U.S. patent number RE38,689 [Application Number 10/191,289] was granted by the patent office on 2005-01-25 for ceramidase gene.
This patent grant is currently assigned to Takara Shuzo Co., Ltd.. Invention is credited to Makoto Ito, Nozomu Okino.
United States Patent |
RE38,689 |
Okino , et al. |
January 25, 2005 |
Ceramidase gene
Abstract
To provide a polypeptide having ceramidase activity, and a gene
encoding thereof useful as an reagent for lipid engineering and as
indices for diagnosis of atopic dermatitis, an easy method for
detecting the polypeptide and method for detecting the gene as well
as a method for detecting atopic dermatitis. A polypeptide having
an amino acid sequence as shown in SEQ ID NO: 1 in Sequence
Listing, or a polypeptide having an amino acid sequence which has
substitution, deletion, addition or insertion of one or more amino
acids in the amino acid sequence of SEQ ID NO: 1 and having
ceramidase activity; a gene encoding the polypeptide; a gene
capable of hybridizing with the above genes under stringent
conditions, and encoding a polypeptide having ceramidase activity;
an oligonucleotide probe or primer, which is capable of hybridizing
under stringent conditions with the above genes or with a gene
having a nucleotide sequence complementary thereto; a method for
detecting a gene encoding a polypeptide having ceramidase activity,
by using the oligonucleotide probe and/or primer; an antibody or a
fragment thereof, which is capable of specifically binding to the
polypeptide; a method for detecting a polypeptide having ceramidase
activity, by using the above antibody or fragment thereof; a method
for detecting atopic dermatitis, by the methods.
Inventors: |
Okino; Nozomu (Fukuoka,
JP), Ito; Makoto (Fukuoka, JP) |
Assignee: |
Takara Shuzo Co., Ltd.
(Kyoto-fu, JP)
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Family
ID: |
16976083 |
Appl.
No.: |
10/191,289 |
Filed: |
July 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
328501 |
Jun 9, 1999 |
06258581 |
Jul 10, 2001 |
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Foreign Application Priority Data
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Aug 20, 1998 [JP] |
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10-234769 |
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Current U.S.
Class: |
435/195;
435/252.3; 435/320.1; 530/350; 536/23.2 |
Current CPC
Class: |
C12N
9/80 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C12N 15/00 (20060101); C07H
1/00 (20060101); C12N 1/20 (20060101); C12N
9/14 (20060101); C07H 21/04 (20060101); C12N
009/14 (); C12N 001/20 (); C12N 015/00 (); C07H
021/04 (); C07H 001/00 () |
Field of
Search: |
;435/195,252.3,320.1
;536/23.2 ;530/350 |
Foreign Patent Documents
Other References
Koch et al. J. Biol. Chem. 271 (51):33110-5, Dec. 20, 1996.* .
N. Okino et al., Purif. and Char. of a Novel Ceramidase from
Pseudomonas aeruginosa, J. Biol. Chem., 273:14368-14373, 1998.*
.
Jurgen Koch et al., Molecular Cloning and Characterization of a
Full-length Complementary DNA Encoding Human Acid Ceramidase, J.
Biol. Chem., 271(51):33110-33115, 1996.* .
Chi-Ming Li et al., Cloning and Cloning and Characterization of the
Full-Length cDNA and Genomic Sequences Encoding Murine Acid
Ceramidase, Genomics 50:267-274, 1998.* .
Suggs et al., PNAS 78 (11):6613-6617, 1981.* .
Edman et al., Eur. J. Biochem. 1:80-91, 1967..
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Primary Examiner: Saidha; Tekchand
Attorney, Agent or Firm: Birch, Stewart, Kolasch &
Birch, LLP
Claims
What is claimed is:
1. An isolated gene encoding a polypeptide having an amino acid
sequence as shown in SEQ ID NO: 1.
2. The isolated gene according to claim 1, wherein the gene has a
nucleotide sequence as shown in SEQ ID NO: 2.
3. An isolated gene capable of hybridizing with the gene of claim 1
under stringent conditions wherein incubation is made at 65.degree.
C. for overnight in a solution containing 6.times.SSC (composition
of 1.times.SSC:0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5%
SDS, 5.times.Denhardt's reagent, and 100 mg/ml herring sperm DNA,
and encoding a polypeptide having ceramidase activity.
4. A transformant harbouring the isolated gene of claim 1.
5. A method for producing a polypeptide having ceramidase activity,
comprising culturing the transformant of claim .[.5.].
.Iadd.4.Iaddend., and collecting a polypeptide having ceramidase
activity from the resulting culture.
6. A transformant harbouring the isolated gene of claim 3.
7. A method for producing a polypeptide having ceramidase activity,
comprising culturing the transformant of claim 6, and collecting a
polypeptide having ceramidase activity from the resulting culture.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a polypeptide having ceramidase
activity, and a gene encoding said polypeptide. More particularly,
the present invention relates to an amino acid sequence of
ceramidase as well as a nucleotide sequence encoding the amino acid
sequence which are useful as an reagent for lipid engineering for
analyzing structure and action of ceramide and as an index for
diagnosis of atopic dermatitis. In addition, the present invention
relates to a method for producing a polypeptide having ceramidase
activity by gene engineering. Further, the present invention
relates to a method for detecting the polypeptide, and a method for
detecting a gene encoding the polypeptide. Further, the present
invention relates to a method for detecting atopic dermatitis by
utilizing the above method for detecting the polypeptide and the
above method for detecting the gene.
2. Discussion of the Related Art
Ceramidase is an enzyme that hydrolyses ceramide, which is one of
sphingolipids, into sphingosine and a fatty acid. Sphingosine
formed by hydrolysis of ceramide by ceramidase has various
physiological activities, such as inhibition of protein kinase C,
activation of phospholipase D, inhibition of a calmodulin-dependent
enzyme, and the like, and is an important substance which is
considered to have a role in regulating cellular functions through
its involvement in cell proliferation and intracellular signal
transduction. The regulation of the level of such sphingosine is an
important role played by ceramidase.
Ceramidases are classified on the basis of its optimum pH into an
acidic ceramidase and a neutral/alkaline ceramidase. There have
been reported that ceramidases having optimum pH in an acidic
region are present in mammalian tissues such as rat brain
[Biochemistry, 8, 1692-1698 (1969)], guinea pig epithelial cell [J.
Biol. Chem., 270, 12677-12684 (1995)], human kidney [Biochim.
Biophys. Acta, 398, 125-131 (1975)], spleen [Biochim. Biophys.
Acta, 1004, 245-251 (1989)], fibroblast [Biochem. J., 205, 419-425
(1982)] and epithelium [FEBS Lett., 268, 110-112 (1990)]; human
urine [J. Biol. Chem., 270, 11098-11102 (1995)], and the like.
Among these ceramidases, the amino acid sequences and the
nucleotide sequences of ceramidase purified from human urine have
been determined [J. Biol. Chem., 271, 33110-33115 (1996)].
Utilizing the homology with this gene, a ceramidase gene of a mouse
has also been obtained [Genomics, 50, 267-274 (1998)]. However, all
these are acidic ceramidases derived from mammals, and no amino
acid sequences or nucleotide sequences of neutral/alkaline
ceramidase or of ceramidases derived from microorganisms have yet
been determined.
On the other hand, a ceramidase-producing microorganism is known to
be present on the epidermis of lesion of atopic dermatitis, and
this enzyme is suggested to be causative of or be involved in the
exacerbation of atopic dermatitis. However, this enzyme is
ceramidase of which optimum pH is within an alkaline region
(hereinafter referred to as alkaline ceramidase) [J. Biol. Chem.,
273, 14368-14373 (1998)].
When a naturally-occurring ceramidase is produced from a
ceramidase-producing microorganism, it is necessary to add an
expensive sphingolipid to a culture medium in order to induce the
production of an enzyme, and in purification step, it is necessary
to separate and remove the remaining sphingolipid or a degradation
product thereof from a fraction containing ceramidase. In addition,
since enzymes other than ceramidases such as sphingomyelinase are
simultaneously produced during the culture, it is difficult to
isolate and purify only a desired ceramidase from these enzymes.
Moreover, the amino acid sequences or the gene structures of
microorganism-derived alkaline ceramidase are completely unknown,
so that the method for producing ceramidase by gene engineering
cannot be utilized.
Furthermore, although the presence of the ceramidase-producing
microorganism is expected to be used as an index for diagnosing
atopic dermatitis and as a method for confirming a therapeutic
effect, no means for simply detecting or identifying such a
microorganism has yet been known. Conventional methods have
necessitated extremely complicated processes that the microorganism
is isolated and then cultured, and thereafter its ceramidase
activity is assayed. Thus, a method for producing a high-purity
ceramidase derived from microorganisms at a lower cost is desired
as well as a method for detecting a ceramidase-producing
microorganism in a simple, non-time-consuming procedure.
SUMMARY OF THE INVENTION
A first object of the present invention is to provide a polypeptide
having ceramidase activity. A second object of the present
invention is to provide a gene encoding the above polypeptide. A
third object of the present invention is to provide a transformant
harbouring the above gene. A fourth object of the present invention
is to provide a production method capable of easily and massively
producing high-purity ceramidase utilizing the above transformant.
A fifth object of the present invention is to provide an
oligonucleotide probe or a primer capable of specifically
hybridizing with the gene of the present invention. A sixth object
of the present invention is to provide an antibody or a fragment
thereof, which is capable of specifically binding to a polypeptide
of the present invention. A seventh object of the present invention
is to provide a method for detecting a ceramidase gene by using the
oligonucleotide probe or the primer of the present invention. An
eighth object of the present invention is to provide a method for
detecting ceramidase by using the antibody or the fragment thereof
of the present invention, and a kit used therefor. Furthermore, a
ninth object of the present invention is to provide a method for
detecting atopic dermatitis by utilizing a method for detecting the
ceramidase gene or method for detecting ceramidase of the present
invention.
As a result of intensive investigation in order to isolate a gene
encoding a polypeptide having ceramidase activity, the present
inventors have succeeded in isolation of a gene encoding a
polypeptide having ceramidase activity and elucidation of the
nucleotide sequence of the gene. In addition, based on such
findings, the present inventors have established a method capable
of easily and massively producing high-purity ceramidase; a method
for detecting a polypeptide having ceramidase activity; and a
method for detecting the ceramidase gene, and the present invention
has been completed thereby. In addition, the correlation between
such ceramidase and atopic dermatitis has been found, whereby a
method for simply detecting atopic dermatitis has been able to be
established.
