Note: Descriptions are shown in the official language in which they were submitted.
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Description
PREPARATION OF PICHIA METHANOLICA AL'XOTROPHIC MUT~.NTS
Back round of the Invention
Methylotrophic yeasts are those yeasts that are able to utilize methanol as a
sole
source of carbon and energy. Species of yeasts that have the biochemical
pathways necessary for
methanol utilization are classified in four genera. Hansenula. Pichia.
Candida. and Torulopsis.
These genera are somewhat artificial, having been based on cell morphology and
growth
characteristics, and do not reflect close genetic relationships (Billon-Grand.
Mvcotaxon 3:201-204,
1989; Kurtzman, Mvcolo~ia 84:72-76. 1992). Furthermore. not all species within
these genera are
capable of utilizing methanol as a source of carbon and energy. As a
consequence of this
classification. there are great differences in physiology and metabolism
between individual species
of a genus.
Methylotrophic yeasts are attractive candidates for use in recombinant protein
production systems. Some methylotrophic yeasts have been shown to grow rapidly
to high biomass
on minimal defined media. Certain genes of methylotrophic veasts are tightly
regulated and highly
2 0 expressed under induced or de-repressed conditions, suggesting that
promoters of these genes might
be useful for producing polypeptides of commercial value. See, for example,
Faber et al., Yeast
11:1331. 1995; Romanos et al., Yeast 8:423, 1992; and Cregg et al.,
Bio/Technoioev 11:905, 1993.
Development of methylotrophic yeasts as hosts for use in recombinant
production
systems has been slow. due in part to a lack of suitable materials (e.g.,
promoters, selectable
markers. and mutant host cells) and methods (e.g.. transformation techniques).
The most highly
developed methylotrophic host systems utilize Pichia pastoris and Hansenula
polvmorpha (Faber et
al., Curr. Gcnet. ''x:305-310, 1994; Cregg et al., ibid.: P,omanos et al.,
ibid.-: L.S. Patent No.
4,855,242: U.S. Patent No. 4,857,467; U.S. Patent No. 4.879.231; and U.S.
Patent No. ~..929,55~).
There remains a need in the art for methods of transforming additional species
of
3 0 methylotrophic yeasts and for using the transformed cells to produce
polypeptides of economic
importance, including industrial enzymes and pharmaceutical proteins. The
present invention
provides compositions and methods useful in these processes as well as other.
related advantages.
Summary of the Invention
3 5 The present invention provides methods for preparing Pichia rnerhanolica
cells
having an auxotrophic mutation. The methods comprise the steps of (a) exposing
P. metjzanolica
cells to mutagenizing conditions; (b) culturing the cells from step (a) in a
rich medium to allow
mutations to become established and replicated in at least a portion of the
cells; ( c 1 culturing the
cells from step (b) in a culture medium deficient in assimilable nitrogen to
deplete cellular nitrogen
4 0 stores; (d) culturing the cells from step (c) in a defined culture medium
comprising an inorganic
nitrogen source and an amount of nystatin sufficient to kill crowing P.
methanolica cells to select
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for cells having a deficiency in a nutritional gene: and (e) culturing the
selected cells from step (dl
in a rich culture medium. Within one embodiment of the invention. the selected
cells from step (e1
are replica plated to a defined medium and cultured to confirm the presence of
an auxotrophic
mutation. Within another embodiment, the selected cells are auxotrophic for
adenine. Within a
related embodiment, the selected cells are deficient in phosphoribosyl-~-
aminoimidazole
carboxylase. Within additional embodiments the mutagenizing conditions
comprise exposure to
ultraviolet light or exposure to a chemical mutagen. Within a further
embodiment. the inorganic
nitrogen source comprises ammonium ions.
These and other aspects of the invention wilt become evident upon reference to
the
following detailed description and the attached drawings.
Brief Description of the Drawings
Fig. 1 illustrates the effects of field strength and pulse duration on
electroporation
efficiency of P. methanolica.
Fig. 2 is a schematic diagram of a recombination event between plasmid pCZR140
and P. metnanolica genomic DNA.
Fig. 3 is a schematic diagram of a recombination event between plasmid pCZR137
and P. methanolica genomic DNA.
2 0 Detailed Description of the Invention
Prior to setting forth the invention in more detail, it will be useful to
define certain
terms used herein:
A "DNA construct" is a DNA molecule, either single- or double-stranded. that
has
been modified through human intervention to contain segments of DNA combined
and juxtaposed
in an arrangement not existing in nature.
"Early log phase growth" is that phase of cellular growh in culture when the
cell
concentration is from ? x 106 cells/ml to 8 x 106 cells/ml.
"Heterologous DNA'' refers to a DNA molecule, or a population of DN:1
molecules.
that does not exist naturally within a given host cell. DNA molecules
heterologous to a particular
3 0 host cell may contain DNA derived from the host cell species so long as
that host DNA is combined
with non-host DNA. For example, a DNA molecule containing a non-host DNA
segment encoding
a polypeptide operably linked to a host DNA segment comprising a transcription
promoter is
considered to be a heterologous DNA molecule.
A "higher eukaryotic" organism is a multicellular eukaryotic organism. The
term
3 5 encompasses both plants and animals.
''Integrative transformants" are cells into which has been introduced
heterologous
DNA, wherein the heterologous DNA has become integrated into the genomic DNA
of the cells.
"Linear DNA" denotes DNA molecules having free ~' and ~' ends. that is non-
circular DNA molecules. Linear DNA can be prepared from closed circular DNA
molecules. such
4 0 as plasmids. by enzymatic digestion or physical disruption.
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The term "operably linked'' indicates that DNA segments are arranged so that
they
function in concert for their intended purposes, e.g., transcription initiates
in the promoter and
proceeds through the coding segment to the terminator.
The term "promoter" is used herein for its art-recognized meaning to denote a
portion
of a gene containing DNA sequences that provide for the binding of RNA
polymerase and initiation
of transcription. Promoter sequences are commonly, but not always. found in
the ~' non-coding
regions of genes. Sequence elements within promoters that function in the
initiation of transcription
are often characterized by consensus nucleotide sequences. These promoter
elements include RNA
polymerase binding sites; TATA sequences; CHAT sequences; differentiation-
specific elements
(DSEs; McGehee et al., Mol. Endocrinol. 7:51-560, 1993); cyclic AMP response
elements (CREs);
serum response elements (SREs; Treisman, Seminars in Cancer Biol. 1:47-58,
1990): glucocorticoid
response elements (GREs); and binding sites for other transcription factors,
such as CRE/ATF
(O'Reilly et al., J. Biol. Chem. 267:19938-19943, 1992), AP2 (Ye et al.. J.
Biol. Chem. 269:25728-
25734. 1994). SP1. cAMP response element binding protein (CREB: Loeken. Gene
Expr. 3:253
264, 1993) and octamer factors. See. in general. Watson et al.. eds.,
Molecular Bioloev ofthe Gene,
4th ed., The Benjamin/Cummings Publishing Company. Inc.. Menlo Park, CA, 1987:
and Lemaigre
and Rousseau, Biochem. J. 303:1-14, 1994.
A "repressing carbon source'' is a metabolizable, carbon-containing compound
that,
when not limited, suppresses the expression in an organism of genes required
for the catablism of
other carbon sources. By "limited'' is meant that the carbon source is
unavailable or becomes
available at such a rate that it is immediately consumed and therefore the
prevailing concentration of
that carbon source in an organism's environment is effectively zero.
Repressing carbon sources that
can be used within the present invention include hexoses and ethanol. Glucose
is particularly
preferred.
"Rich'' culture media are those culture media that are based on complex
sources of
nutrients, typically cell or tissue extracts or protein hydrolysates. Rich
media will vary in
composition from batch to batch due to variations in the composition of the
nutrient sources.
As noted above, the present invention provides methods for preparing Pichia
methanolica cells having an auxotrophic mutation. Auxotrophic mutants of P.
methanolica can be
3 0 transformed with both homologous DNA (DNA from the host species) and
heterologous DNA, and
the resulting transformants can be used within a large number of diverse
biological applications.
The mutant cells of the present invention are particularly well suited for
transformation with
heterologous DNA, which transformed cells can be used for the production of
polypeptides and
proteins. including polypeptides and proteins of higher organisms, including
humans. Auxotrophic
P. methanolica cells can be transformed with other DNA molecules. including
DNA libraries and
synthetic DNA molecules. The invention thus provides host cells that can be
used to express
genetically diverse libraries to produce products that are screened for novel
biological activities. can
be engineered for use as targets for the screening of compound libraries, and
can be genetically
modified to enhance their utiiitv within other processes.
4 0 Cells to be transformed with heterologous DNA will commonly have a
mutation that
can be complemented by a gene (a "selectable marker") on the heterologous DNA
molecule. This
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4
selectable marker allows the transformed cells to grow under conditions in
which untransformed
cells cannot multiply ("selective conditions"). The general principles of
selection are well known in
the art. Commonly used selectable markers are genes that encode enzymes
required for the
synthesis of amino acids or nucleotides. Cells having mutations in these genes
(auxotrophic
mutants) cannot grow in media lacking the specific amino acid or nucleotide
unless the mutation is
complemented by the selectable marker. Use of such "selective" culture media
ensures the stable
maintenance of the heterologous DNA within the host cell. A preferred
selectable marker of this
type for use in Pichia methanolica is a P. methanolica ADE2 gene, which
encodes phosphoribosyl-
~-aminoimidazole carboxylase (AIRC; EC 4.1.1.21 ). The ADE2 gene. when
transformed into an
IO ade2 host cell. allows the cell to grow in the absence of adenine. The
coding strand of a
representative P. methanolica ADE2 gene sequence is shown in SEQ ID NO:1. The
sequence
illustrated includes 1006 nucleotides of 5' non-coding sequence and 442
nucleotides of 3' non-
coding sequence, with the initiation ATG codon at nucleotides 1007-1009.
Within a preferred
embodiment of the invention. a DNA segment comprising nucleotides 407-281 is
used as a
selectable marker. although longer or shorter segments could be used as long
as the coding portion
is operable linked to promoter and terminator sequences. Those skilled in the
art will recognize that
this and other sequences provided herein represent single alleles of the
respective genes, and that
allelic variation is expected to exist. Any functional ADEZ allele can be used
within the present
invention. Other nutritional markers that can be used within the present
invention include the P.
methanolica ADEI. HISS, and LEU2 genes. which allow for selection in the
absence of adenine,
histidine. and leucine, respectively. Heterologous genes, such as genes from
other fungi, can also
be used as selectable markers. For large-scale, industrial processes where it
is desirable to minimize
the use of methanol, it is preferred to use host cells in which both methanol
utilization genes (AUGI
and AUG?l are deleted. For production of secreted proteins. host cells
deficient in vacuolar
protease genes (PEP;I and PRBI) are preferred. Gene-deficient mutants can be
prepared by known
methods, such as site-directed mutagenesis. P. methanolica genes can be cloned
on the basis of
homology with their counterpart Saccharomyces cerevisiae genes. The ADE2 gene
disclosed herein
was given its designation on the basis of such homology.
