Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
METHOD FOR THE PRODUCTION OF GLYCEROL
BY RECOMBINANT ORGANISMS
FIELD OF INVENTION
The present invention relates to the field of molecular biology and the use
of recombinant organisms for the production of desired compounds. More
specifically it describes the expression of cloned genes for glycerol-3-
phosphate
dehydrogenase (G3PDH) and glycerol-3-phosphatase (G3P phosphatase), either
separately or together, for the enhanced production of glycerol.
BACKGROUND
Glycerol is a compound in great demand by industry for use in cosmetics,
liquid soaps, food, pharmaceuticals, lubricants, anti-freeze solutions, and in
numerous other applications. The esters of glycerol are important in the fat
and
oil industry.
Not all organisms have a natural capacity to synthesize glycerol.
However, the biological production of glycerol is known for some species of
bacteria, algae, and yeasts. The bacteria Bacillus licheniformis and
Lactobacillus
lycopersica synthesize glycerol. Glycerol production is found in the
halotolerant
algae Dunaliella sp. and Asteromonas gracilis for protection against high
external
salt concentrations (Ben-Amotz et al., (1982) Experientia 38:49-52).
Similarly,
various osmotolerant yeasts synthesize glycerol as a protective measure. Most
strains of Saccharomyces produce some glycerol during alcoholic fermentation,
and this can be increased physiologically by the application of osmotic stress
(Albertyn et al., (1994) Mol. Cell. Biol. 14,4135-4144). Earlier this century
glycerol was produced commercially with Saccharomyces cultures to which
steering reagents were added such as sulfites or alkalis. Through the
formation of
an inactive complex, the steering agents block or inhibit the conversion of
acetaldehyde to ethanol; thus, excess reducing equivalents (NADH) are
available
to or "steered" towards dihydroxyacetone phosphate (DHAP) for reduction to
produce glycerol. This method is limited by the partial inhibition of yeast
growth
that is due to the sulfites. This limitation can be partially overcome by the
use of
alkalis which create excess NADH equivalents by a different mechanism. In this
practice, the alkalis initiated a Cannizarro disproportionation to yield
ethanol and
acetic acid from two equivalents of acetaldehyde.
The gene encoding glycerol-3-phosphate dehydrogenase (DAR 1,GPD1)
has been cloned and sequenced from Saccharomyces diastaticus (Wang et al.,
(1994),.! Boa. 176:7091-7095). The DAR1 gene was cloned into a shuttle vector
and used to transform E. coli where expression produced active enzyme. Wang et
al., supra, recognizes that DAR1 is regulated by the cellular osmotic
environment
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but does not suggest how the gene might be used to enhance glycerol production
in a recombinant organism.
Other glycerol-3-phosphate dehydrogenase enzymes have been isolated.
For example, sn-glycerol-3-phosphate dehydrogenase has been cloned and
sequenced from S. cerevisiae (Larason et al., (1993) Mol. Microbiol., 10:1101,
(1993)). Albertyn et al., (1994) Mot ('ell. Biol., 14:4135) teach the cloning
of
GPD1 encoding a glycerol-3-phosphate dehydrogenase from S. cerevisiae. Like
Wang et al., both Albertyn et al., and Larason et al. recognize the osmo-
sensitvity
of the regulation of this gene but do not suggest how the gene might be used
in the
production of glycerol in a recombinant organism.
As with G3DPH, glycerol-3-phosphatase has been isolated from
Saccharomyces cerevisiae and the protein identified as being encoded by the
GPP1 and GPP2 genes (Norbeck et al., (1996)J. Biol. ('hem., 271:13875). Like
the genes encoding G3DPFI, it appears that GPP2 is osmotically-induced.
There is no known art that teaches glycerol production from recombinant
organisms with G3PDH/G3P phosphatase expressed together or separately. Nor
is there known art that teaches glycerol production from any wild-type
organism
with these two enzyme activities that does not require applying some stress
(salt
or an osmolyte) to the cell. Eustace ((1987), Can. J. Microbiol., 33:112-117))
teaches away from achieving glycerol production by recombinant DNA
techniques. By selective breeding techniques, these investigators created a
hybridized yeast strain that produced glycerol at greater levels than the
parent
strains; however, the G3PDH activity remained constant or slightly lower.
A microorganism capable of producing glycerol under physiological
conditions is industrially desirable, especially when the glycerol itself will
be used
as a substrate in vivo as part of a more complex catabolic or biosynthetic
pathway
that could be perturbed by osmotic stress or the addition of steering agents.
The problem to be solved, therefore, is how to direct carbon flux towards
glycerol production by the addition or enhancement of certain enzyme
activities,
especially G3PDH and G3P phosphatase which respectively catalyze the
conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate
(G3P) and then to glycerol. This process has not previously been described for
a
recombinant organism and required the isolation of genes encoding the two
enzymes and their subsequent expression. A surprising and unanticipated
difficulty encountered was the toxicity of G3P phosphatase to the host which
required careful control of its expression levels to avoid growth inhibition.
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SUMMARY OF THE INVENTION
The present invention provides a method for the production of glycerol
from a recombinant organism comprising: (i) transforming a suitable host cell
with an expression cassette comprising either or both
(a) a gene encoding a glycerol-3-phosphate dehydrogenase
enzyme;
(b) a gene encoding a glycerol-3-phosphate phosphatase enzyme;
(ii) culturing the transformed host cell in the presence of at least one
carbon
source selected from the group consisting of monosaccharides,
oligosaccharides,
polysaccharides, and single-carbon substrates, or mixtures thereof whereby
glycerol is produced; and (iii) recovering the glycerol. Glucose is the most
preferred carbon source.
The invention further provides transformed host cells comprising
expression cassettes capable of expressing glycerol-3-phosphate dehydrogenase
and glycerol-3-phosphatase activities for the production of glycerol.
BRIEF DESCRIPTION OF BIOLOGICAL
DEPOSITS AND SEQUENCE LISTING
Applicants have made the following biological deposits under the terms of
the Budapest Treaty on the International Recognition of the Deposit of
Micro-organisms for the Purposes of Patent Procedure:
Depositor Identification Intl. Depository
Reference Designation Date of Deposit
Escherichia coli pAH21/DH5a ATCC 98187 26 September 1996
(containing the GPP2 gene)
Escherichia coli (pDAR1A/AA200) ATCC 98248 6 November 1996
(containing the DAR1 gene)
"ATCC" refers to the American Type Culture Collection international
depository located at 12301 Parklawn Drive, Rockville, Is/1D 20852 U.S.A. The
designation is the accession number of the deposited material.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for the biological production of
glycerol from a fermentable carbon source in a recombinant organism. The
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method provides a rapid, inexpensive and environmentally-responsible source of
glycerol useful in the cosmetics and pharmaceutical industries. The method
uses a
microorganism containing cloned homologous or heterologous genes encoding
glycerol-3-phosphate dehydrogenase (G3PDH) and/or glycerol-3-phosphatase
(G3P phosphatase), The microorganism is contacted with a carbon source and
glycerol is isolated from the conditioned media. The genes may be incorporated
into the host microorganism separately or together for the production of
glycerol.
As used herein the following terms may be used for interpretation of the
claims and specification.
The terms "glycerol-3-phosphate dehydrogenase" and "G3PDH" refer to a
polypeptide responsible for an enzyme activity that catalyzes the conversion
of
dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). In vivo
G3PDH may be NADH: NADPH; or FAD-dependent. The NADH-dependent
enzyme (EC 1.1.1.8) is encoded by several genes including GPD1 (GenBank
Z74071x2), or GPD2 (GenBank Z35169x1), or GPD3 (GenBank G984182), or
DAR1 (GenBank Z74071x2). The NADPH-dependent enzyme (EC 1.1.1.94) is
encoded by gpsA (GenBank U321643, (cds 197911-196892) G466746 and
L45246). The FAD-dependent enzyme (EC 1.1.99.5) is encoded by GUT2
(GenBank Z47047x23), or glpD (GenBank 0147838), or glpABC (GenBank
M20938).
The terms "glycerol-3-phosphatase", "sn-glycerol-3-phosphatase", or
"d,l-glycerol phosphatase", and "G3P phosphatase" refer to a polypeptide
responsible for an enzyme activity that catalyzes the conversion of glycerol-3-
phosphate to glycerol. G3P phosphatase is encoded by GPP1 (GenBank
Z47047x125), or GPP2 (GenBank U18813x11).
The term "glycerol kinase" refers to a polypeptide responsible for an
enzyme activity that catalyzes the conversion of glycerol to glycerol-3-
phosphate.
or glycerol-3-phosphate to glycerol, depending on reaction conditions.
Glycerol
kinase is encoded by GUT I (GenBank U11583x19).
The terms "GPD1", "DAR1", "OSG I", "D2830". and "YDL022W" will
be used interchangeably and refer to a gene that encodes a cytosolic glycerol-
3-
phosphate dehydrogenase and is characterized by the base sequence given as
SEQ ID NO:l.
The term "GPD2" refers to a gene that encodes a cytosolic glycerol-3-
phosphate dehydrogenase and is characterized by the base sequence given in
SEQ ID NO:2.
The terms "GUT2" and "YIL155C" are used interchangeably and refer to a
gene that encodes a mitochondrial glycerol-3-phosphate dehydrogenase and is
characterized by the base sequence given in SEQ ID NO:3.
4
=
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The terms "GPP1", "RHR2" and "YIL053W" are used interchangeably
and refer to a gene that encodes a cytosolic glycerol-3-phosphatase and is
characterized by the base sequence given in SEQ ID NO:4.
The terms "GPP2", "HOR2" and "YER062C" are used interchangeably
= 5 and refer to a gene that encodes a cytosolic glycerol-3-
phosphatase and is
characterized by the base sequence given as SEQ ID NO:5.
The term "GUT 1" refers to a gene that encodes a cytosolic glycerol kinase
=
and is characterized by the base sequence given as SEQ ID NO:6.
As used herein, the terms "function" and "enzyme function" refer to the
catalytic activity of an enzyme in altering the energy required to perform a
specific chemical reaction. Such an activity may apply to a reaction in
equilibrium where the production of both product and substrate may be
accomplished under suitable conditions.
The terms "polypeptide" and "protein" are used herein interchangeably.
The terms "carbon substrate" and "carbon source" refer to a carbon source
capable of being metabolized by host organisms of the present invention and
particularly mean carbon sources selected from the group consisting of mono-
saccharides, oligosaccharides, polysaccharides, and one-carbon substrates or
mixtures thereof.
The terms "host cell" and "host organism" refer to a microorganism
capable of receiving foreign or heterologous genes and expressing those genes
to
produce an active gene product.
The terms "foreign gene", "foreign DNA", "heterologous gene", and
"heterologous DNA" all refer to genetic material native to one organism that
has
been placed within a different host organism.
The terms "recombinant organism" and "transformed host" refer to any
organism transformed with heterologous or foreign genes. The recombinant
organisms of the present invention express foreign genes encoding G3PDH and
G3P phosphatase for the production of glycerol from suitable carbon
substrates.
"Gene" refers to a nucleic acid fragment that expresses a specific protein,
including regulatory sequences preceding (5' non-coding) and following (3' non-
coding) the coding region. The terms "native" and "wild-type" gene refer to
the
gene as found in nature with its own regulatory sequences.
As used herein, the terms "encoding" and "coding" refer to the process by
=
which a gene, through the mechanisms of transcription and translation,
produces
an amino acid sequence. The process of encoding a specific amino acid sequence
is meant to include DNA sequences that may involve base changes that do not
cause a change in the encoded amino acid, or which involve base changes which
may alter one or more amino acids, but do not affect the functional properties
of
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the protein encoded by the DNA sequence. Therefore, the invention encompasses
more than the specific exemplary sequences. Modifications to the sequence,
such
as deletions, insertions, or substitutions in the sequence which produce
silent
changes that do not substantially affect the functional properties of the
resulting
protein molecule are also contemplated. For example, alterations in the gene
sequence which reflect the degeneracy of the genetic code, or which result in
the
production of a chemically equivalent amino acid at a given site, are
contemplated: thus. a codon for the amino acid alanine, a hydrophobic amino
acid,
may be substituted by a codon encoding another less hydrophobic residue, such
as
. 10 glycine, or a more hydrophobic residue, such as valine, leucine, or
isoleucine.
Similarly, changes which result in substitution of one negatively charged
residue
for another, such as aspartic acid for glutamic acid, or one positively
charged
residue for another, such as lysine for arginine, can also be expected to
produce a
biologically equivalent product. Nucleotide changes which result in alteration
of
the N-terminal and C-terminal portions of the protein molecule would also not
be
expected to alter the activity of the protein. In some cases, it may in fact
be
desirable to make mutants of the sequence in order to study the effect of
alteration
on the biological activity of the protein. Each of the proposed modifications
is
well within the routine skill in the art, as is determination of retention of
biological activity in the encoded products. Moreover, the skilled artisan
recognizes that sequences encompassed by this invention are also defined by
their
ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65 C),
with the sequences exemplified herein.
The term "expression" refers to the transcription and translation to gene
product from a gene coding for the sequence of the gene product.
The terms "plasmid". "vector", and "cassette" as used herein refer to an
extra chromosomal element often carrying genes which are not part of the
central
metabolism of the cell and are usually in the form of circular double-stranded
DNA
molecules. Such elements may be autonomously replicating sequences, genome
integrating sequences, phage or nucleotide sequences, linear or circular, of a
single- or double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into a unique
construction which is capable of introducing a promoter fragment and DNA
sequence for a selected gene product along with appropriate 3' untranslated
sequence into a cell. "Transformation cassette" refers to a specific vector
containing a foreign gene and having elements in addition to the foreign gene
that
facilitate transformation of a particular host cell. "Expression cassette"
refers to a
specific vector containing a foreign gene and having elements in addition to
the
foreign gene that allow for enhanced expression of that gene in a foreign
host.
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The terms "transformation" and "transfection" refer to the acquisition of
new genes in a cell after the incorporation of nucleic acid. The acquired
genes
may be integrated into chromosomal DNA or introduced as extrachromosomal
replicating sequences. The term "transformant" refers to the cell resulting
from a
transformation.
The term "genetically altered" refers to the process of changing hereditary
material by transformation or mutation.
