Note: Descriptions are shown in the official language in which they were submitted.
WO 2004/026907 CA 02498636 2005-03-10 PCT/EP2003/009812
Use of Saccharomyces cerevisiae erg4 mutants for the expression of glucose
transporters from mammals.
The invention relates to yeast strains in which the human Glut 4 and Glut 1
transporters can be functionally expressed.
Most heterotropic cells transport glucose via special transporter proteins
into the cell
interior. The various organisms have developed different mechanisms mediating
the
transporting of glucose, such as, in particular, proton symport systems, Na'
glucose
transporters, binding protein-dependent systems, phosphotransferase systems,
and
systems for facilitated diffusion. In the eukaryotes, a family of glucose
transporters
which are encoded in mammals by the GLUT genes (GLUT = glucose transporter)
and Saccharomyces cerevisiae by the HXT genes (HXT = hexose transporter)
mediates glucose uptake via facilitated diffusion. Said transporters belong to
a larger
family of sugar transporters. They are characterized by the presence of 12
transmembrane helices and by a plurality of conserved amino acid radicals.
Glucose transport plays an important part in disorders associated with a
defective
glucose homeostasis, such as, for example, diabetes mellitus or Fanconi-Bickel
syndrome. The glucose transport in mammals has therefore been the subject of
numerous studies. To date, thirteen glucose transporter-like proteins have
been
identified (GLUT1 to GLUT12, HMIT - H-myo-inositol transporter)). Said
transporters play key parts which include the uptake of glucose into various
tissues,
its storage in the liver, its insulin-dependent uptake into muscle cells and
adipocytes
and glucose measurement by the t cells of the pancreas.
GLUT1 mediates the transport of glucose into erythrocytes and through the
blood-
brain barrier, but is also expressed in many other tissues, while GLUT4 is
limited to
insulin-dependent tissues, primarily to muscle and fatty tissue. In said
insulin-
dependent tissues, controlling the targeting of GLUT4 transporters through
intracellular compartments or plasma membrane compartments represents an
important mechanism for regulating glucose uptake. In the presence of insulin,
intracellular GLUT 4 is redistributed through the plasma membrane in order to
facilitate glucose uptake. GLUT1 is likewise expressed in said insulin-
dependent
CA 02498636 2005-03-10
2
tissues, and its distribution in the cell is likewise influenced by insulin,
albeit not as
strongly. In addition, the relative efficacy with which GLUTI or GLUT4
catalyze sugar
transport is determined not only by the extent of the targeting of each
transporter to
the cell surface but also by their kinetic properties.
The fact that different glucose transporter isoforms are coexpressed and the
rapid
glucose metabolism have rendered studies on the role and the exact properties
of
each glucose transporter isoform in these insulin-dependent tissues
complicated. In
order to solve these problems, heterologous expression systems such as Xenopus
oocytes, tissue culture cells, insect cells and yeast cells have been used.
However, it
turned out that a number of difficulties appeared in connection with these
systems:
too weak an activity of the heterologously expressed transporters, intrinsic
glucose
transporters in said systems, intracellular retention of a considerable
proportion of
the transporters or even production of inactive transporters.
Naturally occurring GLUT4 protein of mammals, in particular that of humans,
can be
expressed in a functional manner in strains of Saccharomyces cerevisiae under
particular conditions.
Yeast cells are unicell eukaryotic organisms. They are therefore, for some
proteins,
more suitable for expression than bacterial systems, in particular with regard
to
carrying out screen assays for identifying pharmaceutically active substances.
The present invention relates to a purified and isolated polynucleotide
comprising a
DNA sequence which codes for the GLUT4V85M protein.
Said protein contains at position 85 of the amino acid chain of the human
GLUT4
protein an amino acid exchange from valine to methionine. This altered
GLUT4V85M
protein provides further alternatives for expressing a functional GLUT4
protein. A
GLUT4 protein should be regarded as functional in connection with
Saccharomyces
cerevisiae if glucose uptake can be observed in a Saccharomyces cerevisiae
strain
whose glucose transporters in their entirety are inactive (=hxt(-)) after
expression of
I
said GLUT4 protein. Glucose uptake may be determined either by transport
measurements by means of radioactively labeled glucose or by growth on medium
with glucose as sole carbon source.
CA 02498636 2005-03-10
3
In a preferred embodiment, the purified and isolated polynucleotide comprising
a
DNA sequence which calls for a protein GLUT4V85M may include or comprise a
sequence of the following groups:
a) a nucleotide sequence according to Seq ID No. 1,
b) a nucleotide sequence which hybridizes to a sequence of Seq ID No. 1 under
stringent conditions and which codes for a protein GLUT4V85M.
The purified and isolated polynucleotide preferably encodes a GLUT4V85M
protein
which has an amino acid sequence of Seq ID No. 2.
The purified and isolated polynucleotide comprising a DNA sequence which codes
as discussed previously for a protein GLUT4V85M, may be operationally linked
to a
promotor. Suitable promotors are in particular prokaryotic or eukaryotic
promoters
such as, for example, the Lac-, trp-, ADH- or HXT7 promotor. The part of the
polynucleotides, which codes for the protein GLUT4V85M is operationally linked
to a
promotor precisely if a bacterial or eukaryotic organism produces, by means of
said
promotor with the aid of a vector, an mRNA which can be translated into the
protein
GLUT4V85M. An example of such a vector is the vector p4H7GLUT4V85M (Seq ID
No. 3). The protein GLUT4V85M may be expressed in yeast cells by means of said
vector.
The above-described polynucleotide comprising a DNA sequence which codes for a
protein GLUT4V85M is, in a preferred embodiment, suitable for replicating said
polynucleotide in a yeast cell or for expressing the part of the
polynucleotide, which
encodes the protein GLUT4V85M, in a yeast cell to give the protein GLUT 4
V85M.
A yeast cell from Saccharomyces cerevisiae is particularly suitable. For
replication
and expression in a yeast cell, the polynucleotide comprising a DNA sequence
which
calls for a protein GLUT4V85M is present in the form of a yeast vector. The
polynucleotide region coding for the GLUT4V85M protein may be operationally
linked to a yeast cell-specific promotor such as, for example, the ADH
promotor
(alcohol dehydrogenase promotor) or the HXT7 promotor (hexose-transporter
promotor). The yeast sectors are a group of vectors which was developed for
cloning
of DNA in yeasts.
The invention furthermore relates to a yeast cell from Saccharomyces
cerevisiae in
which all glucose transporters are no longer functional (=hxt (-)) and which
contains
CA 02498636 2005-03-10
4
no functional Erg4 protein. Such a yeast cell is preferably a yeast cell
deposited as
Saccharomyces cerevisiae DSM 15187 with the DSMZ (Deutsche Sammlung von
Mikroorganismen and Zelikulturen GmbH, Mascheroder Weg 16, 38124 Brunswick,
Germany).
The invention also relates to a yeast cell in which all glucose transporters
are no
longer functional and which contains no functional Fgyl and no functional Erg4
protein. The lack of an Erg4 protein or of an Fgy1 protein may be attributed
in
particular to an interruption of the corresponding coding genome sections or
to a
partial or complete removal of said coding genome sections.
Preference is given to using as yeast cell which contains no functional
glucose
transporters, no functional Fgyl protein and no functional Erg4 protein, a
yeast cell
as deposited with the DSMZ as Saccharomyces cerevisiae DSM 15184.
A yeast cell as described above is preferably used for expressing a mammalian
GLUT1 protein or a mammalian GLUT4 protein, in particular a protein from rats,
mice, rabbits, pigs, cattle or primates. A preferred embodiment uses the yeast
cell for
expressing a human GLUT4 or GLUTI protein.
A Saccharomyces cerevisiae yeast cell whose glucose transporters in their
entirety
and also the Erg4 protein are no longer functional may contain a
polynucleotide of
the present invention, which comprises a DNA sequence coding for a protein
GLUT4V85M. Said yeast cell can also express the GLUT4V85M protein and thus
contain said protein.
A yeast strain of this kind, containing a polynucleotide which comprises a
DNA,
sequence coding for the GLUT4V85M protein, is preferably the Saccharomyces
cerevisiae DSM 15185 yeast strain which has been deposited with the DMSZ.
