U.S. patent application number 11/122494 was filed with the patent office on 2005-12-29 for starch-inducible promoters, recombinant gene constructs, and methods of regulating gene expression.
Invention is credited to Anderson, Daniel B., Gao, Johnway, Hooker, Brian S., Miller, Keith D., Skeen, Rodney S..
Application Number | 20050289666 11/122494 |
Document ID | / |
Family ID | 27091600 |
Filed Date | 2005-12-29 |
United States Patent
Application |
20050289666 |
Kind Code |
A1 |
Hooker, Brian S. ; et
al. |
December 29, 2005 |
Starch-inducible promoters, recombinant gene constructs, and
methods of regulating gene expression
Abstract
The invention includes an isolated promoter containing
nucleotides 1827 through 2147 of SEQ ID NO.: 1. The invention also
includes methods of regulating expression of a gene by fusing the
isolated promoter to a coding sequence to form a fused construct,
and introducing the fused construct into a host such that the
promoter regulates the expression of the gene product within the
host. The invention additionally includes a recombinant gene
comprising an isolated promoter containing at least nucleotides
1827 through 2147 of SEQ ID NO.: 1, operably linked to a coding
region encoding a gene product of interest. The invention includes
a host cell containing the recombinant gene.
Inventors: |
Hooker, Brian S.;
(Kennewick, WA) ; Miller, Keith D.; (Richland,
WA) ; Gao, Johnway; (Richland, WA) ; Skeen,
Rodney S.; (Pendleton, OR) ; Anderson, Daniel B.;
(Pasco, WA) |
Correspondence
Address: |
WELLS ST. JOHN P.S.
601 W. FIRST AVENUE, SUITE 1300
SPOKANE
WA
99201
US
|
Family ID: |
27091600 |
Appl. No.: |
11/122494 |
Filed: |
May 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11122494 |
May 4, 2005 |
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09921944 |
Aug 2, 2001 |
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6900305 |
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09921944 |
Aug 2, 2001 |
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09632314 |
Aug 4, 2000 |
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Current U.S.
Class: |
800/278 ;
435/254.2; 435/419; 435/468; 435/483 |
Current CPC
Class: |
C07K 14/39 20130101 |
Class at
Publication: |
800/278 ;
435/468; 435/483; 435/419; 435/254.2 |
International
Class: |
A01H 001/00; C12N
015/82; C12P 021/06; C12N 001/18; C12N 015/74; C12N 005/04 |
Goverment Interests
[0002] This invention was made with Government support under
Contract DE-AC06 76RLO 1830 awarded by the United States Department
of Energy. The Government has certain rights in the invention.
Claims
The invention claimed is:
1. A method of regulating expression of a gene product comprising:
providing a coding region comprising a nucleic acid sequence that
encodes a gene product; fusing the coding region with an isolated
promoter to form a fused construct, the isolated promoter
comprising nucleotides 1827 through 2147 of SEQ ID NO.: 1; and
introducing the fused construct into a host such that the promoter
regulates the expression of the gene product within the host.
2. The method of claim 1 wherein the host is a yeast cell, and
wherein the introducing comprises integrating the fused construct
into genomic DNA.
3. The method of claim 2 wherein the yeast is a species other than
Schwanniomyces occidentalis.
4. The method of claim 1 wherein the isolated promoter further
comprises nucleotides 1297 through 1826 of SEQ ID NO.: 1.
5. The method of claim 1 further comprising inducing expression of
the gene providing starch to the host.
6. An isolated gene promoter comprising at least nucleotides 1827
through 2147 of SEQ ID NO.:1.
7. A vector comprising the isolated promoter of claim 6.
8. The vector of claim 7 wherein the vector is a plasmid
vector.
9. A recombinant gene comprising: the isolated promoter of claim 6;
and a coding region comprising a nucleic acid sequence encoding a
gene product other than the Schwanniomyces occidentals ATCC 26077
glucoamylase gene product, the isolated promoter being operably
linked to the coding region.
10. A host cell comprising the recombinant gene of claim 9.
11. A method of regulating expression of a gene product comprising:
providing a coding region comprising a nucleic acid sequence that
encodes a gene product; fusing the coding region with an isolated
promoter to form a fused construct, the isolated promoter
comprising at least nucleotides 647-1297 of SEQ ID NO.: 1; and
introducing the fused construct into a host such that the promoter
regulates the expression of the gene product within the host.
12. A method of expressing a gene product comprising: providing a
starch inducible promoter comprising at least nucleotides 1827
through 2147 of SEQ ID NO.: 1; operably linking the starch
inducible promoter to a coding DNA sequence to form a recombinant
gene, the coding DNA sequence encoding a product of interest;
introducing the recombinant gene into a host cell; and inducing
expression of the recombinant gene.
13. The method of claim 12 wherein the inducing comprises providing
starch into a growth medium.
14. The method of claim 13 wherein the starch is the primary carbon
source within the growth medium.
15. The method of claim 12 wherein the host cell is a yeast.
16. The method of claim 12 wherein the host is a plant cell.
17. The method of claim 12 wherein the host is a plant
protoplast.
18. The method of claim 12 wherein the host is a Nicotiana tabacum
cell.
19. The method of claim 12 wherein the gene product is selected
from the group consisting of an enzyme, an enzyme cofactor, a
ligand, and a receptor.
20. The method of claim 12 wherein the gene product is a structural
protein.
21. A host cell comprising a promoter operably linked to a coding
sequence which encodes a gene product other than Schwanniomyces
occidentalis ATCC 26077 glucoamylase gene product, the promoter
comprising at least nucleotides 1827 through 2147 of SEQ ID NO.:
1.
22. The host cell of claim 21 wherein the host cell is a plant
cell.
23. A method of regulating expression of a gene product comprising:
providing a coding region comprising a nucleic acid sequence that
encodes a gene product; fusing the coding region with an isolated
promoter to form a fused construct, the isolated promoter
comprising nucleotides 486 through 2147 of SEQ ID NO.: 1; and
introducing the fused construct into a host such that the promoter
regulates the expression of the gene product within the host.
24. The method of claim 23 wherein the host is a yeast.
Description
RELATED PATENT DATA
[0001] This patent resulted from a Continuation-In-Part of U.S.
patent application Ser. No. 09/921,944, which was filed Aug. 2,
2001; which is a Continuation-In-Part of U.S. patent application
Ser. No. 09/632,314, filed Aug. 4, 2000, now abandoned. The
entirety of each of these previous applications is hereby
incorporated by reference.
TECHNICAL FIELD
[0003] The invention pertains to isolated promoters, recombinant
polynucleotide constructs, methods of regulating expression of a
gene in recombinant host cells, and methods of expressing a gene
product.
