoaaaClQallCaal~Bal~CaaCaIB''~~=f~aCC'_l~
aflCaag
nL'aactttataccclcaactcccaCllfa~flaCCCecalaacCsCalco2aLaC,_~lCaaaL_'a~c'_'~alCaa
aatttt
tt~cccgaataeaaa~ocaatc~=acc~'c~t~tatccaaaag~lgccaaaacattaaa~~~caa~aac~'g~caa~aa
tggg
ClC
_~a8tgg~Caaa~~ato~aaaaa~ttonoCofoCC~CggCgaaglllaIaCCCCgIC;,'i'CI~C~,VaaCC~'a(:a
lg8gla
~g;ICacatcacacgcagtgcctfgcggClC:IagICaCCaaCCllggBgCCaaaYlCa:ICI~_H~ICII'_':I~CC
~L':la~'lIg
8lgggoCvofvvau[gaCaCCCgICrYCCaCl:lglCaCCaI:gCCgICgICC'EaL'aCBCII~IOY~I;t''lS'~~
'CC CagCagC
IYICIeeaCalll9oCa2CeY~ct'2I2C1_C;ll~e~CeaCCIIL'~°C~_ealfalaaaea'_8_'I:IC
I~IC Oi'C~Ca~IIIC
alltlloaaCCcaa~a~aoa~a~:7Cf~ aaa~_~ClataCYattataaactceaaCCaaactaa2aaaaac:IC_'W
:l'_aatC
'_attaaatctt~cccgtggtcga~~aaacatt~~tctgagactagaaaaaattg'cal=t~_~ttaa~aaaacctcam
gtga
staa~~~atc~=tc~acy't~calga~aagttgcac«aoacccaaaaaactttcctaacta~t~aatcc~t,>a~_«
~'t~'ct
l~aallll~l~lffolll'_Cal~llllCta~a?ClaolCIlIaooflalalECali'Cllal:lC:7Cll;I~;ll'l
la:ll:l:l:ll
l~aBaflCg~Cll~a~lfaYalalLgaatctcaaagllaala«ggCatllg:ICgaaCllualaalac;t:ll
aelaa''"_a
at~~aal~=:lll~_l~aat~aClaC~c'I:I:lal_~a:ltat~'t':7Cll:l:lc:la8~'111:1C112:llla:
lllY:1_';Il;ll'_atle<_'a
~~~CllaCll~'Cgttgftagallgllalacll:Il:ll~:lValal~'c;lal~agcllglg:laleaafc:ClI~c:
I;llallal~_1 '
alalalallgalligglgCllgallilel;ll:ICllglllggggCalgglaaaatacttalatata~taetacat"ai
'_lt
allololCfaaolvofa(Ila'?oolIlaCICoaIaCCalCalgCIlagaglaagClgal
~CCfICCIIIaI~'I~_lCl~la
a~fCtt~aICCIgaYIggICICII:SCaI~':7l~~CaCaapaClOgga~(:~Ial~IC'_~~taaayal~:lC'=at,
_c:alfa:la
~t~al~tlcc~~ttalaamclltet~~ctata~_rtcooetoucaca~ft~l~lac'_tac'_t'_t'_~l'_t~~'_'
_t_'t~cau
~tac~t~t~t(to~ta~calac~t'_t~ea~aac:acetatacac~tat'_c~~~a~t«~t~lt~at~ttet~_'tcca
cc«
tttt~taaa,~ll~~caVlat_'«~"_,_cccaa~~ttcattcal~letcta~ttt~~cl'_calattrt'__'la«
a«ataa_'
rata(salaatac~tttattac~t'm«t~t'_atettl'_at~~alaa_Tlttacctattaa~«!'attaaacccta_'
n'_at~
flat(t~f~cata~accca~ctcaca~a~taatcua~_attaclcal~attclactt~fc«aca~~Jtaacc
ata'_1,'~'~
al2accata'1C~2_aaCacea:lcaclaa_~_aaat'CCC2aaata~~at'_ttltaC_ott'_cttclW
aaa;Itl;llYllal
laf~laaat«taacaa~_~alct~afalaaa~'alalalifltg~~laaclclft~claa~tataa;~matataelat~
'c':1
laaa~I~f«tcllaaHo~CaoCW'aaf_'lllaal9alataaCaccla,?ICl'~l'_f'_'WtaW
';la_Ta;lla(c;u'~aY
nolfoaCaaCCCfa;7Cl~'_Clc'll~Cta_'_la_'_'_a_'_';l~n~lC'_allalaaltlCL'Ifaa'_'IaW
aaalW ;IltlWla;lCC
lClaClaa~ll'_I'_'_IC''?alCalc'll(':ICI~Iai'CCtaaltC~~tc~TllCal'_~CII~ICC_'_>W
:ll:l_1'L,_'C''~:Illc:_Y_Y_Y
'_~C~IIaCaCCIaCCCC'_alCall'_'_2CC'.a'_llli'°_a;l«lCt'Za:IIIIYCaC'_'l:laa
iCi'_';IW li:_'C'1'__'I~a:lr'~:1
1;
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
~tccaa~_t~!ttctgcac~_«aa~"I;f~'a~utc
ct~=attcgaa~~t~cacacafgataaaacttc~_tclcmu~_c«atfa
ctt~aalaaatgtc~~ctttgl«cct~'lct«acat_«cacatagccgaacttfac«lc:ctf~ttcgttcaaaca~'I
c
aaactctttttct«mat~~acaaaataaaaaaaaaaatauagtagctgaactCgtticcga~_ca~_cctac~'tatcc
:al
tlC!?~_galc;aa!_ccacacci'taettc
alyLacataacaaaaaat'=cglgllaCllaaaCalaaa:lf:ItglIl;lY:lttlC:1
CCaC:IIICC;I:I'.T.i'aCgCI~'gaCl'='~CCIi'ICIICIaa;IIgCII(:IC;~CIaCI~IfIfClll:l'=
'_C~C'=C11:~'1'CLI:ILCI
al'nolll:taCClllIgaCIICCIIf'=Cll~':1:1C~'=l''''f::laIICI:IICICaagCIC:II;CaCi'at
l:ll:L'IYC'CL'C;I~CIC'=
ayacctt~laalgaC:llaaalllCIfLCICCICfIlatt~Clllgt:lCCgaaafgCClICtacc~_'~aalgacWll
lC1
CCIIa'_i'al'_CI~aCICi'°Cllll~laCtClatalcalactC<_Clla!'CICCaCIi'CCCaIfIli
'CCa:«CLICC_'faCi
aaattggaccatgaagaattg Ilcgaa~=l~=~'c l ggllgglwacacttcg alC g
~alCa'=allgg:l:l:ll;ll i' g:ll ~'Ca~C
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~;aCllacl
lalgtag«hallggfllllglll:lCC:ICaalCIlgcaggatcaagaCICCgCtaaCIgCalg~llC~aCaCCoaaaC
'=l
ac:aaatatagCttttcatCC:lCItccg~lllgaaCaalac:ag~t'lggCElggtcaaettcgctttaagl:Ig
lCCaaaI~CC
ICtICaCalllCllglCCCa:Iaggaalc:ClllgIllCCI«CagaaIClgaIafaaagglaaagaCll~=ICIZIIga
ICI
t gaaafaa:I:IlggtlCaaagCl gCaatClg:lCCggilaaCC gllglaCIIalIlgaagaaCl«ggagal
~=aCalgIfla
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ctgaatgtacatitcgccagg«ca~tttcattt~=atattgattaagaatttcaaaacattcc«caaal~_cct~'atg
tg
otcttctttcttcaatgacttgaccaacatatt~tctatgtagacttccattgtttttccaagatgctcgtagacatt«
oflgaccaaccgttg~taagttgctcct~ca«clttaaaccaaaaggcattaccttataacaataaatacctclalact
t~acaaatgacgttttttcctogtcatcaagaltlatcatlat«aaalafaacaagagaaggcatccatgaaagltaac
aaclcgttccctgtagtagattccac~~aggceatctatatgtgggagigagaaatatcctttgggcaagcttta«gag
g
tctgtgaagtcgalgcaaacl:ctglcl«tc
l;attc«ctttttcaccacgacgglattt~;ccactaactctg;_ataaa~=
~aL:CICC(:IlalagaCCllat(:IICaaaaY«lglCIaCCIC'=ICgIIIaCCgCCIICgCICffIICI:ICICC'=
ai'a~ltt
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ca
Igtct'=cttccgarcac~=cgaac~=at~=lt«cutttcctcaaaagttcaatcaaggcttcttttattttttcac't
taaa
olagctccaattc~aactgtelgatcf~gtttatcttcltctagtactactttgatltcgacgtcatccactt«tgate
tcgg~_ga«attcattttttcgcgggg«nnnnnnnnnnnnnnnnnnnngcllcaatictcattcla~_acagatgatca
g
ctacaccgttttcaattcccttcuutcaacgatctccatgtcaaactcttggagcaataa~=atccatctcaacatcct
a
vv[Ilggl:llCClll;ltCgcatalatatgC:CICagagCagCalgalCa~lglagaCIaICaCCIaa~'ai'CCCBC
Caggl:l
ecttctaaatttcttgaatgcaaataccaca~~ctagcagttccttctctgtggt~~calaccttac«gtgcatcafcc
a
tcgltctactcgcgtagtagalcacatgca~=c«tttatcaatccgctggcccagaactgctcclactgcataatc(ga
a
~catctcacatgatctcgaat~=gVaagoccagtttggagcltggactattg~=ggcagtgaccaaagc«ccttgatta
a
cttaaaggctgtcaaacattcalcatcaaat~_rga:lclcggtttccttgcacagca~tctggtga~_cg~lc«~'cc
aact
f~gagaaatccttgatgaalcuctgtaaaatcctgcatgtccaagaaaactcctgatglccaagaaaactrmgatgtc
cllgaaa~talllggl~ecfglaact~calcatcacglcaaccttagccttatcaacctctattccctcctca~aaatt
t
tglgccccaaaacgalgccticttlaaccataaaat~=gcatttctccaagttcagcaccaggttt~tclc«cacatct
t
ttgagtaccctgcatag~tlc~acaaacac~_ag~=agaagaaagagccatacacagaaaaatcatccat~
aatacc:tcc:ac
catclcctctatcaaatct~agaaaal~_~aa~lcatacaccgctgaaaagtagvaggt~cattac;lea~'acc~aaa
ggca
tac~cttgtaa~caaag;=tcccataag~acaastaaaagtagtutclc(tggtcalu~g~igaatag!~,'at«~'aa
aa
aatct~=ctata~ccatccaagaa;_caata~'laa~'galggutectaatctttctagcatctgatraat~_aa~_~=
~caat~'~
gaaatggtcttttctaga~=gl a<'ca«t;l;IC:ItI
l:««aalcaatacacatcctat'_tcia~=ttatagtecta~=t~_g~'la
lcag«CaICIllafaatllllHalaaCl''gC:II:ICCICCtItlllagnaCCgca~l~aacfg~a~aIaCC~'aa'!