A yeast cell whose glucose transporters in their entirety and also the Erg4
protein
are no longer functional and which contains a polynucleotide comprising a DNA
sequence which calls for a protein GLUT4V85M may be prepared, for example, by
a) providing a yeast cell whose glucose transporters in their entirety and
also the
Erg4 protein are no longer functional,
b) providing an isolated and purified polynucleotide which comprises a DNA
sequence coding for the GLUT4V85M protein and which can be replicated in
the yeast cell,
c) transforming the yeast cell from a) with the polynucleotide from b),
CA 02498636 2005-03-10
d) selecting a transformed yeast cell,
e) where appropriate expressing the GLUT4V85M protein.
An isolated and purified polynucleotide which comprises a DNA sequence coding
for
5 the GLUT4V85M protein is preferably a vector which can be replicated in a
yeast cell
and in which said DNA sequence was cloned. An example of such a vector is
p4H7GLUT4V85M (Seq ID No. 3).
The invention also relates to a yeast cell whose glucose transporters in their
entirety
and whose proteins for Fgyl and Erg4 are no longer functional and which
contains a
polynucleotide which comprises a DNA sequence coding for the GLUT4V85M
protein. Said yeast cell can also express the GLUT4V85M protein and thus
contain
said protein. A yeast strain of this kind is preferably the Saccharomyces
cerevisiae
DSM 15186 deposited with the DSMZ.
A yeast cell whose glucose transporters in their entirety and also the
proteins Fgyl
and Erg4 are no longer functional and which contains a polynucleotide
comprising a
DNA sequence which codes for the GLUT4V85M protein may be prepared, for
example, by
a) providing a yeast cell whose glucose transporters in their entirety and
also the
proteins Fgyl and Erg4 are no longer functional,
b) providing an isolated and purified polynucleotide which comprises a DNA
sequence coding for the GLUT4V85M protein and which can be replicated in
the yeast cell,
c) transforming the yeast cell from a) with the polynucleotide from b),
d) selecting a transformed yeast cell,
e) where appropriate expressing the GLUT4V85M protein.
The abovementioned isolated and purified polynucleotide which comprises a DNA
sequence coding for the GLUT4V85M protein is preferably a vector which can be
replicated in a yeast cell and in which said DNA sequence was cloned. An
example
of such a vector is p4H7GLUT4V85M (Seq ID No. 3).
CA 02498636 2005-03-10
6
The invention also relates to a yeast cell whose glucose transporters in their
entirety
are no longer functional and which contains a polynucleotide comprising a DNA
sequence which calls for the GLUT4V85M protein.
Said yeast cell can also express the GLUT4V85M protein and thus contain said
protein. A preferred yeast strain of this kind is the Saccharomyces cerevisiae
15188
yeast strain deposited with the DSMZ.
A yeast cell whose glucose transporters in their entirety are no longer
functional and
which contains a polynucleotide comprising a DNA sequence which codes for the
GLUT4 V85M protein may be prepared, for example, by
a) providing a yeast cell whose glucose transporters in their entirety are no
longer functional,
b) providing an isolated and purified polynucleotide which comprises a DNA
sequence coding for the GLUT4V85M protein and which can be replicated in
the yeast cell,
c) transforming the yeast cell from a) with the polynucleotide from b),
d) selecting a transformed yeast cell,
e) where appropriate expressing the GLUT4V85M protein.
An isolated and purified polynucleotide which comprises a DNA sequence coding
for
the GLUT4V85M protein is preferably a vector which can be replicated in a
yeast cell
and in which said DNA sequence was cloned. An example of such a vector is
p4H7GLUT4V85M (Seq ID No. 3).
The invention also relates to a protein having the amino acid sequence
according to
Seq ID No. 2. Said protein is a human GLUT4 protein in which a valine has been
replaced by a methionine in position 85 of the amino acid chain.
The invention also relates to a method for identifying a compound which
stimulates
the activity of a GLUT4 protein, which method comprises the steps
a) providing a yeast cell whose glucose transporters in their entirety and
also
Erg4 protein are no longer functional and which contains a polynucleotide
comprising a DNA sequence which codes for a protein GLUT4V85M,
b) providing a chemical compound,
c) contacting the yeast of a) with the chemical compound of b),
CA 02498636 2005-03-10
7
d) determining glucose uptake by the yeast of c),
e) relating the detected value of the glucose uptake of d) to the detected
value of
glucose uptake in a yeast cell as claimed in a) which has been contacted with
a chemical compound as claimed in b), with a compound which causes an
increase in the amount of glucose taken up in the yeast as claimed in d)
stimulating the activity of said GLUT4 protein. Compounds which stimulate the
activity of the GLUT4V85M protein can be assumed to stimulate also the
GLUT4 activity.
The invention also relates to a pharmaceutical which contains a compound which
has been identified by the method described above and furthermore to additives
and
excipients for formulating a pharmaceutical. Furthermore, the invention
relates to the
use of a compound which has been identified by the method described above for
producing a pharmaceutical for the treatment of type I and/or II diabetes.
The invention also relates to a pharmaceutical comprising a compound which has
been identified by the method described above and to additives and excipients
for
formulating a pharmaceutical. Furthermore, the invention relates to the use of
a
compound identified by the method described above for producing a
pharmaceutical
for the treatment of diabetes.
The invention furthermore relates to the use of a compound identified by a
method
described above for producing a pharmaceutical for the treatment of diabetes.
r'
The present invention also comprises a method for identifying a compound which
inhibits the protein encoded by the Erg4 gene, which method comprises the
steps:
a) providing a yeast cell whose glucose transporters in their entirety and no
longer functional and which contains a polynucleotide comprising a DNA
sequence which codes for the GLUT4V85M protein and can be replicated in a
yeast cell,
b) providing a chemical compound
c) contacting the yeast of a) with the chemical compound of b),
d) determining glucose uptake by the yeast of c),
e) relating the detected value of the glucose uptake of d) to the detected
value of
glucose uptake in a yeast cell as claimed in a) which is not contacted with a
CA 02498636 2005-03-10
8
chemical compound as claimed in b), with a compound which causes an
increase in the amount of glucose taken up in the yeast as claimed in d)
stimulating the activity of a protein Erg4.
The invention furthermore relates to a method for identifying a compound
inhibiting
the corresponding protein of the Fgyl gene, which comprises the steps:
a) providing a yeast cell whose glucose transporters in their entirety and
whose
Erg4 protein are no longer functional and which contains a GLUT4 protein,
b) providing a chemical compound
c) contacting the yeast of a) with the chemical compound of b),
d) determining glucose uptake by the yeast of c),
e) relating the detected value of the glucose uptake of d) to the detected
value of
glucose uptake in a yeast cell as claimed in a) which is not contacted with a
chemical compound as claimed in b), with a compound which causes an
increase in the amount of glucose taken up in the yeast as claimed in d)
stimulating the activity of a protein Fgyl.
The invention also relates to a pharmaceutical comprising a compound which has
been identified by the method described above and to additives and excipients
for
formulating a pharmaceutical.
The invention may be illustrated in more detail below with respect to
technical
details.
r
Hybridization means the assembling of two nucleic acid single strands having
complementary base sequences to double strands. Hybridization may take place
between two DNA strands, one DNA and one RNA strand and between two RNA
strands. In principle, it is possible to prepare hybrid molecules by heating
the nucleic
acids involved which may initially be in double-stranded form, by boiling, for
example, in a waterbath for 10 minutes, until they disintegrate into single-
stranded
molecules without secondary structure. Subsequently, they can be cooled
slowly.
During the cooling phase, complementary chains pair to give double-stranded
hybrid
molecules. Under laboratory conditions, hybridizations are usually carried out
with
the aid of hybridization filters to which single-stranded or denaturable
polynucleotide
CA 02498636 2005-03-10
9
molecules are applied by blotting or electrophoresis. It is possible to
visualize the
hybridization using appropriate complementary polynucleotide molecules by
providing said polynucleotide molecules to be hybridized with a radioactive
fluorescent label. Stringency describes the degree of matching or alignment of
particular conditions. High stringency has higher demands on matching than low
stringency. Depending on the application and objective, particular conditions
with
different stringency are set for the hybridization of nucleic acids. At high
stringency,
the reaction conditions for the hybridization are set in such a way that only
complementary molecules which match very well can hybridize with one another.