Specifically, the gist of the present invention follows: [1] a
polypeptide having an amino acid sequence as shown in SEQ ID NO: 1
in Sequence Listing, or a polypeptide having an amino acid sequence
which has substitution, deletion, addition or insertion of one or
more amino acids in the amino acid sequence of SEQ ID NO: 1 and
having ceramidase activity; [2] a gene encoding the polypeptide of
the above item [1]; [3] the gene according to the above item [2],
wherein the gene has a nucleotide sequence as shown in SEQ ID NO: 2
in Sequence Listing, or a nucleotide sequence which has
substitution, deletion, addition or insertion of one or more bases
in the nucleotide sequence of SEQ ID NO: 2 and encodes a
polypeptide having ceramidase activity; [4] a gene capable of
hybridizing with the gene of the above item [2] under stringent
conditions, and encoding a polypeptide having ceramidase activity;
[5] a transformant harbouring the gene of the above item [2]; [6] a
method for producing a polypeptide having ceramidase activity,
comprising culturing the transformant of the above item [5], and
collecting a polypeptide having ceramidase activity from the
resulting culture; [7] an oligonucleotide probe or a primer, which
is capable of hybridizing under stringent conditions with the gene
of the above item [2] or with a gene having a nucleotide sequence
complementary thereto; [8] a method for detecting a gene encoding a
polypeptide having ceramidase activity, by using the
oligonucleotide probe and/or the primer of the above item [7]; [9]
a kit for detection of a gene encoding a polypeptide having
ceramidase activity, comprising the oligonucleotide probe and/or
the primer of the above item [7]; [10] an antibody or a fragment
thereof, which is capable of specifically binding to the
polypeptide of the above item [1]; [11] a method for detecting a
polypeptide having ceramidase activity, by using the antibody or
the fragment thereof of the above item [10]; [12] a kit for
detection of a polypeptide having ceramidase activity, comprising
the antibody or the fragment thereof of the above item [10]; [13] a
method for detecting atopic dermatitis, comprising detecting a gene
encoding a polypeptide having ceramidase activity by the method of
the above item [8]; and [14] a method for detecting atopic
dermatitis, comprising detecting a polypeptide having ceramidase
activity by the method of the above item [11].
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows a restriction endonuclease map for DNA insert of
plasmids pSCA59 and pGCB38.
DETAILED DESCRIPTION OF THE INVENTION (1) Polypeptide Having
Ceramidase Activity
In the present invention, the term "ceramidase" refers an enzyme
having activity for hydrolyzing ceramide into sphingosine and a
fatty acid as mentioned above.
Ceramidase activity can be determined according to a method
described in, for example, Journal of Biological Chemistry, 273,
14368-14373 (1998).
In the present invention, an origin of ceramidase includes, but not
particularly limited to, for example, ceramidases derived from
microorganisms, such as bacteria, Actinomycetes, yeasts,
filamentous fungi, Ascomycetes and basidiomycetes; or ceramidases
derived from organisms, such as plants, animals and insects.
Concretely, for instance, Pseudomonas aeruginosa strain AN-17,
isolated from skin of patients with atopic dermatitis is preferred
as a source for obtaining the ceramidase of the present invention.
An amino acid sequence of the ceramidase derived from the above
strain is shown in SEQ ID NO: 1 in Sequence Listing. The above
strain AN-17 is deposited under accession number FERM P-15699 with
the National Institute of Bioscience and Human-Technology, Agency
of Industrial Science and Technology, Ministry of International
Trade and Industry [Address: 1-3, Higashi 1-chome, Tsukuba-shi,
Ibaraki-ken Japan (postal code 305-8566)] since Jun. 26, 1996.
Incidentally, in the present specification, the term "polypeptide
having ceramidase activity" (the polypeptide may be simply referred
herein, as ceramidase) refers to a polypeptide having the amino
acid sequence as shown in SEQ ID NO: 1 in Sequence Listing. The
term "polypeptide having ceramidase activity" may further include
not only a polypeptide having an amino acid sequence of
naturally-occurring ceramidase but also a polypeptide having an
amino acid sequence which has a mutation such as substitution,
deletion, addition or insertion of one or more amino acids in the
amino acid sequence as shown in SEQ ID NO: 1, as long as the
polypeptide having an amino acid sequence which has a mutation is a
polypeptide having the same level of ceramidase activity as
determined by the method for determining activity as described the
above. In addition, the polypeptide having an amino acid sequence
may have two or more mutations, as long as the above mutation is a
mutation whereby the resulting polypeptide has ceramidase activity.
As described herein, "an amino acid sequence having a mutation"
includes amino acid sequences in which a naturally-occurring
mutation is introduced as well as those in which an artificial
mutation is introduced. (2) Ceramidase Gene
The ceramidase gene of the present invention refers to a gene
encoding the above polypeptide having ceramidase activity, i.e. a
nucleic acid having a nucleotide sequence encoding an amino acid
sequence of a polypeptide having ceramidase activity. The
ceramidase gene of the present invention includes, for example, a
gene encoding ceramidase derived from the above Pseudomonas
aeruginosa strain AN-17, of which nucleotide sequence is shown in
SEQ ID NO: 2 in Sequence Listing. The ceramidase gene of the
present invention also encompasses a gene encoding an amino acid
sequence of a polypeptide having an amino acid sequence which has
substitution, deletion, addition or insertion of one or more amino
acids in the above amino acid sequence of naturally-occurring
ceramidase, and having ceramidase activity.
The gene of the present invention also encompasses a gene having a
nucleotide sequence which has substitution, deletion, addition or
insertion of one or more bases in the nucleotide sequence as shown
in SEQ ID NO: 2 in Sequence Listing and encodes a polypeptide
having ceramidase activity.
As described herein, "nucleotide sequence which has substitution,
deletion, addition or insertion" includes a nucleic acid sequence
into which a naturally-occurring mutation is introduced or an
artificial mutation is introduced.
Further, the gene of the present invention encompasses a gene
capable of hybridizing with the above-mentioned ceramidase genes
under stringent conditions, and encoding a polypeptide having
ceramidase activity. Hybridization can be performed by a method
described in e.g. Molecular Cloning: A Laboratory Manual 2nd ed.,
published by Cold Spring Harbor Laboratory in 1989, edited by T.
Maniatis et al. The conditions for hybridization include, for
instance, the conditions of incubation with a probe at 65.degree.
C. for overnight in a solution containing 6.times.SSC (composition
of 1.times.SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5%
SDS, 5.times.Denhardt's reagent, and 100 mg/ml herring sperm
DNA.
Since the primary structure and the genetic structure of ceramidase
are clarified by the present invention, there can be obtained a
gene encoding a polypeptide having at least one of deletion,
addition, insertion or substitution of one or more amino acid
residues in an amino acid sequence of naturally-occurring
ceramidase by means of introducing random mutation or site-specific
mutation into the gene of the present invention, by which it is
made possible to obtain genes encoding ceramidases which have
ceramidase activity with slight differences in properties such as
optimum temperature, stable temperature, optimum pH, and stable pH.
In addition, there can be produced these ceramidases by gene
engineering.
The method for introducing random mutation includes, for example, a
method for causing transition mutation, in which cytosine base is
converted into uracil base, by a chemical treatment with sodium
hydrogensulfite [Proc. Natl. Acad. Sci. U.S.A., 79, 1408-1412
(1982)]; a method for lowering fidelity of incorporation of
nucleotides in DNA synthesis by carrying out PCR in a reaction
mixture containing manganese [Anal. Biochem., 224, 347-353 (1995)],
and the like.
The method for introducing site-specific mutation includes, for
example, a method utilizing amber mutation [gapped duplex method;
Nucleic Acids Res., 12, 9441-9456 (1984)]; a method utilizing a
host in which genes dut (dUTPase) and ung (uracil-DNA glycosilase)
are deleted [Kunkel method; Proc. Natl. Acad. Sci. U.S.A., 82,
488-492 (1985)]; a method comprising carrying out PCR by utilizing
amber mutation (WO98/02535), and the like. Various kits for
introducing site-specific mutation into the desired gene by these
methods are commercially available, and by utilizing the above
kits, there can be easily obtained a gene in which a desired
mutation is introduced.
A method for obtaining a microorganism-derived ceramidase gene by
means of hybridization method will be described below.
1) First, a partial amino acid sequence of ceramidase is determined
as information for preparing a probe for cloning a ceramidase gene.
A purified ceramidase is subjected to conventional amino acid
sequencing by Edman degradation without any treatment [J. Biol.
Chem., 256, 7990-7997 (1981)], or a purified peptide fragment
isolated and purified from a peptide mixture obtained by limited
hydrolysis by a highly substrate-specific proteolytic enzyme such
as lysyl endopeptidase and N-tosyl-L-phenylalanyl chloromethyl
ketone (TPCK)-tripsin is subjected to an amino acid sequencing.
Based on the information of the partial amino acid sequence as
elucidated above, an oligonucleotide to be used as a probe for
hybridization is designed, and synthesized chemically.
2) Genomic DNA from a ceramidase-producing microorganism is
prepared, digested completely with an appropriate restriction
enzyme, separated by agarose gel electrophoresis, and then blotted
on a nylon membrane by a usual method. The DNA fragment on the
nylon membrane is hybridized with the synthetic oligonucleotide
probe described above under generally employed conditions. For
example, the nylon membrane is blocked in a pre-hybridization
solution containing salmon sperm DNA and then incubated overnight
together with a .sup.32 P-labeled synthetic oligonucleotide probe.
After washing the nylon membrane, an autoradiogram is prepared, and
a DNA fragment hybridizing with the synthetic oligonucleotide is
detected.
3) A DNA fragment corresponding to signal on the autoradiogram is
extracted and purified from the agarose gel. The DNA fragment thus
obtained is incorporated into a vector such as a plasmid vector by
a usually employed method to prepare a recombinant DNA molecule.
The vector may be any of various commercially available vectors.
Subsequently, the recombinant DNA molecule is introduced into an
appropriate host such as Escherichia coli to obtain a transformant.
The method of transformation may be a usually employed method such
as one selected depending on a vector to be used from those
described in the above Molecular Cloning: A Laboratory Manual, 2nd
Ed., and the like.
4) Subsequently, a transformant harbouring a fragment of a
ceramidase gene is screened. A screening method may be
appropriately selected from colony hybridization, plaque
hybridization and the like depending on the kinds of the vectors.
There may also be employed PCR method using as a sample a colony or
a plaque as it is, or a method employing an expression of
ceramidase activity as an index.
5) A recombinant DNA molecule into which a fragment of a ceramidase
gene is incorporated is prepared from a transformant harbouring the
fragment, and the nucleotide sequence of the fragment is analyzed.
The nucleotide sequence may be determined by a usual method such as
dideoxy method. By comparing the nucleotide sequence thus
determined with the partial amino acid sequence of the ceramidase
obtained previously and the molecular weight of the ceramidase,
there can be determined whether the DNA fragment obtained is the
entire desired ceramidase gene, or a partial sequence thereof.
6) When the DNA fragment obtained does not contain the entire
length of the ceramidase gene, the entire length of the desired
ceramidase gene can be obtained by digesting genomic DNA with other
restriction enzymes using a part of the fragment as a probe; and
performing the hybridization in the same manner to obtain the
deleted region. Based on the DNA fragment comprising a ceramidase
gene thus obtained, the structure of the ceramidase gene and the
entire amino acid sequence of ceramidase are determined.
In addition to the hybridization methods described above, PCR
method may be employed to obtain a ceramidase gene of the present
invention. For example, PCR method using a primer designed based on
the N-terminal amino acid sequence or an internal partial amino
acid sequence of ceramidase or PCR method using a cassette DNA and
a cassette primer in addition to such a primer may be employed to
obtain a ceramidase gene.
Next, ceramidase derived from Pseudomonas aeruginosa AN-17 is taken
as an example and concretely described below. The above ceramidase
is alkaline ceramidase of which optimum pH is in the range of pH
8.0 to 9.0.