BACKGROUND OF THE INVENTION
[0004] Yeast are becoming increasingly utilized as expression
systems for production of proteins and other commercially useful
products. Since yeast are eukaryotic organisms, they can
advantageously be utilized to produce eukaryotic derived proteins
as well as prokaryotic proteins, and can often produce secreted
proteins and/or post-translationally modified proteins which are
difficult or impossible to produce in non-eukaryotic expression
systems.
[0005] Yeast cells can allow relatively inexpensive production of
large amounts of protein as compared to other eukaryotic expression
systems such as mammalian or insect cell systems. As compared to
alternative eukaryotic expression systems, yeast systems can often
allow easy culturing and handling, and can also allow the
protein(s) of interest to be easily purified.
[0006] A helpful tool in production of heterologous proteins in
most expression systems is a strong gene promoter which can allow
high levels of gene expression, resulting in high levels of
production of the protein encoded by the gene. In many instances,
it can be desirable to express high levels of a heterologous
protein at a particular time or over a particular time range, while
a basal level of expression (low or no expression of the
heterologous gene) occurs at other times. Accordingly, an inducible
promoter can be desirable such that expression of the heterologous
protein can be enhanced or triggered by one or more inducing agents
at a chosen time or growth stage of the host.
[0007] In addition to the advantageous of yeast expression systems
set forth above, some yeast promoters can also function to regulate
gene expression in one or more of bacteria, mold, plants and/or
plant cells. However, the number of available promoters having an
ability to function in yeast, or in yeast as well as in prokaryotic
cells, molds and/or plants is limited at the present time. Further
limited is the availability of promoters having the ability to
allow strong and/or inducible expression in yeast, or in both yeast
and alternative systems. Accordingly, it would be desirable to
develop additional promoters for utilization in yeast an
alternative systems, and to develop additional expression
systems.
SUMMARY OF THE INVENTION
[0008] In one aspect the invention encompasses a method of
regulating expression of a gene product. The method includes
providing a coding region which includes a nucleic acid sequence
that encodes a gene product. The coding region is fused with an
isolated promoter to form a fused construct; the isolated promoter
includes nucleotides 1827 through 2147 of SEQ ID. NO.: 1. The
method includes introducing the fused construct into a host such
that the promoter regulates the expression of the gene product
within the host.
[0009] In one aspect the invention encompasses an isolated gene
promoter comprising at least nucleotides 1827 through 2147 of SEQ
ID NO.: 1.
[0010] In one aspect the invention encompasses a recombinant gene
comprising an isolated promoter containing at least nucleotides
1827 through 2147 of SEQ ID NO.: 1, and a coding region comprising
a nucleic acid sequence encoding a gene product other that the
Schwanniomyces occidentalis ATCC 26077 glucoamylase gene product,
the isolated promoter being operably linked to the coding
region.
[0011] In one aspect the invention encompasses a method of
expressing a gene product by providing a starch inducible promoter
including at least nucleotides 1827 through 2147 of SEQ ID NO.: 1
and operably linking the inducible promoter to a coding DNA
sequence to form a recombinant gene. The coding DNA sequence
encodes a protein of interest. The recombinant gene is introduced
into a host cell and expression of the recombinant gene is
induced.
[0012] In one aspect the invention encompasses a host cell
containing a promoter operably linked to a coding sequence which
encodes a gene product other than the Schwanniomyces occidentalis
ATCC 26077 glucoamylase gene product. The promoter comprises at
least nucleotides 1827 through 2147 of SEQ ID NO.: 1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0014] Preferred embodiments of the invention are described below
with reference to the following accompanying drawings.
[0015] FIG. 1 is a flowchart diagram illustrating a particular
aspect of the present invention.
[0016] FIG. 2 shows a schematic illustration of an inverse PCR
method for obtaining an isolated promoter.
[0017] FIG. 3 is a photograph image of a gel separation of PCR
clones of the Schwanniomyces occidentalis glucoamylase
promoter.
[0018] FIG. 4 is a schematic illustration of construction of a
plasmid vector pGA2066.
[0019] FIG. 5 is a schematic illustration of construction of a
plasmid vector pGA2100.
[0020] FIG. 6 is a schematic illustration of construction of a
plasmid vector pGA2101.
[0021] FIG. 7 is a schematic linear representation of a recombinant
DNA construct containing a 1.5 kbp glucoamylase promoter sequence
operably linked to the beta-glucuronidase reporter gene.
[0022] FIG. 8 is a schematic linear representation of a recombinant
DNA construct containing a 1.0 kbp glucoamylase promoter sequence
operably linked to the beta-glucuronidase reporter gene.
[0023] FIG. 9 is a schematic linear representation of a recombinant
DNA construct containing a 0.3 kbp glucoamylase promoter sequence
operably linked to the beta-glucuronidase reporter gene.
[0024] FIG. 10 is a schematic linear representation of a
recombinant DNA construct containing a 1.2 kbp glucoamylase
promoter sequence which lacks the 0.3 kb minimal promoter sequence,
operably linked to the beta-glucuronidase reporter gene.
[0025] FIG. 11 is a schematic linear representation of a
recombinant DNA construct containing a 0.7 kbp glucoamylase
promoter sequence (which lacks the 1.0 kb promoter fragment
sequence) operably linked to the beta-glucuronidase reporter
gene.
[0026] FIG. 12 is a scatter plot of forward scatter height (FSH)
relative to side scatter height (SSH) for light scatter analysis of
cell viability for a control non-transformed cell line and five
transformed cell lines. The five transformants include:
transformant 470, which contains a 1.0 kb GAM promoter fragment;
transformant 478 which contains a 0.3 kb minimal GAM promoter
fragment; transformant 481, which contains a 1.2 kb GAM promoter
fragment; transformant 484 which contains a 0.7 kb fragment of the
GAM promoter; and transformant 469, which contains 1.5 kb of the
GAM promoter.
[0027] FIG. 13 is a histogram depicting fluorescence intensity
relative to the number of cells counted (counts) for five
transformed cell lines shown in FIG. 12, relative to the
non-transformed control.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] This disclosure of the invention is submitted in furtherance
of the constitutional purposes of the U.S. Patent Laws "to promote
the progress of science and useful arts" (Article 1, Section
8).
[0029] The invention encompasses recombinant gene expression
involving isolated polynucleotide promoter sequences. For purposes
of the present description the term "expression" refers to
transcription of a gene to produce corresponding RNA and
translation of the mRNA to produce the gene product (i.e. peptide,
polypeptide or protein), or a portion of the transcription and/or
translation process. The term "isolated" can refer to a naturally
occurring molecule such as, for example, a polynucleotide or a
polypeptide that has been recovered from an organism which produced
it, or alternatively can refer to a synthetic molecule. The
invention also encompasses formation of polynucleotide constructs
containing isolated polynucleotide promoter sequences,
polynucleotide constructs, host cells containing the polynucleotide
constructs, and expression systems which utilize the polynucleotide
constructs.