Representative enzyme pathway
It is contemplated that glycerol may be produced in recombinant
organisms by the manipulation of the glycerol biosynthetic pathway found in
most
microorganisms. Typically, a carbon substrate such as glucose is converted to
glucose-6-phosphate via hexokinase in the presence of ATP. Glucose-phosphate
isomerase catalyzes the conversion of glucose-6-phosphate to fructose-6-
phosphate and then to fructose-1,6-diphosphate through the action of
6-phosphofructokinase. The diphosphate is then taken to dihydroxyacetone
phosphate (DHAP) via aldolase. Finally NADH-dependent G3PDH converts
DHAP to glycerol-3-phosphate which is then dephosphorylated to glycerol by
G3P phosphatase. (Agarwal (1990), Adv. Biochem. Engrg. 41:114).
Alternate pathways for alveerol production
An alternative pathway for glycerol production from DHAP has been
suggested (Wang et al., (1994).1 Boa. 176:7091-7095). In this proposed pathway
DHAP could be dephosphorylated by a specific or non-specific phosphatase to
give dihydroxyacetone, which could then be reduced to glycerol by a dihydroxy-
acetone reductase. Dihydroxyacetone reductase is known in prokaryotes and in
Schizosaccharomyces pombe, and cloning and expression of such activities
together with an appropriate phosphatase could lead to glycerol production.
Another alternative pathway for glycerol production from DHAP has been
suggested (Redkar (1995), Experimental Mycology, 19:241, 1995). In this
pathway DHAP is isomerized to glyceraldehyde-3-phosphate by the common
glycolytic enzyme triose phosphate isomerase. Glyceraldehyde-3-phosphate is
dephosphorylated to glyceraldehyde, which is then reduced by alcohol
dehydrogenase or a NADP-dependent glycerol dehydrogenase activity. The
cloning and expression of the phosphatase and dehydrogenase activities from
Aspergillus nidulans could lead to glycerol production.
=
Genes encoding G3PDH and G3P phosphatase
The present invention provides genes suitable for the expression of
G3PDH and G3P phosphatase activities in a host cell.
Genes encoding G3PDH are known. For example, GPD I has been
isolated from Saccharomyces and has the base sequence given by SEQ ID NO:1,
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encoding the amino acid sequence given in SEQ ID NO:7 (Wang et al., supra).
Similarly, G3PDH activity has also been isolated from Saccharomyces encoded
by GPD2 having the base sequence given in SEQ ID NO:2 encoding the amino
acid sequence given in SEQ ID NO:8 (Eriksson et al.. (1995) MoL Microbiol.,
17:95).
For the purposes of the present invention it is contemplated that any gene
encoding a polypeptide responsible for G3PDH activity is suitable wherein that
activity is capable of catalyzing the conversion of dihydroxyacetone phosphate
(DHAP) to glycerol-3-phosphate (G3P). Further, it is contemplated that any
gene
encoding the amino acid sequence of G3PDH as given by SEQ ID NOS:7, 8, 9,
10, 11 and 12 corresponding to the genes GPD1, GPD2. GUT2, gpsA, glpD, and
the a subunit of glpABC respectively, will be functional in the present
invention
wherein that amino acid sequence may encompass amino acid substitutions,
deletions or additions that do not alter the function of the enzyme. The
skilled
person will appreciate that genes encoding G3PDH isolated from other sources
will also be suitable for use in the present invention. For example, genes
isolated
from prokaryotes include GenBank accessions M34393, M20938, L06231,
U12567, L45246, L45323, L45324, L45325, U32164, U32689, and U39682.
Genes isolated from fungi include GenBank accessions U30625, U30876 and
X56162; genes isolated from insects include GenBank accessions X61223 and
X14179; and genes isolated from mammalian sources include GenBank
accessions U12424, M25558 and X78593.
Genes encoding G3P phosphatase are known. For example, GPP2 has
been isolated from Saccharomyces cerevisiae and has the base sequence given by
SEQ ID NO:5, which encodes the amino acid sequence given in SEQ ID NO:13
(Norbeck et al., (1996), J. Biol. Chem., 271:13875).
For the purposes of the present invention, any gene encoding a G3P
phosphatase activity is suitable for use in the method wherein that activity
is
capable of catalyzing the conversion of glycerol-3-phosphate to glycerol.
Further,
any gene encoding the amino acid sequence of G3P phosphatase as given by
SEQ ID NOS:13 and 14 corresponding to the genes GPP2 and GPP1 respectively,
will be functional in the present invention including any amino acid sequence
that
encompasses amino acid substitutions, deletions or additions that do not alter
the
function of the G3P phosphatase enzyme. The skilled person will appreciate
that
genes encoding G3P phosphatase isolated from other sources will also be
suitable
for use in the present invention. For example, the dephosphorylation of
glycerol-
3-phosphate to yield glycerol may be achieved with one or more of the
following
general or specific phosphatases: alkaline phosphatase (EC 3.1.3.1) [GenBank
M19159, M29663, U02550 or M33965]; acid phosphatase (EC 3.1.3.2) [GenBank
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U51210, U19789, U28658 or L20566]; glycerol-3-phosphatase (EC 3.1.3.-)
[GenBank Z38060 or U18813x11]; glucose-1-phosphatase (EC 3.1.3.10)
[GenBank M33807]; glucose-6-phosphatase (EC 3.1.3.9) [GenBank U00445];
fructose-1,6-bisphosphatase (EC 3.1.3.11) [GenBank X12545 or J03207] or
phosphotidyl glycero phosphate phosphatase (EC 3.1.3.27) [GenBank M23546
=
and M23628].
Genes encoding glycerol kinase are known. For example, GUT1 encoding
the glycerol kinase from Saccharomyces has been isolated and sequenced (Pavlik
et al. (1993), Curr. Genet., 24:21) and the base sequence is given by
SEQ ID NO:6, which encodes the amino acid sequence given in SEQ ID NO:15.
The skilled artisan will appreciate that, although glycerol kinase catalyzes
the
degradation of glycerol in nature, the same enzyme will be able to function in
the
synthesis of glycerol, converting glycerol-3-phosphate to glycerol under the
appropriate reaction energy conditions. Evidence exists for glycerol
production
through a glycerol kinase. Under anaerobic or respiration-inhibited
conditions,
Trypanosoma brucei gives rise to glycerol in the presence of Glycerol-3-P and
ADP. The reaction occurs in the glycosome compartment (Hammond, (1985),
J. Biol. Chem., 260:15646-15654).
Host cells
Suitable host cells for the recombinant production of glycerol by the
expression of G3PDH and G3P phosphatase may be either prokaryotic or
eukaryotic and will be limited only by their ability to express active
enzymes.
Preferred host cells will be those bacteria, yeasts. and filamentous fungi
typically
useful for the production of glycerol such as Citrobacter, Enterobacter,
Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus,
Saccharomyces,
Schizosaccharomyces, Zygosaccharomyces, Pichia. Kluyveromyces. Candida,
Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia,
Salmonella, Bacillus, Streptomyces and Pseudomonas. Preferred in the present
invention are E. coli and Saccharomyces.
Vectors and expression cassettes
The present invention provides a variety of vectors and transformation and
expression cassettes suitable for the cloning, transformation and expression
of
G3PDH and G3P phosphatase into a suitable host cell. Suitable vectors will be
those which are compatible with the bacterium employed. Suitable vectors can
be
derived, for example, from a bacteria, a virus (such as bacteriophage T7 or a
M-13
derived phage), a cosmid, a yeast or a plant. Protocols for obtaining and
using
such vectors are known to those in the art (Sambrook et al.. Molecular
Cloning: A
Laboratory Manual - volumes 1, 2, 3 (Cold Spring Harbor Laboratory: Cold
Spring Harbor, NY, 1989)).
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Typically, the vector or cassette contains sequences directing transcription
and translation of the appropriate gene, a selectable marker, and sequences
allowing autonomous replication or chromosomal integration. Suitable vectors
comprise a region 5' of the gene which harbors transcriptional initiation
controls
and a region 3' of the DNA fragment which controls transcriptional
termination. It
is most preferred when both control regions are derived from genes homologous
to the transformed host cell. Such control regions need not be derived from
the
genes native to the specific species chosen as a production host.
Initiation control regions, or promoters, which are useful to drive
expression of the G3PDH and G3P phosphatase genes in the desired host cell are
numerous and familiar to those skilled in the art. Virtually any promoter
capable
of driving these genes is suitable for the present invention including but not
limited to CYC I, HIS3, GAL I, GAL 10, ADH1, PGK, PH05, GAPDH, ADC,
TRP1, URA3, LEU2, ENO. and TPI (useful for expression in Saccharomyces);
A0X1 (useful for expression in Pichia); and lac, trp, XPL, A.PR, T7, tac, and
trc,
(useful for expression in E. coil).
Termination control regions may also be derived from various genes native
to the preferred hosts. Optionally, a termination site may be unnecessary;
however, it is most preferred if included.
For effective expression of the instant enzymes, DNA encoding the
enzymes are linked operably through initiation codons to selected expression
control regions such that expression results in the formation of the
appropriate
messenger RNA.
Transformation of suitable hosts and expression of G3PDH and G3P phosphatase
for the production of_glycerol
Once suitable cassettes are constructed they are used to transform
appropriate host cells. Introduction of the cassette containing the genes
encoding
G3PDH and/or G3P phosphatase into the host cell may be accomplished by
known procedures such as by transformation, e.g., using calcium-permeabilized
cells, electroporation, or by transfection using a recombinant phage virus
(Sambrook et al., supra).
In the present invention AH21 and DAR1 cassettes were used to transform
the E. coil DH5a as fully described in the GENERAL METHODS and
EXAMPLES.
Media and Carbon Substrates
Fermentation media in the present invention must contain suitable carbon
substrates. Suitable substrates may include but are not limited to
monosaccharides
such as glucose and fructose, oligosaccharides such as lactose or sucrose,
polysaccharides such as starch or cellulose or mixtures thereof and unpurified
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mixtures from renewable feedstocks such as cheese whey permeate, comsteep
liquor, sugar beet molasses. and barley malt. Additionally, the carbon
substrate
may also be one-carbon substrates such as carbon dioxide, or methanol for
which
metabolic conversion into key biochemical intermediates has been demonstrated.
Glycerol production from single carbon sources (e.g., methanol,
formaldehyde or formate) has been reported in methylotrophic yeasts (Yamada
= etal. (1989), Agric. Biol. Chem., 53(2):541-543) and in bacteria (Hunter
et al.
(1985), Biochemistry, 24:4148-4155). These organisms can assimilate single
carbon compounds, ranging in oxidation state from methane to formate, and
produce glycerol. The pathway of carbon assimilation can be through ribulose
monophosphate, through serine, or through xylulose-monophosphate (Gottschalk,
Bacterial Metabolism, Second Edition, Springer-Verlag: New York (1986)). The
ribulose monophosphate pathway involves the condensation of formate with
ribulose-5-phosphate to form a 6 carbon sugar that becomes fructose and
eventually the three carbon product, glyceraldehyde-3-phosphate. Likewise, the
serine pathway assimilates the one-carbon compound into the glycolytic pathway
via methylenetetrahydrofolate.
In addition to one and two carbon substrates. methylotrophic organisms
are also known to utilize a number of other carbon-containing compounds such
as
methylamine, glucosamine and a variety of amino acids for metabolic activity.
=
For example, methylotrophic yeast are known to utilize the carbon from
methylamine to form trehalose or glycerol (Bellion et al. (1993), Microb.
Growth
Cl Compd., [Int. Symp.], 7th, 415-32. Editor(s): Murrell, J. Collin; Kelly,
Don P. Publisher: Intercept, Andover, UK). Similarly, various species of
Candida will metabolize alanine or oleic acid (Sulter et al. (1990), Arch.
Microbiol., 153(5):485-9). Hence, the source of carbon utilized in the present
invention may encompass a wide variety of carbon-containing substrates and
will
only be limited by the choice of organism.
Although all of the above mentioned carbon substrates and mixtures
thereof are suitable in the present invention, preferred carbon substrates are
monosaccharides, oligosaccharides, polysaccharides, single-carbon substrates
or
mixtures thereof. More preferred are sugars such as glucose, fructose,
sucrose,
maltose, lactose and single carbon substrates such as methanol and carbon
= dioxide. Most preferred as a carbon substrate is glucose.
In addition to an appropriate carbon source, fermentation media must
= contain suitable minerals, salts, cofactors, buffers and other
components. known to
those skilled in the art, suitable for the growth of the cultures and
promotion of the
enzymatic pathway necessary for glycerol production.
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Culture Conditions
Typically cells are grown at 30 C in appropriate media. Preferred growth
media are common commercially prepared media such as Luria Bertani (LB)
broth, Sabouraud Dextrose (SD) broth. or Yeast medium (YM) broth. Other
defined or synthetic growth media may also be used and the appropriate medium
for growth of the particular microorganism will be known by one skilled in the
art
of microbiology or fermentation science. The use of agents known to modulate
catabolite repression directly or indirectly, e.g., cyclic adenosine 2':3'-
mono-
phosphate, may also be incorporated into the reaction media. Similarly, the
use of
agents known to modulate enzymatic activities (e.g., sulfites, bisulfites, and
alkalis) that lead to enhancement of glycerol production may be used in
conjunction with or as an alternative to genetic manipulations.
Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0
where the range of pH 6.0 to pH 8.0 is preferred for the initial condition.
Reactions may be performed under aerobic or anaerobic conditions where
anaerobic or microaerobic conditions are preferred.
Identification and purification of G3PDH and G3P phosphatase
The levels of expression of the proteins G3PDH and G3P phosphatase are
measured by enzyme assays. G3PDH activity assay relies on the spectral
properties of the cosubstrate, NADH, in the DHAP conversion to G-3-P. NADH
has intrinsic UV/vis absorption and its consumption can be monitored spectro-
photometrically at 340 rim. G3P phosphatase activity can be measured by any
method of measuring the inorganic phosphate liberated in the reaction. The
most
commonly used detection method uses the visible spectroscopic determination of
a blue-colored phosphomolybdate ammonium complex.
Identification and recovery of glycerol
Glycerol may be identified and quantified by high performance liquid
chromatography (HPLC) and gas chromatography/mass spectroscopy (GC/MS)
analyses on the cell-free extracts. Preferred is a method where the
fermentation
media are analyzed on an analytical ion exchange column using a mobile phase
of
0.01N sulfuric acid in an isocratic fashion.