First, a partial amino acid sequence of ceramidase produced by
Pseudomonas aeruginosa AN-17 is examined. The N-terminal amino acid
sequence of the above ceramidase has a large number in the kinds of
the corresponding codons, and the number of the combinations of the
primer sequences becomes extremely large. In addition, when PCR is
performed using the above primer, a large number of the
non-specific amplified products are formed, and among these
products it is impossible to identify a product derived from a
desired gene. The present inventors have performed peptide mapping
using a proteolytic enzyme or a reagent for limited proteolysis
typically exemplified by cyanogen bromide in order to obtain a
sequence having a less corresponding codon degeneracy as an amino
acid sequence to be used to design a primer, and then tried to
design a primer. However, as described in Examples given below, PCR
is tried in combination of 4 kinds of primers, but no specific
amplified products could be obtained thereby. Accordingly, the
present inventors have further respectively synthesized, based on
the amino acid sequence obtained, synthetic oligonucleotide
primers, i.e., an oligonucleotide primer C-89-1 (SEQ ID NO: 13)
designed from a partial amino acid sequence C-89 (SEQ ID NO: 3) of
the ceramidase and an oligonucleotide primer C-91-cl (SEQ ID NO:
14) designed from a partial amino acid sequence C-91 (SEQ ID NO:
4). PCR is carried out using the above 2 kinds of primers together
with genomic DNA of Pseudomonas aeruginosa strain AN-17 as a
template, whereby obtaining a specifically amplified DNA fragment.
By examining the nucleotide sequence of this fragment and comparing
the results with the partial amino acid sequence of the ceramidase
other than those described above, it is found that the fragment
contains a part of a gene encoding the ceramidase.
This amplified DNA fragment is used as a probe to perform
hybridization or the like, whereby cloning a gene encoding the
entire length of the ceramidase.
The entire nucleotide sequence of a gene encoding ceramidase
derived from Pseudomonas aeruginosa strain AN-17 thus obtained
above is as shown in SEQ ID NO: 16 in Sequence Listing. In
addition, the amino acid sequence of the ceramidase deduced from
the above nucleotide sequence is as shown in SEQ ID NO: 15 in
Sequence Listing. Further, by comparing this amino acid sequence
with the N-terminal amino acid sequence of the ceramidase derived
from Pseudomonas aeruginosa strain AN-17 as shown in SEQ ID NO: 8
in Sequence Listing, it is found that the ceramidase produced by
this strain can be converted after translation to a mature enzyme
in which a polypeptide consisting of N-terminal 24 amino acids is
deleted. This mature ceramidase has an amino acid sequence as shown
in SEQ ID NO: 1 in Sequence Listing, and the nucleotide sequence of
the mature ceramidase-encoding region in the nucleotide sequence of
the ceramidase gene described above is as shown in SEQ ID NO: 2 in
Sequence Listing, respectively. Since the amino acid sequence of
the above ceramidase derived from Pseudomonas aeruginosa strain
AN-17 and the nucleotide sequence encoding it show no homology with
amino acid sequences and nucleotide sequences of known acidic
ceramidases, it is suggested that this enzyme derived from
Pseudomonas aeruginosa strain AN-17 is ceramidase belonging to a
member of a novel family.
By using an entire or a part of a ceramidase gene of which an
entire nucleotide sequence as described above is identified as a
probe for hybridization, a DNA having a high homology with a
ceramidase gene can be screened from genomic DNA libraries or cDNA
libraries obtained from the organisms other than Pseudomonas
aeruginosa strain AN-17. The hybridization may be performed under
generally employed conditions. For example, a nylon membrane in
which genomic DNA libraries or cDNA libraries obtained from the
organisms other than the Pseudomonas aeruginosa strain AN-17 is
immobilized is prepared. The nylon membrane is blocked at
65.degree. C. in a pre-hybridization solution containing
6.times.SSC, 0.5% SDS, 5.times.Denhardt's reagent, 100 mg/ml
herring sperm DNA, and then incubated at 65.degree. C. overnight
with adding the above probe which is .sup.32 P-labeled. This nylon
membrane is firstly washed in 6.times.SSC at room temperature for
10 minutes; subsequently in 2.times.SSC containing 0.1% SDS at room
temperature for 10 minutes; and further in 0.2.times.SSC containing
0.1% SDS at 45.degree. C. for 30 minutes. Thereafter, an
autoradiogram is prepared therefrom to detect DNA hybridizing with
the probe. Incidentally, genes having various homologies can be
obtained by altering conditions, such as washing.
On the other hand, a PCR primer can be designed also based on the
nucleotide sequence of the ceramidase gene of the present
invention. By carrying out PCR using this primer, it is possible to
detect a gene fragment having a high homology with the gene of the
present invention, or to obtain such a gene entirely, and also to
detect an organism which produces ceramidase.
In order to confirm whether or not a gene obtained by hybridization
or PCR described above is a gene which encodes a polypeptide having
ceramidase activity, deduction can be made by comparing the
nucleotide sequence of the gene obtained with the nucleotide
sequence or the amino acid sequence of the ceramidase gene of the
present invention. Alternatively, it is possible to confirm whether
or not the obtained gene is a desired gene by preparing a
polypeptide encoded by the gene obtained followed by assaying the
ceramidase activity by means of using a method as described below.
(3) Transformant Harbouring Ceramidase Gene
The transformant of the present invention is a transformant
harbouring the above ceramidase genes.
The gene of the present invention can be expressed in various hosts
by ligating the gene to known vectors, and the like. Incidentally,
since codon usage is different depending on a host for expressing
the gene of the present invention, the expression level may be
suppressed in the host in some cases. In this case, a codon used in
the gene of the present invention may be used to change into a
codon depending on the host used. In addition, the above expression
vector is not limited only to a plasmid vector, as long as the
vector does not hinder from achieving an object of the present
invention, and there can be used a phage vector, a cosmid vector
and the like. From the viewpoint of easily and massively producing
the polypeptide of the present invention, there are desired a
vector capable of inducing and expressing a foreign gene, a vector
capable of expressing as a fusion protein with a reporter gene
product and the like.
The transformant of the present invention can be obtained by
transforming a host with a vector carrying a ceramidase gene. As a
host, there can be used microorganisms such as Escherichia coli;
animal cells; and plant cells and the like. Subsequently, a
polypeptide having ceramidase activity is produced by culturing the
transformant under the conditions which are usually employed.
The transformant harbouring the ceramidase gene derived from the
above Pseudomonas aeruginosa AN-17 is named and identified as
Escherichia coli JM109/pTCDS7, and deposited with the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, Ministry of International Trade and
Industry [Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken
Japan (postal code 305-8566)] since Aug. 4, 1998 (original
deposition date) [accession number: FERM BP-6728.]
Confirmation of the ceramidase expression can be carried out by
determining ceramidase activity. The determination of the
ceramidase activity can be carried out by using cell extracts of
the transformant as a sample according to a method described in,
for example, Journal of Biological Chemistry, 273, 14368-14373
(1998). In addition, there can be used an antibody against
ceramidase, and in the case where ceramidase is expressed as a
fusion with another kind of a polypeptide, an antibody against the
polypeptide region may be used. When the antibody is used, the
ceramidase can be detected, for instance, by subjecting cell
extracts of the transformant to SDS-polyacrylamide gel
electrophoresis, transferring to polyvinylidene fluoride (PVDF)
membrane, and detecting with an antibody the corresponding
ceramidase on the above membrane. (4) Method for Producing
Polypeptide Having Ceramidase Activity
As one big feature, the method for producing a polypeptide having
ceramidase activity of the present invention comprises culturing
the transformant described above, and collecting a polypeptide
having ceramidase activity from the resulting culture.
In the method of the present invention, in the case where the
transformant is a microorganism or a cultured cell, optimum
conditions for expression of ceramidase in terms of the medium
composition, the pH of a medium, the culture temperature, the
culture time, the amount of an inducer used and the time period of
use may be set, whereby producing the ceramidase efficiently.
The purification of ceramidase from the culture of the transformant
may be carried out by a usual method. When the transformant
accumulates ceramidase in the cells as in the case of Escherichia
coli, the transformant cells are harvested by centrifugation after
termination of culture. The harvested cells are disrupted by
ultrasonic treatment or the like, and thereafter centrifuged to
obtain a cell-free extract. This cell-free extract is used as a
starting material to perform a generally employed protein
purification method such as salting out as well as various
chromatographies such as ion exchange, gel filtration, hydrophobic
and affinity chromatographies. In some transformants used herein,
an expression product is extracellularly secreted, and in such a
case, the product can be purified similarly from the culture
supernatant.
When the ceramidase produced by a transformant is produced in the
cells, various intracellular enzymes and proteins also coexist, but
only in very small amounts as compared to the ceramidase expressed,
thereby making the purification extremely simple. In addition, when
a vector of an extracellular secretory type is used as a vector,
ceramidase is extracellularly secreted, and a ceramidase-containing
fraction also coexists with medium components, and the like.
Nevertheless, since the fraction usually does not substantially
contain protein components which adversely affect the ceramidase
purification, a complicated isolation and purification procedure
which has been required for the purification of, for instance,
ceramidase from the culture of Pseudomonas aeruginosa AN-17 strain,
is not necessitated.
In addition, when Escherichia coli is used as a host, an expressed
product may be developed as an insoluble inclusion body. In this
case, the cells are harvested by centrifugation after termination
of the culture, and the harvested cells are disrupted by, for
example, ultrasonic treatment. Thereafter, the resulting disrupted
cells are centrifuged to collect an insoluble fraction comprising
the inclusion body. The inclusion body is washed and then
solubilized with a usually used solubilizer for protein, such as
urea or guanidine hydrochloride, and if necessary, the solubilized
inclusion body is purified by carrying out various chromatographies
such as ion exchange, gel filtration, hydrophobic and affinity
chromatographies, and then further subjected to a re-folding
procedure by dialysis procedure or dilution procedure, whereby
obtaining a preparation comprising a polypeptide maintaining
ceramidase activity. If necessary, the preparation thus obtained
can be further purified by various chromatographies to obtain a
high-purity polypeptide having ceramidase activity. (5)
Oligonucleotide Probe or Primer
The oligonucleotide probe or the primer of the present invention is
not particularly limited, as long as it is capable of hybridizing
under stringent conditions with the ceramidase gene described above
or a gene having a nucleotide sequence complementary therewith.
The term "stringent conditions" is not particularly limited,
referring to conditions, for instance, where a gene is incubated at
65.degree. C. overnight in a solution containing 6.times.SSC, 0.5%
SDS, 5.times.Denhardt's reagent, and 100 mg/ml herring sperm
DNA.
The oligonucleotide probe described above can be prepared by
chemically synthesizing, for instance, with a usual method
according to a design based on the nucleotide sequence for the
ceramidase gene of the present invention.
The length of the oligonucleotide probe described above is not
particularly limited, and the probe preferably consists of 15 bases
or longer, from the viewpoint of preventing a non-specific
hybridization, and more preferably 18 bases or longer.
Also, the primer of the present invention includes a nucleic acid
having the nucleotide sequence similar to that of the
oligonucleotide probe described above. For example, the primer can
be prepared by chemically synthesizing according to a design based
on the nucleotide sequence for the gene of the present invention.
The length of the primer is not particularly limited, and the
primer can consist of 15 to 40 bases, preferably 17 to 30 bases.
The primer described above can be subjected to various gene
amplification methods such as polymerase chain reaction (PCR)
method, whereby detecting the ceramidase gene of the present
invention.
In addition, as the oligonucleotide probe or the primer described
above, there can be employed a nucleic acid resulting from
fragmentating a nucleic acid encoding naturally-occurring
ceramidase by means of an enzymatic treatment such as a restriction
enzyme treatment or an exonuclease treatment, or a physical
treatment such as an ultrasonic treatment; and subjecting the
fragment thus obtained to isolation and purification process by
various methods for separating a nucleic acid as exemplified by
agarose gel electrophoresis. The nucleic acid thus obtained as
described above is desirably derived from a region having a
sequence peculiar to ceramidase.
The oligonucleotide probe or the primer described above can be
labeled with an appropriate label by a known method, and used to
detect the ceramidase gene of the present invention. The label is
not particularly limited, including, in addition to radioisotopes,
fluorescent substances and ligands such as biotin and digoxigenin.