[0030] Isolated promoters in accordance with the invention comprise
all or a functional portion of the nucleotide sequence set forth in
SEQ ID NO.: 1. SEQ ID NO.: 1 corresponds to a 2182 nucleotide
sequence including the ATG start codon (nucleotides 2148-2150) and
an additional 32 nucleotides (2151-2182) from the coding region of
the Schwanniomyces occidentalis (formerly Schwanniomyces
castellii), ATCC 26077 glucoamylase gene. Nucleotides 1-2147 of SEQ
ID NO.: 1 can be referred to as being "upstream" or 5' relative to
the coding region of the glucoamylase (GAM) coding region. A 1662
base sequence located immediately 5' to the start codon
(corresponding to nucleotides 486-2147 of SEQ ID NO.: 1), is
identified being the GAM promoter region based on open reading
frame analysis. For purposes of the description, the term
"promoter" refers to a nucleic acid sequence located upstream or 5'
to a translational start codon (ATG) of an open reading frame or
coding region of a gene where the promoter region is involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A constitutive promoter is one which is
functional in a native organism or host in an absence of specific
inducing agents. An inducible promoter is a promoter having
promoter activity that is initiated or enhanced by an inducing
agent. For purposes of the description the term "minimal promoter
activity" refers to a detectible ability to initiate, direct or
promote gene transcription. Additionally, the term "minimal
promoter sequence" refers to a minimal nucleic acid sequence
sufficient to direct transcription of a nucleic acid sequence.
[0031] The term "fragment" as used herein can describe a nucleic
acid or a portion of a nucleic acid sequence that is less than a
full length. For example, a promoter fragment can refer to a
portion of a promoter sequence that has promoter activity.
[0032] The Schwanniomyces occidentalis nucleic acid sequences
described herein can be obtained from the American Type Culture
Collection (ATCC), Manassas, Va., under ATCC No. 26077. This yeast
strain has been shown to completely hydrolyze and utilize starch as
a nutrient source. The glucoamylase gene from which the isolated
promoter sequences of the invention are derived is a starch
inducible promoter. The promoters of the invention utilized in
conjunction with the methodology described below can be used to
produce expression systems for expression of recombinant genes to
produce proteins of interest. In particular expression systems of
the invention, the expression of a protein of interest can be
induced by providing starch to the expression host.
[0033] A process encompassed by the present invention is described
generally with reference to the block diagram of FIG. 1. At an
initial step (A) an isolated polynucleotide comprising a promoter
sequence is provided. The isolated promoter sequence can comprise
all or a functional portion of the promoter region of SEQ ID NO.:
1. The terms `functional portion` or "functional fragment" as used
in the present description refers to any portion of a promoter
sequence having at least minimal promoter activity. When utilized
in conjunction with a particular host, a functional promoter is one
that can produce at least minimal transcription within the
particular host.
[0034] In particular embodiments of the present invention, the
isolated promoter utilized in step A of FIG. 1 will contain all or
a portion of nucleotide sequence 1-2147 of SEQ ID NO.: 1. As
indicated above, a 1662 nucleotide sequence containing nucleotides
486-2147 is determined to be the starch inducible promoter region
of the GAM gene of strain 26077. Accordingly, the isolated promoter
is step (A) can comprise an entirety or a functional portion of the
nucleotide sequence from nucleotide 486-2147 of SEQ ID NO.: 1.
Alternatively, an isolated promoter can be provided in step A which
comprises a fragment of SEQ ID NO.: 1 which is unique relative to
other known promoter sequences. The unique fragment can comprise
for example, particular functional portions of SEQ ID NO.: 1 which
are described in more detail below.
[0035] As will be understood by those of ordinary skill in the art,
the promoter sequence set forth in SEQ ID NO.: 1 can be manipulated
by deleting, inserting or modifying one or more nucleic acids
within the sequence utilizing conventional methods to result in a
sequence that retains promoter functionality. The retained
functionality can in certain instances, even be enhanced by such
modification. Accordingly, the invention encompasses utilizing such
modified sequences for the isolated promoter in step (A).
Preferably where a modified isolated promoter utilized in step (A)
the promoter will have at least 70% identity to the corresponding
fragment set forth in SEQ ID NO.: 1. Typically, the promoters of
the invention will include a sequence having at least 80% identity
to a functional fragment of SEQ ID NO.: 1, and can in particular
instances comprises a sequence having at least 90% identity to a
functional fragment of SEQ ID NO.: 1.
[0036] Referring to FIG. 2, an exemplary method for isolation of a
promoter sequence for use in step (A) of FIG. 1 is shown. Genomic
DNA can be isolated from, for example, cultured Schwanniomyces
occidentalis strain ATCC 26077. The isolated genomic DNA can be
purified and used for subsequent isolation of the GAM promoter. For
instance, the genomic DNA (FIG. 2 top) can be digested with
restriction enzymes to produce fragments of the genomic DNA. Such
fragments can be self ligated to form the circular DNA shown in
FIG. 2 (center). Inverse polymerase chain reaction (PCR) can then
be performed to introduce desired restriction enzyme sites into the
fragments. Exemplary reverse primers appropriate for use during
such PCR process are set forth in SEQ ID NOS.: 4 and 5. Exemplary
forward primers for utilization during such inverse PCR process are
set forth in SEQ ID NOS.: 6 and 7.
1TABLE 1 Primer Pairing and Restriction Enzymes for Inverse PCR
Reactions PCR Sample Restriction Enzyme Primer Pairing 1 Bcl I SEQ
ID No.: 4/SEQ ID No.: 7 2 Bcl I SEQ ID No.: 5/SEQ ID No.: 7 3 BstB
I SEQ ID No.: 4/SEQ ID No.: 6 4 BstB I SEQ ID No.: 5/SEQ ID No.: 7
5 Hinc II SEQ ID No.: 4/SEQ ID No.: 7 6 Hinc II SEQ ID No.: 5/SEQ
ID No.: 7 7 Hpa I SEQ ID No.: 4/SEQ ID No.: 7 8 Hpa I SEQ ID No.:
5/SEQ ID No.: 7 9 Sac I SEQ ID No.: 4/SEQ ID No.: 7 10 Xmn I SEQ ID
No.: 4/SEQ ID No.: 7 11 Xmn I SEQ ID No.: 5/SEQ ID No.: 7
[0037] Table 1 shows exemplary restriction enzymes (column 2) that
can be utilized during digestion of genomic DNA, and shows pairing
of the exemplary primers for utilization during inverse PCR
processing. It is to be understood that the invention encompasses
use of alternative or additional restriction enzymes during the
digestion and also encompasses use of alternative primers and
primer pairing.