l~'CIa
fCagagalaggglaaglalltCClIIIICaC;IaCCICIIICaa~ItIgg~IttaatcICCltlga~'~fflaat~Cla
~'av
laggaltCallllC;lagalggetlalatg=gl:~eataa~_ol~g~lgaaatcccctfaatatcatct:laCg:l~I:
IIC;Cl:l1
agCICICCIaIaClfCllga'L'ClCa~llaICagCa~~=If(aCIIr=alCaglagllagClCglC:IllC:lll;Il
~aCagYal
aagtagaglttaglccaaea:laaac~lacctlay=cCCILllggla~cgIIIlcaaalclaclllg=_l~cc
It~:I~IIC:I
gaCCaglCalC
_ggala(:Iaa~=Itr;°C(lCa~a~_~'aggootlofaalCgagtagll(:gglCg:lYlal:lCl~aal~
"=LClfll
lgglCgagIgalglgglCga~talatt~'ataaglglalallllacafgltttgagcatcca«tgtcatl:aal(tagc
at
CatatCalCaa1g1I1CaIaCCa«tCICalCallt~=LCaIC:ICIII:ICaIglllaggal:lgillllgCal_'C:1
1~'llgC:l
lalttgC',,,IlgalIICagi'lL'aIII'=°a~'ll,'llgac ,'arc I:ILCIY~aa~aagC
gg'=CCI g;llCal''aCaaaCC:ICIC
;1CCCCaan'L'lC~_agta~'ga~_ellCa;tYallIC:laCaE,IC:ICII~aCCCC~(:g'_ICga~fa''ai'~a
C:lICaCI~CIICaC
(:aCaICaCICgaI:CCC~a~_'=IC~a'=I~IICIL
aICCCaII:;lI:l:Igacc:atcacllgall:ac;;tICaCIC'CaCCCCL'a~n[C
'_agl'_ICIICaI'CIlCall~'CCaoall:aC'IaCIC,'aCCIlIIC:I(:lClaC(Clggaa~ll:ga:1I;11l
:aCCaIISICaCLaO
IC°_a(:I'_~alaCll~al'_alaa'=LIIIa,'a''CILIC'IIC:IIIC:I;aC:ICII:Gall;agaC
aCll:Yaglal:la'_';:la'aaaa''
aa~aclcca~ttattcactc ~'acCICCeaCIC~aCCICC'~'~Cl:tll:l~
~~'lllCge;l:llafll~~~Ctataa~_t:l~_Cat~=t
aCIICaCaIIIICW
aCaCC;l2llllCWCI:I~_lltt:ILICC~'CaCaCCII~ICIICIa_~alCllll~taaftl;aCatll
CIClalalllallC~olallCa~IaIIC:ICI(lt_'Ilc;ll~aLIICaIIIaCIaII~_ILCaIICIr_IlalCalC
CI_'Cl
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
2flaCaCIgIlgllalCalgilllCaaCll~=lICaaCglIIal~CIfICIglfal~_alglCl~'agfagl~~a.lt;
t~'glllC
llagval~~sna~agta~'tglagaatlClea~=laloCfagglgallgagiat(gallgalagaICCCIIeIYgalIa
gl
t~IICllaalgCCltIII~~ClllalgalC:;I;lCIL':l;lathga~CaCagaCaIIICCgC~=(:CCaa;la'~~'
I'=flC;;al~~aaat
_toga.lccaagglallg~=flgca~~gLa~'C
~'flllggcltlBacllgflgallcglaalRccl~Tlla~=alta_c«tcgt
caat~=~=tgattgclgtctg~_~_acta~=~_ttaamya__~=gtctct_~ttacg~_tagcac«a~_,f«
Ig~_Ilaal~_aacttgtt
PtCtagg~_ttaatttattga'_CalgICaaICaCCI~'I'=aaClt~llgtf'_ag=:IaaCiaa;IClaClCaalla
C CCCaICC
lCgggaatlClllalCtgallgaaltClfl~IIIaCICIaCIgtf~III;ICIgCaICtlglIalClglC«CIIaaIIt
Ct
'_CaatICIIICIIaIIaCICgaCCICaIaCIC~'aCCaCCCagfI'=ICI=gCa.llagaCIgIgCai'It1';li'I
;IIClgtgll
ataCtIICIgICIgIIaCICgaCCIt'ICacIC~=acctcaccca~tgctggcaata~lag(:lot~Illy=tC
~:IyI~ItttaC
t~tttctgtttgattttctgttttctgcat~=atcac«a~~'act~ctagaacacaccaaaalttgtlattgc«agctt
g
acttagtgacttctgatcacatcttgaftgtta~catcacacccatttgga«gacaacctaaaatactataacgacatg
attggtgtttt~_g~ataattgactaaaaacctattalcaatttggcgttgttgccaattgggtgtttgtttgttacat
tt
~agatttcagacttgcttagatcaagttcttg«caa«ttct«tcfgttactaactgtglgtftttcttgttgtc«c
ttgatccaggtacacctctactgtatgcaaactc~atcaacaggcaatcagaacclccttttcaacgacaacalcgacc
g
catagctcgcgaactcagagaaaggaaaaacacagtcaaccttgtaccttagcaaccactcgaaatggctgacgaacag
a
atcaacaaaatggccctaccaacattggt~~ctgeagatgcaccacgtlatcaccgtcagag~_aaag~=aatagcacc
(cct
octalccagaacaacaarttcgagattaagagtggtctcatctcgatgatttaggggaacaaattccatggactgccaa
t
gcaggafccaclcgaccaccttgatgaa«
egataggctctacaacctgacaaagatcaatggtgtcagtgaagacggat
tcaagctccgtttgfttccattctccttgggcgaaaaagcacacatctgegagaagaatltgccccatgactcaatcac
c
acctgggafgattgtaagaaggcttttctatcaaag«c(tctccaatgccagaactgcaa~actgagaaatgagatttc
tggttttacacagaagactggtgaaagc(tatgtgaa~catgg~agcgtttcaag
gg«al:accaaccaatgccctcatc
alggc«factaaagcctttctactcagcactc«tacagaggagtccfaccacgcatea.laatgctictgcataccgcc
agCaalggg:latttcCagaaCaaggat~«~aaYa;lggctg~'~aauggttgagaacctCgctcaatl:agat~~gca
atta
caac~'aggactgtgalaggaccgtcagaggaacagctaactctgatgacaaacaca~_gaa~~~=agatcaaa~cgct
gaatg
acaagctggacaggattcltcttagccagcagaaYCatgtscacttccttgttgatgac~=agcagtatcaaglccaag
at
ggggagggtaaccagttggaagaagtcaactacatcaacaacaaccagagtggclacaaagggtataacaacttcaaaa
c
caacaaccccaacctctcctaccgcagcaccaac~t(gctaatcctcag
gatctagtctatcctccacagcaacaacaag
stcagaacaaaccttttgttccctacaatcaaggttlcgttcctaagcagcaatttctggggaactacca~
ccgccacca
ccacctgggtttacacafcagcaaaaccaaggttcl,'ctgclcctgaggctgatatgaaacaaatgctaeaacagctg
ct
lcacgggcaagcctctagctccatggagallg~caaagaagatatctgagttacataacaagctggactgtagctacaa
tg
atctcaatgtcaagatggaaacactgaataccaaagtccgatac«agayggacattctgcatcttcttca~
ctccaaaa
cagacaagccaactaccatgcaaagcagttcagaatccaaaaaaatatgctcatgctatcacaclgcgtaglggaaaag
c
acttccaactagg
gaggaaccaaagacggtcact~ag~acagtgaagatcaagatgg~~ga~=gattlaaglctcgagaaag
atcaa~ct~acaaaccactcsa~caaccac(c~atcteccacoeacaactuaetcaaccaacaac(c~_acc:latct(
c
ccaoca~catcaacaact~ctccaaaacaa~tt~ct~_'tcaaaaacaaa~'aaaa~~tcitt~tccctcctccctaca
aacc
acagcttccatttcctggccgtcacaagaaa~cltt~_~ca~ataaatatagagctctgtttgc:caagaalattaagg
agg
llg;lgllgCggalaCCICIIgllg;lCgClCIagCYCIaaICCCagaCICICaCaag«tctgaaagacflga«gtgga
g
agaattcaagaagtgcaagggatg~t~=gta«gagtcatgaat,~ca~=tgctatcatacaaaagaagatcaucctaag
aa
octlagt~atccl~~gttcaucactctaccal~ c(c«laggtccalt~=~cutcaatagatgc
ctatgc~'atttaggag
catcagteagtetcatgeegetcict~_tcgecaaaagatt~_g~~,ltcactcuatacaa~~lcctgcaatatawc(c
afe
cta~ct~aca~atca~taa~~atccctcat~e«tec«saaaacctcccaatca~~alc~~t=tt~_'I_"_acalacca
ac
tgactttgta~tcgtggagatggatgaagagcccaa~gaccc«
Igattclagggagacctttctlagccaclgcaggag
ctatgattgatgtcaagaaagggaagattaatctaaatc«g~caaagactttaggataacct(t~=atgtcaaagacgc
g
atgaagaagcctaccatagaag~~caactcttttg~=atcaaagaaata~~alcag«a~cc~=atgaa«actggaagag
ct
oca~aagaagatcatcttaacagt~=ctttaaccaaaa~t~gtgaa~_atggt(ttrlgcatlt~_~~aaacattgg~a
tact
agaagctgtta~actcccataaagcgat~=~'aaaa;ucagaacgc«tgaggaa«~_aatggaccagcaac~'gaggta
atg
~taatgagc~aagaaggglcaactcga~tmamct~mactetr~=age=ac~_lamc~arca~=ccactc~accttgtct
gc
