Note: Descriptions are shown in the official language in which they were submitted.
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EXPRESSION OF FRUCTOSE 1 6 BISPHOSPHATE
ALDOLASE IN TRANSGENIC PLANTS
This invention relates to the expression of fructose 1,6 bisphosphate aldolase
(FDA) in transgenic plants to increase or improve plant growth and
development, yield,
vigor, stress tolerance, carbon allocation and storage into various storage
pools, and
distribution of starch. Transgenic plants expressing FDA have increased carbon
assimilation, export and storage in plant source and sink organs, which
results in growth,
yield and quality improvements in crop plants.
Recent advances in genetic engineering have provided the prerequisite tools to
t0 transform plants to contain alien (often referred to as "heteroiogous") or
improved
endogenous genes. These genes can lead either to an improvement of an already
existing
pathway in plant tissues or to an introduction of a novel pathway to modify
product levels,
increase metabolic efficiency, and or save on energy cost to the cell. It is
presently
possible to produce plants with unique physiological and biochemical traits
and
15 characteristics of high agronomic and crop processing importance. Traits
that play an
essential role in plant growth and development, crop yield potential and
stability, and crop
quality and composition include enhanced carbon assimilation, efficient carbon
storage,
and increased carbon export and partitioning.
Atmospheric carbon fixation (photosynthesis} by plants represents the major
source
20 of energy to support processes in all living organisms. The primary sites
of photosynthetic
activity, generally referred to as "source organs", are mature leaves and, to
a lesser extent,
green stems. The major carbon products of source leaves are starch, which
represents the
transitory storage form of carbohydrate in the chloroplast, and sucrose, which
represents
the predominant form of carbon transport in higher plants. Other plant parts
named "sink
25 organs" (e.g., roots, fruit, flowers, seeds, tubers, and bulbs) are
generally not autotrophic
and depend on import of sucrose or other major translocatable carbohydrates
for their
growth and development. The storage sinks deposit the imported metabolites as
sucrose
and other oligosaccharides, starch and other polysaccharides, proteins, and
triglycerides.
In leaves, the primary products of the Calvin Cycle (the biochemical pathway
30 leading to carbon assimilation) are glyceraldehyde 3-phosphate (G3P) anal
dihydroxyacetone phosphate (DHAP), also known as triose phosphates (triose-P).
The
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condensation of G3P and DHAP into fructose 1,6 bisphosphate (FBP) is catalyzed
reversibly by the enzyme fructose 1,6 bisphosphate aldolase (FDA), and various
isozymes
are known. The acidic isoenzyme appears to be chloroplastic and comprises
about 85% of
the total leaf aldolase activity. The basic isoenzyme is cytosolic. Both
isoenzymes appear
to be encoded by the nuclear genome and are encoded by different genes
(Lebherz et al.,
1984).
In the leaf, the chloroplast FDA is an essential enzyme in the Calvin Cycle,
where
its activity generates metabolites for starch biosynthesis. Removal of more
than 40% of
the plastidic aldolase enzymatic activity by antisense technology reduced leaf
starch
~ o accumulation as well as soluble proteins and chlorophyll levels but also
reduced plant
growth and root formation (Sonnewald et al., 1994). In contrast, the cytosolic
FDA is part
of the sucrose biosynthetic pathway where it catalyzes the reaction of FBP
production.
Moreover, cytosolic FDA is also a key enzyme in the glycolytic and
gluconeogenesis
pathways in both source and sink plant tissues.
~5 In the potato industry, production of higher starch and uniform solids
tubers is
highly desirable and valuable. The current potato varieties that are used for
french fry
production, such as Russet Burbank and Shepody, suffer from a non-uniform
deposition of
solids between the tuber pith (inner core) and the cortex (outer core). French
fry strips that
are taken from pith tissue are higher in water content when compared to outer
cortex
2o french fry strips; cortex tissue typically displays a solids level of
twenty-four percent
whereas pith tissue typically displays a solids level of seventeen percent.
Consequently, in
the french fry production process, the pith strips need to be blanched, dried,
and par-fried
for longer times to eliminate the excess water. Adequate processing of the
pith fries
results in the over-cooking of fries from the high solids cortex. The
blanching, drying, and
2s par frying times of the french fry processor need to be adjusted
accordingly to
accommodate the low solids pith strips and the high solids cortex strips. A
higher solids
potato with a more uniform distribution of starch from pith to cortex would
allow for a
more uttiforrn finished fry product, with higher plant throughput and cost
savings due to
reduced blanch, dry and par-fry times.
3o Although various fructose 1,6 bisphosphate aldolases have been previously
characterized, it has been discovered that overexpression of the enzyme in, a
transgenic
plant provides advantageous results in the plant such as increasing the
assimilation, export
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and storage of carbon, increasing the production of oils and/or proteins in
the plant and
improving tuber solids uniformity.
- The present invention provides structural DNA constructs that encode a
fructose
1,6 bisphosphate aldolase (FDA) enzyme and that are useful in increasing
carbon
assimilation, export, and storage in plants.
In accomplishing the foregoing, there is provided, in accordance with one
aspect of
the present invention, a method of producing genetically transformed plants
that have
elevated carbon assimilation, storage, export, and improved solids uniformity
comprising
the steps of:
to (a) Inserting into the genome of a plant a recombinant, double-stranded DNA
molecule comprising
(i) a promoter that functions in the cells of a target plant tissue,
(ii) a structural DNA sequence that causes the production of an RNA sequence
that encodes a fructose 1,6 bisphosphate aldolase enzyme,
is (iii) a 3' non-translated DNA sequence that functions in plant cells to
cause
transcriptional termination and the addition of polyadenylated nucleotides
to the 3' end of the RNA sequence;
(b) obtaining transformed plant cells; and
(c) regenerating from transformed plant cells genetically transformed plants
that
2o have elevated FDA activity.
In another aspect of the present invention there is provided a recombinant,
doubie-
stranded DNA molecule comprising in sequence
(i) a promoter that functions in the cells of a target plant tissue,
(ii) a structural DNA sequence that causes the production of an RNA sequence
25 that encodes a fructose 1,6 bisphosphate aldolase enzyme,
(iii) a 3' non-translated DNA sequence that functions in plant cells to cause
transcriptional termination and the addition of polyadenylated nucleotides to
the 3' end of the RNA sequence.
In a further aspect of the present invention, the structural DNA sequence that
3o causes the production of an RNA sequence that encodes a fructose i,6
bisphosphate
aldolase enzyme is coupled with a chloroplast transit peptide to facilitate
transport of the
enzyme to the plastid.
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In accordance with the present invention, an improved means for increasing
carbon
assimilation, storage and export in the source tissues of various plants is
provided. Further
means of improved carbon accumulation in sinks (such as roots, tubers, seeds,
stems, and
bulbs) are provided, thus increasing the size of various sinks (larger roots,
tubers, etc.) and
subsequently increasing yield and crop productivity. The increased carbon
availability to
these sinks would also improve composition and use efficiency in the sink
(oil, protein,
starch and/or sucrose production, and/or solids uniformity).
Various advantages may be achieved by the aims of the present invention,
including:
First, increasing the expression of the FDA enzyme in the chloroplast would
increase the flow of carbon through the Calvin Cycle and increase atmospheric
carbon
assimilation during early photoperiod. This would result in an increase in
photosynthetic
efficiency and an increase in chloroplast starch production (a leaf carbon
storage form
degraded during periods when photosynthesis is low or absent). Both of these
responses
would lead to an increase in sucrose production by the leaf and a net increase
in carbon
export during a given photoperiod. This increase in source capacity is a
desirable trait in
crop plants and would lead to increased plant growth, storage ability, yield,
vigor, and
stress tolerance.
Second, increasing FDA expression in the cytosol of photosynthetic cells would
lead to an increase in sucrose production and export out of source leaves.
This increase in
source capacity is a desirable trait in crop plants and would lead to
increased plant growth,
storage ability, yield, vigor, and stress tolerance.
Third, expression of FDA in sink tissues can show several desirable traits,
such as
increased amino acid and/or fatty acid pools via increases in carbon flux
through
glycolysis (and thus pyruvate levels) in seeds or other sinks and increased
starch levels as
result of increased production of glucose 6-phosphate in seeds, roots, stems,
and tubers
. where starch is a major storage nonstructural carbohydrate {reverse
glycolysis). This
increase in sink strength is a desirable trait in crop plants and would lead
to increased plant
growth, storage ability, yield, vigor, and stress tolerance.
Fourth, the invention is particularly desirable for use in the commercial
production
of foods derived from potatoes. Potatoes used for the production of french
fries and other
products suffer from a non-uniform distribution of solids between the tuber
pith (inner
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core) and the cortex (outer core). Thus, french fry strips from the pith
regions of such
tubers have a low solids content and a high water content in comparison to
cortex strips
from the same tubers. Therefore, the french fry processor attempts to adjust
the processing
parameters so that the final inner strips are sufficiently cooked while the
outer cortex strips
s are not overcooked. The results of such adjustments, however, are highly
variable and
may lead to poor quality product. Transgenic potatoes expressing fda will
provide to the
french-fry and potato chip processor a raw product that consistently displays
a higher tuber
solids uniformity with acceptable agronomic traits. In the french fry plant
production
process, inner pith fry strips from higher solids uniformity tubers will
require less time to
1o blanch, less time to dry to a specific solids content, and less time to par-
fry before freezing
and shipping to retail and institutional end-users.
Therefore, with respect to potatoes, the present invention provides 1 ) a
higher
quality, more uniform finish fry product in which french fries from all tuber
regions, when
processed, are nearly the same, 2) a higher through-put in the french fry
processing plant
15 due to lower processing times, and 3) processor cost savings due to lower
energy input
required for lower blanch, dry, and par-fry times. A raw tuber product that
displays a
higher solids uniformity will also produce a potato chip that has a reduced
saddle curl. and
a reduced tendency for center bubble, which are undesirable qualities in the
potato chip
industry. Reduced fat content would also result; this would contribute to
improved
2o consumer appeal and lower oil use (and costs) for the processor. The
increase in solids
uniformity will aiso translate to an increase in overall tuber solids. For
both the french fry
and chipping industries, this overall tuber solids increase will also result
in higher through-
put in the processing plant due to lower processing times, and cost savings
due to lower
energy input for blanching, drying, par-frying, and finish frying.
25 Figure 1 shows the nucleotide sequence and deduced amino acid sequence of a
fructose
1,6 bisphosphate aldolase gene from E. coli (SEQ ID No:l).
Figure 2 shows a plasmid map for plant transformation vector pMON 17524.
3o Figure 3 shows a plasmid map for plant transformation vector pMON17542.
CA 02294525 1999-12-13
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Figure 4 shows the change in diurnal fluctuations of sucrose, glucose, and
starch levels in
tobacco leaves expressing the fda transgene (pMON 17524) and control (pMON
17227).
The light period is from 7:00 to 19:00 hours. Only fully expanded and non-
senescing
leaves were sampled.
Figure 5 shows a plasmid map for plant transformation vector pMON13925.
Figure 6 shows a plasmid map for plant transformation vector pMON 17590.
1 o Figure 7 shows a plasmid map for plant transformation vector pMON 13936.
Figure 8 shows a plasmid map for plant transformation vector pMON 17581.
Figure 9 shows potato tuber cross-sections of improved solids uniformity Segal
Russet
15 Burbank lines (top row) versus unimproved nontransgenic Russet Burbank
(bottom row).
This invention is directed to a method for producing plant cells and plants
demonstrating an increased or improved growth and development, yield, quality,
starch
storage uniformity, vigor, and/or stress tolerance. The method utilizes a DNA
sequence
2o encoding an fda (fructose 1.6 bisphosphate aldolase) gene integrated in the
cellular
genome of a plant as the result of genetic engineering and causes expression
of the FDA
enzyme in the transgenic plant so produced. Plants that overexpress the FDA
enzyme
exhibit increased carbon flow through the Calvin Cycle and increased
atmospheric carbon
assimilation during early photoperiod resulting in an increase in
photosynthetic efficiency
25 and an increase in starch production. Thus, such plants exhibit higher
levels of sucrose
production by the leaf and the ability to achieve a net increase in carbon
export during a
. given photoperiod. This increase in source capacity leads to increased plant
growth that in
turn generates greater biomass andlor increases the size of the sink and
ultimately
providing greater yields of the transgenic plant. This greater biomass or
increased sink
3o size may be evidenced in different ways or plant parts depending on the
particular plant
species or growing conditions of the plant overexpressing the FDA enzyme.
Thus,
increased size resulting from overexpression of FDA may he seen in the seed,
fruit, stem,
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leaf, tuber, bulb or other plant part depending upon the plant species and its
dominant sink
during a particular growth phase and upon the environmental effects caused by
certain
growing conditions, e.g. drought, temperature or other stresses. Transgenic
plants
overexpressing FDA may therefore have increased carbon assimilation, export
and storage
in plant source and sink organs, which results in growth, yield, and
uniformity and quality
improvements.
Plants overexpressing FDA may also exhibit desirable quality traits such as
increased production of starch, oils and/or proteins depending upon the plant
species
overexpressing the FDA. Thus, overexpression of FDA in a particular plant
species may
t o affect or alter the direction of the carbon flux thereby directing
metabolite utilization and
storage either to starch production, protein production or oil production via
the role of
FDA in the glycolysis and gluconeogenesis metabolic pathways.
The mechanism whereby the expression of exogenous FDA modifies carbon
relationships is believed to derive from source-sink relationships. The leaf
tissue is a
15 sucrose source, and if more sucrose resulting from the activity of
increased FDA
expression is transported to a sink, it results in increased storage carbon
(sugars, starch,
oil, protein, etc.) or nitrogen (protein, etc.) per given weight of the sink
tissue.
The expression in a plant of a gene that exists in double-stranded DNA form
involves transcription of messenger RNA (mRNA) from one strand of the DNA by
RNA
20 polymerase enzyme, and the subsequent processing of the mRNA primary
transcript inside
the nucleus. This processing involves a 3' non-translated region, which adds
polyadenylate nucleotides to the 3' end of the RNA. Transcription of DNA into
mRNA is
regulated by a region of DNA usually referred to as the promoter. The promoter
region
contains a sequence of bases that signals RNA polymerase to associate with the
DNA and
25 to initiate the transcription of mRNA using one of the DNA strands as a
template to make
a corresponding complimentary strand of RNA. This RNA is then used as a
template for
. the production of the protein encoded therein by the cells protein
biosynthetic machinery.
A number of promoters that are active in plant cells have been described in
the
literature. These include the nopaline synthase (NOS) and octopine synthase
(OCS)
3o promoters (which are carried on tumor-inducing plasmids of Agrobacterium
tumefaciens),
the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and
35S and
the figwort mosaic virus (FMV) 35S-promoters, the light-inducible promoter
from the
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small subunit of ribulose-1,5-bisphosphate carboxylase (ssRUBISCO), a very
abundant
plant polypeptide, and the chlorophyll alb binding protein gene promoters,
etc. All of
these promoters have been used to create various types of DNA constructs that
have been
expressed in plants; see, e.g., PCT publication WO 84/02913.
Promoters that are known to or are found to cause transcription of DNA in
plant
cells can be used in the present invention. Such promoters may be obtained
from a variety
of sources such as plants and plant viruses and include, but are not limited
to, the enhanced
CaMV35S promoter and promoters isolated from plant genes such as ssRUBISCO
genes.
As described below, it is preferred that the particular promoter selected
should be capable
~o of causing sufficient expression to result in the production of an
effective amount of
fructose 1,6 bisphosphate aldolase enzyme to cause the desired increase in
carbon
assimilation, export or storage. Expression of the double-stranded DNA
molecules of the
present invention can be driven by a constitutive promoter, expressing the DNA
molecule
in all or most of the tissues of the plant. Alternatively, it may be preferred
to cause
15 expression of the fda gene in specific tissues of the plant, such as leaf,
stem, root, tuber,
seed, fruit, etc. The promoter chosen will have the desired tissue and
developmental
specificity. Those skilled in the art will recognize that the amount of
fructose 1,6
bisphosphate aldolase needed to induce the desired increase in carbon
assimilation, export,
or storage may vary with the type of plant. Therefore. promoter function
should be
20 optimized by selecting a promoter with the desired tissue expression
capabilities and
approximate promoter strength and selecting a transformant that produces the
desired
fructose I,6 bisphosphate aldolase activity or the desired change in
metabolism of
carbohydrates in the target tissues. This selection approach from the pool of
transformants
is routinely employed in expression of heterologous structural genes in plants
because
25 there is variation between transformants containing the same heterologous
gene due to the
site of gene insertion within the plant genome (commonly referred to as
"position effect")
In addition to promoters that are known to cause transcription (constitutively
or tissue-
specific) of DNA in plant cells, other promoters may be identified for use in
the current
invention by screening a plant cDNA library for genes that are selectively or
preferably
3o expressed in the target tissues of interest and then isolating the promoter
regions by
methods known in the art. In particular, it may be desirable to use a bundle
sheath cell
specific (or cell enhanced expression) promoter for use with C4 plants such as
corn,
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sorghum, and sugarcane to obtain the yield benefits of overexpression of FDA
and not use
a constitutive promoter or a promoter with mesophyll cell enhanced expression
properties.
