Note: Descriptions are shown in the official language in which they were submitted.
CA 02416347 2003-02-11
IMPROVEMENT IN OR RELATING TO PLANT STARCH COMPOSITION
This application is a division ofI'CT Inteniational Application No.
PCT/GB96/01075
bearing Canadian Application Serial No. 2,217,878 with the international
filing date of
May 3, 1996.
Field of the Invention
This invention relates to novel nucleoride seauences. polypeptides encoded
thereby, vectors
and host cells and host orQa.nisms comprisiria one or more of the novel
sequences, and to
a method of alterin(2 one or more characteristics of an oraanism. The
invention al;so
relates to starch having novel properties and to uses thereof.
Background of the Invention
Starch is the major form of carbon reserve in plants, constitutina 50% or more
of the dry
weiqht of many storaQe organs - e.z. tubers, seeds of cereals. Starch is used
in numerous
food and industrial applications. In many cases, however, it is necessary to
modify the
native starches, via chemical or physical means, in order to produce distinct
properties to
suit particular applications. It would be highly desirable to be able to
produce starches
with the required properties directly in the plant, thereby removing the need
for additional
modification. To achieve this via genetic engineering requires knowledge of
the metabolic
pathwav of starch biosvnthesi.s. This includes characterisation of genes and
encoded gene
products which catalyse the synthesis of starch. Knowledae about the
regulation of starch
biosvnthesis raises the possibility of "re-proarammina" biosynthetic pathways
to create
starches with novel properties that could have new corr.imercial applications.
The commercially useful properties of starch derive frorn the ability of the
native granular
form to swell and absorb water upon suitable treatment. Usuallv heat is
required to cause
granules to swell in a process known as aelatirisation., which has been
defined (W A
Atwell et al, Cereal Foods World 33. 306-311, 1988) as "... the coliapse
(disruption) of
molecular orders within the starch granule manifested in irreversible changes
in properties
such as granular swelling, narive crystallite melting, loss of birefringence,
and starch
solubilisation. The point of initial gelatinisation and the range over which
it occurs is
governed bv srarch concentrarron. method of observation, granule type, and
heterogeneities
within the granule population under obsen%ation". A number of techniques are
available
CA 02416347 2003-02-11
L
for the determination of aelatinisation as induced by heating, a convenient
and accurate
method beinQ differential scanninQ caiorimetrs: . which detects the
temperature ranae and
enthalpy associated with the collapse of molecular orders within the aranule_
To obtain
accurate and meaninaful results, the peak and/or onset temueran:re of the
endotherm
observed bv differential scanninQ calorimetn- is usually determined.
The consequence of the collapse of molecular orders within starch granules is
that the
aranules are capable of taking up water in a process known as pasting, which
has been
defined (W A Atwell et al, Cereal Foods World 33, 306-311, 1988) as "... the
phenomenon following gelatinzsation in the dissolution of starch. It involves
granular
swelling, exudation of molecular comoonents from the granule, and eventualiv,
total
disruption of the granules". The best method of evaluating pastina properties
is
considered to be the viscoamyloaraph (Atwell et al, 1988 cited above) in which
the
viscosity of a stirred starch suspension is monitored under a defined
time/temperature
regime. A typical viscoamylograph profile for potato starch shows an initial
rise in
viscosity, which is considered to be due to aranule swelling. In addition to
the overall
shape of the viscosity response in a viscoamylograph, a convenient
quantitative measure
is the temperature of initial visc;ositv development (onset). Fip-ure 1 shows
such a typical
viscositv profile for potato starch, during and after cooking, and includes
stages A-D
which correspond to viscositv onset (A). maximum viscosi.tv (B), complete
dispersion (C)
and reassociation of molecules (or retroaradation, D). In the fi-aure, the
dotted line
represents viscosity (in stirring number units) of a 10% w/w starch suspension
and the
unbroken line shows the temperature in deorees centiQrade. At a certain point,
defined
by the viscositv peak, aranule swelling is so extensive that the resulting
highly expanded
structures are susceptible to mechanicallv-induced fragmentation under the
stirring
conditions used. With increased heatina and holding at 95 C, further reduction
in
viscositv is observed due to increased fraamentation of swollen aranules. This
veneral
profile has previously always been found for native potato starch.
After heatinQ starches in water to 95 C and holdina at that temperature (for
typically 15
minutes). subseauent coolins to 50 C results in an increase in viscositv due
to the process
of retroQradation or set-back. RetroLyradation (or set-back) is defined
(Atweil et al.. 1988
CA 02416347 2003-02-11
cited above) as ".. . a process which occur s:::':en tite molecules comorising
Qelarinised
starch begin to reassociate in an ordered strucrure... ". At 50 C. it is
primarilv the
amvlose comaonent =hich reassociates. as indicated b%- the increase in
viscoamvlograph
viscositv for starch from normal maize (21.6 amvlose) compared with starch
from waxy
maize (l.? % amvlose) as shown in Figure 2. Fioure 2 is a viscoamvlograph of
l0%wiw
starch suspensions from waxv maize (,solid line), conventional maize (dots and
dashes),
high amylose varietv (hylon 5. dotted line) and a verv high amylose varietv
(hvion 7,
crosses). The temperatur,, protile is also shown by a solid line, as in Figure
1.
The
extent of viscositv increase in the viscoamvlograph on cooling and holding at
50 C
depends on the amount of amvlose which is able to reassociate due to its
exudation from
starch granules during the gelatinisation and pastinQ processes. A
characteristic of
amvlose-rich starches from maize plants is that very little amylose is exuded
from granules
by gelatinisation and pasting up to 95 C, probably due to the restricted
swellina of the
granules. This is illustrated in Figure 2 which shows low viscosities for a
high amvlose
(44.9%) starch (Hvlon 5) from maize during gelatinisation and pasting at 95 C
and little
increase in viscositv on cooling and holding at 50 C. This effect is more
extreme for a
higher amylose content (58%, as in Hylon 7), which shows even lower
viscosities in the
viscoamvlo4raph test (Figure 2). For commercially-available high amylose
starches
(currently available from maize plants. such as those described above),
processing at
greater than 100 C is usuallv necessarv in order to generate the benefits of
high amylose
contents with respect to increased rates and strengths of reassociation, but
use of such high
temperatures is energeticallv un.favourable and c ostiv. Accordingly, there is
an unmet
need for starches of high amvlose content which can be processed below 100 C
and still
show enhanced levels of reassociation, as indicated for example bv
viscoarnvlograph
measurements.
The properties of potato starch are useful in a variety of both food and non-
food (paper.
textiles. adhesives etc.) appiications. However. for many applications.
properties are not
optimum and various chemical and physical modifn.ations well Lnown in the art
are
undertaken in order to improve useful properties. Two types of propertv
manipulation
which would be of use are: the controlled alteration of gelatinisation and
pastina
temperatures: and starches which suffer less granular fragmentation during
pasting than
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conventional starches.
Currentlv the only wavs of manipulating the Qelatinisation and pastinQ
temperatures of
potato starch are bv the inclusion of additives such as suaars. polvhvdroxv
compounds of
salts (Evans & Haisman. Starke 34. 21-4-231. 1982) or by extensive physical or
chemical
pre-treatments (e.Q. Stute, Starke 44. 205-214. 1992). The reduction of
aranule
fraQmentation durinQ pasting can be achieved either bv extensive phvsical
pretreatments
(Stute, Starke 44, 205-214, 1992) or bv chemical cross-linking. Such processes
are
inconvenient and inefficient. It is therefore desirable to obtain plants which
produce starch
which intrinsicallv possesses such advantageous properties.
Starch consists of two main polysaccharides, amvlose and amylopectin. Amvlose
is a
generallv linear polymer containing a-1.4 linked alucose units, while
amylopectin is a
highly branched polymer consistins; of a a-1.4 linked glucan backbone with a-
1,6 linked
zlucan branches. In most plant storage reserves amylopectin constitutes about
75 % of the
starch content. Amylopectin is synthesized by the concetted action of soluble
starch
synthase and starch branching enzyme [a-1,4 glucan: a-1,4 glucan 6-
Qlycosyltransferase,
EC 2.4.1.18]. Starch branching enzyme (SBE) hvdrolvses a-1,4 linkages and
rejoins the
cleaved glucan, via an a-1,6 linkage, to an acceptor chain to produce a
branched structure.
The physical properties of starch are strongly affected by the relative
abundance of
amylose and amvlopeetin, and SBE is therefore a crucial enzvme in determininQ
both the
quantity and quality of starches produced in plant systems.
In most plants studied to date e.g. maize (Bover & Preiss, 1978 Biochem.
Biophys. Res.
Comm. 80, 169-175), rice (Smvth. 1988 Plant Sci. 57, 1-8) and pea (Smith.
Planta 175,
270-279), two forms of SBE have been identified, each encoded by a separate
2ene. A
recent review bv Burton et al.. (1995 The Plant 7ournal 7.. 3-15) has
demonstrated that
the two forms of SBE constitute distinct classes of the enzvme such that, in
oeneral,
enzymes of the same class frotn different plants may exhibit greater
similarity than
enzvmes of different classes from the same plant. In their review. Burton et
al, termed
the vvo respective enzvme families class "A" and class "B". and the reader is
referred
thereto (and to the references cited therein) for a detailed discussion of the
distinctions
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between the two classes. One aPneral distinction of note would appear to be
the presence,
in class A SBE molecules, of a flexible ti-terminal domain. '~rtich is not
found in class
B molecules. The distinctions noted bv Burton er a'. are relied on herein to
define class
A and class B SBE molecules. which terms are to be interpreted accordinQiv.
However in potato, only one isoform ot the SEE molecule (belonging to class B)
has thus
far been reported and only one gene cloned (Blennow & Johansson, 1991
Phytochem. 30.
437-444, and Kof3mann er al., 1991 Liol. Gen. Genet. 230, 39-44). Further,
published
attempts to modify the properties of starch in potato plants (by preventing
expression of
the single known SBE) have aenerally not succeeded (e.2. Muller-Rober &
KoBmann 1994
Plant Cell and Environment 17, 601-613)_
Summary of the Invention
In a first aspect the invention provides a nucleotide sequence encoding an
effective portion
of a class A starch branchinQ, enzyme (SBE) obtainable from potato plants.
Preferably the nucleotide sequence encodes a polypeptide comprising an
effective portion
of the amino acid sequence shown in Figure 5(excluding the sequence MNKRIDL,
which
does not represent part of the SBE molecule), or a functional equivalent
thereof (which
term is discussed below). The amino acid sequence shown in Fi-zttre 5 (Seq ID
No. 15)
includes a leader sequence which directs the polypeptide. when svnthesised in
potato cells,
to the amvloplast. Those skilled in the art will recognise that the leader
sequence is
removed to produce a mature enzyme and that the leader sequence is therefore
not
essential for enzyme activity. Accordingly. ar. "effective portion" of the
polypeptide is
one which possesses sufficient SB:E activitv to complement the brarichin?
enzyme mutation
in E. coli KV 832 cells (described below) and which is active when expressed
in E. coli
in the. phosphorvlation stimulation assay. An example of an incomplete
polvpeptide which
nevertheless constitutes an "effective portion" is the mature enzvme lacking
the leader
sequence. Bv analoav with the pea class A SBE sequence. the potato class A
sequence
shown in Figure ~ probably possesses a leader sequence of about 48 amino acid
residues,
such that the N terminal amino acid sequence is thouaht to commence around the
fflutamic
n the art will appreciate
acid residue (E) at position 49 (EKSSYN.. . etc_). Those skilled 1
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~
that an effective portion of the enzvme may well omit other parts of the
sequence shown
in the fiQure without substantial detrimental effe-_t. For example. the C-
terminal slutamic
acid-rich region could be reduced in lenath, or possiblv deleted entirelv,
without
abolishinQ class A SBE activitv. A comparison with other known SBE sequences,
especiallv other class A SBE sequences (see for example. Burton et al. 1995
cited above),
should indicate those portions which are hi hlv conserved (and thus likely to
be essential
for activity) and those portions which are less well conserved (and thus are
more likely
~ to tolerate sequence changes without substantial loss of enzyme activity).
Conveniently the nucleotide sequence will comprise substantially nucleotides
289 to 2790
of the DNA sequence (Seq ID No. 14) shown in Figure 5 (which nucleotides
encode the
mature enzyme) or a functional equivalent thereof, and may also include
further
nucleotides at the 5' or 3' end. For exampie, for ease of expression, the
sequence will
desirably also comprise an in-frame ATG start codon, and may also encode a
leader
sequence. Thus, in one embodinnzent, the sequence further comprises
nucleotides 145 to
288 of the sequence shown in Figure 5. Other embodiment.s are nucleotides 228
to 2855
of the sequence labelled "psbe2con.seq" in Fieure 8, and nucleotides 57 to
2564 of the
sequence shown in Figure 12 (preferably comprising an in-frame ATG start
codon, such
as the sequence of nucleotides 24 to 56 in the same Figure), or functional
equivalents of
the aforesaid sequences.
The term "functional equivalent" as applied herein to nucleotide sequences is
intended to
encompass those sequences which differ in their nucleotide composition to that
shown in
Figure 5 but which, bv virtue of the de2eneracv of the aenetic code. encode
polypeptides
havinz identical or substantially identical amino acid sequences. It is
intended that the
term should also apply to sequences which are sufficientlv homologous to the
sequence of
the invention that they can hvbridise to the complement thereof under
stringent
hvbridisation conditions - such equivalents will preferably possess at least
85 %, more
preferablv at least 90 %, and most preferablv at least 95% seauence homolo y
with the
sequence of the invention as exemplified by nucleotides 289 to 2790 of the DNA
sequence
shown in FiQure 5. It will be apparent to those skilled in the an that the
nucleotide
sequence of the invention may also find useful application when present as an
"antisense"
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sequence. Accordingly, functionally equivalent sequences will also include
those
sequences which can hvbridise. under strinQent hvbridisation conditions. to
the sequence
of the invention (rather than the complement thereof). Such "antisense"
equivalents will
preferably possess at least 85%. more preferably at least 90 17c, and most
prer"erabiv 95%
sequence homology with the complement of the sequence of the invention as
exemplifed
bv nucleotides 289 to 2790 of the DNA sequence shown in Figure 5. Particular
functional
equivalents are shown, for example. in Figures 8 and 10 (if one disregards the
various
frameshift mutations twted therein).
The invention also provides vectors, particularlv expression vectors,
comprising the
nucleotide sequence of the invention. The vector will typically comprise a
promoter and
one or more reaulatory signals of the type well known to those skilled in the
art. The
invention also includes provision of cells transformed (which term encompasses
transduction and transfection) with a vector comprising the nucleotide
sequence of the
invention.
The invention further provides a class A SBE polypeptide, obtainable from
potato plants.
In particular the invention provides the polypeptide in substantially pure
form, especially
in a form free from other plant-derived (especially pctato plant-derived)
components,
which can be readily accomplished by expression of the relevant nucleotide
sequence in
a suitable non-plant host (such as anv one of the veast strairrs routinely
used for expression
purposes, e. a. Pichia spp. or Sacchar ornvices spp). Typically the enzyme
will substantially
comprise the sequence of amino acid residues 49 to 882 shown in Figure 5
(disregarding
the sequence MNYRIDL., which is not part of the enzvrrie), or a functional
equivalent
thereof. The polypeptide of the invention may be used in a method of modifying
starch
in vitro. comprisine treatinQ starch under suitable conditions (e.g.
appropriate temperature,
pH. etc) with an effective amount of the polvpeptide accordina to the
invention.
The term "functional equivalent". as applied herein to amino acid sequences.
is intended
to encompass amino acid sequerices substantiallv similar to that shown in
Figure 5. such
that the polvpeptide possesses sufficient activitv to complement the branching
enzvme
rnutation in E. coli KV 832 cells (described below) and which is active in E.
coii in the
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phosphorvlation stimuiation assay. Typically such functionally equivalent
amino acid
sequences will preferably possess at least 85 ~, more preferably at least 90
%, and most
preferably at least 95 ',,a sequence identity with the amino acid sequence of
the mature
enzyme (i.e. minus leader sequence) shown in Figure 5. Those skilled in the
art will
appreciate that conservative substitutions mav be made Qenerally throughout
the molecule
without substantiallv affectina the activity of the enzvme. Moreover, some non-
conservative substitutions mav be tolerated, especially in the less highly
conserved regions
of the molecule. S~:ch substitutions may be made, for example, to modify
slightly the
activity of the enzyme. The polypeptide may, if desired, include a leader
sequence, such
as that exemplified by residues 1 to 48 of the amino acid sequence shown in Fi-
aure 5,
althouah other leader sequences and siQnal peptides and the like are known and
may be
included.
A portion of the nucleotide sequence of the invention has been introduced into
a plant and
found to affect the characteristics of the plant. In particular, introduction
of the sequence
of the invention, operably linked in the antisense orientation to a suitable
promoter, was
found to reduce the amount of branched starch molecules in the plant.