caacgaatrt~=~g~a~cc~atcattcc:aact«;I~at~'acl~~ICt~=aacataa~,ycactgaagg«
~atcmaaaccac
«cctaaa~~tctaa~~tavecauc:cit'_,'tccaaaclctactlaccct~toatcattaat~ct~a~ttaaata~l~
at
gaagtgaacctgctattatcttagcttagaaagtata_,~a~a~_c~attg~=ttattcaltatrc~acat«lag''~a
atttc
accta~tttat~cuaccata~~atccatcit~aaaac~aa«
ctattctamatteaaccacaaa<_~'a'~_'ttaaatccca
a(
1g88agaaglaglg;laaaaat:l8a11«i'aaacy_CIl'_al'=CI'='_I~ICaICtaCCCtatctCt~'ataYta
cal~~
gtttctcca~t~'cattgcgtccctaaaaa«~'~_lg~aat~at~«~=teaoaaat~'aaaaa,7at~aacl~=ate
cctactaga
;lcf;1111;1clY~lCal:l~;l:ll~lec;llleallala~~aa_'fl~aal~CtCCatata'_'_aaa'_alCall
lICClltalCall
c:att~accaaat~ctt~aaColtta~_ctaamatccatactalt_'ctttctf~'aleealaca'_(uvtllClllca
aatac
raa« cacc:ctaatealcaa~aaaaaaacamncacatetcc
It:«=_'aactttt_'cttata;l~'a~uat~cca«t~et
?i
CA 02362897 2001-09-18
WO 00/~~325 PCT/US00/07392
(lal'=Caal'=CICCI!?CaaCaIIICa_'a!_!_l~lal_'aCWllalafll(c:aeacttaatcgil'='=agaIC
T'=l~_L':!'~~llll
lal~'~_ate'alIIllC~'~~lClal~_gCCCCICIIICIC~IICaf~ttt«It;aalClt~=~,cap=~'i'tattY
aCla~~'I~'t~:t;l~
a~'avgaatcttgttcteaatt_~g=aaaagtgtcatttcat~=~tyaa_=gaa~~catagtgm~'gatcacaa~_ata
tcaga~'
aa~='~_tata~=«ggtlgacaaa~~gaaaa~_ttgaagtgatgatgcagttc=cagccaccaaaaacggtgaa";_ac
atca~'aa~_
«lCClt'_''lcats'Ct~.'_~.'.gttCtacagaa~'atuatl:lAa°acttclcraa~ata;=CCa~YC
C iTtla:lCla~:lCtall,'I
'caaggaaacagaat«~~aattcgat~'aggaca~'cW
caaamcmuaaaccatcaa~_~al'_CtII~'~=l;ltCl'_CtCC~
CItCIIC~_a~=Coccaaatt~!t'.gaClatc:C:ttlli'ag;llCal~IC'I1'alYCalCa~aItaCi'Caata~
~a;=Cl~tltla';L'
cca_=aaaatapacaagaagCllcal=lcalata«acgcca_=cc~=aaCgllg«algacgc(cagg~Taa~lafatgc
aacaa
ct~_a~~aae~a~cttcta~ct~ttgtatttgcatu~a~=aa~tma~'aa~ctatttggttggatraaa~~taact~tc
tal
acagaccttt~~a~gcatctgtatgccaa~=aa~'gatacaaaaccaa~act~tt~agat~~~atacttttatt~=caa
~~a~ttt
~acatg_~a~ata~tagataaoaaa~~cattgaaaat~~tgca~ct~=accatctotcaa~~_at~a~aatt;~aaaaa
cccct
tcccatagaceattcaatgccacaa~~agcagctcategitgta;=agttcttcggaagga~~ctaca~'tgggaaaga
gttcc
accaactgaatgctgttgaa=gagaatctccatggtatgctgatcatatcaattacttggcgtgcagagta~~agcctc
cc
aacct~acaagttat~aaaggaagaa=ttlttcagagacatacaccattacaactaggatoa~ccttatctttacactc
t
ctgtaaagataagatctacaggagatgtgtcttagaagatgaagtggaaggtatcctgctgcattgccatggctcagca
t
atggtggtcacttcgcgac_ttcaagacagtgtccaagaltcttcaaecaggcttitggtggccaacaatg«taaggac
ectcaggagttlgltltaaagtgtgattcatgccagagaaaaggcaacatcagcaoaagaaat~agatc«l~atgtatg
~~augattttatgoglccattctcatctlcatacggtaataaatatatactggtcgccglaaactacgtatcaaaatgg
gtcgaagctattgclagtcctaccaacgatgcaaaag«gtgctoaagclencaa~~accataatc«
cccaagattlge
aettcccagggla~taatcagt_atggcgggaagtatttcatcaacaag;(ttitgagaacctctt~aa~aa~cat~ga
~1
taaaecacaae~tcsccactccctatcatccaca~acaa~ca~eca~ett~a~atctccaatao~~aeataaaaacaat
t
ctggaaaagactgttgggattacaaggaaagactg~tctocaaagcta~atgat~~cattat~~'gcttact~_aacag
cctt
caagacccccattggtacaaclcctttcaatcitctctatggaaaatcat''tcacctacctgtagagctcgagtacaa
gg
caatgt~~~~'cggtaaaacttctgaacttlgacataaaaact~clgaggagaagtga«aatccaactcagt~'acctc
eat
~agatccgtctagaagcttatgagagctctaaaatctacaaggagagaaccaascttttccatgacaagaa~atcatca
c
taa~gat«cca~~ctg~aeatca~~tgctgctgttcaactctc~ctt~aaactctttcca~
~aaaacicaaamcaaat
~slctggccccttct~tatgactaaggtcc~fctttat~ga~ca~~tcactcta«ctg~taaaagta~a~:tc«cacag
ta
aatgglcaaagactcaagaaatacttaacagatcaaalcc«ccaga~=~tgacatcg~ttcatctcca~gaac«ctt~_
a
tga«aaaggagtaaaggagtcaagcttatgactltaaacaagctcacttggg<tggaagtcccal~actatctcl~taa
a
t;lull111ttattltc«gttatttttgalllglcllggllglglllglgallclC;Ig~=aac~aagEaacaalgto~
a
ata_agtaaaaattcaaaactittactalatagaagacctg°aoatC
~a~t~lataaccc~«~~ai'tacaaaatttt~a
aaatcttctattgcacctagfgaccttacactcgaglac~«
g~=tc~a~tat~t~tgatgtttaaaac,=caaaaaac«t
taaatcatcattttacttgacccacaaaasctacaga~'ac«gta~'ggatttcctgcagtctaaagtg''gtttlcig
caa
aattttcccaetcaacagagt~tg~accac~cact~~a~~ataagatggagagagaatctcaanctcaatcmagcca«
c
clttttacatttcactcgacc«gctatcttctgttatccaccactc~atcaaaacaaattamc~
acctaataatcctt
a~ccaclclcgacclccecc~cttcagca~aaucaaggaamactegaccaatc~alctacttgaccccc~'~~'aatca
c
Ic~acctcc~'cc~cc~tcatcautcYltctatccetc:ictc~acta~acta~atcaatc~~attcctttte'_clal
aetc
raccaattcacccgtclctttcaaaagaagctactc~'aCCIC~IIaCCC
a~_ICtCCta;_CC;cactc~'accgar«ccgcc
tcagccgattlcaacaatt~=actccaccgccgtgaateactc~aW
Itcca~=ccgagtactcctcctc~ttctc;c~'tcg
aatlctca~~gttcaa~atttcactcgacctc~'a~ttttaactc~acctctacaam~zcaactc~'actte«~aaamn
a
ca~a~crct~_aa~_cactstaataoa~~ttactcearct~aasacttcacci'~C;2l~;t:lClCSacatccavaam
caclc~'a
cctccaeeaacaectcaasttactcsastacasaatt~tactc~..'atcaattcaatcta«tcauecctatt~al;ta
caa
ttttgg«tcattgctatctttgagaclaacctattgacatttgagct«gag«ctaaa«tctacc~gagaatcat~_a
~~aattacagtggcagttcttctggttatcctg;lclacaacatggat~agaca~agtcgtcalc«
eaa~=~mca~agaoa
~aac agagagaatatgaaa~cttcagaaggaaa~ct~agata~c:cc~'ag~,~aaga~
a~;c~'atya~ata~'a~=~~tatoa;~cl
ti«a~ac~aagatctggaa~ae~agtacatgcctgaacagacaayagaoctaccaaacttct~'cacaa~=rc:aaaco
tat
l2CClC'CIYaY~aalat~llaY2CllIICtaYCl~aillY:tYllBtCla_'_CaC'_a~~latCCtICCiC~_ac'W
c:aCll2Ca
Ca;tclC~~~lllall~~aa~ati'llla~C:lCCIi'lalL.laa~ll_~tCatCtYS'al:aclCtaat~_'Cllat
tC~_tal~_la_'C
_l:ll'=aa~atgagacaatacaattcclctccatoCl~'caa'_la~'a'lCICtaccaa'"=lal!_aCCfCI<_aa
~Ta!_'lf~i'all
~l~aa~'Yatlg~~aflCllgCoatlClClga~lal'=~'lCal'_a'nata~~ilatCaatcaa<Wi'CIIC'oaa~'
c_all~'ltl
CC'CtICCCCagIV ~aal:gggatctaagccCaa~_lal~ala'_a~aa~a~=lt'=aaa~aCll'=l~~alt'ac
CaII~WCaYCIC
t~_tacc~ct~aattc
26
CA 02362897 2001-09-18
WO OOh~325 PCT/i1S00/07392
SEQ ID N0:186
Artrbitlnp.,is rhtrlitrrrtr
B.aC T6C20
«ast~tttt~y_a~tcaaatatgacta~atgtcat~=tctatgatigactataagaa~ta~aa_cacaacca;atccta
aa
a~ctaaao«gtgtttccatgctagaagatacatagccagatactcataca_acttttat~~;at_tc~~~attcacaca