[0038] As shown in FIG. 2, the restriction enzyme digestion, self
ligation and subsequent inverse PCR can be used to produce the
isolated promoter for utilization in step (A) of FIG. 1.
Alternatively, the isolated promoter obtained can undergo further
processing to produce fragments thereof and/or modify such sequence
for production of alternative isolated promoters which can be
utilized in step (A).
[0039] Referring again to FIG. 1, a polynucleotide sequence that
encodes a protein of interest can be provided in a step (B) and can
be utilized in conjunction with the isolated promoter provided in
step (A) for polynucleotide construct formation step (C). The
encoding polynucleotide provided in step (B) is not limited to any
specific polynucleotide sequence. The coding sequence can comprise
sequence encoding any protein, polypeptide, or peptide of interest,
or can encode two or more proteins/peptides of interest. In
accordance with the invention, a protein of interest can be an
enzyme, a structural protein, an enzyme cofactor, a receptor, a
ligand, a regulatory protein, a peptide, a portion of a protein,
etc. In particular instances, the methodology and expression
systems in accordance with the invention can be useful for
producing therapeutically beneficial proteins/peptides.
[0040] The phylogenetic origin of the coding sequence can be any
desired species, either prokaryotic or eukaryotic. In particular
embodiments, the native origin of the coding sequence can be a
different species than the originating species of the isolated
promoter, can be different species than the originating species of
the eventual host (discussed below), or both.
[0041] Polynucleotide construct formation step (C) can include
forming a recombinant molecule by fusing the isolated promoter
provided in step (A) with the coding sequence provided in step (B).
Preferably, the isolated promoter is operably linked to the
provided coding sequence. For purposes of the description the term
"operably linked" refers to a nucleic acid sequence which is
arranged or joined with a second sequence such that the first
nucleic acid sequence affects the function or processing of the
second nucleic acid sequence. A promoter sequence is operably
linked to a coding sequence if the promoter regulates or mediates
transcription of the coding sequence. Operable linkage of a
polynucleotide to a promoter to form a recombinant polynucleotide
construct in step (C) can allow expression of the polynucleotide
and production of the encoded polypeptide to be controlled by the
promoter. The term "recombinant" as used in the present description
can refer to a nucleic acid molecule which is made at least in part
by artificial combination of two or more segments. The term "gene"
refers to a DNA sequence or molecule which includes an encoding
region and one or more regions involved in regulation of expression
of the coding sequence. The term gene can refer to a native gene
(where native refers to naturally occurring nucleic acid) or can
refer to a DNA molecule having at least some synthetic or
recombinant portion.
[0042] Recombinant DNA construction formation step (C) can
additionally comprise formation of a vector. For purposes of the
present invention, a vector can comprise a plasmid, cosmid, phage
or yeast artificial chromosome (YAC). A particular vector to be
formed in step (C) can be determined based on the intended use of
the vector. For example, vector formation in step (C) can be
determined by appropriateness of the vector for introduction into a
host in step (D) (discussed below).
[0043] Once the desired polynucleotide construct is formed in step
(C), the construct can be utilized for introduction into a host
cell in step (D). Introduction into a host cell is not limited to a
specific method of introduction. In particular embodiments,
introducing a construct into a host cell can comprise
transformation of a host cell. The terms "transgenic" and
"transformed" can refer to any cell tissue, organism or seed into
which foreign or recombinant DNA has been introduced.
Transformation can be achieved utilizing one or more of
electroporation, sonication, T-DNA mediated transformation,
particle-bombardment mediated transformation, microinjection,
virus-mediated transformation, whiskers mediated transformation,
liposome mediated transformation, chemical mediated transformation
and plasma transformation. When referring to plant cells, plant
tissue, whole plants or other plant parts the designated T0 can
refer to the primary transformant and the designation T1 can refer
to the first generation produced from the primary transformant
T0.
[0044] The host cell into which the polynucleotide construct is
introduced in step (D) is not limited to a particular type of cell.
Such host cell can be prokaryotic or eukaryotic. Additionally, the
host cell can be from a different species than the species from
which the isolated promoter has been isolated, can be different
from the species from which the coding polynucleotide is isolated
or can be different from both the originating species of the
promoter and the originating species of the coding
polynucleotide.
[0045] In particular embodiments, the host cell can be a species
belonging to a phylogenetic kingdom that is different than the
phylogenetic kingdom from which at least one of the isolated
promoter and the isolated coding polynucleotide originate. For
example, when the isolated promoter comprises all or a portion of
the promoter region of SEQ ID NO.: 1 isolated from the yeast
(belonging to the fungi kingdom) Schwanniomyces occidentalis 26077,
the host cell can comprise, for example, a cell belonging to a
species within the kingdom Plantae.
[0046] Alternatively, when an isolated promoter is isolated from
Schwanniomyces occidentalis, the host cell can belong to a species
within the same phylogenetic kingdom. When belonging the same
kingdom (Fungi) the host cell species can be the same or can be
different from the species which the promoter is isolated. For
example, when the isolated promoter provided in step (A) is the
Schwanniomyces occidentalis GAM promoter or a portion thereof, the
host cell in step (D) can also be Schwanniomyces occidentalis
(either strain 26077 or an alternative strain), can be a second
species within the Schwanniomyces genus, or can belong to an
independent genus or even an independent phylum within the Fungi
kingdom.
[0047] The invention also encompasses introducing the constructs
formed in step (C) into a prokaryotic host such as bacteria. The
introduction of the construct of the present invention into
bacterial can be useful for manipulation and/or amplification of
the construct. For purposes of the present invention the term
"amplification" of a nucleic acid or nucleic acid sequences refers
to the production of additional copies of the nucleic acid or
sequence. Amplification can utilize host mediated amplification
and/or polymerase chain reaction technology.
[0048] As shown in FIG. 1, once the recombinant polynucleotide
construct has been introduced into the host cell the polypeptide
encoded by the polynucleotide can be expressed by the host cell in
an expression step (E). Alternatively, the host cell can be
utilized to assist transformation of a subsequent host cell or to
amplify or manipulate the polynucleotide construct (not shown).
Expression within a host cell in accordance with the invention can
be constitutive expression or induced expression. In particular
instances, expression can be constitutive at a low level and can be
inducible such that the level of expression increases upon inducing
with an appropriate inducing agent.