Strains of Pichia methanolica are available from the American Type Culture
3 0 Collection (Rockville, MD) and other repositories, and can be used as
starting materials within the
present invention. To prepare auxotrophic mutants of P. methanolica. cells are
first exposed to
mutagenizing conditions, i.e. environmental conditions that cause genetic
mutations in the cells.
Methods for mutagenizing cells are well known in the art and include chemical
treatment, exposure
to ultraviolet light, exposure to x-rays, and retroviral insertional
mutagenesis. Chemical mutagens
include ethylmethane sulfonate (EMS). N-methyl-N'-nitro-N-nitrosoguanidine. ~-
methoxv-6-
chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine2HCl, ~-bromouracil.
acridine. and
aflatoxin. See Lawrence. Methods Enzvmol. 194:273-281. 1991. The proportion of
mutagenized
cells obtained is a function of the strength or amount of mutagenizing agent
to which the cells are
exposed. a low level of mutagen produces a small proportion of mutant cells.
Higher levels of
4 0 mutagen produce a higher proportion of mutant cells. but also kill more
cells. It is therefore
necessaw to balance mutagenesis with killing so that a reasonable number of
mutant cells is
_~~___.. _ ___.~_._
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J
obtained. Balancing is generally done empirically by exposing cells to
different conditions to
establish a killing curve. In general, the cells are exposed to mutagenizing
conditions and cultured
for one day, after which they are tested for viability according to standard
assay methods. Within
the present invention, it is preferred to use a level of mutagenesis that
results in 20-~0°io mortality.
although one skilled in the art will recognize that this value can be adjusted
as necessary, for
example if working with a very large number of cells.
Mutagenized cells are then cultured in a rich medium to allow mutations to
become
established and replicated in at least a portion of the cell population. This
step allows cells in which
the genome has been altered to replicate the mutation and pass it on to their
progeny, thereby
establishing the mutation within the population.
The cells are then transferred to a culture medium deficient in assimilabie
nitrogen so
that cellular nitrogen stores are depleted. By ''deficient in assimilable
nitrogen" it is meant that the
medium lacks an amount of nitrogen sufficient to support growth of the cells.
Depletion of cellular
nitrogen stores will generally require about 12 to 24 hours of incubation.
with 16 hours beine
sufficient under common conditions. Following depletion of nitrogen stores.
the cells are cultured in
a defined culture medium comprising an inorganic nitrogen source and an amount
of an antifungal
antibiotic sufficient to kill growing P. methanolica cells. A preferred
antibiotic is nystatin
(mycostatin). Preferred inorganic nitrogen sources are those comprising
ammonium ions, such as
ammonium sulfate. In general, the medium will contain 10-200 mM ammonium,
preferably about
60 mM ammonium. Nystatin is included at a concentration of 0.1 to i00 mg/l,
preferably 0.5 to 20
mg/L, more preferably about 2 mg/L ( 10 units/L). Treatment with nystatin is
carried out for ten
minutes to six hours. preferably about I hour. Those skilled in the art will
recognize that the actual
antibiotic concentration and exposure time required to kill prototrophic cells
can be readily
determined empirically. and certain adjustments may be necessary to compensate
for variations in
specific activity between individual batches of antibiotic. By depleting
cellular nitrogen stores and
then culturing the cells in a defined medium containing an inorganic nitrogen
source and antibiotic.
cells that are auxotrophic for amino acid or nucleotide biosynthesis remain
alive because then
cannot grow in the defined medium. Growing cells are killed by the antibiotic.
Following the
antibiotic treatment, the cells are transferred to a rich culture medium.
3 0 Auxotrophic mutations are confirmed and characterized by determining the
nutrient
requirements of the treated cells. Replica plating is commonly used for this
determination. Cells
are plated on both rich medium and media lacking specific nutrients. Cells
that do not grow on
particularly plates are auxotrophic for the missing nutrient. Complementation
analysis can be used
for further characterization.
3 5 Heteroiogous DNA can be introduced into P. methanolica cells by any of
several
known methods, including lithium transformation (Hiep et al.. Yeast 9:1189-
1197. 1993: Tarutina
and Tolstorukov, Abst. of the 1 ~th International Specialized Symposium on
Yeasts, Riga (USSR).
1991. 137: Ito et al., J. Bacterioi. 1 X3:163. 1983: Bogdanova et al.. Yeast
11:343. 199~), spheroplast
transformation (Beggs, Nature 275:104, 1978: Hinnen et al., Proc. Natl. Acad.
Sci. USA _7:1929.
40 1978; Cregg et al., Mol. CeII. Biol. 5:3376, 1985), freeze-thaw
polyethylene glycol transformation
(Pichia Expression Kit Instruction Manual, Invitrogen Corp.. San Diego, CA.
Cat. No. K1710-01 ).
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6
or electroporation, the latter method being preferred. Electroporation is the
process of using a
pulsed electric field to transiently permeabilize cell membranes. allowing
macromolecules, such as
DNA, to pass into cells. Electroporation has been described for use with
mammalian {e.g.,
Neumann et al., EMBO J. 1:841-84~, 1982) and fungal (e.g.. Meilhoc et al.:
BioiTechnolo~v 8:223-
227, 19901 host cells. However. the actual mechanism by which DNA is
transferred into the cells is
not well understood. For transformation of P. methanolica. it has been found
that electroporation is
surprisingly efficient when the cells are exposed to an exponentially
decaying, pulsed electric field
having a field strength of from 2.5 to 4.5 kV/cm and a time constant (T) of
from 1 to 40
milliseconds. The time constant T is defined as the time required for the
initial peak voltage Vo to
drop to a value of Vp/e. The time constant can be calculated as the product of
the total resistance
and capacitance of the pulse circuit, i.e., T = R x C. Typically, resistance
and capacitance are either
preset or may be selected by the user, depending on the electroporation
equipment selected. In any
event, the equipment is configured in accordance with the manufacturer's
instructions to provide
field streneth and decay parameters as disclosed above. Electroporation
equipment is available
from commercial suppliers (e.g., BioRad Laboratories. Hercules. CA).
DNA molecules for use in transforming P. methanolica will commonly be prepared
as double-stranded, circular plasmids, which are preferably linearized prior
to transformation. For
polypeptide or protein production, the DNA molecules will include. in addition
to the selectable
marker disclosed above, an expression casette comprising a transcription
promoter. a DNA segment
(e.g., a cDNA) encoding the polypeptide or protein of interest. and a
transcription terminator. These
elements are operably linked to provide for transcription of the DNA segment
of interest. It is
preferred that the promoter and terminator be that of a P. methanohca gene.
Useful promoters
include those from constitutive and methanol-inducible promoters. Promoter
sequences are
generally contained within 1.5 kb upstream of the coding sequence of a gene,
often within 1 kb or
less. In general, regulated promoters are larger than constitutive promoters
due the presence of
regulatory elements. Methanol-inducible promoters, which include both positive
and negative
regulatory elements, may extend more than 1 kb upstream from the initiation
ATG. Promoters are
identified by function and can be cloned according to known methods.
A particularly preferred methanol-inducible promoter is that of a P.
metjZanolica
alcohol utilization gene. A representative coding strand sequence of one such
gene. AUGl. is
shown in SEQ ID N0:2. Within SEQ ID N0:2, the initiation ATG codon is at
nucleotides 13»-
1357. Nucleotides 1-23 of SEQ ID N0:2 are non-AUG1 polylinker sequence. It is
particularly
preferred to utilize as a promoter a segment comprising nucleotides 24-1354 of
SEQ ID N0:2,
although additional upstream sequence can be included. P. methanolica contains
a second alcohol
3 5 utilization gene. A UG2. the promoter of which can be used within the
present invention. A partial
DNA sequence of one AUG2 clone is shown in SEQ ID N0:9. AUG2 promoter segments
used
within the present invention will generally comprise nucleotides 91-169 of SEQ
ID N0:9, although
small truncations at the 3' end would not be expected to negate promoter
function. Other useful
promoters include those of the dihydroxyacetone synthase (DHAS), fotmate
dehvdrogenase (FMD),
4 0 and catalase (CAT) genes. Genes encoding these enzymes from other species
have been described.
and their sequences are available {e.g., Janowicz et al.. Nuc. Acids Res.
13:2043. 198: Hollenberg
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WO 98/02536 PCT/US97/12582
7
and Janowicz. EPO publication 0 299 108; Didion and Roggenkamp. FEBS Left.
303:113, 1992).
Genes encoding these proteins can be cloned by using the known sequences as
probes, or by
aligning known sequences. designing primers based on the alignment. and
amplifying P.
metha»olica DNA by the polymerise chain reaction (PCR).
Constitutive promoters are those that are not activated or inactivated by
environmental conditions; they are always transcriptionally active. Preferred
constitutive promoters
for use within the present invention include those from glyceraldehyde-3-
phosphate dehydrogenase,
triose phosphate isomerase, and phosphoglycerate kinase genes of P.
meths»olica. These genes can
be cloned by complementation in a host cell, such as a Saccharomyces
cerevisiae cell, having a
mutation in the counterpart gene. Mutants of this type are well known in the
art. See, for example,
Kawasaki and Fraenkel, Biochem. Biophvs. Res. Comm. 108:1107-1 I12, 1982;
Mchrtight et al.,
Cell 46:143-147, 1986; Aguilera and Zimmermann, Mol. Gen. Genet. 202:83-89,
1986.
The DNA molecules will further include a selectable marker to allow for
identification. selection, and maintenance of transformants. The DNA molecules
may further
contain additional elements, such an origin of replication and a selectable
marker that allow
amplification and maintenance of the DNA in an alternate host (e.g., E. coli).
To facilitate
integration of the DNA into the host chromosome, it is preferred to have the
entire expression
segment. comprising the promoter--gene of interest--terminator plus selectable
marker, flanked at
both ends by host DNA sequences. This is conveniently accomplished by
including 3' untranslated
2 0 DNA sequence at the downstream end of the expression segment and relying
on the promoter
sequence at the 5' end. When using linear DNA, the expression segment will be
flanked by
cleavage sites to allow for linearization of the molecule and separation of
the expression segment
from other sequences (e.g., a bacterial origin of replication and selectable
marker). Preferred such
cleavage sites are those that are recognized by restriction endonucleases that
cut infrequently within
a DNA sequence. such as those that recognize 8-base target sequences le.g.,
Not I).
Proteins that can be produced in P. melhanolica include proteins of industrial
and
pharmaceutical interest. Such proteins include higher eukaryotic proteins from
plants and animals.
particularly vertebrate animals such as mammals, although certain proteins
from microorganisms
are also of great value. Proteins that can be prepared using methods of the
present invention
3 o include enzymes such as lipases, cellulases, and proteases; enzyme
inhibitors, including protease
inhibitors; growth factors such as platelet derived growth factor, fibroblast
growth factors, and
epidermal growth factor; cvtokines such as erythropoietin and thrombopoietin;
and hormones such
as insulin, leptin. and glucagon .
For production of polypeptides, P. methanolica cells are cultured in a medium
3 5 comprising adequate sources of carbon, nitrogen and trace nutrients at a
temperature of about 25°C
to 35°C. Liquid cultures are provided with sufficient aeration by
conventional means, such as
shaking of small flasks or sparging of fermentors. A preferred culture medium
is ~'EPD (Table 1 ).