ct
caa~tatttatauaut;_tgg
gctaaagclctcaaatacaaaagtacacgacalatatgcataa~=aaaagtataaga~=a
etatatagaaatgtatgaagaattgtacatagaaaatggtatagtattccttatetaaagatc«acalaaaaa~=a~_a
a~_
aaataatc«ttgtgtatttatgagaaaataaraatgtttaagtttaagattt~tg~tca~aatatagaatctcta~ccc
cccttatctig~agtcateacctctcttttatagcctctttccttcttccaatstgccattaattatagaactaattca
t
aaaataactttcacaatcaattcatacacttgaat~acaactcgccaaaatgtgtatcctaactaat~ctcalgcttca
l
cca~clcat~tgcagctcctaattatgaaactgaaactcacgatcaagggacg~ccactcct~ctttgaaccaa~acga
~
attacacc~_ag t~_tteatgaggcttcagctcatcttaactcttttatggctattaccataaaatctg
gggattgtcccaa
tttattttateaatac~actttcccgaaag~~aatttttatttgcccaagggsttttaaaatttgttgg«gt~=tgccc
a
a~=a~~~etttttaaaacttttttccttttcgttgtccataggggttttggaatgacactt:cttaaagtatttocttc
~a
a~tt~'cc
ggatttocaaagtccgtactttttca:laaatiteg~_ccat~tgoctaagagaaaut~~gccat~t~y'ctaa~=
agag~aaatag~ltgatcaaacccc:caaactcaatctttggactttctttgatt~c~agaatt~~t~ctcc~_cctaa
~aagl
atlaeetaeatc~'aclaaacaccgtaaagatctttgttttgcta~tt~'acccctt~tccaa«a~~attaaatccatu
ta
«~actcaaactc~tcagucaaccatgga~ttu~~tatca~aattaaactc~a~ttat~cat~~~a~ctaacrtc~a~
«aa~«gttgc''aagtgaactcgactttcgaatc~'aagctataagcttgagtttagaaagtgtattctgcttata~aa
t
acaaa~gca~_~_ttatt~a~caocaatctagagagaaactgaa~atcttcttccgagctigtactcaaagactly~tc
ata
aatsclt
ott~at~'tgaggtggtatctaaatttgtgaagagcgatctcaacacttagatttcacagagatttat««cc
aa~a~ttggaacttgVaaaaacccttcacgagatagttcagtgaagagceaactcaacacttatagtccaccaaaataa
t
cttatttttccaa~agttgaaacttg~aaaaaccttcttgggat~a~atcta~tttc~t~aa~a~cgaatto~acac«~
aaccccgccgaaattaaccaatattcccaagagt«caactatgatctggtoasacata~ataaa~tcgca~ctlcc:la
~~
uuaa~l~actacatcaalcgcaa~tttctccattataatotaacttaattttg~act~gtt~at~acttgmcal~taa
caaalcg~ctttcactctcttct~tcttca~aaattattactccattaac~gcatgattt~aaaal~a~
at~talaa~ta
caatttttcatygc«caggcttcagagaattagagcaacacttaagtatgtctttagttgaccgaaagcatcttcara
tcutcatccaaclcaactctttgttcccttttagtatttgatagaaaeaaaggcatttatcggct~'actga~_atgtt
aa
cc~~tttaac~ca~taatcctaccaaagagtcttta~~acatcttt«atgttcc«~~~~ctaacat~ttc~'aaaa=ag
cg
«aatcl~tcttg~'gt«gc«cggtttctctcttt«ttgctaa~=taaccgaoaaactctccggatg~=gactcagaat~
t -
ctac«gecaa~atteaacttcatctgaagcttgttcaaoataltaaaacattctcecaa~tcttaaat~t,=accgctc
t
C«u~ac~'at«caC~a';cat~ICatctatata'~aCCtccaCC~lllllCaaYal~all:llC''I1~~1:11~aeaC
:ICCI
aCaf~=ttta~uca(:ac~_~'Caratcotaaaata~tatatacvlC~gll~,vl~ClYaal~:lallalllilCllaa
lC~_tt
a~_~altCatleatatatCCCIafIIIIaCC~llltlaa~talo2lll:l~'~I;lla~~c ttla;a~_IClaalf
~Ilalllal
aCa~ItIlttCla~=tCttIltCa~glClIlaCatgtllta~lalgCaItl~L'aapatlat=~'a~cattct~~
a~taaaac:at
acaatttaa~ct~_aagaaQcaatccagagacgaaaatgeacgta~gtgtceaac~aca~cavccrt~=gt«tcyt~_c
oaca
ccaatacctgac<_~caattgaaa~acgcaacttattgttgttttgaaoatt«tt~aattc~~cccaaaactttctcct
a
t~tttaatgaag~cccaacacgttcttaagacatalttatatagctitgaggttaatgtttagaccctaa~=ctttg~~
a~
_'ctattccc«tttcaattct~tactttgggagcaaaaccctat~~a~actccct~ta~apaa~atccttctaaacctt
a
ttclcctc:«g««attca«eat«cttattcaatcalgt«tg«tllctat~atcattttt~a~taatcttctt~t
ta~~ttta~~gutctcatta~_ggatttagatga«tai'tagattgcccccttoctaagttatct~_laggattctW
am
ma«gllc«aat~cta~ma~a~'tacctaacta~'aat«;_atatta~~taata~~tat~cccaccata~gtaclt
~«a« lgacctaW
al;t~atYa~~cWaa;,cat«aa~aaa~aaca~~ta~~ctcata;_aaa!'~Ct'~t~C~LTIII:IYataCl
a~~ulv;l;lclB;llCaa:lCll~'al8ataalgaat~~Ctttagtatag~aIIICICooCIIIICCggtaatctta!
_ClCl;l;l''1
ata~;l_a~llIlYyY:laCaWaCC~~IIaCICtaaaatcatao111~aollC~'a~aac~~tlccalaalaalCla~'
Cla
t«cl;l~alclaClatC<llCfallIlClga~_aataccctaa~cctaac~CCtltatlalClf~lIlICaCaac:ll
alaal
~lltitl~CllilClIlltYttlCtlllallllClflaClaltltac'lta~_Cllaalll~atta~CCaIIaUICICC
I~C;'l
~'atc:~ ~'a~aac «t~l~aa«
c~atccctaa~_t~'ctycaact=acctct«atu~=aoa~a~t~~~'tctta~gatlaaa«
'aallalalC:l;l;ltll~l:C~=C'=nll!.l:C~=aa~llClClol~allCaccattagactac'utVtil~~:la
l(1;1~';Illaa~llt
IlaIf111CI11Ctlff
~'lIICIC;IC;1C'=tlitClIICIICIIIIICCI.Its'aC:l''~So08aCtaCQ;t~:II~YBttC;CCaaC
CA 02362897 2001-09-18
WO 00/~532~ PCT/US00/07392
aaacagctg;~ca«atttcaac~~aca~tgtrgatc~acaccct~ttggt~tc~ttcaacaccatctgtt;;~gtgtcg
ttcga
caccatctgtt~'~t~'tc~tictacaccatct~ca~aaraaaccagctaccata~~=cgacttcaaca~_a~'ctga~
cttur
lallccaatcgatc:lgcgalcc:lgctacctcctattgcaaga~aa~'as«t~~ctattcaccctgcctactattatc
ttg t
tmacgaggaaagtttc~~a~_~_tcattttgat~=aatcactactagatcatetaga~~~=tcttcgaa~;actta;_t
ytcctcaa
tcaaa~~ca~'atg~=~_~'tacaa~;ctga«atcatcttt«caaactc«cccacactctcic~~ctg~;ca~~a:_~_
aacatcatg;_
ctgcgacaaW
a~'aacccg~_ttcactaaccaattggaglgacaegaagaacgcattcalgatccacticcttc~atgaltc
a~t~=acaaaattactaa~y=~agaa,~atctcttt~ttltctcaa~ctccaacagaaggttt~aa~=gla~~ctt~=~
atcagat
tcaaatcctatcagaggea«~=tccacaacatggtttctaggaaagcaaactaatcaacatattcttccg't~;~=aat
cgat
aagcagtatc~yttatotttt~gtggtgcaa~taatg~caatttcatgacaaagactccaaccgaa~calca~ttctta
t
caacaac~ctctcacaa~tctctccacaaa~~aaata~actatoatc~aaQaaaat«_~ct~a!~~cttcc~acaatc~
ca
~asas~aaeaag~atctacar~_cattgcitctccacctaggaaaeatc~t~gttgagtttactggcaa~=toga«ctc
t
clacacagatcttaatgggaagatcgacaatctgagatctcatatatcttagcc«ctcctacattagcatttatcactg
cagtcacactacgaagt~eaaa~=caactaaacccaattctttagt~t~aacgttcagctcagacctcatctatcccag
tt
gcagaaaaagcctcaatgtcgattgatacatca~ggtgtcgatcgacaccaattgatctt~acggctctgtat«ccttt
atcgtgtggatacgatactttcacagaggaggaggaacttcttcccgatggtgtcgaacgacacccacctcatgtcgat
c
gacaccaataccacataggcaacccagtgctgacgtttgacagagcaggatcgatgccacaca~;tagt~_tcgatcga
cac
tcacctagtg~=c~atcgacacaagacttaaactgccccaatatgccttgcatcmcacctgttactatacaagtctaca
c
accacaa~«ccttgtctactacctcgacagtccaagaaggagattcctgagatgcattgcat~ggtatcat~gatga~a