For the purpose of expressing the.fda gene in source tissues of the plant,
such as
the leaf or stem, it is preferred that the promoters utilized in the double-
stranded DNA
s molecules of the present invention have relatively high expression in these
specific tissues.
For this purpose, one may also choose from a number of promoters for genes
with leaf
specific or leaf-enhanced expression. Examples of such genes known from the
literature
are the chloroplast glutamine synthetase GS2 from pea (Edwards et al., 1990),
the
chloroplast fructose-1,6-bisphosphatase (FBPase) from wheat (Lloyd et al.,
1991), the
to nuclear photosynthetic ST-LS1 from potato (Stockhaus et al., 1989), and the
phenylalanine
ammonia-Iyase (PAL) and chalcone synthase (CHS) genes from Arabidopsis
thaliarra
(Leyva et al., 1995}. Also shown to be active in photosynthetically active
tissues are the
ribulose-1,5-bisphosphate carboxyiase (RUBISCO), isolated from eastern larch
(Larix
laricina) (Campbell et al., 1994); the cab gene, encoding the chlorophyll a/6-
binding
15 protein of PSII, isolated from pine (cab6; Yamamoto et al., 1994), wheat
(Cab-l; Fejes et
al., 1990), spinach (CAB-1; Luebberstedt et al., 1994), and rice (cabl R: Luan
et al., 1992);
the pyruvate orthophosphate dikinase (PPDK) from maize (Matsuoka et al.,
1993); the
tobacco Lhcbl *2 gene (Cerdan et al., 1997); the Arabidopsis thaliaha SUC2
sucrose-H+
symporter gene (Truernit et al., 1995); and the thylacoid membrane proteins,
isolated from
2o spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS; Oelmueller et
al., 1992).
Other chlorophyll alb-binding proteins have been studied and described in the
literature,
such as LhcB and PsbP from white mustard (Sinapis alba; Kretsch et al., 1995).
Homologous promoters to those described here may also be isolated from and
tested in the
target or related crop plant by standard molecular biology procedures.
25 For the purpose of expressing the fda in sink tissues of the plant, for
example the
tuber of the potato plant; the fruit of tomato; or seed of maize, wheat, rice,
or barley, it is
,preferred that the promoters utilized in the double-stranded DNA molecules of
the present
invention have relatively high expression in these specif c tissues. A number
of genes with
tuber-specific or tuber-enhanced expression are known, including the class I
patatin
3o promoter (Bevan et al., 1986; Jefferson et al., 1990); the potato tuber
ADPGPP genes, both
the large and small subunits (Muller et al., 1990); sucrose synthase
(Salangubat and
Belliard, 1987, 1989); the major tuber proteins including the 22 kDa protein
complexes
9
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and proteinase inhibitors {Hannapel, 1990); the granule bound starch synthase
gene
(GBSS) (Rohde et al., 1990); and the other class I and II patatins (Rocha-Sosa
et al., 1989;
Mignery et al., 1988). Other promoters can also be used to express a fructose
1,6
bisphosphate aidolase gene in specific tissues, such as seeds or fruits. The
promoter for 13-
conglycinin (Tierney, 1987) or other seed-specific promoters, such as the
napin and
phaseolin promoters, can be used to over-express an fda gene specifically in
seeds. The
zeros are a group of storage proteins found in maize endosperm. Genomic clones
for zero
genes have been isolated (Pedersen et al., 1982), and the promoters from these
clones,
including the 15 kDa, 16 lcDa, 19 kDa, 22 kDa, 27 kDa, and gamma genes, could
also be
to used to express an fda gene in the seeds of maize and other plants. Other
promoters
known to function in maize, wheat, or rice include the promoters for the
following genes:
waxy, Brittle, Shrunken 2, branching enzymes I and II, starch syntheses,
debranching
enzymes, oleosins, glutelins, and sucrose syntheses. Particularly preferred
promoters for
maize endosperm expression, as well as in wheat and rice, of an fda gene is
the promoter
~5 for a glutelin gene from rice, more particularly the Osgt-i promoter (Zheng
et al., 1993);
the maize granule-bound starch synthase (waxy) gene (zmGBS); the rice small
subunit
ADPGPP promoter (osAGP) ;and the zero promoters, particularly the maize 27 kDa
zero
gene promoter (zm27) (see. generally, Russell et al., 1997). Examples of
promoters
suitable for expression of an_fda gene in wheat include those for the genes
for the
2o ADPglucose pyrophosphorylase (ADPGPP) subunits, for the granule bound and
other
starch syntheses, for the branching and debranching enzymes, for the
embryogenesis-
abundant proteins, for the gliadins, and for the glutenins. Examples of such
promoters in
rice include those for the genes for the ADPGPP subunits, for the granule
bound and other
starch syntheses, for the branching enzymes, for the debranching enzymes, for
sucrose
25 syntheses, and for the glutelins. A particularly preferred promoter is the
promoter for rice
glutelin, Osgt-I. Examples of such promoters for barley include those for the
genes for the
ADPGPP subunits, for the granule bound and other starch syntheses, for the
branching
enzymes, for the debranching enzymes, for sucrose syntheses, for the hordeins,
for the
embryo globulins, and for the aleurone-specific proteins.
3o The solids content of root tissue may be increased by expressing an fda
gene
behind a root-specific promoter. An example of such a promoter is the promoter
from the
acid chitinase gene (Sumac et al., 1990). Expression in root tissue could also
be
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WO 98/58069 PCT/US98/12447
accomplished by utilizing the root-specific subdomains of the CaMV35S promoter
that
have been identified (Benfey et al., 1989).
The RNA produced by a DNA construct of the present invention may also contain
a S' non-translated leader sequence. This sequence can be derived from the
promoter
s selected to express the gene and can be specifically modified so as to
increase translation
of the mRNA. The 5' non-translated regions can also be obtained from viral
RNAs, from
suitable eukaryotic genes, or from a synthetic gene sequence. The present
invention is not
limited to constructs, as presented in the following examples, wherein the non-
translated
region is derived from the 5' non-translated sequence that accompanies the
promoter
1 o sequence. Rather, the non-translated leader sequence can be derived from
an unrelated
promoter or coding sequence.
In monocots, an intron is preferably included in the gene construct to
facilitate or
enhance expression of the coding sequence. Examples of suitable introns
include the
HSP70 intron and the rice actin intron, both of which are known in the art.
Another
15 suitable intron is the castor bean catalase intron (Suzuki et al., 1994)
Polvadenvlation signal
The 3' non-translated region of the chimeric plant gene contains a
polyadenylation
signal that functions in plants to cause the addition of polyadenylate
nucleotides to the
3' end of the RNA. Examples of suitable 3' regions are ( I ) the 3'
transcribed, non-
2o translated regions containing the polyadenylation signal of Agrobacterium
tumor-inducing
(Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2) plant
genes like the
soybean storage protein genes and the small subunit of the ribulose-1,5-
bisphosphate
carboxylase (ssRUBISCO) gene.
Plastid-directed expression of fructose-1 6-bisphosphate aldolase activity_
25 In one embodiment of the invention, the fda gene may be fused to a
chloroplast
transit peptide, in order to target the FDA protein to the plastid. As used
hereinafter,
chloroplast and plastid are intended to include the various forms of plastids
including
amyloplasts. Many plastid-localized proteins are expressed from nuclear genes
as
precursors and are targeted to the plastid by a chloroplast transit peptide
(CTP), which is
3o removed during the import steps. Examples of such chloroplast proteins
include the small
subunit of ribulose-1,5-biphosphate carboxylase (ssRUBISCO, SSU), 5-
enolpyruvateshikimate-3-phosphate synthase (EPSPS), ferredoxin, ferredoxin
11
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WO 98158069 PCT/US98/12447
oxidoreductase, the light-harvesting-complex protein I and protein II, and
thioredoxin F. It
has been demonstrated that non-plastid proteins may be targeted to the
chloroplast by use
of protein fusions with a CTP and that a CTP sequence is sufficient to target
a protein to
the plastid. Those skilled in the art will also recognize that various other
chimeric
constructs can be made that utilize the functionality of a particular plastid
transit peptide to
import the fructose-1,6-diphosphate aldolase enzyme into the plant cell
plastid. The fda
gene could also be targeted to the plastid by transformation of the gene into
the chloroplast
genome (Daniell et al., 1998).
Fructose 1,6 bisphosphate aldolases
to As used herein, the term "fructose 1, 6-bisphophate aldolase" means an
enzyme
(E.C. 4.1.2.13) that catalyzes the reversible cleavage of fructose I,6-
bisphosphate to form
glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP}.
Aldolase
enzymes are divided into two classes, designated class l and class II (Witke
and Gotz,
1993). Various fda genes encoding the enzyme have been sequenced, as have
numerous
proteins, such as the cytosolic enzyme from maize (GenBank Accession
S07789;S10638),
cytosolic enzyme from rice (GenBank Accession JQ0543), cytosolic enzyme from
spinach
(GenBank Accession 531091;S22093), from Arabidopsis thaliana (GenBank
Accession
S11958), from spinach chloroplast (GenBank Accession S3I090;A21815;S22092),
from
yeast (S cerevisiae) (GenBank Accession 507855; S37882; S12945; S39178;
544523;X75781), from Rhodobacter sphaeroides (GenBank Accession
B40767;D41080),
from B. subtilis (GenBank Accession S55426; D32354: E32354; D41835), from
garden
pea (GenBank Accession S29048; S34411 ), from garden pea chloroplast (GenBank
Accession S29047; 534410}, from maize (GenBank Accession 505019), from
Chlamydomonas reinhardtii (GenBank Accession S48639; 558485; S58486; 534367),
from Corynebacterium glutamicum (GenBank Accession 509283; X17313), from
Campylobacter jejuni (GenBank Accession S52413}, from Haemophilus influenzae
(strain
Rd 1~:'V20) (GenBank Accession C64074), from Streptococcus pneumonia (GenBank
Ac Yssion AJ005697), from rice (GenBank Accession X53130), and from the maize
anaerobically regulated gene (GenBank Accession X12872).
3o The class I enzymes may be isolated from higher eukaryotes, such as animals
and
plants, and in some prokaryotes, including Peptococcus aerogens> (Lebherz and
Rutter,
1973), Lactobacillus casei (London and Kline, 1973), Escherichia coli
(Stribling and
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Perham, 1973), Mycobacterium smegmatis (Bai et al., 1975), and most
staphylococcal
species (Gotz et al., 1979). The gene for the FDA enzyme may be obtained by
known
methods and has already been done so for several organisms, such as rabbit
(Lai et al.,
1974), human (Besmond et al., 1983), rat (Tsutsumi et al., 1984), Trypanosoma
brucei
(Clayton, 1985), and Arabidopsis thaliana (Chopra et al., 1990). These class I
enzymes
are invariably tetrameric proteins with a total molecular weight of about 160
kDa and
function by imine formation between the substrate and a lysine residue in the
active site
(Alfounder et al., 1989).
In animal, three class I isozymes, classified as A, B, and C, are expressed in
the
1 o cytosol of muscle, liver, and brain tissue respectively, and they differ
from plant aldolases
in their expression and compartmentation patterns (Joh et al., 1986). In the
leaves of
higher plants, FDA is a class I enzyme, and two different isoenzymes within
the class have
been documented. One is contained in the chloroplast and the other in the
cytosol
(Lebherz et al., i 984). The acidic plant isozyme appear to be chloroplastic
and comprises
IS about 85% of the total leaf aldolase activity. The basic plant isozyme is
cytosolic, and
both isozymes appear to be encoded by the nuclear genome and are encoded by
different
genes (Lebherz et al., 1984).
The class II type aldolases are normally dimeric with molecular mass of
approximately 80 kDa, and their activity depends on divalent metal ions. The
class II
2o enzymes may be isolated from prokaryotes, such as blue-green algae and
bacteria, and
eukaryotic green algae and fungi (Baldwin et al., 1978). The gene for the FDA
class II
enzyme may be obtained by known methods and has already been done so from
several
organisms including Saccharomyces cerevisiae (Jack and Harris, 1971 ),
Bacillus
stearothermophilus (Jack, 1973), and Escherichia coli (Baldwin et al., 1978).
25 It is believed that highly homologous class Ii fructose l, 6-bisphophate
aldolases
with similar catalyzing activity will also be found in other species of
microorganism, such
. as Saccharomyces (Saccharomyces cerevisiae); Bacillus (Bacillus subtilis);
Rhodobacter
(Rhodobacter sphaeroides); Plasmodium (Plasmodium falciparium, Plasmodium
berghei);
Trypanosoma (Trypanosoma brucei); Chlamydomonas (Chlamydomas reinhardtii);
3o Candida (Candida albicans); Corynebacterium (Corynebacterium glutamicum);
Campylobacter (Campylobacter jejuni); and Haemophilus (Haemophilus.influenza).
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Such sequences can be readily isolated by methods well known in the art, for
example by nucleic acid hybridization. The hybridization properties of a given
pair of
nucleic acids are an indication of their similarity or identity. Nucleic acid
sequences can
be selected on the basis of their ability to hybridize with known fda
sequences. Low
stringency conditions may be used to select sequences with less homology or
identity.
One may wish to employ conditions such as about 0.15 M to about 0.9 M sodium
chloride,
at temperatures ranging from about 20°C to about 55°C. High
stringency conditions may
be used to select for nucleic acid sequences with higher degrees of identity
to the disclosed
sequences. Conditions typically employed may include about 0.02 M to about
0.15 M
to sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about
0.1 % N-
iaurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at
hybridization
temperatures between about 50°C and about 70°C. More preferably,
high stringency
conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02%
SDS, about
0.001 M sodium citrate, at a temperature of about 50°C. The skilled
individual will
recognize that numerous variations are possible in the conditions and means by
which
nucleic acid hybridization can be performed to isolate fda sequences having
similarity to
fda sequences known in the art and are not limited to those explicitly
disclosed herein.
Preferably, such an approach is used to isolate fda sequences having greater
than about
60% identity with the disclosed E.coli,fda sequence, more preferably greater
than about
70% identity, most preferably greater than about 80% identity.
Depending on growth conditions Euglena gracilis, Chlamydomonas mundana, and
Chlamydomomas rheinhardi produce either a class I or a class II aldolase
(Cremona, 1968;
Russell and Gibbs, 1967; Guerrini et al., 1971 }.
The isolation of a class II fda gene from E toll is described in the following
examples. Its DNA sequence is given as SEQ ID NO:1 and shown in Figure 1. The
amino acid sequence is shown in SEQ ID N0:2 and shown in Figure 1. This gene
can be
. used as isolated by inserting it into plant expression vectors suitable for
the transformation
method of choice as described. The E. toll FDA enzyme has an app.:xrent pH
optimum
range near pH 7-9 and retains activity in the lower pH range of 5-7 (Baldwin
et al., 1978;
3o Alfounder et al., 1989}.
Thus, many different genes that encode a fructose 1,6 bisphosphate aldolase
activity may be isolated and used in the present invention.
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Synthetic Qene construction
A carbohydrate metabolizing enzyme considered in this invention includes any
sequence of amino acids, such as protein, polypeptide, or peptide fragment,
that
demonstrates the ability to catalyze a reaction involved in the synthesis or
degradation of
starch or sucrose. These can be sequences obtained from a heterologous source,
such as
algae, bacteria, fungi, and protozoa, or endogenous plant sequences, by which
is meant
any sequence that can be naturally found in a plant cell, including native
(indigenous)
plant sequences as well as sequences from plant viruses or plant pathogenic
bacteria.
It will be recognized by one of ordinary skill in the art that carbohydrate
metabolizing enzyme gene sequences may also be modified using standard
techniques
such as site-specific mutation or PCR, or modification of the sequence may be
accomplished by producing a synthetic nucleic acid sequence and will still be
considered a
carbohydrate biosynthesis enzyme nucleic acid sequence of this invention. For
example,
"wobble" positions in codons may be changed such that the nucleic acid
sequence encodes
the same amino acid sequence, or alternatively, codons can be altered such
that
conservative amino acid substitutions result. In either case, the peptide or
protein
maintains the desired enzymatic activity and is thus considered part of this
invention.
A nucleic acid sequence to a carbohydrate metabolizing enzyme may be a DNA or
RNA sequence. derived from genomic DNA, cDNA, mRNA, or may be synthesized in
2o whole or in part. The structural gene sequences may be cloned, for example,
by isolating
genomic DNA from an appropriate source and amplifying and cloning the sequence
of
interest using a polymerase chain reaction (PCR). Alternatively, the gene
sequences may
be synthesized, either completely or in part, especially where it is desirable
to provide
plant-preferred sequences. Thus, all or a portion of the desired structural
gene may be
synthesized using codons preferred by a selected plant host. Plant-preferred
codons may
be determined, for example, from the codons used most frequently in the
proteins
. expressed in a particular plant host species. Other modifications of the
gene sequences
may result in mutants having slightly altered activity.