Additionally, it has
recently been demonstrated in other experimental systems that "sense
suppression" can
aiso occur (i.e. expression of an introduced sequence operably linked in the
sense
orientation can interfere, by some unknown mechanism, with the expression of
the native
Qene), as described by Matzke & Matzke (1995 Plant Physiol. 107, 679-685). Anv
one
of the methods mentioned by Matzke & Matzke could, in theory, be used to
affect the
expression in a host of a homolog-ous SBE gene.
It is believed that antisense methods are mainly operable by the production of
antisense
mRNA which hvbridises to the sense mRh1A, preventing its translation into
functional
polvpeptide, possibly bv causina the hvbrid R\A to be degraded (e.a. Sheehy er
al., 1988
PNAS 85. 8805-8809: %,an der Krol er al.. Mol. Gen. Genet. 220, 204-212).
Sense
suppression also requires homology between the introduced seauence and the
tarQet 'Zene,
but the exact mechanism is unciear. It is apparent however that, in relation
to both
antisense and sense suppression. neither a full length nucleotide sequence.
nor a"native"
sequence is essential. Preferably the "effective portion" used in the method
will comprise
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at least one third of the full len2th sequence, but by simple trial and error
other fraaments
(smaller or larger) may be found which are rsnctional in alterinQ the
characteristics of the
plant.
Thus. in a further aspect the invention orovides a method of altering the
characteristics of
a plant. comprising introducing into the plant an effective portion of the
sequence of the
invention operably linked to a suitabie promoter active in the plant.
Conveniently the
sequence will be linked in the anti-sense orientation to the promoter.
Preferably the plant
is a potato plant. Conveniently, the characteristic altered relates to the
starch content
and/or starch composition of the plant (i.e. amount and/or type of starch
present in the
plant). Preferabty the method of altering the characteristics of the plant
will also comprise
the introduction of one or more further sequences, in addition to an effective
portion of
the sequence of the invention. qhe introduced sequence of the invention and
the one or
more further sequences (which rnay be sense or antisense sequences) may be
operably
linked to a singie promoter (which would ensure both sequences were
transcribed at
essentially the same time), or may be operably linked to separate promoters
(which may
be necessary for optimal expression). Where separate promoters are employed
they may
be identical to each other or different. Suitable promoters are well known to
those skilled
in the art and include both constitutive and inducible types. Examples include
the CaMV
35S promoter (e.a. single or tandem repeat) and the patatin promoter.
Advantaizeously
the promoter will be tissue-specific. Desirably the promoter will cause
expression of the
operably linked sequence at substantial levels only in the tissue of the plant
where starch
svnthesis and/or starch storaae mainlv occurs. Thus, for example, where the
sequence is
introduced into a potato plant. the operably linked promoter may be tuber-
specific, such
as the patatin promoter.
Desirably, for example, the method will also comprise the introduction of an
effective
portlon of a sequence encoding a class B SBE. operably linked in the antisense
orientation
to a suitable promoter active in the plant. Desirably the further sequence
will comprise
an effective portion of the sequence encodina the potato class B SBE molecule.
Convenientiv the fi2rther sequence %vill comprise an effective portion of the
sequence
described by Blennow ~ Johansson (1991 Phytochem. 30, 437-444) or that
disclosed in
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W092/11375. More preferablv. the further sequence will comprise at least an
effe;.tive
portion of the sequence disclosed in Internatiorial Patent Application No. WO
95/26407.
Use of antisense sequences against both class A and class B SBE in combination
has now
been found bv the present inventors to result in the production of starch
havinLy very
greatly altered properties (see below). Those skilled in the art will
appreciate the
possibility that, if the plant already comprises a sense or antisense sequence
which
efficiently inhibits the class B SBE activitv, introduction of a sense or
antisense sequence
to inhibit class A SBE activitv (therebv producing a plant with inhibition of
both class A
and class B activity) miQht alter ?reatly the properties of the starch in the
plant, without
the need for introduction of one or more further sequences. Thus the sequence
of the
invention is convenientlv introduced into plants already having low levels of
class A
and/or class B SBE activity, such that the inhibition resultinLy from the
introduction of the
sequence of the invention is likely io have a more pronounced effect.
The sequence of the invention, and the one or more further sequences if
desired, can be
introduced into the plant by any one of a number of well-known techniques
(e.g.
Agrobacterium-mediated transformation. or by "biolistic" m.ethods). The
sequences are
likely to be most effective in inhibiting SBE activity in potato plants, but
theoretically
could be introduced into any plant. Desirable examples include pea, tomato,
maize,
wheat, rice, barley, sweet potato and cassava plants. Preferably the plant
will comprise
a natural gene encodine an SBE molecule which exhibits reasonable homology
with the
introduced nucleic acid sequence of the invention.
In another aspect, the invention provides a plant cell. or a plant or the
progenv thereof,
which has been altered by the method defined above. The progeny of the altered
plant
mav be obtained. for example, bv veaetative propagation. or by crossing the
altered plant
and reserving the seed so obtained. The inv,,mtion also provides parts of the
altered plant,
such as storase orQans. Convenientlv, for example, the invention provides
tubers
comprisina altered starch. said tubers being obtained from an altered plant or
the proLyeny
thereof. Potato tubers obtained from altered plants (or the progeny thereof)
will be
particularly useful materials in certain industrial applications and for the
preparation andior
processing of foodstuffs and may be used. for example. to prepare low-fat
waffles and
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chips (amvlose L- nerallv being used as a coatina to prevent fat uptake). and
to prepare
mashed potato (especiallv "instant" mashed potato) havin~ particular
characteristics.
In particular relation to potato plants. the invention provides a potato plant
or pan thereof
which, in its wild type possesses an effective SBE A aene, but which plant has
been
altered such that there is no effective expression of an SBE A polypeptide
within the cells
of at least part of the plant. The plant may have been altered by the method
defined
above, or may have beeri selected by conventional breeding to be deleted for
the class A
SBE gene, presence or absence oF which can be readily determined by screening
samples
of the plants with a nucleic acid probe or antibody specific for Ihe potato
class A gene or
sene product respectively.
The invention also provides starch extracted from a plant altered bv the
method defined
above, or the proQeny of such a plant. the starch having altered properties
compared to
starch extracted from equivalent, but unaltered, plants. The invention further
provides a
method of making altered starch, comprising altering a plant by the method
defined above
and extracting therefrom starch having altered properties compared to starch
extracted
from equivalent, but unaltered, plants. Use of nucleotide sequences in
accordance with
the invention has allowed the present inventors to produce potato starches
having a wide
variery of novel properties.
In particular the invention provides the followinJ: a plant (especially a
potato plant) altered
bv the method defined above, containins! starch which. when extracted from the
plant, has
an elevated endotherm peak temperature as judged by DSC, compared to starch
extracted
from a similar, but unaltered, plant: a plant (especially a potato plant)
altered by the
method defined above, containing starch which, when extracted from the plant,
has an
elevated viscosity onset temperature (converuentlv elevated by 10 - 25 C) as
judaed bv
viscoamvloaraph compared to starch extracted from a similar, but unaltered,
plant: a plant
(especiallv a potato plant) altered by the method defined above, curuainina
starch which,
when extracted from the plant. has a decreased peak viscosity (conveniently
decreased by
240 - 700SNUs) as iud2ed bv viscoamvlozraph compared to starch extracted from
a
similar, but unaltered. plant: a plant (especiallv a potato plant) altered by
the method
CA 02416347 2003-02-11
defined above. containinQ starch which, when extracted from the plant, has an
increased
pastinv viscosirv (conveniently increased bv 3 7-?60SNlis) as }udged bv
viscoamylograph
compared to starch extracted from a similar. but unaltered. plant: a plant
(especially a
potato plant) altered bv the method defined above, containinQ starch which,
when extracted
from the plant, has an increased set-back viscosity (convenientlv increased by
224 - 313
SNUs) as judged bv viscoamvloaraph compared to starch extracted from a
similar, but
unaltered, plant: a plant (especially a potato plant) altered by the method
defined above,
containing starch which, when extracted from the plant, has a decreased set-
back viscosity
as judged by viscoamyloaraph compared to starch extracted from a similar, but
un2ltered,
plant; and a plant (especially a potato plant) altered by the method defined
above,
containino starch which, when extracted from the plant, has an elevated
amvlose content
as judged bv iodometric assav (i.e. bv the method of Morrison & Laignelet
1983. cited
above) compared to starch extracted from a similar, but unaltered, plant. The
invention
also provides for starch obtainable or obtained from such plants as aforesaid.
In particular the invention provides for starch which, as extracted from a
potato plant by
wet milling at ambient temperature, has one or more of the following
properties, as judged
by viscoamylograph analysis performed according to the conditions defined
below:
viscosity onset temperature in the range 70-95 C (preferably 75-95 C); peak
viscosity in
the range 500 - 12 stirring number units; pasting viscosity in the range 214 -
434 stirring
number units: set-back viscosity in the ranee 450 - 618 or 14 - 192 stirring
number units;
or displavs no sienificant increase in viscosity during viscoamylograph. Peak,
pasting and
set-back viscosities are defined below. Viscosity onset temperature is the
temperature at
which there is a sudden, marked increase in viscosity from baseline levels
during
viscoamylograph, and is a term well-known to those skilled in the art.
In other particular embodiments. the invention provides starch which as
extracted from a
potato plant bv wet milling at ambient temperature has a peak viscositv in the
range 200 -
500 SNUs and a set-back viscositv in the range 275-618 SNUs as judaed bv
viscoamvlograph accordino to the protocol defined below: and starch which as
extracted
from a potato plant bv wet miliinQ at ambient temperature l:ias a viscositv
which does not
decrease between the start of the heating phase (step 2) and the start of the
final holding
CA 02416347 2003-02-11
phase (step 5) and has a set-back viscosity of 303 SNUs or less as judged by
viscoamvloQraph accordinsi to the protocoI defined below.
For the purposes of the present invention, viscoarnvlovraph conditions are
undcrstood to
pertain to analysis of a 10% (w/w) aqueous suspension of starch at atmospheric
pressure,
using a Newport Scientific Rapid Visco Analyser with a heating profile of:
holding at 50 C
for 2 minutes (step 1), heating from 50 to 95 C at a rate of 1.5 C per minute
(step 2),
holding at 95 C for 15 minutes (step 3),-coolina from 95 to 50 C at a rate of
1.5 C per
minute (step 4), and then holding at 50 C for 15 minutes (step 5). Peak
viscosiry may be
defined for present purposes as the maximum viscositv attained during the
heating phase
(step 2) or the holding phase (step 3) of the viscoamvioaraph. Pasting
viscosity may be
defined as the viscosity attained by the starch suspensions at the end of the
holding phase
(step 3) of the viscoamylograph. Set-back viscosiry may be defined as the
viscosity of the
starch suspension at the end of step 5 of the viscoamvloaraph.
In yet another aspect the invention provides starch from a potato plant having
an apparent
ainylose content (% w/w) of at least 35 %, as judged by iodometric assay
according to the
method described by Morrison & Laignelet (1983 J. Cereal Science 1, 9-20).
Preferably
the starch will have an amylose content of at least 40%, more preferably at
least 50%, and
most preferably at least 66 %. Starch obtained directly from a potato plant
and having
such properties has not hitherto been produced. Indeed, as a result of the
present
invention, it is now possible to generate in vivo potato starch which has some
properties
analogous to the very hiah amvlose starches (e.a. Hylon 7) obtainable from
maize.
Starches with hiRh (at least 35 %) arnvlose contents find commercial
application as,
amonast other reasons. the amylose component of starch reassociates more
strongly and
rapidlv than the amylopectin component during retroaradation processes. This
may result,
for example, in pastes with higher viscosities, aels of greater cohesion, or
films of areater
strensth for starches with high (at least 35%) compared with normal (less than
35%)
amvlose contents. Alternativelv, starches mav be obtained with verv hieh
amvlose
contents, such that the Qranule structure is substantially preserved during
heatins, resulting
in starch suspensions which demonstrate substantiallv no increase in viscosity
during
CA 02416347 2003-02-11
= ,~
cooking (i.e. there is no significant viscosity increase during
viscoamvlooraph conditions
defined above). Such starches typically exhibit a viscosity increase of less
than 10%
(preferably less than 5 %) during viscoamvloQraph under the conditiDns defined
above.
In commerce, these valuable properties are currently obtained from starches of
high
amylose content derived from maize plants. It would be of commercial value to
have an
altetnative source of high amylose starches from potato as other
characteristics such as
granule size, organoleptic properties and textural qualities may distinguish
application
performances of high amylose starches from maize and potato plants.
Thus high amylose starch obtained by the method of the present invention may
find
application in manv different technological fields. which may be broadly
categorised into
two groups: food products and processing; and "Industrial" applications. Under
the
heading of food products, the novel starches of the present invention may find
application
as, for example, films, barriers, coatings or gelling agents. In general, high
amylose
content starches absorb less fat during frying than starches with low amylose
content, thus
the high amylose content starches of the invention may be advantageously used
in
preparing low fat fried products (e.g. potato chips, crisps and the like). The
novel
starches may also be employed with advantage in preparing confectionery and in
granular
and retrograded "resistant" starches. "Resistant" starch is starch which is
resistant to
digestion by a-amylase. As such. resistant starch is not digested by a-
amylases present
in the human small intestine, but passes into the colon where it exhibits
properties similar
to soluble and insoluble dietarv fibre. Resistant starch is thus of great
benefit in foodstuffs
due to its low calorific value and its hiQh dietarv fibre content. Resistant
starch is formed
by the retrogradation (akin to recrvstallization) of amviose from starch gels.
Such
retrogradation is inhibited bv amvlopectin. Accordinalv, the high amvlose
starches of the
present invention are excellent starting materials for the preparation of
resistant starch.
Suitable methods for the preparation of resistant starch are well-known to
those skilled in
the art and include, for example, those described in US 5.051.1-71 and US
5.281.276.
Convenientiv the resistant starches provided by the present invention comprise
at least 5 %
total dietarv fibre, as judged bv the method of Prosky et al.. (1985 J. Assoc.
Off. Anal.
Chem. 68. 677), mentioned in US 5.281. 276.
CA 02416347 2003-02-11
. # . .
~ ~ -
Under the headina of "Industrial" appiications. the novel starches of the
invention mav be
advantageouslv emploved, for example. in corrugating adhesives. in
biodeQradable
products such as loose fill packaaina and foamed shapes, and in the production
of glass
fibers and textiles.
Those skilled in the art will appreciate that the novel starches of the -
invention may, if
desired, be subjected in vitro to conventional enzvmatic, physical andlor
chemical
modificatiori, such as cross-linking, introduction of hydrophobic aroups (e.g.
octenyl
succinic acid, dodecyl succinic acid), or derivatization (e.g. by n:eans of
esterification or
etherification).
In yet another aspect the invention provides high (35% or more) amylose
starches which
Qenerate paste viscosities areater than those obtained from hiQh amylose
starches from
maize plants after processing at temperatures below 100 C. This provides the
advantage
of more economical starch gelatinisation and pasting treat?nents through the
use of lower
processing temperatures than are currently required for high amylose starches
from maize
plants.
The invention will now be further described by way of illustrative example and
with
reference to the drawinas, of which:
Figure 1 shows a typical viscoamylograph for a 10% w/w suspension of potato
starch;
Figure 2 shows vsicoamvlographs for 10% suspensions of starch from various
maize
varieties;
Figure 3 is a schematic representation of the clonina strategy used by the
present
inventors;
Figure 4a shows the amino acid alignment of the C-tetminal portion of starch
branching
enzvme isoforms from various sources; amino acid residues matching the
consensus
CA 02416347 2003-02-11
sequence are shaded;
Fiaure 4b shows the ali2nment of DNA sequences of various starch branchina
enzvme
isoforms which encode a conserved amino acid sequence:
FiQure 5 shows the DNA sequence (Seq ID No. 14) and predicted amino acid
sequence
(Seq ID No. 15) of a full lenEith potato class A SBE cDNA clone obtained bv
PCR;
Figure 6 shows a comparison of the most hiahly conserved part of the amino
acid
sequences of potato class A (uppermost sequence) and class B(lowermost
sequence) SBE
molecules:
FiQure 7 shows a comparison of the amino acid sequence of the full length
potato class A
(uppermost sequence) and pea (lowermost sequence) class A SBE molecules:
FiQure 8 shows a DNA alignment of various full length potato class A SBE
clones
obtained by the inventors;
Figure 9 shows the DNA sequence of a potato class A SBE clone determined by
direct
sequencin; of PCR products. together with the predicted amino acid sequence;
Figure 10 is a multiple DNA alignment of various full length potato SBE A
clones
obtained by the inventors;
Figure 11 is a schematic illustration of the plasmid pSJ64;
Fia-ure 12 shows the DNA sequence and predicted amino acid sequence of the
full length
potato class A SBE clone as present in the plasmid pSJ90: and
Fiaure 13 shows viscoamvioaraphs for 10% w/w suspensions of starch from
various
transgenic potato plants made by the relevant method aspect of the invention.
