Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02273408 1999-06-24
Case 20161
The present invention relates to liquid and dry phytase formulations having an
'
increased stability, preferably thermostability, which is obtained by the
addition of
stabilizing agents, or by crosslinking.
Although a large amount of phosphate is present in feed in form of phytate
phosphorus, monogastric animals, like pigs and poultry, lack the ability to
use this form of
phosphate. The alkali or earth alkali salts of phytic acid occur naturally
mainly in cereals.
Since monogastric animals are not able to use this form of phosphate it is
common practice
to to add phosphate to animal feed.
On the other hand an enzyme called phytase (myo-inositol hexakisphosphate
phosphohydrolase) is known to occur in plants and in some microorganisms.
Since phytase
can be produced by fermentation it is known in the art to use phytase as an
animal feed
additive in order to enhance the nutritive value of plant material by
liberation of inorganic
phosphate from phytic acid (myo-inositol hexakisphosphate). By adding phytase
to the
animal feed the level of phosphorus pollution of the environment can be
reduced since the
animal is able to make use of the phosphate liberated from phytate by the use
of phytase.
For feed application a stable preferably theumostable phytase is of general
interest in
order to avoid problems that may occur during the formulation (e.g. spray
drying,
granulation) and feed treatment processes (e.g. pelleting, extrusion,
expansion) where
temporarily high temperatures (up to 80-120 °C) and shear stress may
affect the protein
structure and lead to an undesired loss of activity.
AB/cb/So 7.5.99
CA 02273408 1999-06-24
-2-
The international patent application WO 93/16175 of Gist-Brocades describes
stabilized liquid formulations of phytase. It is suggested to use as
stabilizing agent urea and
a water-soluble polyol whereby sorbitol, glycerol and polyethylene glycol
having a
molecular weight of 6000 are mentioned.
It is an object of the present invention to improve the stability, preferably
thermostability of phytase whereby stability is defined as the ability to
retain activity under
various conditions. This stability aspect relates to the entire life cycle of
the enzyme which
comprises production (fermentation, downstream processing, formulation and
heat
treatment of feed), distribution (transport and storage) and final
application. For a
to commercially interesting enzyme like phytase it is important to withstand
the high
temperatures reached during various feed treatment processes like pelleting,
extrusion and
expansion (up to 80-120 °C) and to be stable during long-term storage.
The term "stability" as used in the present invention relates to all the
specifications
of an industrial enzyme which comprise aspects such as activity, specificity,
shelf stability,
t5 mechanical stability, microbial stability, toxicity, chemical composition
and physical
parameters such as density, viscosity, hygroscopy, but also colour, odour and
dust. A
preferred aspect of the present invention relates to the stability of phytase
against thermal
inactivation during formulation and feed treatment processes such as
pelleting, extrusion
and expansion.
A major barner to the wide use of phytases is the constraint of thermal
stability (80-
120 °C) required for these enzymes to withstand inactivation during
feed treatment
processes. The currently available industrial phytases all originate from A.
niger and have a
low intrinsic resistance to heat inactivation. As an alternative or in
addition to molecular
biological approaches the present invention enhances the stability, preferably
thermostability of a protein by the addition of different additives and in
another aspect by
the chemical crosslinking of enzyme monomers to oligomers.
The experiments leading to the present invention were also performed with the
so-
called consensus phytase, a phytase developed according to a theoretical
molecular
biological approach which has a higher intrinsic stability compared with
Aspergillus
phytases, see European Patent Application Publication No. 897 985. In the
practice of the
CA 02273408 1999-06-24
.3-
present invention the consensus phytases specifically described in examples 3 -
13 can
also be used.
The present invention discloses the use of different additives which act as
stabilizing
went on the stability, preferably thermostability of the enzyme.
Regarding the temperature dependence of the specific activity of the non-
formulated
phytases which can preferably be used in the present invention three different
groups can
be formed according to their activity maximum. The activity maximum is reached
at the
following temperatures: for A. fumigatus and A. niger phytase at 55 °C,
for A. terreus CBS
and A. nidulans phytase at 45 °C and for consensus phytase at 65
°C. A temperature of 10- '
1 ~ °C above the determined temperature maximum - where the non-
formulated phytases
were completely inactive - was chosen as screening point for studying the
effect of the
stabilizing agents on the thermostability of phytases, i.e. 60 °C for
A. nidulans and
A. terreus CBS phytase, 65 °C for A. niger and A. fumigatus phytase,
and 75 °C for
consensus phytase.
The present invention provides a stabilized, preferably thermostabilized
enzyme
formulation comprising phytase and at least one stabilizing agent selected
from the group
consisting of:
a) polyols containing five carbon atoms, preferably CS sugars, more preferably
2o xylitol or ribitol,
b) polyethylene glycol having a molecular weight of 600 to 4000 Da,
c) the disodium salts of malonic, glutaric and succinic acid,
d) carboxymethylcellulose, and
e) sodium alginate
The present invention also provides a stabilized, preferably thermostabilized
enzyme
formulation comprising phytases which have been crosslinked:
a) by chemical reactions with glutaraldehyde; or by
CA 02273408 1999-06-24
-4-
b) oxidation with sodium periodate and subsequent addition of adipic
acid dihydrazide
Although it would be possible to use other phytases obtained from other
sources than
microorganisms it is preferred to use a phytase which has been produced by
microorganisms. In the present invention preferably such phytases are used
which are
produced by a fungus, and more preferably from the group consisting of
Aspergillus
f ~migatus, Aspergillus nidulans, Aspergillus terreus, and Aspergillus niger.
Another
phytase preferably used in this invention is the so called consensus phytase.
It is, however,
also possible to produce such phytases by genetic engineering whereby the gene
obtained ,
from a fungus is transferred to a host organism like a bacterium (e.g.
E.coli), a yeast or
another fungus, for further details, see e.g. European Patent Application
Publication No.
684313 and European Patent Application Publication No. 897 010.
The term enzyme formulation comprises all liquid and dry formulations in which
the
enzyme phytase may be commerciallized. Preferably, the source of phytase for
such a
formulation is a rather raw, liquid preparation obtained from the fermentation
broth. For
the preparation of a liquid phytase formulation the selected stabilizing
agents are added or
the phytase is crosslinked. To obtain a stabilized, preferably
thermostabilized dry
formulation the phytase is a) spray dried or granulated in the presence of the
selected
stabilizing agents, or b) chemically crosslinking.
2o In one preferred embodiment the liquid enzyme formulation comprises as
stabilizing
agent polyethylene glycol whereby the polyethylene glycol is present in a
concentration of
10-50% (w/w) in the final formulation.
Preferably the enzyme formulation comprises polyethylene glycol having a
molecular
weight of 1000-3350 Da. It is especially preferred to use a polyethylene
glycol having a
molecular weight of about 1450. Polyethylene glycols with molecular weights
slightly
outside of the preferred range (600 Da and 4000 Da, respectively) showed still
reasonable
effect but are less preferred. The stabilizing effect of polyethylene glycol
was shown to be
molecular weight-dependent (see Figures 2 and 3).
In another preferred embodiment of the present invention the stabilizing agent
is
xylitol or ribitol. Both are sugar alcohols having a five carbon atom
structure. Xylitol and
ribitol are preferably used in a concentration of 20 to 60% (w/w) in the final
liquid
CA 02273408 1999-06-24
-5-
formulation. Surprisingly xylitol and ribitol as stabilizing agents of, e. g.
, A. fumigatus
phytase increased the specific activity measured at 65 °C to 11-12 U/mg
at a concentration
of 12.~%, and to 51-90 U/mg at a concentration of 25% of the polyol (see
Figure 4).
In another embodiment of the present invention the liquid enzyme formulation
comprises as stabilizing agent the disodium salts of glutaric, succinic or
malonic acid
whereby the concentration of the salt in the final formulation ranges between
10 and 30%
(w/w). The addition of malonate, succinate and glutarate at a concentration of
25% resulted
in a significant increase in A. fumigatus phytase thermostability with
considerable activity
still being detected at 70 °C for malonate and 65 °C for
succinate and glutarate as can be
1o seen in Figure 6.
In addition thereto the carboxylates stimulated A. fumigatus phytase activity
measured at 37 °C with an approximately 4-fold increase in phytase
activity beefing
obser,~ed in the case of malonate, a 2-fold increase for succinate and minor
effects for
alutarate. Investigation of different concentrations (5, 10 and 25%) of
malonate showed
that thermostabilization of A. fumigatus phytase is concentration-dependent
whereas
stimulation of enzymatic activity, at least in this concentration range, is
not (see Figure 7).
In contrast to these findings different concentrations (5, 10 and 25%) of
sodium acetate
which is a monocarboxylic acid, caused an up to 2-fold increase in specific
activity of
A. fumigatus phytase at 37 °C, but had only minor effects on the
thermostability of the
2o protein (see Figure 8). Therefore, it may be concluded that carboxylate
groups are
responsible for activity modulation whereas bifunctional dicarboxylates
stabilize phytases
possibly by ionic interactions. The sodium malonate and succinate generally
increased the
thermostability of A. nidulans, A. terreus CBS, A. niger and consensus phytase
by S-15 °C.
On the other hand stimulation of phytase activity was only observed for A.
nidulans and
A. fumigatus phytase both having rather low specific activity but not for A.
terreus CBS,
A. niger and consensus phytase (see Figures 9 and 10).
In another embodiment of the present invention the enzyme formulation
comprises
as stabilizing agent the polymers carboxymethylcellulose and/or sodium
alginate whereby
the concentration in the final liquid formulation is between 1 and 20%
preferably 1 and
10% (w/w). The addition of these polymeres to A. fumigatus phytase
preparations resulted
in a significant 5 to 10% increase of phytase thermostability.
CA 02273408 1999-06-24
-6-
In another embodiment of the present invention the enzyme formulation
comprises
as stabilizing agent alginate, preferably sodium alginate and most preferably
in a
concentration of 1 to 10% (w/w) in the final liquid formulation.
In a further embodiment of the present invention the enzyme formulation
comprises
crosslinked phytase. For the preparation of such a stabilized phytase form,
glutaraldehyde
is added to the phyase at a concentration resulting in an oligomerization of
the protein.
In another embodiment the enzyme formulation comprises phytase which has been
crosslinked via its carbohydrate chains. Crosslinking involves as a first step
periodate
oxidation of the carbohydrate residues followed by reaction of the generated
aldehyde
1o groups with adipic acid dihydrazide.
Depending on the conditions employed, the crosslinking reaction can lead to
various
derivatives of an enzyme, namely
a) modified enzyme molecules that have reacted with only one hydrazide
group of adipic acid dihydrazide,
b) intramolecularly crosslinked enzymes, with or without intermolecular
crosslinking, and
c) intermolecularly crosslinked, soluble oligomers or insoluble polymers.
In most cases the reaction results in a mixture of several forms. Crosslinking
of
A. funrigatus and consensus phytase both expressed in Hansenula polymorpha
resulted in
the formation of oligomeric forms. The degree of crosslinking could be
controlled
effectively by changing the degree of enzyme oxidation. An optimal
thermostabilization of
phytase has been observed at a concentration of SO mM sodium periodate applied
to a S
mg/ml phytase solution. For both phytases an increase in thermostability
between 10 and
15 °C has been observed (see Figure 12). It should be noted that the
oxidized phytases
formed a significant amount of dimers, trimers and tetramers even without
addition of
adipic acid dihydrazide (see Figure 11A).
Another aspect of the present invention concerns the use of the listed
stabilizers as
additives for the production of dry/solid phytase formulations. In this
embodiment of the
present invention the addition of stabilizers (1 to 20% (w/w) of
xylitol/ribitol, 1 to 20%
CA 02273408 1999-06-24
(w/w) of polyethylene glycols with a molecular weight preferably between 1000
and 3350
Da and/or 1 to 20% (w/w) of dicarboxylates like malonate, succinate and
glutarate, and/or
1 to 10% (w/w) of the polymers carboxymethlycellulose and/or alignate,
preferably sodium
alginate disolved in 100-200 ml phytase liquid (crosslinked or non-
crosslinked) or added
as solid compounds to the standard granulation mixture (containing
ligninsulfonat as
binder, silica and gipsum as carrier) Such formulation can result in an
increased recovery
(up to 20%) of phytase activity determined after a high shear granulation
process which
included a drying step of the granulates on a fluid bed dryer at 45°C
for 15 min. In addition
such granulates which contain stabilizers can show, when mixed with feed, an
increased
to recovery of enzymatic activity after the feed treatment (e.g. a pelleting
process at 85°C)
compared to granulates without such additives.
Another aspect of the present invention concerns methods of preparing feed
compositions for monogastric animals, whereby the feed is supplemented with a
thermostabilized dry or liquid enzyme formulation according to any of claims
(1-13). The
phytase supplemented feed can be subjected on several methods of feed
processing like
extrusion, expansion and pelleting, where temporarily high temperatures may
occure and
thermostabilization is an advantage.
The stabilized enzyme formulation of the present invention can be appllied for
example on feed pellets. The thermostabilized liquid enzyme formulation may be
diluted
2o with tap water to yield a solution having the desired activity of phytase
(100 - 200 phytase
units/g solution). The feed pellets can be transferred to a mechanical mixer
and the diluted
enzyme formulation is sprayed onto the feed pellets while being agitated in
order to yield a
homogeneous product with an added phytase activity of for example 500 units
phytase/kg
feed pellets. Alternatively the dry or liquid enzyme formulation can be
directly mixed with
the mash feed before this mixture is then subjected to a process such as
pelleting,
expansion or extrusion.
In a further aspect the present invention concerns a method of providing a
monogastric animal with its dietary requirement of phosphorus wherein the
animal is fed
with a feed according to the present invention and whereby no additional
phosphorus is
3o added to the feed.
The results of the experiments of the present invention are shown in the
following
Figures.
CA 02273408 1999-06-24
_8_
Figure 1. Comparison of the temperature dependence of activity of A.
fumigatus, A. nidulans, A. terreus CBS, A. niger and consensus phytase
measured
under standard assay conditions as described in Example 1.
Figure 2. Effect of different polyethylene glycols on the specific activity
ofA.
s fumigatus phytase at 65 °C.
Figure 3. Effect of 50% solutions of polyethylene glycols with different
molecular weights on the thermostability of A. raiger, consensus, A. terreus
CBS and
A. nidulans phytase. The specific activities were measured at 60 °C for
A. terreus
CBS and A. nidulans phytase, at 65 °C for A. niger phytase and at 75
°C for
io consensus phytase.
Figure 4. Effect of 25 and 50% solutions of different polyols on the specific
activity of A. fumigatus phytase at 65 °C.
Figure 5. Temperature dependence of activity of A. niger (A), consensus (B),
A. nidulans (C) and A. terr-eus CBS (D) phytase in the presence of 50% xylitol
as
1s additive.
Figure 6. Temperature dependence of activity of A. fumigatus phytase in the
presence of 25% concentrations of disodium malonate, succinate and glutarate.
Figure 7. Temperature dependence of activity of A. fumigatus phytase in the
presence of 5, 10 and 25% disodium malonate.
2o Figure 8. Temperature dependence of activity of A. fumigatus phytase in the
presence of 5, 10 and 25% sodium acetate.
Figure 9. Temperature dependence of activity of A. niger (A), consensus (B),
A. terreus CBS (C) and A. nidulans (D) phytase in the presence of 25% disodium
malonate.
CA 02273408 1999-06-24
-9-
Figure 10. Temperature dependence of activity of A. niger (A), consensus (B),
A. terreus CBS (C) and A. nidulans (D) phytase in the presence of 25% disodium
succinate.
Figure 11.
s A) SDS-PAGE of A. fumigatus phytase samples after incubation with different
concentrations of sodium periodate.
B) SDS-PAGE of the different oxidized A. fumigatus phytase samples from
(A) after subsequent crosslinking with adipic acid dihydrazide.
Figure 12 Temperature dependence of activity of A. fumigatus phytase and
io consensus phytase before and after crosslinking with periodate/adipic acid
dihydrazide.
Figure 13 Design of the consensus phytase sequence. The letters represent the
amino acid residues in the one-letter code. The following sequences were used
for
the alignment: phyA from Asper-gillus terreus 9A-1 (Mitchell et al, 1997; from
is amino acid (aa) 27), phyA from A. terreus cbs116.46; (van Loon et al.,
1998; from
as 27), phyA from Aspergillus niger var. awamori (Piddington et al, 1993; from
as
27), phyA from A. niger T213; from as 27), phyA from A. niger strain NRRL3135
(van Hartingsveldt et al, 1993; from as 27), phyA from Aspergillus fumigatus
ATCC
13073 (Pasamontes et al, 1993; from as 25), phyA from A. fumigatus ATCC 32722
20 (van Loon et al, 1998; from as 27), plzyA from A. fumigatus ATCC 58128 (van
Loon et al., 1998; from as 27), phyA from A. fumigatus ATCC 26906 (van Loon et
al, 1998; from as 27), phyA from A. fumigatus ATCC 32239 (van Loon et al,
1998;
from as 30), phyA from Emericella nidulans (Pasamontes et al, 1997a; from as
25),
phyA from Talaromyces thermoplzilus (Pasamontes et al, 1997a; from as 24), and
2s phyA from Myceliophthora thermophila (Mitchell et al, 1997; from as 19).
The
alignment was calculated using the program PILEUP. The location of the gaps
was
refined by hand. Capitalized amino acid residues in the alignment at a given
position belong to the amino acid coalition that establish the consensus
residue. In
bold, beneath the calculated consensus sequence, the amino acid sequence of
the
CA 02273408 1999-09-29
-10-
finally constructed consensus phytase (Fcp) (SEQ ID NO:1) is shown. The gaps
in the
calculated consensus sequence were filled by hand according to principals
stated in Example 3.
Figure 14 DNA (SEQ ID N0:2) and amino acid (SEQ ID N0:3) sequence of the
consensus phytase-1 gene (fcp) and of the primers used for the gene
construction. The
calculated amino acid sequence (Figure 13) was converted into a DNA sequence
using
the program BACKTRANSLATE (Devereux et al., 1984) and the codon frequency
table
of highly expressed yeast genes (GCG program package, 9.0). The signal peptide
of the
phytase from A. terreus cbs. 116.46 was fused to the N-terminus. The bold
bases
represent the sequences of the oligonucleotides used to generate the gene. The
names of
the respective oligonucleotides are alternately noted above or below the
sequence. The
underlined bases represent the start and stop codon of the gene. The bases
written in
italics show the two introduced Eco RI sites.
Figure l~ Alignment and consensus sequence of five Basidiomycetes
phytases. The letters represent the amino acid residues in the one-letter
code. The
i5 amino acid sequences of the phytases from Paxillus involutus, phyAl (aa 21)
and
phyA2 (aa 21, WO 98/28409), Trametes pubescens (aa 24, WO 98/28409),
Agrocybe pediades (aa 19, WO 98/28409), and Peniophora lycii (aa 21, WO
98/28409) starting with the amino acid residues mentioned in parentheses, were
used for the alignment and the calculation of the corresponding consensus
sequence
(SEQ ID N0:4) called "Basidio" (Example 4). The alignment was performed by the
program PILEPUP. The location of the gaps was refined by hand. The consensus
sequence was calculated by the program PRETTY. While a vote weight of 0.5 was
assigned to the two P. involutus phytases, all other genes were used with a
vote weight of
1.0 for the consensus sequence calculation. At positions, where the program
was not able
to determine a consensus residues, the Basidio sequence contains a dash and is
shown as
Xaa in the sequence listing. Capitalized amino acid residues in the alignment
at a given
position belong to the amino acid coalition that establish the consensus
residue.
Figure 16 Design of consensus phytase-10 amino acid sequence (SEQ ID NO:S).
Adding the phytase sequence of Thermomyces lanuginosa (Berka et al., 1998) and
the
30 consensus sequence of the phytases from five Basidiomycetes to the
alignment of
CA 02273408 1999-09-29
-11-
Figure 13, an improved consensus sequence was calculated by the program
PRETTY. Additionally, the amino acid sequence of A. niger T213 was omitted,
therefore, using a vote weight of 0.5 for the remaining A. niger phytase
sequences.
For further information see Example 14.
Figure 17 DNA (SEQ ID N0:6) and amino acid (SEQ ID N0:7) sequence of
consensus phytase-10. The amino acid sequence is written above the
corresponding DNA
sequence using the one-letter code. The sequence of the oligonucleotides which
were used to
assemble the gene are in bold letters. The label of oligonucleotides and the
amino acids,
which were changed compared to those for consensus phytase-1, are underlined
and their
io corresponding triplets are highlighted in small cases. The fcpl0 gene was
assembled
from the following oligonucleotides: CP-l, CP-2, CP-3.10, CP-4.10, CP-5.10, CP-
6,
CP-7.10, CP-8.10, CP-9.10, CP-10.10, CP-11.10, CP-12.10, CP-13.10, CP-14.10,
CP-15.10, CP-16.10, CP-17.10, CP18.10, CP-19.10, CP-20.10, CP-21.10, CP-
22.10. The newly synthesized oligonucleotides are additionally marked by
number
is 10. The phytase contains the following 32 exchanges: Y54F, E58A, D69K,
D70G,
A94K, N134Q, I158V, S187A, Q188N, D197N, S204A, T214L, D220E, L234V,
A238P, D246H, T251N, Y259N, E267D, E277Q, A283D, R291I, A320V, R329H,
S364T, I366V, A379K, S396A, G404A, Q415E, A437G, A463E. The mutations
accentuated in bold letters revealed a stabilizing effect on consensus phytase-
1 as
2o tested as single mutation in consensus phytase-1.