The cells may be passaged by dilution into fresh culture medium or stored for
short periods on
plates under refrigeration. For long-term storage. the cells are preferably
kept in a ~0% glycerol
4 0 solution at -70°C.
Table 1
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YEPD
2% D-glucose
?% BactoTM Peptone (Difco Laboratories. Detroit, MI)
I % BactoTM yeast extract (Difco Laboratories)
0.004% adenine
0.006% L-leucine
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9
Table 1. continued
ADE D
0.056% -Ade -Trp -Thr powder
0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200X tryptophan, threonine solution
ADE DS
0.056% -Ade -Trp -Thr powder
0.67% yeast nitrogen base without amino acids
l0 2% D-glucose
0.5% 200X tryptophan, threonine solution
18.22% D-sorbitol
LEU D
0.052% -Leu -Trp -Thr powder
0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200X tryptophan, threonine solution
HIS D
0.052% -His -Trp -Thr powder
2 0 0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200X tryptophan, threonine solution
URA D
0.056% -Ura -Trp -Thr powder
2 5 0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200X tryptophan, threonine solution
URA DS
0.056% -Ura -Trp -Thr powder
3 0 0.67% yeast nitrogen base without amino acids
2% D-glucose
0.5% 200X tryptophan, threonine solution
18.22% D-sorbitol
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Table I. continued
-Leu -Tm -Thr powder
powder made by combining 4.0 g adenine. 3.0 g arginine, ~.0 g aspartic acid.
'_'.0 g histidine.
6.0 g isoleucine. 4.0 g lysine. 2.0 g methionine, 6.0 g phenylalanine. ~.0 g
serine. ~.0 g
5 tyrosine. 4.0 g uracil. and 6.0 g valine (all L- amino acids)
-His -Trp -Thr powder
powder made by combining 4.0 g adenine. 3.0 g arginine. ~.0 g aspartic acid.
6.0 g
isoleucine, 8.0 g leucine, 4.0 g lysine, 2.0 g methionine. 6.0 g
phenylalanine. ~.0 g serine.
5.0 g tyrosine, 4.0 g uracil, and 6.0 g valine (all L- amino acids)
10 -Ura -Trp -Thr powder
powder made by combining 4.0 g adenine, 3.0 g arginine, 5.0 g aspartic acid.
2.0 a histidine.
6.0 g isoleucine. 8.0 g leucine, 4.0 g lysine, 2.0 g methionine, 6.0 g
phenylalanine. 5.0 g
serine, 5.0 g tyrosine, and 6.0 g valine (all L- amino acids)
-Ade -Trp -Thr powder
powder made by combining 3.0 g arginine, ~.0 g aspartic acid. 2.0 g histidine.
6.0 g
isoleucine, 8.0 g leucine, 4.0 g lysine, 2.0 g methionine. 6.0 g
phenylalanine. ~.0 g serine.
5.0 g tyrosine. 4.0 g uracil, and 6.0 g valine (all L- amino acids)
200X trvptophan, threonine solution
3.0% L-threonine, 0.8% L-tryptophan in HBO
2 0 For plates. add 1.8% BactoTM agar (Difco Laboratories)
Electroporation of P. methanolica is preferably carried out on cells in early
log phase
growth. Cells are streaked to single colonies on solid media, preferably solid
YEPD. After about ?
days of growth at 30°C, single colonies from a fresh plate are used to
inoculate the desired volume
of rich culture media ~e.g.. YEPD) to a cell density of about ~ - 10 x 10~
cellsiml. Cells are
incubated at about 2~ - 35°C, preferably 30°C, with vigorous
shaking, until they are in early log
phase. The cells are then harvested, such as by centrifugation at 3000 x g for
2-3 minutes, and
resuspended. Cells are made electrocompetent by reducing disulfide bonds in
the cell walls.
equilibrating them in an ionic solution that is compatible with the
electroporation conditions, and
3 0 chilling them. Cells are typically made electrocompetent by incubating
them in a buffered solution
at pH 6-8 containing a reducing agent. such as dithiothreitol (DTT) or (3-
mercaptoethanol (BMEI. to
reduce cell wall proteins to facilitate subsequent uptake of DNA. A preferred
incubation buffer in
this regard is a fresh solution of 50 mM potassium phosphate buffer. pH 7.5.
containing 2~ mM
DTT. The cells are incubated in this buffer (typically using one-fifth the
original culture volume ~ at
about 30°C for about ~ to 30 minutes, preferably about 1 ~ minutes. The
cells are then harvested and
washed in a suitable electroporation buffer, which is used ice-cold. Suitable
buffers in this regard
include pH 6-8 solutions containing a weak buffer, divalent canons le.g., MgT.
Ca'+) and an
osmotic stabilizer (e.g.. a sugar). After washing. the cells are resuspended
in a small volume of the
buffer, at which time they are electrocompetent and can be used directly or
aliquotted and stored
frozen (preferably at -70°Cl. A preferred electroporation buffer is STM
(270 mM sucrose, 10 mlvl
Tris, pH 7.~. I mM MgCI~). Within a preferred protocol. the cells are
subjected to two washes. first
r
__. _.__..__ _. _ ___ ._.~_ _____. ..__.___..... . ..
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
in the original culture volume of ice-cold buffer. then in one-half the
original volume. Following
the second wash, the cells are harvested and resuspended. typically using
about 3-~ ml of buffer for
an original culture volume of 200 ml.
Electroporation is carried out using a small volume of electrocompetent cells
(typically about 100 ~l) and up to one-tenth volume of linear DNA molecules.
For example, 0.1 ml
of cell suspension in a buffer not exceeding ~0 mM in ionic strength is
combined with 0.1-10 pg of
DNA (vol. _< 10 ~1). This mixture is placed in an ice-cold electroporation
cuvette and subjected to a
pulsed electric field of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm,
and a time constant of
from 1 to 40 milliseconds, preferably 10-30 milliseconds, more preferably 1 S-
25 milliseconds, most
preferably about 20 milliseconds, with exponential decay. The actual equipment
settings used to
achieve the desired pulse parameters will be determined by the equipment used.
When using a
BioRad (Hercules, CA) Gene PulserrM electroporator with a 2 mm electroporation
cuvette,
resistance is set at 600 ohms or greater, preferably "infinite" resistance.
and capacitance is set at 25
uF to obtain the desired field characteristics. After being pulsed. the cells
are diluted approximately
l OX into 1 ml of YEPD broth and incubated at 30°C for one hour.
The cells are then harvested and plated on selective media. Within a preferred
embodiment. the cells are washed once with a small volume (equal to the
diluted volume of the
electroporated cells) of 1X yeast nitrogen base (6.7 g/L yeast nitrogen base
without amino acids;
Difco Laboratories, Detroit, MI), and plated on minimal selective media. Cells
having an ade2
2 0 mutation that have been transformed with an ADE2 selectable marker can be
plated on a minimal
medium that lacks adenine, such as ADE D (Table 1 ) or ADE DS (Table 1 ). In a
typical procedure,
250 ~l aliqouts of cells are plated on 4 separate ADE D or ADE DS plates to
select for Ade+ cells.
P. methanolica recognizes certain infrequently occuring sequences. termed
autonomously replicating sequences (ARS). as origins of DNA replication, and
these sequences
may fortuitously occur within a DNA molecule used for transformation. allowing
the transforming
DNA to be maintained extrachromosomally. However. integrative transformants
are generally
preferred for use in protein production systems. Integrative transformants
have a profound growth
advantage over ARS transformants on selective media containing sorbitol as a
carbon source.
thereby providing a method for selecting integrative transformants from among
a population of
3 0 transformed cells. ARS sequences have been found to exist in the ADE2 gene
and, possibly, the
AUGI gene of P. methanolica. ade2 host cells of Pichia methanolica transformed
with an ADE2
gene can thus become Ade+ by at least two different modes. The ARS within the
ADE2 gene
allows unstable extrachromosomal maintenance of the transforming DNA (Hiep et
al., Yeast
9:1189-1197. 1993). Colonies of such transfotmants are characterized by slower
growth rates and
3 5 pink color due to prolific generation of progeny that are Ade-.
Transforming DNA can also
integrate into the host genome, giving rise to stable transformants that grow
rapidly. are white, and
that fail to give rise to detectable numbers of Ade- progeny. ADE D plates
allow the most rapid
growth of transformed cells, and unstable and stable transformants grow at
roughly the same rates.
After 3-~ days of incubation on ADE D plates at 30°C stable
transformant colonies are white and
4 o roughly twice the size of unstable, pink transfonnants. ADE DS plates are
more selective for stable
transformants, which form large (~5 mm) colonies in 5-7 days, while unstable
(ARS-maintained)
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
12
colonies are much smaller (~ 1 mm ). The more selective ADE DS media is
therefore preferred for
the identification and selection of stable transformants. For some
applications. such as the
screening of genetically diverse libraries for rare combinations of genetic
elements. it is sometimes
desirable to screen larse numbers of unstable transformants, which have been
observed to
outnumber stable transformants by a factor of roughly 100. In such cases,
those skilled in the art
will recognize the utility of plating transformant cells on less selective
media. such as ADE D.
Integrative transformants are preferred for use in protein production
processes. Such
cells can be propagated without continuous selective pressure because DNA is
rarely lost from the
genome. Integration of DNA into the host chromosome can be confirmed by
Southern blot
analysis. Briefly, transformed and untransformed host DNA is digested W th
restriction
endonucleases, separated by electrophoresis, blotted to a support membrane.
and probed with
appropriate host DNA segments. Differences in the patterns of fragments seen
in untransformed
and transformed cells are indicative of integrative transformation.
Restriction enzymes and probes
can be selected to identify transforming DNA segments (e.g., promoter.
terminator. heterologous
DNA, and selectable marker sequences) from among the genomic fragments.
Differences in expression levels of heterologous proteins can result from such
factors
as the site of integration and copy number of the expression cassette and
differences in promoter
activity among individual isolates. It is therefore advantageous to screen a
number of isolates for
expression level prior to selecting a production strain. A variety of suitable
screenins methods are
2 0 available. For example. transformant colonies are grown on plates that are
overlayed with
membranes (e.g., nitrocellulose) that bind protein. Proteins are released from
the cells by secretion
or following lysis. and bind to the membrane. Bound protein can then be
assayed using known
methods, including immunoassays. More accurate analysis of expression levels
can be obtained by
culturing cells in liquid media and analyzing conditioned media or cell
lysates, as appropriate.
Methods for concentrating and purifying proteins from media and lysates will
be determined in part
by the protein of interest. Such methods are readily selected and practiced by
the skilled
practitioner.