CA 02416347 2005-10-26
,.=
17
Examples
Example 1
Cloning of Potato class A SBE
The stratesy for cloning the second form of starch branchin(2 enzyme from
potato is shown
in Figure 3. The small arrowheads represent primers used by the inventors in
PCR and
RACE protocols. The approximate size of the fragments isolated is indicated by
the
numeraIs on the riaht of the Figure. By way of explanation, a comparison of
the amino
acid sequences of several cloned plant starch branching enzymes (SBE) from
maize (class
A), pea (class A), maize (class B), rice (class B) and potato (class B), as
well as human
glvcoeen branching enzyme, allowed the inventors to identify a region in the
carboxy-terminal one third of the protein which is almost completely conserved
(GYLNFMGNEFGHPEWIDFPR) (Figure 4a). A multiple alignment of the DNA
sequences (human, pea class A, potato class B, maize class B, maize class A
and rice class
B, respectivelv) corresponding to this reoion is shown in Figure 4b and was
used to design
an oligo which would potentially hybridize to all known plant starch branching
enzymes:
AATTT(C/T)ATGGGIAA(C/T)GA(A/G)TT(C/T)GG (Seq ID No. 20).
Librarv PCR
The initial isolation of a partial potato class A SBE cDNA clone was from an
amplified
potato tuber cDNA librarv in the XZap vector (Stratagene). One half gL of a
potato
cDNA library (titre 2.3 x 109pfu/mL) was used as template in a 50 uL reaction
containing
100 pmol of a 16 fold degenerate POTSBE primer and 25 pmol of a T7 primer
(present
in the XZap vector 3' to the cDNA sequences - see Figure 3), 100 gM dNTPs, 2.5
U Taq
polymerase and the buffer supplied with the Taq polvmerase (StratageneTm). All
components
except the enzyme were added to a 0.5 mL microcentrifuze tube. covered with
mineral
oil and incubated at 94 C for 7 minutes and then held at C. while the Taq
polvmerase
was added and mixed bv pipettinz. PCR was then performed by incubatinQ for 1
min at
94 C. 1 min at 58 C and 3 minutes at 72 C. for 35 cycles. The PCR products
were
extracted with phenol/chloroform. ethanol precipitated and resuspended in TE
pH 8.0
before clonino into the T/A cloninQ vector pT7BlueR (Invitrogen).
CA 02416347 2003-02-11
Several fraQments between 600 and 1300 bp were amplified. These were isolated
from
an agarose Qei and cloned into the pT7BlueR T-A cloning vector. Restriction
mappina of
24 randomlv selected clones showed that thev belonged to several different
Qroups (based
on size and presencerabsence of restriction sites). Initiallv four clones were
chosen for
sequencing. Of these four. two were found to correspond to the known potato
class B
SBE sequence, however the other two, althoush homologous, differed
significantiv and
were more similar to the pea class A SBE sequence, suggesting that thev
belonged to the
class A family of branching enzymes (Burton er al., 1995 The Plant Journal,
cited Pbove).
The latter two clones (- 800bp) were sequenced fully. They both contained at
the 5' end
the sequence correspondins to the degenerate oligonucleotide used in the PCR
anL' had a
predicted open reading frame of 192 amino acids. The deduced amino acid
sequence was
hiahly homologous to that of the pea class A SBE.
The - 800 bp PCR derived cDNA fraament (corresponding to nucleotides 2281 to
3076
of the psbe2 con.seq sequence shown in Figure 8) was used as a probe to screen
the potato
tuber cDNA library. From one hundred and eighty thousand plaques, seven
positives
were obtained in the primary screen. PCR analysis showed that five of these
clones were
smaller than the original 800 bp cDNA clone, so these were not analysed
further. The
two other clones (designated 3.2.1 and 3.1.1) were approximately 1200 and 1500
bp in
length respectiveltr. These were sequenced from their 5' ends and the combined
consensus
sequence aligned with the sequence from the PCR generated clones. The cDNA
clone
3.2.1 was excised from the phage vector and plasmid DNA was prepared and the
insert
fullv sequenced. Several attempts to obtain longer clones from the library
were
unsuccessful, therefore clones containing the 5' end of the full length gene
were obtained
usinQ RACE (rapid amplification of cDNA ends).
Rapid Amplification of cDNA ends (RACE) and PCR conditions
RA_C.E was performed essentiallv according to Frohman (1992 Amplifications 11-
15).
Two ,cQ of total R.hTA from mature potato tubers was heated to 65 C for 5 min
and quick
cooled on ice. The RNA was then reverse transcribed in a 20 L reaction for 1
hour at
37 C using BRL's M-hILV reverse transcriptase and buffer with 1 mM DTT, 1 mN1
dNTPs. I U/uL RNAsin (Promeaa) and 500 pmol random hexamers (Pharmacia) as
CA 02416347 2003-02-11
primer. Excess primers were removed on a Centricon 100TM_'oiun-in and cDNA was
recovered and precipitated with isopropanol. cDNA was A-tailed in a volume of
20 1
usina 10 units terminal transferase (BRL), 200 ,yl dATP for 10 min at 37 C.
followed
by 5 min at 65 C. The reaction was then diluted to 0.5 ml with TE pH 8 and
stored at
4 C as the cDNA pool. cDNA clones were isolated bv PCR amplification using the
primers RR,dTj7, R and POTSBE24. The PCR was performed in 50 L using a hot
start
technique: 10 L of the cDNA pool was heated to 94 C in water for 5 min with
25 pmol
POTSBE24, 25 pmol R, and 2.5 pmol of R R,dTt, and cooled to 75 C. Five L of
10
x PCR buffer (Stratagene), 200 M dNTPs and 1.25 units of Taq polymerase were
added,
the mixture heated at 45 C for 2 min and 72 C for 40 min follow--d by 35
cycles of 94 C
for 45 sec, 50 C for 25 sec. 72 C for I.5 min and a final incubation at 72 C
for 10 min.
PCR products were separated by electrophoresis on I% low melting aoarose gels
and the
smear covering the ranae 600-800 bp fragments was excised and used in a second
PCR
amplification with 25 pmol of R, and POTSBE25 primers in a 50 uL reaction (28
cycles
of 94 C for 1 min. 50 C 1 min, 72 C 2 min). Products were purified by
chloroform
extraction and cloned into pT7 Blue. PCR was used to screen the colonies and
the longest
clones were sequenced.
The first round of RACE only extended the lenitth of the SBE sequence
approximately 100
bases, therefore a new A-tailed cDNA library was constructed using the class A
SBE
specific oliao POTSBE24 (10 pmol) in an attempt to recover longer RACE
products. The
first and second round PCR reactions were performed using new class A SBE
primers
(POTSBE 28 and 29 respectively) derived from the new sequence data. Conditions
were
as before except that the eloneation step in the first PCR was for 3 min and
the second
PCR consisted of 28 cycles at 94 C for 45 seconds, 55 C for 25 sec and 72 C
for 1
min 45 sec.
Clones raneine in size from 400 bp to 1.4 kb were isolated and sequenced. The
combined
sequence of the longest RACE products and cDNA clones predicted a full length
aene of
about 3150 nucleotides. excludina the polv(A) tail (psbe ?con.seq in Fig. 8).
As the sequence of the 5' half of the Qene was compiled from the sequence of
severai
CA 02416347 2003-02-11
2n
'v
RACE products senerated usinm Taq polvmerase. it was possible that the
compiled
sequence did not represent that of a single mRNA species and/or had nucleotide
sequence
chanQes. The 5' 1600 bases of the 2ene was therefore re-isolated by PCR using
liltrna,
a thermostable DNA polvmerase which. because it possesses a 3*-.5' exonuclease
activitv,
has a lower error rate compared to Taq polymerase. Several PCR products were
cloned
and restriction mapped and found to differ in the number of Hind III. Ssp 1,
and EcoR I
sites. These differences do not represent PCR artefacts as thev were observed
in clones
obtained from independent PCR reactions (data not shown) and indicate that
there are
several forms of the class A SBE aene transcribed in potato tubers.
In order to ensure that the sequence of the full length cDNA clone was derived
from a
sinale mRNA species it was therefore necessarv to PCR the entire Qene in one
piece.
cDNA was prepared accordiniy to the RACE protocol except that the adaptor
oligo
R RIdTõ (5 pmol) was used as a primer and after synthesis the reaction was
diluted to 200
AL with TE pH 8 and stored at 4 C. Two L of the cDNA was used in a PCR
reaction
of 50 l.cL using 25 pmol of class A SBE specific primers PBERI and PBERT (see
below),
and thirty cycles of 94 for 1 min, 60 C for 1 min and 72 C for 3 min. If Taq
polymerase was used the PCR products were cloned into pT7Blue whereas if Ultma
polymerase was used the PCR products were purified bv chloroform extraction,
ethanol
precipitation and kinased in a volume of 20 L (and then cloned into pBSSK IIP
which
had been cut with EcoRV and dephosphorylated). At least four classes of cDNA
were
isolated, which aQain differed in the presence or absence of Hind III. Ssp I
and EcoR I
sites. Three of these clones were sequenced fully, however one clone could not
be
isolated in sufficient quantiry to sequence.
The sequence of one of the clones (number 19) is shown in FiQure 5. The first
methionine
(initiation) codon starts a short open readine frame (ORF) of 7 amino acids
which is out
of frame with the next predicted ORF of 882 amino acids which has a molecular
mass
(Mr) of approximatelv 100 Kd. Nucleotides 6-2996 correspond to SBE sequence -
the rest
of the sequence shown is vector derived. Fizure 6 shows a comparison of the
most hi2h1y
conserved part cf the amino acid sequence of potato class A SBE (residues 180-
871, top,
row) and potato class B SBE (bottom row, residues 98-792): the middle row
indicates the
CA 02416347 2003-02-11
2 l
de2ree of similarity, identical re~idues being denoted by the common letter.
conservative
changes by two dots and neutral changes bv a sir.ale dot. Dashes indicate gaps
introduced
to optimise the aliQnment. The class A SBE protein has 44% identity over the
entire
lenzth with potato class B SBE. and 56% identity therewith in the centra! --
onserved
domain (Fiaure 6), as judQed bv the ":vleLyaliEn" proaram (DNASTAR). However.
Fiaure
7 shows a comparison between potato class A SBE (top row, residues 1-873) and
pea class
A SBE (bottom row, residues 1-861). from which it can be observed that cloned
potato
gene is more homologous to the class A pea enzyrne, where the identity is 70 %
over
nearlv the entire length, and this increases to 83 % over the central
conserved region
(starting at IPPP at position -170). It is clear from this analysis that this
cloned potato
SBE gene belongs to the class A family of SBE genes.
An E. coli culture, containinQ the piasmid pSJ78 (which directs the expression
of a full
length potato SBE Class A gene), has been deposited (on 3rd January 1996)
under the
terms of the Budapest Treaty at The National Collections of Industrial and
Marine Bacteria
Limited (23 St Machar Drive, Aberdeen, AB2 IRY, United Kingdom), under
accession
number NCIMB 40781. Plasmid pSJ78 is equivalent to clone 19 described above.
It
represents a full length SBE A cDNA blunt-end ligated into the vector
pBSSK.IIP.
Polymorphism of class A SBE genes
Sequence analysis of the other two full length class A SBE genes showed that
they contain
frameshift mutations and are therefore unable to encode full length proteins
and indeed
they were unable to complement the branching enzyme deficiency in the KV832
mutant
(described below). An alignment of the full lenath DNA sequences is shown in
Fieure
8: "lOcon.seq" (Seq ID No. 12), "19con.seq" (Seq ID No. 14) and "llcon.seq"
(Seq ID
No. 13) represent the sequence of full lenath clones 10. 19 and 11 obtained by
PCR using
the PBER1 and PBERT primers (see below). whilst "psbe2con.seq" (Seq ID No. 18)
represents the consensus sequence of the RACE clones and cDNA clone 3.2.1.
Those
nucleotides which differ from the overall consensus sequence (not shown) are
shaded.
Dashes indicate gaps introduced to optimise the alignment. Apart from the
frameshift
mutations these ciones are hiahIv homologous. It should be noted that the 5'
sequence of
psbe2con is longer because this is the lonaest RACE product and it also
contains several
CA 02416347 2003-02-11
G ~.
chan(zes compared tD the other clones. The upstream methionine codon is still
present in
this clone but the upstream ORF is shortened to just 3 amino acids and in
addition there
is a 10 base deletion in the 5' untranslated leader.
The other siL7nificant area of variation is in the carboxy terminal re!yzion
of the protein
codinLy region. Closer examination of this area reveals a GAA trinucleotide
repeat
structure which varies in lenath between the four clones. These are typical
characteristics
of a microsatellite repeat region. The most diverLrent clone is #11 which has
only one
GAA triplet whereas clone 19 has eleven perfect repeats and the other two
clones have
five and seven GAA repeats. All of these deletions maintain the ORF but
char.ye the
number of glutamic acid residues at the carboxy terminus of the protein.
Most of the other differences between the clones are single base changes. It
is quite
possible that some of these are PCR errors. To address this question direct
sequencing
of PCR fragments amplified from first strand cDNA was performed. Figure 9
shows the
DNA sequence, and predicted amino acid sequence, obtained by such direct
sequencing.
Certain restriction sites are also marked. Nucleotides which could not be
unambiguously
assigned are indicated using standard IUPAC notation and, where this
uncertainty affects
the predicted amino acid sequence, a question mark is used. Sequence at the
extreme 5'
and 3' ends of the gene could not be determined because ot the heteroQeneity
observed in
the different cloned Qenes in these regions (see previous paragraph). However
this can
be taken as direct evidence that these differences are real and are not PCR or
cloning
artefacts.
There is absolutely no evidence for the frameshift mutations in the PCR
derived sequence
and it would appear that these mutations are an artefact of the cloning
process. resulting
from negative selection pressure in E. coli. This is supported by the fact
that it proved
extremely difficult to clone the full length PCR products intact as many large
deletions
were seen and the full lenath clones obtained were all cloned in one
orientation (away
from the LacZ promoter), perhaps suggesting that expression of the gene is
toxic to the
cells. Difficulties of this nature may have been responsible. at least in pan.
for the
previous failure of other researchers to obtain the present invention.
CA 02416347 2003-02-11
A comparison of all the full lenoth sequences is shown in Fiaure 10. In
addition to clones
10. 11 and 19 are shown the sequences of a Bgl II - Xho I product cloned
directly into the
QE32 expression vector ("86CON.SEQ", Seq ID 'lo. 16) and the consensus
sequence of
the directly sequenced PCR products ("pcrsbe2con.seq". Seq ID No. l;). Those
nucleotides which differ from the consensus sequence (not shown) are shaded,
Dashes
indicate gaps introduced to optimise the alignment. There are 11 nucleotide
differences
predicted to be present in the mRNA population. which are indicated by
asterisks above
and below the sequence The other differences are probably PCR artefacts or
possihlv
sequencincy errors.
Complementation of a branching enzvme deficient E. coli mutant
To determine if the isolated SBE 4ene encodes an active protein i.e. one that
has
branching enzvme activitv, a complementation test was performed in the E. coli
strain
KV832. This strain is unable to make bacterial alvcoaen as the Qene for the
glycoeen
branching enzyme has been deleted (Keil et al., 1987 Mol. Gen. Genet. 207, 294-
301).
When wild type cells are grown in the presence of glucose they svnthesise
glyco;en (a
highlv branched glucose polvmer) which stains a brown colour with iodine,
whereas the
KV832 cells make only a linear chain alueose polymer which stains blueish
green with
iodine. To determine if the cloned SBE aene could restore the ability of the
KV832 cells
to make a branched polymer, the clone pSJ90 (Seq ID No. 19) was used and
constructed
as below. The construct is a PCR-derived. substantiallv full length fragment
(made using
primers PBE 2B and PBE 2X. detailed below), which was cut with Bgl II and K'ho
I and
cloned into the BamH I Sal I sites of the His-tag expression vector pQE32
(Qiagen).
This clone, pSJ86. was sequenced and found to have a frameshift mutation of
two bases
in the 5' half of the Qene. This frameshift was removed by digestion with Nsi
I and SnaB
I and replaced with the correspondinQ fra8ment from a Taq-generated PCR clone
to
produce the plasmid pSJ90 (sequence shown in Fi-aure 12; the first 10 amino
acids are
derived from the expression vector). The polypeptide encoded by pSJ90 would be
predicted to correspond to amino acids 46-882 of the full SBE codinsz
sequence. The
construct pSJ90 was transformed into the branchinQ enzyme deficient KV832
cells and
transformants were arowr, on solid PYG medium (0.85 ','o KH_PO4, 1.19o K,HPO;,
0.6 ','o
veast extract) containinQ 1.0% alucose. To test for compiementation. a loop of
cells was
CA 02416347 2005-10-26
2G
scraped off and resuspended in 150 l of water. to which was added 15kcl
Lugol's solution
(2Q KI and 19 I, per 300m1 water). It was found that the potato SBE fraszment-
transformed KV832 cells now stained a yellow-brown colour with iodine whereas
control
cells containina only the pQE32 vector continued to stain blue-green.