Figure 18 Alignment for the design of consensus phytase-11. In contrast to the
design of consensus phytase-10, for the design of the amino acid sequence (SEQ
ID
N0:8) of consensus phytase-11, all Basidiomycetes phytases were used as
independent
sequences using an assigned vote weight of 0.2 for each Basidiomycetes
sequence.
?s Additionally, the amino acid sequence of A. niger T213 was used in that
alignment,
again.
Figure 19 DNA (SEQ ID N0:9) and amino acid (SEQ ID NO:10) sequence of
consensus phytase-1-thermo[8]-Q50T-K91A. The amino acid sequence is written
above
the corresponding DNA sequence using the one-letter code. The replaced amino
acid
30 residues are underlined. The stop codon of the gene is masked by a star (*)
CA 02273408 1999-09-29
-12-
Figure 20 DNA (SEQ ID NO:11 ) and amino acid (SEQ ID N0:12) sequence of
consensus phytase-10-thermo[3]-QSOT-K91A. The amino acid sequence is written
above
the corresponding DNA sequence using the one-letter code. The replaced amino
acid
residues are underlined. The stop codon of the gene is marked by a star (*)
Figure 21 DNA (SEQ ID N0:13) and amino acid (SEQ ID N0:14) sequence of
A. fumigatus ATCC 13073 phytase a-mutant. The amino acid sequence is written
above
the corresponding DNA sequence using the one-letter code. The replaced amino
acid
residues are underlined. The stop codon of the gene is marked by a star (*)
Figure 22 DNA (SEQ ID NO:15) and amino acid (SEQ ID N0:16) sequence of
to
consensus phytase-7. The amino acids are written above the corresponding DNA
sequence using the one-letter code. The sequence of the oligonucleotides used
to
assemble the gene are in bold letters. Oligonucleotides and amino acids that
were
exchanged are underlined and their corresponding triplets are highlighted in
small cases.
The fcp7 gene was assembled from the following oligonucleotides: CP-l, CP-2,
CP-3,
15 Cp_4.7, CP-5.7, CP-6, CP-7, CP-8.7, CP-9, CP-10.7, CP-11.7, CP-12.7, CP-
13.7, CP-
14.7, CP-15.7, CP-16, CP-17.7, CP-18.7, CP-19.7, CP-20, CP-21, CP-22. The
newly
synthesized oligonucleotides are additionally marked by number 7. The phytase
contains
the following 24 exchanges in comparison to the original consensus phytase:
S89D,
S92G, A94K, D164S, P201S, G203A, G205S, H212P, G224A, D226T, E255T, D256E,
'-o V258T, P265S, Q292H, G300K, Y305H, A314T, S364G, M365I, A397S, S398A,
G-404A, and A405S.
Figure 23 Differential scanning calorimetry (DSC) of consensus phytase-1
and consensus phytase-10. The protein samples were concentrated to ca. 50-60
mg/ml and extensively dialyzed against 10 mM sodium acetate, pH 5Ø A
constant
?s heating rate of 10 °C/min was applied up to 95 °C. DSC of
consensus phytase-10
(upper graph) yielded a melting temperature of 85.4 °C, which is 7.3
°C higher than
the melting point of consensus phytase-1 (78.1 °C, lower graph).
Figure 24 Differential scanning calorimetry (DSC) of consensus phytase-10-
thermo-QSOT and consensus phytase-10-thermo-QSOT-K91A. The protein samples
CA 02273408 1999-06-24
-13-
were concentrated to ca. 50-60 mg/ml and extensively dialyzed against 10 mM
sodium acetate, pH 5Ø A constant heating rate of 10 °C/min was
applied up to 95
°C. DSC of consensus phytase-10-thenno-QSOT (upper graph) yielded a
melting
temperature of 88.6 °C, while the melting point of consensus phytase-10-
thenno
s QSOT-K91A was found at 89.3 °C.
Figure 2~ Comparison of the temperature optimum between consensus
phytase-1, consensus phytase-10 and consensus phytase-10-thermo-QSOT. For the
determination of the temperature optimum, the phytase standard assay was
performed at a series of temperatures between 37 and 86 °C. The diluted
to supernatant of transformed S. cerevisiae strains was used for the
determination. The
other components of the supernatant showed no influence on the determination
of
the temperature optimum: n, consensus phytase-1; 4, consensus phytase-10; ~,
consensus ph5~tase 10-thermo-QSOT.
Figure 26 pH-dependent activity profile and substrate specificity of consensus
~s phytase-10 and its variants thenno-QSOT and thermo-QSOT-K91A. The phytase
activity was determined using the standard assay in appropriate buffers (see
Example 11 ) at different pH-values. Graph ~ a) shows the pH-dependent
activity
profile of consensus phytase-10 (0 ), consensus phytase-10-thermo-QSOT ( . ),
and
consensus ph5~tase-10-thenno-QSOT-K91A (n). Graph b) shows the corresponding
2o substrate specificity tested by replacement of phytate by the indicated
compounds in
the standard assay; open bars, consensus phytase-10 (grey bars, consensus
phytase-
10-thermo-Q~OT; dark bars, consensus phytase-10-thenno-QSOT-K91A). The
numbers correspond to the following compounds: 1, phytate; 2, p-nitrophenyl
phosphate; 3, phenyl phosphate; 4, fructose-1,6-bisphosphate; 5, fructose-6-
25 phosphate; 6, glucose-6-phosphate; 7, ribose-S-phosphate; 8, DL-glycerol-3-
phosphate; 9, glycerol-2-phosphate; 10, 3-phosphoglycerate; 11,
phosphoenolpyruvate; 12, AMP; 13, ADP; 14, ATP.
Figure 27 pH-dependent activity profile and substrate specificity of consensus
phytase-1-thenno[8]-QSOT and of consensus phytase-1-thermo[8]-QSOT-K91A.
CA 02273408 1999-06-24
-14-
The phytase activity was determined using the standard assay in appropriate
buffers
(see Example 11) at different pH-values. Graph a) shows the pH-dependent
activity
profile of the QSOT- ( t ) and the QSOT-K91 A-variant ( . ). Graph b) shows
the
corresponding substrate specificities tested by replacement of phytate by the
s indicated compounds in the standard assay (open bars, consensus phytase-1-
thermo[8]-QSOT; filled bars, consensus phytase-1-thel-lno[8]-QSOT-K91A.). The
substrates are listed in the legend of Figure 26.
Figure 28 Differential scanning calorimetry (DSC) of consensus phytase-1-
thermo[8]-QSOT and consensus phytase-1-thermo[8]-QSOT-K91A. The protein
to samples were concentrated to ca. 50-60 mg/ml and extensively dialyzed
against 10
mM sodium acetate, pH 5Ø A constant heating rate of 10 °C/min was
applied up to
95 °C. DSC of consensus phytase-1-thermo[8]-QSOT (upper graph) showed a
melting temperature of 84.7 °C, while the melting point of consensus
phytase-1-
thermo[8]-QSOT-K91A was found at 85.7 °C.
is Figure 29 Comparison of the temperature optimum between consensus
phytase-1, consensus phytase-1-thermo[3] and consensus phytase-1-thermo[8].
For
the determination of the temperature optimum, the phytase standard assay was
performed at a series of temperatures between 37 and 86 °C. Purified
protein from
the supernatant of transformed S. cerevisiae strains was used for the
determination.
2o O, consensus phytase-1; ~, consensus phytase-1-thermo[3]; _ , consensus
phytase
1-thermo[8].
Figure 30 Comparison of the pH-dependent activity profile and substrate
specificity of consensus phytase-1, consensus phytase-7, and of the phytase
from A.
niger NRRL 3135. The phytase activity was determined using the standard assay
in
2s appropriate buffers (see Example 11 ) at different pH-values. Graph a)
shows the
pH-dependent activity profile of consensus phytase-1 (t), the phytase from A.
niger NRRL 3135 (O), and of consensus phytase-7 (_). Graph b) shows the
corresponding substrate specificity tested by replacement of phytate by the
indicated
compounds in the standard assay (black bars, A. niger NRRL 3135 phytase; grey
CA 02273408 1999-06-24
-15-
bars, consensus phytase-1, dashed bars, consensus phytase-7). The substrates
are
listed in the legend of Figure 26.
Figure 31 Differential scanning calorimetry (DSC) of the phytase from A.
fumigatus ATCC 13073 and of its stabilized a-mutant, which contains the
s following amino acid exchanges FSSY, V100I, F114Y, A243L, S265P, N294D.
The protein samples were concentrated to ca. 50-60 mg/ml and extensively
dialyzed
against 10 mM sodium acetate, pH 5Ø A constant heating rate of 10
°C/min was
applied up to 9~ °C. DSC of consensus A. fumigatus 13073 phytase (upper
graph) .
revealed a melting temperature of 62.5 °C, while the melting point of
the a-mutant
io was found at 67.0 °C
Figure 32 Comparison of the temperature optimum of A. fumigatus 13073
wild-type, its A. fumigatus a-mutant, and a further stabilized a-mutant (E59A-
S 126N-R329H-S364T-G404A). For the determination of the temperature optimum,
the phytase standard assay was performed at a series of temperatures between
37
is and 75 °C. The diluted supernatant of transformed S. cerevisiae
strains was used for
the determination. The other components of the supernatant showed no influence
on the determination of the temperature optimum. O, A. fumigatus ATCC 13073
phytase; _, A. fumigatus ATCC 13073 a-mutant; ~, A. fumigatus ATCC 13073
alpha-mutant-(E59A-S 126N-R329H-S364T-G404A)-Q27T; t , A. fumigatus
2o ATCC 13073 a-mutant-(E59A-S 126N-R329H-S364T-G404A)-Q27T-K68A. Q27T
and K68A corresponds to consensus phytase-1 QSOT and K91A, respectively.
Figure 33 Amino acid sequence of consensus phytase 12 (consphyl2) which
contains a number of active site residues transferred from the "basidio"
consensus
sequence to consensus phytase-10-thermo-QSOT-K91A.
CA 02273408 1999-06-24
-16-
Example 1
a) Materials
Phytic acid (dodecasodium salt) and polyethylene glycols, polyols, sodium
dicarboxylates, sodium periodate, adipic acid dihydrazide and other additives
were
purchased from commercial suppliers. All other chemicals were at least of
analytical grade.
Five-ml HiTrap desalting columns were obtained from Pharmacia. SDS-PAGE gels
(4-
12% NuPAGE Bis-Tris Pre-Cast) and buffers were delivered by NOVEX.
b) Expression and purification of phytases
.~. fumigatus, A. terreus CBS phytase and consensus phytase were overexpressed
in
Hansenula polymorpha. A. niger and A. nidulans phytase were overexpressed in
A. niger.
Cloning, purification and characterization of these phytases was previously
described by
Pasamontes et al. [Appl. Environ. Microbiol. (1997), 63, p. 1696-1700].
Construction,
cloning and purification of consensus phytase were performed according to
European
Patent Application Publication No. 897 985. Non-formulated consensus phytase
had an
increased thermal stability of up to 70 °C and, due to an amino acid
exchange (L at
position 50 for Q), a three-fold higher specific activity compared to A.
fumigatus phytase.
c) Phytase activity assay
For the determination of thermostability the enzymatic activity measurements
with
phytic acid were done at different temperatures by diluting the purified
enzymes to 0.05
U/ml (activities measured at 37 °C) in 0.2 M sodium acetate, pH 5.0 (+/-
additives in %
w/w). An aliquot of the protein solution (250 pl) was preincubated for 5 min
at the desired
temperature, followed by addition of an equal volume of a solution containing
1 % phytic
acid in 0.2 M sodium acetate, pH 5.0 (preincubated as a 10 ml aliquot for 10
min at the
same temperature). After incubation of the sample for 15 min at the desired
temperature
(e.g. at 60 or 65 °C for the screening of additive effects), the
reaction was stopped by
addition of
0.5 ml 15% trichloroacetic acid. Determination of liberated inorganic
phosphate was
performed by standard methods.
CA 02273408 1999-06-24
-17-
d) Evaluation of thermostabilizing additives
In general, the polyols have been dissolved at a concentration of 25 or 50%
(w/w) in
0.2 M sodium acetate, pH 5Ø PEGS have been dissolved at a concentration of
50% with
the exception of PEGs with a molecular weight of 4000, 8000 and 10000 which
were used
at a concentration of 25%. For the screening of PEGs and other polyols, the
preincubation
and reaction temperature was chosen as 60 °C for A. nidulans and A.
terreus CBS phytase,
65 °C for A. fumigatus and A. niger phytase and 75 °C for
consensus phytase.
Disodium malonate, succinate and glutarate were dissolved at concentrations of
5, 10
and 25% and phyase activity was measured after preincubation of enzyme plus
additive
1o and substrate (see above) at the following temperatures: 37, 45, S0, 55,
60, 65, 70, 75, 80,
and 85 °C. In the same way, the temperature dependence of the activity
of different
phytases in the presence of 25% xylitol and ribitol was tested. It should be
noted that the
concentration of the additives was reduced by half after substrate addition.
e) Crosslinking of carbohydrate chains
Crosslinking of phytase carbohydrate chains was performed as described for
invertase by Cesi et al. [Studies in Organic Chemistry 47: Stability and
Stabilization of
Enzymes, Proceedings of an International Symposium held in Maastricht, The
Netherlands, 1992, Elsevier Science Publications B.V., Amsterdam, The
Netherlands].
Phytase samples (S mg protein/ml) were incubated for 2 h at 30 °C in
the presence of
different concentrations (0, 5, 10, 20, 30, 40 and 50 mM) of sodium periodate
in 0.2 M
sodium acetate, pH 5.0, and stored at 4 °C overnight. Each sample was
desalted on a 5-ml
HiTrap desalting column (Pharmacia) connected to an AktaExplorer system
(Pharmacia),
using 0.2 M sodium acetate, pH 5.0, as elution buffer. Crosslinking was
achieved by
adding 100 ul of 0.5 M adipic acid dihydrazide dissolved in 0.2 M sodium
acetate, pH 5.0,
to 900 pl of the desalted oxidation products. Phytase activity measurements
and gel
electrophoresis of the samples were performed after both the oxidation and
crosslinking
steps.
f) High-shear granulation of thermostabilized phytases
100-250 ml of a phytase solution (in total 2500 - 5000 units of crosslinked or
non-
3o crosslinked phytase) were added to 1 kg of a dry mixture of 5-10% calcium
lignosulfonate
CA 02273408 1999-06-24
-18-
(Borregard, Norway), 5-20% silica (Sipernat SOS, Degussa, Germany), 0-20%
thermostabilizing agent and gipsum. During the high-shear granulation process
water was
added until granulates with desired properties were formed. The granulates
were dried in a
fluid bed dryer for 15 min at 45 °C and subsequently fat coated with
natural palm fat
(Palm 46, Florin, Basel, Switzerland).
g) Pelleting stability of thermostabilized dry and liquid phytase formulations
Thermostabilized dry or liquid formulations of phytases (as mentioned above)
were
mixed with feed and subsequently pelleted under steam conditioning at 85
°C. The
pelleting stability of ph~rtase was determined by measurement of the phytase
activity both
in the mash before pelleting and in the delivered pellets.
Example 2
Investigations of the temperature dependence of activity of different fungal
phytases
as described in Example 1 revealed activity maxima at the following
temperatures: 55 °C
for A. fumigatus phytase and A. niger phytase, 45 °C for A. terrreus
CBS phytase and
A. nidulans phytase, and 65 °C for consensus phytase. A temperature 10-
15 °C above the
determined temperature maximum was chosen as screening point for studying the
effects
of polyols, polyethylene glycols, dicarboxylates, carboxymethylcellulose and
sodium
alginate on the thermostability of phytases.
a) Addition of polyethylene glycols of different molecular weights
The addition of 50% or 25% (25% and 12.5% final concentration during the
reaction
period) polyethylene glycol enhanced the specific activity of A. fumigatus
phytase
measured at 65 °C in a molecular weight-dependent fashion, with a
maximum being
observed with PEG 1450 (specific activity 80 U*(mg protein)-') and
considerable activities
also with PEG 1000 (SO U*(mg protein)'') and PEG 3350 (42 U*(mg protein)'').
The
results of this experiment are summarized in Figure 2.
PEGs with molecular weights of 600, 1000, 1450, 3350 and
4000 Da showed similar effects on the other phytases tested. The results of
this experiment
are shown in Figure 3.
CA 02273408 1999-06-24
-19-
b) Addition of polyols
The polyols ribitol, xylitol (CS sugars) and sorbitol (C6 sugar) in
concentrations of 25
and ~0% significantly improved the thermostability of A. fumigatus phytase.
This is shown
in Figure 4.
Erythritol, mannitol, mannoheptulose and mannoheptose were not soluble in 0.2
M
sodium acetate, pH 5.0, at a concentration of 50% (wlw) and, therefore, only
the 25%
values are shown. The specific activities measured at 65 °C were 11, 21
and 11 U*(mg
protein)'' in the presence of 25% ribitol, xylitol and sorbitol, and 51, 90
and 74 U*(mg
protein)'' in the presence of 50% solutions of ribitol, xylitol and sorbitol,
respectively.
1o Polyols containing more than 6 or less than 5 carbon atoms such as glycerol
(C3
sugar), erythritol (C4 sugar), mannoheptose and mannoheptulose (C7 sugars)
showed an
inferior effect on the thermostabilization of A. fumigatus phytase.
Xylitol at a concentration of 50% also increased the temperature optimum of
A. nidulans, A. terreus CBS, A. niger and consensus phytase by 10-15
°C. The results are
shown in Figure ~.
c) Addition of dicarboxylic acids
Malonate, succinate and glutarate at a concentration of 25% (12.5% final
concentration in the activity assay) resulted in a significant increase in A.
fumigatus
2o phytase thermostability with considerable activity still being detected at
70 °C for malonate
and at 65 °C for succinate and glutarate. The results are shown in
Figure 6.
In addition, dicarboxylates stimulated A. fumigatus phytase activity measured
at 37
°C, with an approximately 4-fold increase in phytase activity in the
case of malonate, a 2-
fold increase for succinate and minor effects for glutarate. Investigation of
different
concentrations (~, 10 and 25%) of malonate showed that thermostabilization of
A. fumigatus phytase is concentration-dependent whereas stimulation of
enzymatic
activity, at least in this concentration range, is not. This is shown in
Figure 7.
In contrast to these findings, different concentrations of sodium acetate (S,
10 and
25%), a monocarboxylic acid, caused a 2-fold increase in specific activity of
A. fumigatus
CA 02273408 1999-06-24
-20-
phytase at 37 °C, but had only minor effects on the thermal stability
of the protein. This
can be seen in Figure 8.
Disodium malonate and succinate generally increased the thermostability of
.4. nidulans, A. terreus CBS, A. niger and consensus phytase by 5-15
°C. On the other hand,
stimulation of phy2ase activity was only observed for A. nidulans and A.
fumigatus phytase,
both having a rather low specific activity, but not for A. terreus CBS, A.
niger and
consensus phytase. This is demonstrated in Figures 9 and 10.
d) Effect of crosslinking
In a preliminary experiment, A. fumigatus phytase monomers were crosslinked by
incubation with ~lutaraldehyde. The resulting thermostabilization measured at
60 °C
reached a maximum after
1 hr reaction time but led to activity loss (measured at 37 °C). In a
further set of
experiments, A. fi~migatus phytase monomers were crosslinked via their
carbohydrate
chains. This type of crosslinking was achieved with only minor loss of
specific activity (<
10%) and resulted in the formation of oligomeric forms at sodium periodate
concentrations
above 15 mM. This can be seen from Figure 11.
The extent of thermostabilization was dependent on periodate concentration and
reached a maximum at 50 mM where high specific activities were observed up to
75 °C
(see Figure 12). A pronounced effect of phytase oligomerization on
thermostability was
also observed for consensus phytase crosslinked via its carbohydrate chains.
This can be
seen from Figure 12.
In the present work, we focused our efforts on the thermostabilization effects
of low-
M~ additives - which are highly recommended for stabilization of industrial
enzymes - and
of chemical modification - even though this latter approach is commonly
regarded as less
attractive for technical and economical reasons.
We have found thermostabilization by CS sugars for a range of different
phytases
expressed in filamentous fungi (A. niger) or yeasts (Hansenula polymorpha).
The increase
in thermostability varied to some extent between the different phytases, but
was around 10
°C. The effect of PEGs was molecular weight-dependent. The optimal
thermostabilization
CA 02273408 1999-06-24
-21 -
of all phytases was obtained with PEGs having a molecular weight between 1000
and 3350
Da.
Sodium acetate, a monocarboxylic acid and main component of the standard
phytase
activity assay, caused a concentration-dependent increase in A. fumigatus
phytase activity,
but had no effect on phytase thermostability. Therefore, carboxylate groups
might be
responsible for the activity modulation whereas bifunctional dicarboxylates
possibly
stabilize phytases by ionic interactions.
Example 3
1o Design of the amino acid sequence of consensus phytase-1
Alignment of the amino acid sequences
The alignment was calculated using the program PILEUP from the Sequence
Analysis Package Release 9.0 (Devereux et al., 1984) with the standard
parameter (gap
creation penalty 12, gap extension penalty 4). The location of the gaps was
refined using a
text editor. Table 1 shows the sequences (see Figure 13) without the signal
sequence that
were used for the performance of the alignment starting with the amino acid
(aa) as
mentioned in Table 1.