Low stringency enables molecules also to partially hybridize with relatively
large
sections of unpaired or mispaired bases.
The hybridization conditions are to be understood as being stringent, in
particular, if
the hybridization is carried out in an aqueous solution containing 2 x SSC at
68 C for
at least 2 hours, followed by washing first in 2x SSC/0.1 % SDS at room
temperature
for 5 minutes, then in 1 x SSC/0.1 % SDS at 68 C for 1 hour and then in 0.2%
SSC/0.1% SDS at 68 C for another hour.
A 2 x SSC, 1 x SSC or 0.2 x SSC solution is prepared by diluting a 20 x SSC
solution appropriately. A 20 x SSC solution contains 3 mol/I NaCl and 0.3
mol/I Na
citrate. The pH is 7Ø The skilled worker is familiar with the methods for
hybridizations of polynucleotides under stringent conditions. Appropriate
instructions
can be found in specialist books such as, in particular, Current Protocols in
Molecular Biology (Wiley Interscience; editors: Frederich M. Ausubel, Roger
Brant,
Robert E. Kingston, David J. Moore, J. G. Seidmann, Kevin Struhl; ISBN: 0-471-
50338-X).
The yeast vectors can be divided into different subgroups. Ylp vectors (yeast
integrating plasmids) essentially correspond to the vectors used in bacteria
for
clonings, but contain a selectable yeast gene (e.g. URA3, LEU2).
Only when the foreign DNA integrates into a yeast chromosome after
introduction of
said vector, are these sequences replicated together with the chromosome and,
with
the formation of a clone, stably transferred to all daughter cells.
CA 02498636 2005-03-10
Based on this method, plasmids have been derived which can replicate
autonomously owing to eukaryotic ORIs (origins of replication). Such yeast
vectors
are referred to as YRp vectors (yeast replicating plasmids) or ARS vectors
(autonomously replicating sequence). Furthermore, there are YEp vectors (yeast
5 episomal plasmids) which are derived from the yeast 2 m plasmid and which
contain
a selective marker gene. The class of the YAC vectors (yeast artificial
chromosome)
behave like independent chromosomes.
A yeast vector containing a gene to be expressed is introduced into the yeast
by
10 means of transformation in order for said gene to be able to be expressed.
Examples
of methods suitable for this purpose are electroporation or incubation of
competent
cells with vector DNA. Suitable yeast expression promoters are known to the
skilled
worker, examples being the SODI promotor (superoxide dismutase), ADH promotor
(alcohol dehydrogenase), the promotor of the gene for acidic phosphatase, HXT2
promotor (glucose transporter 2), HXT7 promotor (glucose transporter 7), GAL2
promotor (galactose transporter) and others. The construct comprising a yeast
expression promotor and a gene to be expressed (e.g. GLUT4V85M) is, for the
purpose of expression, part of a yeast vector. To carry out expression, said
yeast
vector may be a self-replicating particle which is independent of the yeast
genome or
may be stably integrated into the yeast genome. A suitable yeast vector is in
principle any polynucleotide sequence which can be propagated in a yeast.
Yeast
vectors which may be used are in particular yeast plasmids or yeast artificial
chromosomes. Yeast vectors usually contain an origin of replication (2 , ars)
or1
starting the replication process and a selection marker which usually
comprises an
auxotrophy marker or an antibiotic resistance gene. Examples of yeast vectors
known to the skilled worker are pBM272, pCS19, pEMBCYe23, pFL26, pG6,
pNN414, pTV3, p426MET25, p4H7 and others.
In accordance with the present invention, selection of a cell means the
specific
concentration thereof, owing to a selection marker such as, for example,
resistance
to an antibiotic or the ability to grow on a particular minimal medium, and
furthermore
the isolation and subsequent cultivation thereof on an agar plate or in
submerged
culture.
CA 02498636 2005-03-10
11
Cultivation, transformation and selection of a transformed yeast cell and also
expression of a protein in a yeast cell are among the methods commonly used by
the
skilled worker. Instructions regarding said methods can be found in standard
text
books, for example in Walker Graeme M.: Yeast Physiology and Biotechnology,
Wiley and Sons, ISBN: 0-471-9446-8 or in Protein Synthesis and Targeting in
Yeast,
Ed. Alistair J. P. Brown, Mick F. Fruite and John E. G. Mc Cartly; Springer
Berlin;
ISBN: 3-540-56521-3 or in "Methods in Yeast Genetics, 1997: A Cold Spring
Harbor
Laboratory Course Manual; Adams Alison (Edt.); Cold Spring Harbor Laboratory;
ISBN: 0-87969-508-0".
The yeast Saccharomyces cerevisiae has 17 known hexose transporters and
additionally three known maltose transporters, which are capable of
transporting
hexoses into said yeast, provided that their expression is sufficiently high.
In one
known strain all transporters suitable for hexose uptake were removed by
deletion.
Said strain contains merely just the two genes MPH2 and MPH3 which are
homologous to maltose transport proteins. The two genes MPH2 and MPH3 are
repressed in the presence of glucose in the medium. Wieczorke et al., FEBS
Left.
464, 123 - 128 (1999) describe the preparation and characterization of this
yeast
strain. Said strain is not able to propagate on a substrate containing glucose
as sole
carbon source. It is possible to select from said strain mutants which
functionally
express GLUT1, starting from a corresponding vector (hxt fgyl -1 strain).
If the yeast strain hxt fgyl-1 is transformed with a plasmid vector which
carries a
GLUT4 gene under control of a yeast promotor, still only very little glucose
is
transported. Functional GLUT4 expression requires further adjustments to this
yeast
strain in order to make possible a significant glucose transport by means of
GLUT4.
Such yeast strains whose cells take up glucose by means of a single glucose
transporter GLUT4 can be isolated on substrates having glucose as sole carbon
source. For this purpose, a yeast hxt fgyl-1 strain carrying a GLUT4 gene
under the
functional control of a yeast promotor is transformed. These yeast cells
transformed
in this way are applied to a nutrient medium containing glucose as sole carbon
source and are incubated thereon. After a few days of incubation at, for
example 30
C, the growth of individual colonies is observed. One of these colonies is
isolated.
The removal of the yeast plasmid from said colony prevents propagation on the
CA 02498636 2005-03-10
12
nutrient medium containing glucose as sole carbon source. If this strain which
no
longer contains a vector plasmid is again transformed with a yeast vector
carrying a
GLUT4 gene under the functional control of a yeast promotor, said strain is
then
again able to propagate on a medium containing glucose as sole carbon source.
The abovementioned yeast strains are the subject matter of International
Application
PCT/EP02/01373, filed on February 9, 2002, which claims the priority of DE
10106718.6 of February 14, 2002.
Yeast strains whose indigenous transporters for hexoses (glucose transporters)
in
their entirety are no longer functional have already been deposited at an
earlier date
in connection with International Application PCT/EP02/01373 with the Deutsche
Sammiung von Mikroorganismen and Zellkulturen GmbH (DSMZ) under the number
DSM 14035, DSM 14036 or DSM 14037.
The polynucleotide and amino acid sequences of GLUT4 are accessible, for
example, via the following entries in gene banks: M20747 (cDNA; human), EMBL:
D28561 (cDNA; rat), EMBL: M23382 (cDNA; mouse), Swissprot: P14672 (protein;
human), Swissprot: P19357 (protein; rat) and Swissprot: P14142 (protein;
mouse).
Polynucleotide sequences and amino acid sequences of GLUT1 are disclosed under
the following code numbers of the databases indicated: EMBL: M20653 (cDNA;
human), EMBL: M13979 (cDNA; rat), EMBL: M23384 (cDNA; mouse), Swissprot:
P11166 (protein; human), Swissprot: P11167 (protein; rat) and Swissprot:
P17809
(protein; mouse).
Pharmaceuticals are dosage forms of pharmacologically active substances for
the
therapy of diseases or bodily malfunctions in humans and animals. Examples of
dosage forms for oral therapy are powders, granules, tablets, pills, lozenges,
sugar-
coated tablets, capsules, liquid extracts, tinctures and syrups. Examples
which are
used for external application are aerosols, sprays, gels, ointments or
powders.