Methods for the recovery of glycerol from fermentation media are known
in the art. For example, glycerol can be obtained from cell media by
subjecting
the reaction mixture to the following sequence of steps: filtration; water
removal;
organic solvent extraction; and fractional distillation (U.S. Patent No.
2,986.495).
=
Selection of transformants by complementation
In the absence of a functional gpsA-encoded G3PDH, E. coli cells are
unable to synthesize G3P, a condition which leads to a block in membrane
biosynthesis. Cells with such a block are auxotrophic, requiring that either
12
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PCT/US97/20293
glycerol or G3P be present in the culture media for synthesis of membrane
phospholipids.
A cloned heterologous wild-type gpsA gene is able to complement the
chromosomal gpsA mutation to allow growth in media lacking glycerol or G3P
= 5 (Wang, et al. (1994), .I. Bad. 176:7091-7095). Based on
this complementation
strategy, growth of gpsA-defective cells on glucose would only occur if they
= possessed a plasmid-encoded gpsA, allowing a selection based on synthesis
of
G3P from DHAP. Cells which lose the recombinant gpsA plasmid during culture
would fail to synthesize G3P and cell growth would subsequently be inhibited.
The complementing G3PDH activity can be expressed not only from gpsA. but
also from other cloned genes expressing G3PDH activity such as GPD1, GPD2,
GPD3, GUT2, glpD, and glpABC. These can be maintained in a gpsA-defective
E. coil strain such as BB20 (Cronan et al. (1974),J. Bad., 118:598),
alleviating
the need to use antibiotic selection and its prohibitive cost in large-scale
fermentations.
A related strategy can be used for expression and selection in
osmoregulatory mutants of S. cerevisiac (Larsson et al. (1993), Mol.
Microbial..
10:1101-1111). These osg I mutants are unable to grow at low water potential
and
show a decreased capacity for glycerol production and reduced G3PDH activity.
The osgl salt sensitivity defect can be complemented by a cloned and expressed
G3PDH gene. Thus, the ability to synthesize glycerol can be used
simultaneously
as a selection marker for the desired glycerol-producing cells.
EXAMPLES
GENERAL METHODS
Procedures for phosphorylations, ligations, and transformations are well
known in the art. Techniques suitable for use in the following examples may be
found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press (1989).
Materials and methods suitable for the maintenance and growth of
bacterial cultures are well known in the art. Techniques suitable for use in
the
following examples may be found in Manual of Methods for General Bacteriology
(Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow. Eugene W. Nester.
Willis
A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for
Microbiology, Washington, DC. (1994) or in Biotechnology: A Textbook of
Industrial Microbiology (Thomas D. Brock, Second Edition (1989) Sinauer
= Associates, Inc., Sunderland, MA). All reagents and materials used for
the growth
and maintenance of bacterial cells were obtained from Aldrich Chemicals
(Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO/BRL (Gaithersburg,
MD), or Sigma Chemical Company (St. Louis. MO) unless otherwise specified.
13
= '
CA 02624764 2008-04-04
WO 98/21340 It..
!Ilia', 11.4l1L7..1
The meaning of abbreviations is as follows: "h" means hour(s), "min"
means minute(s), "sec" means second(s), "d" means day(s), "mL" means
milliliters, "L" means liters.
Cell strains
The following Escherichia coli strains were used for transformation and
expression of G3PDH and G3P phosphatase. Strains were obtained from the
E. coli Genetic Stock Center or from Life Technologies, Gaithersburg, MD).
AA200 (garBIO jhuA22 ompF627.fadL701 re/A] pit-10 spoT1 tpi-1 phoM510
mcrB1) (Anderson et al., (1970),]. Gen. Microbiol., 62:329).
BB20 (tonA22 dphoA8 fadL70.1 relAl glpR2 glpD3 pit-I0 gpsA20 spoT1 T2R)
(Cronan et al., J. Bact., 118:598).
DH5cc (deoR endAl gyrA96 hsdR17 reek' relA I supE44 thi-I daacZYA-
argFV169) phi8OlacZAM15 F-) (Woodcock et al., (1989), Nucl. Acids Res.,
17:3469).
Identification of Glycerol
The conversion of glucose to glycerol was monitored by HPLC and/or GC.
Analyses were performed using standard techniques and materials available to
one
of skill in the art of chromatography. One suitable method utilized a Waters
Maxima 820 HPLC system using UV (210 nm) and RI detection. Samples were
injected onto a ShodexTm SH-1011 column (8 mm x 300 mm; Waters, Milford MA)
equipped with ShodexTmSH-1011P precolumn (6 mm x 50 mm), temperature-
controlled at 50 C, using 0.01 N H2SO4 as mobile phase at a flow rate of
0.5 mL/min. When quantitative analysis was desired, samples were injected onto
a ShodexTm SH-1011 column (8 mm x 300 mm; Waters, Milford, MA) equipped
with a ShodexTM SH-1011P precolumn (6 mm x 50 mm), temperature-controlled at
50 C, using 0.01 N H2SO4 as mobile phase at a flow rate of 0.69 mL/min. When
quantitative analysis was desired, samples were prepared with a known amount
of
trimethylacetic acid as an external standard. Typically, the retention times
of
glycerol (RI detection) and glucose (RI detection) were 17.03 min and 12.66
min,
respectively.
Glycerol was also analyzed by GC/MS. Gas chromatography with mass
spectrometry detection for and quantitation of glycerol was done using a
DB-WAX column (30 m, 0.32 mm I.D., 0.25 urn film thickness, J & W Scientific,
Folsom, CA), at the following conditions: injector: split, 1:15; sample
volume:
1 uL; temperature profile: 150 C intitial temperature with 30 sec hold, 40
C/min
to 180 C, 20 C/min to 240 C, hold for 2.5 min. Detection: El Mass
14
CA 02624764 2008-04-04
WO 98/21340 PCT/US97/20293
Spectrometry (Hewlett Packard 5971, San Fernando, CA), quantitative SIM using
ions 61 m/z and 64 m/z as target ions for glycerol and glycerol-d8, and ion 43
m./z
as qualifier ion for glycerol. Glycerol-d8 was used as an internal standard.
Assay for glycerol-3-phosphatase. GPP
= 5 The assay for enzyme activity was performed by incubating the
extract
with an organic phosphate substrate in a bis-Tris or MES and magnesium buffer,
= pH 6.5. The substrate used was either 1-a-glycerol phosphate. or d,l-a-
glycerol
phosphate. The final concentrations of the reagents in the assay are: buffer
(20 mM, bis-Tris or 50 mM MES); MgC12 (10 mM); and substrate (20 mM). If
the total protein in the sample was low and no visible precipitation occurs
with an
acid quench, the sample was conveniently assayed in the cuvette. This method
involved incubating an enzyme sample in a cuvette that contained 20 mM
substrate (50 4, 200 mM), 50 mM MES, 10 mM MgCl2, pH 6.5 buffer. The
final phosphatase assay volume was 0.5 mL. The enzyme-containing sample was
added to the reaction mixture; the contents of the cuvette were mixed and then
the
cuvette was placed in a circulating water bath at T = 37 C for 5 to 120 min,
the
length of time depending on whether the phosphatase activity in the enzyme
sample ranged from 2 to 0.02 U/mL. The enzymatic reaction was quenched by
the addition of the acid molybdate reagent (0.4 mL). After the Fiske SubbaRow
reagent (0.1 mL) and distilled water (1.5 mL) were added, the solution was
mixed
and allowed to develop. After 10 min, to allow full color development, the
absorbance of the samples was read at 660 nip using a Cary 219 UV/Vis
spectrophotometer. The amount of inorganic phosphate released was compared to
a standard curve that was prepared by using a stock inorganic phosphate
solution
(0.65 mM) and preparing 6 standards with final inorganic phosphate
concentrations ranging from 0.026 to 0.130 gmol/mL.
Spectrophotometric Assay for Glycerol 3-Phosphate Dehydrogenase (G3PDH)
Activity
The following procedure was used as modified below from a method
published by Bell et al. (1975), J. Biol. Chem., 250:7153-8. This method
involved
incubating an enzyme sample in a cuvette that contained 0.2 mM NADH; 2.0 mM
Dihydroxyacetone phosphate (DHAP), and enzyme in 0.1 M Tris/HC1, pH 7.5
buffer with 5 mM DTT,in a total volume of 1.0 mL at 30 C. The
= spectrophotometer was set to monitor absorbance changes at the fixed
wavelength
=
of 340 nm. The instrument was blanked on a cuvette containing buffer only.
After the enzyme was added to the cuvette, an absorbance reading was taken.
The
first substrate, NADH (50 uL 4 mM NADH; absorbance should increase approx
1.25 AU), was added to determine the background rate. The rate should be
followed for at least 3 min. The second substrate, DHAP (50 III. 40 mM DHAP).
= =
CA 02624764 2008-04-04
was then added and the absorbance change over time was monitored for at least
3 min to determine to determine the gross rate. G3PDH activity was defined by
subtracting the background rate from the gross rate.
PLASMID CONSTRUCTION AND STRAIN CONSTRUCTION
Cloning and expression of _glycerol 3-phosphatase for increase of glycerol
production in E. coil
The Saccharomyces cerevisiae chromosomeV lamda clone 6592 ( Gene
Bank, accession U18813x 1 I) was obtained from ATCC. The glycerol
3-phosphate phosphatase (GPP2) gene was cloned by cloning from the lamda
clone as target DNA using synthetic primers (SEQ ID NO:16 with
SEQ ID NO:17) incorporating an BamHI-RBS-XbaI site at the 5' end and a Smal
site at the 3' end. The product was subcloned into pCR-Script (Stratagene,
Madison, WI) at the Sill site to generate the plasmids pAH15 containing GPP2.
The plasmid pAH15 contains the GPP2 gene in the inactive orientation for
expression from the lac promoter in pCR-Script SK+. The BarnHI-SmaI fragment
from pAH15 containing the GPP2 gene was inserted into pBlueScriptII SK+ to
generate plasmid pAH19. The pAH19 contains the GPP2 gene in the correct
orientation for expression from the lac promoter. The XbaI-Pstl fragment from
pAH19 containing the GPP2 gene was inserted into pPHOX2 to create plasmid
pAH21. The pAH21/ DI-15a is the expression plasmid.
Plasmids for the over-expression of DAR1 in E. coli
DAR1 was isolated by PCR cloning from genomic S. cerevisiae DNA
using synthetic primers (SEQ ID NO:18 with SEQ ID NO:19). Successful PCR
cloning places an Ncol site at the 5' end of DAR1 where the ATG within Ncol is
the DAR1 initiator methionine. At the 3' end of DAR1 a BamH1 site is
introduced
following the translation terminator. The PCR fragments were digested with
Ncol
+ BamHI and cloned into the same sites within the expression plasmid pTrc99A
(Pharmacia, Piscataway, NJ) to give pDAR1A.
In order to create a better ribosome binding site at the 5' end of DAR1, an
SpeI-RBS-Ncol linker obtained by annealing synthetic primers (SEQ ID NO:20
with SEQ ID NO:21) was inserted into the NcoI site of pDAR1A to create
pAH40. Plasmid pAH40 contains the new RBS and DAR1 gene in the correct
orientation for expression from the trc promoter of pTrc99A (Phamiacia,
Piscataway, NJ). The Ncol-BamHI fragement from pDAR1A and a second set
of Spel-RBS-NcoI linker obtained by annealing synthetic primers (SEQ ID NO:22
with SEQ ID NO:23) was inserted into the Spei-BamH1 site of pBC-SK+
(Stratagene. Madison, WI) to create plasmid pAH42. The plasmid pAH42
contains a chloramphenicol resistant gene.
16
CA 02624764 2008-04-04
=Y
Construction of expression cassettes for DAR1 and GPP2
Expression cassettes for DAR1 and GPP2 were assembled from the
individual DAR1 and GPP2 subclones described above using standard molecular
biology methods. The BamHI-Pstl fragment from pAH19 containing the
ribosomal binding site (RBS) and GPP2 gene was inserted into pAH40 to create
pAH43. The BamHI-Pstl fragment from pAH19 containing the RBS and GPP2
gene was inserted into pAH42 to create pAH45.
The ribosome binding site at the 5' end of GPP2 was modified as follows.
A BamHI-RBS-SpeI linker, obtained by annealing synthetic primers
GATCCAGGAAACAGA (SEQ ID NO:24) with CTAGTCTGTTTCCTG (SEQ
ID NO:25) to the Xbal-Pstl fragment from pAH19 containing the GPP2 gene, was
inserted into the BamHI-PstI site of pAH40 to create pAH48. Plasmid pAH48
contains the DAR1 gene, the modified RBS, and the GPP2 gene in the correct
orientation for expression from the trc promoter of pTrc99A (Pharmacia,
Piscataway, NJ).
Transformation of E. coli
All the plasmids described here were transformed into E. coli DH5a using
standard molecular biology techniques. The transformants were verified by its
DNA RFLP pattern.
EXAMPLE 1
PRODUCTION OF GLYCEROL FROM E. COLI
TRANSFORMED WITH G3PDH GENE
Media
Synthetic media was used for anaerobic or aerobic production of glycerol
using E. coli cells transformed with pDAR1A. The media contained per liter 6.0
g
Na2HPO4, 3.0 g KH,PO4, 1.0 g NH4C1, 0.5 g NaC1, 1 mL 20% MgSO4.7H20,
8.0 g glucose, 40 mg casamino acids, 0.5 ml 1% thiamine hydrochloride. 100 mg
ampicillin.
Growth Conditions
Strain AA200 harboring pDAR1A or the pTrc99A vector was grown in
aerobic conditions in 50 mL of media shaking at 250 rpm in 250 mL flasks at
37 C. At A600 0.2-0.3 isopropylthio-P-D-galactoside was added to a final
concentration of 1 nilvl and incubation continued for 48 h. For anaerobic
growth
samples of induced cells were used to fill Falcon n4 #2054 tubes which were
capped
and gently mixed by rotation at 37 C for 48 h. Glycerol production was
determined by HPLC analysis of the culture supernatants. Strain
pDAR1A/AA200 produced 0.38 g/L glycerol after 48 h under anaerobic
conditions. and 0.48 g/L under aerobic conditions.