Table l: Origin and vote weight of the phytase amino acid sequences used for
the design of
consensus ~hytase-1
2o - phyA from Aspergillus terreus 9A-1, as 27, vote weight 0.5 (Mitchell et
al., 1997)
- phyA from Aspergillus terreus cbs116.46, as 27, vote weight 0.5 (van Loon et
al., 1998)
- phyA from Asper-gillus niger var. awamori, as 27, vote weight 0.33
(Piddington et al.,
1993)
- phyA from Aspergillus niger T213, as 27, vote weight 0.33
- phyA from Aspergillus niger strain NRRL3135, as 27, vote weight 0.33 (van
Hartingsveldt et al., 1993)
CA 02273408 1999-06-24
-22-
- phyA from Aspergillus fumigatus ATCC 13073, as 26, vote weight 0.2
(Pasamontes et
al., 1997)
phy.A from Aspergillus fumigatus ATCC 32722, as 26, vote weight 0.2 (van Loon
et al.,
1998)
- phyA from Aspergillus fumigatus ATCC 58128, as 26, vote weight 0.2 (van Loon
et al.,
1998)
plzyA from Aspergillus fumigatus ATCC 26906, as 26, vote weight 0.2 (van Loon
et al.,
1998) ,
- phyA from Aspergillus funTigatus ATCC 32239, as 30, vote weight 0.2 (van
Loon et al.,
1998)
- phyA from Emericella nidulans , as 25, vote weight 1.0, Pasamontes et al.,
1997a)
- phyA from Talaromyces thermophilus ATCC 20186, as 24, vote weight 1.0
(Pasamontes
et al., 1997a)
- phyA from Myceliophthora thermophila, as 19, vote weight 1.0 (Mitchell et
al., 1997)
Calculation of the amino acid sequence of consensus
phytase-1
Using the refined alignment as input, the consensus sequence was calculated by
the
program PRETTY from the Sequence Analysis Package Release 9.0 (Devereux et
al.,
1984). PRETTY prints sequences with their columns aligned and can display a
consensus
sequence for an alignment. A vote weight that pays regard to the similarity
between the
amino acid sequences of the phytases aligned was assigned to all sequences.
The vote
weight was set such as the combined impact of all phytases from one sequence
subgroup
(same species, but from different strains), e. g. the amino acid sequences of
all phytases
from A. fumigatus, on the election was set one, that means that each sequence
contributes
with a value of 1 divided by the number of strain sequences (see Table 1). By
this means, it
was possible to prevent that very similar amino acid sequences, e. g. of the
phytases from
different A. fumigatus strains, dominate the calculated consensus sequence.
CA 02273408 1999-06-24
-23-
The program PRETTY was started with the following parameters: The plurality
defining the number of votes below which there is no consensus was set on 2Ø
The
threshold, which determines the scoring matrix value below which an amino acid
residue
may not vote for a coalition of residues, was set on 2. PRETTY used the
PrettyPep.Cmp
consensus scoring matrix for peptides.
Ten positions of the alignment (position 46, 66, 82, 138, 162, 236, 276, 279,
280,
308; Figure 13), for which the program was not able to determine a consensus
residue,
were filled by hand according to the following rules: if a most frequent
residue existed, this
residue was chosen (138, 236, 280); if a prevalent group of similar or
phylogenetically
equivalent residues occurred, the most frequent or, if not available, one
residues of this
croup was selected (46, 66, 82, 162, 276, 308). If there was either a
prevalent residue nor a
prevalent group, one of the occurnng residues was chosen according to common
assumption on their influence on the protein stability (279). Eight other
positions (132,
170, 204, 211, 275, 317, 384, 447; Figure 13) were not filled with the amino
acid residue
selected by the program but normally with amino acids that occur with the same
frequency
as the residues that were chosen by the program. In most cases, the slight
underrating of
the three A. niger sequences (sum of the vote weights: 0.99) was eliminated by
this
corrections.
Conversion of the consensus phytase-1 amino acid sequence to a DNA sequence
The first 26 amino acid residues of A. terreus cbs116.46 phytase were used as
signal
peptide and, therefore, fused to the N-terminus of all consensus phytases. For
this stretch,
we used a special method to calculate the corresponding DNA sequence. Purvis
et al
(1987) proposed that the incorporation of rare codons in a gene has an
influence on the
folding efficiency of the protein. Therefore, at least the distribution of
rare codons in the
signal sequence of A. terreus cbs116.46, which was used for the consensus
phytase and
which is very important for secretion of the protein, but converted into the
S. cerevisiae
codon usage, was transferred into the new signal sequence generated for
expression in S.
cerevisiae. For the remaining parts of the protein, we used the codon
frequency table of
highly expressed S. cerevisiae genes, obtained from the GCG program package,
to translate
the calculated amino acid sequence into a DNA sequence.
The resulting sequence of the fcp gene is shown in Figure 14.
CA 02273408 1999-09-29
-24-
Construction and cloning of the consensus phytase-1 gene
The calculated DNA sequence of consensus phytase-1 (f'cp) was divided into
oligonucleotides of 85 bp, alternately using the sequence of the sense and the
anti-sense
strand. Every oligonucleotide overlaps 20 by with its previous and its
following
oligonucleotide of the opposite strand. The location of all primers, purchased
by
Microsynth, Balgach (Switzerland) and obtained in a PAGE-purified form, is
indicated in
Figure 14.
PCR-Reactions
In three PCR reactions, the synthesized oligonucleotides were composed to the
entire
gene. For the PCR, the High Fidelity Kit from Boehringer Mannheim (Boehringer
Mannheim, Mannheim, Germany) and the thermo cycler The ProtokolTM from AMS
Biotechnology (Europe) Ltd. (Lugano, Switzerland) was used.
Oligonucleotide CP-1 to CP-10 (Mix 1, Figure 14) were mixed to a concentration
of
0.2 p?vlol/~tl of each oligonucleotide. A second oligonucleotide mixture (Mix
2) was
prepared with CP-9 to CP-22 (0.2 pMol/~tl of each oligonucleotide).
Additionally, four
short primers were used in the PCR reactions:
CP-a: Eco RI
5'-TATATGAA TTCATGGGCGTGTTCGTC-3' SEQ ID N0:17
CP-b:
5'-TGAAAAGTTCATTGAAGGTTTC-3' SEQ ID N0:18
CP-c:
5'-TCTTCGAAAGCAGTACAAGTAC-3' SEQ ID N0.19
CP-e: Eco RI
5'-TATATGAATTCTTAAGCGAAAC-3' SEQ ID N0:20
CA 02273408 1999-06-24
-25-
PCR reaction a: 10 ul Mix 1 (2.0 pmol of each oligonucleotide)
2 ~1 nucleotides (10 mM each nucleotide)
2 pl primer CP-a (10 pmol/~tl)
2 pl primer CP-c (10 pmol/~tl)
10,0 pl PCR buffer
0.75 pl polymerase mixture
73.25 ~1 H20
PCR reaction b: 10 ~1 Mix 2 (2.0 pmol of each oligonucleotide)
2 ~1 nucleotides (10 mM each nucleotide)
2 pl primer CP-b (10 pmol/~tl)
2 ~1 primer CP-a (10 pmol/~tl)
10,0 pl PCR buffer
0.75 pl polymerase mixture (2.6 U)
73.25 pl H20
Reaction conditions for PCR reaction a and b:
step 1 2 min - 45C
step 2 30 sec - 72C
step 3 30 sec - 94C
step 4 30 sec - 52C
2o step 5 1 min - 72C
Step 3 to 5 were repeated 40-times.
CA 02273408 1999-06-24
-26-
The PCR products (670 and 905 bp) were purified by an agarose gel
electrophoresis
(0.9%o agarose) and a following gel extraction (QIAEX II Gel Extraction Kit,
Qiagen,
Hilden, Germany). The purified DNA fragments were used for the PCR reaction c.
PCR reaction c: 6 pl PCR product of reaction a (=50 ng)
6 pl PCR product of reaction b (=50 ng)
2 ~tl primer CP-a (10 pmol/~tl)
2 p1 primer CP-a (10 pmol/~tl)
10,0 pl PCR buffer
0.75 pl polymerase mixture (2.6 U)
to 73.25 pl H20
Reaction conditions for PCR reaction c:
step 1 2 min - 94°C
step 2 30 sec - 94°C
step 3 30 sec - 55°C
step 4 1 min - 72°C
Step 2 to 4 were repeated 31-times.
The resulting PCR product (1.4 kb) was purified as mentioned above, digested
with
Eco RI, and ligated in an Eco RI-digested and dephosphorylated pBsk(-)-vector
(Stratagene, La Jolla, CA, USA). 1 ~tl of the ligation mixture was used to
transform E. coli
2o XL-1 competent cells (Stratagene, La Jolla, CA, USA). All standard
procedures were
carned out as described by Sambrook et al. (1987). The DNA sequence of the
constructed
consensus phytase gene (~'cp, Figure 14) was controlled by sequencing as known
in the art.
Example 4
Design of an improved consensus~hytase (consensusphytase-101 amino acid
sequence
The alignments used for the design of consensus phytase-10 were calculated
using
the program PILEUP from the Sequence Analysis Package Release 9.0 (Devereux et
al.,
1984) with the standard parameter (gap creation penalty 12, gap extension
penalty 4). The
location of the gaps was refined using a text editor.
CA 02273408 1999-06-24
-27-
The following sequences were used for the alignment of the Basiodiomycetes
phytases
starting with the amino acid (aa) mentioned in Table 2:
Table 2: Origin and vote weight of five Basidiomycetes p)~tases used for the
calculation of
the correspondins amino acid consensus sequence (basidiol
- ph3Al from Paxillus involutus NN005693, as 21, vote weight 0.5 (WO 98/28409)
- ply:A2 from Paxillus involutus NN005693, as 21, vote weight 0.5 (WO
98/28409)
phyA from Trametes pubescens NN9343, as 24, vote weight 1.0 (WO 98/28409)
- phyA from Agrocybe pediades NN009289, as 19, vote weight 1.0 (WO 98/28409)
- phy:~ from Peniophora lycii NN006113, as 21, vote weight 1.0 (WO 98/28409)
1o The alignment is shown in Figure 3.
In Table 3 the genes, which were used for the performance of the final
alignment, are
arranged. The first amino acid (aa) of the sequence which is used in the
alignment is
mentioned behind the organism designation.
Table 3: Origin and vote weight of the ph ase sequences used for the design of
consensus
phytase 10
- phy~A from Aspergillus terreus 9A-1, as 27, vote weight 0.5 (Mitchell et
al., 1997)
- phy:A from Aspergillus terreus cbs 116.46, as 27, vote weight 0.5 (van Loon
et al., 1998)
- phyA from Aspergillus niger var. awamori, as 27, vote weight 0.5 (Piddington
et al.,
1993)
- phyA from Aspergillus niger strain NRRL3135, as 27, vote weight 0.5 (van
Hartingsveldt
et al., 1993)
- phyA from Aspergillus fumigatus ATCC 13073, as 26, vote weight 0.2
(Pasamontes et
al., 1997)
- phyA from Aspergillus fumigatus ATCC 32722, as 26, vote weight 0.2 (van Loon
et al.,
1998)
- phyA from Aspergillus fumigatus ATCC 58128, as 26, vote weight 0.2 (van Loon
et al.,
1998)
CA 02273408 1999-06-24
- 28 _
- ph~:~ from Aspergillus fumigatus ATCC 26906, as 26, vote weight 0.2 (van
Loon et al.,
1998)
- ph3:-1 from Aspergillus fumigatus ATCC 32239, as 30, vote weight 0.2 (van
Loon et al.,
1998)
- phy~ from Emericella nidulans , as 25, vote weight 1.0, Pasamontes et al.,
1997a)
- phyA from Talaromyces thermophilus ATCC 20186, as 24, vote weight 1.0
(Pasamontes
et al., 1997a)
- phyA from M3~celiophthora thermophila, as 19, vote weight 1.0 (Mitchell et
al., 1997)
- phyA from Thermomyces lanuginosa, as 36, vote weight 1.0 (Berka et al.,
1998)
to - Consensus sequence of five Basidiomycetes phytases, vote weight 1.0
(Basidio, Figure
15)
The corresponding alignment is shown in Figure 16.
Calculation of the amino acid sequence of consensus-10
To improve the alignment, we added the original consensus sequence of five
phytases from four different Basidiomycetes, called Basidio, still containing
the undefined
sequence positions (see Figure 15), nearly all phytase sequences used for
calculation of the
original consensus phytase and one new phytase sequence from the Ascomycete
Thermornyces lanuginosa to a larger alignment. Using the consensus sequence of
the
basidiomycetal phytase sequences, does not pay regard to the diversity among
the five
amino acid sequences, but pays regard to the common and different amino acid
residues
between the phytases from the Ascomycetes and the Basidiomycetes.
We set plurality on 2.0 and threshold on 3. The used vote weight are listed in
Table 3. The
alignment and the corresponding consensus sequence is presented in Figure 16.
The new
consensus phytase sequence has 32 different amino acids in comparison to the
original
consensus phytase. Positions for which the program PRETTY was not able to
calculate a
consensus amino acid residue were filled according to rules mentioned in
Example 3. None
of the residues suggested by the program was replaced.
CA 02273408 1999-06-24
-29-
Furthermore, we included all Basidiomycetes phytases as single amino acid
sequences but
assigning a vote weight of 0.2 in the alignment. The corresponding alignment
is shown in
Figure 18. The calculated consensus amino acid sequence (consensus phytase-11)
has the
following differences to the sequence of consensus phytase-10. Letter X means
that the
program was not able to calculate a consensus amino acid; the amino acid in
parenthesis
corresponds to the amino acid finally included into the consensus phytase-10.
D35X, X(K)69K, X(E)100E, AlOlR, Q134N, X(K)153N, X(H)190H, X(A)204S,
X(E)220D, E222T, V227A, X(R)271R, H287A, X(D)288D, X(K)379K, X(I)389I, E390X,
X(E)415E, X(A)416A, X(R)446L, E463A, whereas the numbering is as in Fig. 17.
~'Ve also checked single amino acid replacements suggested by the improved
consensus
sequences 10 and 11 on their influence on the stability of the original
consensus phytase.
The approach is described in example 5.
Conversion of consensus phytase-10 amino acid sequence to a DNA sequence
The first 26 amino acid residues of A. terreus cbs116.46 phytase were used as
signal
peptide and, therefore, fused to the N terminus of consensus phytase-10. The
used
procedure is further described in Example 3.
The resulting sequence of the fcpl0 gene is shown in Figure 17.
Construction and cloning of the consensus phytase-10 gene (f'cpl0)
2o The calculated DNA sequence of fcpl0 was divided into oligonucleotides of
85 bp,
alternately using the sequence of the sense and the anti-sense strand. Every
oligonucleotide
overlaps 20 by with its previous and its following oligonucleotide of the
opposite strand.
The location of all primers, purchased by Microsynth, Balgach (Switzerland)
and obtained
in a PAGE-purified form, is indicated in Figure 17.
PCR-Reactions
In three PCR reactions, the synthesized oligonucleotides were composed to the
entire
gene. For the PCR, the High Fidelity Kit from Boehringer Mannheim (Boehringer
Mannheim, Mannheim, Germany) and the thermo cycler The Protokol from AMS
CA 02273408 1999-09-29
-30-
Biotechnology (Europe) Ltd. (Lugano, Switzerland) was used. The following
oliQonucleotides were used in a concentration of 0.2 pMol/ml.
~Zix 1.10: CP-1, CP-2, CP-3.10, CP-4.10, CP-5.10, CP-6, CP-7.10, CP-8.10, CP-
9.10, CP-
10.10
'~Zix 2.10: CP-9.10, CP-11.10, CP-12.10, CP-13.10, CP-14.10, CP-15.10, CP-
16.10, CP-
17.10. CP18.10, CP-19.10, CP-20.10, CP-21.10, CP-22.10
The newly synthesized oligonucleotides are marked by number 10. The phytase
contains
the following 32 exchanges, which are underlined in Fijure 17, in comparison
to the v
original consensus phytase: Y~4F, E58A, D69K, D70G, A94K, N134Q, I158V, S187A,
to Q188\T, D197\T, S204A, T214L, D220E, L234V, A238P, D246H, T251N, Y259N,
E267D, E277Q, A283D, R291I, A320V, R329H, S364T, I366V, A379K, S396A, G404A,
Q41~E, A437G, A463E.
Four short PCR primer were used for the assembling of the oligonucleotides:
CP-a: Eco RI
~ 5 5'-TATATGA.=1 TTCATGGGCGTGTTCGTC-3'
CP-b:
5'-TGAAAAGTTCATTGAAGGTTTC-3'
CP-c.10:
5'-TCTTCGAAAGCAGTACACAAAC-3' SEQ ID N0:21
2o CP-e: Eco RI
5'-TATATGAA TTC'ITAAGCGAAAC-3'
PCR reaction a: 10 ~tl Mix 1.10 (2.0 pmol of each oligonucleotide)
2 ~tl nucleotides (10 mM each nucleotide)
2 ~tl primer CP-a (10 pmol/ml)
25 2 ~tl primer CP-c.10 (10 pmol/ml)
10,0 ~tl PCR buffer
0.75 ~tl polymerase mixture
73.25 ~tl H20
CA 02273408 1999-06-24
-31 -
PCR reaction b: 10 ~tl Mix 2.10 (2.0 pmol of each oligonucleotide)
2 ~tl nucleotides (10 mM each nucleotide)
2 ul primer CP-b (10 pmol/ml)
2 ul primer CP-a (10 pmol/ml)
10,0 ~1 PCR buffer
0.75 ~l polymerase mixture (2.6 U)
73.25 ~tl H20
Reaction conditions for PCR reaction a and b:
step 1 2 min - 45 C
1o step 2 30 sec - 72 C
step 3 30 sec - 94 C
step 4 30 sec - 52 C
step 5 1 min - 72 C
Step 3 to 5 were repeated 40-times.
The PCR products (670 and 905 bp) were purified by an agarose gel
electrophoresis
(0.9% agarose) and a following gel extraction (QIAEX II Gel Extraction Kit,
Qiagen,
Hilden, Germany). The purified DNA fragments were used for the PCR reaction c.
PCR reaction c: 6 ~tl PCR product of reaction a =50 ng)
6 pl PCR product of reaction b =50 ng)
2 ~tl primer CP-a (10 pmol/ml)
2 ~tl primer CP-a (10 pmol/ml)
10,0 ~tl PCR buffer
0.75 ~1 polymerase mixture (2.6 U)
73.25 ~tl H20
CA 02273408 1999-06-24
-32-
Reaction conditions for PCR reaction c:
step 1 2 min - 94 °C
step 2 30 sec - 94 °C
step 3 30 sec - 55 °C
step 4 1 min - 72 °C
Step 2 to 4 were repeated 31-times.
The resulting PCR product (1.4 kb) was purified as mentioned above, digested
with
Eco RI, and ligated in an Eco RI-digested and dephosphorylated pBsk(-)-vector
'
(Stratagene, La Jolla, CA, USA). 1 ~tl of the ligation mixture was used to
transform E. coli
XL-1 competent cells (Stratagene, La Jolla, CA, USA). All standard procedures
were
carried out as described by Sambrook et al. (1987). The DNA sequence of the
constructed
gene (~'cpl0) was checked by sequencing as known in the art.
Example 5
Increasing the thermostability of consensus ph ase-1 b~introduction of single
mutations
suQested by the amino acid sequence of consensus phytase-10 and consensusph
ase-11
In order to increase the thermostability of homologous genes, it is also
possible to
test the stability effect of each differing amino acid residue between the
protein of interest
and the calculated consensus sequence and to combine all stabilizing mutations
into the
protein of interest. We used the consensus phytase as protein of interest and
tested the
effect on the protein stability of 34 amino acid residues, differing to
consensus phytase 10
and/or 11 as single mutations.
To construct muteins for expression in A. niger, S. cerevisiae, or H.
polymorpha, the
corresponding expression plasmid containing the consensus phytase gene was
used as
template for site-directed mutagenesis (see Example 8 - 10). Mutations were
introduced
using the "quick exchanges site-directed mutagenesis kit" from Stratagene ( La
Jolla, CA,
USA) following the manufacturer's protocol and using the corresponding
primers. All
mutations made and their corresponding primers are summarized in Table 4.
Plasmids
CA 02273408 1999-09-29
-33-
harboring the desired mutation were identified by DNA sequence analysis as
known in the
art.