If desired, the gene sequence of the fda gene can be changed without changing
the
3o protein sequence in such a manner as may increase expression and thus even
more
positively affect carbohydrate content in transformed plants. A
preferred,manner for
making the changes in the gene sequence is set out in PCT Publication WO
90/10076. A
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gene synthesized by following the methodology set out therein may be
introduced into
plants as described below and result in higher levels of expression of the FDA
enzyme.
This may be particularly useful in monocot5 such as maize, rice, wheat,
sugarcane, and
barley.
Combinations with other transaenes
The effect of fda in transgenic plants may be enhanced by combining it with
other
genes that positively affect carbohydrate assimilation or content, such as a
gene encoding
for a sucrose phosphorylase as described in PCT Publication WO 96/24679, or
ADPGPP
genes such as the E. toll glgC gene and its mutant glgCl6. PCT Publication WO
to 91/19806 discloses how to incorporate the latter gene into many plant
species in order to
increase starch or solids. Another gene that can be combined with fda to
increase carbon
assimilation, export or storage is a gene encoding for sucrose phosphate
synthase (SPS).
PCT Publication WO 92/16631 discloses one such gene and its use in transgenic
plants.
Plant transformation/re~eneration
In developing the nucleic acid constructs of this invention, the various
components
of the construct or fragments thereof will normally be inserted into a
convenient cloning
vector, e.g., a plasmid that is capable of replication in a bacterial host,
e.g., E. toll.
Numerous vectors exist that have been described in the literature. many of
which are
commercially available. After each cloning, the cloning vector with the
desired insert may
2o be isolated and subjected to further manipulation, such as restriction
digestion, insertion of
new fragments or nucleotides, ligation, deletion, mutation, resection, etc. so
as to tailor the
components of the desired sequence. Once the construct has been completed, it
may then
be transferred to an appropriate vector for further manipulation in accordance
with the
manner of transformation of the host cell.
A recombinant DNA molecule of the invention typically includes a selectable
marker so that transformed cells can be easily identified and selected from
non-
transformed cells. Examples of such include, but are not limited to, a
neomycin
phosphotransferase (nptII) gene (Potrykus et al., 1985), which confers
kanamycin
resistance. Cells expressing the nptII gene can be selected using an
appropriate antibiotic
3o such as kanamycin or 6418. Other commonly used selectable markers include
the bar
gene, which confers bialaphos resistance; a mutant EPSP synthase gene (Hinchee
et aL,
1988), which confers glyphosate resistance; a nitrilase gene, which confers
resistance to
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bromoxynil {Stalker et al., 1988); a mutant acetolactate synthase gene (ALS),
which
confers imidazolinone or sulphonylurea resistance (European Patent Application
154,204,
1985); and a methotrexate resistant DHFR gene (Thillet et al., 1988).
Plants that can be made to have enhanced carbon assimilation, increased carbon
export and partitioning by practice of the present invention include, but are
not limited to,
Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus,
avocado, banana,
barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts,
cabbage, canola,
cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus,
clementines,
coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole,
eucalyptus, fennel,
figs, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks,
lemon, lime,
Loblolly pine, mango, melon, mushroom, nut, oat, oil seed rape, okra, onion.
orange, an
ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper,
persimmon, pine,
pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince.
radiata pine,
radicchio, radish, raspberry, rice, rye, sorghum, Southern pine, soybean,
spinach, squash,
strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum,
tangerine, tea,
tobacco, tomato, triticale, turf, a vine, watermelon, wheat, yams, and
zucchini.
A double-stranded DNA molecule of the present invention containing an fda gene
can be inserted into the genome of a plant by any suitable method. Suitable
plant
transformation vectors include those derived from a Ti plasmid of
Agrobacterium
tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella et al.
(1983), Bevan
(1984), Klee et al. (1985) and EPO publication 120,516. In addition to plant
transformation vectors derived from the Ti or root-inducing (Ri) plasmids of
Agrobacterium, alternative methods can be used to insert the DNA constricts of
this
invention into plant cells. Such methods may involve, for example, the use of
liposomes,
electroporation: chemicals that increase free DNA uptake, free DNA delivery
via
microprojectile bombardment, and transformation using viruses or pollen. DNA
may also
be inserted into the chloroplast genome (Daniell et al., 1998).
A plasmid expression vector suitable for the introduction of an fda gene in
monocots using microprojectile bombardment is composed of the following: a
promoter
that is specific or enhanced for expression in the starch storage tissues in
monocots,
generally the endosperm, such as promoters for the zero genes found in the
maize
endosperm (Pedersen et al., 1982); an intron that provides a splice site to
facilitate
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expression of the gene, such as the Hsp70 intron (PCT Publication W093/19189);
and a 3'
polyadenylation sequence such as the nopaline synthase 3' sequence (NOS 3';
Fraley et al.,
1983). This expression cassette may be assembled on high copy replicons
suitable for the
production of large quantities of DNA.
A particularly useful Agrobacterium-based plant transformation vector for use
in
transformation of dicotyledonous plants is plasmid vector pMON530 (Rogers et
al., 1987).
Plasmid pMON530 is a derivative of pMON505 prepared by transferring the 2.3 kb
StuI-
HindIII fragment of pMON316 (Rogers et al., 1987) into pMON526. Plasmid
pMON526
is a simple derivative of pMON505 in which the SmaI site is removed by
digestion with
to XmaI, treatment with Klenow polymerase and ligation. Plasmid pMON530
retains all the
properties of pMON505 and the CaMV35S-NOS expression cassette and now contains
a
unique cleavage site for SmaI between the promoter and polyadenylation signal.
Binary vector pMON505 is a derivative of pMON200 (Rogers et al., 1987) in
which the Ti plasmid homology region, LIH, has been replaced with a 3.8 kb
HindIII to
SmaI segment of the mini RK2 plasmid, pTJS75 (Schmidhauser and Helinski,
1985). This
segment contains the RK2 origin of replication, oriV, and the origin of
transfer, oriT, for
conjugation into Agrobacterium using the tri-parental mating procedure (Horsch
and Klee,
1986). Plasmid pMON505 retains all the important features of pMON200 including
the
synthetic mufti-linker for insertion of desired DNA fragments, the chimeric
2o NOS/NPTIf/NOS gene for kanamycin resistance in plant cells, the
spectinomycinlstreptomycin resistance determinant for selection in E. coli and
A. tumefaciens, an intact nopaline synthase gene for facile scoring of
transformants and
inheritance in progeny, and a pBR322 origin of replication for ease in making
large
amounts of the vector in E coli. Plasmid pMON505 contains a single T-DNA
border
derived from the right end of the pTiT37 nopaline-type T-DNA. Southern blot
analyses
have shown that plasmid pMON505 and any DNA that it carries are integrated
into the
. plant genome, that '°s, the entire plasmid is the T-DNA that is
inserted into the plant
genolrie. One end of the integrated DNA is located between the right border
sequence and
the nopaline synthase gene and the other end is between the border sequence
and the
pBR322 sequences.
Another particularly useful Ti plasmid cassette vector is pMON 17227. This
vector
is described in PCT Publication WO 92/04449 and contains a gene encoding an
enzyme
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WO 98158069 PCT/US98/12447
conferring glyphosate resistance (denominated CP4), which is an excellent
selection
marker gene for many plants, including potato and tomato. The gene is fused to
the
Arabidopsis EPSPS chloroplast transit peptide (CTP2) and expressed from the
FMV
promoter as described therein.
When adequate numbers of cells (or protoplasts) containing the fda gene or
cDNA
are obtained, the cells (or protoplasts) are regenerated into whole plants.
Choice of
methodology for the regeneration step is not critical, with suitable protocols
being
available for hosts from Leguminosae (alfalfa, soybean, clover, etc.),
Umbelliferae (carrot,
celery, parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.),
Cucurbitaceae
to (melons and cucumber), Gramineae (wheat, barley, rice, maize, etc.),
Solanaceae (potato,
tobacco, tomato, peppers}, various floral crops, such as sunflower, and nut-
bearing trees,
such as almonds, cashews, walnuts, and pecans. See, e.g., Ammirato et al. {
1984);
Shimamoto et al. (1989); Fromm (1990); Vasil et al. (1990); Vasii et al.
(1992);
Hayashimoto ( I 990); and Datta et al. { 1990).
15 The following definitions are provided in order to aid those skilled in the
art in
understanding the detailed description of the present invention.
The term "promoter" or "promoter region" refers to a nucleic acid sequence,
usually found upstream (5') to a coding sequence, that controls expression of
the coding
sequence by controlling production of messenger RNA (mRNA) by providing the
20 recognition site for RNA polymerase or other factors necessary for start of
transcription at
the correct site. As contemplated herein, a promoter or promoter region
includes
variations of promoters derived by means of ligation to various regulatory
sequences,
random or controlled mutagenesis, and addition or duplication of enhancer
sequences.
The promoter region disclosed herein, and biologically functional equivalents
thereof, are
25 responsible for driving the transcription of coding sequences under their
control when
introduced into a host as part of a suitable recombinant vector, as
demonstrated by its
ability to produce mRNA.
"Regeneration" refers to the process of growing a plant from a plant cell
(e.g., plant
protoplast or explant).
30 "Transformation" refers to a process of introducing an exogenous nucleic
acid
sequence (e.g., a vector, recombinant nucleic acid molecule) into a cell
or.protoplast in
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which that exogenous nucleic acid is incorporated into a chromosome or is
capable of
autonomous replication.
A "transformed cell" is a cell whose DNA has been altered by the introduction
of
an exogenous nucleic acid molecule into that cell.
The term "gene" refers to chromosomal DNA, plasmid DNA, cDNA, synthetic
DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA
molecule, and
regions flanking the coding sequence involved in the regulation of expression.
"Identity" refers to the degree of similarity between two nucleic acid or
protein
sequences. An alignment of the two sequences is performed by a suitable
computer
t o program. A widely used and accepted computer program for performing
sequence
alignments is CLUSTALW v1.6 (Thompson et al., 1994). The number of matching
bases
or amino acids is divided by the total number of bases or amino acids and
multiplied by
100 to obtain a percent identity. For example, if two 580 base pair sequences
had i45
matched bases, they would be 25 percent identical. If the two compared
sequences are of
15 different lengths, the number of matches is divided by the shorter of the
two lengths. For
example, if there were 100 matched amino acids between 200 and a 400 amino
acid
proteins, they are 50 percent identical with respect to the shorter sequence.
If the shorter
sequence is less than 50 bases or amino acids in length, the number of matches
are divided
by 50 and multiplied by 100 to obtain a percent identity.
20 "C-terminal region" refers to the region of a peptide. polypeptide, or
protein chain
from the middle thereof to the end that carries the amino acid having a free
carboxyl
group.
The phrase "DNA segment heterologous to the promoter region" means that the
coding DNA segment does not exist in nature in the same gene with the promoter
to which
25 it is now attached.
The term "encoding DNA" refers to chromosomal DNA, plasmid DNA, cDNA, or
synthetic DNA that encodes any of the enzymes discussed herein.
The term "genome" as it applies to bacteria encompasses both the chromosome
and
plasmids within a bacterial host cell. Encoding DNAs of the present invention
introduced
3o into bacterial host cells can therefore be either chromosomally integrated
or plasmid-
localized. The term "genome" as it applies to plant cells encompasses not only
chromosomal DNA found within the nucleus, but organelle DNA found within
subcellular
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components of the cell. DNAs of the present invention introduced into plant
cells can
therefore be either chromosomaily integrated or organelle-localized.
The terms "microbe" or "microorganism" refer to algae, bacteria, fungi, and
protozoa.
The term "mutein" refers to a mutant form of a peptide, polypeptide, or
protein.
"N-terminal region" refers to the region of a peptide, polypeptide, or protein
chain
from the amino acid having a free amino group to the middle of the chain.
"Overexpression" refers to the expression of a polypeptide or protein encoded
by a
DNA introduced into a host cell, wherein said polypeptide or protein is either
not normally
present in the host cell, or wherein said polypeptide or protein is present in
said host cell at
a higher Level than that normally expressed from the endogenous gene encoding
said
polypeptide or protein.
The term "plastid" refers to the class of plant cell organelles that includes
amyloplasts, chioroplasts, chromoplasts, elaioplasts, eoplasts, etioplasts,
leucoplasts, and
1s proplastids. These organelles are self replicating and contain what is
commonly referred
to as the "chloroplast genome," a circular DNA molecule that ranges in size
from about
120 kb to about 217 kb, depending upon the plant species, and which usually
contains an
inverted repeat region.
The phrase "simple carbohydrate substrate'' means a monosaccharide or an
oligosaccharide but not a polysaccharide; simple carbohydrate substrate
includes glucose,
fructose, sucrose, lactose. More complex carbohydrate substrates commonly used
in
media such as corn syrup. starch, and molasses can be broken down to simple
carbohydrate substrates.
The term ''solids" refers to the nonaqueous component of a tuber (such as in
potato) or a fruit (such as in tomato) comprised mostly of starch and other
polysaccharides, simple carbohydrates, nonstructural carbohydrated, amino
acids, and
other organic molecules.
The following examples are provided to better elucidate the practice of the
present
invention and should not be interpreted in any way to limit the scope of the
present
3o invention. Those skilled in the art will recognize that various
modifications, truncations,
etc., can be made to the methods and genes described herein while not
departing from the
spirit and scope of the present invention.
21
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EXAMPLES
EXAMPLE 1
cDNA cloning and overexpression
Unless otherwise stated, basic DNA manipulations and genetic techniques, such
as
PCR, agarose electrophoresis, restriction digests, ligations, E. coli
transformations, colony
screens, and Western blots were performed essentially by the protocols
described in
Sambrook et aI. (1989) or Maniatis et al. (1982).
The E. coli fda gene sequence (SEQ ID NO: 1) was obtained from Genbank
(Accession Number X14682) and nucleotide primers with homology to the 5' and
3' end
to were designed for PCR amplification. E. coli chromosomal DNA was extracted
and the E.
coli fda gene was amplified by PCR using the 5' oligonucleotide
5'GGGGCCATGGCTAAGATTTTTGATTTCGTA3' (SEQ ID N0:3) and the 3'
oligonucleotide 5'CCCCGAGCTCTTACAGAACGTCGATCGCGTTCAG3' (SEQ ID
N0:4). The PCR cycling conditions were as follows: 94 C, ~ min (1 cycle);
addition of
polymerase; 94~C, 1 min.. 60 C, 1 min., 72~C, 2 min.30 sec. (35 cycles). The
1.08 kb PCR
product was gel purified and ligated into an E.coli expression vector,
pMON5723, to form
a vector construct that was used for transformation of frozen competent E.coli
JM101
cells. The pMON5723 vector contains the E.coli recA promoter and the T7 genel0
leader
(G10L) sequences, which enable high level expression in E.coli (along et al.,
1988). After
induction of the transformed cells. a distinct protein band of about 40 kDa
was apparent on
an SDS PAGE gel, which correlates with the size of the subunit polypeptide
chain of the
dimeric aldolase II. It was shown that most of the induced protein was present
in the
soluble phase. A gel slice containing the highly induced protein was isolated
and
antibodies were produced in a goat, which was injected with the homogenized
gel slice
(emulsified in Freund's complete adjuvant).
The fda~gene sequence was subsequently cloned into another E.coli expression
vector, under th;. control of the tc:7 promoter. Induction with IPTG of JM101
cells
transformed with this vector showed the same 40 kDa overexpressed protein
band. This
new clone was used in an enzyme assay for FDA activity. Cells transformed with
this
3o vector construct were grown in a liquid culture, induced with IPTG, and
grown for another
3 hours. Subsequently, a 3 mL cell culture was spun down, dissolved in L00mM
Tris and
sonicated. The cell pellet was spun down, and the crude cell extract
supernatant was
22
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WO 98/58069 PCTIUS98/12447
assayed for FDA activity, using a coupled enzymatic assay as described by
Baldwin et al.
(1978). This assay was routinely performed at 30°C.
The reaction was performed in a 1 mL final volume in excess presence of the
enzymes triosephosphate isomerase (TIM) and alpha-glycerophosphate
dehydrogenase
(GDH) in a reaction mixture containing final concentrations of 100mM Tris pH
8.0, 4.75
mM fructose 1,6 bisphosphate, 0.15 mM NADH, S00 U/mL TIM, and 30 U/mL GDH.
The decrease in absorbance at 340nm, after addition of the cell extract
supernatant,
was recorded using an HP diode array spectrophotometer. One international unit
(LU.) of
aldolase activity is that causing the oxidation of 2 ~mol of NADH/min in this
assay
system.