Expression of potato class A SBE in E. coli
Sinsle colonies of KV832. containins! one of the plasmids pQE32. pAGCRl or
pSJ90,
were picked into 50m1 of 2xYT medium contairunz carbenicillin, kanamycin and
streptomycin as appropriate (100, 50 and 25 mg/L. respectively) in a 250m1
flask and
grown for 5 hours, with shaking., at 37 C. IPTG was then added to a final
concentration
of ImM to induce expression and the flasks were further incubated overnight at
25 C.
The cells were harvested bvi centrifusation and resuspended in 50 mM sodium
phosphate
buffer (pH 8.0), containing 300mM NaCI, Ima/ml lysozvme and ImM PMSF and left
on
ice for 1 hour. The cell lysates were then sonicated (3 pulses of 10 seconds
at 40% power
using a microprobe) and cleared bvi centrifuzation at 12,000Q for 10 minutes
at 4 C.
Cleared lysates were concentrated approximately 10 fold in a CentriconT'' 30
filtration
unit. Duplicate 10 1 samples of the resultin; extract were assayed for SBE
activity by the
phosphorylation stimulation method, as described in International Patent
Application No.
WO 95/26407. In brief, the standard assay reaction mixture (0.2m1) was 200mM 2-
(N-morpholino) ethanesulphonic acid (MES) buffer pH6.5, containing 100nCi of
14C
alucose-l-phosphate at 50mM. 0.05 ms rabbit phosphorylase A. and E. coli
lysate. The
reaction mixture was incubated for 60 minutes at 30 C and the reaction
terminated and
szlucan polymer precipitated by the addition of lml of 75 % (v/v) methanol,
1%(w/v)
potassium hvdroxide, and then 0. lml Qlycogen f 10mg/ml). The results are
presented
below:
Construct + SBE Activitv (cpm)
pQE32 (control) 1,829
pSJ90 (potato class A SBE) 14,327
pAGCRI (pea class A SBE) 29,707
The potato class A SBE activity is 7-8 fold above background levels. It was
concluded
therefore that the potato class A SBE aene was able to complement the BE
mutation in the
CA 02416347 2003-02-11
phosphorylation stimulation assav and that the cloned aene does indeed code
for a protein
with branching enzyme activitv.
Oligonucleotides
The following synthetic oligonucleocides (Seq ID No.s 1-I1 respectively) were
used:
RaR1dT17 AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGA(T)17
Ro AAGGATCCGTCGACATC
RI GACATCGATAATACGAC
POTSBE24 CATCCAACCACCATCTCGCA
POTSBE25 TTGAGAGAAGATACCTAAGT
POTSBE28 ATGTTCAGTCCATCT.aAAGT
POTSBE29 AGAACAACAATTCCTAGCTC
PBER 1 GGGGCCTTGAACTCAGCAAT
PBERT CGTCCCAGCATTCGACATAA
PBE 2B CTTGGATCCTTGAACTCAGCAATTTG
PBE2X TAACTCGAGCAACGCGATCACAAGTTCGT
Exampie 2
Production of Transgenic Plants
Construction of plant transformation vectors with antisense starch branching
enzyme
genes
A 1200 bp Sac I - Xho I fraament, encoding approximately the -COOTti half of
the potato
class A SBE (isolated from the rescued XZap clone 3.2.1), was cloned into the
Sac I- Sal
I sites of the plant transformation vector pSJ29 to create plasmid pSJ64,
which is
illustrated schematicallv in Figure 11. In the fiQttre, the black line
represents the DNA
sequence. The broken line represents the bacterial plasmid backbone
(containina the
oriQin of replication and bacterial selection marker), which is not shown in,
full. The filled
trianQles on the line denote the T-DNA borders (RB = right border, LB = left
border).
Relevant restriction sites are shown above the black line, with the
approximate distances
(in kilobases) between the sites (marked bv an asterisk) -aiven bv the
numerals below the
CA 02416347 2003-02-11
line. The thinnest arrows indicate polvadenvlation signals (pAnos = nopaline
synthase,
pAg7 = Aarobacterium Lyene 7), the arrows intermediate in thickness denote
protein
coding reaions (SBE II = potato class A SBE. HYG = hvaromvcin resist.ance
aene) and
the thickest arrows represent promoter regions (P-?x35 = double Ca.VIV 35S
promoter,
Pnos = nopaline svnthase promoter). Thus pSJ64 contained the class A SBE aene
fra2rnent in an antisense orientation between the 2X 35S CaMV promoter and the
nopaline
svnthase polyadenylation siLynal.
For information, pSJ29 is a derivative of the binary vector pGPTV-HYG (Becker
et 31.,
1992 Plant Molecular Biolozy 20, 1195-1197) modified as follows: an
approximately 750
bp (Sac I, T4 DNA polvmerase blunted - Sal I) fragment of pJIT60 (Guerineau et
al.,
1992 Plant Mol. Biol. 18, 815-818) containing the duplicated cauliflower
mosaic virus
(CaMV) 35S promoter (Cabb-JI strain, equivalent to nucleotides 7040 to 7376
duplicated
upstream of 7040 to 7433 . Frank et al., 1980 Cell 21, 285-294) was cloned
into the Hind
III (Klenow polvmerase repaired) - Sal I sites of pGPTV-HYG to create pSJ29.
Plant transformation
Transformation was conducted on two types of potato plant explants; either
wild type
untransformed minitubers (in order to give single transformants containing the
class A
antisense construct alone) or minitubers from three tissue culture lines
(which gave rise
to plants #12, #15, #17 and #18 indicated in Table 1) which had already been
successfully
transformed with the class B (SBE I) antisensz construct containinQ the tandem
35S
promoter (so as to obtain double transformant plants, containing antisense
sequences for
both the class A and class B enzymes).
Details of the method of Aarobacterium transformation. and of the Qrowth of
transformed
plants. are described in International Patent Application No. WO 95/26407,
except that
the medium used contained 3% sucrose (not 1%) until the final transfer and
that the initial
incubation with Aarobacterium (strain 3850) was performed in darkness.
Transformants
containinQ the class A antisense sequence were selected bv arowth in medium
containina
I5maiL hvaromvcin (the class A antisense construct comprisinQ the HYG gene,
i.e.
hvaromvcin phosphotransferase) .
CA 02416347 2005-10-26
Transformation was confirmed in all cases by production of a DNA fragment from
the
antisense sene after PCR in the presence of appropriate primers and a crude
extract of
aenomic DNA from each regenerated shoot.
Characterisation of starch from potato plants
Starch was extracted from plarits as follows: potato tubers were homoaenised
in water for
2 minutes in a WaringTM blender operating at high speed. The homogenate was
washed and
filtered (initiallv throucrr 2mm, then through lmm filters) using about 4
litres of water per,
llOOgms of tubers (6 eYtractions). Washed starch (zranules were finallv
extracted with
acetone and air dried.
Starch extracted from singly transformed potato plants (class A/SBE II
antisense. or class
B/SBE I antisense), or from double transformants (class A/SBE II and class
B/SBE I
antisense), or from untransformed control plants, was partially characterised.
The results
are shown in Table 1. The table shows the amount of SBE activity (units/gram
tissue) in
tubers from each transformed plant. The endotherm peak temperature ( C) of
starch
extracted from several plants was determined by DSC, and the onset temperature
( C) of
pastin; was determined by reference to a viscoamylograph ("RVA"), as described
in WO
95/26407. The viscoamylograph profile was as follows: step 1- 50 C for 2
minutes; step
2 - increase in temperature from 50 C to 95 C at a rate of 1.5 C per minute;
step 3 -
holding at 95 C for 15 minutes: step 4 - cooling from 95 C to 50 C at a rate
of 1.5 C per
minute; and finallv, step 5 - holding at 50 C for 15 minutes. Table 1 shows
the peak,
pasting and set-back viscosities in stirrinc, number units (SNUs), which is a
measure of
the amount of torque required to stir the suspensions. Peak viscosity may be
defined for
present purposes as the maximun viscositv attained durins the heating phase
(step 2)= or
the holding phase (step 3) of the viscoamylograph. Pasting viscosity may be
defined as
the viscosity attained by the starch suspensions at the end of the holding
phase (step 3) of
the viscoamylograph. Set-back viscosity may be defined as the viscosity of the
starch
suspension at the end of step 5 of the viscoamylograph.
A determination of apparent amylose content (7c w/w) was also performed. using
the
iodometric assav method of Morrison & Laipnelet (1983 J. Cereal Sci. 1, 9-20).
The
CA 02416347 2003-02-11
28 -
results (percentaae apparent amylose) are shown in Table 1. The untransformed
and
transformed control plants gave rise to starches having apparent amvlose
contents in the
ranQe 29(+/-3)%.
Generally similar values for amylose content were obtained for starch
extracted from most
of the sin(Ylv transformed plants containina the class A (SBE II) antisense
sequence.
However, some plants (#152, 249) Qave rise to starch having an apparent
amylose content
of 37-38%, notably higher than the control value. Starch extracted from these
plants had
markedly elevated pastini, onset temperatures, and starch from plant 152 also
exhibiteL- an
elevated endotherm peak temperature (starch from plant 249 was not tested bv
DSC).
CA 02416347 2003-02-11
d~
~ ~ t N ~p N ~Np m ~ O OE~ o m o~ ~ ~D O N o a w H
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CA 02416347 2003-02-11
= 29/1
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CA 02416347 2003-02-11
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CA 02416347 2003-02-11
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CA 02416347 2003-02-11
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CA 02416347 2005-10-26
ry It should be noted that. even if other single transfotmants were not to
provide starch with
an altered amvloseiamvlopectin ratio. the starch from such plants might still
have different
properties relative to starch from conventional plants (e. a. different
average molecular
weiQht or different amvlopectin branchin(z patterns). which misht be useful.
Double transforrnant plants. containing antisense sequences for both the class
A and class
B enzymes, had greatly reduced SBE activity (units/gm) compared to
untransformed plants
or single anti-sense class A transformants. (as shown in Table 1). Moreover,
certain of
the double transformant plants contained starch having very significantly
a'tered
properties. For exa.mple, starch extracted from plants #201, 202. 208, 208a,
2'_~6 and
236a had drastically altered amylose/amylopectin ratios, to the extent that
amylose was the
main constituent of starch from these plants. The pasting onset temperatures
of starch
from these plants were also the most areatly increased (by about 25-30 C).
Starch from
plants such as #150, 161, 212. 220 and 230a represented a range of
intermediates, in that
such starch displayed a more modest rise in both amvlose content and pasting
onset
temperature. The results would tend to suggest that there is generally a
correlation
between % amylose content and pasting onset temperature, which is in agreement
with the
known behaviour of starches from other sources, notably maize.
The marked increase in amylose content obtained by inhibition of class A SBE
alone,
compared to inhibition of class B SBE alone (see WO 95/26407) might suggest
that
it would be advantageous to transform plants first with a construct to
suppress class A SBE
expression (probably, in practice, an antisense construct), select those
plants giving rise
to starch with the most altered properties, and then to re-transform with a
construct to
suppress class B SBE expression (again. in practice. probably an antisense
construct), so
as to maximise the deQree of starch modification.
In addition to pastina onset temperatures. other features of the
viscoamylograph profile
e.g. for starches from plants #149, 150, 152. 161, 201. 236 and 236a showed
sisnificant
differences to starches from control plants. as illustrated in Figure 13-3.
Referring to Fiaure
13. a number of viscoamylograph traces are shown. The legend is as follows:
shaded box
- normal potato starch control (29.8 % amylose content): shaded circle -
starch from plant
CA 02416347 2003-02-11
149 (35.6% amv?ose)-. shaded triangle, pointina upwards - plant 152 (37.5%):
shaded
trianzle, pointina downwards - plant 161 (40.9 shaded diamond - plant 150
(53.1 %);
unshaded box - plant 236a (56.7 7,c); unshaded circle - piant 236 t,60.1 ',C);
unshaded
trianQle, pointing upwards - plant 201 (66.4~); unshaded trianzle. pointing
downwards -
Hvlon V starch. from maize (44.9 % amylose). The thin line denotes the heatina
profile.
With increasing amylose content. peak viscosities during processing to 95 C
decrease, and
the drop in viscosity from the peak until the end of the holding period at 95
C also
generally decreases (indeed. for some of the starch samples there is an
increase in
viscosity during this period). Both of these results are indicative of reduced
granule
fraQmentation. and hence increased granule stabilitv during pasting. This
property has not
previously been available in potato starch without extensive prior chemical or
physical
modification. For applications where a maximal viscosity after processinQ to
95 C is
desirable (i.e. correspondina to the viscosity after 47 minutes in the
viscoamylograph test),
starch from piant #152 would be selected as starches with both lower
(Controls, #149) and
higher (#161, #150) amylose contents have lower viscosities following this
gelatinisation
and pasting regime (Figure 13 and Table 1). It is believed that the viscosity
at this stage
is determined by a combination of the extent of granule swelling and the
resistance of
swollen granules to mechanical fragmentation. For any desired viscosity
behaviour, one
skilled in the art would select a potato starch from a range containing
different amylose
contents produced according to the invention by performing suitable standard
viscosiry
tests.
Upon cooling pastes from 95 C to 50 C, potato starches from most plants
transformed
in accordance with the invention showed an increase in viscoamvlograph
viscosity as
expected for partial reassociation of amviose. Starches from plants #149, 152
and 161 all
show viscosities at 50 C significantlv in excess of those for starches from
control plants
(Fiaure 13 and Table 1). This contrasts with the effect of elevated amvlose
contents in
starches from maize plants (Figure 2) which show verv low viscosities
throuQhout the
viscoamvloQraph test. Of particular note is the fact that, for similar amvlose
contents,
starch from potato plant 150 (53 % amvlose) shows markedly increased viscosity
compared
with Hvlon 5 starch (44.9% amvlose) as illustrated in Figur.e 13. This
demonstrates that
CA 02416347 2003-02-11
useful properties which require elevated (35~ or ~reater) amviose ievels can
be obtained
bv processing starches from potato plants below 100 C. whereas more enerL7v-
intensive
processing is required in order to Qenerate similarly useful properties rrom
hiLyh amylose
starches derived from maize plants.
Final viscositv in the viscoamylograph test (set-back viscositv after 92
minutes) is Qreatest
for starch from plant #161 (40.9% amylose) amongst those tested (Figure 13 and
Table
1). Decreasing final viscosities are obtained for starches from plant #152
(37.5%
amylose), #149 (35.6% amylose) and #150 (53.1% amylose). Set-back viscositv
o~,curs
where amylose molecules, exuded from the starch granule durin7 pastinlg, start
r; re-
associate outside the 2ranule and form a viscous gel-like substance. It is
believed that the
set-back viscositv values of starches from transaenic potato plants represent
a balance
between the inherent amvlose content of the starches and the ability of the
amviose
fraction to be exuded from the aranule durinlg pasting and therefore be
available for the
reassociation process which results in viscosity increase. For starches with
low amylose
content, increasinz the amylose content tends to make more amylose available
for re-
association, thus increasin' the set-back viscosity. However, above a
threshold value,
increased amylose content is thought to inhibit granule swelling, thus
preventin-2 exudation
of amvlose from the starch Qranule and reducing the amount of amylose
available for re-
association. This is supported bv the RVA results obtained for the very high
amylose
content potato starches seen in the viscoamvlograph profiles in Figure 13. For
any
desired viscositv behaviour following set-back or retro2radation to any
desired temperature
over anv desired timescale, one skilled in the art would select a potato
starch from a ranae
containina different amylose contents produced according to the invention by
performing
standard viscositv tests.
Further experiments witll starch from plants #201 and 208 showed that this had
an
apparent amvlose content of over 6? r7c (see Table 1). Viscoamylogyraph
studies showed
that starch from these plants had radically altered properties and behaved in
a manner
similar to hvlon 5 starch from maize plants (FiQure 13). Under the conditions
emploved
in the viscoamvloaraph. this starch exhibited extremely limited (nearly
undetectable)
aranule swelling. Thus, for example, unlike starch from control plants, starch
from plants
CA 02416347 2003-02-11
.J 3
201. 208 and 208a did not displav a clearly dPfined pasting viscositv peak
during the
heatinp phase. Microscopic analvsis confirmed that the starch granule
structure underwent
only minor swelling during the experimental heatinQ process. This property may
well be
particularly useful in certain appiications, as will be apparent to those
skilled in the art.