(6) Method for Detecting Gene
One big feature of the method for detecting the gene of the present
invention resides in the detection of a gene in a sample to be
detected by using the oligonucleotide probe and/or the primer
described above.
In the detection method of the present invention, the detection of
a gene may be carried out by hybridization method, or the like with
the oligonucleotide probe described above, or alternatively, the
detection of a gene may be carried out by means of a DNA
amplification method such as PCR method using the primer described
above.
When the oligonucleotide is used, a sample to be detected may, for
example, be samples of a microorganism colony or a tissue segment,
DNA or RNA in these samples immobilized on a membrane, and DNA or
RNA extracted from these samples. Among these samples, the DNA
immobilized on a membrane or the DNA extracted is preferred from
the viewpoint of the stability of the samples.
When the oligonucleotide is used, a gene can be detected by a known
hybridization method such as those described in Molecular Cloning:
A Laboratory Manual, 2nd Ed. and the like.
The conditions of hybridization described above can appropriately
be selected depending on the factors such as the Tm value of the
probe used and the CG content of a target DNA. For example,
conditions described in Molecular Cloning: A Laboratory Manual, 2nd
Ed. can be applied.
When the primer is used, the sample to be detected may, for
example, be microorganism samples such as a culture medium of a
microorganism, a colony of a microorganism, and microorganism cells
as well as a body sample such as skin, tissues and tissue
segments.
The sample to be detected when using the primer described above may
be a microorganism isolated per se, or it may be one in which the
isolated microorganism is further subjected to an appropriate
treatment. A solid sample such as tissues may be used in a form of
an extract or a suspension. In addition, supernatant of such
samples, or one obtained by further subjecting the sample to cell
lysis treatment employing, for example, surfactant treatment as
well as supernatant thereof may also be used. Unless the nucleic
acid to be detected is affected adversely, any operation for
removing other components in the sample may also be carried
out.
When the detection is carried out by PCR method with the primer
described above, PCR conditions can appropriately be selected
depending on the factors such as the Tm value of the primer used
and the length of the amplified region to be detected.
When the primer described above is used, the detection can be
carried out by amplifying with DNA amplification method such as PCR
method; and confirming the presence or absence of a PCR-amplified
product. The method for confirming the presence or absence of
amplification is not particularly limited. For example, the
amplification can be confirmed by subjecting a reaction mixture of
nucleic acid amplification to agarose gel electrophoresis;
thereafter, staining the gel with an appropriate nucleic acid
staining reagent such as ethidium bromide, SYBER Green I or the
like; and detecting the presence or absence of the bands resulting
from irradiation with ultraviolet rays. The bands may be detected
by visual observation, or they may be detected by using, for
example, a fluorescent image analyzer, or the like.
In the detection method of the present invention, the probe and the
primer described above may be used in combination for the purpose
of increasing the detection sensitivity. For example, the primer
described above is used to amplify a ceramidase gene present in a
very small amount in a sample by means of PCR method, and then a
probe is used to hybridize with the gene, whereby achieving highly
sensitive and accurate detection.
When a ceramidase gene is detected by the detection method of the
present invention and the expression level of the gene is further
determined, there can be quantified the intensity of a signal
ascribed to a hybridized probe or a fluorescence intensity of a
band ascribed to a product amplified using a primer, or the like.
(7) Kit for Detecting Gene of the Present Invention
One of the feature of the kit for the detection of the gene of the
present invention resides in that the kit comprises the
oligonucleotide probe and/or the probe described above.
The kit of the present invention may further comprise various
reagents for hybridization typically exemplified by a membrane for
immobilizing a nucleic acid and hybridization buffer; PCR reagents
such as thermostable DNA polymerase, dNTPs mixture and PCR buffer;
reagents for detecting a probe or an amplified DNA; a medium for
microbial growth; and a reagent for extracting a nucleic acid from
a sample. (8) Antibody or Fragment Thereof, Which is Capable of
Specifically Binding to Polypeptide Having Ceramidase Activity
The antibody or a fragment thereof, which is capable of
specifically binding to the polypeptide of the present invention is
not particularly limited, as long as it is capable of specifically
binding to the polypeptide, and may be either of a polyclonal
antibody or a monoclonal antibody. Further, there can be employed
an antibody modified by a known method or a derivative of an
antibody, including, for example, a humanized antibody, a Fab
fragment, a single chain antibody and the like. Furthermore, the
antibody of the present invention can readily be obtained by
immunizing a rabbit or a mouse using an entire or a part of the
polypeptide of the present invention, for example, by a method
described in Current Protocols in Immunology [Ed. by John E.
Coligan, Issued by John Wiley & Sons, Inc. (1992)]. The
antibody can also be produced by means of a gene engineering
technique. In addition, the antibody or a fragment thereof, which
is capable of specifically binding to a certain partial fragment of
a polypeptide can also be encompassed.
Further, a fragment of the antibody is obtained by purifying the
antibody thus obtained, and then treating with a peptidase or the
like. The antibody or a fragment thereof thus obtained may be
applied to detection of a ceramidase-producing microorganism,
affinity chromatography, screening of various libraries (genomic
DNAs or cDNAs), a pharmaceutical, a diagnostic agent, a reagent for
research and development, and the like.
Moreover, the antibody or the fragment thereof of the present
invention may also be modified in various ways for the purpose of
facilitating a detection by enzyme immunoassay, fluorescent
immunoassay, luminescent immunoassay, and the like. (9) Method for
Detecting Polypeptide
One big feature of the method for detecting the polypeptide of the
present invention resides in the detection of a polypeptide having
ceramidase activity by using the antibody or the fragment thereof
described above.
In the present invention, a sample to be detected includes, for
instance, protein samples such as disrupted microbial cells, an
extract or a product obtained by washing tissues such as skin, and
a membrane onto which a protein derived from tissues or
microorganisms is immobilized.
The detection of a specific binding of the antibody or the fragment
thereof to the polypeptide described above may be carried out by a
known method, such as enzyme immunoassay, fluorescent immunoassay,
luminescent immunoassay and the like. (10) Kit for Method for
Detecting Polypeptide of the Present Invention
One feature of the kit for the method for detecting a polypeptide
of the present invention resides in that the kit comprises the
antibody or the fragment thereof described above.
The kit of the present invention may further comprise reaction
buffer, a labeled secondary antibody, a chromogenic reagent, and
the like. (11) Method for Detecting Atopic Dermatitis
As described above, the microorganism which produces ceramidase is
considered to be causative of or to be involved in exacerbation of
atopic dermatitis.
According to the method for detecting atopic dermatitis of the
present invention, atopic dermatitis can be detected by detecting
in a sample from skin a polypeptide having ceramidase activity, or
a gene encoding such a polypeptide.
The sample derived from skin used in the method for detecting
atopic dermatitis of the present invention is not particularly
limited, and may be a scale of skin of an individual to be tested,
a smear of skin obtained by wiping the skin with a cotton swab or a
gauze, a product obtained by washing skin, and the like. The method
for collecting such samples is not particularly limited. Examples
thereof include a method of collecting a scale directly from skin
of an individual to be tested; a method of wiping skin of an
individual to be tested with a cotton swab or a gauze; or a method
of collecting a washing product comprising bringing a cylinder of a
suitable size into close contact with skin of an individual to be
tested, the cylinder being filled with suitable fluid for washing
such as physiological saline or phosphate buffer, and then washing
the skin with the fluid for washing.
These samples thus obtained may directly be used for detection of
the polypeptide described above or for detection of the gene
encoding such a polypeptide. Further, the culture resulting from
culturing a microorganism contained in the sample may be cultured
in an appropriate medium, and used as a sample to be detected,
whereby improving the detection sensitivity. The medium used for
culturing the microorganism is not particularly limited, as long as
the microorganism in the sample from skin can be grown, and the
medium can efficiently produce ceramidase. For example, a medium
containing, as carbon sources and/or nitrogen sources, glycerol,
glucose, sucrose, syrups, yeast extracts, peptone, corn steep
liquor, meat extracts, delipidated soybean, ammonium sulfate,
ammonium nitrate, and the like, is appropriately used. In addition
to those listed above, minerals and metal salts such as sodium
salts, potassium salts, magnesium salts, zinc salts, phosphates,
and the like may also be contained. In addition, to the medium
there may also be added 0.001 to 1% of a lipid such as
sphingomyelin and ceramides, or a surfactant such as
taurodeoxycholates, in order to increase the expression level of
the ceramidase, whereby improving the detection sensitivity. The
culture conditions for the microorganisms are not particularly
limited, it is preferable that the microorganism is cultured at
25.degree. to 37.degree. C. for 1 to 7 days. After termination of
the culture, the culture may directly be used for a detection
procedure, or alternatively, when a polypeptide is detected, a
culture supernatant obtained by centrifuging the culture may be
used as a sample.
The method for detecting atopic dermatitis of the present invention
can be carried out by detecting a gene encoding a polypeptide
having ceramidase activity using the oligonucleotide probe or the
primer described above, or the method may also be carried out by
detecting a polypeptide having ceramidase activity using the
antibody or the fragment thereof described above, which is capable
of specifically binding to the polypeptide having ceramidase
activity. In addition, for the purpose of simply detecting atopic
dermatitis, there may be employed a kit for detecting the gene
encoding a polypeptide having ceramidase activity described above,
or a kit for detecting the polypeptide having ceramidase activity
described above. The kits described above may further comprise a
tool for obtaining a sample described below, including, for
example, a kit further comprising a cotton swab and a culture
medium.
In the method for detecting atopic dermatitis of the present
invention, when the gene encoding a polypeptide having ceramidase
activity is detected by using an oligonucleotide probe, for
example, a nucleic acid prepared by a known method from a sample
from skin of an individual to be tested is hybridized with an
oligonucleotide probe designed based on a nucleotide sequence of
the ceramidase gene under appropriate hybridization conditions for
detecting the formed hybrid. For example, the gene encoding a
polypeptide having ceramidase activity can be detected by dot blot
hybridization where a nucleic acid prepared from a sample from skin
is immobilized on a nitrocellulose membrane or the like.
Alternatively, a sample is immobilized directly on a nitrocellulose
membrane, and the nucleic acid contained in the sample is exposed
by appropriate lysis treatment, and then hybridized, whereby the
formed hybrid can be detected.
In the method for detecting atopic dermatitis of the present
invention, when the gene encoding a polypeptide having ceramidase
activity is detected by a means of gene amplification, a reaction
mixture is prepared from a sample from skin of an individual to be
tested or a nucleic acid prepared from such sample and a primer
designed based on the nucleotide sequence of a ceramidase gene, and
the reaction mixture is subjected to gene amplification reaction.
When PCR method is employed as gene amplification reaction, the
procedures described in Section (6) above are performed to amplify
a DNA, and an amplified DNA is then detected. For example, a
reaction mixture is prepared by adding a pair of primers prepared
based on a nucleotide sequence of a ceramidase gene and a sample
from skin, together with DNA polymerase and dNTPs, and
amplification reaction is performed under conditions appropriate
for the primer sequence and the length of the region expected to be
amplified. After termination of the reaction, the reaction mixture
is analyzed by agarose gel electrophoresis or the like to examine
the presence or absence of an amplification of the DNA fragment
derived from a desired gene.
When a gene encoding a polypeptide having ceramidase activity is
detected in a sample from skin by means of the method described
above, the site of skin where the sample is collected is diagnosed
to have atopic dermatitis.