Cell extracts containing the vector with the fda sequence showed a substantial
increase in aIdolase activity (13.1 LU./mg protein) as compared to cells
transformed with
the control vector (0.15 LU./mg protein). The activity was shown to be
inhibited by
EDTA, known to specifically inhibit class II aldolases.
~ s EXAMPLE 2
Plant transformation and fda expression in tobacco
Targeting of FDA protein
E.coli fructose 1,6 bisphosphate aldolase was targeted to the plastid in
plants in
order to assess its influence on carbohydrate metabolism and starch
biosynthesis in these
2o plant organelles. To accomplish the import of the E.coli aldolase into the
plastids, a vector
was constructed in which the aldolase was fused to the Arabidopsis small
subunit transit
peptide (CTP1) (Stark et al., 1992) or the maize small subunit CTP (Russell et
al., 1993),
creating constructs in which the CTP fda fusion gene was located between the
35S
promoter from the figwort mosaic virus (P-FMV35S; Gowda et al., 1989) and the
3'-
25 nontranslated region of the nopaline synthase gene (NOS 3'; Fraley et al.,
1983)
sequences. The vector construct containing the expression cassette [P-
FMV/CTP1/fda/NOS3'] was subsequently used for tobacco protoplast
transformation,
which W as performed as described in Fromm et al. {1987), with the following
modifications. Tobacco cultivar Xanthi line D (Txd) cell suspensions were
grown in 250-
30 mL flasks, at 25°C and 138 rpm in the dark. For maintenance, a sub-
culture volume of 9
mL was removed and added to 40 mL of fresh Txd media containing MS salts, 3%
sucrose, 0.2 g/L inositol, 0.13 gIL asparagine, 80 ~L of a 50 mg/mL stock of
PCPA, 5 ~L
23
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WO 98/5869 PCTIUS98/12447
of a 1 mg/mL stock of kinetin, and 1 mL of 1000x vitamins (1.3 g/L nicotinic
acid, 0.25
g/L thiamine, 0.25 g/L pyridoxine HCL, and 0.25 g/L calcium pantothenate)
every 3 to 4
days. Protoplasts were isolated from 1-day-old suspension cells that came from
a 2-day-
old culture. Sixteen milliliters of cells were added to 40 mL of fresh Txd
media and
allowed to grow 24 hours prior to digestion and isolation of the protoplasts.
The
centrifugation stage for the enzyme mix has been eliminated. The
electroporation buffer
and protoplast isolation media were filter sterilized rather than autoclaved.
The
electroporation buffer did not have 4 mM CaCl2 added. The suspension cells
were
digested in enzyme for 1 hour. Protoplasts were counted on a hemacytometer,
counting
to only the protoplasts that look intact and circular. Bio-rad Gene Pulser
cuvettes (catalog #
I65-2088) with a 0.4-cm gap and a maximum volume of 0.8 mL were used for the
electroporations. Fifty to 100 p.g of DNA containing the gene of interest
along with 5 ~g
of internal control DNA containing the luciferase gene were added per cuvette.
The final
protoplast density at electroporation was 2x106/mL and electroporater settings
were a 500
~ 5 p,Farad capacitance and I 40 volts on the Bio-rad Gene Pulser. Protoplasts
were put on ice
after resuspension in electroporation buffer and remained on ice in cuvettes
until I O
minutes after electroporation. Protoplasts were added to 7 mL of Txd media +
0.4 M
mannitol and conditioning media after electroporation. At this stage coconut
water was no
longer used. The protoplasts were grown in I- hour day/night photoperiod
regime at 26°C
2o and were spun down and assayed or frozen 20-24 hours after electroporation.
Western blot analysis performed on the protoplast extracts, obtained after
transformation, showed processing into the mature FDA in the tobacco
protoplasts.
Expression was detected of a protein migrating at approximately 40 kDa, which
is the
molecular weight of the aldolase subunit and the size of the protein also
observed after
25 overexpression of the aldolase in E. toll.
The expression cassette (P-FMV/CTPI/fda/NOS3'] was subsequently cloned into
the Noti site of pMON 17227 (described in PCT Publication WO 92/04449), in the
same
orientation as the selectable marker expression cassette, to form the plant
transformation
vector pMON17524, as shown in Figure 2 (SEQ ID NO: 5).
3o An additional construct was made and used for tobacco protoplast
transformation,
fusing the,fda gene to the Arabidopsis EPSPS transit peptide (CTP2), which is
described
in US patent 5,463,175. The transit peptide was cloned (through the SphI site)
into the
24
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WO 98/58069 PCT/US98/12447
SphI site located immediately upstream from the N-terminus of the fda gene
sequence in
the CTP 1 fda fusion (described above). This new CTP2 fda fusion gene was then
cloned
into a vector between the FMV promoter and the NOS 3' sequences. When this
construct
containing the CTP2/fda gene sequences was used for tobacco protoplast
transformation,
expression was detected of a protein migrating at approximately 40 kDa, which
is the
molecular weight of the aldolase subunit and the size of the protein also
observed after
overexpression of the aldolase in E. coli.
The NotI cassette [P-FMV/CTP2/fda/NOS3'] from this construct was then cloned
into the NotI site of pMON 17227, in the same orientation as the selectable
marker
expression cassette, to form the plant transformation vector pMON 17542, which
is shown
in Figure 3 (SEQ ID N0:6).
For cytoplasmic expression of the FDA in tobacco protoplasts, a construct was
made in which the fda gene sequence (without being coupled to a transit
peptide) was
cloned into a vector backbone, between the FMV promoter and the NOS 3'
sequences.
15 Using this construct for tobacco protoplast transformation also showed
expression of a
protein of the same size, migrating at approximately 40 kDa.
fda expression in tobacco plants
Two constructs, containing the fda gene, fused to the Arabidopsis small
subunit
CTP1 (pMON17524) (SEQ ID NO:S, Figure 2) and the Arabidopsis EPSPS (CTP2)
transit
z0 peptide (pMON17542) (SEQ ID N0:6, Figure 3), were used for tobacco plant
transformation, as described in US patent 5,463,175. A vector without the CTP
fda
sequences, pMON 17227 (described in PCT Publication WO 92/04449), was used as
a
negative control. The plant transformation vectors were mobiiized into the ABI
Agrobacterium strain. Mating of the plant vector into the ABI strain was done
by the
25 triparental conjugation system using the helper plasmid pRK2013 (Ditta et
al., 1980).
Growth chamber-grown tobacco transformant lines were generated and first
. screened by Western blot analysis to identify expressors using goat antibody
raised against
E. coli-expressed fda. Subsequently, for pMON 17524-expressing tobacco lines,
leaf
nonstructural carbohydrates were analyzed (sucrose, glucose, and hydrolyzed
starch into
30 glucose) by means of a YSI Instrument, Model 2700 Select Biochemistry
Analyzer.
Starting at flowering stage, leaf samples were also taken from these plants
and analyzed
for diurnal changes in leaf nonstructural carbohydrates.
CA 02294525 1999-12-13
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Five hundred milligrams to 1 g fresh tobacco leaf tissue samples were
harvested
and extracted in S mL of hot Na-phosphate buffer (40 g/L NaH2P04 and 10 g/L
Na2H2P04
in double de-ionized water) by homogenization with a Polytron. Test tubes were
then
placed in an 85°C water bath for 15 minutes. Tubes were centrifuged for
12 minutes at
3000 rpm and the supernatants saved for soluble sugar analysis. The pellet was
resuspended in 5 mL of hot Na-phosphate buffer mixed with a Vortex and
centrifuged as
described above. The supernatant was carefully removed and added to the
previous
supernatant fraction for soluble sugar (sucrose and glucose) analysis by YSI
using
appropriate membranes.
to The starch was extracted from the pellet using the Megazyme Kit (Megazyme,
Australia). To the pellet, 200 ~L of 50% ethanol and 3 mL of thermostable
alpha-amylase
(300U) were added and the mixture vortexed. Samples were then incubated in
boiling
water for 6 minutes and stirred after 2 and 4 minutes. Tubes were placed in
50°C water
bath and 4 mL of 200 mM acetate buffer (pH 4.5) were added followed by 0.1 mL
amyloglucosidase (20 U). Incubation occurred for 1 hour. Test tubes were then
centrifuged
for 15 minutes at 3000 rpm. Aliquots were taken from the supernatant and
analyzed for
glucose by YSI. The free glucose was adjusted to anhydrous glucose (as it
occurs in starch
by multiplying by the ratio 162/ 182). The total volume per tube was 7.1 mL.
As seen in Tablel, expression of the fda gene in tobacco correlated with a
significant increase in leaf starch levels. However, referring to Figure 4,
when a diurnal
profile of starch levels was established in the fda-expressing leaves, this
increase was
apparent mainly early in the photoperiod, which is a phase when leaves are
known to have
peak photosynthetic activity. This increase in starch has no apparent negative
effect on the
plant because the increased starch is turned over during the dark period.
There was no
apparent increase in steady state levels of sucrose or glucose in tobacco
leaves expressing
E.coli fda as compared to the control.
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Table 1
Leaf Carbohydrate Levels of Plants Expressing
the fda Transgenel (pMON17524)
High Expressors Low Expressors Negative
(>0.01 % total protein) (< 0.01 %) Control
(mg/g fresh weight)
STARCH 35.08 2.84 23.25 + 3.20 16.69 + 2.92
to SUCROSE 0.97 0.17 0.86 + 0.25 0.66 + 0.I9
GLUCOSE 1.88 0.17 1.58 + 0.20 1.68 + 0.26
t Leaf samples were harvested at midday.
A second set of transgenic tobacco plants transformed with the construct
pMON17542 were grown in the greenhouse. Tobacco plants containing a vector
without
the CTP fda sequences, pMON17227, were used as negative control. Of all the
pMON17542-lines screened for expression by Western blot analysis, 18 were high
expressors (>0.01 % of the total cellular protein) and 15 lines were low
expressors
{<0.01%). Fifteen plants containing the null vector, pMON17227, were used as
control.
2o Fully expanded leaves from plants expressing the fda transgene and negative
controls were
tested for sucrose export by collecting phloem exudate from excised leaf
systems. The
phloem exudation technique is described in Groussol et al. (1986). Leaves were
harvested
at 11:30 AM and placed in an exudation medium, containing 5 mM EDTA at pH 6.0,
and
allowed to exude for a period of 4 hours under full light and high humidity.
The exudation
solution was immediately analyzed for sucrose level, as described above in the
carbohydrate analysis method. As seen in Table 2, a significant increase in
sucrose export
out of source leaves was observed in plants expressing the fda transgene.
This increase in sucrose export by fda-expressing leaves is an illustration of
an
increase in source capacity, very likely due to an increased carbon flow
through the Calvin
Cycle (in response to increased triose-P utilization) and thus an increase in
net carbon
utilization by the leaf. As seen in Table 2, the increase in sucrose loading
in the phloem
correlates with the level of fda expression.
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Table 2
Levels of Sucrose in Phloem Exudate from
Excised Leaves of,fda Transgenic Tobacco Plants (pMON17542)
Water uptake sucrose in phloem exudate
(pl/g F.Wt./h) (ng/leaf) (ng/g F.Wt.)
fda high expressors 320 ~ 20 330 ~ 60 108 ~ 22
fda low expressors 340 ~ 10 210 ~ 10 77 ~ 3
to
Control 390 ~ 30 160 ~ 10 56 ~ 3
Referring to Table 3, preliminary analysis of plant growth and development
revealed no significant differences in number of leaves or pods per plant,
plant height,
stem diameter, or apparent seed weight per plant, between plants expressing
the fda gene
and the vector control under the specific growing and analysis conditions.
However, as
seen in Table 4, the fda-transgenic plants had a significantly higher root
mass. This may
be an indication that, under these conditions, roots represented a more
dominant sink that
attracted excess carbohydrate produced by the source leaves. Furthermore, the
present
2o illustration shows that the increase in root mass in the presence of the
E.coli fda gene was
accomplished with no apparent negative effect on shoot growth, inflorescence,
or seed set.
Therefore, this increase in root growth and final root dry weight is a
desirable plant trait
because it would lead to a rapid seedling establishment following germination
and greater
plant ability to tolerate drought, cold stress, other environmental
challenges, and
transplanting. In different plants and under different growing conditions,
other plant parts
(such as seed -uit, stem, leaf, tuber, bulb, etc.) are expected to show the
weight increase
obser-~~ed in ~ .ecco roots overexpressing the fda transgene.
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Table 3
Assessment of Certain Plant Growth and Development Parameters in
Tobacco Expressing the fda Transgene~ (pMON 17542)
#pods/plant #leaves/plant Plant height Seed weight
(cm) (g/plant)
high expressors 162 + 40 25.4 + p.g 65.3 + 3.1 18.8 + 2.4
Control 156+28 24.4+0.5 65.8+5.1 17.3+2.6
1 To achieve this analysis, 14 high-expressor lines were compared to IS
control plants.
Measurements were made prior to seed harvest (most pods have reached
maturity). The number of
leaves was confirmed by counting the number of nodes to account for leaf drop.
Table 4
Tobacco Root Dry Weight of Plants Expressing
the E. toll fda Transgene ~ (pMON 17542)
Root Dry Weight
(g/plant)
fda high expressors 64.0 + 3.9
fda low expressors 62.7 + 5.4
Control 31.7 + 1.6
1 Roots from 5 high and 7 low expressing lines and 6 control plants were
excised and washed
carefully then placed in a 65°C drying oven for at least 48 hours.
Roots were removed from the
oven and allowed to equilibrate in the laboratory for 2 hours before dry
weight determination.
2s EXAMPLE 3 -
Plant transformation and fda expression in corn plants
Targeting of FDA protein
Vectors containing the fda gene with and without the plastid targeting peptide
were made for transformation in corn and are also suitable for other monocots,
including
3o rice, wheat, barley, sugarcane, triticale, etc.
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WO 98158069 PCT/US98/12447
For the cytosolic expression of the fda gene in corn plants, a construct was
made
in which the fda gene sequence was fused to the backbone of a vector
containing the
enhanced CaMV 35S promoter (e35S; Kay et al., 1987), the HSP70 intron (L1S
patent
5,593,874), and the NOS3' polyadenylation sequence (Fraley et al., 1983). This
created a
NotI cassette [P-e35SIHSP70 intron/fda/NOS3'] that was cloned into the NotI
site of
pMON30460, a monocot transformation vector, to form the plant transformation
vector
pMON13925, as shown in Figure 5. pMON30460 contains an expression cassette for
the
selectable marker neomycin phosphotransferase typeII gene (nptII) [P-35S/NPTII
/NOS3']
and a unique NotI site for cloning the gene of interest. The final vector
(pMON13925)
to was constructed so that the gene of interest and the selectable marker gene
were cloned in
the same orientation. A vector fragment containing the expression cassettes
for these gene
sequences could be excised from the bacterial selector (Kan) and ori, gel
purif ed, and used
for plant transformation.
For the chloropiast-targeted expression of the fda gene in corn plants, a
construct
~5 was made in which the fda gene sequence, coupled to the maize RUBISCO small
subunit
CTP (Russell et al., 1993}, was fused to the backbone of a vector containing
the enhanced
(CaMV) 35S promoter, the HSP70 intron, and the NOS3' polyadenylation
sequences. This
created a NotI cassette [P-e35S/HSP70 intron/mzSSuCTP/fda/NOS3'] that was
cloned
into the NotI site (in the same orientation as the selectable marker cassette
[P-35S/NPTII
2o /NOS3']) of the monocot transformation vector pMON30460, to form the vector
pMON 17590, as shown in Figure 6. From this vector a fragment containing the
fda gene
expression cassette and the selectable marker cassette could be excised from
the bacterial
selector (Kan) and ori, gel purified, and used for plant transformation.
For the cytosolic endosperm-specific expression of the aldolase gene in corn,
the
25 fda gene sequence was cloned into a vector (in the same orientation as the
selectable
marker cassette[P-35SJNPTII /NOS3']) containing the glutelin gene promoter P-
osgtl
{Zheng et al., 1993), the HSP70 intron, and the NOS3' polyadenylation
sequences to form
the vector pMON13936, as shown in Figure 7. From this vector a fragment
containing the
fda gene expression cassette [P-osgtl/HSP70intron/fda/NOS3'] and the
selectable marker
3o cassette could be excised from the bacterial selector (Kan) and ori, gel
purified, and used
for plant transformation. _
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Maize plant transformation
Transgenic maize plants transformed with the vectors pMON13925 (described
above) or pMON17590 (described above) were produced using microprojectile
bombardment, a procedure well-known to the art (Fromm, 1990; Gordon-Kamm et
aL,
1990; Waiters et al., I 992). Embryogenic callus initiated from immature maize
embryos
was used as a target tissue. Plasmid DNA at 1 mglmL in TE buffer was
precipitated onto
M10 tungsten particles using a calcium chloride / spermidine procedure,
essentially as
described by Klein et al. (1988). In addition to the gene of interest, the
plasmids also
contained the neomycin phosphotransferase II gene (nptlI) driven by the 35S
promoter
to from Cauliflower Mosaic Virus. The embryogenic callus target tissue was
pretreated on
culture medium osmotically buffered with 0.2M mannitol plus 0.2M sorbitol for
approximately four hours prior to bombardment (Vain et al., 1993). Tissue was
bombarded two times with the DNA-coated tungsten particles using the gunpowder
version of the BioRad Particle Delivery System (PDS) 1000 device.