Some re-arown plants have so far been found to increase still further the
apparent amylose
content of starch extracted therefrom. Such increases may be due to:-
i) Growth and development of the first aeneration transformed plants may have
been
affected to some degree by the exogenous growth hormones present in the tissue
culture
svstem, which exoaenoous hormones were not present durin- growth of the second
2eneration plants; and
ii) Subsequent aenerations were arown under field conditions, which may allow
for
attainment of greater maturity than growth under laboratory conditions, it
being generally
held that amylose content of potato starch increases with maturitti of the
potato tuber.
Accordingly, it should be possible to obtain potato plants Qiving rise to
tubers with starch
having an amvlose content in excess of the 66% level so far attained, simply
by analysing
a greater number of transformed plants andlor by re-growing transgenic plants
through one
or more generations under field conditions.
Table 1 shows that another characteristic of starch which is affected by the
presence of
anti-sense sequences to SBE is the phosphorus content. Starch from
untransformed control
plants had a phosphorus content of about 60-710mg/100aram drv weight (as
determined
according to the AOAC Official Methods of Analysis, 15th Edition, Method
948.09
"Phosphorus in Flour"). Introduction into the plant of an anti-sense SBE B
sequence was
found to cause a modest increase (about two-fold) in phosphorus content, which
is in
agreement with the previous findings reported at scientific meetings.
Similarly, anti-sense
to SBE A alone causes only a small rise in phosphorus content relative to
untransformed
controls. However, use of anti-sense to both SBE A and B in combination
results in up
to a four-fold increase in phosphorus content. which is far areater than any
in planta
phosphorus content previously demonstrated for potato starch.
This is useful in that, for certain applications. starch must be
phosphorylated in virro by
CA 02416347 2003-02-11
34 _
chemical modification. The ability to obtain potato starch which, as extracted
from the
plant, alreadv has a high phosphorus content will reduce the amount of in
vitro
phosphorylation required suitabiv to modify the starch. Thus, in another
aspect the
invention provides potato starch which. as extracted from the plant. has a
phosphorus
content in excess of 200mg/ 100Qram drv weiaht starch. Tvpicallv the starch
will have a
phosphorus content in the range 200 - 240ma/ I00gram dr,v weight starch.
CA 02416347 2003-02-11
-35-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
('t) APPLICANT:
(A) NAME: National Starch and Chemical (nvestment
Holding Corporation
(B) STREET: 501 Silverside Road, Suite 27
(C) CITY: Wilmington
(D) STATE: Delaware
(E) COUNTRY: United States of America
(F) POSTAL CODE (ZIP): 19809
(ii) TITLE OF iNVENTIQN: Improvements in or Relating to Plant Starch
Composition
(iii) NUMBER OF SEQUENCES: 20
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disK
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentfn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AAGGATCCGT CGACATCGAT AATACGACTC ACTATAGGGA Ti TTI-TTTTT TTTTTTT 57
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear '
CA 02416347 2003-02-11
-36-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
AAGGATCCGT CGACATC 17
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GACATCGATA ATACGAC 17
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CATCCAACCA CCATCTCGCA 20
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02416347 2003-02-11
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TTGAGAGAAG ATACCTAAGT 20
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ATGTTCAGTC CATCTAAAGT 20
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
AGAACAACAA TTCCTAGCTC 20
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02416347 2003-02-11
. "a , .
-38-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GGGGCCTTGA ACTCAGCAAT 20
(2) INFORMATÃC1'~', FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CGTCCCAGCA TTCGACATAA 20
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucieic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTÃOfj: SEQ ID NO: 10:
CTTGGATCCT TGAACTCAGC AATTTG 26
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02416347 2003-02-11
-39-
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
TAACTCGAGC AACGCGATCA CAAGTTCGT 29
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3003 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GATGGGGCCT TGAACTCAGC AATTTGACAC TCAGTTAGTT ACACTGCCAT CACTTATCAG
ATCTCTATTT TTTCTCTTAA TTCCAACCAA GGAATGAATA AAAAGATAGA TTTGTAAAAA
120
CCCTAAGGAG AGAAGAAGAA AGATGGTGTA TACACTCTCT GGAGTTCGTT TTCCTACTGT
180
TCCATCAGTG TACAAATCTA ATGGATTCAG CAGTAATGGT GATCGGAGGA ATGCTAATAT
240
TTCTGTATTC TTGAAAAAAC ACTCTCTTTC ACGGAAGATC TTGGCTGAAA AGTCTTCTTA
300
CAATTCCGAA TCCCGACCTT-CTACAATTGC AGCATCGGGG AAAGTCCTTG TGCCTGGAAT
360
CCAGAGTGAT AGCTCCTCAT CCTCAACAGA TCAATTTGAG TTCGCTGAGA CATCTCCAGA
420
AAATTCCCCA GCATCAACTG ATGTAGATAG TTCAACAATG GAACACGCTA GCCAGATTAA
480
AACTGAGAAC GATGACGTTG AGCCGTCAAG TGATCTTACA GGAAGTGTTG AAGAGCTGGA
540
TTTTGCTTCA TCACTACAAC TACAAGAAGG TGGTAAACTC GAGGAGTCTA AAACATTAAA
600
CA 02416347 2003-02-11
-40-
TACTTCTGAA GAGACAATTA TTGATGAATC TGATAGGATC AGAGAGAGGG GCATCCCTCC
660
ACCTGGACTT GGTCAGAAGA TTTATGAAAT AGACCCCCTT TTGACAAACT ATCGTCAACA
720
CCTTGATTAC AGGTATTCAC AGTACAAGAA ACTGAGGGAG GCAATTGACA AGTATGAGGG
780
TGGTTTGGAA GCTTTTTCTC GTGGTTATGA AAGAATGGGT TTCACTCGTA GTGCTACAGG
840
TATCACTTAC CGTGAGTGGG CTCCTGGTGC CCAGTCAGCT GCCCTCATTG GGGATTTCAA
900
CAATTGGGAC GCAAATGCTG ACTTTATGAC TCGGAATGAA TTTGGTGTCT GAGAGATTTT
960
TCTGCCAAAT AATGTGGATG GTTCTCCTGC AATTCCTCAT GGGTCCAGAG TGAAGATACG
1020
TATGGACACT CCATCAGGTG TTAAGGATTC CATTCCTGCT TGGATCAACT ACTCTTTACA
1080
GCTTCCTGAT GAAATTCCAT ATAATGGAAT ATATTATGAT CCACCCGAAG-AGGAGAGGTA
1140
TATCTTCCAA CACCCACGGC CAAAGAAACC AAAGTCGGTG AGAATATATG AATCTCATAT
1200
TGGAATGAGT AGTCCGGAGC CTAAAATTAA CTCATACGTG AATTTTAGAG ATGAAGTTCT
1260
TCCTCGCATA AAAAAAGCTT GGGTACAATG CGGTGCAAAT TATGGCTATT CAAGAGCATT
1320
CTTATTATGC TAGTTTTGGT TATCATGTCA CAAATTTTTT TGCACCAAGC AGCCGTTTTG 1380
GAACGCCCGA CGACCTTAAG TCTTTGATTG ATAAAGCTCA TGAGCTAGGA ATTGTTGTTC
1440
TCATGGACAT TGTTCACAGC CATGCATCAA ATAATACTTT AGATGGACTG AACATGTTTG
1500
ACGGCACAGA TAGTTGTTAC TTTCACTCTG GAGCTCGTGG TTATCATTGG ATGTGGGATT
1560
CA 02416347 2003-02-11
-4I -
TCCGCCTCTT TAACTATGGA AACTGGGAGG TACTTAGGTA TCTTCTCTCA AATGCGAGAT
1620
GGTGGTTGGA TGAGTTCAAA TTTGATGGAT TTAGATTTGA TGGTGTGACA TCAATGATGT
1680
GTACTCACCA CGGATTATCG GTGGGATTCA CTGGGAACTA CGAGGAATAC TTTGGACTCG
1740
CAACTGATGT GGATGCTGTT GTGTATCTGA TGCTGGTCAA CGATCTTATT CATGGGCTTT
1800
TCCCAGATGC AATTACCATT GGTGAAGATG TTAGCGGAAT GCCGACATTT TGTGTTCCCG
1860
TTCAAGATGG GGGTGTTGGC TTTGACTATC GGCTGCATAT GGCAATTGCT GATAAATGGA
1920
TTGAGTTGCT CAAGAAACGG GATGAGGATT GGAGAGTGGG TGATATTGTT CATACACTGA
1980
CAAATAGAAG ATGGTCGGAA AAGTGTGTTT CATACGCTGA AAGTCATGAT CAAGCTCTAG
2040
TCGGTGATAA AACTATAGCA TTCTGGCTGA TGGACAAGGA TATGTATGAT TTTATGGCTC
2100 -
TGGATAGACC GTCAACATCA TTAATAGATC GTGGGATAGC ATTACACAAG ATGATTAGGC
2160
TTGTAACTAT GGGATTAGGA GGAGAAGGGT ACCTAAATTT CATGGGAAAT GAATTCGGCC
2220
ACCCTGAGTG GATTGATTTC CCTAGGGCTG AACAACACCT CTCTGATGGC TCAGTAATTC
2280
CCAGAAACCA ATTCAGTTAT GATAAATGCA GACGGAGATT TGACCTGGGA GATGCAGAAT
2340
ATTTAAGATA CCGTGGGTTG CAAGAATTTG ACCGGGCTAT GCAGTATCTT GAAGATAAAT
2400
ATGAGTTTAT GACTTCAGAA CACCAGTTCA TATCACGAAA GGATGAAGGA GATAGGATGA
2460
TTGTATTTGA AAAAGGAAAC CTAGTTTTTG TCTTTAATTT TCACTGGACA AAAGGCTATT
2520
CA 02416347 2003-02-11
, ~ a Il
-42-
CAGACTATCG CATAGGCTGC CTGAAGCCTG GAAAATACAA GGTTGCCTTG GACTCAGATG
2580
ATCCACTTTT TGGTGGCTTC GGGAGAATTG ATCATAATGC CGAATATTTC ACCTTTGAAG
2640
GATGGTATGA TGATCGTCCT CGTTCAATTA TGGTGTATGC ACCTAGTAGA ACAGCAGTGG
2700
TCTATGCACT AGTAGACAAA GAAGAAGAAG AAGAAGAAGA AGTAGCAGTA GTAGAAGAAG
2760
TAGTAGTAGA AGAAGAATGA ACGAACTTGT GATCGCGTTG AAAGATTTGA ACGCCACATA
2820
GAGCTTCTTG ACGTATCTGG CAATATTGCA TTAGTCTTGG CGGAATTTCA TGTGACAACA
2880
GGTTTGCAAT TCTTTCCACT ATTAGTAGTG CAACGATATA CGCAGAGATG AAGTGCTGAA
2940
CAAAAACATA TGTAAAATCG ATGAATTTAT GTCGAATGCT GGGACGATCG AATTCCTGCA
3000
GCC 3003
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2975 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTiON: SEQ ID NO: 13:
TTGATGGGCC TTGAACTCAG CAATTTGACA CTCAGTTAGT TACACTCCTA TCACTTATCA 60
GATCTCTATT TTTTCTCTTA ATTCCAACCA GGGGAATGAA TAAAAGGATA GATTTGTAAA
120
AACCCTAAGG AGAGAAGAAG AAAGATGGTG TATATACTCT CTGGAGTTCG TTTTCCTACT
180
CA 02416347 2003-02-11
-43-
GTTCCATCAG TGTACAAATC TAATGGATTC AGCAGTAATG GTGATCGGAG GAATGCTAAT
240
GTTTCTGTAT TCTTGAAAAA GCACTCTCTT TCACGGAAGA TCTTGGCTGA AAAGTCTTCT
300
TACAATTCCG AATTCCGACC TTCTACAGTT GCAGCATCGG GGAAAGTCCT TGTGCCTGGA
360
ACCCAGAGTG ATAGCTCCTC ATCCTCAACA GACCAATTTG AGTTCACTGA GACATCTCCA
420
GAAAATTCCC CAGCATCAAC TGATGTAGAT AGTi'CAACAA TGGAACACGC TAGCCAGATT
480
AAAACTGAGA ACGATGACGT TGAGCCGTCA AGTGATCTTA CAGGAAGTGT TGAAGAGCTG
540
GATTTTGCTT CATCACTACA ACTACAAGAA GGTGGTAAAC TGGAGGAGTC TAAAACATTA
600
AATACTTCTG AAGAGACAAT TATTGATGAA TCTGATAGGA TCAGAGAGAG GGGCATCCCT
660
CCACCTGGAC TTGGTCAGAA GATTTATGAA ATAGACCCCC TTTTGACAAA CTATCGTCAA
720
CACCTTGATT ACAGGTATTC ACAGTACAAG AAACTGAGGG AGGCAATTGA CAAGTATGAG
780
GGTGGTTTGG AAGCTTTTCT CGTGGTTATG AAAAAATGGG TTTCACTCGT AGTGCTACAG
840
GTATCACTTA CCGTGAGTGG GCTCCTGGTG CCCAGTCAGC TGCCCTCATT GGAGATTTCA
900
ACAATTGGGA CGCAAATGCT GACATTATGA CTCGGAATGA ATTTGGTGTC TGGGAGATTT
960
TTCTGCCAAA TAATGTGGAT GGTTCTCCTG CAATTCCTCA TGGGTCCAGA GTGAAGATAC
1020
GTATGGACAC TCCATCAGGT GTTAAGGATT CCATTCCTGC TTGGATCAAC TACTCTTTAC
1080
AGCTTCCTGA TGAAATTCCA TATAATGGAA TATATTATGA TCCACCCGAA GAGGAGAGGT
1140
~=.