In the method for detecting atopic dermatitis of the present
invention, when the polypeptide having ceramidase activity is
detected, an antibody or a fragment thereof, which is capable of
specifically binding to the polypeptide having ceramidase activity
is used to examine whether or not a polypeptide bound to the
antibody or the fragment thereof described above is present in the
sample from skin of an individual to be tested. The detection of
such a polypeptide can be performed by known immunological
experimental procedures such as immunodiffusion, latex
agglutination method and enzyme immunoassay. From the aspects of
the detection sensitivity and the simplicity of the operation, the
enzyme immunoassay is the most practical method.
When the polypeptide having ceramidase activity is detected in a
sample from skin by the method described above, the site of skin
where the sample is obtained is diagnosed to have atopic
dermatitis. (12) Kit for Detecting Atopic Dermatitis
In order to more simply perform the detection of atopic dermatitis
described above, the kit for detecting the gene encoding a
polypeptide having ceramidase activity described above or the kit
for detecting the polypeptide having ceramidase activity described
above may further comprise a tool for collecting a sample such as a
cotton swab and a culture medium.
According to the present invention, there are provided for the
first time an amino acid sequence of alkaline ceramidase as well as
a nucleotide sequence encoding it, whereby providing gene
engineering method for producing a polypeptide having ceramidase
activity using the above gene, a method for detecting the
ceramidase, and also a method for detecting atopic dermatitis. In
the method for producing ceramidase of the present invention, since
there is no need to add a sphingolipid to a medium for inducing an
expression of ceramidase and there is no contamination of, for
example, enzymes capable of simultaneously inducing, the enzymes
including sphingomyelinase, or there is no admixture of
sphingolipid added to the medium or its degradation products, the
desired ceramidase can readily be purified. Also, there are
provided a probe or a primer, which is capable of hybridizing
specifically with the ceramidase gene of the present invention as
well as an antibody or a fragment thereof, which is capable of
specifically binding to the polypeptide having ceramidase activity
of the present invention. By using the above probe, primer, and
antibody or fragment thereof, ceramidase can be detected simply and
at a high sensitivity. In addition, by using the above probe,
primer, and antibody or fragment thereof, there is exhibited an
excellent effect that the atopic dermatitis can be simply
detected.
EXAMPLES
The present invention is concretely illustrated by means of the
following examples, without intending to restrict the scope of the
present invention thereto.
Example 1
Cloning of Structural Gene of Ceramidase (1) Extraction and
Purification of Genomic DNA
A ceramidase-producing microorganism Pseudomonas aeruginosa strain
AN-17 (FERM P-15699) was inoculated to 200 ml of a PY medium (0.5%
polypeptone, 0.1% yeast extract, 0.5% sodium chloride, pH 7.2) and
cultured at 25.degree. C. for 23 hours. After termination of the
culture, a culture medium obtained was centrifuged to harvest
cells. The harvested cells were suspended in 10 ml of TE buffer (10
mM Tris-HCl, 1 mM EDTA, pH 8.0), and then thereto was added 0.2 ml
of a 50 mg/ml albumin lysozyme solution, and reacted at 30.degree.
C. for 15 minutes. Subsequently, to the solution obtained after
termination of the reaction was added 2 ml of 10% SDS, and the
mixture was stirred gently. Immediately after the solution became
viscous, 10 ml of TE buffer-saturated phenol and 1.5 ml of 5M NaCl
were added to the solution, and stirred gently at room temperature
for 1 hour. The solution obtained after stirring was centrifuged at
2500 rpm for 10 minutes, and supernatant was collected. To the
solution thus obtained was added an equivolume of chloroform and
stirred for 10 minutes, and then centrifuged at 1500 rpm for 10
minutes to collect supernatant. To the supernatant obtained above
was added once again an equivolume of chloroform, stirred, and
centrifuged to collect supernatant (hereinafter a series of these
operations is referred to as phenol-chloroform treatment). To the
supernatant thus collected, an equivolume of isopropanol was added
slowly, and a DNA precipitating on an interface was collected by
winding around a pasteur pipette, and then dissolved in 10 ml of a
TE buffer. To the solution thus obtained was added 20 .mu.l of
RNase A (those prepared by dissolving RNase in 10 mM Tris-HCl, pH
7.5, 15 mM NaCl so as to have a concentration of 10 mg/ml, and then
heat-treating at 100.degree. C. for 15 minutes), and the mixture
was incubated at 50.degree. C. for 1 hour. To the solution after
incubation, 10 .mu.l of a 20 mg/ml protease K solution, 200 .mu.l
of 5M NaCl, and 400 .mu.l of 10% SDS were further added, and the
mixture was incubated at 37.degree. C. for 1 hour, whereby a
reaction is performed. The solution after the reaction was allowed
to cool to room temperature, and subjected to phenol-chloroform
treatment. The treatment was repeated twice, and to an aqueous
phase obtained was added an equivolume of isopropanol and a 1/10
volume of 3M sodium acetate. The mixture was cooled at -20.degree.
C. for 1 hour, and then centrifuged at 10,000 rpm for 10 minutes to
obtain sedimentation. The obtained sediment was rinsed with 70%
ethanol, and dissolved in TE buffer to obtain genomic DNA solution.
By the above procedures, 1.1 mg of genomic DNA was obtained. (2)
Determination of Partial Amino Acid Sequence of Ceramidase
Ceramidase produced by Pseudomonas aeruginosa strain AN-17 (FERM
P-15699) was purified according to a method described in Journal of
Biological Chemistry, 273, No.23, 14368-14373 (1998). About 5 .mu.g
of the ceramidase obtained was applied to SDS-polyacrylamide gel
electrophoresis to be electrophoresed. Subsequently, the proteins
on the gel were transferred to a PVDF membrane ("Immobilon-P,"
manufactured by Millipore) by means of electroblotting. A region
corresponding to a band of ceramidase on this membrane (about 50
pmol) was cut out, and enzyme-digested at 37.degree. C. for 16
hours with shaking in 100 .mu.l of a 20 mM Tris-HCl buffer
containing 0.2 mg of lysyl endopeptidase (0.9 U) and 8%
acetonitrile. This enzyme-digested solution was applied to reverse
phase chromatography to purify a peptide fragment. The peptide
fragment thus obtained was analyzed by Edman degradation using gas
phase peptide sequencer Model 477 (manufactured by Applied
Biosystem) to determine the ceramidase internal partial amino acid
sequences C-89 (SEQ ID NO: 3 in Sequence Listing), C-91 (SEQ ID NO:
4 in Sequence Listing), S-90 (SEQ ID NO: 5 in Sequence Listing),
C-86 (SEQ ID NO: 6 in Sequence Listing) and C-121 (SEQ ID NO: 7 in
Sequence Listing).
On the other hand, apart from the above, the PVDF membrane
described above was applied to a peptide sequencer without a
treatment with lysyl endopeptidase to determine an N-terminal amino
acid sequence N-Term (SEQ ID NO: 8 in Sequence Listing). (3)
Amplification by PCR of Ceramidase Gene-Containing DNA Fragment
Based on the N-terminal amino acid sequence and the internal
partial amino acid sequence determined in Example 1-(2), each
primer as shown in each of SEQ ID NOS: 9 to 11 in Sequence Listing
was designed and synthesized by a DNA synthesizer.
Specifically, a sense mix primer S90-1 as shown in SEQ ID NO: 9 and
a sense mix primer S90-2 as shown in SEQ ID NO: 10, each
corresponding to S-90 as shown in SEQ ID NO: 5; an anti-sense mix
primer C89-cl as shown in SEQ ID NO: 11, an anti-sense mix primer
C89-c2 as shown in SEQ ID NO: 12 and an anti-sense mix primer C89-1
as shown in SEQ ID NO: 13, each corresponding to C-89 as shown in
SEQ ID NO: 3; and an anti-sense mix primer C91-cl as shown in SEQ
ID NO: 14 corresponding to C-91 as shown in SEQ ID NO: 4 were
respectively synthesized.
Each of these primers was subjected to PCR reaction. The PCR was
performed by using Gene Amp Reagent Kit (manufactured by Perkin
Elmer). Forty-reaction cycles of the reaction were carried out,
wherein 1 cycle consists of at 94.degree. C. for 0.5 minutes, at
51.degree. C. for 0.5 minutes, and at 72.degree. C. 1 minute.
In a PCR reaction with the combination of primer C89-1 with primer
C91-cl using the genomic DNA obtained in Example 1-(1) as a
template, amplification of a specific band corresponding to about
350 bp was detected. None of the combinations of S90-1 with C89-cl,
S90-1 with C89-c2, S90-2 with C89-cl, and S90-2 with C89-c2 showed
any amplification of specific bands.
The amplified DNA fragment of about 350 bp obtained above was
collected from a gel after the agarose gel electrophoresis, and the
collected DNA fragment was ligated to pGEM-T easy vector
(manufactured by Promega) to prepare a recombinant plasmid. Using
this plasmid as a template, the nucleotide sequence of the
amplified DNA fragment obtained above was determined by dideoxy
method. As a result, the nucleotide sequence obtained was found to
have a nucleotide sequence which encoded each of the partial amino
acid sequences of the ceramidase, namely, C-89, C-86, C-121 and
C-91, whereby clarifying that this amplified DNA fragment was a
part of the gene encoding a desired ceramidase. (4) Detection of
Ceramidase Gene-Containing DNA Fragment
The PCR amplified DNA fragment obtained in Example 1-(3) was used
as a probe to screen a genomic DNA fragment comprising a ceramidase
gene.
First, 5 .mu.g of the genomic DNA prepared in Example 1-(1) was
digested with 100 U of each of the restriction enzymes SphI, ApaI,
KpnI, XhoI, SalI, HincII, HindIII, EcoRV, EcoRI, PstI, SmaI, BamHI,
NotI and SacII at 37.degree. C. for 22 hours, and the resulting
restriction enzyme-digested DNA was applied to 1% agarose gel
electrophoresis. After the electrophoresis, the DNA was transferred
to a nylon membrane [Hybond-N+, manufactured by Amersham] by means
of Southern blotting. As a hybridization probe, one obtained by
labeling 0.1 .mu.g of the PCR-amplified DNA fragment obtained in
Example 1-(3) with .sup.32 P using DNA labeling kit (ReadyTo Go,
manufactured by Pharmacia) according to the protocol of the kit was
employed.
The nylon membrane described above was pre-hybridized at 65.degree.