Approximately 16
hours following bombardment, the tissue was subcultured onto a medium of the
same
composition except that it contained no mannitol or sorbitol, and it contained
an
appropriate aminoglycoside antibiotic, such as 6418", to select for those
cells that
contained and expressed the 35S/nptII gene. Actively growing tissue sectors
were
transferred to fresh selective medium approximately every 3 weeks. About 3
months after
2o bombardment, plants were regenerated from surviving embryogenic callus
essentially as
described by Duncan and Widholm (1988).
Aldolase activity from transgenic maize
In order to measure leaf aldolase activity, corn leaf samples were taken and
immediately frozen on dry ice. Aldolase enzyme was extracted from the leaf
tissue by
grinding the leaf tissue at 4°C in 1.2 mL of the extraction buffer (100
mM Hepes, pH 8.0,
5 mM MgCl2, 5 mM MnCl2, 100 mM KCI, 10 mM DTT, 1% BSA, 1 mM PMSF, 10
. ~g/mL leupeptin, 10 p,g/mL aprotinin). The extract was centrifuged at 15,000
x g, at 4°C
for 3 minutes, and the non-desalted supernatant was assayed for enzyme
activity. This
extraction method gave about 60% recovery of E. coli FDA activity.
3o Total aldolase activity was determined in 0.98 mL of reaction mixture that
consisted of 100 mM EPPS-NaOH, pH 8.5, 1 mM fructose-bisphosphatezØl mM
NADH,
5 mM MgCl2, 4 units of alpha-glycerophosphate dehydrogenase, and 15 units of
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WO 98/58069 PCT/US98/12447
triosephosphate isomerase. The reaction was initiated by addition of 20 ~L of
leaf extract.
The resulting data, generated from a single experiment, are presented in Table
5.
Table 5
Aldolase Activity from Transgenic Maize Leaves
~ Lines A340/minl20pL Activity
I-I99 (control) 0. i 13 100
pMON 17590 0.233 206
pMON 13925 0.251 222
A phenotype was visible in the primary transformants (RO plants) expressing
the
t o E. coli FDA when the protein was targeted to the chloroplast. The leaves
were chlorotic
but seed set was normal. Rl plants were grown in both field and in greenhouse
experiments. Starch was not detectable in the leaves using an iodine staining
and
pollination was delayed. It is believed that the phenotype in these corn
plants may be the
result of the promoter (e35S) used in both the pMON17590 and pMON13925 vectors
not
being preferred for causing FDA expression in corn. Because e35S is believed
to cause
mesophyll enhanced expression and the Calvin Cycle in a C4 plant such as corn
occurs
predominantly in the bundle sheath cells, the use of a promoter directing
enhanced
expression in the bundle sheath cells (such as the ssRUBISCO promoter) may be
preferred. Vectors containing such a promoter and driving expression of FDA
have been
2o prepared and are being tested in maize.
In particular, the maize RuBISCO small subunit (PmzSSU, a bundle sheath cell-
specific promoter) has been used to construct vectors for cell-specific fda
expression in
maize. A class I aldolase (fdal), an fda without an iron sulfur cluster and
with different
properties from fdall, was utilized to improve carbon metabolism in C4 crops
(e.g. maize)
. The gene for the class I aldolase was amplified from the genome of
Staphylococcus
aurea~s and activity was comfirmed. Transformation vectors were then
constructed to
expreJS both classes of aidolase (fdal and fdall) in a cell-specific manner in
maize. The
following cassettes have been made:
pMON13899: PmzSSU/hsp70/mzSSU CTPlfdal
3o pMON13990PmzSSUIhsp70/mzSSU CTPlfdall
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WO 98/58069 PCT1US98/12447
pMON 13988:P35S/hsp70/fdal.
These vectors were used for corn transformation as described generally above.
The
biochemical and physiological analysis of the primary transformants should
allow for the
identification of aldolase gene overexpression that will lead to increase
growth and
development and yield in maize.
Also, two vectors were used for transformation of corn which would target the
expression of the E. coli fda ll gene in the maize endosperm. The vector pMON
13936
uses the rice gtl promoter to drive expression of aldolase in the cytoplasm of
the
endosperm cells. Another vector (pMON 36416) uses the same promoter with the
maize
to RuBISCO small subunit transit peptide to localize the protein in the
amyloplasts.
Homozygous lines of the cytosolic aldolase transformants have been identified
(Homozygosity of 37 plants was confirmed using western blot analysis) and seed
from
these plants were collected for grain composition analysis (moisture, protein,
starch, and
oil). Of the 53 pMON 36416 primary transformants screened for amylopast-
targeted
IS aldolase expression, 11 were positive. These plants will be tested for
homozygosity
selection/propagation and kernels from the homozygotes will be used for
composition
analysis.
EXAMPLE 4
Plant transformation and fda expression in potato plants
20 Targeting of fda expression
The plant expression vector, pMON 17542 (described earlier}, in which the fda
gene is expressed behind the FMV promoter and the aldolase enzyme is fused to
the
chloroplast transit peptide CTP2, was used for Agrobacterium-mediated potato
transformation.
25 A second potato transformation vector was constructed by cloning the NotI
cassette [P-FMV/CTP2/fda/NOS3'] (described earlier) into the unique NotI site
of
pMON23616. pMON23616 is a potato transformation vector containing the nopaline-
type
T-DNA right border region (Fraley et al., 1985), an expression cassette for
the neomycin
phosphotransferase typeII gene [P-35S/NPTII /NOS3'] (selectable marker), a
unique NotI
30 site for cloning the gene expression cassette of interest, and the T-DNA
left border region
(Barker et al., 1983). Cloning of the NotI cassette [P-FMV/CTP2/fda/NOS3']
(described
earlier) into the NotI site of pMON23616 results in the potato transformation
vector
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WO 981580b9 PCT/US98/12447
pMON17581, as shown in Figure 8. The vector pMON17581 was constructed such
that
the gene of interest and the selectable marker gene were transcribed in the
same direction.
Potato plant transformation
The plant transformation vectors were mobilized into the ABI .4grobacterium
strain. Mating of the plant vector into the ABI strain was done by the
triparental
conjugation system using the helper plasmid pRK2013 (Ditta et al., 1980). The
vector
pMON17542 was used for potato transformation via Agrobacterium transformation
of
Russet Burbank potato callus, following the method described in PCT
Publication WO
96/03513 for glyphosate selection of transformed lines.
to After transformation with the vector pMON17542, transgenic potato plantlets
that
came through selection on glyphosate were screened for expression of E. coli
aldolase by
leaf Western blot analysis. Out of 112 independent lines assayed, 50 fda-
expressing lines
(45%) were identified, with_fda expression levels ranging between 0.12% and
1.2 % of
total extractable protein.
The plant transformation vector PMON 17581 was used for Agrobacterium-
mediated transformation of HS31-638 potato callus. HS3i-638 is a Russet
Burbank potato
line previously transformed with the mutant ADPglucose pyrophosphorylase
(gIgCl6)
gene from E.coli (U.S. Patent 5,498,830). The potato callus was transformed
following the
method described in PCT Publication WO 96/03513, substituting icanamycin
(administered at a concentration of 150-200 mg/L) for glyphosate as a
selective agent.
The transgenic potato plants were screened for expression of the fda gene by
assaying leaf punches from tissue culture plantlets. Western blot analysis
(using antibodies
raised against the E. coli aldolase) of leaf tissue from the pMONI7581-
transformed lines
identified 12 expressing lines out of 56 lines screened. Expression was
detected of a
protein migrating at approximately 40 kDa, which is the molecular weight of
the E. coli
(classII) aldolase subunit and the size of the protein observed after
overexpression of the
aldolase in E. coli.
Specij~c gravity measurements of transgenic potato plants
From the 50 fda-expressing potato lines obtained after transformation with
3o pMON17542, 7 of the highest expressing lines were micropropagated in tissue
culture, and
8 copies of each line were planted in pots 14 inches in diameter and 12 inches
deep,
containing a mixture of: '/2 Metro 350 potting media, '/4 fine sand, '/4 Ready
Earth
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WO 98/58069 PCT1US98/12447
potting media. Wild-type Russet Burbank plantlets from tissue culture were
planted as
controls. All plants were cultivated for approximately 5 months in the
greenhouse in
which daytime temperature was approximately 2I-23°C while nighttime
temperature was
approximately 13°C. Plants were watered every other day throughout
their active growing
period and fertilized with Peter's 20-20-20 commercial fertilizer once a week,
at levels
similar to commercial applications. Fertilization was carned out only for the
first 2 %z
months, at which point fertilization was stopped completely. Plants were
allowed to
naturally senesce, and at approximately 50% senescence, tubers were harvested.
For each line at harvest, al! tubers from all 8 pots were pooled and a total
weight
1o was obtained. Then for each line, tubers 30 g or greater were pooled and
specific gravity
was determined on this group of tubers. Specific gravity is the weight of the
tubers in air
divided by the weight in air minus the weight in water. Results of these
weight
measurements are presented in Table 6.
Table 6
Specific gravity measurements from transgenic potato plants
Line Total CherallCombined% IncreaseCombined WeightSpecific
# in of
Weight% YieldWeight Total WeightTubers over Gravity
30g
Increasof Tubers(Tubers (% of Total
over Weight
a over 30~
30g
RB 6609 4477 67.70% 1.087
40652 5138 neg 1307 neg 25.40% 1.08
40611 7170 8.5% 4533 1.3% 63.20% 1.083
40608 7470 13.0% 1070 neg 14.30% 1.081
40632 7776 21.8% 5453 21.8% 70.1U% 1.088
40614 8688 31.5% 5468 22.2% 62.90% 1.083
40631 8800 33.2% 6188 38.2% 70.30% 1.084
40610 9746 47.0% 7777 73.0% 80% 1.087
This table summarizes the tuber yield and specific gravity for all seven lines
grown in the
greenhouse. The results indicate that, in comparison to the control, all but
one of the fda
lines show an increase in overall tuber yield, and that in four lines, there
is a corresponding
. increase in percentage of tubers that weigh more than 30 g. For combined
tubers over 30
g, the~percent of total weight is near that of the control, and for two lines
is greater than the
control. This indicates that five out of the six of the lines show higher
overall yield and
are not making smaller-tubers. In other words, with the increase in overall
yield, there is a
corresponding increase in percentage of bigger tubers (over 30 g). However,
there is no
increase in specific gravity of the tubers.
CA 02294525 1999-12-13
WO 98158069 PCT/US98/12447
In conclusion, it appears that expression of fda in potato produces greater
numbers
of tubers per plant without a sacrifice in tuber size. This represents a yield
benefit in that
the farmer could potentially be able to produce the same amount of tubers
using Less
acreage. Similar experiments will also be performed by co-expression of fda
with other
carbohydrate metabolizing genes, such as glgCl6, in order to determine how
such
combinations will affect tuber yield, tuber solids deposition and overall
tuber specific
gravity.
Aldolase activity from transgenic potato
After being cultivated for 3 months (post planting) in the greenhouse. leaf
samples
were taken from 6 of the highest,fda-expressing potato lines, obtained after
transformation
with pMON 17542, and assayed for aldolase activity.
In order to measure potato leaf aldolase activity, duplicate leaf samples from
each
line were taken and immediately frozen on dry ice. Aldolase was extracted from
0.2 g of
leaf tissue by grinding at 4°C in 1.2 mL of the extraction buffer: 100
mM Hepes, pH 8.0,
5 mM MgCl2, 5 mM MnClz, 100 mM KCI, 10 mM DTT, 1 % BSA, 1 mM PMSF, 10
~glmL Ieupeptin, 10 pg/mL aprotinin. The extract was assayed for aldolase
activity as
described earlier.
Six independent transgenic potato lines expressing fda were tested for
aldolase
activity. The expression of fda in leaves is an indicator of the expression in
the whole
plant because the FMV promoter used to drive expression of the respective
encoding
DNAs directs gene expression constitutively in most, if not all, tissues of
potato plants.
Table 7 summarizes the quantitative protein expression data for each of the
lines,
and the percent activity for each individual line.
36
CA 02294525 1999-12-13
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Table 7
Aldolase Activity from
Transgenic Russet Burbank Potato Leaves
Exp. # 1 Exp. #2 Average
Lines Act (U/gFW)%Act Act (U/gFW)%Act % Activity
Control 4.461 100 4.732 100 100
40608 6.969 156 8.055 170 163
40610 8.489 190 7.326 155 173
40652 5.812 130 6.367 I35 132
to 40632 5.257 118 4.244 90 104
40631 5.764 129 4.968 105 117
40611 5.715 128 5.836 123 126
Solids uniformity in transgenic potato
Twenty-five Russet Burbank lines expressing fda (potato lines designated
"Maestro"), obtained after transformation with pMON 17542, and fifteen Russet
Burbank
Simple Solid lines, also containing gIgCl6 (PCT Publication WO 91/19806 and US
Patent
5,498,830), expressing fda (potato lines designated "Segal"), obtained after
transformation
with pMON 17581, were field tested at two different sites. For each field
site, 36 plants
2o per line (three repetitions of 12 plants per line) were evaluated for tuber
solids distribution.
At harvest, tubers were pre-sorted at each field site into a ten to twelve
ounce category,
and nine tubers from each replicated plot were analyzed in groups of three.
For a typical 10-12 ounce tuber having a diameter of 7-8 cm, starch
distribution
was evaluated by removing the center longitudinal slice ( 13 mm) from each
tuber. Slices
were then peeled and laid flat on a cutting board where the inner tuber region
(pith region)
was removed by a 14-mm cork punch. The tissue from pith to cortex
{perimedullary
region) was removed by an up-to-a 2-inch cork punch. The remaining cortex
tissue was
approximately an 8-mm wide ring from the outermost region of the slice.
Specif c gravity was then determined by weighing both the pooled pith punches
3o and pooled cortex punches in air and then in water:
Specific gravity = Air Wt./(Air Wt.-Water Wt.)
37
CA 02294525 1999-12-13
WO 98/58069 PCT/US98/12447
After calculating specific gravity, solids levels were determined by the
following equation:
-214.9206 + (218.1852*Sp. Gravity)
The degree of solids uniformity (Solids Uniformity Index) is determined by
calculating the
pith to cortex solids ratio (pith solids divided by cortex solids). The three
groups of three
tubers per plot were averaged, at which point the average of three plot
replications was
calculated per field site.
Analyses of several previous solids uniformity field trials (data not shown)
have
demonstrated nontransgenic, wild-type Russet Burbank potato to have a typical
pith to
cortex tuber solids ratio within the range of 68% to 72%, depending on growing
region
to and agricultural practices. Tables 8-11 provide the pith to cortex solids
ratios by plant line
number, with a higher pith to cortex solids ratio indicating a greater degree
of solids
uniformity.
Tables 8 and 9 represent the data from one field site (site i ) for Segal and
Maestro,
respectively, and illustrate that the majority of Segal and Maestro lines have
higher pith to
cortex solids ratios than that of 68.4% for the Russet Burbank control, with
some lines
approaching an 82% pith to cortex solids ratio.
Tables 10 and 11 represent the data from another field site (site 2) for Segal
and
Maestro, respectively, and also illustrate that the majority of Maestro and
Segal lines have
higher pith to cortex solids ratios than that of the Russet Burbank control,
with some lines
approaching an 88% pith to cortex solids ratio. In the site 2 field trial, the
Russet Burbank
control had an atypical. abnormally high pith-to-cortex solids uniformity
ratio of 79.3%,
which was most likely due to environmental growing conditions. The site 2
results
demonstrate that expression in Russet Burbank potato of E. coli fda, alone or
with co-
expression of glgC 16, increases tuber solids uniformity even in a growing
season when
tuber solids uniformity is already extremely high in nontransgenic Russet
Burbank. That
is, the fda gene continues to perform when agricultural conditions are already
conducive to
an abnormally high solids uniforr. ; level.
38
CA 02294525 1999-12-13
WO 98/58069 PCT/US98112447
Table 8. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio.
Segal Russet Burbank Lines. Site 1
Line Ratio
S-29 79.
I
S-9 75.8
S-20 71.3
S-15 71.3
S-21 70.5
S-S 70.2
to S-18 70.0
RB control 68.4
S-32 68.3
S-16 65.6
is
Table 9. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio.