CA 02416347 2003-02-11
-44-
ATATCTTCCA ACACCCACGG CCAAAGAAAC CAAAGTCGCT GAGAATATAT GAATCTCATA
1200
TTGGAATGAG TAGTCCGGAG CCTAAAATTA ACTCATACGT GAATTTTAGA GATGAAGTTC
1260
TTCCTCGCAT AAAAAAGCTT GGGTACAATG CGCTGCGAAT TATGGCTATT CAAGAGCATT
1320
CTTATTATGC TAGTTTTGGT TATCATGTCA CAAATTTTTT TGCACCAAGC AGCCGTTTTG 1380
CAACGCCCGA CGACCTTAAG TCTTCGATTG ATAAAGCTCA TGAGCTAGGA ATTGTTGTTC
1440
TCATGGACAT CGTTCACAGC CATGCATCAA ATAATACTTT AGATGGACTG AACATGTTTG
1500 -
ACGGCACCGA TAGTTGTTAC TTTCACTCTG GAGCTCGTGG TTATCATTGG ATGTGGGATT
1560
CCGCCTCTTT AACTATGGAA ACTGGGAGGT ACTTAGGTAT CTTCTCTCAA ATGCGAGATG
1620
GTGGTTGGAT GAGTTCAAAT TTGATGGATT TAGATTCGAT GGTGTGACAT CAATGATGTA
1680
TACTCACCAC GGATTATCGG TGGGATTCAC TGGGAACTAC GAGGAATACT TTGGACTCGC
1740
AACTGATGTG GATGCTGTTG TGTATCTGAT GCTGGTCAAC GATCTTATTC ATAGGCTTTT
1800
CCCAGATGCA ATTACCATTG GTGAAGATGT TAGCGGAATG CCGACATTTT GTATTC(-,CGT
1860
TCAAGATGGG GGTGTTGGCT T7GACTATCG GCTGCATATG GCAATTGCTG ATAAATGGAT
1920
TGAGTTGCTC AAGAAACGGG ATGAGGATTG GAGAGTGGGT GATATTGTTC ATACACTGAC
1980
AAATAGAAGA TGGTCGGAAA AGTGTGTTTC ATACGCTGAA AGTCATGATC AAGCTCTAGT
2040
CGGTGATAAA ACTATAGCAT TCT.GGCTGAT GGACAAGGAT ATGTATGATT TTATGGCTCT
2iu0
GGATAGACCG CCAACATCAT TAATAGATCG TGGGATAGCA~TTGCACAAGA TGATTAGGCT
2160
CA 02416347 2003-02-11
-45-
TGTAACTATG GGATTAGGAG GAGAAGGGTA CCTAAATTTC ATGGGAAATG AATTCGGCCA
2220
CCCTGAGTGG ATTGATTTCC CTAGGGCTGA GCCACACCTT TCTGATGGCT CAGTAATTCC
2280
CGGAAACCAA TTCAGTTATG ATAAATGCAG ACGGAGATTT GACCTGGGAG ATGCAGAATA
2340
TTTAAGATAC CATGGGTTAC AAGAATTTGA CTGGGCTATG CAGTAT G, e; G HAGNTAAATA
2400
TGAGTTTATG ACTTCAGAAC ACCAGTTCAT ATCACGAAAG GATGAAGGAG ATAGGATGAT
2460
TGTATTTGAA AGAGGAAACC TAGTTTTCGT CTTTAATTTT CACTGGACAA ATAGCTATTC
2520
AGACTATCGC ATAGGCTGCC TGAAGCCTGG AAAATACAAG GTTGTCTTGG ACTCAGATGA
2580
TCCACTTTTT GGTGGCTTCG GGAGAATTGA TCATAATGCC GAATATTTCA CCTCTGAAGG
2640
ATCGTATGAT GATCGTCCTT GTTCAATTAT GGTGTATGCA CCTAGTAGAA CAGCAGTGGT
2700
CTATGCACTA GTAGACAAAC TAGAAGTAGC AGTAGTAGAA GAACCCATTG AAGAATGAAC
2760
GAACTTGTGA TCGCGTTGAA AGATTTGAAC GTTACTTGGT CATCCACATA GAGCTTCTTG
2820
ACATCAGTCT TGGCGGAATT GCATGTGACA ACAAGGTTTG CAGTTCTTTC CACTATTAGT
2880
AGTCCACCGA TATACGCAGA GATGAAGTGC TGAACAAACA TATGTAAAAT CGATGAATTT
2940
ATGTCGAATG CTGGGACGAT CGAATTCCTG CAGCC 2975
(2) iNFORMAT(ON FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3033 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: singie
(D) TOPOLOGY: linear
CA 02416347 2003-02-11
-46-
(iX) FEATURE:
(A) NAME(KEY: CDS
(B) LOCATION:145..2790
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
TTGATGGGGC CTTGAACTCA GCAATTTGAC ACTCAGTTAG TTACACTCCT ATCACTTATC 60
AGATCTCTAT TTZTTCTCTT AATTCCAACC AAGGAATGAA TAAAAGGATA GATTTGTAAA
120
AACCCTAAGG AGAGAAGAAG AAAG ATG GTG TAT ACA CTC TCT GGA GTT CGT 171
Met Val Tyr Thr Leu Ser GIy Val Arg
1 5
TTT CCT ACT GTT CCA TCA GTG TAC AAA TCT AAT GGA TTC AGC AGT AAT 219
Phe Pro Thr Val Pro Ser Val Tyr Lys Ser Asn Gly Phe Ser Ser Asn
15 20 25
GGT GAT CGG AGG AAT GCT AAT GTT TCT GTA TTC TTG AAA AAG CAC TCT 267
Gly Asp Arg Arg Asn Ala Asn Val Ser Val Phe Leu Lys Lys His Ser
30 35 40 -
CTT TCA CGG AAG ATC TTG GCT GAA AAG TCT TCT TAC AAT TCC GAA TTC 315
Leu Ser Arg Lys lle Leu Ala Glu Lys Ser Ser Tyr Asn Ser Glu Phe
45 50 55
CGA CCT TCT ACA GTT GCA GCA TCG GGG AAA GTC CTT GTG CCT GGA ACC 363
Arg Pro Ser Thr Val Ala Ala Ser Gly Lys Val Leu Val Pro Gly Thr
60 65 70
CAG AGT GAT AGC TCC TCA TCC TCA ACA GAC CAA TTT GAG TTC ACT GAG 411
Gin Ser Asp Ser Ser Ser Ser Ser Thr Asp Gin Phe Glu Phe Thr Glu
75 80 85
ACA TCT CCA GAA AAT TCC CCA GCA TCA ACT GAT GTA GAT AGT TCA ACA 459
Thr Ser Pro Glu Asn Ser Pro Ala Ser Thr Asp Val Asp Ser Ser Thr
90 95 100 105
ATG GAA CAC GCT AGC CAG ATT AAA ACT GAG AAC GAT GAC GTT GAG CCG 507
Met Glu His Ala Ser Gln lie Lys Thr Glu Asn Asp Asp Val Glu Pro
110 115 120
TCA AGT GAT CTT ACA GGA AGT GTT GAA GAG CTG GAT-TTT GCT TCA TCA 555
Ser Ser Asp Leu Thr Gly Ser Val Glu GIu Leu Asp Phe Ala Ser Ser
CA 02416347 2003-02-11
. ' ~
-47-
125 130 135
CTA CAA CTA CAA GAA GGT GGT AAA CTG GAG GAG TCT AAA ACA TTA AAT 603
Leu Gin Leu Gin Glu Gly Gly Lys Leu Glu Glu Ser Lys Thr Leu Asn
140 145 150
ACT TCT GAA GAG ACA AT T A i- Gr",T Cr,A TCT GAT AGG ATC AGA GAG AGG 651
Thr Ser Glu Giu Thr Ile Ile Asp Glu Ser Asp Arg Ile Arg Glu Arg
155 160 165
GGC ATC CCT CCA CCT GGA CTT GGT CAG AAG ATT TAT GAA ATA GAC CCC 699
Giy Ile Pro Pro Pro Gly Leu Giy G1n Lys Ile Tyr Glu Ile Asp Pro
170 175 180 185
CTT TTG ACA AAC TAT CGT CAA CAC CTT GAT TAC AGG TAT TCA CAG TAC 747
Leu Leu Thr Asn Tyr Arg Gin His Leu Asp Tyr Arg Tyr Ser Gln Tyr -
190 195 200
AAG AAA CTG AGG GAG GCA ATT GAC AAG TAT GAG GGT GGT TTG GAA GCC 795
Lys Lys Leu Arg Glu Ala Ile Asp Lys Tyr Giu Gly Gly Leu Giu Ala
205 210 215
TTT TCT CGT GGT TAT GAA AAA ATG GGT TTC ACT CGT AGT GCT ACA GGT 843
Phe Ser Arg Gly Tyr Glu Lys Met Gly Phe Thr Arg Ser Ala Thr Gly
220 225 230
ATC ACT TAC CGT GAG TGG GCT CTT GGT GCC CAG TCA GCT GCC CTC ATT 891
Ile Thr Tyr Arg Glu Trp Ala Leu Gly Ala Gin Ser Ala Ala Leu Ile
235 240 245
GGA GAT TTC AAC AAT TGG GAC GCA AAT GCT GAC ATT ATG ACT CGG AAT 939
Gly Asp Phe Asn Asn Trp Asp Ala Asn Ala Asp Ile Met Thr Arg Asn
250 255 260 265
GAA TTT GGT GTC TGG GAG ATT T'rT CTG CCA AAT AAT GTG GAT GGT TCT 987
Glu Phe Gly Val Trp Giu Ile Phe Leu Pro Asn Asn Val Asp GIy Ser
270 275 280
CCT GCA ATT CCT CAT GGG TCC AGA GTG AAG ATA CGT ATG GAC ACT CCA 1035
Pro Ala Ile Pro His Gly Ser Arg Va) Lys lie Arg Met Asp Thr Pro
285 290 295
TCA GGT GTT AAG GAT TCC ATT CCT GCT TGG ATC AAC TAC TCT TTA CAG 1083
Ser Gly Vai Lys Asp Ser Ile Pro Ala Trp lie Asn Tyr Ser Leu Gin
300 305 310
CTT CCT GAT GAA ATT CCA TAT AAT GGA ATA CAT TAT GAT CCA CCC GAA 1131
Leu Pro Asp Giu Ile Pro Tyr Asn Gly Ile His Tyr Asp Pro Pro Glu
315 320 325
CA 02416347 2003-02-11
-48-
GAG GAG AGG TAT ATC TTC CAA CAC CCA CGG CCA AAG AAA CCA AAG TCG 1179
Glu Glu Arg Tyr Ile Phe Gin His Pra Arg Pro Lys Lys Pro Lys Ser
330 335 340 345
CTG AGA ATA TAT GAA TCT CAT ATT GGA ATG AGT AGT CCG GAG CCT AAA 1227
Leu Arg Ile Tyr Glu Ser His Ile Gly Met Ser Ser Pro Glu Pro Lys
350 355 360
ATT AAC TCA TAC GTG AAT TTT AGA GAT GAA GTT CTT CCT CGC ATA AAA 1275
Ile Asn Ser Tyr Val Asn Phe Arg Asp Glu Val Leu Pro Arg Ile Lys
365 370 375
AAG CTT GGG TAC AAT GCG CTG CAA ATT ATG GCT ATT CAA GAG CAT TCT 1323
Lys Leu Gly Tyr Asn Ala Leu Gin Ile Met Ala lle Gin Giu His Ser
380 385 390 -
TAT TAC GCT AGT TTT GGT TAT CAT GTC ACA AAT TTT TTT GCA CCA AGC 1371
Tyr Tyr Ala Ser Phe GIV Tyr His Val Thr Asn Phe Phe Ala Pro Ser
395 400 405
AGC CGT TTT GGA ACG CCC GAC GAC CTT AAG TCT TTG ATT GAT AAA GCT 1419
Ser Arg Phe Gly Thr Pro Asp Asp Leu Lys Ser Leu lie Asp Lys Ala
410 415 420 425
CAT GAG CTA GGA ATT GTT GTT CTC ATG GAC ATT GTT CAC AGC CAT GCA -1467
His Glu Leu GIV lie Va) Val Leu Met Asp Ile Val His Ser His Ala
430 435 440
TCA AAT AAT ACT TTA GAT GGA CTG AAC ATG T-fT GAC TGC ACC GAT AGT 1515
Ser Asn Asn Thr Leu Asp Gly Leu Asn Met Phe Asp Cys Thr Asp Ser
445 450 455
TGT TAC TTT CAC TCT GGA GCT CGT GGT TAT CAT TGG ATG TGG GAT TCC 1563
Cys Tyr Phe His Ser GIV Ala Arg Gly Tyr His Trp Met Trp Asp Ser
460 465 "- 470
CGC CTC TTT AAC TAT GGA AAC TGG GAG GTA CTT AGG TAT CTT CTC TCA 1611
Arg Leu Phe Asn Tyr Gly Asn Trp Glu Val Leu Arg Tyr Leu Leu Ser
475 480 485
AAT GCG AGA TGG TGG TTG GAT GCG TTC AAA TTT GAT GGA TTT AGA TTT 1659
Asn Ala Arg Trp Trp Leu Asp Ala Phe Lys Phe Asp GIV Phe Arg Phe
490 495 500 505
GAT GGT GTG ACA TCA ATG ATG TAT ATT CAC CAC GGA TTA TCG GTG GGA 1707
Asp GIV Val Thr Ser Met Met Tyr lle His His Giy Leu Ser Val Gly
510 515 520
CA 02416347 2003-02-11
-49-
TTC ACT GGG AAC TAC GAG GAA TAC TTT GGA CTC GCA ACT GAT GTG GAT 1755
Phe Thr Gly Asn Tyr Glu Glu Tyr Phe Gly Leu Ala Thr Asp Val Asp
525 530 535
GCT GTT r,TG TAT CTC .qTe rTr GTC AAC GAT CTT ATT CAT GGG CTT TTC 1803
Ala Val Val Tyr Leu Met Leu Val Asn Asp Leu Ile His Gly Leu Phe
540 545 550
CCA GAT GCA ATT ACC ATT GGT GAA GAT GTT AGC GGA ATG CCG ACA TTT 1851
Pro Asp Ala Ile Thr Iie Giy Glu Asp Vai Ser Gly Met Pro Thr Phe
555 560 565
TGT ATT CCC GTC CAA GAG GGG GGT GTT GGC TTT GAC TAT CGG CTG CAT 1899
Cys Ile Pro Val Gin Glu Gly Gly Val Gly Phe Asp Tyr Arg Leu His
570 575 580 585
ATG GCA ATT GCT GAT AAA CGG ATT GAG TTG CTC AAG AAA CGG GAT GAG 1947
Met Ala Ile Ala Asp Lys Ara Ile Glu Leu Leu Lys Lys Arg Asp Giu
590 595 600
GAT TGG AGA GTG GGT GAT ATT GTT CAT ACA CTG ACA AAT AGA AGA TGG 1995
Asp Trp Arg Val Giy Asp Ile Val His Thr Leu Thr Asn Arg Arg Trp
605 610 615
TCG GAA AAG TGT GTT TCA TAC GCT GAA AGT CAT GAT CAA GCT CTA GTC 2043
Ser G(u Lys Cys Val Ser Tyr Ala Glu Ser His Asp Gln Ala Leu Vai -
620 625 630
GGT GAT AAA ACT ATA GCA TTC TGG CTG ATG GAC AAG GAT ATG TAT GAT 2091
GIy Asp Lys Thr Ile Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp
635 640 645
TTT ATG GCT CTG GAT AGA CCG TCA ACA TCA TTA ATA CAT CGT GGG ATA 2139
Phe Met Ala Leu Asp Arg Pro Ser Thr Ser Leu ile Asp Arg Gly Ife
650 655 660 665
GCA TTG CAC AAG ATG ATT AGG CTT GTA ACT ATG GGA TTA GGA GGA GAA 2187
Ala Leu His Lys Met Ile Arg Leu Val Thr Met Giy Leu Gly Giy Glu
670 675 680
GGG TAC CTA AAT TTC ATG GGA AAT GAA TTC GGC CAC CCT GAG TGG ATT 2235
Gly Tyr Leu Asn Phe Met Gly Asn Glu Phe GIy His Pro Giu Trp Ile
685 690 695
GAT TTC CCT AGG GCT GAA CAA CAC CTC TCT GAT GGC TCA GTA ATC CCC 2283
Asp Phe Pro Arg Ala Glu Gin His Leu Ser Asp Gly Ser Vai ife ~; o
700 705 710
GGA AAC CAA TTC AGT TAT GAT AAA TGC AGA CGG AGA TTT GAC CTG GGA 2331
CA 02416347 2003-02-11
. m _..