[0049] In embodiments of the present invention where the encoded
protein is expressed in the host cell, the expressed protein is not
limited to any particular type, and can be for example, an
intracellular peptide, an excreted or extracellular peptide or a
transmembrane peptide. Alternatively, the expressed peptide can
confer resistance to the host cell. Such conferred resistance can
be resistance to one or more of an antibiotic, a herbicide, a
toxin, a parasite and a pathogen. The invention also encompasses
introducing constructs where the coding sequence codes for a
protein that can regulate or control expression of native genes
within the host cell, or can regulate or control expression of the
introduced (heterologous) genes.
[0050] The invention encompasses recombinant constructs and hosts
containing the constructs where the protein encoded by the
constructs confers a new (non-native) phenotype to the host cell.
Additionally encompassed are constructs and hosts where the
expressed protein has a sequence, a function, or an effect that is
similar to or identical to one or more native polypeptides produced
by the host.
[0051] In one embodiment of the invention, the isolated promoters
of the invention can be utilized to form recombinant genes which
can be introduced and expressed in Schwanniomyces occidentalis. As
indicated above, the native GAM promoter is an inducible promoter.
The native GAM promoter can be induced to initiate or enhance
transcription by agents such as maltose and starch. In native
Schwanniomyces occidentalis the expression level of the
glucoamylase gene can be increased by 100 fold when cells are
shifted from a glucose culture medium to a maltose culture medium.
Accordingly, the use of the isolated promoters of the invention in
recombinant genes can allow host Schwanniomyces occidentalis cells
to be induced to produce a protein of interest. As further
discussed below, the promoter region of SEQ ID NO.: 1 and various
functional fragments thereof can function as constitutive or
inducible promoters in Schwanniomyces occidentalis or in
alternative yeast strains.
[0052] In addition to being useful in yeast expression systems, the
isolated promoters of the invention can also be utilized to form
recombinant molecules for introduction and expression in non-yeast
and even non-fungi systems. In particular, the isolated promoters
of the invention can be utilized for heterologous expression in
plants.
EXAMPLES
Example 1
[0053] Expression utilizing an isolated promoter in a yeast host
cell.
[0054] The glucoamylase gene promoter was isolated from
Schwanniomyces occidentalis strain ATCC 26077 utilizing the
exemplary method discussed above with reference to FIG. 2. The
method included growing Schwanniomyces occidentalis cells overnight
in a culture medium, harvesting the cells and isolating and
purifying genomic DNA utilizing spheroplasting technology. Inverse
PCR methodology was utilized to obtain the sequence shown in SEQ ID
NO.: 1 which is inclusive of the promoter region of the GAM gene.
The PCR methodology utilized the reverse primer set forth in SEQ ID
NOS.: 4 and 5 and the PCR forwarding primers set forth in SEQ ID
NOS.: 6 and 7.
[0055] The isolation included digestion of genomic DNA with
restriction enzymes including Bcl I, Bst B I, Hinc II, Hpa I, Sac I
and Xmn I having restriction sites upstream of the glucoamylase
gene. After digestion, the DNA samples were purified and self
ligated using T4 DNA ligase followed by inverse PCR. Primer pairing
and restriction enzyme for eleven independent samples is indicated
in Table 1.
[0056] Following PCR, the DNA products were separated in an agarose
gel by electrophoresis, the results of which are shown in FIG. 3.
The lane numbers shown in FIG. 3 correspond to the inverse PCR
reactions set forth in Table 1, with lane S being a DNA size
marker. The isolated GAM promoter clones are apparent as dark bands
in the photograph. Lanes 1, 2, 5, 6, 7 and 8 show strong bands
corresponding to ligated DNA samples previously cleaved by Bcl I,
Hinc II, or Hpa I. The sizes of the resulting clones range from
about 0.4 kb to about 4.4 kb with the strongest bands being from
about 1.7 kb to about 2.3 kb.
[0057] For sequencing and analysis, the PCR product from reaction 2
shown in FIG. 3 was initially inserted into transient vector pGEM-T
(Promega, Madison, Wis.) to form pGA2066 as shown in FIG. 4. This
vector was utilized for initial amplification, cloning and
sequencing of the promoter. The determined sequence of the isolated
DNA fragment containing the GAM promoter is set forth in SEQ ID
NO.: 1. The clone has a length of 2182 base pairs which includes
the GAM transcription initiation codon (corresponding to
nucleotides 2148-2150 of SEQ ID No.:1) and an additional 32 bases
within the coding region of the GAM gene nucleotides 2151-2182 of
SEQ ID No.:1). Of the remaining sequence set forth in SEQ ID NO.: 1
(nucleotides 1-2147) a 1662 nucleotide sequence containing
nucleotides 486-2147 was determined to be the GAM promoter and is
referred to as the GAM promoter region.
[0058] Analysis of the 1662 base pair GAM promoter region reveals
seven CAT boxes (nucleotides 1571-1574; nucleotides 1709-1712;
nucleotides 1806-1809; nucleotides 1816-1819; nucleotides
1776-1779; nucleotides 1963-1966; and nucleotides 2015-2018) and
nine TATA boxes (nucleotides 1561-1565; nucleotides 1626-1643;
nucleotides 1730-1734; nucleotides 1864-1868; nucleotides
1884-1887; nucleotides 1937-1943; nucleotides 2034-2043;
nucleotides 2081-2090; and nucleotides 2135-2139). These identified
CAT boxes and TATA boxes are within a 600 base pair fragment within
SEQ ID NO.: 1 relative to the initiation codon (within the sequence
from nucleotide 1548-2147).
[0059] A sequence alignment and comparison was performed to
determine differences between the isolated GAM ATTC 26077 promoter,
and a known sequence corresponding to a fragment of a GAM promoter
from Schwanniomyces occidentalis stain ATCC 26076. The strain 26076
promoter has been shown to lack ability to direct transcription in
Saccharomyces cerevisiae. The comparison utilized a fragment of the
isolated promoter from strain ATCC 26077 set forth in SEQ ID NO.:
2. This fragment corresponds to nucleotides 1823-2105 of SEQ ID
NO.: 1. SEQ ID NO.:2 was aligned with and compared to a 325
nucleotide sequence (SEQ ID NO.:3) from the GAM promoter of strain
ATCC 26076. Comparison revealed difference between the two
sequences at positions corresponding to nucleotides 1984-1986,
1992-1993 and 2113 or SEQ ID NO. 1. Since the isolated GAM promoter
from ATCC 26077 is able to direct transcription in Saccharomyces
(see below), the positions of sequence difference indicates that
these positions are important for providing promoter function,
especially in hosts other than Schwanniomyces occidentalis.