ttctacttcagttgcctccagttgatatagagaagttgtcaccctctcttcaaagtgatatgatacgagatgatgctcc
a
oagaagcaagatgacaagacaaagataatggttgtgtcataacaggtactcagtaacagcaagegtccacatgaggcca
a
tttcagattc~ttgctgaoaatcaatctgta~agat~gtgtt~atc~acaccatcacctgtgtcgatcgacatcccaaa
c
aggctaaaccagaatt~tataat~aa~ctgcttigcaagttcagatcactgct~aaatttgaggacatg«gccacgtca
cccacatcacctctac tccttcatclacctcataaac ~ccaaaootactcatctcctcctcc
taaacctccteatttcat
CaaCa:tgag«taC:l:ltttCaaaa:lactgttttac~gttaaaCa:l~cCaC~agItICICgI(;g;lgCal;Ili'
tICIIICIC
llgatgattgtg~ag~lacaaggagtccaccacccatacctct~tcctgC(:CagClgaCalICClc'a(:IICCItgg
Iaga
CC gcacacacc ~=aclgttaatacgccaccit ggll gaf
gC~gg~ttctactaggcagtatttacIICCICCaIIIoaICC
acct~atccctaaactccaccaaagtcaa~ctattgactctaaacaagc~ctta~tgcgaggcaacccacta~tatggt
t
ltctttttcttgltcttttc««ac~ttttttttttctttttctcgcttactctgaattcgaatgccattggggacaat
~acgtttaagtgt~_ggggaggcagttactaactacgttlttctttttgagtcttattcttattcttatt«tcit«taa
~tcgtt«auga~tcaaaattttgttaggaattatgatctatattctttagattattgcctggtt~~attaat~ctgcag
gt«gactcaatccgacta«ggaaaatctag:uggcaaaccaatagacttgaggatatcrtagtttcc:aacaccaecac
caa~~cte~a~ctaatctcactectcttcctt«acctt«caaac~~tta~tattcata~atctt~actcctlttcata
cct~atattga~=accaat~=t~_ataaaaagaat~~tgttcctct~cccagaaaaaaaaaatgautgatgaatgagag
attga
ta~gattttcagata~totatag~~~taeaaat~tttcttat~atcctattattatacattatttggaat~atcaatta
c
aaaggttggaaaaa~tcaagggtetcoatccagtatgc
gaottcccctttgtttactc~claaaaaaataa~_ttteggag
a~aaagaacaccaaa~aaaaaatattatataagagaccg~tc~atttatcacggtataecacag~=aatea~lctagaa
g~
atcttgaatgttaacugtttgatgagactcagcgatgaaagcctgatagatatagtt~_tggaactta~y_tat~~gaa
tct
ataat~tt~t~_taaectt~m~~aeaaetacaa~nt~~ua;'zattt~~a«aeacti~cta~ccatat~_~'at«a~tt
aoa
al~_atcat~~caata~ ~« a_'a~aata«aecacttcrt~t cI«tcaaacctcttccat~ectW
eacttttet~ct«
_cttga~=ggtaagcaaaa~~ccaagtct~y~~~~a~tt~atatatcc:ctattiltacrattltaactatgtttta~'
gtata~'
~tttta~a~7tctaatt~«at«cta~~agtrttttttagtctttttca~~tctttacatgtttggyatgcatttcgaga
t
tatgg~gcattctg~_cccaaaacatacaacttaagctatagaagcaatct~ga~=acgaaaatgcac~'ta~~'tgtc
gaay'
acaccatccctggtattgaac~=acaccacccct~gtttcgagc~ataccaatacctgacg~caattyaaaaacgtaac
«
attgat~ttttgaagatttcc~aattcgocccagaactltctcclaletttaatgaaggcccaacacgttcttaagaca
t
atttatagttttta~T~ICaat~ttta~acc~taagctttgg~aggclattccctt«tctattctgtacttt~a~;agc
aa
accctgttgagactcccttta;=agaagatccltctaaacctta«c;tcctcttgtt«attcaatlatitcttattcaa
t
catgttttgttcltctat,'atcalgttt~a~taatcttcttgttag~IllagvuttlClC~IIa~~~~_anta«a~~I
altl
a~la~ala2eCCCCII~ClaC2Ilallf~ta2Yattcttcatc«tall9lltataal~Cla2tfcta~a~Tta'_Ctaa
ct
agaacugatattag:«:lataggtatgc;c;aecatuagt;lc«~'Itatttgaccaamalayat~~a,_rcta~_~lc
atuaa~,
a;IFI~;taC::1_.'nt:lYYCll::ll:IL':l:l~?~_;CIC'.II~iIlad;llaClB~~~(~aactaamaaaat
t_~ataataatecat~cttta
_Vlala~_~BIIICIt_~~CIICiCC~~taatctta~ctctaaetata_~a~a<_'IIIC_'_m'~aacaccacca~ll
aCtctaaaa
lC:ll;l_~III~:IYIICY:IYa:IC~YItl~:llaataaCCl:Itl:lalllClaQalllaClalCalc;lallIC~
I~;leaatarlC
l:l:l_2CCti':lt__'CCttl;lll;llCll_2llllC:ICa:IC:IC;;ICa:lit'~':lll~i'l:lll_'c
Il;l Illtl_~I1(CIIllallllCl
ll;lCl~tlll:Illl:lYllt:l:Itlli;lil;t!'CC:III;ICICi:I~I_'c'_~'I_~:lllC_~:1.';laC
lll_'_l'_a:ICI~'~_lClla,'_~alt
asffl~acllal:llc allc;llWtlalll! _~ll~l:lacaa_~al~c
tacltICIICCaa;lllcllc~ilc;_Tl~_alclllCl
C:lllaali~_C:C:~Clll' _':lllalL'aC~tcatc(:altaCtl _'CCl'.
~I:II'!al:llll'LtaaaalCaCIILIIICIICaI Yaoa
y
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/07392
titgttaattggataaccatcc~_atgagtcsttaaataacaagattit~aa~_atlctt!_alctccttgtaaaattt
tcac
accattc~="~'attaaalatttaac~_lattgacaata~au~;at~_~'"alaacctteatcc~!ataaatccat~g~
«ctccaa
ctat~tca
~cct~;gctacttlccattctrtccaagcstactcaa~'aata~ta~atclalcaaacttccaacatcaatcac
aatcttl~'aaacccc~'acglca~'ccae~_tct:lad'~_I~a~~acaaaatcaac~=tcatca~'«ttct~caagg
l~'acgaattt
CIICIICCII~'aal~_llglgaltgctcla~c~'a~llcga~Ill~IC~=CII!'I~=clc~cctttaata~=aacat
icccctaa
aC«Illgaltgla Cl~gtcg;latctCtaCaaaaCttagaCCC'>ItCat~auaC
(lCaatCIICItCllall:lCCaaaa
~~atta~aIiTIIIIa~_~=talltlCtl:lBClaal~al~aaCltII~FICIICC;~~CCIIIICIIC~_I~=~Iiaa
allflCaC;Bac:
ICICC~~aCglCgaggllll~;Cg:l~'_tCaCCtCltlglClgIagClCa~Caa'=:lti'CCICIf'~alal(:g'~
Ial:a~lClllf
~tagtataattatttactctat_gatgttgtcgtatgcctt«care"caacc:at~=tt~'tca~;a~;at~_~=ttgt
clcgatc
ttctcttttccaagagafgglgtaatgccccttacc«~aclatgt~T~_ccggac~=laca~_tctatcga~'lagaat
gcgag
atcaa~;ccaaaaaaat~=caagatcag«cgaccattta~tgtggcaa~lcat~_~_gtaagaaocattgaattg~atc
aagt
ccatggolaaaggaatgactcaccatgagacggalcaag«tagctacaaloc~acaaatca«agaag~tcgaggggaa
tccatttggaca~tga~atatgtgtatgggtcgtgaalggtcgc~=a~Tatt~alc~~_a~aatocg~~ataagt_acg
atagc
tggcttactc~~_tlaatlatggcatggaacgtgttatagtcctaaggaaaccttaa«tgcgtcatggaccctcaaga~
a
cattggaacggaatgtgccgtattaatsttcttgcggtct~gt~aca~atcota~=~tttccccg~aatgaa~aaatgg
ac
ggccaggatlgaaccgtgcctcatgtttatccgtaaagsctcaagcttatccatatttt~gtcatccttatctttttag
g
gtcgtgcttatccattlttggutgatgtttatccgaaaaatggaaggaaccaatcg~~ag~ccgattagtgtcttggag
cg
gccgttggtcgacatttltcacgttttgaltaatccaggacacclcclgc«ecc«ggcc
gacctaatcaccgaccltg
gaccatataaataccccctccgcccatttgattccttagacctaaaaatcgcctagaagagctaagttcacgggagaaa
a
caltcggaagccaaaccgctcagaaatccaagtgagtc«gaettac~'tgaag«lgcacgtgaataggatagtttcltc
a
ttaatttccttaatatagcttaggaggttaattatgaltac~ta~cttaattattyc:«aalccttgattaggatagtt
aatttggttlattaattgttaattaggaata~tgatcattaattacaatta~tatttcctaa~~atoatta~=ggttag
tl
aaaccatgatta~~~~actaaccgctatta~taataatlaa~t~t~=aua~=tt~l«~atta~tagagtttlgcatata
g
gatgaatgcatgatagtatgtgagtctg«gtcatgtlag«aotc«taatta~at~acta~catta~cataaccatga
ggaICCCg lCCgllllglt gICC gllaIgoCHIa(: gaaggCICg:lyuCl~,BaII~~C IaCC