-50-
Gly Asn Gin Phe Ser Tyr Asp Lys Cys Arg Arg Arg Phe Asp Leu GIy
715 720 725
GAT GCA GAA TAT TTA AGA TAC CGT GGG TTG CAA GAA TTT GAC CGG CCT 2379
Asp Ala Glu Tyr Leu Arg Tyr Arg Gly Leu Gin Glu Phe Asp Arg Pro
730 735 740 745
ATG CAG TAT CTT GAA GAT AAA TAT GAG TTT ATG ACT TCA GAA CAC CAG 2427
Met Gin Tyr Leu Giu Asp Lys Tyr Glu Phe Met Thr Ser Glu His Gln
750 755 760
TTC ATA TCA CGA AAG GAT GAA GGA GAT AGG ATG ATT GTA TTT GAA AAA 2475
Phe ile Ser Arg Lys Asp Glu Giy Asp Arg Met i(e Val Phe Glu Lys
765 770 775
GGA AAC CTA GTT TTT GTC TTT AAT TTT CAC TGG ACA AAA AGC TAT TCA - 2523
GIy Asn Leu Val Phe Val P"e Asn Phe His Trp Thr Lys Ser T'yr Ser
780 785 790
GAC TAT CGC ATA GCC TGC CTG AAG CCT GGA AAA TAC AAG GTT GCC TTG 2571
Asp Tyr Arg iie Ala Cys Leu Lys Pro GIy Lys Tyr Lys Val Ala Leu
795 800 805
GAC TCA GAT GAT CCA CTT TTT GGT GGC TTC GGG AGA ATT GAT CAT AAT 2619
Asp Ser Asp Asp Pro Leu Phe Gly Gly Phe Giy Arg lie Asp His Asn
810 815 820 825 -
GCC GAA TAT TTC ACC TTT GAA GGA TGG TAT GAT GAT CGT CCT CGT TCA 2667
Ala Giu Tyr Phe Thr Phe Giu Giy Trp Tyr Asp Asp Arg Pro Arg Ser
830 835 840
ATT ATG GTG TAT GCA CCT TGT AAA ACA GCA GTG GTC TAT GCA CTA GTA 2715
lle Met Val Tyr Ala Pro Cys Lys Thr Ala Val Val Tyr Ala Leu Val
845 850 855
CAC AAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GTA GCA GCA 2763
Asp Lys Glu Glu Glu Ciu Glu Glu Glu Glu Glu Glu Glu Val Ala Ala
860 865 870
GTA GAA GAA GTA GTA GTA GAA GAA GAA TGAACGAACT TGTGATCGCG 2810
Val Giu GIu Val Val Val Glu Glu Giu
875 880
TTGAAAGATT TGAACGCTAC ATAGAGCTTC TTGACGTATC TGGCAATATT GCATCAGTCT
2870
TGGCGGAATT TCATGTGACA CAAGGTTTGC AATTCTTTCC ACTATTAGTA GTGCAACGAT
2930
CA 02416347 2003-02-11
-51-
ATACGCAGAG ATGAAGTGCT GAACAAACAT ATGTAAAATC GATGAATT'i'A TGTCGAATGC
2990
TGGGACGATC GAATTCCTGC AGGCCGGGGG ACCCCTTAGT TCT 3033
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 882 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Met Val Tyr Thr Leu Ser Gly Val Arg Phe Pro Thr Val Pro Ser Val
1 5 10 15
Tyr Lys Ser Asn Giy Phe Ser Ser Asn Gly Asp Arg Arg Asn Ala Asn
20 25 30
Val Ser Val Phe Leu Lys Lys His Ser Leu Ser Arg Lys lie Leu Ala
35 40 45
Glu Lys Ser Ser Tyr Asn Ser Glu Phe Arg Pro Ser Thr Va1 Ala Ala
50 55 60
Ser Gly Lys Val Leu Val Pro GIy Thr Gln Ser Asp Ser Ser Ser Ser
65 70 75 80
Ser Thr Asp Gin Phe Glu Phe Thr Glu Thr Ser Pro GIU Asn Ser Pro
85 90 95
Ala Ser Thr Asp Val Asp Ser Ser Thr Met Glu His Ala Ser Giri lle
100 105 - 11.0
Lys Thr GIu Asn Asp Asp Vai Glu Pro Ser Ser Asp Leu Thr Gly Ser
115 120 125
Val Glu Glu Leu Asp Phe Ala Ser Ser Leu Gin Leu Gin Glu Gly Gly
130 135 140
Lys Leu Glu Glu Ser Lys Thr Leu Asn Thr Ser Glu Glu Thr lle Ile
145 150 155 160
Asp Glu Ser Asp Arg Ile Arg Glu Arg GIy lle Pro Pro Pro Gly Leu
165 170 175 _
CA 02416347 2003-02-11
-52-
Gly Gin Lys lie Tyr GIu lie Asp Pro Leu Leu Thr Asn Tyr Arg Gln
180 185 190
His Leu Asp Tyr Arg Tyr Ser Gin Tyr Lys Lys Leu Arg Glu Ala lie
195 200 205
Asp Lys Tyr Glu Gly Gly L'u Glu Ala Phe Ser Arg Gly Tyr Glu Lys
210 215 220
Met G(y Phe Thr Arg Ser Ala Thr Gly Ile Thr Tyr Arg Glu Trp Ala
225 230 235 240
Leu Gly Ala Gln Ser Ala Ala Leu Ile Gly Asp Phe Asn Asn Trp Asp
245 250 255
Ala Asn Ala Asp lie Met Thr Arg Asn Giu Phe Gly Val Trp Glu Ile
260 265 270
Phe Leu Pro Asn Asn Val Asp Gly Ser Pro Ala 11e Pro His Gly Ser
275 280 285
Arg Val Lys Ile Arg Met Asp Thr Pro Ser Giy Val Lys Asp Ser lie
290 295 300
Pro Ala Trp Ile Asn Tyr Ser Leu Gin Leu Pro Asp Glu lie Pro Tyr
305 310 315 320
Asn Gly lie His Tyr Asp Pro Pro Glu Glu Glu Arg Tyr lie Phe Gin
325 330 335
His Pro Arg Pro Lys Lys Pro Lys Ser Leu Arg Ile Tyr Giu Ser His
340 345 350
Ile Gly Met Ser Ser Pro Glu Pro Lys lie Asn Ser Tyr Val Asn Phe
355 360 365
Arg Asp Glu Val Leu Pro Arg Ile Lys Lys Leu Gly Tyr Asn Ala Leu
370 375 380
G1n iie Met Ala Ile Gin Glu His Ser Tyr Tyr Ala Ser Phe GIy Tyr
385 390 395 400
His Val Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe G1y Thr Pro Asp
405 410 415
Asp Leu Lys Ser Leu (! A.cn N,is !~ His Glu Leu Gly Ile Val Val
420 425 430
Leu Met Asp Ile Val His Ser His Ala Ser Asn Asn Thr Leu Asp Gly
CA 02416347 2003-02-11
-53-
435 440 445
Leu Asn Met Phe Asp Cys Thr Asp Ser Cys Tyr Phe His Ser Gly Ala
450 455 460
Arg GIV Tyr His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Asn
465 470 475 480
Trp Glu Val Leu Arg Tyr Leu Leu Ser Asn Ala Arg Trp Trp Leu Asp
485 490 495
Ala Phe Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met
500 505 510
Tyr lle His His GIV Leu Ser Val Gly Phe Thr GIV Asn Tyr Glu Glu
515 520 525
Tyr Phe GIV Leu Ala Thr Asp Val Asp Ala Val Val Tyr Leu Met Leu
530 535 540
Val Asn Asp Leu Ile His GIV Leu Phe Pro Asp Ala Ile Thr Ile GIV
545 550 555 560
Glu Asp Val Ser Gly Met Pro Thr Phe Cys Ile Pro Val Gin Glu Gly
565 570 575
Gly Val GIV Phe Asp Tyr Arg Leu His Met Ala lle Ala Asp Lys Arg
580 585 590
ife Glu Leu Leu Lys Lys Arg Asp Giu Asp Trp Arg Val GIV Asp Ile
595 600 605
Val His Thr Leu Thr Asn Arg Arg Trp Ser Glu Lys Cys Val Ser Tyr
610 615 620
Ala Glu Ser His Asp G(n Ala-Leu Val Gly Asp Lys Thr {ie Ala Phe
625 630 635 640
Trp Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro
645 650 655
Ser Thr Ser Leu Ile Asp Arg Gly lie Ala Leu His Lys Met lie Arg
660 665 670
Leu Val Thr Met Gly Leu GIV Gly Glu Gly Tyr Leu Asn Phe Met Gly
675 680 685
Asn Giu Phe GIV His Pro Giu Trp Ile Asp Phe Pro Arg Ala Glu Gin
690 695 700
CA 02416347 2003-02-11
-54-
His Leu Ser Asp Gly Ser Val Ile Pro Gly Asn Gin Phe Ser Tyr'Asp
705 710 715 720
Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala GIU Tyr Leu Arg Tyr
725 730 735
Arg Giy Leu Gin G1u Phe Asp Arg Pro Met Gin Tyr Leu Glu Asp Lys
740 745 750
Tyr Glu Phe Met Thr Ser Glu His Gin Phe Ife Ser Arg Lys Asp Glu
755 760 765
Gly Asp Arg Met IIe Val Phe Glu Lys Gly Asn Leu Val Phe Val Phe
770 775 780
Asn Phe His Trp Thr Lys Ser Tyr Ser Asp Tyr Arg lie Ala Cys Leu
785 790 795 800
Lys Pro Giy Lys Tyr Lys Val Ala Leu Asp Ser Asp Asp Pro Leu Phe
805 810 815
Gly Gfy Phe G1y Arg Ile Asp His Asn Ala Glu Tyr Phe Thr Phe Glu
820 825 830
Gly Trp Tyr Asp Asp Arg Pro Arg Ser Ile Met Val Tyr Ala Pro Cys
835 840 845
Lys Thr Ala Val Val Tyr Ala Leu Val Asp Lys Glu Glu Glu Glu Glu
850 855 860
Glu Glu Glu Glu Glu Glu Val Ala Ala Val Glu Glu Va1 Val Val Glu
865 870 875 880
Giu Glu
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2576 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02416347 2003-02-11
-55-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
TCATTAAAGA GGAGAAATTA ACTATGAGAG GATCTCACCA TCACCATCAC CATGGGATCT
TGGCTGAAAA GTCTTCTTAC AATTCCGAAT TCCGACCTTC TACAGTTGCA GCATCGGGGA
120
AAGTCCTTGT GCCTGGAACC CAGAGTGATA GCTCCTCATC CTCAACAAAC CAATTTGAGT
180
TCACTGAGAC ATCTCCAGAA AATTCCCCAG CATCAACTGA TGTAGATAGT TCAACAATGG
240
AACACGCTAG CCAGATTAAA ACTGAGAACG ATGACGTTGA GCCGTCAAGT GATCTTACAG
300
GAAGTGTTGA ACAGCTGGAT TTTGCTTCAT CACTP.Cn,ACT ACPn,CAACGT GGTAAACTGC
360
AGGAGTCTAA AACATTAAAT ACTTCTGAAG AGACAATTAT TGATGAATCT GATAGGATCA
420
GAGAGAGGGG CATCCCTCCA CCTGGACTTG GTCAGAAGAT TTATGAAATA GACCCCCTTT
480
TGACAAACTA TCGTCAACAC CTTGATTACA GGTATTCACA GTACAAGAAA CTGAGGGAGG
540
CAATTGACAA GTATGAGGGT GGTTTGGAAG CTTTTTCTCG TGGTTATGAA AAAATGGGTT
600
TCACTCGTAG TGCTACAGGT ATCACTTACC GTGAGTGGGC TCCTGGTGCC CAGTCAGCTG
660
CCCTCATTGG AGATTTCAAC AATTGGGACG CAAATGCTGA CATTATGACT CGGAATGAAT
720
TTGGTGTCTG GGAGATTTTT CTGCCAAATA ATGTGGATGG TTCTCCTGCA ATTCCTCATG 780
GGTCCAGAGT GAAGATACGT ATGGACACTC CATCAGGTGT TAAGGATTCC ATTCCTGCTT
840
GGATCAACTA CTCTACAGCT TCCTGATGAA ATTCCATATA ATGGAATATA TTATGATCCA
900
CCCGAAGAGG AGAGGTATAT CTTCCAACAC CCACGGCCAA AGAAACCAAA GTCGCTGAGA
960 -
CA 02416347 2003-02-11
g a a _.
-56-
ATATATGAAT CTCATATTGG AATGAGTAGi CCGGAGCCTA AAATTAACTC ATACGTGAAT
1020
TTTAGAGATG AAGTTCTTCC TCGCATAAAA AAGCTTGGGT ACAATGCGCT GCAAATTATG
1080
GCTATTCAAG AGCATTCTTA TTATGCTAGT TTTGGTTATC ATGTCACAAA TTTTTTTGCH 1140
CCAAGCAGCC GTTTTGGAAC GCCCGACGAC CTTAAGTCTT TGATTGATAA AGCTCATGAG
1200
CTAGGAATTG TTGTTCTCAT GGACATTGTT CACAGCCATG CATCAAATAA TACTTTAGAT
1260
GGACTGAACA TGTTTGACGG CACCGATAGT TGTTACTTTC ACTCTGGAGC TCGTGGTTAT
1320
CATTGGATGT GGGATTCCCG CCTTTTTAAC TATGGAAACT GGGAGGTACT TAGGTATCTT
1380
CTCTCAAATG CGAGATGGTG GTTGGATGAG TTCAAATTTG ATGGATTTAG ATTTGATGGT
1440
GTGACATCAA TGATGTATAC TCACCACGGA TTATCGGTGG GATTCACTGG GAACTACGAG
1500
GAATACTTTG GACTCGCAAC TGATGTGGAT GCTGTTGTGT ATCTGATGCT GGTCAACGAT
1560
CTTATTCATG GGCTTTTCCC AGATGCAATT ACCATTGGTG AAGATGTTAG CGGAATGCCG
1620
ACATTTTGTA TTCCCGTTCA 'kGATGGGGGT GTTGGCTTTG ACTATCGGCT GCATATGGCA
1680
ATTGCTGATA AATGGATTGA GTTGCTCAAG AAACGGGATG AGGATTGGAG AGTGGGTGAT
1740
ATTGTTCATA CACTGACAAA TAGAAGATGG TCGGAAAAGT GTGTTTCATA CGCTGAAAGT
1800
CATGATCAAG CTCTAGTCGG TGATAAAACT ATAGCATTCT GGCTGATGGA CAAGGATATG
1860
TATGATTTTA TGGCTCTGGA TAGACCGCCA ACATCATTAA TAGATCGTGG GATAGCATTG
1920
CACAAGATGA TTAGGCTTGT AACTATGGGA TTAGGAGGAG AAGGGTACCT AAATTTCATG
1980
CA 02416347 2003-02-11
-57-
GGAAATGAAT TCGGCCO.CCC TGAGTGGATT GATTTCCCTA GGGCTGAACA ACACCTCTCT
2040
GATGACTCAG TAATTCCCGG AAACCAATTC AGTTATGATA AATGCAGACG GAGATTTGAC
2100
CTGGGAGATG CAGAATATTT AAGATACCGT GGGTTGCAAG AATTTGACCG GGCTATGCAG
2160
TATCTTGAAG ATAAATATGA GTTTATGACT TCAGAACACC AGTTCATATC ACGAAAGGAT
2220
GAAGGAGATA GGATGATTGT ATTTGAAAAA GGAAACCTAG TTTTTGTCTT TAATTTTCAC
2280
TGGACAAAAA GCTATTCAGA CTATCGCATA GGCTGCCTGA AGCCTGGAAA ATACAAGGTT
2340
GCCTTGGACT CAGATGATCC ACTTTTTGGT GGCTTCGGGA GAATTGATCA TAATGCCGAA
2400
TATTTCACCT TTGAAGGATG GTATGATGAT CGTCCTCGTT CAATTATGGT GTATGCACCT
2460
TGTAGAACAG CAGTGGTCTA TGCACTAGTA GACAAAGAAG AAGAAGAAGA AGAAGAAGAA
2520
GAAGAAGTAG CAGTAGTAGA AGAAGTAGTA GTAGAAGAAG AATGAACGAA CTTGTG
2576
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2529 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEO ID NO: 17:
GGATGCTAAT GTTTCTGTAT TCTTGAAAAA GCACTCTCTT TCACGGAAGA TCTTGGCTGA 60
AAAGTCTTCT TACAATTCCG AA-TCCCGACC TTCTACAGTT GCAGCATCGG GGAAAGTCCT
120
CA 02416347 2003-02-11 .,,
-S7a-
TGTGCCTGGA AYCCAGAGTG ATAGCTCCTC ATCCTCAACA GACCAATTTG AGTTCACTGA
180
GACATCTCCA GAAAATTCCC CHGCATCAAC TGATGTAGAT AGTTCAACAA TGGAACACGC
240
TAGCCAGATT AAAACTGAGA ACGATGACGT TGAGCCGTCA AGTGATCTTA CAGGAAGTGT
300
TGAAGAGCTG GATTTTGCTT CATCACTACA ACTACAAGAA GGTGGTAAAC TGGAGGAGTC
360
TAAAACATTA AATACTTCTG AAGAGACAAT TATTGATGAA TCTGATAGGA TCAGAGAGAG