For small-scale protein production (e.g., plate or shake flask production), P.
methanolica transformants that carry an expression cassette comprising a
methanol-regulated
promoter (such as the AUG1 promoter) are grown in the presence of methanol and
the absence of
interfering amounts of other carbon sources (e.g., glucose). For small-scale
experiments, including
preliminary screening of expression levels, transformants may be grown at
30°C on solid media
containing, for example. 20 g/L Bacto-agar {Difco), 6.7 g/L yeast nitrogen
base without amino acids
(Difco). 10 g/L methanol, 0.4 Itg/L biotin, and 0.56 g!L of -Ade -Thr -Trp
powder. Because
3 5 methanol is a volatile carbon source it is readily lost on prolonged
incubation. A continuous supply
of methanol can be provided by placing a solution of 50% methanol in water in
the lids of inverted
plates, whereby the methanol is transferred to the growing cells by
evaporative transfer. In general.
not more than 1 mL of methanol is used per 100-mm plate. Slightly larger scale
experiments can be
carried out using cultures grown in shake flasks. In a typical procedure,
cells are cultivated for two
4 0 days on minimal methanol plates as disclosed above at 30°C, then
colonies are used to inoculate a
small volume of minimal methanol media (6.7 g/L yeast nitrogen base without
amino acids. 10 g~'L
.. _.. . _.. ._~_. ._ _._ ___. .
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
13
methanol. 0.4 ~g/L biotin] at a cell density of about 1 x 106 cells/ml. Cells
are Brown at 30°C.
Cells growing on methanol have a high oxygen requirement, necessitating
vigorous shaking during
cultivation. Methanol is replenished daily (typically 1/100 volume of ~0%
methanol per day).
For production scale culturing, fresh cultures of high producer clones are
prepared in
shake flasks. The resulting cultures are then used to inoculate culture medium
in a fermenter.
Typically, a 500 ml culture in YEPD grown at 30°C for I-2 days with
vigorous agititation is used to
inoculate a S-liter fermenter. The cells are grown in a suitable medium
containing salts, glucose,
biotin, and trace elements at 28°C, pH 5.0, and >30% dissolved 02.
After the initial charge of
glucose is consumed (as indicated by a decrease in oxygen consumption), a
glucoseirnethanol feed
is delivered into the vessel to induce production of the protein of interest.
Because large-scale
fermentation is carried out under conditions of limiting carbon, the presence
of glucose in the feed
does not repress the methanol-inducible promoter. The use of glucose in
combination with
methanol under glucose-limited conditions produces rapid growth, efficient
conversion of carbon to
biomass and rapid changes in physiological growth states, while still
providing full induction of
methanol-inducible gene promoters. In a typical fenmentation run. a cell
density of from about 80 to
about 400 grams of wet cell paste per liter is obtained. "Wet cell paste"
refers to the mass of cells
obtained by harvesting the cells from the fermentor, typically by
centrifugation of the culture.
The invention is further illustrated by the following non-limiting examples.
2 0 Examples
Example 1
P. methanolica cells (strain CBS6515 from American Type Culture Collection,
Rockville, MD) were mutagenized by UV exposure. A killing curve was first
generated by plating
cells onto several plates at approximately 200-250 cells/plate. The plates
were then exposed to UV
radiation using a G8T5 germicidal lamp (Sylvania) suspended 25 cm from the
surfaces of the plates
for periods of time as shown in Table 2. The plates were then protected from
visible light sources
and incubated at 30°C for two days.
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WO 98/02536 PCT/US97/12582
14
Table 2
Viable Cells
Time Plate Plate 2 Average
1
0 sec. 225 229 227
I sec. 200 247 223
2 sec. 176 185 181
4 sec. 149 86 118
8 sec. 20 7 14
16 sec. 0 2 1
Large-scale mutagenesis was then carried out using a 2-second UV exposure to
provide about 20% killing. Cells were plated at approximately 104 cells/plate
onto eight YEPD
plates that were supplemented with 100 mg/L each of uracil. adenine. and
leucine. which were
added to supplement the growth of potential auxotrophs having the cognate
deficiencies. Following
UV exposure the plates were wrapped in foil and incubated overnight at
30°C. The following day
the colonies on the plates 0105 total) were resuspended in water and washed
once with water. An
amount of cell suspension sufficient to give an OD600 of 0.1 - 0.2 was used to
inoculate 500 ml of
minimal broth made with yeast nitrogen base without amino acids or ammonia,
supplemented with
I% glucose and 400 ~g/L biotin. The culture was placed in a 2.8 L baffled Bell
flask and shaken
vigorously overnight at 30°C. The following day the cells had reached
an OD600 of ~1.0 - 2Ø
The cells were pelleted and resuspended in 500 ml of minimal broth
supplemented with 5 g/L
ammonium sulfate. The cell suspension was placed in a 2.8 L baffled Bell flask
and shaken
vigorously at 30°C for 6 hours. 50 ml of the culture was set aside in a
250-ml flask as a control. and
to the remainder of the culture was added 1 mg nystatin (Sigma Chemical Co.,
St. Louis, MO) to
select for auxotrophic mutants (Snow, Nature 211:206-207. 1966). The cultures
were incubated
with shaking for an additional hour. The control and nystatin-treated cells
were then harvested by
centrifugation and washed with water three times. The washed cells were
resuspended to an OD600
of 1.0 in 50% glycerol and frozen. Titering of nystatin-treated cells versus
the control cells for
colony forming units revealed that nystatin enrichment had decreased the
number of viable cells by
3 0 a factor of 104.
10-2 dilutions of nystatin-treated cells were plated on 1 ~ YEPD plates.
Colonies
were replica-plated onto minimal plates (2% agar, I x YNB. 2% glucose. 400
qg/L biotin). The
frequency of auxotrophs was about 2 - 4%. Approximately 180 auxotrophic
colonies were picked
to YEPD + Ade, Leu, Ura plates and replica-plated to various dropout plates.
All of the auxotrophs
3 5 were Ade-. Of these. 30 were noticably pink on dropout plates (LEU D. HIS
D, etc.: see Table I ).
Of the 30 pink mutants. ? I were chosen for further study: the remainder were
either leaky for
growth on ADE D plates or contaminated with wild-type cells.
The Ade- mutants were then subjected to complementation analysis and
phenotypic
testing. To determine the number of loci defined by the mutants. all 21
mutants were mated to a
4 0 single pink. Ade- tester strain (strain #2). Mating was carried out by
mixing cell suspensions
(0D600 - 1 ) and plating the mixtures in 10 u1 aliquots on YEPD plates. The
cells were then
____.___.~_~__..... _~_ .
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
replicated to SPOR media (0.5% Na acetate, 1 % KCI, 1 % glucose. 1 % agar) and
incubated
overnight at 30°C. The cells were then replica-plated to ADE D plates
for scoring of phenotype.
As shown in Table 3. some combinations of mutants failed to give Ade+ colonies
(possibly defining
the same genetic locus as in strain #2), while others gave rise to numerous
Ade+ colonies (possibly
5 defining a separate genetic locus). Because mutant #3 gave Ade+ colonies
when mated to #2,
complementation testing was repeated with mutant #3. If the group of mutants
defined two genetic
loci, then all mutants that failed to give Ade+ colonies when mated to strain
#2 should give Ade+
colonies when mated to #3. Results of the crosses are shown in Table 3.
10 Table 3
Mutant x Mutant #2 x Mutant #3
#1 + _
#3 + _
#10 + _
#15 + _
#18 + _
#24 + _
#28 + _
#30 + _
#2 _ +
#6 _ +
#8 _ +
#9 _ +
#11 - +
#I7 _ +
#19 - +
#20 _ +
Table nued
3. conti
#22 - +
#27 _ +
#4 + +
#12 + +
#16 +
As shown in Table 3. most mutants fell into one of two groups. consistent with
the
idea that there are two adenine biosynthetic genes that, when missing, result
in pink colonies on
I 5 limiting adenine media. Three colonies (#4, # 12, and # 16) may either
define a third locus or exhibit
intragenic complementation. Two intensely pigmented mutants from each of the
two
complementation groups (#3 and #10; #6 and #11 ) were selected for further
characterization.
Additional analysis indicated that Ade- was the only auxotrophy present in
these strains.
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16
A P. methanolica clone bank was constructed in the vector pRS426. a shuttle
vector
comprising 2~ and S. cerevisiae URA3 sequences. allowing it to be propagated
in S. cerevisiae.
Genomic DNA was prepared from strain CBS651 ~ according to standard
procedures. Briefly. cells
were cultured overnight in rich media, spheroplasted with zymolyase. and lysed
with SDS. DNA
was precipitated from the lysate with ethanol and extracted with a
phenol/chloroform mixture, then
precipitated with ammonium acetate and ethanol. Gel electrophoresis of the DNA
preparation
showed the presence of intact, high molecular weight DNA and appreciable
quantities of RNA. The
DNA was partially digested with Sau 3A by incubating the DNA in the presence
of a dilution series
of the enzyme. Samples of the digests were analyzed by electrophoresis to
determine the size
distribution of fragments. DNA migrating between 4 and 12 kb was cut from the
gel and extracted
from the gel slice. The size-fractionated DNA was then ligated to pRS426 that
had been digested
with Bam HI and treated with alkaline phosphatase. Aliquots of the reaction
mixture were
electroporated in E. coli MC1061 cells using a BioRad Gene PulserT'" device as
recommended by
the manufacturer.
The genomic library was used to transform S. cerevisiae strain HBY21A (ade2
ura3)
by electroporation (Becker and Guarente, Methods Enzymol. 194:182-187, 1991).
The cells were
resuspended in 1.2 M sorbitol, and six 300-~1 aliquots were plated onto ADE D,
ADE DS, URA D
and URA DS plates (Table 1). Plates were incubated at 30°C for 4-5
days. No Ade+ colonies were
recovered on the ADE D or ADE DS plates. Colonies from the URA D and URA DS
plates were
2 0 replica-plated to ADE D plates. and two closely spaced, white colonies
were obtained. These
colonies were restreaked and confirmed to be Ura~ and Adey. These two strains.
designated Adel
and Ade6, were streaked onto media containing ~ FOA (5 fluoro orotic acid;
Sikorski and Boeke,
Methods Enzymol. 194:302-318). Ura- colonies were obtained, which were found
to be Ade- upon
replica plating. These results indicate that the Ade+ complementing activity
is genetically linked to
the plasmid-borne URA3 marker. Plasmids obtained from yeast strains Adel and
Ade6 appeared to
be identical by restriction mapping as described below. These genomic clones
were designated
pADEI-1 and pADEI-6, respectively.
Total DNA was isolated from the HBY21 A transformants Ade l and Ade6 and used
to transform E. coli strain MC1061 to AmpR. DNA was prepared from 2 AmpR
colonies of Adel
and 3 AmpR colonies of Ade6. The DNA was digested with Pst I. Sca I, and Pst I
+ Sca I and
analyzed by gel electrophoresis. All five isolates produced the same
restriction pattern.
PCR primers were designed from the published sequence of the P. methanolica
ADE2 gene (also known as ADEl; Hiep et al.. Yeast 9:121-1258. 1993). Primer
9080 (SEQ ID
N0:3) was designed to prime at bases 406-429 of the ADEZ DNA (SEQ ID NO:1 ),
and primer 9079
(SEQ ID N0:4) was designed to prime at bases 282-2829. Both primers included
tails to introduce
Avr Ii and Spe I sites at each end of the amplified sequence. The predicted
size of the resulting
PCR fragment was 2450 bp.