~aCCgggC~'CIaCBC ~aCga1
gta~tataaaggaatgClalgiCICIlalagaClllglgtClaaga~l:ll~~a~lCal';ICCtIgggaYalCBlgga
llC
~tacaaa~ccitaCCta~~It~ataQaa_QCatL'Ctt2catta>~_TtlatCe_tt_~_aatataa_~~aaattC~_a
=ICIaCaI~C
allCl~Cal~!glgCatlataltltgCltglgllgtalgtlgCgl~ttaICIIlgfllat~'ICII~CII~IIaggla~
Ial
tattgttacgtgttgttgctgcttaggggotaaggaag~~ta«aggataacattgtctagatccggggttcactaagta
atactagattacttatgacgtlacttatctttttcaggaaatcttagttgaccaacctlgta~a~ggtgctgatgactt
a
ggacgaactaatgtag~ttttactaagtatattat~tatgtttuttgtaaattcttactaa~tatattat~tatggtll
tcaaaga~gatccggaaactatatacgtttt~gtaaccautctc:gtatgatatatatatatatataaeactcg~attt
c
taaaatttatal~gttatact~actgatatta~tcttgttcgtactatcacaacmac~"cc~c~atagg~«
gaaaagc
cttaggtcacgggcgg~aggctagactcttgcg~ct~aaceactcagec~ aac
~aattcatttt~=~aaacctggattg~_c
cgactgcgg gcgtctaacgt~~acactcctatttc gt«atatttatata~_c ~stata~,
~'ggtcttaca~=at ggtgtctcc
acattattg«aaatg~glllaatccaagtggtttgatctcttlgtt~ccc«taactlt~y~lttccfo~aagggacagt
aaCaallggCat:IglCggCttlCIllIIC«gCgaa~t~3a~C;I:I:IICIICII
~ICIIC'l:lCtlllalCCLIlllgallgC
lCggaataaagCaggtaaactatggaaaaaactcttaaaltccatc~'a:llg:l;lc:lc;a««tceaaga~_ttaga
acllgg
a~aaalctcttclC~at~~aalctaalttcel~aa~aoCaaaClCaaC;ICII:I~_alll:l':1(
Caalt~'aaattcacatctc
catoaa~~tQaataacatt'_eaaa~tt~t~stata~catc~aa~tt~mlltuc
aaaclt_'aatctc«ctt'_tteaca
aat_'tcecatt«~actccn'_aautt~a~l~al:.Tatataaatal~,~actc:ca:lacll<_'Ia_'eaat~attt
atcaatca
c~ttaaetgataat~ct~tataattctcctccuaaga~~cttaactn~_c«talattat«~=~aagat~_cccccacct
~
ttt~gtcttagacatctatgcttttcgtcataacattttgtgtaccuaagat~~a~'acattcct~ttaag~agtaaac
~
t~tacaccata_tt~aacttatc«t~at~cttcatacatsatcteamcaaatU«tt~aeccaatateatcttotcct
gleatg~ctgtaattgtt~aagtcattcgctaatgatttccgaa~aaac,'c
cat~aaaacttctclgtatcattgtgtca
IggCllgaICgatlgaotlaalctta~la~aC(CeaCCll~CllClaCCla; ac
lf~l~_CtCCI:ICIIIg~l:lgglgaC:l:l
ovllvllgIIlC~c:CaCC:IC~a~CI~la~lc:ll~Il~ao~lc c
a~=a~llClll<_!_fi'aaiTlll~l~ta~_Ctaatggg8a
atctcggaacatcalgagacg«gatggcctctttttttttttgttt~'ctutac
~a~tmt~'cltcegagaaaaa'~t
YaaIC~CC:IC(:~(:llltlaCl~'~2l:Illlg3~lCCaIYBCga~~ll:laalC~;II:II;II:I:I;ILIC:Wl
1( ~aaalga~cacaltc
tlccg~tgetcugacatcaaaa~catcinga~=ggatgaaaamuy'ca:l1«I~'a;y';I~maaccmaacacttagat
ttc
accaaaat~aatataattttcaa~_aa~t~~aacu~~aaa:tatcmt~ac
_~ateaa;llcla_'tcal'~teaa~c~coaac
Icaacacttaea«tcacc~aaal~aalala«t«ccaa~a~~maaxlle_'aa;l.ale«t~ae~~al~aaatata~
tctt~t~aa_'a_'c_~aactcaacaclta_~Tatttc~ctaaaaleaatacat«tttc:aa_';1~__~I~_aaactW
'~aaaaatccl
tga'1~_'atgaaalcla~'felt~l~aa~a~c~aacicaacactt;l~=:otIC;IW
I;l:l;tt~;l;It:ll:llltlll:C~:l~'C'g~ln
_~'aaClt~_Y:laaaClIIIIT_a~_~_~_al~_aaatrll~,lCIfIl~aala_'c:aaaeit;J:lC;lc.'11:1
1';IIIICaCC:laaat~aala
laIIIIICCaa'_a 2~l _~'aallf ~aaaaaatcctt~aa~~af ~aaatCla~=iC'If ~'I'_;l:l~;lYC
~a~'Ctaaatoaatata
,y
CA 02362897 2001-09-18
WO 00/5~32> PCT/US00/07392
ttt«ccaaga«gt~aaacttggaaatatcctt~a~~_gatataatctaotc«;_c~~aa~~tal_'aaetcaacactta
~att
tcacl~aaatgaatacat«itccaaga~'gt~!~aacttg~aaaaalccu~a,'~_aal~;taatctaatctt~t~aag
a~r~,
aaclcaaeactt~~autcaccgaaatyattatatttttcaaaga~et~~aamt~'"aaaaatcctl~a~~a~_atgaaa
tt
tagtcctgtgaaga~'cya~_ctte:tcattta~=a;ttcaccgaaataaatala««
maaa:oag~t~~aactcgaaaaaat
cell''a:lggatgaaatettgtctt~~t_aa~_a~=caaactcaacacttaat«ea~aaaaay'aatatatttttcca
a''agg
tayaacttg:la:l;la:uc~_tt~'a~'g~;atgaaatctagtctt~'tgaaga~y'aaelea~cm«
ayatttcmcaaa;ueaa
tatatttttccaaga~';_tgg~=acica_aaaatgtttt~~agggatga:talclt~'lclt~'laaae;lac,'a~'
ctcaacactta
tctttcaccgtaatgaatatatttttttccga~_aggtggaactt~!gaaaa;mc«~=a~~~'galgaaatctaatc«~
~tg'a
agllaa~a~'Ct(:a:lC;lcttagatttCaecaaaat~aataCattttlt:aaaaa ~_ ~=t ~~ ~'aac t t
Y=a:laaatactt~ ai'°~L'al
~aaatctactctt=:1~'aagagc«aactcaacactgg=~atticacc~2laal~'c;:lt:ltattt«tcaa~~aggt
tgaacttgg
aaaaatcc«gaggy~t~,aaat«a~tc«~=t~at~agcaagttcaacactla«Imeac~gaaataaatatatttttc
cs~a~=aggtasaactttgaaaaatacttgaggoat~aaatctagtc«gtgaaga_c:aaa«caacatttaaatttcac
cg
aaatgaatatatttttccaa~aggtggaacttggaaaaatctttgagggal~aaatcta~tctt~~t~aatagcgaact
ca
acactaacatttcacaaaaatgaatacattttttcaagaggtggaacttg~~aaaaatccttgagggatgaaatctaat
ct
tgtgaaaagcgaactcaacacttggatttcaccgaaatgattatatitalctaaga'
gtggaactaggaaaaatccttga
~gggtgaaatctagtcttgtgataagcgagttcaacacttagatttcaccoaaat~'aatatat«ttccga~a~gtgga
a
cttggataaatccttgacggatcaaatctagttttgtgaagagctaactcaacatlta~atttcacc~aaat~aatata
t
t«tccaagaggt~gaacttg~aaaaatctttgaocgatgaaatctagtctt~tgaaaa~=c~a~ctcaacacttaaatt
t
cgctgaaatgataatattttttcaaga~gt~_aaacttgeaaaaattcttgagVeat~aaatatagtcttgtgaagagc
ga
actcaacacttaaattttaccgaaatg~atatatttttccgagaggtogaacttggaaaaatcc«gagagatgaaacct
agttttgt~aagagcaaactcaacacttagatttcaacaaaatgaatatatttltc:c:aa~a~'~~tegaacttg~aa
aaatc
cttgagggatgaaatctggtctggtgaagagcgaacacaacgcttaaattmalcaaaat_aatalatt«tccaaga~_~
=
tggaac«ggaaaaatccttgacggatgaaatctaglc«gtgaagcgcaaacmaacartlagatt«tccaaaattaa
tatatttutcaa~aaat~~aactt~_~aaaaatctitaa~_'_at~_'caalcta~
lcll_'l<_aaaa~ceaartcaacactta
oatttcacc~aaalgaatatatttttccaagag~t~gaacttnaaaaact«tt~a~~~=al~aaatrtt~tcttgtgaa
t
agcaaactcaacatttagatttaaccaaaaatgaatatatttttccaa~a~gtg~aamtga;la:laalccttgaagga
l~_
aaatcta~tcatgtgaaga~cgagctaaatgaatttatttutcgagag~l~ga:tcll~'a;laatatcc«~agg~atg
aa
atctagttttctgaagagc~aactcaacacttagatttc~ctgaaatgaatatat«t«aa~~=a~~t~'aaacttggaa
a
aatc«tgagg~atgaaatctagtcttgtgaagagcgaacgcaacacttagat«caccgaaatgaatalattttccaao
aggtgaaacttggaaaaattctteag~gatgaaatctagtcttgtgaa~a~c«aactcaacactta~atttcaccgaaa
t
gatlatattttt«ccgagag_tggaacttggaaaaatcatt~ac~oat~aaatcta~tctt~tgaagtgcgaactcaal
acttagatttcaccgaaat«aatatatttttccaagaagggcaac«ggaa:laalccttga=ggatgaaatccagtctt