420
GGGCATCCCT CCACCTGGAC TTGGTCAGAA GATTTATGAA ATAGACCCCC TTTTGACAAA
480
CTATCGTCAA CACCTTGATT ACAGGTATTC ACAGTACAAG AAACTGAGGG AGGCAATTGA
540
CAAGTATGAG GGTGGTTTGG AAGCTTTTTC TCGTGGTTAT GAAAAAATGG GTTTCACTCG
600
TAGTGCTACA GGTATCACTT ACCGTGAGTG GGCTCCTGGT GCCCAGTCAG-CTGCCCTCAT
660
TGGAGATTTC AACAATTGGG ACGCAAATGC TGACATTATG ACTCGGAATG AATTTGGTGT
720
CTGGGAGATT TTTCTGCCAA ATAATGTGGA TGGTTCTCCT GCAATTCCTC ATGGGTCCAG
780
AGTGAAGATA CGYATGGACA CTCCATCAGG TGTTAAGGAT TCCATTCCTG CTTGGATCAA
840
CTACTCTTTA CAGCTTCCTG ATGAAATTCC ATATAATGGA ATATATTATG ATCCACCCGA
900
AGAGGAGAGG TATRTCTTCC AACACCCACG GCCAAAGAAA CCAAAGTCGC TGAGAATATA
960
TGAATCTCAT ATTGGAATGA GTAGTCCGGA GCCTAAAATT AACTCATACG TGAATTTTAG
1020
AGATGAAGTT CTTCCTCGCA TAAAAAASCT TGGGTACAAT GCGGTGCAAA TTATGGCTAT
1080 - -
CA 02416347 2003-02-11
-57b-
TCAAGAGCAT TCTTATTATG CTAGTTTTGG TTATCATGTC ACAAATTTTT TTGCACCAAG
1140
CAGCCGTTTT GGAACGCCCG ACGACCTTAA GTCTTTGATT GATAAAGCTC ATGACCTAGG
1200
AATTGTTGTT CTCATGGACA TTGTTCACAG CCATGCATCA AATAATACTT TAGATGGACT
"1260
GAACATGTTT GACGGCACAG ATAGTTGTTA CTTTCACTCT GGAGCTCGTG GTTATCATTG
1320
GATGTGGGAT TCCCGCCTCT TTAACTATGG AAAC ~'GGGAG GTACTTAGGT ATCTTCTCTC
1380
AAATGCGAGA TGGTGGTTGG ATGAGTTCAA ATTTGATGGA TTTAGATTTG ATGGTGTGAC
1440
ATCAATGATG TATACTCACC ACGGATTATC GGTGGGATTC ACTGGGAACT ACGAGGAATA
1500
CTTTGGACTC GCAACTGATG TGGATGCTGT TGTGTATCTG ATGCTGGTCA ACGATCTTAT
1560
TCACGGGCTT TTCCCAGATG CAATTACCAT TGGTGAAGAT GTTAGCGGAA TGCCGACATT
1620 - -
TTGTATTCCC GTTCAAGATG GGGGTGTTGG CTTTGACTAT CGGCTGCATA TGGCAATTGC
1680
TGATAAATGG ATTGAGTTGC TCAAGAAACG GGATGAGGAT TGGAGAGTGG GTGATATTGT
1740
TCATACACTG ACAAATAGAA GATGGTCGGA AAAGTGTGTT TCATMCGCTG AAAGTCATGA
1800
TCAAGCTCTA GTCGGTGATA AAACTATAGC ATYCTGGCTG ATGGACAAGG ATATGTATGA
1860
TTTTATGGCT CTGGATAGAC CGYCAACAYC ATTAATAGAT CGTGGGATAG CATTGCACAA
1920
GATGATTAGG CTTGTAACTA TGGGATTAGG AGGAGAAGGG TACCTAAATT TCATGGGAAA
1980
TGAATTCGGC CACCCTGAGT GGATTGATTT CCCTAGGGCT GARCAACACC TCTCTGATGG
2040
CA 02416347 2003-02-11
-57c-
CTCAGTAATT CCCGGAAACC AATTCAGTTA TGATAAATGC AGACGGAGAT TTGACCTGGG
2100
AGATGCAGAA TATTTAAGAT ACCATGGGTT GCAAGAATTT GACCGGGCTA TGCAGTATCT
2160
TGAAGATAAA TATGAGTTTA TGACTTCAGA ACACCAGTTC ATATCACGAA AGGATGAAGG
2220
AGATAGGATG ATTGTATTTG AAAi-\r-,CC :CCT" GTi T T T GTCTTTAATT TTCACTGGAC
2280
AAATAGCTAT TCAGACTATC GCATAGGCTG CCTGAAGCCT GGAAAATACA AGGTTGGCTT
2340
GGACTCAGAT GATCCACTTT TTGGTGGCTT CGGGAGAATT GATCATAATG CCGAATATTT
2400
CACCTCTGAA GGATCGTATG ATGATCGTCC TCGTTCAATT ATGGTGTATG CACCTAGTAG
2460
AACAGCAGTG GTCTATGCAC TAGTAGACAA ANTAGAAGNA GAAGAAGAAG AAGAANCCGN
2520
NGAAGAATT 2529
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3231 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: iinear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GATTTAATAC GACTCACTAT AGGGATTTTT TTl?TT T TTT TTTTAAAAAC CTCCTCCACT 60
CAGTCTTGGG ATCTCTCTCT CTCTTCACGC TTCTCTTGGG GCCTTGAACT CAGCAATTTG 120
ACACTCAGTT AGTTACACTC CTATCACTCA TCAGATCTCT ATTTTTTCTC TTAATTCCAA 180
CCAAGGAATG AATTAAAAGA TTAGATTTGA AGGAGAGAAG AAGAAAGATG GTGTATACAC
240 - :
CA 02416347 2003-02-11
-57d-
TCTCTGGAGT TCGTTTTCCT ACTGTTCCAT CAGTGTACAA ATCTAATGGA TTCAGCAGTA 300
ATGGTGATCG GAGGAATGCT AATGTTTCTG TATTCTTGAA AAAGCACTCT CTTTCACGGA
360
AGATCTTGGC TGAAAAGTCT TCTTACGATT CCGAATCCCG ACCTTCTACA GTTGCAGCAT
420
CGGGGAAAGT CCTTGTACCT GGA.-~,TCCAGA GTGATAGCTC CTCATCCTCA ACAGACCAAT
480
TTGAGTTCAC TGAGACAGCT CCAGAAAATT CCCCAGCATC AACTGATGTG GATAGTTCAA
540
CAATGGAACA CGCTAGCCAG ATTAAAACTG AGAACGATGA CGTTGAGCCG TCAAGTGATC
600
TTACAGGAAG TGTTGAAGAG TTGGATTTTG CTTCATCACT ACAACTACAA GAAGGTGGTA
660
AACTGGAGGA GTCTAAAACA TTAAATACTT CTGAAGAGAC AATTATTGAT GAATCTGATA
720
GGATCAGAGA GAGGGGCATC CCTCCACCTG GACTTGGTCA GAAGATTTAT GAAATAGACC
780
CCCTTTTGAC AAACTATCGT CAACACCTTG ATTACAGGTA TTCACAGTAC AAGAAAATGA
840
GGGAGGCAAT TGACAAGTAT GAGGGTGGTT TGGAAGCTTT TTCTCGTGGT TATGAAAAAA
900
TGGGTTTCAC TCGTAGTGCT ACAGGTATCA CTTACCGTGA GTGGGCTCCT GGTGCCCAGT
960
CAGCTGCTCT CATTGGAGAT TTCAACAATT GGGACGCAAA TGCTGACATT ATGACTCGGA
1020
ATGAATTTGG TGTCTGGGAG ATTTTTCTGC CAAATAATGT GGATGGTTCT CCTGCAATTC
1080
CTCATGGGTC CAGAGTGAAG ATACGCATGG ACACTTCATC AGGTGTTAAG GATTCCATTC
1140
CTGCTTGGAT CAACTACTCT TTACAGCTTC CTGATGAAAT TCCATATAAT GGAATATATT
1200
ATGATCCACC CGAAGAGGAG-AGGTATGTCT TCCAACACCC ACGGCCAAAG AAACCAAAGT
1260
CA 02416347 2003-02-11
-57e-
CGCTGAGAAT ATATGAATCT CATATTGGAA TGAGTAGTCC GGAGCCTAAA ATTAACTCAT
1320
ACGTGAATTT TAGAGATGAA GTTCTTCCTC GCATAAAAAA CCTTGGGTAC AATGCGGTGC
1380
AAATTATGGC TATTCAAGAG CATTCTTATT ATGCTAGTTT TGGTTATCAT GTCACAAATT
1440
TTTTTGCACC AAGCAGCCGT TTTGGAACGC CCGACGACCT TAAGTCTTTG ATTGATAAAG
1500
CTCATGAGCT AGGAATTGTT GTTCTCATGG ACATTGTTCA CAGCCATGCA TCAAATAATA
1560
CTTTAGATGG ACTGAACATG TTTGACGGCA CAGATAGTTG TTACTTTCAC TCTGGAGCTC
1620
GTGGTTATCA TTGGATGTGG GATTCCCGCC TCTTTAACTA TGGAAACTGG GAGGTACTTA
1680
GGTATCTTCT CTCAAATGCG AGATGGTGGT TGGATGAGTG CAAATTTGRT GGATTTAGAT
1740
TTGATGGTGT GACATCAATG ATGTATACTC ACCACGGATT ATCGGTGGGATTCACTGGGA
1800
ACTACGAGGA ATACTTTGGA CTCGCAACTG ATGTRGATGC TGCCGTGTAT CTGATGCTGG
1860
CCAACGATCT TATTCATGGG CTTTTCCCAG ATGCAATTAC CATTGGTGAA GATGTTAGCG
1920
GAATGCCGAC ATTTTGTATT..CCCGTTCAAG ATGGGGGTGT TGGCTTTGAC TATCGGCTGC
1980 '
ATATGGCAAT TGCTGATAAA TGGATTGAGT TGCTCAAGAA ACGGGATGAG GATTGGAGAG
2040
TGGGTGATAT TGTTCATACA CTGACAAATA GAAGATGGTC GGAAAAGTGT GTTTCATACG
2100
CTGAAAGTCA TGATCAAGCT CTAGTCGGTG ATAAAACTAT AGCATTCTGG CTGATGGACA
2160
AGGATATGTA TGATTTTATG GCTTTGGATA GACCGTCAAC ATCATTAATA GATCGTGGGA
2220 - -
CA 02416347 2003-02-11
-57f-
TAGCATTGCA CAAGATGATT AGGCTTGTAA CTATGGGATT AGGAGGAGAA GGGTACCTAA
2280
ATTTCATGGG AAATGAATTC GGCCACCCTG AGTGGATTGA TTTCCCTAGG GCTGAACAAC
2340
ACCTCTCTGA TGGCTCAGTA ATTCCCGGAA ACCAATTCAG TTATGATAAA TGCAGACGGA
2400
GATTTGA.CCT ~rr,AGATGrA GA.ATATTTAA GATACCGTGG GTTGCAAGAA TTTGACCGGG
2460
CTATGCAGTA TCTTGAAGAT AAATATGAGT TTATGACTTC AGAACACCAG TTCATATCAC
2520
GAAAGGATGA AGGAGATAGG ATGATTGTAT TTGAAAAAGG AAACCTAGTT TTTGTCTTTA
2580
ATTTTCACTG GACAAAAAGC TATTCAGACT ATCGCATAGG CTGGCTGAAG CCTGGAAAAT
2640
ACAAGGTTGC CTTGGACTCA GATGATCCAC TTTTTGGTGG CTTCGGGAGA ATTGATCATA
2700
ATGCCGAATG TTTCACCTTT GAAGGATGGT ATGATGATCG TCCTCGTTCA ATTATGGTGT
2760 - -
ATGCACCTAG TAGAACAGCA GTGGTCTATG CACTAGTAGA CAAAGAAGAA GAAGAAGAAG
2820
AAGTAGCAGT AGTAGAAGAA GTAGTAGTAG AAGAAGAATG AACGAACTTG TGATCGCGTT
2880
GAAAGATTTG AACGCTACAT AGAGCTTCTT GACGTATCTG GCAATATTGC ATCAGTCTTG
2940
GCGGAATTTC ATGTGACAAA AGGTTTGCAA TTCTTTCCAC TATTAGTAGT GCAACGATAT
3000
ACGCAGAGAT GAAGTGCTGA ACAAACATAT GTAAAATCGA TGAATTTATG TCGAATGCTG
3060
r-r n nr, r-r-r1rr i=n r=rn rrTrT T/~.rTTr:Tr./1 (:TTrT(~.Td QA, TTrT('ATCTC
TTTA._ TGTA
NA
3120
CAGCCCACTA GAAATCAATT ATGTGAGACC TAAAAAACAA TAACCATAAA ATGGAAATAG
3180
TGCTGATCTA ATGATGTTTT AANCCNNNNA AAAAAAAAAA AAAAACTCGA G 3231
CA 02416347 2003-02-11
-57g-
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2578 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
TCATTAAAGA GGAGAAATTA ACTATGAGAG GATCTCACCA TCACCATCAC CATGGGATCT
TGGCTGAAAA GTCTTCTTAC AATTCCGAAT TCCGACCTTC TACAGTTGCA GCATCGGGGA
120
AAGTCC T TG T GCCTGGAACC CAGAGTGATA GCTCCTCATC CTCAACAAAC CAATTTGAGT
180
TCACTGAGAC ATCTCCAGAA AATTCCCCAG CATCAACTGA TGTAGATAGT TCAACAATGG
240 '
AACACGCTAG CCAGATTAAA ACTGAGAACG ATGACGTTGA GCCGTCAAGT GATCTTACAG
300
GAAGTGTTGA AGAGCTGGAT TTTGCTTCAT CACTACAACT ACAAGAAGGT GGTAAACTGG
360
AGGAGTCTAA AACATTAAAT ACTTCTGAAG AGACAATTAT TGATGAATCT GATAGGATCA
420
GAGAGAGGGG CATCCCTCCA CCTGGACTTG GTCAGAAGAT TTATGAAATA GACCCCCTTT
480
TGACAAACTA TCGTCAACAC CTTGATTACA GGTATTCACA GTACAAGAAA CTGAGGGAGG
540
CAATTGACAA GTATGAGGGT GGTTTGGAAG CTTTTTCTCG TGGTTATGAA AAAATGGGTT
600
TCACTCGTAG TGCTACAGGT ATCACTTACC GTGAGTGGGC TCCTGGTGCC CAGTCAGCTG
660
CA 02416347 2003-02-11
-57h-
CCCTCATTGG AGATTTCAAC AATTGGGACG CAAATGCTGA CATTATGACT CGGAATGAAT
720
TTGGTGTCTG GGAGATTTTT CTGCCAAATA ATGTGGATGG TTCTCCTGCA ATTCCTCATG 780
GGTCCAGAGT GAAGATACGT ATGGACACTC CATCAGGTGT TAAGGATTCC ATTCCTGCTT
840
GGATCAACTA CTCTTCACAG CTTCCTGATG AAATTCCATA TAATGGAATA TATTATGATC
900
CACCCGAAGA GGAGAGGTAT ATCTTCCAAC ACCCACGGCC AAAGAAACCA AAGTCGCTGA
960
GAATATATGA ATCTCATATT GGAATGAGTA GTCCGGAGCC TAAAATTAAC TCATACGTGA
1020
-
ATTTTAGAGA TGAAGTTCTT CCTCGCATAA AAAAGCTTGG GTACAATGCG GTGCAAATTA
1080
TGGCTATTCA AGAGCATTCT TATTATGCTA GTTTTGGTTA TCATGTCACA AATTTTTTTG 1140
CACCAAGCAG CCGTTTTGGA ACGCCCGACG ACCTTAAGTC TTTGATTGAT AAAGCTCATG
1200
AGCTAGGAAT TGTTGTTCTC ATGGACATTG TTCACAGCCA TGCATCAAAT AATACTTTAG
1260
ATGGACTGAA CATGTTTGAC GGCACCGATA GTTGTTACTT TCACTCTGGA GCTCGTGGTT
1320
ATCATTGGAT GTGGGATTCC CGCCTTTTTA ACTATGGAAA CT'GGGAGGTA CTTAGGTATC
1380
TTCTCTCAAA TGCGAGATGG TGGTTGGATG AGTTCAAATT TGATGGATTT AGATTTGATG
1440 -
GTGTGACATC AATGATGTAT ACTCACCACG GATTATCGGT GGGATTCACT GGGAACTACG
1500
AGGAATACTT TGGACTCGCA ACTGATGTGG ATGCTGTTGT GTATCTGATG CTGGTCAACG
1560
ATCTTATTCA TGGGCTTTTC CCAGATGCAA TTACCATTGG TGAAGATGTT AGCGGAATGC
1620
CGACATTTTG TATTCCCGTT CAAGATGGGG GTGTTGGCTT TGACTATCGG CTGCATATGG
1680
CA 02416347 2003-02-11
-57i-
CAATTGCTGA TAAATGGATT GAGTTGCTCA AGAAACGGGA TGAGGATTGG AGAGTGGGTG
1740
ATATTGTTCA TACACTGACA AATAGAAGAT GGTCGGAAAA GTGTGTTTCA TACGCTGAAA
1800
GTCATGATCA AGCTCTAGTC GGTGATAAAA CTATAGCATT CTGGCTGATG GACAAGGATA
1860
TGTATGATTT TATGGCTCTG GATAGACCGC CAACATCATT AATAGATCGT GGGATAGCAT
1920
TGCACAAGAT GATTAGGCTT GTAACTATGG GATTAGGAGG AGAAGGGTAC CTAAATTTCA
1980
TGGGAAATGA ATTCGGCCAC CCTGAGTGGA TTGATTTCCC TAGGGCTGAA CAAGACCTCT
2040
CTGATGACTC AGTAATTCCC GGAAACCAAT TCAGTTATGA TAAATGCAGA CGGAGATTTG
2100
ACCTGGGAGA TGCAGAATAT TTAAGATACC GTGGGTTGCA AGAATTTGAC CGGGCTATGC
2160
AGTATCTTGA AGATAAATAT GAGTTTATGA CTTCAGAACA CCAGTTCATA TCACGAAAGG
2220 - -
ATGAAGGAGA TAGGATGATT GTATTTGAAA AAGGAAACCT AGTT?TTGTC TTTAATTTTC
2280
ACTGGACAAA AAGCTATTCA GACTATCGCA TAGGCTGCCT GAAGCCTGGA AAATACAAGG
2340
TTGCCTTGGA CTCAGATGAT CCACTTiTTG GTGGCTTCGG GAGAATTGAT CATAATGCCG
2400
AATATTTCAC CTTTGAAGGA TGGTATGATG ATCGTCCTCG TTCAATTATG GTGTAi'GCAC
2460
CTTGTAGAAC AGCAGTGGTC TATGCACTAG TAGACAAAGA AGAAGAAGAA GAAGAAGAAG
2520
AAGAAGAAGT AGCAGTAGTA GAAGAAGTAG TAGTAGAAGA AGAATGAACG AACTTGTG
2578
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
CA 02416347 2003-02-11
-57j-
(B) TYPE: nucieic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xi) SEQUENCE DESCRlPT(ON: SEQ ID NO: 20:
AATTTYATGG GNAAYGARTT YGG 23