PCR was carried out using plasmid DNA from the five putative ADE2 clones as
template DNA. The 100 ~l reaction mixtures contained 1x Taq PCR buffer
(Boehringer Mannheim.
Indianapolis. IN), 10-100 ng of plasmid DNA. 0.2~ mM dNTPs. 100 pmol of each
primer. and 1 ~I
Taq polymerase (Boehringer Mannheim). PCR was run for 30 cycles of 30 seconds
at 94°C. 60
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
17
seconds at 50°C, and 120 seconds at 72°C. Each of the five
putative ADE2 genomic clones yielded
a PCR product of the expected size (2.4 kb). Restriction mapping of the DNA
fragment from one
reaction gave the expected size fragments when digested with Bgl II or Sal I.
The positive PCR reactions were pooled and digested with Spe I. Vector pRS426
was digested with Spe I and treated with calf intestinal phosphatase. Four ~1
of PCR fragment and
1 ~l of vector DNA were combined in a 10 ~1 reaction mix using conventional
ligation conditions.
The ligated DNA was analyzed by gel electrophoresis. Spe I digests were
analyzed to identify
plasmids carrying a subclone of the ADE2 gene within pRS426. The correct
plasmid was
designated pCZR118.
1o Because the ADE2 gene in pCZR118 had been amplified by PCR, it was possible
that mutations that disabled the functional character of the gene could have
been generated. To test
for such mutations, subclones with the desired insert were transformed singly
into Saccharomyces
cerevisiae strain HBY21A. Cells were made electrocompetent and transformed
according to
standard procedures. Transformants were plated on URA D and ADE D plates.
Three phenotypic
groups were identified. Clones l, 2, 11, and 12 gave robust growth of many
transfotmants on ADE
D. The transformation frequency was comparable to the frequency of Ura+
transformants. Clones
6, 8, 10, and 14 also gave a high efficiency of transformation to both Ura+
and Ade", but the Ade+
colonies were somewhat smaller than those in the first group. Clone 3 gave
many Ura~, colonies,
but no Ade+ colonies, suggesting it carried a non-functional ade2 mutation.
Clones 1. 2, 11, and 12
2 o were pooled.
To identify the P. methanolica ade2 complementation group, two representative
mutants from each complementation group (#3 and # 10; #6 and # 11 ), which
were selected on the
basis of deep red pigmentation when grown on limiting adenine, were
transformed with the cloned
ADE gene. Two hundred ml cultures of early log phase cells were harvested by
centrifugation at
2 5 3000 x g for 3 minutes and resuspended in 20 ml of fresh KD buffer (50 mM
potassium phosphate
buffer, pH 7.5, containing 25 mM DTT). The cells were incubated in this buffer
at 30°C for 1 ~
minutes. The cells were then harvested and resuspended in 200 ml of ice-cold
STM (270 mM
sucrose, 10 mM Tris, pH 7.5, 1 mM MgCl2). The cells were harvested and
resuspended in 100 ml
of ice-cold STM. The cells were again harvested and resuspended in 3-S ml of
ice-cold STM. 100-
3 0 ~1 aliquouts of electrocompetent cells from each culture were then mixed
with Not I-digested
pADEI-1 DNA. The cell/DNA mixture was placed in a 2 mm electroporation cuvette
and subjected
to a pulsed electric field of 5 kV/cm using a BioRad Gene PulserTM set to
100052 resistance and
capacitance of 25 ~F. After being pulsed, the cells were diluted by addition
of 1 ml YEPD and
incubated at 30°C for one hour. The cells were then harvested by gentle
centrifugation and
3 5 resuspended in 400 ~l minimal selective media lacking adenine (ADE D). The
resuspended
samples were split into 200-p.l aliqouts and plated onto ADE D and ADE DS
plates. Plates were
incubated at 30°C for 4-5 days. Mutants #6 and #I1 gave Adet
transformants. No Adel
transformants were observed when DNA was omitted. hence the two isolates
appeared to define the
ade2 complementation group. The ADE2 sequence is shown in SEQ ID NO:1.
Example 2
CA 02261020 2001-09-10
09110/2eA1 23:44 4167030331 CYNTHIA J LEDGLEY PAGE 641e5
1$
The P. merhanolica clone bark disclosed in fixample 1 was used as a source for
cloning the Alcohol Utilization Gene (Ai'JGl). The clone bank was stored as
independent pools,
each representing about 200-250 individual genomic clones. 0.1 ~1 of
"miniprep" DNA from each
pool was used as a template in a polymerise chain reaction with PCR primers
(8784, SEQ ID N~:S;
8787, S'EQ Ib N0:6) that were designed from an alignment of conserved
sequences in alcohol
oxidise genes from Hansenula polymorplza, Candida boidini, and Pichia
pastoris. 'rhe
amplif cation reaction was run for 30 cycles of 94°C, 30 seconds;
50°G, 30 seconds; 72°G, 60
seconds; followed by a 7 minute incubation at 7Z°C. One pool (#5) gave
a ---500 by band. DNA
sequencing of this PCR product revealed that it encoded an amino acid sequence
with --70°I°
1 o sequence identity with the Pichfa pa$toris alcohol oxidise encoded by the
A~Xl gene and about
$S% sequence identity with the f>f'ansenula polymorpha alcohol oxidise encoded
by the M~X'1
gene. The sequence of the cloned ,,~ UG1 gene Xs sb.4wrz izx SEA 1D N02.
Sub-pools of pool #5 were analyzed by PCR using the sapae prunars used in tlm
.
iizitial amplification. One positive sub-pool was further broken down to
identify a positive colony.
This positive colony was streaked on plates, and 17NA was prepared from
individual colonies.
Thzee colonies gave identical patterns after digestion with Cla T.
Restriction mapping of the genamic clone and ~'C8 product revealed that the
AUGT
gene lay on a 7.5 kb genomic insert and chat sites within the PCR fragment
could be uniquely
identified within the genomic insert. Eecause tire oxletntatiozr of the gene
within the PCft fragment
2 o was known, the lattex infarxzxation provided the approximate location and
direction of transcription
of the ,4 U~l gene witl» ttze genvmic insert. DNA sequencing within this
region revealed a gene
with very high sequence similarity at the azx~.ino acid level to other known
alcohol oxidise genes.
Example 3
25 ade~ mutant P. metharrolica cells ate trartsfvrmed by electroporation
essentially as
disclosed above.with an expression vector comprising the AUC'rl promoter and
terrr~:i~tor, human
GAD65 Ia~T.P; ~C,arlsen et al., Proc. Nail. Acid. Sci. USA 58:8337-8341,
199T), and ADEZ
selectable marker. Colonies are patched to agar minimal methanol plates (10 to
100 colonies per
100-mm plate) containing 20 glL BactoTM-agar (»ifco), 6.7 g2, yeast nitrogen
bask without amino
3 o acids (Difco), 10 g/L, methanol, and 0.4 llg/L biotin. The agar is
overlayed with nitrocellulose, and
the plates are inverted owes lids containing 1 ml of 50% methanol in water and
incubated for 3 to 5
days at 30°C. The membrane is then transferred to a alter soaked in 0.?
M NaOI~, 0_1% SDS, 35
rzu'vf dithiothreitol to lyse the adhered cells. After 30 minutes, cell debris
is rinsed from the filter
with distilled water, and the filter is neutralir~ed by rinsing it for 30
minutes in 0.1 M acetic acid.
3 5 The filters are then assayed for adhered protein. Unoccupied binding sites
are
blocked by rinsing in TTBS NF1~1 (Z0 mM Tris ply 7.4, 0.1% Tween 20, 1&0 mlvl
NaCI, 5%
powdered nonfat milk) for 30 minutes at room temperature. The filters are then
transferred to a
solution containing G.4.D6 monoclonal antibody (Charm and Gottlieb. J.
Neurosci. 8:2123-2130,
1988), diluted 1:1000 in TTBS-NFM. 'rhe filters are incubated in the antibody
solution with gentle
40 agitation for at lest one hour, then washed with TTBS (20 mvt Tris pH 7.4,
0-1%'fween 20, lu0
m1-1 NaClj two times for five minutes each. The iiltcrs ale then incubated in
goat anti-mouse
ttrade-mark
'li:? lU/U9/~UU1 y~:~~a~ ~-~>41b'fU3U3:i1 ~i received
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
19
antibody conjugated to horseradish peroxidase {I pg/ml in TTBS-NFM) for at
least one hour, then
washed three times, ~ minutes per wash with T'TBS. The filters are then
exposed to commercially
available chemiluminescence reagents (ECLTM; Amersham Inc., Arlington Heights,
IL). Light
generated from positive patches is detected on X-ray film.
To more accurately detect the level of GAD65 expression, candidate clones are
cultured in shake flask cultures. Colonies are grown for two days on minimal
methanol plates at 30
°C as disclosed above. The colonies are used to inoculate 20 ml of
minimal methanol media (6.7
g/L yeast nitrogen base without amino acids, 10 g/L methanol, 0.4 pg/L biotin)
at a cell density of 1
x 106 cells/ml. The cultures are grown for 1-2 days at 30°C with
vigorous shaking. 0.2 ml of 50%
methanol is added to each culture daily. Cells are harvested by centrifugation
and suspended in ice-
cold lysis buffer (20 mM Tris pH 8.0, 40 mM NaCI, ? mM PMSF, 1 mM EDTA, 1
ug/ml
leupeptin, 1 p,g/ml pepstatin, 1 pg/ml aprotinin) at 10 ml final volume per 1
g cell paste. 2.5 ml of
the resulting suspension is added to 2.5 ml of 400-600 micron, ice-cold, acid-
washed glass beads in
a 15-ml vessel, and the mixture is vigorously agitated for one minute, then
incubated on ice for 1
minute. The procedure is repeated until the cells have been agitated for a
total of five minutes.
Large debris and unbroken cells are removed by centrifugation at 1000 x g for
~ minutes. The
clarified lysate is then decanted to a clean container. The cleared lysate is
diluted in sample buffer
(5% SDS, 8 M urea, 100 mM Tris pH 6.8, 10% glycerol, 2 mM EDTA, 0.01%
bromphenol glue)
and electrophoresed on a 4-20% acrylamide gradient gel (Novex, San Diego, CA).
Proteins are
2 0 blotted to nitrocellose and detected with GAD6 antibody as disclosed
above.
Clones exhibiting the highest levels of methanol-induced expression of foreign
protein in shake flask culture are more extensively analyzed under high cell
density fermentation
conditions. Cells are first cultivated in 0.5 liter of YEPD broth at
30°C for 1 - 2 days with vigorous
agitation. then used to inoculate a 5-liter fermentation apparatus {e.g.,
BioFlow III; New Brunswick
2 5 Scientific Co., Inc., Edison, NJ). The fermentation vessel is first
charged with mineral salts by the
addition of 57.8 g (NH4)2S04, 68 g KH2P04, 30.8 g MgS04~7H20. 8.6 g
CaS04~2H20, 2.0 g
NaCI, and 10 ml antifoam (PPG). H20 is added to bring the volume to 2.5 L, and
the solution is
autoclaved 40 minutes. After cooling, 350 ml of 50% glucose, 250 ml 10 X trace
elements (Table
4), 25 ml of 200 ~g/ml biotin, and 250 ml cell inoculum are added.