Table 4~ Primers used for site-directed mutaaenesis of consensus phyase
(Exchanged bases are highlighted in bold. The introduction of a restriction
site is marked
above the sequence. When a restriction site is written in parenthesis, the
mentioned site
was destroyed by introduction of the mutation.)
mutation Primer set
Kpy
I
to QSOT 5'-CACTTGTGGGGTACCTACTCTCCATACTTCTC-3' SEQ ID N0:22
5'-GAGAAGTATGGAGAGTAGGTACCCCACAAGTG-3' SEQ ID N0:23
Y54F 5'-GGTCAATACTCTCCATTCTTCTCTTTGGAAG-3' SEQ ID N0:24
5'-CTTCCAAAGAGAAGAATGGAGAGTATTGACC-3' SEQ ID N0:25
E58A ~'-CATACTTCTCTTTGGCAGACGAATCTGC-3' SEQ ID N0:26
5'-GCAGATTCGTCTGCCAAAGAGAAGTATG-3' SEQ ID N0:27
Aat II
D69K 5'-CTCCAGACGTCCCAAAGGACTGTAGAGTTAC-3' SEQ ID N0:28
5'-GTAACTCTACAGTCCTTTGGGACGTCTGGAG-3' SEQ ID N0:29
Aat II
D70G ~'-CTCCAGACGTCCCAGACGGCTGTAGAGTTAC-3' SEQ ID N0:30
5'-GTAACTCTACAGCCGTCTGGGACGTCTGGAG-3' SEQ ID N0:31
K91A 5'-GATACCCAACTTCTTCTGCGTCTAAGGCTTACTCTG-3' SEQ ID N0:32
5'-CAGAGTAAGCCTTAGACGCAGAAGAAGTTGGGTATC-3' SEQ ID N0:33
CA 02273408 1999-09-29
-34-
ScaI
A94K 5'-CTTCTAAGTCTAAGAAGTACTCTGCTTTG-3' SEQ ID N0:34
S'-CAAAGCAGAGTACTTCTTAGACTTAGAAG-3' SEQ ID N0:35
AIOIR 5'-GCTTACTCTGCTTTGATTGAACGGATTCAAAAGAACGCTAC-3' SEQIDN0:36
5'-GTAGCGTTCTTTTGAATCCGTTCAATCAAAGCAGAGTAAGC-3' SEQ ID N0:37
\T13~Q ~'-CCATTCGGTGAACAGCAAATGGTTAACTC-3' SEQ ID N0:38
5'-GAGTTAACCATTTGCTGTTCACCGAATGG-3' SEQ ID N0:39
A~ru I
K1~3N ~'-GATACAAGGCTCTCGCGAGAAACATTGTTC -3' SEQ ID N0:40
~'-GGAACAATGTTTCTCGCGAGAGCCTTGTATC-3' SEQ ID N0:41
Bss HI
I158V S'-GATTGTTCCATTCGTGCGCGCTTCTGGTTC-3' SEQ ID N0:42
5'-GAACCAGAAGCGCGCACGAATGGAACAATC-3' SEQ ID N0:43
Bcl I
D 197N 5'-CTCCAGTTATTAACGTGA TCATTCCAGAAGG-3' SEQ ID N0:44
5'-CCTTCTGGAATGATCACGTTAATAACTGGAG-3' SEQ ID N0:45
Apa I
?o S 187A 5'-GGCTGACCCAGGGGCCCAACCACACCAAGC-3' SEQ ID N0:46
~'-GCTTGGTGTGGTTGGGCCCCTGGGTCAGCC-3' SEQ ID N0:47
A~co I
T214L 5'-CACTTTGGACCA TGGTCTTTGTACTGCTTTCG-3' SEQ ID N0:48
5'-CGAAAGCAGTACAAAGACCATGGTCCAAAGTG-3' SEQ ID N0:49
~5 Avr II
E222T S'-GCTTTCGAAGACTCTACCCTAGGTGACGACGTTG-3' SEQ ID NO:50
5'-CAACGTCGTCACCTA GGGTAGAGTCTTCGAAAGC-3' SEQ ID NO:51
CA 02273408 1999-09-29
-35-
V227A 5'-GGTGACGACGCTGAAGCTAACTTCAC-3' SEQ ID N0:52
5'-GTGAAGTTAGCTTCAGCGTCGTCACC-3' SEQ ID N0:53
Sac lI
L234~' S'-CTAACTTCACCGCGGTGTTCGCTCCAG-3' SEQ ID N0:54
S'-CTGGAGCGAACACCGCGGTGAAGTTAG-3' SEQ ID NO:55
A238P ~'-GCTTTGTTCGCTCCACCTATTAGAGCTAGATTGG-3' SEQ ID N0:56
~'-CCAATCTAGCTCTAATAGGTGGAGCGAACAAAGC-3' SEQ ID N0:57
1 o Hpa I
T251:~ ~'-GCCAGGTGTTAACTTGACTGACGAAG-3' SEQ ID N0:58
~'-TTCGTCAGTCAAGTTAACACCTGGC-3' SEQ ID N0:59
Aat II
Y259N ~'-GACGAAGACGTCGTTAACTTGATGGAC-3' SEQ ID N0:60
i5 ~'-GTCCATCAAGTTAACGACGTCTTCGTC-3' SEQ ID N0:61
Asp I
E267D 5'-GTCCATTCGACACTGTCGCTAGAACTT C-3' SEQ ID N0:62
5'-GAAGTTCTAGCGACAGTGTCGAATGGAC-3' SEQ ID N0:63
~o E277Q ~'-CTGACGCTACTCAGCTGTCTCCATTC-3' SEQ ID N0:64
~'-GAATGGAGACAGCTGAGTAGCGTCAG-3' SEQ ID N0:65
A283D ~'-GTCTCCATTCTGTGATTTGTTCACTCAC-3' SEQ ID N0:66
~'-GTGAGTGAACAAATCACAGAATGGAGAC-3' SEQ ID N0:67
2s Ksp I
H287A 5'-GCTTTGTTCACCGCGGACGAATGGAG-3' SEQ ID N0:68
5'-CTCCATTCGTCCGCGGTGAACAAAGC-3' SEQ ID N0:69
CA 02273408 1999-09-29
-36-
Bam HI
R291I ~'-CACGACGAATGGA TCCAATACGACTAC-3' SEQ ID N0:70
5'-GTAGTCGTATTGGA TCCATTCGTCGTG-3' SEQ ID N0:71
Bsi WI
6'-GACGAATGGAGAGCGTACGACTACTTG-3' SEQ ID N0:72
~'-CAAGTAGTCGTACGCTCTCCATTCGTC-3' SEQ ID N0:73
Hpa I
A320V 5'-GGTGTTGGTTTCGTTAACGAATTGATTGC-3' SEQ ID N0:74
5'-GCAATCAATTCGTTAACGAAACCAACACC-3' SEQ ID N0:75
(Bgl II)
R329H ~'-GCTAGATTGACTCACTCTCCAGTTCAAG-3' SEQ ID N0:76
~'-CTTGAACTGGAGAGTGAGTCAATCTAGC-3' SEQ ID N0:77
Eco RV
S364T 5'-CTCACGACAACACTATGATATCTATTT'TCTTC-3' SEQ ID N0:78
5'-GAAGAAAATAGA TA TCATAGTGTTGTCGTGAG-3' SEQ ID N0:79
Nco I
I366v 5'-CGACAACTCCATGGTTTCTATTTTCTTCGC-3' SEQ ID N0:80
~'-GCGAAGAAAATAGAAACCA TGGAGTTGTCG-3' SEQ ID N0:81
Kpn I
3o A379K 5'-GTACAACGGTACCAAGCCATTGTCTAC-3' SEQ ID N0:82
5'-GTAGACAATGGCTTGGTACCGTTGTAC-3' SEQ ID N0:83
S396A S'-CTGACGGTTACGCTGCTTCTTGGAC-3' SEQ ID N0:84
S'-GTCCAAGAAGCAGCGTAACCGTCAG-3' SEQ ID N0:85
CA 02273408 1999-09-29
-37-
G40~A 5'-CTGTTCCATTCGCTGCTAGAGCTTAC-3' SEQ ID N0:86
5'-GTAAGCTCTAGCAGCGAATGGAACAG-3' SEQ ID N0:87
Q41~E 5'-GATGCAATGTGAAGCTGAAAAGGAACC-3' SEQ ID N0:88
~'-GGTTCCTT I"TCAGCTTCACATTGCATC-3' SEQ ID N0:89
Sal I
A.~37G 5'-CACGGTTGTGGTGTCGACAAGTTGGG-3' SEQ ID N0:90
5'-CCCAACTTGTCGACACCACAACCGTG-3' SEQ ID N0:91
Mini I
io .~463E 5'-GATCTGGTGGCAATTGGGAGGAATGTTTCG-3' SEQIDN0:92
5'-CGAAACATTCCTCCCAA TTGCCACCAGATC-3' SEQ ID N0:93
and accordinjly for other mutations.
The temperature optimum of the purified phytases, expressed in Saccharomyces
cereoisiae
15 (Example 9), was determined as outlined in Example 11. Table 5 shows the
effect on the
stability of consensus phytase for each mutation introduced.
CA 02273408 1999-06-24
-38-
Table 5: Stability effect of the individual amino acid replacements in
consensus nhytase-1
(+
or
-
means
a
positive,
respectively,
negative
effect
on
the
protein
stability
up
to
1
C,
++
and
--
means
a
positive,
respectively,
negative
effect
on
the
protein
stability
between
1
and
3
C:
the
number
or
11
corresponds
to
the
consensus
phytase
sequence
that
suggests
the
5
amino
acid
replacement.)
stabilizing
neutral
destabilizing
mutation effect mutation effect mutation effect
E58A (10) + D69A Y54F (10) -
D69K (11) + D70G (10) V73I -
D197N (10) + N134Q (10) A94K (10) -
T214L (10) ++ G186H AIOIR (11) -
E222T (11) ++ S187A (10) K153N (11) -
E267D (10) + T214V I158V (10) --
R291I* + T251N (10) G203A - -
R329H (10) + Y259N (10) G205S -
S364T (10) + + A283D (10) A217V -
A379K (11) + A320V (10) V227A (11) --
G404A ( 10) + + K445T L234V ( 10) -
A463E (10) A238P (10) --
E277Q (10) -
H287A (11) -
Q292A ( 10) -
CA 02273408 1999-06-24
-39-
~'Ve combined eight positive mutations (E58A, D197N, E267D, R291I, R329H,
S364T,
A379K, G404A) in the consensus phytase using the primers and the technique
mentioned
above in this example. Furthermore, the mutations QSOT and K91A were
introduced which
mainly influence the catalytical characteristics of the phytase (see European
Patent
Application Publication No. 897 985 as well as Example 11). The DNA and amino
acid
sequence of the resulting phytase gene (consensus phytase-thermo[8]-QSOT-K91A)
is
shown in Figure 19. In this way, the temperature optimum and the melting point
of the
consensus phytase was increased by 7 °C (Figure 27, 28, 29).
Using the results of Table 5, we further improved the thermostability of
consensus phytase
10 by the following back mutations K94A, V 158I, and A396S that revealed a
strong
negative influence on the stability of consensus phytase. The resulting
protein is phytase-
10-thermo [3]. Furthermore, we introduced the mutations QSOT and K91A which
mainly
influence the catalytical characteristics of consensus phytase (see patent
application EP
Publication No. 897 985 as well as Example 11 and Figure 26 and 27). The
resulting DNA
and amino acid sequence is shown in Figure 20. The optimized phytase showed a
4 °C
higher temperature optimum and melting point than consensus phytase 10 (Figure
24 and
25). Furthermore, the phytase has also a strongly increased specific activity
with phytate as
substrate of 250 U/mg at pH 5.5 (Figure 26).
": This amino acid replacement was found in another round of mutations.
CA 02273408 1999-06-24
-40-
Example 6
Stabilization of the phytase of A. ,fumigatus ATCC 13073 by replacement of
amino acid
residues with the corresponding consensus~hytase-1 and consensus phytase-10
residues
At six typical positions where the A. fumigatus 13073 is the only or nearly
the only
ph~2ase in the alignment of Figure 13 that does not contain the corresponding
consensus
ph~~tase amino acid residue, the non-consensus amino acid residue was replaced
by the
consensus one. In a first round, the following amino acids were substituted in
A. fumigatus
13073 phytase, containing the Q27T substitution and the signal sequence of A.
terreus
cbs.116.46 ph~~tase (see Figure 21):
F~5(28)Y, V 100(73)I, F114(87)Y, A243(220)L, S265(242)P, N294(282)D.
The numbers in parentheses confer to the numbering of Figure 13.
In a second round, four of the seven stabilizing amino acid exchanges (E59A,
R329H,
S364T, G404A) found in the consensus phytase-10 sequence and, tested as single
mutation
in consensus phytase-1 (Table 5), were additionally introduced into the A.
fumigatus a-
mutant. Furthermore, the amino acid replacement S126N, shown to reduce the
protease
susceptibility of the phytase, was introduced.
The mutations were introduced as described in example 5 (see Table 6) and
expressed as
described in example 8 to 10. The resulting A. fumigatus 13073 phytase
variants were
called a-mutant and a-mutant-E59A-S 126N-R329H-S364T-G404A.
The temperature optimum (60 °C, Figure 32) and the melting point (67.0
°C, Figure 31) of
the .4. fumigatus 13073 phytase a-mutant was increased by 5 °C in
comparison to the
values of the wild-type (temperature optimum: 55 °C, T,": 60
°C). The five additional
amino acid replacements further increased the temperature optimum by 3
°C (Figure 32).
CA 02273408 1999-09-29
-41 -
Table 6' Mutaaenesis primers for stabilization of A fumiQams nhytase ATCC
13073
Mutation Primer
F~~y ~'-CACGTACTCGCCATACTTTTCGCTCGAG-3' SEQ ID N0:94
~'-CTCGAGCGAAAAGTATGGCGAGTACGTG-3' SEQ ID N0:95
(~7zo I)
E58 A ~'-CCATACTTTTCGCTCGCGGACGAGCTGTCCGTG-3' SEQ ID N0:96
~'-CACGGACAGCTCGTCCGCGAGCGAAAAGTAGG-3' SEQ ID N0:97
V 100I ~'-GTATAAGAAGCTTATTACGGCGATCCAGGCC-3' SEQ ID N0:98
io 5'-GGCCTGGATCGCCGTAATAAGCTTCTTATAC-3' SEQ ID N0:99
F114Y ~'-CTTCAAGGGCAAGTACGCCTTTTTGAAGACG-3' SEQ ID NO:100
5'-CGTCTTCAAAAAGGCGTACTTGCCCTTGAAG-3' SEQ ID NO:101
15 A243L 5'-CATCCGAGCTCGCCTCGAGAAGCATCTTC-3' SEQ ID N0:102
5'-GAAGATGCTTCTCGAGGCGAGCTCGGATG-3' SEQ ID N0:103
S?6~P 5'-CTAATGGATGTGTCCGTTTGATACGGTAG-3' SEQ ID N0:104
5'-CTACCGTATCAAACGGACACATGTCCATTAG-3' SEQ ID NO:105
\T29-1D 5'-GTGGAAGAAGTACGACTACCTTCAGTC-3' SEQ ID N0:106
5'-GACTGAAGGTAGTCGTACTTCTTCCAC-3' SEQ ID N0:107
(Mlu I)
R329H 5'-GCCCGGTTGACGCATTCGCCAGTGCAGG-3' SEQ ID N0:108
2s 5'-CCTGCACTGGCGAA TGCGTCAACCGGGC-3' SEQ ID N0:109
CA 02273408 1999-09-29
-42-
I~'co I
S3G.IT 5'-CACACGACAACACCATGGT"TTCCATCTTC-3' SEQ ID NO:110
~'-GAAGATGGAAACCATGGTGTTGTCGTGTG-3' SEQ ID NO:I 11
(Bss HI )
G40~A ~'-GTGGTGCCTTTCGCCGCGCGAGCCTACTTC-3' SEQ ID N0:112
~'-GAAGTAGGCTCGCGCGGCGAAAGGCACCAC-3' SEQ ID N0:113
CA 02273408 1999-06-24
- 43 -
Example 7
Introduction of the active site amino acid residues of the A. niger NRRL 3135
phytase into
the consensus phvtase-1
~'Ve used the crystal structure of the Aspergillus niger NRRL 3135 phytase to
define
all active site amino acid residues (see Reference Example and EP 897 010).
Using the
alignment of Figure 13, we replaced the following active site residues and
additionally the
not identical adjacent ones of the consensus phytase by that of the A. niger
phytase:
S89D, S92G, A94K, D164S, P201S, G203A, G205S, H212P, G224A, D226T, E255T,
D256E, V258T, P265S, Q292H, G300K, Y305H, A314T, S364G, M365I, A397S, S398A,
1o G40-iA, and A40~S
The new protein sequence consensus phytase -7 was backtranslated into a DNA
sequence
(Figure 22) as described in Example 3. The corresponding gene (fcp~ was
generated as
described in Example 3 using the following oligonucleotide mixes:
Mix 1.7: CP-1, CP-2, CP-3, CP-4.7, CP-5.7, CP-6, CP-7, CP-8.7, CP-9, CP-10.7
Mix 2.7: CP-9, CP-10.7, CP-11.7, CP-12.7, CP-13.7, CP-14.7, CP-15.7, CP-16, CP-
17.7,
CP-18.7, CP-19.7, CP-20, CP-21, CP-22.
The DNA sequences of the oligonucleotides are indicated in Figure 15 The newly
synthesized oligonucleotides are additionally marked by number 7. After
assembling of the
oligonucleotides using the same PCR primers as mentioned in Example 3, the
gene was
cloned into an expression vector as described in Examples 8 - 10.
The pH-profile determined after expression in H. polymorpha and purification
was shifted
into the acidic range of the pH-spectrum showing an optimum at pH 4.5-S.0 (see
Figure
30). The enzyme had a broad pH-optimum reaching at least 60% of its maximum
activity
from pH 2.5 to pH 6Ø Up to pH 5.0, the profile resembled the profile of the
A. niger
NRRL 3135 phytase. However, below pH 5.0 it lacked the typical low at pH 4.0
of the
profile of A. niger phytase.
CA 02273408 1999-06-24
-44-
Example 8
Expression of the consensus phytase Qenes in Hansenula polymorpha
The phytase expression vectors, used to transform H. polymorpha RB 11
(Gellissen et
al., 1994), was constructed by inserting the Eco RI fragment of pBsk fcp or
variants
thereof into the multiple cloning site of the H. polymorpha expression vector
pFPMT121,
which is based on an ura3 selection marker from S. cerevisiae, a formate
dehydrogenase
(FMD) promoter element and a methanol oxidase (MO) termimator element from H.
polymorpha. The 5' end of the fcp gene is fused to the FMD promoter, the 3'
end to the
.1TOX terminator (Gellissen et al., 1996; EP 0299 108 B). The resulting
expression vector
1o are designated pFPMTfcp, pFPMTfcplO, pFPMTfcp7.
The constructed plasmids were propagated in E. coli. Plasmid DNA was purified
using standard state of the art procedures. The expression plasmids were
transformed into
the H. polymoipha strain RP11 deficient in orotidine-5'-phosphate
decarboxylase (ura3)
using the procedure for preparation of competent cells and for transformation
of yeast as
described in Gelissen et al. (1996). Each transformation mixture was plated on
YNB
(0.14% w/v Difco YNB and 0.5% ammonium sulfate) containing 2% glucose and 1.8%
agar and incubated at 37 °C. After 4 to 5 days individual transformant
colonies were picked
and grown in the liquid medium described above for 2 days at 37 °C.
Subsequently, an
aliquot of this culture was used to inoculate fresh vials with YNB-medium
containing 2%
glucose. After seven further passages in selective medium, the expression
vector integrates
into the yeast genome in multimeric form. Subsequently, mitotically stable
transformants
were obtained by two additional cultivation steps in 3 ml non-selective liquid
medium
(YPD, 2% glucose, 10 g yeast extract, and 20 g peptone). In order to obtain
genetically
homogeneous recombinant strains an aliquot from the last stabilization culture
was plated
on a selective plate. Single colonies were isolated for analysis of phytase
expression in
YNB containing 2% glycerol instead of glucose to derepress the find promoter.
Purification
of the consensus phytases was done as described in Example 9.
CA 02273408 1999-06-24
- 45 -
Example 9
Expression of the consensus phytase Qenes in Saccharomyces cerevisiae and
purification
of the phytases from culture supernatant
The consensus phytase genes were isolated from the corresponding Bluescript-
plasmid (pBsk fcp, pBSK fcpl0, pBsk fcp~ and ligated into the Eco RI sites of
the
expression cassette of the Saccharomyces cerevisiae expression vector pYES2
(Invitrogen,
San Diego, CA, USA) or subcloned between the shortened GAPFL (glyceraldhyde-3-
phosphate dehydrogenase) promoter and the pho~ terminator as described by
Japes et al.
( 1990). The correct orientation of the gene was checked by PCR.
Transformation of S
cerevisiae strains. e. g. INVSc 1 (Invitrogen, San Diego, CA, USA) was done
according to
Hinnen et al. (1978). Single colonies harboring the phytase gene under the
control of the
GAPFL promoter were picked and cultivated in S ml selection medium (SD-uracil,
Sherman et al., 1986) at 30°C under vigorous shaking (250 rpm) for one
day. The
preculture was then added to S00 ml YPD medium (Sherman et al., 1986) and
grown
~5 under the same conditions. Induction of the gall promoter was done
according to
manufacturer's instruction. After four days of incubation cell broth was
centrifuged (7000
rpm, GS3 rotor, 15 min, 5°C) to remove the cells and the supernatant
was concentrated by
way of ultrafiltration in Amicon 8400 cells (PM30 membranes) and ultrafree-15
centrifugal filter devices (Biomax-30K, Millipore, Bedford, MA, USA). The
concentrate
(10 ml) was desalted on a 40 ml Sephadex G25 Superfine column (Pharmacia
Biotech,
Freiburg, Germany), with 10 mM sodium acetate, pH 5.0, serving as elution
buffer. The
desalted sample was brought to 2 M (NH4)2S04 and directly loaded onto a 1 ml
Butyl
Sepharose 4 Fast Flow hydrophobic interaction chromatography column (Pharmacia
Biotech, Feiburg, Germany) which was eluted with a linear gradient from 2 M to
0 M
(NH4)2S04 in 10 mM sodium acetate, pH 5Ø Phytase was eluted in the break-
through,
concentrated and loaded on a 120 ml Sephacryl S-300 gel permeation
chromatography
column (Pharmacia Biotech, Freiburg, Germany). Consensus phytase and consensus
phytase -7 eluted as a homogeneous symmetrical peak and was shown by SDS-PAGE
to be
approx. 95% pure.
o CA 02273408 1999-09-29
-46-
Examnle 10
Expression of the consensus phytase genes in AsperQillus niQer
The Bluescript-plasmids pBsk fcp, pBSK fcpl0, and pBsk fcp7 were used as
template
for the introduction of a Bsp HI-site upstream of the start codon of the genes
and an Eco
RV-site downstream of the stop codon. The ExpandTM High Fidelity PCR Kit
(Boehringer Mannheim, Mannheim, Germany) was used with the following primers:
Primer Asp-1:
Bsp HI
5'-TATATCATGAGCGTGTTCGTCGTGCTACTGTTC-3' SEQ ID N0:114
Primer Asp-2 used for cloning of fcp and fcp7:
Eco RV
3'-ACCCGACTTACAAAGCGAATTCTATAGATATAT-5' SEQ ID NO:115
Primer Asp-3 used for cloning of fcpl0:
Eco RV
3'-ACCCTTCTTACAAAGCGAATTCTATAGATATAT-5' SEQ ID NO:116
The reaction was performed as described by the supplier. The PCR-amplified fcp-
genes had a new Bsp HI site at the start codon, introduced by primer Asp-l,
which resulted
in a replacement of the second amino acid residue glycine by serine.