C. for 1 hour or longer in a hybridization solution containing 0.5M
Church-Method phosphate buffer [Proc. Natl. Acad. Sci. U.S.A., 81,
1991-1995 (1984)], pH 7.0, 7% SDS and 1 mM EDTA, and then the
labeled probe described above was added thereto so as to have a
concentration of 6 fmol/ml, and the hybridization was carried out
at 65.degree. C. overnight. Subsequently, the nylon membrane was
washed three times each at 65.degree. C. for 15 minutes with a
washing solution previously warmed to 65.degree. C. (composition:
1% SDS, 40 mM sodium phosphate buffer). After removal of excessive
water from the nylon membrane, the nylon membrane was brought into
contact with imaging plate (manufactured by Fuji Photo Film Co.,
Ltd.) for 20 minutes to be photosensitized. After the
photosensitization, imaging plate was analyzed by BAS 1000 Imaging
Analyzer (manufactured by Fuji Photo Film Co., Ltd.) to detect a
probe on the nylon membrane. As a result, the digested products by
SphI, ApaI, KpnI, XhoI, SalI, HincII, HindIII, EcoRV, EcoRI, PstI,
SmaI, BamHI, NotI and SacII were found to have the signals ascribed
to the probes hybridized at the sites of approximately 7.1 kb, 2.8
kb, 12.6 kb, 5.8 kb, 1.8 kb, 1.8 kb, 13.7 kb, 12.6 kb, 9 kb, 6 kb,
3.5 kb, 6.7 kb, 11.8 kb and 1.5 kb, respectively. (5) Ceramidase
Gene Cloning
Based on the results of Example 1-(4), an about 2.8 kb ApaI
fragment was cloned. Ten milligrams of ApaI-digested genomic DNA
was separated by 1% agarose gel electrophoresis, and an agarose gel
at the position corresponding to a size of about 2.8 kb was cut
out. Using Sephaglas BandPrep Kit (manufactured by Amersham
Pharmacia), a DNA fragment was extracted from the gel and purified,
and this DNA fragment was inserted into pBluescript II SK
(manufactured by Toyobo Co., Ltd.) at ApaI site to prepare a
recombinant plasmid. Escherichia coli strain JM109 was transformed
with this plasmid, and cultured overnight on L plate of an agar
medium (composition: 1% tryptone, 0.5% yeast extract, 1% NaCl, pH
7.2) containing 100 .mu.g/ml of ampicillin, and from the colonies
formed 300 colonies were selected and inoculated onto a nylon
membrane (Hybond-N, manufactured by Amersham) placed on the same
agar plate. After culturing at 37.degree. C. for 16 hours, the
nylon membrane was treated for 5 minutes on a filter paper immersed
in an alkaline denaturation solution (composition: 0.5M NaOH, 1.5M
NaCl) (denaturation), and for 5 minutes on a filter paper immersed
in a neutralization solution [composition: 0.5M Tris-HCl (pH 7.5),
3M NaCl] (neutralization), and then rinsed with 2.times.SSC
(composition of 1.times.SSC: 0.15M NaCl, 0.015M sodium citrate, pH
7.0).
This nylon membrane was hybridized under the conditions described
above using as a probe the PCR-amplified DNA fragment obtained in
Example 1-(3), and 2 colonies exhibited the signals. One of these
colonies was used to prepare a plasmid, which was named pSCA59 and
used to determine the nucleotide sequence of the inserted DNA
fragment in this plasmid. As a result, the nucleotide sequences
corresponding to the amino acid sequence obtained in Example 1-(2),
C-89, C-86, C-121 and C-91 were found together with the stop codons
in the same frames, while no nucleotide sequence corresponding to
an N-terminal amino acid sequence N-Term or S-90 was found. In
other words, it was clarified that this plasmid contained 3'
terminal (C-terminal region) containing a stop codon of a
ceramidase gene, but not 5' terminal (N-terminal region).
Subsequently, in order to obtain the entire length of a ceramidase
gene, a DNA fragment encoding the region near the N-terminal which
was deleted in pSCA59 was screened by Southern hybridization method
in the same manner as described above. The probe used herein was a
KpnI/SacII-digested fragment of pSCA59 having a size of about 560
bp which contained the sequence near the 5' terminal of the
ceramidase gene. A KpnI/SacII fragment in a size of about 560 bp
isolated by agarose gel electrophoresis was labeled with .sup.32 P
in the same manner as described above, and subjected to Southern
hybridization in the same manner as described above. As a result,
each of the probes was hybridized with an SphI-digested fragment at
a site of about 7.1 kb, with an ApaI-digested fragment at a site of
about 2.8 kb, with an XhoI-digested fragment at a site of about 5.8
kb, with an EcoRI-digested fragment at a site of about 9 kb, with a
PstI-digested fragment at a site of about 6 kb, with an
SmaI-digested fragment at a site of about 3.5 kb, with a
BamHI-digested fragment at a site of about 2.1 kb, and with an
SacII-digested fragment at a site of about 2.5 kb,
respectively.
Based on the above results, an about 2.1 kb BamHI fragment was
cloned. Ten milligrams of a BamHI-digested genomic DNA was
separated by 1% agarose gel electrophoresis, and an agarose gel was
cut out at the position corresponding to a size of about 2.1 kb,
and a DNA fragment extracted and purified from the gel using
Sephaglas BandPrep Kit was inserted into pGEM-3Zf(+) (manufactured
by Promega) at a BamHI site. Nine positive colonies were obtained
by transforming Escherichia coli strain JM109 with this plasmid,
and performing colony hybridization in the same manner as described
above. A plasmid was prepared from one of these colonies, and named
pGCB38. Thereafter, the nucleotide sequence of the inserted DNA
fragment in the above pGCB38 was determined. First, various
deletion mutants were prepared by digesting this plasmid with
restriction enzymes PstI and XbaI, and then treating with
Exonuclease III (manufactured by Nippon Gene) and Mung Bean
Nuclease (manufactured by Nippon Gene) according to a standard
method. The nucleotide sequence of the ceramidase gene inserted in
pGCB38 was determined by analyzing the nucleotide sequences of
these mutants and pGCB38 with dideoxy method. As a result, the
nucleotide sequences corresponding to a ceramidase N-terminal amino
acid sequence N-Term and S-90 were found, whereby clarifying that
pGCB38 contained the region encoding the N-terminal of the
ceramidase gene.
A restriction map of the inserted DNA fragment of plasmids pSCA59
and pGCB 28 is shown in FIG. 1. Based on the results of the
analysis of the nucleotide sequences of the both plasmids, the
entire nucleotide sequence of the ceramidase gene and the amino
acid sequence of the ceramidase were determined. The nucleotide
sequence of open reading frame (ORF) encoding the ceramidase
produced by Pseudomonas aeruginosa strain AN-17 is as shown in SEQ
ID NO: 16 in Sequence Listing, and the amino acid sequence encoded
by this ORF is as shown in SEQ ID NO: 15 in Sequence Listing. A
nucleotide sequence encoding mature ceramidase, which was clarified
by an N-terminal amino acid sequence N-Term of the ceramidase
obtained in Example 1-(2) (SEQ ID NO: 8 in Sequence Listing), is
shown in SEQ ID NO: 2 in Sequence Listing. The amino acid sequence
of mature ceramidase encoded by this nucleotide sequence is shown
in SEQ ID NO: 1 in Sequence Listing.
Example 2
Construction of Plasmid Expressing Ceramidase Polypeptide
A plasmid expressing ceramidase was constructed by following the
procedures shown below.
Deletion mutants of plasmid pGCB38 prepared in Example 1-(5),
pGCB38-D13, has an insertion of up to 15 bases upstream of an
initiation codon of the ORF encoding ceramidase. A DNA fragment of
about 1.3 kb resulting from digestion of this plasmid with
restriction enzymes HindIII and BamHI was isolated and purified by
1% agarose gel electrophoresis to obtain DNA Fragment-1. On the
other hand, a DNA fragment of about 1.6 kb resulting from digestion
of plasmid pSCA59 with restriction enzymes HindIII and BamHI was
isolated and purified by 1% agarose gel electrophoresis to obtain
DNA Fragment-2. Subsequently, a HindIII-digested plasmid pTV119N
(manufactured by Takara Shuzo Co., Ltd.) was ligated with a mixture
of DNA Fragment-1 and DNA Fragment-2 obtained above. Thereafter, a
recombinant plasmid in which the insertion fragments were properly
inserted in the order of DNA Fragment-1 and DNA Fragment-2
downstream of a lac promoter of the pTV119N was selected, and named
pTCD11. Further, the pTCD11 was digested with a restriction enzyme
SmaI to prepare plasmid pTCDS7 in which a 500 bp SmaI fragment was
deleted. The Escherichia coli strain JM109 transformed with this
plasmid was named and indicated-as JM109/pTCDS7 under accession
number FERM BP-6728 and deposited to National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology, Ministry of International Trade and Industry.
Example 3
Expression of Ceramidase in Transformed E. coli
The Escherichia coli strain JM109/pTCDS7 obtained in Example 2 was
inoculated in 5 ml of L medium containing 100 .mu.g/ml of
ampicillin, and cultured with shaking at 37.degree. C. overnight,
and a 1 ml portion of the culture medium was inoculated to 200 ml
of the same L medium. After culturing at 37.degree. C. until the
turbidity (absorbance at 600 nm) reached about 0.6, to the
resulting culture was added isopropyl-.beta.-D-thiogalactoside
(IPTG) so as to give a final concentration of 0.1 mM, and then
further cultured with shaking at 37.degree. C. for 8 hours. After
termination of the culture, the cells were harvested by
centrifugation of the culture medium, suspended in 3 ml of a 10 mM
Tris-HCl buffer (pH 8.0) containing 0.5 mM
4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and treated
ultrasonically to disrupt the cells. The disrupted cell fluid thus
obtained was centrifuged to collect supernatant, which was used as
a crude enzyme solution.
This crude enzyme solution was assayed for its ceramidase activity
using C12-NBD-ceramide as a substrate according to the method
described in Journal of Biological Chemistry, 273, 14368-14373
(1998). Specifically, a reaction solution was prepared by
dissolving 550 pmol of C12-NBD-ceramide, 2.5 mM calcium chloride,
0.25% (w/v) Triton X-100 and an appropriate amount of the crude
enzyme solution in 20 .mu.l of a 25 mM Tris-HCl buffer (pH 8.5),
and the reaction solution was incubated at 37.degree. C. for 20
minutes. The resulting reaction mixture was incubated in a boiling
water for 5 minutes to terminate the reaction, and the reaction
mixture was further dried under reduced pressure. The dried product
was dissolved in chloroform/methanol=2/1 (weight ratio) solution,
and subjected to silica gel thin layer chromatography [eluent:
chloroform/methanol/25% aqueous ammonia=90/20/0.5 (volume ratio)].
After the development, the amounts of C12-NBD-ceramide and its
degradation product C12-NBD-fatty acid at the excitation wavelength
of 475 nm and the fluorescence wavelength of 525 nm were assayed
with a CS-9300 chromatoscanner (manufactured by Shimadzu
Corporation). One unit of this enzyme was defined as an amount of
enzyme required for the formation of 1 .mu.mol of C12-NBD-fatty
acid per one minute under the conditions described above. As a
result, it was shown that the Escherichia coli strain JM109/pTCDS7
harbouring the ceramidase gene according to the present invention
produced about 32 units of the ceramidase per 1 L of the culture
medium.
Example 4
Detection of Atopic Dermatitis
Based on the nucleotide sequence of the ceramidase gene described
above, primer 382U, a primer for upstream, and primer 884L, a
primer for downstream, were designed and synthesized as primers for
detecting a ceramidase gene. The nucleotide sequences of primer
382U and primer 884L are as shown in SEQ ID NOS: 17 and 18 in
Sequence Listing. When a PCR was performed using these two primers
and a ceramidase gene of the nucleotide sequence shown in SEQ ID
NO: 2 in Sequence Listing as a template, a DNA fragment of 523 bp
was amplified.
Skin-derived samples were collected from skin of each of 24
patients with atopic dermatitis and 8 normal individuals in the
following procedures. Skin of an individual to be tested was wiped
with a sterilized cotton swab, and the swab was immersed in 200
.mu.l of a sterilized distilled water, and stirred vigorously prior
to collecting a sample. The collected sample was heated at
100.degree. C. for 5 minutes to obtain a sample solution to be used
for a PCR.