Table 4
10 X trace elements:
FeS04~7Hz0 100mM 27.8
g/L
CuS04~5H20 2mM 0.5 g/L
3 5 ZnCl2 8mM 1.09
g/L
MnS04~H20 8mM 1.35
giZ
CoCl~~6H20 2mM 0.48
g/L
Na~Mo04~2H~0 1mM 0.24
g/L
H3B03 8mM 0.5 g/L
KI O.Smm 0.08
g/L
biotin jmg/f,
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
thiamine 0.5 g/L
Add 1-2 mls H2S04 per liter to bring compounds into solution.
The fermentation vessel is set to run at 28°C, pH 5.0, and >30%
dissolved 02. The
5 cells will consume the initial charge of glucose, as indicated by a sharp
demand for oxygen during
glucose consumption followed by a decrease in oxygen consumption after glucose
is exhausted.
After exhaustion of the initial glucose charge, a glucose-methanol feed
supplemented with NH.~+
and trace elements is delivered into the vessel at 0.2% {w/v) glucose, 0.2%
(w/v) methanol for 5
hours followed by 0.1 % (w/v) glucose, 0.4% {w/v) methanol for 25 hours. A
total of 550 grams of
10 methanol is supplied through one port of the vessel as pure methanol using
an initial delivery rate of
12.5 ml/hr and a final rate of 25 ml/hr. Glucose is supplied through a second
port using a 700 ml
solution containing 175 grams glucose, 250 ml lOX trace elements, and 99 g
(NH4)ZS04. Under
these conditions the glucose and methanol are simultaneously utilized, with
the induction of
GAD65 expression upon commencement of the glucose-methanol feed. Cells from
the
15 fermentation vessel are analyzed for GAD65 expression as described above
for shake flask cultures.
Cells are removed from the fermentation vessel at certain time intervals and
subsequently analyzed. Little GAD65 expression is observed during growth on
glucose.
Exhaustion of glucose leads to low level expression of the GAD65 protein;
expression is enhanced
by the addition of MeOH during feeding of the fermentation culture. The
addition of methanol has
2 0 a clear stimulatory effect of the expresion of human GAD65 driven by the
methanol-responsive
A UGH promoter.
Example 4
Transformation conditions were investigated to determine the electric field
conditions. DNA topology, and DNA concentration that were optimal for
efficient transformation of
P. methanolica. All experiments used P. methanolica ade2 strain #1l. Competent
cells were
prepared as previously described. Electroporation was carried out using a
BioRad Gene PulserTM.
Three field parameters influence transformation efficiency by electroporation:
capacitance, field strength, and pulse duration. Field strength is determined
by the voltage of the
3 0 electric pulse, while the pulse duration is determined by the resistance
setting of the instrument.
Within this set of experiments, a matrix of field strength settings at various
resistances was
examined. In all experiments, the highest capacitance setting (25 ~F) of the
instrument was used.
100 ~1 aliquots of electrocompetent cells were mixed on ice with 10 Itl of DNA
that contaist~ed
approximately 1 ~g of the ADE2 plasmid pCZR133 that had been linearized with
the restricaatt
3 5 enzyme Not I. Cells and DNA were transferred to 2 mm electroporation
cuvettes (BTX Corp., ::.n
Diego, CA) and electropulsed at field strengths of 0.5 kV (2.5 kV/cm), 0.75 kV
(3.75 kV/cm}, 1.0
kV (5.0 kVlcm), 1.25 kV (6.25 kV/cm), and 1.5 kV (7.5 kV/cm). These field
strength conditions
were examined at various pulse durations. Pulse duration was manipulated by
varying the
instrument setting resistances to 200 ohms, 600 ohms, or "infinite" ohms.
Pulsed cells were
4 0 suspended in YEPD and incubated at 30°C for one hour, harvested,
resuspended, and plated. Three
separate sets of experiments were conducted. In each set, electroporation
conditions of 0.75 kV
____.__ ___._.._ _._ _._ ..__.. ___.__~......_~.__.
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
21
(3.75 kV/cm) at a resistance of "infinite" ohms was found to give a
dramatically higher
transformation efficiency than other conditions tested (see Fig. 1 ).
After the optimal pulse conditions were established, the influence of DNA
topology
on transformation efficiency was investigated. Electrocompetent cells were
mixed with 1 ug of
uncut, circular pCZR133 or with 1 ~g of Not I-digested pCZR133. In three
separate experiments,
an average of roughly 25 transformants were recovered with circular DNA while
linear DNA
yielded an average of nearly 1 x 104 transformants. These data indicate that
linear DNA transforms
P. methanolica with much greater efficiency than circular DNA.
Finally, the relationship between DNA concentration and transformation
efficiency
to was investigated. Aliquots of linear pCZR133 DNA (1 ng, 10 ng, 100 ng and 1
pg in 10 ~1 H20)
were mixed with 100 ~I electrocompetent cells, and electroporation was carried
out at 3.75 kV/cm
and "infinite" ohms. The number of transformants varied from about 10 ( 1 ng
DNA) to 104 ( 1 pg
DNA) and was found to be proportional to the DNA concentration.
Example ~
Integration of transforming DNA into the genome of P. methanolica was detected
by
comparison of DNA from wild-type cells and stable, white transformant
colonies. Two classes of
integrative transformants were identified. In the first, transforming DNA was
found to have
integrated into a homologous site. In the second class, transforming DNA was
found to have
2o replaced the endogenous AUGI open reading frame. While not wishing.to be
bound by theory, this
second transformant is believed to have arisen by a "transplacement
recombination event"
(Rothstein, Methods Enzvmol. 194:281-301, 1991) whereby the transforming DNA
replaces the
endogenous DNA via a double recombination event.
P. methanolica ade2 strain #11 was transformed to Ade+ with Asp I-digested
pCZR140, a Bluescript~ (Stratagene Cloning Systems, La 3olla, CA)-based vector
containing the P.
methanolica ADE2 gene and a mutant of AUGI in which the entire open reading
frame between the
promoter and terminator regions has been deleted (Fig. 2). Genomic DNA was
prepared from wild
type and transformant cells grown for two days on YEPD plates at 30°C.
About 100-200 ~l of cells
was suspended in 1 ml H20, then centrifuged in a microcentrifuge for 30
seconds. The cell pellet
3 o was recovered and resuspended in 400 p1 of SCE + DTT + zymolyase ( 1.2 M
sorbitol, 10 mM Na
citrate, 10 mM EDTA, 10 mM DTT, 1-2 rng/ml rymolyase 100T) and incubated at
37°C for 10-15
minutes. 400 p1 of I% SDS was added, and the solution was mixed until clear.
300 p1 of 5 M
potassium acetate, pH 8.9 was added, and the solution was mixed and
centrifuged at top speed in a
microcentrifuge for five minutes. 750 p1 of the supernatant was transferred to
a new tube and
3 5 extracted with an equal volume of phenol/chloroform. 600 ~l of the
resulting supernatant was
recovered, and DNA was precipitated by the addition of 2 volumes of ethanol
and centrifugation for
15 minutes in the cold. The DNA pellet was resuspended in 50 ml TE (10 mM Tris
pH 8. 1 mM
EDTA) + 100 ~g/mI RNAase for about 1 hour at 65°C. 10-pl DNA samples
were digested with
Eco RI (5 ~1) in a 100 p1 reaction volume at 37°C overnight. DNA was
precipitated with ethanol.
4 o recovered by centrifugation, and resuspended in 7.5 ~1 TE + 2.5 ~1 SX
loading dye. The entire I 0
ml volume was applied to one lane of a 0.7% agarose in 0.5 X TBE ( 10 X TBE is
108 g/L Tris base
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
22
7-9, 5~ g/L boric acid. 8.3 g/L disodium EDTA) gel. The gel was run at 100 V
in 0.5 X TBE
containing ethidium bromide. The gel was photographed, and DNA was
electrophoreticallv
transferred to a positively derivatized nylon membrane (Nytran~ N+, Schleicher
& Schuell, Keene,
NH) at 400 mA, 20 mV for 30 minutes. The membrane was then rinsed in 2 X SSC.
blotted onto
denaturation solution for five minutes, neutralized in 2 X SSC, then cross-
linked damp in a W
crosslinker (Stratalinker~, Stratagene Cloning Systems) on automatic setting.
The blot was
hybridized to a PCR-generated AUGI promoter probe using a commercially
available kit (ECLTM
kit, Amersham Corp., Arlington Heights, IL). Results indicated that the
transforming DNA altered
the structure of the AUGI promoter DNA, consistent with a homologous
integration event (Fig. 2).
In a second experiment, P. methanolica ade 2 strain #11 was transformed to
Ade+
with Not I-digested pCZR137, a vector containing a human GAD65 cDNA between
the AUGI
promoter and terminator (Fig. 3). Genomic DNA was prepared as described above
from wild-type
cells and a stable, white, Ade+ transformant and digested with Eco RI. The
digested DNA was
separated by electrophoresis and blotted to a membrane. The blot was probed
with a PCR-
generated probe corresponding to either the AUGI open reading frame or the
AUGI promoter.
Results demonstrated that the AUGI open reading frame DNA was absent from the
transformant
strain, and that the AUGI promoter region had undergone a significant
rearrangement. These
results are consistent with a double recombination event (transplacement)
between the transforming
DNA and the host genome (Fig. 3).
Example 6
An AUGl strain of P. methanolica is grown in high-density fermentation
conditions.
The fermentation vessel is charged with mineral salts by the addition of 57.8
g (NH4)2S04, 46.6 g
KCI, 30.8 g MgS04~7H20, 8.6 g CaS04~2H20, 2.0 g NaCI, and 10 ml antifoam
(PPG). H20 is
added to bring the volume to 2.5 L, and the solution is autoclaved 40 minutes.
After cooling. 350
ml of 50% glucose, 250 ml 10 X trace elements (Table 4), 210 ml of 30%
NaPhosphate, 25 ml 200
g,g/ml biotin, and 250 ml cell inoculum are added. Cells are batch-fed glucose
or glucose/methanol
in three phases. In phase l, the cells receive 0.4%/L/hour glucose (w/v final
fermentation volume)
for 25 hours using 750 g glucose, 110 g (NH4)2S04, and 278 ml 10 X trace
elements per 1.5 liter.
3 0 The cells are then given a transition feed of 0.2% glucose, 0.2%
methanol/L/hour for ~ hours. The
final glucose-supplemented methanol feed contains 0.1 % glucose, 0.4%
methanol/L/hr for 25 hours.
Final biomass is about 300 g/L cell paste.
CA 02261020 2001-10-16
23
Example 7
For fermentation of a P. methanolica augl d strain, the fermentation vessel is
initially charged with mineral salts, glucose, phosphate, trace elements and
biotin as disclosed in
Example 6. 250 ml of cell inoculum is added. A glucose feed is prepared using
600 g glucose, 108
g (NH4)zS04, and 273 ml 10 X trace elements per 1.2 liter. The cells are batch-
fed in three phases.