Injectable or infusible solutions allow parenteral administration, using
vials, bottles or
prefilled syringes. These and other pharmaceuticals are known to the skilled
worker
in the field of pharmaceutical technology.
CA 02498636 2005-03-10
13
Excipients for formulating a pharmaceutical made possible the preparation of
the
active substance with the purpose of optimizing the application, distribution
and
development of action of the active ingredient for the particular application.
Examples of such excipients are fillers, binders, disintegrants or glidants,
such as
lactose, sucrose, mannitol, sorbitol, cellulose, starch, dicalcium phosphate,
polyglycols, alginates, polyvinylpyrrolidone, carboxymethylcellulose, talc or
silicon
dioxide.
Diabetes manifests itself by the excretion of glucose together with the urine
with an
abnormal increase in the blood glucose level (hyperglycaemia), owing to a
chronic
metabolic condition due to insulin deficiency or reduced insulin action. The
lack of, or
reduced, insulin action leads to insufficient absorption and conversion by the
cells of
the glucose taken up into the blood. In fatty tissue, insulin-antagonistic
hormones
have the effect of increasing lypolysis accompanied by an increase in the free
fatty
acid levels in the blood.
Adiposity (obesity) is the abnormal weight gain owing to an energy imbalance
due to
excessive intake of calories, which constitutes a health risk.
The amount of a hexose which is taken up by a provided yeast strain as
described
just above can be determined by means of uptake studies using radioactively
labeled
glucose. For this purpose, a particular concentration of the yeast cells is
suspended
in, for example, 100 I of a buffer, for example at a concentration of 60 mg
(wet
weight) per ml, and admixed with a defined amount of 14C- or 3H-labeled
glucose as
sole carbon source. The cells are incubated, and defined amounts thereof are
removed at specific times. The amount of glucose taken up is determined with
the
aid of LSC (Liquid Scintillation Counting). The amount of a hexose which is
taken up
by a yeast strain provided and as just described above may, however, also be
determined by means of a growth assay on media containing glucose as sole
carbon
source. For this purpose, the rate of growth of the strain is determined,
after addition
of the compound, for example by measuring the optical density of the culture
at
CA 02498636 2005-03-10
14
600 nm at regular intervals, and this value is compared with the rate of
growth of a
control strain (e.g. yeast wild-type strain).
A compound is provided, in particular, by chemical synthesis or by isolating
chemical
substances from biological organisms. It is also possible to carry out
chemical
synthesis in an automated manner. The compounds obtained by synthesis or
isolation can be dissolved in a suitable solvent. Suitable solvents are in
particular
aqueous solutions which contain a specific proportion of an organic solvent
such as,
for example, DMSO (dimethylsulfoxide).
Conducting a strain of the yeast with a compound for identifying a compound in
accordance with an invention mentioned above is done in particular in
automated
laboratory systems provided therefor. Such systems may comprise specifically
prepared chambers with depressions, or microtiter plates, Eppendorf tubes or
laboratory glassware. Automated laboratory systems are usually designed for
high
throughput rates. A method such as the one mentioned above, carried out with
the
aid of an automated laboratory system, is therefore also referred to as HTS
(High
Throughput Screening).
Seq ID No. 1 discloses a polynucleotide sequence comprising the coding region
of
the GLUT4V85M protein. Seq ID No. 2 discloses the amino acid sequence of the
GLUT4V85M protein. Seq ID No. 3 discloses the polynucleotide sequence of the
p4H7GLUT4V85M vector.
Examples
Use of yeast strains
All of the yeast strains described herein were derived from strain CEN-PK2-1 C
(MATa leu2-3, 112 ura3-52 trpl-289 his3-A1 MAL2-8 SUC2). The preparation of a
yeast strain having deletions in the hexose transporter genes (HXT) has been
described by Wieczorke et al., FEBS Lett. 464, 123 - 128 (1999): EBY-18ga
(MATa
Ahxt1-17 Aga12 Dagt1 Asti1 leu2-3, 112 ura3-52 trpl -289 his3-M1 MAL2-8c
SUC2),
EBY.VW4000 (MATa Ohxt1-17 Aga12 Eagt1 Amph2 Amph3 OstI1 Ieu2-3, 112 ura3-52
CA 02498636 2005-03-10
trpl-289 his3-01 MAL2-8` SUC2). The media were based on 1% yeast extract and
2% peptone (YP), while the minimal media were composed of 0.67% Difco yeast
nitrogen base without amino acids (YNB) and contained additives required for
auxotrophy and different carbon sources. The yeast cells were grown under
aerobic
5 conditions at 30 C on a rotary shaker or on agar plates. Cell growth was
monitored
by measuring the optical density at 600 nm (OD600) or by determining the
diameter of
yeast colonies.
10 Determination of glucose uptake
Glucose transport was measured as uptake of D-[U-14C1-glucoses (Amersham) and
the kinetic parameters were determined from Eadie-Hofstee plots. The cells
were
removed by centrifugation, washed with phosphate-buffer and resuspended in
phosphate buffer at a concentration of 60 mg (wet weight) per ml. Glucose
uptake
15 was determined for glucose concentrations between 0.2 and 100 mM, and the
specific activity of the substrate was between 0.1 and 55.5 kBq mol"1. The
cells and
the glucose solutions were preincubated at 30 C for 5 minutes. Glucose uptake
was
started by adding radioactive glucose to the cells. After incubation for 5
seconds,
10 ml of ice-cold stop buffer (0.1 M KiPO4, pH 6.5, 500 mM glucose) were added
and
the cells were removed quickly by filtering on glass fiber filters (0 = 24 mm,
Whatman). The filters were quickly washed three times with ice-cold buffer and
the
radioactivity incorporated was measured using a liquid scintillation counter.
An
addition by cytochalasin B (final concentration 20 M, dissolved in ethanol)
was,-
measured in a 15-second uptake assay with 50 mM or 100 mM radioactive glucose,
after the cells had been incubated in the presence of the inhibitor or of only
the
solvent for 15 minutes.
A novel heterologous expression system for glucose transporters from mammalian
cells has been developed. The system is based on an S. cerevisiae strain from
which all endogenous glucose transporters have been removed destroying the
encoding genes. Said strain is no longer able to take up glucose via the
plasma
membrane and to grow with glucose as sole carbon source. In order to integrate
the
heterologous glucose transporters of humans or of other mammals, GLUT1 and
CA 02498636 2005-03-10
16
GLUT4 in an active form into the yeast plasma membrane, additional mutations
had
to be introduced into the yeast strain. GLUT1 is active only in an fgyl-1
mutant strain
and GLUT4 only in fgyl-1 fgy4-X double mutants.
The FGY1 gene has been cloned. It is the S. cerevisiae ORF YMR212c. With
respect to the function, the results indicate that either Fgyl or a product
generated
by Fgyl inhibits the activity of human glucose transporters or is involved in
fusing the
GLUT-transporting vesicles to the plasma membrane.
In contrast to GLUT1 and similarly to mammalian cells, a large proportion of
the
GLUT4 proteins in the yeast is located in intracellular structures. A total of
nine
recessive mutants were isolated (fgy4-1 to fgy4-9) in which GLUT4 is now
directed
further to the plasma membrane and, in the case of a concomitant fgyl-1
mutation,
becomes active there.
The insertion gene bank described by Bruns et al. (Genes Dev. 1994; 8: 1087-
105)
was used for complementation analysis. The hxt fgyl -1 strain was transformed
first
with a GLUT4 plasmid and then with the mobilized insertion gene bank. This was
followed by screening for transformants which were able to grow on glucose
medium. It turned out that in one of the mutants studied the ERG4 gene had
been
destroyed. ERG4 codes for an enzyme (oxidoreductase) of ergosterol
biosynthesis.
This enzyme, sterol C-24(28)-reductase catalyzes the last step of
ergosterolbiosynthesis and converts ergosta-5,7,22,24,(28)-tetraenol to the
final
product ergosterol. The Erg4 protein presently contains eight transmembrane
domains and is located in the endoplasmic reticulum. An erg4 mutant is viable,
since
incorporation of the ergosterol precursors into the yeast membranes
compensates
for the loss of ergosterol.