Subsequently, the
DNA-fragment was digested with Bsp HI and Eco RV and ligated into the Nco I
site
downstream of the glucoamylase promoter of Aspergillus niger (glaA) anal the
Eco RV site
upstream of the Aspergillus nidularzs tryptophan C terminator (trpC) (Mullaney
et al.,
1985). After this cloning step, the genes were sequenced to detect possible
failures
introduced by PCR. The resulting expression plasmids which basically
corresponds to the
pGLAC vector as described in Example 9 of EP 684 313, contained the orotidine-
5'-
phosphate decarboxylase gene (pyr4) of Neurospora crassa as a selection
marker.
Transformation of Aspergillus niger and expression of the consensus phytase
genes was
done as described in EP 684 313. The consensus phytases were purified as
described in
Example 9.
CA 02273408 1999-06-24
- 47 -
Examnle 11
Determination of phytase activi~ and of temperature optimum
Phytase activity was determined basically as described by Mitchell et al
(1997). The
activity was measured in an assay mixture containing 0.5% phytic acid (=5 mM)
in 200
mM sodium acetate, pH 5Ø After 15 min of incubation at 37 °C, the
reaction was stopped
by addition of an equal volume of 15% trichloroacetic acid. The liberated
phosphate was
quantified by mixing 100 ~tl of the assay mixture with 900 ~tl H20 and 1 ml of
0.6 M
H~SO~, 2% ascorbic acid and 0.5% ammonium molybdate. Standard solutions of
potassium phosphate were used as reference. One unit of enzyme activity was
defined as
to the amount of enzyme that releases 1 ~tmol phosphate per minute at 37
°C. The protein
concentration was determined using the enzyme extinction coefficient at 280 nm
calculated
according to Pace et al (1995): consensus phytase, 1.101; consensus phytase 7,
1.068;
consensus ph~rtase 10, 1.039.
In case of pH-optimum curves, purified enzymes were diluted in 10 mM sodium
acetate,
pH 5Ø Incubations were started by mixing aliquots of the diluted protein
with an equal
volume of 1% phytic acid (=10 mM) in a series of different buffers: 0.4 M
glycine/HCI, pH
2.5; 0.4 M acetate/NaOH, pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5; 0.4 M imidazole/HCI,
pH 6.0, 6.5;
0.4 M Tris/HC1 pH 7.0, 7.5, 8.0, 8.5, 9Ø Control experiments showed that pH
was only
slightly affected by the mixing step. Incubations were performed for 15 min at
37 °C as
2o described above.
For determinations of the substrate specificities of the phytases, phytic acid
in the
assay mixture was replaced by 5 mM concentrations of the respective phosphate
compounds. The activity tests were performed as described above.
For determination of the temperature optimum, enzyme (100 ~tl) and substrate
solution (100 ~tl) were pre-incubated for 5 min at the given temperature. The
reaction was
started by addition of the substrate solution to the enzyme. After 15 min
incubation, the
reaction was stopped with trichloroacetic acid and the amount of phosphate
released was
determined.
The pH-optimum of the original consensus phytase was around pH 6.0-6.5 (70
3o U/mg). By introduction of the QSOT mutation, the pH-optimum shifted to pH
6.0 (130
CA 02273408 1999-06-24
- 48 -
LT/mg). After introduction of K91A, the pH optimum shifted one pH-unit into
the acidic
pH-range showing a higher specific activity between pH 2.5 and pH 6Ø That
was shown
for the stabilized mutants and for consensus phytase-10, too (Figure 26 and
27).
Consensus phytase-7, which was constructed to transfer the catalytic
characteristics of the
.-1. niger phytase NRRL 3135 into the consensus phytase, had a pH-profile
which is shifted
into the acidic range of the pH-spectrum showing an optimum between pH 4.5 and
5.0 (see
Figure 31). The enzyme had a broad pH-optimum reaching at least 60% of its
increased
maximum activity from pH 2.5 to pH 6Ø The substrate spectrum, too, resemble
more to
that of the A. niger NRRL 3135 phytase than to the consensus phytase-1.
The temperature optimum of consensus phytase-1 (71 °C) was 16-26
°C higher than
the temperature optimum of the wild-type phytases (45-55 °C, Table 7)
which were used to
calculate the consensus sequence. The improved consensus phytase-10 showed a
further
increase of its temperature optimum to 80 °C (Figure 33). The
temperature optimum of the
consensus phytase-1-thermo[8] was found in the same range (78 °C) using
the supernatant
of an overproducing S. cerevisiae strain. The highest temperature optimum
reached of 82 °
C was determined for consensus phytase-10-thermo-QSOT-K91A.
Table 7: Temperature optimum and Tm value of consensus phytase and of the
phytases
from A. jumigatus, A. niger, E. nidulans, and M. therrnophila. The
determination of the
temperature optimum was performed as described in Example 11 The Tm values
were
determined by differential scanning calorimetry as described in Example 12.
temperature ~ Tm
phytase I optimum [°C] ~ [°C]
Consensus phytase-10-thermo- 82 89.3
QSOT-K91 A
Consensus phytase-10-thermo- 82 88.6
QSOT
Consensus phytase-10 ~ 80 ~ 85.4
Consensus phytase-1-thermo[8]- ~ 78 ~ 84.7
CA 02273408 1999-06-24
- 49 -
QSOT
Consensus phytase-1-thermo[8]-78 85.7
QSOT-K91 A
Consensus phytase-1 71 78.1
A. niger NRRL3135 55 63.3
A. fumigatus 13073 55 62.5
f
A. fumigatus 13073 60 67.0
i
OC-mutant
'. A. fumigatus 13073 63 -
Oc,-mutant (optimized)
A. terreus 9A-1 49 57.5
A. terreus cbs.116.46 45 58.5
i
~ E. nidulans 45 55.7
I
M. tlrermophila 55 n. d.
T. thermophilus 45 n. d.
Example 12
Determination of the melting point by differential scanning calorimetrvIDSC
In order to determine the unfolding temperature of the phytases, differential
scanning
calorimetry was applied as previously published by Brugger et al (1997).
Solutions of 50-
60 mg/ml homogeneous phytase were used for the tests. A constant heating rate
of 10 °
C/min was applied up to 90-95 °C.
The determined melting points reflect the results obtained for the temperature
optimums (Table 7). The most stable consensus phytase designed is consensus
phytase-10-
CA 02273408 1999-06-24
-$0-
thermo-Q$OT-K91A showing a melting temperature under the choosen condition of
89.3 °
C. This is 26 to 33.6 °C higher than the melting point of the wild-type
phytases used.
Example 13
Transfer of basidiomycete ~h~rtase active site into consensus phytase-10-
thermo-O$OT-
K91 A
As described previously (Example $), mutations derived from the basidiomycete
phytase active site were introduced into the consensus phytase 10. The
following five
constructs a) to e) were prepared: '
This construct is called consensus phytase 12, and it comprises a selected
number of active
1o site residues of the basidio consensus sequence, its amino acid sequence
(consphyl2) is
shown in Fig. 33 (the first 26 amino acids forms the signal peptide, amended
positions are
underlined);
a cluster of mutations (Cluster II) was transferred to the consensus 10
sequence, viz.:
S80Q, Y86F, S90G, K91A, S92A, K93T, A94R, Y9$I;
1$ analogously, another cluster of mutations (Cluster III) was transferred,
viz.: T129V,
E133A, Q143N, M136S, V137S, N138Q, S139A;
analogously, a further cluster of mutations (Cluster IV) was transferred,
viz.: A168D,
E171T, K172N, F173W;
and finally, a further cluster of mutations (Cluster V) was transferred, viz.:
Q297G, S298D,
2o G300D, Y30$T.
These constructs were expressed as described in Examples 8 to 10.
CA 02273408 1999-06-24
-51 -
References:
Akanuma, S., Yamagishi, A., Tanaka, N. & Oshima, T. (1998). Serial increase in
the
thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by
experimental evolution. Prot. Sci. 7, 698-705.
Arase, A., Yomo, T., Urabe, L, Hata, Y., Katsube, Y. & Okada, H. (1993).
Stabilization of
xylanase by random mutagenesis. FEBS Lett. 316, 123-127.
Berka, R. M., Rey, M. W., Brown, K. M., Byun, T. & Klotz, A. V. (1998).
Molecular
characterization and expression of a phytase gene from the thermophilic fungus
Thermomyces lanuginosus. Appl. Environ. Microbiol. 64, 4423-4427.
1o Blaber, M., Lindstrom, J. D., Gassner, N., Xu, J., Heinz, D. W. & Matthews,
B. W. (1993).
Energetic cost and structural consequences of burying a hydroxyl group within
the core of
a protein determined from Ala'Ser and Val'Thr substitutions in T4 lysozyme.
Biochemistry
32, 11363-11373.
Brugger, R., Mascarello, F., Augem, S., van Loon, A. P. G. M. & Wyss, M.
(1997).
Thermal denaturation of phytases and pH 2.5 acid phosphatase studied by
differential
scanning calorimetry. In The Biochemistry of phytate and plrytase (eds.
Rasmussen, S. K;
Raboy, V.; Dalboge, H. and Loewus, F.; Kluwer Academic Publishers.
Cosgrove, D.J. (1980) Inositol phosphates - their chemistry, biochemistry and
physiology:
studies in organic chemistry, chapter 4. Elsevier Scientific Publishing
Company,
Amsterdam, Oxford, New York.
Devereux, J., Haeberli, P.& Smithies, O. (1984) A comprehensive set of
sequence analysis
programs for the VAX. Nucleic Acids Res. 12, 387-395.
Gellissen, G., Hollenberg, C. P., Janowicz, Z. A. (1994) Gene expression in
methylotrophic yeasts . In: Smith, A. (ed.) Gene expression in recombinant
microorganisms. Dekker, New York, 395-439.
Gellissen, G., Piontek, M., Dahlems, U., Jenzelewski, V., Gavagan, J. E.,
DiCosimo, R.,
Anton, D. I. & Janowicz, Z. A. (1996) Recombinant Hansenula polymorpha as a
biocatalyst: coexpression of the spinach glycolate oxidase (GO) and the S.
cerevisiae
catalase T (CTTI ) gene. Appl. Microbiol. Biotechnol. 46, 46-54.
CA 02273408 1999-06-24
-52-
Gerber, P. and Miiller, K. (1995) Moloc molecular modeling software. J.
Comput. Aided
.'l~ol. Des. 9, 251-268
Hinnen, A., Hicks, J. B. & Fink, G, R. (1978) Transformation of yeast. Proc.
Natl. Acad.
Sci. LSA 75, 1929-1933.
Imanaka, T., Shibazaki, M. & Takagi, M. (1986). A new way of enhancing the
thermostability of proteases. A'ature 324, 695-697.
Janes, M., Meyhack, B., Zimmermann, W. & Hinnen, A. (1990) The influence of
GAP
promoter variants on hirudine production, average plasmid copy number and cell
growth in
Saccharomyces cerevisiae. Curr. Genet. 18, 97-103.
1o Karpusas, M., Baase, W. A., Matsumura, M. & Matthews, B. W. (1989).
Hydrophobic
packing in T4 lysozyme probed by cavity-filling mutants. Proc. Natl. Acad.
Sci. (USA) 86,
8237-8241.
Margarit, L, Campagnoli, S., Frigerio, F., Grandi, G., Fillipis, V. D. &
Fontana, A. (1992).
Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus
subtilis neutral
protease. Prot. Eng. 5, 543-550.
Matthews, B. W. (1987a). Genetic and structural analysis of the protein
stability problem.
Biochemistry 26, 6885-6888.
Matthews, B. W. (1993). Structural and genetic analysis of protein stability.
Annu. Rev.
Biochem. 62, 139-160.
2o Matthews, B. W., Nicholson, H. & Becktel, W. (1987). Enhanced protein
thermostability
from site-directed mutations that decrease the entropy of unfolding. Proc.
Natl. Acad Sci.
(USA) 84, 6663-6667.
Mitchell, D. B., Vogel, K., Weimann, B. J., Pasamontes, L. & van Loon, A. P.
G. M.
(1997) The phytase subfamily of histidine acid phosphatases: isolation of
genes for two
novel phytases from the fungi Aspergillus terreus and Myceliophthora
thermophila,
Microbiology 143, 245-252.
CA 02273408 1999-06-24
-53-
Mullaney, E. J., Hamer, J. E., Roberti, K. A., Yelton, M. M. & Timberlake, W.
E. (1985)
Primary structure of the trpC gene from Aspergillus nidulans. Mol. Gen. Genet.
199, 37-
46.
Munoz, V. & Senano, L. (1995). Helix design, prediction and stability. Curr.
Opin.
Biotechnol. 6, 382-386.
Pace, N. C., Vajdos, F., Fee, L., Grimsley, G. & Gray, T. (1995). How to
measure and
predict the molar absorption coefficient of a protein. Prot. Sci. 4, 2411-
2423.
Pantoliano, M. W., Landner, R. C., Brian, P. N., Rollence, M. L., Wood, J. F.
& Poulos, T.
L. (1987). Protein engineering of subtilisin BPN': enhanced stabilization
through the
introduction of two cysteines to form a disulfide bond. Biochemistry 26, 2077-
2082.
Pasamontes, L., Haiker, M., Henriquez-Huecas, M., Mitchell, D. B. & van Loon,
A. P. G.
M. (1997a). Cloning of the phytases from Emericella nidulans and the
thermophilic fungus
Talaromyces thermophilus. Biochin:. Biophys. Acta 1353, 217-223.
Pasamontes, L., Haiker, M., Wyss, M., Tessier, M. & van Loon, A. P. G. M.
(1997)
Cloning, purification and characterization of a heat stable phytase from the
fungus
Aspergillus fumigatus, Appl. Environ. Microbiol. 63, 1696-1700.
Piddington, C. S., Houston, C. S., Paloheimo, M., Cantrell, M., Miettinen-
Oinonen, A.
Nevalainen, H., & Rambosek, J. (1993) The cloning and sequencing of the genes
encoding
phytase (phy) and pH 2.5-optimum acid phosphatase (aph) from Aspergillus niger
var.
mvamori. Gene 133, 55-62.
Purvis, I. J., Bettany, A. J. E., Santiago, T. C., Coggins, J. R., Duncan, K.,
Eason, R. &
Brown, A. J. P. (1987). The efficiency of folding of some proteins is
increased by
controlled rates of translation in vivo. J. Mol. Biol. 193, 413-417.
Risse, B., Stempfer, G., Rudolph, R., Schumacher, G. & Jaenicke, R. (1992).
Characterization of the stability effect of point mutations of pyruvate
oxidase from
Lactobacillus plantarum: protection of the native state by modulating coenzyme
binding
and subunit interaction. Prot. Sci. 1, 1710-1718.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A
Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
CA 02273408 1999-06-24
-54-
Sauer, R., Hehir, K., Stearman, R., Weiss, M., Jeitler-Nilsson, A., Suchanek,
E. & Pabo, C.
(1986). An engineered intersubunit disulfide enhances the stability and DNA
binding of
the N-terminal domain of 1-repressor. Biochemistry 25, 5992-5999.
Serrano, L., Day, A. G. & Fersht, A. R. (1993). Step-wise mutation of barnase
to binase. A
procedure for engineering increased stability of proteins and an experimental
analysis of
the evolution of protein stability. J. Mol. Biol. 233, 305-312.
Sheman, J. P., Finck, G. R. & Hicks, J. B. (1986) Laboratory course manual for
methods in
yeast genetics. Cold Spring Harbor University.
Steipe, B., Schiller, B., Plueckthun, A. & Steinbach, S. (1994). Sequence
statistics reliably
predict stabilizing mutations in a protein domain. J. Mol. Biol. 240, 188-192.
van den Burg, B., Vriend, G., Veltman, O. R., Venema & G., Eijsink, V. G. H.
(1998).
Engineering an enzyme to resist boiling. Proc. Natl. Acad Sci. (USA) 95, 2056-
2060.
Van Etten, R.L. (1982) Human prostatic acid phosphatase: a histidine
phosphatase. Ann.
NYAcad. Sci. 390,27-50
van Hartingsveldt, W., van Zeijl, C. M. F., Harteveld, G. M., Gouka, R. J.,
Suykerbuyk, M.
E. G., Luiten, R. G. M., van Paridon, P. A., Selten, G. C. M., Veenstra, A.
E., van Gorcom,
R. F. M., & van den Hondel, C. A. M. J. J. (1993) Cloning, characterization
and
overexpression of the phytase-encoding gene (pl~yA) of Aspergillus niger. Gene
127, 87-
94.
van Loon, A. P. G. M., Simoes-Nunes, C., Wyss, M., Tomschy, A., Hug, D.,
Vogel,
K. & Pasamontes, L. (1998). A heat resistant phytase of Aspergillus fumigatus
with
superior performance in animal experiments. Phytase optimization and natural
variability.
In the Biochemistry ofphytate and phytases. Kluwer Academic Press, s.a.