In the first phase, the cells receive glucose for 12 to 25 hours at
0.4%/L/hour. The cells are then
induced with a bolus addition of 1% methanol by weight and transitioned to
methanol utilization
with a mixed 0.2% glucose/0.1 % methanol feed for 10 hours. In the third
phase, a mixed feed of
O.a!% glucose, 0.2% methanol is delivered for 15 hours.
Example 8
P. methanolica cells in which the AUGI gene had been disrupted by insertion of
a
-"~ G~~D65 expression construct retained the ability to grow on methanol,
indicating that a second
alcohol oxidase gene was present. The second gene, designated AUG2, was
identified by PCR.
Seque~~ce analysis of the 5' coding region of the gene showed that the N-
terminus of the encoded
protein was similar to those of known alcohol oxidase genes.
Strain MC GADB, a transformant that grew very poorly on minimal methanol
broth,
w;ts used as a source for cloning the AL~G2 gene. Genomic DNA was prepared
from MC GAD8
anal amplified with sense and antisense PCR primers specific for the AUGI open
reading frame
(8784, SEQ ID N0:5; 8787, SEQ ID NO:6). A product identical in size to the
AUGI product but
showing very low intensity on an anal5rtical gel was obtained.
The putative AUG2 PCR product was digested with a battery of restriction
enzymes.
Partial digestion by Eco RI and Pvu I, and the presence of several Bgl II
sites suggested that the
DNA was contaminated with small amounts of AUGl. To remove the contaminating
AUG1 DNA,
2 5 the PCR mixture was cut with Eco RI and gel purified. Since the MC GAD 8
product did not
appear to have an Eco RI site, it was unaffected. The resulting gel-purified
DNA was reamplified
and again analyzed by restriction digestion. The DNA gave a different
restriction map from that of
th.e AUGI PCR product.
Southern blot analysis was performed on genomic DNA from MC GAD8 and wild
3 0 type cells using either A UGI or A UG2 open reading frame PCR fragments as
probes. The A UG2
probe hybridized at low stringency to the AUGl locus and at both low and high
stringency to a
second locus. The AUGI probe bound to both loci at low stringency, but bound
predominantly to
the AUGI locus at high stringency. These data indicated that the new PCR
product from MC
C~ADB was similar to but distinct from .A UGl. Sequence analysis showed an 83%
identity between
3 S A UGI and A UG2 gene products.
To clone the AUG2 genomic locus, PC:R primers were designed from the original
A UG2 PCR fragment. Primers 9885 (SI=:Q ID N0:7) and 9883 (SEQ ID N0:8) wrere
used to screen
a P. methanolica genomic library. :'~ positive clone bank pool was then probed
with the original
MC GAD8 PCR product. Cells were plated on 10 plates at about 5000
colonies/plate and grown
40 overnight, then the plates were overlaved w-ith tiller discs (l~ybond-N':
Amersham Corh., Arlington
heights, IL). Colonies were denatured. neutralized. and LlV cross-linked.
Bacterial debris w,is
%~trade-mark
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
24
washed from the filters with SX SSC, and the filters were again cross-linked.
Blots were pre-
hybridized in pairs at 42°C for 1 hour in 25 ml hybridization buffer.
Approximately 250 ng of
probe was then added to each pair of filters. Hybridization was conducted at
42°C for four hours.
The blots were then washed in 500 ml of 0.1 X SSC, 6M urea. 0.4% SDS at
42°C for 10 minutes,
four times. The blots were then neutralized with 500 ml of 2 X SSC at room
temperature for 5
minutes, two rinses. The blots were then immersed in 100 ml development
reagent (ECL,
Amersham Corp.).
Positive colonies were picked and amplified using PCR primers 9885 (SEQ ID
N0:7) and 9883 (SEQ ID N0:8) to confirm their identity. Positive pools were
streaked on plates,
and single colonies were rescreened by PCR. One colony was selected for
further analysis
(restriction mapping and sequencing). A partial sequence of the AUG2 gene is
shown in SEQ ID
N0:9. As shown in SEQ ID N0:9, the A UG2 sequence begins at the HindIII site a
nucleotide 91.
Nucleotides upstream from this position are vector sequence. The coding
sequence begins at
nucleotide 170.
Disruption of the AUG2 gene had little effect on cell growth on methanol.
Cells
lacking both functional AUGI and AUG2 gene products did not grow on methanol.
Subsequent
analysis showed that the AUGI gene product is the only detectable alcohol
oxidase in cells grown in
a fermentor.
2 o From the foregoing, it will be appreciated that, although specific
embodiments of the
invention have been described herein for purposes of illustration, various
modifications may be
made without deviating from the spirit and scope of the invention.
Accordingly, the invention is not
limited except as by the appended claims.
____._~__._._.__ __ _ .~._.r.. __...
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle, Washington 98102
United States of America
(ii) TITLE OF INVENTION: PREPARATION OF PICHIA METHANOLICA AUXOTROPHIC
MUTANTS
(iii) NUMBER OF SEQUENCES: 9
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: ZymoGenetics, Inc.
(B) STREET: 1201 Eastlake Avenue East
(C) CITY: Seattle
(D) STATE: WA
(E) COUNTRY: USA
(F) ZIP: 98102
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Parker, Gary E
(B) REGISTRATION NUMBER: 31-648
(C) REFERENCE/DOCKET NUMBER: 96-17
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 206-442-6673
(B) TELEFAX: 206-442-6678
(2) INFORMATION FOR SEQ ID N0:1:
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
26
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3077 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
CAGCTGCTCT GCTCCTTGAT TCGTAATTAA TGTTATCCTT TTACTTTGAA CTCTTGTCGG 60
TCCCCAACAG GGATTCCAAT CGGTGCTCAG CGGGATTTCC CATGAGGTTT TTGACAACTT 120
TATTGATGCT GCAAAAACTT TTTTAGCCGG GTTTAAGTAA CTGGGCAATA TTTCCAAAGG 180
CTGTGGGCGT TCCACACTCC TTGCTTTTCA TAATCTCTGT GTATTGTTTT ATTCGCATTT 240
TGATTCTCTT ATTACCAGTT ATGTAGAAAG ATCGGCAAAC AAAATATCAA CTTTTATCTT 300
GAACGCTGAC CCACGGTTTC AAATAACTAT CAGAACTCTA TAGCTATAGG GGAAGTTTAC 360
TGCTTGCTTA AAGCGGCTAA AAAGTGTTTG GCAAATTAAA AAAGCTGTGA CAAGTAGGAA 420
CTCCTGTAAA GGGCCGATTC GACTTCGAAA GAGCCTAAAA ACAGTGACTA TTGGTGACGG 480
AAAATTGCTA AAGGAGTACT AGGGCTGTAG TAATAAATAA TGGAACAGTG GTACAACAAT 540
AAAAGAATGA CGCTGTATGT CGTAGCCTGC ACGAGTAGCT CAGTGGTAGA GCAGCAGATT 600
GCAAATCTGT TGGTCACCGG TTCGATCCGG TCTCGGGCTT CCTTTTTTGC TTTTTCGATA 660
TTTGCGGGTA GGAAGCAAGG TCTAGTTTTC GTCGTT1~CGG ATGGTTTACG AAAGTATCAG 720
CCATGAGTGT TTCCCTCTGG CTACCTAATA TATTTATTGA TCGGTCTCTC ATGTGAATGT 780
TTCTTTCCAA GTTCGGCTTT CAGCTCGTAA ATGTGCAAGA AATATTTGAC TCCAGCGACC 840
TTTCAGAGTC AAATTAATTT TCGCTAACAA TTTGTGTTTT TCTGGAGAAA CCTAAAGATT 900
-__ ..~._~.~_____~ _ _._.~____ ~_.e..
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
27
TAACTGATAAGTCGAATCAACATCTTTAAA AAGATCTCTGCAGCGGCCAG960
TCCTTTAGTT
TATTAACCAATAGCATATTCACAGGCATCACATCGGAACATTCAGAATGGACTCGCAAAC1020
TGTCGGGATTTTAGGTGGTGGCCAACTTGGTCGTATGATCGTTGAAGCTGCACACAGATT1080
GAATATCAAAACTGTGATTCTCGAAAATGGAGACCAGGCTCCAGCAAAGCAAATCAACGC1140
TTTAGATGACCATATTGACGGCTCATTCAATGATCCAAAAGCAATTGCCGAATTGGCTGC1200
CAAGTGTGATGTTTTAACCGTTGAGATTGAACATGTTGACACTGATGCGTTGGTTGAAGT1260
TCAAAAGGCAACTGGCATCAAAATCTTCCCATCACCAGAAACTATTTCATTGATCAAAGA1320
TAAATACTTGCAAAAAGAGCATTTGATTAAGAATGGCATTGCTGTTGCCGAATCTTGTAG1380
TGTTGAAAGTAGCGCAGCATCTTTAGAAGAAGTTGGTGCCAAATACGGCTTCCCATACAT1440
GCTAAAATCTAGAACAATGGCCTATGACGGAAGAGGTAATTTTGTTGTCAAAGACAAGTC1500