[0060] The promoter activity of various fragments of SEQ ID NO.: 1
was analyzed by fusing various promoter fragments to a bacterial
glucuronidase gene. Initially two fragments were utilized. A first
fragment contains a 1.5 kb nucleotide sequence which includes a
portion of the GAM promoter corresponding to nucleotides 647-2147
of SEQ ID NO.: 1, and a second 1.0 kb fragment including
nucleotides 1297-2147 of SEQ ID. NO.: 1. These two clones (referred
to as GAM15 and GAM10 respectively) were cloned from pGA2066
utilizing forward primers having the sequence set forth in SEQ ID
NOS.: 8 and 9, with the primer sequence set forth in SEQ ID NO.: 8
being utilized for the 1.5 kb GAM promoter and the sequence set
forth in SEQ ID NO.: 9 being utilized as a forward primer to obtain
the 1.0 kb GAM promoter. The cloning additionally utilized a
reverse primer having the sequence set forth in SEQ ID NO.: 10
which was utilized for each of the 1.5 kb and 1.0 kb promoters. The
specified forward and reverse primers introduced a Spe I
restriction enzyme site at the 5' end of the promoter sequence and
introduced a Hind III restriction enzyme site at the 3' end of the
promoter sequences.
[0061] The forward and reverse primers above were used in
conjunction with PCR to produce polynucleotides corresponding to
the 1.5 kb and 1.0 kb promoters having appropriate ends to allow
insertion into vector pGA2028D to form plasmid vectors pGA2100
containing the 1.5 kb GAM promoter as illustrated in FIG. 5, and
the vector pGA2101 containing the 1.0 kb GAM promoter as
illustrated in FIG. 6. Each of the resulting vectors has a DNA
replicon designated `2 micron` for plasmid replication in
Saccharomyces strains. The vector region designated `ColE1` is the
origin for plasmid replication during gene manipulation in E. coli
strains, `f1 ori` is the phage origin and `gus` is the bacterial
glucuronidase gene. Tcyc1 is the transcription terminator and
Zeocin is the Zeocin resistance gene. Formation of each of the two
plasmids including operably linking the isolated promoter fragment
to the coding portion of the E. coli beta-glucuronidase (GUS)
gene.
[0062] Plasmid vectors pGA2100 and pGA2101 were independently
utilized to transform a Saccharomyces host. Each plasmid was
introduced into a Saccharomyces hybrid yeast strain (obtained from
James R. Mattoon of University of Colorado) utilizing plasma
transformation of the host. After transformation, the Saccharomyces
cells were plated onto appropriate selective medium containing
glucose, and incubated at 30.degree. C. for 4 days.
[0063] Transformed colonies were chosen for determining activity of
the introduced GAM promoter sequences utilizing glucuronidase (GUS)
activity analysis. For this analysis, protein samples were
collected from isolated colonies by suspending a single transformed
colony and subsequently disrupting the cells. After disruption, the
sample was centrifuged and the supernatant containing protein was
analyzed for protein content and GUS activity.
[0064] The determined GUS activity for individual colonies
containing either the GAM15 or GAM10 promoters are set forth in
Table 2. The GUS specific activity (units of glucuronidase activity
per milligram of total protein, where 1 unit of glucuronidase
activity is the amount of glucuronidase that converts 1 pmole of
4-methylumbelleiferul-beta-D-glucu- ronide (MUG) to
4-methylumbelliferone) is reported for two isolated colonies
containing the 1.5 kb fragment and four isolated colonies
containing the 1.0 kb fragment. The measured GUS activity was
compared to a control assay which utilized non-transformed cells
(C*).
[0065] The GUS analysis indicate that the 1.5 kb fragment and the
1.0 kb fragment function as promoters to direct transcription of
the recombinant GUS gene. These results additionally indicate that
the GAM promoter fragments isolated from Schwanniomyces
occidentalis are able to function as promoters in other fungal
species.
2TABLE 2 GUS-activity analysis of transformants grown in glucose
medium Isolated GUS specific activity Average activity Transformant
Promoter (unit/mg) (units/mg) C* N/A 6 6 1 GAM15 54 60 2 GAM15 66 3
GAM10 100 97 .+-. 15 4 GAM10 99 5 GAM10 111 6 GAM10 76 C*
Non-transformed control
Example 2
[0066] Inducible expression from isolated promoters in yeast host
cells.
[0067] Transformed Saccharomyces colonies containing either the 1.5
kb promoter fragment or the 1.0 kb promoter fragment were chosen
for determining activity and inducibility of the introduced
promoter utilizing the glucuronidase activity analysis described
above. Transformed colonies were first grown in a medium containing
glucose. Cells were washed and subsequently transferred into
culture medium containing 2% potato starch for GUS gene expression
analysis. The results of the GUS activity assays are summarized in
Table 3.
[0068] The GUS specific activity is reported for six isolated
colonies transformed with the 1.5 kb fragment and six isolated
transformed colonies containing the 1.0 kb GAM promoter fragment.
The specific activities are reported relative to a control host
cell which has not been transformed with the GUS expression
vector.
3TABLE 3 GUS-activity analysis of transformants grown in starch
medium Isolated GUS specific activity Average activity Transformant
Promoter (unit/mg) (units/mg) C* N/A 0.0 0.0 1 GAM15 1394 890 .+-.
350 2 GAM15 1405 3 GAM15 582 4 GAM15 854 5 GAM15 685 6 GAM15 963 7
GAM10 1398 1521 .+-. 327 8 GAM10 1645 9 GAM10 2123 10 GAM10 1250 11
GAM10 1432 12 GAM10 1277 C* Non-transformed control
[0069] Comparing the results presented in Table 3 obtained for
colonies grown in starch medium with the results presented in Table
2 for colonies grown in glucose medium, the results indicate that
the two promoter fragments are each highly induced by starch and
maintain their starch inducibility even in a non-Schwanniomyces
host. Accordingly, the promoters of the invention can
advantageously allow use of starch as an inducing agent in
alternative expression systems to allow controlled and highly
inducible expression utilizing a relatively inexpensive inducing
agent.
Example 3
[0070] Promoter Deletion Analysis
[0071] The glucoamylase promoter-gus reporter gene construct
containing the 1.5 kb promoter fragment described above was
utilized for promoter deletion analysis. Restriction endonuclease
digestion of the GAM15 construct plasmid was utilized to produce
four deletion constructs to determine the functionality of shorter
fragments and particular regions within the 1.5 kb promoter
fragment.
[0072] As indicated above, the 1.5 kb promoter fragment contains
nucleotide sequence corresponding to nucleotides 647-2147 of SEQ ID
NO.: 1. A portion of the plasmid containing the 1.5 kb glucoamylase
promoter fragment with the plasmid being designated as the 469
plasmid is shown in FIG. 7. Four additional constructs were derived
from this plasmid by restriction endonuclease digestion. A 1.0 kb
glucoamylase promoter fragment operably linked to the GUS reported
gene was formed by digestion with Nco I/Nae I to produce plasmid
470 depicted in FIG. 8. The 1.0 kb construct includes GAM promoter
sequence corresponding to nucleotides 1297-2147 of SEQ ID NO.:1
Digestion of plasmid 469 with Nae I/Bgl II was utilized to form a
0.3 kb `minimal glucoamylase promoter` operably linked to the GUS
reporter gene in a plasmid construct designated 478 shown in FIG.