The PCR was performed using EXTaq (manufactured by Takara Shuzo
Co., Ltd.). 100 .mu.l of a PCR reaction solution containing 5 .mu.l
of the sample solution, each. 0.2 .mu.M of primer 382U and primer
884L, each 0.2 mM of dATP, dCTP, dGTP and dTTP, a 2.5 U of EXTaq
was prepared using a reaction buffer appended to EXTaq for 40
cycles, wherein one cycle consists of 1 minute at 94.degree. C., 1
minute at 60.degree. C., and 1 minute at 72.degree. C. A part of
the resulting reaction mixture was subjected to an agarose gel
electrophoresis, and the resulting gel after electrophoresis was
stained with ethidium bromide to examine the presence or absence of
an amplification of the DNA fragment.
As a result, in samples of 15 out of 24 patients with atopic
dermatitis, a specific amplification of DNA fragment of about 500
bp, an index for the presence of a ceramidase gene was found. On
the other hand, no such amplification of the fragment was noted in
any of 8 samples derived from the normal individuals.
EQUIVALENT
The invention may be embodied in other specific forms without
departing from the spirit or essential characteristics thereof. The
present embodiment is therefore to be considered in all respects as
illustrative and not restrictive, the scope of the invention being
indicated by the appended claims rather than by the foregoing
description and all changes which come within the meaning and range
of equivalency of the claims are therefore intended to be embraced
therein.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 18 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 646 <212> TYPE:
PRT <213> ORGANISM: Pseudomonas aeruginosa <400>
SEQUENCE: 1 Asp Asp Leu Pro Tyr Arg Phe Gly Leu Gly Lys Ala Asp Ile
Thr Gly 1 5 10 15 Glu Ala Ala Glu Val Gly Met Met Gly Tyr Ser Ser
Leu Glu Gln Lys 20 25 30 Thr Ala Gly Ile His Met Arg Gln Trp Ala
Arg Ala Phe Val Ile Glu 35 40 45 Glu Ala Ala Ser Gly Arg Arg Leu
Val Tyr Val Asn Thr Asp Leu Gly 50 55 60 Met Thr Phe Gln Ala Val
His Leu Lys Val Leu Ala Arg Leu Lys Ala 65 70 75 80 Lys Tyr Pro Gly
Val Tyr Asp Glu Asn Asn Val Met Leu Ala Ala Thr 85 90 95 His Thr
His Ser Gly Pro Gly Gly Phe Ser His Tyr Ala Met Tyr Asn 100 105 110
Leu Ser Val Leu Gly Phe Gln Glu Lys Thr Phe Asn Ala Ile Val Asp 115
120 125 Gly Ile Val Arg Ser Ile Glu Arg Ala Gln Ala Arg Leu Gln Pro
Gly 130 135 140 Arg Leu Phe Tyr Gly Ser Gly Glu Leu Arg Asn Ala Ser
Arg Asn Arg 145 150 155 160 Ser Leu Leu Ser His Leu Lys Asn Pro Asp
Ile Ala Gly Tyr Glu Asp 165 170 175 Gly Ile Asp Pro Gln Met Ser Val
Leu Ser Phe Val Asp Ala Asn Gly 180 185 190 Glu Leu Ala Gly Ala Ile
Ser Trp Phe Pro Val His Ser Thr Ser Met 195 200 205 Thr Asn Ala Asn
His Leu Ile Ser Pro Asp Asn Lys Gly Tyr Ala Ser 210 215 220 Tyr His
Trp Glu His Asp Val Ser Arg Lys Ser Gly Phe Val Ala Ala 225 230 235
240 Phe Ala Gln Thr Asn Ala Gly Asn Leu Ser Pro Asn Leu Asn Leu Lys
245 250 255 Pro Gly Ser Gly Pro Phe Asp Asn Glu Phe Asp Asn Thr Arg
Glu Ile 260 265 270 Gly Leu Arg Gln Phe Ala Lys Ala Tyr Glu Ile Ala
Gly Gln Ala Gln 275 280 285 Glu Glu Val Leu Gly Glu Leu Asp Ser Arg
Phe Arg Phe Val Asp Phe 290 295 300 Thr Arg Leu Pro Ile Arg Pro Glu
Phe Thr Asp Gly Gln Pro Arg Gln 305 310 315 320 Leu Cys Thr Ala Ala
Ile Gly Thr Ser Leu Ala Ala Gly Ser Thr Glu 325 330 335 Asp Gly Pro
Gly Pro Leu Gly Leu Glu Glu Gly Asn Asn Pro Phe Leu 340 345 350 Ser
Ala Leu Gly Gly Leu Leu Thr Gly Val Pro Pro Gln Glu Leu Val 355 360
365 Gln Cys Gln Ala Glu Lys Thr Ile Leu Ala Asp Thr Gly Asn Lys Lys
370 375 380 Pro Tyr Pro Trp Thr Pro Thr Val Leu Pro Ile Gln Met Phe
Arg Ile 385 390 395 400 Gly Gln Leu Glu Leu Leu Gly Ala Pro Ala Glu
Phe Thr Val Met Ala 405 410 415 Gly Val Arg Ile Arg Arg Ala Val Gln
Ala Ala Ser Glu Ala Ala Gly 420 425 430 Ile Arg His Val Val Phe Asn
Gly Tyr Ala Asn Ala Tyr Ala Ser Tyr 435 440 445 Val Thr Thr Arg Glu
Glu Tyr Ala Ala Gln Glu Tyr Glu Gly Gly Ser 450 455 460 Thr Leu Tyr
Gly Pro Trp Thr Gln Ala Ala Tyr Gln Gln Leu Phe Val 465 470 475 480
Asp Met Ala Val Ala Leu Arg Glu Arg Leu Pro Val Glu Thr Ser Ala 485
490 495 Ile Ala Pro Asp Leu Ser Cys Cys Gln Met Asn Phe Gln Thr Gly
Val 500 505 510 Val Ala Asp Asp Pro Tyr Ile Gly Lys Ser Phe Gly Asp
Val Leu Gln 515 520 525 Gln Pro Arg Glu Ser Tyr Arg Ile Gly Asp Lys
Val Thr Val Ala Phe 530 535 540 Val Thr Gly His Pro Lys Asn Asp Leu
Arg Thr Glu Lys Thr Phe Leu 545 550 555 560 Glu Val Val Asn Ile Gly
Lys Asp Gly Lys Gln Thr Pro Val Thr Val 565 570 575 Ala Thr Asp Asn
Asp Trp Asp Thr Gln Tyr Arg Trp Glu Arg Val Gly 580 585 590 Ile Ser
Ala Ser Lys Ala Thr Ile Ser Trp Ser Ile Pro Pro Gly Thr 595 600 605
Glu Pro Gly His Tyr Tyr Ile Arg His Tyr Gly Asn Ala Lys Asn Phe 610
615 620 Trp Thr Gln Lys Ile Ser Glu Ile Gly Gly Ser Thr Arg Ser Phe
Glu 625 630 635 640 Val Leu Gly Thr Thr Pro 645 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211>
LENGTH: 1941 <212> TYPE: DNA <213> ORGANISM:
Pseudomonas aeruginosa <400> SEQUENCE: 2 gacgacctgc
cctaccgctt cggcctgggc aaggcggaca tcaccggcga agccgccgaa 60
gtcggcatga tgggttactc ctccctcgaa cagaagaccg ccggcatcca catgcgccag
120 tgggcgcgtg ccttcgtgat cgaggaagcg gccagcggac gtcgcctggt
ctacgtcaac 180 accgacctgg ggatgacctt ccaggccgtg cacctgaagg
tcctggcccg gctcaaggcg 240 aagtaccccg gtgtctacga cgagaacaac
gtgatgctcg ccgccaccca cacccactcc 300 ggtccgggcg gcttctccca
ctacgcgatg tacaacctgt cggtgctcgg cttccaggaa 360 aagaccttca
acgccatcgt cgacggcatc gtccgctcca tcgagcgggc ccaggccagg 420
ttgcagcccg gccgcctgtt ctacggcagc ggcgagctgc gcaacgccag ccgcaaccgt
480 tcgctgctgt cgcacctgaa gaatccggac atcgccggct acgaggatgg
catcgacccg 540 cagatgagcg tgctcagctt cgtcgacgcc aacggcgagc
tggccggcgc gatcagttgg 600 ttcccggtgc acagcacctc gatgaccaac
gccaatcacc tgatctcccc ggacaacaag 660 ggctacgcct cctatcactg
ggagcacgac gtcagccgca agagcggttt cgtcgccgcc 720 ttcgcccaga
ccaatgccgg caacctgtcg cccaacctga acctgaagcc cggctccggt 780
cccttcgaca acgagttcga caacacccgc gagatcggtc tgcgccaatt cgccaaggcc
840 tacgagatcg ccggccaggc ccaggaggaa gtgctcggcg aactggattc
gcgcttccgt 900 ttcgtcgact tcacccgcct gccgatccgc ccggagttca
ccgacggcca gccgcgccag 960 ttgtgcaccg cggccatcgg caccagcctg
gccgccggta gcaccgaaga cggtccaggc 1020 ccgctggggc tggaggaagg
caacaatccg ttcctctcgg cccttggcgg gttgctcacc 1080 ggcgtgccgc
cgcaggaact ggtgcaatgc caggcggaaa agaccatcct cgccgacacc 1140
ggcaacaaga aaccctaccc ctggacgccg acggtgctgc cgatccagat gttccgcatc
1200 ggccagttgg aactgctcgg cgcccccgcc gagttcaccg tgatggccgg
ggtgcggatc 1260 cgccgcgcgg tgcaggcggc cagcgaagcg gccggtatcc
gccatgtggt cttcaatggc 1320 tacgcgaatg cctatgccag ctacgtcacc
acccgcgagg aatacgccgc ccaggaatac 1380 gaaggcggct cgaccctcta
cggcccctgg acccaggccg cctaccagca gttgttcgtc 1440 gacatggcgg
tggcgctgcg cgaacgcctg ccggtggaaa cctcggcgat agcgccggac 1500
ctgtcctgct gccagatgaa cttccagacc ggagtagtgg ccgatgatcc ctatatcggc
1560 aagtccttcg gcgacgtgtt gcaacaaccc agggaaagtt atcgcatcgg
cgacaaggtg 1620 accgtcgctt tcgtgaccgg acatccgaag aatgacttgc
gcaccgagaa gactttcctg 1680 gaagtggtga atatcggcaa ggatggcaaa
cagacgcccg tgaccgttgc caccgataat 1740 gactgggata cccaataccg
ctgggagaga gtgggtatat ctgcctcgaa agcgactatc 1800 agctggtcca
ttccaccagg gaccgagccc ggccattact acatcaggca ctatggcaac 1860
gcgaagaact tctggaccca gaagatcagc gaaatcggcg gctcgacccg ctccttcgag
1920 gtgctcggca ccactcccta g 1941 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa
<400> SEQUENCE: 3 Ser Phe Gly Asp Val Leu Gln Gln Pro Arg Glu
Ser Tyr Arg Ile Gly 1 5 10 15 Asp Lys <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa
<400> SEQUENCE: 4 Ile Ser Glu Ile Gly Gly Ser Thr Arg Ser Phe
Glu Val Leu Gly Thr 1 5 10 15 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 5 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Pseudomonas aeruginosa <400>
SEQUENCE: 5 Asp Asp Leu Pro Tyr Arg Phe Gly Leu Gly Lys 1 5 10
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Pseudomonas aeruginosa <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (3)..(3) <223> OTHER
INFORMATION: Xaa equals any amino acid <400> SEQUENCE: 6 Val
Thr Xaa Ala Phe Val Thr Gly His Pro Lys 1 5 10 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Pseudomonas aeruginosa