g
tgaagagcgaactcaacacttcoalIlCaCCgaaatgaatatattttctcaaactCllY~aCt_C~a~'actaag~aat
~a
tatctccg~tttcacacaaacaaataatgagactttctgtgaugcatg~~a~c~cucaa~~~«accaoac~caat~tc
ctcatcalg~tttctccaaagcttcacttclcagcactctctacagag~t~tcctlcccaa~
ata=~at~cttcttgat
accgc«ctaacgggaacttcctcaacaaagacgtlgaagaa~~at~g_~agctg~la~=a~aactt~~cacagtcggat
gg
caactacaatgaagattacgata_aagcatcc~ccaca~ctct~attctoat<_a~aa~caccacao~~aaatgaaagc
ta
taaateacaaact~gacaagctactcctt~t~_caaca«aagcacattcat«tct~=~~t~al~at~'a~'ac~_ttcc
aagtc
ca~_gat~
gg~_atactctgtaglcagaaga~=gtcaactal''IgCagaaOC:L:I~'~;lirvlla~'aacaaaga«W
aacaac«
eaagca~=aaccatcccaatctgtcttaca~=aa~tacaaat~=ttgcaaaccc ;te:l~'~'ae~aa
~tclac:ccctctca~_ca~'r
tgaataaacccaa~cccttt~=ttccatacaaccaaggtca:=~=~~tat~«celaa~'c;a~_c
~~tama~~~~caactatcc~,
ccecaacttccarcacctg~gttcacaca~caocaacuaca:tcca~ctlcaacaamcaca«
c;aaacttoaa~aacat
sttaca~caaatactcca~eeacaa~ca~caggg~caatggatctcyccaacaaeal~~>cayaaatccataacaag~t
cg
attgcactttcaacgatctgaacattaa:lcttgaggcactcacctcaaa~gtca~'atacat~=oaagoacaaactgc
gtcy
acctclgctcccaaagtaagaggacttccaggaaagttcatacagaaccy=aa~=~~auac~_ccacc~ctcacgctat
cac
catctgtcat~atc:ga~a~ll~cclallcgacat~tctccacatcaalcarc~=a~y_aca~t"alettca~gacggg
aa~~
cttctactcaUattgaaatttcaglt~tt~gactc~accatttagct~
~atccc~tt«caaamca~ztccaacctagac
~agaaagca~ccatcattgaVa~'g:ltggtaaaacgaucaagccagcaccattacrmac~_lgctcuccatggaaa«
cag~aaa~'cats'g.uagaaa~_atacaattctc«~ca~a~=aa~_ca~ctt~'ate'a~at~'_aayc~~t~;y'c:
cmaata~_
aaguctcaacct~atcccg~atccteaeaaa~at~'tya~'aaattcaattct=~>aaa~~:ltcaa~=;uuatc
aagattca
_~~aa~algattgt~a~~ct;latccatrtaygccact~=ttaa=a~aa~tetmaagaaa:tac't~~aa~atc;cl~g
aacm
c:acaClaCC:llgllca:allg~=Il:lat(g~=IIIICaoCaaflgll:lal~l~alt('=~'''a'_ellC:l~l:
l<'tUCll;t;ll~lCaC
lCtlc;al~~(::la~~aagClgYaBlICallla~tacaa~cc«ycoaCel~_aClll~'alCCllC'CL~al;Wvlll
l(:a8~~~
aa:ICC;CIII~=~CCI~CI:IC:la~alCl'?Cl:agtaateattaal'_'=a'il'=~Taa~'IaC'ilaC:l~:ll
lli ~ll~l'=Cll~a~;ll
_~:laaCCa~il:ll:l:laau~alLCWaatccta~_~aanaCCalIClla~(:C'llC~llY~'Y;t_'Ci':IIY:I
ISIY:Iti'!(:;laa':aCa
a~a~_a;Ilaa~ICll'aaCCl(~°naayaCalCaa~LI;=Ca~lll~'aC:IICa:W i';laaefl
YC:1;1:1~~'aCB~CI'_la';:la
CA 02362897 2001-09-18
WO 00/5532, PCT/US00/07392
~aaaaaatca~~_~'mcaocctcaaccttcggattcaatcaccagaccaagcacaacctctacacctgac«
gcgagatct
caaaaa~~aaatct~at~'a;_caa;~aa~aaaccata~=a~aa~cta~ctcagaca~u~a~'~_aacuaa~=ac=taa
acl~=~atc
a~at~caa~'a!_aaa~=elcaamaaaat~y_~~~att~alactatcccoagaaaaaagtttact(caa~'at;~,~lc
t~ta~~'a~'
ata~attatcracca~=aa~~agaaagag~~cctatttc~=a~aaaa~aaoaattaa~,tattct~ctac~'catctet
caa;aya
=gatgct~aatat~=at~al~'a~'atta~_a~_a~~~actalgca~~alcclctctalracccamtcllcttaata;l
~=t~t~'a~'
_'a~caamta~=a~_ac
tta:tacaa;cteactt~c~_any'aattcccaa~act,'«tctataaataaaat(ttt:utttct
tgtta«t«gar(t~=ttttt~;~ctgtgut~_l~=attclca~~~aaaatagaaaca~=c~'tg~'a'~ta~'a,'taa
aaatttta
aaatlttactctaca~_agcaaca~'~"~_atc
gagcat~_tca~t~taaagaaattcaagaa«tgaaaaaag«rtg«gca
atcagagaccatgagatcga~'ta~ttt~glc~~a~tatta~lt~~at=attttaaaaacecaaaatittgaaatcata
tttat
actc~accaaca'~aa~ctacaaa~acttaca~a~a~tttatcaa~tttaca~a_'~attaca'~aa~~~«rtaa~aca
ttcc
tagfcaaca~a~aaca~tgcttcaggacaaaca~aca~agtot~_gcccaccacctclracctttgitcccacatgcgt
tt
tta~_a~ataac~aaatcccaa_';tctacttctccaccactc~atctcaccctatcctticccacc~_acatcatctc
tttlc
ctctcca«cactcgacctc~'cagavacattctccgtcacgtctctcactc~accaaacatcactc~=acatctctcic
ac
cgcctctct«cactcgatc~aaacgcctcfcctctctattctccgcctactctacc~cca~accttcacc~ICtcaac~
_
tcgcctcctcttcacmgacctcgcc'=tcccaactlcaccalccctcaccacttcgtcaactttclcactcgaccaaaa
t
tcagctttcatcgctcacgccactgccttctccctctrttccactcaacgacgggacceolttcatcalctctcacc~=
ct
ctc~cctcctc«cactceacc'_cac~ascacctcaacctctactcceatttctttttctcacctctccatactcaacc
~
ctactcgacctcatctccg«ccctclcttttactcgaccgccggaccggcttcaccatctctcaactatccaccgltca
ctcgaccicgccattcattgcgcctccgtttatctcttcactcgaccgctcctcaaaccgccamglcttctctccattc
~cc~ucactcgaccac~catttac:cgtclctcattcgtcttcactctaccgctaaactcgaaaccataatttcactgt
a
ctcgaccgtaatactcgaccgtgtac«g:iccgg«lagtgt«gcatttatttggactaacatattg'ac~_t«~octtt
sa~tlacattctt«tca~~=aaatcaatatgagtaactacagtggcgaatcctccatg'~at~c~~attaeaacatc~a
t~
aa~ct~aatctto'_maact<_'_~acca~_as_a~~aea~'c~acaa~cttat~a'_aecttca~a'_c'_oa~accc
aac~ctcaeta
actt~ac~caat~a~'a~~a~a~ct~a~att,'ctagaggaaagagagcaatgaccagcagatat~_a~'tt~ategac
~atga
tall~acglC~a~lal~a~cclga~tcat~~cata~agagaceaaecl~ll~aala~~CCla:IlYa;l~'ICC:Ca~l
~~auv
agtaCaIC:IL'aCIIIICYa~CtYaaC~aCIICIQ~geaacoaaotacccctattatca~act«aoccca~mI~~CQC
Ia
CCggaggaCgIaCaBCaCCIaIIC ~'a~'a;IglgICalCtg gagacactgatgtc«acccgIaCgIC
i'CIIilCaagaag ga
aataaga~a~titctclceactct~_caa~tg~agatgtatcagg~acttaca~ca~at~a~ct~~aoa~tgaa~g~tt
«~
ogttcttgactttttCC~I'_a~C'~agCa~tgttaccagctBlClelCa~za~Cll~~aa~~alfotlt~c'CIIaCC
Ca~I
ovaaagi'HaaCIaaaCCCaagIIagagagggaagagttgaaggatttglootta;IClal[~uvaaCvatalopCvCl
Caa
CICI~C~BC~ICC;la~ayaama~attc~aagccct~l=aICf~L'CIaCtatcaacgcictala~c~aalZItCI~L'
IaCI
crag' oaalctacagg«tec
~t~_ICtaacacagacatggagalgattaattctgcactcaag~_~=cattctccgtagaaca
aa~~~caaoaa~_'ICCt~aa~~~~c~acctcaat~at~caccacca~ttat~cttct~ttcatccacct'_tet~~at
acao
saagtg~~gc=cacaccaacgggaagaagagggcgcgaggagccctttotgtaggt~'gtgttgtsacaccaa«ctgat
ta
cal~Ie~l~~tacclttCaC~ICICC:I~~~~lllgalCCgavv:lltalggalilagalCnc:ll~'colc~'II~I~
a~'lllCl~
~a~cac~acatg~tt~_~=cgatlmatcgctacaaa«tgagcaclcctt~accc~aaca~ccaacatm~=cttccclg
caccgaggccacaaccatac«ra~_~~c~~aaaacattgac«caa~cct~c~cgt~attacctctacttt~a~'a~cgc
tc
cacc~act~'al~acaac~tccctacaaa~aasctaeccaa~cts?a'_att~'ctaacaca'_;1t'_a_'~ala'~~
~'a~~'a~~a~~t