17
CA 02624764 2008-04-04
EXAMPLE 2
PRODUCTION OF GLYCEROL FROM E. COLI
TRANSFORMED WITH G3P PHOSPHATASE GENE (GPP2)
Media
Synthetic phoA media was used in shake flasks to demonstrate the
increase of glyceol by GPP2 expression in E. coli. The phoA medium contained
per liter: Amisoy, 12 g; ammonium sulfate, 0.62 g; MOPS, 10.5 g; Na-citrate,
1.2 g; NaOH (1 M), 10 mL: I M MgSO4, 12 mL; 100X trace elements, 12 mL;
50% glucose, 10 mL; 1% thiamine, 10 mL; 100 mg/mL L-proline, 10 mL;
2.5 mM FeC13, 5 mL; mixed phosphates buffer, 2 mL (5 mL 0.2 M NaH2PO4+
9 mL 0.2 M K1HPO4), and pH to 7Ø The 100X traces elements for phoA
medium /L contained: ZnSO4 .7 H20, 0.58 g; MnSO4.H20, 0.34 g; CuSO4.5
1-170, 0.49 g; CoCl2.6 I-120, 0.47 g: H3B03, 0.12 g, NaMo04.2 H20, 0.48 g.
Shake Flasks Experiments
The strains pAH21/DH5a (containing GPP2 gene) and pPHOX2/DH5ct
(control) were grown in 45 mL of media (phoA media, 50 ug/mL carbenicillin,
and 1 ug/mL vitamin Bp) in a 250 mL shake flask at 37 C. The cultures were
grown under aerobic condition (250 rpm shaking) for 24 h. Glycerol production
was determined by HPLC analysis of the culture supernatant. pAH2I/DH5ot
produced 0.2 g/L glycerol after 24 h.
EXAMPLE 3
Production of glycerol from D-elucose using
recombinant E. coli containing both GPP2 and DAR1
Growth for demonstration of increased glycerol production by E. coli
D1-15a-containing pAH43 proceeds aerobically at 37 C in shake-flask cultures
(Erlenmeyer flasks, liquid volume 1/5th of total volume).
Cultures in minimal media/1% glucose shake-flasks are started by
inoculation from overnight LB/1% glucose culture with antibiotic selection.
Minimal media are: filter-sterilized defined media, final pH 6.8 (NCI),
contained
per liter: 12.6 g (NH4)2SO4, 13.7 g K2HPO4, 0.2 g yeast extract (Difco), 1 g
NaHCO3, 5 mg vitamin B12, 5 mL Modified Balch's Trace-Element Solution (the
composition of which can be found in Methods for General and Molecular
Bacteriology (P. Gerhardt et al., eds, p. 158, American Society for
Microbiology,
Washington, DC (1994)). The shake-flasks are incubated at 37 C with vigorous
shaking for overnight, after which they are sampled for GC analysis of the
supernatant. The pAH43/DH5a showed glycerol production of 3.8 g/L after 24 h.
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EXAMPLE 4
Production of glycerol from D-glucose using
recombinant E. coli containing Both GPP2 and DAR1
Example 4 illustrates the production of glucose from the recombinant
E. coli DH5a/pAH48, containing both the GPP2 and DAR1 genes.
The strain DH5a/pAH48 was constructed as described above in the
GENERAL METHODS.
Pre-Culture
DH5a/pAH48 were pre-cultured for seeding into a fermentation run.
Components and protocols for the pre-culture are listed below.
Pre-Culture Media
KH2PO4 30.0 g/L
Citric acid 2.0 g/L
MgSO4=7H20 2.0 g/L
98% H2SO4 2.0 mL/L
Ferric ammonium citrate 0.3 g/L
CaC12.21-120 0.2 g/L
Yeast extract 5.0 g/L
Trace metals 5.0 mL/L
Glucose 10.0 g/L
Carbenicillin 100.0 mg/L
The above media components were mixed together and the pH adjusted to
6.8 with NH40H. The media was then filter sterilized.
Trace metals were used according to the following recipe:
/5 Citric acid, monohydrate 4.0 g/L
MgSO4=71420 3.0 g/L
MnSO4=H20 0.5 g/L
NaC1 1.0 g/L
FeSO4=7H20 0.1 g/L
CoC12.6H10 0.1 g/L
CaCl2 0.1 g/L
ZnSO4=7H20 0.1 g/L
CuSO4=5 1120 10 mg/L
A1K(SO4)2=12H20 10 mg/L
H3B03 10 mg/L
=
Na2Mo04=2H20 10 mg/L
NiSO4.61-I20 10 mg/L
Na2Se03 10 mg/L
Na2W04-2H20 10 mg/L
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Cultures were started from seed culture inoculated from 50 pl frozen
stock (15% glycerol as cryoprotectant) to 600 mL medium in a 2-L Erlenmeyer
flask. Cultures were grown at 30 C in a shaker at 250 rpm for approximately
12 h and then used to seed the fennenter.
Fermentation growth
Vessel
15-L stirred tank fermenter
Medium
K1-12PO4 6.8 g/L
Citric acid 2.0 g/L
MgSO4=7H20 2.0 g/L
98% H2SO4 2.0 mL/L
Ferric ammonium citrate 0.3 g/L
CaC1r2H,0 0.2 g/L
Mazu DF204 antifoam 1.0 mL/L
The above components were sterilized together in the ferrnenter vessel.
The pH was raised to 6.7 with NH4OH. Yeast extract (5 g/L) and trace metals
solution (5 mL/L) were added aseptically from filter sterilized stock
solutions.
Glucose was added from 60% feed to give final concentration of 10 g/L.
Carbenicillin was added at 100 mg/L. Volume after inoculation was 6 L.
Environmental Conditions For Fermentation
The temperature was controlled at 36 C and the air flow rate was
controlled at 6 standard liters per minute. Back pressure was controlled at
0.5 bar.
The agitator was set at 350 rpm. Aqueous ammonia was used to control pH at
6.7.
The glucose feed (60% glucose monohydrate) rate was controlled to maintain
excess glucose.
Results
The results of the fermentation run are given in Table I.
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Table 1
EFT 0D550 [Glucose] [Glycerol] Total Glucose Total
Glycerol
(hr) (AU) (g/L) (g/L) Fed (g) Produced (g)
0 0.8 9.3 25
= 6 4.7 4.0 2.0 49 14
8 5.4 0 3.6 71 25
. 10 6.7 0.0 4.7 116 33
12 7.4 2.1 7.0 157 49
14.2 10.4 0.3 10.0 230 70
16.2 18.1 9.7 15.5 259 106
18.2 12.4 14.5 305
20.2 11.8 17.4 17.7 353 119
22.2 11.0 12.6 382
24.2 10.8 6.5 26.6 404 178
26.2 10.9 6.8 442
28.2 10.4 10.3 31.5 463 216
30.2 10.2 13.1 30.4 493 213
32.2 10.1 8.1 28.2 512 196
34.2 10.2 3.5 33.4 530 223
36.2 10.1 5.8 548 .
38.2 9.8 5.1 36.1 512 233
21
CA 02624764 2008-11-13
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: E.I. DU PONT DE NEMOURS AND COMPANY
GENENCOR INTERNATIONAL, INC.
(ii) TITLE OF INVENTION: METHOD FOR THE
PRODUCTION OF
GLYCEROL BY RECOMBINANT
ORGANISMS
(iii) NUMBER OF SEQUENCES: 25
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: E. I. DU PONT DE NEMOURS AND COMPANY
(B) STREET: 1007 MARKET STREET
(C) CITY: WILMINGTON
(D) STATE: DELAWARE
(E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 19898
(A) ADDRESSEE: GENENCOR INTERNATIONAL, INC.
(B) STREET: 4 CAMBRIDGE PLACE
1870 SOUTH WINTON ROAD
(C) CITY: ROCHESTER
(D) STATE: NEW YORK
(E) COUNTRY: U.S.A.
(F) POSTAL CODE (ZIP): 14618
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: DISKETTE, 3.5 INCH
(B) COMPUTER: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: MICROSOFT WINDOWS 95
(D) SOFTWARE: MICROSOFT WORD VERSION 7.0A
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,624,764
(B) FILING DATE: NOVEMBER 10, 1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
00 APPLICATION NUMBER: US 60/030,602
(B) FILING DATE: NOVEMBER 13, 1996
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: ALLEGRO, LISA
(B) REFERENCE/DOCKET NUMBER: 34711-3083
(ix) TELECOMMUNICATIONS INFORMATION:
(A) TELEPHONE: 416-865-0040
(B) TELEFAX: 416-865-7380
22
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VM) 98/21340 PCT/US97/20293
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1380 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CTTTAATTTT CTTTTATCTT ACTCTCCTAC ATAAGACATC AAGAAACAAT TGTATATTGT 60
ACACCCCCCC CCTCCACAAA CACAAATATT GATAATATAA AGATGTCTGC TGCTGCTGAT 120
AGATTAAACT TAACTTCCGG CCACTTGAAT GCTGGTAGAA AGAGAAGTTC CTCTTCTGTT 180
TCTTTGAAGG CTGCCGAAAA GCCTTTCAAG GTTACTGTGA TTGGATCTGG TAACTGGGGT 240
ACTACTATTG CCAAGGTGGT TGCCGAAAAT TGTAAGGGAT ACCCAGAAGT TTTCGCTCCA 300
ATAGTACAAA TGTGGGTGTT CGAAGAAGAG ATCAATGGTG AAAAATTGAC TGAAATCATA 360
AATACTAGAC ATCAAAACGT GAAATACTTG CCTGGCATCA CTCTACCCGA CAATTTGGTT 420
GCTAATCCAG ACTTGATTGA TTCAGTCAAG GATGTCGACA TCATCGTTTT CAACATTCCA 480
CATCAATTTT TGCCCCGTAT CTGTAGCCAA TTGAAAGGTC ATGTTGATTC ACACGTCAGA 540
GCTATCTCCT GTCTAAAGGG TTTTGAAGTT GGTGCTAAAG GTGTCCAATT GCTATCCTCT 600 .
TACATCACTG AGGAACTAGG TATTCAATGT GGTGCTCTAT CTGGTGCTAA CATTGCCACC 660
GAAGTCGCTC AAGAACACTG GTCTGAAACA ACAGTTGCTT ACCACATTCC AAAGGATTTC 720
AGAGGCGAGG GCAAGGACGT CGACCATAAG GTTCTAAAGG CCTTGTTCCA CAGACCTTAC 780
TTCCACGTTA GTGTCATCGA AGATGTTGCT GGTATCTCCA TCTGTGGTGC TTTGAAGAAC 840
GTTGTTGCCT TAGGTTGTGG TTTCGTCGAA GGTCTAGGCT GGGGTAACAA CGCTTCTGCT 900
GCCATCCAAA GAGTCGGTTT GGGTGAGATC ATCAGATTCG GTCAAATGTT TTTCCCAGAA 960
TCTAGAGAAG AAACATACTA CCAAGAGTCT GCTGGTGTTG CTGATTTGAT CACCACCTGC 1020
GCTGGTGGTA GAAACGTCAA GGTTGCTAGG CTAATGGCTA CTTCTGGTAA GGACGCCTGG 1080
:
GAATGTGAAA AGGAGTTGTT GAATGGCCAA TCCGCTCAAG GTTTAATTAC CTGCAAAGAA 1140
GTTCACGAAT GGTTGGAAAC ATGTGGCTCT GTCGAAGACT TCCCATTATT TGAAGCCGTA 1200
TACCAAATCG TTTACAACAA CTACCCAATG AAGAACCTGC CGGACATGAT TGAAGAATTA 1260
GATCTACATG AAGATTAGAT TTATTGGAGA AAGATAACAT ATCATACTTC CCCCACTTTT 1320
TTCGAGGCTC TTCTATATCA TATTCATAAA TTAGCATTAT GTCATTTCTC ATAACTACTT 1380
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PCI7US97/20293
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2946 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID 11O:2:
GAATTCGAGC CTGAAGTGCT GATTACCTTC AGGTAGACTT CATCTTGACC CATCAACCCC 60
AGCGTCAATC CTGCAAATAC ACCACCCAGC AGCACTAGGA TGATAGAGAT AATATAGTAC .120
GTGGTAACGC TTGCCTCATC ACCTACGCTA TGGCCGGAAT CGGCAACATC CCTAGAATTG 180
AGTACGTGTG ATCCGGATAA CAACGGCAGT GAATATATCT TCGGTATCGT AAAGATGTGA 240
TATAAGATGA TGTATACCCA ATGAGGAGCG CCTGATCGTG ACCTAGACCT TAGTGGCAAA 300
AACGACATAT CTATTATAGT GGGGAGAGTT TCGTGCAAAT AACAGACGCA GCAGCAAGTA 360
ACTGTGACGA TATCAACTCT TTTTTTATTA TGTAATAAGC AAACAAGCAC GAATGGGGAA 420
AGCCTATGTG CAATCACCAA GGTCGTCCCT TTTTTCCCAT TTGCTAATTT AGAATTTAAA 480
GAAACCAAAA GAATGAAGAA AGAAAACAAA TACTAGCCCT AACCCTGACT TCGTTTCTAT 540
GATAATACCC TGCTTTAATG AACGGTATGC CCTAGGGTAT ATCTCACTCT GTACGTTACA 600
AACTCCGGTT ATTTTATCGG AACATCCGAG CACCCGCGCC TTCCTCAACC CAGGCACCGC 660
CCCAGGTAAC CGTGCGCGAT GAGCTAATCC TGAGCCATCA CCCACCCCAC CCGTTGATGA 720
CAGCAATTCG GGAGGGCGAA AATAAAACTG GAGCAAGGAA TTACCATCAC CGTCACCATC 780
ACCATCATAT CGCCTTAGCC TCTAGCCATA GCCATCATGC AAGCGTGTAT CTTCTAAGAT 840
TCAGTCATCA TCATTACCGA GTTTGTTTTC CTTCACATGA TGAAGAAGGT TTGAGTATGC 900
. .