9. The 0.3 kb minimal promoter corresponds to nucleotides 1826-2147
of SEQ ID NO.: 1. Bgl Il/Hind III digestion of the 469 plasmid was
utilized to form a 1.2 kb promoter fragment which lacks the 300 bp
minimal promoter sequence. The 1.2 kb fragment corresponds to
nucleotides 647-1826 of SEQ ID NO.:1, and is operably linked to the
GUS reporter gene in a plasmid construct 481 depicted in FIG. 10. A
0.7 kb promoter fragment was prepared by digestion of plasmid 469
with Nco I/Hind III to produce the plasmid construct 484 shown in
FIG. 11. The 0.7 kb promoter sequence contains nucleotides 647-1297
of SEQ ID NO.:1 (and lacks the 1.0 kb promoter fragment
sequence).
[0073] The newly constructed deletion plasmids were isolated and
purified and were used to transform Saccharomyces as described
above with respect to the 1.5 and previous 1.0 kb promoter fragment
transformants. Transformants were selected by growth on Zeocin
plates. Individual colonies were then chosen, each containing one
of the five plasmids and were grown in liquid culture overnight at
30.degree. C. Non-transformed yeast was utilized as a control.
[0074] After growth overnight, the yeast were pelleted and
re-suspended in medium containing 0.2% soluble starch for four
hours at 25.degree. C. to induce GUS expression. In order to
analyze GUS activity of the transformed cell line, the yeast were
pelleted and resuspended in a staining buffer containing 1%
fluorescein diglucuronide (FDGIcU, available from Molecular Probes,
Eugene Oreg.) and incubated at room temperature for 15 minutes.
[0075] After incubation, the cells were again pelleted by
centrifugation and were subsequently resuspended in a non-FDGIcU
buffer. The cells in the resulting suspension were analyzed by flow
cytometry on the FL1 channel (indicated as myc/GaM488 in FIG. 13.).
The intact FDGIcU molecule does not fluoresce. However, upon being
cleaved by the enzyme .beta.-glucuronidase, fluorescein moiety is
fluorescent. Accordingly, fluorescence intensity of the transformed
yeast is proportional to the amount of glucuronidase enzyme present
within the yeast cells.
[0076] Referring to FIG. 12, yeast cells were sorted in the R1
sort-gate using light scatter analysis to assess cell viability.
Populations of intact viable cells were obtained by sorting and
were analyzed using cytometry. The fluorescence intensity relative
to the number of cells (counts) is presented in FIG. 13 for each of
the five transformants relative to a control (non-transformed) cell
line. The results of the GUS analysis are summarized in Table
4.
4TABLE 4 GUS-activity analysis of GAM promoter fragment
transformants Geometric Mean Yeast line GAM Promoter fragment
Fluorescence Intensity Control N/A 4.82 478 0.3 kb (nucleotides
1827-2147 13.91 of SEQ ID NO.: 1) 469 1.5 kb (nucleotides 647-2147
16.1 of SEQ ID NO.: 1) 481 1.2 kb (nucleotides 647-1826 13.82 of
SEQ ID NO.: 1) 470 1.0 kb (nucleotides 1297-2147 16.55 of SEQ ID
NO.: 1) 484 0.7 kb (nucleotides 647-1297 15.09 of SEQ ID NO.:
1)
[0077] The results above indicate that the promoters corresponding
to the 1.5 kb GAM promoter fragment and the 1.0 kb GAM promoter
fragment have the highest promoter activity. The 300 base pair
fragment shows measurable activity above the control indicating
that such fragment can serve as a functional promoter. The 0.7 kb
fragment which lacks the sequence of the 1.0 kb promoter fragment,
is additionally functional as a promoter having activity between
that of the 300 kb minimal promoter region and the 1.0 and 1.5 kb
fragments. The lowest activity is observed for the 1.2 kb promoter
fragment which lacks the 300 kb minimal promoter region.
Accordingly, promoters of the invention and various constructs and
expression systems in accordance with the invention can preferably
comprise at least the 300 kb fragment, corresponding to nucleotides
1827-2147 of SEQ ID NO.: 1. Alternatively, or in addition to
nucleotides 1827-2147 of SEQ ID NO.: 1, isolated promoters of the
invention can comprise a promoter fragment corresponding to
nucleotides 647-1297 of SEQ ID NO.: 1. In some applications, an
isolated promoter of the invention can comprise at least
nucleotides 1297-2147 of SEQ ID NO.: 1.
Example 4
[0078] Expression in a plant cell protoplast host utilizing an
isolated yeast promoter.
[0079] Protoplasts were prepared from Nicotiana tabacum cell
suspension cultures. Super-coiled plasmid DNA, either pGA2100 or
pGA2101 described above, was combined with salmon sperm DNA as a
carrier, and was utilized for electroporation transformation of the
protoplasts. The protoplasts were subsequently cultured for 48
hours at 28.degree. C. in modified Murashige and Skoog (MS) medium
in the presence of sucrose. The protoplasts were then tested for
GUS activity.
[0080] The GUS activity of the protoplasts was analyzed by
extracting protein samples from the protoplasts using sonication of
suspended protoplasts, centrifugation, and collection of the
protein in the resulting supernatant. The supernatant was analyzed
for GUS activity as described above in previous examples. Four
transformant protoplast samples were compared to a control,
non-transformed protoplasts samples. The results of the GUS
activity analysis on the transformed protoplasts is presented in
Table 5. Samples 1 and 2 correspond to transformants containing the
1.5 kb GAM fragment and samples 3 and 4 correspond to transformant
protoplasts containing the 1.0 kb promoter fragment.
5TABLE 5 GUS-activity analysis of transformants grown in glucose
medium Isolated GUS specific Transformant Promoter Culture medium
activity (unit/mg) C* N/A sucrose 10.2 1 GAM15 sucrose 49.2 2 GAM15
sucrose 36.0 3 GAM10 sucrose 60.0 4 GAM10 sucrose 51.2 C*
Non-transformed control protoplasts
[0081] As indicated by the results shown in Table 5, the isolated
Schwanniomyces occidentalis promoter and fragments thereof are able
to function as a promoter in plant protoplast cultures.
[0082] The combined results of the examples set forth above
indicate that the GAM promoter isolated from Schwanniomyces
occidentalis and fragments thereof as small as 300 kb (nucleotides
1827-2147 of SEQ ID NO.: 1) can be utilized as a functional
promoter in other fungal species and also in species belonging to
other phylogenetic kingdoms including plants.