CA 02273408 1999-09-29
54/ 1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: F. Hoffmann-La Roche AG
(B) STREET: 124 Grenzacherstrasse
(C) CITY: Basle
(D) STATE: n/a
(E) COUNTRY: Switzerland
(F) POSTAL CODE (ZIP): CH-4070
(ii) TITLE OF INVENTION: Phytage Formulation
(iii) NUMBER OF SEQUENCES: 116
(iv) CORRESPONDENCE ADDRESS
(A) NAME: COWLING, STRATHY & HENDERSON
(B) STREET: 160 ELGIN STREET, SUITE 2600
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 1C3
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,273,408
(B) FILING DATE: 24-JUNE-1999
(V11) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EPO 98111960.5
(B) FILING DATE: 29-JUN-1998
(viii) ATTORNEY/AGENT INFORMATION
(A) NAME: COWLING, STRATHY & HENDERSON
(B) REFERENCE NUMBER: 08-883678CA
(ix) TELECOMMUNICATION INFORMATION
(A) TELEPHONE: 613-233-1781
(B) TELEFAX: 613-563-9869
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 471 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02273408 1999-09-29
54/2
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Asn Ser His Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro
1 5 10 15
Glu Ile Ser His Leu Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu
20 25 30
Asp Glu Ser Ala Ile Ser Pro Asp Val Pro Asp Asp Cys Arg Val Thr
35 40 45
Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Ala Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala
65 70 75 80
Thr Ala Phe Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile
115 120 125
Val Pro Phe Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala
130 135 140
Glu Lys Phe Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly
145 150 155 160
Ser Gln Pro His Gln Ala Ser Pro Val Ile Asp Val Ile Ile Pro Glu
165 170 175
Gly Ser Gly Tyr Asn Asn Thr Leu Asp His Gly Thr Cys Thr Ala Phe
180 185 190
Glu Asp Xaa Xaa Xaa Ser Glu Leu Gly Asp Asp Val Glu Ala Asn Phe
195 200 205
Thr Ala Leu Phe Ala Pro Ala Ile Arg Ala Arg Leu Glu Ala Asp Leu
210 215 220
Pro Gly Val Thr Leu Thr Asp Glu Asp Val Val Tyr Leu Met Asp Met
225 230 235 240
Cys Pro Phe Glu Thr Val Ala Arg Thr Ser Xaa Xaa Xaa Xaa Xaa Xaa
245 250 255
Xaa Xaa Xaa Xaa Xaa Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu
260 265 270
CA 02273408 1999-09-29
54/3
Phe Thr His Asp Glu Trp Arg Gln Tyr Asp Tyr Leu Gln Ser Leu Gly
275 280 285
Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly
290 295 300
Val Gly Phe Ala Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val
305 310 315 320
Gln Asp His Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr
325 330 335
Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Ser
340 345 350
Met Ile Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro
355 360 365
Leu Ser Thr Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser
370 375 380
Ala Ser Trp Thr Val Pro Phe Gly Ala Arg Ala Tyr Val Glu Met Met
385 390 395 400
Gln Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln
405 410 415
Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val
420 425 430
Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp
435 440 445
Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala
450 455 460
Glu Cys Phe Ala Xaa Xaa Xaa
465 470
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1426 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: sig~eptide
(B) LOCATION:12..89
CA 02273408 1999-09-29
54/4
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:12..1412
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
TATATGAATT C ATG GGC GTG TTC GTC GTG CTA CTG TCC ATT GCC ACC TTG 50
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu
1 5 10
TTC GGT TCC ACA TCC GGT ACC GCC TTG GGT CCT CGT GGT AAT TCT CAC 98
Phe Gly Ser Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His
15 20 25
TCT TGT GAC ACT GTT GAC GGT GGT TAC CAA TGT TTC CCA GAA ATT TCT 146
Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser
30 35 40 45
CAC TTG TGG GGT CAA TAC TCT CCA TAC TTC TCT TTG GAA GAC GAA TCT 194
His Leu Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Ser
50 55 60
GCT ATT TCT CCA GAC GTT CCA GAC GAC TGT AGA GTT ACT TTC GTT CAA 242
Ala Ile Ser Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln
65 70 75
GTT TTG TCT AGA CAC GGT GCT AGA TAC CCA ACT TCT TCT AAG TCT AAG 290
Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys
80 85 90
GCT TAC TCT GCT TTG ATT GAA GCT ATT CAA AAG AAC GCT ACT GCT TTC 338
Ala Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe
g5 100 105
AAG GGT AAG TAC GCT TTC TTG AAG ACT TAC AAC TAC ACT TTG GGT GCT 386
Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala
110 115 120 125
GAC GAC TTG ACT CCA TTC GGT GAA AAC CAA ATG GTT AAC TCT GGT ATT 434
Asp Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile
130 135 140
AAG TTC TAC AGA AGA TAC AAG GCT TTG GCT AGA AAG ATT GTT CCA TTC 482
Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe
145 150 155
ATT AGA GCT TCT GGT TCT GAC AGA GTT ATT GCT TCT GCT GAA AAG TTC 530
Ile Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe
160 165 170
ATT GAA GGT TTC CAA TCT GCT AAG TTG GCT GAC CCA GGT TCT CAA CCA 57B
Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro
175 180 185
CA 02273408 1999-09-29
54/5
CAC CAA GCT TCT CCA GTT ATT GAC GTT ATT ATT CCA GAA GGA TCC GGT 626
His Gln Ala Ser Pro Val Ile Asp Val Ile Ile Pro Glu Gly Ser Gly
190 195 200 205
TAC AAC AAC ACT TTG GAC CAC GGT ACT TGT ACT GCT TTC GAA GAC TCT 674
Tyr Asn Asn Thr Leu Asp His Gly Thr Cys Thr Ala Phe Glu Asp Ser
210 215 220
GAA TTG GGT GAC GAC GTT GAA GCT AAC TTC ACT GCT TTG TTC GCT CCA 722
Glu Leu Gly Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro
225 230 235
GCTATT AGAGCT AGATTGGAA GACTTG CCAGGTGTT ACTTTGACT 770
GCT
AlaIle ArgAla ArgLeuGlu AlaAspLeu ProGlyVal ThrLeuThr
240 245 250
GACGAA GACGTT GTTTACTTG ATGGACATG TGTCCATTC GAAACTGTT 818
AspGlu AspVal ValTyrLeu MetAspMet CysProPhe GluThrVal
255 260 265
GCTAGA ACTTCT GACGCTACT GAATTGTCT CCATTCTGT GCTTTGTTC 866
AlaArg ThrSer AspAlaThr GluLeuSer ProPheCys AlaLeuPhe
270 275 280 285
ACTCAC GACGAA TGGAGACAA TACGACTAC TTGCAATCT TTGGGTAAG 914
ThrHis AspGlu TrpArgGln TyrAspTyr LeuGlnSer LeuGlyLys
290 295 300
TACTAC GGTTAC GGTGCTGGT AACCCATTG GGTCCAGCT CAAGGTGTT 962
TyrTyr GlyTyr GlyAlaGly AsnProLeu GlyProAla GlnGlyVal
305 310 315
GGTTTC GCTAACGAA ATT GCTAGATTG ACTAGATCT CCAGTTCAA 1010
TTG
GlyPhe AlaAsnGlu LeuIle AlaArgLeu ThrArgSer ProValGln
320 325 330
GACCAC ACTTCTACT AACCAC ACTTTGGAC TCTAACCCA GCTACTTTC 1058
AspHis ThrSerThr AsnHis ThrLeuAsp SerAsnPro AlaThrPhe
335 340 345
CCATTG AACGCTACT TTGTAC GCTGACTTC TCTCACGAC AACTCTATG 1106
ProLeu AsnAlaThr LeuTyr AlaAspPhe SerHisAsp AsnSerMet
350 355 360 365
ATTTCT ATTTTCTTC GCTTTG GGTTTGTAC AACGGTACT GCTCCATTG 1154
IleSer IlePhePhe AlaLeu GlyLeuTyr AsnGlyThr AlaProLeu
370 375 380
TCTACT ACTTCTGTT GAATCT ATTGAAGAA ACTGACGGT TACTCTGCT 1202
SerThr ThrSerVal GluSer IleGluGlu ThrAspGly TyrSerAla
385 390 395
TCTTGG ACTGTTCCA TTCGGT GCTAGAGCT TACGTTGAA ATGATGCAA 1250
SerTrp ThrValPro PheGly AlaArgAla TyrValGlu MetMetGln
400 405 410
CA 02273408 1999-09-29
54/6
TGT CAA GCT GAA AAG GAA CCA TTG GTT AGA GTT TTG GTT AAC GAC AGA 1298
Cys Gln Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg
415 420 425
GTT GTT CCA TTG CAC GGT TGT GCT GTT GAC AAG TTG GGT AGA TGT AAG 1346
Val Val Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys
430 435 440 445
AGA GAC GAC TTC GTT GAA GGT TTG TCT TTC GCT AGA TCT GGT GGT AAC 1394
Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn
450 455 460
TGG GCT GAA TGT TTC GCT TAAGAATTCA TATA 1426
Trp Ala Glu Cys Phe Ala
465
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
1 5 10 15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
20 25 30
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
35 40 45
Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Ser Ala Ile Ser
50 55 60
Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
65 70 75 80
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Ala Tyr Ser
85 90 95
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
100 105 110
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
115 120 125
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
130 135 140
CA 02273408 1999-09-29
54/7
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
145 150 155 160
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
165 170 175
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
180 185 190
Ser Pro Val Ile Asp Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
195 200 205
Thr Leu Asp His Gly Thr Cys Thr Ala Phe Glu Asp Ser Glu Leu Gly
210 215 220
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
225 230 235 240
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
245 250 255
Val Val Tyr Leu Met Asp Met Cys Pro Phe Glu Thr Val Ala Arg Thr
260 265 270
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
275 280 285
Glu Trp Arg Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
290 295 300
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
305 310 315 320
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr
325 330 335
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
340 345 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Ser Met Ile Ser Ile
355 360 365
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu Ser Thr Thr
370 375 380
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
385 390 395 400
Val Pro Phe Gly Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
405 410 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
420 425 430
CA 02273408 1999-09-29
54/8
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
435 440 445
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
450 455 460
Cys Phe Ala
465
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Ser Xaa Pro Xaa Arg Xaa Thr Ala Ala Gln Leu Pro Ile Pro Xaa Gln
1 5 10 15
Xaa Gln Xaa Xaa Trp Ser Pro Tyr Ser Pro Tyr Phe Pro Val Ala Xaa
20 25 30
Tyr Xaa Ala Pro Pro Ala Gly Cys Gln Ile Xaa Gln Val Asn Ile Ile
35 40 45
Gln Arg His Gly Ala Arg Phe Pro Thr Ser Gly Ala Ala Thr Arg Ile
50 55 60
Gln Ala Ala Val Ala Lys Leu Gln Ser Ala Xaa Xaa Xaa Thr Asp Pro
65 70 75 80
Lys Leu Asp Phe Leu Xaa Asn Xaa Thr Tyr Xaa Leu Gly Xaa Asp Asp
85 90 95
Leu Val Pro Phe Gly Ala Xaa Gln Ser Ser Gln Ala Gly Gln Glu Ala
100 105 110
Phe Thr Arg Tyr Ser Xaa Leu Val Ser Xaa Asp Asn Leu Pro Phe Val
115 120 125
Arg Ala Ser Gly Ser Asp Arg Val Val Asp Ser Ala Thr Asn Trp Thr
130 135 140
Ala Gly Phe Ala Xaa Ala Ser Xaa Asn Thr Xaa Xaa Pro Xaa Leu Xaa
145 150 155 160
Val Ile Leu Ser Glu Xaa Gly Asn Asp Thr Leu Asp Asp Asn Met Cys
165 170 175
CA 02273408 1999-09-29
54/9
Pro Xaa Ala Gly Asp Ser Asp Pro Gln Xaa Asn Xaa Trp Leu Ala Val
180 185 190
Phe Ala Pro Pro Ile Thr Ala Arg Leu Asn Ala Ala Ala Pro Gly Ala
195 200 205
Asn Leu Thr Asp Xaa Asp Ala Xaa Asn Leu Xaa Xaa Leu Cys Pro Phe
210 215 220
Glu Thr Val Ser Xaa Glu Xaa Xaa Ser Xaa Phe Cys Asp Leu Phe Glu
225 230 235 240
Xaa Xaa Pro Glu Glu Phe Xaa Ala Phe Xaa Tyr Xaa Gly Asp Leu Asp
245 250 255
Lys Phe Tyr Gly Thr Gly Tyr Gly Gln Pro Leu Gly Pro Val Gln Gly
260 265 270
Val Gly Tyr Ile Asn Glu Leu Leu Ala Arg Leu Thr Xaa Gln Ala Val
275 280 285
Arg Asp Asn Thr Gln Thr Asn Arg Thr Leu Asp Ser Ser Pro Xaa Thr
290 295 300
Phe Pro Leu Asn Arg Thr Phe Tyr Ala Asp Phe Ser His Asp Asn Gln
305 310 315 320
Met Val Ala Ile Phe Ser Ala Met Gly Leu Phe Asn Gln Ser Ala Pro
325 330 335
Leu Asp Pro Ser Xaa Pro Asp Pro Asn Arg Thr Trp Val Thr Ser Lys
340 345 350
Leu Val Pro Phe Ser Ala Arg Met Val Val Glu Arg Leu Xaa Cys Xaa
355 360 365
Xaa Xaa Gly Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
370 375 380
Xaa Xaa Xaa Xaa Xaa Val Arg Val Leu Val Asn Asp Ala Val Gln Pro
385 390 395 400
Leu Glu Phe Cys Gly Gly Asp Xaa Asp Gly Xaa Cys Thr Leu Asp Ala
405 410 415
Phe Val Glu Ser Gln Xaa Tyr Ala Arg Glu Asp Gly Gln Gly Asp Phe
420 425 430
Glu Lys Cys Phe Ala Thr Pro Xaa Xaa
435 440
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 482 amino acids
CA 02273408 1999-09-29
54/10
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Asn Ser His Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro
1 5 10 15
Glu Ile Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala
20 25 30
Asp Glu Ser Ala Ile Ser Pro Asp Val Pro Lys Gly Cys Arg Val Thr
35 40 45
Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Lys Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala
65 70 75 80
Thr Ala Phe Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
g5 g0 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Met Val Asn
100 105 110
Ser Gly Ile Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Xaa Ala Arg Lys
115 120 125
Ile Val Pro Phe Val Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser
130 135 140
Ala Glu Lys Phe Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro
145 150 155 160
Gly Ala Asn Pro His Gln Ala Ser Pro Val Ile Asn Val Ile Ile Pro
165 170 175
Glu Gly Ala Gly Tyr Asn Asn Thr Leu Asp His Gly Leu Cys Thr Ala
180 185 190
Phe Glu Glu Xaa Xaa Xaa Ser Glu Leu Gly Asp Asp Val Glu Ala Asn
195 200 205
Phe Thr Ala Val Phe Ala Pro Pro Ile Arg Ala Arg Leu Glu Ala His
210 215 220
Leu Pro Gly Val Asn Leu Thr Asp Glu Asp Val Val Asn Leu Met Asp
225 230 235 240
CA 02273408 1999-09-29
54/11
Met Cys Pro Phe Asp Thr Val Ala Arg Thr Ser Asp Xaa Xaa Ala Thr
245 250 255
Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Ser Pro Phe Cys Asp
260 265 270
Leu Phe Thr His Xaa Xaa Asp Glu Trp Ile Gln Tyr Asp Tyr Leu Gln
275 280 285
Ser Leu Gly Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro
290 295 300
Ala Gln Gly Val Gly Phe Val Asn Glu Leu Ile Ala Arg Leu Thr His
305 310 315 320
Ser Xaa Pro Val Gln Asp His Thr Ser Thr Asn His Thr Leu Asp Ser
325 330 335
Asn Pro Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser
340 345 350
His Asp Asn Thr Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn
355 360 365
Gly Thr Lys Pro Leu Ser Thr Thr Ser Val Glu Xaa Xaa Ser Ile Glu
370 375 380
Glu Thr Asp Gly Tyr Ala Ala Ser Trp Thr Val Pro Phe Ala Ala Arg
385 390 395 400
Ala Tyr Val Glu Met Met Gln Cys Xaa Xaa Glu Ala Xaa Xaa Xaa Xaa
405 410 415
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Lys Glu Pro Leu Val Arg
420 425 430
Val Leu Val Asn Asp Arg Val Val Pro Leu His Gly Cys Gly Val Asp
435 440 445
Lys Leu Gly Arg Cys Lys Arg Asp Asp Phe Val Glu Gly Leu Ser Phe
450 455 460
Ala Arg Ser Gly Xaa Xaa Gly Asn Trp Glu Glu Cys Phe Ala Xaa Xaa
465 470 475 480
Xaa Xaa
(2) INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1426 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
CA 02273408 1999-09-29
54/12
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: sig peptide
(B) LOCATION:12..89
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:12..1412
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
TATATGAATT C ATG GGC GTG TTC GTC GTG CTA CTG TCC ATT GCC ACC TTG 50
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu
470 475 480
TTC GGT TCC ACA TCC GGT ACC GCC TTG GGT CCT CGT GGT AAT TCT CAC 98
Phe Gly Ser Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His
485 490 495
TCT TGT GAC ACT GTT GAC GGT GGT TAC CAA TGT TTC CCA GAA ATT TCT 146
Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser
500 505 510
CAC TTG TGG GGT CAA TAC TCT CCA TTC TTC TCT TTG GCT GAC GAA TCT 194
His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser
515 520 525
GCT ATT TCT CCA GAC GTT CCA AAG GGT TGT AGA GTT ACT TTC GTT CAA 242
Ala Ile Ser Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln
530 535 540
GTT TTG TCT AGA CAC GGT GCT AGA TAC CCA ACT TCT TCT AAG TCT AAG 290
Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys
545 550 555 560
AAG TAC TCT GCT TTG ATT GAA GCT ATT CAA AAG AAC GCT ACT GCT TTC 338
Lys Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe
565 570 575
AAG GGT AAG TAC GCT TTC TTG AAG ACT TAC AAC TAC ACT TTG GGT GCT 386
Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala
580 585 590
GAC GAC TTG ACT CCA TTC GGT GAA CAA CAA ATG GTT AAC TCT GGT ATT 434
Asp Asp Leu Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile
595 600 .605
AAG TTC TAC AGA AGA TAC AAG GCT TTG GCT AGA AAG ATT GTT CCA TTC 482
Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe
610 615 620
GTT AGA GCT TCT GGT TCT GAC AGA GTT ATT GCT TCT GCT GAA AAG TTC 530
CA 02273408 1999-09-29
54/ 13
Val Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe
625 630 635 640
ATTGAA GGTTTCCAA TCTGCTAAG TTGGCTGAC CCAGGT GCTAACCCA 578
IleGlu GlyPheGln SerAlaLys LeuAlaAsp ProGly AlaAsnPro
645 650 655
CACCAA GCTTCTCCA GTTATTAAC GTTATTATT CCAGAA GGTGCTGGT 626
HisGln AlaSerPro ValIleAsn ValIleIle ProGlu GlyAlaGly
660 665 670
TACAAC AACACTTTG GACCACGGT TTGTGTACT GCTTTC GAAGAATCT 674
TyrAsn AsnThrLeu AspHisGly LeuCysThr AlaPhe GluGluSer
675 680 685
GAATTG GGTGACGAC GTTGAAGCT AACTTCACT GCTGTT TTCGCTCCA 722
GluLeu GlyAspAsp ValGluAla AsnPheThr AlaVal PheAlaPro
690 695 700
CCTATT AGAGCTAGA TTGGAAGCT CACTTGCCA GGTGTT AACTTGACT 770
ProIle ArgAlaArg LeuGluAla HisLeuPro GlyVal AsnLeuThr
705 710 715 720
GACGAA GACGTTGTT AACTTGATG GACATGTGT CCATTC GACACTGTT 818
AspGlu AspValVal AsnLeuMet AspMetCys ProPhe AspThrVal
725 730 735
GCTAGA ACTTCTGAC GCTACTCAA TTGTCTCCA TTCTGT GACTTGTTC 866
AlaArg ThrSerAsp AlaThrGln LeuSerPro PheCys AspLeuPhe
740 745 750
ACTCAC GACGAATGG ATTCAATAC GACTACTTG CAATCT TTGGGTAAG 914
ThrHis AspGluTrp IleGlnTyr AspTyrLeu GlnSer LeuGlyLys
755 760 765
TACTAC GGTTACGGT GCTGGTAAC CCATTGGGT CCAGCT CAAGGTGTT 962
TyrTyr GlyTyrGly AlaGlyAsn ProLeuGly ProAla GlnGlyVal
770 775 780
GGTTTC GTTAACGAA TTGATTGCT AGATTGACT CACTCT CCAGTTCAA 1010
GlyPhe ValAsnGlu LeuIleAla ArgLeuThr HisSer ProValGln
785 790 795 800
GACCAC ACTTCTACT AACCACACT TTGGACTCT AACCCA GCTACTTTC 1058
AspHis ThrSerThr AsnHisThr LeuAspSer AsnPro AlaThrPhe
805 810 815
CCATTG AACGCTACT TTGTACGCT GACTTCTCT CACGAC AACACTATG 1106
ProLeu AsnAlaThr LeuTyrAla AspPheSer HisAsp AsnThrMet
820 825 830
GTTTCT ATTTTCTTC GCTTTGGGT TTGTACAAC GGTACT AAGCCATTG 1154
ValSer IlePhePhe AlaLeuGly LeuTyrAsn GlyThr LysProLeu
835 840 845
CA 02273408 1999-09-29
54/14
TCT ACT ACT TCT GTT GAA TCT ATT GAA GAA ACT GAC GGT TAC GCT GCT 1202
Ser Thr Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ala Ala
850 855 860
TCT TGG ACT GTT CCA TTC GCT GCT AGA GCT TAC GTT GAA ATG ATG CAA 1250
Ser Trp Thr Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln
865 870 875 880
TGT GAA GCT GAA AAG GAA CCA TTG GTT AGA GTT TTG GTT AAC GAC AGA 1298
Cys Glu Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg
885 890 895
GTT GTT CCA TTG CAC GGT TGT GGT GTT GAC AAG TTG GGT AGA TGT AAG 1346
Val Val Pro Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys
900 905 910
AGA GAC GAC TTC GTT GAA GGT TTG TCT TTC GCT AGA TCT GGT GGT AAC 1394
Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn
915 920 925
TGG GAA GAA TGT TTC GCT TAAGAATTCA TATA 1426
Trp Glu Glu Cys Phe Ala
930
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
1 5 10 15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
20 25 30
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
35 40 45
Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
50 55 60
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
65 70 75 80
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Lys Tyr Ser
85 90 95
CA 02273408 1999-09-29
54/15
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
100 105 110
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
115 120 125
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
130 135 140
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Val Arg Ala
145 150 155 160
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
165 170 175
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
180 185 190
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
195 200 205
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Glu Leu Gly
210 215 220
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
225 230 235 240
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
245 250 255
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
260 265 270
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
275 280 285
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
290 295 300
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
305 310 315 320
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
325 330 335
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
340 345 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
355 360 365
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
370 375 3B0
CA 02273408 1999-09-29
54/16
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ala Ala Ser Trp Thr
385 390 395 400
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
405 410 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
420 425 430
Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
435 440 445
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
450 455 460
Cys Phe Ala
465