TCGAAACAATAAGACGACGA TGGCTCTGCC ATTGGTTATA TTACGCTTTT GCGGCGAGGT 960
GCCGATGGGT TGCTGAGGGG AAGAGTGTTT AGCTTACGGA CCTATTGCCA TTGTTATTCC 1020
GATTAATCTA TTGTTCAGCA GCTCTTCTCT ACCCTGTCAT TCTAGTATTT TTTTTTTTTT 1080 '
TTTTTGGTTT TACTTTTTTT TCTTCTTGCC TTTTTTTCTT GTTACTTTTT TTCTAGTTTT 1140
TTTTCCTTCC ACTAAGCTTT TTCCTTGATT TATCCTTGGG TTCTTCTTTC TACTCCTTTA 1200
. .
GATTTTTTTT TTATATATTA ATTTTTAAGT TTATGTATTT TGGTAGATTC AATTCTCTTT 1260
CCCTTTCCTT TTCCTTCGCT CCCCTTCCTT ATCAATGCTT GCTGTCAGAA GATTAACAAG 1320
. .
ATACACATTC CTTAAGCGAA CGCATCCGGT GTTATATACT CGTCGTGCAT ATAAAATTTT 1380
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GCCTTCAAGA TCTACTTTCC TAAGAAGATC ATTATTACAA ACACAACTGC ACTCAAAGAT 1440
GACTGCTCAT ACTAATATCA AACAGCACAA ACACTGTCAT GAGGACCATC CTATCAGAAG 1500
ATCGGACTCT GCCGTGTCAA TTGTACATTT GAAACGTGCG CCCTTCAAGG TTACAGTGAT 1560
TGGTTCTGGT AACTGGGGGA CCACCATCGC CAAAGTCATT GCGGAAAACA CAGAATTGCA 1620
TTCCCATATC TTCGAGCCAG AGGTGAGAAT GTGGGTTTTT GATGAAAAGA TCGGCGACGA 1680
AAATCTGACG GATATCATAA ATACAAGACA CCAGAACGTT AAATATCTAC CCAATATTGA 1740
CCTGCCCCAT AATCTAGTGG CCGATCCTGA TCTTTTACAC TCCATCAAGG GTGCTGACAT 1800
CCTTGTTTTC AACATCCCTC ATCAATTTTT ACCAAACATA GTCAAACAAT TGCAAGGCCA 1860
CGTGGCCCCT CATGTAAGGG CCATCTCGTG TCTAAAAGGG TTCGAGTTGG GCTCCAAGGG 1920
TGTGCAATTG CTATCCTCCT ATGTTACTGA TGAGTTAGGA ATCCAATGTG GCGCACTATC 1980
TGGTGCAAAC TTGGCACCGG AAGTGGCCAA GGAGCATTGG TCCGAAACCA CCGTGGCTTA 2040
CCAACTACCA AAGGATTATC AAGGTGATGG CAAGGATGTA GATCATAAGA TTTTGAAATT 2100
GCTGTTCCAC AGACCTTACT TCCACGTCAA TGTCATCGAT GATGTTGCTG GTATATCCAT 2160
TGCCGGTGCC TTGAAGAACG TCGTGGCACT TGCATGTGGT TTCGTAGAAG GTATGGGATG 2220
GGGTAACAAT GCCTCCGCAG CCATTCAAAG GCTGGGTTTA GGTGAAATTA tCAAGTTCGG 2280
TAGAATGTTT TTCCCAGAAT CCAAAGTCGA GACCTACTAT CAAGAATCCG CTGGTGTTGC 2340
AGATCTGATC ACCACCTGCT CAGGCGGTAG AAACGTCAAG GTTGCCACAT ACATGGCCAA 2400
GACCGGTAAG TCAGCCTTGG AAGCAGAAAA GGAATTGCTT AACGGTCAAT CCGCCCAAGG 2460
GATAATCACA TGCAGAGAAG TTCACGAGTG GCTACAAACA TGTGAGTTGA CCCAAGAATT 2520
CCCAATTATT CGAGGCAGTC TACCAGATAG TCTACAACAA CGTCCGCATGCAAGACCTAC 2580
CGGAGATGAT TGAAGAGCTA GACATCGATG ACGAATAGAC ACTCTCCCCC CCCCTCCCCC 2640
TCTGATCTTT CCTGTTGCCT CTTTTTCCCC CAACCAATTT ATCATTATAC ACAAGTTCTA 2700
CAACTACTAC TAGTAACATT.ACTACAGTTA TTATAATTTT CTATTCTCTT TTTCTTTAAG 2760
AATCTATCAT TAACGTTAAT TTCTATATAT ACATAACTAC CATTATACAC GCTATTATCG 9820
.TTTACATATC ACATCACCGT TAATGAAAGA TACGACACCC TGTACACTAA CACAATTAAA 2380
,TAATCGCCAT AACCTTTTCT CTTATCTATA GCCCTTAAAG CTGTTTCTTC GAGCTTTTCA 2940 =
=
CTGCAG 2946'
(2) INFORMATION FOR SEQ ID NO:3:
(1) SEQUENCE CHARACTERISTICS:
. (A) LENGTH: 3178 base pairs
CA 02624764 2008-04-04
WO 98/21340
PC171097/20293
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CTGCAGAACT TCGTCTGCTC TGTGCCCATC CTCGCGGTTA GAAAGAAGCT GAATTGTTTC 60
ATGCGCAAGG GCATCAGCGA GTGACCAATA ATCACTGCAC TAATTCCTTT TTAGCAACAC 120
ATACTTATAT ACAGCACCAG ACCTTATGTC TTTTCTCTGC TCCGATACGT TATCCCACCC 180
AACTTTTATT TCAGTTTTGG CAGGGGAAAT TTCACAACCC CGCACGCTAA AAATCGTATT 240
TAAACTTAAA AGAGAACAGC CACAAATAGG GAACTTTGGT CTAAACGAAG GACTCTCCCT 300
CCCTTATCTT GACCGTGCTA TTGCCATCAC TGCTACAAGA CTAAATACGT ACTAATATAT 360
GTTTTCGGTA ACGAGAAGAA GAGCTGCCGG TGCAGCTGCT GCCATGGCCA CAGCCACGGG 420
GACGCTGTAC TGGATGACTA GCCAAGGTGA TAGGCCGTTA GTGCACAATG ACCCGAGCTA 480
CATGGTGCAA TTCCCCACCG CCGCTCCACC GGCAGGTCTC TAGACGAGAC CTGCTGGACC 540
GTCTGGACAA GACGCATCAA TTCGACGTGT TGATCATCGG TGGCGGGGCC ACGGGGACAG 600
GATGTGCCCT AGATGCTGCG, ACCAGGGGAC TCAATGTGGC CCTTGTTGAA AAGGGGGATT 660
TTGCCTCGGG AACGTCGTCC AAATCTACCA AGATGATTCA CGGTGGGGTG CGGTACTTAG 720
AGAAGGCCTT CTGGGAGTTC TCCAAGGCAC AACTGGATCT GGTCATCGAG GCACTCAACG 780
AGCGTAAACA TCTTATCAAC ACTGCCCCTC ACCTGTGCAC GGTGCTACCA ATTCTGATCC 840
CCATCTACAG CACCTGGCAG GTCCCGTACA TCTATATGGG CTGTAAATTC TACGATTTCT 900
TTGGCGGTTC CCAAAACTTG AAAAAATCAT ACCTACTGTC CAAATCCGCC ACCGTGGAGA 960
AGGCTCCCAT GCTTACCACA GACAATTTAA AGGCCTCGCT TGTGTACCAT GATGGGTCCT 1020
TTAACGACTC GCGTTTGAAC GCCACTTTAG CCATCACGGG TGTGGAGAAC GGCGCTACCG 1080
TCTTGATCTA TGTCGAGGTA CAAAAATTGA TCAAAGACCC AACTTCTGGT AAGGTTATCG 1140
GTGCCGAGGC CCGGGACGTT GAGACTAATG AGCTTGTCAG AATCAACGCT AAATGTGTGG 1200
TCAATGCCAC GGGCCCATAC AGTGACGCCA TTTTGCAAAT GGACCGCAAC CCATCCGGTC 1260
TGCCGGACTC CCCGCTAAAC GACAACTCCA AGATCAAGTC GACTTTCAAT CAAATCTCCG 1320
TCATGGACCC GAAAATGGTC ATCCCATCTA TTGGCGTTCA CATCGTATTG CCCTCTTTTT 1380
ACTCCCCGAA GGATATGGGT TTGTTGGACG TCAGAACCTC TGATGGCAGA GTGATGTTCT 1440
TTTTACCTTG GCAGGGCAAA GTCCTTGCCG GCACCACAGA CATCCCACTA AAGCAAGTCC 1500
26
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VA) 98/21340 PCT/US97/20293
CAGAAAACCC TATGCCTACA GAGGCTGATA TTCAAGATAT CTTGAAAGAA CTACAGCACT 1560 1 '
ATATCGAATT CCCCGTGAAA AGAGAAGACG TGCTAAGTGC ATGGGCTGGT GTCAGACCTT 1620
TGGTCAGAGA TCCACGTACA ATCCCCGCAG ACGGGAAGAA GGGCTCTGCC ACTCAGGGCG 1680
TGGTAAGATC CCACTTCTTG TTCACTTCGG ATAATGGCCT AATTACTATT GCAGGTGGTA 1740
AATGGACTAC TTACAGACAA ATGGCTGAGG AAACAGTCGA CAAAGTTGTC GAAGTTGGCG 1800
GATTCCACAA CCTGAAACCT TGTCACACAA GAGATATTAA GCTTGCTGGT GCAGAAGAAT 1860
GGACGCAAAA CTATGTGGCT TTATTGGCTC AAAACTACCA TTTATCATCA AAAATGTtCA 1920
ACTACTTGGT TCAAAACTAC GGAACCCGTT CCTCTATCAT TTGCGAATTT TTCAAAGAAT 1980
CCATGGAAAA TAAACTGCCT TTGTCCTTAG CCGACAAGGA AAATAACGTA ATCTACTCTA 2040
GCGAGGAGAA CAACTTGGTC AATTTTGATA CTTTCAGATA TCCATTCACA ATCGGTGAGT" 2100
TAAAGTATTC CATGCAGTAC GAATATTGTA GAACTCCCTT GGACTTCCTT TTAAGAAGAA 2160 -
CAAGATTCGC CTTCTTGGAC GCCAAGGAAG CTTTGAATGC CGTGCATGCC ACCGTCAAAG 2220
TTATGGGTGA TGAGTTCAAT TGGTCGGAGA AAAAGAGGCA GTGGGAACTT GAAAAAACTG 2280 .
TGAACTTCAT CCAAGGACGT TTCGGTGTCT AAATCGATCA TGATAGTTAA GGGTGACAAA 2340
GATAACATTC ACAAGAGTAA TAATAATGGT AATGATCATA ATAATAATAA TGATAGTAAT 2400.
AACAATAATA ATAATGGTGG TAATGGCAAT GAAATCGCTA TTATTACCTA TTTTCCTTAA 2460
TGGAAGAGTT AAAGTAAACT AAAAAAACTA CAAAAATATA TGAAGAAAAA AAAAAAAAGA 2520
GGTAATAGAC TCTACTACTA CAATTGATCT TCAAATTATG ACCTTCCTAG TGTTTATATT 2580
CTATTTCCAA TACATAATAT AATCTATATA ATCATTGCTG GTAGACTTCC GTTTTAATAT 2640
CGTTTTAATT ATCCCCTTTA TCTCTAGTCT AGTTTTATCA TAAAATATAG AAACACTAAA 2700
TAATATTCTT CAAACGGTCC TGGTGCATAC GCAATACATA TTTATGGTGC AAAAAAAAAA 2760
ATGGAAAATT TTGCTAGTCA TAAACCCTTT CATAAAACAA TACGTAGACA TCGCTACTTG 2820
AAATTTTCAA GTTTTTATCA GATCCATGTT TCCTATCTGC CTTGACAACC TCATCGTCGA 2880'
AATAGTACCA TTTAGAACGC CCAATATTCA CATTGTGTTC AAGGTCTTTA TTCACCAGTG 2940 '
ACGTGTAATG GCCATGATTA ATGTGCCTGT ATGGTTAACC ACTCCAAATA GCTTATATTT 3000
_
CATAGTGTCA TTGTTTTTCA ATATAATGTT TAGTATCAAT GGATATGTTA CGACGGTGTT 3060
ATTTTTCTTG GTCAAATCGT AATAAAATCT CGATAAATGG ATGACTAAGA 7TTTTGGTAA 3120
AGTTACAAAA TTTATCGTTT TCACTGTTCT CAATTTTTTG TTCTTGTAAT CACTCGAG 3178
= .
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(2) INFORMATION FOR SEQ ID NO:4:
.(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 816 base pairs
õ
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
ATGAAACGTT TCAATGTTTT AAAATATATC AGAACAACAA AAGCAAATAT ACAAACCATC 60
GCAATGCCTT TGACCACAAA ACCTTTATCT TTGAAAATCA ACGCCGCTCT ATTCGATGTT 120
GACGGTACCA TCATCATCTC TCAACCAGCC ATTGCTGCTT TCTGGAGAGA TTTCGGTAAA 180
GACAAGCCTT ACTTCGATGC CGAACACGTT ATTCACATCT CTCACGGTTG GAGAACTTAC 240
GATGCCATTG CCAAGTTCGC TCCAGACTTT GCTGATGAAG AATACGTTAA CAAGCTAGAA 300
GGTGAAATCC CAGAAAAGTA CGGTGAACAC TCCATCGAAG TTCCAGGTGC TGTCAAGTTG 360
TGTAATGCTT TGAACGCCTT GCCAAAGGAA AAATGGGCTG TCGCCACCTC TGGTACCCGT 420.