[0083] In compliance with the statute, the invention has been
described in language more or less specific as to structural and
methodical features. It is to be understood, however, that the
invention is not limited to the specific features shown and
described, since the means herein disclosed comprise preferred
forms of putting the invention into effect. The invention is,
therefore, claimed in any of its forms or modifications within the
proper scope of the appended claims appropriately interpreted in
accordance with the doctrine of equivalents.
Sequence CWU 1
1
10 1 2182 DNA Schwanniomyces castellii 1 tgatcatctt gaagttaaat
ccaagttatt caagtaattt aaagttgaat aatgtagtta 60 tttcagtggc
cttaaaccag tccatcgaga cgacttcagc ctcttcgaga ccacaaggtt 120
cgtttaataa ggaaatgaat agaatcacct ggagatattc gcagccatta atactatcaa
180 gtgaaaatcc tgaagaaaaa ttaattgcaa gatttttgac taatagtaaa
ggtagtgaac 240 atgaaagtgg tattcaagtt aaatttttga ttaatgatcc
tccactgaaa ttttctaagg 300 ctttatattt tgatgatgaa tcaacagagg
ttccttgtgt aaggaatctt attagtggaa 360 gctacagcag tcattcttaa
acatgattaa tgtctagatt tattggttat ttaggcattc 420 ttttttttaa
aatatttttt gttaatatct ttgagtttat gttttttgtt cgttttatct 480
tttaaagtag tgtttatagt tttagtattg ttaacctttt tttcctaaat gttagtatgc
540 atgcttaaaa tgatgtcaga ggtagagtat gaattaattc cttttataaa
tgctgttttg 600 tgagatcttt taaaattatc tatctttctc tttaaaggat
atgttttgat ttctgattga 660 tttgagttcc aacgacaatc gaatgtattc
atatagtagt tactacctta aacacaatcc 720 agatggttta accaactgat
gcctaagttt catgtggtgc tctttaacat cctttttgtc 780 ttcaaatttc
aatgccatta gttcacatgt atatacgcca agagagtttt gtgaccaact 840
tacatttact agcaagtatt atctacaaag caaaaattac gacatatttg tgttggatcc
900 atcaactgtg gacacgaata acaagttccc aggattccta attattcaac
tgccagataa 960 ataacatata tccaaaggtt caacattatt taccaaattc
aaagttggat tttgttaaat 1020 ggaatgacaa tagaaattgg ttgggtttat
gtgcaaaaga atctaatttt gcatatattt 1080 tcgtaaactt caattcctaa
aatcttgcga aacttctctt tagaggaaat tggttccatt 1140 ctaccttcta
tcaaactact ccaaatacaa gcggcttaaa atctacatgt aaatacctta 1200
ctgttacaat tattctccct tgaattgacc aacctgacca tgaaaccttt ttggaatcag
1260 cctatttaca ctaataattt ttatcctaag tgccatggaa gctattatat
aagttttacc 1320 agtgagagag gatcttgact tgacgaacaa catttcaact
agaatgctct atatcttcct 1380 ccgggaaaag cggccgctac catttgtttt
acactctcac catcacaaaa gtgccattca 1440 acggattttt gtccgcgatc
tctcggtaaa atgtgttctc gaaatgtgcc ttattgccaa 1500 aaaataaaaa
ataaaaaata atgtgggggt ggcatccttc aacttgtcgg atttattgcg 1560
taatagattt caatcaacat gatcttaatc catactggct tatgctctct tagaggctta
1620 tctcttaata attttattat atatctattc taactattga aaaactattg
aatatgcttt 1680 aaaactggct atgctgtatt tgacttctca atgcaaaatt
caacacttct ataatgtaac 1740 acactaaaaa tttttcagaa tcggaatagt
cgagacaatt gattttccga actattgcga 1800 aatccaatgg agcaacaatg
agagatctac attttaaacc ccagtctact ccagatattg 1860 gagtataacc
ccattcttac cgttatatcc atgacccgca tcgaaatttt caaaggattt 1920
cgaggaaatt ctttcctaaa atacgaagtg ttattggtga ttcaattact acggaaacta
1980 ctccattatg gatgtagagt tggtgaatgt agcgcaattg taatttgcga
agttatagta 2040 atagtttggc aaactggaga atttttcatt attgggaaaa
tataaataaa ggcaagtatc 2100 cattgaaatt ttaaaaatga actcatgact
gtattataac aagcaagatg atttttctga 2160 agctgattaa aagtatagta at 2182
2 328 DNA Schwanniomyces castellii 2 agatctacat tttaaacccc
agtctactcc agatattgga gtataacccc attcttaccg 60 ttatatccat
gacccgcatc gaaattttca aaggatttcg aggaaattct ttcctaaaat 120
acgaagtgtt attggtgatt caattactac ggaaactact ccattatgga tgtagagttg
180 gtgaatgtag cgcaattgta atttgcgaag ttatagtaat agtttggcaa
actggagaat 240 ttttcattat tgggaaaata taaataaagg caagtatcca
ttgaaatttt aaaaatgaac 300 tcatgactgt attataacaa gcaagatg 328 3 325
DNA Schwanniomyces castellii 3 agatctacat tttaaacccc agtctactcc
agatattgga gtataacccc attcttaccg 60 ttatatccat gacccgcatc
gaaattttca aaggatttcg aggaaattct ttcctaaaat 120 acgaagtgtt
attggtgatt caattactac ggaaactact catatggtag tagagttggt 180
gaatgtagcg caattgtaat ttgcgaagtt atagtaatag tttggcaaac tggagaattt
240 ttcattattg ggaaaatata aataaaggca agtatccatt gaaattttaa
aatgaactca 300 tgactgtatt ataacaagca agatg 325 4 35 DNA artificial
sequence oligonucleotide primer 4 gctctagaca tatgatgagt ttccgtagta
attga 35 5 35 DNA Artificial Sequence oligonucleotide primer 5
gctctagaat tactatactt ttaatcagct tcaga 35 6 36 DNA Artificial
Sequence Oligonucleotide primer 6 gatgcatgct atctttaatg actctgctgt
cgatgc 36 7 36 DNA Artificial Sequence Oligonucleotide primer 7
gatgcatgct agttgttaaa ccactggtgg aaggtg 36 8 33 DNA Artificial
Sequence Oligonucleotide primer 8 tctagaacta gtgatttctg attgatttga
gtt 33 9 33 DNA Artificial Sequence Oligonuceotide primer 9
tctagaacta gttctatcaa actactccaa ata 33 10 33 DNA Artificial
Sequence Oligonucleotide primer 10 ggtaccaagc ttcttgcttg ttataataca
gtc 33
* * * * *