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 482 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Asn Ser His Ser Cys Asp Thr Val Asp Xaa Gly Tyr Gln Cys Xaa Pro
1 5 10 15
Glu Ile Ser His Leu Trp Gly Gln Tyr Ser Pro Phe Phe Ser Leu Ala
20 25 30
Asp Glu Ser Ala Ile Ser Pro Asp Val Pro Lys Gly Cys Arg Val Thr
35 40 45
Phe Val Gln Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser
50 55 60
Lys Ser Lys Lys Tyr Ser Ala Leu Ile Glu Arg Ile Gln Lys Asn Ala
65 70 75 80
Thr Xaa Phe Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr
85 90 95
Leu Gly Ala Asp Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Val Asn
100 105 110
CA 02273408 1999-09-29
54/17
Ser Gly Ile Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Xaa Ala Arg Asn
115 120 125
Ile Val Pro Phe Val Arg Ala Ser Gly Ser Asp Arg Val Ile Ala Ser
130 135 140
Ala Glu Lys Phe Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro
145 150 155 160
Xaa Ala Xaa Xaa His Gln Ala Ser Pro Val Ile Asn Val Ile Ile Pro
165 170 175
Glu Gly Ser Gly Tyr Asn Asn Thr Leu Asp His Gly Leu Cys Thr Ala
180 185 190
Phe Glu Asp Xaa Xaa Xaa Ser Thr Leu Gly Asp Asp Ala Glu Ala Asn
195 200 205
Phe Thr Ala Val Phe Ala Pro Pro Ile Arg Ala Arg Leu Glu Ala Xaa
210 215 220
Leu Pro Gly Val Asn Leu Thr Asp Glu Asp Val Val Asn Leu Met Asp
225 230 235 240
Met Cys Pro Phe Asp Thr Val Ala Arg Thr Ser Asp Xaa Xaa Ala Thr
245 250 255
Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Ser Pro Phe Cys Asp
260 265 270
Leu Phe Xaa Xaa Thr Ala Asp Glu Trp Xaa Gln Tyr Asp Tyr Leu Gln
275 280 285
Ser Leu Xaa Lys Tyr Tyr Gly Tyr Gly Ala Gly Asn Pro Leu Gly Pro
290 295 300
Ala Gln Gly Val Gly Phe Xaa Asn Glu Leu Ile Ala Arg Leu Thr His
305 310 315 320
Ser Xaa Pro Val Gln Asp His Thr Ser Thr Asn His Thr Leu Asp Ser
325 330 335
Asn Pro Ala Thr Phe Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser
340 345 350
His Asp Asn Thr Met Val Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn
355 360 365
Gly Thr Lys Pro Leu Ser Thr Thr Ser Val Glu Ser Ile Xaa Xaa Xaa
370 375 380
Glu Thr Asp Gly Tyr Ala Ala Ser Trp Thr Val Pro Phe Ala Ala Arg
385 390 395 400
CA 02273408 1999-09-29
54/ 18
Ala Tyr Val Glu Met Met Gln Cys Glu Ala Gly Gly Xaa Gly Xaa Gly
405 410 415
Gly Xaa Glu Gly Xaa Xaa Glu Lys Xaa Xaa Xaa Glu Pro Leu Val Arg
420 425 430
Val Leu Val Asn Asp Arg Val Val Pro Leu His Gly Cys Gly Val Asp
435 440 445
Lys Leu Gly Arg Cys Lys Leu Asp Asp Phe Val Glu Gly Leu Ser Phe
450 455 460
Ala Arg Ser Gly Xaa Xaa Gly Asn Trp Ala Glu Cys Phe Ala Xaa Xaa
465 470 475 480
Xaa Xaa
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1404 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: sigJpeptide
(B) LOCATION:1..78
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..1401
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATG GGC GTG TTC GTC GTG CTA CTG TCC ATT GCC ACC TTG TTC GGT TCC 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
470 475 480
ACA TCC GGT ACC GCC TTG GGT CCT CGT GGT AAT TCT CAC TCT TGT GAC 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
485 490 495
ACT GTT GAC GGT GGT TAC CAA TGT TTC CCA GAA ATT TCT CAC TTG TGG 144
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
500 505 510 515
GGT ACC TAC TCT CCA TAC TTC TCT TTG GCA GAC GAA TCT GCT ATT TCT 192
Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
520 525 530
CA 02273408 1999-09-29
54/ 19
CCA GAC GTT CCA GAC GAC TGT AGA GTT ACT TTC GTT CAA GTT TTG TCT 240
Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
535 540 545
AGA CAC GGT GCT AGA TAC CCA ACT TCT TCT GCG TCT AAG GCT TAC TCT 288
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
550 555 560
GCT TTG ATT GAA GCT ATT CAA AAG AAC GCT ACT GCT TTC AAG GGT AAG 336
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
565 570 575
TAC GCT TTC TTG AAG ACT TAC AAC TAC ACT TTG GGT GCT GAC GAC TTG 384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
580 585 590 595
ACT CCA TTC GGT GAA AAC CAA ATG GTT AAC TCT GGT ATT AAG TTC TAC 432
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
600 605 610
AGA AGA TAC AAG GCT TTG GCT AGA AAG ATT GTT CCA TTC ATT AGA GCT 480
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
615 620 625
TCT GGT TCT GAC AGA GTT ATT GCT TCT GCT GAA AAG TTC ATT GAA GGT 528
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
630 635 640
TTC CAA TCT GCT AAG TTG GCT GAC CCA GGT TCT CAA CCA CAC CAA GCT 576
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
645 650 655
TCT CCA GTT ATT AAC GTG ATC ATT CCA GAA GGA TCC GGT TAC AAC AAC 624
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
660 665 670 675
ACT TTG GAC CAC GGT ACT TGT ACT GCT TTC GAA GAC TCT GAA TTA GGT 672
Thr Leu Asp His Gly Thr Cys Thr Ala Phe Glu Asp Ser Glu Leu Gly
680 685 690
GAC GAC GTT GAA GCT AAC TTC ACT GCT TTG TTC GCT CCA GCT ATT AGA 720
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
695 700 705
GCT AGA TTG GAA GCT GAC TTG CCA GGT GTT ACT TTG ACT GAC GAA GAC 768
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
710 715 720
GTT GTT TAC TTG ATG GAC ATG TGT CCA TTC GAC ACT GTC GCT AGA ACT 816
Val Val Tyr Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
725 730 735
TCT GAC GCT ACT GAA TTG TCT CCA TTC TGT GCT TTG TTC ACT CAC GAC 864
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
740 745 750 755
CA 02273408 1999-09-29
54/20
GAA TGG ATC CAA TAC GAC TAC TTG CAA AGC TTG GGT AAG TAC TAC GGT 912
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
760 765 770
TAC GGT GCT GGT AAC CCA TTG GGT CCA GCT CAA GGT GTT GGT TTC GCT 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
775 780 785
AAC GAA TTG ATT GCT AGA TTG ACT CAC TCT CCA GTT CAA GAC CAC ACT 1008
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
790 795 800
TCT ACT AAC CAC ACT TTG GAC TCT AAC CCA GCT ACT TTC CCA TTG AAC 1056
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
805 810 815
GCT ACT TTG TAC GCT GAC TTC TCT CAC GAC AAC ACT ATG ATA TCT ATT 1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Ile Ser Ile
820 825 830 835
TTC TTC GCT TTG GGT TTG TAC AAC GGT ACC AAG CCA TTG TCT ACT ACT 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
840 845 850
TCT GTT GAA TCT ATT GAA GAA ACT GAC GGT TAC TCT GCT TCT TGG ACT 1200
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
855 860 865
GTT CCA TTC GCT GCT AGA GCT TAC GTT GAA ATG ATG CAA TGT CAA GCT 1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
870 875 880
GAA AAG GAA CCA TTG GTT AGA GTT TTG GTT AAC GAC AGA GTT GTT CCA 1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
885 890 895
TTG CAC GGT TGT GCT GTT GAC AAG TTG GGT AGA TGT AAG AGA GAC GAC 1344
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
900 905 910 915
TTC GTT GAA GGT TTG TCT TTC GCT AGA TCT GGT GGT AAC TGG GCT GAA 1392
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
920 925 930
TGT TTC GCT TAA 1404
Cys Phe Ala
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02273408 1999-09-29
54/21
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
1 5 10 15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
20 25 30
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
35 40 45
Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
50 55 60
Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
65 70 75 80
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
85 90 95
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
100 105 110
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
115 120 125
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
130 135 140
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
145 150 155 160
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
165 170 175
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
180 185 190
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ser Gly Tyr Asn Asn
195 200 205
Thr Leu Asp His Gly Thr Cys Thr Ala Phe Glu Asp Ser Glu Leu Gly
210 215 220
Asp Asp Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
225 230 235 240
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
245 250 255
Val Val Tyr Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
260 265 270
CA 02273408 1999-09-29
54/22
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
275 280 285
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
290 295 300
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Ala
305 310 315 320
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
325 330 335
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
340 345 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Ile Ser Ile
355 360 365
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
370 375 380
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
385 390 395 400
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
405 410 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
420 425 430
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
435 440 445
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
450 455 460
Cys Phe Ala
465
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1404 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(v) FRAGMENT TYPE: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..1401
CA 02273408 1999-09-29
54/23
(ix) FEATURE:
(A) NAME/KEY: sig~eptide
(B) LOCATION:1..78
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ATG GGC GTG TTC GTC GTG CTA CTG TCC ATT GCC ACC TTG TTC GGT TCC 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
470 475 480
ACA TCC GGT ACC GCC TTG GGT CCT CGT GGT AAC TCT CAC TCT TGT GAC 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
485 490 495
ACT GTT GAC GGT GGT TAC CAA TGT TTC CCA GAA ATT TCT CAC TTG TGG 144
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
500 505 510 515
GGT ACA TAC TCT CCA TTC TTC TCT TTG GCT GAC GAA TCT GCT ATT TCT 192
Gly Thr Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
520 525 530
CCA GAC GTT CCA AAG GGT TGT AGA GTT ACT TTC GTT CAA GTT TTG TCT 240
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
535 540 545
AGA CAC GGT GCT AGA TAC CCA ACT TCT TCT GCG TCT AAG GCG TAC TCT 288
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
550 555 560
GCT TTG ATT GAA GCT ATT CAA AAG AAC GCT ACT GCT TTC AAG GGT AAG 336
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
565 570 575
TAC GCT TTC TTG AAG ACT TAC AAC TAC ACT TTG GGT GCT GAC GAC TTG 384
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
580 585 590 595
ACT CCA TTC GGT GAA CAA CAA ATG GTT AAC TCT GGT ATT AAG TTC TAC 432
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
600 605 610
AGA AGA TAC AAG GCT TTG GCT AGA AAG ATT GTT CCA TTC ATT AGA GCT 480
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
615 620 625
TCT GGT TCT GAC AGA GTT ATT GCT TCT GCT GAA AAG TTC ATT GAA GGT 528
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
630 635 640
TTC CAA TCT GCT AAG TTG GCT GAC CCA GGT GCT AAC CCA CAC CAA GCT 576
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
645 650 655
CA 02273408 1999-09-29
54/24
TCT CCA GTT ATT AAC GTT ATT ATT CCA GAA GGT GCT GGT TAC AAC AAC 624
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
660 665 670 675
ACT TTG GAC CAC GGT TTG TGT ACT GCT TTC GAA GAA TCT GAA TTG GGT 672
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Glu Leu Gly
680 685 690
GAC GAC GTT GAA GCT AAC TTC ACT GCT GTT TTC GCT CCA CCA ATT AGA 720
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
695 700 705
GCT AGA TTG GAA GCT CAC TTG CCA GGT GTT AAC TTG ACT GAC GAA GAC 768
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
710 715 720
GTT GTT AAC TTG ATG GAC ATG TGT CCA TTC GAC ACT GTT GCT AGA ACT 816
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
725 730 735
TCT GAC GCT ACT CAA TTG TCT CCA TTC TGT GAC TTG TTC ACT CAC GAC 864
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
740 745 750 755
GAA TGG ATT CAA TAC GAC TAC TTG CAA TCT TTG GGT AAG TAC TAC GGT 912
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
760 765 770
TAC GGT GCT GGT AAC CCA TTG GGT CCA GCT CAA GGT GTT GGT TTC GTT 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
775 780 785
AAC GAA TTG ATT GCT AGA TTG ACT CAC TCT CCA GTT CAA GAC CAC ACT 1008
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
790 795 800
TCT ACT AAC CAC ACT TTG GAC TCT AAC CCA GCT ACT TTC CCA TTG AAC 1056
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
805 810 815
GCT ACT TTG TAC GCT GAC TTC TCT CAC GAC AAC ACT ATG GTT TCT ATT 1104
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
820 825 830 835
TTC TTC GCT TTG GGT TTG TAC AAC GGT ACT AAG CCA TTG TCT ACT ACT 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
840 845 850
TCT GTT GAA TCT ATT GAA GAA ACT GAC GGT TAC TCT GCT TCT TGG ACT 1200
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
855 860 865
GTT CCA TTC GCT GCT AGA GCT TAC GTT GAA ATG ATG CAA TGT GAA GCT 1248
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
870 875 880
CA 02273408 1999-09-29
54/25
GAA AAG GAA CCA TTG GTT AGA GTT TTG GTT AAC GAC AGA GTT GTT CCA 1296
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
885 890 895
TTG CAC GGT TGT GGT GTT GAC AAG TTG GGT AGA TGT AAG AGA GAC GAC 1344
Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
900 905 910 915
TTC GTT GAA GGT TTG TCT TTC GCT AGA TCT GGT GGT AAC TGG GAA GAA 1392
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
920 925 930
TGT TTC GCT TAA 1404
Cys Phe Ala
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
1 5 10 15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
20 25 30
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
35 40 45
Gly Thr Tyr Ser Pro Phe Phe Ser Leu Ala Asp Glu Ser Ala Ile Ser
50 55 60
Pro Asp Val Pro Lys Gly Cys Arg Val Thr Phe Val Gln Val Leu Ser
65 70 75 80
Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Ala Ser Lys Ala Tyr Ser
85 90 95
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
100 105 110
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
115 120 125
Thr Pro Phe Gly Glu Gln Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
130 135 140
CA 02273408 1999-09-29
54/26
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
145 150 155 160
Ser Gly Ser Asp Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
165 170 175
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ala Asn Pro His Gln Ala
180 185 190
Ser Pro Val Ile Asn Val Ile Ile Pro Glu Gly Ala Gly Tyr Asn Asn
195 200 205
Thr Leu Asp His Gly Leu Cys Thr Ala Phe Glu Glu Ser Glu Leu Gly
210 215 220
Asp Asp Val Glu Ala Asn Phe Thr Ala Val Phe Ala Pro Pro Ile Arg
225 230 235 240
Ala Arg Leu Glu Ala His Leu Pro Gly Val Asn Leu Thr Asp Glu Asp
245 250 255
Val Val Asn Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
260 265 270
Ser Asp Ala Thr Gln Leu Ser Pro Phe Cys Asp Leu Phe Thr His Asp
275 280 285
Glu Trp Ile Gln Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
290 295 300
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Val Gly Phe Val
305 310 315 320
Asn Glu Leu Ile Ala Arg Leu Thr His Ser Pro Val Gln Asp His Thr
325 330 335
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
340 345 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Thr Met Val Ser Ile
355 360 365
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Thr Thr
370 375 380
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ala Ser Trp Thr
385 390 395 400
Val Pro Phe Ala Ala Arg Ala Tyr Val Glu Met Met Gln Cys Glu Ala
405 410 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
420 425 430
CA 02273408 1999-09-29
54/27
Leu His Gly Cys Gly Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
435 440 445
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Glu Glu
450 455 460
Cys Phe Ala
465
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1404 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATOIN:1..1401
(ix) FEATURE:
(A) NAME/KEY: sig~eptide
(B) LOCATION:1..78
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ATG GGG GTT TTC GTC GTT CTA TTA TCT ATC GCG ACT CTG TTC GGC AGC 48
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
470 475 480
ACA TCG GGC ACT GCG CTG GGC CCC CGT GGA AAT CAC TCC AAG TCC TGC 96
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn His Ser Lys Ser Cys
485 490 495
GAT ACG GTA GAC CTA GGG TAC CAG TGC TCC CCT GCG ACT TCT CAT CTA 144
Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro Ala Thr Ser His Leu
500 505 510 515
TGG GGC ACG TAC TCG CCA TAC TTT TCG CTC GAG GAC GAG CTG TCC GTG 192
Trp Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Leu Ser Val
520 525 530
TCG AGT AAG CTT CCC AAG GAT TGC CGG ATC ACC TTG GTA CAG GTG CTA 240
Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr Leu Val Gln Val Leu
535 540 545
TCG CGC CAT GGA GCG CGG TAC CCA ACC AGC TCC AAG AGC AAA AAG TAT 288
Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Lys Tyr
550 555 560
CA 02273408 1999-09-29
54/28
AAG AAG CTT ATT ACG GCG ATC CAG GCC AAT GCC ACC GAC TTC AAG GGC 336
Lys Lys Leu Ile Thr Ala Ile Gln Ala Asn Ala Thr Asp Phe Lys Gly
565 570 575
AAG TAC GCC TTT TTG AAG ACG TAC AAC TAT ACT CTG GGT GCG GAT GAC 384
Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp
580 585 590 595
CTC ACT CCC TTT GGG GAG CAG CAG CTG GTG AAC TCG GGC ATC AAG TTC 432
Leu Thr Pro Phe Gly Glu Gln Gln Leu Val Asn Ser Gly Ile Lys Phe
600 605 610
TAC CAG AGG TAC AAG GCT CTG GCG CGC AGT GTG GTG CCG TTT ATT CGC 480
Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val Val Pro Phe Ile Arg
615 620 625
GCC TCA GGC TCG GAC CGG GTT ATT GCT TCG GGA GAG AAG TTC ATC GAG 528
Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly Glu Lys Phe Ile Glu
630 635 640
GGG TTC CAG CAG GCG AAG CTG GCT GAT CCT GGC GCG ACG AAC CGC GCC 576
Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly Ala Thr Asn Arg Ala
645 650 655
GCT CCG GCG ATT AGT GTG ATT ATT CCG GAG AGC GAG ACG TTC AAC AAT 624
Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser Glu Thr Phe Asn Asn
660 665 670 675
ACG CTG GAC CAC GGT GTG TGC ACG AAG TTT GAG GCG AGT CAG CTG GGA 672
Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu Ala Ser Gln Leu Gly
680 685 690
GAT GAG GTT GCG GCC AAT TTC ACT GCG CTC TTT GCA CCC GAC ATC CGA 720
Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe Ala Pro Asp Ile Arg
695 700 705
GCT CGC CTC GAG AAG CAT CTT CCT GGC GTG ACG CTG ACA GAC GAG GAC 768
Ala Arg Leu Glu Lys His Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
710 715 720
GTT GTC AGT CTA ATG GAC ATG TGT CCG TTT GAT ACG GTA GCG CGC ACC 816
Val Val Ser Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
725 730 735
AGC GAC GCA AGT CAG CTG TCA CCG TTC TGT CAA CTC TTC ACT CAC AAT 864
Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln Leu Phe Thr His Asn
740 745 750 755
GAG TGG AAG AAG TAC GAC TAC CTT CAG TCC TTG GGC AAG TAC TAC GGC 912
Glu Trp Lys Lys Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
760 765 770
TAC GGC GCA GGC AAC CCT CTG GGA CCG GCT CAG GGG ATA GGG TTC ACC 960
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Ile Gly Phe Thr
775 780 785
CA 02273408 1999-09-29
54/29
AAC GAG CTG ATT GCC CGG TTG ACG CGT TCG CCA GTG CAG GAC CAC ACC 1008
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr
790 795 800
AGC ACT AAC TCG ACT CTA GTC TCC AAC CCG GCC ACC TTC CCG TTG AAC 1056
Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala Thr Phe Pro Leu Asn
805 810 815
GCT ACC ATG TAC GTC GAC TTT TCA CAC GAC AAC AGC ATG GTT TCC ATC 1104
Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn Ser Met Val Ser Ile
820 825 830 835
TTC TTT GCA TTG GGC CTG TAC AAC GGC ACT GAA CCC TTG TCC CGG ACC 1152
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu Pro Leu Ser Arg Thr
840 845 850
TCG GTG GAA AGC GCC AAG GAA TTG GAT GGG TAT TCT GCA TCC TGG GTG 1200
Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr Ser Ala Ser Trp Val
855 860 865
GTG CCT TTC GGC GCG CGA GCC TAC TTC GAG ACG ATG CAA TGC AAG TCG 1248
Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr Met Gln Cys Lys Ser
870 875 880
GAA AAG GAG CCT CTT GTT CGC GCT TTG ATT AAT GAC CGG GTT GTG CCA 1296
Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn Asp Arg Val Val Pro
885 890 895
CTG CAT GGC TGC GAT GTG GAC AAG CTG GGG CGA TGC AAG CTG AAT GAC 1344
Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg Cys Lys Leu Asn Asp
900 905 910 915
TTT GTC AAG GGA TTG AGT TGG GCC AGA TCT GGG GGC AAC TGG GGA GAG 1392
Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly Gly Asn Trp Gly Glu
920 925 930
TGC TTT AGT TGA 1404
Cys Phe Ser
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
1 5 10 15
CA 02273408 1999-09-29
54/30
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn His Ser Lys Ser Cys
20 25 30
Asp Thr Val Asp Leu Gly Tyr Gln Cys Ser Pro Ala Thr Ser His Leu
35 40 45
Trp Gly Thr Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Leu Ser Val
50 55 60
Ser Ser Lys Leu Pro Lys Asp Cys Arg Ile Thr Leu Val Gln Val Leu
65 70 75 80
Ser Arg His Gly Ala Arg Tyr Pro Thr Ser Ser Lys Ser Lys Lys Tyr
85 90 95