ayatac~yal~tatcatttca~'tyaocat~tacctccaac~ag~=~a~~a~caa~=a~'ctt~a~_c~aa~_cmaca~
aaac
aaca~taa~«~ma__'a_~~t~et_'caa~aaacaaoata~~ctactcatcaaet'_c«caa~~_ccalcaa'_t«ct~
aca~a
caagctaagCl'=CICCII:II(:laW
1C;1'=CgafIlCgCagggBgagCCICCICa~?gacat~CCCIC~a~'Ea~alal'=aCg
cgcca~;aeccaactc~CCaCa~~CCI~a~CCBa~IC:I(:CaCaIgCCI~a~CCIa~I~aCC'la~la~lCICaCaa~
ICCCI
2C gtggCatICalCatlC gagCC lC Yg ~a'=l:lC~~gagaaaeaa~ila~gC I~~I:;IC lC ~CIC
~~lC I'L' ~C;1~'C a~~a~laC
aC~aCIICICCa~ICCC~IagCltaCgCgalCgC~glgCl~;°CC~Ca~CB~aa:laa~a~aY;IIC~':IY
I:IIC:IIC:I~ag('g
_~IoCI~~CC'=C''?aWaagga''C;t''a~~It~,aolaCCCCCag~oggaagC;lgagaC;ICa;ll:a~:1'':t
'';IIICIIC~BI~CCC
Igggagcattcaca~=~C~_~Cl~fl~af~_aCCaalICCgCICCIlCIICCaCI~ag~lilaLCaCCICaC:ICCa(C
allgla
atataccatctcrl'_111Ita1IlIVIIICI_~'l~al_~IoIIII~IC;Cf_pa'_laClClC«CCe:lalll~~lC
;iCaCa~I~'
~aCl~l~l~atuaa~t«~~o~~~=~_a~_~~'Clca~oaagtgl~l~IIQCoIl~lalalaalCll~'e~lCl,_C:II
IC:IICIaa
~_'C;lla~Taa;laaaCCaaaaaaa;laal(!_aaaaatlll::l~aaaalgatticacaaaaaml~~;l~'1,'tlC
alata~ll~Call
oCallla~oalC~a~ICIa~a'_l~_lllCalIIai'~alCall~_catat~cata_QQo_'aCaal~al~aL'al:l'
_cCtIQia:l~
l:ltlll!',!_~IIC:ICCaVataaaW
.1'_I~cCClc~lt~lla~ll~icl~al~cata~lcaaf~:laall_'a:l~laaaaCIaC~
tlCCal~CCla~alt~Cl(aaCIC'_ayaCaC'=aCf'_lla'=~alCl~ataccauccctalC;ntlll~':laWl_a
etCl~'a
tttaeaattatcat~tctt~~calceaatu_'aactcat~~ataccctaaaatactt~~at«tcatacicallllaaca
actctt~tlaatccaa~laect~_ammotattata~ca~(taacccaaacccaaacctaaaetitr(t(caa~_ccct
:llalC:IC(l~l~':l~lYlll~'l!_;t~'~_ICII;IIIIC~=atl~a~Clt~~?fa8aaa~l~lla~=~'IlC
ila;lC "JCa;=a~=ataL'I
~ICllal~'la~IlCI;IYIIC~C~IIIIIC
~_T;.IC:le~ata~~acta~~t'_~_~C_'Clla~alC;tl~'_'_IILS','_al'_I~Ilta
CA 02362897 2001-09-18
WO 00/55325 PCT/US00/()7392
aaa~=aaaa~~~Tgt~aa«catt~;u;_Tataa~~aaag~~aaa~aattctag~_~~aa~'tuayctaaaaaa~=ttag
aaaaaaaa
tcta~_taaa~,Ttttt~~~a;u~ttaaa=a:laa~aat~=ao~ttcu~tta~ttaaa~aagaa~~'~=ttaaaa~_cc
tttt~tt
taaaaaattaaaaaca~~aaccna~tt~'uaaa~aaatccaaatcc~cta~atgtatcaaa~t~ll~'a~Taaa~c«ct
c
cta~=a~ttaa;;Ta~Taaaa~_aaaa;=aat~'atatyaaaaa~=a~'ttt;aaagatlcatgagt~'caaa~y=gta
~a~=ttaa~ttct
I'_t:Ilt~ooaCta~a~lt'=~'~;u;l;teC:lll;lta;W
llCall'_llal:ICfat~L'~=l:l!_al~'''~':llClfalClClYlal~Ca
Ia~3Cll°°°aCllaCClll;!'~Cattctactaaa~'ctcaatcattclt~'a~aoa
llCC:CIL'il:lc'llaa'~CCI:IIICt~Ia
a~=~;~taccatlatt~tctctt~=accttcacctta~ccaaat~a~ttca«~_at~~;lt~catt~Tc«eattcacgt
trta~aa
ctaat~aat~«aaa~~«a«get,eatttgaaaecatstatag~Ttcgagtataa~Taa'acaoatt~att~'ataacaa
~~c
at~ectaac~itt«ea~taaaat«aatc;ltatc~catctta~aactaccaactt~_~acatt~attttattt~ctcta
c
ctgat~c«t~~~ttct~a~tceccaccltcaaacctctcc«caactatgtc« chat«~cug:l~'V~caa~caaa_a
ctaa~tttt~~~T~a~ttaatat~tctalaal«~catgttttcagt~tccattcatcatcatttc~a~~tcaa~_tttt
ota
tcattcatcact~ttttatatca«tctcatcattctlocatactttgcat~atta~gataocut~~catacatatt~ca
tttct~a~tt_ttttca~~t~at«~~a~ctgttt~cgaecaaatta~aagaaac_agcca~~aaccagaa~Tatataet
c~
accaccatgtcgtgt~catccaac~ccatactc~accccctgotcgagtgact«ogagccattcttcccatctactgga
ccccca~atcgagtaacctca~ctcag~ccactc~at~acgccactcgaccccct~~lc~aotalcac«cgccaaacca
cctgaccacactcggccg«cactctaccace«actcgacca~toggtcgaetatcatcactcaccaccaacaccacta
ctcgaccgggcactcgatcacatcttctlagtctactcaaatccocactcaaccaoacaa~_c~~agcacaa~~aagag
aa
_a~gagaagaceaagt~tttg~aaec''gcctogacctccatcegatcacgaaocccatctc~gcccattatcactcta
tg
ggtcgggcgateaggnattggcctetctcctatcattttatttcettttgcataaatagatgtcttt~gY«ctetcct
gagacatctaglcgaca«eEg«t«gc«cag««attttclgt«tacictggctgcgccgc««Yc«ctacaa
CCtttaattc~aYatttttccaa~uattea~attccoCattlQatttcattt~ttatctt~tttctctaCICt«atCt
ctttacttatccaatattatcatttatct~_c~=ltlat~tctttga~catattgtct~a~tagtgac«a~,aucttaa
~
gatg~gatagagtagttotg~aatceelagtctatagaat~~ttaagtttta~aatt~att~_aatccctica~~acta
~l
lgt~tttactgcttatttctttctoatcaact~=oaattc~alcccaa~cattccc~caacca'=aa~=gt~_tlc~at
~~aat
~cttYatccacta~ticct~agatattcgtctctatcccaa~g~att~gccgtttaga~c~tttattgactttatca~t
c
I~ItCltaIt~CCI~L'Cala~ffa~BllCeoll;tet~ovaatla~lCl~iT~oClagl:l(lgCIIY:I~~TaIIIC
I:IICaCo~~
agt_aatt~atctatt~tt~=aacat~«otctaggYataec«~att~cecttgttaaccatly~ata~gctaggatac
cactttaltcoattaccccatcc«ag~=aat«ctc:~tctat«~atttctct~ttttatcgttatc~=c«aaltgttct
catt~~catgtctcttc~ttttt~caattactcyatcaacttactc~accacactca~t~tct~~:caaca~actatgc
agt
cgagtacagtt~tcatatttcctetct~=ttactc~accatatcttacttctggcatca~cct~tt~togttga~_t~a
ttt
tagtaattct~ttau~«lctgtt« c;tec:at~ttc~ctta~gactgttagaaaccccaaaact
~ttattgcttv~ctt
~acttagt~acttctgatcacatctcatctgt«gcatcacacctattt~gattgacaacctaaaaulctacaac~acat
=att=~tgtttttg~ataattgactaaaaacc
ta«atcacttaacactta~atticaccgaaataaaalatit«ccaa
~atgtsaaacll~~aaac««t~a~=;;~ateaantc«gtctt~tsaatagcaaacacaacatttagattlcargaaaat
saatatatttttccaagaggt~_taacu~aaaaaatccttgas~~at~aaatcta~ic«~'tgat~a_cgaacicaaca
c
«agatttcaccaaaat~aatalatttttcraa~a~~t~~gartc~aaaaaattctt~=ac~~atgaaatc«ytc«gt~
aata~caaacacaacattta~at«cac~=aaaat~aatatatttttccaa~a~ototaacttgaaaaaatcc«ga~gga
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~>:laal,~aal;IlalllllCt
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tctt
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~7
CA 02362897 2001-09-18
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTS PARTIE DE CETTE DEMANDS OU CE BREVET
COMPREND PLUS D'UN TOME.
CECI EST LE TOME _ 1 DE S
NOTE: Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
:. I r.
JUMBO APPLICATIONS/PAi'~1VTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE
THIS IS VOLUME ( _ OF s
WOTE:.For additional voiumes-please contaci'the Canadian Patent Ofif~cQ -
~,: ' ~ :,.
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