ATATATACCTGAAGCTTTGAAAGTTTTAGATGACAGGCCGTTATACGCCGAGAAATGGGC1560
TCCATTTTCAAAGGAGTTAGCTGTTATGGTTGTGAGATCAATCGATGGCCAAGTTTATTC1620
CTACCCAACTGTTGAAACCATCCACCAAAACAACATCTGTCACACTGTCTTTGCTCCAGC1680
TAGAGTTAACGATACTGTCCAAAAGAAGGCCCAAATTTTGGCTGACAACGCTGTCAAATC1740
TTTCCCAGGTGCTGGTATCTTTGGTGTTGAAATGTTTTTATTACAAAATGGTGACTTATT1800
AGTCAACGAAATTGCCCCAAGACCTCACAATTCTGGTCACTATACCATCGACGCTTGTGT1860
CACCTCGCAATTTGAAGCTCATGTTAGGGCCATTACTGGTCTACCCATGCCGAAGAACTT1920
CACTTGTTTGTCGACTCCATCTACCCAAGCTATTATGTTGAACGTTTTAGGTGGCGATGA1980
GCAAAACGGTGAGTTCAAGATGTGTAAAAGAGCACTAGAAACTCCTCATGCTTCTGTTTA2040
CTTATACGGTAAGACTACAAGACCAGGCAGAAAAATGGGTCACATTAATATAGTTTCTCA2100
ATCAATGACTGACTGTGAGCGTAGATTACATTACATAGAAGGTACGACTAACAGCATCCC2160
TCTCGAAGAACAGTACACTACAGATTCCATTCCGGGCACTTCAAGCAAGCCATTAGTCGG2220
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
28
TGTCATCATG GGTTCCGATT CGGACCTACC AGTCATGTCT CTAGGTTGTA ATATATTGAA 2280
GCAATTTAAC GTTCCATTTG AAGTCACTAT CGTTTCCGCT CATAGAACCC CACAAAGAAT 2340
GGCCAAGTAT GCCATTGATG CTCCAAAGAG AGGGTTGAAG TGCATCATTG CTGGTGCTGG 2400
TGGTGCCGCT CATTTACCGG GAATGGTTGC GGCGATGACG CCGCTGCCTG TTATTGGTGT 2460
CCCTGTTAAA GGCTCTACTT TGGATGGTGT TGATTCACTA CACTCCATCG TTCAAATGCC 2520
AAGAGGTATT CCTGTTGCTA CTGTGGCTAT TAACAATGCT ACTAACGCTG CCTTGCTAGC 2580
TATCACAATC TTAGGTGCCG GCGATCCAAA TACTTGTCTG CAATGGAAGT TTATATGAAC 2640
AATATGGAAA ATGAAGTTTT GGGCAAGGCT GAAAAATTGG AAAATGGTGG ATATGAAGAA 2700
TACTTGAGTA CATACAAGAA GTAGAACCTT TTATATTTGA TATAGTACTT ACTCAAAGTC 2760
TTAATTGTTC TAACTGTTAA TTTCTGCTTT GCATTTCTGA AAAGTTTAAG ACAAGAAATC 2820
TTGAAATTTC TAGTTGCTCG TAAGAGGAAA CTTGCATTCA AATAACATTA ACAATAAATG 2880
ACAATAATAT AT-fATTTCAA CACTGCTATA TGGTAGTTTT ATAGGTTTGG TTAGGA1TTG 2940
AGATATTGCT AGCGCTTATC ATTATCCTTA ATTGTTCATC GACGCAAATC GACGCATTTC 3000
CACAAAAATT TTCCGAACCT GTTTTTCACT TCTCCAGATC TTGGTTTAGT ATAGCTTTTG 3060
ACACCTAATA CCTGCAG 3077
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3386 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
__._.w......_ _. _..._._
CA 02261020 1999-O1-14
WO 98/02536 PCT/US97/12582
29
(xi) SEQUENCE SEQ ID
DESCRIPTION: N0:2:
GAATTCCTGCAGCCCGGGGGATCGGGTAGTGGAATGCACGGTTATACCCACTCCAAATAA60
AAGTGTAGTAGCCGGACTGAAAGGTTTTAGGAGTCTGTTTGTTTGTTCATGTGCATCATT120
CCCTAATCTGTTAACAGTCTCGGAGTATACAAAAAAGTAAGTCAAATATCAAGGTGGCCG180
GGGGCAGCATCGAGACTCGAGATGGTACATACTTAAAAGCTGCCATATTGAGGAACTTCA240
AAGTTTTATCTGTTTTTAGAATTAAAAGACGATTGTTGTAACAAAACGTTGTGCCTACAT300
AAACTCAAATTAATGGAAATAGCCTGTTTTGAAAAATACACCTTCTTAAGTACTGACAAA360
GTTTTGTTAAATGACTATCGAACAAGCCATGAAATAGCACATTTCTGCCAGTCACTTTTA420
ACACTTTCCTGCTTGCTGGTTGACTCTCCTCATACAAACACCCAAAAGGGAAACTTTCAG480
TGTGGGGACACTTGACATCTCACATGCACCCCAGATTAATTTCCCCAGACGATGCGGAGA540
CAAGACAAAACAACCCTTTGTCCTGCTCTTTTCTTTCTCACACCGCGTGGGTGTGTGCGC~
600
AGGCAGGCAGGCAGGCAGCGGGCTGCCTGCCATCTCTAATCGCTGCTCCTCCCCCCTGGC660
TTCAAATAACAGCCTGCTGCTATCTGTGACCAGATTGGGACACCCCCCTCCCCTCCGAAT720
GATCCATCACCTTTTGTCGTACTCCGACAATGATCCTTCCCTGTCATCTTCTGGCAATCA780
GCTCCTTCAATAATTAAATCAAATAAGCATAAATAGTAAAATCGCATACAAACGTCATGA840
AAAGTTTTATCTCTATGGCCAACGGATAGTCTATCTGCTTAATTCCATCCACTTTGGGAA900
CCGCTCTCTCTTTACCCCAGATTCTCAAAGCTAATATCTGCCCCTTGTCTATTGTCCTTT960
CTCCGTGTACAAGCGGAGCTTTTGCCTCCCATCCTC~f-fGCTTTGTTTCGGTTATTTT1TT1020
TTCTTTTGAAACTCTTGGTCAAATCAAATCAAACAAAACCAAACCTTCTATTCCATCAGA1080
TCAACCTTGTTCAACATTCTATAAATCGATATAAATATAACCTTATCCCTCCCTTGTTTT1140
TTACCAATTAATCAATCTTCAAATTTCAAATATTTTCTACTTGCTTTATTACTCAGTATT1200
AACATTTGTTTAAACCAACTATAACTTTTAACTGGCTTTAGAAGTTTTATTTAACATCAG1260
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TTTCAATTTACATCTTTATTTATTAACGAAATCTTTACGAATTAACTCAA 1320
TCAAAACTTT
TACGAAAAAAAAATCTTACTATTAATTTCTCAAAATGGCTATTCCAGATGAATTTGATAT1380
TATTGTTGTCGGTGGTGGTTCCACCGGTTGTGCTCTTGCTGGTAGATTAGGTAACTTGGA1440
CGAAAACGTCACAGTTGCTTTAATCGAAGGTGGTGAAAACAACATCAACAACCCATGGGT1500
TTACTTACCAGGTGTTTATCCAAGAAACATGAGATTAGACTCAAAGACTGCTACTTTTfA1560
CTCTTCAAGACCATCACCACACTTGAACGGTAGAAGAGCTATTGTTCCATGTGCTAACAT1620
C~I~fGGGTGGTGGTTCTTCCATCAACTTCTTGATGTACACCAGAGCCTCTGCCTCCGATTA1680
CGATGATfGGGAATCTGAAGGTTGGACTACCGATGAATTATTACCACTAATGAAGAAGAT1740
TGAAACTTATCAAAGACCATGTAACAACAGAGAATTGCACGGTTTCGATGGTCCAATfAA1800
GGTTTCATTTGGTAACTATACTTATCCAAACGGTCAAGATTTCATTAGAGCTGCCGAATC1860
TCAAGGTATTCCATTTGTTGATGATGCTGAAGATTTGAAATGTTCCCACGGTGCTGAGCA1920
CTGGTTGAAGTGGATCAACAGAGACTTAGGTAGAAGATCCGATTCTGCTCATGCTTACAT1980
TCACCCAACCATGAGAAACAAGCAAAACTTGTTCTTGATTACTTCCACCAAGTGTGAAAA2040
GATTATCATTGAAAACGGTGTTGCTACTGGTGTTAAGACTGTTCCAATGAAGCCAACTGG2100
TTCTCCAAAGACCCAAGTTGCTAGAACTTTCAAGGCTAGAAAGCAAATTATTGITfCTTG2160
TGGTACTATCTCATCACCATTAGTTTTGCAAAGATCTGGTATCGGTTCCGCTCACAAGTT2220
GAGACAAGTTGGTATTAAACCAATTGTTGACTTACCAGGTGTTGGTATGAACTTCCAAGA2280
TCACTACTGTTTCTTCACTCCATACCATGTCAAGCCAGATACTCCATCATTCGATGACTT2340
TGTTAGAGGTGATAAAGCTGTTCAAAAATCTGCTTTCGACCAATGGTATGCTAACAAGGA2400
TGGTCCATTAACCACTAATGGTATTGAGGCAGGTGTTAAGATTAGACCAACTGAAGAAGA2460
ATTAGCCACTGCTGATGACGAATTCAGAGCTGCTTATGATGACTACTTTGGTAACAAGCC2520
AGATAAGCCATTAATGCACTACTCTCTAATTTCTGGTTTCTTTGGTGACCACACCAAGAT2580
T _~ ..___.._ __. ___ ___ .. _..._
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TCCAAACGGT AAGTACATGT GCATGTTCCA CTTCTTGGAA TATCCATTCT CCAGAGGTTT 2640
CGTTCACGTT GTTTCTCCAA ACCCATACGA TGCTCCTGAC TTTGATCCAG GTTTCATGAA 2700
CGATCCAAGA GATATGTGGC CAATGGTTTG GTCTTACAAG AAGTCCAGAG AAACTGCCAG 2760
AAGAATGGAC TGTTTTGCCG GTGAAGTTAC TTCTCACCAC CCACACTACC CATACGACTC 2820
ACCAGCCAGA GCTGCTGACA TGGACTTGGA AACTACTAAA GCTTATGCTG GTCCAGACCA 2880
CTTTACTGCT AACTTGTACC ACGGTTCATG GACTGTTCCA ATTGAAAAGC CAACTCCAAA 2940
GAACGCTGCT CACGTTACTT CTAACCAAGT TGAAAAACAT CGTGACATCG AATACACCAA 3000
GGAGGATGAT GCTGCTATCG AAGATTACAT CAGAGAACAC ACTGAAACCA CATGGCATTG 3060
TCTTGGTACT TGTTCAATGG CTCCAAGAGA AGGT1-CTAAG GTTGTCCCAA CTGGTGGTGT 3120
TGTTGACTCC AGATTAAACG TTTACGGTGT TGAAAAGTTG AAGGTTGCTG ATTTATCAAT 3180
TTGCCCAGAT AATGTTGGTT GTAACACTTA CTCTACTGCT TTGTTAATCG GTGAAAAGGC 3240
TTCTACCTTA GTTGCTGAAG ACTTGGGCTA CTCTGGTGAT GCTTTGAAGA TGACTGTTCC 3300
AAACTTCAAA TTGGGTACTT ATGAAGAAGC TGGTCTAGCT AGATTCTAGG GCTGCCTGTT 3360
TGGATATTTf TATAATTTTT GAGAGT 3386
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
TGATCACCTA GGACTAGTGA CAAGTAGGAA CTCCTGTA 38
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(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
CAGCTGCCTA GGACTAGTTT CCTCTTACGA GCAACTAGA 39
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
TGGTTGAAGT GGATCAA 17
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
GTGTGGTCAC CGAAGAA 17
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
_.. _._ . . _ ___ _ ~ _ .._ __.
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(vii) IMMEDIATE SOURCE:
(B) CLONE: ZC9885
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GTTGTTCCTT CCAAACCATT GAAC 24
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(vii) IMMEDIATE SOURCE:
(B) CLONE: ZC9883
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
AAAGTAAGAA GCGTAGCCTA GTTG 24
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 329 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GACCATGATT ACGCCAAGCG CGCAATTAAC CCTCACTAAA GGGAACAAAA GCTGGGTACC 60
GGGCCCCCCC TCGAGGTCGA CGGTATCGAT AAGCTTTATT ATAACATTAA TATACTATTT 120
TATAACAGGA TTGAAAATTA TATTTATCTA TCTAAAACTA AAATTCAAAA TGGCTATTCC 180
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TGAAGAATTC GATATCATTG TTGTCGGTGG TGGTTCTGCC GGCTGTCCTA CTGCTGGTAG 240
ATTGGCTAAC TTAGACCCAA ATTTAACTGT TGCTTTAATC GAAGCTGGTG AAAACAACAT 300
TAACAACCCA TGGGTCTACT TACCAGGCG 329