Lys Lys Leu Ile Thr Ala Ile Gln Ala Asn Ala Thr Asp Phe Lys Gly
100 105 110
Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp
115 120 125
Leu Thr Pro Phe Gly Glu Gln Gln Leu Val Asn Ser Gly Ile Lys Phe
130 135 140
Tyr Gln Arg Tyr Lys Ala Leu Ala Arg Ser Val Val Pro Phe Ile Arg
145 150 155 160
Ala Ser Gly Ser Asp Arg Val Ile Ala Ser Gly Glu Lys Phe Ile Glu
165 170 175
Gly Phe Gln Gln Ala Lys Leu Ala Asp Pro Gly Ala Thr Asn Arg Ala
180 185 190
Ala Pro Ala Ile Ser Val Ile Ile Pro Glu Ser Glu Thr Phe Asn Asn
195 200 205
Thr Leu Asp His Gly Val Cys Thr Lys Phe Glu Ala Ser Gln Leu Gly
210 215 220
Asp Glu Val Ala Ala Asn Phe Thr Ala Leu Phe Ala Pro Asp Ile Arg
225 230 235 240
Ala Arg Leu Glu Lys His Leu Pro Gly Val Thr Leu Thr Asp Glu Asp
245 250 255
Val Val Ser Leu Met Asp Met Cys Pro Phe Asp Thr Val Ala Arg Thr
260 265 270
Ser Asp Ala Ser Gln Leu Ser Pro Phe Cys Gln Leu Phe Thr His Asn
275 280 285
Glu Trp Lys Lys Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Tyr Tyr Gly
290 295 300
CA 02273408 1999-09-29
54/31
Tyr Gly Ala Gly Asn Pro Leu Gly Pro Ala Gln Gly Ile Gly Phe Thr
305 310 315 320
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr
325 330 335
Ser Thr Asn Ser Thr Leu Val Ser Asn Pro Ala Thr Phe Pro Leu Asn
340 345 350
Ala Thr Met Tyr Val Asp Phe Ser His Asp Asn Ser Met Val Ser Ile
355 360 365
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Glu Pro Leu Ser Arg Thr
370 375 380
Ser Val Glu Ser Ala Lys Glu Leu Asp Gly Tyr Ser Ala Ser Trp Val
385 390 395 400
Val Pro Phe Gly Ala Arg Ala Tyr Phe Glu Thr Met Gln Cys Lys Ser
405 410 415
Glu Lys Glu Pro Leu Val Arg Ala Leu Ile Asn Asp Arg Val Val Pro
420 425 430
Leu His Gly Cys Asp Val Asp Lys Leu Gly Arg Cys Lys Leu Asn Asp
435 440 445
Phe Val Lys Gly Leu Ser Trp Ala Arg Ser Gly Gly Asn Trp Gly Glu
450 455 460
Cys Phe Ser
465
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1426 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(v) FRAGMENT TYPE: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:12..1412
(ix) FEATURE:
(A) NAME/KEY: sig~eptide
(B) LOCATION:12..89
CA 02273408 1999-09-29
54/32
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 15:
TATATGAATT C ATG GGC GTG TTC GTC GTG CTA CTG TCC ATT GCC ACC TTG 50
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu
470 475 480
TTC GGT TCC ACA TCC GGT ACC GCC TTG GGT CCT CGT GGT AAT TCT CAC 98
Phe Gly Ser Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His
485 490 495
TCT TGT GAC ACT GTT GAC GGT GGT TAC CAA TGT TTC CCA GAA ATT TCT 146
Ser Cys Asp Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser
500 505 510
CAC TTG TGG GGT CAA TAC TCT CCA TAC TTC TCT TTG GAA GAC GAA TCT 194
His Leu Trp Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Ser
515 520 525
GCT ATT TCT CCA GAC GTT CCA GAC GAC TGT AGA GTT ACT TTC GTT CAA 242
Ala Ile Ser Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln
530 535 540
GTT TTG TCT AGA CAC GGT GCT AGA TAC CCA ACT GAC TCT AAG GGT AAG 290
Val Leu Ser Arg His Gly Ala Arg Tyr Pro Thr Asp Ser Lys Gly Lys
545 550 555 560
AAG TAC TCT GCT TTG ATT GAA GCT ATT CAA AAG AAC GCT ACT GCT TTC 338
Lys Tyr Ser Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe
565 570 575
AAG GGT AAG TAC GCT TTC TTG AAG ACT TAC AAC TAC ACT TTG GGT GCT 386
Lys Gly Lys Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala
580 585 590
GAC GAC TTG ACT CCA TTC GGT GAA AAC CAA ATG GTT AAC TCT GGT ATT 434
Asp Asp Leu Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile
595 600 605
AAG TTC TAC AGA AGA TAC AAG GCT TTG GCT AGA AAG ATT GTT CCA TTC 482
Lys Phe Tyr Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe
610 615 620
ATT AGA GCT TCT GGT TCT TCT AGA GTT ATT GCT TCT GCT GAA AAG TTC 530
Ile Arg Ala Ser Gly Ser Ser Arg Val Ile Ala Ser Ala Glu Lys Phe
625 630 635 640
ATT GAA GGT TTC CAA TCT GCT AAG TTG GCT GAC CCA GGT TCT CAA CCA 578
Ile Glu Gly Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro
645 650 655
CAC CAA GCT TCT CCA GTT ATT GAC GTT ATT ATT TCT GAC GCT TCT TCT 626
His Gln Ala Ser Pro Val Ile Asp Val Ile Ile Ser Asp Ala Ser Ser
660 665 670
TAC AAC AAC ACT TTG GAC CCA GGT ACT TGT ACT GCT TTC GAA GAC TCT 674
CA 02273408 1999-09-29
54/33
Tyr Asn Asn Thr Leu Asp Pro Gly Thr Cys Thr Ala Phe Glu Asp Ser
675 680 685
GAA TTG GCT GAC ACT GTT GAA GCT AAC TTC ACT GCT TTG TTC GCT CCA 722
Glu Leu Ala Asp Thr Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro
690 695 700
GCT ATT AGA GCT AGA TTG GAA GCT GAC TTG CCA GGT GTT ACT TTG ACT 770
Ala Ile Arg Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr
705 710 715 720
GAC ACT GAA GTT ACT TAC TTG ATG GAC ATG TGT TCT TTC GAA ACT GTT 818
Asp Thr Glu Val Thr Tyr Leu Met Asp Met Cys Ser Phe Glu Thr Val
725 730 735
GCT AGA ACT TCT GAC GCT ACT GAA TTG TCT CCA TTC TGT GCT TTG TTC 866
Ala Arg Thr Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe
740 745 750
ACT CAC GAC GAA TGG AGA CAC TAC GAC TAC TTG CAA TCT TTG AAG AAG 914
Thr His Asp Glu Trp Arg His Tyr Asp Tyr Leu Gln Ser Leu Lys Lys
755 760 765
TAC TAC GGT CAC GGT GCT GGT AAC CCA TTG GGT CCA ACT CAA GGT GTT 962
Tyr Tyr Gly His Gly Ala Gly Asn Pro Leu Gly Pro Thr Gln Gly Val
770 775 780
GGT TTC GCT AAC GAA TTG ATT GCT AGA TTG ACT AGA TCT CCA GTT CAA 1010
Gly Phe Ala Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln
785 790 795 800
GAC CAC ACT TCT ACT AAC CAC ACT TTG GAC TCT AAC CCA GCT ACT TTC 1058
Asp His Thr Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe
805 810 815
CCA TTG AAC GCT ACT TTG TAC GCT GAC TTC TCT CAC GAC AAC GGT ATT 1106
Pro Leu Asn Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Gly Ile
820 825 830
ATT TCT ATT TTC TTC GCT TTG GGT TTG TAC AAC GGT ACT GCT CCA TTG 1154
Ile Ser Ile Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu
835 840 845
TCT ACT ACT TCT GTT GAA TCT ATT GAA GAA ACT GAC GGT TAC TCT TCT 1202
Ser Thr Thr Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ser
850 855 860
GCT TGG ACT GTT CCA TTC GCT TCT AGA GCT TAC GTT GAA ATG ATG CAA 1250
Ala Trp Thr Val Pro Phe Ala Ser Arg Ala Tyr Val Glu Met Met Gln
865 870 875 880
TGT CAA GCT GAA AAG GAA CCA TTG GTT AGA GTT TTG GTT AAC GAC AGA 1298
Cys Gln Ala Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg
885 890 895
CA 02273408 1999-09-29
54/34
GTT GTT CCA TTG CAC GGT TGT GCT GTT GAC AAG TTG GGT AGA TGT AAG 1346
Val Val Pro Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys
900 905 910
AGA GAC GAC TTC GTT GAA GGT TTG TCT TTC GCT AGA TCT GGT GGT AAC 1394
Arg Asp Asp Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn
915 920 925
TGG GCT GAA TGT TTC GCT TAAGAATTCA TATA 1426
Trp Ala Glu Cys Phe Ala
930
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Gly Val Phe Val Val Leu Leu Ser Ile Ala Thr Leu Phe Gly Ser
1 5 10 15
Thr Ser Gly Thr Ala Leu Gly Pro Arg Gly Asn Ser His Ser Cys Asp
20 25 30
Thr Val Asp Gly Gly Tyr Gln Cys Phe Pro Glu Ile Ser His Leu Trp
35 40 45
Gly Gln Tyr Ser Pro Tyr Phe Ser Leu Glu Asp Glu Ser Ala Ile Ser
50 55 60
Pro Asp Val Pro Asp Asp Cys Arg Val Thr Phe Val Gln Val Leu Ser
65 70 75 80
Arg His Gly Ala Arg Tyr Pro Thr Asp Ser Lys Gly Lys Lys Tyr Ser
85 90 95
Ala Leu Ile Glu Ala Ile Gln Lys Asn Ala Thr Ala Phe Lys Gly Lys
100 105 110
Tyr Ala Phe Leu Lys Thr Tyr Asn Tyr Thr Leu Gly Ala Asp Asp Leu
115 120 125
Thr Pro Phe Gly Glu Asn Gln Met Val Asn Ser Gly Ile Lys Phe Tyr
130 135 140
Arg Arg Tyr Lys Ala Leu Ala Arg Lys Ile Val Pro Phe Ile Arg Ala
145 150 155 160
Ser Gly Ser Ser Arg Val Ile Ala Ser Ala Glu Lys Phe Ile Glu Gly
165 170 175
CA 02273408 1999-09-29
54/35
Phe Gln Ser Ala Lys Leu Ala Asp Pro Gly Ser Gln Pro His Gln Ala
180 185 190
Ser Pro Val Ile Asp Val Ile Ile Ser Asp Ala Ser Ser Tyr Asn Asn
195 200 205
Thr Leu Asp Pro Gly Thr Cys Thr Ala Phe Glu Asp Ser Glu Leu Ala
210 215 220
Asp Thr Val Glu Ala Asn Phe Thr Ala Leu Phe Ala Pro Ala Ile Arg
225 230 235 240
Ala Arg Leu Glu Ala Asp Leu Pro Gly Val Thr Leu Thr Asp Thr Glu
245 250 255
Val Thr Tyr Leu Met Asp Met Cys Ser Phe Glu Thr Val Ala Arg Thr
260 265 270
Ser Asp Ala Thr Glu Leu Ser Pro Phe Cys Ala Leu Phe Thr His Asp
275 280 285
Glu Trp Arg His Tyr Asp Tyr Leu Gln Ser Leu Lys Lys Tyr Tyr Gly
290 295 300
His Gly Ala Gly Asn Pro Leu Gly Pro Thr Gln Gly Val Gly Phe Ala
305 310 315 320
Asn Glu Leu Ile Ala Arg Leu Thr Arg Ser Pro Val Gln Asp His Thr
325 330 335
Ser Thr Asn His Thr Leu Asp Ser Asn Pro Ala Thr Phe Pro Leu Asn
340 345 350
Ala Thr Leu Tyr Ala Asp Phe Ser His Asp Asn Gly Ile Ile Ser Ile
355 360 365
Phe Phe Ala Leu Gly Leu Tyr Asn Gly Thr Ala Pro Leu Ser Thr Thr
370 375 380
Ser Val Glu Ser Ile Glu Glu Thr Asp Gly Tyr Ser Ser Ala Trp Thr
385 390 395 400
Val Pro Phe Ala Ser Arg Ala Tyr Val Glu Met Met Gln Cys Gln Ala
405 410 415
Glu Lys Glu Pro Leu Val Arg Val Leu Val Asn Asp Arg Val Val Pro
420 425 430
Leu His Gly Cys Ala Val Asp Lys Leu Gly Arg Cys Lys Arg Asp Asp
435 440 445
Phe Val Glu Gly Leu Ser Phe Ala Arg Ser Gly Gly Asn Trp Ala Glu
450 455 460
CA 02273408 1999-09-29
54/36
Cys Phe Ala
465
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TATATGAATT CATGGGCGTG TTCGTC 26
(2) INFORMATION FOR SEQ ID N0: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
TGAAAAGTTC ATTGAAGGTT TC 22
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
CA 02273408 1999-09-29
54/37
TCTTCGAAAG CAGTACAAGT AC 22
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
TATATGAATT CTTAAGCGAA AC 22
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
TCTTCGAAAG CAGTACACAA AC 22
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
CACTTGTGGG GTACCTACTC TCCATACTTC TC 32
CA 02273408 1999-09-29
54/38
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
GAGAAGTATG GAGAGTAGGT ACCCCACAAG TG 32
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 24:
GGTCAATACT CTCCATTCTT CTCTTTGGAA G 31
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
CTTCCAAAGA GAAGAATGGA GAGTATTGAC C 31
(2) INFORMATION FOR SEQ ID NO: 26:
CA 02273408 1999-09-29
54/39
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
CATACTTCTC TTTGGCAGAC GAATCTGC 28
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 27:
GCAGATTCGT CTGCCAAAGA GAAGTATG 28
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 28:
CTCCAGACGT CCCAAAGGAC TGTAGAGTTA C 31
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
CA 02273408 1999-09-29
54/40
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
GTAACTCTAC AGTCCTTTGG GACGTCTGGA G 31
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 30:
CTCCAGACGT CCCAGACGGC TGTAGAGTTA C 31
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
GTAACTCTAC AGCCGTCTGG GACGTCTGGA G 31
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
CA 02273408 1999-09-29
54/41
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 32:
GATACCCAAC TTCTTCTGCG TCTAAGGCTT ACTCTG 36
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
CAGAGTAAGC CTTAGACGCA GAAGAAGTTG GGTATC 36
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
CTTCTAAGTC TAAGAAGTAC TCTGCTTTG 29
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02273408 1999-09-29
54/42
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CAAAGCAGAG TACTTCTTAG ACTTAGAAG 29
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
GCTTACTCTG CTTTGATTGA ACGGATTCAA AAGAACGCTA C 41
(2) INFORMATION FOR SEQ ID N0: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
GTAGCGTTCT TTTGAATCCG TTCAATCAAA GCAGAGTAAG C 41
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02273408 1999-09-29
54/43
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
CCATTCGGTG AACAGCAAAT GGTTAACTC 29
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GAGTTAACCA TTTGCTGTTC ACCGAATGG 29
(2) INFORMATION FOR SEQ ID N0: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
GATACAAGGC TCTCGCGAGA AACATTGTTC 30
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
CA 02273408 1999-09-29
54/44
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
GGAACAATGT TTCTCGCGAG AGCCTTGTAT C 31
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GATTGTTCCA TTCGTGCGCG CTTCTGGTTC 30
(2) INFORMATION FOR SEQ ID N0: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
GAACCAGAAG CGCGCACGAA TGGAACAATC 30
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
CA 02273408 1999-09-29
54/45
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
CTCCAGTTAT TAACGTGATC ATTCCAGAAG G 31
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
CCTTCTGGAA TGATCACGTT AATAACTGGA G 31
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
GGCTGACCCA GGGGCCCAAC CACACCAAGC 30
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
CA 02273408 1999-09-29
54/46
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
GCTTGGTGTG GTTGGGCCCC TGGGTCAGCC 30
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
CACTTTGGAC CATGGTCTTT GTACTGCTTT CG 32
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
CGAAAGCAGT ACAAAGACCA TGGTCCAAAG TG 32
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
CA 02273408 1999-09-29
54/47
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
GCTTTCGAAG ACTCTACCCT AGGTGACGAC GTTG 34
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 51:
CAACGTCGTC ACCTAGGGTA GAGTCTTCGA AAGC 34
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
GGTGACGACG CTGAAGCTAA CTTCAC 26
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
CA 02273408 1999-09-29
54/48
GTGAAGTTAG CTTCAGCGTC GTCACC 26
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
CTAACTTCAC CGCGGTGTTC GCTCCAG 27
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
{xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
CTGGAGCGAA CACCGCGGTG AAGTTAG 27
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
GCTTTGTTCG CTCCACCTAT TAGAGCTAGA TTGG 34
CA 02273408 1999-09-29
54/49
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 57:
CCAATCTAGC TCTAATAGGT GGAGCGAACA AAGC 34
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
GCCAGGTGTT AACTTGACTG ACGAAG 26
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
TTCGTCAGTC AAGTTAACAC CTGGC 25
(2) INFORMATION FOR SEQ ID NO: 60:
CA 02273408 1999-09-29
54/50
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
GACGAAGACG TCGTTAACTT GATGGAC , 27
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
GTCCATCAAG TTAACGACGT CTTCGTC 27
(2) INFORMATION FOR SEQ ID N0: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
GTCCATTCGA CACTGTCGCT AGAACTTC 28
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
CA 02273408 1999-09-29
54/51
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
GAAGTTCTAG CGACAGTGTC GAATGGAC 28
(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
CTGACGCTAC TCAGCTGTCT CCATTC 26
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 65:
GAATGGAGAC AGCTGAGTAG CGTCAG 26
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
CA 02273408 1999-09-29
54/52
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
GTCTCCATTC TGTGATTTGT TCACTCAC 28
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 67:
GTGAGTGAAC AAATCACAGA ATGGAGAC 28
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
GCTTTGTTCA CCGCGGACGA ATGGAG 26
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
CA 02273408 1999-09-29
54/53
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
CTCCATTCGT CCGCGGTGAA CAAAGC 26
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
CACGACGAAT GGATCCAATA CGACTAC 27
(2) INFORMATION FOR SEQ ID N0: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
GTAGTCGTAT TGGATCCATT CGTCGTG 27
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02273408 1999-09-29
54/54
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
GACGAATGGA GAGCGTACGA CTACTTG 27
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
CAAGTAGTCG TACGCTCTCC ATTCGTC 27
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
GGTGTTGGTT TCGTTAACGA ATTGATTGC 29
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02273408 1999-09-29
54/55
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
GCAATCAATT CGTTAACGAA ACCAACACC 29
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
GCTAGATTGA CTCACTCTCC AGTTCAAG 28
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
CTTGAACTGG AGAGTGAGTC AATCTAGC 28
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
CA 02273408 1999-09-29
54/56
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
CTCACGACAA CACTATGATA TCTATTTTCT TC 32
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
GAAGAAAATA GATATCATAG TGTTGTCGTG AG 32
(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 80:
CGACAACTCC ATGGTTTCTA TTTTCTTCGC 30
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
CA 02273408 1999-09-29
54/57
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
GCGAAGAAAA TAGAAACCAT GGAGTTGTCG 30
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
GTACAACGGT ACCAAGCCAT TGTCTAC 27
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
GTAGACAATG GCTTGGTACC GTTGTAC 27
(2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
CA 02273408 1999-09-29
54/58
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
CTGACGGTTA CGCTGCTTCT TGGAC 25
(2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
GTCCAAGAAG CAGCGTAACC GTCAG 25
(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
CTGTTCCATT CGCTGCTAGA GCTTAC 26
(2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
CA 02273408 1999-09-29
54/59
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 87:
GTAAGCTCTA GCAGCGAATG GAACAG 26
(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
GATGCAATGT GAAGCTGAAA AGGAACC 27
(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
GGTTCCTTTT CAGCTTCACA TTGCATC 27
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
CA 02273408 1999-09-29
54/60
CACGGTTGTG GTGTCGACAA GTTGGG 26
(2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 91:
CCCAACTTGT CGACACCACA ACCGTG 26
(2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
GATCTGGTGG CAATTGGGAG GAATGTTTCG 30
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
CGAAACATTC CTCCCAATTG CCACCAGATC 30
CA 02273408 1999-09-29
54/61
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
CACGTACTCG CCATACTTTT CGCTCGAG 28
(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
CTCGAGCGAA AAGTATGGCG AGTACGTG 28
(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
CCATACTTTT CGCTCGCGGA CGAGCTGTCC GTG 33
(2) INFORMATION FOR SEQ ID NO: 97:
CA 02273408 1999-09-29
54/62
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
CACGGACAGC TCGTCCGCGA GCGAAAAGTA GG 32
(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
GTATAAGAAG CTTATTACGG CGATCCAGGC C 31
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
GGCCTGGATC GCCGTAATAA GCTTCTTATA C 31
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
CA 02273408 1999-09-29
54/63
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
CTTCAAGGGC AAGTACGCCT TTTTGAAGAC G 31
(2) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
CGTCTTCAAA AAGGCGTACT TGCCCTTGAA G 31
(2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
CATCCGAGCT CGCCTCGAGA AGCATCTTC 29
(2) INFORMATION FOR SEQ ID NO: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
CA 02273408 1999-09-29
54/64
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
GAAGATGCTT CTCGAGGCGA GCTCGGATG 29
(2) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:
CTAATGGATG TGTCCGTTTG ATACGGTAG 29
(2) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:
CTACCGTATC AAACGGACAC ATGTCCATTA G 31
(2) INFORMATION FOR SEQ ID NO: 106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02273408 1999-09-29
54/65
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:
GTGGAAGAAG TACGACTACC TTCAGTC 27
(2) INFORMATION FOR SEQ ID NO: 107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
GACTGAAGGT AGTCGTACTT CTTCCAC 27
(2) INFORMATION FOR SEQ ID NO: 108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:
GCCCGGTTGA CGCATTCGCC AGTGCAGG 28
(2) INFORMATION FOR SEQ ID NO: 109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02273408 1999-09-29
54/66
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:
CCTGCACTGG CGAATGCGTC AACCGGGC 28
(2) INFORMATION FOR SEQ ID NO: 110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:
CACACGACAA CACCATGGTT TCCATCTTC 29
(2) INFORMATION FOR SEQ ID N0: 111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:
GAAGATGGAA ACCATGGTGT TGTCGTGTG 29
(2) INFORMATION FOR SEQ ID NO: 112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
CA 02273408 1999-09-29
54/67
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
GTGGTGCCTT TCGCCGCGCG AGCCTACTTC 30
(2) INFORMATION FOR SEQ ID NO: 113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:
GAAGTAGGCT CGCGCGGCGA AAGGCACCAC 30
(2) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:
TATATCATGA GCGTGTTCGT CGTGCTACTG TTC 33
(2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
CA 02273408 1999-09-29
54/68
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:
ACCCGACTTA CAAAGCGAAT TCTATAGATA TAT 33
(2) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116:
ACCCTTCTTA CAAAGCGAAT TCTATAGATA TAT 33