The inhibiting influence of Erg4 on GLUT4 functionality was confirmed by
specific
deletion of erg4 in the hxt fgyl-1 strain. The resulting strain (hxt fgyl-1
Aerg4) was
referred to as SDY022.
Protein interaction assays with the aid of the split-ubiquitin system showed
that
human GLUT4 directly interacts with yeast Erg4. It can therefore be assumed
that
CA 02498636 2005-03-10
17
the yeast Erg4 protein in the endoplasmic reticulum either directly prevents
further
translocation of GLUT4 or modifies GLUT4 in some way which is important for
translocation and/or function.
Likewise, it was shown that deletion of ERG4 in the hxt null strain alone,
i.e. despite
functional FGY1, activates GLUT1, but not GLUT4. The results of the growth
assay
are summarized in Table 1.
In order to rule out that Ergosterol itself exerts a negative influence on
GLUT4,
growth assays were carried out on agar plates containing Ergosterol under
aerobic
conditions. Any yeast strains transformed with GLUT4 were unable to grow under
these conditions (Table 2). The GLUT1 transformants in the hxt fgyl-1 strain
showed, in contrast to aerobic growth, no growth on glucose under anaerobic
conditions. GLUT1 transformants were able to grow only after deletion of ERG4.
The exchange of Va185 for Met by in vitro mutagenesis rendered GLUT4
independent of the fgyl -1 mutation and resulted in GLUT4V85M being functional
even in an hxt erg4 strain. This observation indicates that Fgy1 acts directly
or
indirectly on this position which is located within the second transmembrane
helix of
GLUT transporters.
Table 3 displays the descriptions of the yeast strains deposited in connection
with
the present patent application with the Deutsche Sammlung von Mikroorganisnen
and Zellkulturen (DSMZ) - Mascheroder Weg 1 b 38124 Brunswick, Germany.
Table 1: Growth of GLUTI and GLUT4 transformants on glucose medium.
Genotype I % Glucose I % Glucose
Ahxt fgy1-1 GLUT4 - GLUT1 ++
Ahxt fgyl-1 Aerg4 GLUT4 ++ GLUT1 ++
Ohxt fgyl-1 Derg4 Vector - Vector -
Ohxt fgyl-1 Aerg5 GLUT4 - GLUT1 ++
Ohxt fgyl-1 Aerg4 Derg5 GLUT4 + GLUT1 ++
CA 02498636 2005-03-10
18
Ahxt Eerg4 GLUT4 - GLUT1
\hxt oerg5 GLUT4 - GLUT1 -
Table 2: Growth of GLUT1 and GLUT4 transformants on glucose medium with or
without ergosterol under anaerobic conditions
1 % Glucose+
Genotype 1 % Glucose
33 mg/I Ergosterol
Ahxt fgyl-1 GLUT1 - -
GLUT4 - -
Ahxt fgyl-1 derg4 GLUT1 - ++
GLUT4 - -
Ahxt fgy1-1 Aerg5 GLUT1 - -
GLUT4 - -
Lihxt fgyl-1 Aerg4 Derg5 GLUT1 - ++
GLUT4 - -
Ahxt Derg4 GLUT1 - (+)
GLUT4 - -
Ahxt ierg5 GLUT1 - -
GLUT4 - -
Table 3: Features of the deposited yeast strains (Saccharomyces cerevisiae)
Number of Genotype Phenotype Plasmid
deposit with
the DSMZ
DSM 15187 MATa Ahxtl-17 AgaI2 Strain growth with -
Dagt1 Ostll omph2 1 % maltose as
omph3 Lerg4 leu2-3, carbon source;
112 ura3-52 trpl-289 auxotrophic for
CA 02498636 2005-03-10
19
his3-01 MAL2-8c SUC2 glucose, leucine,
tryptophan,
histidine and uracil
DSM 15184 MATa Ahxtl -17 Aga12 Strain growth with -
oagtl ostl1 Aerg4 fgyl - 1 % maltose as
1 leu2-3, 112 ura3-52 carbon source;
trpl-289 his3-01 MAL2- auxotrophic for
8c SUC2 glucose, leucine,
tryptophan,
histidine and uracil
DSM 15185 MATa Ohxt1-17 Aga12 Strain growth with p4H7GLUT4V85M
Dagtl ostll Amph2 1 % maltose as (Selection marker
Amph3 Derg4 leu2-3, carbon source; URA3),
112 ura3-52 trpl-289 auxotrophic for = Seq ID No. 3
his3-M1 MAL2-8c SUC2 glucose, leucine,
tryptophan and
histidine
DSM 15186 MATa Ohxt1-17 Aga12 Strain growth with p4H7GLUT4V85M
Dagtl Estil Aerg4 fgyl- 1% maltose as (Selection marker
1 leu2-3, 112 ura3-52 carbon source; URA3)
trpl-289 his3-o1 MAL2- auxotrophic for = Seq ID No. 3
8c SUC2 glucose, leucine,
tryptophan and
histidine
DSM 15188 MATa Ohxt1-17 Aga12 Strain growth with p4H7GLUT4V85M
Aagtl AstIl Omph2 1 % maltose as (Selection marker
omph3 leu2-3, 112 carbon source; URA3)
ura3-52 trpl -289 his3- auxotrophic for = Seq ID No. 3
Al MAL2-8c SUC2 glucose, leucine,
tryptophan and
histidine
CA 02498636 2005-03-10
Basic medium: 0.67% Yeast Nitrogen Base without amino acids (Difco); pH 6.2.
Auxotrophy supplementation: Leucine (0.44 mM), tryptophan (0.19 mM), histidine
(0.25 mM9, uracil (0.44 mM). Maltose may be between 1-2%.
CA 02498636 2005-03-10
21
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark H6chst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT
D-65926 Frankfurt issued pursuant to Rule 7.1 by
the INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the ACCESSION NUMBER issued by
DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY:
DSMefg DSM 15185
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified in section I was accompanied by:
a scientific description
a proposed taxonomic designation
(Indicate as applicable)
III. RECEIPT AND ACCEPTANCE
The present International Depositary Authority accepts the microorganism
identified in section I, which it received on
2002-09-03 (date of the original deposit)'
IV. RECEIPT OF A REQUEST FOR CONVERSION
The present International Depositary Authority received the microorganism
identified under section I on
(date of the original deposit) and received a request for conversion of the
original deposit to a deposit conforming to the
Budapest Treaty on (date of receipt of the request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN GmbH the International Depositary Authority,
or of authorized
Address: Mascheroder Weg 1b official(s)
(illegible signature)
D-38124 Brunswick
Date: 2002-09-10
' If Rule 6.4.d) applies, this date shall be the date on which the status of
the international depositary authority was
acquired.
Form DSMZ-BP/4 (single page) 12/2001
CA 02498636 2005-03-10
22
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark HOchst VIABILITY STATEMENT
D-65926 Frankfurt issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Aventis Pharma Deutschland GmbH
Industriepark HOchst ACCESSION NUMBER given by the
INTERNATIONAL DEPOSITARY AUTHORITY:
Address: D-65926 Frankfurt DSM 15185
Date of the deposit or of the transfer':
2002-09-03
III. VIABILITY STATEMENT
The viability of the microorganism identified under section II above was
tested on 2002-09-062. On that date, the said
microorganism was
3 viable
^3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN GmbH the International Depositary Authority,
or of authorized
official(s)
Address: Mascheroder Weg 1 b
D-38124 Brunswick (illegible signature)
Date: 2002-09-10
' Indicate the date of the original deposit or, where a new deposit or a
transfer has been made, the date of the most
recent relevant new deposit or transfer.
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most
recent viability test.
3 Mark with a cross the applicable box.
4 Fill in if the information has been requested and if the results of the test
were negative.