<400> SEQUENCE: 7 Thr Phe Leu Glu Val Val Asn Ile Gly Lys 1 5
10 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Pseudomonas aeruginosa <400> SEQUENCE: 8 Asp Asp Leu Pro Tyr
Arg Phe Gly Leu Gly Lys Ala Asp Ile Thr Gly 1 5 10 15 Glu Ala Ala
Glu Val Gly Met Met Gly 20 25 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 9 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Designed oligonucleotide based on
the amino acid sequence represented in SEQ ID NO5 <400>
SEQUENCE: 9 gaygayctnc cntaymg 17 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 10 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Designed
oligonucleotide based on the amino acid sequence represented in SEQ
ID NO5 <400> SEQUENCE: 10 gaygayttrc cntaymg 17 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 11 <211>
LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Designed oligonucleotide based on the amino acid sequence
represented in SEQ ID NO3 <400> SEQUENCE: 11 ccratnckrt
asgaytc 17 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 12 <211> LENGTH: 17 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Designed oligonucleotide based on the amino acid
sequence represented in SEQ ID NO3 <400> SEQUENCE: 12
ccratnckrt agctytc 17 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 13 <211> LENGTH: 17 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Designed oligonucleotide based on
the amino acid sequence represented in SEQ ID NO3 <400>
SEQUENCE: 13 gaygtsytsc arcarcc 17 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 14 <211> LENGTH: 17
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Designed
oligonucleotide based on the amino acid sequence represented in SEQ
ID NO4 <400> SEQUENCE: 14 aagctycasr asccvtg 17 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15 <211>
LENGTH: 670 <212> TYPE: PRT <213> ORGANISM: Pseudomonas
aeruginosa <400> SEQUENCE: 15
Met Ser Arg Ser Ala Phe Thr Ala Leu Leu Leu Ser Cys Val Leu Leu 1 5
10 15 Ala Leu Ser Met Pro Ala Arg Ala Asp Asp Leu Pro Tyr Arg Phe
Gly 20 25 30 Leu Gly Lys Ala Asp Ile Thr Gly Glu Ala Ala Glu Val
Gly Met Met 35 40 45 Gly Tyr Ser Ser Leu Glu Gln Lys Thr Ala Gly
Ile His Met Arg Gln 50 55 60 Trp Ala Arg Ala Phe Val Ile Glu Glu
Ala Ala Ser Gly Arg Arg Leu 65 70 75 80 Val Tyr Val Asn Thr Asp Leu
Gly Met Thr Phe Gln Ala Val His Leu 85 90 95 Lys Val Leu Ala Arg
Leu Lys Ala Lys Tyr Pro Gly Val Tyr Asp Glu 100 105 110 Asn Asn Val
Met Leu Ala Ala Thr His Thr His Ser Gly Pro Gly Gly 115 120 125 Phe
Ser His Tyr Ala Met Tyr Asn Leu Ser Val Leu Gly Phe Gln Glu 130 135
140 Lys Thr Phe Asn Ala Ile Val Asp Gly Ile Val Arg Ser Ile Glu Arg
145 150 155 160 Ala Gln Ala Arg Leu Gln Pro Gly Arg Leu Phe Tyr Gly
Ser Gly Glu 165 170 175 Leu Arg Asn Ala Ser Arg Asn Arg Ser Leu Leu
Ser His Leu Lys Asn 180 185 190 Pro Asp Ile Ala Gly Tyr Glu Asp Gly
Ile Asp Pro Gln Met Ser Val 195 200 205 Leu Ser Phe Val Asp Ala Asn
Gly Glu Leu Ala Gly Ala Ile Ser Trp 210 215 220 Phe Pro Val His Ser
Thr Ser Met Thr Asn Ala Asn His Leu Ile Ser 225 230 235 240 Pro Asp
Asn Lys Gly Tyr Ala Ser Tyr His Trp Glu His Asp Val Ser 245 250 255
Arg Lys Ser Gly Phe Val Ala Ala Phe Ala Gln Thr Asn Ala Gly Asn 260
265 270 Leu Ser Pro Asn Leu Asn Leu Lys Pro Gly Ser Gly Pro Phe Asp
Asn 275 280 285 Glu Phe Asp Asn Thr Arg Glu Ile Gly Leu Arg Gln Phe
Ala Lys Ala 290 295 300 Tyr Glu Ile Ala Gly Gln Ala Gln Glu Glu Val
Leu Gly Glu Leu Asp 305 310 315 320 Ser Arg Phe Arg Phe Val Asp Phe
Thr Arg Leu Pro Ile Arg Pro Glu 325 330 335 Phe Thr Asp Gly Gln Pro
Arg Gln Leu Cys Thr Ala Ala Ile Gly Thr 340 345 350 Ser Leu Ala Ala
Gly Ser Thr Glu Asp Gly Pro Gly Pro Leu Gly Leu 355 360 365 Glu Glu
Gly Asn Asn Pro Phe Leu Ser Ala Leu Gly Gly Leu Leu Thr 370 375 380
Gly Val Pro Pro Gln Glu Leu Val Gln Cys Gln Ala Glu Lys Thr Ile 385
390 395 400 Leu Ala Asp Thr Gly Asn Lys Lys Pro Tyr Pro Trp Thr Pro
Thr Val 405 410 415 Leu Pro Ile Gln Met Phe Arg Ile Gly Gln Leu Glu
Leu Leu Gly Ala 420 425 430 Pro Ala Glu Phe Thr Val Met Ala Gly Val
Arg Ile Arg Arg Ala Val 435 440 445 Gln Ala Ala Ser Glu Ala Ala Gly
Ile Arg His Val Val Phe Asn Gly 450 455 460 Tyr Ala Asn Ala Tyr Ala
Ser Tyr Val Thr Thr Arg Glu Glu Tyr Ala 465 470 475 480 Ala Gln Glu
Tyr Glu Gly Gly Ser Thr Leu Tyr Gly Pro Trp Thr Gln 485 490 495 Ala
Ala Tyr Gln Gln Leu Phe Val Asp Met Ala Val Ala Leu Arg Glu 500 505
510 Arg Leu Pro Val Glu Thr Ser Ala Ile Ala Pro Asp Leu Ser Cys Cys
515 520 525 Gln Met Asn Phe Gln Thr Gly Val Val Ala Asp Asp Pro Tyr
Ile Gly 530 535 540 Lys Ser Phe Gly Asp Val Leu Gln Gln Pro Arg Glu
Ser Tyr Arg Ile 545 550 555 560 Gly Asp Lys Val Thr Val Ala Phe Val
Thr Gly His Pro Lys Asn Asp 565 570 575 Leu Arg Thr Glu Lys Thr Phe
Leu Glu Val Val Asn Ile Gly Lys Asp 580 585 590 Gly Lys Gln Thr Pro
Val Thr Val Ala Thr Asp Asn Asp Trp Asp Thr 595 600 605 Gln Tyr Arg
Trp Glu Arg Val Gly Ile Ser Ala Ser Lys Ala Thr Ile 610 615 620 Ser
Trp Ser Ile Pro Pro Gly Thr Glu Pro Gly His Tyr Tyr Ile Arg 625 630
635 640 His Tyr Gly Asn Ala Lys Asn Phe Trp Thr Gln Lys Ile Ser Glu
Ile 645 650 655 Gly Gly Ser Thr Arg Ser Phe Glu Val Leu Gly Thr Thr
Pro 660 665 670 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 16 <211> LENGTH: 2013 <212> TYPE: DNA
<213> ORGANISM: Pseudomonas aeruginosa <400> SEQUENCE:
16 atgtcacgtt ccgcattcac cgcgctcttg ctgtcctgcg tcctgctggc
gctctccatg 60 cctgccaggg ccgacgacct gccctaccgc ttcggcctgg
gcaaggcgga catcaccggc 120 gaagccgccg aagtcggcat gatgggttac
tcctccctcg aacagaagac cgccggcatc 180 cacatgcgcc agtgggcgcg
tgccttcgtg atcgaggaag cggccagcgg acgtcgcctg 240 gtctacgtca
acaccgacct ggggatgacc ttccaggccg tgcacctgaa ggtcctggcc 300
cggctcaagg cgaagtaccc cggtgtctac gacgagaaca acgtgatgct cgccgccacc
360 cacacccact ccggtccggg cggcttctcc cactacgcga tgtacaacct
gtcggtgctc 420 ggcttccagg aaaagacctt caacgccatc gtcgacggca
tcgtccgctc catcgagcgg 480 gcccaggcca ggttgcagcc cggccgcctg
ttctacggca gcggcgagct gcgcaacgcc 540 agccgcaacc gttcgctgct
gtcgcacctg aagaatccgg acatcgccgg ctacgaggat 600 ggcatcgacc
cgcagatgag cgtgctcagc ttcgtcgacg ccaacggcga gctggccggc 660
gcgatcagtt ggttcccggt gcacagcacc tcgatgacca acgccaatca cctgatctcc
720 ccggacaaca agggctacgc ctcctatcac tgggagcacg acgtcagccg
caagagcggt 780 ttcgtcgccg ccttcgccca gaccaatgcc ggcaacctgt
cgcccaacct gaacctgaag 840 cccggctccg gtcccttcga caacgagttc
gacaacaccc gcgagatcgg tctgcgccaa 900 ttcgccaagg cctacgagat
cgccggccag gcccaggagg aagtgctcgg cgaactggat 960 tcgcgcttcc
gtttcgtcga cttcacccgc ctgccgatcc gcccggagtt caccgacggc 1020
cagccgcgcc agttgtgcac cgcggccatc ggcaccagcc tggccgccgg tagcaccgaa
1080 gacggtccag gcccgctggg gctggaggaa ggcaacaatc cgttcctctc
ggcccttggc 1140 gggttgctca ccggcgtgcc gccgcaggaa ctggtgcaat
gccaggcgga aaagaccatc 1200 ctcgccgaca ccggcaacaa gaaaccctac
ccctggacgc cgacggtgct gccgatccag 1260 atgttccgca tcggccagtt
ggaactgctc ggcgcccccg ccgagttcac cgtgatggcc 1320 ggggtgcgga
tccgccgcgc ggtgcaggcg gccagcgaag cggccggtat ccgccatgtg 1380
gtcttcaatg gctacgcgaa tgcctatgcc agctacgtca ccacccgcga ggaatacgcc
1440 gcccaggaat acgaaggcgg ctcgaccctc tacggcccct ggacccaggc
cgcctaccag 1500 cagttgttcg tcgacatggc ggtggcgctg cgcgaacgcc
tgccggtgga aacctcggcg 1560 atagcgccgg acctgtcctg ctgccagatg
aacttccaga ccggagtagt ggccgatgat 1620 ccctatatcg gcaagtcctt
cggcgacgtg ttgcaacaac ccagggaaag ttatcgcatc 1680 ggcgacaagg
tgaccgtcgc tttcgtgacc ggacatccga agaatgactt gcgcaccgag 1740
aagactttcc tggaagtggt gaatatcggc aaggatggca aacagacgcc cgtgaccgtt
1800 gccaccgata atgactggga tacccaatac cgctgggaga gagtgggtat
atctgcctcg 1860 aaagcgacta tcagctggtc cattccacca gggaccgagc
ccggccatta ctacatcagg 1920 cactatggca acgcgaagaa cttctggacc
cagaagatca gcgaaatcgg cggctcgacc 1980 cgctccttcg aggtgctcgg
caccactccc tag 2013 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 17 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer 382U derived from Pseudomonas
aeruginosa <400> SEQUENCE: 17 ggcttctccc actacgcgat g 21
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 18
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer 884L derived from Pseudomonas aeruginosa
<400> SEQUENCE: 18 cgaattggcg cagaccgatc t 21
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