Maestro Russet Burbank Lines. Site 1
Line Ratio
2o M-13 74.0
M-12 73.6
M-I 73.4
M-3 73.0
M-6 72.4
25 M-9 71.2
M-11 70.6
M-18 70.5
M-17 69.9
M- I 9 69.4
3o M-5 69.3
M-20 68.9
RB control 68.4
M-8 68.3
M-43 67.7
35 M-23 67.3
M-7 67.0
M-39 . 66.6
M-22 66.0
M-10 65.4
40 . M-27 61.4
39
CA 02294525 1999-12-13
WO 98158069 PCT/US98/12447
Table 10. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio
Segal Russet Burbank Lines. Site 2
Line Ratio
S-33 87.4
S-54 87.1
S-OS 86.8
S-29 85.1
S-21 84.3
S-16 83.2
S-20 81.5
S-18 80.7
S-32 80.6
RB control 79.3
S-09 79.0
Table lI. Solids Uniformity Index: Pith Solids to Cortex Solids Ratio
Maestro Russet Burbank Lines. Site 2
2o Line Ratio
M-04 87.7
M-18 83.9
M-17 83.8
M-03 83.7
M-09 83.4
M-15 83.2
M-29 82.9
M-44 82.3
M-08 82.2
3o M-43 81.6
M-22 81.1
M-OS 80.8
M-O 1 80.5
M-20 80.2
M-45 79.6
M-39 79.5
M-27 . 79.5
RB control 79.3
M-13 78.9
.
. M-22 78.8
M-19 78.7
M-07 78.2
M-12 77.9
M-23 77.3
M-06 76.5
M-10 75.0
M-11 74.1
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WO 98/58069 PCT/US98/12447
The effect of aldolase on pith to cortex solids ratios in the Segal lines is
slightly
more dramatic than in Maestro lines. We believe this phenotype is due to
expression of
fda in a background in which the Russet Burbank host expresses g1gC16 at a
relatively low
to moderate level, and that the combination of fda plus glgC 16 provides
improved
s benefits. Cross sectional tuber slices (Figure 9) of three Segal lines with
improved solids
uniformity illustrate a greater deposition of starch within the inner regions
of the tuber.
Specifically, an increase in cortex volume accompanied by relocation of the
xylem ring
towards the center of the tuber, plus a more opaque pith tissue due to an
increase in starch
density, are evident in the transgenic lines. This physiological alteration
may be due to an
t o increase in sucrose translocation from source to sink, which may influence
phloem
element distribution during tuber development or sucrose availability for
starch
biosynthesis across the tuber.
Example 5
Plant transformation and FDA expression in cotton plants
is The E. toll fda vectors pMON17524 [FMV/CTP1/fda] (Figure 2) and
pMON17542 [FMV/CTP2/fda] (Figure 3) were transformed into cotton using
Agrobacterium as described by Umbeck et al. ( 1987) and in US Patent 5004863.
The
protein was targeted to the chloroplast using either the Arabidopsis SSU CTP 1
(pMON17524) or the Arabidopsis EPSPS (pMON17542) chloroplast transit peptide.
20 Aldolase expression in cotton
Five-week-old calli transformed with both vectors were analyzed by Western
blot
analyses and by aldolase assays. Western blot analysis indicated a large
amount of protein
at the position of the full-length FDA standard and a lesser amount at the
same position in
the control callus extracts. It appeared that the protein was fully processed.
To verify that
25 FDA was expressed in the tissue and for comparison of activity, calli
transformed with the
two vectors were extracted in a buffer that would prevent loss of activity of
the transgene
. product. BSA was added to final concentration of 1 mg/mL, which limited the
analysis of
processing on import by Western blot. Aldolase assays were performed plus or
minus 25
mM EDTA, which inhibits the E. toll enzyme but not the plant enzyme. The
results of the
3o assays are shown in Table 12.
41
CA 02294525 1999-12-13
WO 98158069 PCT/US98/12447
Table 12
Aldolase Activity in Cotton Calli and Cotton Leaf
0 A340 e-3/mg protein/5 min
Colony -EDTA +EDTA Fold Increase
Controls
Cotton Leaf (Coker) 4.0 4.2 -
Uninoculated Calli 7.7 5.6 1.3X
Inoculated Calli (E35S/GUS) #1 6.8 6.1 -
#2 3.5 4.0 -
1o FDA calli
pMON 17542 # 1 3.5 2.3 1.SX
#3 5.5 2.6 2.1X
#5 9.2 3.8 2.4X
#4 19.8 3.6 S.SX
~ 5 pMON 17524 #2 15.2 5.8 2.6X
#3 12.5 4.0 3.1X
#S 14.4 2.9 4.9X
#6 4.1 1.2 3.SX
2o The results indicate that there is good expression of the,fila gene in
cotton callus. Almost all
calli had at least twofold higher aldolase activity, and the increase was
sensitive to inhibition by
EDTA. Processing appeared complete by Western blot analysis using these
samples.
42
CA 02294525 1999-12-13
WO 98158069 PCT/US98/12447
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Gerard Barry
NordineCheikh
Ganesh Kishore
(ii) TITLE OF INVENTION: Expression of Fructose 1,6 Bisphosphate
Aldolase in Transgenic Plants
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Arnold, White & Durkee
(B) STREET: P.O. Box 4433
(C) CITY: Houston
(D) STATE: Texas
(E) COUNTRY: United States of America
(F) ZIP: 77210-4433
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US Unknown
(B) FILING DATE: Concurrently Herewith
(C) CLASSIFICATION: Unknown
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US Prov. App. Serial No.
60/049,995
(B) FILING DATE: June 17, 1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Patricia A. Kammerer
(B) REGISTRATION NUMBER: 29,775
(Cy) REFERENCE/DOCKET NUMBER: MOBT086
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (713) 787-1400
.
(B) TELEFAX: (713) 787-1440
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1080 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
47
ICA 02294525 1999-12-13
WO 98/58069 PCTIUS98/12447
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
ATGTCTAAGA TTTTTGATTT CGTAAAACCT GGCGTAATCA CTGGTGATGA CGTACAGAAA
60
GTTTTCCAGG TAGCAAAAGA AAACAACTTC GCACTGCCAG CAGTAAACTG CGTCGGTACT
120
GACTCCATCA ACGCCGTACT GGAAACCGCT GCTAAAGTTA AAGCGCCGGT TATCGTTCAG
180
TTCTCCAACG GTGGTGCTTC CTTTATCGCT GGTAAAGGCG TGAAATCTGA CGTTCCGCAG
240
GGTGCTGCTA TCCTGGGCGC GATCTCTGGT GCGCATCACG TTCACCAGAT GGCTGAACAT
300
TATGGTGTTC CGGTTATCCT GCACACTGAC CACTGCGCGA AGAAACTGCT GCCGTGGATC
360
GACGGTCTGT TGGACGCGGG TGAAAAACAC TTCGCAGCTA CCGGTAAGCC GCTGTTCTCT
420
TCTCACATGA TCGACCTGTC TGAAGAATCT CTGCAAGAGA ACATCGAAAT CTGCTCTAAA
480
TACCTGGAGC GCATGTCCAA AATCGGCATG ACTCTGGAAA TCGAACTGGG TTGCACCGGT
540
GGTGAAGAAG ACGGCGTGGA CAACAGCCAC ATGGACGCTT CTGCACTGTA CACCCAGCCG
600
GAAGACGTTG ATTACGCATA CACCGAACTG AGCAAAATCA GCCCGCGTTT CACCATCGCA
660
GCGTCCTTCG GTAACGTACA CGGTGTTTAC AAGCCGGGTA ACGTGGTTCT GACTCCGACC
720
ATCCTGCGTG ATTCTCAGGA ATATGTTTCC AAGAAACACA ACCTGCCGCA CAACAGCCTG
780
. AACTTCGTAT TCCACGGTGG TTCCGGTTCT ACTGCTCAGG AAATCAAAGA CTCCGTAAGC
840
TACGGCGTAG TAAAAATGAA CATCGATACC GATACCCAAT GGGCAACCTG GGAAGGCGTT
900
CTGAACTACT ACAAAGCGAA CGAAGCTTAT CTGCAGGGTC AGCTGGGTAA CCCGAAAGGC
96 0
GAAGATCAGC CGAACAAGAA ATACTACGAT CCGCGCGTAT GGCTGCGTGC CGGTCAGACT
1020
48
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TCGATGATCG CTCGTCTGGA GAAAGCATTC CAGGAACTGA ACGCGATCGA CGTTCTGTAA
1080
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A)LENGTH:
359amino
acids
(B)TYPE:
amino
(C)STRANDEDNESS:
(D)TOPOLOGY:
Linear
(xi) SEQUENCE DESCRIPTION:SEQ N0:2
ID
Met SerLys Ile Phe Phe LysPro GlyValIleThr Gly
Asp Val
5 10 15
Asp AspVal Gln Lys Phe ValAla LysGluAsnAsn Phe
Val Gln
20 25 30
Ala LeuPro Ala Val Cys GlyThr AspSerIleAsn Ala
Asn Val
35 40 45
Val LeuGlu Thr Ala Lys LysAla ProValIleVal Gln
Ala Val
50 55 60
Phe SerAsn Gly Gly Ser IleAla GlyLysGlyVal Lys
Ala Phe
65 70 75
Ser AspVal Pro Gln Ala IleLeu GlyAlaIleSer Gly
Gly Ala
80 85 90
Ala HisHis Val His Met GluHis TyrGlyValPro Val
Gln Ala
95 100 105
Ile LeuHis Thr Asp Cys LysLys LeuLeuProTrp Ile
His Ala
110 115 120
Asp Gly Leu Leu Asp Ala Gly Glu Lys His Phe Ala Ala Thr Gly
125 120 135
Lys Pro Leu Phe Ser Ser His Met Ile Asp Leu Ser Glu Glu Ser
- 140 145 150
Leu Gln Glu Asn Ile Glu Ile Cys Ser Lys Tyr Leu Glu Arg Met
I55 160 165
~Ser Lys Ile Gly Met Thr Leu Glu Ile Glu Leu Gly Cys Thr Gly
170 175 180
Gly Glu Glu Asp Gly Val Asp Asn Ser His Met Asp Ala Ser Ala
185 190 195
Leu Tyr Thr Gln Pro Glu Asp Val Asp Tyr Ala Tyr Thr Glu Leu
200 205 210
49
ICA 02294525 1999-12-13
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Ser Lys Ile Ser Pro Arg Phe Thr Ile Ala Ala Ser Phe Gly Asn
215 220 225
Val His Gly Val Tyr Lys Pro Gly Asn Val Val Leu Thr Pro Thr
230 235 240
Ile Leu Arg Asp Ser Gln Glu Tyr Val Ser Lys Lys His Asn Leu
245 250 255
Pro His Asn Ser Leu Asn Phe Val Phe His Gly Gly Ser Gly Ser
260 265 270
Thr Ala Gln Glu Ile Lys Asp Ser Val Ser Tyr Gly Val Val Lys
275 2B0 285
Met Asn Ile Asp Thr Asp Thr Gln Trp Ala Thr Trp Glu Gly Val
290 295 300
Leu Asn Tyr Tyr Lys Ala Asn Glu Ala Tyr Leu Gln Gly Gln Leu
305 310 315
Gly Asn Pro Lys Gly Glu Asp Gln Pro Asn Lys Lys Tyr Tyr Asp
320 325 330
Pro Arg Val Trp Leu Arg Ala Gly Gln Thr Ser Met Ile Ala Arg
335 340 345
Leu Glu Lys Ala Phe Gln Glu Leu Asn Ala Ile Asp Val Leu
350 355
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GGGGCCATGG CTAAGATTTT TGATTTCGTA
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
. (C) STRANDEDNESS: single
(D) TOPOLOGY: Linear
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
CCCCGAGCTC TTACAGAACG TCGATCGCGT TCAG
{2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
CA 02294525 1999-12-13
WO 98/58069 PCT/US98/12447
(A) LENGTH: 10847 base pairs
IB) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
1 CGATAAGCTTGATGTAATTGGAGGAAGATC ATCCCCATTC
AAAATTTTCA
51 TTCGATTGCTTCAATTGAAGTTTCTCCGATGGCGCAAGTTAGCAGAATCT
101 GCAATGGTGTGCAGAACCCATCTCTTATCTCCAATCTCTCGAAATCCAGT
151 CAACGCAAATCTCCCTTATCGGTTTCTCTGAAGACGCAGCAGCATCCACG
201 AGCTTATCCGATTTCGTCGTCGTGGGGATTGAAGAAGAGTGGGATGACGT
251 TAATTGGCTCTGAGCTTCGTCCTCTTAAGGTCATGTCTTCTGTTTCCACG
301 GCGTGCATGCTTCACGGTGCAAGCAGCCGTCCAGCAACTGCTCGTAAGTC
351 CTCTGGTCTTTCTGGAACCGTCCGTATTCCAGGTGACAAGTCTATCTCCC
401 ACAGGTCCTTCATGTTTGGAGGTCTCGCTAGCGGTGAAACTCGTATCACC
451 GGTCTTTTGGAAGGTGAAGATGTTATCAACACTGGTAAGGCTATGCAAGC
501 TATGGGTGCCAGAATCCGTAAGGAAGGTGATACTTGGATCATTGATGGTG
551 TTGGTAACGGTGGACTCCTTGCTCCTGAGGCTCCTCTCGATTTCGGTAAC
601 GCTGCAACTGGTTGCCGTTTGACTATGGGTCTTGTTGGTGTTTACGATTT
651 CGATAGCACTTTCATTGGTGACGCTTCTCTCACTAAGCGTCCAATGGGTC
701 GTGTGTTGAACCCACTTCGCGAAATGGGTGTGCAGGTGAAGTCTGAAGAC
75i GGTGATCGTCTTCCAGTTACCTTGCGTGGACCAAAGACTCCAACGCCAAT
801 CACCTACAGGGTACCTATGGCTTCCGCTCAAGTGAAGTCCGCTGTTCTGC
851 TTGCTGGTCTCAACACCCCAGGTATCACCACTGTTATCGAGCCAATCATG
901 ACTCGTGACCACACTGAAAAGATGCTTCAAGGTTTTGGTGCTAACCTTAC
951 CGTTGAGACTGATGCTGACGGTGTGCGTACCATCCGTCTTGAAGGTCGTG
1001 GTAAGCTCACCGGTCAAGTGATTGATGTTCCAGGTGATCCATCCTCTACT
1051 GCTTTCCCATTGGTTGCTGCCTTGCTTGTTCCAGGTTCCGACGTCACCAT
1101 CCTTAACGTTTTGATGAACCCAACCCGTACTGGTCTCATCTTGACTCTGC
1151 AGGAAATGGGTGCCGACATCGAAGTGATCAACCCACGTCTTGCTGGTGGA
1202 GAAGACGTGGCTGACTTGCGTGTTCGTTCTTCTACTTTGAAGGGTGTTAC
1251 TGTTCCAGAAGACCGTGCTCCTTCTATGATCGACGAGTATCCAATTCTCG
1301 CTGTTGCAGCTGCATTCGCTGAAGGTGCTACCGTTATGAACGGTTTGGAA
1351 GAACTCCGTGTTAAGGAAAGCGACCGTCTTTCTGCTGTCGCAAACGGTCT
1401 CAAGCTCAACGGTGTTGATTGCGATGAAGGTGAGACTTCTCTCGTCGTGC
1451 GTGGTCGTCCTGACGGTAAGGGTCTCGGTAACGCTTCTGGAGCAGCTGTC
1501 GCTACCCACCTCGATCACCGTATCGCTATGAGCTTCCTCGTTATGGGTCT
1551 CGTTTCTGAAAACCCTGTTACTGTTGATGATGCTACTATGATCGCTACTA
1601 GCTTCCCAGAGTTCATGGATTTGATGGCTGGTCTTGGAGCTAAGATCGAA
1651 CTCTCCGACACTAAGGCTGCTTGATGAGCTCAAGAATTCGAGCTCGGTAC
1701 CGGATCCAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTT
1751 CAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTCGAGT
1801 ATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTA
1851 ATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAA
1901 TGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAA
1951 TTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATT
. ATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTG
2001
2051,GCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTAC
2101 CATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGC
2151 TGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTAT
2201 GAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATT
2251 GCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATA
2301 TTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGCATCAAT
2351 CGACCTGCAGCCACTCGAAGCGGCCGCGTTCAAGCTTGAGCTCAGGATTT
2401 AGCAGCATTCCAGATTGGGTTCAATCAACAAGGTACGAGCCATATCACTT
2451 TATTCAAATTGGTATCGCCAAAACCAAGAAGGAACTCCCATCCTCAAAGG