Form DSMZ-BP/9 (single page) 12/2001
CA 02498636 2005-03-10
23
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sanunlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark H6chst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT
D-65926 Frankfurt issued pursuant to Rule 7.1 by
the INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the ACCESSION NUMBER issued by
DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY:
DSMhij DSM 15186
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified in section I was accompanied by:
a scientific description
a proposed taxonomic designation
(Indicate as applicable)
III. RECEIPT AND ACCEPTANCE
The present International Depositary Authority accepts the microorganism
identified in section I, which it received on
2002-09-03 (date of the original deposit)'
IV. RECEIPT OF A REQUEST FOR CONVERSION
The present International Depositary Authority received the microorganism
identified under section I on
(date of the original deposit) and received a request for conversion of the
original deposit to a deposit conforming to the
Budapest Treaty on (date of receipt of the request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of
authorized
GmbH official(s)
Address: Mascheroder Weg 1b (illegible signature)
D-38124 Brunswick
Date: 2002-09-10
If Rule 6.4.d) applies, this date shall be the date on which the status of the
international depositary authority was
acquired.
Form DSMZ-BP/4 (single page) 12/2001
CA 02498636 2005-03-10
24
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sanunlunc,
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark H6chst VIABILITY STATEMENT
D-65926 Frankfurt issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Aventis Pharma Deutschland GmbH
Industriepark H6chst ACCESSION NUMBER given by the
INTERNATIONAL DEPOSITARY AUTHORITY:
Address: D-65926 Frankfurt DSM 15186
Date of the deposit or of the transfer:
2002-09-03
III. VIABILITY STATEMENT
The viability of the microorganism identified under section II above was
tested on 2002-09-062. On that date, the said
microorganism was
3 viable
^3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of
authorized
GmbH official(s)
Address: Mascheroder Weg 1 b (illegible signature)
D-38124 Brunswick
Date: 2002-09-10
1 Indicate the date of the original deposit or, where a new deposit or a
transfer has been made, the date of the most
recent relevant new deposit or transfer.
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most
recent viability test.
3 Mark with a cross the applicable box.
4 Fill in if the information has been requested and if the results of the test
were negative.
Form DSMZ-BP/9 (single page) 12/2001
CA 02498636 2005-03-10
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark H6chst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT
D-65926 Frankfurt issued pursuant to Rule 7.1 by
the INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the ACCESSION NUMBER issued by
DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY:
DSMsyz DSM 15187
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified in section I was accompanied by:
a scientific description
a proposed taxonomic designation
(Indicate as applicable)
Ill. RECEIPT AND ACCEPTANCE
The present International Depositary Authority accepts the microorganism
identified in section I, which it received on
2002-09-03 (date of the original deposit)'
IV. RECEIPT OF A REQUEST FOR CONVERSION
The present International Depositary Authority received the microorganism
identified under section I on
(date of the original deposit) and received a request for conversion of the
original deposit to a deposit conforming to the
Budapest Treaty on (date of receipt of the request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of
authorized
GmbH official(s)
Address: Mascheroder Weg 1b (illegible signature)
D-38124 Brunswick
Date: 2002-09-10
If Rule 6.4.d) applies, this date shall be the date on which the status of the
international depositary authority was
acquired.
Form DSMZ-BP/4 (single page) 12/2001
CA 02498636 2005-03-10
26
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark Hochst VIABILITY STATEMENT
D-65926 Frankfurt issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Aventis Pharma Deutschland GmbH
Industriepark Hochst ACCESSION NUMBER given by the
INTERNATIONAL DEPOSITARY AUTHORITY:
Address: D-65926 Frankfurt DSM 15187
Date of the deposit or of the transfer:
2002-09-03
III. VIABILITY STATEMENT
The viability of the microorganism identified under section II above was
tested on 2002-09-062. On that date, the said
microorganism was
3 viable
^3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED"
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of
authorized
GmbH official(s)
Address: Mascheroder Wag 1b (illegible signature)
D-38124 Brunswick
Date: 2002-09-10
Indicate the date of the original deposit or, where a new deposit or a
transfer has been made, the date of the most
recent relevant new deposit or transfer.
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most
recent viability test.
3 Mark with a cross the applicable box.
4 Fill in if the information has been requested and if the results of the test
were negative.
Form DSMZ-BP/9 (single page) 12/2001
CA 02498636 2005-03-10
27
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark H6chst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT
D-65926 Frankfurt issued pursuant to Rule 7.1 by
the INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the ACCESSION NUMBER issued by
DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY:
DSMuvw DSM 15188
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified in section I was accompanied by:
a scientific description
a proposed taxonomic designation
(Indicate as applicable)
III. RECEIPT AND ACCEPTANCE
The present International Depositary Authority accepts the microorganism
identified in section I, which it received on
2002-09-03 (date of the original deposit)'
IV. RECEIPT OF A REQUEST FOR CONVERSION
The present International Depositary Authority received the microorganism
identified under section I on
(date of the original deposit) and received a request for conversion of the
original deposit to a deposit conforming to the
Budapest Treaty on (date of receipt of the request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of
authorized
GmbH official(s)
Address: Mascheroder Weg 1b (illegible signature)
D-38124 Brunswick
Date: 2002-09-10
If Rule 6.4.d) applies, this date shall be the date on which the status of the
international depositary authority was
acquired.
Form DSMZ-BP/4 (single page) 12/2001
CA 02498636 2005-03-10
28
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Phamia Deutschland GmbH
Industriepark HBchst VIABILITY STATEMENT
D-65926 Frankfurt issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Aventis Pharma Deutschland GmbH
Industriepark H6chst ACCESSION NUMBER given by the
INTERNATIONAL DEPOSITARY AUTHORITY:
Address: D-65926 Frankfurt DSM 15188
Date of the deposit or of the transfer:
2002-09-03
III. VIABILITY STATEMENT
The viability of the microorganism identified under section 11 above was
tested on 2002-09-062. On that date, the said
microorganism was
3 viable
^3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED"
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of
authorized
GmbH official(s)
Address: Mascheroder Weg 1 b (illegible signature)
D-38124 Brunswick
Date: 2002-09-10
1 Indicate the date of the original deposit or, where a new deposit or a
transfer has been made, the date of the most
recent relevant new deposit or transfer.
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most
recent viability test.
3 Mark with a cross the applicable box.
4 Fill in if the information has been requested and if the results of the test
were negative.
Form DSMZ-BP/9 (single page) 12/2001
CA 02498636 2005-03-10
29
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark Hbchst RECEIPT IN THE EVENT OF AN ORIGINAL DEPOSIT
D-65926 Frankfurt issued pursuant to Rule 7.1 by
the INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by the ACCESSION NUMBER issued by
DEPOSITOR: the INTERNATIONAL DEPOSITARY AUTHORITY:
DSMdef DSM 15184
II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified in section I was accompanied by:
a scientific description
a proposed taxonomic designation
(Indicate as applicable)
III. RECEIPT AND ACCEPTANCE
The present International Depositary Authority accepts the microorganism
identified in section I, which it received on
2002-09-03 (date of the original deposit)'
IV. RECEIPT OF A REQUEST FOR CONVERSION
The present International Depositary Authority received the microorganism
identified under section I on
(date of the original deposit) and received a request for conversion of the
original deposit to a deposit conforming to the
Budapest Treaty on (date of receipt of the request for conversion)
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of
authorized
GmbH official(s)
Address: Mascheroder Weg 1b (illegible signature)
D-38124 Brunswick
Date: 2002-09-10
' If Rule 6.4.d) applies, this date shall be the date on which the status of
the international depositary authority was
acquired.
Form DSMZ-BP/4 (single page) 12/2001
CA 02498636 2005-03-10
BUDAPEST TREATY ON THE INTERNATIONAL DSMZ
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung
FOR THE PURPOSES OF PATENT PROCEDURE von Mikroorganismen
and Zellkulturen GmbH
INTERNATIONAL FORM
Aventis Pharma Deutschland GmbH
Industriepark H6chst VIABILITY STATEMENT
D-65926 Frankfurt issued pursuant to Rule 10.2 by the
INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page
1. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Aventis Pharma Deutschland GmbH
Industriepark HOchst ACCESSION NUMBER given by the
INTERNATIONAL DEPOSITARY AUTHORITY:
Address: D-65926 Frankfurt DSM 15184
Date of the deposit or of the transfer:
2002-09-03
III. VIABILITY STATEMENT
The viability of the microorganism identified under section 11 above was
tested on 2002-09-062. On that date, the said
microorganism was
3 viable
^3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED4
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of the person(s) having the
power to represent
MIKROORGANISMEN UND ZELLKULTUREN the International Depositary Authority, or of
authorized
GmbH official(s)
Address: Mascheroder Weg 1b (illegible signature)
D-38124 Brunswick
Date: 2002-09-10
1 Indicate the date of the original deposit or, where a new deposit or a
transfer has been made, the date of the most
recent relevant new deposit or transfer.