GACATGGCCA AGAAATGGTT CGACATTTTG AAGATCAAGA GACCAGAATA CTTCATCACC 480
GCCAATGATG TCAAGCAAGG TAAGCCTCAC CCAGAACCAT ACTTAAAGGG TAGAAACGGT 540
TTGGGTTTCC CAATTAATGA ACAAGACCCA TCCAAATCTA AGGTTGTTGT CTTTGAAGAC 600
GCACCAGCTG GTATTGCTGC TGGTAAGGCT GCTGGCTGTA AAATCGTTGG TATTGCTACC 660
ACTTTCGATT TGGACTTCTT GAAGGAAAAG GGTTGTGACA TCATTGTCAA GAACCACGAA 720
TCTATCAGAG TCGGTGAATA CAACGCTGAA ACCGATGAAG TCGAATTGAT CTTTGATGAC 780
TACTTATACG CTAAGGATGA CTTGTTGAAA TGGTAA 816
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 753 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:'
ATGGGATTGA CTACTAAACC TCTATCTTTG AAAGTTAACG CCGCTTTGTT CGACGTCGAC 60
GGTACCATTA TCATCTCTCA ACCAGCCATT GCTGCATTCT GGAGGGATTT CGGTAAGGAC 120
AAACCTTATT TCGATGCTGA ACACGTTATC CAAGTCTCGC ATGGTTGGAG AACGTTTGAT 180
28
=
=
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GCCATTGCTA AGTTCGCTCC AGACTTTGCC AATGAAGAGT ATGTTAACAA ATTAGAAGCT 240
GAAATTCCGG TCAAGTACGG TGAAAAATCC ATTGAAGTCC CAGGTGCAGT TAAGCTGTGC 30,0
AACGCTTTGA ACGCTCTACC AAAAGAGAAA TGGGCTGTGG CAACTTCCGG TACCCGTGAT 360
ATGGCACAAA AATGGTTCGA GCATCTGGGA ATCAGGAGAC CAAAGTACTT CATTACCGCT 420
AATGATGTCA AACAGGGTAA GCCTCATCCA GAACCATATC TGAAGGGCAG GAATGGCTTA 480
GGATATCCGA TCAATGAGCA AGACCCTTCC AAATCTAAGG TAGTAGTATT TGAAGACGCT 540
CCAGCAGGTA TTGCCGCCGG,AAAAGCCGCC GGTTGTAAGA TCATTGGTAT TGCCACTACT 600
TTCGACTTGG ACTTCCTAAA GGAAAAAGGC TGTGACATCA TTGTCAAAAA CCACGAATCC 660
ATCAGAGTTG GCGGCTACAA TGCCGAAACA GACGAAGTTG AATTCATTTT TGACGACTAC 720
TTATATGCTA AGGACGATCT GTTGAAATGG TAA 753
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2520 base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TGTATTGGCC ACGATAACCA CCCTTTGTAT ACTGTTTTTG TTTTTCACAT GGTAAATAAC 60
GACTTTTATT AAACAACGTA TGTAAAAACA TAACAAGAAT CTACCCATAC AGGCCATTTC 120
GTAATTCTTC TCTTCTAATT GGAGTAAAAC CATCAATTAA AGGGTGTGGA GTAGCATAGT 180
GAGGGGCTGA CTGCATTGAC AAAAAAATTG AAAAAAAAAA AGGAAAAGGA AAGGAAAAAA 240
AGACAGCCAA GACTTTTAGA ACGGATAAGG TGTAATAAAA TGTGGGGGGA TGCCTGTTCT 300
CGAACCATAT AAAATATACC ATGTGGTTTG AGTTGTGGCC GGAACTATAC AAATAGTTAT 360
ATGTTTCCCT CTCTCTTCCG ACTTGTAGTA TTCTCCAAAC GTTACATATT CCGATCAAGC 420
CAGCGCCTTT ACACTAGTTT AAAACAAGAA CAGAGCCGTA TGTCCAAAAT AATGGAAGAT 480
TTACGAAGTG ACTACGTCCC GCTTATCGCC AGTATTGATG TAGGAACGAC CTCATCCAGA 540
TGCATTCTGT TCAACAGATG GGGCCAGGAC GTTTCAAAAC ACCAAATTGA ATATTCAACT 600
TCAGCATCGA AGGGCAAGAT TGGGGTGTCT GGCCTAAGGA GACCCTCTAC AGCCCCAGCT 660
CGTGAAACAC CAAACGCCGG.TGACATCAAA ACCAGCGGAA AGCCCATCTT TTCTGCAGAA 720
GGCTATGCCA TTCAAGAAAC CAAATTCCTA AAAATCGAGG AATTGGACTT GGACTTCCAT 780
,9
õ
CA 02624764 2008-04-04
WO 98/21340 PCT/US97/20293
AACGAACCCA CGTTGAAGTT CCCCAAACCG GGTTGGGTTG AGTGCCATCC GCAGAAATTA 840 '
CTGGTGAACG TCGTCCAATG CCTTGCCTCA AGTTTGCTCT CTCTGCAGAC TATCAACAGC 900
=
GAACGTGTAG CAAACGGTCT CCCACCTTAC AAGGTAATAT'GCATGGGTAT AGCAAACATG 960
AGAGAAACCA CAATTCTGTG GTCCCGCCGC ACAGGAAAAC CAATTGTTAA CTACGGTATT 1020
GTTTGGAACG ACACCAGAAC GATCAAAATC GTTAGAGACA AATGGCAAAA CACTAGCGTC 1080
, GATAGGCAAC TGCAGCTTAG ACAGAAGACT GGATTGCCAT TGCTCTCCAC GTATTTCTCC 1140
TGTTCCAAGC TGCGCTGGTT CCTCGACAAT GAGCCTCTGT GTACCAAGGC GTATGAGGAG 1200
AACGACCTGA TGTTCGGCAC TGTGGACAGA TGGCTGATTT ACCAATTAAC TAAACAAAAG 1260
GCGTTCGTTT CTGACGTAAC CAACGCTTCC AGAACTGGAT TTATGAACCT CTCCACTTTA 1320
AAGTACGACA ACGAGTTGCT GGAATTTTGG GGTATTGACA AGAACCTGAT TCACATGCCC 1380
GAAATTGTGT CCTCATCTCA ATACTACGGT GACTTTGGCA TTCCTGATTG GATAATGGAA 1440
AAGCTACACG ATTCGCCAAA AACAGTACTG CGAGATCTAG TCAAGAGAAA CCTGCCCATA 1500
CAGGGCTGTC TGGGCGACCA AAGCGCATCC ATGGTGGGGC AACTCGCTTA CAAACCCGGT 1560
GCTGCAAAAT GTACTTATGG TACCGGTTGC TTTTTACTGT ACAATACGGG GACCAAAAAA 1620
TTGATCTCCC AACATGGCGC ACTGACGACT CTAGCATTTT GGTTCCCACA TTTGCAAGAG 1680
TACGGTGGCC AAAAACCAGA ATTGAGCAAG CCACATTTTG CATTAGAGGG TTCCGTCGCT 1740
GTGGCTGGTG CTGTGGTCCA ATGGCTACGT GATAATTTAC GATTGATCGA TAAATCAGAG 1800
GATGTCGGAC CGATTGCATC TACGGTTCCT GATTCTGGTG GCGTAGTTTT CdTCCCCGCA 1860
TTTAGTGGCC TATTCGCTCC CTATTGGGAC CCAGATGCCA GAGCCACCAT AATGGGGATG 1920
TCTCAATTCA CTACTGCCTC CCACATCGCC AGAGCTGCCG TGGAAGGTGT TTGCTTTCAA 1980
GCCAGGGCTA TCTTGAAGGC AATGAGTTCT GACGCGTTTG GTGAAGGTTC CAAAGACAGG 2040
GACTTTTTAG AGGAAATTTC CGACGTCACA TATGAAAAGT CGCCCCTGTC GdTTCTGGCA 2100
GTGGATGGCG GGATGTCGAG GTCTAATGAA GTCATGCAAA TTCAAGCCGA TATCCTAGGT 2160
'
CCCTGTGTCA AAGTCAGAAG GTCTCCGACA GCGGAATGTA CCGCATTGGG GGCAGCCATT 2220
=
GCAGCCAATA TGGCTTTCAA GGATGTGAAC GAGCGCCCAT TATGGAAGGA CCTACACtAT 2260
GTTAAGAAAT GGGTCTTTTA CAATGGAATG GAGAAAAACG AACAAATATC ACCAGAGGCT 2340
, CATCCAAACC TTAAGATATT CAGAAGTGAA TCCGACGATG CTGAAAGGAG AAAGCATTGG 2400
AAGTATTGGG AAGTTGCCGT GGAAAGATCC AAAGGTTGGC TGAAGGACAT AGAAGGTGAA 2460 4
CACGAACAGG TTCTAGAAAA CTTCCAATAA CAACATAAAT AATTTCTATT AACAATGTAA 2520
CA 02624764 2008-04-04
VVC) 98/21340 PCl/US97/20293
(2) INFORMATION FOR SEQ ID NO:?:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 391 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:?:
Met Ser Ala Ala Ala Asp Arg Leu Asn Leu Thr Ser Gly His Leu Asn
1 5 10 15
Ala Gly Arg Lys Arg Ser Ser Ser Ser Val Ser Leu Lys Ala Ala Glu
=
20 25 30
Lys,Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr Thr
35 40 45
Ile Ala Lys Vol Val Ala Glu Asn Cys Lys Gly Tyr Pro Glu Val Phe
50 55 60
Ala Pro Ile Val Gin Met Trp Val Phe Glu Glu Glu Ile Asn Gly Glu
65 70 75 80
Lys Leu Thr Glu Ile Ile Asn Thr Arg His Gin Asn Vol Lys Tyr Leu
85 90 95
Pro Gly Ile Thr Leu Pro Asp Asn Leu Val Ala Asn Pro Asp Leu Ile
100 105 110
Asp Ser Val Lys Asp Val Asp Ile Ile Val Phe Asn Ile Pro His Gin
115 120 125
Phe Leu Pro Arg Ile Cys Ser Gin Leu Lys Gly. His Val Asp Ser His
130 135 140
Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Val Gly Ala Lys Gly
145 150 155 160
. Val Gin Leu Leu Ser Ser Tyr Ile Thr Glu Glu Leu Gly Ile Gin Cys
165 170 175
Gly Ala Leu Ser Gly Ala Asn Ile Ala Thr Glu Val Ala Gin Glu His
180 185 190 '
Trp Ser Glu Thr Thr Val Ala Tyr His Ile Pro Lys /kap Phe Arg Gly '
195 200 205
Glu Gly Lys Asp Val Asp His Lys Vol Lou Lys Ala Leu Phe His Arg . .
.
210 215 22.0
, Pro Tyr Phe His Val Ser Val Ile Glu Asp Val Ala Gly Ile Ser Ile
225 230 235 240
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Cys Gly Ala Leu Lys Asn Val Val Ala Lou Gly Cys Gly Phe Vol Glu
245 250 255
Gly Leu Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gin Arg Val Gly
260 265 270
Leu Gly Glu Ile Ile Arg Phe Gly Gin Met Phe Phe Pro Glu Ser Arg
275 280 285
Glu Glu Thr Tyr Tyr Gin Glu Ser Ala Gly Vol Ala Asp Lou Ile Thr
290 295 300
Thr Cys Ala Gly Gly Arg Asn Val Lys Val Ala Arg Leu Met Ala Thr
305 310 315 320
Ser Gly Lys Asp Ala Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gin
325 330 335
Ser Ala Gin Gly Leu Ile Thr Cys Lys Glu Val His Glu Trp Leu Glu
340 345 350
Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Vol Tyr Gin
355 360 365
Ile Vol Tyr Asn Asn Tyr Pro Met Lys Asn Leu Pro Aso Met Ile Glu
370 375 380
Glu Leu Asp Lou His Glu Asp
385 390
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Thr Ala His Thr Asn Ile Lys Gin His Lys His Cys His Glu Asp
1 5 10 15
His Pro Ile Arg Arg Ser Asp Ser Ala Val Ser Ile Val His Leu Lys
20 25 30
Arg Ala Pro Phe Lys Vol Thr Vol Ile Gly Ser Gly Asn Trp Gly Thr
35 40 45
Thr Ile Ala Lys Vol Ile Ala Glu Asn Thr Glu Leu His Ser His Ile
50 55 60
Phe Glu Pro Glu Vol Arq Met Trp Val Phe Asp Glu Lys Ile Gly Asp
65 70 75 60
3?
:
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Glu Asn Leu Thr Asp Ile Ile Asn Thr Arg His Gin Asn Val Lys Tyr
85 90 95
Leu Pro Asn Ile Asp Leu Pre His Asn Leu Vol Ala Asp Pro Asp Leu
100 105 110
Leu His Ser Ile Lys Gly Ala Asp Ile Leu Val Phe Asn Ile Pro His
115 120 125
Gin Phe Leu Pro Asn Ile Val Lys Gin Leu Gin Gly His Val Ala Pro
130 135 140
His Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Leu Gly Ser Lys
145 150 155 160
Gly Val Gin Leu Leu Ser Ser Tyr Val Thr Asp Glu Leu Gly Ile Gin
165 170 175
Cys Gly Ala Leu Ser Gly Ala Asn Leu Ala Pro Glu Vol Ala Lys Glu
180 185 190
His Trp Ser Giu Thr Thr Vol Ala Tyr Gin Leu Pro Lys Asp Tyr Gin
195 200 205
Gly Asp Gly Lys Asp Val Asp His Lys Ile Leu Lys Leu Leu Phe His
210 215 220
Arg Pro Tyr Phe His Val Asn Val Ile Asp Asp Val Ala Gly Ile Ser
225 230 235 240
Ile Ala Gly Ala Leu Lys Asn Val Val Ala Leu Ala Cys Gly Phe Val
245 250 255
Glu Gly Met Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gin Arg Leu
260 265 270
Gly Leu Gly Glu Ile Ile Lys Phe Gly Arg Met Phe Phe Pro Glu Ser.