2501 TTTGTAAGGAAGAATTCTCAGTCCAAAGCCTCAACAAGGTCAGGGTACAG
$1
CA 02294525 1999-12-13
WO 98158069 PCT/US98/12447
2551 AGTCTCCAAA GAGATCAATG
CCATTAGCCA AAGAATCTTC
AAAGCTACAG
2601 AATCAAAGTA CATCATGGTC
AACTACTGTT AGTAAGTTTC
CCAGCACATG
2651 AGAAAAAGACATCCACCGAA GACTTAAAGTTAGTGGGCATCTTTGAAAGT
2701 AATCTTGTCAACATCGAGCA GCTGGCTTGTGGGGACCAGACP~AAAAAGGA
2751 ATGGTGCAGAATTGTTAGGC GCACCTACCAAAAGCATCTTTGCCTTTATT
2801 GCAAAGATAAAGCAGATTCC TCTAGTACAAGTGGGGAACA
AAATAACGTG
2851 GAAAAGAGCTGTCCTGACAG CCCACTCACTAATGCGTATGACGAACGCAG
2901 TGACGACCACAAAAGAATTC CCTCTATATAAGAAGGCATTCATTCCCATT
2951 TGAAGGATCATCAGATACTG AACCAATCCTTCTAGAAGATCTCCACAATG
3001 GCTTCCTCTATGCTCTCTTC CGCTACTATGGTTGCCTCTCCGGCTCAGGC
3051 CACTATGGTCGCTCCTTTCA ACGGACTTAAGTCCTCCGCTGCCTTCCCAG
3101 CCACCCGCAAGGCTAACAAC GACATTACTTCCATCACAAGCAACGGCGGA
3151 AGAGTTAACTGCATGCAGGT GTGGCCTCCGATTGGAAAGAAGAAGTTTGA
3201 GACTCTCTCTTACCTTCCTG ACCTTACCGATTCCGGTGGTCGCGTCAACT
3251 GCATGCAGGCCATGGCTAAG ATTTTTGATTTCGTAAAACCTGGCGTAATC
3301 ACTGGTGATGACGTACAGAA AGTTTTCCAGGTAGCAAAAGAAAACAACTT
3351 CGCACTGCCAGCAGTAAACT GCGTCGGTACTGACTCCATCAACGCCGTAC
3401 TGGAAACCGCTGCTAAAGTT AAAGCGCCGGTTATCGTTCAGTTCTCCAAC
3451 GGTGGTGCTTCCTTTATCGC TGGTAAAGGCGTGAAATCTGACGTTCCGCA
3501 GGGTGCTGCTATCCTGGGCG CGATCTCTGGTGCGCATCACGTTCACCAGA
3551 TGGCTGAACATTATGGTGTT CCGGTTATCCTGCACACTGACCACTGCGCG
3601 AAGAAACTGCTGCCGTGGAT CGACGGTCTGTTGGACGCGGGTGAAAAACA
3651 CTTCGCAGCTACCGGTAAGC CGCTGTTCTCTTCTCACATGATCGACCTGT
3701 CTGAAGAATCTCTGCAAGAG AACATCGAAATCTGCTCTAAATACCTGGAG
3751 CGCATGTCCAAAATCGGCAT GACTCTGGAAATCGAACTGGGTTGCACCGG
3801 TGGTGAAGAAGACGGCGTGG ACAACAGCCACATGGACGCTTCTGCACTGT
3851 ACACCCAGCCGGAAGACGTT GATTACGCATACACCGAACTGAGCAAAATC
3901 AGCCCGCGTTTCACCATCGC AGCGTCCTTCGGTAACGTACACGGTGTTTA
3951 CAAGCCGGGTAACGTGGTTC TGACTCCGACCATCCTGCGTGATTCTCAGG
4001 AATATGTTTCCAAGAAACAC AACCTGCCGCACAACAGCCTGAACTTCGTA
4051 TTCCACGGTGGTTCCGGTTC TACTGCTCAGGAAATCAAAGACTCCGTAAG
4101 CTACGGCGTAGTAAAAATGA ACATCGATACCGATACCCAATGGGCAACCT
4151 GGGAAGGCGTTCTGAACTAC TACAAAGCGAACGAAGCTTATCTGCAGGGT
4201 CAGCTGGGTAACCCGAAAGG CGAAGATCAGCCGAACAAGAAATACTACGA
4251 TCCGCGCGTATGGCTGCGTG CCGGTCAGACTTCGATGATCGCTCGTCTGG
4301 AGAAAGCATTCCAGGAACTG AACGCGATCGACGTTCTGTAAGAGCTCGGT
4351 ACCGGATCCAATTCCCGATC GTTCAAACATTTGGCAATAAAGTTTCTTAA
4401 GATTGAATCCTGTTGCCGGT CTTGCGATGATTATCATATAATTTCTGTTG
4451 AATTACGTTAAGCATGTAAT AATTAACATGTAATGCATGACGTTATTTAT
4501 GAGATGGGTTTTTATGATTA GAGTCCCGCAATTATACATTTAATACGCGA
4551 TAGAAAACAAAATATAGCGC GCAAACTAGGATAAATTATCGCGCGCGGTG
4601 TCATCTATGTTACTAGATCG GGGATCGATCCCCGGGCGGCCGCCACTCGA
4651 GTGGTGGCCGCATCGATCGT GAAGTTTCTCATCTAAGCCCCCATTTGGAC
4701 GTGAATGTAGACACGTCGAA ATAAAGATTTCCGAATTAGAATAATTTGTT
4751 TATTGCTTTCGCCTATAAAT ACGACGGATCGTAATTTGTCGTTTTATCAA
4801 AATGTACTTTCATTTTATAA TAACGCTGCGGACATCTACATTTTTGAATT
4851 GAAAAAAAATTGGTAATTAC TCTTTCTT?'"'TCTCCATATTGACCATCATA
4901 CTCATTGCTGATCCATGTAG ATTTCCC'CATS '~GCCATTTACAATTG
4951 AATATATCCTGCCGCCGCTG CCGCTT".CCCC'"GGAGCTTGCATGTT
5001 GGTTTCTACGCAGAACTGAG CCGGTTAAGA"~.ATTTCCATTGAGAAC
5051 TGAGCCATGTGCACCTTCCC CCCAACAC.~TGAGCGACGGGGCAACGGAG
5101 TGATCCACATGGGACTTTTc CTAGCTTGGCTGCCATTTTTGGGGTGAGGC
5151 CGTTCGCGCGGGGCGCCAGC TGGGGGGATGGGAGGCCCGCGTTACCGGGA
5201 GGGTTCGAGA TCACGCGCCA
AGGGGGGGCA
CCCCCCTTCG
GCGTGCGCGG
5251 GGGCGCAGCCCTGGTTAAAA ACAAGGTTTA TTTAAAAGCA
TAAATATTGG
5301 GGTTAAAAGA
CAGGTTAGCG
GTGGCCGAAA
AACGGGCGGA
AACCCTTGCA
5351 AATGCTGGAT AATAGGTGCG
TTTCTGCCTG
TGGACAGCCC
CTCAAATGTC
5401 CCCCTCATCT
GTCATCACTC
TGCCCCTCAA
GTGTCAAGGA
TCGCGCCCCT
52
CA 02294525 1999-12-13
WO 98/58069 PCT/US98/12447
5451 CATCTGTCAGTAGTCGCGCCCCTCAAGTGTCAATACCGCAGGGCACTTAT
5501 CCCCAGGCTTGTCCACATCATCTGTGGGAAACTCGCGTAAAATCAGGCGT
5551 TTTCGCCGATTTGCGAGGCTGGCCAGCTCCACGTCGCCGGCCGAAATCGA
5601 GCCTGCCCCTCATCTGTCAACGCCGCGCCGGGTGAGTCGGCCCCTCAAGT
5651 GTCAACGTCCGCCCCTCATCTGTCAGTGAGGGCCAAGTTTTCCGCGTGGT
5701 ATCCACAACGCCGGCGGCCGGCCGCGGTGTCTCGCACACGGCTTCGACGG
5751 CGTTTCTGGCGCGTTTGCAGGGCCATAGACGGCCGCCAGCCCAGCGGCGA
5801 GGGCAACCAGCCCGGTGAGCGTCGGAAAGGGTCGATCGACCGATGCCCTT
5851 GAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTA
5901 TCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAG
5951 GTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAG
6001 CGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCC
6051 TCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAG
6101 CAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCT
6151 GGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCG
6201 CTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAG
6251 GTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTAC
6301 CAGCCTAACTTCGATCACTGGACCGCTGATCGTCACGGCGATTTATGCCG
6351 CCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTA
6401 TACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCAC
6451 CTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCC
6501 AAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCA
6551 ACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACG
6601 CGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGT
6651 GCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTT
6701 AGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGC
6751 AAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGT
6801 TTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCC
6851 GGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCT
6901 GTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCG
6951 CCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGG
7001 CATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATC
?051 GGTATCATTACCCCCATGAACAGAAATTCCCCCTTACACGGAGGCATCAA
7101 GTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAG
7151 CCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAAC
7201 AGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGC
7251 AGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCA
7301 GCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGAC
7351 AAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCC
7401 ATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCG
7451 GCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACC
7501 GCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCT
7551 CGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCA
7601 GCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC
7651 AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAA
7701 AGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT
7751 CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA
7801 AAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC
7851. CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC
7901 GTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT
7951 CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC
8001 GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC
8051 GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG
8101 GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT
8151 ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACC
8201 TTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG
8251 TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAG
8301 GATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG
53
CA 02294525 1999-12-13
WO 98/58069 PCT/US98112447
8351 AACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGAT
8401 CTTCACCTAG ATTAAAAATGAAGTTTTAAATCAATCTAAA
ATCCTTTTAA
8451 GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG
8501 GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT
8551 CCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCA
8601 GTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCA
8651 GCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAAC
8701 TTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAA
8751 GTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGT
8801 CGGGAGCACAGGATGACGCCTAACAATTCATTCAAGCCGACACCGCTTCG
8851 CGGCGCGGCTTAATTCAGGAGTTAAACATCATGAGGGAAGCGGTGATCGC
8901 CGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATC
8951 TCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGC
9001 GGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAG
9051 GCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTT
9101 CGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATT
9152 GTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACT
9201 GCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGC
9251 CAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAA
9301 CATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGT
9351 TCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGA
9401 ACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTG
9451 TCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGT
9501 CGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCA
9551 TACTTGAAGCTAGGCAGGCTTATCTTGGACAAGAAGATCGCTTGGCCTCG
9601 CGCGCAGATCAGTTGGAAGAATTTGTTCACTACGTGAAAGGCGAGATCAC
9651 CAAGGTAGTCGGCAAATAATGTCTAACAATTCGTTCAAGCCGACGCCGCT
9701 TCGCGGCGCGGCTTAACTCAAGCGTTAGATGCTGCAGGCATCGTGGTGTC
9751 ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA
9801 GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC
9851 GGTCCTCCGATCGAGGATTTTTCGGCGCTGCGCTACGTCCGCKACCGCGT
9901 TGAGGGATCAAGCCACAGCAGCCCACTCGACCTCTAGCCGACCCAGACGA
9951 GCCAAGGGATCTTTTTGGAATGCTGCTCCGTCGTCAGGCTTTCCGACGTT
10001 TGGGTGGTTGAACAGAAGTCATTATCGTACGGAATGCCAAGCACTCCCGA
10051 GGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACC
10101 TTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCT
10151 TAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTGAA
10201 GGCGGGAAACGACAATCTGATCCCCATCAAGCTTGAGCTCAGGATTTAGC
10251 AGCATTCCAGATTGGGTTCAATCAACAAGGTACGAGCCATATCACTTTAT
10301 TCAAATTGGTATCGCCAAAACCAAGAAGGAACTCCCATCCTCAAAGGTTT
10351 GTAAGGAAGAATTCTCAGTCCAAAGCCTCAACAAGGTCAGGGTACAGAGT
10401 CTCCAAACCATTAGCCAAAAGCTACAGGAGATCAATGAAGAATCTTCAAT
10451 CAAAGTAAACTACTGTTCCAGCACATGCATCATGGTCAGTAAGTTTCAGA
10501 AAAAGACATCCACCGAAGACTTAAAGTTAGTGGGCATCTTTGAAAGTAAT
10551 CTTGTCAACATCGAGCAGCTGGCTTGTGGGGACCAGACAA
AAAAGGAATG
10601 GTGCAGAATTGTTAGGCGCACCTACCAAAA CTTTATTGCA
GCATCTTTGC
10651 AAGAT.':AAGCAGATTCCTCTAGTACAAGTGGGGAACAAAA
TAACGTGGAA
.10701AAGAG~iGTCCTGACAGCCCACTCACTAATGCGTATGACG
AACGCAGTGA
10751 , CGACC.~s'dAAAAGAATTCCCT
CTATATAAGA
AGGCATTCAT
TCCCATTTGA
10801 AGGATCATCAGATACTGAAC
CAATCCTTCT
AGAAGATCTA
AGCTTAT
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10901 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: Linear
54
CA 02294525 1999-12-13
WO 98/58069 PCT/US98/12447
(x1) SEQUENCE DESCRIPTION: SEQ ID N0:6:
1 CGATAAGCTTGATGTAATTGGAGGAAGATC ATCCCCATTC
AAAATTTTCA
51 TTCGATTGCTTCAATTGAAGTTTCTCCGATGGCGCAAGTTAGCAGAATCT
101 GCAATGGTGTGCAGAACCCATCTCTTATCTCCAATCTCTCGAAATCCAGT
151 CAACGCAAATCTCCCTTATCGGTTTCTCTGAAGACGCAGCAGCATCCACG
201 AGCTTATCCGATTTCGTCGTCGTGGGGATTGAAGAAGAGTGGGATGACGT
251 TAATTGGCTCTGAGCTTCGTCCTCTTAAGGTCATGTCTTCTGTTTCCACG
301 GCGTGCATGCTTCACGGTGCAAGCAGCCGTCCAGCAACTGCTCGTAAGTC
351 CTCTGGTCTTTCTGGAACCGTCCGTATTCCAGGTGACAAGTCTATCTCCC
401 ACAGGTCCTTCATGTTTGGAGGTCTCGCTAGCGGTGAAACTCGTATCACC
451 GGTCTTTTGGAAGGTGAAGATGTTATCAACACTGGTAAGGCTATGCAAGC
501 TATGGGTGCCAGAATCCGTAAGGAAGGTGATACTTGGATCATTGATGGTG
l5 551 TTGGTAACGGTGGACTCCTTGCTCCTGAGGCTCCTCTCGATTTCGGTAAC
601 GCTGCAACTGGTTGCCGTTTGACTATGGGTCTTGTTGGTGTTTACGATTT
651 CGATAGCACTTTCATTGGTGACGCTTCTCTCACTAAGCGTCCAATGGGTC
701 GTGTGTTGAACCCACTTCGCGAAATGGGTGTGCAGGTGAAGTCTGAAGAC
751 GGTGATCGTCTTCCAGTTACCTTGCGTGGACCAAAGACTCCAACGCCAAT
801 CACCTACAGGGTACCTATGGCTTCCGCTCAAGTGAAGTCCGCTGTTCTGC
851 TTGCTGGTCTCAACACCCCAGGTATCACCACTGTTATCGAGCCAATCATG
901 ACTCGTGACCACACTGAAAAGATGCTTCAAGGTTTTGGTGCTAACCTTAC
951 CGTTGAGACTGATGCTGACGGTGTGCGTACCATCCGTCTTGAAGGTCGTG
1001 GTAAGCTCACCGGTCAAGTGATTGATGTTCCAGGTGATCCATCCTCTACT
1051 GCTTTCCCATTGGTTGCTGCCTTGCTTGTTCCAGGTTCCGACGTCACCAT
1101 CCTTAACGTTTTGATGAACCCAACCCGTACTGGTCTCATCTTGACTCTGC
1151 AGGAAATGGGTGCCGACATCGAAGTGATCAACCCACGTCTTGCTGGTGGA
1201 GAAGACGTGGCTGACTTGCGTGTTCGTTCTTCTACTTTGAAGGGTGTTAC
1251 TGTTCCAGAAGACCGTGCTCCTTCTATGATCGACGAGTATCCAATTCTCG
1301 CTGTTGCAGCTGCATTCGCTGAAGGTGCTACCGTTATGAACGGTTTGGAA