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most
recent viability test.
3 Mark with a cross the applicable box.
Fill in if the information has been requested and if the results of the test
were negative.
Form DSMZ-BP/9 (single page) 12/2001
CA 02498636 2006-09-01
31
SEQUENCE LISTING
<110> Sanofi-Aventis Deutschland GmbH
<120> Use of saccharomyces cerevisiae ergo mutants for the
expression of glucose transporters from mammals
<130> 9982-875
<140> CA 2,498,636
<141> 2003-09-04
<150> 10242763.1
<151> 2002-09-14
<160> 3
<170> Patentln Ver. 2.1
<210> 1
<211> 1530
<212> DNA
<213> Homo sapiens
<400> 1
atgccgtcgg gcttccaaca gataggctcc gaagatgggg aaccccctca gcagcgagtg 60
actgggaccc tggtccttgc tgtgttctct gcggtgcttg gctccctgca gtttgggtac 120
aacattgggg tcatcaatgc ccctcagaag gtgattgaac agagctacaa tgagacgtgg 180
ctggggaggc aggggcctga gggacccagc tacatccctc caggcaccct caccaccctc 240
tgggccctct ccatggccat cttttccgtg ggcggcatga tttcctcctt cctcattggt 300
CA 02498636 2006-09-01
32
atcatctctc agtggcttgg aaggaaaagg gccatgctgg tcaacaatgt cctggcggtg 360
ctggggggca gcctcatggg cctggccaac gctgctgcct cctatgaaat gctcatcctt 420
ggacgattcc tcattggcgc ctactcaggg ctgacatcag ggctggtgcc catgtacgtg 480
ggggagattg ctcccactca cctgcggggc gccctgggga cgctcaacca actggccatt 540
gttatcggca ttctgatcgc ccaggtgctg ggcttggagt ccctcctggg cactgccagc 600
ctgtggccac tgctcctggg cctcacagtg ctacctgccc tcctgcagct ggtcctgctg 660
cccttctgtc ccgagagccc ccgctacctc tacatcatcc agaatctcga ggggcctgcc 720
agaaagagtc tgaagcgcct gacaggctgg gccgatgttt ctggagtgct ggctgagctg 780
aaggatgaga agcggaagct ggagcgtgag cggccactgt ccctgctcca gctcctgggc 840
agccgtaccc accggcagcc cctgatcatt gcggtcgtgc tgcagctgag ccagcagctc 900
tctggcatca atgctgtttt ctattattcg accagcatct tcgagacagc aggggtaggc 960
cagcctgcct atgccaccat aggagctggt gtggtcaaca cagtcttcac cttggtctcg 1020
gtgttgttgg tggagcgggc ggggcgccgg atgctccatc tcctgggoet ggcgggcatg 1080
tgtggctgtg ccatcctgat gactgtggct ctgctcctgc tggagcgagt tccagccatg 1140
agctacgtct ccattgtggc catctttggc ttcgtggcat tttttgagat tggccctggc 1200
cccattcctt ggttcatcgt ggccgagctc ttcagccagg gaccccgccc ggcagccatg 1260
gctgtggctg gtttctccaa ctggacgagc aacttcatca ttggcatggg tttccagtat 1320
gttgcggagg ctatggggcc ctacgtcttc cttctatttg cggtcctcct gctgggcttc 1380
ttcatcttca ccttcttaag agtacctgaa actcgaggcc ggacgtttga ccagatctca 1440
gctgccttcc accggacacc ctctctttta gagcaggagg tgaaacccag cacagaactt 1500
gagtatttag ggccagatga gaacgactga 1530
<210> 2
<211> 509
<212> PRT
<213> Homo sapiens
<400> 2
Met Pro Ser Gly Phe Gln Gln Ile Gly Ser Glu Asp Gly Glu Pro Pro
CA 02498636 2006-09-01
3s
1 5 10 15
Gln Gln Arg Val Thr Gly Thr Leu Val Lou Ala Val Phe Ser Ala Val
20 25 30
Leu Gly Ser Lou Gln Phe Gly Tyr Asn Ile Gly Val Ile Asn Ala Pro
35 40 45
Gln Lys Val Ile Glu Gln Ser Tyr Asn Glu Thr Trp Leu Gly Arg Gln
50 55 60
Gly Pro Glu Gly Pro Ser Ser Ile Pro Pro Gly Thr Leu Thr Thr Lou
65 70 75 80
Trp Ala Leu Ser Met Ala Ile Phe Ser Val Gly Gly Met Ile Ser Ser
85 90 95
Phe Leu Ile Gly Ile Ile Ser Gln Trp Leu Gly Arg Lys Arg Ala Met
100 105. 110
Leu Val Asn Asn Val Leu Ala Val Leu Gly Gly Ser Leu Met Gly Lou
115 120 125
Ala Asn Ala Ala Ala Ser Tyr Glu Met.Leu Ile Lou Gly Arg Phe Lou
130 135 140
Ile Gly Ala Tyr Ser Gly Leu Thr Ser Gly Leu Val Pro Met Tyr Val
145 150 155 160
Gly Glu Ile Ala Pro Thr His Lou Arg Gly Ala Lou Gly Thr Leu Asn
CA 02498636 2006-09-01
3Lt
165 170 175
Gln Leu Ala Ile Val Ile Gly Ile Leu Ile Ala Gln Val Leu Gly Leu
180 185 190
Glu Ser Leu Leu Gly Thr Ala Ser Leu Trp Pro Leu Leu Leu Gly Leu
195 200 205
Thr Val Leu Pro Ala Leu Leu Gln Leu Val Leu Leu Pro Phe Cys Pro
210 215 220
Glu Ser Pro Arg Tyr Leu Tyr Ile Ile Gln Asn Leu Glu Gly Pro Ala
225 230 235 240
Arg Lys Ser Leu Lys Arg Leu Thr Gly Trp Ala Asp Val Ser Gly Val
245 250 255
Leu Ala Glu Leu Lys Asp Glu Lys Arg Lys Leu Glu Arg Glu Arg Pro
260 265 270
Leu Ser Leu Leu Gln Leu Leu Gly Ser Arg Thr His Arg Gln Pro Leu
275 280 285
Ile Ile Ala Val Val Leu Gln Leu Ser Gln Gln Leu Ser Gly Ile Asn
290 295 300
Ala Val Phe Tyr Tyr Ser Thr Ser Ile.Phe Glu Thr Ala Gly Val Gly
305 310 315 320
Gln Pro Ala Tyr Ala Thr Ile Gly Ala Gly Val Val Asn Thr Val Phe
CA 02498636 2006-09-01
325 330 335
Thr Leu Val Ser Val Leu Leu Val Glu Arg Ala Gly Arg Arg Thr Leu
340 345 350
His Leu Leu Gly Leu Ala Gly Met Cys'Gly Cys Ala Ile Leu Met Thr
355 360 365
Val Ala Leu Leu Leu Leu Glu Arg Val Pro Ala Met Ser Tyr Val Ser
370 375 380
Ile Val Ala Ile Phe Gly Phe Val Ala Phe Phe Glu Ile Gly Pro Gly
385 390 395 400
Pro Ile Pro Trp Phe Ile Val Ala Glu Leu Phe Ser Gln Gly Pro Arg
405 410 415
Pro Ala Ala Met Ala Val Ala Gly Phe Ser Asn Trp Thr Ser Asn Phe
420 425 430
Ile Ile Gly Met Gly Phe Gln Tyr Val Ala Glu Ala Met Gly Pro Tyr
435 440 445
Val Phe Leu Leu Phe Ala Val Leu Leu Leu Gly Phe Phe Ile Phe Thr
450 455 460
Phe Leu Arg Val Pro Glu Thr Arg Gly Arg Thr Phe Asp Gln Ile Ser
465 470 475 480
Ala Ala Phe His Arg Thr Pro Ser Leu Leu Glu Gln Glu Val Lys Pro
CA 02498636 2006-09-01
3(
485 490 495
Ser Thr Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Asp
500 505
<210> 3
<211> 7809
<212> DNA
<213> Saccharomyces cerevisiae
<400> 3
cgtaggaaca aattcgggcc cctgcgtgtt cttctgaggt tcatctttta catttgcttc 60
tgctggataa ttttcagagg caacaaggaa aaattagatg gcaaaaagtc gtctttcaag 120
gaaaaatccc caccatcttt cgagatcccc tgtaacttat tggcaactga aagaatgaaa 180
aggaggaaaa tacaaaatat actagaactg aaaaaaaaaa agtataaata gagacgatat 240
atgccaatac ttcacaatgt tcgaatctat tcttcatttg cagctattgt aaaataataa 300
aacatcaaga acaaacaagc tcaacttgtc ttttctaaga acaaagaata aacacaaaaa 360
caaaaagttt ttttaatttt aatcaaaaaa tgccgtcggg cttccaacag ataggctccg 420
aagatgggga accccctcag cagcgagtga ctgggaccct ggtccttgct gtgttctctg 480
cggtgcttgg ctccctgcag tttgggtaca acattggggt catcaatgcc cctcaggagg 540
tgattgaaca gagctacaat gagacgtggc tggggaggca ggggcctgag ggacccagct 600
ccatccctcc aggcaccctc accaccctct gggccctctc catggccatc ttttccgtgg 660
gcggcatgat ttcctccttc ctcattggta tcatctctca gtggcttgga aggaaaaggg 720
ccatgctggt caacaatgtc ctggcggtgc tggggggcag cctcatgggc ctggccaacg 780
ctgctgcctc ctatgaaatg ctcatccttg gacgattcct cattggcgcc tactcagggc 840
tgacatcagg gctggtgccc atgtacgtgg gggagattgc tcccactcac ctgcggggcg 900
ccctggggac gctcaaccaa ctggccattg ttatcggcat tctgatcgcc caggtgctgg 960
gcttggagtc cctcctgggc actgccagcc tgtggccact gctcctgggc ctcacagtgc 1020
CA 02498636 2006-09-01
=37
tacctgccct cctgcagctg gtcctgctgc ccttctgtcc cgagagcccc cgctacctct 1080
acatcatcca gaatctcgag gggcctgcca gaaagagtct gaagcgcctg acaggctggg 1140
ccgatgtttc tggagtgctg gctgagctga aggatgagaa gcggaagctg gagcgtgagc 1200
ggccactgtc cctgctccag ctcctgggca gcggtaccca ccggcagccc ctgatcattg 1260
cggtcgtgct gcagctgagc cagcagctct ctggcatcaa tgctgttttc tattattcga 1320
ccagcatctt cgagacagca ggggtaggcc agcctgccta tgccaccata ggagctggtg 1380
tggtcaacac agtcttcacc ttggtctcgg tgttgttggt ggagcgggcg gggcgccgga 1440
cgctccatct cctgggcctg gcgggcatgt gtggctgtgc catcctgatg actgtggctc 1500
tgctcctgct ggagcgagtt ccagccatga gctacgtctc cattgtggcc atctttggct 1560