275 280 285
Lys Val Glu Thr Tyr Tyr Gin Glu Ser Ala Gly Val Ala Asp Leu Ile
290 295 300
Thr Thr Cys Ser Gly Gly Arg Asn Val Lys Val Ala Thr Tyr Met Ala
305 310 315 320
Lys Thr Gly Lys Ser Ala Leu Glu Ala Glu Lys Glu LOU =Leu Asn Gly
325 330 335
Gin Ser Ala Gin Gly Ile Ile-Tht Cys Arg Glu Val His Glu Trp Leu
340 345 350
Gin Thr Cys Glq Leu Thr Gin Glu Phe Pro Ile Ile Arg Gly Ser Lou
355 360 365
=
. Pro Asp Ser Leu Gin Gln Arg Pro His Gly Arg Pro Thr Gly Asp Asp
370 375 380
33
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;2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 614 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Thr Arg Ala Thr Trp Cys Asn Ser Pro Pro Pro Leu His Arg Gin
1 5 10 15
Val Ser Arg Arg Asp Leu Leu Asp Arg Leu Asp Lys Thr His Gin Phe
20 25 30
Asp Val Leu Ile Ile Gly Gly Gly Ala Thr Gly Thr Gly Cys Ala Leu
35 40 45
Asp Ala Ala Thr Arg Gly Leu Asn Val Ala Leu Val Glu Lys Gly Asp
50 55 60
Phe Ala Ser Gly Thr Ser Ser Lys Ser Thr Lys Met Ile His Giy Gly
65 70 75 80
Val Arg Tyr Leu Glu Lys Ala Phe Trp Glu Phe Ser Lys Ala Gin Leu
85 90 95
Asp Leu Val Ile Glu Ala Leu Asn Glu Arg Lys His Leu Ile Asn Thr
100 105 110
Ala Pro His Leu Cys Thr Val Leu Pro Ile Leu Ile Pro Ile Tyr Ser
115 120 125
Thr Trp Gin Val Pro Tyr Ile Tyr Met Gly Cys Lys Phe Tyr Asp Phe
130 135 140
Phe Gly Gly Ser Gin Asn Leu Lys Lys Ser Tyr Leu Leu Ser Lys Ser
145 150 155 160
Ala Thr Val Glu Lys Ala Pro Met Leu Thr Thr Asp Asn Leu Lys Ala
165 170 175
Ser Leu Val Tyr His Asp Gly Ser Phe Asn Asp Ser Arg Leu Asn Ala
180 185 190
Thr Leu Ala Ile Thr Gly Val Glu Asn Gly Ala Thr Val Leu Ile Tyr
195 200 205
Val Glu Val Gin Lys Leu Ile Lys Asp Pro Thr Sr Gly Lys Val Ile
210 215 220
Gly Ala Glu Ala Arg Asp Val Glu Thr Asn Glu Leu Val Arg Ile Asn
225 230 235 240
34
_ .
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PCT/US97/20293
Ala Lys Cys Val Val Asn Ala Thr Gly Pro Tyr Ser Asp Ala Ile Leu
245 250 . 255. = .
Gin Met Asp Arg Asn Pro Ser Gly Leu Pro Asp Ser Pro Leu Asn Asp
260 265 270
Asn Ser Lys Ile Lys Ser Thr Phe Asn Gin Ile Ser Val Met Asp Pro
275 280 285
Lys Met Val Ile Pro Ser Ile Gly Val His Ile Val Leu Pro Ser Phe
290 295 300
Tyr Ser Pro Lys Asp Met Gly Leu Leu Asp Val Arg Thr Ser Asp Gly
305 310 315 320
Arg Val Met Phe Phe Leu Pro Trp Gin Gly Lys Val Leu Ala Gly Thr
325 330 335
Thr Asp Ile Pro Leu Lys Gin Val Pro Glu Asn Pro Met Pro Thr Glu
340 345 350
Ala Asp Ile Gin Asp Ile Leu Lys Glu Leu Gin His Tyr Ile Glu Phe
355 360 365
Pro Val Lys Arg Glu Asp Val Leu Ser Ala Trp Ala Gly Val Arg Pro
370 375 380
Leu Val Arg Asp Pro Arg Thr Ile Pro Ala Asp Gly Lys Lys Gly Ser
385 390 395 400
Ala Thr Gin Gly Val Val Arg Ser His Phe Leu Phe Thr Ser Asp Asn
405 410 415
Gly Leu Ile Thr Ile Ala Gly Gly Lys Trp Thr Thr Tyr Arg Gin Met
420 425 430
Ala Glu Glu Thr Val Asp Lys Val Val Glu Val Gly Gly Phe His Asn
435 440 445
Leu Lys Pro Cys His Thr Arg Asp Ile Lys Leu Ala Gly Ala Glu Glu
450 455 460
Trp Thr Gln Asn Tyr Val Ala Leu Leu Ala Gin Asn Tyr His Leu Ser
465 470 475 480
Ser Lys Met Ser Asn Tyr Leu Val Gin Asn Tyr Gly Thr Arg Ser Ser
485 490 495
Ile Ile Cys Glu Phe Phe Lys Glu Ser Met Glu Asn Lys Leu Pro Leu
500 505 510
Ser Leu Ala Asp Lys Glu Asn Asn Val Ile Tyr Ser Ser Glu Glu Asn
515 520 525
Asn Leu Val Asn Phe Asp Thr Phe Arg Tyr Pro Phe Thr Ile Gly Glu
530 535 540
_ .
CA 02624764 2008-04-04
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PCT/US97/20293
Leu Lys Tyr Ser Met Gln Tyr Glu Tyr Cys Arg Thr Pro Leu Asp Phe
545 550 555 560
Leu Leu Arg Arg Thr Arg Phe Ala Phe Leu Asp Ala Lys Glu Ala Leu
565 570 575
Asn Ala Val His Ala Thr Val Lys Val Met Gly Asp Glu Phe Asn Trp
580 585 590
Ser Glu Lys Lys Arg Gln Trp Glu Leu Glu Lys Thr Val Asn Phe Ile
595 600 . 605
Gln Gly Arg Phe Gly Val
610
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 339 amino acids
(B) TYPE: amino acid
(C) STRANDEDEESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE
DESCRIPTION: SEQ ID NO:10:
Met Asn Gln Arg Asn Ala Ser Met Thr Val Ile Gly Ala Gly Ser Tyr
1 5 10 15
Gly Thr Ala Leu Ala Ile Thr Leu Ala Arg Asn Gly His Glu Val Val
20 25 30
Leu Trp Gly His Asp Pro Glu His Ile Ala Thr Leu Glu Arg Asp Arg
35 40 45
. .
Cys Asn Ala Ala Phe Leu Pro Asp Val Pro Phe Pro Asp Thr Leu His
50 55 60
Leu Glu Ser Asp Leu Ala Thr Ala Leu Ala Ala Ser Arg Asn Ile Leu
65 70 75 80
Val Val Val Pro Ser His Val Phe Gly Glu Val Leu Arg Gln Ile Lys
= 85 90 95
Pro Leu Met Arg Pro Asp Ala Arg Leu Val Trp Ala Thr Lys Gly Leu
100 105 110
Glu Ala Glu Thr Gly Arg Leu Leu Gln Asp Val Ala Arg Glu Ala Leu
115 120 125
Gly Asp Gln Ile Pro Leu Ala Val Ile Ser Gly Pro Thr Phe Ala Lys
130 135 140
Glu Leu Ala Ala Gly Leu Pro Thr Ala Ile Ser Leu Ala Ser Thr Asp
145 150 155 160
36
.
.
. .
CA 02624764 2008-04-04
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Gin Thr Phe Ala Asp Asp Leu Gin Gin Leu Leu His Cys Gly Lys Ser
165 170 175
Phe Arg Val Tyr Ser Asn Pro Asp Phe Ile Gly Val Gin Leu Gly Gly
180 185 190
Ala Val Lys Asn Val Ile Ala Ile Gly Ala Gly Met Ser Asp Gly Ile
195 200 205
= Gly Phe Gly Ala Asn Ala Arg Thr Ala Leu Ile Thr Arg Gly Leu Ala
210 215 220
Glu Met Ser Arg Leu Gly Ala Ala Leu Gly Ala Asp Pro Ala Thr Phe
225 230 235 240
Met Gly Met Ala Gly Leu Gly Asp Leu Val Leu Thr Cys Thr Asp Asn
245 250 255
Gin Ser Arg Asn Arg Arg Phe Gly Met Met Leu Gly Gin Gly Met Asp
260 265 270
Val Gin Ser Ala Gin Glu Lys Ile Gly Gin Val Val Glu Gly Tyr Arg
275 280 285
Asn Thr Lys Glu Val Arg Glu Leu Ala His Arg Phe Gly Val Glu Met.
290 295 300
Pro Ile Thr Glu Glu Ile Tyr Gin Val Leu Tyr Cys Gly Lys Ash Ala
305 310 315 320
Arg Glu Ala Ala Leu Thr Leu Leu Gly Arg Ala Arg Lys Asp Glu Arg
325 330 335
Ser Ser His
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Glu Thr Lys Asp Leu Ile Val Ile Gly Gly Gly Ile Asn Gly Ala
1 5 10 15
Gly Ile Ala Ala Asp Ala Ala Gly Arg Gly Leu ger Val Leu Met Leu
20 25 30
= Glu Ala Gin Asp Leu Ala Cys Ala Thr Ser Ser Ala Ser Ser Lys Leu
35 40 45
Ile His Gly Gly Leu Arg Tyr Leu Glu His Tyr Glu Phe Arg Leu Val
50 55 60
37
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PC171A97/20293
.
.
Ser Glu Ala Leu Ala Glu Arg Glu Val Leu Leu Lys Met Ala Pro His
65 70 75 80
Ile Ala Phe Pro Met Arg Phe Arg Leu Pro His Arg Pro His Leu Arg
85 90 95
Pro Ala Trp Met Ile Arg Ile Gly Leu Phe Met Tyr Asp His Leu Gly
100 105 110
Lys Arg Thr Ser Leu Pro Gly Ser Thr Gly Leu Arg Phe Gly Ala Asn
115 120 125
Ser Val Leu Lys Pro Glu Ile Lys Arg Gly Phe Glu Tyr Ser. Asp Cys
130 135 140
Trp Val Asp Asp Ala Arg Leu Val Leu Ala Asn Ala Gin Met Val Val
145 150 155 160
Arg Lys Gly Gly Glu Val Leu Thr Arg Thr Arg Ala Thr Ser Ala Arg
165 170 175
Arg Glu Asn Gly Lou Trp Ile Val Glu Ala Glu Asp Ile Asp Thr Gly
180 185 190
Lys Lys Tyr Ser Trp Gin Ala Arg Gly Leu Val Asn Ala Thr Gly Pro
195 200 205
Trp Val Lys Gin Phe Phe Asp Asp Gly Met His Leu Pro Ser Pro Tyr
210 215 220
Gly Ile Arg Leu Ile Lys Gly Ser His Ile Val Val Pro Arg Val His
225 230 235 240
Thr Gin Lys Gin Ala Tyr Ile Leu Gin Asn Glu Asp Lys Arg Ile Val
245 250 255
Phe Val Ile Pro Trp Met Asp Glu Phe Ser Ile Ile Gly Thr Thr Asp
260 265 270
Val Glu Tyr Lys Gly Asp Pro Lys Ala Val Lys Ile Glu Glu Ser Glu
275 280 285
Ile Asn Tyr Leu Leu Asn Val Tyr Asn Thr His Phe Lys Lys Gin' Lou
290 295 300
Ser Arg Asp Asp Ile Val Trp Thr Tyr Ser Gly Val Arg Pro Leu Cys
.305 310 315 320
Asp Asp Glu Ser Asp Ser Pro Gin Ala Ile Thr Arg Asp Tyr Thr Leu
325 330 335
Asp Ile His Asp Glu Asn Gly Lys Ala Pro Lou Leu Ser Val Phe Gly
340 345 350
=
Gly Lys Lou Thr Thr Tyr Arg Lys Leu Ala Glu His Ala Leu G1u Lys
355 360 365
38
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PCI1US97/20293
Leu Thr Pro Tyr Tyr Gin Gly Ile Gly Pro Ala Trp Thr Lys Glu Ser
=
370 375 380
Val Leu Pro Gly Gly Ala Ile Glu Gly Asp Arg Asp Asp Tyr Ala Ala
385 390 . = 395 400
= Arg Leu Arg Arg Arg Tyr Pro Phe Leu Thr Glu Ser Leu Ala Arg His
405 410 415
Tyr Ala Arg Thr Tyr Gly Ser Asn Ser Glu Lou Lou Leu Gly Asn Ala
=
420 425 430
Gly Thr Val Ser Asp Leu Gly Glu Asp Phe Gly His Glu Phe Tyr Glu
435 440 445
Ala Glu Leu Lys Tyr Leu Vol Asp His Glu Trp Vol Arg Arg Ala Asp
450 455 460
Asp Ala Leu Trp Arg Arg Thr Lys Gin Gly Met Trp Leu Asn Ala Asp
465 470 475 480
Gin Gln Ser Arg Val Ser Gin Trp Leu Val Glu Tyr Thr Gin Gin Arg
485 490 495
Leu Ser Leu Ala Ser
500
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 542 amino acids
(3) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Lys Thr Arg Asp Ser Gin Ser Ser Asp Val Ile Ile Ile Gly Gly
1 5 10 15
Gly Ala Thr Gly Ala Gly Ile Ala Arg Asp Cys Ala Leu Arg Gly Leu
20 25 30
Arg Val Ile Leu Val Glu Arg His Asp Ile Ala Thr Gly Ala Thr Gly
35 40 45
Arg Asn His Gly Leu Lou His Ser Gly Ala Arg Tyr Ala Vol Thr Asp
50 55 60
Ala Glu Ser Ala Arg Glu Cys Ile Ser Glu Asn Gin Ile Lou Lys Arg
65 70 75 80 -
Ile Ala Arg His Cys Val Glu Pro Thr Asn Gly Leu Phe Ile Thr Leu
85 90 95
39
. . .
.