1351 GAACTCCGTGTTAAGGAAAGCGACCGTCTTTCTGCTGTCGCAAACGGTCT
1401 CAAGCTCAACGGTGTTGATTGCGATGAAGGTGAGACTTCTCTCGTCGTGC
1451 GTGGTCGTCCTGACGGTAAGGGTCTCGGTAACGCTTCTGGAGCAGCTGTC
1501 GCTACCCACCTCGATCACCGTATCGCTATGAGCTTCCTCGTTATGGGTCT
1551 CGTTTCTGAAAACCCTGTTACTGTTGATGATGCTACTATGATCGCTACTA
1601 GCTTCCCAGAGTTCATGGATTTGATGGCTGGTCTTGGAGCTAAGATCGAA
1651 CTCTCCGACACTAAGGCTGCTTGATGAGCTCAAGAATTCGAGCTCGGTAC
1701 CGGATCCAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTT
1751 CAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTCGAGT
1801 ATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTA
1851 ATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAA
1901 TGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAA
1951 TTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATT
2001 ATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTG
2051 GCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTAC
2101 CATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGC
2151 TGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTAT
2201 GAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATT
2251 GCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATA
2301 TTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGCATCAAT
2351 CGACCTGCAGCCACTCGAAGCGGCCGCGTTCAAGCTTGAGCTCAGGATTT
2401 AGCAGCATTCCAGATTGGGTTCAATCAACAAGGTACGAGCCATATCACTT
2451 TATTCAAATTGGTATCGCCAAAACCAAGAAGGAACTCCCATCCTCAAAGG
2501 TTTGTAAGGAAGAATTCTCAGTCCAAAGCCTCAACAAGGTCAGGGTACAG
2551 AGTCTCCAAACCATTAGCCAAAAGCTACAGGAGATCAATGAAGAATCTTC
2601 AATCAAAGTAAACTACTGTTCCAGCACATGCATCATGGTCAGTAAGTTTC
2651 AGAAAAAGACATCCACCGAAGACTTAAAGTTAGTGGGCATCTTTGAAAGT
2701 AATCTTGTCAACATCGAGCAGCTGGCTTGTGGGGACCAGACAAAAAAGGA
SS
CA 02294525 1999-12-13
WO 98/58069 PCT/US98/12447
2751 ATGGTGCAGA ATTGTTAGGC GCACCTACCA AAAGCATCTT
TGCCTTTATT
2801 GCAAAGATAA AGCAGATTCC TCTAGTACAA GTGGGGAACA
AAATAACGTG
2851 GAAAAGAGCT GTCCTGACAG CCCACTCACT AATGCGTATG
ACGAACGCAG
2901 TGACGACCAC AAAAGAATTC CCTCTATATA AGAAGGCATT
CATTCCCATT
2951 TGAAGGATCA TCAGATACTG AACCAATCCT TCTAGAAGAT
CTAAGCTTAT
3001 CGATAAGCTT GATGTAATTG GAGGAAGATC AAAATTTTCA
ATCCCCATTC
3051 TTCGATTGCT TCAATTGAAG TTTCTCCGAT GGCGCAAGTT
AGCAGAATCT
3101 GCAATGGTGT GCAGAACCCA TCTCTTATCT CCAATCTCTC
GAAATCCAGT
3151 CAACGCAAAT CTCCCTTATC GGTTTCTCTG AAGACGCAGC
AGCATCCACG
3201 AGCTTATCCG ATTTCGTCGT CGTGGGGATT GAAGAAGAGT
GGGATGACGT
3251 TAATTGGCTC TGAGCTTCGT CCTCTTAAGG TCATGTCTTC
TGTTTCCACG
3301 GCGTGCATGC AGGCcatggC TAAGATTTTT GATTTCGTAA
AACCTGGCGT
3351 AATCACTGGT GATGACGTAC AGAAAGTTTT CCAGGTAGCA
AAAGAAAACA
3401 ACTTCGCACT GCCAGCAGTA AACTGCGTCG GTACTGACTC
CATCAACGCC
3451 GTACTGGAAA CCGCTGCTAA AGTTAAAGCG CCGGTTATCG
TTCAGTTCTC
3501 CAACGGTGGT GCTTCCTTTA TCGCTGGTAA AGGCGTGAAA
TCTGACGTTC
3551 CGCAGGGTGC TGCTATCCTG GGCGCGATCT CTGGTGCGCA
TCACGTTCAC
3601 CAGATGGCTG AACATTATGG TGTTCCGGTT ATCCTGCACA
CTGACCACTG
3651 CGCGAAGAAA CTGCTGCCGT GGATCGACGG TCTGTTGGAC
GCGGGTGAAA
3701 AACACTTCGC AGCTACCGGT AAGCCGCTGT TCTCTTCTCA
CATGATCGAC
3751 CTGTCTGAAG AATCTCTGCA AGAGAACATC GAAATCTGCT
CTAAATACCT
3801 GGAGCGCATG TCCAAAATCG GCATGACTCT GGAAATCGAA
CTGGGTTGCA
3851 CCGGTGGTGA AGAAGACGGC GTGGACAACA GCCACATGGA
CGCTTCTGCA
3901 CTGTACACCC AGCCGGAAGA CGTTGATTAC GCATACACCG
AACTGAGCAA
3951 AATCAGCCCG CGTTTCACCA TCGCAGCGTC CTTCGGTAAC
GTACACGGTG
4001 TTTACAAGCC GGGTAACGTG GTTCTGACTC CGACCATCCT
GCGTGATTCT
4051 CAGGAATATG TTTCCAAGAA ACACAACCTG CCGCACAACA
GCCTGAACTT
4101 CGTATTCCAC GGTGGTTCCG GTTCTACTGC TCAGGAAATC
AAAGACTCCG
4151 TAAGCTACGG CGTAGTAAAA ATGAACATCG ATACCGATAC
CCAATGGGCA
4201 ACCTGGGAAG GCGTTCTGAA CTACTACAAA GCGAACGAAG
CTTATCTGCA
4251 GGGTCAGCTG GGTAACCCGA AAGGCGAAGA TCAGCCGAAC
AAGAAATACT
4301 ACGATCCGCG CGTATGGCTG CGTGCCGGTC AGACTTCGAT
GATCGCTCGT
4351 CTGGAGAAAG CATTCCAGGA ACTGAACGCG ATCGACGTTC
TGTAAGAGCT
4401 CGGTACCGGA TCCAATTccc GATCGTTCAA ACATTTGGCA
ATAAAGTTTC
4451 TTAAGATTGA ATCCTGTTGC CGGTCTTGCG ATGATTATCA
TATAATTTCT
4501 GTTGAATTAC GTTAAGCATG TAATAATTAA CATGTAATGC
ATGACGTTAT
4551 TTATGAGATG GGTTTTTATG ATTAGAGTCC CGCAATTATA
CATTTAATAC
4601 GCGATAGAAA ACAAAATATA GCGCGCAAAC TAGGATAAAT
TATCGCGCGC
4651 GGTGTCATCT ATGTTACTAG ATCGGGGATC GATCCCCGGG
CGGCCGCCAC
4701 TCGAGTGGTG GCCGCATCGA TCGTGAAGTT TCTCATCTAA
GCCCCCATTT
4751 GGACGTGAAT GTAGACACGT CGAAATAAAG ATTTCCGAAT
TAGAATAATT
4801 TGTTTATTGC TTTCGCCTAT AAATACGACG GATCGTAATT
TGTCGTTTTA
4851 TCAAAATGTA CTTTCATTTT ATAATAACGC TGCGGACATC
TACATTTTTG
4901 AATTGAAAAA AAATTGGTAA TTACTCTTTC TTTTTCTCCA
TATTGACCAT
4951 CATACTCATT GCTGATCCAT GTAGATTTCC CGGACATGAA
GCCATTTACA
5001 ATTGAATATA TCCTGCCGCC GCTGCCGCTT TGCACCCGGT
GGAGCTTGCA
5051 TGTTGGTTTC TACGCAGAAC TGAG~~"GGTT AGGCAGATAA
TTTCCATTGA
5101 GAACTGAGCC ATGTGCACCT TCCC: ~AAC ACGGTGAGCG
ACGGGGCAAC
515 GGAGTGATCC ACATGGGACT TTTCC %..GCT TGGCTGCCAT
TTTTGGGGTG
5201 AGGCCGTTCG CGCGGGGCGC CAGCTGGGGG GATGGGAGGC
CCGCGTTACC
5251 GGGAGGGTTC GAGAAGGGGG GGCACCCCCC TTCGGCGTGC
GCGGTCACGC
5301 GCCAGGGCGC AGCCCTGGTT AAAAACAAGG TTTATAAATA
TTGGTTTAAA
5351 AGCAGGTTAA AAGACAGGTT AGCGGTGGCC GAAAAACGGG
CGGAAACCCT
5401 TGCAAATGCT GGATTTTCTG CCTGTGGACA GCCCCTCAAA
TGTCAATAGG
5451 TGCGCCCCTC ATCTGTCATC ACTCTGCCCC TCAAGTGTCA
AGGATCGCGC
5501 CCCTCATCTG TCAGTAGTCG CGCCCCTCAA GTGTCAATAC
CGCAGGGCAC
5551 TTATCCCCAG GCTTGTCCAC ATCATCTGTG GGAAACTCGC
GTAAAATCAG
5601 GCGTTTTCGC CGATTTGCGA GGCTGGCCAG CTCCACGTCG
CCGGCCGAAA
56
CA 02294525 1999-12-13
WO 98/58069 PCT/US98/12447
5651 TCGAGCCTGCCCCTCATCTGTCAACGCCGCGCCGGGTGAGTCGGCCCCTC
5701 AAGTGTCAACGTCCGCCCCTCATCTGTCAGTGAGGGCCAAGTTTTCCGCG
5751 TGGTATCCACAACGCCGGCGGCCGGCCGCGGTGTCTCGCACACGGCTTCG
5801 ACGGCGTTTCTGGCGCGTTTGCAGGGCCATAGACGGCCGCCAGCCCAGCG
5851 GCGAGGGCAACCAGCCCGGTGAGCGTCGGAAAGGGTCGATCGACCGATGC
5901 CCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATG
5951 ACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGG
6001 ACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCT
6051 GGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCAC
6101 GCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGA
6151 GAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCT
6201 TGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTT
6251 CTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAG
6301 GCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTC
6351 TTACCAGCCTAACTTCGATCACTGGACCGCTGATCGTCACGGCGATTTAT
6401 GCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGC
6451 CCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGG
6501 CCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCA
6551 CTCCAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAA
6601 ACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCG
6651 CACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGA
6701 TCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACT
6751 GGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTG
6801 CTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCC
6851 GTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATG
6901 TTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTAC
6951 ATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGT
7001 CCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAAC
7051 CGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTT
7101 CATCGGTATCATTACCCCCATGAACAGAAATTCCCCCTTACACGGAGGCA
7152 TCAAGTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCA
7201 GAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGAT
7251 GAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTA
7301 CCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACA
7351 TGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGC
7401 AGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGC
7451 AGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTA
7501 TGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAA
7551 TACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCT
7601 TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT
7651 ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATA
7701 ACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT
7751 AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA
7801 GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGAC
7851 TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCT
7901 GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG
7951 AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT
8001 AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC
8051.GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG
8101 ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG
8151 CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC
8201 GGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT
8251 TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG
8301 CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA
8351 AAAGGATCTC TTTGATCTTTTCTACGGGGTCTGACGCTCA
AAGAAGATCC
8401 GTGGAACGAA AAGGGATTTTGGTCATGAGATTATCAAAAA
AACTCACGTT
8451 GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATC
8501 TAAAGTATAT TTGGTCTGACAGTTACCAATGCTTAATCAG
ATGAGTAAAC
57
CA 02294525 1999-12-13
WO 98/58069 PCT/US98/12447
8551 TGAGGCACCT ATCTCAGCGA TCTGTCTATT
TCGTTCATCC ATAGTTGCCT
8601 GACTCCCCGT CGTGTAGATA ACTACGATAC
GGGAGGGCTT ACCATCTGGC
8651 CCCAGTGCTG CAATGATACC GCGAGACCCA CTCCAGATTT
CGCTCACCGG
8701 ATCAGCAATA AACCAGCCAG CCGGAAGGGC
CGAGCGCAGA AGTGGTCCTG
8751 CAACTTTATC CGCCTCCATC CAGTCTATTA GGAAGCTAGA
ATTGTTGCCG
8801 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC CCATTGCTGC
AACGTTGTTG
8851 AGGTCGGGAG CACAGGATGA CGCCTAACAA CCGACACCGC
TTCATTCAAG
8901 TTCGCGGCGC GGCTTAATTC AGGAGTTAAA GAAGCGGTGA
CATCATGAGG
8951 TCGCCGAAGT ATCGACTCAA CTATCAGAGG CATCGAGCGC
TAGTTGGCGT
9001 CATCTCGAAC CGACGTTGCT GGCCGTACAT CCGCAGTGGA
TTGTACGGCT
9051 TGGCGGCCTG AAGCCACACA GTGATATTGA ACGGTGACCG
TTTGCTGGTT
9101 TAAGGCTTGA TGAAACAACG CGGCGAGCTT CCTTTTGGAA
TGATCAACGA
9151 ACTTCGGCTT CCCCTGGAGA GAGCGAGATT TAGAAGTCAC
CTCCGCGCTG
9201 CATTGTTGTG CACGACGACA TCATTCCGTG GCTAAGCGCG
GCGTTATCCA
9251 AACTGCAATT TGGAGAATGG CAGCGCAATG AGGTATCTTC
ACATTCTTGC
9301 GAGCCAGCCA CGATCGACAT TGATCTGGCT CAAAAGCAAG
ATCTTGCTGA
9351 AGAACATAGC GTTGCCTTGG TAGGTCCAGC CTCTTTGATC
GGCGGAGGAA
9401 CGGTTCCTGA ACAGGATCTA TTTGAGGCGC CTTAACGCTA
TAAATGAAAC
9451 TGGAACTCGC CGCCCGACTG GGCTGGCGAT TAGTGCTTAC
GAGCGAAATG
9501 GTTGTCCCGC ATTTGGTACA GCGCAGTAAC GCGCCGAAGG
CGGCAAAATC
9551 ATGTCGCTGC CGACTGGGCA ATGGAGCGCC GTATCAGCCC
TGCCGGCCCA
9601 GTCATACTTG AAGCTAGGCA GGCTTATCTT ATCGCTTGGC
GGACAAGAAG
9651 CTCGCGCGCA GATCAGTTGG AAGAATTTGT AAAGGCGAGA
TCACTACGTG
9701 TCACCAAGGT AGTCGGCAAA TAATGTCTAA AAGCCGACGC
CAATTCGTTC
9751 CGCTTCGCGG CGCGGCTTAA CTCAAGCGTT GGCATCGTGG
AGATGCTGCA
9801 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TTCCCAACGA
TCAGCTCCGG
9851 TCAAGGCGAG TTACATGATC CCCCATGTTG CGGTTAGCTC
TGCAHAAAAG
9901 CTTCGGTCCT CCGATCGAGG ATTTTTCGGC GTCCGCKACC
GCTGCGCTAC
9951 GCGTTGAGGG ATCAAGCCAC AGCAGCCCAC GCCGACCCAG
TCGACCTCTA
10001 ACGAGCCAAG GGATCTTTTT GGAATGCTGC GGCTTTCCGA
TCCGTCGTCA
10051 CGTTTGGGTG GTTGAACAGA AGTCATTATC CCAAGCACTC
GTACGGAATG
10101 CCGAGGGGAA CCCTGTGGTT GGCATGCACA CGAACGGATA
TACAAATGGA
10151 AACCTTTTCA CGCCCTTTTA AATATCCGTT ACGCTCTTTT
ATTCTAATAA
10201 CTCTTAGGTT TACCCGCCAA TATATCCTGT TAGTTTAAAC
CAAACACTGA
10251 TGAAGGCGGG AAACGACAAT CTGATCCCCA GCTCAGGATT
TCAAGCTTGA
10301 TAGCAGCATT CCAGATTGGG TTCAATCAAC CCATATCACT
AAGGTACGAG
10351 TTATTCAAAT TGGTATCGCC AAAACCAAGA ATCCTCAAAG
AGGAACTCCC
10401 GTTTGTAAGG AAGAATTCTC AGTCCAAAGC TCAGGGTACA
CTCAACAAGG
10451 GAGTCTCCAA ACCATTAGCC AAAAGCTACA GAAGAATCTT
GGAGATCAAT
10501 CAATCAAAGT AAACTACTGT TCCAGCACAT CAGTAAGTTT
GCATCATGGT
10551 CAGAAAAAGA CATCCACCGA AGACTTAAAG TCTTTGAAAG
TTAGTGGGCA
10601 TAATCTTGTC AACATCGAGC AGCTGGCTTG ACAAAAAAGG
TGGGGACCAG
10651 AATGGTGCAG AATTGTTAGG CGCACCTACC TTGCCTTTAT
AAAAGCATCT
10701 TGCAAAGATA AAGCAGATTC CTCTAGTACA AAAATAACGT
AGTGGGGAAC
10751 GGAAAAGAGC TGTCCTGACA GCCCACTCAC GACGAACGCA
TAATGCGTAT
10801 GTGACGACCA CAAAAGAATT CCCTCTATAT TCATTCCCAT
AAGAAGGCAT
10851 TTGAAGGATC ATCAGATACT GAACCAATCC
TTCTAGAAGA TCTAAGCTTA
. 10901T
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