tcgtggcatt ttttgagatt ggccctggcc ccattccttg gttcatcgtg gccgagctct 1620
tcagccaggg acgccgcccg gcagccatgg ctgtggctgg tttctccaac tggacgagca 1680
acttcatcat tggcatgggt ttccagtatg ttgcggaggc tatggggccc tacgtcttcc 1740
ttctatttgc ggtcctcctg ctgggcttct tcatcttcac cttcttaaga gtacctgaaa 1800
ctcgaggccg gacgtttgac cagatctcag ctgccttcca ccggacaccc tctcttttag 1860
agcaggaggt gaaacccagc acagaacttg agtatttagg gccagatgag aacgactgac 1920
tcgagtcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct 1980
aaccgaaaag gaaggagtta gaaaacctga agtctaggtc cctatttatt tttttatagt 2040
tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagaog 2100
cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa 2160
ggctttaatt tgcggccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca 2220
ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 2280
cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 2340
ccttcccaac agttgcgcag cctgaatggc gaatggcgcg acgcgccctg tagcggcgca 2400
ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 2460
gcgcccgctc ctttcgcttt cttcccttcc tttctcgcaa cgttcgccgg ctttccccgt 2520
caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 2580
cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 2640
tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 2700
acaacactca acectatctc ggtctattct tttgatttat aagggatttt gccgatttcg 2760
gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 2820
CA 02498636 2006-09-01
38
ttaacgttta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 2880
accgcatagg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata 2940
catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct 3000
tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat 3060
agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta 3120
tactgttgac ccaatgcgtc tcccttgtca tcaaaaccca caccgggtgt cataatcaac 3180
caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa 3240
tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag 3300
cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct 3360
gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg 3420
tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca 3480
atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt 3540
agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt 3600
aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca 3660
tccaatgaag cacacaagtt tgtttgcttt tcgtgoatga tattaaatag cttggcagca 3720
acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt 3780
cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt 3840
tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct 3900
tccttctgtt cggagattac cgaatcaaaa aaatttcaaa gaaaccgaaa tcaaaaaaaa 3960
gaatacaaaa aaaatgatga attgaattga aaagctgtgg tatggtgcac tctcagtaca 4020
atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 4080
ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 4140
agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 4200
gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gtatgatcca 4260
atatcaaagg aaatgatagc attgaaggat gagactaatc caattgagga gtggcagcat 4320
atagaacagc taaagggtag tgctgaagga agcatacgat accccgcatg gaatgggata 4380
atatcacagg aggtactaga ctacctttca tcctacataa atagacgcat ataagtacgc 4440
atttaagcat aaacacgcac tatgccgttc ttctcatgta tatatatata caggcaacac 4500
gcagatatag gtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg 4560
gaagcgctcg ttttcggaaa cgctttgaag ttcctattcc gaagttccta ttctctagaa 4620
CA 02498636 2006-09-01
'3 R
agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc tctgaagacg cactttcaaa 4680
aaaccaaaaa cgcaccggac tgtaaggagc tactaaaata ttgcgaatac cgcttccaca 4740
aacattgctc aaaagtatct ctttgctata tatctctgtg ctatatccct atataaccta 4800
cccatccacc tttcgctcat tgaacttgca tctaaactcg acctctacat tttttatgtt 4860
tatctctagt attactcttt agacaaaaaa attgtagtaa gaactattca tagagtgaat 4920
cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt atatagagac aaaatagaag 4980
aaaccgttca taattttctg accaatgaag aatcatcaac gctatcactt tctgttcaca 5040
aagtatgcgc aatccacatc ggtatagaat ataatcgggg atgcctttat cttgaaaaaa 5100
tgcacccgca gcttcgctag taatcagtaa acgcgggaag tggagtcagg ctttttttat 5160
ggaagagaaa atagacacca aagtagcctt cttctaacct taacggacct acagtgcaaa 5220
aagttatcaa gagactgcat tatagagcgc acaaaggaga aaaaaagtaa tctaagatgc 5280
tttgttagaa aaatagcgct ctcgggatgc atttttgtag aacaaaaaag aagtatagat 5340
tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt tctgtaaaaa tgcagctcag 5400
attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttttacaa aaatgaagca 5460
cagattcttc gttggtaaaa tagcgctttc gcgttgcatt tctgttctgt aaaaatgcag 5520
ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttc tacaaaatga 5580
agcacagatg cttcgttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 5640
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 5700
gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 5760
tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 5820
aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 5880
cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 5940
agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 6000
ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 6060
tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 6120
tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 6180
caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 6240
accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 6300
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 6360
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 6420
CA 02498636 2006-09-01
Yo
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 6480
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 6540
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 6600
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 6660
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 6720
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 6780
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 6840
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 6900
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 6960
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 7020
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 7080
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 7140
acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 7200
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 7260
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 7320
ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 7380
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 7440
taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 7500
cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 7560
gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 7620
tgagcgcaac gcaattaatg tgagttacct cactcattag gcaccccagg ctttacactt 7680
tatgcttccg gctcctatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 7740
cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct 7800
ggagctttt 7809