'
CA 02624764 2008-04-04
VM 911121340 PCMS97/20293
Pro Glu Asp Asp Leu Ser Phe Gin Ala Thr Phe Ile Arg Ala Cys Glu
100 105 110
.Glu Ala Gly Ile Ser Ala Glu Ala Ile Asp Pro Gin Gin Ala Arg Ile
115 120 125
Ile Glu Pro Ala Val Asn Pro Ala Lou Ile Gly Ala Val Lys Vol Pro
130 135 140
Asp Gly Thr Vol Asp Pro Phe Arg Lou Thr Ala Ala Asn Met Leu Asp
145 150 155 160
Ala Lys Giu His Gly Ala Val Ile Lou Thr Ala His Glu Val Thr Gly
165 170 175
Leu Ile Arg Glu Gly Ala Thr Val Cys Gly Val Arg Val Arg Asn His
180 185 190
Leu Thr Gly Glu Thr Gin Ala Lou His Ala Pro Val Val Val Asn Ala
195 200 205
Ala Gly Ile Trp Gly Gin His Ile Ala Glu Tyr Ala Asp Leu Arg. Ile
210 215 220
Arg Met Phe Pro Ala Lys Gly Ser Leu Leu Ile Met Asp His Arg Ile
225 230 235 240
Asn Gin His Val Ile Asn Arg Cys Arg Lys Pro Ser Asp Ala Asp Ile
245 250 255
Leu Val Pro Gly Asp Thr Ile Ser Leu Ile Gly Thr Thr Ser Leu Arg
260 265 270
Ile Asp Tyr Asn Glu Ile Asp Asp Asn Arg Val Thr Ala Glu Glu Val
275 280 285
Asp Ile Leu Leu Arg Glu Gly Glu Lys Leu Ala Pro Val Met Ala Lys
290 295 300
Thr Arg Ile Leu Arg Ala Tyr Ser Gly Val Arg Pro Leu Val Ala Ser
305 310 315 320
Asp Asp Asp Pro Ser Gly Arg Asn Lou Ser Arg Gly Ile Val Leu Leu
325 330 335
Asp His Ala Glu Arg Asp Gly Leu Asp Gly Phe Ile Thr Ile Thr Gly
340 345 350
Gly Lys Leu Net Thr Tyr Arg Leu Met Ala Glu Trp Ala Thr Asp Ala
355 360 365
Val Cys Arg Lys Leu Gly Asn Thr Arg Pro Cys Thr Thr Ala Asp Leu
370 375 380
Ala Leu Pro Gly Ser Gin Glu Pro Ala Glu Val Thr Leu Arg Lys Val
385 390 395 400
CA 02624764 2008-04-04
WO 98/21340 PC17/US97a0293
Ile Ser Leu Pro Ala Pro Leu Arg Sly Ser Ala Val Tyr Arg His Gly
405 410 415
Asp Arg Thr Pro Ala Trp Leu Ser Glu Gly Arg Lou His Arg Ser Leu
420 425 430
Val Cys Glu Cys Glu Ala Val Thr Ala Gly Glu Val Gin Tyr Ala Val
435 440 445
Glu Asn Leu Asn Val Asn Ser Leu Leu Asp Leu Arg Arg Arg Thr Arg
=
450 455 460
Val Gly Met Gly Thr Cys Gin Gly Glu Leu Cys Ala Cys Arg Ala Ala
465 470 475 480
Gly Leu Leu Gin Arg Phe Asn Val Thr Thr Ser Ala Gin Ser Ile Glu
485 490 495
Gin Leu Ser Thr Phe Leu Asn Glu Arg Trp Lys Gly Val Gin Pro Ile
500 505 510
Ala Trp Gly Asp Ala Leu Arg Glu Ser Glu Phe Thr Arg Trp Val Tyr
515 520 325
Gin Gly Leu Cys Gly Leu Glu Lys Glu Gin Lys Asp Ala Leu
530 535 540
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 250 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Gly Leu Thr Thr Lys Pro Leu Ser Leu Lys Val Asn Ala Ala Leu
1 5 10 15
Phe Asp Val Asp Gly Thr Ile Ile Ile Ser Gin Pro Ala Ile Ala Ala
20 . 25 30
Phe Trp Arg Asp Phe Gly Lys Asp Lys Pro Tyr Phe Asp Ala Glu His
35 40 45
Val Ile Gin Val Ser His Gly Trp Arg Thr Phe Asp Ala Ile Ala Lys
50 55 60
Phe Ala Pro Asp Phe Ala Asn Glu Glu Tyr Val Asn Lys Lou Glu Ala
65 70 75 80
Glu Ile Pro Val Lys Tyr Gly Glu Lys Ser Ile Glu Val Pro Gly Ala
85 90 95
41
õ -
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PCUUS97/20293
Val Lys Leu Cys Asn Ala Leu Asn Ala Leu Pro Lys Glu Lys Trp Ala
100 105 110
Val Ala Thr Ser Gly Thr Arg Asp Met Ala Gin Lys Trp Phe Glu His
115 120 125
Leu Gly Ile Arg Arg Pro Lys Tyr Phe Ile Thr Ala Asn Asp Val Lys
130 135 140
Gin Gly Lys Pro His Pro Glu Pro Tyr Leu Lys Gly Arg Asn Gly Leu
145 150 155 160
Gly Tyr Pro Ile Asn Glu Gin Asp Pro Ser Lys Ser Lys Val Val Val
165 170 175
Phe Glu Asp Ala Pro Ala Gly Ile Ala Ala Gly Lys Ala Ala Gly Cys
180 165 190
Lys Ile Ile Gly Ile Ala Thr Thr Phe Asp Leu Asp Phe Leu Lys Glu
195 200 205
Lys Gly Cys Asp Ile Ile Vol Lys Aan His Glu Ser Ile Arg Val'Cly
210 215 220
Gly Tyr Asn Ala Glu Thr Asp Glu Val Glu Phe Ile Phe Asp Asp Tyr
225 230 235 240
Leu Tyr Ala Lys Asp Asp Leu Leu Lys Trp
245 250
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Lys Arg Phe Asn Val Leu Lys Tyr Ile Arg Thr Thr Lys Ala Asn
1 1 5 10 15
Ile Gin Thr Ile Ala Met Fro Leu Thr Thr Lys Pro Leu Ser Leu Lys
20 25 30
Ile Asn Ala Ala Leu Phe Asp Val Asp Gly Thr Ile Ile Ile Ser Gin
35 40 45
Pro Ala Ile Ala Ala Phe Trp Arg Asp Phe Gly Lys Asp Lys Pro Tyr
50 55 60
Phe Asp Ala Glu His Val Ile His Ile Ser His Gly Trp Arg Thr Tyr
65 70 75 BO
42
_
CA 02624764 2008-04-04
=
WO 98/21340 ¨ PCTIUS9/a0293
Asp. Ala Ile Ala Lys Phe Ala Pro Asp Phe Ala Asp Glu Glu Tyr Val
85 90 95
Asn Lys Leu Glu Gly Glu Ile Pro Glu Lys Tyr Gly Glu His Ser Ile
100 105 110
= Glu Val Pro Gly Ala Val Lys Leu Cys Asn Ala Leu Asn Ala Leu Pro
115 120 125
= Lys Glu Lys Trp Ala Val Ala Thr Ser Gly Thr Arg Asp Met Ala Lys
130 135 140
Lys Trp Phe Asp Ile Leu Lys Ile Lys Arg Pro Glu Tyr Phe Ile Thr
145 150 155 160
Ala Asn Asp Val Lys Gin Gly Lys Pro His Pro Glu Pro Tyr Leu Lys
165 170 175
Gly Arg Asn Gly Leu Gly Phe Pro Ile Asn Glu Gin Asp Pro Ser Lys
180 185 190
Ser Lys Val Val Val Phe Glu Asp Ala Pro Ala Gly Ile Ala Ala Gly
195 200 205
Lys Ala Ala Gly Cys Lys Ile Val Gly Ile Ala Thr Thr Phe Asp Leu
210 215 220
Asp Phe Leu Lys Glu Lys Gly Cys Asp Ile Ile Val Lys Asn His Glu
225 230 235 240
Ser Ile Arg Val Gly Glu Tyr Asn Ala Glu Thr Asp Glu Val Glu Leu
245 250 255
Ile Phe Asp Asp Tyr Leu Tyr Ala Lys Asp Asp Leu Leu Lys Trp
260 265 270
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 709 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
. Met Phe Pro Ser Leu Phe Arg Leu Val Val Phe Ser Lys Arg Tyr Ile
1 5 10 15
Phe Arg Ser Ser Gin Arg Leu Tyr Thr Ser Leu Lys Gin Glu Gin Ser
20 25 30
Arg Met Ser Lys Ile Met Glu Asp Leu Arg Ser Asp Tyr Val Pro Leu
35 40 45
43
=
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Ile Ala Ser Ile Asp Val Giy Thr Thr Ser Ser Arg Cys Ile Leu Phe
50 55 60
Asn Arg Trp Gly Gin Asp Val Ser Lys His Gin Ile Glu Tyr Ser Thr
65 70 75 80
Ser Ala Ser Lys Gly Lys Ile Gly Val Ser Gly Lou Arg Arg Pro Ser
85 90 95
Thr Ala Pro Ala Arg Glu Thr Pro Asn Ala Gly Asp Ile Lys Thr Ser
100 105 110
Gly Lys Pro Ile Phe Ser Ala Glu Gly Tyr Ala Ile Gin Glu Thr Lys
115 120 125
Phe Leu Lys Ile Glu Glu Leu Asp Leu Asp Phe His Asn Glu Pro Thr
130 135 140
Leu Lys Phe Pro Lys Pro Gly Trp Val Glu Cys His Pro Gin Lys Lou
145 150 155 160
Leu Val Asn Val Val Gin Cys Lou Ala Ser Ser Leu Leu Set Lou Gin
165 170 175
Thr Ile Asn Ser Glu Arg Val Ala Asn Gly Leu Pro Pro Tyr Lys Val
180 185 190
Ile Cys Met Gly Ile Ala Asn Met Arg Glu Thr Thr Ile Leu Trp Ser
195 200 205
Arg Arg Thr Gly Lys Pro Ile Val Asn Tyr Gly Ile Val Trp Asn Asp
210 215 220
Thr Arg Thr Ile Lys Ile Val Arg Asp Lys Trp Gin Asn Thr Ser Val
225 230 235 240
Asp Arg Gin Leu Gin Leu Arg Gin Lys Thr Gly Leu Pro Leu Lou Ser
245 250 255
Thr Tyr Phe Ser Cys Ser Lys Leu Arg Trp Phe Leu Asp Asn Glu Pro
260 265 270
Lou Cys Thr Lys Ala Tyr Glu Glu Asn Asp Leu Met Phe Gly Thr Val
275 280 285
Asp Thr Trp Leu Ile Tyr Gin Leu Thr Lys Gin Lys Ala Phe Val Ser
290 295 300
Asp Val Thr Asn Ala Ser Arg Thr Gly Phe Met Asn Lou Ser Thr Leu
305 310 315 320
Lys Tyr Asp Asn Glu Leu Lou Glu Phe Trp Glv Ile Asp Lys Asn Leu
325 330 335
Ile His Met Pro Glu Ile Val Ser Ser Ser Gin Tyr Tyr Gly Asp Phe
340 345 350
44
CA 02624764 2008-04-04
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Gly Ile Pro Asp Trp Ile Met Glu Lys Leu His Asp Ser Pro Lys Thr
355 360 365
Val Leu Arg Asp Leu Val Lys Arg Asn Leu Pro Ile Gin Gly Cys Leu
370 375 380
Gly Asp Gin Ser Ala Ser Met Val Gly Gin Leu Ala Tyr Lys Pro Gly
385 390 395 400
=
Ala Ala Lys Cys Thr Tyr Gly Thr Gly Cys Phe Leu Leu Tyr Asn Thr
405 410 415
Gly Thr Lys Lys Leu Ile Ser Gln His Gly Ala Leu Thr Thr Leu Ala
420 425 430
Phe Trp Phe Pro His Leu Gin Glu Tyr Gly Gly Gin Lys Pro Glu Leu
435 440 445
Ser Lys Pro His Phe Ala Leu Glu Gly Ser Val Ala Val Ala Gly Ala
450 455 460
Val Val Gin Trp Leu Arq Asp Asn Leu Arg Leu Ile Asp Lys Ser Glu
465 470 475 480
Asp Val Gly Pro Ile Ala Ser Thr Val Pro Asp Ser Gly Gly Val Val
485 490 495
Phe Val Pro Ala Phe Ser Gly Leu Phe Ala Pro Tyr Trp Asp Pro Asp
500 505 510
Ala Arg Ala Thr Ile Met Gly Met Ser Gin Phe Thr Thr Ala Ser His
515 520 525
Ile Ala Arg Ala Ala Val Glu Gly Val Cys Phe Gin Ala Arg Ala Ile
530 535 540
Leu Lys Ala Met Ser Ser Asp Ala Phe Gly Glu Gly Ser Lys Asp Arg
545 550 555 560
Asp Phe Leu Glu Glu Ile Ser Asp Val Thr Tyr Glu Lys Ser Pro Leu
565 570 575
Ser Val Leu Ala Val Asp Gly Gly Met Ser Arg Ser Asn Glu Val Met
580 585 590
Gin Ile Gin Ala Asp Ile Leu Gly Pro Cys Val Lys Val Arg Arg Ser
595 600 605
Pro Thr Ala Glu Cys Thr Ala Leu Gly Ala Ala Ile Ala Ala Asn Met
610 615 620
Ala Phe Lys Asp Val Asn Glu Arg Pro Leu Trp Lys Asp Leu His Asp .
625 630 635 640
Val Lys Lys Trp Val Phe Tyr Asn Gly Met Glu Lys Asn Glu Gin Ile
645 650 655
. _
CA 02624764 2008-04-04
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PCT/US97/20293
Ser Pro Glu Ala His Pro Asn Leu Lys Ile Phe Arg Ser Glu Ser Asp '
660 665 670
Asp Ala Glu Arg Arg Lys His Trp Lys Tyr Trp Glu Val Ala Val Glu
675 680 685
Arg Ser Lys Gly Trp Leu Lys Asp Ile Glu Gly Glu His Glu Gin Val
690 695 700
Leu Glu Asn Phe Gin
705
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GCGCGGATCC AGGAGTCTAG AATTATGGGA TTGACTACTA AACCTCTATC T 51
(2) INFORMATION FOR SEQ ID NO:17:
(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:17:
GATACGCCCG GGTTACCATT TCAACAGATC GTCCTT 36
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TTGATAATAT AACCATGGCT GCTGCTGCTG ATAG 34
46
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(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NC:19:
GTATGATATG TTATCTTGGA TCCAATAAAT CTAATCTTC 39
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs =
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CATGACTAGT AAGGAGGACA ATTC 24
(2) INFORMATION FOR SEQ ID NO':21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CATGGAATTG TCCTCCTTAC TAGT '24
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
=
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CTAGTAAGGA GGACAATTC 19
47
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(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CATGGAATTG TCCTCCTTA 19
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GATCCAGGAA ACAGA 15
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CTAGTCTGTT TCCTG 15
48
